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Novel antimicrobial bolisin peptides

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Title: Novel antimicrobial bolisin peptides.
Abstract: The invention relates to antibiotically effective peptides which are prepared for medical and commercial use by using biotechnological methods and chemical synthesis. The antibiotically effective peptides can be used in a suitable galenic formulation as medicaments/animal medicaments, food additives or as preservatives for the prevention of microbial contaminations of cosmetics, medical products and requisites. ...


USPTO Applicaton #: #20090149391 - Class: 514 13 (USPTO) - 06/11/09 - Class 514 
Drug, Bio-affecting And Body Treating Compositions > Designated Organic Active Ingredient Containing (doai) >Peptide Containing (e.g., Protein, Peptones, Fibrinogen, Etc.) Doai >Cyclopeptides >16 To 24 Peptide Repeating Units In Known Peptide Chain

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The Patent Description & Claims data below is from USPTO Patent Application 20090149391, Novel antimicrobial bolisin peptides.

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US 20090149390 A1 20090611 1 76 1 243 PRT Homo sapiens MISC_FEATURE (8)..(8) Xaa is tryptophan or an oxidant resistant amino acid 1 Asp Glu Pro Pro Gln Ser Gln Xaa Asp Arg Val Lys Asp Leu Ala Thr 1 5 10 15 Val Tyr Val Asp Val Leu Lys Asp Ser Gly Arg Asp Tyr Val Ser Gln 20 25 30 Phe Glu Gly Ser Ala Leu Gly Lys Gln Leu Asn Leu Lys Leu Leu Asp 35 40 45 Asn Xaa Asp Ser Val Thr Ser Thr Phe Ser Lys Leu Arg Glu Gln Leu 50 55 60 Gly Pro Val Thr Gln Glu Phe Xaa Asp Asn Leu Glu Lys Glu Thr Glu 65 70 75 80 Gly Leu Arg Gln Glu Met Ser Lys Asp Leu Glu Glu Val Lys Ala Lys 85 90 95 Val Gln Pro Tyr Leu Asp Asp Phe Gln Lys Lys Xaa Gln Glu Glu Met 100 105 110 Glu Leu Tyr Arg Gln Lys Val Glu Pro Leu Arg Ala Glu Leu Gln Glu 115 120 125 Gly Ala Arg Gln Lys Leu His Glu Leu Gln Glu Lys Leu Ser Pro Leu 130 135 140 Gly Glu Glu Met Arg Asp Arg Ala Arg Ala His Val Asp Ala Leu Arg 145 150 155 160 Thr His Leu Ala Pro Tyr Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala 165 170 175 Arg Leu Glu Ala Leu Lys Glu Asn Gly Gly Ala Arg Leu Ala Glu Tyr 180 185 190 His Ala Lys Ala Thr Glu His Leu Ser Thr Leu Ser Glu Lys Ala Lys 195 200 205 Pro Ala Leu Glu Asp Leu Arg Gln Gly Leu Leu Pro Val Leu Glu Ser 210 215 220 Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu 225 230 235 240 Asn Thr Gln 2 267 PRT Homo sapiens MISC_FEATURE (22)..(22) Xaa is tryptophan or an oxidant resistant amino acid 2 Met Lys Ala Ala Val Leu Thr Leu Ala Val Leu Phe Leu Thr Gly Ser 1 5 10 15 Gln Ala Arg His Phe Xaa Gln Gln Asp Glu Pro Pro Gln Ser Pro Xaa 20 25 30 Asp Arg Val Lys Asp Leu Ala Thr Val Tyr Val Asp Val Leu Lys Asp 35 40 45 Ser Gly Arg Asp Tyr Val Ser Gln Phe Glu Gly Ser Ala Leu Gly Lys 50 55 60 Gln Leu Asn Leu Lys Leu Leu Asp Asn Xaa Asp Ser Val Thr Ser Thr 65 70 75 80 Phe Ser Lys Leu Arg Glu Gln Leu Gly Pro Val Thr Gln Glu Phe Xaa 85 90 95 Asp Asn Leu Glu Lys Glu Thr Glu Gly Leu Arg Gln Glu Met Ser Lys 100 105 110 Asp Leu Glu Glu Val Lys Ala Lys Val Gln Pro Tyr Leu Asp Asp Phe 115 120 125 Gln Lys Lys Xaa Gln Glu Glu Met Glu Leu Tyr Arg Gln Lys Val Glu 130 135 140 Pro Leu Arg Ala Glu Leu Gln Glu Gly Ala Arg Gln Lys Leu His Glu 145 150 155 160 Leu Gln Glu Lys Leu Ser Pro Leu Gly Glu Glu Met Arg Asp Arg Ala 165 170 175 Arg Ala His Val Asp Ala Leu Cys Thr His Leu Ala Pro Tyr Ser Asp 180 185 190 Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu Ala Leu Lys Glu Asn 195 200 205 Gly Gly Ala Arg Leu Ala Glu Tyr His Ala Lys Ala Thr Glu His Leu 210 215 220 Ser Thr Leu Ser Glu Lys Ala Lys Pro Ala Leu Glu Asp Leu Arg Gln 225 230 235 240 Gly Leu Leu Pro Val Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala 245 250 255 Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln 260 265 3 267 PRT Homo sapiens MISC_FEATURE (22)..(22) Xaa is tryptophan or an oxidant resistant amino acid 3 Met Lys Ala Ala Val Leu Thr Leu Ala Val Leu Phe Leu Thr Gly Ser 1 5 10 15 Gln Ala Arg His Phe Xaa Gln Gln Asp Glu Pro Pro Gln Ser Pro Xaa 20 25 30 Asp Arg Val Lys Asp Leu Ala Thr Val Tyr Val Asp Val Leu Lys Asp 35 40 45 Ser Gly Arg Asp Tyr Val Ser Gln Phe Glu Gly Ser Ala Leu Gly Lys 50 55 60 Gln Leu Asn Leu Lys Leu Leu Asp Asn Xaa Asp Ser Val Thr Ser Thr 65 70 75 80 Phe Ser Lys Leu Arg Glu Gln Leu Gly Pro Val Thr Gln Glu Phe Xaa 85 90 95 Asp Asn Leu Glu Lys Glu Thr Glu Gly Leu Arg Gln Glu Met Ser Lys 100 105 110 Asp Leu Glu Glu Val Lys Ala Lys Val Gln Pro Tyr Leu Asp Asp Phe 115 120 125 Gln Lys Lys Xaa Gln Glu Glu Met Glu Leu Tyr Arg Gln Lys Val Glu 130 135 140 Pro Leu Arg Ala Glu Leu Gln Glu Gly Ala Arg Gln Lys Leu His Glu 145 150 155 160 Leu Gln Glu Lys Leu Cys Pro Leu Gly Glu Glu Met Arg Asp Arg Ala 165 170 175 Arg Ala His Val Asp Ala Leu Cys Thr His Leu Ala Pro Tyr Ser Asp 180 185 190 Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu Ala Leu Lys Glu Asn 195 200 205 Gly Gly Ala Arg Leu Ala Glu Tyr His Ala Lys Ala Thr Glu His Leu 210 215 220 Ser Thr Leu Ser Glu Lys Ala Lys Pro Ala Leu Glu Asp Leu Arg Gln 225 230 235 240 Gly Leu Leu Pro Val Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala 245 250 255 Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln 260 265 4 18 PRT Homo sapiens MISC_FEATURE (2)..(2) X is an oxidant resistant amino acid 4 Asp Xaa Leu Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu Lys Glu 1 5 10 15 Ala Phe 5 18 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is an oxidant resistant amino acid 5 Asp Xaa Phe Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu Lys Glu 1 5 10 15 Ala Phe 6 18 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is an oxidant resistant amino acid 6 Asp Xaa Leu Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Phe Lys Glu 1 5 10 15 Ala Phe 7 18 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is an oxidant resistant amino acid 7 Asp Xaa Phe Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Phe Lys Glu 1 5 10 15 Ala Phe 8 18 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is an oxidant resistant amino acid 8 Asp Xaa Leu Lys Ala Phe Tyr Asp Lys Val Phe Glu Lys Phe Lys Glu 1 5 10 15 Phe Phe 9 18 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is an oxidant resistant amino acid 9 Asp Xaa Leu Lys Ala Phe Tyr Asp Lys Phe Phe Glu Lys Phe Lys Glu 1 5 10 15 Phe Phe 10 18 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is an oxidant resistant amino acid 10 Asp Xaa Phe Lys Ala Phe Tyr Asp Lys Phe Phe Glu Lys Phe Lys Glu 1 5 10 15 Phe Phe 11 18 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is an oxidant resistant amino acid 11 Asp Xaa Leu Lys Ala Phe Tyr Asp Lys Val Phe Glu Lys Phe Lys Glu 1 5 10 15 Ala Phe 12 18 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is an oxidant resistant acid 12 Asp Xaa Leu Lys Ala Phe Tyr Asp Lys Val Phe Glu Lys Phe Lys Glu 1 5 10 15 Ala Phe 13 18 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is an oxidant resistant amino acid 13 Asp Xaa Leu Lys Ala Phe Tyr Asp Lys Val Phe Glu Lys Leu Lys Glu 1 5 10 15 Phe Phe 14 18 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is an oxidant resistant amino acid 14 Asp Xaa Leu Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Phe Lys Glu 1 5 10 15 Phe Phe 15 18 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is an oxidant resistant amino acid 15 Asp Xaa Leu Lys Ala Phe Tyr Asp Lys Val Phe Glu Lys Phe Lys Glu 1 5 10 15 Phe Phe 16 18 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is an oxidant resistant amino acid 16 Glu Xaa Leu Lys Leu Phe Tyr Glu Lys Val Leu Glu Lys Phe Lys Glu 1 5 10 15 Ala Phe 17 18 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is an oxidant resistant amino acid 17 Glu Xaa Leu Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Phe Lys Glu 1 5 10 15 Ala Phe 18 18 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is an oxidant resistant amino acid 18 Glu Xaa Leu Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu Lys Glu 1 5 10 15 Phe Phe 19 18 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is an oxidant resistant amino acid 19 Glu Xaa Leu Lys Ala Phe Tyr Asp Lys Val Phe Glu Lys Phe Lys Glu 1 5 10 15 Ala Phe 20 18 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is an oxidant resistant amino acid 20 Glu Xaa Leu Lys Ala Phe Tyr Asp Lys Val Phe Glu Lys Leu Lys Glu 1 5 10 15 Phe Phe 21 18 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is an oxidant resistant amino acid 21 Glu Xaa Leu Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Phe Lys Glu 1 5 10 15 Phe Phe 22 18 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is an oxidant resistant amino acid 22 Glu Xaa Leu Lys Ala Phe Tyr Asp Lys Val Phe Glu Lys Phe Lys Glu 1 5 10 15 Phe Phe 23 18 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is an oxidant resistant amino acid 23 Asp Xaa Leu Lys Ala Leu Tyr Asp Lys Val Ala Glu Lys Leu Lys Glu 1 5 10 15 Ala Leu 24 18 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is an oxidant resistant amino acid 24 Asp Xaa Phe Lys Ala Phe Tyr Glu Lys Val Ala Glu Lys Leu Lys Glu 1 5 10 15 Phe Phe 25 18 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is an oxidant resistant amino acid 25 Asp Xaa Phe Lys Ala Phe Tyr Glu Lys Phe Phe Glu Lys Phe Lys Glu 1 5 10 15 Phe Phe 26 18 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is an oxidant resistant amino acid 26 Glu Xaa Leu Lys Ala Leu Tyr Glu Lys Val Ala Glu Lys Leu Lys Glu 1 5 10 15 Ala Leu 27 18 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is an oxidant resistant amino acid 27 Glu Xaa Leu Lys Ala Phe Tyr Glu Lys Val Ala Glu Lys Leu Lys Glu 1 5 10 15 Ala Phe 28 18 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is an oxidant resistant amino acid 28 Glu Xaa Phe Lys Ala Phe Tyr Glu Lys Val Ala Glu Lys Leu Lys Glu 1 5 10 15 Phe Phe 29 18 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is an oxidant resistant amino acid 29 Glu Xaa Leu Lys Ala Phe Tyr Glu Lys Val Phe Glu Lys Phe Lys Glu 1 5 10 15 Phe Phe 30 18 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is an oxidant resistant amino acid 30 Glu Xaa Leu Lys Ala Phe Tyr Glu Lys Phe Phe Glu Lys Phe Lys Glu 1 5 10 15 Phe Phe 31 18 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is an oxidant resistant amino acid 31 Glu Xaa Phe Lys Ala Phe Tyr Glu Lys Phe Phe Glu Lys Phe Lys Glu 1 5 10 15 Phe Phe 32 18 PRT Homo sapiens MISC_FEATURE (6)..(6) Xaa is tryptophan or an oxidant resistant amino acid 32 Asp Phe Leu Lys Ala Xaa Tyr Asp Lys Val Ala Glu Lys Leu Lys Glu 1 5 10 15 Ala Xaa 33 18 PRT Homo sapiens MISC_FEATURE (6)..(6) Xaa is tryptophan or an oxidant resistant amino acid 33 Glu Phe Leu Lys Ala Xaa Tyr Glu Lys Val Ala Glu Lys Leu Lys Glu 1 5 10 15 Ala Xaa 34 18 PRT Homo sapiens MISC_FEATURE (3)..(3) Xaa is tryptophan or an oxidant resistant amino acid 34 Asp Phe Xaa Lys Ala Xaa Tyr Asp Lys Val Ala Glu Lys Leu Lys Glu 1 5 10 15 Xaa Xaa 35 18 PRT Homo sapiens MISC_FEATURE (3)..(3) Xaa is tryptophan or oxidant resistant amino acid 35 Glu Phe Xaa Lys Ala Xaa Tyr Glu Lys Val Ala Glu Lys Leu Lys Glu 1 5 10 15 Xaa Xaa 36 18 PRT Homo sapiens MISC_FEATURE (13)..(13) Xaa is an oxidant resistant amino acid 36 Asp Lys Leu Lys Ala Phe Tyr Asp Lys Val Phe Glu Xaa Ala Lys Glu 1 5 10 15 Ala Phe 37 18 PRT Homo sapiens MISC_FEATURE (3)..(3) Xaa is an oxidant resistant amino acid 37 Asp Lys Xaa Lys Ala Val Tyr Asp Lys Phe Ala Glu Ala Phe Lys Glu 1 5 10 15 Phe Leu 38 18 PRT Homo sapiens MISC_FEATURE (13)..(13) Xaa is an oxidant resistant amino acid 38 Glu Lys Leu Lys Ala Phe Tyr Glu Lys Val Phe Glu Xaa Ala Lys Glu 1 5 10 15 Ala Phe 39 18 PRT Homo sapiens MISC_FEATURE (3)..(3) Xaa is an oxidant resistant amino acid 39 Glu Lys Xaa Lys Ala Val Tyr Glu Lys Phe Ala Glu Ala Phe Lys Glu 1 5 10 15 Phe Leu 40 18 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is an oxidant resistant amino acid 40 Asp Xaa Leu Lys Ala Phe Val Asp Lys Phe Ala Glu Lys Phe Lys Glu 1 5 10 15 Ala Tyr 41 18 PRT Homo sapiens MISC_FEATURE (3)..(3) Xaa is an oxidant resistant amino acid 41 Glu Lys Xaa Lys Ala Val Tyr Glu Lys Phe Ala Glu Ala Phe Lys Glu 1 5 10 15 Phe Leu 42 18 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is an oxidant resistant amino acid 42 Asp Xaa Leu Lys Ala Phe Val Tyr Asp Lys Val Phe Lys Leu Lys Glu 1 5 10 15 Phe Phe 43 18 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is an oxidant resistant amino acid 43 Glu Xaa Leu Lys Ala Phe Val Tyr Glu Lys Val Phe Lys Leu Lys Glu 1 5 10 15 Phe Phe 44 18 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is an oxidant resistant amino acid 44 Asp Xaa Leu Arg Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu Lys Glu 1 5 10 15 Ala Phe 45 18 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is an oxidant resistant amino acid 45 Glu Xaa Leu Arg Ala Phe Tyr Glu Lys Val Ala Glu Lys Leu Lys Glu 1 5 10 15 Ala Phe 46 18 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is an oxidant resistant amino acid 46 Asp Xaa Leu Lys Ala Phe Tyr Asp Arg Val Ala Glu Lys Leu Lys Glu 1 5 10 15 Ala Phe 47 18 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is an oxidant resistant amino acid 47 Glu Xaa Leu Lys Ala Phe Tyr Glu Arg Val Ala Glu Lys Leu Lys Glu 1 5 10 15 Ala Phe 48 18 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is an oxidant resistant amino acid 48 Asp Xaa Leu Lys Ala Phe Tyr Asp Lys Val Ala Glu Arg Leu Lys Glu 1 5 10 15 Ala Phe 49 18 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is an oxidant resistant amino acid 49 Glu Xaa Leu Lys Ala Phe Tyr Glu Lys Val Ala Glu Arg Leu Lys Glu 1 5 10 15 Ala Phe 50 18 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is an oxidant resistant amino acid 50 Asp Xaa Leu Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu Arg Glu 1 5 10 15 Ala Phe 51 18 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is an oxidant resistant amino acid 51 Glu Xaa Leu Lys Ala Phe Tyr Glu Lys Val Ala Glu Lys Leu Arg Glu 1 5 10 15 Ala Phe 52 18 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is an oxidant resistant amino acid 52 Asp Xaa Leu Lys Ala Phe Tyr Asp Arg Val Ala Glu Arg Leu Lys Glu 1 5 10 15 Ala Phe 53 18 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is an oxidant resistant amino acid 53 Glu Xaa Leu Lys Ala Phe Tyr Glu Arg Val Ala Glu Arg Leu Lys Glu 1 5 10 15 Ala Phe 54 18 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is an oxidant resistant amino acid 54 Asp Xaa Leu Arg Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu Arg Glu 1 5 10 15 Ala Phe 55 18 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is an oxidant resistant amino acid 55 Glu Xaa Leu Arg Ala Phe Tyr Glu Lys Val Ala Glu Lys Leu Arg Glu 1 5 10 15 Ala Phe 56 18 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is an oxidant resistant amino acid 56 Asp Xaa Leu Arg Ala Phe Tyr Asp Arg Val Ala Glu Lys Leu Lys Glu 1 5 10 15 Ala Phe 57 17 PRT Homo sapiens MISC_FEATURE (1)..(1) Xaa is an oxidant resistant amino acid 57 Xaa Leu Arg Ala Phe Tyr Glu Arg Val Ala Glu Lys Leu Lys Glu Ala 1 5 10 15 Phe 58 18 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is an oxidant resistant amino acid 58 Asp Xaa Leu Lys Ala Phe Tyr Asp Lys Val Ala Glu Arg Leu Arg Glu 1 5 10 15 Ala Phe 59 18 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is an oxidant resistant amino acid 59 Glu Xaa Leu Lys Ala Phe Tyr Glu Lys Val Ala Glu Arg Leu Arg Glu 1 5 10 15 Ala Phe 60 18 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is an oxidant resistant amino acid 60 Asp Xaa Leu Arg Ala Phe Tyr Asp Lys Val Ala Glu Arg Leu Lys Glu 1 5 10 15 Ala Phe 61 18 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is an oxidant resistant amino acid 61 Glu Xaa Leu Arg Ala Phe Tyr Glu Lys Val Ala Glu Arg Leu Lys Glu 1 5 10 15 Ala Phe 62 37 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is tryptophan or an oxidant resistant amino acid 62 Asp Xaa Leu Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu Lys Glu 1 5 10 15 Ala Phe Pro Asp Xaa Leu Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys 20 25 30 Leu Lys Glu Ala Phe 35 63 37 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is tryptophan or an oxidant resistant amino acid 63 Asp Xaa Leu Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu Lys Glu 1 5 10 15 Phe Phe Pro Asp Xaa Leu Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys 20 25 30 Leu Lys Glu Phe Phe 35 64 37 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is tryptophan or an oxidant resistant amino acid 64 Asp Xaa Phe Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu Lys Glu 1 5 10 15 Ala Phe Pro Asp Xaa Phe Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys 20 25 30 Leu Lys Glu Ala Phe 35 65 37 PRT Homo sapiens MISC_FEATURE (13)..(13) Xaa is tryptophan or an oxidant resistant amino acid 65 Asp Lys Leu Lys Ala Phe Tyr Asp Lys Val Phe Glu Xaa Ala Lys Glu 1 5 10 15 Ala Phe Pro Asp Lys Leu Lys Ala Phe Tyr Asp Lys Val Phe Glu Xaa 20 25 30 Leu Lys Glu Ala Phe 35 66 37 PRT Homo sapiens MISC_FEATURE (3)..(3) Xaa is tryptophan or an oxidant resistant amino acid 66 Asp Lys Xaa Lys Ala Val Tyr Asp Lys Phe Ala Glu Ala Phe Lys Glu 1 5 10 15 Phe Leu Pro Asp Lys Xaa Lys Ala Val Tyr Asp Lys Phe Ala Glu Ala 20 25 30 Phe Lys Glu Phe Leu 35 67 37 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is tryptophan or an oxidant resistant amino acid 67 Asp Xaa Phe Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Phe Lys Glu 1 5 10 15 Ala Phe Pro Asp Xaa Phe Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys 20 25 30 Phe Lys Glu Ala Phe 35 68 37 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is tryptophan or an oxidant resistant amino acid 68 Asp Xaa Leu Lys Ala Phe Val Tyr Asp Lys Val Phe Lys Leu Lys Glu 1 5 10 15 Phe Phe Pro Asp Xaa Leu Lys Ala Phe Val Tyr Asp Lys Val Phe Lys 20 25 30 Leu Lys Glu Phe Phe 35 69 37 PRT Homo sapiens MISC_FEATURE (2)..(2) Xaa is tryptophan or an oxidant resistant amino acid 69 Asp Xaa Leu Lys Ala Phe Tyr Asp Lys Phe Ala Glu Lys Phe Lys Glu 1 5 10 15 Phe Phe Pro Asp Xaa Leu Lys Ala Phe Tyr Asp Lys Phe Ala Glu Lys 20 25 30 Phe Lys Glu Phe Phe 35 70 10 PRT Homo sapiens 70 Asp Glu Pro Pro Gln Ser Pro Trp Asp Arg 1 5 10 71 14 PRT Homo sapiens 71 Leu Leu Asp Asn Trp Asp Ser Val Thr Ser Thr Phe Ser Lys 1 5 10 72 16 PRT Homo sapiens 72 Glu Gln Leu Gly Pro Val Thr Gln Glu Phe Trp Asp Asn Leu Glu Lys 1 5 10 15 73 9 PRT Homo sapiens 73 Trp Gln Glu Glu Met Glu Leu Tyr Arg 1 5 74 9 PRT Homo sapiens 74 Trp Gln Glu Glu Met Glu Leu Tyr Arg 1 5 75 9 PRT Homo sapiens 75 Leu Ser Pro Leu Gly Glu Glu Met Arg 1 5 76 18 PRT Homo sapiens 76 Asp Trp Phe Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Phe Lys Glu 1 5 10 15 Ala Phe US 20090149391 A1 20090611 US 12289180 20081022 12 DE 01129094.7 20011207 20060101 A
A
61 K 38 10 F I 20090611 US B H
20060101 A
C
07 K 7 08 L I 20090611 US B H
20060101 A
C
12 P 21 00 L I 20090611 US B H
20060101 A
A
61 P 31 04 L I 20090611 US B H
US 514 13 530326 514 14 435 711 435 712 Novel antimicrobial bolisin peptides US 10497909 00 20040607 US 7449545 A WO PCT/EP02/13724 20021204 US 12289180 Liepke Cornelia
Hannover DE
omitted DE
Baxmann Susann
Lehrte DE
omitted DE
Adermann Knut
Hannover DE
omitted DE
JACOBSON HOLMAN PLLC
400 SEVENTH STREET N.W., SUITE 600 WASHINGTON DC 20004 US
IPF Pharmaceuticals GmbH. 03

The invention relates to antibiotically effective peptides which are prepared for medical and commercial use by using biotechnological methods and chemical synthesis. The antibiotically effective peptides can be used in a suitable galenic formulation as medicaments/animal medicaments, food additives or as preservatives for the prevention of microbial contaminations of cosmetics, medical products and requisites.

The invention relates to peptides having a highly effective antimicrobial activity, processes for the preparation thereof, and the use thereof.

It is known that naturally occurring peptides may be antibiotically effective. Thus, WO 97/35877 describes the occurrence and purification of antibiotic peptides from cow's milk, and PCT/EP 01/06518 describes the recovery of antimicrobial peptides from human placenta extract and from bovine thymus extract, and their application.

One of the peptides purified from bovine thymus extract is bolisin (PCT/EP 01/06518, SEQ ID No. 5). Bolisin is a fragment of the bovine mitochondrial proteolipid comprising 17 amino acids, and it inhibits the growth of pathogenic microorganisms in low-salt nutrient media.

It is an object of the invention to provide bolisin-based peptides which have a particularly high specific activity under different conditions. It is also an object of the invention to provide peptides which inhibit the growth of microorganisms in environments with physiological salt concentrations and at the same time have as low as possible a hemolytic activity.

Surprisingly, this object is achieved by providing peptides of the sequence


R1-ZX3X3X1BX3RX2X3X4BRX2BX3X3B—R2,

wherein

R1 represents an amino group (NH2), an amino acid or a peptide having up to 10 amino acids, and R2 represents COOH, CONH2, an amino acid or a peptide having up to 10 amino acids;

Z represents an aromatic amino acid (W, Y, F), preferably tyrosine (Y) or an amino acid mono- or polyhalogenated at the aromatic moiety, preferably tyrosine (Y);

X1 represents arginine (R) or an aromatic amino acid (W, Y, F), preferably tryptophan (W) or an amino acid mono- or polyhalogenated at the aromatic moiety, preferably tryptophan;

X2 represents serine (S), a basic amino acid (R, K), preferably arginine (R), or an aromatic amino acid (W, Y, F), preferably tryptophan (W) or an amino acid mono- or polyhalogenated at the aromatic moiety, preferably tryptophan;

X3 represents threonine (T), a hydrophobic amino acid (I, V, A, L) or arginine (R);

X4 represents aspartic acid (D), proline (P), a basic amino acid (K, R), preferably arginine (R), or an aromatic amino acid (W, Y, F), preferably tryptophan (W) or an amino acid mono- or polyhalogenated at the aromatic moiety, preferably tryptophan; and

B represents a basic amino acid (K, R).

The invention also relates to derivatives and/or fragments of the peptides according to the invention having antimicrobial activity, especially the derivatives and/or fragments which are amidated, acylated, acetylated, alkylated, sulfated, phosphorylated, halogenated, especially halogenated at aromatic amino acid side chains, glycosylated, oxidized, modified by esterification or lactone formation, and/or cyclized. Halogenated peptides are preferably halogenated at aromatic residues of amino acid side chains. The amino acids are designated with the one-letter code.

Preferably, the peptides according to the invention have the following sequences:

R1-ZIX3X1BX3RX2ADBRX2BALB-R2 (Sequence ID Nos.: 2, 4, 8, 12, 13, 15) R1-ZIX3X1BX3RX2APBRX2BALB-R2 (Sequence ID No.: 11) R1-ZIX3X1BX3RX2ABBRX2BALB-R2 (Sequence ID Nos.: 1, 3, 5, 6, 7, 9, 10, 18, 21-24, 28, 29, 31, 32, 35, 38, 41, 45, 47, 48, 51) R1-ZIX3X1BX3RX2AZBRX2BALB-R2 (Sequence ID Nos.: 14, 16, 17, 19, 20, 25-27, 30, 33, 34, 36, 37, 42-44, 46, 49, 50, 52, 53, 54) R1-ZIX3X1BX3RX2TZBRX2BALB-R2 (Sequence ID No.: 55)

wherein X3 represents a hydrophobic amino acid (I, V) or arginine (R), and R1, R2, Z, X1, X2 and B have the meanings as stated above. Preferred peptides of the invention are listed in Table 1. The invention also relates to their derivatives and/or fragments having antimicrobial activity, especially the derivatives and/or fragments which are amidated, acylated, acetylated, alkylated, sulfated, phosphorylated, halogenated, glycosylated, oxidized, modified by esterification or lactone formation, and/or cyclized.

The invention comprises the peptides modified by esterification or lactone formation as well as the peptides according to the invention which have been reacted at a free amino group with activated polyoxyalkylene glycol or a conjugate of a polyoxyalkylene glycol and, for example, a fatty acid. Preferred are fatty acids having a chain length of from 12 to 18 carbon atoms and polyoxyethylene as the polyoxyalkylene glycol having a chain length of from 2 to 100. More preferred are activated polysorbate esters and their derivatives. The peptide conjugates are prepared for improving the pharmacological properties of the peptides.

In addition to the derivatives mentioned, the peptides may also comprise the corresponding D-amino acids instead of the natural L-amino acids, as well as iminoamino acids and rare amino acids, such as hydroxylysine, homoserine and ornithine. Further, the invention comprises the retro, inverso and retro-inverso peptides. “Retro peptides” means the peptides according to the invention having a reverse amino acid sequence, “inverso peptides” means the peptides according to the invention which consist of D-amino acids, and “retro-inverso peptides” means those peptides which have a reverse amino acid sequence formed from D-amino acids.

The invention also relates to peptide mimetics of the peptides according to the invention. These are characterized by a modification of one or more peptide bonds, for example, by a reverse peptide bond or by an ester bond.

The bolisin analogues according to the invention are cationic peptides having antibiotic activity. The bolisin analogues contain at least 9 and at most 37 amino acid residues, preferably not more than 20 amino acid residues.

The peptide analogues of bolisin according to the invention have a clearly increased antimicrobial activity. The peptides described in the present invention also effectively inhibit the growth of pathogenic germs in nutrient media which have a physiological salt concentration and therefore, in contrast to the naturally occurring bolisin, can be used for a wide variety of applications. Human pathogenic germs with antibiotic resistance are also killed at low peptide concentrations.

As the fragments, there may be used, in particular, N-terminally or C-terminally truncated peptides, but also peptides in which individual amino acids are deleted, preferably not more than 10% of the amino acids. The invention relates to these fragments to the extent where they have antimicrobial properties. “Antimicrobial properties” of the fragment means that the fragment has at least 50% of the activity of the parent peptide, i.e., at most requires double the inhibitory concentration. The test microorganisms employed are either Staphylococcus aureus or Escherichia coli, depending on against which microorganism the peptide exhibits a better activity. The microorganisms are characterized as follows:

Staphylococcus aureus: Gram-positive cocci, ATCC 25923;

Escherichia coli: Gram-negative rods, ATCC 25922.

The invention also comprises nucleic acids which code for the peptides according to the invention, and vectors and plasmids which contain such nucleic acids.

The invention also relates to a medicament which contains one or more peptides according to the invention.

The peptides according to the invention are suitable for the treatment of diseases caused by misplaced microbial colonizations, such as infections, inflammations, microbially induced tumors, microbially caused degenerative diseases, diarrheic diseases, colics, deviations in the oral, intestinal and vaginal floras, caries, sepsis, toxic shock conditions. The misplaced microbial colonization may be caused, for example, by bacteria, fungi, yeasts, protists, viruses, mycoplasmas, filariae and/or plasmodiums. They may be employed in both acute and chronic diseases of humans and animals.

The peptides according to the invention are preferably used in medicinal formulations. The medicinal formulation contains one or more of the peptides according to the invention, or a physiologically acceptable salt of the peptides. Medicinal formulations can contain pharmaceutically usual auxiliary agents which contribute, for example, to the solubility, stability or sterility of the medicament or increase the efficiency of uptake into the body. Further, other antibiotic substances may be contained, for example, from the group of penicillins, cephalosporins, carbacephems, cephamycins, carbapenems, quinolones, tetracyclines, aminoglycosides, macrolides, glycopeptides, chloroamphenicols, glycylcyclines, licosamides, fluoroquinolones and peptide antibiotics.

The form and composition of the medicament which contains the peptides depends on the route of administration. Preferably, galenic formulations and application forms are selected in which the peptides arrive at the target site in a non-degraded condition. The peptides can be administered enterally, parenterally, intranasally, orally and by inhalation. Preferably, the peptides are packaged with an injection preparation either as a solution or as a lyophilizate for dissolution immediately before use. The peptides can be, for example, injected intravenously, injected or implanted intraperitoneally, injected or implanted subcutaneously, injected intradermally, injected or implanted intramuscularly, injected intrathecally, inhaled (aerosol, spray) or applied topically (e.g., creams, ointments, eyedrops, eardrops, shampoos). Bolisin analogues can be administered locally as injection, drops, spray, tablets, suppositories, cream, ointments, gel etc. It is possible to perform the administration as a bolus or repeatedly over a period of time.

The dose of the peptides to be administered depends on the indication and the application of particular derivatives. Preferably, the peptides according to the invention are employed in amounts of from 0.1 to 100 mg/kg of body weight.

The invention also relates to a diagnostic agent which contains peptides or nucleic acids according to the invention.

The peptides according to the invention may also be used as additives to food-stuffs. They may be used in the preparation of foodstuffs as auxiliary agents, especially for foodstuffs which are prepared by fermentation and other bacterial processes. The preparations according to the patent are natural preservatives. Further possibilities of the application of the peptides according to the invention are the use as disinfectants for all situations in which microorganisms are not desired. Examples thereof are the application for the surface disinfection or as a coating of medicinal products, air filters and requisites, such as clothing and jewels. “Medicinal products” means all medical auxiliaries which are applied to the patient, such as implants, prostheses, stents, tubes, cannulas, catheters, synthetic vascular systems, artificial heart valves and needles. The term “medicinal products” further includes containers for the storage of blood or blood components as well as containers for organ shipping etc. Further, it is possible to use the peptides in water processing, or as a component of shampoos and soaps, as food additives, cosmetic preservatives, additives for the preservation of cell culture media etc. and as a plant protection agent.

EXAMPLES Chemical Synthesis of Bolisin Analogues

The peptide synthesis of bolisin analogues is based on the standard method of solid-phase Fmoc strategy. For the automated synthesis, an ABI 433 A Synthesizer (Applied Biosystems, Weiterstadt, Germany) was used. An Fmoc-Lys(boc) Wang resin served as the solid phase. During the peptide synthesis, the individual coupling steps were checked to ensure that each amino acid was incorporated in a high yield. Peptides were cleaved from the resin with 94% trifluoroacetic acid (TFA), 3% ethanedithiol and 3% water and subsequently purified by chromatography.

For the chromatographic purification, reverse phase (RP) HPLC was applied. A preparative RP C18 column (PrepPAK 500 C18, 47×300 mm) served as the solid phase, and acetonitrile served as eluant (mobile solvent A: 0.07% TFA in water; mobile solvent B: 80% acetonitrile/0.07% TFA in water). At a flow rate of 40 ml/min, the peptides were eluted from the solid phase by the gradient method (increment of 0.5% mobile solvent B per min in the eluant) and detected at a wavelength of 215 nm.

The synthetic peptides were analyzed by mass spectrometry. The purity of the peptides was checked by capillary zone gel electrophoresis and analytical reverse-phase HPLC (purity ≧95%). The amino acid sequence of the peptides was verified by automated Edman degradation.

The amino acid sequences of the synthesized bolisin analogues according to the invention are stated in Table 1 using the one-letter code.

TABLE 1 Name SEQ ID No. Sequence BOL-6 1 YIVYKIRSARKRSKALK BOL-7 2 YIVYKIRSADKRRKALK BOL-11 3 YIVYKRRSARKRSKALK BOL-12 4 YIVYKRRSADKRRKALK BOL-13 5 YIVYKIRSARKRRKALK BOL-14 6 YIRYKIRSARKRRKALK BOL-15 7 YIVYKRRSARKRRKALK BOL-16 8 YIRYKRRSADKRRKALK BOL-17 9 YIRYKIRSARKRRKALK BOL-18 10 YIRYKRRSARKRRKALK BOL-21 11 YIVYKIRSAPKRSKALK BOL-25 12 YIVWKIRSADKRSKALK BOL-27 13 YIVYKIRWADKRSKALK BOL-29 14 YIVYKIRSAWKRSKALK BOL-30 15 YIVYKIRSADKRWKALK BOL-44 16 YIVYKIRSAWKRRKALK BOL-45 17 YIVYKIRSAWKRWKALK BOL-46 18 YIVYKIRSARKRWKALK BOL-47 19 YIVYKIRWAWKRSKALK BOL-48 20 YIVYKIRWAWKRRKALK BOL-49 21 YIVYKIRWARKRSKALK BOL-50 22 YIVYKIRWARKRRKALK BOL-51 23 YIVYKIRWARKRWKALK BOL-52 24 YIVYKIRRARKRRKALK BOL-53 25 YIVYKIRRAWKRSKALK BOL-54 26 YIVYKIRRAWKRRKALK BOL-55 27 YIVYKIRRAWKRWKALK BOL-56 28 YIVYKIRSAKKRKKALK BOL-57 29 YIVYKIRSAKKRWKALK BOL-58 30 YIVYKIRSAWKRKKALK BOL-59 31 YIVYKIRWAKKRKKALK BOL-60 32 YIVYKIRWAKKRWKALK BOL-61 33 YIVYKIRWAWKRKKALK BOL-67 34 YIVYKIRSAWKRWKAL BOL-68 35 YIVYKIRSARKRWKAL BOL-69 36 YIVYKIRSAWKRRK BOL-70 37 YIVYKIRSAWKRWK BOL-71 38 YIVYKIRSARKRWK BOL-72 39 YIVYKIRWARKRRKAL BOL-73 40 YIVYKIRWARKRRK BOL-74 41 YIVWKIRSARKRRKALK BOL-75 42 YIVWKIRSAWKRSKALK BOL-79 43 YIVRKIRSAWKRWKALK BOL-80 44 YIVRKIRSAWKRRKALK BOL-81 45 YIVWKIRSARKRWKALK BOL-82 46 YIVWKIRSAWKRWKALK BOL-83 47 YIVRKIRSARKRRKALK BOL-84 48 KYIVYKIRSARKRRKALK BOL-85 49 KYIVYKIRSAWKRSKALK BOL-86 50 KYIVYKIRSAWKRRKALK BOL-87 52 KYIVYKIRWARKRRKALK BOL-91 52 YIVYKIRFAFKRSKAL BOL-92 53 YIVYKIRFAFKRRKAL BOL-93 54 YIVYKIRWAWKRSKAL BOL-97 55 YIVYKIRWTWKRSKAL BOL-103 56 KLAKRRKWAWRIKYVIY

Examination of the Antimicrobial Effectiveness of Bolisin Analogues

The bolisin analogues of the present invention were examined for their suitability as antibiotic therapeutical agents by means of various biological tests. Thus, the chemically synthesized peptides were examined for their antimicrobial effectiveness against three Gram-positive and three Gram-negative bacterial strains as well as against a yeast.

The following microorganisms were used:

Gram-positive: Staphylococcus aureus ATCC 25923 Enterococcus faecalis ATCC 51299 (vancomycin-resistant) Streptococcus pneumoniae DSM 11865 (penicillin-resistant) Gram-negative: Escherichia coli ATCC 25922 Pseudomonas aeruginosa ATCC 9027 Klebsiella pneumoniae ATCC 10031 Yeast: Candida albicans ATCC 10231

The antibiotic activity of bolisin analogues was examined by determining the minimum inhibition concentration. The minimum inhibition concentration of an analogue designates the lowest peptide concentration which is required to inhibit the growth of microorganisms completely after an incubation time of 18±2 h. This method allows to obtain a quantitative result about the antimicrobial potency of the peptides. These examinations were performed by analogy with the recommendations (M7-A3) edited by the NCCLS (National Committee for Clinical Laboratory Standards) by applying a dilution test in a liquid medium using chemically synthesized peptides (peptide sequences according to claims 1 to 3). Mueller Hinton Broth was used as the test medium according to the NCCLS recommendations unless indicated otherwise. Table 2 shows the antimicrobial activity of the peptide analogues, which is clearly improved as compared to the parent peptide bolisin. The minimum inhibition concentrations are stated in [μg/ml].

TABLE 2 Micro- organisms 1 2 3 4 5 6 7 Bolisin* >300 >300 >300 >300 >300   >300 nd Bolisin >300 >300 >300 >300 >300   >300 >300   Bolisin** 25 300 300 6.25 150 150 nd BOL-6* 37.5 200 150 37.5     (9.375) 37.5 nd BOL-7* 75 >300 >300 150  (75) 300 nd BOL-11* 37.5 >300 >300 37.5 (150) 150 nd BOL-12* 18.75 >300 >300 18.75 (300) 200 nd BOL-13* 18.75 75 150 18.75     (9.375) 18.75 nd BOL-14* 50 >300 >300 37.5 (150) 75 nd BOL-15* 18.75 >300 >300 18.75 (150) 75 nd BOL-16* 100 >300 >300 75 (300) 150 nd BOL-17* 37.5 200 300 37.5 200 9.375 nd BOL-18* 18.75 >300 >300 18.75 300 37.5 nd BOL-21* 75 >300 >300 150 (150) 200 nd BOL-25* 150 >300 >300 150 (150) 200 nd BOL-27* 75 300 300 75 (150) 150 nd BOL-29* 9.375 75 150 9.375    (18.75) 50 nd BOL-30* 100 >300 >300 150 (300) 300 nd BOL-44 150 >300 200 200  (75) 75   37.5 BOL-45 18.75 100 100 12.5  (25) 4.7    18.75 BOL-46 300 200 200 200 (100) 150 150 BOL-47 18.75 25 18.75 18.75  (25) 9.375    18.75 BOL-48 9.375 12.5 9.375 18.75 200 4.7     9.375    (6.5) BOL-49 75 25 18.75 50   (12.5) 18.75    18.75 BOL-50 37.5 25 18.75 50    (6.25) 9.375     9.375 BOL-51 37.5 25 12.5 50 300 18.75    (18.75)  (25) BOL-52 300 200 200 >300   (37.5) 75   (37.5) BOL-53 150 200 200 200  (75) 100   (37.5) BOL-54 200 100 75 100  (25) 18.75    18.75 BOL-55 12.5 50 37.5 12.5     (9.375) 3.125     9.375 BOL-56 >300 >300 >300 >300 (200) 300 (300) BOL-57 >300 >300 >300 >300 (200) 300 300 BOL-58 >300 >300 300 >300 (100) 100  (75) BOL-59 50 50 50 150    (18.75) 18.75     (9.375) BOL-60 50 25 50 100    (18.75) 18.75    (18.75) BOL-61 37.5 25 25 25 300 18.75     9.375  (75) BOL-67 25 100 100 25  (50) 37.5    18.75 BOL-68 200 200 300 200 (200) 300 200 BOL-69 300 150 200 150 (200) 300   37.5 BOL-70 100 150 150 100 (100) 75   37.5 BOL-71 200 200 300 300 (200) 300 300 BOL-72 75 25 18.75 100   (12.5) 18.75    18.75 BOL-73 37.5 25 18.75 50     (9.375) 18.75     9.375 BOL-74 300 150 150 300   (12.5) 18.75  75 BOL-75 150 200 150 75   (37.5) 75   37.5 BOL-79 9.375 >300 300 3.125    (4.7) 2.35    4.7 BOL-80 >300 >300 >300 >300    (18.75) 150   37.5 BOL-81 300 150 75 300    (18.75) 25  75 BOL-82 18.75 75 37.5 18.75     (9.375) 12.5     9.375 BOL-83 >300 >300 >300 >300 >300   >300 300 BOL-84 >300 >300 300 >300  (75) 75   37.5 BOL-85 >300 300 300 >300   (37.5) 150  75 BOL-86 75 200 150 150    (18.75) 50    18.75 BOL-87 9.375 37.5 9.375 18.75     (9.375) 9.375     9.375 BOL-91 25 37.5 37.5 25 (150) 18.75 nd BOL-92 18.75 18.75 25 18.75   (37.5) 12.5 nd BOL-93 25 37.5 25 25   (37.5) 25 nd BOL-97 12.5 18.75 18.75 18.75   (37.5) 18.75 nd BOL-103 18.75 37.5 37.5 18.75    (9.4) 18.75 nd *Test medium: half-concentrated Mueller Hinton Broth **Test medium: 3 g/l Tryptic Soy Broth (low ion strength medium)

Values in parentheses designate a 75% inhibition of the growth of the microorganisms at the peptide concentration stated. A complete growth inhibition could not be achieved in this case. For determining the minimum inhibition concentration, the following microorganisms were used:

1. Staphylococcus aureus ATCC 25923

2. Enterococcus faecalis ATCC 51299 (vancomycin-resistant)

3. Streptococcus pneumoniae DSM 11865 (penicillin-resistant)

4. Escherichia coli ATCC 25922

5. Pseudomonas aeruginosa ATCC 9027

6. Klebsiella pneumoniae ATCC 10031

7. Candida albicans ATCC 10231

The peptides according to the invention effectively inhibit the growth of both Gram-positive and Gram-negative bacteria as well as of yeasts. Human pathogens, such as Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae, were effectively killed. Of particular importance is the very effective growth inhibition of bacteria having an existing antibiotic-resistance, such as Streptococcus pneumoniae (penicillin-resistant) and Enterococcus faecalis (vancomycin-resistant). The potency of action of the bolisin analogues is manifoldly better than that of the parent peptide bolisin.

Examination of the Effectiveness at Increased Salt Concentration

Also for these examinations, the minimum inhibition concentration of the peptides against Gram-positive and Gram-negative microorganisms (microorganisms 1-6) was determined. Tryptic Soy Broth (3 g/l) with an addition of salt in physiological concentration (150 mM sodium chloride) was used as the test medium. Table 3 shows the antimicrobial activity of the peptide analogues in a salt-containing medium, which is also significantly improved over that of the parent peptide bolisin.

TABLE 3 Micro- organisms 1 2 3 4 6 Bolisin >300   >300   >300   >300 >300   BOL-44 200 (300) >300   150 >300   BOL-45   37.5 200 200 75    4.7 BOL-46 >300   >300   >300   >300 >300   BOL-47    18.75  50   37.5 25     9.375 BOL-48    18.75    18.75  25 9.375 9.375 b BOL-49  75  75 100 50  75 BOL-50   37.5  50  50 37.5   37.5 BOL-51  75   37.5   37.5 37.5  25 BOL-52 >300   >300   >300   >300 (300) BOL-53 300 >300   >300 200 >300   BOL-54 300 300 300 200 (200) BOL-55    18.75 150 150 b 12.5    4.7 BOL-56 >300   >300   >300   >300 >300   BOL-57 >300   >300   >300   >300 >300   BOL-58 >300   >300   >300   >300 >300   BOL-59  75 200 200 100 150 BOL-60 100  75  75 75  50 BOL-61    18.75  25   37.5 75    6.25 BOL-67   37.5 150 150 75 9.375 BOL-68 >300   >300   >300   >300 >300   BOL-69 300 >300   >300   150 >300   BOL-70 150 200 300 100 100 BOL-71 >300   (300) >300   >300 >300   BOL-72  75  50  50 75  75 BOL-73   37.5   37.5  75 b 50 150 BOL-74 >300   300 >300   >300  75 BOL-75 300 300 b 300 200 300 BOL-79    4.7 >300   >300   4.7   <=1.56 BOL-80 >300   >300   >300   >300 >300   BOL-81 (300) 200  (75) >300  50 BOL-82    18.75  75  75 9.375    6.25 BOL-83 >300   >300   >300   >300 >300   BOL-84 >300   >300   >300   >300 300 BOL-85 >300   >300   >300   >300 >300   BOL-86 (300) >300   (300) >300 200 BOL-87   37.5   37.5   37.5 25   37.5

Values in parentheses designate a 75% inhibition of the growth of the microorganisms at the peptide concentration stated. A complete growth inhibition could not be achieved in this case.

Examination of the cytotoxic activity of bolisin analogues

Bolisin analogues having a good antimicrobial activity against at least one of the seven strains of microorganisms tested (MIC ≦37.5 μg/ml) were selected for checking their cytotoxic activity.

In many cases, the ability of antimicrobial peptides to deposit on plasma membranes and permeabilize them is considered the mechanism of action of these substances. It is desirable that bacterial membranes are selectively damaged, whereas the cells of the host should remain unaffected. As a simple model for the examination of the cytotoxic effect of a peptide on eukaryotic cells, the hemolysis test is used in which the lysing and thus toxic activity of the peptides against red blood cells (erythrocytes) is examined. The procedure for determining the hemolytic activity of the peptides was performed by analogy with Helmerhorst et al. (Helmerhorst et al., 1999, FEBS Lett. 449: 105-110).

Erythrocytes were isolated from the citrate-containing whole blood of a healthy subject by centrifugation (1500×g, 20° C., 10 min) and diluted 200 fold with test medium. Peptides were added at various concentrations to a 96 well microtitration plate with V-shaped bottoms and incubated with the diluted erythrocyte suspension at 37° C. for 1 h. The erythrocytes were separated by a subsequent centrifugation (1000×g for 5 min). The supernatants colored from released hemoglobin were transferred to a 96 well microtitration plate with flat bottoms, and its absorption was determined at a wavelength of λ=450 nm in a microtitration plate reader. Incubation with a 1% solution of the surfactant Tween-20 served as a reference value for 100% hemolysis. Erythrocytes incubated only with test medium served as the negative control.

The hemolytic activity of bolisin analogues was stated on the basis of the total hemolysis by Tween-20 and calculated by the following formula:


Hemolysis [%]=[A450 nm (peptide)−A450 nm (negative control)]/[A450 nm (1% Tween 20)−A450 nm (negative control)]*100

TSB medium with 287 mM glucose served as the test medium. The isotonic glucose concentration is to prevent non-specific hemolysis in a hypotensive medium (osmoprotection).

It is desirable that the peptides according to the invention exhibit no or but moderate lytic properties against erythrocytes. “Moderate lytic properties” means a lysis of less than 30% of the erythrocytes for the application of antimicrobially effective peptide concentrations.

FIG. 1 shows that bolisin analogues in antimicrobially effective concentrations do not cause any hemolysis worth mentioning (<5%). Even at high concentrations (500 μg/ml), only a moderate lytic activity can be established (<30%). For comparison, the hemolytic activity of the antimicrobial peptide MBI-28 (Gough et al., 1996, Infect. Immun. 64(12): 4922-7) was plotted.

1-19. (canceled) 20: Artificial peptide selected from the group consisting of SEQ ID NOS: 1-42 and 44-56. 21: The peptide of claim 20 conjugated with a polymer. 22: The peptide of claim 20 conjugated with polysorbate. 23: A medicament comprising an antibiotically effective amount of the peptide of claim 20 in combination with a pharmacologically acceptable carrier or diluent. 24: A medicament comprising an antibiotically effective amount of the peptide of claim 21 in combination with a pharmacologically acceptable carrier or diluent. 25: A medicament comprising an antibiotically effective amount of the peptide of claim 22 in combination with a pharmacologically acceptable carrier or diluent. 26: The medicament according to claim 23 further comprising at least one ingredient, wherein the at least one ingredient is (i) an additional antibiotically effective ingredient (ii) an antivirally effective ingredient, an antiparasitically effective ingredient, or an antifungally effective ingredient. 27: The medicament according to claim 23 in an application form selected from the group consisting of an infusion, ointment, tablet, spray, and slow release capsule. 28: The medicament according to claim 24 in an application form selected from the group consisting of an infusion, ointment, tablet, spray, and slow release capsule. 29: A diagnostic agent comprising an effective amount of the peptide of claim 20 in combination with a pharmacologically acceptable carrier or diluent. 30: A diagnostic agent comprising an effective amount of the peptide of claim 21 in combination with a pharmacologically acceptable carrier or diluent. 31: A diagnostic agent comprising an effective amount of the peptide of claim 22 in combination with a pharmacologically acceptable carrier or diluent. 32: A foodstuff incorporating the peptide of claim 20. 33: A method for inhibiting the growth of a microorganism comprising contacting the microorganism with an antimicrobially effective amount of the peptide of claim 20. 34: A method for inhibiting the growth of a microorganism comprising contacting the microorganism with an antimicrobially effective amount of the peptide of claim 21. 35: A method for inhibiting the growth of a microorganism comprising contacting the microorganism with an antimicrobially effective amount of the peptide of claim 22. 36: A process for preparing the peptide according to claim 20 comprising expressing the protein in a prokaryotic or eukaryotic organism or chemically synthesizing the protein. 37: A method of using the peptide of claim 20 comprising administering an antimicrobially effective amount of the peptide to a patient in need thereof.


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stats Patent Info
Application #
US 20090149391 A1
Publish Date
06/11/2009
Document #
File Date
09/23/2014
USPTO Class
Other USPTO Classes
International Class
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Food Additives
Galen


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