AU2013204065C1 - Selectively targeted antimicrobial peptides and the use thereof - Google Patents
Selectively targeted antimicrobial peptides and the use thereof Download PDFInfo
- Publication number
- AU2013204065C1 AU2013204065C1 AU2013204065A AU2013204065A AU2013204065C1 AU 2013204065 C1 AU2013204065 C1 AU 2013204065C1 AU 2013204065 A AU2013204065 A AU 2013204065A AU 2013204065 A AU2013204065 A AU 2013204065A AU 2013204065 C1 AU2013204065 C1 AU 2013204065C1
- Authority
- AU
- Australia
- Prior art keywords
- seq
- peptide
- stamp
- antimicrobial
- composition
- 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.)
- Ceased
Links
- 102000044503 Antimicrobial Peptides Human genes 0.000 title claims abstract description 110
- 108700042778 Antimicrobial Peptides Proteins 0.000 title claims abstract description 110
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 285
- 230000008685 targeting Effects 0.000 claims abstract description 138
- 241000194019 Streptococcus mutans Species 0.000 claims abstract description 100
- 239000000203 mixture Substances 0.000 claims abstract description 98
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 76
- 230000000813 microbial effect Effects 0.000 claims abstract description 54
- 230000002147 killing effect Effects 0.000 claims abstract description 47
- 238000000034 method Methods 0.000 claims abstract description 27
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 16
- 239000003910 polypeptide antibiotic agent Substances 0.000 claims description 86
- 230000000694 effects Effects 0.000 claims description 64
- 150000001413 amino acids Chemical group 0.000 claims description 60
- NLVFBUXFDBBNBW-PBSUHMDJSA-N tobramycin Chemical compound N[C@@H]1C[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N NLVFBUXFDBBNBW-PBSUHMDJSA-N 0.000 claims description 58
- 229960000707 tobramycin Drugs 0.000 claims description 58
- 239000003795 chemical substances by application Substances 0.000 claims description 43
- 101710082261 Competence-stimulating peptide Proteins 0.000 claims description 21
- 108010067396 dornase alfa Proteins 0.000 claims description 17
- 239000012634 fragment Substances 0.000 claims description 14
- 239000003242 anti bacterial agent Substances 0.000 claims description 13
- 230000003115 biocidal effect Effects 0.000 claims description 13
- 239000000137 peptide hydrolase inhibitor Substances 0.000 claims description 11
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 claims description 8
- 239000000758 substrate Substances 0.000 claims description 7
- MYSWGUAQZAJSOK-UHFFFAOYSA-N ciprofloxacin Chemical compound C12=CC(N3CCNCC3)=C(F)C=C2C(=O)C(C(=O)O)=CN1C1CC1 MYSWGUAQZAJSOK-UHFFFAOYSA-N 0.000 claims description 6
- ULGZDMOVFRHVEP-RWJQBGPGSA-N Erythromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 ULGZDMOVFRHVEP-RWJQBGPGSA-N 0.000 claims description 4
- 229960003405 ciprofloxacin Drugs 0.000 claims description 3
- VCOPTHOUUNAYKQ-WBTCAYNUSA-N (3s)-3,6-diamino-n-[[(2s,5s,8e,11s,15s)-15-amino-11-[(6r)-2-amino-1,4,5,6-tetrahydropyrimidin-6-yl]-8-[(carbamoylamino)methylidene]-2-(hydroxymethyl)-3,6,9,12,16-pentaoxo-1,4,7,10,13-pentazacyclohexadec-5-yl]methyl]hexanamide;(3s)-3,6-diamino-n-[[(2s,5s,8 Chemical compound N1C(=O)\C(=C/NC(N)=O)NC(=O)[C@H](CNC(=O)C[C@@H](N)CCCN)NC(=O)[C@H](C)NC(=O)[C@@H](N)CNC(=O)[C@@H]1[C@@H]1NC(N)=NCC1.N1C(=O)\C(=C/NC(N)=O)NC(=O)[C@H](CNC(=O)C[C@@H](N)CCCN)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CNC(=O)[C@@H]1[C@@H]1NC(N)=NCC1 VCOPTHOUUNAYKQ-WBTCAYNUSA-N 0.000 claims description 2
- SGKRLCUYIXIAHR-AKNGSSGZSA-N (4s,4ar,5s,5ar,6r,12ar)-4-(dimethylamino)-1,5,10,11,12a-pentahydroxy-6-methyl-3,12-dioxo-4a,5,5a,6-tetrahydro-4h-tetracene-2-carboxamide Chemical compound C1=CC=C2[C@H](C)[C@@H]([C@H](O)[C@@H]3[C@](C(O)=C(C(N)=O)C(=O)[C@H]3N(C)C)(O)C3=O)C3=C(O)C2=C1O SGKRLCUYIXIAHR-AKNGSSGZSA-N 0.000 claims description 2
- FFTVPQUHLQBXQZ-KVUCHLLUSA-N (4s,4as,5ar,12ar)-4,7-bis(dimethylamino)-1,10,11,12a-tetrahydroxy-3,12-dioxo-4a,5,5a,6-tetrahydro-4h-tetracene-2-carboxamide Chemical compound C1C2=C(N(C)C)C=CC(O)=C2C(O)=C2[C@@H]1C[C@H]1[C@H](N(C)C)C(=O)C(C(N)=O)=C(O)[C@@]1(O)C2=O FFTVPQUHLQBXQZ-KVUCHLLUSA-N 0.000 claims description 2
- 108010001478 Bacitracin Proteins 0.000 claims description 2
- 108010065839 Capreomycin Proteins 0.000 claims description 2
- IECPWNUMDGFDKC-UHFFFAOYSA-N Fusicsaeure Natural products C12C(O)CC3C(=C(CCC=C(C)C)C(O)=O)C(OC(C)=O)CC3(C)C1(C)CCC1C2(C)CCC(O)C1C IECPWNUMDGFDKC-UHFFFAOYSA-N 0.000 claims description 2
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 claims description 2
- 229930182566 Gentamicin Natural products 0.000 claims description 2
- 108010026389 Gramicidin Proteins 0.000 claims description 2
- OJMMVQQUTAEWLP-UHFFFAOYSA-N Lincomycin Natural products CN1CC(CCC)CC1C(=O)NC(C(C)O)C1C(O)C(O)C(O)C(SC)O1 OJMMVQQUTAEWLP-UHFFFAOYSA-N 0.000 claims description 2
- YJQPYGGHQPGBLI-UHFFFAOYSA-N Novobiocin Natural products O1C(C)(C)C(OC)C(OC(N)=O)C(O)C1OC1=CC=C(C(O)=C(NC(=O)C=2C=C(CC=C(C)C)C(O)=CC=2)C(=O)O2)C2=C1C YJQPYGGHQPGBLI-UHFFFAOYSA-N 0.000 claims description 2
- 229930189077 Rifamycin Natural products 0.000 claims description 2
- 239000004098 Tetracycline Substances 0.000 claims description 2
- 108010059993 Vancomycin Proteins 0.000 claims description 2
- RRDRHWJDBOGQHN-JWCTVYNTSA-N [2-[(2s,5r,8s,11s,14r,17s,22s)-17-[(1r)-1-hydroxyethyl]-22-[[(2s)-2-[[(2s,3r)-3-hydroxy-2-[[(2s)-2-[6-methyloctanoyl(sulfomethyl)amino]-4-(sulfomethylamino)butanoyl]amino]butyl]amino]-4-(sulfomethylamino)butanoyl]amino]-5,8-bis(2-methylpropyl)-3,6,9,12,15 Chemical compound CCC(C)CCCCC(=O)N(CS(O)(=O)=O)[C@@H](CCNCS(O)(=O)=O)C(=O)N[C@H]([C@@H](C)O)CN[C@@H](CCNCS(O)(=O)=O)C(=O)N[C@H]1CCNC(=O)[C@H]([C@@H](C)O)NC(=O)[C@@H](CCNCS(O)(=O)=O)NC(=O)[C@H](CCNCS(O)(=O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](CC(C)C)NC(=O)[C@H](CCNCS(O)(=O)=O)NC1=O RRDRHWJDBOGQHN-JWCTVYNTSA-N 0.000 claims description 2
- 229960003022 amoxicillin Drugs 0.000 claims description 2
- LSQZJLSUYDQPKJ-NJBDSQKTSA-N amoxicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=C(O)C=C1 LSQZJLSUYDQPKJ-NJBDSQKTSA-N 0.000 claims description 2
- 229960003071 bacitracin Drugs 0.000 claims description 2
- 229930184125 bacitracin Natural products 0.000 claims description 2
- CLKOFPXJLQSYAH-ABRJDSQDSA-N bacitracin A Chemical compound C1SC([C@@H](N)[C@@H](C)CC)=N[C@@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]1C(=O)N[C@H](CCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2N=CNC=2)C(=O)N[C@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)NCCCC1 CLKOFPXJLQSYAH-ABRJDSQDSA-N 0.000 claims description 2
- 239000003782 beta lactam antibiotic agent Substances 0.000 claims description 2
- 229960004602 capreomycin Drugs 0.000 claims description 2
- 229960005091 chloramphenicol Drugs 0.000 claims description 2
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 claims description 2
- 229960002227 clindamycin Drugs 0.000 claims description 2
- KDLRVYVGXIQJDK-AWPVFWJPSA-N clindamycin Chemical compound CN1C[C@H](CCC)C[C@H]1C(=O)N[C@H]([C@H](C)Cl)[C@@H]1[C@H](O)[C@H](O)[C@@H](O)[C@@H](SC)O1 KDLRVYVGXIQJDK-AWPVFWJPSA-N 0.000 claims description 2
- 229940108538 colistimethate Drugs 0.000 claims description 2
- 108700028201 colistinmethanesulfonic acid Proteins 0.000 claims description 2
- 229960003722 doxycycline Drugs 0.000 claims description 2
- 229960003276 erythromycin Drugs 0.000 claims description 2
- 229960000308 fosfomycin Drugs 0.000 claims description 2
- YMDXZJFXQJVXBF-STHAYSLISA-N fosfomycin Chemical compound C[C@@H]1O[C@@H]1P(O)(O)=O YMDXZJFXQJVXBF-STHAYSLISA-N 0.000 claims description 2
- 229940083579 fusidate sodium Drugs 0.000 claims description 2
- 229960004675 fusidic acid Drugs 0.000 claims description 2
- IECPWNUMDGFDKC-MZJAQBGESA-N fusidic acid Chemical compound O[C@@H]([C@@H]12)C[C@H]3\C(=C(/CCC=C(C)C)C(O)=O)[C@@H](OC(C)=O)C[C@]3(C)[C@@]2(C)CC[C@@H]2[C@]1(C)CC[C@@H](O)[C@H]2C IECPWNUMDGFDKC-MZJAQBGESA-N 0.000 claims description 2
- 229960004905 gramicidin Drugs 0.000 claims description 2
- ZWCXYZRRTRDGQE-SORVKSEFSA-N gramicidina Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@@H](CC(C)C)NC(=O)[C@H](CC=3C4=CC=CC=C4NC=3)NC(=O)[C@@H](CC(C)C)NC(=O)[C@H](CC=3C4=CC=CC=C4NC=3)NC(=O)[C@@H](CC(C)C)NC(=O)[C@H](CC=3C4=CC=CC=C4NC=3)NC(=O)[C@H](C(C)C)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](C(C)C)NC(=O)[C@H](C)NC(=O)[C@H](NC(=O)[C@H](C)NC(=O)CNC(=O)[C@@H](NC=O)C(C)C)CC(C)C)C(=O)NCCO)=CNC2=C1 ZWCXYZRRTRDGQE-SORVKSEFSA-N 0.000 claims description 2
- OJMMVQQUTAEWLP-KIDUDLJLSA-N lincomycin Chemical compound CN1C[C@H](CCC)C[C@H]1C(=O)N[C@H]([C@@H](C)O)[C@@H]1[C@H](O)[C@H](O)[C@@H](O)[C@@H](SC)O1 OJMMVQQUTAEWLP-KIDUDLJLSA-N 0.000 claims description 2
- 229960005287 lincomycin Drugs 0.000 claims description 2
- 239000003120 macrolide antibiotic agent Substances 0.000 claims description 2
- 229940041033 macrolides Drugs 0.000 claims description 2
- 229960004023 minocycline Drugs 0.000 claims description 2
- 229940041009 monobactams Drugs 0.000 claims description 2
- 229960000210 nalidixic acid Drugs 0.000 claims description 2
- MHWLWQUZZRMNGJ-UHFFFAOYSA-N nalidixic acid Chemical compound C1=C(C)N=C2N(CC)C=C(C(O)=O)C(=O)C2=C1 MHWLWQUZZRMNGJ-UHFFFAOYSA-N 0.000 claims description 2
- 229960002950 novobiocin Drugs 0.000 claims description 2
- YJQPYGGHQPGBLI-KGSXXDOSSA-N novobiocin Chemical compound O1C(C)(C)[C@H](OC)[C@@H](OC(N)=O)[C@@H](O)[C@@H]1OC1=CC=C(C(O)=C(NC(=O)C=2C=C(CC=C(C)C)C(O)=CC=2)C(=O)O2)C2=C1C YJQPYGGHQPGBLI-KGSXXDOSSA-N 0.000 claims description 2
- LSQZJLSUYDQPKJ-UHFFFAOYSA-N p-Hydroxyampicillin Natural products O=C1N2C(C(O)=O)C(C)(C)SC2C1NC(=O)C(N)C1=CC=C(O)C=C1 LSQZJLSUYDQPKJ-UHFFFAOYSA-N 0.000 claims description 2
- BTVYFIMKUHNOBZ-QXMMDKDBSA-N rifamycin s Chemical class O=C1C(C(O)=C2C)=C3C(=O)C=C1NC(=O)\C(C)=C/C=C\C(C)C(O)C(C)C(O)C(C)C(OC(C)=O)C(C)C(OC)\C=C/OC1(C)OC2=C3C1=O BTVYFIMKUHNOBZ-QXMMDKDBSA-N 0.000 claims description 2
- 229940081192 rifamycins Drugs 0.000 claims description 2
- HJHVQCXHVMGZNC-JCJNLNMISA-M sodium;(2z)-2-[(3r,4s,5s,8s,9s,10s,11r,13r,14s,16s)-16-acetyloxy-3,11-dihydroxy-4,8,10,14-tetramethyl-2,3,4,5,6,7,9,11,12,13,15,16-dodecahydro-1h-cyclopenta[a]phenanthren-17-ylidene]-6-methylhept-5-enoate Chemical compound [Na+].O[C@@H]([C@@H]12)C[C@H]3\C(=C(/CCC=C(C)C)C([O-])=O)[C@@H](OC(C)=O)C[C@]3(C)[C@@]2(C)CC[C@@H]2[C@]1(C)CC[C@@H](O)[C@H]2C HJHVQCXHVMGZNC-JCJNLNMISA-M 0.000 claims description 2
- 235000019364 tetracycline Nutrition 0.000 claims description 2
- 150000003522 tetracyclines Chemical class 0.000 claims description 2
- 229940040944 tetracyclines Drugs 0.000 claims description 2
- IEDVJHCEMCRBQM-UHFFFAOYSA-N trimethoprim Chemical compound COC1=C(OC)C(OC)=CC(CC=2C(=NC(N)=NC=2)N)=C1 IEDVJHCEMCRBQM-UHFFFAOYSA-N 0.000 claims description 2
- 229960001082 trimethoprim Drugs 0.000 claims description 2
- MYPYJXKWCTUITO-LYRMYLQWSA-N vancomycin Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)NC)[C@H]1C[C@](C)(N)[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-N 0.000 claims description 2
- 229960003165 vancomycin Drugs 0.000 claims description 2
- MYPYJXKWCTUITO-UHFFFAOYSA-N vancomycin Natural products O1C(C(=C2)Cl)=CC=C2C(O)C(C(NC(C2=CC(O)=CC(O)=C2C=2C(O)=CC=C3C=2)C(O)=O)=O)NC(=O)C3NC(=O)C2NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CC(C)C)NC)C(O)C(C=C3Cl)=CC=C3OC3=CC2=CC1=C3OC1OC(CO)C(O)C(O)C1OC1CC(C)(N)C(O)C(C)O1 MYPYJXKWCTUITO-UHFFFAOYSA-N 0.000 claims description 2
- 239000002132 β-lactam antibiotic Substances 0.000 claims description 2
- 229940124586 β-lactam antibiotics Drugs 0.000 claims description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims 1
- 230000000845 anti-microbial effect Effects 0.000 abstract description 56
- 230000027455 binding Effects 0.000 abstract description 30
- 206010036790 Productive cough Diseases 0.000 description 44
- 208000024794 sputum Diseases 0.000 description 44
- 210000003802 sputum Anatomy 0.000 description 43
- 210000004027 cell Anatomy 0.000 description 31
- 241000894006 Bacteria Species 0.000 description 29
- -1 L-amino acid enantiomers Chemical class 0.000 description 29
- 208000015181 infectious disease Diseases 0.000 description 29
- 238000011282 treatment Methods 0.000 description 26
- 238000002474 experimental method Methods 0.000 description 24
- 235000001014 amino acid Nutrition 0.000 description 23
- 238000009472 formulation Methods 0.000 description 18
- 241000194023 Streptococcus sanguinis Species 0.000 description 17
- 238000003556 assay Methods 0.000 description 17
- 230000001580 bacterial effect Effects 0.000 description 16
- 239000000243 solution Substances 0.000 description 15
- 210000001519 tissue Anatomy 0.000 description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 13
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 13
- 239000002953 phosphate buffered saline Substances 0.000 description 13
- 241000589516 Pseudomonas Species 0.000 description 12
- 241000194026 Streptococcus gordonii Species 0.000 description 12
- 238000004128 high performance liquid chromatography Methods 0.000 description 12
- 239000002609 medium Substances 0.000 description 12
- 230000001717 pathogenic effect Effects 0.000 description 12
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 12
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 12
- 210000003296 saliva Anatomy 0.000 description 12
- 210000004899 c-terminal region Anatomy 0.000 description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 11
- 241000282414 Homo sapiens Species 0.000 description 10
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 10
- 229930006000 Sucrose Natural products 0.000 description 10
- 201000010099 disease Diseases 0.000 description 10
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 10
- 239000005720 sucrose Substances 0.000 description 10
- 230000002195 synergetic effect Effects 0.000 description 10
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 9
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 9
- 230000000844 anti-bacterial effect Effects 0.000 description 9
- 239000003937 drug carrier Substances 0.000 description 9
- 239000000975 dye Substances 0.000 description 9
- 239000012528 membrane Substances 0.000 description 9
- 244000005700 microbiome Species 0.000 description 9
- 239000012071 phase Substances 0.000 description 9
- 239000000126 substance Substances 0.000 description 9
- 108060001084 Luciferase Proteins 0.000 description 8
- 102000035195 Peptidases Human genes 0.000 description 8
- 108091005804 Peptidases Proteins 0.000 description 8
- 239000004365 Protease Substances 0.000 description 8
- 238000002835 absorbance Methods 0.000 description 8
- 229940088710 antibiotic agent Drugs 0.000 description 8
- 230000001013 cariogenic effect Effects 0.000 description 8
- 238000012217 deletion Methods 0.000 description 8
- 238000011534 incubation Methods 0.000 description 8
- 210000004379 membrane Anatomy 0.000 description 8
- 244000052769 pathogen Species 0.000 description 8
- 239000000546 pharmaceutical excipient Substances 0.000 description 8
- 239000003016 pheromone Substances 0.000 description 8
- 238000007747 plating Methods 0.000 description 8
- 229920001223 polyethylene glycol Polymers 0.000 description 8
- 239000000843 powder Substances 0.000 description 8
- 230000002829 reductive effect Effects 0.000 description 8
- 239000005089 Luciferase Substances 0.000 description 7
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 7
- 229940126575 aminoglycoside Drugs 0.000 description 7
- 230000037430 deletion Effects 0.000 description 7
- 238000010790 dilution Methods 0.000 description 7
- 239000012895 dilution Substances 0.000 description 7
- 210000000214 mouth Anatomy 0.000 description 7
- 235000000346 sugar Nutrition 0.000 description 7
- 150000008163 sugars Chemical class 0.000 description 7
- 241000222122 Candida albicans Species 0.000 description 6
- 239000000872 buffer Substances 0.000 description 6
- 239000000969 carrier Substances 0.000 description 6
- 239000003814 drug Substances 0.000 description 6
- 235000019441 ethanol Nutrition 0.000 description 6
- 239000012737 fresh medium Substances 0.000 description 6
- 230000006870 function Effects 0.000 description 6
- 239000001963 growth medium Substances 0.000 description 6
- 230000002209 hydrophobic effect Effects 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 6
- 238000004949 mass spectrometry Methods 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 108010043167 novispirin G10 Proteins 0.000 description 6
- WVUSYUYFHJGZMG-GBMWXUEWSA-N novispirin g 10 Chemical compound C([C@H](NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](N)CCCCN)[C@@H](C)CC)[C@@H](C)CC)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)NCC(O)=O)C1=CN=CN1 WVUSYUYFHJGZMG-GBMWXUEWSA-N 0.000 description 6
- 210000000056 organ Anatomy 0.000 description 6
- 150000003384 small molecules Chemical class 0.000 description 6
- 239000007787 solid Substances 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 239000000725 suspension Substances 0.000 description 6
- 102100023659 Coiled-coil domain-containing protein 120 Human genes 0.000 description 5
- 150000008574 D-amino acids Chemical class 0.000 description 5
- 108010010803 Gelatin Proteins 0.000 description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 5
- 108010019494 Histatins Proteins 0.000 description 5
- 102000006492 Histatins Human genes 0.000 description 5
- 101000978260 Homo sapiens Coiled-coil domain-containing protein 120 Proteins 0.000 description 5
- 239000006142 Luria-Bertani Agar Substances 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 5
- 229920002472 Starch Polymers 0.000 description 5
- 239000000443 aerosol Substances 0.000 description 5
- 235000010443 alginic acid Nutrition 0.000 description 5
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 210000001124 body fluid Anatomy 0.000 description 5
- 239000010839 body fluid Substances 0.000 description 5
- 230000015556 catabolic process Effects 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 238000011278 co-treatment Methods 0.000 description 5
- 238000010276 construction Methods 0.000 description 5
- 238000006731 degradation reaction Methods 0.000 description 5
- 208000002925 dental caries Diseases 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 235000013305 food Nutrition 0.000 description 5
- 210000001035 gastrointestinal tract Anatomy 0.000 description 5
- 239000000499 gel Substances 0.000 description 5
- 239000008273 gelatin Substances 0.000 description 5
- 229920000159 gelatin Polymers 0.000 description 5
- 235000019322 gelatine Nutrition 0.000 description 5
- 235000011852 gelatine desserts Nutrition 0.000 description 5
- 235000011187 glycerol Nutrition 0.000 description 5
- 239000007943 implant Substances 0.000 description 5
- 239000002773 nucleotide Substances 0.000 description 5
- 125000003729 nucleotide group Chemical group 0.000 description 5
- 239000003921 oil Substances 0.000 description 5
- 238000007911 parenteral administration Methods 0.000 description 5
- 239000002245 particle Substances 0.000 description 5
- 239000006187 pill Substances 0.000 description 5
- 229920000642 polymer Polymers 0.000 description 5
- 235000018102 proteins Nutrition 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 230000001603 reducing effect Effects 0.000 description 5
- 238000013207 serial dilution Methods 0.000 description 5
- 241000894007 species Species 0.000 description 5
- 235000019698 starch Nutrition 0.000 description 5
- 230000004083 survival effect Effects 0.000 description 5
- 239000003826 tablet Substances 0.000 description 5
- 229920001817 Agar Polymers 0.000 description 4
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 4
- 241000416162 Astragalus gummifer Species 0.000 description 4
- 201000003883 Cystic fibrosis Diseases 0.000 description 4
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 4
- 108010002069 Defensins Proteins 0.000 description 4
- 102000000541 Defensins Human genes 0.000 description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 4
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 4
- 108700026244 Open Reading Frames Proteins 0.000 description 4
- 239000002202 Polyethylene glycol Substances 0.000 description 4
- 102000012479 Serine Proteases Human genes 0.000 description 4
- 108010022999 Serine Proteases Proteins 0.000 description 4
- 241000191967 Staphylococcus aureus Species 0.000 description 4
- 229920001615 Tragacanth Polymers 0.000 description 4
- 239000000783 alginic acid Substances 0.000 description 4
- 239000004599 antimicrobial Substances 0.000 description 4
- 102000005936 beta-Galactosidase Human genes 0.000 description 4
- 108010005774 beta-Galactosidase Proteins 0.000 description 4
- 229940095731 candida albicans Drugs 0.000 description 4
- 239000002775 capsule Substances 0.000 description 4
- POIUWJQBRNEFGX-XAMSXPGMSA-N cathelicidin Chemical compound C([C@@H](C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC(C)C)C1=CC=CC=C1 POIUWJQBRNEFGX-XAMSXPGMSA-N 0.000 description 4
- 239000006285 cell suspension Substances 0.000 description 4
- 238000013461 design Methods 0.000 description 4
- 229940039227 diagnostic agent Drugs 0.000 description 4
- 239000000032 diagnostic agent Substances 0.000 description 4
- 210000003527 eukaryotic cell Anatomy 0.000 description 4
- 239000007850 fluorescent dye Substances 0.000 description 4
- 239000008103 glucose Substances 0.000 description 4
- 230000036541 health Effects 0.000 description 4
- 230000006872 improvement Effects 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 239000004615 ingredient Substances 0.000 description 4
- 230000003993 interaction Effects 0.000 description 4
- 239000008101 lactose Substances 0.000 description 4
- 230000007246 mechanism Effects 0.000 description 4
- 238000012544 monitoring process Methods 0.000 description 4
- 239000002324 mouth wash Substances 0.000 description 4
- 235000019198 oils Nutrition 0.000 description 4
- 239000006072 paste Substances 0.000 description 4
- 239000003755 preservative agent Substances 0.000 description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- 238000001228 spectrum Methods 0.000 description 4
- 239000007921 spray Substances 0.000 description 4
- 238000010186 staining Methods 0.000 description 4
- 238000006467 substitution reaction Methods 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 239000000454 talc Substances 0.000 description 4
- 235000012222 talc Nutrition 0.000 description 4
- 229910052623 talc Inorganic materials 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 230000001225 therapeutic effect Effects 0.000 description 4
- 235000010487 tragacanth Nutrition 0.000 description 4
- 239000000196 tragacanth Substances 0.000 description 4
- 229940116362 tragacanth Drugs 0.000 description 4
- 239000001993 wax Substances 0.000 description 4
- 210000005253 yeast cell Anatomy 0.000 description 4
- JNYAEWCLZODPBN-JGWLITMVSA-N (2r,3r,4s)-2-[(1r)-1,2-dihydroxyethyl]oxolane-3,4-diol Chemical compound OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O JNYAEWCLZODPBN-JGWLITMVSA-N 0.000 description 3
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 3
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 3
- 101710107631 Andropin Proteins 0.000 description 3
- 101800003484 Apidaecin Proteins 0.000 description 3
- 101100441251 Arabidopsis thaliana CSP2 gene Proteins 0.000 description 3
- 101100494773 Caenorhabditis elegans ctl-2 gene Proteins 0.000 description 3
- 241000589875 Campylobacter jejuni Species 0.000 description 3
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 3
- 206010007134 Candida infections Diseases 0.000 description 3
- 108050004290 Cecropin Proteins 0.000 description 3
- 108010053770 Deoxyribonucleases Proteins 0.000 description 3
- 102000016911 Deoxyribonucleases Human genes 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 101100112369 Fasciola hepatica Cat-1 gene Proteins 0.000 description 3
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 3
- 235000010643 Leucaena leucocephala Nutrition 0.000 description 3
- 240000007472 Leucaena leucocephala Species 0.000 description 3
- 108060003100 Magainin Proteins 0.000 description 3
- 101100005271 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) cat-1 gene Proteins 0.000 description 3
- 108010067902 Peptide Library Proteins 0.000 description 3
- 241000607142 Salmonella Species 0.000 description 3
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 description 3
- 241000607768 Shigella Species 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 241000295644 Staphylococcaceae Species 0.000 description 3
- 241001521783 Streptococcus mutans UA159 Species 0.000 description 3
- 241000193998 Streptococcus pneumoniae Species 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 239000000654 additive Substances 0.000 description 3
- 235000010419 agar Nutrition 0.000 description 3
- 229920000615 alginic acid Polymers 0.000 description 3
- 230000009435 amidation Effects 0.000 description 3
- 238000007112 amidation reaction Methods 0.000 description 3
- 230000000843 anti-fungal effect Effects 0.000 description 3
- 229940121375 antifungal agent Drugs 0.000 description 3
- NUTHXVZQNRZFPR-FHDGIMILSA-N apidaecin Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCC(N)=O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)[C@@H](NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)CN)C(C)C)C1=CC=C(O)C=C1 NUTHXVZQNRZFPR-FHDGIMILSA-N 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 235000012216 bentonite Nutrition 0.000 description 3
- 230000032770 biofilm formation Effects 0.000 description 3
- 201000003984 candidiasis Diseases 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 238000000576 coating method Methods 0.000 description 3
- 239000006071 cream Substances 0.000 description 3
- 238000009792 diffusion process Methods 0.000 description 3
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 3
- 239000006185 dispersion Substances 0.000 description 3
- 239000003995 emulsifying agent Substances 0.000 description 3
- 239000000839 emulsion Substances 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 239000000945 filler Substances 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 238000000799 fluorescence microscopy Methods 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 239000008187 granular material Substances 0.000 description 3
- USSYUMHVHQSYNA-SLDJZXPVSA-N indolicidin Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)N[C@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(N)=O)CC1=CNC2=CC=CC=C12 USSYUMHVHQSYNA-SLDJZXPVSA-N 0.000 description 3
- 239000003701 inert diluent Substances 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000002372 labelling Methods 0.000 description 3
- 230000014759 maintenance of location Effects 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- 230000001404 mediated effect Effects 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 239000004006 olive oil Substances 0.000 description 3
- 239000000816 peptidomimetic Substances 0.000 description 3
- 102000013415 peroxidase activity proteins Human genes 0.000 description 3
- 108040007629 peroxidase activity proteins Proteins 0.000 description 3
- 239000008194 pharmaceutical composition Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000003380 propellant Substances 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- RMAQACBXLXPBSY-UHFFFAOYSA-N silicic acid Chemical compound O[Si](O)(O)O RMAQACBXLXPBSY-UHFFFAOYSA-N 0.000 description 3
- 235000012239 silicon dioxide Nutrition 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 230000009870 specific binding Effects 0.000 description 3
- 229960000268 spectinomycin Drugs 0.000 description 3
- 239000003381 stabilizer Substances 0.000 description 3
- 239000008107 starch Substances 0.000 description 3
- 229940032147 starch Drugs 0.000 description 3
- 229940031000 streptococcus pneumoniae Drugs 0.000 description 3
- 239000000829 suppository Substances 0.000 description 3
- 239000004094 surface-active agent Substances 0.000 description 3
- 239000000375 suspending agent Substances 0.000 description 3
- 239000001974 tryptic soy broth Substances 0.000 description 3
- 108010050327 trypticase-soy broth Proteins 0.000 description 3
- 239000000080 wetting agent Substances 0.000 description 3
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 3
- PUPZLCDOIYMWBV-UHFFFAOYSA-N (+/-)-1,3-Butanediol Chemical compound CC(O)CCO PUPZLCDOIYMWBV-UHFFFAOYSA-N 0.000 description 2
- BUMACWDGTIFQAZ-WIXHNTGMSA-N (2s)-2-[[(2s,3s)-2-[[2-[[(2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-[[(2s,3r)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-[[2-[[(2s)-2-[[(2s)-2-[[(2s)-1-[2-[[(2s)-2-[[(2s,3s)-2-amino-3-methylpentanoyl]amino]-4-methylpentanoyl]amino]acetyl]pyrrolidine-2-carbonyl Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(O)=O BUMACWDGTIFQAZ-WIXHNTGMSA-N 0.000 description 2
- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 description 2
- VBICKXHEKHSIBG-UHFFFAOYSA-N 1-monostearoylglycerol Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(O)CO VBICKXHEKHSIBG-UHFFFAOYSA-N 0.000 description 2
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 2
- BZTDTCNHAFUJOG-UHFFFAOYSA-N 6-carboxyfluorescein Chemical compound C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C11OC(=O)C2=CC=C(C(=O)O)C=C21 BZTDTCNHAFUJOG-UHFFFAOYSA-N 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- 101100055070 Agrocybe aegerita Agr10 gene Proteins 0.000 description 2
- 108010033863 Alexomycin Proteins 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 108010062877 Bacteriocins Proteins 0.000 description 2
- 101100441244 Caenorhabditis elegans csp-1 gene Proteins 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- 241000606153 Chlamydia trachomatis Species 0.000 description 2
- 241000193163 Clostridioides difficile Species 0.000 description 2
- 108091035707 Consensus sequence Proteins 0.000 description 2
- 201000007336 Cryptococcosis Diseases 0.000 description 2
- 241000221204 Cryptococcus neoformans Species 0.000 description 2
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 2
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 description 2
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 2
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 2
- 208000002064 Dental Plaque Diseases 0.000 description 2
- 206010012735 Diarrhoea Diseases 0.000 description 2
- LVGKNOAMLMIIKO-UHFFFAOYSA-N Elaidinsaeure-aethylester Natural products CCCCCCCCC=CCCCCCCCC(=O)OCC LVGKNOAMLMIIKO-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 206010017533 Fungal infection Diseases 0.000 description 2
- 241000206672 Gelidium Species 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 241000590002 Helicobacter pylori Species 0.000 description 2
- 229930195725 Mannitol Natural products 0.000 description 2
- 108010036176 Melitten Proteins 0.000 description 2
- 241000588655 Moraxella catarrhalis Species 0.000 description 2
- 241000588652 Neisseria gonorrhoeae Species 0.000 description 2
- 108010053775 Nisin Proteins 0.000 description 2
- NVNLLIYOARQCIX-MSHCCFNRSA-N Nisin Chemical compound N1C(=O)[C@@H](CC(C)C)NC(=O)C(=C)NC(=O)[C@@H]([C@H](C)CC)NC(=O)[C@@H](NC(=O)C(=C/C)/NC(=O)[C@H](N)[C@H](C)CC)CSC[C@@H]1C(=O)N[C@@H]1C(=O)N2CCC[C@@H]2C(=O)NCC(=O)N[C@@H](C(=O)N[C@H](CCCCN)C(=O)N[C@@H]2C(NCC(=O)N[C@H](C)C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCSC)C(=O)NCC(=O)N[C@H](CS[C@@H]2C)C(=O)N[C@H](CC(N)=O)C(=O)N[C@H](CCSC)C(=O)N[C@H](CCCCN)C(=O)N[C@@H]2C(N[C@H](C)C(=O)N[C@@H]3C(=O)N[C@@H](C(N[C@H](CC=4NC=NC=4)C(=O)N[C@H](CS[C@@H]3C)C(=O)N[C@H](CO)C(=O)N[C@H]([C@H](C)CC)C(=O)N[C@H](CC=3NC=NC=3)C(=O)N[C@H](C(C)C)C(=O)NC(=C)C(=O)N[C@H](CCCCN)C(O)=O)=O)CS[C@@H]2C)=O)=O)CS[C@@H]1C NVNLLIYOARQCIX-MSHCCFNRSA-N 0.000 description 2
- 206010031252 Osteomyelitis Diseases 0.000 description 2
- 102000004316 Oxidoreductases Human genes 0.000 description 2
- 108090000854 Oxidoreductases Proteins 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 241000235645 Pichia kudriavzevii Species 0.000 description 2
- ATUOYWHBWRKTHZ-UHFFFAOYSA-N Propane Chemical compound CCC ATUOYWHBWRKTHZ-UHFFFAOYSA-N 0.000 description 2
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 2
- 206010040070 Septic Shock Diseases 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- 241000191940 Staphylococcus Species 0.000 description 2
- 108010070741 Tachypleus tridentatus tachyplesin peptide Proteins 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- XLOMVQKBTHCTTD-UHFFFAOYSA-N Zinc monoxide Chemical compound [Zn]=O XLOMVQKBTHCTTD-UHFFFAOYSA-N 0.000 description 2
- 206010000269 abscess Diseases 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 229960001126 alginic acid Drugs 0.000 description 2
- 150000004781 alginic acids Chemical class 0.000 description 2
- 102000018568 alpha-Defensin Human genes 0.000 description 2
- 108050007802 alpha-defensin Proteins 0.000 description 2
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 2
- 229960000723 ampicillin Drugs 0.000 description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
- 239000005557 antagonist Substances 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 108010016341 bactenecin Proteins 0.000 description 2
- RHISNKCGUDDGEG-UHFFFAOYSA-N bactenecin Chemical compound CCC(C)C1NC(=O)C(C(C)C)NC(=O)C(C(C)C)NC(=O)C(C(C)CC)NC(=O)C(CCCN=C(N)N)NC(=O)C(NC(=O)C(CC(C)C)NC(=O)C(N)CCCN=C(N)N)CSSCC(C(=O)NC(CCCN=C(N)N)C(O)=O)NC(=O)C(C(C)C)NC(=O)C(CCCN=C(N)N)NC1=O RHISNKCGUDDGEG-UHFFFAOYSA-N 0.000 description 2
- 239000000440 bentonite Substances 0.000 description 2
- 229910000278 bentonite Inorganic materials 0.000 description 2
- SVPXDRXYRYOSEX-UHFFFAOYSA-N bentoquatam Chemical compound O.O=[Si]=O.O=[Al]O[Al]=O SVPXDRXYRYOSEX-UHFFFAOYSA-N 0.000 description 2
- SESFRYSPDFLNCH-UHFFFAOYSA-N benzyl benzoate Chemical compound C=1C=CC=CC=1C(=O)OCC1=CC=CC=C1 SESFRYSPDFLNCH-UHFFFAOYSA-N 0.000 description 2
- 102000012265 beta-defensin Human genes 0.000 description 2
- 108050002883 beta-defensin Proteins 0.000 description 2
- 239000011230 binding agent Substances 0.000 description 2
- 229920002988 biodegradable polymer Polymers 0.000 description 2
- 239000004621 biodegradable polymer Substances 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 239000001768 carboxy methyl cellulose Substances 0.000 description 2
- 230000000747 cardiac effect Effects 0.000 description 2
- 239000012876 carrier material Substances 0.000 description 2
- 108060001132 cathelicidin Proteins 0.000 description 2
- 102000014509 cathelicidin Human genes 0.000 description 2
- 230000022534 cell killing Effects 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 235000010980 cellulose Nutrition 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 229940038705 chlamydia trachomatis Drugs 0.000 description 2
- OSASVXMJTNOKOY-UHFFFAOYSA-N chlorobutanol Chemical compound CC(C)(O)C(Cl)(Cl)Cl OSASVXMJTNOKOY-UHFFFAOYSA-N 0.000 description 2
- 239000003593 chromogenic compound Substances 0.000 description 2
- 230000001684 chronic effect Effects 0.000 description 2
- 238000011260 co-administration Methods 0.000 description 2
- 229940110456 cocoa butter Drugs 0.000 description 2
- 235000019868 cocoa butter Nutrition 0.000 description 2
- 239000003086 colorant Substances 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 235000012343 cottonseed oil Nutrition 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 239000007857 degradation product Substances 0.000 description 2
- 238000003795 desorption Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 235000005911 diet Nutrition 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 239000000539 dimer Substances 0.000 description 2
- 239000002270 dispersing agent Substances 0.000 description 2
- 239000002552 dosage form Substances 0.000 description 2
- 239000008298 dragée Substances 0.000 description 2
- 238000009510 drug design Methods 0.000 description 2
- 206010014665 endocarditis Diseases 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- MMXKVMNBHPAILY-UHFFFAOYSA-N ethyl laurate Chemical compound CCCCCCCCCCCC(=O)OCC MMXKVMNBHPAILY-UHFFFAOYSA-N 0.000 description 2
- LVGKNOAMLMIIKO-QXMHVHEDSA-N ethyl oleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC LVGKNOAMLMIIKO-QXMHVHEDSA-N 0.000 description 2
- 229940093471 ethyl oleate Drugs 0.000 description 2
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 2
- 230000004907 flux Effects 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 229940037467 helicobacter pylori Drugs 0.000 description 2
- KSXBMTJGDUPBBN-VPKNIDFUSA-N histatin 5 Chemical compound C([C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(O)=O)C1=CN=CN1 KSXBMTJGDUPBBN-VPKNIDFUSA-N 0.000 description 2
- 244000052637 human pathogen Species 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 125000004435 hydrogen atom Chemical class [H]* 0.000 description 2
- 235000010979 hydroxypropyl methyl cellulose Nutrition 0.000 description 2
- 239000001866 hydroxypropyl methyl cellulose Substances 0.000 description 2
- 229920003088 hydroxypropyl methyl cellulose Polymers 0.000 description 2
- UFVKGYZPFZQRLF-UHFFFAOYSA-N hydroxypropyl methyl cellulose Chemical compound OC1C(O)C(OC)OC(CO)C1OC1C(O)C(O)C(OC2C(C(O)C(OC3C(C(O)C(O)C(CO)O3)O)C(CO)O2)O)C(CO)O1 UFVKGYZPFZQRLF-UHFFFAOYSA-N 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 208000022760 infectious otitis media Diseases 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 230000002452 interceptive effect Effects 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 235000010445 lecithin Nutrition 0.000 description 2
- 239000000787 lecithin Substances 0.000 description 2
- 229940067606 lecithin Drugs 0.000 description 2
- 239000002502 liposome Substances 0.000 description 2
- 239000008297 liquid dosage form Substances 0.000 description 2
- 239000007937 lozenge Substances 0.000 description 2
- 239000000314 lubricant Substances 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 2
- 239000000594 mannitol Substances 0.000 description 2
- 235000010355 mannitol Nutrition 0.000 description 2
- VDXZNPDIRNWWCW-JFTDCZMZSA-N melittin Chemical compound NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(N)=O)CC1=CNC2=CC=CC=C12 VDXZNPDIRNWWCW-JFTDCZMZSA-N 0.000 description 2
- 239000004530 micro-emulsion Substances 0.000 description 2
- 244000005706 microflora Species 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
- 239000008267 milk Substances 0.000 description 2
- 210000004080 milk Anatomy 0.000 description 2
- 238000000465 moulding Methods 0.000 description 2
- 239000004309 nisin Substances 0.000 description 2
- 235000010297 nisin Nutrition 0.000 description 2
- 239000002674 ointment Substances 0.000 description 2
- 235000008390 olive oil Nutrition 0.000 description 2
- 244000039328 opportunistic pathogen Species 0.000 description 2
- 238000004091 panning Methods 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 238000002823 phage display Methods 0.000 description 2
- 229960002292 piperacillin Drugs 0.000 description 2
- WCMIIGXFCMNQDS-IDYPWDAWSA-M piperacillin sodium Chemical compound [Na+].O=C1C(=O)N(CC)CCN1C(=O)N[C@H](C=1C=CC=CC=1)C(=O)N[C@@H]1C(=O)N2[C@@H](C([O-])=O)C(C)(C)S[C@@H]21 WCMIIGXFCMNQDS-IDYPWDAWSA-M 0.000 description 2
- 229920005862 polyol Polymers 0.000 description 2
- 150000003077 polyols Chemical class 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- 150000004804 polysaccharides Chemical class 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 108010047804 ranalexin Proteins 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 238000004007 reversed phase HPLC Methods 0.000 description 2
- 239000008159 sesame oil Substances 0.000 description 2
- 235000011803 sesame oil Nutrition 0.000 description 2
- 235000002639 sodium chloride Nutrition 0.000 description 2
- 239000007909 solid dosage form Substances 0.000 description 2
- 239000008247 solid mixture Substances 0.000 description 2
- 239000007790 solid phase Substances 0.000 description 2
- 238000010532 solid phase synthesis reaction Methods 0.000 description 2
- 235000010356 sorbitol Nutrition 0.000 description 2
- 239000000600 sorbitol Substances 0.000 description 2
- UNFWWIHTNXNPBV-WXKVUWSESA-N spectinomycin Chemical compound O([C@@H]1[C@@H](NC)[C@@H](O)[C@H]([C@@H]([C@H]1O1)O)NC)[C@]2(O)[C@H]1O[C@H](C)CC2=O UNFWWIHTNXNPBV-WXKVUWSESA-N 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 239000006188 syrup Substances 0.000 description 2
- 235000020357 syrup Nutrition 0.000 description 2
- ZJQFYZCNRTZAIM-PMXBASNASA-N tachyplesin Chemical compound C([C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@H](C(N[C@H]2CSSC[C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC=3C=CC=CC=3)NC(=O)[C@@H](NC(=O)[C@H](CC=3C4=CC=CC=C4NC=3)NC(=O)[C@@H](N)CCCCN)CSSC[C@H](NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC=3C=CC(O)=CC=3)NC2=O)C(=O)N[C@@H](CCCNC(N)=N)C(N)=O)C(=O)N1)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 ZJQFYZCNRTZAIM-PMXBASNASA-N 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 230000000699 topical effect Effects 0.000 description 2
- 239000013638 trimer Substances 0.000 description 2
- 210000001635 urinary tract Anatomy 0.000 description 2
- 210000002700 urine Anatomy 0.000 description 2
- 210000001215 vagina Anatomy 0.000 description 2
- 230000002792 vascular Effects 0.000 description 2
- 230000035899 viability Effects 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- BVIYGXUQVXBHQS-IUYQGCFVSA-N (2R,4S)-2-methyltetrahydrofuran-2,3,3,4-tetrol Chemical compound C[C@@]1(O)OC[C@H](O)C1(O)O BVIYGXUQVXBHQS-IUYQGCFVSA-N 0.000 description 1
- UKVZSPHYQJNTOU-GQJPYGCMSA-N (2S)-6-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[2-[[(2S)-2-[[(2S)-1-[(2S)-2-[[(2S)-5-amino-2-[[(2S)-2-[[2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S,3R)-2-amino-3-hydroxybutanoyl]amino]-5-carbamimidamidopentanoyl]amino]-3-hydroxypropanoyl]amino]-3-hydroxypropanoyl]amino]-5-carbamimidamidopentanoyl]amino]propanoyl]amino]acetyl]amino]-4-methylpentanoyl]amino]-5-oxopentanoyl]amino]-3-phenylpropanoyl]pyrrolidine-2-carbonyl]amino]-3-methylbutanoyl]amino]acetyl]amino]-5-carbamimidamidopentanoyl]amino]-3-methylbutanoyl]amino]-3-(1H-imidazol-5-yl)propanoyl]amino]-5-carbamimidamidopentanoyl]amino]-4-methylpentanoyl]amino]-4-methylpentanoyl]amino]-5-carbamimidamidopentanoyl]amino]hexanoic acid Chemical compound C([C@H](NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H](N)[C@@H](C)O)CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(O)=O)C1=CC=CC=C1 UKVZSPHYQJNTOU-GQJPYGCMSA-N 0.000 description 1
- SWZCTMTWRHEBIN-QFIPXVFZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-(4-hydroxyphenyl)propanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)C1=CC=C(O)C=C1 SWZCTMTWRHEBIN-QFIPXVFZSA-N 0.000 description 1
- NSOITZIGPJTCMQ-KCXFZFQESA-N (4r,7s,10s,13s,16s,19s,22r)-22-[[(2s,3s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-1-[(2s)-2-[[(2s,3s)-2-[[(2s)-6-amino-2-[[(2s,3s)-2-[[(2s)-2-[[2-[[2-[[(2s)-2-[[(2s)-2-amino-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]acetyl]amino]acetyl]amino]-4-methylpentanoyl]a Chemical compound C([C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]1C(N[C@@H](C)C(=O)N[C@H](C(=O)N[C@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CSSC1)C(O)=O)[C@@H](C)O)C(C)C)=O)C1=CC=CC=C1 NSOITZIGPJTCMQ-KCXFZFQESA-N 0.000 description 1
- QYAPHLRPFNSDNH-MRFRVZCGSA-N (4s,4as,5as,6s,12ar)-7-chloro-4-(dimethylamino)-1,6,10,11,12a-pentahydroxy-6-methyl-3,12-dioxo-4,4a,5,5a-tetrahydrotetracene-2-carboxamide;hydrochloride Chemical compound Cl.C1=CC(Cl)=C2[C@](O)(C)[C@H]3C[C@H]4[C@H](N(C)C)C(=O)C(C(N)=O)=C(O)[C@@]4(O)C(=O)C3=C(O)C2=C1O QYAPHLRPFNSDNH-MRFRVZCGSA-N 0.000 description 1
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 description 1
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 1
- 229940058015 1,3-butylene glycol Drugs 0.000 description 1
- KGLPWQKSKUVKMJ-UHFFFAOYSA-N 2,3-dihydrophthalazine-1,4-dione Chemical class C1=CC=C2C(=O)NNC(=O)C2=C1 KGLPWQKSKUVKMJ-UHFFFAOYSA-N 0.000 description 1
- JNODDICFTDYODH-UHFFFAOYSA-N 2-hydroxytetrahydrofuran Chemical compound OC1CCCO1 JNODDICFTDYODH-UHFFFAOYSA-N 0.000 description 1
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 description 1
- OBWSOTREAMFOCQ-UHFFFAOYSA-N 4-(4-amino-3,5-dimethylphenyl)-2,6-dimethylaniline;hydrochloride Chemical compound Cl.CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 OBWSOTREAMFOCQ-UHFFFAOYSA-N 0.000 description 1
- XZKIHKMTEMTJQX-UHFFFAOYSA-N 4-Nitrophenyl Phosphate Chemical compound OP(O)(=O)OC1=CC=C([N+]([O-])=O)C=C1 XZKIHKMTEMTJQX-UHFFFAOYSA-N 0.000 description 1
- FJKROLUGYXJWQN-UHFFFAOYSA-N 4-hydroxybenzoic acid Chemical compound OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 1
- 102100031126 6-phosphogluconolactonase Human genes 0.000 description 1
- 108010029731 6-phosphogluconolactonase Proteins 0.000 description 1
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 description 1
- 101710110983 Abaecin Proteins 0.000 description 1
- 241000186046 Actinomyces Species 0.000 description 1
- 239000005995 Aluminium silicate Substances 0.000 description 1
- 244000153158 Ammi visnaga Species 0.000 description 1
- 235000010585 Ammi visnaga Nutrition 0.000 description 1
- WZPBZJONDBGPKJ-UHFFFAOYSA-N Antibiotic SQ 26917 Natural products O=C1N(S(O)(=O)=O)C(C)C1NC(=O)C(=NOC(C)(C)C(O)=O)C1=CSC(N)=N1 WZPBZJONDBGPKJ-UHFFFAOYSA-N 0.000 description 1
- 101710081722 Antitrypsin Proteins 0.000 description 1
- 235000003276 Apios tuberosa Nutrition 0.000 description 1
- 241000256844 Apis mellifera Species 0.000 description 1
- 244000105624 Arachis hypogaea Species 0.000 description 1
- 235000010777 Arachis hypogaea Nutrition 0.000 description 1
- 235000010744 Arachis villosulicarpa Nutrition 0.000 description 1
- 101710192393 Attachment protein G3P Proteins 0.000 description 1
- 208000004429 Bacillary Dysentery Diseases 0.000 description 1
- 208000031729 Bacteremia Diseases 0.000 description 1
- 108090000363 Bacterial Luciferases Proteins 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 101710125965 Bacteriocin leucocin-A Proteins 0.000 description 1
- 241000606125 Bacteroides Species 0.000 description 1
- 101710114744 Bombinin Proteins 0.000 description 1
- 208000009079 Bronchial Spasm Diseases 0.000 description 1
- 208000014181 Bronchial disease Diseases 0.000 description 1
- 206010006482 Bronchospasm Diseases 0.000 description 1
- 241000589513 Burkholderia cepacia Species 0.000 description 1
- 125000001433 C-terminal amino-acid group Chemical group 0.000 description 1
- 102100027557 Calcipressin-1 Human genes 0.000 description 1
- 241000589876 Campylobacter Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 1
- 101710115643 Cathelicidin-1 Proteins 0.000 description 1
- 108010022172 Chitinases Proteins 0.000 description 1
- 102000012286 Chitinases Human genes 0.000 description 1
- 241000606161 Chlamydia Species 0.000 description 1
- 206010008631 Cholera Diseases 0.000 description 1
- 101000749287 Clitocybe nebularis Clitocypin Proteins 0.000 description 1
- 101000767029 Clitocybe nebularis Clitocypin-1 Proteins 0.000 description 1
- 208000037384 Clostridium Infections Diseases 0.000 description 1
- 206010009657 Clostridium difficile colitis Diseases 0.000 description 1
- 206010054236 Clostridium difficile infection Diseases 0.000 description 1
- 206010010356 Congenital anomaly Diseases 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- 229920002785 Croscarmellose sodium Polymers 0.000 description 1
- 229940094664 Cysteine protease inhibitor Drugs 0.000 description 1
- 102400000361 DCD-1 Human genes 0.000 description 1
- 101800003946 DCD-1 Proteins 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- CYCGRDQQIOGCKX-UHFFFAOYSA-N Dehydro-luciferin Natural products OC(=O)C1=CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 CYCGRDQQIOGCKX-UHFFFAOYSA-N 0.000 description 1
- 235000019739 Dicalciumphosphate Nutrition 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- JWCSIUVGFCSJCK-CAVRMKNVSA-N Disodium Moxalactam Chemical compound N([C@]1(OC)C(N2C(=C(CSC=3N(N=NN=3)C)CO[C@@H]21)C(O)=O)=O)C(=O)C(C(O)=O)C1=CC=C(O)C=C1 JWCSIUVGFCSJCK-CAVRMKNVSA-N 0.000 description 1
- 208000000059 Dyspnea Diseases 0.000 description 1
- 206010013975 Dyspnoeas Diseases 0.000 description 1
- 238000004435 EPR spectroscopy Methods 0.000 description 1
- 241000588722 Escherichia Species 0.000 description 1
- 239000001856 Ethyl cellulose Substances 0.000 description 1
- ZZSNKZQZMQGXPY-UHFFFAOYSA-N Ethyl cellulose Chemical compound CCOCC1OC(OC)C(OCC)C(OCC)C1OC1C(O)C(O)C(OC)C(CO)O1 ZZSNKZQZMQGXPY-UHFFFAOYSA-N 0.000 description 1
- 229910052693 Europium Inorganic materials 0.000 description 1
- 208000010201 Exanthema Diseases 0.000 description 1
- 241000724791 Filamentous phage Species 0.000 description 1
- 239000004606 Fillers/Extenders Substances 0.000 description 1
- 108090000331 Firefly luciferases Proteins 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- BJGNCJDXODQBOB-UHFFFAOYSA-N Fivefly Luciferin Natural products OC(=O)C1CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 BJGNCJDXODQBOB-UHFFFAOYSA-N 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 108010015133 Galactose oxidase Proteins 0.000 description 1
- 229920001503 Glucan Polymers 0.000 description 1
- 108010073178 Glucan 1,4-alpha-Glucosidase Proteins 0.000 description 1
- 102100022624 Glucoamylase Human genes 0.000 description 1
- 239000004366 Glucose oxidase Substances 0.000 description 1
- 108010015776 Glucose oxidase Proteins 0.000 description 1
- 108010018962 Glucosephosphate Dehydrogenase Proteins 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- AEMRFAOFKBGASW-UHFFFAOYSA-N Glycolic acid Polymers OCC(O)=O AEMRFAOFKBGASW-UHFFFAOYSA-N 0.000 description 1
- 206010018612 Gonorrhoea Diseases 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 1
- 241000606790 Haemophilus Species 0.000 description 1
- 241000589989 Helicobacter Species 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 239000004705 High-molecular-weight polyethylene Substances 0.000 description 1
- 102400000777 His3-(20-43)-peptide Human genes 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000898505 Homo sapiens Histatin-3 Proteins 0.000 description 1
- 101000918983 Homo sapiens Neutrophil defensin 1 Proteins 0.000 description 1
- 101100247605 Homo sapiens RCAN1 gene Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 108700023483 L-lactate dehydrogenases Proteins 0.000 description 1
- 108010023244 Lactoperoxidase Proteins 0.000 description 1
- 102000045576 Lactoperoxidases Human genes 0.000 description 1
- 108090001090 Lectins Proteins 0.000 description 1
- 102000004856 Lectins Human genes 0.000 description 1
- 108010028275 Leukocyte Elastase Proteins 0.000 description 1
- 102000016799 Leukocyte elastase Human genes 0.000 description 1
- DDWFXDSYGUXRAY-UHFFFAOYSA-N Luciferin Natural products CCc1c(C)c(CC2NC(=O)C(=C2C=C)C)[nH]c1Cc3[nH]c4C(=C5/NC(CC(=O)O)C(C)C5CC(=O)O)CC(=O)c4c3C DDWFXDSYGUXRAY-UHFFFAOYSA-N 0.000 description 1
- 102000013460 Malate Dehydrogenase Human genes 0.000 description 1
- 108010026217 Malate Dehydrogenase Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 240000003183 Manihot esculenta Species 0.000 description 1
- 235000016735 Manihot esculenta subsp esculenta Nutrition 0.000 description 1
- 102000005741 Metalloproteases Human genes 0.000 description 1
- 108010006035 Metalloproteases Proteins 0.000 description 1
- 229920000168 Microcrystalline cellulose Polymers 0.000 description 1
- 241000588621 Moraxella Species 0.000 description 1
- 101710200033 Moricin Proteins 0.000 description 1
- 206010065764 Mucosal infection Diseases 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- GXCLVBGFBYZDAG-UHFFFAOYSA-N N-[2-(1H-indol-3-yl)ethyl]-N-methylprop-2-en-1-amine Chemical compound CN(CCC1=CNC2=C1C=CC=C2)CC=C GXCLVBGFBYZDAG-UHFFFAOYSA-N 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 240000007817 Olea europaea Species 0.000 description 1
- 239000005642 Oleic acid Substances 0.000 description 1
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 description 1
- 206010033078 Otitis media Diseases 0.000 description 1
- 235000019483 Peanut oil Nutrition 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 208000029082 Pelvic Inflammatory Disease Diseases 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 208000037581 Persistent Infection Diseases 0.000 description 1
- 108010004729 Phycoerythrin Proteins 0.000 description 1
- 241000269908 Platichthys flesus Species 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 1
- 239000004952 Polyamide Substances 0.000 description 1
- 229920002732 Polyanhydride Polymers 0.000 description 1
- 229920000954 Polyglycolide Polymers 0.000 description 1
- 229920001710 Polyorthoester Polymers 0.000 description 1
- 241000605862 Porphyromonas gingivalis Species 0.000 description 1
- 208000032536 Pseudomonas Infections Diseases 0.000 description 1
- 241000589755 Pseudomonas mendocina Species 0.000 description 1
- 208000035415 Reinfection Diseases 0.000 description 1
- 206010057190 Respiratory tract infections Diseases 0.000 description 1
- 235000004443 Ricinus communis Nutrition 0.000 description 1
- 241000606701 Rickettsia Species 0.000 description 1
- 235000019485 Safflower oil Nutrition 0.000 description 1
- 206010039438 Salmonella Infections Diseases 0.000 description 1
- 241001354013 Salmonella enterica subsp. enterica serovar Enteritidis Species 0.000 description 1
- 235000017276 Salvia Nutrition 0.000 description 1
- 240000007164 Salvia officinalis Species 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 229940122055 Serine protease inhibitor Drugs 0.000 description 1
- 101710102218 Serine protease inhibitor Proteins 0.000 description 1
- 108010071390 Serum Albumin Proteins 0.000 description 1
- 102000007562 Serum Albumin Human genes 0.000 description 1
- 208000019802 Sexually transmitted disease Diseases 0.000 description 1
- 241000607764 Shigella dysenteriae Species 0.000 description 1
- 206010040550 Shigella infections Diseases 0.000 description 1
- 241000700584 Simplexvirus Species 0.000 description 1
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 241000565302 Sphyrapicus Species 0.000 description 1
- 241000589970 Spirochaetales Species 0.000 description 1
- 206010051017 Staphylococcal bacteraemia Diseases 0.000 description 1
- 241000191963 Staphylococcus epidermidis Species 0.000 description 1
- 241001147691 Staphylococcus saprophyticus Species 0.000 description 1
- 208000007107 Stomach Ulcer Diseases 0.000 description 1
- 241000194017 Streptococcus Species 0.000 description 1
- 241001147754 Streptococcus gordonii str. Challis Species 0.000 description 1
- 241001134658 Streptococcus mitis Species 0.000 description 1
- 241000193987 Streptococcus sobrinus Species 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- 241000255588 Tephritidae Species 0.000 description 1
- WKDDRNSBRWANNC-UHFFFAOYSA-N Thienamycin Natural products C1C(SCCN)=C(C(O)=O)N2C(=O)C(C(O)C)C21 WKDDRNSBRWANNC-UHFFFAOYSA-N 0.000 description 1
- 206010044248 Toxic shock syndrome Diseases 0.000 description 1
- 231100000650 Toxic shock syndrome Toxicity 0.000 description 1
- 208000005448 Trichomonas Infections Diseases 0.000 description 1
- 206010044620 Trichomoniasis Diseases 0.000 description 1
- 108010092464 Urate Oxidase Proteins 0.000 description 1
- 108010046334 Urease Proteins 0.000 description 1
- 238000005411 Van der Waals force Methods 0.000 description 1
- 101000859429 Vespa magnifica Vespid chemotactic peptide 5h Proteins 0.000 description 1
- 241000607626 Vibrio cholerae Species 0.000 description 1
- 201000007096 Vulvovaginal Candidiasis Diseases 0.000 description 1
- 239000003875 Wang resin Substances 0.000 description 1
- 108010093894 Xanthine oxidase Proteins 0.000 description 1
- 102100033220 Xanthine oxidase Human genes 0.000 description 1
- 101500009721 Xenopus laevis Magainin-2 Proteins 0.000 description 1
- 101000998548 Yersinia ruckeri Alkaline proteinase inhibitor Proteins 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 239000003655 absorption accelerator Substances 0.000 description 1
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000004103 aerobic respiration Effects 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- 235000012211 aluminium silicate Nutrition 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 230000000675 anti-caries Effects 0.000 description 1
- 230000001475 anti-trypsic effect Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 239000003429 antifungal agent Substances 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 238000011203 antimicrobial therapy Methods 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 239000007900 aqueous suspension Substances 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- WZPBZJONDBGPKJ-VEHQQRBSSA-N aztreonam Chemical compound O=C1N(S([O-])(=O)=O)[C@@H](C)[C@@H]1NC(=O)C(=N/OC(C)(C)C(O)=O)\C1=CSC([NH3+])=N1 WZPBZJONDBGPKJ-VEHQQRBSSA-N 0.000 description 1
- 229960003644 aztreonam Drugs 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- PXXJHWLDUBFPOL-UHFFFAOYSA-N benzamidine Chemical compound NC(=N)C1=CC=CC=C1 PXXJHWLDUBFPOL-UHFFFAOYSA-N 0.000 description 1
- 229960002903 benzyl benzoate Drugs 0.000 description 1
- 239000003139 biocide Substances 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000036760 body temperature Effects 0.000 description 1
- 235000008429 bread Nutrition 0.000 description 1
- 238000000339 bright-field microscopy Methods 0.000 description 1
- 239000006172 buffering agent Substances 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 108010025307 buforin II Proteins 0.000 description 1
- 239000001273 butane Substances 0.000 description 1
- 235000019437 butane-1,3-diol Nutrition 0.000 description 1
- 230000000981 bystander Effects 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 235000010216 calcium carbonate Nutrition 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 235000012241 calcium silicate Nutrition 0.000 description 1
- CJZGTCYPCWQAJB-UHFFFAOYSA-L calcium stearate Chemical compound [Ca+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O CJZGTCYPCWQAJB-UHFFFAOYSA-L 0.000 description 1
- 235000013539 calcium stearate Nutrition 0.000 description 1
- 239000008116 calcium stearate Substances 0.000 description 1
- 229960003669 carbenicillin Drugs 0.000 description 1
- FPPNZSSZRUTDAP-UWFZAAFLSA-N carbenicillin Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)C(C(O)=O)C1=CC=CC=C1 FPPNZSSZRUTDAP-UWFZAAFLSA-N 0.000 description 1
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 1
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 1
- 239000004359 castor oil Substances 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- CZTQZXZIADLWOZ-CRAIPNDOSA-N cefaloridine Chemical compound O=C([C@@H](NC(=O)CC=1SC=CC=1)[C@H]1SC2)N1C(C(=O)[O-])=C2C[N+]1=CC=CC=C1 CZTQZXZIADLWOZ-CRAIPNDOSA-N 0.000 description 1
- 229960003866 cefaloridine Drugs 0.000 description 1
- 229960000603 cefalotin Drugs 0.000 description 1
- GCFBRXLSHGKWDP-XCGNWRKASA-N cefoperazone Chemical compound O=C1C(=O)N(CC)CCN1C(=O)N[C@H](C=1C=CC(O)=CC=1)C(=O)N[C@@H]1C(=O)N2C(C(O)=O)=C(CSC=3N(N=NN=3)C)CS[C@@H]21 GCFBRXLSHGKWDP-XCGNWRKASA-N 0.000 description 1
- 229960004682 cefoperazone Drugs 0.000 description 1
- 229960000484 ceftazidime Drugs 0.000 description 1
- ORFOPKXBNMVMKC-DWVKKRMSSA-N ceftazidime Chemical compound S([C@@H]1[C@@H](C(N1C=1C([O-])=O)=O)NC(=O)\C(=N/OC(C)(C)C(O)=O)C=2N=C(N)SC=2)CC=1C[N+]1=CC=CC=C1 ORFOPKXBNMVMKC-DWVKKRMSSA-N 0.000 description 1
- 229960004755 ceftriaxone Drugs 0.000 description 1
- VAAUVRVFOQPIGI-SPQHTLEESA-N ceftriaxone Chemical compound S([C@@H]1[C@@H](C(N1C=1C(O)=O)=O)NC(=O)\C(=N/OC)C=2N=C(N)SC=2)CC=1CSC1=NC(=O)C(=O)NN1C VAAUVRVFOQPIGI-SPQHTLEESA-N 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 238000003570 cell viability assay Methods 0.000 description 1
- 230000035567 cellular accumulation Effects 0.000 description 1
- 230000004700 cellular uptake Effects 0.000 description 1
- 229920002301 cellulose acetate Polymers 0.000 description 1
- VUFGUVLLDPOSBC-XRZFDKQNSA-M cephalothin sodium Chemical compound [Na+].N([C@H]1[C@@H]2N(C1=O)C(=C(CS2)COC(=O)C)C([O-])=O)C(=O)CC1=CC=CS1 VUFGUVLLDPOSBC-XRZFDKQNSA-M 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- UKVZSPHYQJNTOU-IVBHRGSNSA-N chembl1240717 Chemical compound C([C@H](NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H](N)[C@H](C)O)CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(O)=O)C1=CC=CC=C1 UKVZSPHYQJNTOU-IVBHRGSNSA-N 0.000 description 1
- XVVTWELITATBSB-UEDJBKKJSA-N chembl3221603 Chemical compound C([C@H](NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)[C@H](CCCNC(N)=N)NC(=O)CNC(=O)CNC(=O)[C@@H](N)CCCNC(N)=N)CC(C)C)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C1=CC=C(O)C=C1 XVVTWELITATBSB-UEDJBKKJSA-N 0.000 description 1
- 235000015218 chewing gum Nutrition 0.000 description 1
- 229960004926 chlorobutanol Drugs 0.000 description 1
- 150000005827 chlorofluoro hydrocarbons Chemical class 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 108010043681 clavanin A Proteins 0.000 description 1
- 239000004927 clay Substances 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 229960003326 cloxacillin Drugs 0.000 description 1
- LQOLIRLGBULYKD-JKIFEVAISA-N cloxacillin Chemical compound N([C@@H]1C(N2[C@H](C(C)(C)S[C@@H]21)C(O)=O)=O)C(=O)C1=C(C)ON=C1C1=CC=CC=C1Cl LQOLIRLGBULYKD-JKIFEVAISA-N 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 238000002648 combination therapy Methods 0.000 description 1
- 239000007891 compressed tablet Substances 0.000 description 1
- 238000007906 compression Methods 0.000 description 1
- 230000006835 compression Effects 0.000 description 1
- 230000001010 compromised effect Effects 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 230000001268 conjugating effect Effects 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000013270 controlled release Methods 0.000 description 1
- 235000014510 cooky Nutrition 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 239000002385 cottonseed oil Substances 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 235000010947 crosslinked sodium carboxy methyl cellulose Nutrition 0.000 description 1
- 239000001767 crosslinked sodium carboxy methyl cellulose Substances 0.000 description 1
- 101150064416 csp1 gene Proteins 0.000 description 1
- 229940127089 cytotoxic agent Drugs 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 125000001295 dansyl group Chemical group [H]C1=C([H])C(N(C([H])([H])[H])C([H])([H])[H])=C2C([H])=C([H])C([H])=C(C2=C1[H])S(*)(=O)=O 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 239000004053 dental implant Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- NEFBYIFKOOEVPA-UHFFFAOYSA-K dicalcium phosphate Chemical compound [Ca+2].[Ca+2].[O-]P([O-])([O-])=O NEFBYIFKOOEVPA-UHFFFAOYSA-K 0.000 description 1
- 229940038472 dicalcium phosphate Drugs 0.000 description 1
- 229910000390 dicalcium phosphate Inorganic materials 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 230000000378 dietary effect Effects 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- UGMCXQCYOVCMTB-UHFFFAOYSA-K dihydroxy(stearato)aluminium Chemical compound CCCCCCCCCCCCCCCCCC(=O)O[Al](O)O UGMCXQCYOVCMTB-UHFFFAOYSA-K 0.000 description 1
- 230000003292 diminished effect Effects 0.000 description 1
- 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 1
- 239000007884 disintegrant Substances 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- ICZZXLGKHXWNSU-ULVOZOJHSA-N drosocin Chemical compound NC(=N)NCCC[C@@H](C(=O)N[C@@H](C(C)C)C(O)=O)NC(=O)[C@H](C(C)CC)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H]1N(C(=O)[C@H](CC=2N=CNC=2)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H]2N(CCC2)C(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H]2N(CCC2)C(=O)[C@H](CO)NC(=O)[C@H](CC=2C=CC(O)=CC=2)NC(=O)[C@H]2N(CCC2)C(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H]2N(CCC2)C(=O)[C@H](CCCCN)NC(=O)CN)C(C)O[C@@H]2[C@@H]([C@@H](O[C@@H]3[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O3)O)[C@@H](O)[C@@H](CO)O2)NC(C)=O)CCC1 ICZZXLGKHXWNSU-ULVOZOJHSA-N 0.000 description 1
- 208000000718 duodenal ulcer Diseases 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 210000000959 ear middle Anatomy 0.000 description 1
- 230000001804 emulsifying effect Effects 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 239000002702 enteric coating Substances 0.000 description 1
- 238000009505 enteric coating Methods 0.000 description 1
- 230000000688 enterotoxigenic effect Effects 0.000 description 1
- 230000007515 enzymatic degradation Effects 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- BEFDCLMNVWHSGT-UHFFFAOYSA-N ethenylcyclopentane Chemical compound C=CC1CCCC1 BEFDCLMNVWHSGT-UHFFFAOYSA-N 0.000 description 1
- 229940093499 ethyl acetate Drugs 0.000 description 1
- 235000019325 ethyl cellulose Nutrition 0.000 description 1
- 229920001249 ethyl cellulose Polymers 0.000 description 1
- OGPBJKLSAFTDLK-UHFFFAOYSA-N europium atom Chemical compound [Eu] OGPBJKLSAFTDLK-UHFFFAOYSA-N 0.000 description 1
- 230000005713 exacerbation Effects 0.000 description 1
- 201000005884 exanthem Diseases 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 239000003172 expectorant agent Substances 0.000 description 1
- 239000003885 eye ointment Substances 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 210000003495 flagella Anatomy 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 238000002073 fluorescence micrograph Methods 0.000 description 1
- 238000001215 fluorescent labelling Methods 0.000 description 1
- NBVXSUQYWXRMNV-UHFFFAOYSA-N fluoromethane Chemical compound FC NBVXSUQYWXRMNV-UHFFFAOYSA-N 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- 235000012631 food intake Nutrition 0.000 description 1
- 235000013350 formula milk Nutrition 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 239000007903 gelatin capsule Substances 0.000 description 1
- 208000007565 gingivitis Diseases 0.000 description 1
- 108090001082 glucan-binding proteins Proteins 0.000 description 1
- 229940116332 glucose oxidase Drugs 0.000 description 1
- 235000019420 glucose oxidase Nutrition 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- YQEMORVAKMFKLG-UHFFFAOYSA-N glycerine monostearate Natural products CCCCCCCCCCCCCCCCCC(=O)OC(CO)CO YQEMORVAKMFKLG-UHFFFAOYSA-N 0.000 description 1
- SVUQHVRAGMNPLW-UHFFFAOYSA-N glycerol monostearate Natural products CCCCCCCCCCCCCCCCC(=O)OCC(O)CO SVUQHVRAGMNPLW-UHFFFAOYSA-N 0.000 description 1
- 150000002334 glycols Chemical class 0.000 description 1
- NRRWQFGMQBIGRJ-AXGRSTHOSA-N glycyl-l-lysyl-l-prolyl-l-arginyl-l-prolyl-l-tyrosyl-l-seryl-l-prolyl-l-arginyl-l-prolyl-l-threonyl-l-seryl-l-histidyl-l-prolyl-l-arginyl-l-prolyl-l-isoleucyl-l-arginyl-l-valine Chemical compound NC(=N)NCCC[C@@H](C(=O)N[C@@H](C(C)C)C(O)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H]1N(C(=O)[C@H](CC=2NC=NC=2)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H]2N(CCC2)C(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H]2N(CCC2)C(=O)[C@H](CO)NC(=O)[C@H](CC=2C=CC(O)=CC=2)NC(=O)[C@H]2N(CCC2)C(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H]2N(CCC2)C(=O)[C@H](CCCCN)NC(=O)CN)[C@@H](C)O)CCC1 NRRWQFGMQBIGRJ-AXGRSTHOSA-N 0.000 description 1
- 208000001786 gonorrhea Diseases 0.000 description 1
- 239000005090 green fluorescent protein Substances 0.000 description 1
- 238000001631 haemodialysis Methods 0.000 description 1
- 210000003709 heart valve Anatomy 0.000 description 1
- 230000000322 hemodialysis Effects 0.000 description 1
- 125000000623 heterocyclic group Chemical group 0.000 description 1
- 102000018474 human neutrophil peptide 1 Human genes 0.000 description 1
- 239000003906 humectant Substances 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 150000002430 hydrocarbons Chemical class 0.000 description 1
- 125000001165 hydrophobic group Chemical group 0.000 description 1
- 229960002182 imipenem Drugs 0.000 description 1
- ZSKVGTPCRGIANV-ZXFLCMHBSA-N imipenem Chemical compound C1C(SCC\N=C\N)=C(C(O)=O)N2C(=O)[C@H]([C@H](O)C)[C@H]21 ZSKVGTPCRGIANV-ZXFLCMHBSA-N 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 206010022000 influenza Diseases 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 239000007972 injectable composition Substances 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 239000002198 insoluble material Substances 0.000 description 1
- 238000009830 intercalation Methods 0.000 description 1
- 230000002687 intercalation Effects 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 238000001361 intraarterial administration Methods 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000007913 intrathecal administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 230000002262 irrigation Effects 0.000 description 1
- 238000003973 irrigation Methods 0.000 description 1
- 230000007794 irritation Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 description 1
- 239000000644 isotonic solution Substances 0.000 description 1
- 239000007951 isotonicity adjuster Substances 0.000 description 1
- NLYAJNPCOHFWQQ-UHFFFAOYSA-N kaolin Chemical compound O.O.O=[Al]O[Si](=O)O[Si](=O)O[Al]=O NLYAJNPCOHFWQQ-UHFFFAOYSA-N 0.000 description 1
- 229940057428 lactoperoxidase Drugs 0.000 description 1
- 229960000433 latamoxef Drugs 0.000 description 1
- 239000002523 lectin Substances 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- JCCNYMKQOSZNPW-UHFFFAOYSA-N loratadine Chemical compound C1CN(C(=O)OCC)CCC1=C1C2=NC=CC=C2CCC2=CC(Cl)=CC=C21 JCCNYMKQOSZNPW-UHFFFAOYSA-N 0.000 description 1
- 239000006210 lotion Substances 0.000 description 1
- 101150078797 luxS gene Proteins 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- MGIUUAHJVPPFEV-ABXDCCGRSA-N magainin ii Chemical compound C([C@H](NC(=O)[C@H](CCCCN)NC(=O)CNC(=O)[C@@H](NC(=O)CN)[C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O)C1=CC=CC=C1 MGIUUAHJVPPFEV-ABXDCCGRSA-N 0.000 description 1
- VTHJTEIRLNZDEV-UHFFFAOYSA-L magnesium dihydroxide Chemical compound [OH-].[OH-].[Mg+2] VTHJTEIRLNZDEV-UHFFFAOYSA-L 0.000 description 1
- 239000000347 magnesium hydroxide Substances 0.000 description 1
- 229910001862 magnesium hydroxide Inorganic materials 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000001869 matrix assisted laser desorption--ionisation mass spectrum Methods 0.000 description 1
- 229960002260 meropenem Drugs 0.000 description 1
- DMJNNHOOLUXYBV-PQTSNVLCSA-N meropenem Chemical compound C=1([C@H](C)[C@@H]2[C@H](C(N2C=1C(O)=O)=O)[C@H](O)C)S[C@@H]1CN[C@H](C(=O)N(C)C)C1 DMJNNHOOLUXYBV-PQTSNVLCSA-N 0.000 description 1
- 239000003475 metalloproteinase inhibitor Substances 0.000 description 1
- 230000001394 metastastic effect Effects 0.000 description 1
- 206010061289 metastatic neoplasm Diseases 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 239000003094 microcapsule Substances 0.000 description 1
- 235000019813 microcrystalline cellulose Nutrition 0.000 description 1
- 239000008108 microcrystalline cellulose Substances 0.000 description 1
- 229940016286 microcrystalline cellulose Drugs 0.000 description 1
- 108010029942 microperoxidase Proteins 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 108010070373 modelin 1 Proteins 0.000 description 1
- 108091005601 modified peptides Proteins 0.000 description 1
- 239000007932 molded tablet Substances 0.000 description 1
- 230000004001 molecular interaction Effects 0.000 description 1
- 238000009343 monoculture Methods 0.000 description 1
- CQDGTJPVBWZJAZ-UHFFFAOYSA-N monoethyl carbonate Chemical compound CCOC(O)=O CQDGTJPVBWZJAZ-UHFFFAOYSA-N 0.000 description 1
- 229940051866 mouthwash Drugs 0.000 description 1
- 229940066491 mucolytics Drugs 0.000 description 1
- 210000004400 mucous membrane Anatomy 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- UPSFMJHZUCSEHU-JYGUBCOQSA-N n-[(2s,3r,4r,5s,6r)-2-[(2r,3s,4r,5r,6s)-5-acetamido-4-hydroxy-2-(hydroxymethyl)-6-(4-methyl-2-oxochromen-7-yl)oxyoxan-3-yl]oxy-4,5-dihydroxy-6-(hydroxymethyl)oxan-3-yl]acetamide Chemical compound CC(=O)N[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@H]1[C@H](O)[C@@H](NC(C)=O)[C@H](OC=2C=C3OC(=O)C=C(C)C3=CC=2)O[C@@H]1CO UPSFMJHZUCSEHU-JYGUBCOQSA-N 0.000 description 1
- IJDNQMDRQITEOD-UHFFFAOYSA-N n-butane Chemical compound CCCC IJDNQMDRQITEOD-UHFFFAOYSA-N 0.000 description 1
- OFBQJSOFQDEBGM-UHFFFAOYSA-N n-pentane Natural products CCCCC OFBQJSOFQDEBGM-UHFFFAOYSA-N 0.000 description 1
- 230000003533 narcotic effect Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 208000004235 neutropenia Diseases 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 231100000344 non-irritating Toxicity 0.000 description 1
- 239000012457 nonaqueous media Substances 0.000 description 1
- 239000002736 nonionic surfactant Substances 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 1
- 235000021313 oleic acid Nutrition 0.000 description 1
- 150000002895 organic esters Chemical class 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 235000010603 pastilles Nutrition 0.000 description 1
- 239000000312 peanut oil Substances 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 230000007030 peptide scission Effects 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 239000002304 perfume Substances 0.000 description 1
- 208000028169 periodontal disease Diseases 0.000 description 1
- 229940021222 peritoneal dialysis isotonic solution Drugs 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- ZIJCGKYVZPZELZ-SSZNUFPPSA-N pgya Chemical compound C([C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@@H](NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(C)C)NC(=O)CN)[C@@H](C)CC)C1=CC=CC=C1 ZIJCGKYVZPZELZ-SSZNUFPPSA-N 0.000 description 1
- 230000003285 pharmacodynamic effect Effects 0.000 description 1
- WVDDGKGOMKODPV-ZQBYOMGUSA-N phenyl(114C)methanol Chemical compound O[14CH2]C1=CC=CC=C1 WVDDGKGOMKODPV-ZQBYOMGUSA-N 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- ZPWVBRGFVGXRKZ-DMZHRTQBSA-N pmap-23 Chemical compound C([C@H](NC(=O)[C@H](CCCCN)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](NC(=O)[C@@H](N)CCCNC(N)=N)[C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(N)=O)C1=CC=CC=C1 ZPWVBRGFVGXRKZ-DMZHRTQBSA-N 0.000 description 1
- 229920001983 poloxamer Polymers 0.000 description 1
- 229920002647 polyamide Polymers 0.000 description 1
- 239000004417 polycarbonate Substances 0.000 description 1
- 229920000515 polycarbonate Polymers 0.000 description 1
- 229920001296 polysiloxane Polymers 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 108010027884 porcine myeloid antibacterial peptide 23 Proteins 0.000 description 1
- 229920001592 potato starch Polymers 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 230000002028 premature Effects 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 239000006041 probiotic Substances 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 239000001294 propane Substances 0.000 description 1
- 229960004063 propylene glycol Drugs 0.000 description 1
- 235000013772 propylene glycol Nutrition 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 108010032966 protegrin-1 Proteins 0.000 description 1
- 238000000159 protein binding assay Methods 0.000 description 1
- 230000006337 proteolytic cleavage Effects 0.000 description 1
- 230000002685 pulmonary effect Effects 0.000 description 1
- 230000009325 pulmonary function Effects 0.000 description 1
- 229940107568 pulmozyme Drugs 0.000 description 1
- 150000003856 quaternary ammonium compounds Chemical class 0.000 description 1
- 229910052761 rare earth metal Inorganic materials 0.000 description 1
- 150000002910 rare earth metals Chemical class 0.000 description 1
- 206010037844 rash Diseases 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 210000000664 rectum Anatomy 0.000 description 1
- 230000000306 recurrent effect Effects 0.000 description 1
- 108010054624 red fluorescent protein Proteins 0.000 description 1
- 210000005000 reproductive tract Anatomy 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 208000020029 respiratory tract infectious disease Diseases 0.000 description 1
- 230000004043 responsiveness Effects 0.000 description 1
- 239000003340 retarding agent Substances 0.000 description 1
- 230000000979 retarding effect Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 1
- JQXXHWHPUNPDRT-WLSIYKJHSA-N rifampicin Chemical compound O([C@](C1=O)(C)O/C=C/[C@@H]([C@H]([C@@H](OC(C)=O)[C@H](C)[C@H](O)[C@H](C)[C@@H](O)[C@@H](C)\C=C\C=C(C)/C(=O)NC=2C(O)=C3C([O-])=C4C)C)OC)C4=C1C3=C(O)C=2\C=N\N1CC[NH+](C)CC1 JQXXHWHPUNPDRT-WLSIYKJHSA-N 0.000 description 1
- 229960001225 rifampicin Drugs 0.000 description 1
- 235000005713 safflower oil Nutrition 0.000 description 1
- 239000003813 safflower oil Substances 0.000 description 1
- YGSDEFSMJLZEOE-UHFFFAOYSA-M salicylate Chemical compound OC1=CC=CC=C1C([O-])=O YGSDEFSMJLZEOE-UHFFFAOYSA-M 0.000 description 1
- 229960001860 salicylate Drugs 0.000 description 1
- 206010039447 salmonellosis Diseases 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000003345 scintillation counting Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 230000036303 septic shock Effects 0.000 description 1
- 239000003001 serine protease inhibitor Substances 0.000 description 1
- 238000007493 shaping process Methods 0.000 description 1
- 229940007046 shigella dysenteriae Drugs 0.000 description 1
- 201000005113 shigellosis Diseases 0.000 description 1
- 150000004760 silicates Chemical class 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 231100000046 skin rash Toxicity 0.000 description 1
- WTJDAUWOECZENF-OZWITMHCSA-N smap-29 Chemical compound NC(=O)[C@H](C)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@@H]1CCCN1C(=O)CNC(=O)[C@@H](NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCNC(N)=N)NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@@H](N)CCCNC(N)=N)[C@@H](C)CC)C(C)C)CC1=CC=C(O)C=C1 WTJDAUWOECZENF-OZWITMHCSA-N 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 description 1
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 1
- 239000008109 sodium starch glycolate Substances 0.000 description 1
- 229940079832 sodium starch glycolate Drugs 0.000 description 1
- 229920003109 sodium starch glycolate Polymers 0.000 description 1
- 210000004872 soft tissue Anatomy 0.000 description 1
- 235000010199 sorbic acid Nutrition 0.000 description 1
- 239000004334 sorbic acid Substances 0.000 description 1
- 229940075582 sorbic acid Drugs 0.000 description 1
- 239000003549 soybean oil Substances 0.000 description 1
- 235000012424 soybean oil Nutrition 0.000 description 1
- 238000009987 spinning Methods 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 239000003206 sterilizing agent Substances 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 150000005846 sugar alcohols Chemical class 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 238000013268 sustained release Methods 0.000 description 1
- 239000012730 sustained-release form Substances 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 208000006379 syphilis Diseases 0.000 description 1
- 229960001114 temocillin Drugs 0.000 description 1
- BVCKFLJARNKCSS-DWPRYXJFSA-N temocillin Chemical compound N([C@]1(OC)C(N2[C@H](C(C)(C)S[C@@H]21)C(O)=O)=O)C(=O)C(C(O)=O)C=1C=CSC=1 BVCKFLJARNKCSS-DWPRYXJFSA-N 0.000 description 1
- MPLHNVLQVRSVEE-UHFFFAOYSA-N texas red Chemical compound [O-]S(=O)(=O)C1=CC(S(Cl)(=O)=O)=CC=C1C(C1=CC=2CCCN3CCCC(C=23)=C1O1)=C2C1=C(CCC1)C3=[N+]1CCCC3=C2 MPLHNVLQVRSVEE-UHFFFAOYSA-N 0.000 description 1
- WROMPOXWARCANT-UHFFFAOYSA-N tfa trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.OC(=O)C(F)(F)F WROMPOXWARCANT-UHFFFAOYSA-N 0.000 description 1
- 239000002562 thickening agent Substances 0.000 description 1
- 229960004659 ticarcillin Drugs 0.000 description 1
- OHKOGUYZJXTSFX-KZFFXBSXSA-N ticarcillin Chemical compound C=1([C@@H](C(O)=O)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)C=CSC=1 OHKOGUYZJXTSFX-KZFFXBSXSA-N 0.000 description 1
- 238000010610 time kill assay Methods 0.000 description 1
- 239000000606 toothpaste Substances 0.000 description 1
- 229940034610 toothpaste Drugs 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- 210000003437 trachea Anatomy 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000008733 trauma Effects 0.000 description 1
- 239000002753 trypsin inhibitor Substances 0.000 description 1
- 208000019206 urinary tract infection Diseases 0.000 description 1
- 239000002966 varnish Substances 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 235000019871 vegetable fat Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 230000002861 ventricular Effects 0.000 description 1
- 229940118696 vibrio cholerae Drugs 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
- 229940126572 wide-spectrum antibiotic Drugs 0.000 description 1
- 108091005957 yellow fluorescent proteins Proteins 0.000 description 1
- 239000011787 zinc oxide Substances 0.000 description 1
- 235000014692 zinc oxide Nutrition 0.000 description 1
- KRJOFJHOZZPBKI-KSWODRSDSA-N α-defensin-1 Chemical compound C([C@H]1C(=O)N[C@H]2CSSC[C@H]3C(=O)N[C@H](C(N[C@@H](C)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)NCC(=O)N[C@H](C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC=4C=CC(O)=CC=4)NC(=O)[C@H](CSSC[C@H](NC2=O)C(O)=O)NC(=O)[C@H](C)N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](C)C(=O)N3)C(=O)N[C@H](C(=O)N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=2C3=CC=CC=C3NC=2)C(=O)N[C@@H](C)C(=O)N1)[C@@H](C)CC)[C@@H](C)O)=O)[C@@H](C)CC)C1=CC=CC=C1 KRJOFJHOZZPBKI-KSWODRSDSA-N 0.000 description 1
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
SELECTIVELY TARGETED ANTIMICROBIAL PEPTIDES AND THE USE The present invention relates to targeting peptides capable of specifically binding to microbial organisms (e.g., P. aeruginosa or S. mutans), antimicrobial peptides having antimicrobial activities, and specifically/selectively targeted antimicrobial peptides (STAMPs). In addition, the present invention provides methods of selectively killing or inhibiting microbial organisms by using the peptides or compositions provided by the present invention.
Description
SELECTIVELY TARGETED ANTIMICROBIAL PEPTIDES AND THE USE 2013204065 11 Apr 2013
THEREOF
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. Provisional Application Serial No. 60/842,871, filed on September 6, 2006, which is incorporated herein by reference.
FIELD OF INVENTION
[0002] The present invention relates to the field of antimicrobial compositions and treatment.
BACKGROUND OF THE INVENTION
[0003] The indigenous microflora found at human mucosal surfaces are critical for acquiring nutrients and providing protective colonization against pathogenic microorganisms. When the normal flora are disrupted by any number of factors, the result is often microbial infections at the mucosal surface, many of which affect populations worldwide. The lack of a robust immune response at mucosal surfaces has limited the prescribing clinician to conventional antibiotics or antimicrobials for treatment of mucosal infections. Unfortunately for the normal flora, most small molecule antibiotics have broad spectrum of activity, killing benign and pathogenic organisms indiscriminately. This effect often leads to severe antibiotic associated infections due to the vacated niche available for pathogen colonization. Clostridium difficile, Candida albicans and Staphylococcus aureus are examples of classical opportunistic pathogens that take advantage of increased niche size after antibiotic treatment. The problems resulting from wide-spectrum antibiotic use, combined with the emergence of drug-resistant strains, highlight the fundamental need for new “targeted” antibiotic therapies to combat mucosal pathogens with a minimal impact on normal microflora. 1 2 2013204065 13 Apr 2016 [0004] Previous efforts toward achieving target-specific antimicrobial therapy consisted of conjugating antibiotics to monoclonal antibodies or constructing large fusion proteins with bactericidal and bacterial recognition domains (Qiu et aL 2005). Neither method has yet to result in functional, effective therapeutics due to the low efficiency of chemical conjugation, instability of large proteins, or high cost of production.
[0005] Although GIOKHc, a specifically targeted antimicrobial peptide (STAMP), has been developed and demonstrated increased killing potency, selectivity and kinetics against targeted bacteria (Eckert et al„ 2006), there is a need to develop novel STAMPs that are capable of specifically or selectively killing or inhibiting the growth of undesirable target microorganisms.
Summary of the Invention [0006] One aspect of the present invention relates to a selectively/specifically targeted antimicrobial peptide (STAMP) which comprises a targeting peptide and an antimicrobial peptide. The STAMP further comprises a linker peptide.
[0007] In one embodiment, the targeting peptide is selected from the group consisting of C16 or CSPCK>(TFFRLFNRSFTQALGK, SEQ ID NO. 2), M8 or CSPM* (TFFRLFNR, SEQ ID NO 5), and peptide 1903 (NIFEYFLE, SEQ ID NO 10).
[0008] In another embodiment, the linker peptide is selected from the group consisting of GGG (SEQ ID NO 17 ), AAA (SEQ ID NO 18), SAT (SEQ ID NO 19), ASA (SEQ ID NO 20), SGG (SEQ ID NO 21), PYP (SEQ ID NO 22), SGS (SEQ ID NO 23), GGS (SEQ ID NO 24), SPS(SEQ ID NO 25), PSGSP(SEQ ID NO 26), PSPSP(SEQ ID NO 27), GGSGGS (SEQ ID NO 28) or a combination (a multimer) of any two (dimer), three (trimer), four (tetramer), five (pentamer) or more than five thereof.
AH25( 11201776_2):GCC 2a 2013204065 27 May 2016 [0008a] In another aspect, there is provided a composition comprising a targeting peptide that binds Streptococcus mutans attached to a detectable agent or to an antimicrobial peptide, wherein the amino acid sequence of said targeting peptide consists of a fragment of the competence stimulating peptide (CSP) ranging in length from 6 to 20 amino acids.
[0008b] In another aspect, there is provided a STAMP composition comprising: a) a targeting peptide, wherein the targeting peptide consists of a fragment of the competence stimulating peptide (CSP) ranging in length from 6 to 20 amino acids; b) a linker peptide, wherein one terminus of the linker peptide is linked to the targeting peptide; and c) an antimicrobial peptide, wherein the antimicrobial peptide is linked to the other terminus of the linker peptide.
[0009] In another embodiment, the antimicrobial peptide is selected from the group consisting of G2 (a derivative of novispirin G10f KNLRRHRKGIHilKKY * as shown in SEQ ID NO 3) (* denotes C-terminal amidation), S6L3-33 having an amino acid 11361051:gcc sequence of FKKFWKWFRRF (SEQ ID NO 7) and BD2.21 having an amino acid sequence of KLFKFLRKHLL (SEQ ID NO 11). 2013204065 11 Apr 2013 [0010] In another embodiment, the STAMP comprises a targeting peptide and an antimicrobial peptide, wherein the targeting peptide is covalently linked to the antimicrobial peptide via a peptide bond, wherein the targeting peptide is selected from the group consisting of C16 (SEQ ID NO 2), M8 (SEQ ID NO 5), and 1903 (SEQ ID NO 10); and wherein the antimicrobial peptide is selected from the group consisting of G2 (SEQ ID NO 3), S6L3-33 (SEQ ID NO 7) and BD2.21 (SEQ ID NO 11).
[0011] In another embodiment, the STAMP comprises a targeting peptide which is covalently linked to a linker peptide via a peptide bond and an antimicrobial peptide which is covalently linked to the linker peptide via a peptide bond, wherein the targeting peptide is selected from the group consisting of C16 (SEQ ID NO 2), M8 (SEQ ID NO 5), and 1903 (SEQ ID NO 10); wherein the antimicrobial peptide is selected from the group consisting of G2 (SEQ ID NO 3), S6L3-33 (SEQ ID NO 7) and BD2.21 (SEQ ID NO 11); and wherein the peptide linker is selected from the group consisting of GGG (SEQ ID NO 17), AAA (SEQ ID NO 18), SAT (SEQ ID NO 19), ASA (SEQ ID NO 20), SGG (SEQ ID NO 21), PYP (SEQ ID NO 22), SGS (SEQ ID NO 23), GGS (SEQ ID NO 24), SPS(SEQ ID NO 25), PSGSP(SEQ ID NO 26), PSPSP(SEQ ID NO 27), and GGS GGS (SEQ ID NO 28).
[0012] In another embodiment, the STAMP is selected from the group consisting of C16G2 (SEQ ID NO. 4); C16-33 (SEQ ID NO. 8); C16-BD2.21 (SEQ ID NO. 14); M8G2 (SEQ ID NO. 15); M8-33 (SEQ ID NO. 9); M8-BD2.21 (SEQ ID NO.6); 1903-G2 (SEQ ID NO. 12);1903-33 (SEQ ID NO. 16); and 1903-BD2.21 (SEQ ID NO. 13).
[0013] In another embodiment, the amino acids in the STAMP are D-amino acid enantiomer.
[0014] Another aspect of the present invention relates to a STAMP composition comprising a STAMP and an antibiotic, wherein the STAMP composition shows a 3 synergistic antimicrobial effect in killing or reducing the growth of a target microbial organism. In one embodiment, the STAMP is GlOKHc (SEQ ID NO 36). In another embodiment, the antibiotic is tobramycin. In a preferred embodiment, the STAMP composition comprises GlOKHc (SEQ ID NO 36) and tobramycin. 2013204065 11 Apr 2013 [0015] Another aspect of the present invention relates to a STAMP composition comprising a STAMP and an agent which can enhance, maintain, or facilitate the function or activity of the STAMP. In one embodiment, the agent is a protease inhibitor or rhDNase. In a preferred embodiment, the STAMP composition comprises GlOKHc (SEQ ID NO 36) and a protease inhibitor and/or rhDNase.
[0016] Another aspect of the present invention is a diagnostic agent comprising a targeting peptide and a detectable agent. In one embodiment, the targeting peptide is conjugated to the detectable agent.
[0017] Another aspect of the present invention relates to the use of a composition of the present invention (e.g., the STAMP or the STAMP composition) in selectively killing, inhibiting or reducing the growth of a target microbial organism in a subject or on a biofilm or treating a disease associated with a target microbial organism.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1: Selective killing activity of Cl6G2 against S. mutans. S. mutans, S. sanguinis, and S. gordonii planktonic cells were exposed to 25 μΜ of the STAMP C16G2, or its untargeted parent antimicrobial peptide G2, for 1 min. Surviving cfu/mL were detected and compared. Data represent averages from at least 3 independent experiments.
[0019] Figure 2: Inhibitory activity of G2 and C16G2 against single-species biofilms. S. gordonii (A), S. sanguinis (B) and S. mutans (C) monoculture biofilms were grown and then exposed for 1 min to 25 μΜ STAMP or STAMP component (as indicated in the figure), washed, and regrown with fresh medium. Biofilm recovery was monitored over time by OD60o- Data represent averages from 3 independent experiments. 4 [0020] Figure 3: C16G2 activity against S. mutans within a multi-species biofilm. Mixed cultures of S. mutans, S. sanguinis, and S. gordonii were allowed to form a biofilm in saliva and were then exposed to 25 μΜ C16G2, CSPCi6, or G2. After washing, the biofilms were allowed to recover in fresh medium/saliva. The regrowth of the biofilm over time was monitored by measuring absorbance at OD60o, while the health of the S. mutans within the biofilm was measured by luciferase activity (RLU production). The data were plotted as RLU/OD60o and represent averages of at least 3 independent experiments. 2013204065 11 Apr 2013 [0021] Figure 4: Activity of M8G2 against oral bacteria in biofilms. S. mutans (A) or S. sanguinis (B) single-species biofilms were mock-treated or exposed to 25 μΜ M8G2 (specified in the figure). After removal of the STAMP and the addition of fresh medium, biofilm recovery was monitored over time by monitoring absorbance at OD600· The data represent the average of 3 independent experiments.
[0022] Figure 5: Biofilm inhibitory activity of S6L3-33 and S6L3-33-containing STAMPs. Single-species biofilms of S. mutans (A) or S. sanguinis (B) were treated with M8-33, C16-33 or S6L3-33 alone (specified in the figure) for 1 min. After agent removal and stringent washing, the regrowth of the biofilms was tracked over 4 h by measuring absorbance at OD6oo after the addition of fresh medium. The data represent an average value obtained from at least 3 independent assays.
[0023] Figure 6: HPLC and MALDI spectra for GlOKHc. The quality of purified GlOKHc was assessed by HPLC (a) and MALDI mass spectrometry (b). By monitoring UV 215, a single peak was detected during HPLC (at 10.06 mL) that had the correct mass for GlOKHc (4267.44).
[0024] Figure 7: Antimicrobial kinetics of GlOKHc, G10, and tobramycin. P. aeruginosa strain ATCC 15692 was either mock treated or challenged with the STAMP GlOKHc, untargeted G10, or tobramycin (10 μΜ) and the surviving cfu/mL quantitated after 1 min, 5 min, 30 min and 2h. The assay was conducted in 30% mouse serum and represents the average of at least three independent experiments. 5 [0025] Figure 8: Time-kill assay against high-density planktonic P. aeruginosa. Cultures (1x10s cfu/mL) were exposed to 5 μΜ GlOKHc or G10 with and without equal molar tobramycin co-treatment, as well as administered tobramycin alone. After 24 h, surviving cfu/mL were determined by plating. Data points represent the averages of three independent experiments. 2013204065 11 Apr 2013 [0026] Figure 9: Enhanced antimicrobial activity of GlOKHc and tobramycin against biofilm P. aeruginosa. Biofilms were grown on disk reactors and challenged with 100 pg/mL of agent as indicated. After 4 and 24 h, surviving bacteria were harvested and plated for quantitation from at least 3 independent experiments. * indicates that the number of cfu/mL from GlOKHc/tobramycin treated cultures was too small to appear on the log scale.
[0027] Figure 10: Dye uptake mediated by sub-inhibitory concentrations of GlOKHc. (a) P. aeruginosa were treated with medium (left column) or 2 pM GlOKHc (right column) for 5 min followed by PI dye addition. Bright-field (upper panel) and fluorescence (lower panel) images of the same field were collected and evaluated for intracellular dye accumulation (red fluorescence), (b) Surviving cfu/mL from untreated (dye only) and GlOKHc-treated cultures were quantitated after visualization and plated as 5-fold serial dilution.
[0028] Figure 11: Activity and stability of GlOKHc and GlOKHc-D in sputum. (A) Exogenously added P. aeruginosa were challenged with 25 pM GlOKHc (with or without PMSF), 25 pM GlOKHc-D, or left untreated (specified in the figure), and the surviving cfu/mL rescued and quantitated 4 h after agent addition. Rescued cfu/mL were expressed as the average of three independent experiments with standard deviations. (B) GlOKHc (with and without PMSF, specified in the figure) was added to sputum for specific durations and peptide stability (milli-absorbance units, mAU) was monitored by HPLC. The increasing mobile phase linear gradient is shown in black. (**) Intact GlOKHc identified by MALDI mass spectrometry at retention volume 10.29 mL. (*) Fractions collected for antimicrobial analysis (Table 1). 6 [0029] Figure 12: Effect of rhDNase on GlOKHc and GlOKHc-D activity in concentrated sputum. Minimally diluted pooled sputum samples with exogenously added P. aeruginosa were treated with 25 μΜ GlOKHc (with PMSF) or 50 μΜ GlOKHc-D for 1 h after pretreatment with or without rhDNase. Rescued cells were quantitated and expressed as the percentage of input cfu/mL. The data represent the average of at least 3 independent experiments with standard deviation. 2013204065 11 Apr 2013 [0030] Figure 13: Killing of single-species Streptococcus mutans mature biofilms with C16-BD2.21 and 1903-BD2.21. The figure indicates that C16-BD2.21 and 1903-BD2.21 can kill 33% and 15% of the viable S. mutans within the mature biofilm (grown 18-24 h) respectively, after the biofilm was treated by the peptides for only 20 min.
[0031] Figure 14: Impact of C16-BD2.21 and 1903-BD2.21 on multi-species biofilm of oral Streptococci. (A) shows that C16-BD2.21 has no impact on the total cfu/mL population and 1903-BD2.21 reduced total population by about 30%. (B) shows that ratio of surviving S. mutans to total Streptococci was 0.075 under C16-BD2.21 treatment and the ratio was about 0.2 under 1903-BD2.21 treatment.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0032] One aspect of the present invention relates to selectively/specifically targeted antimicrobial peptides (STAMPs) and the use thereof.
[0033] The term "selectively/specifically targeted antimicrobial peptide" or "STAMP" refers to a chimeric polypeptide which comprises a targeting peptide and an antimicrobial peptide, wherein the targeting peptide is covalently linked or conjugated (e.g., via a peptide bond) to the antimicrobial peptide either at the C-terminal or N-terminal of the targeting peptide. For example, one STAMP may comprise one of the following two structures: 1) a targeting peptide with its C-terminal covalently linked to the N-terminal of an antimicrobial peptide [Amino terminus-targeting peptide -peptide bond-antimicrobial peptide-carboxyl terminus], and 2) an antimicrobial peptide with its C-terminal covalently linked to the N-terminal of a targeting peptide 7 [Amino terminus-antimicrobial peptide-peptide bond-targeting peptide-carboxyl terminus], 2013204065 11 Apr 2013 [0034] In one embodiment of the present invention, the STAMP further comprises a peptide linker by which the targeting peptide is covalently linked or conjugated to the antimicrobial peptide. In this case, a STAMP may comprise one of the following two structures: 1) a targeting peptide with its C-terminal covalently linked to the N-terminal of a linker peptide and an antimicrobial peptide with its N-terminal covalently linked to the C-terminal of the linker peptide (Amino terminus-targeting peptide -peptide bond-linker peptide-antimicrobial peptide-peptide bond-carboxyl terminus) and 2) a targeting peptide with its N-terminal covalently linked to the C-terminal of a linker peptide and an antimicrobial peptide with its C-terminal covalently linked to the N-terminal of the linker peptide (Amino terminus-antimicrobial peptide -peptide bond-linker peptide-peptide bond-targeting peptide-carboxyl terminus).
[0035] According to the present invention, a targeting peptide can be any suitable peptide which recognizes or binds to a target (e.g., a target cell, a target tissue, a target microbial organism). Particularly, a targeting peptide specifically interacts with or specifically recognizes a target, through, for example, the cell surface appendages such as flagella and pili, and surface exposed proteins, lipids and polysaccharides of a target. In one embodiment, a targeting peptide specifically recognizes or interacts with only one or a few target(s) while minimally recognizing or interacting with non-targets). In another embodiment, a targeting peptide can be a peptide capable of specifically binding to a microorganism, e.g., a target microbial organism.
[0036] In one embodiment, the targeting peptide provided by the present invention can be identified via screening peptide libraries. For example, a phage display peptide library can be screened against a target microbial organism or a desired antigen or epitope thereof. In particular, phage display peptide libraries (e.g., Ph.D 7, Ph.D.12, Ph.D C7C libraries from New England Biolabs) that contain >109 unique random-peptide-sequence-containing phage clones. The Ph.D.-C7C library displays 7-mer 8 peptides with disulfide linkages, while the Ph.D.-7 and Ph.D.-12 libraries contain completely randomized 7-mer and 12-mer residues, respectively. The M13 filamentous phage used for the procedure carried the random insert as an N-terminal fusion to the minor coat protein pill. In screening a targeting peptide that specifically recognizes a target or target microbial organism, 1010 pfu/ml of phage library was incubated with 109 microbial organisms (e.g., bacterial cells) for which targeting peptides are desired. After centrifugation, unbound phage was removed by aspiration. The pellet, which contained the target microbial organisms with the bound phage, was washed several times using buffers containing mild detergent to remove loosely bound phage particles, and the tightly bound phage particles were eluted. This process is termed panning. The eluted phage was amplified by infecting E. coli F+ strains. After 3-4 rounds of panning and amplification, a phage pool was obtained, which contained clones with high binding affinity for the bacteria that it was panned against. Ten to twenty phage clones from this pool were randomly picked for DNA sequencing, from which the amino acid sequence of the peptide insert was determined. By aligning the amino acid sequence of multiple clones from the same phage pool, a consensus sequence for the binding/targeting peptide was constructed. This consensus sequence represents one of the binding/targeting peptides specific for the particular microbial organism. To confirm the binding specificity of the consensus peptide, the peptide was chemically synthesized and conjugated to a dye (e.g., FITC, a green fluorescence dye). The labeled peptide was incubated with the microbial organism and analyzed by fluorescent microscopy for target microbial species-specific binding. This methodology ensured that peptides selected from phage display exhibit the same binding specificity as a free peptide independent of the Ml3 phage particle. 2013204065 11 Apr 2013 [0037] The targeting peptide of the present invention can also be a peptide obtained based on rational design. For example, one can design a targeting peptide based on the biochemical and biophysical characteristics of amino acids and the surfaces of microorganisms. In general, positively charged peptides are likely to bind negatively charged components on the cell surface and vice versa. Similarly, hydrophobic peptides may bind to hydrophobic pockets on the cell surface based on hydrophobic 9 interactions while the secondary or tertiary structure of a peptide may fit into certain structures on the surface of a microorganism. 2013204065 11 Apr 2013 [0038] A peptide identified through a screening or design method can be used as a targeting peptide for specifically recognizing a target microbial organism. Examples of such targeting peptide are disclosed in U.S. Pat. Appl. No. 10/706, 391 (U.S. Pub. No. 20040137482), which include, for example, 1) targeting peptides capable of specifically binding to or recognizing Pseudomonas, especially P.aeruginosa (e.g., targeting peptidesl2:l, 12:2, 12:3, 12:4, 12:5, 12:6, 12:7, 12:8; 12:9, and 12:10); 2) targeting peptides capable of specifically binding to Staphylococcus, especially S. aureus (e.g., targeting peptides SA5:1, SA5:3, SA5:4, SA5:5, SA5:6, SA5:7, SA5:8, SA5:9, SA5:10, SA2:2, SA2:4, SA2:5, SA2:6, SA2:7, SA2:8, SA2:9, SA2:10, and SA2:11); and 3) targeting peptides capable of specifically binding to E. coli (e.g., targeting peptides DH5.1, DH5.2, DH5.3, DH5.4, DH5.5, DH5.6, DH5.7, DH5.8, and DH5.9).
[0039] In one embodiment of the present invention, targeting peptides specifically binding to or recognizing Pseudomonas, especially P.aeruginosa, include, for example, cat-1 (or KH) domain, KKHRKHRKHRKH (SEQ ID NO 31). Targeting peptides specifically binding to or recognizing Streptococci include, for example, bacterial pheromones such as CSP (SGSLSTFFRLFNRSFTQALGK, SEQ ID NO 1), CSP 1 (EMRES KFFRDFIEQRKK, SEQ ID NO 29) and CSP2 (EMRISRIILDFLFLRKK, SEQ ID NO 30) and fragments thereof. Further, targeting peptides specifically binding to or recognizing S. pneumoniae include, for example, CSP1 and CSP2. Targeting peptides specifically binding to or recognizing S. mutans include, for example, CSP, C16 or CSPCi6 (TFFRLFNRSFTQALGK, SEQ ID NO 2), M8 or CSPM8 (TFFRLFNR, SEQ ID NO 5), and peptide 1903 (NIFEYFLE, SEQ ID NO 10).
[0040] The targeting peptides provided by the present invention can be naturally or non-naturally occurring peptides. For example, the targeting peptides provided by the present invention can be recombinantly made, chemically synthesized, or naturally 10 existing. In one embodiment, the targeting peptide contains an amino acid sequence that naturally exists (e.g., CSP, CSP 1 and CSP2). In another embodiment, the targeting peptide contains an amino acid sequence that constitutes an internal part of a naturally occurring polypeptide (e.g., C16 and M8 in the present invention). In another embodiment, the targeting peptide contains an amino acid sequence encoded by a sequence naturally existing in a genome and such amino acid sequence is not adjacent to any amino acid sequence naturally adjacent to it, e.g., such amino acid sequence is adjacent to a heterologous sequence in the targeting peptide. 2013204065 11 Apr 2013 [0041] The targeting peptide provided by the present invention can also include a peptide having an amino acid sequence that is derived or modified from a targeting amino acid sequence specifically illustrated in the present invention, provided that the derived or modified sequence still maintains or has an enhanced specificity with respect to its target microbial organism. For example, the targeting amino acid sequence can be structurally modified via deletion, mutation, addition of amino acids or other structural entities, or any other structural changes as long as these changes do not alter or adversely affect the binding ability of the targeting amino acid sequence to its target microbial organism.
[0042] The targeting peptide according to the present invention specifically interacts with or binds to the target organism (e.g., through the external surface of the organism) via different molecular interactions such as ionic interaction, Vander Waals forces, ligand-receptor interaction, or hydrophobic interaction. For example, the targeting peptide of the present invention can also be a peptide ligand, receptor, or fragment thereof that specifically recognizes a target microbial organism. In one example, the targeting peptide of the present invention can be a glucan binding protein of Streptococcus mutans that can specifically bind insoluble glucans on the surface of S. mutans. For another example, the targeting peptides can be a bacterial pheromone or a fragment thereof.
[0043] The targeting peptide according to the present invention comprises about 4 to about 40 amino acids, from about 5 to about 30, or from about 6 to about 20. In 11 one preferred embodiment, the targeting peptide has a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. 2013204065 11 Apr 2013 [0044] It is contemplated that the targeting peptides includes peptides which specifically bind a target cell or tissue (e.g., a plant call, an animal cell, or fungal organism). The examples of these targeting peptides include Chitinase, Lectins, and targeting fragments thereof.
[0045] The targeting peptide according to the present invention can be produced by any suitable method known to one skilled in the art by itself or in combination with a linker peptide and an antimicrobial peptide. For example, the targeting peptides can be chemically synthesized via a synthesizer or recombinantly made using an expression system, e.g., a bacterial, yeast, or eukaryotic cell expression system. In the chemical synthesis, the targeting peptide can be made by L-amino acid enantiomers or D-amino acid enantiomers. It is observed that the targeting peptide consisting of D-enantiomers increases the stability without compromising the activity of the targeting peptide.
[0046] The linker peptide according to the present invention is a peptide that can be used to connect a targeting peptide to an antimicrobial peptide without interfering or reducing the activity of the targeting peptide or the antimicrobial peptide. The peptide linker is from about 2 to 20 amino acids, from 2 to 12, or from 3 to 12 amino acids. Examples of the peptide linkers include, for example, GGG (SEQ ID NO 17), AAA (SEQ ID NO 18), SAT (SEQ ID NO 19), ASA (SEQ ID NO 20), SGG (SEQ ID NO 21), PYP (SEQ ID NO 22), SGS (SEQ ID NO 23), GGS (SEQ ID NO 24), SPS(SEQ ID NO 25), PSGSP(SEQ ID NO 26), PSPSP(SEQ ID NO 27), or a combination (a multimer) of any two (dimer), three (trimer), four (tetramer), five (pentamer) or more than five of the listed peptide linkers. In one embodiment, the linker peptide is GGG (SEQ ID NO 17). In another embodiment, the linker peptide is a dimmer of GGS (SEQ ID NO 24), which is GGSGGS (SEQ ID NO 28).
[0047] The linker peptide according to the present invention can be produced by any suitable method known to one skilled in the art by itself or in combination with a 12 targeting peptide and an antimicrobial peptide. For example, the linker peptides can be chemically synthesized via a synthesizer or recombinantly made using an expression system, e.g., a bacterial, yeast, or eukaryotic cell expression system. In the chemical synthesis, the linker peptide can be made by L-amino acid enantiomers or D-amino acid enantiomers. It is observed that peptides consisting of D-enantiomers increase the stability without comprising the activity of the peptides. 2013204065 11 Apr 2013 [0048] The antimicrobial peptide according to the present invention is a peptide capable of killing a microbial organism or inhibiting its growth. The antimicrobial activities of the antimicrobial peptides of the present invention include, without limitation, antibacterial, antiviral, or antifungal activities. Antimicrobial peptides include various classes of peptides, e.g., peptides originally isolated from plants as well as animals. In animals, antimicrobial peptides are usually expressed by various cells including neutrophils and epithelial cells. In mammals including human, antimicrobial peptides are usually found on the surface of the tongue, trachea, and upper intestine. Naturally occurring antimicrobial peptides are generally amphipathic molecules that contain fewer than 100 amino acids. Many of these peptides generally have a net positive charge (i.e., cationic) and most form helical structures.
[0049] In one embodiment, the antimicrobial peptide according to the present invention comprises about 2 to about 100 amino acids, from about 5 to about 50, or from about 7 to about 20. In one preferred embodiment, the targeting peptide has a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids.
[0050] In another embodiment, the antimicrobial peptide has the antimicrobial activity with a minimum inhibitory concentration (MIC) of no more than about 40μΜ, no more than about 30 μΜ, no more than 20 μΜ, or no more than 10 μΜ.
[0051] In another embodiment, the antimicrobial peptides include those listed in Table 7 (SEQ ID Nos 34-35 and 54-97). In another embodiment, the antimicrobial peptide contains one or more antimicrobial peptides including, without limitation, alexomycin, andropin, apidaecin, bacteriocin, β-pleated sheet bacteriocin, bactenecin, 13 buforin, cathelicidin, α-helical clavanin, cecropin, dodecapeptide, defensin, β-defensin, α-defensin, gaegurin, histatin, indolicidin, magainin, melittin, nisin, novispirin G10, protegrin, ranalexin, tachyplesin, and derivatives thereof. 2013204065 11 Apr 2013 [0052] Among these known antimicrobial peptides, tachyplesins are known to have antifungal and antibacterial activities. Andropin, apidaecin, bactencin, clavanin, dodecappeptide, defensin, and indolicidin are antimicrobial peptides having antibacterial activities. Buforin, nisin and cecropin peptides have been demonstrated to have antimicrobial effects on Escherichia, coli, Shigella disenteriae, Salmonella typhimurium, Streptococcus pneumoniae, Staphylococcus aureus, and Pseudomonas aeroginosa. Magainin and ranalexin peptides have been demonstrated to have antimicrobial effects on the same organisms, and in addition have such effects on Candida albicans, Cryptococcus neoformans, Candida krusei, and Helicobacter pylori. Magainin has also been demonstrated to have antimicrobial effects on herpes simplex virus. Alexomycin peptides have been demonstrated to have antimicrobial effects on Campylobacter jejuni, Moraxella catarrhalis and Haemophilus inflluenzae while defensin and β-pleated sheet defensin peptides have been shown to have antimicrobial effects on Streptococcus pneumoneae. Histatin peptides and the derivatives thereof are another class of antimicrobial peptides, which have antifungal and antibacterial activities against a variety of organisms including Streptococcus mutans (MacKay, B. J. et al., Infect. Immun. 44:695-701 (1984); Xu, et al., J. Dent. Res. 69:239 (1990)).
[0053] In one embodiment, the antimicrobial peptide of the present invention contains one or more antimicrobial peptides from a class of histatin peptides and the derivatives thereof. For example, the antimicrobial peptide of the present invention contains one or more derivatives of histatin including, without limitation, histatin 5 having an amino acid sequence of DSHAKRHHGY KRKFHEKHHS HRGY (SEQ ID NO 32) or dhvar-1 having an amino acid sequence of KRLFKELKFS LRKY (SEQ ID NO 33).
[0054] In another embodiment, the antimicrobial peptide of the present invention contains one or more antimicrobial peptides from a class of protegrins and the 14 derivatives thereof. For example, the antimicrobial peptide of the present invention contains protegrin PG-1 having an amino acid sequence of 2013204065 11 Apr 2013 RGGRLCYCRRRFCVCVGR (SEQ ID NO 3334). The protegrin peptides have been shown to have antimicrobial effects on Streptococcus mutans, Neisseria gonorrhoeae, Chlamydia trachomatis and Haempohilus influenzae. Protegrin peptides are described in U.S. Pat. Nos. 5,693,486, 5,708,145, 5,804,558, 5,994,306, and 6,159,936, all of which are incorporated herein by reference.
[0055] In yet another embodiment, the antimicrobial peptide of the present invention contains one or more antimicrobial peptides from a class of novispirin and the derivatives thereof for treating cariogenic organisms, e.g., Streptococcus mutans or Pseudomonas aeroginosa. For example, the antimicrobial peptide of the present invention includes novispirin G10 having an amino acid sequence KNLRRIIRKGIHIIKKYG (SEQ ID NO 35) and G2 (a derivative of novispirin G10) which has one C-terminal amino acid deletion, and one internal deletion from G10 and an amidated C-terminus having the amino acid sequence of KNLRIIRKGIHIIKKY* (SEQ ID NO 3) (* denotes C-terminal amidation).
[0056] In yet another embodiment, the antimicrobial peptide of the present invention contains peptides rationally designed and tested to possess the antimicrobial activity against a microbial organism (e.g., Streptococcus mutans). The examples of these peptides include, without any limitation, S6L3-33 having an amino acid sequence of FKKFWKWFRRF (SEQ ID NO 7) and BD2.21 having an amino acid sequence of KLFKFLRKHLL (SEQ ID NO 11).
[0057] The antimicrobial peptide according to the present invention can be produced by any suitable method known to one skilled in the art by itself or in combination with a targeting peptide and a linker peptide. For example, the antimicrobial peptides can be chemically synthesized via a synthesizer or recombinantly made using an expression system, e.g., a bacterial, yeast, or eukaryotic cell expression system. In the chemical synthesis, the antimicrobial peptide can be made by L-amino acid enantiomers or D-amino acid enantiomers. 15 [0058] In yet another embodiment, the STAMP comprises a targeting peptide and an antimicrobial peptide, wherein the targeting peptide is covalently linked to the antimicrobial peptide via a peptide bond, wherein the targeting peptide is selected from the group consisting of C16 (SEQ ID NO 2), M8 (SEQ ID NO 5), and 1903 (SEQ ID NO 10); and wherein the antimicrobial peptide is selected from the group consisting of G2 (SEQ ID NO 3), S6L3-33 (SEQ ID NO 7) and BD2.21 (SEQ ID NO 11). 2013204065 11 Apr 2013 [0059] In yet another embodiment, the STAMP comprises a targeting peptide which is covalently linked to a linker peptide via a peptide bond and an antimicrobial peptide which is covalently linked to the linker peptide via a peptide bond, wherein the targeting peptide is selected from the group consisting of C16 (SEQ ID NO 2), M8 (SEQ ID NO 5), and 1903 (SEQ ID NO 10); wherein the antimicrobial peptide is selected from the group consisting of G2 (SEQ ID NO 3), S6L3-33 (SEQ ID NO 7) and BD2.21 (SEQ ID NO 11). In yet another embodiment, the peptide linker is selected from the group consisting of GGG (SEQ ID NO 17), AAA (SEQ ID NO 18), SAT (SEQ ID NO 19), ASA (SEQ ID NO 20), SGG (SEQ ID NO 21), PYP (SEQ ID NO 22), SGS (SEQ ID NO 23), GGS (SEQ ID NO 24), SPS(SEQ ID NO 25), PSGSP(SEQ ID NO 26), PSPSP(SEQ ID NO 27), and GGSGGS (SEQ ID NO 28). Examples of such STAMPs include but are not limited to the STAMPS listed in Table 1: C16G2 (SEQ ID NO 4); C16-33 (SEQ ID NO 8); C16-BD2.21 (SEQ ID NO 14); M8G2 (SEQ ID NO 6); M8-33 (SEQ ID NO 9); M8-BD2.21 (SEQ ID NO 15); 1903-G2 (SEQ ID NO 12);1903-33 (SEQ ID NO 16); and 1903-BD2.21 (SEQ ID NO 13).
[0060] Another aspect of the present invention relates to a composition comprising a plurality of STAMPS, wherein the composition comprises a first STAMP and a second STAMP and the first STAMP is different from the second STAMP. In one embodiment, the first STAMP comprises a first targeting peptide covalently linked to a first antimicrobial peptide via a peptide bond. The second STAMP comprises a second targeting peptide covalently linked to a second antimicrobial peptide via a peptide bond. The difference between the first STAMP and the second STAMP is such that at least one corresponding moiety of the two STAMPs is different from each 16 other. For example, in one embodiment, the first targeting peptide is different from the second targeting peptide or the first antimicrobial peptide is different from the second antimicrobial peptide. In another embodiment, the first targeting peptide is different from the second targeting peptide and the first antimicrobial peptide is different from the second antimicrobial peptide. 2013204065 11 Apr 2013 [0061] In another embodiment, the first STAMP comprises a first targeting peptide which is covalently linked to a first linker peptide via a peptide bond and a first antimicrobial peptide which is covalently linked to the first linker peptide via a peptide bond. The second STAMP comprises a second targeting peptide which is covalently linked to a second linker peptide via a peptide bond and a second antimicrobial peptide which is covalently linked to the second linker peptide via a peptide bond. The difference between the first STAMP and the second STAMP is such that at least one corresponding moiety of the two STAMPs is different from each other. For example, in one embodiment, the first targeting peptide is different from the second targeting peptide (the first linker peptide is the same as the second linker peptide and the first antimicrobial peptide is the same as the second antimicrobial peptide); or the first linker peptide is different from the second peptide linker; or the first antimicrobial peptide is different from the second antimicrobial peptide. In another embodiment, the first targeting peptide is the same as the second targeting peptide (the first linker peptide is different from the second linker peptide and the first antimicrobial peptide is different from the second antimicrobial peptide); or the first peptide linker is the same as the second peptide linker; or the first antimicrobial peptide is the same the second antimicrobial peptide. In another embodiment, the first targeting peptide is different from the second targeting peptide, the first linker peptide is different from the second peptide linker; and the first antimicrobial peptide is different from the second antimicrobial peptide.
[0062] The STAMP of the present invention can be made by any suitable means known to one skilled in the art. In one embodiment, a nucleotide sequence encoding the STAMP can be synthesized through a DNA synthesizer or a nucleotide sequence encoding a targeting peptide can be ligated to a nucleotide sequence encoding an 17 antimicrobial peptide moiety, either directly or via a nucleotide sequence encoding a peptide linker. The nucleotide can be expressed in an appropriate expression system, e.g., a commercially available bacterial, yeast, or eukaryotic cell expression system. In the chemical synthesis, the STAMP can be made by L-amino acid enantiomers or D-amino acid enantiomers. It is observed that the STAMP consisting of D-enantiomers increases the stability without comprising the activity of the STAMP. 2013204065 11 Apr 2013 [0063] Another aspect of the present invention relates to a STAMP composition comprising a STAMP and an antibiotic. A synergistic antimicrobial effect has been unexpectedly observed when a STAMP is co-administered with an antibiotic in killing or reducing the growth of a target microbial organism. Antibiotics suitable for coadministration with the STAMP include substances, produced synthetically or naturally, which can inhibit the growth of or kill microbial organisms. Such antibiotics include, without any limitation, β-lactam antibiotics (e.g., ampicillin, aziocillin, aztreonam, carbenicillin, cefoperazone, ceftriaxone, cephaloridine, cephalothin, cloxacillin, moxalactam, penicillin, piperacillin, and ticarcillin), amoxicillin, bacitracin, chloramphenicol, clindamycin, capreomycin, colistimethate, ciprofloxacin, doxycycline, erythromycin, fusidic acid, fosfomycin, fusidate sodium, gramicidin, gentamycin, , lincomycin, minocycline, macrolides, monobactams, nalidixic acid, novobiocin, ofloxcin, rifamycins, tetracyclines, vancomycin, tobramycin, and trimethoprim. In one example, the STAMP composition comprises a GlOKHc STAMP (SEQ ID NO 36) and tobramycin and exhibits a synergistic enhancement of antimicrobial activity to P. aeruginosa in both biofilms and planktonic cultures.
[0064] Another aspect of the present invention relates to a STAMP composition comprising a STAMP and an agent which can enhance, maintain, or facilitate the function or activity of the STAMP. In one embodiment, the chemical is a protease inhibitor. The STAMP composition is exposed to a protease-present environment where the presence of the protease may reduce the antimicrobial activity of the STAMP via, for example, enzymatic degradation. The combination of a protease inhibitor and a STAMP stabilizes the STAMP from the protease degradation and thus enhance the activity of the STAMP. The protease-present environment includes, for 18 example, body fluid (e.g., urine, blood, serum, salvia, sputum, mucosal fluid). The protease includes, for example, neutrophil elastase, proteinase-3, cycteine protease, metalloprotease, serine-protease, or other proteases derived from bacteria and/or hosts. The protease inhibitor includes, for example, BMF, EDTA, PMFS, benzamidine, and/or recombinant a-1 antitrypsin (rAAT). 2013204065 11 Apr 2013 [0065] In yet another embodiment, the agent is human DNase. One example of the STAMP composition is the combination of a STAMP (GlOKHc (SEQ ID NO 36) and a DNase. The composition was used to reduce P.areruginosa in sputum and exhibited enhanced antimicrobial activity of GlOKHc, as the DNase reduced sputum viscosity and enhanced the STAMP diffusion.
[0066] Another aspect of the present invention relates to a pharmaceutical composition comprising a STAMP and a suitable pharmaceutical carrier. The term "pharmaceutically acceptable carrier" as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a STAMP from one location, body fluid, tissue, organ, or portion of the body, to another location, body fluid, tissue, organ, or portion of the body. Each carrier must be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients, e.g., a STAMP, of the formulation and suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit/risk ratio. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; ( 1) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering 19 agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations. 2013204065 11 Apr 2013 [0067] Another aspect of the present invention is a diagnostic agent comprising a targeting peptide and a detectable agent. The diagnostic agent of this invention can be useful in diagnostic assays, e.g., for detecting the presence or amount of a target or target microbial organism in a place where the target organism is susceptible to exist (e.g., tissues, organs, body fluid, sputum, surface of a body or organ, mucosal surface, implant, biofilm, or serum, device, air, fluid, cell culture, surface of an industry article or a device), or for detecting the onset, development, or remission of a condition (e.g., an infection or a disease) associated with the target microorganism.
[0068] In one embodiment, the targeting peptide typically will be labeled with or conjugated to a detectable agent. Numerous detectable agents are available which can be generally grouped into the following categories: [0069] (a) Radioisotopes, such as 35S, 14C, 13C, 15N, 1251,3H, and 131I. The peptide can be labeled with the radioisotope using the techniques known in the art and radioactivity can be measured using scintillation counting; in addition, the peptide can be spin labeled for electron paramagnetic resonance for carbon and nitrogen labeling.
[0070] (b) Fluorescent agents such as BODIPY, BODIPY analogs, rare earth chelates (europium chelates), fluorescein and its derivatives, FITC, 5,6 carboxyfluorescein, rhodamine and its derivatives, dansyl, Lissamine, phycoerythrin, green fluorescent protein, yellow fluorescent protein, red fluorescent protein and Texas Red. Fluorescence can be quantified using a fluorometer.
[0071] (c) Various enzyme-substrate agents, such luciferases (e.g., firefly luciferase and bacterial luciferase), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), 20 heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like. Examples of enzyme-substrate combinations include, for example: (i) Horseradish peroxidase (HRPO) with hydrogen peroxidase as a substrate, wherein the hydrogen peroxidase oxidizes a dye precursor (e.g., orthophenylene diamine (OPD) or 3,3',5,5'-tetramethyl benzidine hydrochloride (TMB)); (ii) alkaline phosphatase (AP) with para-Nitrophenyl phosphate as chromogenic substrate; and (iii) β-D-galactosidase (β -D-Gal) with a chromogenic substrate (e.g., p-nitrophenyl- β- D-galactosidase) or fluorogenic substrate 4-methylumbelliferyl- β- D-galactosidase. 2013204065 11 Apr 2013 [0072] In another embodiment, the detectable agent is not necessarily conjugated to the targeting peptide but is capable of recognizing the presence of the targeting peptide and the agent can be detected. For example, the detectable agent is an antibody that specifically binds to the targeting peptide. The antibody can then be detected or quantified through various methods known in the art (See Harlow & Lane, Antibodies- A Laboratory Manual (1988)).
[0073] In another embodiment, the diagnostic agent of the present invention can be provided in a kit, i.e., a packaged combination of reagents in predetermined amounts with instructions for performing the diagnostic assay. Where the targeting peptide is labeled with an enzyme, the kit will include substrates and cofactors required by the enzyme (e.g., a substrate precursor which provides the detectable chromophore or fluorophore). In addition, other additives may be included such as stabilizers, buffers (e.g., a block buffer or lysis buffer) and the like. The relative amounts of the various reagents may be varied widely to provide for concentrations in solution of the reagents which substantially optimize the sensitivity of the assay. Particularly, the reagents may be provided as dry powders, usually lyophilized, including excipients which on dissolution will provide a reagent solution having the appropriate concentration.
[0074] According to another aspect of the present invention, the compositions (e.g., the STAMPs or the STAMP compositions) of the present invention can be used to 21 kill, inhibit or reduce the growth of a target microbial organism to which the targeting peptide specifically binds. 2013204065 11 Apr 2013 [0075] In one embodiment, the compositions of the present invention provide antimicrobial effect to a target microbial organism and can be used to treat a disease or infection associated with the target microbial organism. An antimicrobial effect includes inhibiting the growth or killing of the target microbial organisms, or interfering with any biological functions of the target microbial organisms. In general, the compositions of the present invention can be used to treat a disease or infection at any place in a host, e.g., at any tissue including surfaces of any tissue or implant. In one embodiment, the compositions are used to specifically kill or inhibit planktonic target microbial organisms in body fluid (e.g., blood, sputum). In one embodiment, the compositions of the present invention are used to treat a disease or infection on a mucosal surface or a surface containing a biofilm.
[0076] The term "biofilm" refers to an accumulation of microbial organisms that produce an extracellular polysaccharide and proteinaceous material that act as a natural glue to immobilize or embed the organisms. Biofilms may form on solid biological or non-biological surfaces. A biofilm consisting essentially of non-harmful, non-pathogenic, commensal microbial organisms is essential for maintaining a healthy and normal microbial flora to prevent the invasion and establishment of other pathogenic microbial organisms, e.g., yeast infection. However, if the microorganism population in a biofilm is disturbed by overpopulation of pathogenic microbial organisms (e.g., cariogenic organisms like Streptococcus mutans), the resulting biofilm may lead to biofilm-associated microbial infection. Examples of biofilm-associated microbial infections include infections of oral soft tissues, teeth and dental implants; middle ear; gastrointestinal tract; urogenital tract; airway/lung tissue; eye; urinary tract prostheses; peritoneal membrane and peritoneal dialysis catheters, indwelling catheters for hemodialysis and for chronic administration of chemotherapeutic agents (Hickman catheters); cardiac implants such as pacemakers, prosthetic heart valves, ventricular assist devices, and synthetic vascular grafts and stents; prostheses, internal fixation devices, and percutaneous sutures; and tracheal 22 and ventilator tubing. Both indwelling and subcutaneous biomedical implants or devices are potential sites for microbial or boilfilm-based infections and represent important targets for the control of infection, inflammation, and the immune response. Biomedical systems such as blood oxygenators, tracheal lavage, dental water units, and dialyzers are also susceptible to bacterial contamination and biofilm formation. 2013204065 11 Apr 2013 [0077] In yet another embodiment, the composition of present invention can be used to disturb the balance of pathogen-containing biofilm (e.g., a biofilm overpopulated by pathogenic microbial organisms) such that undesirable, pathogenic microbial organisms (target microbial organisms) are selectively killed, inhibited or reduced and the desirable, non-pathogenic microbial populations (non-target microbial organisms) are minimally impacted. The composition can be used in many places in an animal or human body which have mucosal surfaces colonized by multiple species microbial biofilms. Examples of these places include mouth, vagina, gastrointestinal (GI) tract, esophageal tract, respiratory tract, implants. For example, in human mouth there usually exist many different microbes including yeasts and bacteria. Most of the bacteria are non-harmful commensal bacteria. Administering the composition of the present invention targets specifically to, for example, cariogenic organisms (e.g. Streptococcus mutans) and will have minimum effect on non-targeted microbial organisms, and thus will not have an undesirable effect on non-targeted microbial organisms.
[0078] The composition of the present invention can also be used to inhibit target microbial organisms or apply to various biofilm surfaces outside of a human body, e.g., industrial articles or applications. For example, in food processing industry the composition of the present invention can be administered to food processing equipments or food itself to prevent infections related to food consumption, e.g., Salmonella in a poultry processing facility.
[0079] The target microbial organism of the present invention can be any bacteria, rickettsia, fungi, yeasts, protozoa, or parasites. In one embodiment, the target microbial organism is a cariogenic organism, e.g., Streptococcus mutans. In another 23 embodiment, the target microbial organisms of the present invention include, without limitation, Escherichia coli, Candida, Salmonella, Staphylococcus, and Pseudomonas, especially Campylobacter jejuni, Candida albicans, Candida krusei, Chlamydia trachomatis, Clostridium difficile, Cryptococcus neoformans, Haempohilus influenzae, Helicobacter pylor, Moraxella catarrhalis, Neisseria gonorrhoeae, Pseudomonas aeroginosa, Salmonella typhimurium, Shigella disenteriae, Staphylococcus aureus, and Streptococcus pneumoniae. 2013204065 11 Apr 2013 [0080] For example, S. mutans infection is commonly found in mouth and causes dental caries. Porphyromonas gingivalis, various Actinomyces species, spirochetes, and black-pigmented bacteroides are commonly associated with infections of gingival and surrounding connective tissues, which cause periodontal diseases. Streptococcus pneumoniae, Haemophilius influenza, or Moraxella cararrhalis infections are commonly found in acute otitis media (AOM) and otitis media effusion (OME) as complications of upper respiratory infections in young children.
[0081] Helicobacter pylori (H. pylori) bacteria are found in the gastric mucous layer or adherent to the epithelial lining of the stomach, and cause more than 90% of duodenal ulcers and up to 80% of gastric ulcers. Other GI tract infections include, without limitation, Campylobacter bacterial infection, primarily Campylobacter jejuni associated with diarrhea, cholera caused by Vibrio cholerae serogroups, salmonellosis caused by bacteria salmonella such as S. typhimurium and S. enteritidis, shigellosis caused by bacteria Shigella, e.g., Shigella dysenteriae and traveler's diarrhea caused by enterotoxigenic Escherichia coli (ETEC). Clostridium difficile infection is also commonly found in the gastrointestinal tract or esophageal tract.
[0082] Pseudomonas organisms have been associated with common-source nosocomial outbreaks; in addition, they have been incriminated in bacteremia, endocarditis, and osteomyelitis in narcotic addicts. Infections with Pseudomonas organisms can also occur in the ear, lung, skin, or urinary tract of patients, often after the primary pathogen has been eradicated by antibiotics. Serious infections are almost invariably associated with damage to local tissue or with diminished host resistance. 24
Patients compromised by cystic fibrosis and those with neutropenia appear at particular risk to severe infection with P. aeruginosa. Premature infants; children with congenital anomalies and patients with leukemia; patients with bums; and geriatric patients with debilitating diseases are likely to develop Pseudomonas infections. The organism is prevalent in urine receptacles and on catheters, and on the hands of hospital staff. 2013204065 11 Apr 2013 [0083] The staphylococci, of which Staphylococcus aureus is the most important human pathogen, are hardy, gram-positive bacteria that colonize the skin of most human beings. If the skin or mucous membranes are disrupted by surgery or trauma, staphylococci may gain access to and proliferate in the underlying tissues, giving rise to a typically localized, superficial abscess. Although these cutaneous infections are most commonly harmless, the multiplying organisms may invade the lymphatics and the blood, leading to the potentially serious complications of staphylococcal bacteremia.
[0084] These complications include septic shock and serious metastatic infections, including endocarditis, arthritis, osteomyelitis, pneumonia, and abscesses in virtually any organ. Certain strains of S. aureus produce toxins that cause skin rashes or that mediate multisystem dysfunction, as in toxic shock syndrome. Coagulase-negative staphylococci, particularly S. epidermidis, are important nosocomial pathogens, with a particular predilection for infecting vascular catheters and prosthetic devices. S. saprophyticus is a common cause of urinary tract infection.
[0085] Yeast or Candida infections (Candidiasis) typically occur either orally (Oropharyngeal Candida or OPC) or vaginally (Vulvovaginal Candida or VVC). Candidiasis is caused by a shift in the local environment that allows Candida strains (most commonly Candida albicans) already present on skin and on mucosal surfaces such as mouth and vagina to multiply unchecked. Gonorrhea, chlamydia, syphilis, and trichomoniasis are infections in the reproductive tract, which cause sexually transmitted diseases, e.g., pelvic inflammatory disease. 25 [0086] Administration of the compositions according to the present invention. The STAMP or the STAMP composition can be administered to a subject by any administration route known in the art, including without limitation, oral, enteral, buccal, nasal, topical, rectal, vaginal, aerosol, transmucosal, epidermal, transdermal, ophthalmic, pulmonary, and/or parenteral administration. A parenteral administration refers to an administration route that typically relates to injection which includes but is not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intra cardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and/or intrastemal injection and/or infusion. 2013204065 11 Apr 2013 [0087] The STAMP or the STAMP composition can be given to a subject in the form of formulations or preparations suitable for each administration route. The formulations useful in the methods of the present invention include one or more STAMPs, one or more pharmaceutically acceptable carriers therefor, and optionally other therapeutic ingredients. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated and the particular mode of administration. The amount of a STAMP which can be combined with a carrier material to produce a pharmaceutically effective dose will generally be that amount of a STAMP which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of the STAMP, preferably from about 5 percent to about 70 percent.
[0088] Methods of preparing these formulations or compositions include the step of bringing into association a STAMP with one or more pharmaceutically acceptable carriers and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a STAMP with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product. 26 [0089] Formulations suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a STAMP as an active ingredient. A compound may also be administered as a bolus, electuary, or paste. For example, in one embodiment, the compositions of the present invention are used to treat or prevent cariogenic organism infections, e.g., S. mutans infection associated with dental caries and are prepared as additives to food or any products having direct contact to an oral environment, especially an oral environment susceptible to dental caries. To treat or prevent dental caries one or more compositions of the present invention can be formulated into a baby formula, mouthwash, lozenges, gel, varnish, toothpaste, toothpicks, tooth brushes, or other tooth cleansing devices, localized delivery devices such as sustained release polymers or microcapsules, oral irrigation solutions of any kind whether mechanically delivered or as oral rinses, pacifiers, and any food including, without limitation, chewing gums, candies, drinks, breads, cookies, and milk. 2013204065 11 Apr 2013 [0090] In solid dosage forms for oral administration (e. g., capsules, tablets, pills, dragees, powders, granules and the like), the STAMP is mixed with one or more pharmaceutically-acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, (5) solution retarding agents, such as paraffin, (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, acetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid 27 polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like. 2013204065 11 Apr 2013 [0091] A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered peptide or peptidomimetic moistened with an inert liquid diluent. Tablets, and other solid dosage forms, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of a STAMP therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain pacifying agents and may be of a composition that they release the STAMP(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The STAMP can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.
[0092] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In 28 addition to the STAMP, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents. 2013204065 11 Apr 2013 [0093] Suspensions, in addition to the STAMP, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
[0094] Formulations for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more STAMPs with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active agent. Formulations which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.
[0095] Formulations for the topical or transdermal or epidermal administration of a STAMP composition include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active component may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required. The ointments, pastes, creams and gels may contain, in addition to the STAMP composition, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, 29 polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof. Powders and sprays can contain, in addition to the STAMP composition, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane. 2013204065 11 Apr 2013 [0096] The STAMP composition can be alternatively administered by aerosol. This is accomplished by preparing an aqueous aerosol, liposomal preparation or solid particles containing the STAMPs. A nonaqueous (e. g., fluorocarbon propellant) suspension could be used. Sonic nebulizers can also be used. An aqueous aerosol is made by formulating an aqueous solution or suspension of the agent together with conventional pharmaceutically acceptable carriers and stabilizers. The carriers and stabilizers vary with the requirements of the particular compound, but typically include nonionic surfactants (Tweens, Pluronics, or polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols. Aerosols generally are prepared from isotonic solutions.
[0097] Transdermal patches can also be used to deliver STAMP compositions to an infection site. Such formulations can be made by dissolving or dispersing the agent in the proper medium. Absorption enhancers can also be used to increase the flux of the peptidomimetic across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the peptidomimetic in a polymer matrix or gel.
[0098] Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of this invention.
[0099] Formulations suitable for parenteral administration comprise a STAMP in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, 30 which may contain antioxidants, buffers, bacterostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents. 2013204065 11 Apr 2013 [00100] Examples of suitable aqueous and nonaqueous carriers which may be employed in the formulations suitable for parenteral administration include water, ethanol, polyols (e. g., such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
[00101] Formulations suitable for parenteral administration may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
[00102] Injectable depot forms are made by forming microencapsule matrices of a STAMP or in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of the STAMP to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Depot injectable formulations are also prepared by entrapping the STAMP in liposomes or microemulsions which are compatible with body tissue.
[00103] In a preferred embodiment of the invention, a STAMP composition is delivered to a disease or infection site in a therapeutically effective dose. As is known in the art of pharmacology, the precise amount of the pharmaceutically effective dose 31 of a STAMP that will yield the most effective results in terms of efficacy of treatment in a given patient will depend upon, for example, the activity, the particular nature, pharmacokinetics, pharmacodynamics, and bioavailability of a particular STAMP, physiological condition of the subject (including race, age, sex, weight, diet, disease type and stage, general physical condition, responsiveness to a given dosage and type of medication), the nature of pharmaceutically acceptable carriers in a formulation, the route and frequency of administration being used, and the severity or propensity of a disease caused by pathogenic target microbial organisms, to name a few. However, the above guidelines can be used as the basis for fine-tuning the treatment, e. g., determining the optimum dose of administration, which will require no more than routine experimentation consisting of monitoring the subject and adjusting the dosage. Remington: The Science and Practice of Pharmacy (Gennaro ed. 20.sup.th edition, Williams & Wilkins PA, USA) (2000). 2013204065 11 Apr 2013
Examples
Example 1. Targeted killing of Streptococcus mutans by a list of antimicrobial peptides [00104] 1.1 Design and construction of STAMPs used in the example and STAMP components (e.g., selectively targeting domains/peptides, antimicrobial peptides, and linker peptides). STAMPs and their components designed and synthesized in the examples are listed in Table 1. An initial STAMP was constructed by synthesizing full length S. mutans-specific competence stimulating peptide (CSP, 21 amino acids, SEQ ID NO 1, pheromone produced by S. mutans) with the antimicrobial peptide G2 (SEQ ID N03) (16 amino acids, derived from the wide spectrum antimicrobial peptide novispirin G10 (SEQ ID NO 35)(Eckert et al., 2006) at either the C-terminus or the N-terminus. Biological testing of these STAMPs did not reveal any antimicrobial activity. The C-terminal 16 amino acids of CSP, called CSPCi6 (SEQ ID NO 2), which in previous studies was shown to still have pheromone activity (Qi et al., 2005)), was used as a substitute for CSP. Peptides containing CSPCi6 at either the N or C-terminus of G2, with different linker regions of flexible amino acids in 32 between, were then synthesized and screened for their antimicrobial activities (data not shown). From among the potential STAMPs, C16G2 (SEQ ID NO 4) which consisted of (from N to C terminus) CSPCi6, a short linker peptide (GGG) and G2 (Table 1), was selected for further study due to its improved minimum inhibitory concentration (MIC), greatly enhanced killing kinetics and selectivity against S. mutans (when compared to G2 alone), as disclosed in detail below. 2013204065 11 Apr 2013 [00105] Table 1 - Peptide sequences (single-letter amino acid code) of selected STAMPs, and STAMP components
Peptide Properties Amino-acid sequence SEQ ID CSP Pheromone SGSLSTFFRLFNRSFTQALGK 1 C16 (CSPcie) Targeting TFFRLFNRSFTQALGK 2 G2 Antimicrobial KNLRIIRKGIHIIKK Y * 3 C16G2 STAMP TFFRLFNRSFTQALGKGGGKNLRIIRKGIHIIKKY* 4 M8 or cspM8 Targeting TFFRLFNR 5 M8G2 STAMP TFFRLFNRGGGKNLRIIRKGIHIIKKY * 6 S6L3-33 Antimicrobial FKKFWKWFRRF 7 C16-33 STAMP TRRRLFNRSFTOALGKSGGGFKKFWKWFRRF 8 M8-33 STAMP TFFRLFNRSGGGFKKFWKWFRRF 9 1903 Targeting NIFEYFLE 10 BD2.21 Antimicrobial KLFKFLRKHLL 11 1903-G2 STAMP NIFEYFLEGGGKNLRIIRKGIHIIKKY 12 1903- BD2.21 STAMP NIFEYFLEGGGKLFKFLRKHLL 13 C16- BD2.21 STAMP TFFRLFNRSFTQALGKGGGKLFKFLRKHLL 14 M8- BD2.21 STAMP TFFRLFNRGGGKLFKFLRKHLL 15 1903-33 STAMP NIFEYFLEGGGFKKFWKWFRRF 17 *Denotes peptide C-terminal amidation 33
Linker regions between targeting and killing peptides are underlined 2013204065 11 Apr 2013 [00106] To determine whether there was a region within the CSPCi6 sequence that was responsible for S. mutans-specific binding, we synthesized a series of fluorescently labeled CSPCi6 fragments, and analyzed their ability to bind to S. mutans. The following strategies were utilized in dissecting the CSPCi6 sequence (Table 2): First, a series of fragments were constructed by generating deletions of 3 or 4 amino acids, from the N to C terminus, across the CSPCi6 sequence (Cl6-1 to Cl6-5). Peptides lacking larger portions of the C or N termini of CSPCi6 were also synthesized (Cl6-6 to Cl6-12). Additionally, peptides with Arg to Asn (a positive to negative change in charge) (Cl6-4) or Phe to Gly substitutions (for a general decrease in hydrophobicity) (Cl6-3), as well as peptides representing a 4-residue Ala scan of the C16 sequence were constructed (Cl6-15 to Cl6-18). Binding assays were performed as described previously (15), and the results summarized in Table 2. CSPCi6 and any peptides containing Thr6 through Argl3 (TFFRLFNR, SEQ ID NO 5) of CSP were detected as bound to S. mutans UA159 or comD cells while any interruption to this region via deletion, substitution or Ala scanning reduced the detected fluorescent binding compared to CSPCi6- Some peptides, such as Cl6-3, -11, -16 and -17, which contained only Thr6-Phell and Phe7-Phell, showed binding but at a weaker intensity than CSPCi6 or any other peptides with the complete Thr6-Argl3 region. Additionally, we observed that Arg to Asn or Phe to Gly substitutions were deleterious to cell binding, suggesting that these residues within TFFRLFNR (SEQ ID NO 5, called M8 or CSPMs) are required for binding to S. mutans. CSPMs exhibited little or no binding to the other oral streptococci listed in Table 3, indicating that CSPM8 may also be capable of specifically binding to S. mutans surfaces. In general, the peptides listed in Table that showed positive binding to S. mutans can be used as targeting peptides against S. mutans. These peptides include C16 (SEQ ID NO 1), Cl6-3 (SEQ ID NO 39), Cl6-4 (SEQ ID NO 40), Cl6-5 (SEQ ID NO 41), Cl6-6 (SEQ ID NO 42), Cl6-11 (SEQ ID NO 47), Cl6-12 (SEQ ID NO 5), Cl6-16 (SEQ ID NO 51), Cl6-17 (SEQ ID NO 52), and Cl6-18 (SEQ ID NO 53). 34 [00107] Table 2 - Binding of CSP-fragment peptides to S. mutans. 2013204065 11 Apr 2013
Reported relative binding represents results from both UA159 and comD.
Relative S. mutans
Peptide_Amino acid sequence_binding C16 (SEQ ID NO 1) T F F R F F N R S F T Q A L G K +++ 3 to 4 amino acid internal deletions Cl6-1 (SEQ ID NO 37) - - R F F N R s F T Q A L G K Cl6-2 (SEQ ID NO 38) T F F - - - N R s F T Q A L G K - Cl6-3 (SEQ ID NO 39) T F F R F F - - - - T Q A L G K ++ Cl6-4 (SEQ ID NO 40) T F F R F F N R s - - - A L G K +++ Cl6-5 (SEQ ID NO 41) T F F R F F N R s F T Q - - - K +++ Terminal deletions Cl6-6 (SEQ ID NO 42) T F F R F F N R s +++ Cl6-7 (SEQ ID NO 43) - - - R F F N R s F T Q A - - - - Cl6-8 (SEQ ID NO 44) R s F T Q A L G K - Cl6-9 (SEQ ID NO 45) T F F - Cl6-10 (SEQ ID NO 46) T F F R - Cl6-11 (SEQ ID NO 47) T F F R F + 06-12 (SEQ ID NO 5) (CSPms) T F F R F F N R +++ Substitutions 06-13 (SEQ ID NO 48) T G G R F G N R s G T Q A L G K 06-14 (SEQ ID NO 49) T F F N F F N N s F T Q A L G K - Alanine-scannine 06-15 (SEQ ID NO 50) A A A A F F N R s F T Q A L G K 06-16 (SEQ ID NO 51) T F F R A A A A s F T Q A L G K + 06-17 (SEQ ID NO 52) T F F R F F N R A A A A A L G K ++ 06-18 (SEQ ID NO 53) T F F R F F N R s F T Q A A A A +++ [00108] Targeting peptides specific to S. mutans 1903 (SEQ ID NO 10) and antimicrobial peptides S6L3-33 (SEQ ID NO 7) and BD2.21 (SEQ IN NO 13) were developed in the inventors' laboratory (See Example 4). Targeting peptides were conjugated to antimicrobial peptides via a linker GGG (SEQ ID NO 17) to yield the STAMPS C16-33 (SEQ ID NO 8), M8-33(SEQ ID NO 9), 1903-BD2.21(SEQ ID NO. 13), and C16-BD2.21(SEQ ID NO 14), all of which were tested in the similar manner as C16G2 and M8G2.
[00109] All peptides listed in Tables 1 and 3 were synthesized using double-coupling cycles by standard 9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase synthesis methods (431A Peptide Synthesizer, Applied Biosciences or Apex396, Advanced Chemtech) as described previously (Eckert et al., 2006). Completed peptides were cleaved from the resin with 95% trifluoroacetic acid (TFA) with appropriate 35 scavengers and purified by reverse-phase high performance liquid chromatography (RP-HPLC) (ACTA Purifier, Amersham) to 90-95%. Peptide molecular mass was determined by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Peptides C16G2, G2, and M8G2 were synthesized with amidated C-termini using Fmoc-Tyr(tBu)-Rink Amide MBHA resin (Anaspec). All other peptides were synthesized with the appropriately-substituted Wang resins. 2013204065 11 Apr 2013 [00110] 1.2 Fluorescent labeling of peptides and fluorescence microscopy. Aliquots of CSPCi6 (SEQ ID NO 2), CSP-fragment peptides (Table 2), and C16G2 (SEQ ID NO 4) were labeled with carboxyfluorescein (Sigma) as described previously (Eckert et al., 2006). After peptide cleavage but prior to the bacterial labeling assay, fluorescence intensity per μΜ peptide was checked fluorimetrically (kex=488nm, kem=520nrn VersaFluor, BioRad) and found to be relatively similar (data not shown). To evaluate the level of peptide binding to bacteria, streptococci from an overnight culture (OD6oo of 0.7-1.0) were washed with phosphate buffered saline (1XPBS), diluted 1:2 into lxPBS, and exposed to peptide (16 μΜ) for 5 min at 25 °C. After incubation with peptide, unbound agent was removed from the bacteria by three cycles of centrifugation (5 min, 16,000 χ g) and resuspension in lxPBS. Labeling of oral streptococci was evaluated using brightfield and fluorescence microscopy (Nikon E400) at a 40x magnification. The digital images utilized for the semi-quantitative binding assessment were acquired with the factory-supplied software (SPOT, Diagnostics).
[00111] 1.3 Determination of antimicrobial activity. The general antimicrobial activity of peptides against planktonic bacteria was determined by a MIC assay in TH broth (all oral streptococci) (Qi et al., 2005).
[00112] S. mutans, S. gordonii Challis (DL1), and S. sanguinis NY101 strains were grown in Todd Hewitt (TH, Fisher) broth medium at 37 °C under anaerobic conditions (80% N2, 10% C02, and 10% H2). S. mutans strains UA159 (Ajdic et al.,2002), ATCC 25175, and T8 (Rogers, 1975), are wild-type clinical isolates, while comD is a knockout mutant that was constructed previously from the wild-type UA140 36 background (Qi et al., 2005). Luciferase expressing S. mutans strain JM11 was constructed from UA140 as described (Merritt et al., 2005). Exponentially growing bacterial cells were diluted to ~ 1 x 105 cfu/mL in TH and placed into 96-well plates (Fisher). Peptides were then serially diluted and added to the bacteria. MIC was determined by identifying the concentration of peptide that completely inhibited bacterial growth after ~24 h incubation. 2013204065 11 Apr 2013 [00113] 1.4 Determination of bactericidal kinetics. To determine the short term killing rate and selectivity of C16G2 and G2 we performed time-kill experiments, essentially as described previously (Eckert et al.,2006). S. mutans UA159, S. gordonii, or S. sanguinis were grown to log phase and diluted to ~lxl05 cfu/mL in growth medium. Under aerobic conditions, 25 μΜ G2 or C16G2 was added to the cell suspension and incubated at 25 °C. At 1 min, 10 pL of cell suspension was removed, rescued by dilution into growth medium (1:50) and kept on ice. For plating, 20-500 pL of rescued cells were spread on growth medium agar plates and colonies were counted after overnight incubation at 37 °C under anaerobic conditions. We considered 60 cfu/mL as the detection limit for this assay. Values of surviving cfu/mL were expressed as the ratio of survivors from C16G2-treated cultures to cfu/mL from samples exposed to G2.
[00114] 1.5 Examination of antimicrobial activity against single-species biofilms. To initiate biofilm formation, ~ 1 χ 107 bacteria per well (from overnight cultures) were seeded in TH medium (100 pL) to a 96-well flat-bottom plate. For all streptococci except S. mutans, the medium was supplemented with 0.5% (w/v) mannose and glucose. S. mutans UA159 biofilms were grown with 0.5% (w/v) sucrose. Plates were then centrifuged briefly to pellet the cells, and bacteria were incubated for 3-4 hours at 37 °C for biofilm formation. After incubation, the supernatant was carefully removed and biofilms were treated with 25 pM peptide in lx PBS or lx PBS alone for 1 min. The peptide solution was then removed and 100 pL TH was added to further dilute any remaining peptide. To minimize biofilm loss, cells were briefly centrifuged after TH addition, after which the supernatants were removed and fresh medium plus 37 appropriate sugars were returned. Cells were then incubated anaerobically at 37 °C and biofilm growth was monitored over time by measuring absorbance at OD600 with a microplate spectrophotometer (Benchmark Plus, BioRad). 2013204065 11 Apr 2013 [00115] 1.6 Evaluation of antimicrobial activity against bacterial biofilm in saliva. For these experiments, we employed methods similar to those previously described (Bleher et al., 2003). A day prior to the assay, saliva was collected and pooled from 5 adult volunteers in the laboratory, diluted 1:4 in TH broth and centrifuged 2,000xg for 10 min. The supernatant was then filter sterilized (0.2 pm filter, Nunc) and stored at 4 °C. A portion of pooled saliva was also diluted 1:2 in lxPBS and processed as before. On the day of the assay, overnight cultures of JM11 and other oral streptococci were normalized to OD600 1 0 and ~3 x 106 cfu/mL of each species was added to 10 mL of the TH-diluted saliva. Sucrose, mannose, and glucose (1% w/v each) were then added and the solution mixed. Aliquots (500 pL) of the saliva and bacteria mixture were then placed into 1.5 mL Eppendorf tubes (Fisher). After a brief centrifugation (4,000xg, 2 min), the tubes were incubated for 3-4 hours at 37 °C to form multispecies biofilms. The supernatants were then removed and the spent media replaced with 100 pL PBS-diluted saliva (1:2) plus 25 pM (freshly added) peptide. After biofilms were exposed to the agent for 5 minutes, the PBS-saliva was removed, cells briefly centrifuged, and 500 pL fresh TH-saliva with sugars was returned. At each time point, total biofilm growth was measured by reading absorbance at OD60o, and the health of S. mutans within the population examined by relative luciferase expression (relative light unit, (RLU) production), as described previously (Merritt et al., 2005). Briefly, biofilms were resuspended by vortex and aspiration and 100 pL of each sample transferred to a new Eppendorf tube with 25 pL 1 mM D-luciferin (Sigma) solution suspended in 0.1 M citrate buffer, pH 6.0. For the 2 h timepoint, biofilms were stimulated after resuspension by the addition of 1% sucrose 30 min prior to recording luciferase activity. RLU production was measured using a TD 20/20 luminometer (Turner Biosystems) and reported values were obtained from the average of 3 independent samples. The data were plotted as the RLU/OD600 over time. 38 [00116] 1.7 C16G2 has enhanced antimicrobial activity and specificity against planktonic S. mutans cells. To evaluate the antimicrobial activity and general specificity of C16G2, minimum inhibitory concentration (MIC) tests were performed against a panel of bacterial species including various strains of S. mutans and closely related oral streptococci (Gilmore et al., 1987). As shown in Table 3, The MIC values of C16G2 ranged from 3-5 DM for all S. mutans strains tested, a 4-5 fold increase in antimicrobial activity over the parental antimicrobial peptide G2 (12-20 □ M). In comparison, we observed little difference in susceptibility between G2 and C16G2 (2 fold or less) against S. gordonii and S. sanguinis. 2013204065 11 Apr 2013 [00117] GlOKHc (SEQ ID No 36) did not show much improvement in MIC after 24 h incubation, but displayed greatly enhanced killing kinetics and specificity against the targeted bacteria during short time exposure (when compared to the untargeted parental antimicrobial peptide (Eckert et al., 2006). Therefore, comparative experiments were performed to examine the killing ability C16G2 and G2 against its targeted and untargeted bacteria after a short time exposure. As shown in Figure 1, with 1 min exposure, C16G2 was over 20-fold more active against its targeted bacterium S. mutans, in comparison to G2, while it exhibited a similar level of activity as G2 against other oral streptococci tested. These findings provided the first indications that the addition of the CSPCi6 targeting domain to G2 had resulted in an antimicrobial with selective activity against S. mutans, and not other closely related oral streptococci.
[00118] Table 3 - MIC of G2-containing STAMPs and STAMP components against bacteria. MICs represent averages of at least 3 independent experiments with standard deviations. MIC (aM) of:
Strains_CSP CSPqe G2_C16G2 CSPM8 M8G2 S. mutans 39 2013204065 11 Apr 2013 UA159 50.8±9.3 >60 12.1±4.5 3.011.6 >60 3.2511.9 25175 >60 >60 14.812.0 3.810.3 >60 3.510.5 T8 >60 >60 14.211.5 3.710.2 >60 nt comD >60 >60 15.314.2 5.112.4 >60 4.0+2.0 Non-mutans streptococci S. gordonii >60 >60 41.3114.0 23.517.8 >60 20+5.0 S. sanguinis >60 >60 33.617.5 19.1+4.0 >60 15+2.5 [00119] 1.8 C16G2 is also active against biofilm cells. S. mutans predominantly exist in a biofilm growth state in vivo. It is known in the art that biofilm-associated cells are 100-1000 fold more resistant to antibiotics (Donlan et al., 2002). To test whether C16G2 still has activity against S. mutans biofilms in vitro, biofilm-associated S. mutans, S. gordonii, or S. sanguinis, were treated with 25 DM C16G2, G2, CSP, CSPCi6, or lxPBS, for 1 min, washed, and their re-growth was monitored over time. As shown in Figure 2, S. gordonii or S. sanguinis biofilms exposed to any of the peptides tested grew similarly to untreated biofilms after peptide addition and removal (Fig. 2A-B). In contrast, S. mutans strains UA159 (Fig. 2C) as well as T8 and 25175 (data not shown) were severely inhibited by treatment with C16G2, but were unaffected by treatment with the other peptides. These results indicate that C16G2 can function as an anti-S. mutans STAMP in a biofilm environment with only a short period of exposure (1 min), a time-frame which is relevant for clinical treatments in the oral cavity (Axelsson & Lindhe, 1987).
[00120] 1.9 C16G2 can selectively eliminate S. mutans from a mixed species biofilm. In addition to growing as biofilm in vivo, S. mutans are also constantly bathed in saliva as they adhere to the tooth surface. To examine whether C16G2 could selectively kill S. mutans under these conditions, 2 species of non-cariogenic oral streptococci (S. gordonii and S. sanguinis), were mixed with S. mutans JM11, a strain harboring a transcriptional fusion between luciferase (luc) and the promoter for 40 the constitutively active gene lactate dehydrogenase (ldh), which has the same susceptibility to C16G2 as the wild type UA159. JM11 has been previously utilized to measure the fitness of S. mutans populations, and decreasing relative light unit (RLU) production was shown to strongly correlate with reduced cell viability (Merritt et al., 2005). The mixed-species biofilms were formed with saliva, and then peptides (25 μΜ) suspended in saliva were added for 5 min and removed, and the posttreatment growth of the biofilm was further monitored. The number of viable S. mutans cells within the biofilm was quantified in parallel by luciferase expression. It was found that C16G2 was able to dramatically reduce the S. mutans population within the mixture (reflected in the low luciferase activity) after 5 min exposure, compared to CSPCi6 and G2 (Fig. 3). Interestingly, even after 120 min post treatment, the total number of S. mutans within the mixture remained low (Fig. 3). Taken together, these results indicate that a short exposure of C16G2 is capable of selectively inhibiting the growth of S. mutans within a multi-species biofilm and in the presence of saliva for a minimum of 2 h without harming bystander bacteria or affecting the overall health of the biofilm. 2013204065 11 Apr 2013 [00121] 1.10 Enhanced antimicrobial activity of C16G2 is related to targeted ComD-independent binding of CSPCi6 to S. mutans. To further explore the mechanism of C16G2 enhanced activity against S. mutans, CSPCi6 and C16G2 were fluorescently labeled and tested their ability to bind S. mutans and other streptococci. Consistent with observed killing activity, it was found that CSPCi6 and C16G2 could specifically bind to S. mutans with a very short time exposure (1-2 min), but not to other oral streptococci (data not shown). Previous genetic studies suggested that CSP may interact with ComD to activate DNA competence in S. mutans (Li et al, 2001). It was unexpected to find that a similar MIC was observed for UA159 and the comD strain (Table 3). Consistent with this observation, it was also found that fluorescent labeled CSPCi6 and C16G2 bound to UA159 and the comD mutant in a similar manner, indicating that the specific binding ability of CSP to S. mutans is independent of ComD. 41 [00122] 1 .11 M8G2 has similar anti-S. mutans activity as C16G2. Based on the data above, it was hypothesized that CSPMs would be sufficient to function as an alternative targeting domain for an anti-S. mutans STAMP. To test this hypothesis, CSPM8 and G2 were synthesized together to form the STAMP M8G2 (Table 1). As shown in Table 3, M8G2 displayed similar MICs against S. mutans and other oral streptococci when compared to C16G2. Furthermore, single-species biofilm inhibition assays showed that M8G2, like C16G2, was capable of inhibiting the recovery of S. mutans biofilms (Fig. 4A), but not those of S. sanguinis (Fig. 4B), after 1 min exposure. Since the CSPMS domain is much smaller than CSPCi6 and consequently easier to chemically synthesize, these results provide a basis for a future design of shorter anti-S. mutans STAMPs based on CSPMs· 2013204065 11 Apr 2013 [00123] 1.12 CSPCi6/CSPM8-guided STAMPs are functional with an alternative killing domain. Since the targeting and antimicrobial components of a STAMP are functionally independent, despite being synthesized as one peptide (Eckert et al, 2006), it was reasoned that a combination of CSPCi6 or CSPMs with a different general antimicrobial peptide could also result in increased killing activity and selectivity towards S. mutans when compared with the untargeted killing peptide alone. Therefore, both targeting peptides were conjugated to S6L3-33, a model wide-spectrum antimicrobial peptide, in a similar arrangement as C16G2 and M8G2, to yield the STAMPs C16-33 and M8-33 (Table 1). As shown in Table 4, a 2-3 fold difference in MIC between S6L3-33 and the derived STAMPs was observed against S. mutans and the other oral streptococci tested. However, when single-species biofilm studies were conducted (shown in Fig. 5), the S. mutans-selective activity of the STAMPs was readily apparent: both Cl6-33 and M8-33 were capable of retarding S. mutans biofilm growth after a short exposure (Fig. 5A), while cultures of S. sanguinis were not affected by STAMP administration (Fig. 5B). These results indicate a clear enhancement of STAMP activity selective for S. mutans biofilms.
[00124] Table 4 - MIC of STAMPs constructed with the S6L3-33 antimicrobial region. 42 MICs represent averages of at least 3 independent experiments with standard deviations. MIC (uMl
Peptide UA159 comD S. sanguinis S. gordonii S6L3-33 7.0±3.0 6.5±2.5 40±7.5 20±5.0 C16-33 2.5±2.1 2.2±0.5 13.3±5.8 14.6±5.0 M8-33 2.5±2.0 2.5±2.0 20±2.0 10±2.5 2013204065 11 Apr 2013 [00125] In general, a series of STAMPs which exhibited specificity for S. mutans and not other oral streptococci were synthesized and evaluated. The STAMPs were designed for S. mutans-selective activity by incorporating portions of a natural pheromone produced by these cariogenic bacteria (CSP) as the targeting domain within the linear STAMP peptide. By exclusively utilizing short (<3 kD) linear peptides for the targeting and antimicrobial regions, we were able to rapidly synthesize and isolate the complete STAMP molecule in once piece via solid-phase chemical methods, a distinct advantage over the recombinant expression and difficult purification routes necessary to construct the large (>70 kD) protein-based targeted antimicrobials that have been described (Qiu et al. 2005). Additionally, the flexibility provided by synthetic routes enabled us to easily increase STAMP diversity by switching between different combinations of targeting domains (CSPMs and CSPcie) and killing domains (G2 and S6L3-33) when constructing STAMPs against S. mutans, a task that would otherwise require tedious cloning procedures.
[00126] As shown in Figure 5, CSPcie and CSPMs were able to be conjugated to an alternative antimicrobial peptide (S6L3-33) without the loss of S. mutans selective killing ability. This finding further validates the notion that the STAMP targeting and antimicrobial domains function independently, and are capable of being linked in different combinations without the loss of activity. This suggests that future STAMP construction will be an unlimited “tunable” process whereby a myriad of 43 combinations of antimicrobial, linker and targeting domains can be synthesized in order to select a STAMP with the best specific activity. Furthermore, bacterial STAMP resistance (should it evolve) (Perron et al, 2006) could be easily overcome by switching to alternative, functionally analogous STAMP components, as was done with G2 and S6L3-33 in this study. Additionally, peptide pheromones are widely utilized by pathogenic bacteria especially Gram-positive organisms, and therefore represent a large and growing pool from which future targeting peptides could be selected for STAMP construction. 2013204065 11 Apr 2013 [00127] C16G2, M8G2, C16-33 and M8-33 displayed robust specific activity against targeted S. mutans bacteria in planktonic cultures and in biofilms with both single and multi-species, suggesting that we were able to construct a set of functional STAMPs that can discriminate between S. mutans and other non-cariogenic oral streptococci. This selective activity, combined the low cytotoxicity of these peptides (Eckert, et al, unpublished data) indicates that they are useful for anti-caries therapeutic development. Currently, treatments for S. mutans infection include abstinence from dietary sugars, mechanical removal of the dental plaque, and general biocide mouthwashes. While all are temporarily effective to varied degrees, the unavoidable loss of normal flora that occurs with mechanical removal or general antibiotic treatment allows S. mutans to re-establish a niche in the oral cavity without difficulty (Caufield et al., 2000). Therefore, a STAMP with a pathogen-selective (e.g., S. mutans-selective) killing ability is an ideal solution which selectively kills or reduces the pathogen (e.g., S. mutans) in the flora and allows the normal flora to outgrow affected S. mutans populations. Such an “antibiotic-probiotic” therapeutic will help prevent dental caries progression and the high health care costs associated with this disease (Anderson & Shi, 2006).
Example 2. Enhancement of antimicrobial activity against Pseudomonas aeruginosa by co-administration of GlOKHc and tobramycin.
[00128] 2.1 Pseudomonas aeruginosa is a common opportunistic human pathogen that is associated with life-threatening acute infections and chronic airway 44 colonization during cystic fibrosis. In the US Patent Application Publication NO. 20040137482, novispirin G10, a wide-spectrum antimicrobial peptide was converted into a selectively-targeted antimicrobial peptide (STAMP), GlOKHc. Compared to novispirin G10, the GlOKHc STAMP had an enhanced killing ability against Pseudomonas mendocina. In this experiment, we explored the antimicrobial activity of GlOKHc against P. aeruginosa and a synergistic enhancement in killing activity when the GlOKHc STAMP was co-administered with tobramycin. 2013204065 11 Apr 2013 [00129] 2.2 The GlOKHc STAMP and its components. GlOHc STAMP has the following sequence and components:
GlOKHc [targeting peptide-linker peptide-antimicrobial peptide, the linker is underlined]: KKHRKHRKHRKHGGSGGSKNLRRIIRKGIHIIKKYG (SEQ ID NO 36) G10 (Novispirin) antimicrobial peptide: KNLRRIIRKGIHIIKKYG (SEQ ID NO 35)
Cat-1 (also called KH) targeting peptide: KKHRKHRKHRKH (SEQ ID NO 31) Linker peptide: GGSGGS (SEQ ID NO 28).
Solid-phase peptide synthesis of G10 (KNLRRIIRKGIHIIKKYG, SEQ ID NO
35) and GlOKHc (KKHRKHRKHRKH-GGSGGS-KNLRRIIRKGIHIIKKYG, SEQ ID NO 36) was carried out using the Fast-Fmoc (9-fluorenylmethoxycarbonyl) methodology on a 431A Peptide Synthesizer (Applied Biosciences). Completed peptides were cleaved from the resin using 95% TFA with the appropriate scavengers.
Peptide mass was confirmed by matrix-assisted laser desorption/ionization (MALDI) mass spectroscopy (Voyager System 4291, Applied Biosystems) and crude peptides
purified by reverse-phase high-pressure liquid chromatography (HPLC, ACTA 45
Purifier, Amersham) while monitoring UV 215. The mobile phase during HPLC consisted of water/acetonitrile (with 0.1% trifluorocetic acid) at a flow rate of 0.5 mL/min (Source 15 RPC column, Amersham). The HPLC and MALDI profiles for purified GlOKHc are shown in Figure 6. Specifically, after purification, a single peak for GlOKHc was observed at retention volume 10.06 mL (Fig. 6A), which was found to have the expected mass for GlOKHc (predicted 4267.08, observed 4267.44) as shown in Figure 6B. 2013204065 11 Apr 2013 [00130] 2.3 Antimicrobial Activities. The general antimicrobial activities of GlOKHc, G10, and tobramycin against clinical isolates of P. aeruginosa were evaluated by minimum inhibitory concentration (MIC) assay as previously described (Eckert et al., 2006) and shown in Table 5. MICs are reported in μΜ, though for familiarity, 1 μΜ tobramycin = 0.468 μg/mL. P. aeruginosa were grown to log phase and adjusted to ~lxl05 cfu/mL in Mueller-Hinton (MH) broth and added to 96-well plates. Two-fold serial dilutions of peptide were then added to bacteria and the plates incubated for 18-24 hours at 37 °C. MIC was determined as the concentration of peptide present in the last clear well (no growth). As expected, GlOKHc was significantly more active against the P. aeruginosa clinical isolates when compared with G10 alone (Student’s t test, p = 0.001): the MICs for GlOKHc ranged from 0.5 to 29 μΜ (mean 6.22 μΜ), compared with the MICs for G10, which ranged from 10 to 60 μΜ (mean 23.4 μΜ). Since the KH domain (or the Cat-1 peptide) itself does not have any antimicrobial activity, the increased anti-P. aeruginosa activity of GlOKHc is likely due to the targeted binding ability of KH to Pseudomonas spp, as previously reported (Eckert et al., 2006). In contrast to tobramycin, GlOKHc was also effective against aminoglycoside and multiple-antibiotic resistant P. aeruginosa isolated from CF patients (AGR10, MR15). Additionally, as mucoid P. aeruginosa are often associated with reduced susceptibility to antimicrobial agents, we were encouraged to find that GlOKHc was active against one such strain, PDO300. Overall, GlOKHc was 46 not as active as tobramycin against the sensitive isolates examined (typically 1-2 dilution steps less effective). 2013204065 11 Apr 2013 [00131] Table 5 - MICs of tobramycin, G10, and GlOKHc against P. aeruginosa lab and clinical isolates. The average MIC from at least 3 independent experiments is shown. The KH targeting domain alone does not have any antimicrobial activity (data not shown). For reference, 1 μΜ tobramycin = 0.468 pg/mL.
Strain GlOKHc MIC (uM) novispirin G10 tobramyi PAOl 6 23 2.5 PA 14 5.5 10 0.7 PAK 5.5 13 2.12 PDO300* 6 45 nt ATCC 15692 6 16 3.05 ATCC 27583 6 16 1.75 ATCC 10145 4.5 14.5 1.75 ATCC 9027 5.5 15 2.12 AGR10 1.1 18 55 MR15 0.5 14 55 S40 29 60 0.4 S60 3.13 30 0.4 S100 1.1 30 3.5 *mucoid phenotype, nt: not tested [00132] Time-Kill (killing kinetics) experiments were performed essentially as described previously (Eckert et al., 2006). Briefly, P. aeruginosa were grown to log phase and diluted to ~lxl05 cfu/mL (moderate density planktonic cultures) in LB with 30% mouse serum (MP Biomedicals) prior to the addition of 10 μΜ tobramycin, G10 or GlOKHc to the cell suspensions. At each time point, 10 pL of the culture was removed and P. aeruginosa cells rescued by dilution in 500 pL LB and kept on ice until plating. Surviving cfu/mL were quantitated after plating on LB agar and incubating overnight at 37 °C under aerobic conditions.
[00133] As shown in Lig. 7, the killing kinetics assay revealed that GlOKHc had an obvious improvement in killing versus G10 against P. aeruginosa: 10 pM GlOKHc treatment of the cultures was associated with a decrease in viable P. aeruginosa (to under 100 cfu/mL by 30 min), while G10 was ineffective over the time course 47 examined. The rate of GlOKHc antimicrobial activity was similar to an equimolar dosage of tobramycin (4.68 pg/mL). These results suggest that GlOKHc and tobramycin have similar potency against clinical isolates as well as lab strains, and that GlOKHc can inhibit the growth of drug-resistant P. aeruginosa. Furthermore, the data indicate that GlOKHc appears to require the KH Pseudomonas spp targeting domain for effective P. aeruginosa cell killing: G10 alone showed poor activity unless incubated 18-24 h (Table 5). 2013204065 11 Apr 2013 [00134] 2.4 Synergistic killing effect of GlOKHc and tobramycin. For evaluation of enhanced activity between GlOKHc and tobramycin against high density planktonic cultures, ATCC 15692 were grown overnight were adjusted to ~lxl08 cfu/mL in ddH20 (pH 7.4) and exposed to 5 μΜ tobramycin, 5 μΜ GlOKHc or a combination of both agents (a combination of 5 pM tobramycin (2.34 pg/mL) and 5 pM GlOKHc or G10). 10 pL of the treated cultures was rescued by dilution after 24 h and the surviving cfu/mL plated on LB and counted after growth on LB agar.
[00135] As shown in Figure 8, we observed a clear enhancement in killing activity when tobramycin and the STAMP (but not G10) were co-administered. Surviving cfu/mL from co-treated cultures (~1 x 103 cfu/mL) were 5 logi0 lower than the level recovered from untreated cultures (~1 x 108 cfu/mL) or those exposed to either tobramycin or GlOKHc (1 x 107 cfu/mL and ~1 x 108 cfu/mL, respectively). These results suggest that when applied together, these agents are markedly more effective against planktonic P. aeruginosa than either constituent singly and can eliminate nearly all of a high cell-density culture by 24 hours, even when GlOKHc was administered at a concentration below the MIC for the tested strain.
[00136] 2.5 Synergistic killing effect of GlOKHc and tobramycin on biofilm. A synergistic killing effect between tobramycin and GlOKHc was also observed against biofilm-associated P. aeruginosa. In this experiment, a rotating-disk biofilm reactor system was used for generating quantitative data on biofilm susceptibility to tobramycin, G10 and GlOKHc. The system consisted of a reactor vessel containing 250 mL of diluted trypticase-soy broth (TSB) (1:100) medium. Reactors were 48 inoculated with overnight cultures (1%, v/v). After static overnight growth in TSB, a flow of fresh medium was initiated (dilution rate, 0.7 h '). After 24 h in a flow of medium, the polycarbonate chips with attached biofilm bacteria were aseptically removed from the spinning disk and washed three times in ddH20 (pH 7.4) and incubated in 1 mL ddH20. G10 (100 pg/mL), GlOKHc (100 pg/mL), tobramycin (100 pg/mL), or a combination of the two was added as indicated. The chips were then incubated for 4 or 24 h in 24-well tissue culture plates (Falcon no. 353047; Becton Dickinson Labware, Franklin Lakes, NJ). To estimate the number of viable P. aeruginosa remaining, the disks were placed in 1 mL PBS and the cells were dispersed using a tissue homogenizer (Brinkmann Instruments, Westbury, NY) and the total cfu per chip was determined by serial dilution and plating on LB agar. 2013204065 11 Apr 2013 [00137] As shown in Figure 9, 100 pg/mL GlOKHc or 100 pg/mL tobramycin alone had very limited killing effects against P. aeruginosa biofilms after 4 h or even 24 h. However, the combination of 100 pg/mL GlOKHc and 100 pg/mL tobramycin dramatically reduced the level of surviving cfu/mL after 4 h, a 4 logi0 improvement in killing ability when compared to either agent alone. More strikingly, no cfu/mL were recovered when the combined agents were co-incubated with P. aeruginosa for 24 h (a decrease of nearly 5 logio from individual applications). These data indicate a strong enhancement in killing activity when GlOKHc and tobramycin are used against in vitro P. aeruginosa biofilms. Additionally, these results were consistent with Figure 8, suggesting that GlOKHc and tobramycin may be synergistic against P. aeruginosa in planktonic or biofilm modes of growth, though further experiments are necessary to fully establish synergistic activity.
[00138] 2.6 GlOKHc mediated membrane permeability. The results from Figures 8 and 9 suggest that the rate of tobramycin cell killing could be increased by GlOKHc co-treatment. In the absence of peptide, robust bacterial uptake of tobramycin is an active process that requires an intact ΔΨ gradient (electric potential of the proton motive force) which is maximized during aerobic respiration. This process may be slowed or eliminated in anoxic environments, (such as the interior of biofilms), 49 suggesting that tobramycin diffusion across P. aeruginosa membranes (or lack thereof) is critical to at least one mechanism of aminoglycoside tolerance in these bacteria (14, 33, 40). Therefore, due to the membrane-disrupting AMP domain in GlOKHc and its previously described anti-outer membrane activity (11), it was contemplated that GlOKHc was permeabilizing the P. aeruginosa outer and inner membranes, enabling increased tobramycin uptake and leading to the observed synergy. 2013204065 11 Apr 2013 [00139] In order to confirm that GlOKHc-membrane disruption could mediate cellular accumulation of a small molecule, overnight cultures of P. aeruginosa were diluted 1:50 in LB and grown to log phase (3-4 h, ~lxl05 cfu/mL) prior to mock treatment or treatment with 2 μΜ GlOKHc. After 5 min, membrane-compromised cells were stained with propidium iodide (PI) (LIVE/DEAD Baclight Viable Stain, Invitrogen) in the presence or absence of sub-lethal GlOKHc concentrations (2 μΜ) in accordance with the manufacturer’s protocol. PI is a small molecule dye that binds double stranded DNA and fluoresces red upon excitation and was used as a surrogate for tobramycin as the internalization of an aminoglycoside is not easily assayable. The dye cannot cross an intact cytoplasmic membrane and is commonly used for cell viability analysis. Dye intercalation into DNA (red stain), was detected by fluorescence microscopy (Nikon E400) at a 40x magnification. Brightfield and red fluorescence images were collected using the factory default settings (SPOT, Diagnostics). To determine bactericidal activity after peptide treatment and PI staining, samples prepared in parallel to visualized cultures were plated on LB agar after 1:5 serial dilutions. Images of surviving cfu/mL were taken with a GelDoc (BioRad) using QuantityOne software.
[00140] It was expected that GlOKHc-induced membrane disruption would lead to an increase in nucleic acid staining when compared to PI alone. As shown in Figure 10, bacteria treated with PI alone remained unstained. In comparison, intracellular PI staining was clearly visible in cultures exposed to PI and GlOKHc. Additionally, the amount of red fluorescence observed was proportional to the amount and length of GlOKHc treatment (data not shown). To ensure that we were not simply staining P. aeruginosa killed by GlOKHc, the viable cfu/mL were evaluated from the visualized 50 cultures. From the serial dilutions shown below the images in Figure 10, it was clear the number of viable P. aeruginosa recovered was similar between cultures treated with PI alone or PI/G10KHc. Overall, these data suggest that a sub-lethal dosage of GlOKHc can induce membrane damage and promote the uptake of small molecules, such as tobramycin or PI into metabolically active P. aeruginosa cells. 2013204065 11 Apr 2013 [00141] 2.7 Conclusion. In general, In this study, we explored the antimicrobial activity of GlOKHc against P. aeruginosa. GlOKHc was found to be highly active (equal to tobramycin) against P. aeruginosa clinical isolates. Most interestingly, we observed a synergistic-like enhancement in killing activity when biofilms and planktonic cultures of P. aeruginosa were co-treated with GlOKHc and tobramycin. The data indicate that the mechanism of enhanced activity may involve increased tobramycin uptake due to GlOKHc-mediated cell membrane disruption. These results suggest that GlOKHc may be useful against P. aeruginosa during acute and chronic infection states, especially when co-administered with tobramycin.
[00142] P. aeruginosa is a persistent and recurrent opportunistic pathogen responsible for life-threatening recurrent infections during CF. Frequent isolation of antibiotic-resistant P. aeruginosa suggests that it is critical that new therapies be developed to inhibit and treat P. aeruginosa colonization of airway mucosal surfaces before currently-prescribed treatment options are no longer effective.
[00143] This experiment shows that GlOKHc is markedly improved in comparison to its wide-spectrum parent peptide G10, and is similar to that of tobramycin. Additionally, GlOKHc is effective against high density planktonic cultures and P. aeruginosa biofilms in vitro (Fig. 3-4). When compared to tobramycin, GlOKHc was nearly 10-fold more effective per μΜ at reducing biofilm viability (100 pg/mL tobramycin = 213 μΜ, 100 pg/mL GlOKHc = 23.5 μΜ). Against high-density planktonic cells, however, 5 μΜ (2.34 pg/mL) tobramycin alone was markedly more bactericidal than either 5 μΜ G10 or GlOKHc after 24 hours (1-2 logio improvement). The difference in tobramycin activity may be linked to the anaerobic environment 51 found at the interior of P. aeruginosa biofilms, which inhibits robust aminoglycoside cellular uptake. 2013204065 11 Apr 2013 [00144] The highest level of anti-P. aeruginosa activity observed in planktonic or biofilm cultures occurred when both agents were applied together. Co-administered tobramycin and GlOKHc resulted in a marked enhancement of killing activity: nearly 10,000-fold more bacteria were eliminated by co-treatment than by either agent alone in planktonic and biofilm cultures. Though additive and synergistic anti-P. aeruginosa activity has been described between an antimicrobial peptide and tobramycin (Saiman et al., 2001), as well as tobramycin plus numerous other conventional small-molecule antibiotic (Bonacorsi et al., 1999), the current experiment represents the first reported example of biofilm-associated P. aeruginosa being synergistically or additively eliminated by an aminoglycoside/peptide combination.
[00145] Aerosolized tobramycin has been approved for the control of P. aeruginosa infections in CF patients, and not unexpectedly, tobramycin and aminoglycoside-resistant strains of P. aeruginosa and other organisms have been isolated from CF sputum. This fact, combined with the relatively high rate of unpleasant post-treatment dyspnea, bronchospasm, and increased cough, suggests that tobramycin may be best utilized in a smaller dosage size in combination with another agent. It is concluded that GlOKHc can be a candidate for co-administration due to its engineered Pseudomonas selectivity, and potent antimicrobial effects against P. aeruginosa biofilms and multi-drug and aminoglycoside-resistant strains.
Example 3. Enhanced stability and activity of the GlOKHc STAMP by using D-amino acid enantiomer to synthesize the STAMP and/or chemical antagonists (e.g., rhDNase).
[00146] 3.1 Material preparation and methods. GlOKHc (KKHRKHRKHRKH-GGSGGS-KNLRRIIRKGIHIIKKYG, SEQ ID NO 36, [targeting domain-linker-antimicrobial domain]), and the D-enantiomer GlOKHc-D were synthesized by Fast-52
Fmoc (9-fluorenylmethoxycarbonyl) methodology on a 431A Peptide Synthesizer (Applied Biosciences), as described previously (Eckert et al., 2006), using D-amino acids for GlOKHc-D purchased from Anaspec (San Jose, CA). 2013204065 11 Apr 2013 [00147] Eight expectorated sputum samples from different patients were obtained from patients with CF at Children's Hospital Los Angeles (Los Angeles, CA, USA) during routine clinical practice and stored at -80 °C within 1 h of collection. Sputum sample collection for this study was approved by the institutional review board at Children's Hospital Los Angeles (CCI #05-00040). All personal identifiers, including age, gender and prognosis, were unknown to our laboratory.
[00148] 3.2 Activity and stability of GlOKHc and GlOKHc-D in sputum. Determination of peptide antimicrobial effects or activity in sputum was investigated in a similar manner to previous reports (Sajjan et al., 2001). To assay the activity of GlOKHc and GlOKHc-D against exogenous P. aeruginosa in sputum, collected sputum samples were diluted 1:10 in 10 mM PBS (PBS) and pooled (referred to as pooled sputum). In 100 pL pooled sputum, ATCC 15692 was added to a final concentration of ~5xl06 cfu/mL prior to 25 μΜ peptide addition.
[00149] In samples examining the effect of protease inhibitors on peptide activity, pooled sputum samples were pre-treated for 30 min with 1 mM protease inhibitor, either phenylmethylsulphonylfluoride (PMSF), beta-mercaptoethanol (BME), or ethylenediaminetetraacetic acid (EDTA), (all acquired from Sigma-Aldrich), followed by addition of 25 μΜ GlOKHc and ATCC 15692 (~5xl06 cfu/mL).
[00150] Bacteria surviving peptide treatment were rescued by dilution (1:50) in growth media at 4 h and kept on ice before appropriate dilution and plating on LB agar supplemented with ampicillin (25 pg/mL). After overnight incubation at 37 °C, colonies were counted and the surviving cfu/mL quantitated. 100 cfu/mL was considered as the countable limit for all plating procedures. Endogenous organisms already present in the pooled sputum were observed, but were a minority of the population compared to the exogenously added cells (less than 1%, data not shown). 53
As a result endogenous and exogenous cfu/mL were not differentiated for these cultures. 2013204065 11 Apr 2013 [00151] The general antimicrobial effect of GlOKHc in sputum samples from CF patients was evaluated by a killing kinetics assay. As shown in Figure 11 A, at 4 h post-peptide addition GlOKHc was not active against exogenously added P. aeruginosa when mixed with pooled sputum samples. This finding was in contrast to GlOKHc activity in growth medium, were the GlOKHc STAMP was found to reduce the recoverable cfu/mL over 90% after 30 min of exposure, and eliminate all P. aeruginosa cfu/mL by 2 h of treatment (See Experiment 2).
[00152] Considering it likely that the loss of GlOKHc activity was due to degradation, the GlOKHc STAMP stability in sputum was examined over time. Stability of peptides in sputum was monitored by HPLC. Briefly, pooled sputum samples were diluted 1:10 in PBS and centrifuged repeatedly to remove insoluble materials. GlOKHc or GlOKHc-D (100 μΜ) was then added to 100 pL diluted pooled sputum (with or without 1 mM PMSF pretreatment for 1 h) and mixed at room temperature. At the indicated timepoints, 20 pL 10% HC1 was added to stop peptide degradation and the sample was filtered twice (0.2 pm nylon, Nunc) prior to injection to the column (Source 15 RPC, Amersham). Water/acetonitrile with 0.1% trifluorocetic acid was used as the mobile phase and samples were eluted with a linear gradient of increasing acetonitrile composition (from 10% to -35%) at a flow rate of 0.25 mL/min, 11.5 mL of mobile phase per run. Intact GlOKHc and degradation products were monitored by UV (215 nm) and fractions collected where indicated. Collected fractions from successive runs were pooled and lyophilized overnight prior to evaluation of antimicrobial activity by the MIC assay described above. HPLC profiles were obtained using the manufacturers protocol (Unicorn, Amersham), and differentially colored and overlayed using Photoshop 7.0 (Adobe) for construction of Figure 11B.
[00153] As shown in Figure 11B, the signature peak (retention volume 10.29) for GlOKHc was almost entirely degraded after 30 min of exposure to sputum. Several of 54 the resultant fractions were collected and possible degradation products were identified by mass spectrometry, none of which showed an MIC below 100 μΜ against several clinical P. aeruginosa isolates (Table 6). 2013204065 11 Apr 2013 [00154] Table 6 - MIC (μΜ) of GlOKHc, GlOKHc-D and sputum-digested products
MIC (uMT
Synthesized Peptides: ATCC 15692 ATCC 9027 ATCC 27853 G10KHcb 6 5.5 6 GlOKHc-D 15 15 10 HPLC collected fractionsc: 10.29 (GlOKHc) 6 6 6 8.50 >100 >100 >100 7.49 >100 >100 >100 4.04 >100 >100 >100
aMIC range of 3 independent experiments b data previously reported included for comparison (6) factions shown in Figure IB
[00155] It is known in the art that the increased levels of serine proteases present in CF sputum. Thus, it was contemplated that we hypothesized that this class of proteases were responsible for the rapid GlOKHc degradation observed, and that protease inhibitors could stabilize GlOKHc in sputum and restore antimicrobial function. To examine this possibility, P. aeruginosa and GlOKHc were added to pooled sputum samples pre-treated with a variety of protease inhibitors, and the surviving bacteria were quantitated. Samples treated with BME (a cysteine protease inhibitor) or EDTA (metalloprotease inhibitor) were ineffective in rescuing GlOKHc 55 activity or stability (data not shown). As shown in Figure 11 A, however, P. aeruginosa were effectively killed (a reduction of over 3 logi0 in surviving cfu/mL) by the GlOKHc STAMP in samples pre-treated with the general serine protease inhibitor PMSF. The inhibitor alone had only a small affect on P. aemginosa viability. Accordingly, the GlOKHc signal from PMSF-treated sputum remained high through 4 h when examined by HPLC (Figure 1 IB). Overall these data indicate that GlOKHc is active against P. aeruginosa in sputum when protected from serine protease degradation. 2013204065 11 Apr 2013 [00156] 3.3 D-enantiomer GlOKHc STAMP and its activity. Because of the chiral requirement of most serine proteases (Milton et al., 1992), an all D-amino acid enantiomer of GlOKHc, GlOKHc-D, was synthesized as an alternative means to circumvent protease activity without the use of inhibitors. GlOKHc-D was synthesized by standard solid phase methods as mentioned and confirmed by mass spectrometry.
[00157] As shown in Figure 11 A, GlOKHc-D reduced the level of recovered P. aeruginosa 3-4 logi0 (compared to untreated samples) after 4 h of peptide exposure, indicating that GlOKHc-D has a level of activity in sputum similar to that of L-GlOKHc when stabilized. However, the enantiomer was less affective against P. aeruginosa in growth medium after 24 h (as evaluated by MIC, Table 6), suggesting that GlOKHc-D and L-GlOKHc do not have completely identical activities.
[00158] 3.4 Effect of rhDNase on STAMP activity in sputum. Recombinant human DNase, which is commonly used during treatment of CF to reduce sputum viscosity and promote airway clearing (Fuchs et al., 1994), was added to pooled, concentrated sputum samples to determine if GlOKHc/PMSF and GlOKHc-D activity could be improved during co-treatment under these conditions. To determine the effect of rhDNase (Genentech, San Francisco, CA) on STAMP killing ability, individual sputum samples were diluted 1:2 in 100 pg/mL rhDNase, briefly vortexed, and incubated at room temperature for 10 min. Treated samples were then pooled, followed by 1 mM PMSF addition, where appropriate, and incubation for 1 h. 25 μΜ 56
GlOKHc or GlOKHc-D was then added with ~5xl06 cfu/mL ATCC 15692 and incubated 4 h. Survivors were then rescued and quantitated by plating as described above. 2013204065 11 Apr 2013 [00159] As shown in Figure 12, we observed a clear enhancement of antimicrobial activity when rhDNase was utilized in conjunction with GlOKHc/PMSF or GlOKHc -D (fewer than 5% of untreated cfu/mL remaining), when compared to samples not treated with rhDNase (-30% of untreated cfu/mL recovered). P. aeruginosa were not affected by rhDNase treatment alone. These results suggest that the killing effects of GlOKHc/PMSF or GlOKHc-D in sputum can be further enhanced by co-treatment with a sputum mucolytic agent, which may reduce sputum viscosity and enhance peptide diffusion.
[00160] 3.5 Conclusions. In general, the activity of GlOKHc can be extended in expectorated sputum when protected from proteolytic cleavage, either by constructing D-version peptides and/or by co-administering a protease inhibitor and/or in combination with rhDNase. In particular, it was found that robust GlOKHc STAMP activity could be maintained in expectorated sputum if serine protease-dependant digestion associated with this fluid was inhibited, either by chemical antagonists or by the construction of a D-amino acid enantiomer of GlOKHc. Further it was revealed that STAMP activity in sputum can be further enhanced when samples were treated with a combination of peptide and rhDNase. The results illustrates the importance of exploring a combination therapy to treat CF, especially if protease-sensitive peptide-based agents, such as GlOKHc, are used as alternatives to, or in conjunction with, conventional small-molecule antibiotics.
Experiment 4. Identification of peptide 1903 and BD2.21 and the STAMPs thereof.
[00161] The targeting peptide 1903 was obtained by scanning the genomic sequence of S. mutans UA140. The predicted open reading frames (ORFs) of the publicly- available genome were examined and those ORFs that encoded for proteins under 50 amino acids were noted and re-examined after scanning the entire genome. A number 57 of peptides predicted to be encoded by these ORFs were selected, synthesized with fluorescent labels, tested for binding to S. mutans biofilms. Peptide 1903 showed the binding activity to S. mutans and then was used to synthesize 1903 based STAMPs. 2013204065 11 Apr 2013 [00162] BD2.21 was rationally-designed as part of the “Beta-deletion 2” antimicrobial peptide library. A common alpha helical residue arrangement, HHCCHHCHHH(n), was replaced using mostly positive (C) and hydrophobic (H) residues at the positions indicated. There is some variability in the pattern of residues we used, and some non-hydrophobic and uncharged residues were incorporated. The replacement was limited to 3-5 cationic amino acids per peptide, and 4-7 hydrophobic residues (9 to 12 total). The antimicrobial affects of modified peptides were tested and BD2.21 showed an MIC of 5.5 uM against planktonic S mutans. BD2.21 was then used to synthesize BD2.21 based STAMP such as 1903-BD2.21 having an amino acid sequence of NIFEYFLE-GGG-KLFKFLRKHLL as shown in SEQ ID NO. 13 and C16-BD2.21 having an amino acid sequence of TFFRLFNRSFTQALGK-GGG-KLFKFLRKHLL as shown in SEQ ID NO 14.
[00163] Killing of single-species Streptococcus mutans mature biofilms. S. mutans biofilms were seeded with 10Λ5 cells/well and grown overnight with 1 % sucrose (TH medium) in 48-well plates (final volume 400 pL). After incubation, the supernatant was removed from the biofilms and replaced with 200 pL PBS with 50 pM STAMP. PBS alone was used for the negative control (100% survival) with ethanol as the control for complete killing of the biofilms (0% survival). Biofilms were treated with peptide for 20 min, then washed lx with PBS. To measure biofilm survival, 20 pL CellTiterBlue diluted into 160pL TH medium was added per well. After 3-5 min, the supernatants were removed to a 96 well plate and the absorbance read at 570 nm. High absorbance at 570 indicated more substrate reduction by viable cells remaining in the biofilm. As shown in Figure 13, the results indicate that C16-BD2.21 and 1903-BD2.21 can kill 66% and 85% of the viable S. mutans within the biofilm, respectively, after a treatment time of only 20 min. 58 [00164] Selectivity of STAMPs against multi-species biofilms of oral streptococci. To measure selectivity of STAMPs, mixed biofilms were seeded to 48-well plates. Streptococcus mitis, S. sobrinus, S. gordonii, and S. sanguinis were mixed with S. mutans strain JM11 (spectinomycin resistant) at a 1:1:1:1:10 ratio, total of 10Λ5 cells/well. Biofilms were grown overnight in TH medium with 1% sucrose, 1% dextrose, and 1% mannose, for 18-24 h. Mature biofilms were treated with PBS plus STAMP as described for the single-species biofilm assay, with the same controls. After treatment, biofilms were washed 2x in PBS and then physically disrupted with a sterile pipette tip in 100 pL PBS per well. Cell suspensions were then serially diluted 10-fold to 10A-6. Diluted suspensions were then plated on TH medium and TH supplemented with spectinomycin, 800 pg/mL. The total amount of biofilm killing (all streptococci) was determined by counting colonies from TH-only plates. Controls: 100% of untreated corresponds to the number of cfu/mL recorded from untreated biofilms, 0% survival was obtained from ethanol sterilized samples. S. mutans killing was determined from quantitating colonies on TH-spectinomycin plates, and combined total cfu/mL, was utilized to calculate the ratio of S. mutans:total population. 1:1 ratio indicates no selectivity. 2013204065 11 Apr 2013 [00165] As shown in Figure 14, C16-BD2.21 has no impact on the total cfu/mL population, suggesting that non-S. mutans streptococci are not affected by the STAMP to a significant degree (See Figure 14(A)). This is confirmed by the observed ratio of surviving S. mutans to total streptococci, which is .075 (See Figure 14 (B)). 1903-BD2.21 also had a selective ratio (well under 1, see Figure 14 (B)), though had some impact on other oral streptococci (See Figure 14 (A)).
[00166] Table 7. Antimicrobial Peptides
Andropin (SEQ ID NO 54)
VFIDILDKMENAIHKAAQAGIGIAKPIEKMILPK
Apidaecin(SEQ ID NO 55)
GNRPVYIPPPRPPHPRL
Bacteriocin leucocin A (SEQ ID NO 56) 59
KYYGNGVHCTKSGCSVNWGEAFSAGVHRLANGGNGFW 2013204065 11 Apr 2013 bactenecin (SEQ ID NO 57)
RLCRIV VIR V CR
Buforin II (SEQ ID NO 58) TRSSRAGLQFPVGRVHRLLRK
Cathelicidin (human LL-37) (SEQ ID NO 59) LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES
Clavanin A (SEQ ID NO 60) VFQFLGKIIHHVGNFVHGFSHVF
Cecropin (SEQ ID NO 61)
RWKIFKKIEKV GQNIRDGIVKAGP A V A V V GQ A ATI
Cyclic Dodecapeptide (SEQ ID NO 62)
RICRIIFLRVCR
β-defensin I (human) (SEQ ID NO 63) DFASCHTNGGICLPNRCPGHMIQIGICFRPRVKCCRSW α-defensin (HNP-1) (SEQ ID NO 64)
ACY CRIPACIAGERRYGTCIY QGRLWAFCC
Gaegurin (SEQ ID NO 65) SLFSLIKAGAKFLGKNLLKQGACYAACKASKQC Histatin (SEQ ID NO 66)
DSHEERHHGRHGHHKYGRKFHEKHHSHRGYRSNYLYDN
Indolicidin (SEQ ID NO 67)
ILPWKWPWWPWRR
Magainin II (SEQ ID NO 68) GIGKFLHSAKKFGKAFVGEIMNS
Melittin B (SEQ ID NO 69) GIGAVLKVLTTGLPALISWIKRKRQQ
Nisin A (SEQ ID NO 70)
ITSISLCTPGCKTGALMGCNMKTATCHCSIHVSK novispirin G10 (SEQ ID NO 35)
KNLRRIIRKGIHIIKKYG
Protegrin (SEQ ID N034) RGGRLCY CRRRFCVC VGR 60 2013204065 11 Apr 2013
Ranalexin (SEQ ID NO 71) LGGLIKIVPAMICAVTKKC
Tachyplesin (SEQ ID NO 72) KWCFRVCYRGICYRRCR
Maximin H5 (amphibians) (SEQ ID NO 73)
ILGPVLGLVSDTLDDVLGIL
Surfactant Extract 1 (SEQ ID NO 74)
DDDDDD DCD-1 (SEQ ID NO 75)
SSLLEKGLDGAKKAVGGLGKLGKDAVEDLESVGKGAVHDVKDVLDSV SSL-25 (SEQ ID NO 76)
S SLLEKGLDG AKKA V GGLGKLGKD A SSL-23 (SEQ ID NO 77)
S SLLEKGLDG AKKA V GGLGKLGK
DermaseptinDS5 (SEQ ID NO 78)
GLW S KIKT AGKS V AKA A AKA A VKA VTN A V
Moricin (insect) (SEQ ID NO 79)
AKIPIKAIKTVGKAVGKGLRAINIASTANDVFNFLKPKKRKA
Bombinin (frog) (SEQ ID NO 80) GIGALSAKGALKGLAKGLAEHFAN
Pleurocidin (white flounder) (SEQ ID NO 81) GWGSFFKKAAHVGKHVGKAALHTYL
SMAP29 (sheep) (SEQ ID NO 81) RGLRRLGRKIAHGVKKY GPTVLRIIRIAG
PMAP-23 (pig) (SEQ ID NO 83) RIIDLLWRVRRPQKPKFVTVWVR VCP-5h (wasp) (SEQ ID NO 84) FLPIIGKLLSGLL-NH2
Abaecin (honeybee) (SEQ ID NO 85) YVPLPNVPQPGRRPFPTFPGQGPFNPKIKWPQGY
Drosocin (fruitfly) (SEQ ID NO 86)
GKPRPY SPRPTSHPRPIRV 61 2013204065 11 Apr 2013
Pyrrohocoricin (sap-sucker bug) (SEQ ID NO 87) VDKGSYLPRPTPPRPIYNRN
L15K7 (SEQ ID NO 88) KLLKLLLKLLKLLLKLLLKLLK KLApep (SEQ ID NO 89) KLALKLALKAWKAALKLA-NH2
D2A2i (SEQ ID NO 90) FAKKFAKKFKKFAKKFAKFAFAF
Modelin-1 (SEQ ID NO 91)
KFWKKW AKKWFKFWKA W FARE (SEQ ID NO 92) Ac-FARFFARFFARF-Ac YFK-P (SEQ ID NO 93) YKFFKFFFPKFKGFFFKF-NH2 KSF2 (SEQ ID NO 94) KKVVFKFKFK-NH2 CAM 135 (SEQ ID NO 95) GWRFIKKIFRVFKGF-NH2 PGAa (SEQ ID NO 96) GIFSKFGKAFKKAAKHAAKA-NH2 PGYa (SEQ ID NO 97) GFFRRFRDFFKKIGEKFKKIGY-NH2 62
References in the disclosures are listed below and are incorporated in their entirety. Ajdic, D., W. M. McShan, R. E. McLaughlin, G. Savic, J. Chang, Μ. B. Carson, C. Primeaux, R. Tian, S. Kenton, H. Jia, S. Lin, Y. Qian, S. Li, H. Zhu, L. Najar, 2013204065 11 Apr 2013 H. Lai, J. White, B. A. Roe, and J. J. Lerretti. 2002. Genome sequence of Streptococcus mutans UA159, a cariogenic dental pathogen. Proc. Natl. Acad. Sci. US A 99:14434-9.
Anderson, Μ. H., and W. Shi. 2006. A probiotic approach to caries management. Pediatic Dentistry In Press.
Axelsson, P., and J. Lindhe. 1987. Efficacy of mouthrinses in inhibiting dental plaque and gingivitis in man. J. Clin. Periodontol. 14:205-12.
Blehert, D. S., R. J. Palmer, Jr., J. B. Xavier, J. S. Almeida, and P. E. Kolenbrander. 2003. Autoinducer 2 production by Streptococcus gordonii DL1 and the biofilm phenotype of a luxS mutant are influenced by nutritional conditions. J. Bacteriol. 185:4851-60.
Bonacorsi, S., L. Litoussi, S. Lhopital, and E. Bingen. 1999. Comparative in vitro activities of meropenem, imipenem, temocillin, piperacillin, and ceftazidime in combination with tobramycin, rifampin, or ciprofloxacin against Burkholderia cepacia isolates from patients with cystic fibrosis. Antimicrob. Agents Chemother. 43:213-217.
Caufield, P. W., A. P. Dasanayake, Y. Li, Y. Pan, J. Hsu, and J. M. Hardin. 2000. Natural history of Streptococcus sanguinis in the oral cavity of infants: evidence for a discrete window of infectivity. Infect. Immun. 68:4018-23. Donlan, R. M., and J. W. Costerton. 2002. Biofilms: survival mechanisms of clinically relevant microorganisms. Clin. Microbiol. Rev. 15:167-93.
Eckert, R., L. Qi, D. K. Yarbrough, J. He, Μ. H. Anderson, W. Shi. 2006. Adding selectivity to antimicrobial peptides: rational design of a multidomain peptide against Pseudomonas spp. Antimicrob. Agents. Chemother. 50:1480-1488. Luchs, H. J., D. S. Borowitz, D. H. Christiansen, E. M. Morris, M. L. Nash, B. W. Ramsey, B. J. Rosenstein, A. L. Smith, and Μ. E. Wohl. 1994. Effect of aerosolized recombinant human DNase on exacerbations of respiratory 63 symptoms and on pulmonary function in patients with cystic fibrosis. The Pulmozyme Study Group. N Engl J Med 331:637-42. 2013204065 11 Apr 2013
Gilmour, Μ. N., T. S. Whittam, M. Kilian, and R. K. Selander. 1987. Genetic relationships among the oral streptococci. J. Bacteriol. 169:5247-57.
Li, Y. Η., P. C. Lau, J. H. Lee, R. P. Ellen, and D. G. Cvitkovitch. 2001. Natural genetic transformation of Streptococcus mutans growing in biofilms. J. Bacteriol. 183:897-908.
Merritt, J., J. Kreth, F. Qi, R. Sullivan, and W. Shi. 2005. Non-disruptive, real-time analyses of the metabolic status and viability of Streptococcus mutans cells in response to antimicrobial treatments. J. Microbiol. Methods 61:161-70.
Milton, R. C., S. C. Milton, and S. B. Kent. 1992. Total chemical synthesis of a D-enzyme: the enantiomers of HIV-1 protease show reciprocal chiral substrate specificity [corrected]. Science 256:1445-8.
Perron, G. G., M. Zasloff, and G. Bell. 2006. Experimental evolution of resistance to an antimicrobial peptide. Proc. Biol. Sci. 273:251-6.
Qi, F., J. Kreth, C. M. Levesque, O. Kay, R. W. Mair, W. Shi, D. G. Cvitkovitch, and S. D. Goodman. 2005. Peptide pheromone induced cell death of Streptococcus mutans. FEMS Microbiol. Lett. 251:321-6.
Qiu, X. Q., J. Zhang, H. Wang, G. Y. Wu. 2005. A novel engineered peptide, a narrow-spectrum antibiotic, is effective against vancomycin-resistant Enterococcus faecalis. Antimicrob. Agents Chemother. 49:1184-1189.
Rogers, A. H. 1975. Bacteriocin types of Streptococcus mutans in human mouths.
Arch. Oral Biol. 20:853-858.
Saiman, L., S. Tabibi, T. D. Starner, P. San Gabriel, P. L. Winokur, Η. P. Jia, P. B. McCray, Jr., and B. F. Tack. 2001. Cathelicidin peptides inhibit multiply antibiotic-resistant pathogens from patients with cystic fibrosis. Antimicrob. Agents Chemother. 45:2838-44.
Sajjan, U. S., L. T. Tran, N. Sole, C. Rovaldi, A. Akiyama, P. M. Friden, J. F. Forstner, and D. M. Rothstein. 2001. P-113D, an antimicrobial peptide active against 64 2013204065 11 Apr 2013
Pseudomonas aeruginosa, retains activity in the presence of sputum from cystic fibrosis patients. Antimicrob Agents Chemother 45:3437-44. 65
Claims (23)
1. A composition comprising a targeting peptide that binds Streptococcus mutans attached to a detectable agent or to an antimicrobial peptide, wherein the amino acid sequence of said targeting peptide comprises a fragment of the competence stimulating peptide (CSP) ranging in length from 6 to 20 amino acids.
2. The composition of claim 1, wherein said fragment comprises the amino acid sequence TFFRLFNR (SEQ ID NO: 5).
3. The composition of claim 1, wherein said fragment comprises the amino acid sequence TFFRLFNRSFTQALGK (SEQ ID NO: 2).
4. The composition of claim 1 wherein said targeting peptide is attached to an antimicrobial peptide.
5. The composition of claim 4 wherein the targeting peptide is attached to the antimicrobial peptide by a linker peptide, where the amino acid sequence of said linker peptide is selected from the group consisting of GGG (SEQ ID NO: 17), AAA (SEQ ID NO:18), SAT (SEQ ID NO:19), ASA (SEQ ID NO:20), SGG (SEQ ID NO: 21), PYP (SEQ ID NO:22), SGS (SEQ ID NO:23), GGS (SEQ ID NO:24), SPS(SEQ ID NO:25), PSGSP (SEQ ID NO:26), PSPSP (SEQ ID NO:27), and GGSGGS (SEQ ID NO:28) .
6. The composition of claim 4 wherein the amino acid sequence of said antimicrobial peptide is selected from the group consisting of SEQ ID NOs 34-35 and SEQ ID NOs 54-97.
7. The composition of claim 4 wherein the amino acid sequence of said antimicrobial peptide is selected from the group consisting of SEQ ID NOs 3, 7 and 11.
8. The composition of claim 4, wherein the carboxyl terminus of said antimicrobial peptide is amidated.
9. The composition of claim 1 wherein said targeting peptide is attached to a detectable agent.
10. The composition of claim 9 wherein the targeting peptide is conjugated to the detectable agent.
11. The composition of claim 9 wherein the detectable agent is selected from the group consisting of a radioisotope, a fluorescent agent, and an enzyme-substrate agent.
12. A STAMP composition comprising: a) a targeting peptide, wherein the targeting peptide comprises a fragment of the competence stimulating peptide (CSP) ranging in length from 6 to 20 amino acids; b) a linker peptide, wherein one terminus of the linker peptide is linked to the targeting peptide; and c) an antimicrobial peptide, wherein the antimicrobial peptide is linked to the other terminus of the linker peptide.
13. The STAMP composition of claim 12 wherein the amino acid sequence of said linker peptide is selected from the group consisting of SEQ ID NOs 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 and 28.
14. The STAMP composition of claim 12 wherein the amino acid sequence of said antimicrobial peptide is selected from the group consisting of SEQ ID NOs 34-35 and SEQ ID NOs 54-97.
15. The STAMP composition of claim 12 wherein the amino acid sequence of said antimicrobial peptide is selected from the group consisting of SEQ ID NOs 3, 7 and 11.
16. The STAMP composition of claim 15 wherein the STAMP comprises an amino acid sequence selected from the group consisting of SEQ ID Nos 4, 6, 8, 9, 12, 13, 14, 15, and 16.
17. The STAMP composition of claim 12, further comprising an antibiotic.
18. The STAMP composition of claim 17 wherein the antibiotic is selected from the group consisting of β-lactam antibiotics, amoxicillin, bacitracin, chloramphenicol, clindamycin, capreomycin, colistimethate, ciprofloxacin, doxycycline, erythromycin, fusidic acid, fosfomycin, fusidate sodium, gramicidin, gentamycin, lincomycin, minocycline, macrolides, monobactams, nalidixic acid, novobiocin, ofloxcin, rifamycins, tetracyclines, vancomycin, tobramycin, and trimethoprim.
19. The STAMP composition of claim 12, further comprising an agent that enhances or maintains the activity of the said peptides.
20. The STAMP composition of claim 19 wherein the agent is a protease inhibitor or rhDNase.
21. A method of selectively killing or inhibiting a target microbial organism in a subject comprising the step of administering the STAMP of claim 12, 17, or 19 to the subject, wherein the targeting peptide specifically binds to the target microbial organism.
22. A method of selectively killing or inhibiting a target microbial organism in a biofilm comprising the step of administering the STAMP of claim 12, 17, or 19 to the biofilm, wherein the targeting peptide specifically binds to the target microbial organism.
23. The STAMP composition of claim 12 wherein the targeting peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 2 and 5.
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU2013204065A AU2013204065C1 (en) | 2006-09-06 | 2013-04-11 | Selectively targeted antimicrobial peptides and the use thereof |
AU2016228293A AU2016228293B2 (en) | 2006-09-06 | 2016-09-16 | Selectively targeted antimicrobial peptides and the use thereof |
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US84287106P | 2006-09-06 | 2006-09-06 | |
US60/842,871 | 2006-09-06 | ||
AU2007292221A AU2007292221B2 (en) | 2006-09-06 | 2007-09-06 | Selectively targeted antimicrobial peptides and the use thereof |
PCT/US2007/077795 WO2008030988A2 (en) | 2006-09-06 | 2007-09-06 | Selectively targeted antimicrobial peptides and the use thereof |
AU2013204065A AU2013204065C1 (en) | 2006-09-06 | 2013-04-11 | Selectively targeted antimicrobial peptides and the use thereof |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
AU2007292221A Division AU2007292221B2 (en) | 2006-09-06 | 2007-09-06 | Selectively targeted antimicrobial peptides and the use thereof |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
AU2016228293A Division AU2016228293B2 (en) | 2006-09-06 | 2016-09-16 | Selectively targeted antimicrobial peptides and the use thereof |
Publications (3)
Publication Number | Publication Date |
---|---|
AU2013204065A1 AU2013204065A1 (en) | 2013-05-02 |
AU2013204065B2 AU2013204065B2 (en) | 2016-06-16 |
AU2013204065C1 true AU2013204065C1 (en) | 2017-05-25 |
Family
ID=48446870
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
AU2013204065A Ceased AU2013204065C1 (en) | 2006-09-06 | 2013-04-11 | Selectively targeted antimicrobial peptides and the use thereof |
Country Status (1)
Country | Link |
---|---|
AU (1) | AU2013204065C1 (en) |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7087228B2 (en) * | 2002-07-03 | 2006-08-08 | University Of Southern California | Preventing tooth decay and infective endocarditis using natural oligopeptides |
-
2013
- 2013-04-11 AU AU2013204065A patent/AU2013204065C1/en not_active Ceased
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7087228B2 (en) * | 2002-07-03 | 2006-08-08 | University Of Southern California | Preventing tooth decay and infective endocarditis using natural oligopeptides |
Non-Patent Citations (1)
Title |
---|
Qi, F. et al. "Peptide pheromone induced cell death of Streptococcus mutans" FEMS Microbiology Letters (2005) 251: 321-326 * |
Also Published As
Publication number | Publication date |
---|---|
AU2013204065A1 (en) | 2013-05-02 |
AU2013204065B2 (en) | 2016-06-16 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US10111926B2 (en) | Selectively targeted antimicrobial peptides and the use thereof | |
US9072793B2 (en) | Antibacterial and antifungal peptides | |
Upert et al. | Emerging peptide antibiotics with therapeutic potential | |
EP2518080B1 (en) | Antibiotic peptides | |
KR101502453B1 (en) | Polymyxin derivatives and uses thereof | |
Sharma et al. | Short antimicrobial peptides | |
JP2001520631A (en) | Antifungal peptide | |
PT750506E (en) | ANTIMICROBIAL COMPOUNDS OF LONG SPECTRUM AND METHODS FOR THEIR USE | |
US20130053305A1 (en) | Peptide compounds that can be used as antibacterial agents | |
AU2016228293B2 (en) | Selectively targeted antimicrobial peptides and the use thereof | |
AU2013204065C1 (en) | Selectively targeted antimicrobial peptides and the use thereof | |
US20190375791A1 (en) | New d-configured cateslytin peptide | |
JP2005532784A (en) | New antibacterial voricin peptide |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
FGA | Letters patent sealed or granted (standard patent) | ||
DA2 | Applications for amendment section 104 |
Free format text: THE NATURE OF THE AMENDMENT IS AS SHOWN IN THE STATEMENT(S) FILED 14 DEC 2016 |
|
DA3 | Amendments made section 104 |
Free format text: THE NATURE OF THE AMENDMENT IS AS SHOWN IN THE STATEMENT(S) FILED 14 DEC 2016 |
|
HB | Alteration of name in register |
Owner name: THE REGENTS OF THE UNIVERSITY OF CALIFORNIA Free format text: FORMER NAME(S): C3-JIAN, INC.; THE REGENTS OF THE UNIVERSITY OF CALIFORNIA Owner name: C3 JIAN, LLC Free format text: FORMER NAME(S): C3-JIAN, INC.; THE REGENTS OF THE UNIVERSITY OF CALIFORNIA |
|
MK14 | Patent ceased section 143(a) (annual fees not paid) or expired |