WO2023081691A9 - Method of treating and preventing bone and joint infections - Google Patents
Method of treating and preventing bone and joint infections Download PDFInfo
- Publication number
- WO2023081691A9 WO2023081691A9 PCT/US2022/079130 US2022079130W WO2023081691A9 WO 2023081691 A9 WO2023081691 A9 WO 2023081691A9 US 2022079130 W US2022079130 W US 2022079130W WO 2023081691 A9 WO2023081691 A9 WO 2023081691A9
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- bone
- gram
- lysin
- antibiotic
- osteomyelitis
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims abstract description 89
- 206010005940 Bone and joint infections Diseases 0.000 title description 6
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 claims abstract description 128
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims abstract description 124
- 210000000988 bone and bone Anatomy 0.000 claims abstract description 109
- 239000007943 implant Substances 0.000 claims abstract description 79
- 208000015181 infectious disease Diseases 0.000 claims abstract description 63
- 241000192125 Firmicutes Species 0.000 claims abstract description 41
- 239000000203 mixture Substances 0.000 claims abstract description 23
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 86
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 76
- 229920001184 polypeptide Polymers 0.000 claims description 73
- 239000003242 anti bacterial agent Substances 0.000 claims description 63
- 230000003115 biocidal effect Effects 0.000 claims description 56
- 229940088710 antibiotic agent Drugs 0.000 claims description 52
- 206010031252 Osteomyelitis Diseases 0.000 claims description 51
- 108010013198 Daptomycin Proteins 0.000 claims description 41
- DOAKLVKFURWEDJ-QCMAZARJSA-N daptomycin Chemical compound C([C@H]1C(=O)O[C@H](C)[C@@H](C(NCC(=O)N[C@@H](CCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@H](CO)C(=O)N[C@H](C(=O)N1)[C@H](C)CC(O)=O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](CC(N)=O)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)CCCCCCCCC)C(=O)C1=CC=CC=C1N DOAKLVKFURWEDJ-QCMAZARJSA-N 0.000 claims description 41
- 229960005484 daptomycin Drugs 0.000 claims description 41
- 206010060968 Arthritis infective Diseases 0.000 claims description 34
- 150000001413 amino acids Chemical group 0.000 claims description 31
- 230000000844 anti-bacterial effect Effects 0.000 claims description 24
- 230000003385 bacteriostatic effect Effects 0.000 claims description 17
- 210000004027 cell Anatomy 0.000 claims description 17
- 241000191963 Staphylococcus epidermidis Species 0.000 claims description 13
- 241000191967 Staphylococcus aureus Species 0.000 claims description 12
- 241000191940 Staphylococcus Species 0.000 claims description 8
- 230000002147 killing effect Effects 0.000 claims description 8
- 230000002195 synergetic effect Effects 0.000 claims description 7
- 108010059993 Vancomycin Proteins 0.000 claims description 6
- 229960003165 vancomycin Drugs 0.000 claims description 6
- 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 6
- 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 6
- RJQXTJLFIWVMTO-TYNCELHUSA-N Methicillin Chemical compound COC1=CC=CC(OC)=C1C(=O)N[C@@H]1C(=O)N2[C@@H](C(O)=O)C(C)(C)S[C@@H]21 RJQXTJLFIWVMTO-TYNCELHUSA-N 0.000 claims description 5
- 229930182555 Penicillin Natural products 0.000 claims description 5
- 229960000308 fosfomycin Drugs 0.000 claims description 5
- YMDXZJFXQJVXBF-STHAYSLISA-N fosfomycin Chemical compound C[C@@H]1O[C@@H]1P(O)(O)=O YMDXZJFXQJVXBF-STHAYSLISA-N 0.000 claims description 5
- 210000003127 knee Anatomy 0.000 claims description 5
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 claims description 5
- 229960003085 meticillin Drugs 0.000 claims description 5
- 241000194033 Enterococcus Species 0.000 claims description 4
- 206010031253 Osteomyelitis acute Diseases 0.000 claims description 4
- 206010031256 Osteomyelitis chronic Diseases 0.000 claims description 4
- 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 claims description 4
- 229940126575 aminoglycoside Drugs 0.000 claims description 4
- 210000003423 ankle Anatomy 0.000 claims description 4
- 206010012601 diabetes mellitus Diseases 0.000 claims description 4
- 210000001624 hip Anatomy 0.000 claims description 4
- 229960000282 metronidazole Drugs 0.000 claims description 4
- VAOCPAMSLUNLGC-UHFFFAOYSA-N metronidazole Chemical compound CC1=NC=C([N+]([O-])=O)N1CCO VAOCPAMSLUNLGC-UHFFFAOYSA-N 0.000 claims description 4
- 229940049954 penicillin Drugs 0.000 claims description 4
- LITBAYYWXZOHAW-XDZRHBBOSA-N (2s,5r,6r)-6-[[(2r)-2-[(4-ethyl-2,3-dioxopiperazine-1-carbonyl)amino]-2-phenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid;(2s,3s,5r)-3-methyl-4,4,7-trioxo-3-(triazol-1-ylmethyl)-4$l^{6}-thia-1-azabicyclo[3.2.0]hept Chemical compound C([C@]1(C)S([C@H]2N(C(C2)=O)[C@H]1C(O)=O)(=O)=O)N1C=CN=N1.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 LITBAYYWXZOHAW-XDZRHBBOSA-N 0.000 claims description 3
- 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 3
- WZRJTRPJURQBRM-UHFFFAOYSA-N 4-amino-n-(5-methyl-1,2-oxazol-3-yl)benzenesulfonamide;5-[(3,4,5-trimethoxyphenyl)methyl]pyrimidine-2,4-diamine Chemical compound O1C(C)=CC(NS(=O)(=O)C=2C=CC(N)=CC=2)=N1.COC1=C(OC)C(OC)=CC(CC=2C(=NC(N)=NC=2)N)=C1 WZRJTRPJURQBRM-UHFFFAOYSA-N 0.000 claims description 3
- 229930186147 Cephalosporin Natural products 0.000 claims description 3
- UIOFUWFRIANQPC-JKIFEVAISA-N Floxacillin 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=C(F)C=CC=C1Cl UIOFUWFRIANQPC-JKIFEVAISA-N 0.000 claims description 3
- 208000008589 Obesity Diseases 0.000 claims description 3
- 241000194017 Streptococcus Species 0.000 claims description 3
- 229940124587 cephalosporin Drugs 0.000 claims description 3
- 150000001780 cephalosporins Chemical class 0.000 claims description 3
- 229960002227 clindamycin Drugs 0.000 claims description 3
- 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 3
- 229940047766 co-trimoxazole Drugs 0.000 claims description 3
- 229960002488 dalbavancin Drugs 0.000 claims description 3
- 108700009376 dalbavancin Proteins 0.000 claims description 3
- 229960003722 doxycycline Drugs 0.000 claims description 3
- 229960004273 floxacillin Drugs 0.000 claims description 3
- 229940124307 fluoroquinolone Drugs 0.000 claims description 3
- 238000001990 intravenous administration Methods 0.000 claims description 3
- 229960003907 linezolid Drugs 0.000 claims description 3
- TYZROVQLWOKYKF-ZDUSSCGKSA-N linezolid Chemical compound O=C1O[C@@H](CNC(=O)C)CN1C(C=C1F)=CC=C1N1CCOCC1 TYZROVQLWOKYKF-ZDUSSCGKSA-N 0.000 claims description 3
- 235000020824 obesity Nutrition 0.000 claims description 3
- VHFGEBVPHAGQPI-MYYQHNLBSA-N oritavancin Chemical compound O([C@@H]1C2=CC=C(C(=C2)Cl)OC=2C=C3C=C(C=2O[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O[C@@H]2O[C@@H](C)[C@H](O)[C@@](C)(NCC=4C=CC(=CC=4)C=4C=CC(Cl)=CC=4)C2)OC2=CC=C(C=C2Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]2C(=O)N[C@@H]1C(N[C@H](C1=CC(O)=CC(O)=C1C=1C(O)=CC=C2C=1)C(O)=O)=O)=O)NC(=O)[C@@H](CC(C)C)NC)[C@H]1C[C@](C)(N)[C@@H](O)[C@H](C)O1 VHFGEBVPHAGQPI-MYYQHNLBSA-N 0.000 claims description 3
- 229960001607 oritavancin Drugs 0.000 claims description 3
- 108010006945 oritavancin Proteins 0.000 claims description 3
- 210000000963 osteoblast Anatomy 0.000 claims description 3
- 229940104641 piperacillin / tazobactam Drugs 0.000 claims description 3
- 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 claims description 3
- 229960001225 rifampicin Drugs 0.000 claims description 3
- 238000007910 systemic administration Methods 0.000 claims description 3
- KGPGQDLTDHGEGT-JCIKCJKQSA-N zeven Chemical compound C=1C([C@@H]2C(=O)N[C@H](C(N[C@H](C3=CC(O)=C4)C(=O)NCCCN(C)C)=O)[C@H](O)C5=CC=C(C(=C5)Cl)OC=5C=C6C=C(C=5O[C@H]5[C@@H]([C@@H](O)[C@H](O)[C@H](O5)C(O)=O)NC(=O)CCCCCCCCC(C)C)OC5=CC=C(C=C5)C[C@@H]5C(=O)N[C@H](C(N[C@H]6C(=O)N2)=O)C=2C(Cl)=C(O)C=C(C=2)OC=2C(O)=CC=C(C=2)[C@H](C(N5)=O)NC)=CC=C(O)C=1C3=C4O[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]1O KGPGQDLTDHGEGT-JCIKCJKQSA-N 0.000 claims description 3
- IZXIZTKNFFYFOF-UHFFFAOYSA-N 2-Oxazolidone Chemical compound O=C1NCCO1 IZXIZTKNFFYFOF-UHFFFAOYSA-N 0.000 claims description 2
- 108010015899 Glycopeptides Proteins 0.000 claims description 2
- 102000002068 Glycopeptides Human genes 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
- 108010028921 Lipopeptides Proteins 0.000 claims description 2
- 241001147695 Staphylococcus caprae Species 0.000 claims description 2
- 241001134656 Staphylococcus lugdunensis Species 0.000 claims description 2
- 241000191978 Staphylococcus simulans Species 0.000 claims description 2
- 239000004098 Tetracycline Substances 0.000 claims description 2
- 210000001513 elbow Anatomy 0.000 claims description 2
- 229910052751 metal Inorganic materials 0.000 claims description 2
- 239000002184 metal Substances 0.000 claims description 2
- 230000000149 penetrating effect Effects 0.000 claims description 2
- 206010039073 rheumatoid arthritis Diseases 0.000 claims description 2
- 210000002832 shoulder Anatomy 0.000 claims description 2
- 229940037648 staphylococcus simulans Drugs 0.000 claims description 2
- 229940124530 sulfonamide Drugs 0.000 claims description 2
- 150000003456 sulfonamides Chemical class 0.000 claims description 2
- 235000019364 tetracycline Nutrition 0.000 claims description 2
- 150000003522 tetracyclines Chemical class 0.000 claims description 2
- 241000295644 Staphylococcaceae Species 0.000 claims 2
- YZBQHRLRFGPBSL-RXMQYKEDSA-N carbapenem Chemical compound C1C=CN2C(=O)C[C@H]21 YZBQHRLRFGPBSL-RXMQYKEDSA-N 0.000 claims 2
- DQJCDTNMLBYVAY-ZXXIYAEKSA-N (2S,5R,10R,13R)-16-{[(2R,3S,4R,5R)-3-{[(2S,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}-5-(ethylamino)-6-hydroxy-2-(hydroxymethyl)oxan-4-yl]oxy}-5-(4-aminobutyl)-10-carbamoyl-2,13-dimethyl-4,7,12,15-tetraoxo-3,6,11,14-tetraazaheptadecan-1-oic acid Chemical compound NCCCC[C@H](C(=O)N[C@@H](C)C(O)=O)NC(=O)CC[C@H](C(N)=O)NC(=O)[C@@H](C)NC(=O)C(C)O[C@@H]1[C@@H](NCC)C(O)O[C@H](CO)[C@H]1O[C@H]1[C@H](NC(C)=O)[C@@H](O)[C@H](O)[C@@H](CO)O1 DQJCDTNMLBYVAY-ZXXIYAEKSA-N 0.000 claims 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 claims 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 claims 1
- 229960003644 aztreonam Drugs 0.000 claims 1
- 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 1
- 229960005287 lincomycin Drugs 0.000 claims 1
- 229960002180 tetracycline Drugs 0.000 claims 1
- 229930101283 tetracycline Natural products 0.000 claims 1
- 150000003952 β-lactams Chemical class 0.000 claims 1
- 101710126949 Lysin Proteins 0.000 description 112
- 239000012634 fragment Substances 0.000 description 65
- 241000894006 Bacteria Species 0.000 description 42
- 230000000694 effects Effects 0.000 description 32
- 235000001014 amino acid Nutrition 0.000 description 29
- 229940024606 amino acid Drugs 0.000 description 26
- 238000011282 treatment Methods 0.000 description 25
- 108090000623 proteins and genes Proteins 0.000 description 23
- 108700010690 exebacase Proteins 0.000 description 21
- 230000001580 bacterial effect Effects 0.000 description 20
- 229950009605 exebacase Drugs 0.000 description 20
- 235000018102 proteins Nutrition 0.000 description 20
- 102000004169 proteins and genes Human genes 0.000 description 20
- 125000003275 alpha amino acid group Chemical group 0.000 description 19
- 239000003814 drug Substances 0.000 description 18
- 210000001179 synovial fluid Anatomy 0.000 description 16
- 241001465754 Metazoa Species 0.000 description 15
- 230000012010 growth Effects 0.000 description 15
- 239000008194 pharmaceutical composition Substances 0.000 description 15
- 238000006467 substitution reaction Methods 0.000 description 15
- 230000009885 systemic effect Effects 0.000 description 15
- 125000000539 amino acid group Chemical group 0.000 description 13
- -1 coatings Substances 0.000 description 13
- 229940079593 drug Drugs 0.000 description 13
- 230000009467 reduction Effects 0.000 description 12
- 210000002303 tibia Anatomy 0.000 description 12
- 241000283973 Oryctolagus cuniculus Species 0.000 description 11
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
- 230000004927 fusion Effects 0.000 description 10
- 210000001519 tissue Anatomy 0.000 description 10
- 239000003795 chemical substances by application Substances 0.000 description 9
- 238000010790 dilution Methods 0.000 description 9
- 239000012895 dilution Substances 0.000 description 9
- 230000002401 inhibitory effect Effects 0.000 description 9
- 239000003937 drug carrier Substances 0.000 description 8
- 239000012530 fluid Substances 0.000 description 8
- 230000006870 function Effects 0.000 description 8
- 239000011780 sodium chloride Substances 0.000 description 8
- 230000032770 biofilm formation Effects 0.000 description 7
- 238000000338 in vitro Methods 0.000 description 7
- 244000005700 microbiome Species 0.000 description 7
- 230000004048 modification Effects 0.000 description 7
- 238000012986 modification Methods 0.000 description 7
- 210000000440 neutrophil Anatomy 0.000 description 7
- 239000000546 pharmaceutical excipient Substances 0.000 description 7
- 210000002966 serum Anatomy 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 239000000126 substance Substances 0.000 description 7
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 6
- 201000010099 disease Diseases 0.000 description 6
- 238000009472 formulation Methods 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 239000000843 powder Substances 0.000 description 6
- 230000002265 prevention Effects 0.000 description 6
- 238000001356 surgical procedure Methods 0.000 description 6
- 239000003826 tablet Substances 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 230000004520 agglutination Effects 0.000 description 5
- 239000006161 blood agar Substances 0.000 description 5
- 210000004899 c-terminal region Anatomy 0.000 description 5
- 210000002421 cell wall Anatomy 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- 238000011260 co-administration Methods 0.000 description 5
- 208000035475 disorder Diseases 0.000 description 5
- 239000002552 dosage form Substances 0.000 description 5
- 238000002347 injection Methods 0.000 description 5
- 239000007924 injection Substances 0.000 description 5
- 210000001503 joint Anatomy 0.000 description 5
- 230000000399 orthopedic effect Effects 0.000 description 5
- 244000052769 pathogen Species 0.000 description 5
- 239000003380 propellant Substances 0.000 description 5
- 230000002829 reductive effect Effects 0.000 description 5
- 239000004094 surface-active agent Substances 0.000 description 5
- 208000024891 symptom Diseases 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 238000002560 therapeutic procedure Methods 0.000 description 5
- 239000001974 tryptic soy broth Substances 0.000 description 5
- 108010050327 trypticase-soy broth Proteins 0.000 description 5
- 108020004705 Codon Proteins 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 4
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 4
- 239000004480 active ingredient Substances 0.000 description 4
- 239000004599 antimicrobial Substances 0.000 description 4
- 230000004071 biological effect Effects 0.000 description 4
- 238000002815 broth microdilution Methods 0.000 description 4
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 4
- 235000018417 cysteine Nutrition 0.000 description 4
- 238000003745 diagnosis Methods 0.000 description 4
- 239000000839 emulsion Substances 0.000 description 4
- 230000014509 gene expression Effects 0.000 description 4
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 4
- 210000000629 knee joint Anatomy 0.000 description 4
- 230000035772 mutation Effects 0.000 description 4
- 230000001717 pathogenic effect Effects 0.000 description 4
- 230000006320 pegylation Effects 0.000 description 4
- 239000006187 pill Substances 0.000 description 4
- 230000002035 prolonged effect Effects 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 239000004474 valine Substances 0.000 description 4
- 239000003981 vehicle Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 208000035143 Bacterial infection Diseases 0.000 description 3
- 238000009631 Broth culture Methods 0.000 description 3
- 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 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 3
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 3
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 3
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 3
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 3
- 230000001154 acute effect Effects 0.000 description 3
- 239000000654 additive Substances 0.000 description 3
- 239000000443 aerosol Substances 0.000 description 3
- 230000003466 anti-cipated effect Effects 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 208000022362 bacterial infectious disease Diseases 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 239000002775 capsule Substances 0.000 description 3
- 229910002092 carbon dioxide Inorganic materials 0.000 description 3
- 238000010611 checkerboard assay Methods 0.000 description 3
- 238000000576 coating method Methods 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 238000011109 contamination Methods 0.000 description 3
- 230000001054 cortical effect Effects 0.000 description 3
- 239000013078 crystal Substances 0.000 description 3
- 230000009089 cytolysis Effects 0.000 description 3
- 238000001804 debridement Methods 0.000 description 3
- 102000037865 fusion proteins Human genes 0.000 description 3
- 108020001507 fusion proteins Proteins 0.000 description 3
- 239000005090 green fluorescent protein Substances 0.000 description 3
- 230000036541 health Effects 0.000 description 3
- 230000002101 lytic effect Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 150000004667 medium chain fatty acids Chemical class 0.000 description 3
- 210000003205 muscle Anatomy 0.000 description 3
- 150000007523 nucleic acids Chemical class 0.000 description 3
- 239000002674 ointment Substances 0.000 description 3
- 108091033319 polynucleotide Proteins 0.000 description 3
- 102000040430 polynucleotide Human genes 0.000 description 3
- 239000002157 polynucleotide Substances 0.000 description 3
- 238000012552 review Methods 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 239000007921 spray Substances 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- 238000012384 transportation and delivery Methods 0.000 description 3
- 241001515965 unidentified phage Species 0.000 description 3
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 2
- 206010053555 Arthritis bacterial Diseases 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 208000031729 Bacteremia Diseases 0.000 description 2
- 208000034309 Bacterial disease carrier Diseases 0.000 description 2
- 108010074051 C-Reactive Protein Proteins 0.000 description 2
- 102100032752 C-reactive protein Human genes 0.000 description 2
- 241000282472 Canis lupus familiaris Species 0.000 description 2
- 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 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 description 2
- 229930182566 Gentamicin Natural products 0.000 description 2
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 208000004575 Infectious Arthritis Diseases 0.000 description 2
- 238000012313 Kruskal-Wallis test Methods 0.000 description 2
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 2
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 2
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 2
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 2
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 2
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 2
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 2
- 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 2
- 241000124008 Mammalia Species 0.000 description 2
- 238000000585 Mann–Whitney U test Methods 0.000 description 2
- MSFSPUZXLOGKHJ-UHFFFAOYSA-N Muraminsaeure Natural products OC(=O)C(C)OC1C(N)C(O)OC(CO)C1O MSFSPUZXLOGKHJ-UHFFFAOYSA-N 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 241001494479 Pecora Species 0.000 description 2
- 108010013639 Peptidoglycan Proteins 0.000 description 2
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- 239000004743 Polypropylene Substances 0.000 description 2
- 206010040047 Sepsis Diseases 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 241000282887 Suidae Species 0.000 description 2
- 208000031650 Surgical Wound Infection Diseases 0.000 description 2
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 2
- 239000004473 Threonine Substances 0.000 description 2
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 2
- 206010052428 Wound Diseases 0.000 description 2
- 239000003070 absorption delaying agent Substances 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 230000000996 additive effect Effects 0.000 description 2
- 230000002411 adverse Effects 0.000 description 2
- 235000004279 alanine Nutrition 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 230000000845 anti-microbial effect Effects 0.000 description 2
- 230000000941 anti-staphylcoccal effect Effects 0.000 description 2
- 238000011882 arthroplasty Methods 0.000 description 2
- 238000011881 arthroscopic irrigation Methods 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
- 230000008827 biological function Effects 0.000 description 2
- 229920001400 block copolymer Polymers 0.000 description 2
- 210000001185 bone marrow Anatomy 0.000 description 2
- 239000012876 carrier material Substances 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000003915 cell function Effects 0.000 description 2
- 230000001684 chronic effect Effects 0.000 description 2
- 108010082025 cyan fluorescent protein Proteins 0.000 description 2
- 230000003111 delayed effect Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 231100000676 disease causative agent Toxicity 0.000 description 2
- 239000002612 dispersion medium Substances 0.000 description 2
- 239000012055 enteric layer Substances 0.000 description 2
- 210000003195 fascia Anatomy 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 210000001035 gastrointestinal tract Anatomy 0.000 description 2
- 229960002518 gentamicin Drugs 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- BXWNKGSJHAJOGX-UHFFFAOYSA-N hexadecan-1-ol Chemical compound CCCCCCCCCCCCCCCCO BXWNKGSJHAJOGX-UHFFFAOYSA-N 0.000 description 2
- 210000004394 hip joint Anatomy 0.000 description 2
- 230000003301 hydrolyzing effect Effects 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 230000002458 infectious effect Effects 0.000 description 2
- 208000014674 injury Diseases 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 2
- 229960000310 isoleucine Drugs 0.000 description 2
- 239000007951 isotonicity adjuster Substances 0.000 description 2
- 239000008101 lactose Substances 0.000 description 2
- 210000002414 leg Anatomy 0.000 description 2
- 210000000265 leukocyte Anatomy 0.000 description 2
- 239000007937 lozenge Substances 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
- 230000014759 maintenance of location Effects 0.000 description 2
- 229930182817 methionine Natural products 0.000 description 2
- DNIAPMSPPWPWGF-UHFFFAOYSA-N monopropylene glycol Natural products CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 239000003921 oil Substances 0.000 description 2
- 235000019198 oils Nutrition 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 229960001019 oxacillin Drugs 0.000 description 2
- UWYHMGVUTGAWSP-JKIFEVAISA-N oxacillin 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=C1 UWYHMGVUTGAWSP-JKIFEVAISA-N 0.000 description 2
- 230000002085 persistent effect Effects 0.000 description 2
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 2
- 239000008055 phosphate buffer solution Substances 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 229920001155 polypropylene Polymers 0.000 description 2
- 230000002980 postoperative effect Effects 0.000 description 2
- 239000003755 preservative agent Substances 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 238000011555 rabbit model Methods 0.000 description 2
- 108010054624 red fluorescent protein Proteins 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000008261 resistance mechanism Effects 0.000 description 2
- 201000001223 septic arthritis Diseases 0.000 description 2
- 229910001220 stainless steel Inorganic materials 0.000 description 2
- 239000010935 stainless steel Substances 0.000 description 2
- 238000010561 standard procedure Methods 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 238000013268 sustained release Methods 0.000 description 2
- 239000012730 sustained-release form Substances 0.000 description 2
- 238000010610 time kill assay Methods 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 230000008733 trauma Effects 0.000 description 2
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 2
- 230000001018 virulence Effects 0.000 description 2
- 108091005957 yellow fluorescent proteins Proteins 0.000 description 2
- NOOLISFMXDJSKH-UTLUCORTSA-N (+)-Neomenthol Chemical compound CC(C)[C@@H]1CC[C@@H](C)C[C@@H]1O NOOLISFMXDJSKH-UTLUCORTSA-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
- FFJCNSLCJOQHKM-CLFAGFIQSA-N (z)-1-[(z)-octadec-9-enoxy]octadec-9-ene Chemical compound CCCCCCCC\C=C/CCCCCCCCOCCCCCCCC\C=C/CCCCCCCC FFJCNSLCJOQHKM-CLFAGFIQSA-N 0.000 description 1
- TXGPGHBYAPBDAG-UHFFFAOYSA-N 1,1,2,2,3,3-hexafluoro-4,4-bis(trifluoromethyl)cyclobutane Chemical compound FC(F)(F)C1(C(F)(F)F)C(F)(F)C(F)(F)C1(F)F TXGPGHBYAPBDAG-UHFFFAOYSA-N 0.000 description 1
- DDMOUSALMHHKOS-UHFFFAOYSA-N 1,2-dichloro-1,1,2,2-tetrafluoroethane Chemical compound FC(F)(Cl)C(F)(F)Cl DDMOUSALMHHKOS-UHFFFAOYSA-N 0.000 description 1
- CPKVUHPKYQGHMW-UHFFFAOYSA-N 1-ethenylpyrrolidin-2-one;molecular iodine Chemical compound II.C=CN1CCCC1=O CPKVUHPKYQGHMW-UHFFFAOYSA-N 0.000 description 1
- HBXWUCXDUUJDRB-UHFFFAOYSA-N 1-octadecoxyoctadecane Chemical compound CCCCCCCCCCCCCCCCCCOCCCCCCCCCCCCCCCCCC HBXWUCXDUUJDRB-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
- LCSKNASZPVZHEG-UHFFFAOYSA-N 3,6-dimethyl-1,4-dioxane-2,5-dione;1,4-dioxane-2,5-dione Chemical compound O=C1COC(=O)CO1.CC1OC(=O)C(C)OC1=O LCSKNASZPVZHEG-UHFFFAOYSA-N 0.000 description 1
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 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 1
- 108090000531 Amidohydrolases Proteins 0.000 description 1
- 102000004092 Amidohydrolases Human genes 0.000 description 1
- 101800002011 Amphipathic peptide Proteins 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 108700042778 Antimicrobial Peptides Proteins 0.000 description 1
- 102000044503 Antimicrobial Peptides Human genes 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 201000006082 Chickenpox Diseases 0.000 description 1
- 208000003322 Coinfection Diseases 0.000 description 1
- NOOLISFMXDJSKH-UHFFFAOYSA-N DL-menthol Natural products CC(C)C1CCC(C)CC1O NOOLISFMXDJSKH-UHFFFAOYSA-N 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 108010002069 Defensins Proteins 0.000 description 1
- 206010064687 Device related infection Diseases 0.000 description 1
- 239000004338 Dichlorodifluoromethane Substances 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 206010017553 Furuncle Diseases 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 206010021531 Impetigo Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- GSDSWSVVBLHKDQ-JTQLQIEISA-N Levofloxacin Chemical compound C([C@@H](N1C2=C(C(C(C(O)=O)=C1)=O)C=C1F)C)OC2=C1N1CCN(C)CC1 GSDSWSVVBLHKDQ-JTQLQIEISA-N 0.000 description 1
- 241000186781 Listeria Species 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 241000736262 Microbiota 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
- 208000002565 Open Fractures Diseases 0.000 description 1
- 235000019483 Peanut oil Nutrition 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- 102000007079 Peptide Fragments Human genes 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 229920001214 Polysorbate 60 Polymers 0.000 description 1
- 229920000153 Povidone-iodine Polymers 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 206010061926 Purulence Diseases 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 208000035415 Reinfection Diseases 0.000 description 1
- 229930189077 Rifamycin Natural products 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 229920001800 Shellac Polymers 0.000 description 1
- 206010062255 Soft tissue infection Diseases 0.000 description 1
- 239000004147 Sorbitan trioleate Substances 0.000 description 1
- PRXRUNOAOLTIEF-ADSICKODSA-N Sorbitan trioleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@@H](OC(=O)CCCCCCC\C=C/CCCCCCCC)[C@H]1OC[C@H](O)[C@H]1OC(=O)CCCCCCC\C=C/CCCCCCCC PRXRUNOAOLTIEF-ADSICKODSA-N 0.000 description 1
- 241001288016 Streptococcus gallolyticus Species 0.000 description 1
- 201000005010 Streptococcus pneumonia Diseases 0.000 description 1
- 241000193998 Streptococcus pneumoniae Species 0.000 description 1
- 241000194021 Streptococcus suis Species 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 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 1
- 108010053950 Teicoplanin Proteins 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
- RTAQQCXQSZGOHL-UHFFFAOYSA-N Titanium Chemical compound [Ti] RTAQQCXQSZGOHL-UHFFFAOYSA-N 0.000 description 1
- 206010046980 Varicella Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- ZWBTYMGEBZUQTK-PVLSIAFMSA-N [(7S,9E,11S,12R,13S,14R,15R,16R,17S,18S,19E,21Z)-2,15,17,32-tetrahydroxy-11-methoxy-3,7,12,14,16,18,22-heptamethyl-1'-(2-methylpropyl)-6,23-dioxospiro[8,33-dioxa-24,27,29-triazapentacyclo[23.6.1.14,7.05,31.026,30]tritriaconta-1(32),2,4,9,19,21,24,26,30-nonaene-28,4'-piperidine]-13-yl] acetate Chemical compound CO[C@H]1\C=C\O[C@@]2(C)Oc3c(C2=O)c2c4NC5(CCN(CC(C)C)CC5)N=c4c(=NC(=O)\C(C)=C/C=C/[C@H](C)[C@H](O)[C@@H](C)[C@@H](O)[C@@H](C)[C@H](OC(C)=O)[C@@H]1C)c(O)c2c(O)c3C ZWBTYMGEBZUQTK-PVLSIAFMSA-N 0.000 description 1
- 206010000269 abscess Diseases 0.000 description 1
- 230000021736 acetylation Effects 0.000 description 1
- 238000006640 acetylation reaction Methods 0.000 description 1
- 229940081735 acetylcellulose Drugs 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 239000000783 alginic acid Substances 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 229960001126 alginic acid Drugs 0.000 description 1
- 150000004781 alginic acids Chemical class 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 239000012080 ambient air Substances 0.000 description 1
- 230000009435 amidation Effects 0.000 description 1
- 238000007112 amidation reaction Methods 0.000 description 1
- 229960004821 amikacin Drugs 0.000 description 1
- LKCWBDHBTVXHDL-RMDFUYIESA-N amikacin Chemical compound O([C@@H]1[C@@H](N)C[C@H]([C@@H]([C@H]1O)O[C@@H]1[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O1)O)NC(=O)[C@@H](O)CCN)[C@H]1O[C@H](CN)[C@@H](O)[C@H](O)[C@H]1O LKCWBDHBTVXHDL-RMDFUYIESA-N 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 230000036592 analgesia Effects 0.000 description 1
- 230000003214 anti-biofilm Effects 0.000 description 1
- 238000009635 antibiotic susceptibility testing Methods 0.000 description 1
- 238000011203 antimicrobial therapy Methods 0.000 description 1
- 210000001765 aortic valve Anatomy 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 210000001188 articular cartilage Anatomy 0.000 description 1
- 210000000544 articulatio talocruralis Anatomy 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 229960004099 azithromycin Drugs 0.000 description 1
- MQTOSJVFKKJCRP-BICOPXKESA-N azithromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)N(C)C[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 MQTOSJVFKKJCRP-BICOPXKESA-N 0.000 description 1
- 239000003899 bactericide agent Substances 0.000 description 1
- 230000001420 bacteriolytic effect Effects 0.000 description 1
- 239000000022 bacteriostatic agent Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000009640 blood culture Methods 0.000 description 1
- 229940041011 carbapenems Drugs 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229960004424 carbon dioxide Drugs 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000004359 castor oil Substances 0.000 description 1
- 235000019438 castor oil Nutrition 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 229920002301 cellulose acetate Polymers 0.000 description 1
- 229940106164 cephalexin Drugs 0.000 description 1
- ZAIPMKNFIOOWCQ-UEKVPHQBSA-N cephalexin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@@H]3N(C2=O)C(=C(CS3)C)C(O)=O)=CC=CC=C1 ZAIPMKNFIOOWCQ-UEKVPHQBSA-N 0.000 description 1
- 229960000541 cetyl alcohol Drugs 0.000 description 1
- YMKDRGPMQRFJGP-UHFFFAOYSA-M cetylpyridinium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCC[N+]1=CC=CC=C1 YMKDRGPMQRFJGP-UHFFFAOYSA-M 0.000 description 1
- 229960001927 cetylpyridinium chloride Drugs 0.000 description 1
- DDTDNCYHLGRFBM-YZEKDTGTSA-N chembl2367892 Chemical compound CC(=O)N[C@H]1[C@@H](O)[C@H](O)[C@H](CO)O[C@H]1O[C@@H]([C@H]1C(N[C@@H](C2=CC(O)=CC(O[C@@H]3[C@H]([C@H](O)[C@H](O)[C@@H](CO)O3)O)=C2C=2C(O)=CC=C(C=2)[C@@H](NC(=O)[C@@H]2NC(=O)[C@@H]3C=4C=C(O)C=C(C=4)OC=4C(O)=CC=C(C=4)[C@@H](N)C(=O)N[C@H](CC=4C=C(Cl)C(O5)=CC=4)C(=O)N3)C(=O)N1)C(O)=O)=O)C(C=C1Cl)=CC=C1OC1=C(O[C@H]3[C@H]([C@@H](O)[C@H](O)[C@H](CO)O3)NC(C)=O)C5=CC2=C1 DDTDNCYHLGRFBM-YZEKDTGTSA-N 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 235000015218 chewing gum Nutrition 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 230000001332 colony forming effect Effects 0.000 description 1
- 230000002301 combined effect Effects 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- DTPCFIHYWYONMD-UHFFFAOYSA-N decaethylene glycol Polymers OCCOCCOCCOCCOCCOCCOCCOCCOCCOCCO DTPCFIHYWYONMD-UHFFFAOYSA-N 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- PXBRQCKWGAHEHS-UHFFFAOYSA-N dichlorodifluoromethane Chemical compound FC(F)(Cl)Cl PXBRQCKWGAHEHS-UHFFFAOYSA-N 0.000 description 1
- 235000019404 dichlorodifluoromethane Nutrition 0.000 description 1
- 229940042935 dichlorodifluoromethane Drugs 0.000 description 1
- 229940087091 dichlorotetrafluoroethane Drugs 0.000 description 1
- XXJWXESWEXIICW-UHFFFAOYSA-N diethylene glycol monoethyl ether Chemical compound CCOCCOCCO XXJWXESWEXIICW-UHFFFAOYSA-N 0.000 description 1
- 229940075557 diethylene glycol monoethyl ether Drugs 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
- 239000006185 dispersion Substances 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000005553 drilling Methods 0.000 description 1
- 239000000890 drug combination Substances 0.000 description 1
- 210000001198 duodenum Anatomy 0.000 description 1
- 230000004522 effect on agglutination Effects 0.000 description 1
- 210000002310 elbow joint Anatomy 0.000 description 1
- 206010014665 endocarditis Diseases 0.000 description 1
- 239000002702 enteric coating Substances 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 229960003276 erythromycin Drugs 0.000 description 1
- 229940044949 eucalyptus oil Drugs 0.000 description 1
- 239000010642 eucalyptus oil Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 108091006047 fluorescent proteins Proteins 0.000 description 1
- 102000034287 fluorescent proteins Human genes 0.000 description 1
- 239000013022 formulation composition Substances 0.000 description 1
- 230000005714 functional activity Effects 0.000 description 1
- 208000003512 furunculosis Diseases 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 125000005456 glyceride group Chemical group 0.000 description 1
- ZEMPKEQAKRGZGQ-XOQCFJPHSA-N glycerol triricinoleate Natural products CCCCCC[C@@H](O)CC=CCCCCCCCC(=O)OC[C@@H](COC(=O)CCCCCCCC=CC[C@@H](O)CCCCCC)OC(=O)CCCCCCCC=CC[C@H](O)CCCCCC ZEMPKEQAKRGZGQ-XOQCFJPHSA-N 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 208000027136 gram-positive bacterial infections Diseases 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 125000000487 histidyl group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 description 1
- 238000010231 histologic analysis Methods 0.000 description 1
- 210000002758 humerus Anatomy 0.000 description 1
- 230000002209 hydrophobic effect Effects 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
- 230000001771 impaired effect Effects 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000002452 interceptive effect 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
- 238000011005 laboratory method Methods 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 239000010410 layer Substances 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 231100000225 lethality Toxicity 0.000 description 1
- 108010000849 leukocyte esterase Proteins 0.000 description 1
- 229960003376 levofloxacin Drugs 0.000 description 1
- 125000005647 linker group Chemical group 0.000 description 1
- 230000033001 locomotion Effects 0.000 description 1
- 210000004705 lumbosacral region Anatomy 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 239000003120 macrolide antibiotic agent Substances 0.000 description 1
- 229940041033 macrolides Drugs 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000002483 medication Methods 0.000 description 1
- 229940041616 menthol Drugs 0.000 description 1
- 239000004530 micro-emulsion Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 239000002480 mineral oil Substances 0.000 description 1
- 235000010446 mineral oil Nutrition 0.000 description 1
- 238000001823 molecular biology technique Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 239000002324 mouth wash Substances 0.000 description 1
- 239000007923 nasal drop Substances 0.000 description 1
- 229940100662 nasal drops Drugs 0.000 description 1
- 239000007922 nasal spray Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- BCCOBQSFUDVTJQ-UHFFFAOYSA-N octafluorocyclobutane Chemical compound FC1(F)C(F)(F)C(F)(F)C1(F)F BCCOBQSFUDVTJQ-UHFFFAOYSA-N 0.000 description 1
- 235000019407 octafluorocyclobutane Nutrition 0.000 description 1
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 239000000312 peanut oil Substances 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 210000004197 pelvis Anatomy 0.000 description 1
- 230000009518 penetrating injury Effects 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 230000002688 persistence Effects 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 229940124531 pharmaceutical excipient Drugs 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 239000002953 phosphate buffered saline Substances 0.000 description 1
- 229960002292 piperacillin Drugs 0.000 description 1
- 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 1
- 230000036470 plasma concentration Effects 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 239000004417 polycarbonate Substances 0.000 description 1
- 229920000515 polycarbonate Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 239000003910 polypeptide antibiotic agent Substances 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 150000004804 polysaccharides Chemical class 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 230000001323 posttranslational effect Effects 0.000 description 1
- 229920001592 potato starch Polymers 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 229960001621 povidone-iodine Drugs 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 208000011354 prosthesis-related infectious disease Diseases 0.000 description 1
- 235000019833 protease Nutrition 0.000 description 1
- 125000006239 protecting group Chemical group 0.000 description 1
- 230000004224 protection Effects 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 210000002320 radius Anatomy 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 238000009877 rendering Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 229960000885 rifabutin Drugs 0.000 description 1
- 229960003292 rifamycin Drugs 0.000 description 1
- HJYYPODYNSCCOU-ODRIEIDWSA-N rifamycin SV Chemical compound OC1=C(C(O)=C2C)C3=C(O)C=C1NC(=O)\C(C)=C/C=C/[C@H](C)[C@H](O)[C@@H](C)[C@@H](O)[C@@H](C)[C@H](OC(C)=O)[C@H](C)[C@@H](OC)\C=C\O[C@@]1(C)OC2=C3C1=O HJYYPODYNSCCOU-ODRIEIDWSA-N 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000001878 scanning electron micrograph Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000008159 sesame oil Substances 0.000 description 1
- 235000011803 sesame oil Nutrition 0.000 description 1
- 239000004208 shellac Substances 0.000 description 1
- ZLGIYFNHBLSMPS-ATJNOEHPSA-N shellac Chemical compound OCCCCCC(O)C(O)CCCCCCCC(O)=O.C1C23[C@H](C(O)=O)CCC2[C@](C)(CO)[C@@H]1C(C(O)=O)=C[C@@H]3O ZLGIYFNHBLSMPS-ATJNOEHPSA-N 0.000 description 1
- 229940113147 shellac Drugs 0.000 description 1
- 235000013874 shellac Nutrition 0.000 description 1
- 235000021391 short chain fatty acids Nutrition 0.000 description 1
- 150000004666 short chain fatty acids Chemical class 0.000 description 1
- 210000000323 shoulder joint Anatomy 0.000 description 1
- 238000009097 single-agent therapy Methods 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 230000000391 smoking effect Effects 0.000 description 1
- 210000004872 soft tissue Anatomy 0.000 description 1
- 239000008247 solid mixture Substances 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 235000019337 sorbitan trioleate Nutrition 0.000 description 1
- 229960000391 sorbitan trioleate Drugs 0.000 description 1
- 239000003549 soybean oil Substances 0.000 description 1
- 235000012424 soybean oil Nutrition 0.000 description 1
- 239000008347 soybean phospholipid Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 229960005404 sulfamethoxazole Drugs 0.000 description 1
- JLKIGFTWXXRPMT-UHFFFAOYSA-N sulphamethoxazole Chemical compound O1C(C)=CC(NS(=O)(=O)C=2C=CC(N)=CC=2)=N1 JLKIGFTWXXRPMT-UHFFFAOYSA-N 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 238000011477 surgical intervention Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012385 systemic delivery Methods 0.000 description 1
- LPQZKKCYTLCDGQ-WEDXCCLWSA-N tazobactam Chemical compound C([C@]1(C)S([C@H]2N(C(C2)=O)[C@H]1C(O)=O)(=O)=O)N1C=CN=N1 LPQZKKCYTLCDGQ-WEDXCCLWSA-N 0.000 description 1
- 229960003865 tazobactam Drugs 0.000 description 1
- 229960003879 tedizolid Drugs 0.000 description 1
- XFALPSLJIHVRKE-GFCCVEGCSA-N tedizolid Chemical compound CN1N=NC(C=2N=CC(=CC=2)C=2C(=CC(=CC=2)N2C(O[C@@H](CO)C2)=O)F)=N1 XFALPSLJIHVRKE-GFCCVEGCSA-N 0.000 description 1
- 229960001608 teicoplanin Drugs 0.000 description 1
- 125000005931 tert-butyloxycarbonyl group Chemical group [H]C([H])([H])C(OC(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 229940040944 tetracyclines Drugs 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 239000010936 titanium Substances 0.000 description 1
- 229910052719 titanium Inorganic materials 0.000 description 1
- 229960000707 tobramycin Drugs 0.000 description 1
- 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 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 150000003626 triacylglycerols Chemical class 0.000 description 1
- CYRMSUTZVYGINF-UHFFFAOYSA-N trichlorofluoromethane Chemical compound FC(Cl)(Cl)Cl CYRMSUTZVYGINF-UHFFFAOYSA-N 0.000 description 1
- 229940029284 trichlorofluoromethane Drugs 0.000 description 1
- 210000000689 upper leg Anatomy 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 208000019206 urinary tract infection Diseases 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
- 210000003857 wrist joint Anatomy 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/14—Peptides containing saccharide radicals; Derivatives thereof, e.g. bleomycin, phleomycin, muramylpeptides or vancomycin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
Definitions
- the present disclosure relates generally to the treatment and prevention of bone and/or joint and/or implant-associated infections, particularly osteomyelitis and prosthetic joint infections due to Gram-positive bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), using lysin(s) and optionally one or more antibiotics by locally delivering the lysins.
- MRSA methicillin-resistant Staphylococcus aureus
- Bone and joint infections including osteomyelitis and implant-associated orthopedic infections are complex and difficult to treat.
- Staphylococcus aureus the most common bacterium recovered in orthopedic infection, forms biofilms, which are protective extracellular polymeric matrices embedding clusters of sessile bacteria on implants as well as in/on bone.
- Staphylococcus aureus can exist as persister cells and small colony variants (SCVs) and enter and survive intracellularly in cells such as fibroblasts and osteoblasts.
- Lysins are cell wall hydrolytic enzymes, which are produced from bacteriophage, viruses that infect and kill bacteria. When phages replicate, lysins accumulate in the bacterial cytoplasm before translocating to the cell wall, hydrolyzing peptidoglycan, and rapidly killing bacteria. Lysins can be recombinantly produced and purified and can be administered to attack cell walls from the outside of cells, thus providing a potentially rapid bactericidal treatment option.
- the present disclosure is also directed to a composition
- a composition comprising a therapeutically effective amount of a PlySs2 lysin formulated for local administration to a subject with a bone or joint or implant-associated infection comprising Gram-positive bacteria, such as local administration to a bone of a subject with a bone or joint infection, comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof having at least 80% identity to SEQ ID NO: 1, optionally with one or more antibiotic(s), wherein the PlySs2 variant comprises bactericidal and/or bacteriostatic activity against Gram-positive bacteria.
- the local administration comprises intramedullary local administration.
- FIG. 1 depicts the amino acid sequence of a lysin (SEQ ID NO: 1) and a polynucleotide (SEQ ID NO: 18) encoding the lysin as described in the detailed description.
- SEQ ID NO: 1 represents a 245 amino acid polypeptide, including the initial methionine residue which is removed during post-translational processing, leaving a 244-amino acid polypeptide.
- FIG. 2 depicts the rapid in vitro bactericidal activity of the lysins, exebacase and CF-296, on infected implants, as described in the Examples.
- FIG. 3 depicts a rabbit foreign body osteomyelitis screw model as described in the Examples.
- the left panel depicts a scanning electron microscope image showing bacteria colonizing the implant (screw) surface.
- the right panel depicts an X-ray of the medial (left) and superior (right) view of the left leg of a supine rabbit.
- FIGS. 4a-4j depict the surgical procedure used to insert an infected implant into bone and to locally treat the infection as described in the Examples.
- FIG 4a depicts the exposure of a smooth flat portion of the tibia.
- FIGS. 4b and 4c depict the creation of a hole through the cortical bone into the medullary cavity using a micro drill.
- FIGS. 4d and 4e depict the hole after or during injection with water (FIG. 4d) or lysin or lysin carrier (FIG. 4e).
- FIG. 4f depicts insertion of an infected implant into the hole.
- FIG. 4g depicts the use of a screwdriver to tighten a screw used to secure the infected implant.
- FIG. 4h depicts confirmation that the implant is secure.
- FIGS. 4i and 4j depict the post-surgical view, above (FIG. 4i) and parallel to (FIG. 4j), the medial tibia of a supine rabbit.
- FIG. 5 depicts the treatments used to treat the infected bone and implant as described in the Examples.
- FIG. 6 depicts the amount of bacteria recovered from bone and the screw implant 5 days after lysin treatment as described in the Examples.
- Carrier refers to a solvent, additive, excipient, dispersion medium, solubilizing agent, coating, preservative, isotonic and absorption delaying agent, surfactant, propellant, diluent, vehicle and the like with which an active compound is administered.
- Such carriers can be sterile liquids, such as water, saline solutions, aqueous dextrose solutions, aqueous glycerol solutions, and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
- Non-limiting examples of pharmaceutically acceptable carriers or excipients include any of the standard pharmaceutical carriers such as phosphate buffered saline solutions, water, and emulsions such as oil/water emulsions and microemulsions. Suitable pharmaceutical carriers are described, for example, in “Remington's Pharmaceutical Sciences” by E.W. Martin, 18th Edition.
- the pharmaceutically acceptable carrier may be a carrier that does not exist in nature.
- Bacteriostatic or “bacteriostatic activity” refers to the property of causing the death of bacteria or capable of killing bacteria to an extent of at least a 3-loglO (99.9%) or a better reduction among an initial population of bacteria over an 18-24 hour period.
- Bacteriostatic activity refers to the property of inhibiting bacterial growth, including inhibiting growing bacterial cells, thus causing a 2-loglO (99%) or better and up to just under a 3-log reduction among an initial population of bacteria over an 18-24 hour period.
- Antibacterial refers to both bacteriostatic and bactericidal agents.
- Drug resistant refers generally to a bacterium that is resistant to the antibacterial activity of a drug. When used in certain ways, drug resistance may specifically refer to antibiotic resistance. In some cases, a bacterium that is generally susceptible to a particular antibiotic can develop resistance to the antibiotic, thereby becoming a drug resistant microbe or strain.
- MDR multidrug resistant
- a “multidrug resistant” (“MDR”) pathogen is one that has developed resistance to at least two classes of antimicrobial drugs, each used as monotherapy. For example, certain strains of S. aureus have been found to be resistant to several antibiotics including methicillin and/or vancomycin (Antibiotic Resistant Threats in the United States, 2013, U.S. Department of Health and Services, Centers for Disease Control and Prevention).
- MDR multidrug resistant
- One skilled in the art can readily determine if a bacterium is drug resistant using routine laboratory techniques that determine the susceptibility or resistance of a bacterium to a drug or antibiotic.
- Co-administer refers to the administration of two agents, such as a lysin, and an antibiotic or any other antibacterial agent in a sequential manner, as well as administration of these agents in a substantially simultaneous manner, such as in a single mixture/composition or in doses given separately, but nonetheless administered substantially simultaneously to the subject, for example at different times in the same day or 24-hour period.
- Such co-administration of two agents, such as a lysin with one or more additional antibacterial agents can be provided as a continuous treatment lasting up to days, weeks, or months. Additionally, depending on the use, the co-administration need not be continuous or coextensive.
- Subject refers to a mammal, a plant, a lower animal, a single cell organism or a cell culture.
- the term “subject” is intended to include organisms, e.g., prokaryotes and eukaryotes, which are susceptible to or afflicted with bacterial infections, for example Grampositive bacterial infections.
- subjects include mammals, e.g., humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals.
- the subject is a human, e.g., a human suffering from, at risk of suffering from, or susceptible to infection by Gram-positive bacteria, whether such infection be systemic, topical or otherwise concentrated or confined to a particular organ or tissue.
- a particular lysin polypeptide for example, can be, e.g., derived or removed from a native protein by enzymatic or chemical cleavage, or can be prepared using conventional peptide synthesis techniques (e.g., solid phase synthesis) or molecular biology techniques (such as those disclosed in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989)) or can be strategically truncated or segmented yielding active fragments, maintaining e.g., lysin activity against the same or at least one common target bacterium.
- conventional peptide synthesis techniques e.g., solid phase synthesis
- molecular biology techniques such as those disclosed in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989)
- active fragments maintaining e.g., lysin activity against the same or at least one common target
- Active fragment refers to a portion of a polypeptide that retains one or more functions or biological activities of the isolated polypeptide from which the fragment was taken, for example bactericidal activity against one or more Gram-positive bacteria, such as S. aureus or S. epidermidis.
- “Synergistic” or “Superadditive” refers to a beneficial effect brought about by two substances in combination that exceeds the sum of the effects of the two agents working independently. In certain embodiments the synergistic or superadditive effect significantly, z.e., statistically significantly, exceeds the sum of the effects of the two agents working independently.
- One or both active ingredients may be employed at a sub-threshold level, z.e., a level at which if the active substance is employed individually produces no or a very limited effect. The effect can be measured by assays such as the checkerboard assay, described here.
- Treatment refers to any process, action, application, therapy, or the like, wherein a subject, including a human being, is subjected to medical aid with the object of curing a disorder, eradicating a pathogen, or improving the subject's condition, directly or indirectly. Treatment also refers to reducing incidence, alleviating symptoms, eliminating recurrence, preventing recurrence, preventing incidence, reducing the risk of incidence, improving symptoms, improving prognosis or combinations thereof. “Treatment” may further encompass reducing the population, growth rate or virulence of the bacteria in the subject and thereby controlling or reducing a bacterial infection in a subject or bacterial contamination of an organ, tissue or environment.
- treatment that reduces incidence may, for example, be effective to inhibit growth of at least one Gram-positive bacterium in a particular milieu, whether it be a subject or an environment.
- treatment of an already established infection refers to reducing the population, killing, inhibiting the growth, and/or eradicating, the Gram-positive bacteria responsible for an infection or contamination.
- Preventing refers to the prevention of the incidence, recurrence, spread, onset or establishment of a disorder such as a bacterial infection. It is not intended that the present disclosure be limited to complete prevention or to prevention of establishment of an infection. In some embodiments, the onset is delayed, or the severity of a subsequently contracted disease or the chance of contracting the disease is reduced, and such constitutes examples of prevention.
- Constracted diseases refers to diseases manifesting with clinical or subclinical symptoms, such as the detection of fever, sepsis or bacteremia, as well as diseases that may be detected by growth of a bacterial pathogen (e.g., in culture) when symptoms associated with such pathology are not yet manifest.
- a bacterial pathogen e.g., in culture
- “Derivative,” in the context of a peptide or polypeptide or active fragment thereof, is intended to encompass, for example, a polypeptide modified to contain one or more-chemical moieties other than an amino acid that do not substantially adversely impact or destroy the polypeptide’s activity, such as lysin activity.
- the chemical moiety can be linked covalently to the peptide, e.g., via an amino terminal amino acid residue, a carboxy terminal amino acid residue, or at an internal amino acid residue. Such modifications may be natural or non-natural.
- a non-natural modification may include the addition of a protective or capping group on a reactive moiety, addition of a detectable label, such as an antibody and/or fluorescent label, addition or modification of glycosylation, or addition of a bulking group such as PEG (pegylation) and other changes known to those skilled in the art.
- the non- natural modification may be a capping modification, such as N-terminal acetylations and C- terminal amidations.
- Exemplary protective groups that may be added to lysin polypeptides include, but are not limited to t-Boc and Fmoc.
- fluorescent label proteins such as, but not limited to, green fluorescent protein (GFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) and mCherry, are compact proteins that can be bound covalently or noncovalently to a polypeptide or fused to a polypeptide without interfering with normal functions of cellular proteins.
- a polynucleotide encoding a fluorescent protein is inserted upstream or downstream of the polynucleotide sequence. This will produce a fusion protein (e.g., Lysin Polypeptide:: GFP) that does not interfere with cellular function or function of a polypeptide to which it is attached.
- Polyethylene glycol (PEG) conjugation to proteins has been used as a method for extending the circulating half-life of many pharmaceutical proteins.
- polypeptide derivatives such as lysin polypeptide derivatives
- derivative encompasses polypeptides, such as lysin polypeptides, chemically modified by covalent attachment of one or more PEG molecules. It is anticipated that lysin polypeptides, such as pegylated lysins, will exhibit prolonged circulation half-life compared to unpegylated polypeptides, while retaining biological and therapeutic activity.
- Percent amino acid sequence identity refers to the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, such as a lysin polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as a part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for example, using publicly available software such as BLAST or software available commercially for example from DNASTAR. Two or more polypeptide sequences can be anywhere from 0-100% identical, or any integer value there between.
- two polypeptides are “substantially identical” when at least 80% of the amino acid residues (typically at least about 85%, at least about 90%, and typically at least about 95%, at least about 98%, or at least 99%) are identical.
- the term “percent (%) amino acid sequence identity” as described herein applies to peptides as well.
- substantially identical will encompass mutated, truncated, fused, or otherwise sequence-modified variants of isolated polypeptides and peptides, such as those described herein, and active fragments thereof, as well as polypeptides with substantial sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95% identity, at least 98% identity, or at least 99% identity as measured for example by one or more methods referenced above) as compared to the reference (wild type or other intact) polypeptide.
- substantial sequence identity e.g., at least 80%, at least 85%, at least 90%, at least 95% identity, at least 98% identity, or at least 99% identity as measured for example by one or more methods referenced above
- Two amino acid sequences are “substantially homologous” when at least about 80% of the amino acid residues (typically at least about 85%, at least about 90%, at least about 95%, at least about 98% identity, or at least about 99% identity) are identical, or represent conservative substitutions.
- sequences of polypeptides of the present disclosure are substantially homologous when one or more, or several, or up to 10%, or up to 15%, or up to 20% of the amino acids of the polypeptide, such as the lysin polypeptides described herein, are substituted with a similar or conservative amino acid substitution, and wherein the resulting polypeptide, such as the lysins described herein, have at least one activity, antibacterial effects, and/or bacterial specificities of the reference polypeptide, such as the lysins described herein.
- a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art.
- amino acids with basic side chains e.g., lysine, arginine, histidine
- acidic side chains e.g., aspartic acid, glutamic acid
- uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
- nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
- beta-branched side chains e.g., threonine, valine, isoleucine
- aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
- Biofilm refers to an aggregate of microorganisms in which cells adhere to each other on a biotic or abiotic surface. These adherent cells are frequently embedded within a matrix comprised of, but not limited to, extracellular polymeric substance (EPS).
- EPS extracellular polymeric substance
- Biofilm EPS which is also referred to as slime (although not everything described as slime is a biofilm) or plaque, is a polymeric conglomeration generally composed of extracellular DNA, proteins, and polysaccharides.
- Persister cells refer to a subpopulation of bacteria that survive high concentrations of antibiotics without any specific resistance mechanism. The presence of persisters within a population is indicated by killing data that show most cells in a population dying, with a subpopulation (0.1-10%) persisting, even in the presence of a high concentration of antibiotic. Persisters pre-exist in a population and arise independently of the use of antibiotics. Persisters survive high concentrations of antibiotics by overexpression of genes such as the chromosomal toxin -antitoxin modules that shut down cellular functions and hence antibiotic targets, resulting in a dormant cell that is tolerant to the lethal action of antibiotics,
- SCVs Small Colony Variants
- SCVs refer to the generation of resistant phenotypic variants, which occur during bacterial growth. SCVs may occur in cases of e.g., soft tissue infections, osteomyelitis, and device-related infections, including those associated with prosthetic joints. SCVs differ from the normal phenotype in e.g.. their small colony size, reduced growth rate, their tendency to persist and their greater resistance to antimicrobials. These variants may represent a stable, inheritable change or a transient colony type.
- Suitable in the context of an antibiotic being suitable for use against certain bacteria refers to an antibiotic that was found to be effective against those bacteria even if resistance subsequently developed.
- lysins may be administered locally, e.g. to a bone of a subject, to kill, inhibit or prevent biofilm-forming bacteria, or to kill, inhibit or prevent biofilms, that may cause or are associated with bone and /or joint and/or implant-associated infections due to antibiotic resistant and/or multiple drug resistant (MDR) bacteria, such as Staphylococcus aureus including methicillin-resistant Staphylococcus aureus (MRSA) and multiple drug resistant (MDR) Staphylococcus epidermidis.
- MDR multiple drug resistant
- the lysins described herein are able to disrupt, eradicate and/or prevent biofilms formed by, e.g.
- Staphylococcus epidermidis or Staphylococcus aureus in bone.
- the lysins of the present disclosure may be used against bacteria from e.g., Streptococcus, Staphylococcus, Enterococcus and/or other genera as described herein.
- the present disclosure is directed to a method of treating or preventing a bone and/or joint and/or implant-associated infection, which method comprises: locally administering a therapeutically effective amount of a PlySs2 lysin as described herein to a subject in need thereof, such as locally administering an effective amount of a PlySs2 lysin to a bone, such as intramedullary local administration, wherein the bone or joint infection comprises a Gram-positive bacteria.
- the bone infection to be prevented or treated is osteomyelitis, i.e., an inflammatory reaction and destruction of bone due to bacterial colonization of the bone itself, the bone marrow and/or the surrounding tissue. Osteomyelitis can occur by local spread of bacteria from an adjacent, contaminating source caused by trauma or bone surgery, for example.
- the bone infection to be prevented or treated is due to Grampositive bacteria, such as Staphylococcus aureus or MRSA.
- the Grampositive bacteria have the ability to form biofilms and/or enter into and survive within osteoblasts, thus allowing the Gram-positive bacteria to evade the immune system and many traditional antibiotics.
- the bone infection to be prevented or treated is acute osteomyelitis.
- the bone infection is chronic osteomyelitis.
- the chronic osteomyelitis is osteomyelitis wherein the delay between infection and efficacious treatment exceeds 4-6 weeks.
- chronic osteomyelitis occurs in patients who suffered from acute osteomyelitis in the pre- antibiotic era or in their childhood. Such infections can recur after a symptom-free interval of several decades due to, e.g., the asymptomatic persistence of a biofilm adhering on dead bone.
- the bone infection is exogenous osteomyelitis.
- the exogenous osteomyelitis may occur when bone extends out from the skin, allowing a potentially infectious organism to enter from an abscess or bum, a puncture wound, or other trauma such as an open fracture.
- the osteomyelitis comprises an infection of a long bone, such as the femur, tibia, humerus, and radius.
- the osteomyelitis comprises an infection of the vertebral column, in particular the lumbar spine, the sacrum, and the pelvis.
- children develop osteomyelitis in long bone and adults develop osteomyelitis in the vertebral column.
- the osteomyelitis to be prevented or treated is haematogenous osteomyelitis.
- Haematogenous osteomyelitis may be acquired from the spread of organisms from preexisting infections e.g., impetigo, furunculosis (persistent boils), infected lesions of varicella (chickenpox), and sinus, ear, dental, soft tissue, respiratory, and genitourinary infections.
- a genitourinary infection can lead to osteomyelitis of the sacrum or iliac.
- the bone infection to be prevented or treated comprises an implant- associated infection.
- the implant is a mechanical device, such as a metal plate, pin, rod, wire or screw, which may be used, e.g. to stabilize and/or join the ends of fractured bones.
- implant-associated infection becomes chronic wherein only antibiotics are used to treat the infection.
- the lysins of the present methods are used to treat or prevent a joint infection.
- Infected joints may include infected hip, knee, ankle, shoulder, elbow or wrist joints.
- the infected joint is a knee joint or a hip joint.
- the infected joint is a native joint.
- Infection of a native joint (also referred to herein as septic arthritis of a native joint) may occur when a penetrating injury, such as a puncture wound, occurs near or above a joint, allowing bacteria to directly enter the joint.
- the joint infection occurs when bacteria from a distant infection spreads through the bloodstream to the native joint.
- the infected joint is a prosthetic joint, including, for example, septic arthritis of a prosthetic joint.
- the prosthetic joints may include hip, knee, shoulder, elbow, and ankle prostheses.
- the prosthetic joint is a prosthetic hip or knee.
- the prosthetic joint infection occurs within 1 year of surgery.
- Such an infection can be initiated through the introduction of microorganisms at the time of surgery. This can occur through either direct contact or aerosolized contamination of the prosthesis or periprosthetic tissue. Once in contact with the surface of the implant, microorganisms may colonize the surface.
- the prosthetic joint infections occur due to the spread of an infection from an adjacent site.
- a superficial surgical site infection can progress to involve the prosthesis.
- the prosthetic joint infection occurs due to the spread of organisms from a remote site of infection via the bloodstream.
- the prosthetic joint infection is recurring.
- the joint infection is a relapsing multiple drug resistant infection, such as a relapsing multiple drug resistant S. aureus or S. epidermidis prosthetic knee infection (PKI).
- PKI prosthetic knee infection
- a prosthetic joint infection is indicated when a pathogen is isolated by culture from at least two separate tissue or fluid samples obtained from the affected prosthetic joint or when four of the following six criteria exist: elevated serum erythrocyte sedimentation rate (ESR) and serum C-reactive protein (CRP) concentration, elevated synovial leukocyte count, elevated synovial neutrophil percentage (PMN%), presence of purulence in the affected joint, isolation of a microorganism in one culture of periprosthetic tissue or fluid, or greater than five neutrophils per high-power field in five high-power fields observed from histologic analysis of periprosthetic tissue at x400 magnification.
- ESR serum erythrocyte sedimentation rate
- CRP serum C-reactive protein
- PMN synovial neutrophil percentage
- synovial fluid is a viscous fluid found in the cavities of synovial joints. The principal role of synovial fluid is to reduce friction between the articular cartilage of synovial joints during movement.
- a synovial fluid sample can be obtained by aspiration.
- the aspirant may be assessed for total nucleated cell counts and neutrophil percentages as an indicator of prosthetic joint infection.
- the amount of total nucleated cells per microliter and/or the percentage of neutrophils is greater in a synovial fluid obtained from a subject suffering from prosthetic joint infection in comparison to that of a subject who is not suffering from a prosthetic joint infection.
- a threshold of 1,100 total nucleated cells per microliter and/or a threshold of 64% neutrophils in a synovial fluid from a subject with a prosthetic joint indicates a prosthetic joint infection, such as a prosthetic knee joint infection.
- an enzyme present in neutrophils may be assessed using, e.g., colorimetric strips that are widely available for determining pyruia for the diagnosis of urinary tract infection as described in Parvizi et al., “Diagnosis of periprosthetic joint infection: the utility of a simple yet unappreciated enzyme.”, J. Bone Joint Surg. Am., 2011, 93:2242-2248, which is herein incorporated by reference in its entirety.
- synovial fluid sample is cultured to determine whether or not a diagnosis of prosthetic joint infection is indicated and to identify the infecting pathogen(s). This information can also inform the choice of antibiotics if used during treatment.
- aspirated synovial fluid can be either inoculated into blood culture bottles at the time of collection or transported to a microbiology laboratory and inoculated onto solid and/or liquid media. See, e.g., Fehring et al., “Aspiration as a guide to sepsis in revision total hip arthroplasty,” 1996, J. Arthroplasty, 11:543-547, which is herein incorporated by reference in its entirety.
- the present bone and/or joint and/or implant-associated infections are caused by Gram-positive bacteria, such as a Streptococcus species including Streptococcus gallolyticus and Streptococcus pneumonia. More typically, however, the bone and/or joint and/or implant-associated infections is caused by a Staphylococcus species e.g. S. aureus or S. epidermidis. In other embodiments, the Staphylococcus species is a coagulase-negative Staphylococcus species such as Staphylococcus epidermidis, Staphylococcus simulans, Staphylococcus caprae, Staphylococcus lugdunensis or a combination thereof. Typically, Staphylococcus epidermidis is the coagulase-negative Staphylococcus species identified in bone and/or joint and/or implant-associated infections.
- the present bone and/or joint and/or implant-associated infections are caused by Gram-positive bacterial species from the Enterococcus genus or the Listeria genus.
- the present bone and/or joint and/or implant-associated infections are caused by a polymicrobial infection.
- a combination of Enterococcus species and Staphylococcus species may be identified as causative agents of a bone and/or joint and/or implant- associated infections. Examples of causative microorganisms, typically associated with specific infected structures are shown below in Table 1.
- the methods of the present disclosure comprise directly (locally) administering a lysin or active fragment thereof or a variant or derivative thereof as described herein to a bone, typically via local intramedullary administration, i.e., directly inside the bone to treat or prevent bone, joint and/or implant-associated infections.
- the lysin of the present disclosure is directly administered to the medullary cavity, i.e. a cavity of a bone marrow.
- the locally administered lysin or active fragment thereof or a variant or derivative thereof penetrates through bone and into the synovial space.
- the lysin of the present disclosure is directly administered to the implant.
- the present methods for treating and/or preventing bone and/or joint and/or implant- associated infections and/or inhibiting, preventing, disrupting or eradicating biofilms in a subject comprise administering a lysin or active fragment thereof or a variant or derivative thereof as described herein to a subject in need thereof, typically by local administration, typically directly inside of a bone, as described herein, and optionally in combination with one or more antibiotics as also herein described.
- the present lysins or active fragments thereof or variants or derivatives thereof exhibit bactericidal and/or bacteriostatic activity against Grampositive bacteria.
- the present lysins or active fragments thereof or variants or derivatives thereof also exhibit a low propensity for resistance, suppress antibiotic resistance and/or exhibit synergy with conventional antibiotics.
- the present lysins or active fragments thereof or variants or derivatives thereof inhibit bacterial agglutination, biofilm formation and/or reduce or eradicate biofilm, including biofilm in a subject with a bone, joint and/or implant-associated infection.
- the bactericidal activity of the present lysins or active fragments thereof or variants or derivatives thereof may be determined using any method known in the art.
- the present lysins or active fragments thereof or variants or derivatives thereof may be assessed in vitro using time kill assays as described, for example, in Mueller, et al., 2004, Antimicrob Agents Chemotherapy, 48:369-377, which is herein incorporated by reference in its entirety.
- the bacteriostatic activity of the present lysins or active fragments thereof or variants or derivatives thereof may also be assessed using any art-known method.
- growth curves may be performed in e.g., cation adjusted Mueller Hinton II Broth supplemented in human serum (caMHB/50% HuS) to a final concentration of 50% or in 100% serum or in a non-standard medium (caMHB supplemented to 25% with horse serum and 0.5 mM with DTT (caMHB-HSD)).
- the Gram-positive bacteria may be suspended with lysin and culture turbidity can be measured at an optical density at 600 nm using, e.g.
- SPECTRAMAX® M3 Multi-Mode Microplate reader (Molecular Devices) with e.g., readings every 1 minute for 11 hours at 24°C with agitation. Doubling times can be calculated in the logarithmic-phase of cultures grown in flasks with aeration according to the method described in Saito et al, 2014, Antimicrob Agents Chemother 58:5024- 5025, which is herein incorporated by reference in its entirety and compared to the doubling times of cultures in the absence of the present lysins or active fragments thereof or variants or derivatives thereof.
- the present lysins or active fragments thereof or variants or derivatives thereof exhibit lysin activity in the presence of synovial fluid, such as human synovial fluid.
- synovial fluid such as human synovial fluid.
- Suitable methods for assessing the activity of a lysin in synovial fluid are known in the art and described in the examples. Briefly, a MIC value (i.e., the minimum concentration of peptide sufficient to suppress at least 80% of the bacterial growth compared to control) may be determined for a lysin in a synovial fluid and its MIC value compared to, e.g., a parent lysin or the absence of lysin.
- MIC values for a lysin may be determined against e.g., S. epidermidis or S. aureus in e.g., Mueller-Hinton broth (MHB) supplemented with physiological salt concentrations and synovial fluid, such as human synovial fluid.
- Minimum Inhibitory Concentrations (MICs) of a lysin against e.g., S. epidermidis may be determined using broth microdilution (BMD) following Clinical and Laboratory Standards Institute (CLSI) methodology (M07-A11, 2018, which is herein incorporated by reference in its entirety) in a non-standard medium (caMHB supplemented to 50% with human synovial fluid (caMHB-HSF)).
- BMD broth microdilution
- CLSI Clinical and Laboratory Standards Institute
- the present isolated polypeptides comprising lysins, variant lysins, active fragments thereof or derivatives reduce the minimum inhibitory concentration (MIC) of an antibiotic needed to inhibit bacteria in the presence of e.g., human serum or synovial fluid. Any known method to assess this MIC may be used.
- a checkerboard assay is used to determine the effect of a lysin on antibiotic concentration. The checkerboard assay is based on a modification of the CLSI method for MIC determination by broth microdilution (See CLSI. 2015. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard- 10th Edition. Clinical and Laboratory Standards Institute, Wayne, PA, which is herein incorporated by reference in its entirety and Ceri et al. 1999. J. Clin. Microbiol. 37: 1771-1776, which is also herein incorporated by reference in its entirety).
- Checkerboards are constructed by first preparing columns of e.g., a 96-well polypropylene microtiter plate, wherein each well has the same amount of antibiotic diluted 2-fold along the horizontal axis. In a separate plate, comparable rows are prepared in which each well has the same amount of lysin diluted e.g., 2-fold along the vertical axis. The lysin and antibiotic dilutions are then combined, so that each column has a constant amount of antibiotic and doubling dilutions of lysin, while each row has a constant amount of lysin and doubling dilutions of antibiotic. Each well thus has a unique combination of lysin and antibiotic.
- Bacteria are added to the drug combinations at concentrations of 1 x 10 5 CFU/ml in caMHB-HSF, for example.
- the MIC of each drug, alone and in combination, is then recorded after e.g., 16 hours at 37°C in ambient air.
- Summation fractional inhibitory concentrations ( ⁇ FICs) are calculated for each drug and the minimum FIC value ( ⁇ FlCmin) is used to determine the effect of the lysin/antibiotic combination.
- Inhibition of bacterial agglutination may be assessed using any method known in the art.
- the method described in Walker et al. may be used, i.e., Walker et al., 2013, PLoS Pathog, 9:el003819, which is herein incorporated by reference in its entirety.
- Methods for assessing the ability of the lysins or active fragments thereof or variants or derivatives thereof to inhibit or reduce biofilm formation in vitro are well known in the art and include a variation of the broth microdilution minimum Inhibitory Concentration (MIC) method with modifications (See Ceri et al. 1999. J. Clin Microbial. ' T. l r l - l r 16, which is herein incorporated by reference in its entirety and Schuch et al., 2017, Antimicrob. Agents Chemother. 61, pages 1-18, which is herein incorporated by reference in its entirety.) In this method for assessing the Minimal Biofilm Eradicating Concentration (MBEC), fresh colonies of e.g., an S.
- MIC broth microdilution minimum Inhibitory Concentration
- aureus strain or an S. epidermidis strain are suspended in medium, e.g., phosphate buffer solution (PBS) diluted e.g. ,1:100 in TSBg (tryptic soy broth supplemented with 0.2% glucose), added as e.g., 0.15 ml aliquots, to a Calgary Biofilm Device (96-well plate with a lid bearing 96 polycarbonate pegs; Innovotech Inc.) and incubated e.g., 24 hours at 37°C. Biofilms are then washed and treated with e.g., a 2-fold dilution series of the lysin in e.g., TSBg at e.g., 37°C for 24 hours.
- PBS phosphate buffer solution
- TSBg tryptic soy broth supplemented with 0.2% glucose
- the biofilms are destained in e.g., 33% acetic acid and the OD600 of e.g., extracted crystal violet is determined.
- the MBEC of each sample is the minimum lysin concentration required to remove >95% of the biofilm biomass assessed by crystal violet quantitation.
- the present lysins, variant lysins and fragments thereof are assessed against a Gram-positive bacterial lysate obtained from a subject with a bone and/or joint and/or implant-associated infection as described herein.
- Methods for obtaining such isolates are well known in the art and described, for example, in Schmidt-Malan et al., Diag. Microbiol. Infect. Dis. 85:77-79, which is herein incorporated by reference in its entirety.
- Suitable lysins for use with the present method include the PlySs2 lysins as described in WO 2013/170015 and WO 2013/170022, each of which is herein incorporated by reference in its entirety.
- the terms “PlySs2 lysin”,“PlySs2 lysins”, “PlySs2” “Exebacase” and “CF-301” are used interchangeably and encompass the PlySs2 lysin set forth herein as SEQ ID NO: 1 (with or without initial methionine residue) or an active fragment thereof or variants or derivatives thereof as described in WO 2013/170015 and WO 2013/170022.
- PlySs2 which was identified as an anti- staphylococcal lysin encoded within a prophage of the Streptococcus suis genome, exhibits bactericidal and bacteriostatic activity against the bacteria described below in Table 2.
- a lysin suitable for use with the present method is the PlySs2 lysin of SEQ ID NO: 1.
- the PlySs2 lysin of SEQ ID NO: 1 has a domain arrangement characteristic of most bacteriophage lysins, defined by a catalytic N-terminal domain (FIG. 1) linked to a cell wallbinding C-terminal domain (FIG. 1).
- the N-terminal domain belongs to the cysteine-histidine- dependent amidohydrolases/peptidases (CHAP) family common among lysins and other bacterial cell wall-modifying enzymes.
- the C-terminal domain belongs to the SH3b family that often forms the cell wall-binding element of lysins.
- FIG. 1 depicts the PlySs2 lysin of SEQ ID NO: 1 with the N- and C-terminal domains shown as shaded regions.
- the N-terminal CHAP domain corresponds to the first shaded amino acid sequence region starting with LNN and the C-terminal SH3b domain corresponds to the second shaded region starting with RSY.
- a lysin suitable for use with the methods disclosed herein comprises one or more of the following lysins: pp55 (SEQ ID NO: 3), pp61 (SEQ ID NO: 4), pp65 (SEQ ID NO: 5), pp296 (SEQ ID NO: 6 also referred to herein as CF-296), pp324 (SEQ ID NO: 7), pp325 (SEQ ID NO: 8), pp338 (SEQ ID NO: 9), pp341 (SEQ ID NO: 10), pp388 (SEQ ID NO: 11), pp400 (SEQ ID NO: 12), pp616 (SEQ ID NO: 13), pp619 (SEQ ID NO: 14), pp628 (SEQ ID NO: 15), pp632 (SEQ ID NO: 16), and pp642 (SEQ ID NO: 17).
- the present methods comprise the administration of a variant lysin to a subject in need thereof.
- Suitable lysin variants for use with the present method include those polypeptides having at least one substitution, insertion and/or deletion in reference to SEQ ID NO: 1 that retain at least one biological function of the reference lysin.
- the variant lysins exhibit antibacterial activity including a bacteriolytic and/or bacteriostatic effect against a broad range of Gram-positive bacteria, including S. aureus and S. epidermidis and an ability to inhibit agglutination, inhibit biofilm formation and/or reduce biofilm.
- the present lysin variants render Gram-positive bacteria more susceptible to antibiotics.
- a lysin variant suitable for use with the present methods includes an isolated polypeptide sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98%, such as at least 99% or such as at least 99.5% sequence identity with SEQ ID NO: 1, wherein the variant lysin retains one or more biological activities of the PlySs21ysin having the amino acid sequence of SEQ ID NO: 1 as described herein.
- Lysin variants may be formed by any method known in the art and as described in WO 2013/170015, which is herein incorporated by reference in its entirety, e.g., by modifying the PlySs2 lysin of SEQ ID NO: 1 through site-directed mutagenesis or via mutations in hosts that produce the PlySs2 lysin of SEQ ID NO: 1, and which retain one or more of the biological functions as described herein.
- substitutions or replacements to, e.g., the CHAP domain and/or the SH3b domain of the PlySs2 lysin of SEQ ID NO: 1.
- Sequence comparisons to the Genbank database can be made with either or both of the CHAP and/or SH3b domain sequences or with the PlySs2 lysin full amino acid sequence of SEQ ID NO: 1, for instance, to identify amino acids for substitution.
- a mutant or variant having an alanine replaced for valine at valine amino acid residue 19 in the PlySs2 amino acid sequence of SEQ ID NO: 1 is active and capable of killing Gram-positive bacteria in a manner similar to and as effective as the SEQ ID NO: 1 PlySs2 lysin.
- the CHAP domain contains conserved cysteine and histidine amino acid sequences (the first cysteine and histidine in the CHAP domain) which are characteristic and conserved in CHAP domains of different polypeptides. It is reasonable to predict, for example, that the conserved cysteine and histidine residues should be maintained in a mutant or variant of PlySs2 so as to maintain activity or capability. Accordingly, particularly desirable residues to retain in a lysin variant of the present disclosure include active-site residues Cys26, Hisio2, duns, and Asnno in the CHAP domain of SEQ ID NO: 1.
- Particularly desirable substitutions include: Lys for Arg and vice versa such that a positive charge may be maintained, Glu for Asp and vice versa such that a negative charge may be maintained, Ser for Thr such that a free -OH can be maintained and Gin for Asn such that a free NH2 can be maintained.
- Suitable variant lysins are described in PCT Published Application No. WO 2019/165454 (International Application No.: PCT/US2019/019638), which is herein incorporated by reference in its entirety.
- suitable variant lysins include those set forth herein as SEQ ID NOS: 3-17 as well as variant lysins having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98%, such as at least 99%, such as at least 99.5% sequence identity with any one of SEQ ID NOS: 3-17 (with or without the initial methionine), wherein the variant lysin retains one or more biological activities of the PlySs2 lysin having the amino acid sequence of SEQ ID NO: 1 as described herein.
- a particularly preferred PlySs2 lysin variant is SEQ ID NO: 6.
- SEQ ID NOs: 3-17 are modified lysin polypeptides having at least one amino acid substitution relative to a counterpart wild-type PlySs2 lysin SEQ ID NO: 1, while preserving antibacterial activity and effectiveness.
- SEQ ID NOs: 3-17 may be described by reference to their amino acid substitutions with respect to SEQ ID NO: 1, as shown below in Table A.
- the amino acid sequences of the modified lysin polypeptides (referencing differences from SEQ ID NO: 1 and the positions of its amino acid residues) are summarized using one-letter amino acid codes as follows: Table A
- the present method includes administering an active fragment of a lysin to a subject in need thereof.
- Suitable active fragments include those that retain a biologically active portion of a protein or peptide fragment of the lysin embodiments, as described herein.
- Such variants include polypeptides comprising amino acid sequences that include fewer amino acids than the full-length protein of the lysin protein and exhibit at least one activity of the corresponding full-length protein.
- biologically active portions comprise a domain or motif with at least one activity of the corresponding protein.
- An exemplary domain sequence for the N-terminal CHAP domain of the PlySs2 lysin is provided in FIG. 1.
- a biologically active portion of a protein or protein fragment of the disclosure can be a polypeptide which is, for example, 10, 25, 50, 100 amino acids in length.
- Other biologically active portions, in which other regions of the protein are deleted can be prepared by recombinant techniques and evaluated for one or more of the functional activities of the native form of a polypeptide of the embodiments.
- suitable active fragments include those having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98%, such as at least 99% or such as at least 99.5% sequence identity with the active fragments described herein, wherein the active fragment thereof retains at least one activity of a CHAP and/or the SH3b domain, e.g., as shown in FIG. 1.
- a lysin or active fragment thereof or variant or derivative thereof as described herein for use in the present method may be produced by a bacterial organism after being infected with a particular bacteriophage or may be produced or prepared recombinantly or synthetically.
- the present lysins may be produced via the isolated gene for the lysin from the phage genome, putting the gene into a transfer vector, and cloning said transfer vector into an expression system, using standard methods of the art, as described for example in WO 2013/170015, which is herein incorporated by reference in its entirety.
- present lysin variants may be truncated, chimeric, shuffled or “natural,” and may be in combination as described, for example, in International Patent Publication No. WO 2013/17002, which is incorporated herein in its entirety by reference.
- Mutations can be made in the amino acid sequences, or in nucleic acid sequences encoding the polypeptides and lysins described herein, including in the lysin sequence set forth in SEQ ID NO: 1, or in active fragments or truncations thereof, such that a particular codon is changed to a codon which codes for a different amino acid, an amino acid is substituted for another amino acid, or one or more amino acids are deleted.
- Such a mutation is generally made by making the fewest nucleotide changes possible.
- a substitution mutation of this sort can be made to change an amino acid in the resulting protein in a non-conservative manner (for example, by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to another grouping) or in a conservative manner (for example, by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to the same grouping).
- Such a conservative change generally leads to less change in the structure and function of the resulting protein.
- a non-conservative change is more likely to alter the structure, activity or function of the resulting protein.
- the present disclosure should be considered to include sequences containing conservative changes which do not significantly alter the activity or binding characteristics of the resulting protein.
- amino acid changes or substitutions in the lysin polypeptide sequence can be made to replace or substitute one or more, one or a few, one or several, one to five, one to ten, or such other number of amino acids in the sequence of the lysin(s) provided herein to generate mutants or variants thereof.
- mutants or variants thereof may be predicted for function or tested for function or capability for anti-bacterial activity as described herein against, e.g., Staphylococcal, Streptococcal, or Enterococcal bacteria, and/or for having comparable activity to the lysin(s) as described and particularly provided herein.
- changes made to the sequence of lysin, and mutants or variants described herein can be tested using the assays and methods known in the art and described herein.
- One of skill in the art on the basis of the domain structure of the lysin(s) hereof can predict one or more, one or several amino acids suitable for substitution or replacement and/or one or more amino acids which are not suitable for substitution or replacement, including reasonable conservative or non-conservative substitutions.
- the methods of treating or preventing bone, joint and/or implant - associated infections as described herein comprise co-administering a therapeutically effect amount of one or more antibiotics and a PlySs2 lysin.
- co-administration of a lysin or active fragment thereof or variant or derivative thereof and one or more antibiotic as described herein results in a synergistic bactericidal and/or bacteriostatic effect on Gram-positive bacteria such as S. aureus or S. epidermidis.
- the co-administration is used to suppress virulence phenotypes including biofilm formation and/or agglutination.
- the co-administration is used to reduce an amount of biofilm in a subject.
- the present lysins or active fragments thereof or variants or derivatives thereof are administered locally as described herein and the antibiotics are administered systemically, such as orally, more typically intravenously.
- antibiotics for use with the present methods include antibiotics that are capable of penetrating bone.
- antibiotics include those of different types and classes, such as betalactams penicillins (e.g. methicillin, oxacillin, piperacillin, tazobactam, flucloxacillin, cioxacillin), cephalosporins (e.g. cefalexin and cefactor), carbapenems (e.g. imipenem and entapenem); macrolides (e.g. erythromycin, azithromycin), aminoglycosides (e.g.
- glycopeptides e.g., vancomycin, teicoplanin, dalbavancin, oritavancin
- oxazolidinones e.g linezolid and tedizolid
- lipopeptides e.g. daptomycin
- sulfonamides e.g.
- sulfamethoxazole tetracyclines (e.g., doxycycline), lincomycins (e.g., clindamycin), fluoroquinolones (e.g., levofloxacin), phosphonic antibiotics (e.g., fosfomycin) and/or a rifamycin antibiotic, such as rifampin or rifabutin.
- suitable antibiotics include particular antibiotic combinations such as piperacillin/tazobactam, trimethoprim/sulfamethoxazole, and fosfomycin.
- the antibiotics are administered systemically as described herein for at least two weeks, such as at least four weeks, such as at least six weeks, e.g., 6-24 weeks.
- the antibiotic is an antibiotic typically used to treat osteomyelitis, such as acute osteomyelitis, such as vancomycin or daptomycin, typically daptomycin.
- the present methods exclude antibiotics that poorly penetrate bone, such antibiotics are known in the art and include, e.g. penicillin and metronidazole.
- the present disclosure is directed to a method of preventing or treating a bone, joint and/or implant-associated infection due to Gram-positive bacteria as described herein, which method comprises: locally administering, as described herein, a therapeutically effective amount of a PlySs2 lysin, active fragment thereof or variant or derivative thereof as described herein to a subject in need thereof in conjunction with Debridement and Implant Retention (DAIR).
- DAIR Debridement and Implant Retention
- an antibiotic as described herein is co-administered with the PlySs2 lysin.
- debridement of infected and potentially infected tissues around e.g., an implant is typically performed followed by arthroscopic irrigation of involved tissues with copious volumes of fluid, such as sterile saline.
- a PlySs2 lysin, active fragment thereof or variant or derivative thereof as described herein is administered locally to the bone, typically inside of the bone as described herein, by drilling a hole in the bone as described in the examples, before, during or after arthroscopic irrigation.
- bonepenetrating antibiotics as described herein such as fosfomycin, vancomycin or daptomycin, are subsequently systemically administered, e.g., orally or intravenously administered, to the subject for e.g., at least two weeks, such as 6-24 weeks.
- the antibiotic is administered intravenously.
- the subject to be administered a lysin of the disclosure is elderly or suffers from a condition associated with a higher risk of a bone and/or joint and/or implant- associated infection.
- the subject at risk for a bone, joint and/or implant-associated infection may suffer from obesity, e.g., a body mass index (BMI) threshold of 35.
- BMI body mass index
- An elderly subject for example, is at least 65 years, such as 65-90 years, 75-90 years, or 79-89 years.
- possible reasons for the increased risk of bone, joint and/or implant- associated infections, such as prosthetic bone or joint infections, with obesity include prolonged operative duration and/or the presence of other comorbidities.
- the subject at risk for a bone, joint and/or implant-associated infection, particularly a prosthetic joint infection suffers from diabetes mellitus.
- the risk associated with diabetes may be due to increased biofilm formation in the presence of elevated levels of glucose, impaired leukocyte function, or microvascular changes in subjects with diabetes mellitus, which may influence wound healing and the development of superficial surgical site infections.
- Other risk factors for bone and/or joint and/or implant-associated infections include rheumatoid arthritis, male gender and smoking.
- a diagnosis of bacteremia in the year preceding an implant surgery is also a risk factor for a bone and/or joint infection, such as a prosthetic joint infection.
- Dosages of the present lysins or active fragments thereof or variants or derivatives thereof that are administered to a subject in need thereof depend on a number of factors including the activity of infection being treated, the age, health and general physical condition of the subject to be treated, the mode of administration, the activity of a particular lysin or active fragment thereof or variant or derivative thereof, the nature and activity of the antibiotic, if any, with which a lysin or active fragment thereof or variant or derivative thereof according to the present disclosure is being paired and the combined effect of such pairing.
- lysins or active fragments thereof or variants or derivatives thereof to be administered systemically are anticipated to fall within the range of the range of 0.00001-200 mg/kg, such as 0.2 mg/kg to about 0.3 mg/kg, such as 0.25 mg/kg, such as, 1-150 mg/kg, such as 40 mg/kg to 100 mg/kg and are administered 1-4 times daily for a period up to 14 days.
- the antibiotic may be administered at standard dosing regimens or in lower amounts in view of e.g., synergy. All such dosages and regimens however (whether of the lysin or active fragment thereof or variant or derivative thereof or any antibiotic administered in conjunction therewith) are subject to optimization.
- Optimal dosages can be determined by performing in vitro and in vivo pilot efficacy experiments as is within the skill of the art but taking the present disclosure into account.
- the present lysins or active fragments thereof or variants or derivatives thereof are administered locally, e.g., directly into bone.
- effective amounts of the present lysins or active fragments thereof or variants or derivatives thereof to be administered for local administration, such as into bone are anticipated to fall within the range of 0.00001-200 mg/mL.
- 1 mg/mL to about 100 mg/mL, such as 75 mg/mL, such as, 1-150 mg/mL, such as 40 mg/mL to 100 mg/mL may be administered.
- the amount is 10-15 mg/ml, e.g., 10-11 mg/ml, such as 10 mg/ml.
- the lysins or active fragments thereof or variants or derivatives thereof and the one or more antibiotics of the present disclosure may be administered by the same mode of administration or by different modes of administration, and may be administered once, twice or multiple times, one or more in combination or individually.
- the present lysins or active fragments thereof or variants or derivatives thereof may be administered in an initial dose followed by a subsequent dose or doses, particularly depending on the response, e.g., the bactericidal and/or bacteriostatic effects and/or the effect on agglutination and/or biofilm formation or reduction and may be combined or alternated with antibiotic dose(s).
- the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs.
- the animal model can also be used to achieve a desirable concentration range and route of administration. Obtained information can then be used to determine the effective doses, as well as routes of administration in humans.
- direct, local administration is local administration inside of bone.
- the present lysins or active fragments thereof or variants or derivatives thereof and one or more antibiotics as described herein, such as daptomycin are administered simultaneously.
- the present lysins or active fragments thereof or variants or derivatives thereof and the one or more antibiotics of the present method, such as daptomycin are administered in series, such as sequentially, in any order.
- the lysin is administered during or subsequent to administration of a standard of care antibiotic treatment, e.g., a two-week course of oxacillin and gentamicin or daptomycin.
- the lysin or active fragment thereof or variant or derivative thereof of the present disclosure is administered locally, e.g., inside the bone of a subject followed by a conventional, systemic regimen, e.g., standard of care (SOC) dosages, of one or more antibiotics of the present disclosure, such as daptomycin.
- SOC standard of care
- one or more antibiotics of the present disclosure, such as daptomycin is systemically administered to a subject followed by direct administration, typically inside of bone, of a lysin or active fragment thereof or variant or derivative thereof of the present disclosure, followed by additional dosages of the one or more antibiotics of the present disclosure at conventional systemic dosages, such as daptomycin.
- the lysins or active fragments thereof or variants or derivatives thereof or the one or more antibiotics may be administered at sub-MIC levels, e.g., at sub-MIC levels ranging from 0.9X MIC to 0.0001X MIC.
- the present lysins or active fragments thereof or variants or derivatives thereof, alone or in combination with sub-MIC levels of antibiotics are typically used to inhibit the growth of Gram-positive bacteria, reduce agglutination, and/or inhibit biofilm formation or to reduce or eradicate biofilm.
- the present lysin or active fragment thereof or variant or derivative thereof or the one or more antibiotics of the present disclosure are administered to a subject in need thereof at the MIC level or greater than the MIC level, such as IX MIC, 2X MIC, 3X MIC, 4X MIC, 10MIC or 20MIC.
- the combination of the present lysin or active fragment thereof or variant or derivative thereof and the one or more antibiotics of the present disclosure exhibit a synergistic killing or synergistic bacteriostatic effect on Gram-positive lysins.
- the present lysin or active fragment thereof or variant or derivative thereof are administered locally inside of a bone at concentrations greater than the MIC value and the antibiotics are administered systemically at dosages below conventional dosages.
- daptomycin may be administered at dosages of less than the conventional human dose of 6 mg/kg, such as 3 mg/kg.
- the lysin or active fragment thereof or variant or derivatives thereof of the present disclosure, optionally administered either alone or in combination or in series, with the one or more antibiotics described herein may each be included in a single pharmaceutical formulation or be separately formulated in the form of a solution, a suspension, an emulsion, an inhalable powder, an aerosol, or a spray, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, tampon applications emulsions, aerosols, sprays, suspensions, lozenges, troches, candies, injectants, chewing gums, ointments, smears, time-release patches, liquid absorbed wipes, and combinations thereof.
- administration of the pharmaceutical formulations may include systemic administration.
- Systemic administration can be enteral or oral, i.e., a substance is given via the digestive tract, parenteral, i.e., a substance is given by other routes than the digestive tract such as by injection or inhalation.
- the lysins or active fragments thereof or variants or derivatives thereof and optionally the one or more antibiotics of the present disclosure can be administered to a subject orally, parenterally, by inhalation, topically, rectally, nasally, buccally or via an implanted reservoir or by any other known method.
- the lysins or active fragments thereof or variants or derivatives thereof and/or the one or more antibiotics of the present disclosure can also be administered by means of sustained release dosage forms.
- the lysins or active fragments thereof or variants or derivatives thereof and optionally, the one or more antibiotics of the present disclosure can be formulated into solid or liquid preparations, for example tablets, capsules, powders, solutions, suspensions and dispersions.
- the lysins or active fragments thereof or variants or derivatives thereof and/or the one or more antibiotics of the present disclosure can be formulated with excipients such as, e.g., lactose, sucrose, com starch, gelatin, potato starch, alginic acid and/or magnesium stearate.
- the lysins or active fragments thereof or variants or derivatives thereof and/or the one or more antibiotics of the present disclosure is mixed with a pharmaceutical excipient to form a solid pre-formulation composition.
- tablets may be sugar coated or enteric coated by standard techniques.
- the tablets or pills may be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action.
- the tablet or pill can include an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former.
- the two dosage components can be separated by an enteric layer, which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release.
- enteric layers or coatings such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.
- the pharmaceutical formulations of the present disclosure are formulated as inhalable compositions.
- the present pharmaceutical formulations are advantageously formulated as a dry, inhalable powder.
- the present pharmaceutical formulations may further be formulated with a propellant for aerosol delivery.
- suitable propellants include, but are not limited to: dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane and carbon dioxide.
- the formulations may be nebulized.
- the inhalable pharmaceutical formulations include excipients.
- suitable excipients include, but are not limited to: lactose, starch, propylene glycol diesters of medium chain fatty acids; triglyceride esters of medium chain fatty acids, short chains, or long chains, or any combination thereof; perfluorodimethylcyclobutane; perfluorocyclobutane; polyethylene glycol; menthol; lauroglycol; diethylene glycol monoethylether; polyglycolized glycerides of medium chain fatty acids; alcohols; eucalyptus oil; short chain fatty acids; and combinations thereof.
- a surfactant can be added to an inhalable pharmaceutical formulation of the present disclosure in order to lower the surface and interfacial tension between the medicaments and the propellant.
- the surfactant may be any suitable, non-toxic compound which is non-reactive with the present polypeptides.
- surfactants include, but are not limited to: oleic acid; sorbitan trioleate; cetyl pyridinium chloride; soya lecithin; polyoxyethylene(20) sorbitan monolaurate; polyoxyethylene (10) stearyl ether; polyoxyethylene (2) oleyl ether; poly oxypropylene-poly oxy ethylene ethylene diamine block copolymers; poly oxy ethylene(20) sorbitan monostearate; polyoxyethylene(20) sorbitan monooleate; polyoxypropylenepolyoxyethylene block copolymers; castor oil ethoxylate; and combinations thereof.
- the pharmaceutical formulations of the present disclosure comprise nasal formulations.
- Nasal formulations include, for instance, nasal sprays, nasal drops, nasal ointments, nasal washes, nasal injections, nasal packings, bronchial sprays and inhalers, or indirectly through use of throat lozenges, mouthwashes or gargles, or through the use of ointments applied to the nasal nares, or the face or any combination of these and similar methods of application.
- the pharmaceutical formulations of the present disclosure are more typically administered locally, such as directly inside of a bone as described herein.
- the pharmaceutical formulations comprise a therapeutically effective amount of a PlySs2 lysin active fragments thereof or variants or derivatives thereof formulated for local administration, such as to a bone, comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof having at least 80% identity to SEQ ID NO: 1, wherein the PlySs2 variant comprises bactericidal and/or bacteriostatic activity against Gram-positive bacteria and wherein the formulation for local administration optionally comprises intramedullary local administration.
- the pharmaceutical formulations may include a pharmaceutically acceptable carrier such as distilled water, a saline solution, albumin, a serum, or any combinations thereof.
- a pharmaceutically acceptable carrier such as distilled water, a saline solution, albumin, a serum, or any combinations thereof.
- lysin-containing pharmaceutical formulations are co-administered with conventional antibiotics, more typically conventional antibiotics that are known to penetrate bone well as described herein.
- pharmaceutical formulations comprising conventional antibiotics are administered systemically as described herein.
- the antibiotic pharmaceutical formulations do not include penicillin and/or metronidazole.
- the pharmaceutical formulations of the present disclosure may be presented in unit dosage form and may be prepared by any methods well known in the art.
- the amount of active ingredients which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the duration of exposure of the recipient to the infectious bacteria, the size and weight of the subject, and the particular mode of administration.
- the amount of active ingredients that can be combined with a carrier material to produce a single dosage form will generally be that amount of each compound which produces a therapeutic effect. Generally, out of one hundred percent, the total amount will range from about 1 percent to about ninety-nine percent of active ingredients, typically from about 5 percent to about 70 percent, most typically from about 10 percent to about 30 percent.
- the experimental surgical model (FIG. 4) was developed and performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Mayo Clinic as well as the Animal Care and Use Review Office of the United States Army Medical Research and Development Command Office of Research Protections. In conducting research using animals, the investigators adhered to the laws of the United States and regulations of the Department of Agriculture.
- lysin or lysin carrier (vehicle) was injected in the same fashion, inserting the feeding tube to the hub, towards the distal tibia, and slowly drawing the feeding tube out of the bone as the lysin was injected (FIG. 4e).
- an implant was placed into the hole with screw forceps and secured with a screwdriver (FIGS. 4f, 4g and 4h).
- systemic treatment of either daptomycin (6 mg/kg) or saline for injection was delivered intravenously (FIG. 5).
- the muscle, fascia, and skin were closed in three separate layers using 3-0 coated VICRYL® sutures (Ethicon Inc, Sommerville, NJ) to secure tissues over the screw head.
- daptomycin (6 mg/kg) or saline for injection was administered daily for the next three days via an intravenous catheter placed in the marginal ear vein.
- a rabbit dose of 12 mg/kg of daptomycin results in a similar area under the plasma concentration curve to human dosing of 6 mg/kg. See, for example, Chambers el al., “Relationship between susceptibility to daptomycin in vitro and activity in vivo in a rabbit model of aortic valve endocarditis,” 2009, Antimicrob. Agents Chemother., 43, 1463-1467.
- the daptomycin dose of 6 mg/kg was selected to allow for study of combination activity.
- Catheters were flushed with heparinized saline twice daily and after treatment.
- rabbits were euthanized, and tibiae and implants aseptically collected. Specifically, an incision was made over the tibia from the knee to the ankle. The ankle was manually separated from the distal tibia. The muscle was dissected away from the implant and the implant removed and placed into a sterile culture tube. The tibia was fully separated from the knee joint with a bone cutting forceps, crushed into small fragments with a sterile bone cutting forceps, placed into a sterile culture tube, and frozen at -80°C.
- Implants were vortexed 30 seconds in 1 mL saline, sonicated (40 kHz, 0.22 w/cm 2 ) for 5 minutes and vortexed for an additional 30 seconds.
- the resultant sonicate fluid was titrated in 1:10 dilutions and 0.1 mL of each dilution plated on blood agar plates, which were incubated 24 hours in 5% CO2 at 37°C following which recovered colonies were counted and reported as log 10 cfu/implant. If no colonies were recovered, sonicate fluid was incubated with tryptic soy broth for any additional 24 hours. If broth culture was positive, confirmed by plating broth onto blood agar, quantities were reported as 0.5 logio cfu/implant; if negative, quantities were reported as 0.1 logio cfu/implant, the limit of detection of the method.
- a novel model of acute MRSA implant-associated osteomyelitis was used to test local delivery of antistaphylococcal lysin in additional to systemic delivery of daptomycin. All treatment groups had significantly reduced amounts of MRSA recovered from both bone and implants, with the most active treatments being locally delivered exebacase alone and systemic daptomycin with either of the lysins delivered locally. There was no difference between the activity of the two lysins delivered locally when administered with systemic daptomycin. Lysins, administered locally in addition to traditional therapies, may offer a potential strategy for combating Staphylococcus aureus implant-associated infections.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Epidemiology (AREA)
- Immunology (AREA)
- Gastroenterology & Hepatology (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- General Engineering & Computer Science (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Oncology (AREA)
- Communicable Diseases (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The present disclosure is directed to a method of treating or preventing a bone and/or joint and/or implant-associated infection comprising a Gram-positive bacteria, which method comprises: locally administering to a subject in need thereof, such as a bone of a subject, a therapeutically effective amount of a PlySs2 lysin or a variant thereof. Compositions comprising the PlySs2 lysin or variant thereof formulated for local administration are also provided.
Description
METHOD OF TREATING AND PREVENTING BONE AND JOINT INFECTIONS
CROSS-REFERENCE TO RELATED APPLICATIONS
[1] This application claims the benefit of, and relies on the filing date of, U.S. provisional patent application number 63/275,750, filed 4 November 2022, the entire disclosure of which is herein incorporated by reference in its entirety.
SEQUENCE LISTING
[2] The instant application contains a Sequence Listing which has been submitted electronically in ASCI format and is hereby incorporated by reference in its entirety. Said XML copy, created on 31 October 2022, is named 0341-0033-PCT October 31, 2022 and is 27 kilobytes in size.
FIELD OF THE DISCLOSURE
[3] The present disclosure relates generally to the treatment and prevention of bone and/or joint and/or implant-associated infections, particularly osteomyelitis and prosthetic joint infections due to Gram-positive bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), using lysin(s) and optionally one or more antibiotics by locally delivering the lysins.
BACKGROUND
[4] Bone and joint infections including osteomyelitis and implant-associated orthopedic infections are complex and difficult to treat. For example, Staphylococcus aureus, the most common bacterium recovered in orthopedic infection, forms biofilms, which are protective extracellular polymeric matrices embedding clusters of sessile bacteria on implants as well as in/on bone. In addition, Staphylococcus aureus can exist as persister cells and small colony variants (SCVs) and enter and survive intracellularly in cells such as fibroblasts and osteoblasts. These factors render it difficult for the immune system and antimicrobial agents to come into contact with bacteria; it can also be challenging for systemically delivered therapeutics to reach communities of bacteria in bone and on implants. Consequently, bone and joint infections, including implant-associated orthopedic infection, often require surgical intervention. Depending
on the clinical presentation, duration of infection (acute versus chronic) and patient’s health, debridement, irrigation and device retention may be considered, or implant removal and subsequent reimplantation may be needed, accompanied by a prolonged duration of systemic antibiotic therapy. In some cases, surgery or device removal is not an option, with treatment based solely on antimicrobial therapy, outcomes of such an approach are generally poor. Systemic antibiotics may be associated with toxicity, microbiome disturbances, selection of resistance in target or commensal microbiota, and/or interactions with other medications. Further, many patients do not complete their prescribed course of antibiotics, which can lead to recurrent infection and antimicrobial resistance.
[5] As a result of emerging antibiotic resistance and recognition of the poor anti-biofilm activity of many available antibiotics, there is renewed interest in new medical modalities for treating bone and joint infections, including the use of lysins as therapeutic agents. Lysins are cell wall hydrolytic enzymes, which are produced from bacteriophage, viruses that infect and kill bacteria. When phages replicate, lysins accumulate in the bacterial cytoplasm before translocating to the cell wall, hydrolyzing peptidoglycan, and rapidly killing bacteria. Lysins can be recombinantly produced and purified and can be administered to attack cell walls from the outside of cells, thus providing a potentially rapid bactericidal treatment option.
[6] Currently there are no FDA approved antimicrobials for the treatment of orthopedic implants or osteomyelitis. Thus, there is an urgent, unmet need for improved methods using antimicrobials, such as lysins, which are fast acting and target biofilms to help manage these devastating infections.
SUMMARY
[7] In one aspect, the present disclosure is directed to a method of treating or preventing a bone and/or joint and/or implant-associated infection comprising a Gram-positive bacteria, which method comprises: locally administering to a subject in need thereof, such as locally administering to a bone of a subject, a therapeutically effective amount of a PlySs2 lysin comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof having at least 80% identity to SEQ ID NO: 1, wherein the variant comprises bactericidal and/or bacteriostatic activity against the Grampositive bacteria. In one embodiment of this aspect, the local administration comprises intramedullary local administration.
[8] The present disclosure is also directed to a composition comprising a therapeutically effective amount of a PlySs2 lysin formulated for local administration to a subject with a bone or joint or implant-associated infection comprising Gram-positive bacteria, such as local administration to a bone of a subject with a bone or joint infection, comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof having at least 80% identity to SEQ ID NO: 1, optionally with one or more antibiotic(s), wherein the PlySs2 variant comprises bactericidal and/or bacteriostatic activity against Gram-positive bacteria. In one embodiment of this aspect, the local administration comprises intramedullary local administration.
BRIEF DESCRIPTION OF THE DRAWINGS
[9] FIG. 1 depicts the amino acid sequence of a lysin (SEQ ID NO: 1) and a polynucleotide (SEQ ID NO: 18) encoding the lysin as described in the detailed description. SEQ ID NO: 1 represents a 245 amino acid polypeptide, including the initial methionine residue which is removed during post-translational processing, leaving a 244-amino acid polypeptide.
[10] FIG. 2 depicts the rapid in vitro bactericidal activity of the lysins, exebacase and CF-296, on infected implants, as described in the Examples.
[11] FIG. 3 depicts a rabbit foreign body osteomyelitis screw model as described in the Examples. The left panel depicts a scanning electron microscope image showing bacteria colonizing the implant (screw) surface. The right panel depicts an X-ray of the medial (left) and superior (right) view of the left leg of a supine rabbit.
[12] FIGS. 4a-4j depict the surgical procedure used to insert an infected implant into bone and to locally treat the infection as described in the Examples. FIG 4a depicts the exposure of a smooth flat portion of the tibia. FIGS. 4b and 4c depict the creation of a hole through the cortical bone into the medullary cavity using a micro drill. FIGS. 4d and 4e depict the hole after or during injection with water (FIG. 4d) or lysin or lysin carrier (FIG. 4e). FIG. 4f depicts insertion of an infected implant into the hole. FIG. 4g depicts the use of a screwdriver to tighten a screw used to secure the infected implant. FIG. 4h depicts confirmation that the implant is secure. FIGS. 4i and 4j depict the post-surgical view, above (FIG. 4i) and parallel to (FIG. 4j), the medial tibia of a supine rabbit.
[13] FIG. 5 depicts the treatments used to treat the infected bone and implant as described in the Examples.
[14] FIG. 6 depicts the amount of bacteria recovered from bone and the screw implant 5 days after lysin treatment as described in the Examples.
DETAILED DESCRIPTION
Definitions
[15] As used herein, the following terms and cognates thereof shall have the following meanings unless the context clearly indicates otherwise:
[16] “Carrier” refers to a solvent, additive, excipient, dispersion medium, solubilizing agent, coating, preservative, isotonic and absorption delaying agent, surfactant, propellant, diluent, vehicle and the like with which an active compound is administered. Such carriers can be sterile liquids, such as water, saline solutions, aqueous dextrose solutions, aqueous glycerol solutions, and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
[17] “Pharmaceutically acceptable carrier” refers to any and all solvents, additives, excipients, dispersion media, solubilizing agents, coatings, preservatives, isotonic and absorption delaying agents, surfactants, propellants, diluents, vehicles and the like that are physiologically compatible. The carrier(s) must be “acceptable” in the sense of not being deleterious to the subject to be treated in amounts typically used in medicaments. Pharmaceutically acceptable carriers are compatible with the other ingredients of the composition without rendering the composition unsuitable for its intended purpose. Furthermore, pharmaceutically acceptable carriers are suitable for use with subjects as provided herein without undue adverse side effects (such as toxicity, irritation, and allergic response). Side effects are “undue” when their risk outweighs the benefit provided by the composition. Non-limiting examples of pharmaceutically acceptable carriers or excipients include any of the standard pharmaceutical carriers such as phosphate buffered saline solutions, water, and emulsions such as oil/water emulsions and microemulsions. Suitable pharmaceutical carriers are described, for example, in “Remington's Pharmaceutical Sciences” by E.W. Martin, 18th Edition. The pharmaceutically acceptable carrier may be a carrier that does not exist in nature.
[18] “Bactericidal” or “bactericidal activity” refers to the property of causing the death of bacteria or capable of killing bacteria to an extent of at least a 3-loglO (99.9%) or a better reduction among an initial population of bacteria over an 18-24 hour period.
[19] “Bacteriostatic” or “bacteriostatic activity” refers to the property of inhibiting bacterial growth, including inhibiting growing bacterial cells, thus causing a 2-loglO (99%) or better and up to just under a 3-log reduction among an initial population of bacteria over an 18-24 hour period.
[20] “Antibacterial” refers to both bacteriostatic and bactericidal agents.
[21] “Antibiotic” refers to a compound having properties that have a negative effect on bacteria, such as lethality or reduction of growth. An antibiotic can have a negative effect on Gram-positive bacteria, Gram-negative bacteria, or both. By way of example, an antibiotic can affect cell wall peptidoglycan biosynthesis, cell membrane integrity or DNA or protein synthesis in bacteria.
[22] “Drug resistant” refers generally to a bacterium that is resistant to the antibacterial activity of a drug. When used in certain ways, drug resistance may specifically refer to antibiotic resistance. In some cases, a bacterium that is generally susceptible to a particular antibiotic can develop resistance to the antibiotic, thereby becoming a drug resistant microbe or strain. A “multidrug resistant” (“MDR”) pathogen is one that has developed resistance to at least two classes of antimicrobial drugs, each used as monotherapy. For example, certain strains of S. aureus have been found to be resistant to several antibiotics including methicillin and/or vancomycin (Antibiotic Resistant Threats in the United States, 2013, U.S. Department of Health and Services, Centers for Disease Control and Prevention). One skilled in the art can readily determine if a bacterium is drug resistant using routine laboratory techniques that determine the susceptibility or resistance of a bacterium to a drug or antibiotic.
[23] “Effective amount” refers to an amount which, when applied or administered in an appropriate frequency or dosing regimen, is sufficient to prevent, reduce, inhibit or eliminate bacterial growth or bacterial burden or prevent, reduce or ameliorate the onset, severity, duration or progression of the disorder being treated (here Gram-positive bacterial pathogen growth or infection), prevent the advancement of the disorder being treated, cause the regression of the disorder being treated, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy, such as antibiotic or bacteriostatic therapy.
[24] “Co-administer” refers to the administration of two agents, such as a lysin, and an antibiotic or any other antibacterial agent in a sequential manner, as well as administration of these agents in a substantially simultaneous manner, such as in a single mixture/composition or in doses given separately, but nonetheless administered substantially simultaneously to the subject, for example at different times in the same day or 24-hour period. Such co-administration of two
agents, such as a lysin with one or more additional antibacterial agents, can be provided as a continuous treatment lasting up to days, weeks, or months. Additionally, depending on the use, the co-administration need not be continuous or coextensive. For example, if the use were as a systemic antibacterial agent to treat, e.g., a joint or bone or implant-associated infection, the lysin, could be administered only initially within 24 hours of an additional antibiotic use and then the additional antibiotic use may continue without further administration of the lysin.
[25] “Subject” refers to a mammal, a plant, a lower animal, a single cell organism or a cell culture. For example, the term “subject” is intended to include organisms, e.g., prokaryotes and eukaryotes, which are susceptible to or afflicted with bacterial infections, for example Grampositive bacterial infections. Examples of subjects include mammals, e.g., humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In certain embodiments, the subject is a human, e.g., a human suffering from, at risk of suffering from, or susceptible to infection by Gram-positive bacteria, whether such infection be systemic, topical or otherwise concentrated or confined to a particular organ or tissue.
[26] “Polypeptide” refers to a polymer made from amino acid residues and generally having at least about 30 amino acid residues. The term “polypeptide” is used herein interchangeably with the term “protein” and “peptide.” The term includes not only polypeptides in isolated form, but also active fragments and derivatives thereof. The term “polypeptide” also encompasses fusion proteins or fusion polypeptides comprising a lysin polypeptide, and maintaining, for example, a lysin function. Depending on context, a polypeptide or protein or peptide can be a naturally occurring polypeptide or a recombinant, engineered or synthetically produced polypeptide. A particular lysin polypeptide, for example, can be, e.g., derived or removed from a native protein by enzymatic or chemical cleavage, or can be prepared using conventional peptide synthesis techniques (e.g., solid phase synthesis) or molecular biology techniques (such as those disclosed in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989)) or can be strategically truncated or segmented yielding active fragments, maintaining e.g., lysin activity against the same or at least one common target bacterium.
[27] “Fusion polypeptide” refers to an expression product resulting from the fusion of two or more nucleic acid segments, resulting in a fused expression product typically having two or more domains or segments, which typically have different properties or functionality. In a more
particular sense, the term “fusion polypeptide” also refers to a polypeptide or peptide comprising two or more heterologous polypeptides or peptides covalently linked, either directly or via an amino acid or peptide linker. The polypeptides forming the fusion polypeptide are typically linked C-terminus to N-terminus, although they can also be linked C-terminus to C-terminus, N-terminus to N-terminus, or N-terminus to C-terminus. The term “fusion polypeptide” can be used interchangeably with the term “fusion protein. Thus, the open-ended expression “a polypeptide comprising” a certain structure includes larger molecules than the recited structure such as fusion polypeptides.
[28] “Heterologous” refers to nucleotide or polypeptide sequences that are not naturally contiguous. For example, in the context of the present disclosure, the term “heterologous” can be used to describe a combination or fusion of two or more polypeptides wherein the fusion polypeptide is not normally found in nature, such as for example a lysin polypeptide and a cationic and/or a polycationic peptide, an amphipathic peptide, a sushi peptide (Ding et al. Cell Mol Life Sci., 65(7-8): 1202- 19 (2008)), a defensin peptide (Ganz, T. Nature Reviews Immunology 3, 710- 720 (2003)), a hydrophobic peptide, and/or an antimicrobial peptide which may have enhanced lytic activity. Included in this definition are two or more lysin polypeptides or active fragments thereof. These can be used to make a fusion polypeptide with lytic activity.
[29] “Active fragment” refers to a portion of a polypeptide that retains one or more functions or biological activities of the isolated polypeptide from which the fragment was taken, for example bactericidal activity against one or more Gram-positive bacteria, such as S. aureus or S. epidermidis.
[30] “Synergistic” or “Superadditive” refers to a beneficial effect brought about by two substances in combination that exceeds the sum of the effects of the two agents working independently. In certain embodiments the synergistic or superadditive effect significantly, z.e., statistically significantly, exceeds the sum of the effects of the two agents working independently. One or both active ingredients may be employed at a sub-threshold level, z.e., a level at which if the active substance is employed individually produces no or a very limited effect. The effect can be measured by assays such as the checkerboard assay, described here.
[31] “Treatment” refers to any process, action, application, therapy, or the like, wherein a subject, including a human being, is subjected to medical aid with the object of curing a disorder, eradicating a pathogen, or improving the subject's condition, directly or indirectly. Treatment also
refers to reducing incidence, alleviating symptoms, eliminating recurrence, preventing recurrence, preventing incidence, reducing the risk of incidence, improving symptoms, improving prognosis or combinations thereof. “Treatment” may further encompass reducing the population, growth rate or virulence of the bacteria in the subject and thereby controlling or reducing a bacterial infection in a subject or bacterial contamination of an organ, tissue or environment. Thus, “treatment” that reduces incidence may, for example, be effective to inhibit growth of at least one Gram-positive bacterium in a particular milieu, whether it be a subject or an environment. On the other hand “treatment” of an already established infection refers to reducing the population, killing, inhibiting the growth, and/or eradicating, the Gram-positive bacteria responsible for an infection or contamination.
[32] “Preventing” refers to the prevention of the incidence, recurrence, spread, onset or establishment of a disorder such as a bacterial infection. It is not intended that the present disclosure be limited to complete prevention or to prevention of establishment of an infection. In some embodiments, the onset is delayed, or the severity of a subsequently contracted disease or the chance of contracting the disease is reduced, and such constitutes examples of prevention.
[33] “Contracted diseases” refers to diseases manifesting with clinical or subclinical symptoms, such as the detection of fever, sepsis or bacteremia, as well as diseases that may be detected by growth of a bacterial pathogen (e.g., in culture) when symptoms associated with such pathology are not yet manifest.
[34] “Derivative,” in the context of a peptide or polypeptide or active fragment thereof, is intended to encompass, for example, a polypeptide modified to contain one or more-chemical moieties other than an amino acid that do not substantially adversely impact or destroy the polypeptide’s activity, such as lysin activity. The chemical moiety can be linked covalently to the peptide, e.g., via an amino terminal amino acid residue, a carboxy terminal amino acid residue, or at an internal amino acid residue. Such modifications may be natural or non-natural. In certain embodiments, a non-natural modification may include the addition of a protective or capping group on a reactive moiety, addition of a detectable label, such as an antibody and/or fluorescent label, addition or modification of glycosylation, or addition of a bulking group such as PEG (pegylation) and other changes known to those skilled in the art. In certain embodiments, the non- natural modification may be a capping modification, such as N-terminal acetylations and C- terminal amidations. Exemplary protective groups that may be added to lysin polypeptides
include, but are not limited to t-Boc and Fmoc. Commonly used fluorescent label proteins such as, but not limited to, green fluorescent protein (GFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) and mCherry, are compact proteins that can be bound covalently or noncovalently to a polypeptide or fused to a polypeptide without interfering with normal functions of cellular proteins. In certain embodiments, a polynucleotide encoding a fluorescent protein is inserted upstream or downstream of the polynucleotide sequence. This will produce a fusion protein (e.g., Lysin Polypeptide:: GFP) that does not interfere with cellular function or function of a polypeptide to which it is attached. Polyethylene glycol (PEG) conjugation to proteins has been used as a method for extending the circulating half-life of many pharmaceutical proteins. Thus, in the context of polypeptide derivatives, such as lysin polypeptide derivatives, the term “derivative” encompasses polypeptides, such as lysin polypeptides, chemically modified by covalent attachment of one or more PEG molecules. It is anticipated that lysin polypeptides, such as pegylated lysins, will exhibit prolonged circulation half-life compared to unpegylated polypeptides, while retaining biological and therapeutic activity.
[35] “Percent amino acid sequence identity” refers to the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, such as a lysin polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as a part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for example, using publicly available software such as BLAST or software available commercially for example from DNASTAR. Two or more polypeptide sequences can be anywhere from 0-100% identical, or any integer value there between. In the context of the present disclosure, two polypeptides are “substantially identical” when at least 80% of the amino acid residues (typically at least about 85%, at least about 90%, and typically at least about 95%, at least about 98%, or at least 99%) are identical. The term “percent (%) amino acid sequence identity” as described herein applies to peptides as well. Thus, the term “Substantially identical” will encompass mutated, truncated, fused, or otherwise sequence-modified variants of isolated polypeptides and peptides, such as those described herein, and active fragments thereof, as well as polypeptides with substantial sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95% identity, at least 98% identity, or at least 99% identity as measured for example by one
or more methods referenced above) as compared to the reference (wild type or other intact) polypeptide. Two amino acid sequences are “substantially homologous” when at least about 80% of the amino acid residues (typically at least about 85%, at least about 90%, at least about 95%, at least about 98% identity, or at least about 99% identity) are identical, or represent conservative substitutions. The sequences of polypeptides of the present disclosure, are substantially homologous when one or more, or several, or up to 10%, or up to 15%, or up to 20% of the amino acids of the polypeptide, such as the lysin polypeptides described herein, are substituted with a similar or conservative amino acid substitution, and wherein the resulting polypeptide, such as the lysins described herein, have at least one activity, antibacterial effects, and/or bacterial specificities of the reference polypeptide, such as the lysins described herein.
[36] As used herein, a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
[37] “Biofilm” refers to an aggregate of microorganisms in which cells adhere to each other on a biotic or abiotic surface. These adherent cells are frequently embedded within a matrix comprised of, but not limited to, extracellular polymeric substance (EPS). Biofilm EPS, which is also referred to as slime (although not everything described as slime is a biofilm) or plaque, is a polymeric conglomeration generally composed of extracellular DNA, proteins, and polysaccharides.
[38] “Persister cells” refer to a subpopulation of bacteria that survive high concentrations of antibiotics without any specific resistance mechanism. The presence of persisters within a population is indicated by killing data that show most cells in a population dying, with a subpopulation (0.1-10%) persisting, even in the presence of a high concentration of antibiotic. Persisters pre-exist in a population and arise independently of the use of antibiotics. Persisters survive high concentrations of antibiotics by overexpression of genes such as the chromosomal
toxin -antitoxin modules that shut down cellular functions and hence antibiotic targets, resulting in a dormant cell that is tolerant to the lethal action of antibiotics,
[39] “Small Colony Variants (SCVs)” refer to the generation of resistant phenotypic variants, which occur during bacterial growth. SCVs may occur in cases of e.g., soft tissue infections, osteomyelitis, and device-related infections, including those associated with prosthetic joints. SCVs differ from the normal phenotype in e.g.. their small colony size, reduced growth rate, their tendency to persist and their greater resistance to antimicrobials. These variants may represent a stable, inheritable change or a transient colony type.
[40] “Suitable” in the context of an antibiotic being suitable for use against certain bacteria refers to an antibiotic that was found to be effective against those bacteria even if resistance subsequently developed.
Bone and Joint Infections
[41] The present inventor has surprisingly recognized that certain biologies, i.e., lysins, may be administered locally, e.g. to a bone of a subject, to kill, inhibit or prevent biofilm-forming bacteria, or to kill, inhibit or prevent biofilms, that may cause or are associated with bone and /or joint and/or implant-associated infections due to antibiotic resistant and/or multiple drug resistant (MDR) bacteria, such as Staphylococcus aureus including methicillin-resistant Staphylococcus aureus (MRSA) and multiple drug resistant (MDR) Staphylococcus epidermidis. The lysins described herein are able to disrupt, eradicate and/or prevent biofilms formed by, e.g. Staphylococcus epidermidis or Staphylococcus aureus, in bone. The lysins of the present disclosure may be used against bacteria from e.g., Streptococcus, Staphylococcus, Enterococcus and/or other genera as described herein.
[42] In one aspect, the present disclosure is directed to a method of treating or preventing a bone and/or joint and/or implant-associated infection, which method comprises: locally administering a therapeutically effective amount of a PlySs2 lysin as described herein to a subject in need thereof, such as locally administering an effective amount of a PlySs2 lysin to a bone, such as intramedullary local administration, wherein the bone or joint infection comprises a Gram-positive bacteria.
[43] In some embodiments, the bone infection to be prevented or treated is osteomyelitis, i.e., an inflammatory reaction and destruction of bone due to bacterial colonization of the bone itself,
the bone marrow and/or the surrounding tissue. Osteomyelitis can occur by local spread of bacteria from an adjacent, contaminating source caused by trauma or bone surgery, for example. In some embodiments, the bone infection to be prevented or treated, such as osteomyelitis, is due to Grampositive bacteria, such as Staphylococcus aureus or MRSA. In some embodiments, the Grampositive bacteria have the ability to form biofilms and/or enter into and survive within osteoblasts, thus allowing the Gram-positive bacteria to evade the immune system and many traditional antibiotics.
[44] In some embodiments, the bone infection to be prevented or treated is acute osteomyelitis. In other embodiments, the bone infection is chronic osteomyelitis. In a particular embodiment, the chronic osteomyelitis is osteomyelitis wherein the delay between infection and efficacious treatment exceeds 4-6 weeks.
[45] In some embodiments, chronic osteomyelitis occurs in patients who suffered from acute osteomyelitis in the pre- antibiotic era or in their childhood. Such infections can recur after a symptom-free interval of several decades due to, e.g., the asymptomatic persistence of a biofilm adhering on dead bone.
[46] In some embodiments, the bone infection is exogenous osteomyelitis. In these embodiments, the exogenous osteomyelitis may occur when bone extends out from the skin, allowing a potentially infectious organism to enter from an abscess or bum, a puncture wound, or other trauma such as an open fracture.
[47] In some embodiments, the osteomyelitis comprises an infection of a long bone, such as the femur, tibia, humerus, and radius. In other embodiments, the osteomyelitis comprises an infection of the vertebral column, in particular the lumbar spine, the sacrum, and the pelvis. Typically, children develop osteomyelitis in long bone and adults develop osteomyelitis in the vertebral column.
[48] In some embodiments, the osteomyelitis to be prevented or treated is haematogenous osteomyelitis. Haematogenous osteomyelitis may be acquired from the spread of organisms from preexisting infections e.g., impetigo, furunculosis (persistent boils), infected lesions of varicella (chickenpox), and sinus, ear, dental, soft tissue, respiratory, and genitourinary infections. In some embodiments, a genitourinary infection can lead to osteomyelitis of the sacrum or iliac.
[49] In some embodiments, the bone infection to be prevented or treated comprises an implant- associated infection. Typically, the implant is a mechanical device, such as a metal plate, pin, rod,
wire or screw, which may be used, e.g. to stabilize and/or join the ends of fractured bones. In some embodiments, implant-associated infection becomes chronic wherein only antibiotics are used to treat the infection.
[50] In other embodiments, the lysins of the present methods are used to treat or prevent a joint infection. Infected joints may include infected hip, knee, ankle, shoulder, elbow or wrist joints. Typically, the infected joint is a knee joint or a hip joint.
[51] In some embodiments, the infected joint is a native joint. Infection of a native joint (also referred to herein as septic arthritis of a native joint) may occur when a penetrating injury, such as a puncture wound, occurs near or above a joint, allowing bacteria to directly enter the joint. In other embodiments, the joint infection occurs when bacteria from a distant infection spreads through the bloodstream to the native joint.
[52] In other embodiments, the infected joint is a prosthetic joint, including, for example, septic arthritis of a prosthetic joint. The prosthetic joints may include hip, knee, shoulder, elbow, and ankle prostheses. Typically, the prosthetic joint is a prosthetic hip or knee.
[53] In some embodiments, the prosthetic joint infection, such as an implant-associated infection, occurs within 1 year of surgery. Such an infection can be initiated through the introduction of microorganisms at the time of surgery. This can occur through either direct contact or aerosolized contamination of the prosthesis or periprosthetic tissue. Once in contact with the surface of the implant, microorganisms may colonize the surface.
[54] In other embodiments, the prosthetic joint infections occur due to the spread of an infection from an adjacent site. For example, in the early postoperative time period, a superficial surgical site infection can progress to involve the prosthesis. In other embodiments, the prosthetic joint infection occurs due to the spread of organisms from a remote site of infection via the bloodstream.
[55] In some embodiments, the prosthetic joint infection is recurring. For example, in some embodiments, the joint infection is a relapsing multiple drug resistant infection, such as a relapsing multiple drug resistant S. aureus or S. epidermidis prosthetic knee infection (PKI).
[56] In one embodiment, a prosthetic joint infection is indicated when a pathogen is isolated by culture from at least two separate tissue or fluid samples obtained from the affected prosthetic joint or when four of the following six criteria exist: elevated serum erythrocyte sedimentation rate (ESR) and serum C-reactive protein (CRP) concentration, elevated synovial leukocyte count, elevated synovial neutrophil percentage (PMN%), presence of purulence in the affected joint,
isolation of a microorganism in one culture of periprosthetic tissue or fluid, or greater than five neutrophils per high-power field in five high-power fields observed from histologic analysis of periprosthetic tissue at x400 magnification.
[57] Typically, the fluid obtained from a prosthetic joint to assess for pathogens is synovial fluid. As used herein, “synovial fluid” is a viscous fluid found in the cavities of synovial joints. The principal role of synovial fluid is to reduce friction between the articular cartilage of synovial joints during movement.
[58] In some embodiments, a synovial fluid sample can be obtained by aspiration. The aspirant may be assessed for total nucleated cell counts and neutrophil percentages as an indicator of prosthetic joint infection. Typically, the amount of total nucleated cells per microliter and/or the percentage of neutrophils is greater in a synovial fluid obtained from a subject suffering from prosthetic joint infection in comparison to that of a subject who is not suffering from a prosthetic joint infection. For example, in some embodiments, a threshold of 1,100 total nucleated cells per microliter and/or a threshold of 64% neutrophils in a synovial fluid from a subject with a prosthetic joint indicates a prosthetic joint infection, such as a prosthetic knee joint infection.
[59] In some embodiments, instead of or in addition to determining an amount of neutrophils, a level of leukocyte esterase, an enzyme present in neutrophils, may be assessed using, e.g., colorimetric strips that are widely available for determining pyruia for the diagnosis of urinary tract infection as described in Parvizi et al., “Diagnosis of periprosthetic joint infection: the utility of a simple yet unappreciated enzyme.”, J. Bone Joint Surg. Am., 2011, 93:2242-2248, which is herein incorporated by reference in its entirety.
[60] More typically, however, a synovial fluid sample is cultured to determine whether or not a diagnosis of prosthetic joint infection is indicated and to identify the infecting pathogen(s). This information can also inform the choice of antibiotics if used during treatment. In these embodiments, aspirated synovial fluid can be either inoculated into blood culture bottles at the time of collection or transported to a microbiology laboratory and inoculated onto solid and/or liquid media. See, e.g., Fehring et al., “Aspiration as a guide to sepsis in revision total hip arthroplasty,” 1996, J. Arthroplasty, 11:543-547, which is herein incorporated by reference in its entirety.
Causative Microorganisms
[61] In some embodiments, the present bone and/or joint and/or implant-associated infections are caused by Gram-positive bacteria, such as a Streptococcus species including Streptococcus gallolyticus and Streptococcus pneumonia. More typically, however, the bone and/or joint and/or implant-associated infections is caused by a Staphylococcus species e.g. S. aureus or S. epidermidis. In other embodiments, the Staphylococcus species is a coagulase-negative Staphylococcus species such as Staphylococcus epidermidis, Staphylococcus simulans, Staphylococcus caprae, Staphylococcus lugdunensis or a combination thereof. Typically, Staphylococcus epidermidis is the coagulase-negative Staphylococcus species identified in bone and/or joint and/or implant-associated infections.
[62] In some embodiments, the present bone and/or joint and/or implant-associated infections are caused by Gram-positive bacterial species from the Enterococcus genus or the Listeria genus.
[63] In some embodiments, the present bone and/or joint and/or implant-associated infections are caused by a polymicrobial infection. For example, a combination of Enterococcus species and Staphylococcus species may be identified as causative agents of a bone and/or joint and/or implant- associated infections. Examples of causative microorganisms, typically associated with specific infected structures are shown below in Table 1.
Table 1. Gram-positive bacterial causative agents of bone and/or joint and/or implant- associated infections
[64] In some embodiments, the methods of the present disclosure comprise directly (locally) administering a lysin or active fragment thereof or a variant or derivative thereof as described herein to a bone, typically via local intramedullary administration, i.e., directly inside the bone to treat or prevent bone, joint and/or implant-associated infections. In some embodiments, the lysin of the present disclosure is directly administered to the medullary cavity, i.e. a cavity of a bone marrow. In some embodiments, the locally administered lysin or active fragment thereof or a variant or derivative thereof penetrates through bone and into the synovial space. In still another embodiment, the lysin of the present disclosure is directly administered to the implant.
Lysins
[65] The present methods for treating and/or preventing bone and/or joint and/or implant- associated infections and/or inhibiting, preventing, disrupting or eradicating biofilms in a subject, comprise administering a lysin or active fragment thereof or a variant or derivative thereof as described herein to a subject in need thereof, typically by local administration, typically directly inside of a bone, as described herein, and optionally in combination with one or more antibiotics as also herein described. In some embodiments, the present lysins or active fragments thereof or variants or derivatives thereof exhibit bactericidal and/or bacteriostatic activity against Grampositive bacteria. In some embodiments, the present lysins or active fragments thereof or variants or derivatives thereof also exhibit a low propensity for resistance, suppress antibiotic resistance and/or exhibit synergy with conventional antibiotics. In other embodiments, the present lysins or active fragments thereof or variants or derivatives thereof inhibit bacterial agglutination, biofilm formation and/or reduce or eradicate biofilm, including biofilm in a subject with a bone, joint and/or implant-associated infection.
[66] The bactericidal activity of the present lysins or active fragments thereof or variants or derivatives thereof may be determined using any method known in the art. For example, the
present lysins or active fragments thereof or variants or derivatives thereof may be assessed in vitro using time kill assays as described, for example, in Mueller, et al., 2004, Antimicrob Agents Chemotherapy, 48:369-377, which is herein incorporated by reference in its entirety.
[67] The bacteriostatic activity of the present lysins or active fragments thereof or variants or derivatives thereof may also be assessed using any art-known method. For example, growth curves may be performed in e.g., cation adjusted Mueller Hinton II Broth supplemented in human serum (caMHB/50% HuS) to a final concentration of 50% or in 100% serum or in a non-standard medium (caMHB supplemented to 25% with horse serum and 0.5 mM with DTT (caMHB-HSD)). The Gram-positive bacteria may be suspended with lysin and culture turbidity can be measured at an optical density at 600 nm using, e.g. a SPECTRAMAX® M3 Multi-Mode Microplate reader (Molecular Devices) with e.g., readings every 1 minute for 11 hours at 24°C with agitation. Doubling times can be calculated in the logarithmic-phase of cultures grown in flasks with aeration according to the method described in Saito et al, 2014, Antimicrob Agents Chemother 58:5024- 5025, which is herein incorporated by reference in its entirety and compared to the doubling times of cultures in the absence of the present lysins or active fragments thereof or variants or derivatives thereof.
[68] In some embodiments, the present lysins or active fragments thereof or variants or derivatives thereof exhibit lysin activity in the presence of synovial fluid, such as human synovial fluid. Suitable methods for assessing the activity of a lysin in synovial fluid are known in the art and described in the examples. Briefly, a MIC value (i.e., the minimum concentration of peptide sufficient to suppress at least 80% of the bacterial growth compared to control) may be determined for a lysin in a synovial fluid and its MIC value compared to, e.g., a parent lysin or the absence of lysin.
[69] More particularly MIC values for a lysin may be determined against e.g., S. epidermidis or S. aureus in e.g., Mueller-Hinton broth (MHB) supplemented with physiological salt concentrations and synovial fluid, such as human synovial fluid. Minimum Inhibitory Concentrations (MICs) of a lysin against e.g., S. epidermidis may be determined using broth microdilution (BMD) following Clinical and Laboratory Standards Institute (CLSI) methodology (M07-A11, 2018, which is herein incorporated by reference in its entirety) in a non-standard medium (caMHB supplemented to 50% with human synovial fluid (caMHB-HSF)).
[70] In some embodiments, the present isolated polypeptides comprising lysins, variant lysins, active fragments thereof or derivatives reduce the minimum inhibitory concentration (MIC) of an antibiotic needed to inhibit bacteria in the presence of e.g., human serum or synovial fluid. Any known method to assess this MIC may be used. In some embodiments, a checkerboard assay is used to determine the effect of a lysin on antibiotic concentration. The checkerboard assay is based on a modification of the CLSI method for MIC determination by broth microdilution (See CLSI. 2015. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard- 10th Edition. Clinical and Laboratory Standards Institute, Wayne, PA, which is herein incorporated by reference in its entirety and Ceri et al. 1999. J. Clin. Microbiol. 37: 1771-1776, which is also herein incorporated by reference in its entirety).
[71] Checkerboards are constructed by first preparing columns of e.g., a 96-well polypropylene microtiter plate, wherein each well has the same amount of antibiotic diluted 2-fold along the horizontal axis. In a separate plate, comparable rows are prepared in which each well has the same amount of lysin diluted e.g., 2-fold along the vertical axis. The lysin and antibiotic dilutions are then combined, so that each column has a constant amount of antibiotic and doubling dilutions of lysin, while each row has a constant amount of lysin and doubling dilutions of antibiotic. Each well thus has a unique combination of lysin and antibiotic. Bacteria are added to the drug combinations at concentrations of 1 x 105 CFU/ml in caMHB-HSF, for example. The MIC of each drug, alone and in combination, is then recorded after e.g., 16 hours at 37°C in ambient air. Summation fractional inhibitory concentrations (^FICs) are calculated for each drug and the minimum FIC value (^FlCmin) is used to determine the effect of the lysin/antibiotic combination.
[72] Inhibition of bacterial agglutination may be assessed using any method known in the art. For example, the method described in Walker et al. may be used, i.e., Walker et al., 2013, PLoS Pathog, 9:el003819, which is herein incorporated by reference in its entirety.
[73] Methods for assessing the ability of the lysins or active fragments thereof or variants or derivatives thereof to inhibit or reduce biofilm formation in vitro are well known in the art and include a variation of the broth microdilution minimum Inhibitory Concentration (MIC) method with modifications (See Ceri et al. 1999. J. Clin Microbial. ' T. lrl - lr16, which is herein incorporated by reference in its entirety and Schuch et al., 2017, Antimicrob. Agents Chemother. 61, pages 1-18, which is herein incorporated by reference in its entirety.) In this method for
assessing the Minimal Biofilm Eradicating Concentration (MBEC), fresh colonies of e.g., an S. aureus strain or an S. epidermidis strain, are suspended in medium, e.g., phosphate buffer solution (PBS) diluted e.g. ,1:100 in TSBg (tryptic soy broth supplemented with 0.2% glucose), added as e.g., 0.15 ml aliquots, to a Calgary Biofilm Device (96-well plate with a lid bearing 96 polycarbonate pegs; Innovotech Inc.) and incubated e.g., 24 hours at 37°C. Biofilms are then washed and treated with e.g., a 2-fold dilution series of the lysin in e.g., TSBg at e.g., 37°C for 24 hours. After treatment, wells are washed, air-dried at e.g., 37°C and stained with e.g., 0.05% crystal violet for 10 minutes. After staining, the biofilms are destained in e.g., 33% acetic acid and the OD600 of e.g., extracted crystal violet is determined. The MBEC of each sample is the minimum lysin concentration required to remove >95% of the biofilm biomass assessed by crystal violet quantitation.
[74] In some embodiments, the present lysins, variant lysins and fragments thereof are assessed against a Gram-positive bacterial lysate obtained from a subject with a bone and/or joint and/or implant-associated infection as described herein. Methods for obtaining such isolates are well known in the art and described, for example, in Schmidt-Malan et al., Diag. Microbiol. Infect. Dis. 85:77-79, which is herein incorporated by reference in its entirety.
[75] Suitable lysins for use with the present method include the PlySs2 lysins as described in WO 2013/170015 and WO 2013/170022, each of which is herein incorporated by reference in its entirety. As used herein, the terms “PlySs2 lysin”,“PlySs2 lysins”, “PlySs2” “Exebacase” and “CF-301” are used interchangeably and encompass the PlySs2 lysin set forth herein as SEQ ID NO: 1 (with or without initial methionine residue) or an active fragment thereof or variants or derivatives thereof as described in WO 2013/170015 and WO 2013/170022. PlySs2, which was identified as an anti- staphylococcal lysin encoded within a prophage of the Streptococcus suis genome, exhibits bactericidal and bacteriostatic activity against the bacteria described below in Table 2.
Table 2. Reduction in Growth of Different Bacteria and Relative kill with a lysin, PlySs2 (partial listing).
[76] In some embodiments, a lysin suitable for use with the present method is the PlySs2 lysin of SEQ ID NO: 1. The PlySs2 lysin of SEQ ID NO: 1 has a domain arrangement characteristic of most bacteriophage lysins, defined by a catalytic N-terminal domain (FIG. 1) linked to a cell wallbinding C-terminal domain (FIG. 1). The N-terminal domain belongs to the cysteine-histidine- dependent amidohydrolases/peptidases (CHAP) family common among lysins and other bacterial cell wall-modifying enzymes. The C-terminal domain belongs to the SH3b family that often forms the cell wall-binding element of lysins. FIG. 1 depicts the PlySs2 lysin of SEQ ID NO: 1 with the
N- and C-terminal domains shown as shaded regions. The N-terminal CHAP domain corresponds to the first shaded amino acid sequence region starting with LNN and the C-terminal SH3b domain corresponds to the second shaded region starting with RSY.
[77] In some embodiments, a lysin suitable for use with the methods disclosed herein comprises one or more of the following lysins: pp55 (SEQ ID NO: 3), pp61 (SEQ ID NO: 4), pp65 (SEQ ID NO: 5), pp296 (SEQ ID NO: 6 also referred to herein as CF-296), pp324 (SEQ ID NO: 7), pp325 (SEQ ID NO: 8), pp338 (SEQ ID NO: 9), pp341 (SEQ ID NO: 10), pp388 (SEQ ID NO: 11), pp400 (SEQ ID NO: 12), pp616 (SEQ ID NO: 13), pp619 (SEQ ID NO: 14), pp628 (SEQ ID NO: 15), pp632 (SEQ ID NO: 16), and pp642 (SEQ ID NO: 17).
[78] In some embodiments, the present methods comprise the administration of a variant lysin to a subject in need thereof. Suitable lysin variants for use with the present method include those polypeptides having at least one substitution, insertion and/or deletion in reference to SEQ ID NO: 1 that retain at least one biological function of the reference lysin. In some embodiments, the variant lysins exhibit antibacterial activity including a bacteriolytic and/or bacteriostatic effect against a broad range of Gram-positive bacteria, including S. aureus and S. epidermidis and an ability to inhibit agglutination, inhibit biofilm formation and/or reduce biofilm. In some embodiments, the present lysin variants render Gram-positive bacteria more susceptible to antibiotics.
[79] In some embodiments, a lysin variant suitable for use with the present methods includes an isolated polypeptide sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98%, such as at least 99% or such as at least 99.5% sequence identity with SEQ ID NO: 1, wherein the variant lysin retains one or more biological activities of the PlySs21ysin having the amino acid sequence of SEQ ID NO: 1 as described herein.
[80] Lysin variants may be formed by any method known in the art and as described in WO 2013/170015, which is herein incorporated by reference in its entirety, e.g., by modifying the PlySs2 lysin of SEQ ID NO: 1 through site-directed mutagenesis or via mutations in hosts that produce the PlySs2 lysin of SEQ ID NO: 1, and which retain one or more of the biological functions as described herein. For example, one of skill in the art can reasonably make and test substitutions or replacements to, e.g., the CHAP domain and/or the SH3b domain of the PlySs2 lysin of SEQ ID NO: 1. Sequence comparisons to the Genbank database can be made with either or both of the CHAP and/or SH3b domain sequences or with the PlySs2 lysin full amino acid
sequence of SEQ ID NO: 1, for instance, to identify amino acids for substitution. For example, a mutant or variant having an alanine replaced for valine at valine amino acid residue 19 in the PlySs2 amino acid sequence of SEQ ID NO: 1 is active and capable of killing Gram-positive bacteria in a manner similar to and as effective as the SEQ ID NO: 1 PlySs2 lysin.
[81] Further, as indicated in FIG. 1, the CHAP domain contains conserved cysteine and histidine amino acid sequences (the first cysteine and histidine in the CHAP domain) which are characteristic and conserved in CHAP domains of different polypeptides. It is reasonable to predict, for example, that the conserved cysteine and histidine residues should be maintained in a mutant or variant of PlySs2 so as to maintain activity or capability. Accordingly, particularly desirable residues to retain in a lysin variant of the present disclosure include active-site residues Cys26, Hisio2, duns, and Asnno in the CHAP domain of SEQ ID NO: 1. Particularly desirable substitutions include: Lys for Arg and vice versa such that a positive charge may be maintained, Glu for Asp and vice versa such that a negative charge may be maintained, Ser for Thr such that a free -OH can be maintained and Gin for Asn such that a free NH2 can be maintained.
[82] Suitable variant lysins are described in PCT Published Application No. WO 2019/165454 (International Application No.: PCT/US2019/019638), which is herein incorporated by reference in its entirety. Particularly, suitable variant lysins include those set forth herein as SEQ ID NOS: 3-17 as well as variant lysins having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98%, such as at least 99%, such as at least 99.5% sequence identity with any one of SEQ ID NOS: 3-17 (with or without the initial methionine), wherein the variant lysin retains one or more biological activities of the PlySs2 lysin having the amino acid sequence of SEQ ID NO: 1 as described herein. A particularly preferred PlySs2 lysin variant is SEQ ID NO: 6.
[83] SEQ ID NOs: 3-17 are modified lysin polypeptides having at least one amino acid substitution relative to a counterpart wild-type PlySs2 lysin SEQ ID NO: 1, while preserving antibacterial activity and effectiveness. SEQ ID NOs: 3-17 may be described by reference to their amino acid substitutions with respect to SEQ ID NO: 1, as shown below in Table A. The amino acid sequences of the modified lysin polypeptides (referencing differences from SEQ ID NO: 1 and the positions of its amino acid residues) are summarized using one-letter amino acid codes as follows:
Table A
[84] In some embodiments, the present method includes administering an active fragment of a lysin to a subject in need thereof. Suitable active fragments include those that retain a biologically active portion of a protein or peptide fragment of the lysin embodiments, as described herein. Such variants include polypeptides comprising amino acid sequences that include fewer amino acids than the full-length protein of the lysin protein and exhibit at least one activity of the corresponding full-length protein. Typically, biologically active portions comprise a domain or motif with at least one activity of the corresponding protein. An exemplary domain sequence for the N-terminal CHAP domain of the PlySs2 lysin is provided in FIG. 1. An exemplary domain sequence for the C terminal SH3b domain of the PlySs21ysin is also provided in FIG. 1. A biologically active portion of a protein or protein fragment of the disclosure can be a polypeptide which is, for example, 10, 25, 50, 100 amino acids in length. Other biologically active portions, in which other regions of the protein are deleted can be prepared by recombinant techniques and evaluated for one or more of the functional activities of the native form of a polypeptide of the embodiments.
[85] In some embodiments, suitable active fragments include those having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98%, such as at least 99% or such as at least 99.5% sequence identity with the active fragments described herein,
wherein the active fragment thereof retains at least one activity of a CHAP and/or the SH3b domain, e.g., as shown in FIG. 1.
[86] A lysin or active fragment thereof or variant or derivative thereof as described herein for use in the present method may be produced by a bacterial organism after being infected with a particular bacteriophage or may be produced or prepared recombinantly or synthetically. In as much the lysin polypeptide sequences and nucleic acids encoding the lysin polypeptides are described and referenced herein, the present lysins may be produced via the isolated gene for the lysin from the phage genome, putting the gene into a transfer vector, and cloning said transfer vector into an expression system, using standard methods of the art, as described for example in WO 2013/170015, which is herein incorporated by reference in its entirety. The present lysin variants may be truncated, chimeric, shuffled or “natural,” and may be in combination as described, for example, in International Patent Publication No. WO 2013/17002, which is incorporated herein in its entirety by reference.
[87] Mutations can be made in the amino acid sequences, or in nucleic acid sequences encoding the polypeptides and lysins described herein, including in the lysin sequence set forth in SEQ ID NO: 1, or in active fragments or truncations thereof, such that a particular codon is changed to a codon which codes for a different amino acid, an amino acid is substituted for another amino acid, or one or more amino acids are deleted.
[88] Such a mutation is generally made by making the fewest nucleotide changes possible. A substitution mutation of this sort can be made to change an amino acid in the resulting protein in a non-conservative manner (for example, by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to another grouping) or in a conservative manner (for example, by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to the same grouping). Such a conservative change generally leads to less change in the structure and function of the resulting protein. A non-conservative change is more likely to alter the structure, activity or function of the resulting protein. The present disclosure should be considered to include sequences containing conservative changes which do not significantly alter the activity or binding characteristics of the resulting protein. Thus, one of skill in the art, based on a review of the sequence of the PlySs2 lysin polypeptide provided herein and on their knowledge and the public information available for other lysin polypeptides, can make amino acid
changes or substitutions in the lysin polypeptide sequence. Amino acid changes can be made to replace or substitute one or more, one or a few, one or several, one to five, one to ten, or such other number of amino acids in the sequence of the lysin(s) provided herein to generate mutants or variants thereof. Such mutants or variants thereof may be predicted for function or tested for function or capability for anti-bacterial activity as described herein against, e.g., Staphylococcal, Streptococcal, or Enterococcal bacteria, and/or for having comparable activity to the lysin(s) as described and particularly provided herein. Thus, changes made to the sequence of lysin, and mutants or variants described herein can be tested using the assays and methods known in the art and described herein. One of skill in the art, on the basis of the domain structure of the lysin(s) hereof can predict one or more, one or several amino acids suitable for substitution or replacement and/or one or more amino acids which are not suitable for substitution or replacement, including reasonable conservative or non-conservative substitutions.
Antibiotics
[89] In some embodiments, the methods of treating or preventing bone, joint and/or implant - associated infections as described herein comprise co-administering a therapeutically effect amount of one or more antibiotics and a PlySs2 lysin. In some embodiments, co-administration of a lysin or active fragment thereof or variant or derivative thereof and one or more antibiotic as described herein results in a synergistic bactericidal and/or bacteriostatic effect on Gram-positive bacteria such as S. aureus or S. epidermidis. In other embodiments, the co-administration is used to suppress virulence phenotypes including biofilm formation and/or agglutination. In some embodiments, the co-administration is used to reduce an amount of biofilm in a subject.
[90] In some embodiments, the present lysins or active fragments thereof or variants or derivatives thereof are administered locally as described herein and the antibiotics are administered systemically, such as orally, more typically intravenously.
[91] Suitable antibiotics for use with the present methods include antibiotics that are capable of penetrating bone. Such antibiotics include those of different types and classes, such as betalactams penicillins (e.g. methicillin, oxacillin, piperacillin, tazobactam, flucloxacillin, cioxacillin), cephalosporins (e.g. cefalexin and cefactor), carbapenems (e.g. imipenem and entapenem); macrolides (e.g. erythromycin, azithromycin), aminoglycosides (e.g. gentamicin, tobramycin, amikacin), glycopeptides (e.g., vancomycin, teicoplanin, dalbavancin, oritavancin),
oxazolidinones (e.g linezolid and tedizolid), lipopeptides (e.g. daptomycin), sulfonamides (e.g. sulfamethoxazole), tetracyclines (e.g., doxycycline), lincomycins (e.g., clindamycin), fluoroquinolones (e.g., levofloxacin), phosphonic antibiotics (e.g., fosfomycin) and/or a rifamycin antibiotic, such as rifampin or rifabutin. Other suitable antibiotics include particular antibiotic combinations such as piperacillin/tazobactam, trimethoprim/sulfamethoxazole, and fosfomycin.
[92] In some embodiments the antibiotics are administered systemically as described herein for at least two weeks, such as at least four weeks, such as at least six weeks, e.g., 6-24 weeks.
[93] In some embodiments, the antibiotic is an antibiotic typically used to treat osteomyelitis, such as acute osteomyelitis, such as vancomycin or daptomycin, typically daptomycin.
[94] In some embodiments, the present methods exclude antibiotics that poorly penetrate bone, such antibiotics are known in the art and include, e.g. penicillin and metronidazole.
[95] In another aspect, the present disclosure is directed to a method of preventing or treating a bone, joint and/or implant-associated infection due to Gram-positive bacteria as described herein, which method comprises: locally administering, as described herein, a therapeutically effective amount of a PlySs2 lysin, active fragment thereof or variant or derivative thereof as described herein to a subject in need thereof in conjunction with Debridement and Implant Retention (DAIR). Optionally, an antibiotic as described herein is co-administered with the PlySs2 lysin.
[96] In some embodiments, debridement of infected and potentially infected tissues around e.g., an implant, is typically performed followed by arthroscopic irrigation of involved tissues with copious volumes of fluid, such as sterile saline. In some embodiments, a PlySs2 lysin, active fragment thereof or variant or derivative thereof as described herein is administered locally to the bone, typically inside of the bone as described herein, by drilling a hole in the bone as described in the examples, before, during or after arthroscopic irrigation. In some embodiments, bonepenetrating antibiotics as described herein, such as fosfomycin, vancomycin or daptomycin, are subsequently systemically administered, e.g., orally or intravenously administered, to the subject for e.g., at least two weeks, such as 6-24 weeks. In one embodiment, the antibiotic is administered intravenously.
[97] In some embodiments, the subject to be administered a lysin of the disclosure is elderly or suffers from a condition associated with a higher risk of a bone and/or joint and/or implant- associated infection. For example, the subject at risk for a bone, joint and/or implant-associated infection may suffer from obesity, e.g., a body mass index (BMI) threshold of 35. An elderly
subject, for example, is at least 65 years, such as 65-90 years, 75-90 years, or 79-89 years. Without being limited by theory, possible reasons for the increased risk of bone, joint and/or implant- associated infections, such as prosthetic bone or joint infections, with obesity include prolonged operative duration and/or the presence of other comorbidities.
[98] In some embodiments, the subject at risk for a bone, joint and/or implant-associated infection, particularly a prosthetic joint infection, suffers from diabetes mellitus. Without being limited by theory, the risk associated with diabetes may be due to increased biofilm formation in the presence of elevated levels of glucose, impaired leukocyte function, or microvascular changes in subjects with diabetes mellitus, which may influence wound healing and the development of superficial surgical site infections.
[99] Other risk factors for bone and/or joint and/or implant-associated infections include rheumatoid arthritis, male gender and smoking. In addition, a diagnosis of bacteremia in the year preceding an implant surgery is also a risk factor for a bone and/or joint infection, such as a prosthetic joint infection.
Dosages and Administration
[100] Dosages of the present lysins or active fragments thereof or variants or derivatives thereof that are administered to a subject in need thereof depend on a number of factors including the activity of infection being treated, the age, health and general physical condition of the subject to be treated, the mode of administration, the activity of a particular lysin or active fragment thereof or variant or derivative thereof, the nature and activity of the antibiotic, if any, with which a lysin or active fragment thereof or variant or derivative thereof according to the present disclosure is being paired and the combined effect of such pairing. Generally, effective amounts of the present lysins or active fragments thereof or variants or derivatives thereof to be administered systemically are anticipated to fall within the range of the range of 0.00001-200 mg/kg, such as 0.2 mg/kg to about 0.3 mg/kg, such as 0.25 mg/kg, such as, 1-150 mg/kg, such as 40 mg/kg to 100 mg/kg and are administered 1-4 times daily for a period up to 14 days. The antibiotic may be administered at standard dosing regimens or in lower amounts in view of e.g., synergy. All such dosages and regimens however (whether of the lysin or active fragment thereof or variant or derivative thereof or any antibiotic administered in conjunction therewith) are subject to optimization. Optimal
dosages can be determined by performing in vitro and in vivo pilot efficacy experiments as is within the skill of the art but taking the present disclosure into account.
[101] More typically the present lysins or active fragments thereof or variants or derivatives thereof are administered locally, e.g., directly into bone. Generally, effective amounts of the present lysins or active fragments thereof or variants or derivatives thereof to be administered for local administration, such as into bone, are anticipated to fall within the range of 0.00001-200 mg/mL. In one embodiment, 1 mg/mL to about 100 mg/mL, such as 75 mg/mL, such as, 1-150 mg/mL, such as 40 mg/mL to 100 mg/mL may be administered. In another embodiment, the amount is 10-15 mg/ml, e.g., 10-11 mg/ml, such as 10 mg/ml.
[102] The lysins or active fragments thereof or variants or derivatives thereof and the one or more antibiotics of the present disclosure may be administered by the same mode of administration or by different modes of administration, and may be administered once, twice or multiple times, one or more in combination or individually. Thus, the present lysins or active fragments thereof or variants or derivatives thereof may be administered in an initial dose followed by a subsequent dose or doses, particularly depending on the response, e.g., the bactericidal and/or bacteriostatic effects and/or the effect on agglutination and/or biofilm formation or reduction and may be combined or alternated with antibiotic dose(s). Typically, the lysins or active fragments thereof or variants or derivatives thereof are locally administered, e.g., directly to the bone, such as inside of the bone, followed by conventional doses and administration modes, e.g., systemically, of the one or more antibiotics of the present disclosure.
[103] It is contemplated that the present lysins or active fragments thereof or variants or derivatives thereof provide a bactericidal and, when used in smaller amounts, a bacteriostatic effect, and are active against a range of antibiotic -resistant bacteria and are not associated with evolving resistance. Based on the present disclosure, in a clinical setting, the present lysins or active fragments thereof or variants or derivatives thereof are a potent alternative (or additive or component) of compositions for treating or preventing bone and/or joint and/or implant-associated infections arising from drug- and multidrug-resistant bacteria when combined with certain antibiotics (even antibiotics to which resistance has developed). Existing resistance mechanisms for Gram-positive bacteria should not affect sensitivity to the lytic activity of the present polypeptides.
[104] For any lysin of the present disclosure as described herein or active fragments thereof or variants or derivatives thereof, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model can also be used to achieve a desirable concentration range and route of administration. Obtained information can then be used to determine the effective doses, as well as routes of administration in humans. In one embodiment, direct, local administration is local administration inside of bone.
[105] In some embodiments, the present lysins or active fragments thereof or variants or derivatives thereof and one or more antibiotics as described herein, such as daptomycin, are administered simultaneously. In other embodiments, the present lysins or active fragments thereof or variants or derivatives thereof and the one or more antibiotics of the present method, such as daptomycin, are administered in series, such as sequentially, in any order. In some embodiments, the lysin is administered during or subsequent to administration of a standard of care antibiotic treatment, e.g., a two-week course of oxacillin and gentamicin or daptomycin.
[106] In more typical embodiments, the lysin or active fragment thereof or variant or derivative thereof of the present disclosure is administered locally, e.g., inside the bone of a subject followed by a conventional, systemic regimen, e.g., standard of care (SOC) dosages, of one or more antibiotics of the present disclosure, such as daptomycin. In other typical embodiments, one or more antibiotics of the present disclosure, such as daptomycin, is systemically administered to a subject followed by direct administration, typically inside of bone, of a lysin or active fragment thereof or variant or derivative thereof of the present disclosure, followed by additional dosages of the one or more antibiotics of the present disclosure at conventional systemic dosages, such as daptomycin.
[107] In some embodiments, the lysins or active fragments thereof or variants or derivatives thereof or the one or more antibiotics may be administered at sub-MIC levels, e.g., at sub-MIC levels ranging from 0.9X MIC to 0.0001X MIC. At such sub-MIC levels, the present lysins or active fragments thereof or variants or derivatives thereof, alone or in combination with sub-MIC levels of antibiotics, are typically used to inhibit the growth of Gram-positive bacteria, reduce agglutination, and/or inhibit biofilm formation or to reduce or eradicate biofilm.
[108] In some embodiments, the present lysin or active fragment thereof or variant or derivative thereof or the one or more antibiotics of the present disclosure are administered to a subject in need thereof at the MIC level or greater than the MIC level, such as IX MIC, 2X MIC, 3X MIC, 4X
MIC, 10MIC or 20MIC. In some embodiments, the combination of the present lysin or active fragment thereof or variant or derivative thereof and the one or more antibiotics of the present disclosure exhibit a synergistic killing or synergistic bacteriostatic effect on Gram-positive lysins. Typically, the present lysin or active fragment thereof or variant or derivative thereof are administered locally inside of a bone at concentrations greater than the MIC value and the antibiotics are administered systemically at dosages below conventional dosages. For example, daptomycin may be administered at dosages of less than the conventional human dose of 6 mg/kg, such as 3 mg/kg.
Formulations
[109] The lysin or active fragment thereof or variant or derivatives thereof of the present disclosure, optionally administered either alone or in combination or in series, with the one or more antibiotics described herein may each be included in a single pharmaceutical formulation or be separately formulated in the form of a solution, a suspension, an emulsion, an inhalable powder, an aerosol, or a spray, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, tampon applications emulsions, aerosols, sprays, suspensions, lozenges, troches, candies, injectants, chewing gums, ointments, smears, time-release patches, liquid absorbed wipes, and combinations thereof.
[110] In some embodiments, administration of the pharmaceutical formulations may include systemic administration. Systemic administration can be enteral or oral, i.e., a substance is given via the digestive tract, parenteral, i.e., a substance is given by other routes than the digestive tract such as by injection or inhalation. Thus, the lysins or active fragments thereof or variants or derivatives thereof and optionally the one or more antibiotics of the present disclosure can be administered to a subject orally, parenterally, by inhalation, topically, rectally, nasally, buccally or via an implanted reservoir or by any other known method. The lysins or active fragments thereof or variants or derivatives thereof and/or the one or more antibiotics of the present disclosure can also be administered by means of sustained release dosage forms.
[111] For oral administration, the lysins or active fragments thereof or variants or derivatives thereof and optionally, the one or more antibiotics of the present disclosure can be formulated into solid or liquid preparations, for example tablets, capsules, powders, solutions, suspensions and dispersions. In some embodiments, the lysins or active fragments thereof or variants or derivatives
thereof and/or the one or more antibiotics of the present disclosure can be formulated with excipients such as, e.g., lactose, sucrose, com starch, gelatin, potato starch, alginic acid and/or magnesium stearate.
[112] For preparing solid compositions such as tablets and pills, the lysins or active fragments thereof or variants or derivatives thereof and/or the one or more antibiotics of the present disclosure is mixed with a pharmaceutical excipient to form a solid pre-formulation composition. If desired, tablets may be sugar coated or enteric coated by standard techniques. The tablets or pills may be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can include an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two dosage components can be separated by an enteric layer, which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.
[113] In another embodiment, the pharmaceutical formulations of the present disclosure are formulated as inhalable compositions. In some embodiments, the present pharmaceutical formulations are advantageously formulated as a dry, inhalable powder. In specific embodiments, the present pharmaceutical formulations may further be formulated with a propellant for aerosol delivery. Examples of suitable propellants include, but are not limited to: dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane and carbon dioxide. In certain embodiments, the formulations may be nebulized.
[114] In some embodiments, the inhalable pharmaceutical formulations include excipients. Examples of suitable excipients include, but are not limited to: lactose, starch, propylene glycol diesters of medium chain fatty acids; triglyceride esters of medium chain fatty acids, short chains, or long chains, or any combination thereof; perfluorodimethylcyclobutane; perfluorocyclobutane; polyethylene glycol; menthol; lauroglycol; diethylene glycol monoethylether; polyglycolized glycerides of medium chain fatty acids; alcohols; eucalyptus oil; short chain fatty acids; and combinations thereof.
[115] A surfactant can be added to an inhalable pharmaceutical formulation of the present disclosure in order to lower the surface and interfacial tension between the medicaments and the
propellant. The surfactant may be any suitable, non-toxic compound which is non-reactive with the present polypeptides. Examples of suitable surfactants include, but are not limited to: oleic acid; sorbitan trioleate; cetyl pyridinium chloride; soya lecithin; polyoxyethylene(20) sorbitan monolaurate; polyoxyethylene (10) stearyl ether; polyoxyethylene (2) oleyl ether; poly oxypropylene-poly oxy ethylene ethylene diamine block copolymers; poly oxy ethylene(20) sorbitan monostearate; polyoxyethylene(20) sorbitan monooleate; polyoxypropylenepolyoxyethylene block copolymers; castor oil ethoxylate; and combinations thereof.
[116] In some embodiments, the pharmaceutical formulations of the present disclosure comprise nasal formulations. Nasal formulations include, for instance, nasal sprays, nasal drops, nasal ointments, nasal washes, nasal injections, nasal packings, bronchial sprays and inhalers, or indirectly through use of throat lozenges, mouthwashes or gargles, or through the use of ointments applied to the nasal nares, or the face or any combination of these and similar methods of application.
[117] The pharmaceutical formulations of the present disclosure, however, are more typically administered locally, such as directly inside of a bone as described herein. Typically, the pharmaceutical formulations comprise a therapeutically effective amount of a PlySs2 lysin active fragments thereof or variants or derivatives thereof formulated for local administration, such as to a bone, comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof having at least 80% identity to SEQ ID NO: 1, wherein the PlySs2 variant comprises bactericidal and/or bacteriostatic activity against Gram-positive bacteria and wherein the formulation for local administration optionally comprises intramedullary local administration. The pharmaceutical formulations may include a pharmaceutically acceptable carrier such as distilled water, a saline solution, albumin, a serum, or any combinations thereof. Typically, such lysin-containing pharmaceutical formulations are co-administered with conventional antibiotics, more typically conventional antibiotics that are known to penetrate bone well as described herein. In some embodiments, pharmaceutical formulations comprising conventional antibiotics are administered systemically as described herein. In some embodiments, the antibiotic pharmaceutical formulations do not include penicillin and/or metronidazole.
[118] The pharmaceutical formulations of the present disclosure may be presented in unit dosage form and may be prepared by any methods well known in the art. The amount of active ingredients which can be combined with a carrier material to produce a single dosage form will vary depending
upon the host being treated, the duration of exposure of the recipient to the infectious bacteria, the size and weight of the subject, and the particular mode of administration. The amount of active ingredients that can be combined with a carrier material to produce a single dosage form will generally be that amount of each compound which produces a therapeutic effect. Generally, out of one hundred percent, the total amount will range from about 1 percent to about ninety-nine percent of active ingredients, typically from about 5 percent to about 70 percent, most typically from about 10 percent to about 30 percent.
EXAMPLES
Example 1. In vitro time-kill studies
In vitro time-kill studies were performed using cortical screws grown overnight with the MRSA IDRL-6169 and then placed in 0.1 ml of lysin carrier, exebacase (10.64 mg/ml) or CF-296 (10.16 mg/ml). After 1, 2, 4, or 8 hours, three screws were processed for quantitative culture. The results were reported as logio colony forming units (cfu)/implant.
As shown in FIG. 2, reductions of 4.72 and 3.86 logio cfu/implant were observed after 1 hour for exebacase and CF-296, respectively. After 8 hours, the logio cfu/implant were reduced by 5.69 for exebacase and 4.41 for CF-296. Accordingly, the time kill assays demonstrated rapid bactericidal activity for both exebacase and CF-296.
Example 2. Treatment of a Rabbit Foreign Body Osteomyelitis Screw Model
An acute rabbit model of implant-associated MRSA osteomyelitis with intramedullary local delivery of exebacase (10.64 mg/mL) or CF-296 (10.16 mg/mL), with and without systemic daptomycin was developed. The minimum inhibitory concentration (MIC) of exebacase, CF-296 and daptomycin was 0.5 pg/mL. The tested lysins were supplied in the carrier solution described above in Example 1. Daptomycin (Xellia Pharmaceuticals USA Inc., North Wales, PA) was supplied as 500 milligram (mg) powder and reconstituted in 10 mL sterile 0.9% sodium chloride.
Implant Seeding
1.5 x 7 millimeter (mm) titanium cortex screws (DePuy Synthes, Monument, CO)), were individually placed into 0.2 milliliter (mL) microcentrifuge tubes and autoclaved. 37 pl of a 104 cfu/mL suspension of MRSA IDRL-6169 in trypticase soy broth (TSB) was added to the tubes,
each containing individual implants. Subsequently, the microcentrifuge tubes were placed on an orbital shaker at 37°C for ~16 hours. The CDC-NIH Guide for Biosafety in Microbiological and Biomedical Laboratories was followed.
On each surgical day, one or two implants were quantitatively cultured by sonication followed by quantitative culture of the resultant sonicate fluid on sheep blood agar to confirm bacterial colonization. Representative scanning electron microscopy (SEM) images of a MRSA IDRL-6169 seeded orthopedic implant, prior to surgical implantation, are shown in FIG. 3.
Surgical Methods
The experimental surgical model (FIG. 4) was developed and performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Mayo Clinic as well as the Animal Care and Use Review Office of the United States Army Medical Research and Development Command Office of Research Protections. In conducting research using animals, the investigators adhered to the laws of the United States and regulations of the Department of Agriculture.
Sixty New Zealand male and female rabbits were studied. All animals tolerated the procedures well and there was no unintentional loss of life throughout the study. Rabbits were administered anesthesia and analgesia. Once fully sedated, the left leg was shaved and cleansed with a combination of ChloraPrep™ (BD, Franklin Lakes, NJ), PDI® Povidone-Iodine 3’s Swabsticks (Orangeburg, NY) and alcohol swabsticks (Medline, Northfield, IL). A 1.5 centimeter (cm) incision was made on the proximal portion of the medial left tibia. The fascia and muscle were cleared to expose a smooth flat portion of the tibia (FIG. 4a). Using a Microtorque II micro drill (Harvard Apparatus, Holliston, MA) with a 1.4 mm carbide round burr (Roboz Surgical Instrument Co., Inc., Gaithersburg, MD), a hole was punched through the cortical bone into the medullary cavity (FIGS. 4b and 4c). To remove any bone fragments that may have entered the medullary cavity, 2 liters (L) of water was flushed into the tibia with a 20 gauge, 1-inch polypropylene feeding tube (#FTP-20-30, Instech, Plymouth Meeting, PA) (FIG. 4d). 0.6 mL of lysin or lysin carrier (vehicle) was injected in the same fashion, inserting the feeding tube to the hub, towards the distal tibia, and slowly drawing the feeding tube out of the bone as the lysin was injected (FIG. 4e). Immediately after local treatment was delivered, an implant was placed into the hole with screw forceps and secured with a screwdriver (FIGS. 4f, 4g and 4h). Concurrently,
systemic treatment of either daptomycin (6 mg/kg) or saline for injection was delivered intravenously (FIG. 5). Using the simple interrupted suturing technique, the muscle, fascia, and skin were closed in three separate layers using 3-0 coated VICRYL® sutures (Ethicon Inc, Sommerville, NJ) to secure tissues over the screw head.
Post-Operative Methods
As depicted in FIG. 5, daptomycin (6 mg/kg) or saline for injection was administered daily for the next three days via an intravenous catheter placed in the marginal ear vein. A rabbit dose of 12 mg/kg of daptomycin results in a similar area under the plasma concentration curve to human dosing of 6 mg/kg. See, for example, Chambers el al., “Relationship between susceptibility to daptomycin in vitro and activity in vivo in a rabbit model of aortic valve endocarditis,” 2009, Antimicrob. Agents Chemother., 43, 1463-1467.
The daptomycin dose of 6 mg/kg was selected to allow for study of combination activity. Catheters were flushed with heparinized saline twice daily and after treatment. On day 5, rabbits were euthanized, and tibiae and implants aseptically collected. Specifically, an incision was made over the tibia from the knee to the ankle. The ankle was manually separated from the distal tibia. The muscle was dissected away from the implant and the implant removed and placed into a sterile culture tube. The tibia was fully separated from the knee joint with a bone cutting forceps, crushed into small fragments with a sterile bone cutting forceps, placed into a sterile culture tube, and frozen at -80°C.
The implants and bones were processed separately. Implants were vortexed 30 seconds in 1 mL saline, sonicated (40 kHz, 0.22 w/cm2) for 5 minutes and vortexed for an additional 30 seconds. The resultant sonicate fluid was titrated in 1:10 dilutions and 0.1 mL of each dilution plated on blood agar plates, which were incubated 24 hours in 5% CO2 at 37°C following which recovered colonies were counted and reported as log 10 cfu/implant. If no colonies were recovered, sonicate fluid was incubated with tryptic soy broth for any additional 24 hours. If broth culture was positive, confirmed by plating broth onto blood agar, quantities were reported as 0.5 logio cfu/implant; if negative, quantities were reported as 0.1 logio cfu/implant, the limit of detection of the method.
Whole tibiae bones were cryopulverized with a Mixer Mill 400 (Retsch, Newtown, PA). Specifically, crushed bones were placed into 50 mL stainless steel grinding jars containing a single
20 mm stainless steel ball and frozen in liquid nitrogen for 30 seconds. Jars were placed in a Mixer Mill and pulverized at a frequency of 28 Hz for 90 seconds. Resultant bone powder was placed into a sterile culture tube, weighed and 10 mL sterile saline added. Tubes were vortexed for 30 seconds, sonicated for 5 minutes and vortexed an additional 30 seconds. Sonicated bone homogenate was titrated in 1:10 dilutions and 0.1 ml of each dilution plated on blood agar plates, which were incubated a minimum of 24 hours in 5% CO2 and 37°C. Bone homogenates were stored at -80°C. If no growth was observed, the homogenate was thawed, and 30 mL of TSB added to the tube, which was incubated on an orbital shaker at 37°C. After 24 hours, broth cultures were subcultured onto blood agar to confirm the presence of Staphylococcus aureus. Results were report as logio cfu/gram of bone. If broth cultures were positive, they were reported as 0.5 logio cfu/gram of bone; if negative, they were reported as 0.1 logio cfu/gram of bone.
Statistical Analysis
Descriptive summaries were reported as median (minimum, maximum). Comparisons among the six groups were first performed using the Kruskal Wallis test. Due to statistically significant differences between the groups, further comparisons between the groups were performed in a pairwise manner using the Wilcoxon rank sum test. Non-parametric tests were used due to small sample size and non-normally distributed data. No adjustment was done for multiple comparisons. All tests were two sided with p-values less than 0.05 considered statistically significant. Analysis was performed using SAS software version 9.4 (SAS Inc, Cary, NC).
Results
A summary of the treatment groups as described above and the bacterial densities on bone and implant on day five are shown below in Tables 1 and 2, respectively.
1 SD (single dose)
2 DI (Day 1)
3 DAP (daptomycin)
4 IV (intravenous)
5 EXE (exebacase)
1, 2 three animals had no detectable bacteria in either sample type studied.
3, 4 Logw cfu reductions were defined as median logio cfu/gram of bone or implant of untreated groups minus median logio cfu/gram of bone or implant of treated groups. Bolded values represent greater than 3 logio cfu reductions, indicating bactericidal activity.
5 EXE (exebacase)
6 DAP (daptomycin)
As shown in FIG. 6 and Table 2, above, all treatments studied exhibited activity compared to control, whether based on bone or implant cultures (P<0.0025). For bone and implant cultures, there were >3 and >4 logio cfu reductions, respectively, for locally delivered exebacase alone, locally delivered exebacase with systemically delivered daptomycin, and locally delivered CF-296 with systemically delivered daptomycin compared to vehicle control animals. Compared to bones of systematically delivered daptomycin-treated animals, there were 1.15, 1.46, and 1.70 logio cfu reductions in bones of animals receiving locally delivered exebacase alone, locally delivered exebacase with systemically delivered daptomycin, and locally delivered CF-296 with systemic
daptomycin, respectively (FIG. 6, Table 2). In addition, bacterial densities in bones were less than 1 logio cfu in 50, 40, and 70% of animals in those same groups, respectively. Locally delivered CF-296 with systemically delivered daptomycin resulted in significant reductions in Staphylococcus aureus recovered from bones and implants compared to daptomycin (P=0.0098 and P=0.0064, respectively) or CF-296 alone (P=0.0040 and P=0.0154, respectively). Bacterial densities on implants were reduced by 3.87 (P=0.007), 3.48 (P=0.0015) and 3.17 (P=0.0064) logio cfu for animals treated with local exebacase-, local exebacase with systemic daptomycin-, and local CF-296 with systemically delivered daptomycin-treated animals, respectively, compared to systemic daptomycin-treated animals (FIG. 6, Table 2). In these groups, 60, 50, and 75% of the animals, respectively, had <1 logio cfu recovered from their implant. Locally delivered exebacase alone resulted in greater cfu reductions on implants than locally delivered CF-296 alone (P=0.0015); there was no difference between the two lysins’ activity when delivered locally in conjunction with systemic daptomycin, whether based on bone or implant cultures. Overall, locally delivered exebacase alone or with systemic daptomycin and locally delivered CF-296 with systemically delivered daptomycin showed the most activity.
Individual P-values are shown below in Table 3. Comparisons among the six groups were first performed using the Kruskal Wallis test. Due to statistically significant differences between the groups, further comparisons were performed in a pairwise manner using the Wilcoxon rank sum test. All tests were two sided with p-values less than 0.05 considered statistically significant (highlighted). Analysis was performed using SAS software version 9.4 (SAS Inc, Cary, NC).
Table 3. Statistical comparison of bacterial counts from bone and implant cultures for each treatment group.
In summary, a novel model of acute MRSA implant-associated osteomyelitis was used to test local delivery of antistaphylococcal lysin in additional to systemic delivery of daptomycin. All treatment groups had significantly reduced amounts of MRSA recovered from both bone and implants, with the most active treatments being locally delivered exebacase alone and systemic daptomycin with either of the lysins delivered locally. There was no difference between the activity of the two lysins delivered locally when administered with systemic daptomycin. Lysins, administered locally in addition to traditional therapies, may offer a potential strategy for combating Staphylococcus aureus implant-associated infections.
Claims
1. A method of treating or preventing a bone and/or joint and/or implant-associated infection comprising a Gram-positive bacteria, which method comprises: locally administering to a subject in need thereof, such as locally administering to a bone of a subject, a therapeutically effective amount of a PlySs2 lysin comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof having at least 80% identity to SEQ ID NO: 1, wherein the variant comprises bactericidal and/or bacteriostatic activity against the Grampositive bacteria.
2. The method of claim 1, wherein, the local administration comprises intramedullary local administration.
3. The method of claim 1 or claim 2, wherein the PlySs2 or variant thereof reduces an amount of Gram-positive bacteria on an implant.
4. The method of claim 1 or claim 2, wherein the PlySs2 or variant thereof reduces an amount of Gram-positive bacteria in bone.
5. The method of any one of the preceding claims, wherein the bone and/or joint infection comprises persister cells.
6. The method of any one of the preceding claims, wherein the bone and/or joint infection comprises small colony variants.
7. The method of any one of the preceding claims, wherein the bone and/or joint infection comprises a biofilm.
8. The method of any one of the preceding claims, wherein the bone and/or joint infection comprises osteomyelitis, a prosthetic joint infection or a native joint infection.
9. The method of claim 8, wherein the osteomyelitis is chronic osteomyelitis or the prosthetic joint infection is a recurring prosthetic joint infection.
10. The method of any one of the preceding claims, wherein the bone and/or joint infection comprises osteomyelitis and wherein the osteomyelitis is acute osteomyelitis, exogenous osteomyelitis, implant-associated osteomyelitis or haematogenous osteomyelitis.
11. The method of claim 10, wherein the osteomyelitis is implant-associated osteomyelitis.
12. The method of claim 11, wherein the implant- associated osteomyelitis is from an implant comprising a metal plate, a pin, a rod, a wire and/or a screw.
13. The method of any one of the preceding claims, wherein the prosthetic joint infection comprises a prosthetic hip, shoulder, elbow, ankle, or knee infection.
14. The method of any one of the preceding claims, wherein the subject suffers from obesity, diabetes mellitus, rheumatoid arthritis or is elderly.
15. The method of any one of the preceding claims, wherein the administering step further comprises co-administering a therapeutically effective amount of one or more antibiotic(s) capable of penetrating bone.
16. The method of any one of the preceding claims, wherein the one or more antibiotic(s) is administered systemically.
17. The method of claim 16, wherein the systemic administration comprises intravenous and/or oral administration.
18. The method of any one of claims 15-17, wherein the one or more antibiotic(s) is/are selected from the group consisting of a beta-lactam, an aminoglycoside, a glycopeptide, an oxazolidinone, a lipopeptide, a cephalosporin, a carbapenem, a tetracycline, a lincomycin, a phosphonic antibiotic and a sulfonamide.
19. The method of any one of claims 15-18, wherein the one or more antibiotic(s) comprises vancomycin, daptomycin, piperacillin/tazobactam, flucloxacillin, cioxacillin, an aminoglycoside, a fluoroquinolone, doxycycline, linezolid, clindamycin, trimethoprim/sulfamethoxazole, fosfomycin, rifampin, dalbavancin, and/or oritavancin.
20. The method of any one of claims 15-19, wherein the one or more antibiotic(s) comprises daptomycin.
21. The method of any one of claims 15-20, wherein the one or more antibiotic(s) does not comprise penicillin and/or metronidazole.
22. The method of any one of the preceding claims, wherein the one or more antibiotic(s) and the PlySs2 lysin or variant thereof demonstrate synergistic killing of the Gram-positive bacteria.
23. The method of any one of the preceding claims, wherein the Gram-positive bacteria comprise Staphylococcus bacteria, Enterococcus bacteria and/or Streptococcus bacteria.
24. The method of claim 23, wherein the Staphylococcus bacteria comprises Staphylococcus aureus.
25. The method of any one of the preceding claims, wherein the Gram-positive bacteria are antibiotic -resistant Gram-positive bacteria.
26. The method of claim 25, wherein the Gram-positive bacteria comprise Methicillin-resistant Staphylococcus aureus.
27. The method of any one of claims 1-23 or 25, wherein the Gram-positive bacteria comprise coagulase-negative staphylococci.
28. The method of claim 27, wherein the coagulase-negative staphylococci comprise at least one of Staphylococcus simulans, Staphylococcus caprae, Staphylococcus lugdunensis and/or Staphylococcus epidermidis.
29. The method of any one of the preceding claims, wherein the PlySs2 lysin comprises the amino acid sequence of SEQ ID NO: 1 without the initial methionine residue.
30. The method of any one of the preceding claims, wherein the PlySs2 lysin variant comprises at least one of the following amino acid sequences: SEQ ID NO: 3-17, optionally without the initial methionine residue.
31. The method of claim 30, wherein the PlySs2 lysin variant comprises the amino acid sequence of SEQ ID NO: 6.
32. The method of any one of the preceding claims, wherein the PlySs2 lysin has at least 90%, such as 95%, such as 99.5% identity to the polypeptide of SEQ ID NO: 1.
33. The method of any one of the preceding claims, wherein the Gram-positive bacteria comprises multidrug-resistant Gram-positive bacteria.
34. The method of any one of the preceding claims, wherein the Gram-positive bacteria has entered into an osteoblast.
35. A composition comprising a therapeutically effective amount of a PlySs2 lysin formulated for local administration to a subject with a bone or joint infection comprising Gram-positive bacteria, such as local administration to a bone of a subject with a bone or joint infection, comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof having at least 80% identity to SEQ ID NO: 1, optionally with one or more antibiotic(s),
wherein the PlySs2 variant comprises bactericidal and/or bacteriostatic activity against Gram-positive bacteria.
36. The composition of claim 35, wherein the local administration comprises intramedullary local administration.
37. The composition of claim 35 or claim 36, wherein the composition comprises the one or more antibiotics, and wherein the combination of the one more antibiotics and the PlySs2 lysin or variant thereof exhibits synergistic killing of the Gram-positive bacteria.
38. The composition of any one of the preceding claims, wherein the PlySs2 lysin comprises SEQ ID NO: 1 without the initial methionine residue.
39. The composition of any one of the preceding claims, wherein the one or more antibiotic(s) does not comprise penicillin and/or metronidazole.
40. The composition of any one of the preceding claims, wherein the one or more antibiotic(s) comprises vancomycin, daptomycin, piperacillin/tazobactam, flucloxacillin, cioxacillin, a cephalosporin, a carbapenem, aztreonam, an aminoglycoside, a fluoroquinolone, doxycycline, linezolid, clindamycin, trimethoprim/sulfamethoxazole, fosfomycin, rifampin, dalbavancin, and/or oritavancin.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US202163275750P | 2021-11-04 | 2021-11-04 | |
US63/275,750 | 2021-11-04 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2023081691A1 WO2023081691A1 (en) | 2023-05-11 |
WO2023081691A9 true WO2023081691A9 (en) | 2024-06-20 |
Family
ID=86242008
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2022/079130 WO2023081691A1 (en) | 2021-11-04 | 2022-11-02 | Method of treating and preventing bone and joint infections |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2023081691A1 (en) |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20100310626A1 (en) * | 2008-01-24 | 2010-12-09 | Remedor Biomed Ltd. | Erythropoietin and fibronectin compositions for bone regeneration |
WO2012145573A2 (en) * | 2011-04-21 | 2012-10-26 | Universiteit Utrecht Holding Bv | Streptococcus bacteriophage lysins for treatment of gram positive bacteria in companion animals and livestock |
MX2020008860A (en) * | 2018-02-26 | 2020-12-10 | Contrafect Corp | Modified plyss2 lysins and uses thereof. |
CN114025783A (en) * | 2019-04-11 | 2022-02-08 | 康特拉费克特公司 | Method for treating and preventing bone and joint infections |
-
2022
- 2022-11-02 WO PCT/US2022/079130 patent/WO2023081691A1/en unknown
Also Published As
Publication number | Publication date |
---|---|
WO2023081691A1 (en) | 2023-05-11 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
RU2234940C2 (en) | Method for treatment of staphylococcus infection in mammal (variant) | |
IL97344A (en) | Synergistic compositions containing béta-lactam antibiotics and cationic oligopeptides for treating infections | |
US10499655B2 (en) | Reagents and methods for inhibiting or disrupting biofilm | |
JP2021527699A (en) | Cytolysin and its derivatives resensitize Staphylococcus aureus and Gram-positive bacteria to antibiotics | |
JP2021513865A (en) | Modified LySs2 lysin and its use | |
CA2341699A1 (en) | A method for the treatment of staphylococcal disease | |
US20220193186A1 (en) | Method of treating and preventing bone and joint infections | |
WO2023081691A9 (en) | Method of treating and preventing bone and joint infections | |
US20220160842A1 (en) | Method of treating infective endocarditis | |
WO2016146037A1 (en) | Inhibitor used to inhibit/disintegrate biofilm and application thereof | |
CN107847502A (en) | Bactericidal composition | |
CN111569079B (en) | Antibody and antibiotic combined preparation for resisting staphylococcus infection | |
EP1671644A2 (en) | Pharmaceutical compositions containing Lysostaphin alone or in combination with an antibiiotic for the treatment of Staphylococcal infections | |
Safarpour-Dehkordi et al. | A comprehensive investigation of the medicinal efficacy of antimicrobial fusion peptides expressed in probiotic bacteria for the treatment of pan drug-resistant (PDR) infections | |
WO2017017177A1 (en) | Antimicrobial peptide for nosocomial infections | |
WO2022261360A1 (en) | Plyss2 lysins and variants thereof for use against multidrug resistant gram-positive bacteria | |
Lee et al. | Achromobacter xylosoxidans infection following total knee arthroplasty | |
CN115702160A (en) | Novel antibacterial peptide or peptide analogue and use thereof | |
JP2023526386A (en) | Modified PlySs2 Lysin and Antibiotic Combinations Used Against Gram-Positive Bacteria |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 22890996 Country of ref document: EP Kind code of ref document: A1 |
|
NENP | Non-entry into the national phase |
Ref country code: DE |