Compositions and methods for determining the presence of activated white blood cells using an electrochemical assay - Patents.com

Abstract

A substrate composition for leukocyte esterase comprising a monoester, the monoester being one of an α-amino acid ester or an α-hydroxy acid ester, specific for leukocyte esterase, characterized in that the monoester has a first portion for participating in a redox reaction and a second portion with an amine or alcohol protecting group.

Description

Field of the Technology

[0001] (Cross - Reference to Related Applications) This application claims priority to U.S. Patent Application No. 16 / 145,014, filed on September 27, 2018, the entire disclosure of which is incorporated herein by reference.

[0002] (Technical Field) The present disclosure relates to a novel application of an electrochemical assay for determining the activity of white blood cells in a test sample. More specifically, the present disclosure relates to novel methods and kits for determining the activity of enzymes released by activated white blood cells, particularly leukocyte esterase and human neutrophil elastase, in patients at risk of developing an infectious disease.

Background Art

[0003] The presence of an abnormally large number of white blood cells in urine is a commonly used indicator of an infectious process. Historically, technicians have relied on manual visual counting under a microscope, but this visual approach has been largely replaced by dipstick assays for detecting genitourinary infections. In the majority of such commercially available "dipstick" assays, the activity of the enzyme leukocyte esterase ("LE") is used as a surrogate for the presence of activated white blood cells. Assays for human neutrophil elastase ("HNE") have also been reported to show high sensitivity in the diagnosis of male urinary tract infections.

[0004] Known leukocyte esterase assays are colorimetric, meaning that the presence of enzyme activity is reported based on a change in color. Typically, color test strips can be matched to a color chart (none to 2+ / 3+) where the color intensity increases by 3-4 increments, representing LE concentrations from 30 ng / mL to over 1500 ng / mL. However, colorimetric assays have clear drawbacks. Because there are only 3-4 increments of color intensity available, it can be very difficult to determine differences in leukocyte esterase concentration. Furthermore, even inter- and intra-rater reliability in classifying such color increments can be insufficient. This is especially true when the dipstick result is not very clear (trace or 1+). In such cases, the test result may be too unreliable to make treatment decisions. Therefore, the usefulness of dipstick results is limited to cases where leukocyte esterase activity is very high. In addition, substances that change the color of urine (nitrofurantoin, phenazopyridine, etc.) also affect the dipstick measurement.

[0005] In recent years, physicians have shown interest in using leukocyte esterase testing on serous fluids from joints, lungs, abdomen, and even middle ear exudate. While the results are very promising for diagnosing peri-prosthetic joint infections (PJI), the colorimetric test is impractical in 17–29% of samples due to the presence of blood and debris. The same applies to other body cavities, as aspiration does not always yield a clear fluid. Furthermore, the colorimetric leukocyte esterase test cannot be performed on serum samples.

[0006] More recently, lactate ester substrates have been demonstrated to improve the sensitivity and rate of LE assays. The alcohol portion is released as a hydroxylpyrrole compound, which then reacts with a diazonium salt to produce a purple azo dye. However, such assays have limited utility in bloody or turbid fluid states and would require expensive optical sensors to provide accurate and quantitative measurements. Therefore, improved substrates and assays for detecting leukocytes and leukocyte enzymes in samples are urgently needed. [Overview of the project]

[0007] A simplified summary of the present invention is provided below to provide a basic understanding of some aspects of the invention. This summary is not a comprehensive overview of the invention. It is not intended to identify essential means / key elements of the invention or to describe 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 explanations that will be presented later.

[0008] In one embodiment, the disclosure is directed to a method for screening, detecting, and confirming infection in patients at risk of infection or patients already exhibiting symptoms associated with infection. In one embodiment, the method follows the steps of obtaining a sample from a subject of interest, detecting the presence or absence of a leukocyte marker in the sample, and determining a treatment based on the degree and presence of the leukocyte marker in the sample.

[0009] In some embodiments, the leukocyte marker may be one or any combination of markers such as cytokines, chemokines, oxygen and nitrogen radicals, leukocyte elastase, leukocyte esterase, neutrophil elastase, gelatinase, IL-1β, metalloproteinases (MMPs), cathepsins such as cathepsin A and cathepsin B, and phospholipases such as phospholipase A and phospholipase B.

[0010] In one embodiment, the present disclosure relates to a composition comprising a leukocyte enzyme or, more 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 alternative embodiments, the composition comprises both an LE substrate and an HNE substrate. In yet another embodiment, the composition may comprise additional substrates specific to other enzymes or biomarkers other than LE and HNE.

[0011] In some embodiments, the substrate exhibits specificity for LE or HNE. In one embodiment, the substrate comprises a monoester, which is one of α-amino acid esters such as alanine ester or α-hydroxy acid esters such as lactate ester, which have specificity for leukocyte esterase, and the monoester has a first portion for participating in the redox reaction and a second portion containing an amine or alcohol protecting group that masks the functional group (i.e., amine or alcohol) to prevent undesirable chemical reactivity.

[0012] In some embodiments, the substrate can be represented by formula 1 shown below. formula 1 Here, A comprises an ether group (i.e., -O-) or an amine group (i.e., NRa, where Ra is 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 protecting 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, B bonded to oxygen in formula 1 is substituted with an amino group. In further embodiments, B comprises an aminophenyl. In some embodiments, B comprises a substituted aminophenyl.

[0013] In some embodiments, the LE substrate includes a compound such as that described in Formula 2 below. formula 2

[0014] X1 and X2 are independently O, S, or NRa. Ra is H, an alkyl, or an aryl group. Both X1 and X2 can be oxygen, or both can be NRa. Alternatively, one of X1 and X2 is oxygen and the other is NRa.

[0015] Y1 and Y2 are independently O or NRa. Ra is as described above. Both Y1 and Y2 can be oxygen, or both can be NRa. Alternatively, one of Y1 and Y2 is oxygen and the other is NRa.

[0016] R1 and R2 are independently alkyl or aryl groups or substituted alkyl, substituted aryl, or protecting groups. In some embodiments, both R1 and R2 are methyl. In some embodiments, R1 and R2 may be tosyl. In some embodiments, R2 may be tosyl.

[0017] R3 and R4 are independently alkyl groups, protecting groups, or peptide moieties. Protecting groups include tosyl, benzoyl, benzyl, trimethylsilyl, [bis-(4-methoxyphenyl)phenylmethyl], carbobenzyloxy, and tert-butyloxycarbonyl and 9-fluorenylmethyloxycarbonyl. In one embodiment, R4 may be tosyl. The peptide moiety may include any combination of natural and / or non-natural amino acids.

[0018] Each of the R5 groups on the ring is independently a halogen atom; hydroxyl group; C1-C6 alkyl group; C3-C6 cycloalkyl group; C3-C6 cycloalkyl C1-C6 alkyl group; C2-C6 alkenyl group; C2-C6 alkynyl group; C1-C6 haloalkyl group (including trifluoro C1-C6 alkyl group); C2-C6 haloalkenyl group; C2-C6 haloalkynyl group; C3-C6 halocycloalkyl group; C3-C6 halocycloalkyl C1-C6 alkyl group; C1-C6 alkoxy group; C 3-C6 cycloalkyloxy group; C2-C6 alkenyloxy group; C2-C6 alkynyloxy group; C1-C6 alkylcarbonyloxy group; C1-C6 haloalkoxy group; C1-C6 alkylthio group; C1-C6 alkylsulfinyl group; C1-C6 alkylsulfonyl group; C1-C6 haloalkylthio group; C1-C6 haloalkylsulfinyl group; C1-C6 haloalkylsulfonyl group; amino group; C1-C6 alkylcarbonylamino group; mono(C1-C6 alkyl)amino group ;di(C1-C6alkyl)amino group;hydroxy C1-C6 alkyl;C1-C6 alkoxy C1-C6 alkyl;C1-C6 alkylthio C1-C6 alkyl;C1-C6 alkylsulfinyl C1-C6 alkyl;C1-C6 alkylsulfonyl C1-C6 alkyl;C1-C6 haloalkylthio C1-C6 alkyl;C1-C6 haloalkylsulfinyl C1-C6 alkyl;C1-C6 haloalkylsulfonyl C1-C6 alkyl;cyano C1-C6 alkyl The group is a C1-C6 alkoxy group; a C1-C6 cycloalkyl group; a C1-C6 alkyloxy group; a C1-C6 haloalkoxy 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.

[0019] In some embodiments, the LE substrate comprises 4-((tosyl-L-alanyl)oxy)phenyltosyl-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)phenyltosyl-L-alaninate and 4-(((S)-2-(tosyloxy)propanoyl)oxy)phenyl(S)-2-(tosyloxy)propanoate.

[0020] In some embodiments, the HNE substrate includes a compound such as that described in Formula 3 below. formula 3

[0021] Here, A1-A2-A3-A4 represent a core tetrapeptide scaffold sequence that functions as an enzyme active site, B contains a moiety capable of participating in the redox reaction, and C contains an acyl group. In some embodiments, A1-A2-A3-A4 contains AAPV (SEQ ID NO: 1). In some embodiments, SEQ ID NO: 1 has a conservative substitution. In some embodiments, B contains a redox-active alcohol intermediate. In some embodiments, B contains a phenol derivative. B contains a quinone. In some embodiments, B contains a hydroquinone. In some embodiments, B contains a substituted quinone or a substituted hydroquinone. In some embodiments, C contains N-methioxysuccinyl.

[0022] In some embodiments, the HNE substrate comprises 3-{[(1S)-1-{[(2S)-1-(5-{[(1S)-1-({4-[(2S)-2-({1-[(2S)-2-[(2S)-2-(3-carboxypropanamide)propanamide]propanoyl]pyrrolidine-2-yl}formamide)-3-methylbutanamide]phenyl}carbamoyl)-2-methylpropyl]carbamoyl}imidazolidined-1-yl)-1-oxopropan-2-yl]carbamoyl}ethyl]carbamoyl}propanoic acid.

[0023] In some embodiments, a leukocyte enzyme substrate is included in the assay. In some embodiments, the assay includes an electrochemical assay. In alternative embodiments, the assay may include a colorimetric step combined with the electrochemical assay. In some embodiments, the electrochemical assay includes an internally calibrated electrochemical continuous enzyme assay ("ICECEA"). In some embodiments, the electrochemical assay includes the leukocyte substrate and electrochemical measuring device of this disclosure. In some embodiments, the electrochemical measuring device includes a working electrode, a reference electrode, and an auxiliary electrode.

[0024] In some embodiments, the Disclosure relates to a method for detecting the presence of leukocyte enzymes in a sample and determining a treatment plan. In some embodiments, the presence of leukocyte enzymes in a sample indicates the presence of leukocytes in the sample. In some embodiments, the leukocyte enzyme includes LE. In some embodiments, the leukocyte enzyme includes human neutrophil elastase HNE. In some embodiments, the leukocyte enzyme is detected by contacting the enzyme with an enzyme substrate. In some embodiments, the substrate is any LE substrate of the Disclosure. In some embodiments, the substrate is any HNE substrate of the Disclosure.

[0025] In some embodiments, the amount of leukocyte enzymes present in the sample is quantified. In some embodiments, the presence of leukocytes in the sample indicates an infection. In some embodiments, the infection includes a urinary tract infection ("UTI"). In some embodiments, the infection includes a peri-prosthetic joint infection ("PJI"). In some embodiments, the infection includes spontaneous bacterial peritonitis ("SBP"). In some embodiments, the sample includes a biological sample. In some embodiments, the biological sample includes one of urine, saliva, synovial fluid, pleural fluid, pericardial fluid, ascites, cerebrospinal fluid ("CSF"), and middle ear fluid.

[0026] In some embodiments, a method of screening a patient at risk of developing an infectious disease is included, following the step of detecting the presence of a leukocyte enzyme in a sample by contacting the leukocyte enzyme with a substrate in an assay. In some embodiments, the assay includes an electrochemical assay. In some embodiments, the electrochemical assay includes an internally calibrated electrochemical continuous enzyme assay ("ICECEA").

[0027] In some embodiments, the method of detecting the presence of a leukocyte enzyme in an electrochemical assay includes adding a first aliquot of a reactant or product of the leukocyte enzyme to the substrate of the leukocyte enzyme. In some embodiments, the leukocyte enzyme substrate is in an electrolyte solution. In some embodiments, the method includes measuring the current flowing through the electrodes of the electrochemical assay. In some embodiments, the method includes adding at least one additional aliquot of a reactant or product of the leukocyte enzyme to the substrate of the leukocyte enzyme. In some embodiments, the method includes measuring the current flowing through the electrodes of the electrochemical assay a second time. In some embodiments, the method includes adding the leukocyte enzyme to the substrate of the leukocyte enzyme. In some embodiments, the method includes measuring the current flowing through the electrodes of the electrochemical assay a third time.

[0028] In some embodiments, a method of screening an infected patient by detecting the presence of a leukocyte enzyme in an electrochemical assay following a process that includes adding a first aliquot of the 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 includes measuring a current flowing through an electrode of the electrochemical assay. In some embodiments, the method includes adding at least one additional aliquot of the leukocyte enzyme to the substrate of the leukocyte enzyme. In some embodiments, the method includes measuring a current flowing through an electrode of the electrochemical assay a second time. In some embodiments, the method includes adding a product or reactant of the leukocyte enzyme to the substrate of the leukocyte enzyme. In some embodiments, the method includes measuring a current flowing through an electrode of the electrochemical assay a third time.

[0029] In another aspect, the present disclosure is directed to a kit that includes a suitable substrate, directions for optimizing results, and optionally, a patient-specific therapy based on the observed results. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] [Figure 1] FIG. 1 depicts a first hydroquinone substrate and a first ester hydrolysis step.

[0031] [Figure 2] FIG. 2 depicts a semiquinone intermediate and a second ester hydrolysis step.

[0032] [Figure 3] FIG. 3 depicts a final benzoquinone oxidation product.

[0033] [Figure 4] Figure 4 shows the results of using 4-((tosyl-L-alanyl)oxy)phenyltosyl-L-alaninate ("TAPTA") in an internally calibrated electrochemical continuous enzyme assay (ICECEA).

[0034] [Figure 5] Figure 5 shows the NMR spectrum of 4-((tosyl-L-alanyl)oxy)phenyltosyl-L-alaninate ("TAPTA").

[0035] [Figure 6] Figure 6 is a schematic diagram of the cleavage mechanism of an embodiment of the monoester of the present invention.

[0036] [Figure 7] Figure 7 is a voltammogram showing a decrease peak that increases with higher LE concentrations for an electrode screen-printed with one embodiment of the substrate of the present invention. [Detailed Description of the Invention]

[0037] As used herein and in the appended claims, the singular forms "a," "and," and "the" include plural references unless the context explicitly indicates otherwise.

[0038] As used herein, “white blood cells” may refer to any white blood cell (“WBC”). White blood cells are cells of the immune system that are involved in protecting the body from infections and the invasion of pathogens. All white blood cells / WBCs are classified into five classes based on morphological characteristics that distinguish them from one another. These include neutrophils, eosinophils, basophils, monocytes, and lymphocytes. Neutrophils make up about 40–75% of white blood cells, eosinophils make up about 1–6%, basophils make up less than 1%, monocytes make up about 2–10%, and lymphocytes (such as B lymphocytes and T lymphocytes) make up about 20–45%.

[0039] As used herein, the term “patient” may refer to a biological system to which treatment can be administered. A biological system may include, for example, individual cells, sets of cells (e.g., cell cultures), organs, tissues, or multicellular organisms. “Patient” may refer to a human patient or a non-human patient. In preferred embodiments, the patient is a human patient.

[0040] As used herein, the terms “effective dose” or “therapeutic dose” may refer to the amount of compound or drug that can produce a medically desirable outcome in the treated subject. Treatments may be carried out alone or in combination with other drugs or therapies, in vivo or ex vivo. A therapeutic dose may be administered in one or more doses, topically, or in a prescribed amount, and is not intended to be limited to a specific formulation or route of administration.

[0041] The terms “treating” a disease or “treatment” refer to implementing a protocol that may involve administering one or more drugs to a patient (human or other) to alleviate the signs or symptoms of the disease. Alleviation may occur before or after the signs or symptoms of the disease appear. Thus, “treating” or “treatment” includes “preventing” a disease or “prevention” of a disease. The terms “prevent” or “prevention” refer to preventive and / or preventive measures whose purpose is to prevent or slow the targeted pathological condition or impairment.

[0042] This disclosure relates to compositions and methods for the rapid detection (including determination of relative activity) of leukocyte enzymes released by active leukocytes, particularly enzymes released by active leukocytes such as leukocyte esterase ("LE") and human neutrophil elastase ("HNE").

[0043] In at least one aspect of the present disclosure, a method for screening a subject for infection is described, the method comprising: (a) obtaining a tissue or body fluid sample from a subject at risk of developing an infection; (b) applying the sample to a detector device, the detector device comprising at least one substrate specific to at least one of LE and / or HNE, the at least one substrate being adapted to detect at least one threshold level of LE and / or HNE, the threshold level being associated with the presence of infection; and (c) determining the threshold levels of LE and / or HNE present in the sample, and further determining if the respective concentrations of LE and / or HNE exceed the threshold level, the method further comprising the measurement being a positive screening for infection.

[0044] This disclosure provides a method for determining that an infection is a periarthritis of a prosthesis (PJI). In some embodiments, the threshold level of leukocyte esterase (LE) for the detection of PJI is at least about 20 pg / ml of leukocyte esterase in the synovial fluid sample.

[0045] Compositions and methods for rapid detection utilize specific substrates called LE substrates and HNE substrates for detecting leukocyte enzymes such as LE and HNE. Compositions and methods for rapid detection may, but are not limited to, electrochemical assays for detecting leukocyte enzymes, particularly internally calibrated electrochemical continuous enzyme assays ("ICECEA").

[0046] In some embodiments, the substrate can detect LE. Such substrates can be readily hydrolyzed by LE to produce redox intermediates, which can provide a detectable electrochemical response. In some embodiments, the substrate for detecting LE (i.e., the "LE substrate") is represented by the following formula 1. formula 1

[0047] Here, A is the part that determines the identity of the acyl group, such as alanine or lactate, at the ester cleavage site which has enzyme specificity for leukocyte esterases, and B is the part that can be involved in a redox reaction that can be detected using an electrochemical assay (e.g., by using ICECEA or a screen-printed electrochemical sensor).

[0048] In some embodiments, A comprises an amino group (i.e., -NRa, where Ra is an alkyl, aryl, or aralkyl group optionally substituted with H), or A comprises an ether group (i.e., -O-).

[0049] The acyl group defined by A is protected using any effective amine or alcohol protecting group C (e.g., a tosyl group). The part B, which is an alcohol intermediate of the ester released during hydrolysis by an esterase, is a redox substrate and participates in the redox reaction. Furthermore, the oxygen-bonded B in formula 1 may be substituted with an -NH bond (i.e., the ester group presented in formula 1 may be substituted with an amide group), and this is still within the scope of the disclosure.

[0050] The amine or alcohol protecting group C is acetyl (Ac), benzoyl (Bz), benzyl (Bn), β-methoxyethoxymethyl (MEM), dimethoxytrityl (DMT), methiooxymethyl (MOM), methoxytrityl[(4-methoxyphenyl)diphenylmethyl](MMT), p-methoxybenzyl (PMB), methylthiomethyl, pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trityl (Tr), silly (e.g., TMS, TBDMS, TOM), It may contain any of the following (e.g., 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).

[0051] In some embodiments, redox moiety B is a phenol derivative that can form an ester via its hydroxyl group. Such intermediates can be oxidized and release electrons. For example, hydroquinone, a phenol derivative, contains two hydroxyl groups in paraconformation. Each hydroxyl group can bind to independently form distinct lactate esters that are substrates for leukocyte esterases (Figure 1). The resulting double-stranded substrate has two potential target sites for leukocyte esterase activity, and the degradation of the substrate is stepwise. Ester hydrolysis by leukocyte esterase at the first target occurs relatively slowly due to molecular damage to the active site, while hydrolysis at the subsequent second active site occurs more rapidly. This can effectively improve the specificity of the electrochemical assay because nonspecific hydrolysis is less likely to initiate a cascade. After the first ester hydrolysis step, the oxidation reaction can remove a hydrogen atom and release electrons, forming a semiquinone lactate ester intermediate (Figure 2). After subsequent hydrolysis of the remaining ester, a quinone-based intermediate is released, which can be further oxidized to form parabenzoquine. Parabenzoquine is reduced at low potential, which can minimize interference from other redox species in the sample and improve assay selectivity. The final product is shown in Figure 3.

[0052] In other embodiments, methods for treating patients exhibiting positive signs of LE and HNE are described. In one embodiment, serious infections caused by Gram-positive bacteria are currently difficult to treat because many of these pathogens are now resistant to standard antimicrobial agents. For this reason, at least one aspect of the disclosure is to treat patients prophylactically before invasive surgery to minimize the risk of infection. In at least one embodiment, a patient identified as having an infection can initiate a comprehensive treatment plan in active infection control, including the administration of antimicrobial agents such as penicillin, cephalosporins, tetracyclines, daptomycin, tigecycline, linezolid, quinupristin / dalfopristin, and dalbavancin. In other embodiments, there are methods for screening or detecting the risk of PJI by developing those useful for treating infections caused by drug-resistant Gram-positive and Gram-negative bacteria.

[0053] 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-bonded B of formula 2 is substituted with an amino group. In further embodiments, B comprises an aminophenyl. In some embodiments, B comprises a substituted aminophenyl.

[0054] Two specific, explicitly non-limiting examples of substrates for detecting leukocyte esterases ("LE") within the range of Formula 1 include 4-((tosyl-L-alanyl)oxy)phenyltosyl-L-alaninate (hereinafter referred to as Compound A) and 4-(((S)-2-(tosyloxy)propanoyl)oxy)phenyl(S)-2-(tosyloxy)propanoate (hereinafter referred to as Compound B). Compound A is also referred to herein as "TAPTA". The NMR of Compound A is shown in Figure 5, showing the structure of the tosyl moiety and its binding. Phenylethylenediamine variants of Compound A and Compound B (i.e., in which the para-oxygen is replaced by an NH linker) should also be considered within the scope of this disclosure and are similarly suitable for inclusion in electrochemical assays of this disclosure (e.g., ICECEA). Compound A Compound B

[0055] In some embodiments, the LE substrate comprises a composition such as that described in Formula 2 below. formula 2

[0056] X1 and X2 are independently O, S, or NRa. Ra is H, an alkyl, or an aryl group. Both X1 and X2 can be oxygen, or both can be NRa. Alternatively, one of X1 and X2 is oxygen and the other is NRa.

[0057] Y1 and Y2 are independently O, S, or NRa. Ra is as described above. Y1 and Y2 can both be oxygen, or both be NRa. Alternatively, one of Y1 and Y2 is oxygen and the other is NRa.

[0058] R1 and R2 are independently alkyl or aryl groups or substituted alkyl, substituted aryl, or protecting groups. In some embodiments, R1 or R2, or both thereof, are methyl. In some embodiments, R1 or R2, or both thereof, may be tosyl. In some embodiments, R2 is tosyl.

[0059] R3 and R4 are, independently, alkyl, protecting group, or peptide moieties such as tosyl, benzoyl, benzyl, trimethylsilyl, [bis-(4-methoxyphenyl)phenylmethyl], carbobenzyloxy, and tert-butyloxycarbonyl, 9-fluorenylmethyloxycarbonyl. In one embodiment, R4 is tosyl. The peptide moiety may include any combination of natural and / or non-natural amino acids.

[0060] R2 and R4 also include acetyl (Ac), benzoyl (Bz), benzyl (Bn), β-methoxyethoxymethyl (MEM), dimethoxytrityl (DMT), methiooxymethyl (MOM), methoxytrityl [(4-methoxyphenyl)diphenylmethyl] (MMT), p-methoxybenzyl (PMB), methylthiomethyl, pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trityl (Tr), silyl (e.g., TMS, TBDMS, TOM), The protecting group may include TIPS), methyl, 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, Nps). In one embodiment, the protecting group may be any one of tosyl, benzoyl, benzyl, trimethylsilyl, [bis-(4-methoxyphenyl)phenylmethyl], carbobenzyloxy, tert-butyloxycarbonyl, or 9-fluorenylmethyloxycarbonyl.

[0061] Each of the R5 groups on the ring is independently a halogen atom; hydroxyl group; C1-C6 alkyl group; C3-C6 cycloalkyl group; C3-C6 cycloalkyl C1-C6 alkyl group; C2-C6 alkenyl group; C2-C6 alkynyl group; C1-C6 haloalkyl group (including trifluoro C1-C6 alkyl group); C2-C6 haloalkenyl group; C2-C6 haloalkynyl group; C3-C6 halocycloalkyl group; C3-C6 halocycloalkyl C1-C6 alkyl group; C1-C6 alkoxy group; C 3-C6 cycloalkyloxy group; C2-C6 alkenyloxy group; C2-C6 alkynyloxy group; C1-C6 alkylcarbonyloxy group; C1-C6 haloalkoxy group; C1-C6 alkylthio group; C1-C6 alkylsulfinyl group; C1-C6 alkylsulfonyl group; C1-C6 haloalkylthio group; C1-C6 haloalkylsulfinyl group; C1-C6 haloalkylsulfonyl group; amino group; C1-C6 alkylcarbonylamino group; mono(C1-C6 alkyl)amino group ;di(C1-C6alkyl)amino group;hydroxy C1-C6 alkyl;C1-C6 alkoxy C1-C6 alkyl;C1-C6 alkylthio C1-C6 alkyl;C1-C6 alkylsulfinyl C1-C6 alkyl;C1-C6 alkylsulfonyl C1-C6 alkyl;C1-C6 haloalkylthio C1-C6 alkyl;C1-C6 haloalkylsulfinyl C1-C6 alkyl;C1-C6 haloalkylsulfonyl C1-C6 alkyl;cyano C1-C6 alkyl The group is a C1-C6 alkoxy group; a C1-C6 alkoxy group; a C1-C6 cycloalkyl group; a C1-C6 alkyloxy group; a C1-C6 haloalkoxy 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 H, alkyl, aryl, or aralkyl group. X1 and X2 can both be oxygen, or both be 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.

[0062] In some embodiments, the substrate detects human neutrophil elastase ("HNE"). In some embodiments, the substrate for detecting HNE (i.e., the "HNE substrate") can be represented by Equation 3, as shown below. formula 3

[0063] A1 to A4 (i.e., A1-A2-A3-A4) represent core tetrapeptide scaffold sequences that function as the enzyme active site (i.e., the active site of human neutrophil elastase / HNE). The tetrapeptide sequence of Ala-Ala-Pro-Val (AAPV) (SEQ ID NO: 1) is the most common, but any of the four peptide sites can be substituted with native or non-native amino acids to improve the substrate sensitivity of HNE. For example, a conservative substitution can be made to SEQ ID NO: 1 and is still within the scope of this disclosure. As used herein, a “conservative substitution” is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are 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), non-charged 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).

[0064] In formula 3, B represents a redox moiety similar to that of the LE substrates represented in formula 1 above. For example, B may include a derivative of phenol that can form an ester via its hydroxyl group, e.g., a redox-active alcohol intermediate. This may include, for example, a hydroquinone intermediate or a hydroquinone-based redox group. In formula 3, C represents an acyl group, e.g., N-methoxysuccinyl. Acyl groups may help improve the substrate sensitivity of HNEs, and some acyl groups, e.g., N-methoxysuccinyl, may also increase the solubility of the substrate.

[0065] A specific, explicitly non-limiting example of a substrate for detecting HNEs within the range of Equation 3 is compound C, which is 3-{[(1S)-1-{[(2S)-1-(5-{[(1S)-1-({4-[(2S)-2-({1-[(2S)-2-[(2S)-2-(3-carboxypropanamide)propanamide]propanoyl]pyrrolidine-2-yl}formamide)-3-methylbutanamide]phenyl}carbamoyl)-2-methylpropyl]carbamoyl}imidazolidine-1-yl)-1-oxopropan-2-yl]carbamoyl}ethyl]carbamoyl}propanoic acid. Compound C

[0066] As described above in relation to the embodiments in Figures 1 and 2 and formulas 2 and 3, since the double-stranded substrate obtained for cleavage by leukocyte esterase has two potential target sites, there was reason to believe that diesters (consisting of two symmetric or asymmetric α-amino or α-hydroxy acid esters) would be more effective substrates. Furthermore, it was expected that the degradation of the substrate would likely be stepwise, and that ester hydrolysis by leukocyte esterase at the initial active site would be slower or more deliberate due to steric hindrance caused by the double substrate. Since nonspecific hydrolysis is less likely to initiate a cascade of stepwise hydrolysis, it was initially thought that this could improve the specificity of the electrochemical assay. Surprisingly, however, the applicant found that diesters were less effective than monoesters. Even in mixtures of diesters and monoesters where monoesters were present at very low concentrations (e.g., about 1%), the effectiveness of the monoesters prevailed and determined the effectiveness of the overall composition. In fact, the effectiveness of the monoesters was not discovered until the diester composition was purified to the point where the concentration of monoesters was reduced to less than 1%. At that point, the effectiveness of the diester composition decreased sharply, indicating that the monoester was a more effective substrate for reacting with leukocyte esterase enzymes.

[0067] Therefore, in one embodiment, the substrate of the present invention comprises a monoester, which is one of α-amino acid esters such as alanine ester or α-hydroxy acid esters such as lactate ester, having specificity for leukocyte esterases. The monoester has a first portion for participating in the redox reaction and a second portion containing an amine or alcohol blocking group.

[0068] In one embodiment, the composition comprises a monoester represented by formula I, where A comprises oxygen (O) or NRa, where Ra is H or optionally substituted alkyl, aryl, or aralkyl group, so that A determines the identity of the acyl group of the ester, such that A is O when the monoester is an α-hydroxy acid ester (i.e., a lactate ester), or NRa when the monoester is an α-amino acid ester (i.e., alanine ester). B is the first part, and C is the second part.

[0069] In one embodiment, any oxygen linking group connecting the first and / or second parts can be replaced with a nitrogen linking group, and a nitrogen linking group can be replaced with an oxygen linking group.

[0070] In one embodiment, the first portion (B) comprises one of the following: a substituted or unsubstituted derivative of phenol, a substituted or unsubstituted hydroxyanthracene, a substituted or unsubstituted aminophenol, or a substituted or unsubstituted hydroxyphenanthroline.

[0071] In one embodiment, the second part (C) is acetyl (Ac), benzoyl (Bz), benzyl (Bn), β-methoxyethoxymethyl (MEM), dimethoxytrityl (DMT), methiooxymethyl (MOM), methoxytrityl [(4-methoxyphenyl)diphenylmethyl] (MMT), p-methoxybenzyl (PMB), methylthiomethyl, pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trityl (Tr), silyl (e.g., TMS, TBDMS, T It comprises one of the following: OM, TIPS), methyl, 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, Nps, etc.).

[0072] In one embodiment, the second portion is a sulfonyl group having a substituted or unsubstituted heterocyclic or heteroaryl ring.

[0073] In one embodiment, formula 1 is further refined to the general structure shown in formula 4. formula 4 Here, the first part B is 4-hydroxyphenyl ( ) comprises, where A is oxygen or NRa, where Ra is H or optionally substituted alkyl, aryl, or aralkyl group, thereby determining the identity of the acyl group of the ester, R1, R2 and R3 are independently hydrogen or optionally substituted alkyl groups (if A is oxygen, R3 is absent), and R4 is a substituted or unsubstituted heterocyclic or heteroaryl group.

[0074] In one embodiment, R4 is pyridinyl, pyridadinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, quinolyl, isoquinolyl, 1,2,3,4-tetrahydroquinolyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, indolyl, benimidazolyl, benzofuranil, sinnolinyl, indazolyl, indolidinyl, phthalazinyl, triazinyl, thiadiazolyl, oxadiazolyl, purinyl, 1-oxoisoindolyl, 1,2,4-trizynyl, 1,3,4-triazinyl, isoindolyl It is one of the following: benzothiophenyl, benzotriazolyl, benzothiazolyl, benzoxazolyl, tetrahydroquinolyl, dihydroquinolyl, naphthilidinyl, quinoxalinyl, quinazolinyl, dihydroisoquinolyl, tetrahydroisoquinolyl, benzofuryl, phlopyridinyl, pyrrolopyridimidinyl, or azaindolyl.

[0075] In one embodiment, R4 is a pyridine with or without the addition of a substituted or unsubstituted polar group.

[0076] In one embodiment, R4 is a pyridine selected from one of pyridine (1), methoxypyridine (2), and (methoxycarbonyl)pyridine (3), as shown below. In one particular embodiment, R4 is (methoxycarbonyl)pyridine(3).

[0077] In certain embodiments, the composition of the monoester substrate is 4-hydroxyphenyl(N-(3-(methoxycarbonyl)pyridine-5-sulfonyl)-L-alaninate.

[0078] In yet another specific embodiment, the composition of the monoester is represented by formula 5. formula 5

[0079] Referring to Figure 6, the cleavage mechanism of Equation 5 is shown. Specifically, leukocyte esterase (LE) cleaves the monoester substrate at the oxygen site during ester hydrolysis.

[0080] In one embodiment, the substrate for the leukocyte esterase enzyme is screen-printed onto the surface of an electrode sensor strip using known commercially available techniques and materials.

[0081] As described herein, leukocytes can produce leukocyte enzymes that can be detected and / or quantified by the electrochemical assay (i.e., ICECEA) of this disclosure.

[0082] Leukocyte enzymes may include, for example, those described in WO2010 / 036930, which are incorporated herein by reference in their entirety, such as IL-1β, leukocyte elastase, leukocyte esterase, and / or gelatinase B, as well as human neutrophil elastase. Leukocyte esterase ("LE") is an esterase produced by leukocytes (white blood cells). LE is the subject of urinalysis for the presence of, for example, leukocyte / WBC and other abnormalities associated with infection. Human neutrophil elastase ("HNE"), also known as human leukocyte elastase ("HLE"), is a serine protease. It belongs to the same family as chymotrypsin and has broad substrate activity. HNE is secreted by neutrophils and macrophages, two of the five classifications of leukocytes described herein. HNE is 218 amino acids long and has two asparagine-linked carbohydrate chains. HNE has two forms: IIa and IIb.

[0083] As used herein, the term “sample” may refer to a biological sample, including a sample of biological tissue or fluid origin obtained in vivo or in vitro. A biological sample may be, but is not limited to, body fluids (e.g., serous fluid, blood, plasma, serum, or urine), organs, tissues, fractions, and cells isolated from mammals, including, for example, humans. A biological sample may also include a section of a biological sample containing tissue. A biological sample may also include extracts from a biological sample, such as a biological fluid (e.g., blood, serum, ascites, and / or urine). Of particular interest, but clearly not limited, are urine, sputum (e.g., from a patient diagnosed with cystic fibrosis), ascites (e.g., from a patient with cirrhosis and ascites), and other serous fluids, such as synovial fluid, pleural fluid, pericardial fluid, cerebrospinal fluid ("CSF"), and middle ear fluid.

[0084] In some embodiments, the presence of leukocytes, i.e., determined by detecting and / or quantifying the amount of leukocyte enzymes (e.g., LE and / or HNE) present in a biological sample, may indicate the presence of infection in a subject. Such embodiments may utilize the LE and / or HNE substrates of this disclosure in electrochemical assays, particularly ICECEA as described herein. For example, the presence of LE and / or HNE in urine may indicate that a subject has a urinary tract infection ("UTI"). Similarly, the presence of LE and / or HNE in synovial fluid may indicate that a subject has a joint infection, such as, for example, periarthritis of a prosthesis ("PJI"), but is not necessarily limited. These examples indicating the presence of infection are merely illustrative uses of the substrates of this disclosure and are not limited in themselves, as they may or may not be utilized in electrochemical assays, such as ICECEA.

[0085] In some embodiments, the substrates of this disclosure are used to indicate that a subject has periarthritis of the joint (PJI). PJI is a devastating complication following total joint replacement and poses challenges for surgeons in both diagnosis and treatment. Establishing an accurate and timely diagnosis of PJI is crucial for making treatment decisions. In patients presenting with painful prostheses, it is important to complete a thorough examination to rule out or diagnose the presence of infection. In most cases, serological tests such as erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) are the first screening tests chosen. In patients with elevated serological markers or those strongly suspected of infection, the next step is to perform joint aspiration for synovial fluid examination. Traditionally, bacterial culture of synovial fluid has been used to diagnose PJI. Because bacterial culture itself is not sufficiently sensitive, with 30% of infections being culture-negative, orthopedic surgeons make a diagnosis considering serological test results, the percentage of leukocytes and polymorphonuclears in the synovial fluid, and histological analysis. Unfortunately, however, bacterial cultures and conventional synovial fluid tests can take several days to over a week to yield results.

[0086] Therefore, in some embodiments, synovial fluid aspirated from a painful joint would be tested for LE and / or HNE activity using the enzyme substrates of this disclosure. For example, this can be achieved by using the ICECEA assay described herein. In such embodiments, the activity of LE and / or HNE would be reported as a series of absolute concentrations. This can be performed in an office or operating room and yield results in minutes for point-of-care decision-making.

[0087] Based on accumulated population data, levels of LE and / or HNE activity can be combined with additional metrics to predict the likelihood of infection. Additional metrics may include joint type, history of previous infections, and serological test results (ESR and CRP). The surgeon can determine the most appropriate treatment algorithm for the patient, taking into account the likelihood of infection. If infection is likely, treatment for PJI may be considered, based on factors such as the extent of infection, including prosthesis removal, placement of an antibiotic spacer, incision or debridement, and long-term antibiotic restriction. Finally, if infection is unlikely or has been largely ruled out, other etiologies of painful prostheses may be considered.

[0088] In addition to making an initial diagnosis of infection, the substrate of current disclosure, e.g., assays (e.g., ICECEA), can be used to establish resolution of PJI with the aim of determining the correct timing for re-implantation of a new prosthesis. In addition to serological markers and other synovial fluid tests, levels of LE and / or HNE activity can be used to determine the success of treatment as discussed above. In patients with persistently elevated LE and / or HNE, the surgeon may try continuing intravenous administration of antibiotics or attempting to replace the antibiotic spacer to improve the prospect of complete resolution of the infection.

[0089] In some embodiments, the substrates of this disclosure are used to indicate that a subject has spontaneous bacterial peritonitis (SBP). SBP is a serious, life-threatening complication that is relatively common in patients with cirrhosis and ascites. For patients with this complication, rapid diagnosis and early administration of antibiotics are crucial for survival, as the in-hospital mortality rate can be as high as 20%. For patients with ascites, fever, altered mental status, and abdominal tenderness are frequent signs of SBP. In such cases, a diagnostic paracentesis is performed, and the diagnosis is made based on an absolute neutrophil count greater than 250 cells / mm3 and / or bacterial culture.

[0090] Therefore, in some embodiments, peritoneal fluid obtained from a diagnostic puncture will be tested for LE and / or HNE activity using the enzyme substrates of this disclosure. For example, this can be achieved by using the ICECEA assay described herein. Using the ICECEA assay, LE or HNE activity is reported as a series of absolute concentration measurements. Based on the accumulation of population data collected from many patients, the absolute concentrations of LE and / or HNE are compared to absolute criteria for diagnostic standards to provide a calculation of the probability of SBP presence. The likelihood of infection can be used to inform the treating physician about the most appropriate treatment algorithm. The measured levels of LE or HNE can also provide important prognostic information, with higher levels indicating a worse prognosis.

[0091] In some embodiments, the substrates of this disclosure are used to indicate that the subject has a urinary tract infection (UTI), also known as a genitourinary tract infection. In healthy women with classic UTI symptoms such as dysuria or frequent urination, but without vaginal discharge or irritation, a diagnosis of UTI can usually be made based on clinical symptoms alone. On the other hand, women with undefined symptoms, asymptomatic pregnant women, elderly patients, and children have a much lower pre-test probability of having a UTI. This disclosure is not limited to testing women for UTI. The absolute criteria for diagnosing a UTI are an intermediate urine culture (>10³–10⁵ microorganisms) or pyuria (more than 10⁴ leukocytes per ml).

[0092] Therefore, in some embodiments, midstream urine from symptomatic patients will be tested for leukocyte esterase ("LE") and / or human neutrophil elastase ("HNE") activity using the enzyme substrates of this disclosure. For example, this can be achieved by using the ICECEA assay described herein. Based on population data, the likelihood of infection can be determined based on both the measurement of LE and / or HNE activity and additional factors such as the presence of specific symptoms and patient characteristics (age, sex, pregnancy, etc.). Depending on the likelihood of infection, a physician may decide whether to administer oral antibiotics.

[0093] Population data for the clinical applications of this disclosure (i.e., in indicating a patient as having an infection, but not limited to, for example, PJI, SBP, and / or UTI) can be used to translate measurements of LE and / or HNE activity to predictability of the presence of infection. The test device itself can be used as a medium for collecting and distributing such population-based data. For example, an electrochemical biosensor connected to a smartphone (or similar device) can allow a physician to provide selected information to a centralized database that can be used to continuously improve the calculation of the likelihood of infection. The biosensor can also report the likelihood of infection for an individual patient to a surgeon based on LE and / or HNE activity and additional metrics that can be used to refine treatment algorithms.

[0094] In some embodiments, substrates for detecting leukocyte enzymes, such as LE and / or HNE substrates, are incorporated into the assay. Such assays may include, for example, electrochemical assays. Electrochemical assays are cost-effective, highly sensitive, and simplify the calibration process. Furthermore, such methods are equally effective in bloody or turbid liquids. Preferred electrochemical assays include internally calibrated electrochemical continuous enzyme assays ("ICECEA"). The use of the LE substrate of this disclosure ("TAPTA") in ICECEA is described in Example 1 below. ICECEA is generally disclosed in PCT / US2014 / 03713 and US2016 / 0040209, and the entirety of that disclosure is incorporated herein. ICECEA utilizes the integration of enzyme-free pre-assay calibration and electrochemical enzyme assay in a continuous experiment. This is thought to result in a uniquely shaped amperometric trace that enables selective and highly sensitive measurement of enzymes such as LE and HNE.

[0095] ICECEA generally follows the method described in US2016 / 0040209. First, the enzyme substrate (e.g., LE and / or HNE substrates of this disclosure) is placed in the background electrolyte. Next, the reactants or products of the enzyme's enzymatic reaction are added to the first enzyme substrate / background electrolyte, creating what is called the “first assay mixture.” The current flowing through the electrodes of the electrochemical assay is measured after the first assay mixture has been formed. Next, the enzyme (e.g., LE and / or HNE) is added to the “first assay mixture” to create the “second assay mixture,” and the current is measured again over a predetermined period. The enzyme activity is determined based on the change in current over time caused by the addition of the enzyme. Optimally, the enzyme is added after the reactants / products are added to the enzyme substrate, but the order can be reversed, i.e., the enzyme is added to the substrate first, followed by the reactants / products.

[0096] ICECEA includes an electrochemical analyzer. The electrochemical analyzer includes a working electrode, a reference electrode, and an auxiliary electrode. Current is measured through the working electrode. The working electrode may be a noble metal electrode, a 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. An electrochemical assay system may also consist only of a working electrode and a reference electrode. To measure changes in current, an amperometric trace of the current is collected.

[0097] Generally, in ICECEA, adding reactants / products to the enzyme substrate (in the electrolyte) of an electrochemical assay system involves the following steps: First, the first aliquot of the reactant / product is added to the enzyme substrate (in the electrolyte). The current flowing through the electrodes of the electrochemical assay system is measured after the first aliquot has been added. One or more additional aliquots of the reactant / product are added to the mixture, and the current flowing through the electrodes of the electrochemical assay system is measured again. Preferably, at least three aliquots of the reactant / product are added to the enzyme substrate (in the electrolyte) before the enzyme is added to the mixture. Alternatively, aliquots of the reactant / product are added to the substrate (in the electrolyte) after the enzyme has been added to the mixture.

[0098] The enzymatic activity of an enzyme can be determined from the slope of a line created by measuring the current flowing through the working electrode of an electrochemical assay system at predetermined intervals over a predetermined period of time, after the reactants / products have been added to the substrate (before the enzyme has been added, or vice versa). The advantage of this method is that the addition of reactants / products to the substrate (in the electrolyte) and the addition of the enzyme are performed in the same container using the same electrode.

[0099] In at least one embodiment, a customized kit is described comprising a solution of the enzyme substrate and other required reactants in a background electrolyte, a solution of the redox active component of the enzyme reaction, and a solution of the assayed enzyme. Thus, the amperometric measurement is performed using any electrochemical analyzer equipped with an amperometric method and a conventional electrochemical cell equipped with a working electrode, a reference electrode, and a counter electrode immersed in the solution containing the enzyme substrate. The working electrode is held at a potential E versus the potential of the reference electrode. Potential E is suitable for either the oxidation or reduction of a chemical species present in the solution containing the redox active component of the enzyme reaction. The experiment is performed by spiking one or more known aliquots of the redox activity-containing solution, followed by one aliquot of the solution containing the assayed enzyme, into a stirred solution containing the enzyme substrate and other required reactants, and measuring the current flowing through the working electrode.

[0100] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure belongs. Any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of this disclosure, but preferred methods and materials are described herein. All publications referenced herein are incorporated herein by reference in their entirety.

[0101] The publications disclosed herein are provided solely for disclosure prior to the filing date of the present invention. Furthermore, the publication dates provided may differ from the actual publication dates, which need to be verified individually.

[0102] Each application and patent cited in this text, as well as each document or reference, patent or non-patent document (including those in the examination of each issued patent, “Application Reference Documents”) cited in each application and patent, and each PCT and foreign application or patent corresponding to and / or claiming priority to any of these applications and patents, as well as each document cited or referenced in each Application Reference Document, are expressly incorporated herein by reference. More generally, each document or reference cited in this text and each document or reference cited in each of the references cited herein (including manufacturer specifications, instructions, etc.) are expressly incorporated herein by reference.

[0103] The following non-limiting embodiments may help illustrate this disclosure further. 6. Examples

[0104] 1. Use of 4-((tosyl-L-alanyl)oxy)phenyltosyl-L-alaninate in an internally calibrated electrochemical continuous enzyme assay (ICECEA).

[0105] The activity of leukocyte esterases (LE) was measured in an internally calibrated electrochemical continuous enzyme assay (ICECEA) using the substrate 4-((tosyl-L-alanyl)oxy)phenyltosyl-L-alaninate, compound A (also known as "TAPTA"). The measurement results are shown in Figure 4. Compound A ICECEA was performed as generally described in US2016 / 0040209 and the detailed description above. Briefly, the pre-assay stage involved performing three distinct calibration steps by spiked a solution of the redox active component of the enzymatic reaction into a solution of the enzyme substrate ("TAPTA") and the required reactants. These three distinct calibration steps are indicated by bold "a" in Figure 4. After calibration, the assay stage was initiated by spiked one aliquot of the assay enzyme (LE) into the enzyme substrate solution. This step is indicated by bold "b" in Figure 4. Following the enzymatic reaction, the current flowing through the working electrode was measured. The enzyme assay was calibrated for LE concentrations in the range of 0–250 μg / L. The enzymatic activity of LE showed a linear response to LE concentration and predicted infection. 2. Synthesis of the monoester of Equation 5 Example 2a. 4-Hydroxyphenyl(N-(3-(methoxycarbonyl)pyridine-5-sulfonyl)-L-alaninate (monoester) can be prepared by partial hydrolysis of 1,4-phenylenebis(N-(3-(methoxycarbonyl)pyridine-5-sulfonyl)-L-alaninate (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, and amines. Example 2b. Hydrolysis with NaOH 1,4-phenylenebis(N-(3-(methoxycarbonyl)pyridine-5-sulfonyl)-L-alaninate (21 mg, 0.032 mmol) was dissolved in THF and treated with 1 M NaOH (0.045 mL, 139 mol%) at 30°C for 4 days. The solvent was evaporated, the residue was dissolved in dichloromethane, rinsed with 1 M HCl, and then dried over MgSO4 to obtain a colorless glass product. Example 2c. Hydrolysis with triethylamine 1,4-phenylenebis(N-(3-(methoxycarbonyl)pyridine-5-sulfonyl)-L-alaninate (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. Then, 1 M was obtained. HCl was added to adjust the pH to 1. Next, the layers were separated, and the aqueous layer was extracted twice with dichloromethane. The organic extract was dried over MgSO4 and evaporated to obtain pink foam. Chromatography on silica gel using dichloromethane-ethyl acetate (70:30) yielded 4-hydroxyphenyl (N-(3-(methoxycarbonyl)pyridine-5-sulfonyl)-L-alaninate (36 mg, yield 63%, melting point: 113-116°C) as a white crystalline solid. 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(MH)-. Example 2d. 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 obtain N-(3-(methoxycarbonyl)pyridine-5-sulfonyl)-L-alanine. This intermediate was also prepared by condensing L-alanine benzyl ester with methyl 5-(chlorosulfonyl)pyridine-3-carboxylate, followed by hydrogenation in toluene on 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 obtain hydroxyphenyl(N-(3-(methoxycarbonyl)pyridine-5-sulfonyl)-L-alanine). Alternatively, N-(3-(methoxycarbonyl)pyridine-5-sulfonyl)-L-alanine was condensed with hydroquinone monobenzyl ether or mono-BOC-hydroquinone [tert-butyl 4-(hydroxyphenyl) carbonate], and then hydrogenated on palladium in acetic acid or hydrolyzed with HCl(g) to obtain 4-hydroxyphenyl(N-(3-(methoxycarbonyl)pyridine-5-sulfonyl)-L-alanine salt.

[0106] 3. Use of 4-hydroxyphenyl(N-(3-(methoxycarbonyl)pyridine-5-sulfonyl)-L-alaninate in screen-printed electrodes

[0107] Referring to Figure 7, the effectiveness of using the substrate of the present invention on a screen-printed electrode strip is demonstrated. Specifically, an electrode strip was screen-printed with a substrate having the structure shown in Equation 5. The strip was then exposed to various concentrations of LE (i.e., LE from 0.125 U / ml to 0.35 U / ml).

[0108] The plot clearly shows a significant reduction peak at approximately -0.17V, which corresponds to the redox reaction of hydroquinone molecules released during cleavage of the monoester by leukocyte esterases at the ester active site. Furthermore, since the reduction peak was found to be directly related to LE activity in the sample in a dose-dependent manner, the screen-printed electrode strip was not only confirmed to detect the presence of LE, but could also provide a quantitative measurement of the activity level or concentration of LE in the sample, which directly corresponds to the enzymatic cleavage of the monoester. Based on this measured level, it is possible to determine whether the patient's LE level is high enough to indicate infection.

[0109] The above-mentioned examples and descriptions of preferred embodiments should be construed as illustrative rather than limiting the disclosure as defined by the claims. For ease of understanding, numerous variations and combinations of the above features can be utilized without departing from the disclosure as defined in the claims. Such variations are not considered departures from the scope of the disclosure, and all such variations are intended to fall within the following scope:

Claims

1. 1. A substrate composition for leukocyte esterase comprising a monoester, the monoester is one of an α-amino acid ester or an α-hydroxy acid ester and is specific for leukocyte esterase; The substrate composition, wherein the monoester has a first portion for participating in a redox reaction and a second portion with an amine or alcohol protecting group.

2. 10. The matrix composition of claim 1, wherein the matrix composition has the general structure represented by the formula: wherein the first moiety B is 4-hydroxyphenyl ( ), A comprises oxygen or NRa, where Ra is H or a substituted alkyl, aryl, or aralkyl group, R1, R2, and R3 are independently hydrogen or an optionally substituted alkyl group (when A is oxygen, R3 is absent), and R4 is a substituted or unsubstituted heterocycle or heteroaryl.

3. The R4 may be pyridinyl, pyridazinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, quinolyl, isoquinolyl, 1,2,3,4-tetrahydroquinolyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, indolyl, benimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, triazinyl, thiadiazolyl, oxadiazolyl, purinyl, 1-oxoisoindolyl, 1,2,4-triazinyl, 1,3,4-triazinyl, isoindolyl, 3. The substrate composition of claim 2, which is one of benzothiophenyl, benzotriazolyl, benzothiazolyl, benzoxazolyl, tetrahydroquinolyl, dihydroquinolyl, naphthyridinyl, quinoxalinyl, quinazolinyl, dihydroisoquinolyl, tetrahydroisoquinolyl, benzofuryl, furopyridinyl, pyrrolopyridimidinyl, or azaindolyl.

4. 4. The substrate composition of claim 3, wherein R4 is pyridine with or without the addition of a substituted or unsubstituted polar group.

5. 5. The substrate composition of claim 4, wherein R4 is a pyridine selected from one of pyridine (1), methoxypyridine (2), and (methoxycarbonyl)pyridine (3), as shown below.

6. 6. The substrate composition of claim 5, wherein R4 is (methoxycarbonyl)pyridine (3).

7. 2. The substrate composition of claim 1, wherein the monoester is 4-hydroxyphenyl(N-(3-(methoxycarbonyl)pyridine-5-sulfonyl)-L-alaninate.

8. 2. The substrate composition of claim 1, wherein the monoester has the structure:

9. 10. An electrochemical device for detecting leukocyte esterase, comprising a substrate having the substrate composition of claim 1.

10. 10. The electrochemical device of claim 9, wherein the substrate composition is screen printed onto the electrode sensor.

11. 1. A method for screening patients at risk for infection, comprising detecting the presence of leukocyte esterase enzyme in an electrochemical assay, applying or contacting a biological sample to a screen-printed electrode sensor strip comprising the substrate composition of claim 1; measuring the current or voltage across the electrodes using a potentiometric or voltammetric reader; determining the activity level of leukocyte esterase based on the current or voltage in the electrochemical assay; and determining the likelihood of infection based on said activity level.

12. 12. The method of claim 11, wherein the biological sample comprises one of urine, saliva, synovial fluid, pleural fluid, pericardial fluid, peritoneal fluid, cerebrospinal fluid ("CSF"), and middle ear fluid.

13. 12. The method of claim 11, wherein the activity level indicates the presence of activated leukocytes in the sample, which is indicative of an infection.

14. 14. The method of claim 13, wherein the infection comprises one of a periprosthetic joint infection (PJI), a native joint infection, a urinary tract infection (UTI), bacterial peritonitis, meningitis, and endometritis.

15. A composition characterized by having the general structure represented by the formula: wherein the first moiety B is 4-hydroxyphenyl ( ), A comprises oxygen or NRa, where Ra is H or a substituted alkyl, aryl, or aralkyl group, R1, R2, and R3 are independently hydrogen or an optionally substituted alkyl group (when A is oxygen, R3 is absent), and R4 is a substituted or unsubstituted heterocycle or heteroaryl.

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