US20210371898A1 - Compositions and methods for determining the presence of active leukocyte cells using an electrochemical assay - Google Patents
Compositions and methods for determining the presence of active leukocyte cells using an electrochemical assay Download PDFInfo
- Publication number
- US20210371898A1 US20210371898A1 US17/342,077 US202117342077A US2021371898A1 US 20210371898 A1 US20210371898 A1 US 20210371898A1 US 202117342077 A US202117342077 A US 202117342077A US 2021371898 A1 US2021371898 A1 US 2021371898A1
- Authority
- US
- United States
- Prior art keywords
- optionally substituted
- aryl
- alkyl
- hydrogen
- substituted alkyl
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000000203 mixture Substances 0.000 title claims abstract description 43
- 238000000034 method Methods 0.000 title claims abstract description 41
- 238000007812 electrochemical assay Methods 0.000 title claims abstract description 36
- 210000000265 leukocyte Anatomy 0.000 title abstract description 73
- 239000000758 substrate Substances 0.000 claims abstract description 122
- 238000012360 testing method Methods 0.000 claims abstract description 18
- 108010000849 leukocyte esterase Proteins 0.000 claims description 104
- -1 β-methoxyethoxymethyl Chemical group 0.000 claims description 70
- 125000003118 aryl group Chemical group 0.000 claims description 51
- 208000015181 infectious disease Diseases 0.000 claims description 50
- 125000000547 substituted alkyl group Chemical group 0.000 claims description 49
- 229910052739 hydrogen Inorganic materials 0.000 claims description 33
- 239000001257 hydrogen Substances 0.000 claims description 33
- 229910052760 oxygen Inorganic materials 0.000 claims description 31
- 239000001301 oxygen Substances 0.000 claims description 28
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 27
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 26
- 239000000523 sample Substances 0.000 claims description 23
- 125000000217 alkyl group Chemical group 0.000 claims description 21
- QIGBRXMKCJKVMJ-UHFFFAOYSA-N 1,4-Benzenediol Natural products OC1=CC=C(O)C=C1 QIGBRXMKCJKVMJ-UHFFFAOYSA-N 0.000 claims description 18
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 18
- 125000002088 tosyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1C([H])([H])[H])S(*)(=O)=O 0.000 claims description 18
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 15
- 229910052757 nitrogen Inorganic materials 0.000 claims description 14
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 13
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims description 13
- 125000003368 amide group Chemical group 0.000 claims description 13
- 125000003710 aryl alkyl group Chemical group 0.000 claims description 13
- 230000000903 blocking effect Effects 0.000 claims description 13
- 150000001412 amines Chemical class 0.000 claims description 11
- 125000001584 benzyloxycarbonyl group Chemical group C(=O)(OCC1=CC=CC=C1)* 0.000 claims description 11
- 238000003745 diagnosis Methods 0.000 claims description 11
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 claims description 10
- 239000012472 biological sample Substances 0.000 claims description 10
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 9
- 238000006479 redox reaction Methods 0.000 claims description 9
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 9
- 150000002148 esters Chemical class 0.000 claims description 8
- 125000005448 ethoxyethyl group Chemical group [H]C([H])([H])C([H])([H])OC([H])([H])C([H])([H])* 0.000 claims description 8
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 8
- 150000002989 phenols Chemical class 0.000 claims description 8
- 125000001412 tetrahydropyranyl group Chemical group 0.000 claims description 8
- 125000003236 benzoyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C(*)=O 0.000 claims description 7
- 125000005931 tert-butyloxycarbonyl group Chemical group [H]C([H])([H])C(OC(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 7
- 238000003776 cleavage reaction Methods 0.000 claims description 5
- 125000001072 heteroaryl group Chemical group 0.000 claims description 5
- 125000000623 heterocyclic group Chemical group 0.000 claims description 5
- 230000007017 scission Effects 0.000 claims description 5
- LJCZNYWLQZZIOS-UHFFFAOYSA-N 2,2,2-trichlorethoxycarbonyl chloride Chemical compound ClC(=O)OCC(Cl)(Cl)Cl LJCZNYWLQZZIOS-UHFFFAOYSA-N 0.000 claims description 4
- YQTCQNIPQMJNTI-UHFFFAOYSA-N 2,2-dimethylpropan-1-one Chemical group CC(C)(C)[C]=O YQTCQNIPQMJNTI-UHFFFAOYSA-N 0.000 claims description 4
- 125000002774 3,4-dimethoxybenzyl group Chemical group [H]C1=C([H])C(=C([H])C(OC([H])([H])[H])=C1OC([H])([H])[H])C([H])([H])* 0.000 claims description 4
- 125000004172 4-methoxyphenyl group Chemical group [H]C1=C([H])C(OC([H])([H])[H])=C([H])C([H])=C1* 0.000 claims description 4
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 claims description 4
- 125000003545 alkoxy group Chemical group 0.000 claims description 4
- 125000004390 alkyl sulfonyl group Chemical group 0.000 claims description 4
- 125000004391 aryl sulfonyl group Chemical group 0.000 claims description 4
- GPKUICFDWYEPTK-UHFFFAOYSA-N methoxycyclohexatriene Chemical compound COC1=CC=C=C[CH]1 GPKUICFDWYEPTK-UHFFFAOYSA-N 0.000 claims description 4
- 125000004092 methylthiomethyl group Chemical group [H]C([H])([H])SC([H])([H])* 0.000 claims description 4
- 125000001736 nosyl group Chemical group S(=O)(=O)(C1=CC=C([N+](=O)[O-])C=C1)* 0.000 claims description 4
- UYWQUFXKFGHYNT-UHFFFAOYSA-N phenylmethyl ester of formic acid Natural products O=COCC1=CC=CC=C1 UYWQUFXKFGHYNT-UHFFFAOYSA-N 0.000 claims description 4
- 239000000126 substance Substances 0.000 claims description 4
- 125000001981 tert-butyldimethylsilyl group Chemical group [H]C([H])([H])[Si]([H])(C([H])([H])[H])[*]C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 4
- 125000002221 trityl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1C([*])(C1=C(C(=C(C(=C1[H])[H])[H])[H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 claims description 4
- 125000000472 sulfonyl group Chemical group *S(*)(=O)=O 0.000 claims description 2
- 125000004453 alkoxycarbonyl group Chemical group 0.000 claims 14
- 125000005161 aryl oxy carbonyl group Chemical group 0.000 claims 14
- ORTFAQDWJHRMNX-UHFFFAOYSA-N hydroxidooxidocarbon(.) Chemical group O[C]=O ORTFAQDWJHRMNX-UHFFFAOYSA-N 0.000 claims 14
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims 14
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims 12
- JCXJVPUVTGWSNB-UHFFFAOYSA-N Nitrogen dioxide Chemical compound O=[N]=O JCXJVPUVTGWSNB-UHFFFAOYSA-N 0.000 claims 12
- 125000003282 alkyl amino group Chemical group 0.000 claims 12
- 125000004414 alkyl thio group Chemical group 0.000 claims 12
- 125000001769 aryl amino group Chemical group 0.000 claims 12
- 125000005110 aryl thio group Chemical group 0.000 claims 12
- 229910052799 carbon Inorganic materials 0.000 claims 12
- 125000002485 formyl group Chemical group [H]C(*)=O 0.000 claims 12
- 229910052736 halogen Inorganic materials 0.000 claims 12
- 150000002367 halogens Chemical class 0.000 claims 12
- 150000002431 hydrogen Chemical class 0.000 claims 12
- 125000002768 hydroxyalkyl group Chemical group 0.000 claims 12
- 125000005027 hydroxyaryl group Chemical group 0.000 claims 12
- 125000005415 substituted alkoxy group Chemical group 0.000 claims 12
- 125000000446 sulfanediyl group Chemical group *S* 0.000 claims 12
- 125000000687 hydroquinonyl group Chemical class C1(O)=C(C=C(O)C=C1)* 0.000 claims 3
- 125000004104 aryloxy group Chemical group 0.000 claims 2
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 claims 2
- 102000004190 Enzymes Human genes 0.000 description 79
- 108090000790 Enzymes Proteins 0.000 description 79
- 229940088598 enzyme Drugs 0.000 description 79
- 101000851058 Homo sapiens Neutrophil elastase Proteins 0.000 description 61
- 102100033174 Neutrophil elastase Human genes 0.000 description 51
- 125000004169 (C1-C6) alkyl group Chemical group 0.000 description 28
- 238000003556 assay Methods 0.000 description 27
- 230000000694 effects Effects 0.000 description 23
- 0 [H][C@@](C)(*C)C(=O)OB Chemical compound [H][C@@](C)(*C)C(=O)OB 0.000 description 22
- 239000000376 reactant Substances 0.000 description 16
- 239000000047 product Substances 0.000 description 15
- 125000004191 (C1-C6) alkoxy group Chemical group 0.000 description 12
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 12
- 208000019206 urinary tract infection Diseases 0.000 description 12
- 125000003277 amino group Chemical group 0.000 description 11
- 239000000243 solution Substances 0.000 description 11
- 125000003917 carbamoyl group Chemical group [H]N([H])C(*)=O 0.000 description 10
- 230000007062 hydrolysis Effects 0.000 description 10
- 238000006460 hydrolysis reaction Methods 0.000 description 10
- 210000001179 synovial fluid Anatomy 0.000 description 10
- AZQWKYJCGOJGHM-UHFFFAOYSA-N 1,4-benzoquinone Chemical compound O=C1C=CC(=O)C=C1 AZQWKYJCGOJGHM-UHFFFAOYSA-N 0.000 description 9
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 9
- 238000001952 enzyme assay Methods 0.000 description 9
- 102000052502 human ELANE Human genes 0.000 description 9
- 210000002700 urine Anatomy 0.000 description 9
- 206010062070 Peritonitis bacterial Diseases 0.000 description 8
- 125000002252 acyl group Chemical group 0.000 description 8
- 229960003767 alanine Drugs 0.000 description 8
- 239000003792 electrolyte Substances 0.000 description 8
- 125000004203 4-hydroxyphenyl group Chemical group [H]OC1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 7
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 7
- 208000024891 symptom Diseases 0.000 description 7
- 125000005913 (C3-C6) cycloalkyl group Chemical group 0.000 description 6
- 206010060968 Arthritis infective Diseases 0.000 description 6
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical group C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 6
- 125000004093 cyano group Chemical group *C#N 0.000 description 6
- 238000010931 ester hydrolysis Methods 0.000 description 6
- 239000012530 fluid Substances 0.000 description 6
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 6
- 238000005259 measurement Methods 0.000 description 6
- 125000006239 protecting group Chemical group 0.000 description 6
- 238000012216 screening Methods 0.000 description 6
- 229940126062 Compound A Drugs 0.000 description 5
- NLDMNSXOCDLTTB-UHFFFAOYSA-N Heterophylliin A Natural products O1C2COC(=O)C3=CC(O)=C(O)C(O)=C3C3=C(O)C(O)=C(O)C=C3C(=O)OC2C(OC(=O)C=2C=C(O)C(O)=C(O)C=2)C(O)C1OC(=O)C1=CC(O)=C(O)C(O)=C1 NLDMNSXOCDLTTB-UHFFFAOYSA-N 0.000 description 5
- 235000004279 alanine Nutrition 0.000 description 5
- 229940024606 amino acid Drugs 0.000 description 5
- 235000001014 amino acid Nutrition 0.000 description 5
- 150000001413 amino acids Chemical class 0.000 description 5
- 150000001875 compounds Chemical class 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 150000005690 diesters Chemical class 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 238000006911 enzymatic reaction Methods 0.000 description 5
- 210000000440 neutrophil Anatomy 0.000 description 5
- 108090000765 processed proteins & peptides Chemical group 0.000 description 5
- 230000000405 serological effect Effects 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- 125000004738 (C1-C6) alkyl sulfinyl group Chemical group 0.000 description 4
- 125000004739 (C1-C6) alkylsulfonyl group Chemical group 0.000 description 4
- 125000006700 (C1-C6) alkylthio group Chemical group 0.000 description 4
- 125000004737 (C1-C6) haloalkoxy group Chemical group 0.000 description 4
- 125000004749 (C1-C6) haloalkylsulfinyl group Chemical group 0.000 description 4
- 125000004741 (C1-C6) haloalkylsulfonyl group Chemical group 0.000 description 4
- 125000006771 (C1-C6) haloalkylthio group Chemical group 0.000 description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 description 4
- 229940061720 alpha hydroxy acid Drugs 0.000 description 4
- 210000003567 ascitic fluid Anatomy 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 125000005347 halocycloalkyl group Chemical group 0.000 description 4
- 125000005647 linker group Chemical group 0.000 description 4
- 238000007254 oxidation reaction Methods 0.000 description 4
- 230000009467 reduction Effects 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 206010003445 Ascites Diseases 0.000 description 3
- 108010074051 C-Reactive Protein Proteins 0.000 description 3
- 102100032752 C-reactive protein Human genes 0.000 description 3
- 108010028275 Leukocyte Elastase Proteins 0.000 description 3
- 102000016799 Leukocyte elastase Human genes 0.000 description 3
- 229910003813 NRa Inorganic materials 0.000 description 3
- AMNPXXIGUOKIPP-UHFFFAOYSA-N [4-(carbamothioylamino)phenyl]thiourea Chemical compound NC(=S)NC1=CC=C(NC(N)=S)C=C1 AMNPXXIGUOKIPP-UHFFFAOYSA-N 0.000 description 3
- XFMGWUKGHCDTMD-PMACEKPBSA-N [4-[(2S)-2-(4-methylphenyl)sulfonyloxypropanoyl]oxyphenyl] (2S)-2-(4-methylphenyl)sulfonyloxypropanoate Chemical compound S(=O)(=O)(C1=CC=C(C)C=C1)O[C@H](C(=O)OC1=CC=C(C=C1)OC([C@H](C)OS(=O)(=O)C1=CC=C(C)C=C1)=O)C XFMGWUKGHCDTMD-PMACEKPBSA-N 0.000 description 3
- 235000008206 alpha-amino acids Nutrition 0.000 description 3
- 125000000539 amino acid group Chemical group 0.000 description 3
- 230000003115 biocidal effect Effects 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 125000001033 ether group Chemical group 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 230000003647 oxidation Effects 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 3
- 210000004911 serous fluid Anatomy 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 238000012421 spiking Methods 0.000 description 3
- 238000006467 substitution reaction Methods 0.000 description 3
- 229910052717 sulfur Inorganic materials 0.000 description 3
- 125000000026 trimethylsilyl group Chemical group [H]C([H])([H])[Si]([*])(C([H])([H])[H])C([H])([H])[H] 0.000 description 3
- 125000006823 (C1-C6) acyl group Chemical group 0.000 description 2
- 125000004454 (C1-C6) alkoxycarbonyl group Chemical group 0.000 description 2
- 125000004844 (C1-C6) alkoxyimino group Chemical group 0.000 description 2
- 125000000171 (C1-C6) haloalkyl group Chemical group 0.000 description 2
- 125000006766 (C2-C6) alkynyloxy group Chemical group 0.000 description 2
- 125000006643 (C2-C6) haloalkenyl group Chemical group 0.000 description 2
- 125000006644 (C2-C6) haloalkynyl group Chemical group 0.000 description 2
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 2
- ZEQNFZGURXXSDL-UHFFFAOYSA-N BNC(=O)CCCCC Chemical compound BNC(=O)CCCCC ZEQNFZGURXXSDL-UHFFFAOYSA-N 0.000 description 2
- 125000000882 C2-C6 alkenyl group Chemical group 0.000 description 2
- 125000003320 C2-C6 alkenyloxy group Chemical group 0.000 description 2
- 125000003601 C2-C6 alkynyl group Chemical group 0.000 description 2
- WLAJKJUTHGJVCW-LLVKDONJSA-N COC(=O)C1=CN=CC(S(=O)(=O)C[C@@H](C)C(=O)OC2=CC=C(O)C=C2)=C1 Chemical compound COC(=O)C1=CN=CC(S(=O)(=O)C[C@@H](C)C(=O)OC2=CC=C(O)C=C2)=C1 WLAJKJUTHGJVCW-LLVKDONJSA-N 0.000 description 2
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 2
- 108090000371 Esterases Proteins 0.000 description 2
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 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
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-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
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 2
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-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
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 2
- NMMIHXMBOZYNET-UHFFFAOYSA-N Methyl picolinate Chemical compound COC(=O)C1=CC=CC=N1 NMMIHXMBOZYNET-UHFFFAOYSA-N 0.000 description 2
- 208000005228 Pericardial Effusion Diseases 0.000 description 2
- 108010064785 Phospholipases Proteins 0.000 description 2
- 102000015439 Phospholipases Human genes 0.000 description 2
- 206010036790 Productive cough Diseases 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 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
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 125000003806 alkyl carbonyl amino group Chemical group 0.000 description 2
- 125000005196 alkyl carbonyloxy group Chemical group 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 239000004599 antimicrobial Substances 0.000 description 2
- 235000009582 asparagine Nutrition 0.000 description 2
- 229960001230 asparagine Drugs 0.000 description 2
- 210000003651 basophil Anatomy 0.000 description 2
- YGYLYUIRSJSFJS-QMMMGPOBSA-N benzyl (2s)-2-aminopropanoate Chemical compound C[C@H](N)C(=O)OCC1=CC=CC=C1 YGYLYUIRSJSFJS-QMMMGPOBSA-N 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 210000001124 body fluid Anatomy 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 208000019425 cirrhosis of liver Diseases 0.000 description 2
- 125000000000 cycloalkoxy group Chemical group 0.000 description 2
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 2
- 210000000959 ear middle Anatomy 0.000 description 2
- 239000008151 electrolyte solution Substances 0.000 description 2
- 210000003979 eosinophil Anatomy 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 125000005843 halogen group Chemical group 0.000 description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 2
- 238000005984 hydrogenation reaction Methods 0.000 description 2
- 125000004356 hydroxy functional group Chemical group O* 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 229960000310 isoleucine Drugs 0.000 description 2
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- MRZZIJUGMGGPLK-UHFFFAOYSA-N methyl 5-chlorosulfonylpyridine-3-carboxylate Chemical compound COC(=O)C1=CN=CC(S(Cl)(=O)=O)=C1 MRZZIJUGMGGPLK-UHFFFAOYSA-N 0.000 description 2
- 210000001616 monocyte Anatomy 0.000 description 2
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 210000004912 pericardial fluid Anatomy 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
- 210000004910 pleural fluid Anatomy 0.000 description 2
- 235000019260 propionic acid Nutrition 0.000 description 2
- 150000004053 quinones Chemical class 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 125000006850 spacer group Chemical group 0.000 description 2
- 210000003802 sputum Anatomy 0.000 description 2
- 208000024794 sputum Diseases 0.000 description 2
- 125000003107 substituted aryl group Chemical group 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- TZHVYFBSLOMRCU-YFKPBYRVSA-N tert-butyl (2s)-2-aminopropanoate Chemical compound C[C@H](N)C(=O)OC(C)(C)C TZHVYFBSLOMRCU-YFKPBYRVSA-N 0.000 description 2
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 2
- 238000011285 therapeutic regimen Methods 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
- 239000004474 valine Substances 0.000 description 2
- 230000000007 visual effect Effects 0.000 description 2
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 1
- SOVUOXKZCCAWOJ-HJYUBDRYSA-N (4s,4as,5ar,12ar)-9-[[2-(tert-butylamino)acetyl]amino]-4,7-bis(dimethylamino)-1,10,11,12a-tetrahydroxy-3,12-dioxo-4a,5,5a,6-tetrahydro-4h-tetracene-2-carboxamide Chemical compound C1C2=C(N(C)C)C=C(NC(=O)CNC(C)(C)C)C(O)=C2C(O)=C2[C@@H]1C[C@H]1[C@H](N(C)C)C(=O)C(C(N)=O)=C(O)[C@@]1(O)C2=O SOVUOXKZCCAWOJ-HJYUBDRYSA-N 0.000 description 1
- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 description 1
- 150000005208 1,4-dihydroxybenzenes Chemical class 0.000 description 1
- MUVQKFGNPGZBII-UHFFFAOYSA-N 1-anthrol Chemical class C1=CC=C2C=C3C(O)=CC=CC3=CC2=C1 MUVQKFGNPGZBII-UHFFFAOYSA-N 0.000 description 1
- FBKKYZMTAAQTJO-UHFFFAOYSA-N 1h-1,10-phenanthrolin-2-one Chemical class C1=CC=NC2=C(NC(=O)C=C3)C3=CC=C21 FBKKYZMTAAQTJO-UHFFFAOYSA-N 0.000 description 1
- ZIIUUSVHCHPIQD-UHFFFAOYSA-N 2,4,6-trimethyl-N-[3-(trifluoromethyl)phenyl]benzenesulfonamide Chemical compound CC1=CC(C)=CC(C)=C1S(=O)(=O)NC1=CC=CC(C(F)(F)F)=C1 ZIIUUSVHCHPIQD-UHFFFAOYSA-N 0.000 description 1
- CDAWCLOXVUBKRW-UHFFFAOYSA-N 2-aminophenol Chemical class NC1=CC=CC=C1O CDAWCLOXVUBKRW-UHFFFAOYSA-N 0.000 description 1
- IWTFOFMTUOBLHG-UHFFFAOYSA-N 2-methoxypyridine Chemical compound COC1=CC=CC=N1 IWTFOFMTUOBLHG-UHFFFAOYSA-N 0.000 description 1
- 229960000549 4-dimethylaminophenol Drugs 0.000 description 1
- 206010000097 Abdominal tenderness Diseases 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 208000006820 Arthralgia Diseases 0.000 description 1
- BBBIYJQXXSUBPD-MFNIIGHSSA-N CC(C)C(CC(=O)C1CCCN1C(=O)[C@H](C)NC(=O)[C@H](C)CC(=O)CCC(=O)O)C(=O)NC1=CC=C(NC(=O)[C@@H](NC(=O)C2CCCN2C(=O)[C@H](C)CC(=O)[C@H](C)NC(=O)CCC(=O)O)C(C)C)C=C1 Chemical compound CC(C)C(CC(=O)C1CCCN1C(=O)[C@H](C)NC(=O)[C@H](C)CC(=O)CCC(=O)O)C(=O)NC1=CC=C(NC(=O)[C@@H](NC(=O)C2CCCN2C(=O)[C@H](C)CC(=O)[C@H](C)NC(=O)CCC(=O)O)C(C)C)C=C1 BBBIYJQXXSUBPD-MFNIIGHSSA-N 0.000 description 1
- YQUQWHNMBPIWGK-UHFFFAOYSA-N CC(C)C1=CC=C(O)C=C1 Chemical compound CC(C)C1=CC=C(O)C=C1 YQUQWHNMBPIWGK-UHFFFAOYSA-N 0.000 description 1
- UMFMAUGZTMNSMW-UHFFFAOYSA-N CC(C)C1=CC=CN=C1.CC1=CC=C(C(C)C)C=N1.CC1=CN=CC(C(C)C)=C1 Chemical compound CC(C)C1=CC=CN=C1.CC1=CC=C(C(C)C)C=N1.CC1=CN=CC(C(C)C)=C1 UMFMAUGZTMNSMW-UHFFFAOYSA-N 0.000 description 1
- DKMAFFCLBPUKOE-PMACEKPBSA-N CC1=CC=C(S(=O)(=O)N[C@@H](C)C(=O)OC2=CC=C(OC(=O)[C@H](C)NS(=O)(=O)C3=CC=C(C)C=C3)C=C2)C=C1 Chemical compound CC1=CC=C(S(=O)(=O)N[C@@H](C)C(=O)OC2=CC=C(OC(=O)[C@H](C)NS(=O)(=O)C3=CC=C(C)C=C3)C=C2)C=C1 DKMAFFCLBPUKOE-PMACEKPBSA-N 0.000 description 1
- OVYGYLMYODHQHA-SDIJQETESA-N CC1=CC=C(S(=O)(=O)N[C@@H](C)C(=O)OC2=CC=C(OC(=O)[C@H](C)NS(=O)(=O)C3=CC=C(C)C=C3)C=C2)C=C1.CC1=CC=C(S(=O)(=O)O[C@@H](C)C(=O)OC2=CC=C(OC(=O)[C@H](C)OS(=O)(=O)C3=CC=C(C)C=C3)C=C2)C=C1 Chemical compound CC1=CC=C(S(=O)(=O)N[C@@H](C)C(=O)OC2=CC=C(OC(=O)[C@H](C)NS(=O)(=O)C3=CC=C(C)C=C3)C=C2)C=C1.CC1=CC=C(S(=O)(=O)O[C@@H](C)C(=O)OC2=CC=C(OC(=O)[C@H](C)OS(=O)(=O)C3=CC=C(C)C=C3)C=C2)C=C1 OVYGYLMYODHQHA-SDIJQETESA-N 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 102000005572 Cathepsin A Human genes 0.000 description 1
- 108010059081 Cathepsin A Proteins 0.000 description 1
- 102000004225 Cathepsin B Human genes 0.000 description 1
- 108090000712 Cathepsin B Proteins 0.000 description 1
- 102000005600 Cathepsins Human genes 0.000 description 1
- 108010084457 Cathepsins Proteins 0.000 description 1
- 229930186147 Cephalosporin Natural products 0.000 description 1
- 102000019034 Chemokines Human genes 0.000 description 1
- 108010012236 Chemokines Proteins 0.000 description 1
- 108090000317 Chymotrypsin Proteins 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 201000003883 Cystic fibrosis Diseases 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108010013198 Daptomycin Proteins 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 102000013382 Gelatinases Human genes 0.000 description 1
- 108010026132 Gelatinases Proteins 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 108090000174 Interleukin-10 Proteins 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 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
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 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
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 102000011720 Lysophospholipase Human genes 0.000 description 1
- 108020002496 Lysophospholipase Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 102000002274 Matrix Metalloproteinases Human genes 0.000 description 1
- 108010000684 Matrix Metalloproteinases Proteins 0.000 description 1
- 102000001776 Matrix metalloproteinase-9 Human genes 0.000 description 1
- 108010015302 Matrix metalloproteinase-9 Proteins 0.000 description 1
- 102000005741 Metalloproteases Human genes 0.000 description 1
- 108010006035 Metalloproteases Proteins 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- QPFYXYFORQJZEC-FOCLMDBBSA-N Phenazopyridine Chemical compound NC1=NC(N)=CC=C1\N=N\C1=CC=CC=C1 QPFYXYFORQJZEC-FOCLMDBBSA-N 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 208000008718 Pyuria Diseases 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 102000012479 Serine Proteases Human genes 0.000 description 1
- 108010022999 Serine Proteases Proteins 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 229910021607 Silver chloride Inorganic materials 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- 206010047786 Vulvovaginal discomfort Diseases 0.000 description 1
- 230000003187 abdominal effect Effects 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 108010087049 alanyl-alanyl-prolyl-valine Proteins 0.000 description 1
- 229910001854 alkali hydroxide Inorganic materials 0.000 description 1
- 150000008044 alkali metal hydroxides Chemical class 0.000 description 1
- 150000001370 alpha-amino acid derivatives Chemical group 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 238000004082 amperometric method Methods 0.000 description 1
- 238000004458 analytical method 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
- 238000011882 arthroplasty Methods 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 125000005334 azaindolyl group Chemical group N1N=C(C2=CC=CC=C12)* 0.000 description 1
- 239000000987 azo dye Substances 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 125000000499 benzofuranyl group Chemical group O1C(=CC2=C1C=CC=C2)* 0.000 description 1
- 125000004601 benzofurazanyl group Chemical group N1=C2C(=NO1)C(=CC=C2)* 0.000 description 1
- 125000004618 benzofuryl group Chemical group O1C(=CC2=C1C=CC=C2)* 0.000 description 1
- 125000005872 benzooxazolyl group Chemical group 0.000 description 1
- 125000001164 benzothiazolyl group Chemical group S1C(=NC2=C1C=CC=C2)* 0.000 description 1
- 125000004196 benzothienyl group Chemical group S1C(=CC2=C1C=CC=C2)* 0.000 description 1
- 125000003354 benzotriazolyl group Chemical group N1N=NC2=C1C=CC=C2* 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
- 239000013060 biological fluid Substances 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 238000004820 blood count Methods 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- PPKJUHVNTMYXOD-PZGPJMECSA-N c49ws9n75l Chemical compound O=C([C@@H]1N(C2=O)CC[C@H]1S(=O)(=O)CCN(CC)CC)O[C@H](C(C)C)[C@H](C)\C=C\C(=O)NC\C=C\C(\C)=C\[C@@H](O)CC(=O)CC1=NC2=CO1.N([C@@H]1C(=O)N[C@@H](C(N2CCC[C@H]2C(=O)N(C)[C@@H](CC=2C=CC(=CC=2)N(C)C)C(=O)N2C[C@@H](CS[C@H]3C4CCN(CC4)C3)C(=O)C[C@H]2C(=O)N[C@H](C(=O)O[C@@H]1C)C=1C=CC=CC=1)=O)CC)C(=O)C1=NC=CC=C1O PPKJUHVNTMYXOD-PZGPJMECSA-N 0.000 description 1
- 125000000837 carbohydrate group Chemical group 0.000 description 1
- 229910021387 carbon allotrope Inorganic materials 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 229940124587 cephalosporin Drugs 0.000 description 1
- 150000001780 cephalosporins Chemical class 0.000 description 1
- 210000003756 cervix mucus Anatomy 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 229960002376 chymotrypsin Drugs 0.000 description 1
- 125000000259 cinnolinyl group Chemical group N1=NC(=CC2=CC=CC=C12)* 0.000 description 1
- 238000009535 clinical urine test Methods 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 238000007398 colorimetric assay Methods 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 229960002488 dalbavancin Drugs 0.000 description 1
- 108700009376 dalbavancin Proteins 0.000 description 1
- 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 description 1
- 229960005484 daptomycin Drugs 0.000 description 1
- 238000001804 debridement Methods 0.000 description 1
- 238000007257 deesterification reaction Methods 0.000 description 1
- 239000012954 diazonium Substances 0.000 description 1
- 150000001989 diazonium salts Chemical class 0.000 description 1
- WBKFWQBXFREOFH-UHFFFAOYSA-N dichloromethane;ethyl acetate Chemical compound ClCCl.CCOC(C)=O WBKFWQBXFREOFH-UHFFFAOYSA-N 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 206010013990 dysuria Diseases 0.000 description 1
- 238000002848 electrochemical method Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 125000004185 ester group Chemical group 0.000 description 1
- NLFBCYMMUAKCPC-KQQUZDAGSA-N ethyl (e)-3-[3-amino-2-cyano-1-[(e)-3-ethoxy-3-oxoprop-1-enyl]sulfanyl-3-oxoprop-1-enyl]sulfanylprop-2-enoate Chemical compound CCOC(=O)\C=C\SC(=C(C#N)C(N)=O)S\C=C\C(=O)OCC NLFBCYMMUAKCPC-KQQUZDAGSA-N 0.000 description 1
- 235000019439 ethyl acetate Nutrition 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 125000003838 furazanyl group Chemical group 0.000 description 1
- 125000004612 furopyridinyl group Chemical group O1C(=CC2=C1C=CC=N2)* 0.000 description 1
- 125000002541 furyl group Chemical group 0.000 description 1
- 239000011521 glass Substances 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
- 239000008241 heterogeneous mixture Substances 0.000 description 1
- 150000004679 hydroxides Chemical class 0.000 description 1
- 125000004464 hydroxyphenyl group Chemical group 0.000 description 1
- 125000002883 imidazolyl group Chemical group 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 125000003453 indazolyl group Chemical group N1N=C(C2=C1C=CC=C2)* 0.000 description 1
- 125000003406 indolizinyl group Chemical group C=1(C=CN2C=CC=CC12)* 0.000 description 1
- 125000001041 indolyl group Chemical group 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 230000007794 irritation Effects 0.000 description 1
- 125000000904 isoindolyl group Chemical group C=1(NC=C2C=CC=CC12)* 0.000 description 1
- 125000005956 isoquinolyl group Chemical group 0.000 description 1
- 125000001786 isothiazolyl group Chemical group 0.000 description 1
- TYZROVQLWOKYKF-ZDUSSCGKSA-N linezolid Chemical compound O=C1O[C@@H](CNC(=O)C)CN1C(C=C1F)=CC=C1N1CCOCC1 TYZROVQLWOKYKF-ZDUSSCGKSA-N 0.000 description 1
- 229960003907 linezolid Drugs 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 230000003340 mental effect Effects 0.000 description 1
- 229910044991 metal oxide Inorganic materials 0.000 description 1
- 150000004706 metal oxides Chemical class 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- VYQNWZOUAUKGHI-UHFFFAOYSA-N monobenzone Chemical compound C1=CC(O)=CC=C1OCC1=CC=CC=C1 VYQNWZOUAUKGHI-UHFFFAOYSA-N 0.000 description 1
- 229960000990 monobenzone Drugs 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- OCIDXARMXNJACB-UHFFFAOYSA-N n'-phenylethane-1,2-diamine Chemical compound NCCNC1=CC=CC=C1 OCIDXARMXNJACB-UHFFFAOYSA-N 0.000 description 1
- 125000004593 naphthyridinyl group Chemical group N1=C(C=CC2=CC=CN=C12)* 0.000 description 1
- 229960000564 nitrofurantoin Drugs 0.000 description 1
- NXFQHRVNIOXGAQ-YCRREMRBSA-N nitrofurantoin Chemical compound O1C([N+](=O)[O-])=CC=C1\C=N\N1C(=O)NC(=O)C1 NXFQHRVNIOXGAQ-YCRREMRBSA-N 0.000 description 1
- 150000002831 nitrogen free-radicals Chemical class 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 229910000510 noble metal Inorganic materials 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 229940127249 oral antibiotic Drugs 0.000 description 1
- 230000000399 orthopedic effect Effects 0.000 description 1
- 208000005923 otitis media with effusion Diseases 0.000 description 1
- 125000001715 oxadiazolyl group Chemical group 0.000 description 1
- 125000002971 oxazolyl group Chemical group 0.000 description 1
- 229910052763 palladium Inorganic materials 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 150000002960 penicillins Chemical class 0.000 description 1
- 229960001181 phenazopyridine Drugs 0.000 description 1
- 125000004592 phthalazinyl group Chemical group C1(=NN=CC2=CC=CC=C12)* 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- LVTJOONKWUXEFR-FZRMHRINSA-N protoneodioscin Natural products O(C[C@@H](CC[C@]1(O)[C@H](C)[C@@H]2[C@]3(C)[C@H]([C@H]4[C@@H]([C@]5(C)C(=CC4)C[C@@H](O[C@@H]4[C@H](O[C@H]6[C@@H](O)[C@@H](O)[C@@H](O)[C@H](C)O6)[C@@H](O)[C@H](O[C@H]6[C@@H](O)[C@@H](O)[C@@H](O)[C@H](C)O6)[C@H](CO)O4)CC5)CC3)C[C@@H]2O1)C)[C@H]1[C@H](O)[C@H](O)[C@H](O)[C@@H](CO)O1 LVTJOONKWUXEFR-FZRMHRINSA-N 0.000 description 1
- 125000000561 purinyl group Chemical group N1=C(N=C2N=CNC2=C1)* 0.000 description 1
- 125000003373 pyrazinyl group Chemical group 0.000 description 1
- 125000003226 pyrazolyl group Chemical group 0.000 description 1
- 125000002098 pyridazinyl group Chemical group 0.000 description 1
- 125000004076 pyridyl group Chemical group 0.000 description 1
- 125000000714 pyrimidinyl group Chemical group 0.000 description 1
- 125000000168 pyrrolyl group Chemical group 0.000 description 1
- 125000002294 quinazolinyl group Chemical group N1=C(N=CC2=CC=CC=C12)* 0.000 description 1
- 125000005493 quinolyl group Chemical group 0.000 description 1
- 125000001567 quinoxalinyl group Chemical group N1=C(C=NC2=CC=CC=C12)* 0.000 description 1
- 229940052337 quinupristin/dalfopristin Drugs 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- HKZLPVFGJNLROG-UHFFFAOYSA-M silver monochloride Chemical compound [Cl-].[Ag+] HKZLPVFGJNLROG-UHFFFAOYSA-M 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- VQGTYFGVHQDLGM-UHFFFAOYSA-N tert-butyl 2,5-dihydroxybenzoate Chemical compound CC(C)(C)OC(=O)C1=CC(O)=CC=C1O VQGTYFGVHQDLGM-UHFFFAOYSA-N 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- 229940040944 tetracyclines Drugs 0.000 description 1
- 125000003831 tetrazolyl group Chemical group 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 125000001113 thiadiazolyl group Chemical group 0.000 description 1
- 125000000335 thiazolyl group Chemical group 0.000 description 1
- 125000001544 thienyl group Chemical group 0.000 description 1
- 229960004089 tigecycline Drugs 0.000 description 1
- 125000004306 triazinyl group Chemical group 0.000 description 1
- 125000001425 triazolyl group Chemical group 0.000 description 1
- 208000000143 urethritis Diseases 0.000 description 1
- 206010046901 vaginal discharge Diseases 0.000 description 1
- 238000001075 voltammogram Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000010626 work up procedure Methods 0.000 description 1
- 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 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
- C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
- C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
- C07D213/79—Acids; Esters
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
- C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
- C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
- C07D213/79—Acids; Esters
- C07D213/80—Acids; Esters in position 3
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/001—Enzyme electrodes
- C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/483—Physical analysis of biological material
- G01N33/487—Physical analysis of biological material of liquid biological material
- G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
- G01N33/48721—Investigating individual macromolecules, e.g. by translocation through nanopores
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/24—Immunology or allergic disorders
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/26—Infectious diseases, e.g. generalised sepsis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/50—Determining the risk of developing a disease
Definitions
- the present disclosure relates to a novel application of an electrochemical assay for the determination of the activity of leukocyte cells within a test sample. More particularly, the present disclosure relates to novel methods and kits for determining the activity of enzymes released by active leukocyte cells, especially leukocyte esterase and human neutrophil elastase, in a patient at risk of developing an infection.
- Known assays for LE are chromogenic, in that the presence of enzyme activity is reported based upon a color change.
- a color test strip can be matched to a color chart with 3-4 increments of increasing color intensity (from none to 2+/3+), which represents a LE concentration of 30 ng/mL to greater than 1500 ng/mL.
- 3-4 increments of increasing color intensity from none to 2+/3+
- LE concentration of 30 ng/mL to greater than 1500 ng/mL.
- there are clear disadvantages to a colorimetric assay With only 3-4 available color intensity increments, resolution of differences in leukocyte esterase concentration may be quite difficult. In addition, inter-rater and even intra-rater reliability in classifying such color increments may be poor.
- dipstick results are less definitive (trace or 1+); test results, in such cases, may be too unreliable for making treatment decisions.
- the utility of dipstick results is limited to cases in which leukocyte esterase activity is exceedingly high. Any substance that changes the color of urine (e.g. nitrofurantoin, phenazopyridine) also affects dipstick readings.
- the disclosure is directed towards a method for screening, detecting and confirming an infection in patients at risk of an infection or those patients who have already exhibited symptoms associated with an infection.
- the method follows the step of obtaining a sample from the subject in need, detecting the presence or absence of leukocyte markers in the sample, and instituting a therapeutic regimen based on the degree and presence of the leukocyte markers in the sample.
- the leukocyte markers can be one or any combinations of such markers as cytokines, chemokines, oxygen and nitrogen radicals, leukocyte elastase, leukocyte esterase, neutrophil elastase, gelatinases, IL-1 ⁇ , metalloproteinases (MMPs), cathepsins, such as cathepsin A and cathepsin B, phospholipases, such as, for example, phospholipase A and phospholipase B.
- MMPs metalloproteinases
- cathepsins such as cathepsin A and cathepsin B
- phospholipases such as, for example, phospholipase A and phospholipase B.
- the present disclosure is directed to a composition comprising a leukocyte enzyme or specifically a neutrophil enzyme substrate.
- the leukocyte enzyme comprises leukocyte esterase (“LE”).
- the leukocyte enzyme substrate comprises an LE substrate.
- the leukocyte enzyme comprises human neutrophil elastase (“HNE”).
- the leukocyte enzyme substrate comprises an HNE substrate.
- the composition comprises both an LE substrate and a HNE substrate.
- the composition may contain additional substrates specific to other enzymes or biomarkers than LE and HNE.
- the substrates demonstrate specificity for LE or HNE.
- the substrate comprises a monoester, the monoester being one of an ⁇ -amino acid ester, such as an alanine ester, or an ⁇ -hydroxy acid ester, such as a lactate ester, with specificity for leukocyte esterases, the monoester having a first moiety for participating in a redox reaction, and a second moiety comprising an amine or alcohol blocking group, which masks the functional group (i.e., amine or alcohol) to prevent undesirable chemical reactivity.
- an ⁇ -amino acid ester such as an alanine ester
- an ⁇ -hydroxy acid ester such as a lactate ester
- the substrates may follow Formula I as depicted below:
- A comprises an ether group (i.e. —O—) or an amine group (i.e., NR a , where R a is a H or an optionally substituted alkyl, aryl, or aralkyl group)
- B comprises a moiety capable of participating in a redox reaction
- C comprises an alcohol or amine blocking group.
- A comprises an amino group.
- A comprises an ether group.
- B comprises a redox active alcohol intermediate.
- B comprises a phenol.
- B comprises a substituted phenol.
- C comprises a tosyl protecting group.
- the oxygen linking B in Formula I is substituted with an amino group.
- B comprises aminophenyl.
- B comprises a substituted aminophenyl.
- the LE substrate comprises a compound as described in Formula II below:
- X1 and X2 are independently O, S or NRa.
- Ra is an H, an alkyl or an aryl group.
- X1 and X2 can be both oxygen or both NRa.
- one of X1 and X2 is oxygen and the other is NRa.
- Y1 and Y2 are independently O or NRa. Ra is as described above. Y1 and Y2 can be both oxygen or both NRa. Alternatively, one of Y1 and Y2 is oxygen and the other is NRa.
- R1 and R2 are independently an alkyl or an aryl group or a substituted alkyl, a substituted aryl or a protecting group. In some embodiments, R1 and R2 are both methyl. In some embodiments, R1 and R2 may be a tosyl. In some embodiments, R2 may be a tosyl.
- R3 and R4 are independently an alkyl, a protecting group or a peptide moiety.
- a protecting group includes tosyl, benzoyl, benzyl, trimethylsilyl, [bis-(4-methoxyphenyl)phenylmethyl], carbobenzyloxy, and tert-Butyloxycarbonyl, 9-Fluorenylmethyloxycarbonyl.
- R4 may be a tosyl.
- the peptide moiety can include any combination of natural and/or non-natural amino acids.
- Each of the R5 on the ring is independently a halogen atom; a hydroxyl group; a C1-C6 alkyl group; a C3-C6 cycloalkyl group; a C3-C6 cycloalkyl C1-C6 alkyl group; a C2-C6 alkenyl group; a C2-C6 alkynyl group; a C1-C6 haloalkyl group (including trifluoro C1-C6alkyl); a C2-C6 haloalkenyl group; a C2-C6 haloalkynyl group; a C3-C6 halocycloalkyl group; a C3-C6 halocycloalkyl C1-C6 alkyl group; a C1-C6 alkoxy group; a C3-C6 cycloalkyloxy group; a C2-C6 alkenyloxy group; a C 2 -C 6 alky
- the LE substrate comprises 4-((tosyl-L-alanyl)oxy)phenyl tosyl-L-alaninate. In some embodiments, the LE substrate comprises 4-(((S)-2-(tosyloxy)propanoyl)oxy)phenyl (S)-2-(tosyloxy)propanoate. In some embodiments, the LE substrate comprises a phenylenediamine variant of one of 4-((tosyl-L-alanyl)oxy)phenyl tosyl-L-alaninate and 4-(((S)-2-(tosyloxy)propanoyl)oxy)phenyl (S)-2-(tosyloxy)propanoate.
- the HNE substrate comprises a compound as described in Formula III below:
- a 1 -A 2 -A 3 -A 4 represent a core tetrapeptide scaffold sequence which serves as the enzyme active site
- B comprises a moiety capable of participating in a redox reaction
- C comprises an acyl group.
- a 1 -A 2 -A 3 -A 4 comprise AAPV (SEQ ID NO: 1).
- SEQ ID NO: 1 has conservative substitutions.
- B comprises a redox active alcohol intermediate.
- B comprises a derivative of phenol.
- B comprises a quinone.
- B comprises a hydroquinone.
- B comprises a substituted quinone or a substituted hydroquinone.
- C comprises N-methyoxysuccinyl.
- the HNE substrate comprises 3- ⁇ [(1S)-1- ⁇ [(2S)-1-(5- ⁇ [(1S)-1-( ⁇ 4-[(2S)-2-( ⁇ 1-[(2S)-2-[(2S)-2-(3-carboxypropanamido)propanamido]propanoyl]pyrrolidin-2-yl ⁇ formamido)-3-methylbutanamido]phenyl ⁇ carbamoyl)-2-methylpropyl]carbamoyl ⁇ imidazolidin-1-yl)-1-oxopropan-2-yl]carbamoyl ⁇ ethyl]carbamoyl ⁇ propanoic acid.
- the leukocyte enzyme substrate is included in an assay.
- the assay comprises an electrochemical assay.
- the assay may include a colorimetric step in combination with the electrochemical assay.
- the electrochemical assay comprises an internally calibrated electrochemical continuous enzyme assay (“ICECEA”).
- the electrochemical assay comprises a leukocyte substrate of the present disclosure and an electrochemical measuring device.
- the electrochemical measuring device includes a working electrode, a reference electrode, and an auxiliary electrode.
- the present disclosure is directed to a method of detecting the presence of a leukocyte enzyme in a sample and instituting a therapeutic plan.
- the presence of a leukocyte enzyme in the sample indicates the presence of a leukocyte in the sample.
- the leukocyte enzyme comprises LE.
- the leukocyte enzyme comprises human neutrophil elastase HNE.
- the leukocyte enzyme is detected by contacting the enzyme with a substrate of the enzyme.
- the substrate is any LE substrate of the present disclosure.
- the substrate is any HNE substrate of the present disclosure.
- the amount of leukocyte enzyme present in the sample is quantified. In some embodiments, the presence of a leukocyte in the sample is indicative of an infection.
- the infection comprises a urinary tract infection (“UTI”). In some embodiments, the infection comprises a periprosthetic joint infection (“PJI”). In some embodiments, the infection comprises spontaneous bacterial peritonitis (“SBP”). In some embodiments, the sample comprises a biological sample. In some embodiments, the biological sample comprises one of urine, sputum, synovial fluid, pleural fluid, pericardial fluid, peritoneal fluid, cerebrospinal fluid (“CSF”) and middle ear fluid.
- CSF cerebrospinal fluid
- the method of screening a patient at risk of developing an infection following the steps of detecting the presence of a leukocyte enzyme in a sample by contacting a leukocyte enzyme with a substrate in an assay.
- the assay comprises an electrochemical assay.
- the electrochemical assay comprises an internally calibrated electrochemical continuous enzyme assay (“ICECEA”).
- the method of detecting the presence of a leukocyte enzyme in an electrochemical assay comprises a step of adding a first aliquot of a reactant or product of a leukocyte enzyme to a substrate of the leukocyte enzyme.
- the leukocyte enzyme substrate is in an electrolyte solution.
- the method comprises a step of measuring current flowing through an electrode of the electrochemical assay.
- the method comprises a step of adding at least one additional aliquot of the reactant or product of a leukocyte enzyme to the substrate of the leukocyte enzyme.
- the method comprises a step of measuring current flowing through an electrode of the electrochemical assay for a second time.
- the method comprises a step of adding the leukocyte enzyme to the substrate of the leukocyte enzyme.
- the method comprises a step of measuring current flowing through an electrode of the electrochemical assay for a third time.
- the leukocyte enzyme substrate is in an electrolyte solution.
- the method comprises a step of measuring current flowing through an electrode of the electrochemical assay.
- the method comprises a step of adding at least one additional aliquot of the leukocyte enzyme to the substrate of the leukocyte enzyme.
- the method comprises a step of measuring current flowing through an electrode of the electrochemical assay for a second time.
- the method comprises a step of adding a product or reactant of a leukocyte enzyme to the substrate of the leukocyte enzyme. In some embodiments, the method comprises a step of measuring current flowing through an electrode of the electrochemical assay for a third time.
- kits containing suitable substrate direction for optimizing the results and optionally providing patient specific therapeutic regimen based on the observed results.
- FIG. 1 represents an initial hydroquinone substrate and first ester hydrolysis step.
- FIG. 2 represents a semiquinone intermediate and second ester hydrolysis step.
- FIG. 3 represents a final benzoquinone oxidation product.
- FIG. 4 represents the results of using 4-((tosyl-L-alanyl)oxy)phenyl tosyl-L-alaninate (“TAPTA”) in an internally calibrated electrochemical continuous enzyme assay (ICECEA).
- TAPTA 4-((tosyl-L-alanyl)oxy)phenyl tosyl-L-alaninate
- ICECEA internally calibrated electrochemical continuous enzyme assay
- FIG. 5 represents the NMR of 4-((tosyl-L-alanyl)oxy)phenyl tosyl-L-alaninate (“TAPTA”).
- FIG. 6 is a schematic of the cleavage mechanism of a monoester embodiment of the present invention.
- FIG. 7 are voltammograms showing increasing reduction peaks with higher LE concentration for an electrode screen-printed with one embodiment of a substrate of the present invention.
- leukocyte may refer to any white blood cell (“WBC”).
- WBC white blood cell
- Leukocytes are cells of the immune system that are involved in protecting the body against infectious disease and invading pathogens. All leukocytes/WBCs are divided into five classes based on morphological characteristics that differentiate themselves from one another. They include neutrophils, eosinophils, basophils, monocytes, and lymphocytes. Neutrophils comprise approximately 40-75% of leukocytes, eosinophils comprise approximately 1-6% of leukocytes, basophils comprise less than 1% of leukocytes, monocytes comprise approximately 2-10% of leukocytes, and lymphocytes (e.g. B lymphocytes and T lymphocytes) comprise approximately 20-45% of leukocytes.
- WBC white blood cell
- a biological system can include, for example, an individual cell, a set of cells (e.g. a cell culture), an organ, a tissue, or multi-cellular organism.
- a “patient” can refer to a human patient or a non-human patient. In preferred embodiments, the patient is a human patient.
- an effective amount or “therapeutically effective amount” as used herein may refer to an amount of the compound or agent that is capable of producing a medically desirable result in a treated subject.
- the treatment method can be performed in vivo or ex vivo, alone or in conjunction with other drugs or therapy.
- a therapeutically effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route.
- treating refers to executing a protocol, which may include administering one or more drugs to a patient (human or otherwise), in an effort to alleviate signs or symptoms of the disease. Alleviation can occur prior to signs or symptoms of the disease appearing as well as after their appearance. Thus, “treating” or “treatment” includes “preventing” or “prevention” of disease.
- the terms “prevent” or “preventing” refer to prophylactic and/or preventative measures, wherein the object is to prevent or slow down the targeted pathologic condition or disorder.
- the present disclosure relates to compositions and methods for rapid detection (including determining the relative activity) of enzymes released by active leukocyte cells, e.g. leukocyte enzymes released by active leukocyte cells, in particular leukocyte esterase (“LE”) and human neutrophil elastase (“HNE”).
- active leukocyte cells e.g. leukocyte enzymes released by active leukocyte cells, in particular leukocyte esterase (“LE”) and human neutrophil elastase (“HNE”).
- LE leukocyte esterase
- HNE human neutrophil elastase
- a method of screening a subject for infection comprising the steps of (a) obtaining a sample of tissue or bodily fluid from a subject at risk of developing an infection, (b) applying the sample to a detector device, wherein the detector device comprises at least one substrate which is specific for at least one of LE and/or HNE, wherein at least one substrate is adapted to detect a threshold level at least one of LE and/or HNE, the threshold level correlated with a presence of infection; (c) ascertaining the threshold levels of LE and/or HNE present in the sample, wherein if the concentration each of LE and/or HNE exceeds the threshold level, and further wherein such measurement is a positive screen for infection.
- the disclosure provides a method wherein the infection is a periprosthetic joint infection (PJI).
- the threshold level of leukocyte esterase (LE) for detection of PJI is at least about 20 pg/ml of leukocyte esterase in a synovial fluid sample.
- compositions and methods for rapid detection utilize specific substrates for detecting leukocyte enzymes, e.g. LE and HNE, referred to as LE substrates and HNE substrates respectively.
- the compositions and methods for rapid detection may utilize electrochemical assays to detect the leukocyte enzymes, in particular, internally calibrated electrochemical continuous enzyme assay (“ICECEA”), but are not necessarily limited as such.
- ICECEA internally calibrated electrochemical continuous enzyme assay
- the substrates are capable of detecting LE. Such substrates are readily hydrolyzed by LE to generate a redox intermediate, which can provide a detectable electrochemical response.
- the substrates for detecting LE i.e. “LE substrates” may follow Formula I as depicted below:
- A determines the identity of the acyl group, e.g. an alanine or lactate, at the ester cleavage site with enzyme specificity for leukocyte esterase and B is a moiety capable of participating in a redox reaction, which can be detected using an electrochemical assay (e.g. by using ICECEA or screen-printed electrochemical sensors).
- an electrochemical assay e.g. by using ICECEA or screen-printed electrochemical sensors.
- A comprises an amino group (i.e., —NR a , where R a is a H or an optionally substituted alkyl, aryl, or aralkyl group), or A comprises an ether group (i.e. —O—).
- the acyl group defined by A is protected using any effective amine or alcohol blocking group C (e.g. a tosyl group).
- the alcohol intermediate of the ester, moiety B, to be released upon hydrolysis by the esterase is a redox substrate and participates in a redox reaction.
- the oxygen linking B in Formula I may be substituted with an —NH linking moiety (i.e. the ester group presented in Formula I may be substituted with an amido group) and still be within the scope of the present disclosure.
- the amine or alcohol blocking group C may comprise any of the following: acetyl (Ac), benzoyl (Bz), benzyl (Bn), ⁇ -methoxyethoxymethyl (MEM), dimethoxytrityl (DMT), methyoxymethyl (MOM), methoxytrityl [(4-methoxyphenyl)diphenylmethyl] (MMT), p-Methoxybenzyl (PMB), methylthiomethyl, pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trityl (Tr), sily (e.g.
- the redox moiety B is a derivate of phenol, which may form an ester through its hydroxyl group. Such an intermediate may undergo oxidation to release an electron.
- one phenol derivative, hydroquinone contains two hydroxyl groups in a para conformation. Each hydroxyl group can be bound to form a distinct lactate ester, which is independently a substrate of leukocyte esterase ( FIG. 1 ).
- the resulting duplex substrate has two potential target sites for leukocyte esterase activity, and breakdown of the substrate is stepwise. Ester hydrolysis with leukocyte esterase at the first target will occur relatively slow due to molecular hindrance of the active sites; however, subsequent hydrolysis of the second active site will occur more quickly.
- an oxidation reaction can release an electron with removal of a hydrogen atom forming a semiquinone lactate ester intermediate ( FIG. 2 ).
- the quinone-based intermediate is released and can be further oxidized to form para-benzoquine.
- Para-benzoquine is reduced at low potentials, which minimizes interference from other redox active species within the sample and may improve assay selectivity.
- the final product is shown in FIG. 3 .
- At least one aspect of the disclosure is to prophylactically treat a patient prior to any invasive operation to minimize risk of infection.
- patients identified as suffering from an infection may be initiated a comprehensive treatment plan including administering antimicrobial agent, such as penicillins, cephalosporins, tetracyclines, daptomycin, tigecycline, linezolid, quinupristin/dalfopristin and dalbavancin and the like that may be useful in combating an active infection.
- antimicrobial agent such as penicillins, cephalosporins, tetracyclines, daptomycin, tigecycline, linezolid, quinupristin/dalfopristin and dalbavancin and the like that may be useful in combating an active infection.
- methods of screening or detecting risk of PJI by developing useful for the treatment of infections due to drug-resistant Gram-positives and Gram-negatives.
- B comprises a quinone. In some embodiments, B comprises a phenol. In some embodiments, B comprises a substituted quinone or a substituted phenol. In some embodiments, C comprises a tosyl protecting group. In some embodiments, the oxygen linking B in Formula II is substituted with an amino group. In further embodiments, B comprises aminophenyl. In some embodiments, B comprises substituted aminophenyl.
- substrates for detecting leukocyte esterase (“LE”) that are within the scope of Formula I include 4-((tosyl-L-alanyl)oxy)phenyl tosyl-L-alaninate (Compound A below) and 4-(((S)-2-(tosyloxy)propanoyl)oxy)phenyl (S)-2-(tosyloxy)propanoate (Compound B below).
- Compound A is also referred to herein as “TAPTA.”
- An NMR of Compound A is shown in FIG. 5 , illustrating the tosyl moiety structure and its attachment.
- Phenylethylenediamine variants of Compound A and Compound B i.e. the para-oxygens are replaced with NH linkers
- the LE substrate comprises a composition as described in Formula II below:
- X 1 and X 2 are independently O, S or NR a .
- R a is an H, an alkyl or an aryl group.
- X 1 and X 2 can be both oxygen or both NR a .
- one of X 1 and X 2 is oxygen and the other is NR a .
- Y 1 and Y 2 are independently O, S or NR a .
- R a is as described above.
- Y 1 and Y 2 can be both oxygen or both NR a .
- one of Y 1 and Y 2 is oxygen and the other is NR a .
- R 1 and R 2 are independently an alkyl or an aryl group or a substituted alkyl, a substituted aryl or a protecting group. In some embodiments, R 1 or R 2 or both is methyl. In some embodiments, R 1 or R 2 or both may be a tosyl. In one embodiment, R 2 is a tosyl.
- R 3 and R 4 are independently an alkyl, a protecting group such as tosyl, benzoyl, benzyl, trimethylsilyl, [bis-(4-methoxyphenyl)phenylmethyl], carbobenzyloxy, tert-Butyloxycarbonyl, 9-Fluorenylmethyloxycarbonyl, or a peptide moiety.
- R 4 is a tosyl.
- the peptide moiety can include any combination of natural and/or non-natural amino acids.
- R2 and R4 may also comprise any of the following: acetyl (Ac), benzoyl (Bz), benzyl (Bn), ⁇ -methoxyethoxymethyl (MEM), dimethoxytrityl (DMT), methyoxymethyl (MOM), methoxytrityl [(4-methoxyphenyl)diphenylmethyl] (MMT), p-Methoxybenzyl (PMB), methylthiomethyl, pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trityl (Tr), sily (e.g.
- protecting group can be any one of tosyl, benzoyl, benzyl, trimethylsilyl, [bis-(4-methoxyphenyl)phenylmethyl], carbobenzyloxy, tert-Butyloxycarbonyl, 9-Fluorenylmethyloxycarbonyl.
- Each of the R 5 on the ring is independently a halogen atom; a hydroxyl group; a C 1 -C 6 alkyl group; a C 3 -C 6 cycloalkyl group; a C 3 -C 6 cycloalkyl C 1 -C 6 alkyl group; a C 2 -C 6 alkenyl group; a C 2 -C 6 alkynyl group; a C 1 -C 6 haloalkyl group (including trifluoro C 1 -C 6 alkyl); a C 2 -C 6 haloalkenyl group; a C 2 -C 6 haloalkynyl group; a C 3 -C 6 halocycloalkyl group; a C 3 -C 6 halocycloalkyl C 1 -C 6 alkyl group; a C 1 -C 6 alkoxy group; a C 3 -C 6 cycloalkyloxy group; a C 2
- n 0, 1, 2, 3, or 4.
- X 1 and X 2 are independently O or NR a .
- R a is a H, an alkyl, an aryl, or aralkyl group.
- X 1 and X 2 can be both oxygen or both NR a .
- one of X 1 and X 2 is oxygen and the other is NR a
- Y 1 and Y 2 are independently O or NR a .
- the substrates detect human neutrophil elastase (“HNE”).
- HNE substrates may follow Formula III as depicted below:
- a 1 through A 4 represent a core tetrapeptide scaffold sequence, which serves as the enzyme active site (i.e. the active site for human neutrophil elastase/HNE).
- a tetrapeptide sequence of Ala-Ala-Pro-Val (AAPV) (SEQ ID NO: 1) is most common, but natural or unnatural amino acids may be substituted at any of the four peptide sites in order to improve substrate sensitivity for HNE. For example, conservative substitutions may be made for SEQ ID NO: 1 and still be within the scope of the present disclosure.
- “conservative substitutions” are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
- Families of amino acid residues having similar side chains 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, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
- B in Formula III represents a redox moiety, similar to the LE substrate displayed in Formula I above.
- B may comprise derivate of phenol, which may form an ester through its hydroxyl group, e.g., a redox active alcohol intermediate. This may comprise, for example, a hydroquinone intermediate or hydroquinone-based redox groups.
- C in Formula III represents an acyl group, for example, N-methyoxysuccinyl. The acyl group may serve to improve substrate sensitivity for HNE, and some acyl groups, for example N-methoxysuccinyl, may also increase substrate solubility.
- One specific, explicitly non-limiting example of a substrate for detecting HNE that is within the scope of Formula III includes 3- ⁇ [(1S)-1- ⁇ [(2S)-1-(5- ⁇ [(1S)-1-( ⁇ 4-[(2S)-2-( ⁇ 1-[(2S)-2-[(2S)-2-(3-carboxypropanamido)propanamido]propanoyl]pyrrolidin-2-yl ⁇ formamido)-3-methylbutanamido]phenyl ⁇ carbamoyl)-2-methylpropyl]carbamoyl ⁇ imidazolidin-1-yl)-1-oxopropan-2-yl]carbamoyl ⁇ ethyl]carbamoyl ⁇ propanoic acid, Compound C below.
- the diester was less effective than the monoester. Even in mixtures of diester and monoester in which the monoester was present in very low concentration (e.g. about 1%), the effectiveness of the monoester was predominant and dictated the effectiveness of the composition as a whole. Indeed, the effectiveness of the monoester was not discovered until the diester composition was purified to the point that the concentration of the monoester fell to below 1%. At that point, the effectiveness of the diester composition dropped precipitously, thereby indicating that the monoester was a more effective substrate for reacting with leukocyte esterase enzymes.
- the substrate of the present invention comprises a monoester, the monoester being one of an ⁇ -amino acid ester, such as an alanine ester, or an ⁇ -hydroxy acid ester, such as a lactate ester, with specificity for leukocyte esterases.
- the monoester has a first moiety for participating in a redox reaction, and a second moiety comprising an amine or alcohol blocking group.
- the composition comprises a monoester as depicted in Formula I, wherein A comprises oxygen (O) or NR a , where R a is a H or an optionally substituted alkyl, aryl, or aralkyl group, whereby A determines the identity of the acyl group of the ester, in that A is O if said monoester is an ⁇ -hydroxy acid ester (i.e. lactate ester) or A is NR a if said monoester is an ⁇ -amino acid ester (i.e. alanine ester).
- B is the first moiety and C is the second moiety.
- any oxygen linking group linking the first and/or second moiety can be substituted by nitrogen linking groups, and nitrogen linking groups can be substituted by oxygen linking groups.
- the first moiety (B) comprises one of a substituted or unsubstituted derivative of phenol, substituted or unsubstituted hydroxyanthracene, substituted or unsubstituted aminophenol, or substituted or unsubstituted hydroxyphenanthroline.
- the second moiety (C) comprises one of the following: acetyl (Ac), benzoyl (Bz), benzyl (Bn), ⁇ -methoxyethoxymethyl (MEM), dimethoxytrityl (DMT), methyoxymethyl (MOM), methoxytrityl [(4-methoxyphenyl)diphenylmethyl] (MMT), p-Methoxybenzyl (PMB), methylthiomethyl, pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trityl (Tr), sily (e.g.
- the second moiety is a sulfonyl group with a substituted or unsubstituted heterocycle or heteroaryl ring.
- Formula 1 is further refined to the general structure depicted in Formula IV:
- first moiety B comprises 4-hydroxyphenyl
- A comprises oxygen or NR a , where R a is a H or an optionally substituted alkyl, aryl, or aralkyl group, whereby A determines the identity of the acyl group of the ester, R1, R2, and R3 are independently hydrogen or optionally substituted alkyl groups (R3 is absent if A is oxygen), and R4 is a substituted or unsubstituted heterocycle or heteroaryl.
- R4 is one of pyridinyl, pyridazinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, quinolyl, isoquinolyl, 1,2,3,4-tetrahydroquinolyl, tetrazolyl, furyl, thienyl, isooxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, indolyl, benimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, triazinyl, thiadiazolyl, oxadiazolyl, purinyl, 1-oxoisoindolyl, 1,2,4-trizainyl, 1,3,4-triazinyl, isoindolyl, furazanyl, benzof
- R4 is a pyridine with or without the addition of substituted or unsubstituted polar groups.
- R4 is a pyridine selected from one of the following: pyridine (I), methoxypyridine (II), and (methoxycarbonyl)pyridine (III) as represented below:
- R4 is (methoxycarbonyl)pyridine (III).
- the composition of the monoester substrate is 4-Hydroxyphenyl (N-(3-(methoxycarbonyl)pyridine-5-sulfonyl)-L-alaninate.
- composition of the monoester is depicted in Formula V:
- the cleaving mechanism for Formula V is shown. Specifically, the leukocyte esterase (LE) cleaves the monoester substrate at the oxygen site upon ester hydrolysis.
- LE leukocyte esterase
- the substrate of the leukocyte esterase enzyme is screen-printed onto the surfaces of an electrode sensor strips using known and commercially-available techniques and materials.
- leukocytes are capable of producing leukocyte enzymes that are able to be detected and/or quantified by the electrochemical assays (i.e. ICECEA) of the present disclosure.
- electrochemical assays i.e. ICECEA
- Leukocyte enzymes may include, for example, those described in WO 2010/036930, hereby incorporated by reference in its entirety, such as, for example, IL-10, leukocyte elastase, leukocyte esterase, and/or gelatinase B, along with human neutrophil elastase.
- Leukocyte esterase (“LE”) is an esterase produced by leukocytes (white blood cells). LE is the subject of, for example, urine tests for the presence of leukocytes/WBCs and other abnormalities associated with infection.
- Human neutrophil elastase (“HNE”) also known as human leukocyte elastase (“HLE”), is a serine protease.
- HNE is secreted by neutrophils and macrophages, two of the five classes of leukocytes as described herein. HNE is 218 amino acids long and has two asparagine-linked carbohydrate chains. There are two forms of HNE, deemed IIa and IIb.
- sample may refer to a biological sample, including a sample of biological tissue or fluid origin obtained in vivo or in vitro.
- Biological samples can be, but are not limited to, body fluid (e.g., serous fluid, blood, blood plasma, serum, or urine), organs, tissues, fractions, and cells isolated from mammals including, for example, humans.
- body fluid e.g., serous fluid, blood, blood plasma, serum, or urine
- Biological samples also may include sections of the biological sample including tissues.
- Biological samples may also include extracts from a biological sample, for example, a biological fluid (e.g., blood, serum, peritoneal fluid, and/or urine).
- a biological fluid e.g., blood, serum, peritoneal fluid, and/or urine.
- urine for example, in a patient diagnosed with cystic fibrosis
- peritoneal fluid for example, in a patient with liver cirrhosis and ascites
- other serous fluids including but not limited to, for example, synovial fluid, pleural fluid, pericardial fluid, cerebrospinal fluid (“CSF”) and middle ear fluid.
- CSF cerebrospinal fluid
- the presence of leukocytes i.e. as determined by detecting and/or quantifying the amount of a leukocyte enzyme (e.g. LE and/or HNE) present in the biological sample may indicate the presence of an infection in a subject.
- a leukocyte enzyme e.g. LE and/or HNE
- Such embodiments may utilize the LE and/or HNE substrates of the present disclosure in an electrochemical assay, in particular ICECEA as described herein.
- the presence of LE and/or HNE in urine may indicate a subject as having a urinary tract infection (“UTI”).
- the presence of LE and/or HNE in synovial fluid may indicate a subject as having a joint infection, for example but not necessarily limited to a periprosthetic joint infection (“PJI”).
- PJI periprosthetic joint infection
- the substrates of the present disclosure are used to indicate a subject as having periprosthetic joint infection (PJI).
- PJI is a devastating complication following total joint arthroplasty, which remains a challenge for surgeons both diagnostically and therapeutically.
- Establishing an accurate and timely diagnosis of PJI is of critical importance for making treatment decisions. For patients presenting with a painful prosthesis, it is important to complete a work-up to either rule out or diagnose the presence of infection.
- serological testing including erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) is the initial screening test of choice. In patients with elevated serological markers or even just a high suspicion of infection, the next step is to perform joint aspiration for testing of synovial fluid.
- ESR erythrocyte sedimentation rate
- CRP C-reactive protein
- bacterial culture of synovial fluid has been used to make the diagnosis of PJI.
- bacterial culture is not in itself sufficiently sensitive, with as many as 30% of infections being culture negative, orthopedic surgeons also consider the results of serological testing, synovial fluid white blood cell count and polymorphonuclear percentage, and histological analysis to make a diagnosis.
- bacterial culture and traditional synovial fluid testing can require days to more than a week to yield a result.
- synovial fluid aspirated from a painful joint would be tested for LE and/or HNE activity using an enzyme substrate of the present disclosure.
- this may be accomplished through use of an ICECEA assay as described herein.
- the activity of LE and/or HNE would be reported as a continuous measurement of absolute concentration. This could be performed in the office or operating room to yield a result in minutes for point-of-care decision-making.
- the level of LE and/or HNE activity can be combined with additional metrics to predict the likelihood that an infection is present.
- Additional metrics may include the type of joint, a history of prior infection, and the results of serological testing (ESR and CRP).
- ESR and CRP results of serological testing
- Surgeons can consider the likelihood that an infection is present to determine the most appropriate treatment algorithm for their patient.
- treatment for PJI such as prosthesis extraction and antibiotic spacer placement, incision and debridement, or long-term antibiotic suppression, could be considered based on the acuity of the infection, among other factors.
- a surgeon could consider initiating treatment or waiting for additional diagnostic results.
- other etiologies for a painful prosthesis may be considered in cases for which the likelihood of the presence of infection is low or for which infection has largely been ruled out.
- the substrates of the current disclosure may be used to establish the resolution of PJI in order to determine the correct timing for re-implantation of a new prosthesis.
- the level of LE and/or HNE activity may be used in addition to serological markers and other synovial fluid tests to determine the success of treatment, such as discussed supra.
- surgeons may elect to continue intravenous antibiotics or attempt an exchange of the antibiotic spacer to improve prospects of complete resolution of infection.
- the substrates of the present disclosure are used to indicate a subject as having spontaneous bacterial peritonitis (SBP).
- SBP spontaneous bacterial peritonitis
- SBP is a serious and life-threatening complication that is relativity common in patients with liver cirrhosis and ascites.
- a rapid diagnosis and early administration of antibiotics is critical for survival, and in-hospital mortality can be as high as 20%.
- presenting symptoms of fever, change in mental status, and abdominal tenderness are frequent signs of SBP.
- a diagnostic paracentesis is performed, and a diagnosis is made based on an absolute neutrophil count above 250 cells/mm 3 and/or bacterial culture.
- ascitic fluid obtained from diagnostic paracentesis would be tested for LE and/or HNE activity using an enzyme substrate of the present disclosure.
- this may be accomplished through use of an ICECEA assay as described herein.
- the activity of LE or HNE would be reported as a continuous measurement of absolute concentration.
- the absolute concentration of LE and/or HNE would be compared to gold standard diagnostic criteria to provide a calculation of the probability that SBP is present.
- the likelihood of infection can be used to inform the treating physician as to the most appropriate treatment algorithm.
- the measured level of LE or HNE could also provide important prognostic information, with a higher level indicating a worse prognosis.
- the substrates of the present disclosure are used to indicate a subject as having a urinary tract infection (UTI), also known as a urogenital infection.
- UTI urinary tract infection
- a diagnosis of UTI can typically be made on clinical symptoms alone.
- women with poorly defined symptoms, asymptomatic pregnant females, elderly patients, and children have a much lower pre-test probability for UTI.
- the present disclosure is not limited to testing women for UTI.
- the gold standard for diagnosis of UTI is mid-stream urine culture (with >10 3 -10 5 organisms) or pyuria (greater than 10 4 leukocytes per ml).
- mid-stream urine for symptomatic patients would be tested for leukocyte esterase (“LE”) and/or human neutrophil elastase (“HNE”) activity using an enzyme substrate of the present disclosure.
- L leukocyte esterase
- HNE human neutrophil elastase
- this may be accomplished through use of an ICECEA assay as described herein.
- likelihood of infection can be determined based on both measurement of LE and/or HNE activity and additional factors, such as the presence of specific symptoms and patient characteristics (i.e. age, gender, pregnancy).
- a physician can decide whether or not to administer oral antibiotics.
- Population data for the clinical applications of the present disclosure can be used to convert the measure of LE and/or HNE activity to a predictive probability for the presence of infection.
- the test device itself can be used as a medium to both collect and distribute such population-based data.
- a smartphone (or similar device) connected electrochemical biosensor can allow physicians to provide selected information to a centralized database, which may then be used to continuously improve the calculation of infection likelihood.
- the biosensor may also report back to surgeons the likelihood of infection for their individual patient based upon LE and/or HNE activity and additional metrics that can be used to hone their treatment algorithm.
- the substrates for detecting leukocyte enzymes are incorporated into an assay.
- an assay may comprise, for example, an electrochemical assay.
- Electrochemical assays are cost-effective, highly sensitive, and simplify the calibration process. Furthermore, such methods would be just as effective in bloody or turbid fluid.
- a preferred electrochemical assay comprises an internally calibrated electrochemical continuous enzyme assay (“ICECEA”).
- ICECEA Use of a LE substrate of the present disclosure (“TAPTA”) in an ICECEA is described in Example 1, infra.
- ICECEAs are generally disclosed in PCT/US2014/03713 and U.S. 2016/0040209, the disclosure of which is hereby incorporated in its entirety.
- ICECEA utilizes integration of an enzyme-free pre-assay calibration with an electrochemical enzyme assay in a continuous experiment. This is believed to result in a uniquely shaped amperometric trace that allows for selective and sensitive determination of enzymes, e.g. LE and HNE, present in a sample.
- enzymes e.g. LE and HNE
- ICECEAs generally follow the following method as described in U.S. 2016/0040209.
- an enzyme substrate e.g. an LE and/or HNE substrate of the present disclosure
- a reactant or product of an enzymatic reaction of the enzyme is added to the first enzyme substrate/background electrolyte, which creates what is described as a “first assay mixture.”
- Current flowing through an electrode of the electrochemical assay is then measured after the first assay mixture is formed.
- the enzyme e.g. LE and/or HNE
- the enzyme is added to the “first assay mixture” to create a “second assay mixture,” and the current is measured again over a predetermined time period.
- Enzyme activity is determined based on the change in current over time caused by the addition of the enzyme. While optimally the enzyme is added after the reactant/product is added to the enzyme substrate, the order can be switched, i.e. the enzyme is added to the substrate first and then the reactant/product is added.
- the ICECEA includes an electrochemical measuring device.
- the electrochemical measuring device includes a working electrode, a reference electrode, and an auxiliary electrode.
- the current is measured through the working electrode.
- the working electrode may be a noble metal electrode, metal oxide electrode, an electrode made of a carbon allotrope, or a modified electrode.
- the auxiliary electrode may be a platinum wire.
- the reference electrode may be Ag/AgCl/NaCl or any other reference electrode.
- the electrochemical assay system can also be made of only a working electrode and a reference electrode. Measuring the changes in current may be done by collecting an amperometric trace of the current.
- adding the reactant/product to the enzyme substrate (in electrolyte) in the electrochemical assay system includes the following steps. First, a first aliquot of the reactant/product is added to the enzyme substrate (in electrolyte). Current flowing through an electrode of the electrochemical assay system is measured after the first aliquot is added. One or more additional aliquots of the reactant/product are added to the mixture and current flowing through an electrode of the electrochemical assay system is measured again. Preferably, at least three aliquots of the reactant/product are added to the enzyme substrate (in electrolyte) before the enzyme is added to the mixture. Alternatively, the aliquots of the reactant/product are added to the substrate (in electrolyte) after the enzyme is added to the mixture.
- the enzymatic activity of the enzyme may be determined from the slope of a line created from measuring the current flowing through a working electrode of the electrochemical assay system after the reactant/product is added to the substrate (before the enzyme is added, or vice versa as described herein) at predetermined intervals over a predetermined time period.
- a customized kit containing a solution of enzyme substrate and other necessary reactants in a background electrolyte; a solution of redox active component of enzymatic reaction; and a solution of assayed enzyme.
- an amperometric measurement is done by using any electrochemical measurement device with amperometric method and a conventional electrochemical cell with the working, reference, and counter electrodes immersed in a solution containing the enzyme substrate.
- the working electrode is held at a potential E vs. the potential of the reference electrode.
- the potential E is adequate for either the oxidation or reduction of species present in the solution containing the redox active component of the enzymatic reaction.
- the experiment is performed by spiking one or more known aliquots of a redox active containing solution followed by one aliquot of a solution containing assayed enzyme into a stirred solution that contains enzyme substrate and other necessary reactants and measuring the current flowing through the working electrode.
- the substrate 4-((tosyl-L-alanyl)oxy)phenyl tosyl-L-alaninate, Compound A below (also referred to as “TAPTA”) was used as a substrate to measure the activity of leukocyte esterase (LE) in an internally calibrated electrochemical continuous enzyme assay (ICECEA). The results are indicated in FIG. 4 .
- the ICECEA was conducted as generally described in U.S. 2016/0040209 as well as in the detailed description supra. Briefly, in the pre-assay phase, three (3) distinct calibration steps were performed by spiking a solution of enzyme substrate (“TAPTA”) and necessary reactants with a solution of the redox active component of the enzymatic reaction. These three distinct calibration steps are denoted by a bold “a” in FIG. 4 .
- the assay phase was commenced by spiking one aliquot of assayed enzyme (LE) into the enzyme substrate solution. This step is denoted by a bold “b” in FIG. 4 .
- the enzymatic reaction was followed by measuring current flowing through the working electrode.
- the enzyme assay was calibrated for LE concentrations ranging from 0-250 ⁇ g/L. The enzyme activity of LE demonstrated a linear response relative to LE concentration and predictive of an infection.
- 4-Hydroxyphenyl N-(3-(methoxycarbonyl)pyridine-5-sulfonyl)-L-alaninate (Monoester) can be prepared by partial hydrolysis of 1,4-Phenylene bis(N-(3-(methoxycarbonyl)pyridine-5-sulfonyl)-L-alanininate (synthesized by a modification of the procedure for Compound III described in Hanson et al., Chembiochem 2018, 19, https://www.ncbi.nlm.nih.gov/pubmed/29679431).
- Suitable bases include alkali hydroxides, alkaline earth hydroxides, ammonia, amines, etc.
- 1,4-Phenylene bis(N-(3-(methoxycarbonyl)pyridine-5-sulfonyl)-L-alanininate (21 mg, 0.032 mmol) was dissolved in THF and treated with 1M NaOH (0.045 mL, 139 mol %), at 30° C. for 4 days. The solvent was evaporated, the residue was dissolved in dichloromethane, rinsed with 1M HCl, and dried over MgSO 4 to give the product as a colorless glass.
- electrode strips were screen printed with the substrate having the structure as depicted in Formula V.
- the strips were contacted with varying concentrations of LE (i.e. 0.125 U/ml to 0.35 U/ml LE).
- the plot clearly indicates significant reduction peaks at about ⁇ 0.17 V, which correspond to the redox reaction of hydroquinone molecules that are released upon cleavage of the monoester by leukocyte esterases at the ester active site.
- the screen-printed electrode strips not only were confirmed to detect the presence of LE, but also can provide a quantitative measure as to the activity level or concentration of LE in a sample that directly corresponds to the enzymes cleavage of the monoester. Based on this measured level, a determination can be made as to whether the patient's level of LE is high enough to indicate infection.
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Immunology (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Genetics & Genomics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Pathology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- General Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Nanotechnology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Medicinal Chemistry (AREA)
- Electrochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The present disclosure relates to compositions, methods and test devices for determining the presence of active leukocyte cells, for example, by using novel LE and/or HNE substrates in an electrochemical assay.
Description
- This application is a continuation of U.S. application Ser. No. 16/145,014, filed Sep. 27, 2018, which is a continuation-in-part of application Ser. No. 16/087411, filed Sep. 21, 2018, a national stage application of International Application Number PCT/US2017/022976, which claims priority to U.S. Provisional Patent Application Ser. No. 62/311,405, filed Mar. 22, 2016, and to U.S. Provisional Patent Application Ser. No. 62/352,560, filed Jun. 21, 2016. The disclosure of each of the applications identified above are hereby incorporated by reference in their entirety.
- The present disclosure relates to a novel application of an electrochemical assay for the determination of the activity of leukocyte cells within a test sample. More particularly, the present disclosure relates to novel methods and kits for determining the activity of enzymes released by active leukocyte cells, especially leukocyte esterase and human neutrophil elastase, in a patient at risk of developing an infection.
- The presence of an abnormally high number of leukocyte cells in urine is a commonly used indicator of an infectious process. Historically, technicians have relied on manual visual count under a microscope. This visual technique has been largely replaced by a dipstick assay for detection of urogenital infections. In a large majority of such commercial ‘dipstick’ assays, activity of the enzyme leukocyte esterase (“LE”) is used as a proxy for the presence of active leukocyte cells. An assay for human neutrophil elastase (“HNE”) has also been reported to have great sensitivity for the diagnosis of urethral infections in men.
- Known assays for LE are chromogenic, in that the presence of enzyme activity is reported based upon a color change. Typically, a color test strip can be matched to a color chart with 3-4 increments of increasing color intensity (from none to 2+/3+), which represents a LE concentration of 30 ng/mL to greater than 1500 ng/mL. However, there are clear disadvantages to a colorimetric assay. With only 3-4 available color intensity increments, resolution of differences in leukocyte esterase concentration may be quite difficult. In addition, inter-rater and even intra-rater reliability in classifying such color increments may be poor. This is especially true for instances in which dipstick results are less definitive (trace or 1+); test results, in such cases, may be too unreliable for making treatment decisions. Thus, the utility of dipstick results is limited to cases in which leukocyte esterase activity is exceedingly high. Any substance that changes the color of urine (e.g. nitrofurantoin, phenazopyridine) also affects dipstick readings.
- In recent years, leukocyte esterase testing has piqued the interest of physicians for applications using serous fluid, such as that from joint, lung, abdominal, or even middle ear effusions. While results have been quite promising for the diagnosis of periprosthetic joint infection (PJI), a colorimetric test is rendered impractical in as many as 17-29% of samples due to the presence of blood or debris. The same would be true for other body cavities, for which aspiration often does not yet often always yield clear fluid. Further, a colorimetric leukocyte esterase test cannot be attempted on serum samples.
- More recently, a lactate ester substrate has been demonstrated to have improvement in terms of LE assay sensitivity and speed. The alcohol portion is released as a hydroxyl-pyrrole compound, which then reacts with diazonium salt to produce a purple azo dye. However, such an assay has limited utility in bloody or turbid fluid conditions and would require expensive optical sensors to provide a precise, quantitative measurement. Accordingly, there is an urgent need for improved substrates and assays to detect leukocytes and leukocyte enzymes in a sample.
- The following presents a simplified summary of the invention in order to provide a basic understanding of some aspects of the invention. This summary is not an extensive overview of the invention. It is not intended to identify key/critical elements of the invention or to delineate the scope of the invention. Its sole purpose is to present some concepts of the invention in a simplified form as a prelude to the more detailed description that is presented later.
- In one aspect, the disclosure is directed towards a method for screening, detecting and confirming an infection in patients at risk of an infection or those patients who have already exhibited symptoms associated with an infection. In one embodiment, the method follows the step of obtaining a sample from the subject in need, detecting the presence or absence of leukocyte markers in the sample, and instituting a therapeutic regimen based on the degree and presence of the leukocyte markers in the sample.
- In some embodiments, the leukocyte markers can be one or any combinations of such markers as cytokines, chemokines, oxygen and nitrogen radicals, leukocyte elastase, leukocyte esterase, neutrophil elastase, gelatinases, IL-1β, metalloproteinases (MMPs), cathepsins, such as cathepsin A and cathepsin B, phospholipases, such as, for example, phospholipase A and phospholipase B.
- In one aspect, the present disclosure is directed to a composition comprising a leukocyte enzyme or specifically a neutrophil enzyme substrate. In some embodiments, the leukocyte enzyme comprises leukocyte esterase (“LE”). In some embodiments, the leukocyte enzyme substrate comprises an LE substrate. In some embodiments, the leukocyte enzyme comprises human neutrophil elastase (“HNE”). In some embodiments, the leukocyte enzyme substrate comprises an HNE substrate. In an alternative embodiment, the composition comprises both an LE substrate and a HNE substrate. In yet another embodiment, the composition may contain additional substrates specific to other enzymes or biomarkers than LE and HNE.
- In some embodiments, the substrates demonstrate specificity for LE or HNE. In one embodiment, the substrate comprises a monoester, the monoester being one of an α-amino acid ester, such as an alanine ester, or an α-hydroxy acid ester, such as a lactate ester, with specificity for leukocyte esterases, the monoester having a first moiety for participating in a redox reaction, and a second moiety comprising an amine or alcohol blocking group, which masks the functional group (i.e., amine or alcohol) to prevent undesirable chemical reactivity.
- In some embodiments, the substrates may follow Formula I as depicted below:
- wherein A comprises an ether group (i.e. —O—) or an amine group (i.e., NRa, where Ra is a H or an optionally substituted alkyl, aryl, or aralkyl group), B comprises a moiety capable of participating in a redox reaction, and C comprises an alcohol or amine blocking group. In some embodiments, A comprises an amino group. In some embodiments, A comprises an ether group. In some embodiments, B comprises a redox active alcohol intermediate. In some embodiments, B comprises a phenol. In some embodiments, B comprises a substituted phenol. In some embodiments, C comprises a tosyl protecting group. In some embodiments, the oxygen linking B in Formula I is substituted with an amino group. In further embodiments, B comprises aminophenyl. In some embodiments, B comprises a substituted aminophenyl.
- In some embodiments, the LE substrate comprises a compound as described in Formula II below:
- X1 and X2 are independently O, S or NRa. Ra is an H, an alkyl or an aryl group. X1 and X2 can be both oxygen or both NRa. Alternatively, one of X1 and X2 is oxygen and the other is NRa.
- Y1 and Y2 are independently O or NRa. Ra is as described above. Y1 and Y2 can be both oxygen or both NRa. Alternatively, one of Y1 and Y2 is oxygen and the other is NRa.
- R1 and R2 are independently an alkyl or an aryl group or a substituted alkyl, a substituted aryl or a protecting group. In some embodiments, R1 and R2 are both methyl. In some embodiments, R1 and R2 may be a tosyl. In some embodiments, R2 may be a tosyl.
- R3 and R4 are independently an alkyl, a protecting group or a peptide moiety. Example of a protecting group includes tosyl, benzoyl, benzyl, trimethylsilyl, [bis-(4-methoxyphenyl)phenylmethyl], carbobenzyloxy, and tert-Butyloxycarbonyl, 9-Fluorenylmethyloxycarbonyl. In one embodiment, R4 may be a tosyl. The peptide moiety can include any combination of natural and/or non-natural amino acids.
- Each of the R5 on the ring is independently a halogen atom; a hydroxyl group; a C1-C6 alkyl group; a C3-C6 cycloalkyl group; a C3-C6 cycloalkyl C1-C6 alkyl group; a C2-C6 alkenyl group; a C2-C6 alkynyl group; a C1-C6 haloalkyl group (including trifluoro C1-C6alkyl); a C2-C6 haloalkenyl group; a C2-C6 haloalkynyl group; a C3-C6 halocycloalkyl group; a C3-C6 halocycloalkyl C1-C6 alkyl group; a C1-C6 alkoxy group; a C3-C6 cycloalkyloxy group; a C2-C6 alkenyloxy group; a C2-C6 alkynyloxy group; a C1-C6 alkylcarbonyloxy group; a C1-C6 haloalkoxy group; a C1-C6 alkylthio group; a C1-C6 alkylsulfinyl group; a C1-C6 alkylsulfonyl group; a C1-C6 haloalkylthio group; a C1-C6 haloalkylsulfinyl group; a C1-C6 haloalkylsulfonyl group; an amino group; a C1-C6 alkylcarbonylamino group; a mono(C1-C6 alkyl)amino group; a di(C1-C6 alkyl)amino group; a hydroxy C1-C6 alkyl group; a C1-C6 alkoxy C1-C6 alkyl group; a C1-C6 alkylthio C1-C6 alkyl group; a C1-C6 alkylsulfinyl C1-C6 alkyl group; a C1-C6 alkylsulfonyl C1-C6 alkyl group; a C1-C6 haloalkylthio C1-C6 alkyl group; a C1-C6 haloalkylsulfinyl C1-C6 alkyl group; a C1-C6 haloalkylsulfonyl C1-C6 alkyl group; a cyano C1-C6 alkyl group; a C1-C6 alkoxy C1-C6 alkoxy group; a C3-C6 cycloalkyl C1-C6 alkyloxy group; a C1-C6 haloalkoxy C1-C6 alkoxy group; a cyano C1-C6 alkoxy group; a C1-C6 acyl group; a C1-C6 alkoxyimino C1-C6 alkyl group; a carboxyl group; a C1-C6 alkoxycarbonyl group; a carbamoyl group; a mono(C1-C6 alkyl)aminocarbonyl group; a di(C1-C6 alkyl)aminocarbonyl group; a nitro group; or a cyano group. n is 0, 1, 2, 3, or 4.
- In some embodiments, the LE substrate comprises 4-((tosyl-L-alanyl)oxy)phenyl tosyl-L-alaninate. In some embodiments, the LE substrate comprises 4-(((S)-2-(tosyloxy)propanoyl)oxy)phenyl (S)-2-(tosyloxy)propanoate. In some embodiments, the LE substrate comprises a phenylenediamine variant of one of 4-((tosyl-L-alanyl)oxy)phenyl tosyl-L-alaninate and 4-(((S)-2-(tosyloxy)propanoyl)oxy)phenyl (S)-2-(tosyloxy)propanoate.
- In some embodiments, the HNE substrate comprises a compound as described in Formula III below:
- wherein A1-A2-A3-A4 represent a core tetrapeptide scaffold sequence which serves as the enzyme active site, B comprises a moiety capable of participating in a redox reaction, and C comprises an acyl group. In some embodiments, A1-A2-A3-A4 comprise AAPV (SEQ ID NO: 1). In some embodiments, SEQ ID NO: 1 has conservative substitutions. In some embodiments, B comprises a redox active alcohol intermediate. In some embodiments, B comprises a derivative of phenol. B comprises a quinone. In some embodiments, B comprises a hydroquinone. In some embodiments, B comprises a substituted quinone or a substituted hydroquinone. In some embodiments, C comprises N-methyoxysuccinyl.
- In some embodiments, the HNE substrate comprises 3-{[(1S)-1-{[(2S)-1-(5-{[(1S)-1-({4-[(2S)-2-({1-[(2S)-2-[(2S)-2-(3-carboxypropanamido)propanamido]propanoyl]pyrrolidin-2-yl}formamido)-3-methylbutanamido]phenyl}carbamoyl)-2-methylpropyl]carbamoyl}imidazolidin-1-yl)-1-oxopropan-2-yl]carbamoyl}ethyl]carbamoyl}propanoic acid.
- In some embodiments, the leukocyte enzyme substrate is included in an assay. In some embodiments, the assay comprises an electrochemical assay. In an alternative embodiment, the assay may include a colorimetric step in combination with the electrochemical assay. In some embodiments, the electrochemical assay comprises an internally calibrated electrochemical continuous enzyme assay (“ICECEA”). In some embodiments, the electrochemical assay comprises a leukocyte substrate of the present disclosure and an electrochemical measuring device. In some embodiments, the electrochemical measuring device includes a working electrode, a reference electrode, and an auxiliary electrode.
- In some embodiments, the present disclosure is directed to a method of detecting the presence of a leukocyte enzyme in a sample and instituting a therapeutic plan. In some embodiments, the presence of a leukocyte enzyme in the sample indicates the presence of a leukocyte in the sample. In some embodiments, the leukocyte enzyme comprises LE. In some embodiments, the leukocyte enzyme comprises human neutrophil elastase HNE. In some embodiments, the leukocyte enzyme is detected by contacting the enzyme with a substrate of the enzyme. In some embodiments, the substrate is any LE substrate of the present disclosure. In some embodiments, the substrate is any HNE substrate of the present disclosure.
- In some embodiments, the amount of leukocyte enzyme present in the sample is quantified. In some embodiments, the presence of a leukocyte in the sample is indicative of an infection. In some embodiments, the infection comprises a urinary tract infection (“UTI”). In some embodiments, the infection comprises a periprosthetic joint infection (“PJI”). In some embodiments, the infection comprises spontaneous bacterial peritonitis (“SBP”). In some embodiments, the sample comprises a biological sample. In some embodiments, the biological sample comprises one of urine, sputum, synovial fluid, pleural fluid, pericardial fluid, peritoneal fluid, cerebrospinal fluid (“CSF”) and middle ear fluid.
- In some embodiments, the method of screening a patient at risk of developing an infection following the steps of detecting the presence of a leukocyte enzyme in a sample by contacting a leukocyte enzyme with a substrate in an assay. In some embodiments, the assay comprises an electrochemical assay. In some embodiments, the electrochemical assay comprises an internally calibrated electrochemical continuous enzyme assay (“ICECEA”).
- In some embodiments, the method of detecting the presence of a leukocyte enzyme in an electrochemical assay comprises a step of adding a first aliquot of a reactant or product of a leukocyte enzyme to a substrate of the leukocyte enzyme. In some embodiments, the leukocyte enzyme substrate is in an electrolyte solution. In some embodiments, the method comprises a step of measuring current flowing through an electrode of the electrochemical assay. In some embodiments, the method comprises a step of adding at least one additional aliquot of the reactant or product of a leukocyte enzyme to the substrate of the leukocyte enzyme. In some embodiments, the method comprises a step of measuring current flowing through an electrode of the electrochemical assay for a second time. In some embodiments, the method comprises a step of adding the leukocyte enzyme to the substrate of the leukocyte enzyme. In some embodiments, the method comprises a step of measuring current flowing through an electrode of the electrochemical assay for a third time.
- In some embodiments, the method of screening a patient for infection by detecting the presence of a leukocyte enzyme in an electrochemical assay following a process including a step of adding a first aliquot of a leukocyte enzyme to a substrate of the leukocyte enzyme. In some embodiments, the leukocyte enzyme substrate is in an electrolyte solution. In some embodiments, the method comprises a step of measuring current flowing through an electrode of the electrochemical assay. In some embodiments, the method comprises a step of adding at least one additional aliquot of the leukocyte enzyme to the substrate of the leukocyte enzyme. In some embodiments, the method comprises a step of measuring current flowing through an electrode of the electrochemical assay for a second time. In some embodiments, the method comprises a step of adding a product or reactant of a leukocyte enzyme to the substrate of the leukocyte enzyme. In some embodiments, the method comprises a step of measuring current flowing through an electrode of the electrochemical assay for a third time.
- In another aspect, the present disclosure is directed to kits containing suitable substrate, direction for optimizing the results and optionally providing patient specific therapeutic regimen based on the observed results.
-
FIG. 1 represents an initial hydroquinone substrate and first ester hydrolysis step. -
FIG. 2 represents a semiquinone intermediate and second ester hydrolysis step. -
FIG. 3 represents a final benzoquinone oxidation product. -
FIG. 4 represents the results of using 4-((tosyl-L-alanyl)oxy)phenyl tosyl-L-alaninate (“TAPTA”) in an internally calibrated electrochemical continuous enzyme assay (ICECEA). -
FIG. 5 represents the NMR of 4-((tosyl-L-alanyl)oxy)phenyl tosyl-L-alaninate (“TAPTA”). -
FIG. 6 is a schematic of the cleavage mechanism of a monoester embodiment of the present invention. -
FIG. 7 are voltammograms showing increasing reduction peaks with higher LE concentration for an electrode screen-printed with one embodiment of a substrate of the present invention. - As used herein and in the appended claims, the singular forms “a”, “and” and “the” include plural references unless the context clearly dictates otherwise.
- As used herein, “leukocyte” may refer to any white blood cell (“WBC”). Leukocytes are cells of the immune system that are involved in protecting the body against infectious disease and invading pathogens. All leukocytes/WBCs are divided into five classes based on morphological characteristics that differentiate themselves from one another. They include neutrophils, eosinophils, basophils, monocytes, and lymphocytes. Neutrophils comprise approximately 40-75% of leukocytes, eosinophils comprise approximately 1-6% of leukocytes, basophils comprise less than 1% of leukocytes, monocytes comprise approximately 2-10% of leukocytes, and lymphocytes (e.g. B lymphocytes and T lymphocytes) comprise approximately 20-45% of leukocytes.
- The term “patient” as used herein may refer to a biological system to which a treatment can be administered. A biological system can include, for example, an individual cell, a set of cells (e.g. a cell culture), an organ, a tissue, or multi-cellular organism. A “patient” can refer to a human patient or a non-human patient. In preferred embodiments, the patient is a human patient.
- The terms “effective amount” or “therapeutically effective amount” as used herein may refer to an amount of the compound or agent that is capable of producing a medically desirable result in a treated subject. The treatment method can be performed in vivo or ex vivo, alone or in conjunction with other drugs or therapy. A therapeutically effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route.
- The term “treating” or “treatment” of a disease refers to executing a protocol, which may include administering one or more drugs to a patient (human or otherwise), in an effort to alleviate signs or symptoms of the disease. Alleviation can occur prior to signs or symptoms of the disease appearing as well as after their appearance. Thus, “treating” or “treatment” includes “preventing” or “prevention” of disease. The terms “prevent” or “preventing” refer to prophylactic and/or preventative measures, wherein the object is to prevent or slow down the targeted pathologic condition or disorder.
- The present disclosure relates to compositions and methods for rapid detection (including determining the relative activity) of enzymes released by active leukocyte cells, e.g. leukocyte enzymes released by active leukocyte cells, in particular leukocyte esterase (“LE”) and human neutrophil elastase (“HNE”).
- In at least one aspect of the present disclosure, a method of screening a subject for infection is described, the method comprising the steps of (a) obtaining a sample of tissue or bodily fluid from a subject at risk of developing an infection, (b) applying the sample to a detector device, wherein the detector device comprises at least one substrate which is specific for at least one of LE and/or HNE, wherein at least one substrate is adapted to detect a threshold level at least one of LE and/or HNE, the threshold level correlated with a presence of infection; (c) ascertaining the threshold levels of LE and/or HNE present in the sample, wherein if the concentration each of LE and/or HNE exceeds the threshold level, and further wherein such measurement is a positive screen for infection.
- The disclosure provides a method wherein the infection is a periprosthetic joint infection (PJI). In some embodiments, the threshold level of leukocyte esterase (LE) for detection of PJI is at least about 20 pg/ml of leukocyte esterase in a synovial fluid sample.
- The compositions and methods for rapid detection utilize specific substrates for detecting leukocyte enzymes, e.g. LE and HNE, referred to as LE substrates and HNE substrates respectively. The compositions and methods for rapid detection may utilize electrochemical assays to detect the leukocyte enzymes, in particular, internally calibrated electrochemical continuous enzyme assay (“ICECEA”), but are not necessarily limited as such.
- In some embodiments, the substrates are capable of detecting LE. Such substrates are readily hydrolyzed by LE to generate a redox intermediate, which can provide a detectable electrochemical response. In some embodiments, the substrates for detecting LE (i.e. “LE substrates”) may follow Formula I as depicted below:
- Where A determines the identity of the acyl group, e.g. an alanine or lactate, at the ester cleavage site with enzyme specificity for leukocyte esterase and B is a moiety capable of participating in a redox reaction, which can be detected using an electrochemical assay (e.g. by using ICECEA or screen-printed electrochemical sensors).
- In some embodiments, A comprises an amino group (i.e., —NRa, where Ra is a H or an optionally substituted alkyl, aryl, or aralkyl group), or A comprises an ether group (i.e. —O—).
- The acyl group defined by A is protected using any effective amine or alcohol blocking group C (e.g. a tosyl group). The alcohol intermediate of the ester, moiety B, to be released upon hydrolysis by the esterase is a redox substrate and participates in a redox reaction. Additionally, the oxygen linking B in Formula I may be substituted with an —NH linking moiety (i.e. the ester group presented in Formula I may be substituted with an amido group) and still be within the scope of the present disclosure.
- The amine or alcohol blocking group C may comprise any of the following: acetyl (Ac), benzoyl (Bz), benzyl (Bn), β-methoxyethoxymethyl (MEM), dimethoxytrityl (DMT), methyoxymethyl (MOM), methoxytrityl [(4-methoxyphenyl)diphenylmethyl] (MMT), p-Methoxybenzyl (PMB), methylthiomethyl, pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trityl (Tr), sily (e.g. TMS, TBDMS, TOM, TIPS), methyl, and ethoxyethyl (EE), benzyloxycarbonyl (Cbz); p-methoxybenzylcarbonyl (Moz or MeOZ), tert-butoxycarbonyl (BOC), 9-fluorenylmethyloxycarbonyl (FMOC), 3,4-Dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP), tosyl (Ts), trichloroethoxycarbonyl (Troc), arylsulfonyl, or alkylsulfonyl (e.g.Nosyl and Nps).
- In some embodiments, the redox moiety B is a derivate of phenol, which may form an ester through its hydroxyl group. Such an intermediate may undergo oxidation to release an electron. For example, but not necessarily limited to, one phenol derivative, hydroquinone, contains two hydroxyl groups in a para conformation. Each hydroxyl group can be bound to form a distinct lactate ester, which is independently a substrate of leukocyte esterase (
FIG. 1 ). The resulting duplex substrate has two potential target sites for leukocyte esterase activity, and breakdown of the substrate is stepwise. Ester hydrolysis with leukocyte esterase at the first target will occur relatively slow due to molecular hindrance of the active sites; however, subsequent hydrolysis of the second active site will occur more quickly. This may effectively improve the specificity of an electrochemical assay, as non-specific hydrolysis would be less likely to begin the cascade. After the first ester hydrolysis step, an oxidation reaction can release an electron with removal of a hydrogen atom forming a semiquinone lactate ester intermediate (FIG. 2 ). After subsequent hydrolysis of the remaining ester, the quinone-based intermediate is released and can be further oxidized to form para-benzoquine. Para-benzoquine is reduced at low potentials, which minimizes interference from other redox active species within the sample and may improve assay selectivity. The final product is shown inFIG. 3 . - In other aspects, methods of treating a patient with positive indication of LE and HNE is described. In one embodiment, the serious infections caused by Gram-positive bacteria are currently difficult to treat because many of these pathogens are now resistant to standard antimicrobial agents. To that end, at least one aspect of the disclosure is to prophylactically treat a patient prior to any invasive operation to minimize risk of infection. In at least one embodiment, patients identified as suffering from an infection may be initiated a comprehensive treatment plan including administering antimicrobial agent, such as penicillins, cephalosporins, tetracyclines, daptomycin, tigecycline, linezolid, quinupristin/dalfopristin and dalbavancin and the like that may be useful in combating an active infection. In other embodiments, methods of screening or detecting risk of PJI, by developing useful for the treatment of infections due to drug-resistant Gram-positives and Gram-negatives.
- In some embodiments, B comprises a quinone. In some embodiments, B comprises a phenol. In some embodiments, B comprises a substituted quinone or a substituted phenol. In some embodiments, C comprises a tosyl protecting group. In some embodiments, the oxygen linking B in Formula II is substituted with an amino group. In further embodiments, B comprises aminophenyl. In some embodiments, B comprises substituted aminophenyl.
- Two specific, explicitly non-limiting examples of substrates for detecting leukocyte esterase (“LE”) that are within the scope of Formula I include 4-((tosyl-L-alanyl)oxy)phenyl tosyl-L-alaninate (Compound A below) and 4-(((S)-2-(tosyloxy)propanoyl)oxy)phenyl (S)-2-(tosyloxy)propanoate (Compound B below). Compound A is also referred to herein as “TAPTA.” An NMR of Compound A is shown in
FIG. 5 , illustrating the tosyl moiety structure and its attachment. Phenylethylenediamine variants of Compound A and Compound B (i.e. the para-oxygens are replaced with NH linkers) are also to be considered within the scope of the present disclosure and are likewise suitable for inclusion in electrochemical assays of the present disclosure (e.g. in ICECEA). - In some embodiments, the LE substrate comprises a composition as described in Formula II below:
- X1 and X2 are independently O, S or NRa. Ra is an H, an alkyl or an aryl group. X1 and X2 can be both oxygen or both NRa. Alternatively, one of X1 and X2 is oxygen and the other is NRa.
- Y1 and Y2 are independently O, S or NRa. Ra is as described above. Y1 and Y2 can be both oxygen or both NRa. Alternatively, one of Y1 and Y2 is oxygen and the other is NRa.
- R1 and R2 are independently an alkyl or an aryl group or a substituted alkyl, a substituted aryl or a protecting group. In some embodiments, R1 or R2 or both is methyl. In some embodiments, R1 or R2 or both may be a tosyl. In one embodiment, R2 is a tosyl.
- R3 and R4 are independently an alkyl, a protecting group such as tosyl, benzoyl, benzyl, trimethylsilyl, [bis-(4-methoxyphenyl)phenylmethyl], carbobenzyloxy, tert-Butyloxycarbonyl, 9-Fluorenylmethyloxycarbonyl, or a peptide moiety. In one embodiment, R4 is a tosyl. The peptide moiety can include any combination of natural and/or non-natural amino acids.
- R2 and R4 may also comprise any of the following: acetyl (Ac), benzoyl (Bz), benzyl (Bn), β-methoxyethoxymethyl (MEM), dimethoxytrityl (DMT), methyoxymethyl (MOM), methoxytrityl [(4-methoxyphenyl)diphenylmethyl] (MMT), p-Methoxybenzyl (PMB), methylthiomethyl, pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trityl (Tr), sily (e.g. TMS, TBDMS, TOM, TIPS), methyl, and ethoxyethyl (EE), benzyloxycarbonyl (Cbz); p-methoxybenzylcarbonyl (Moz or MeOZ), tert-butoxycarbonyl (BOC), 9-fluorenylmethyloxycarbonyl (FMOC), 3,4-Dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP), tosyl (Ts), trichloroethoxycarbonyl (Troc), arylsulfonyl, or alkylsulfonyl (e.g.Nosyl and Nps). In one embodiment, protecting group can be any one of tosyl, benzoyl, benzyl, trimethylsilyl, [bis-(4-methoxyphenyl)phenylmethyl], carbobenzyloxy, tert-Butyloxycarbonyl, 9-Fluorenylmethyloxycarbonyl.
- Each of the R5 on the ring is independently a halogen atom; a hydroxyl group; a C1-C6 alkyl group; a C3-C6 cycloalkyl group; a C3-C6 cycloalkyl C1-C6 alkyl group; a C2-C6 alkenyl group; a C2-C6 alkynyl group; a C1-C6 haloalkyl group (including trifluoro C1-C6alkyl); a C2-C6 haloalkenyl group; a C2-C6 haloalkynyl group; a C3-C6 halocycloalkyl group; a C3-C6 halocycloalkyl C1-C6 alkyl group; a C1-C6 alkoxy group; a C3-C6 cycloalkyloxy group; a C2-C6 alkenyloxy group; a C2-C6 alkynyloxy group; a C1-C6 alkylcarbonyloxy group; a C1-C6 haloalkoxy group; a C1-C6 alkylthio group; a C1-C6 alkylsulfinyl group; a C1-C6 alkylsulfonyl group; a C1-C6 haloalkylthio group; a C1-C6 haloalkylsulfinyl group; a C1-C6 haloalkylsulfonyl group; an amino group; a C1-C6 alkylcarbonylamino group; a mono(C1-C6 alkyl)amino group; a di(C1-C6 alkyl)amino group; a hydroxy C1-C6 alkyl group; a C1-C6 alkoxy C1-C6 alkyl group; a C1-C6 alkylthio C1-C6 alkyl group; a C1-C6 alkylsulfinyl C1-C6 alkyl group; a C1-C6 alkylsulfonyl C1-C6 alkyl group; a C1-C6 haloalkylthio C1-C6 alkyl group; a C1-C6 haloalkylsulfinyl C1-C6 alkyl group; a C1-C6 haloalkylsulfonyl C1-C6 alkyl group; a cyano C1-C6 alkyl group; a C1-C6 alkoxy C1-C6 alkoxy group; a C3-C6 cycloalkyl C1-C6 alkyloxy group; a C1-C6 haloalkoxy C1-C6 alkoxy group; a cyano C1-C6 alkoxy group; a C1-C6 acyl group; a C1-C6 alkoxyimino C1-C6 alkyl group; a carboxyl group; a C1-C6 alkoxycarbonyl group; a carbamoyl group; a mono(C1-C6 alkyl)aminocarbonyl group; a di(C1-C6 alkyl)aminocarbonyl group; a nitro group; or a cyano group. n is 0, 1, 2, 3, or 4. In at least one embodiment, X1 and X2 are independently O or NRa. Ra is a H, an alkyl, an aryl, or aralkyl group. X1 and X2 can be both oxygen or both NRa. Alternatively, one of X1 and X2 is oxygen and the other is NRa, in yet another embodiment, Y1 and Y2 are independently O or NRa.
- In some embodiments, the substrates detect human neutrophil elastase (“HNE”). In some embodiments, the substrates for detecting HNE (i.e. “HNE substrates”) may follow Formula III as depicted below:
- A1 through A4 (i.e. A1-A2-A3-A4) represent a core tetrapeptide scaffold sequence, which serves as the enzyme active site (i.e. the active site for human neutrophil elastase/HNE). A tetrapeptide sequence of Ala-Ala-Pro-Val (AAPV) (SEQ ID NO: 1) is most common, but natural or unnatural amino acids may be substituted at any of the four peptide sites in order to improve substrate sensitivity for HNE. For example, conservative substitutions may be made for SEQ ID NO: 1 and still be within the scope of the present disclosure. As used herein, “conservative substitutions” are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains 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, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
- B in Formula III represents a redox moiety, similar to the LE substrate displayed in Formula I above. For example, B may comprise derivate of phenol, which may form an ester through its hydroxyl group, e.g., a redox active alcohol intermediate. This may comprise, for example, a hydroquinone intermediate or hydroquinone-based redox groups. C in Formula III represents an acyl group, for example, N-methyoxysuccinyl. The acyl group may serve to improve substrate sensitivity for HNE, and some acyl groups, for example N-methoxysuccinyl, may also increase substrate solubility.
- One specific, explicitly non-limiting example of a substrate for detecting HNE that is within the scope of Formula III includes 3-{[(1S)-1-{[(2S)-1-(5-{[(1S)-1-({4-[(2S)-2-({1-[(2S)-2-[(2S)-2-(3-carboxypropanamido)propanamido]propanoyl]pyrrolidin-2-yl}formamido)-3-methylbutanamido]phenyl}carbamoyl)-2-methylpropyl]carbamoyl}imidazolidin-1-yl)-1-oxopropan-2-yl]carbamoyl}ethyl]carbamoyl}propanoic acid, Compound C below.
- As described above in connection with the embodiments of
FIGS. 1 and 2 , and Formulas II and III, there was reason to believe that a diester (consisting of two symmetric or asymmetric α-amino or α-hydroxy acid esters) would be a more effective substrate as the resulting duplex substrate would have two potential target sites for cleavage by leukocyte esterases. Further, the breakdown of the substrate would likely be stepwise such that ester hydrolysis with leukocyte esterase at the first active site would be slower, or more deliberate, due to the steric hindrance caused by the dual substrates. The initial though was that this may improve the specificity of an electrochemical assay, as non-specific hydrolysis would be less likely to begin the cascade of stepwise hydrolysis. However, Applicants found, surprisingly, that the diester was less effective than the monoester. Even in mixtures of diester and monoester in which the monoester was present in very low concentration (e.g. about 1%), the effectiveness of the monoester was predominant and dictated the effectiveness of the composition as a whole. Indeed, the effectiveness of the monoester was not discovered until the diester composition was purified to the point that the concentration of the monoester fell to below 1%. At that point, the effectiveness of the diester composition dropped precipitously, thereby indicating that the monoester was a more effective substrate for reacting with leukocyte esterase enzymes. - Accordingly, in one embodiment, the substrate of the present invention comprises a monoester, the monoester being one of an α-amino acid ester, such as an alanine ester, or an α-hydroxy acid ester, such as a lactate ester, with specificity for leukocyte esterases. The monoester has a first moiety for participating in a redox reaction, and a second moiety comprising an amine or alcohol blocking group.
- In one embodiment, the composition comprises a monoester as depicted in Formula I, wherein A comprises oxygen (O) or NRa, where Ra is a H or an optionally substituted alkyl, aryl, or aralkyl group, whereby A determines the identity of the acyl group of the ester, in that A is O if said monoester is an α-hydroxy acid ester (i.e. lactate ester) or A is NRa if said monoester is an α-amino acid ester (i.e. alanine ester). B is the first moiety and C is the second moiety.
- In one embodiment, any oxygen linking group linking the first and/or second moiety can be substituted by nitrogen linking groups, and nitrogen linking groups can be substituted by oxygen linking groups.
- In one embodiment, the first moiety (B) comprises one of a substituted or unsubstituted derivative of phenol, substituted or unsubstituted hydroxyanthracene, substituted or unsubstituted aminophenol, or substituted or unsubstituted hydroxyphenanthroline.
- In one embodiment, the second moiety (C) comprises one of the following: acetyl (Ac), benzoyl (Bz), benzyl (Bn), β-methoxyethoxymethyl (MEM), dimethoxytrityl (DMT), methyoxymethyl (MOM), methoxytrityl [(4-methoxyphenyl)diphenylmethyl] (MMT), p-Methoxybenzyl (PMB), methylthiomethyl, pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trityl (Tr), sily (e.g. TMS, TBDMS, TOM, TIPS), methyl, and ethoxyethyl (EE), benzyloxycarbonyl (Cbz); p-methoxybenzylcarbonyl (Moz or MeOZ), tert-butoxycarbonyl (BOC), 9-fluorenylmethyloxycarbonyl (FMOC), 3,4-Dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP), tosyl (Ts), trichloroethoxycarbonyl (Troc), arylsulfonyl, or alkylsulfonyl (e.g.Nosyl and Nps).
- In one embodiment, the second moiety is a sulfonyl group with a substituted or unsubstituted heterocycle or heteroaryl ring.
- In one embodiment, Formula 1 is further refined to the general structure depicted in Formula IV:
- wherein the first moiety B comprises 4-hydroxyphenyl
- A comprises oxygen or NRa, where Ra is a H or an optionally substituted alkyl, aryl, or aralkyl group, whereby A determines the identity of the acyl group of the ester, R1, R2, and R3 are independently hydrogen or optionally substituted alkyl groups (R3 is absent if A is oxygen), and R4 is a substituted or unsubstituted heterocycle or heteroaryl.
- In one embodiment, R4 is one of pyridinyl, pyridazinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, quinolyl, isoquinolyl, 1,2,3,4-tetrahydroquinolyl, tetrazolyl, furyl, thienyl, isooxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, indolyl, benimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, triazinyl, thiadiazolyl, oxadiazolyl, purinyl, 1-oxoisoindolyl, 1,2,4-trizainyl, 1,3,4-triazinyl, isoindolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzotriazolyl, benzothiazolyl, benzooxazolyl, tetrahydroquinolyl, dihydroquinolyl, naphthyridinyl, quinoxalinyl, quinazolinyl, dihydroisoquinolyl, tetrahydroisoquinolyl, benzofuryl, furopyridinyl, pyrrolopyridimidinyl, or azaindolyl.
- In one embodiment, R4 is a pyridine with or without the addition of substituted or unsubstituted polar groups.
- In one embodiment, R4 is a pyridine selected from one of the following: pyridine (I), methoxypyridine (II), and (methoxycarbonyl)pyridine (III) as represented below:
- In one particular embodiment, R4 is (methoxycarbonyl)pyridine (III).
- In a particular embodiment, the composition of the monoester substrate is 4-Hydroxyphenyl (N-(3-(methoxycarbonyl)pyridine-5-sulfonyl)-L-alaninate.
- In yet another particular embodiment, the composition of the monoester is depicted in Formula V:
- Referring to
FIG. 6 , the cleaving mechanism for Formula V is shown. Specifically, the leukocyte esterase (LE) cleaves the monoester substrate at the oxygen site upon ester hydrolysis. - In one embodiment, the substrate of the leukocyte esterase enzyme is screen-printed onto the surfaces of an electrode sensor strips using known and commercially-available techniques and materials.
- As described herein, leukocytes are capable of producing leukocyte enzymes that are able to be detected and/or quantified by the electrochemical assays (i.e. ICECEA) of the present disclosure.
- Leukocyte enzymes may include, for example, those described in WO 2010/036930, hereby incorporated by reference in its entirety, such as, for example, IL-10, leukocyte elastase, leukocyte esterase, and/or gelatinase B, along with human neutrophil elastase. Leukocyte esterase (“LE”) is an esterase produced by leukocytes (white blood cells). LE is the subject of, for example, urine tests for the presence of leukocytes/WBCs and other abnormalities associated with infection. Human neutrophil elastase (“HNE”), also known as human leukocyte elastase (“HLE”), is a serine protease. It is in the same family as chymotrypsin and possesses broad substrate activity. HNE is secreted by neutrophils and macrophages, two of the five classes of leukocytes as described herein. HNE is 218 amino acids long and has two asparagine-linked carbohydrate chains. There are two forms of HNE, deemed IIa and IIb.
- The term “sample” as used herein may refer to a biological sample, including a sample of biological tissue or fluid origin obtained in vivo or in vitro. Biological samples can be, but are not limited to, body fluid (e.g., serous fluid, blood, blood plasma, serum, or urine), organs, tissues, fractions, and cells isolated from mammals including, for example, humans. Biological samples also may include sections of the biological sample including tissues. Biological samples may also include extracts from a biological sample, for example, a biological fluid (e.g., blood, serum, peritoneal fluid, and/or urine). Of particular interest, but explicitly non-limiting, are urine, sputum (for example, in a patient diagnosed with cystic fibrosis), peritoneal fluid (for example, in a patient with liver cirrhosis and ascites) and other serous fluids, including but not limited to, for example, synovial fluid, pleural fluid, pericardial fluid, cerebrospinal fluid (“CSF”) and middle ear fluid.
- In some embodiments, the presence of leukocytes, i.e. as determined by detecting and/or quantifying the amount of a leukocyte enzyme (e.g. LE and/or HNE) present in the biological sample may indicate the presence of an infection in a subject. Such embodiments may utilize the LE and/or HNE substrates of the present disclosure in an electrochemical assay, in particular ICECEA as described herein. For example, the presence of LE and/or HNE in urine may indicate a subject as having a urinary tract infection (“UTI”). Similarly, the presence of LE and/or HNE in synovial fluid may indicate a subject as having a joint infection, for example but not necessarily limited to a periprosthetic joint infection (“PJI”). These examples of indicating the presence of infection are not limited as such, as these are merely exemplary uses of the substrates of the present disclosure, and they may or may not be utilized in an electrochemical assay, for example, in an ICECEA.
- In some embodiments, the substrates of the present disclosure are used to indicate a subject as having periprosthetic joint infection (PJI). PJI is a devastating complication following total joint arthroplasty, which remains a challenge for surgeons both diagnostically and therapeutically. Establishing an accurate and timely diagnosis of PJI is of critical importance for making treatment decisions. For patients presenting with a painful prosthesis, it is important to complete a work-up to either rule out or diagnose the presence of infection. In most cases, serological testing, including erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP), is the initial screening test of choice. In patients with elevated serological markers or even just a high suspicion of infection, the next step is to perform joint aspiration for testing of synovial fluid. Classically, bacterial culture of synovial fluid has been used to make the diagnosis of PJI. As bacterial culture is not in itself sufficiently sensitive, with as many as 30% of infections being culture negative, orthopedic surgeons also consider the results of serological testing, synovial fluid white blood cell count and polymorphonuclear percentage, and histological analysis to make a diagnosis. Unfortunately, bacterial culture and traditional synovial fluid testing can require days to more than a week to yield a result.
- Thus, in some embodiments, synovial fluid aspirated from a painful joint would be tested for LE and/or HNE activity using an enzyme substrate of the present disclosure. For example, this may be accomplished through use of an ICECEA assay as described herein. In such embodiments, the activity of LE and/or HNE would be reported as a continuous measurement of absolute concentration. This could be performed in the office or operating room to yield a result in minutes for point-of-care decision-making.
- Based on an accumulation of population data, the level of LE and/or HNE activity can be combined with additional metrics to predict the likelihood that an infection is present. Additional metrics may include the type of joint, a history of prior infection, and the results of serological testing (ESR and CRP). Surgeons can consider the likelihood that an infection is present to determine the most appropriate treatment algorithm for their patient. In cases with a high likelihood that infection is present, treatment for PJI, such as prosthesis extraction and antibiotic spacer placement, incision and debridement, or long-term antibiotic suppression, could be considered based on the acuity of the infection, among other factors. In cases in which there is a moderate likelihood that infection is present, a surgeon could consider initiating treatment or waiting for additional diagnostic results. Finally, other etiologies for a painful prosthesis may be considered in cases for which the likelihood of the presence of infection is low or for which infection has largely been ruled out.
- In addition to making an initial diagnosis of infection, the substrates of the current disclosure, e.g. as used in an assay (such as, for example, an ICECEA) may be used to establish the resolution of PJI in order to determine the correct timing for re-implantation of a new prosthesis. The level of LE and/or HNE activity may be used in addition to serological markers and other synovial fluid tests to determine the success of treatment, such as discussed supra. For patients with a persistently elevated LE and/or HNE, surgeons may elect to continue intravenous antibiotics or attempt an exchange of the antibiotic spacer to improve prospects of complete resolution of infection.
- In some embodiments, the substrates of the present disclosure are used to indicate a subject as having spontaneous bacterial peritonitis (SBP). SBP is a serious and life-threatening complication that is relativity common in patients with liver cirrhosis and ascites. For patients with this complication, a rapid diagnosis and early administration of antibiotics is critical for survival, and in-hospital mortality can be as high as 20%. For patients with ascites, presenting symptoms of fever, change in mental status, and abdominal tenderness are frequent signs of SBP. In such cases, a diagnostic paracentesis is performed, and a diagnosis is made based on an absolute neutrophil count above 250 cells/mm3 and/or bacterial culture.
- Thus, in some embodiments, ascitic fluid obtained from diagnostic paracentesis would be tested for LE and/or HNE activity using an enzyme substrate of the present disclosure. For example, this may be accomplished through use of an ICECEA assay as described herein. Using an ICECEA assay, the activity of LE or HNE would be reported as a continuous measurement of absolute concentration. Based on an accumulation of population data collected from many patients, the absolute concentration of LE and/or HNE would be compared to gold standard diagnostic criteria to provide a calculation of the probability that SBP is present. The likelihood of infection can be used to inform the treating physician as to the most appropriate treatment algorithm. The measured level of LE or HNE could also provide important prognostic information, with a higher level indicating a worse prognosis.
- In some embodiments, the substrates of the present disclosure are used to indicate a subject as having a urinary tract infection (UTI), also known as a urogenital infection. For healthy women with classic UTI symptoms, such as dysuria and frequency, and no vaginal discharge or irritation, a diagnosis of UTI can typically be made on clinical symptoms alone. On the contrary, women with poorly defined symptoms, asymptomatic pregnant females, elderly patients, and children have a much lower pre-test probability for UTI. The present disclosure is not limited to testing women for UTI. The gold standard for diagnosis of UTI is mid-stream urine culture (with >103-105 organisms) or pyuria (greater than 104 leukocytes per ml).
- Thus, in some embodiments, mid-stream urine for symptomatic patients would be tested for leukocyte esterase (“LE”) and/or human neutrophil elastase (“HNE”) activity using an enzyme substrate of the present disclosure. For example, this may be accomplished through use of an ICECEA assay as described herein. Based on population data, likelihood of infection can be determined based on both measurement of LE and/or HNE activity and additional factors, such as the presence of specific symptoms and patient characteristics (i.e. age, gender, pregnancy). Depending on the likelihood of infection, a physician can decide whether or not to administer oral antibiotics.
- Population data for the clinical applications of the present disclosure (i.e. in indicating a patient as having an infection, for example, but not limited to, PJI, SBP, and/or UTI) can be used to convert the measure of LE and/or HNE activity to a predictive probability for the presence of infection. The test device itself can be used as a medium to both collect and distribute such population-based data. For example, a smartphone (or similar device) connected electrochemical biosensor can allow physicians to provide selected information to a centralized database, which may then be used to continuously improve the calculation of infection likelihood. The biosensor may also report back to surgeons the likelihood of infection for their individual patient based upon LE and/or HNE activity and additional metrics that can be used to hone their treatment algorithm.
- In some embodiments, the substrates for detecting leukocyte enzymes, e.g. LE and/or HNE substrates, are incorporated into an assay. Such an assay may comprise, for example, an electrochemical assay. Electrochemical assays are cost-effective, highly sensitive, and simplify the calibration process. Furthermore, such methods would be just as effective in bloody or turbid fluid. A preferred electrochemical assay comprises an internally calibrated electrochemical continuous enzyme assay (“ICECEA”). Use of a LE substrate of the present disclosure (“TAPTA”) in an ICECEA is described in Example 1, infra. ICECEAs are generally disclosed in PCT/US2014/03713 and U.S. 2016/0040209, the disclosure of which is hereby incorporated in its entirety. ICECEA utilizes integration of an enzyme-free pre-assay calibration with an electrochemical enzyme assay in a continuous experiment. This is believed to result in a uniquely shaped amperometric trace that allows for selective and sensitive determination of enzymes, e.g. LE and HNE, present in a sample.
- ICECEAs generally follow the following method as described in U.S. 2016/0040209. First, an enzyme substrate (e.g. an LE and/or HNE substrate of the present disclosure) is placed in a background electrolyte. Next, a reactant or product of an enzymatic reaction of the enzyme is added to the first enzyme substrate/background electrolyte, which creates what is described as a “first assay mixture.” Current flowing through an electrode of the electrochemical assay is then measured after the first assay mixture is formed. Next, the enzyme (e.g. LE and/or HNE) is added to the “first assay mixture” to create a “second assay mixture,” and the current is measured again over a predetermined time period. Enzyme activity is determined based on the change in current over time caused by the addition of the enzyme. While optimally the enzyme is added after the reactant/product is added to the enzyme substrate, the order can be switched, i.e. the enzyme is added to the substrate first and then the reactant/product is added.
- The ICECEA includes an electrochemical measuring device. The electrochemical measuring device includes a working electrode, a reference electrode, and an auxiliary electrode. The current is measured through the working electrode. The working electrode may be a noble metal electrode, metal oxide electrode, an electrode made of a carbon allotrope, or a modified electrode. The auxiliary electrode may be a platinum wire. The reference electrode may be Ag/AgCl/NaCl or any other reference electrode. The electrochemical assay system can also be made of only a working electrode and a reference electrode. Measuring the changes in current may be done by collecting an amperometric trace of the current.
- Generally, in an ICECEA, adding the reactant/product to the enzyme substrate (in electrolyte) in the electrochemical assay system includes the following steps. First, a first aliquot of the reactant/product is added to the enzyme substrate (in electrolyte). Current flowing through an electrode of the electrochemical assay system is measured after the first aliquot is added. One or more additional aliquots of the reactant/product are added to the mixture and current flowing through an electrode of the electrochemical assay system is measured again. Preferably, at least three aliquots of the reactant/product are added to the enzyme substrate (in electrolyte) before the enzyme is added to the mixture. Alternatively, the aliquots of the reactant/product are added to the substrate (in electrolyte) after the enzyme is added to the mixture.
- The enzymatic activity of the enzyme may be determined from the slope of a line created from measuring the current flowing through a working electrode of the electrochemical assay system after the reactant/product is added to the substrate (before the enzyme is added, or vice versa as described herein) at predetermined intervals over a predetermined time period. An advantage of this method is that the addition of the reactant/product to the substrate (in electrolyte) and the addition of the enzyme are performed in the same container using the same electrode.
- In at least one embodiment, a customized kit is described containing a solution of enzyme substrate and other necessary reactants in a background electrolyte; a solution of redox active component of enzymatic reaction; and a solution of assayed enzyme. As such, an amperometric measurement is done by using any electrochemical measurement device with amperometric method and a conventional electrochemical cell with the working, reference, and counter electrodes immersed in a solution containing the enzyme substrate. The working electrode is held at a potential E vs. the potential of the reference electrode. The potential E is adequate for either the oxidation or reduction of species present in the solution containing the redox active component of the enzymatic reaction. The experiment is performed by spiking one or more known aliquots of a redox active containing solution followed by one aliquot of a solution containing assayed enzyme into a stirred solution that contains enzyme substrate and other necessary reactants and measuring the current flowing through the working electrode.
- Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference in their entireties.
- Publications disclosed herein are provided solely for their disclosure prior to the filing date of the present invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.
- Each of the applications and patents cited in this text, as well as each document or reference, patent or non-patent literature, cited in each of the applications and patents (including during the prosecution of each issued patent; “application cited documents”), and each of the PCT and foreign applications or patents corresponding to and/or claiming priority from any of these applications and patents, and each of the documents cited or referenced in each of the application cited documents, are hereby expressly incorporated herein by reference in their entirety. More generally, documents or references are cited in this text, as well as each document or reference cited in each of the herein-cited references (including any manufacturer's specifications, instructions, etc.), is hereby expressly incorporated herein by reference.
- The following non-limiting examples serve to further illustrate the present disclosure.
- 1. Use of 4-((Tosyl-L-Alanyl)Oxy)Phenyl Tosyl-L-Alaninate in an Internally Calibrated Electrochemical Continuous Enzyme Assay (ICECEA)
- The substrate 4-((tosyl-L-alanyl)oxy)phenyl tosyl-L-alaninate, Compound A below (also referred to as “TAPTA”) was used as a substrate to measure the activity of leukocyte esterase (LE) in an internally calibrated electrochemical continuous enzyme assay (ICECEA). The results are indicated in
FIG. 4 . - The ICECEA was conducted as generally described in U.S. 2016/0040209 as well as in the detailed description supra. Briefly, in the pre-assay phase, three (3) distinct calibration steps were performed by spiking a solution of enzyme substrate (“TAPTA”) and necessary reactants with a solution of the redox active component of the enzymatic reaction. These three distinct calibration steps are denoted by a bold “a” in
FIG. 4 . After calibration, the assay phase was commenced by spiking one aliquot of assayed enzyme (LE) into the enzyme substrate solution. This step is denoted by a bold “b” inFIG. 4 . The enzymatic reaction was followed by measuring current flowing through the working electrode. The enzyme assay was calibrated for LE concentrations ranging from 0-250 μg/L. The enzyme activity of LE demonstrated a linear response relative to LE concentration and predictive of an infection. - 2. Synthesis of Monoester of Formula V
- 4-Hydroxyphenyl (N-(3-(methoxycarbonyl)pyridine-5-sulfonyl)-L-alaninate (Monoester) can be prepared by partial hydrolysis of 1,4-Phenylene bis(N-(3-(methoxycarbonyl)pyridine-5-sulfonyl)-L-alanininate (synthesized by a modification of the procedure for Compound III described in Hanson et al., Chembiochem 2018, 19, https://www.ncbi.nlm.nih.gov/pubmed/29679431). Suitable bases include alkali hydroxides, alkaline earth hydroxides, ammonia, amines, etc.
- Hydrolysis with NaOH. 1,4-Phenylene bis(N-(3-(methoxycarbonyl)pyridine-5-sulfonyl)-L-alanininate (21 mg, 0.032 mmol) was dissolved in THF and treated with 1M NaOH (0.045 mL, 139 mol %), at 30° C. for 4 days. The solvent was evaporated, the residue was dissolved in dichloromethane, rinsed with 1M HCl, and dried over MgSO4 to give the product as a colorless glass.
- Hydrolysis with triethylamine. 1,4-Phenylene bis(N-(3-(methoxycarbonyl)pyridine-5-sulfonyl)-L-alanininate (98 mg, 0.151 mmol) was dissolved in dichloromethane (2 mL) and triethylamine (28 mg, 0.277 mmol, 184 mol %). Water (62 mg) was added and the heterogeneous mixture was stirred at 30° C. for 3 days. 1M HCl was added to pH 1. The layers were separated, and the aqueous layer was extracted twice with dichloromethane. The combined organic extracts were dried over MgSO4 and evaporated to give a pink foam. Chromatography on silica gel with dichloromethane-ethyl acetate (70:30) afforded 4-Hydroxyphenyl (N-(3-(methoxycarbonyl)pyridine-5-sulfonyl)-L-alaninate (36 mg, 63% yield) as a white crystalline solid, mp 113-116° C. NMR (DMSO-d6) δ 1.37 (3H, d), 3.91 (3H, s), 4.31 (1H, q), 6.68 (4H, Abq),8.59 (1H, t), 8.96 (1H, br), 9.18 (1H, d), 9.22 (1H, d), 9.47 (1H, s); ms+ 381 (M+H)+; ms− 379 (M−H)−.
- Synthesis of 4-Hydroxyphenyl (N-(3-(methoxycarbonyl)pyridine-5-sulfonyl)-L-alaninate from L-Alanine tert-Butyl ester or L-Alanine benzyl ester. L-Alanine tert-butyl ester was condensed with methyl 5-(chlorosulfonyl)pyridine-3-carboxylate in the presence of triethylamine. The tert-butyl group was removed by treatment with HCl (g) in dichloromethane to give N-(3-(methoxycarbonyl)pyridine-5-sulfonyl)-L-alanine. This intermediate was also prepared by condensation of L-alanine benzyl ester with methyl 5-(chlorosulfonyl)pyridine-3-carboxylate followed by hydrogenation in EtOAc over Pd/C.
- N-(3-(methoxycarbonyl)pyridine-5-sulfonyl)-L-alanine was condensed with excess hydroquinone in acetonitrile in the presence of DCC and DMAP to afford Hydroxyphenyl (N-(3-(methoxycarbonyl)pyridine-5-sulfonyl)-L-alaninate. Alternatively, N-(3-(methoxycarbonyl)pyridine-5-sulfonyl)-L-alanine was condensed with hydroquinone monobenzyl ether or mono-BOC-hydroquinone [tert-butyl 4-(hydroxyphenyl) carbonate] followed by hydrogenation over palladium in acetic acid or hydrolysis with HCl (g) respectively to afford 4-Hydroxyphenyl (N-(3-(methoxycarbonyl)pyridine-5-sulfonyl)-L-alaninate.
- 3. Use of 4-Hydroxyphenyl (N-(3-(Methoxycarbonyl)Pyridine-5-Sulfonyl)-L-Alaninate on Screen-Printed Electrode.
- Referring to
FIG. 7 , the effectiveness of using the substrate of the present invention on a screen-printed electrode strip is demonstrated. Specifically, electrode strips were screen printed with the substrate having the structure as depicted in Formula V. The strips were contacted with varying concentrations of LE (i.e. 0.125 U/ml to 0.35 U/ml LE). - The plot clearly indicates significant reduction peaks at about −0.17 V, which correspond to the redox reaction of hydroquinone molecules that are released upon cleavage of the monoester by leukocyte esterases at the ester active site. Moreover, because the reduction peaks were seen to be directly related to the LE activity within the sample in a dose dependent manner, the screen-printed electrode strips not only were confirmed to detect the presence of LE, but also can provide a quantitative measure as to the activity level or concentration of LE in a sample that directly corresponds to the enzymes cleavage of the monoester. Based on this measured level, a determination can be made as to whether the patient's level of LE is high enough to indicate infection.
- The foregoing examples and description of the preferred embodiments should be taken as illustrating, rather than as limiting the present disclosure as defined by the claims. As will be readily appreciated, numerous variations and combinations of the features set forth above can be utilized without departing from the present disclosure as set forth in the claims. Such variations are not regarded as a departure from the scope of the disclosure, and all such variations are intended to be included within the scope of the following.
Claims (18)
1. A substrate composition with a specificity for leukocyte esterases having a first moiety for participating in a redox reaction and a second moiety comprising an amine blocking group or alcohol blocking group, wherein said substrate has a chemical formula of Formula I:
Wherein “A” comprises oxygen (O) or NRa, where Ra is hydrogen (H) or optionally substituted alkyl, aryl, or aralkyl, “B” has the general structure
where X, Y, and L are the
same or different and are independently nitrogen (N) or carbon, R5 and R6are the same or different and are independently hydrogen (H), a halogen, (OH)2P(O)2, optionally substituted alkyl or aryl, halogenated alkyl or halogenated aryl, optionally substituted alkoxy (but not at position 4), OH, amino, optionally substituted alkylamino or arylamino, NO2, thio, optionally substituted alkylthio or arylthio, hydroxycarbonyl, formyl, optionally substituted alkoxycarbonyl or aryloxycarbonyl, amido, optionally substituted alkyl or aryl amido, or optionally substituted hydroxyalkyl or hydroxyaryl, and “C” is an amine blocking group or alcohol blocking group.
2 The substrate composition of claim 1 , wherein moiety B is selected from one of the following:
Where X, Y, and Z are the same or different and are independently nitrogen (N) or carbon, R′ is hydrogen (H) or optionally substituted alkyl or aryl, and R6 is hydrogen (H), a halogen, (OH)2P(O)2, optionally substituted alkyl or aryl, halogenated alkyl or halogenated aryl, optionally substituted alkoxy (but not at position 4), OH, amino, optionally substituted alkylamino or arylamino, NO2, thio, optionally substituted alkylthio or arylthio, hydroxycarbonyl, formyl, optionally substituted alkoxycarbonyl or aryloxycarbonyl, amido, optionally substituted alkyl or aryl amido, or optionally substituted hydroxyalkyl or hydroxyaryl.
3. The substrate composition of claim 1 , wherein said second moiety C comprises one of the following: acetyl (Ac), benzoyl (Bz), benzyl (Bn), β-methoxyethoxymethyl (MEM), dimethoxytrityl (DMT), methyoxymethyl (MOM), methoxytrityl [(4-methoxyphenyl)diphenylmethyl] (MMT), p-Methoxybenzyl (PMB), methylthiomethyl, pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trityl (Tr), sily (e.g. TMS, TBDMS, TOM, TIPS), methyl, and ethoxyethyl (EE), benzyloxycarbonyl (Cbz); p-methoxybenzylcarbonyl (Moz or MeOZ), tert-butoxycarbonyl (BOC), 9-fluorenylmethyloxycarbonyl (FMOC), 3,4-Dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP), tosyl (Ts), trichloroethoxycarbonyl (Troc), arylsulfonyl, or alkylsulfonyl (e.g.Nosyl and Nps).
4. The substrate composition of claim 1 , wherein said second moiety C comprises a sulfonyl group with a substituted or unsubstituted aryl, heteroaryl, or heterocycle
5. The substrate composition of claim 1 , wherein said second moiety C is one of Tosyl, pyridine-sulfonyl, methoxypyridine-sulfonyl, or (methoxycarbonyl)pyridine-sulfonyl
6. A substrate composition for leukocyte esterase with the general structure depicted in Formula IV:
Wherein “A” comprises oxygen (O) or NRa, where Ra is hydrogen (H) or optionally substituted alkyl, aryl, or aralkyl, R1 and R2 are either the same or different and are independently hydrogen or optionally substituted alkyl “B” has the
general structure
where X, Y, and Z are the same or different and are independently nitrogen (N) or carbon, R5 and R6are the same or different and are independently hydrogen (H), a halogen, (OH)2P(O)2, optionally substituted alkyl or aryl, halogenated alkyl or halogenated aryl, optionally substituted alkoxy (but not at position 4), OH, amino, optionally substituted alkylamino or arylamino, NO2, thio, optionally substituted alkylthio or arylthio, hydroxycarbonyl, formyl, optionally substituted alkoxycarbonyl or aryloxycarbonyl, amido, optionally substituted alkyl or aryl amido, or optionally substituted hydroxyalkyl or hydroxyaryl, and R4 is a substituted or unsubstituted aryl, heteroaryl, or heterocycle.
7. The substrate composition of claim 6 , wherein moiety B is selected from one of the following:
Where X, Y, and Z are the same or different and are independently nitrogen (N) or carbon, R′ is hydrogen (H) or optionally substituted alkyl or aryl, and R6 is hydrogen (H), a halogen, (OH)2P(O)2, optionally substituted alkyl or aryl, halogenated alkyl or halogenated aryl, optionally substituted alkoxy (but not at position 4), OH, amino, optionally substituted alkylamino or arylamino, NO2, thio, optionally substituted alkylthio or arylthio, hydroxycarbonyl, formyl, optionally substituted alkoxycarbonyl or aryloxycarbonyl, amido, optionally substituted alkyl or aryl amido, or optionally substituted hydroxyalkyl or hydroxyaryl.
8. The substrate composition of claim 6 , wherein said composition has the structure:
Wherein “A” comprises oxygen (O) or NRa, where Ra is hydrogen (H) or optionally substituted alkyl, aryl, or aralkyl, R1 and R2 are either the same or different and are independently hydrogen or optionally substituted alkyl, where W, X, Y, and Z are the same or different and are independently nitrogen (N) or carbon, R5 and R6are the same or different and are independently hydrogen (H), a halogen, (OH)2P(O)2, optionally substituted alkyl or aryl, halogenated alkyl or halogenated aryl, optionally substituted alkoxy (but not at position 4), OH, amino, optionally substituted alkylamino or arylamino, NO2, thio, optionally substituted alkylthio or arylthio, hydroxycarbonyl, formyl, optionally substituted alkoxycarbonyl or aryloxycarbonyl, amido, optionally substituted alkyl or aryl amido, or optionally substituted hydroxyalkyl or hydroxyaryl, and R7 is hydrogen (H), OH, amino, alkyl, aryl, alkoxy, aryloxy, hydroxycarbonyl, alkoxycarbonyl, or aryloxycarbonyl.
9. The substrate composition of claim 6 , wherein said composition has the structure:
Wherein “A” comprises oxygen (O) or NRa, where Ra is hydrogen (H) or optionally substituted alkyl, aryl, or aralkyl, R1 and R2 are either the same or different and are independently hydrogen or optionally substituted alkyl, where W, X, Y, and Z are the same or different and are independently nitrogen (N) or carbon, R5 and R6are the same or different and are independently hydrogen (H), a halogen, (OH)2P(O)2, optionally substituted alkyl or aryl, halogenated alkyl or halogenated aryl, optionally substituted alkoxy (but not at position 4), OH, amino, optionally substituted alkylamino or arylamino, NO2, thio, optionally substituted alkylthio or arylthio, hydroxycarbonyl, formyl, optionally substituted alkoxycarbonyl or aryloxycarbonyl, amido, optionally substituted alkyl or aryl amido, or optionally substituted hydroxyalkyl or hydroxyaryl, and R7 is H, CH3, OCH3, or CO2CH3.
10. The substrate composition of claim 8 , wherein said composition has the structure:
Wherein “A” comprises oxygen (O) or NRa, where Ra is hydrogen (H) or optionally substituted alkyl, aryl, or aralkyl, R1 and R2 are either the same or different and are independently hydrogen or optionally substituted alkyl, where W, X, Y, and Z are the same or different and are independently nitrogen (N) or carbon, R6 is hydrogen (H), a halogen, (OH)2P(O)2, optionally substituted alkyl or aryl, halogenated alkyl or halogenated aryl, optionally substituted alkoxy (but not at position 4), OH, amino, optionally substituted alkylamino or arylamino, NO2, thio, optionally substituted alkylthio or arylthio, hydroxycarbonyl, formyl, optionally substituted alkoxycarbonyl or aryloxycarbonyl, amido, optionally substituted alkyl or aryl amido, or optionally substituted hydroxyalkyl or hydroxyaryl, and R7 is hydrogen (H), OH, amino, alkyl, aryl, alkoxy, aryloxy, hydroxycarbonyl, alkoxycarbonyl, or aryloxycarbonyl.
11. The substrate composition of claim 8 , wherein said composition has the structure:
Wherein “A” comprises oxygen (O) or NRa, where Ra is hydrogen (H) or optionally substituted alkyl, aryl, or aralkyl, R1 and R2 are either the same or different and are independently hydrogen or optionally substituted alkyl, where W, X, Y, and Z are the same or different and are independently nitrogen (N) or carbon, R6 is hydrogen (H), a halogen, (OH)2P(O)2, optionally substituted alkyl or aryl, halogenated alkyl or halogenated aryl, optionally substituted alkoxy (but not at position 4), OH, amino, optionally substituted alkylamino or arylamino, NO2, thio, optionally substituted alkylthio or arylthio, hydroxycarbonyl, formyl, optionally substituted alkoxycarbonyl or aryloxycarbonyl, amido, optionally substituted alkyl or aryl amido, or optionally substituted hydroxyalkyl or hydroxyaryl, and R7 is H, CH3, OCH3, or CO2CH3.
12. A substrate composition with a specificity for leukocyte esterases having a first moiety for participating in a redox reaction and a second moiety comprising an amine blocking or alcohol blocking group, wherein said substrate has a chemical formula of Formula I:
Wherein “A” comprises oxygen (O) or NRa, where Ra is hydrogen (H) or optionally substituted alkyl, aryl, or aralkyl, “B” has the general structure
where X, Y, and Z are the same or different and are independently nitrogen (N) or carbon, R5 and R6are the same or different and are independently hydrogen (H), a halogen, (OH)2P(O)2, optionally substituted alkyl or aryl, halogenated alkyl or halogenated aryl, optionally substituted alkoxy, OH, amino, optionally substituted alkylamino or arylamino, NO2, thio, optionally substituted alkylthio or arylthio, hydroxycarbonyl, formyl, optionally substituted alkoxycarbonyl or aryloxycarbonyl, amido, optionally substituted alkyl or aryl amido, or optionally substituted hydroxyalkyl or hydroxyaryl, and “C” is an amine blocking group or alcohol blocking group.
13. The substrate composition of claim 12 , wherein moiety B is the following
Wherein X, Y, and Z are the same or different and are independently nitrogen (N) or carbon, R′ is hydrogen (H) or optionally substituted alkyl or aryl, and R6 is hydrogen (H), a halogen, (OH)2P(O)2, optionally substituted alkyl or aryl, halogenated alkyl or halogenated aryl, optionally substituted alkoxy, OH, amino, optionally substituted alkylamino or arylamino, NO2, thio, optionally substituted alkylthio or arylthio, hydroxycarbonyl, formyl, optionally substituted alkoxycarbonyl or aryloxycarbonyl, amido, optionally substituted alkyl or aryl amido, or optionally substituted hydroxyalkyl or hydroxyaryl.
14. A substrate composition for leukocyte esterase with the general structure depicted in Formula IV:
Wherein “A” comprises oxygen (O) or NRa, where Ra is hydrogen (H) or optionally substituted alkyl, aryl, or aralkyl, R1 and R2 are either the same or different and are independently hydrogen or optionally substituted alkyl “B” has
the general structure,
where X, Y, and Z are the same or different and are independently nitrogen (N) or carbon, R5 and R6 are the same or different and are independently hydrogen (H), a halogen, (OH)2P(O)2, optionally substituted alkyl or aryl, halogenated alkyl or halogenated aryl, optionally substituted alkoxy, OH, amino, optionally substituted alkylamino or arylamino, NO2, thio, optionally substituted alkylthio or arylthio, hydroxycarbonyl, formyl, optionally substituted alkoxycarbonyl or aryloxycarbonyl, amido, optionally substituted alkyl or aryl amido, or optionally substituted hydroxyalkyl or hydroxyaryl, and R4 is a substituted or unsubstituted aryl, heteroaryl, or heterocycle.
15. The substrate composition of claim 6 , wherein moiety B is the following:
Wherein X, Y, and Z are the same or different and are independently nitrogen (N) or carbon, R′ is hydrogen (H) or optionally substituted alkyl or aryl, and R6 is hydrogen (H), a halogen, (OH)2P(O)2, optionally substituted alkyl or aryl, halogenated alkyl or halogenated aryl, optionally substituted alkoxy, OH, amino, optionally substituted alkylamino or arylamino, NO2, thio, optionally substituted alkylthio or arylthio, hydroxycarbonyl, formyl, optionally substituted alkoxycarbonyl or aryloxycarbonyl, amido, optionally substituted alkyl or aryl amido, or optionally substituted hydroxyalkyl or hydroxyaryl.
16. A device for detecting leukocyte esterase comprising a substrate that releases one of phenol, a derivative of phenol, or optionally substituted hydroquinone as the electrochemical mediator upon cleavage of an ester linkage by leukocyte esterase.
17. A method for detecting leukocyte esterase in a biological sample comprising:
detecting release of one phenol, a derivative of phenol, or optionally substituted hydroquinone as an electrochemical mediator in an electrochemical assay.
18. A method for diagnosis of infection comprising:
determining the level of leukocyte esterase in a test sample based on release of one of phenol, a derivative of phenol, or an optionally substituted hydroquinone as the electrochemical mediator in an electrochemical assay.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US17/342,077 US20210371898A1 (en) | 2016-03-22 | 2021-06-08 | Compositions and methods for determining the presence of active leukocyte cells using an electrochemical assay |
Applications Claiming Priority (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201662311405P | 2016-03-22 | 2016-03-22 | |
US201662352560P | 2016-06-21 | 2016-06-21 | |
PCT/US2017/022976 WO2017165222A1 (en) | 2016-03-22 | 2017-03-17 | Compositions and methods for derermining the presence of active leukocyte cells using an electrochemical assay |
US201816087411A | 2018-09-21 | 2018-09-21 | |
US16/145,014 US11104933B1 (en) | 2016-03-22 | 2018-09-27 | Compositions and methods for determining the presence of active leukocyte cells using an electrochemical assay |
US17/342,077 US20210371898A1 (en) | 2016-03-22 | 2021-06-08 | Compositions and methods for determining the presence of active leukocyte cells using an electrochemical assay |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/145,014 Continuation US11104933B1 (en) | 2016-03-22 | 2018-09-27 | Compositions and methods for determining the presence of active leukocyte cells using an electrochemical assay |
Publications (1)
Publication Number | Publication Date |
---|---|
US20210371898A1 true US20210371898A1 (en) | 2021-12-02 |
Family
ID=69950791
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/145,014 Active 2037-07-24 US11104933B1 (en) | 2016-03-22 | 2018-09-27 | Compositions and methods for determining the presence of active leukocyte cells using an electrochemical assay |
US17/342,077 Pending US20210371898A1 (en) | 2016-03-22 | 2021-06-08 | Compositions and methods for determining the presence of active leukocyte cells using an electrochemical assay |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/145,014 Active 2037-07-24 US11104933B1 (en) | 2016-03-22 | 2018-09-27 | Compositions and methods for determining the presence of active leukocyte cells using an electrochemical assay |
Country Status (7)
Country | Link |
---|---|
US (2) | US11104933B1 (en) |
EP (1) | EP3856710A4 (en) |
JP (1) | JP2022502059A (en) |
CN (1) | CN113242851A (en) |
AU (1) | AU2019348034A1 (en) |
CA (1) | CA3115920A1 (en) |
WO (1) | WO2020069176A1 (en) |
Family Cites Families (37)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE2854987A1 (en) * | 1978-12-20 | 1980-06-26 | Boehringer Mannheim Gmbh | DIAGNOSTIC AGENTS FOR DETECTING PROTEOLYTIC ENZYMS AND CHROMOGENS SUITABLE FOR THIS |
DE2905531A1 (en) | 1979-02-14 | 1981-01-08 | Boehringer Mannheim Gmbh | DIAGNOSTIC AGENT FOR DETECTING LEUCOCYTES IN BODY LIQUIDS |
DE3104078C2 (en) | 1981-02-06 | 1983-07-21 | Max Planck Gesellschaft zur Förderung der Wissenschaften e.V., 3400 Göttingen | Method for determining the pH value inside a cell; 1,4-dibutyryloxy-2,3-dicyanobenzene; 1,4-di (-tert-butyloxycarbonyl-1-alanyloxy) -2-3-dicyanobenzene |
US4657855A (en) | 1984-04-06 | 1987-04-14 | Miles Laboratories, Inc. | Composition and test device for determining the presence of leukocytes, esterase and protease in a test sample |
US4637979A (en) | 1984-04-06 | 1987-01-20 | Miles Laboratories, Inc. | Composition and test device for determining the presence of leukocytes containing a zwitterion coupling agent |
US4897444A (en) | 1985-05-31 | 1990-01-30 | The Research Foundation Of The State University Of New York | Immobilized fluorogenic substrates for enzymes; and processes for their preparation |
JPH03244396A (en) | 1990-02-23 | 1991-10-31 | Terumo Corp | Detection of hydrolase and detection tool therefor |
DE4331803A1 (en) | 1993-09-18 | 1995-03-23 | Bosch Gmbh Robert | Electronically commutated electric motor |
US5464739A (en) | 1994-08-22 | 1995-11-07 | Bayer Corporation | Composition method for determining the presence of leukocyte cells, esterase or protease in a test sample |
CA2161574A1 (en) | 1994-11-15 | 1996-05-16 | James Noffsinger | Methodology for colorimetrically determining the concentration of white blood cells in a biological fluid |
US6893868B2 (en) | 1997-02-20 | 2005-05-17 | Onco Immunin, Inc. | Homo-doubly labeled compositions for the detection of enzyme activity in biological samples |
DE19829707C2 (en) | 1998-07-03 | 2002-07-18 | Macherey Nagel Gmbh & Co Hg | Diagnostic agent for the detection of leukocytes, elastases, esterases and proteases |
US6528652B1 (en) | 1999-01-21 | 2003-03-04 | Chronimed | Composition and device for detecting leukocytes in urine |
US6348324B1 (en) | 1999-01-21 | 2002-02-19 | Hypoguard America Limited | Composition and device for detecting leukocytes in urine |
US6750053B1 (en) | 1999-11-15 | 2004-06-15 | I-Stat Corporation | Apparatus and method for assaying coagulation in fluid samples |
EP1482972A4 (en) | 2001-11-20 | 2005-11-23 | Seattle Genetics Inc | Treatment of immunological disorders using anti-cd30 antibodies |
US20100226931A1 (en) | 2004-06-24 | 2010-09-09 | Nicholas Valiante | Compounds for immunopotentiation |
US20090258348A1 (en) | 2005-02-02 | 2009-10-15 | Universität Bayreuth | Esterases for monitoring protein biosynthesis in vitro |
US7504235B2 (en) | 2005-08-31 | 2009-03-17 | Kimberly-Clark Worldwide, Inc. | Enzyme detection technique |
US7935538B2 (en) | 2006-12-15 | 2011-05-03 | Kimberly-Clark Worldwide, Inc. | Indicator immobilization on assay devices |
US8180421B2 (en) | 2007-12-12 | 2012-05-15 | Kimberly-Clark Worldwide, Inc. | Resonance energy transfer based detection of nosocomial infection |
US9103796B2 (en) | 2007-12-14 | 2015-08-11 | Kimberly-Clark Worldwide, Inc. | Multi-layered devices for analyte detection |
EP2231137B1 (en) | 2007-12-19 | 2017-01-25 | Versitech Limited | Method of modulating membrane potential of a cell |
WO2010022281A1 (en) | 2008-08-20 | 2010-02-25 | Greystone Pharmaceuticals, Inc. | Methods for using human neutrophil elastase as an indicator of active wound infection |
WO2010036930A1 (en) | 2008-09-26 | 2010-04-01 | Javad Parvizi | Methods and kits for detecting joint infection |
US10577639B2 (en) | 2009-06-26 | 2020-03-03 | University Of Florida Research Foundation, Inc. | Rapid bed-side measurement of neutrophil elastase activity in biological fluids |
KR101433473B1 (en) | 2011-12-16 | 2014-08-22 | 부산대학교 산학협력단 | Biosensors using redox cycling |
CA2862499C (en) | 2012-01-24 | 2020-10-27 | Cd Diagnostics, Inc. | System for detecting infection in synovial fluid |
US10073096B2 (en) | 2012-10-05 | 2018-09-11 | Life Technologies Corporation | Markers capable of distinguishing cell pluripotency and uses thereof |
US20160040209A1 (en) | 2013-04-02 | 2016-02-11 | Board Of Regents, The University Of Texas System | Fast quantification of enzyme activity by electroanalysis |
KR101633473B1 (en) | 2014-05-12 | 2016-06-27 | 부산대학교 산학협력단 | Protease detection sensor |
EP3149212B1 (en) * | 2014-05-30 | 2020-08-26 | Georgia State University Research Foundation, Inc. | Electrochemical methods and compounds for the detection of enzymes |
US10337047B2 (en) | 2015-08-05 | 2019-07-02 | Alfaisal University | Assay for early detection of a disease using a magnetic nanoparticle biosensor |
EP3432873A4 (en) | 2016-03-22 | 2020-01-08 | Parvizi Surgical Innovation, LLC | Compositions and methods for determining the presence of active leukocyte cells using an electrochemical assay |
US20190064165A1 (en) * | 2016-03-22 | 2019-02-28 | Parvizi Surgical Innovation, Llc | Compositions and methods for determining the presence of active leukocyte cells using an electrochemical assay |
CN106824251A (en) * | 2016-12-23 | 2017-06-13 | 吉林大学 | The combined material and catalysis treatment method of water and soil are polluted in a kind of catalytic treatment |
WO2019094575A1 (en) | 2017-11-08 | 2019-05-16 | Gorski Waldemar | Redox substrates for leukocyte esterase |
-
2018
- 2018-09-27 US US16/145,014 patent/US11104933B1/en active Active
-
2019
- 2019-09-26 AU AU2019348034A patent/AU2019348034A1/en active Pending
- 2019-09-26 CN CN201980077453.5A patent/CN113242851A/en active Pending
- 2019-09-26 JP JP2021517697A patent/JP2022502059A/en active Pending
- 2019-09-26 WO PCT/US2019/053224 patent/WO2020069176A1/en unknown
- 2019-09-26 EP EP19864492.4A patent/EP3856710A4/en active Pending
- 2019-09-26 CA CA3115920A patent/CA3115920A1/en active Pending
-
2021
- 2021-06-08 US US17/342,077 patent/US20210371898A1/en active Pending
Also Published As
Publication number | Publication date |
---|---|
EP3856710A4 (en) | 2022-06-15 |
CN113242851A (en) | 2021-08-10 |
EP3856710A1 (en) | 2021-08-04 |
JP2022502059A (en) | 2022-01-11 |
AU2019348034A1 (en) | 2021-05-20 |
WO2020069176A1 (en) | 2020-04-02 |
US11104933B1 (en) | 2021-08-31 |
CA3115920A1 (en) | 2020-04-02 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP4503182B2 (en) | Proteasome inhibitor drug monitoring method | |
US11372005B2 (en) | Method and compositions for the treatment and detection of endothelin-1 related kidney diseases | |
MX2010010417A (en) | Biosensor with improved analyte specificity. | |
US20190064165A1 (en) | Compositions and methods for determining the presence of active leukocyte cells using an electrochemical assay | |
US8173360B2 (en) | Cell death inhibitor | |
CA3018776A1 (en) | Compositions and methods for determining the presence of active leukocyte cells using an electrochemical assay | |
US20240201190A1 (en) | Compositions and methods of diagnosing pancreatic cancer | |
EP2454387A1 (en) | Urinary trypsin inhibitors as diagnostic aid for interstitial cystitis | |
US20210371898A1 (en) | Compositions and methods for determining the presence of active leukocyte cells using an electrochemical assay | |
US20220017942A1 (en) | Compositions and methods for determining the presence of active leukocyte cells using an electrochemical assay | |
WO2020247952A2 (en) | Systems comprising substrates and methods of using the same for detection of pancreatic cancers | |
WO1998005970A1 (en) | Method for examining chronic rejection reactions following organ transplantation and method for determining urine components | |
US20230203560A1 (en) | Glucosyl esters for infection screening | |
EP0760482A2 (en) | Method for judging eradication of H. pylori based on rates of changes in the pepsinogen I/II ratio | |
US6528652B1 (en) | Composition and device for detecting leukocytes in urine | |
Morton et al. | Urinary bladder fungus ball. | |
RU2291440C1 (en) | Method for diagnosing hepatitis transit into chronic stage | |
US10400263B2 (en) | Tripeptide rhodamine compound | |
CA2607726A1 (en) | Method of treating lymphangioleiomyomatosis (lam) | |
RU2316770C1 (en) | Differential diagnosis method for diagnosing abacterial prostatitis cases | |
Polito et al. | Evaluation of renal damage by urinary enzyme dosage | |
US20100003712A1 (en) | Biomarker for assessing response to chymase treatment | |
Coron et al. | Early Detection of Acute Graft-Versus-Host Disease by Wireless Capsule Endoscopy Combined With Probe-Based Confocal Laser Endomicroscopy: Time to Change the Diagnostic Algorithm? |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: CLEU DIAGNOSTICS, LLC, PENNSYLVANIA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:FLEISCHMAN, ANDREW NEIL;PARVIZI, JAVAD;BIHOVSKY, RON H.;SIGNING DATES FROM 20191001 TO 20191025;REEL/FRAME:057623/0018 |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |