WO2010022281A1 - Procédés d'utilisation d'élastase neutrophile humaine en tant qu'indicateur d'infection de plaie active - Google Patents

Procédés d'utilisation d'élastase neutrophile humaine en tant qu'indicateur d'infection de plaie active Download PDF

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WO2010022281A1
WO2010022281A1 PCT/US2009/054532 US2009054532W WO2010022281A1 WO 2010022281 A1 WO2010022281 A1 WO 2010022281A1 US 2009054532 W US2009054532 W US 2009054532W WO 2010022281 A1 WO2010022281 A1 WO 2010022281A1
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seq
wound
bodipy
hne
neutrophil elastase
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PCT/US2009/054532
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English (en)
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Mark R. Wardell
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Greystone Pharmaceuticals, Inc.
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    • 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
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • 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
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
    • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
    • G01N2333/96433—Serine endopeptidases (3.4.21)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/12—Pulmonary diseases
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/18—Dental and oral 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
    • G01N2800/20—Dermatological disorders

Definitions

  • the present invention provides for a rapid point-of-care diagnostic system which measures the level of Human Neutrophil Elastase (HNE) activity in biological fluid, including chronic wound fluid to provide a quantitative indication of the level of "Active" Infection in a chronic wound.
  • HNE Human Neutrophil Elastase
  • the HNE is secreted by host neutrophils which migrate to the wound site to neutralize the microbiological agents that are the primary cause of the active infection.
  • Simply measuring microbiological agents such as bacteria, fungi or the expressions, metabolites, toxins, or cellular components or agents emitting from such microorganisms, or components of the biofilm matrix in which bacteria and microorganisms reside will not provide a meaningful indication of whether the existence of these microorganisms is contributing to the failure of chronic wounds to heal.
  • an "Active” infection is an infection caused by microorganisms, whether planktonic or associated in biofilm communities that are maintaining a chronic state of wound inflammation or contributing to wound deterioration and preventing wound healing by continuing to stimulate the host to send large numbers of neutrophils to the wound site to eliminate the threat.
  • the present invention may also be applied to the diagnosis of other active infections, for example, chronic lung infection or in chronic periodontal disease such as gingivitis, by measuring the levels of HNE activity in sputum or gingival crevicular fluid, respectively.
  • the present invention teaches that high levels of HNE in wounds are directly proportional to the massive recruitment of neutrophils to the wound (see Fig. 1) that have been activated to trigger the oxidative burst and release of protease granules, including the azurophilic granules that contain HNE, by stimuli emanating from microbial agents and colonies that are actively parasitizing wounds.
  • the measurement of high HNE levels therefore, will provide a useful indicator to wound care physicians of a problematical microbial presence that needs to be immediately addressed within the treatment options before the wound will progress towards healing.
  • High levels of HNE are a host response to "Active" infection, and the present invention further recognizes that these high levels of HNE contribute significantly to the further destruction of host tissue and the failure of wounds to heal.
  • Treatment options therefore include strategies that will lower HNE activity, but this is treating the effect of the infection.
  • a more logical way to treat the problem would be to treat the cause of the problem by eliminating the "Active" infection. Examples of how this is done today include antibiotic treatment and sharp debridement.
  • Antibiotic treatment is only effective against planktonic bacteria and sharp debridement is a surgical procedure that must be repeatedly performed by a qualified health professional and involves removal of biofilm (along with other components of "slough” and necrotic tissue) that can re-establish within 24-48 hours.
  • Wolcott' s group at the Southwest Regional Wound Care Center and Medical Biofilm Research Institute in Lubbock Texas is one group actively investigating new mechanisms of controlling biofilm growth in chronic inflammatory diseases, including wounds.
  • Various agents are under investigation as anti- biofilm therapeutics, including Lactoferrin, Xylitol, EDTA, Gallium, Dispersin B, Farnesol, RNA-III Inhibitory Peptide, Furanone C30 (Wolcott and Rhoads, 2008; A study of biofilm- based wound management in subjects with critical limb ischemia.
  • FIGURE 1 Agents emitted from "Active" Infection recruit neutrophils to the site of infection. Neutrophils get activated to trigger the oxidative burst, the release of proteases and the phagocytic mode to eliminate the causative agent of the "Active" infection. With the constant recruitment and activation of neutrophils, the levels of HNE become higher than the levels of natural inhibitors (e.g. ⁇ r protease inhibitor) that normally inactivate the excess HNE such that this excess HNE also contributes to the disease by degrading host tissue (collagens, elastin, proteoglycans, growth factors) thereby stalling wound healing or causing even more host tissue damage.
  • inhibitors e.g. ⁇ r protease inhibitor
  • FIGURE 2 Example Diagnostic Device System in Which the HNE Activity in Biological Fluid May be Measured.
  • the system consists of a sterile swab for biological fluid collection (b), a plastic tube device (available from Medical Packaging Corporation) in which the chemistry of the test takes place (a), and a fluorometer (c) to measure the amount of fluorescent product released during the test.
  • FIGURE 3 Steps in the Use of the Example HNE Activity Detector.
  • FIGURE 4 Schematic Representations of HNE-Specific FRET Peptide Substrates, (a) FRET peptide substrate containing a single fluorophore and a single quencher, (b) FRET peptide substrate containing a single fluorophore and two quenchers with two HNE-specific cleavage sites.
  • the present invention utilizes the discovery that increased levels of Human Neutrophil Elastase (HNE) activity provides a quantitative indication of the level of "Active" infection in a chronic wound.
  • HNE Human Neutrophil Elastase
  • One embodiment incorporates this phenomenon in providing a rapid, point-of-care diagnostic as described herein.
  • a FRET peptide substrate containing an HNE-specific amino acid sequence for example, Quencher-APEEIMRRQ-Fluorophore (SEQ ID NO: 1) or Quencher-AAPV- Fluorophore (SEQ ID NO: 2), will be incubated with wound fluid at ambient temperature for around 10 or more minutes.
  • an HNE-specific substrate peptide is represented schematically in Figures 2 and 3.
  • the practitioner opens the end of the sterile swab package (Fig. 2, b) opposite from the sample collection bulb. The practitioner then removes the top of the plastic device (Fig. 2, a; available from Medical Packaging Corporation) which has a slot on its underside for the hollow shaft of the sterile swab to insert into. Once the swab is inserted into the top, the wound care provider swabs the affected area of the patient and then inserts the swab into the plastic tube, replacing the top on the tube [ Figure 3 (a)].
  • the buffer in which the HNE-specific FRET substrate peptide is dissolved in the top bulb of the device could be any buffer system conducive to the proteolysis reaction, including but not limited to Tris, phosphate, acetate, citrate, borate, cacodylate, TAPS, Bicine, Tricine HEPES, TES, MOPS, PIPES, and MES. It may contain a number of additives conducive to peptide stability of the proteolytic reaction. For example, it might contain an antioxidant to protect methionine or cysteine or fluorophore or quencher oxidation, should these amino acids or oxidisable fluorophore/quencher be included in the FRET peptide substrate. It might contain a detergent such as Brij-35 or other compound to promote solubility or proteolytic reaction. Its volume and the peptide concentration within it will be optimized to meet the specific kinetic reaction conditions that facilitate a 10 minute point-of- care diagnostic system.
  • the test will be designed to be done at "room temperature", which is defined as the approximate temperature of the room in which the test is performed.
  • room temperature is defined as the approximate temperature of the room in which the test is performed.
  • a typical range of temperatures that would be found at different times of the year in hospital clinics in the various countries where this test would be sold would be 65-85 0 F (-18-30 0 C).
  • This is also the temperature range within which regulatory agencies like to see robust test performance. It is not anticipated that temperature will need to be controlled for successful performance of the test, but the practitioner may be directed to perform the test within the above as the range of temperature.
  • One embodiment of the test would be a rapid point-of-care test that the wound care professional would perform during the patient consultation to help guide their immediate decision about treatment strategy. The 10 minute time for completion of the test reflects this goal.
  • test performance it is a goal to also design into the capability of the test several alternative options for test performance to make it more flexible for the practitioner, which would mean the test would take longer to complete.
  • the practitioner may only have time to collect the sample and might choose to run the test at a later time and the option to collect a sample on a swab and run the test up to several hours later may be possible.
  • the test will include a "stop" step, where the enzymatic reaction is stopped (by the addition of a neutralizing antibody and/or protease inhibitor at a certain time point) and the practitioner will have the option to complete the test up to that point and come back up to several hours later to read the result.
  • the central objective is to provide a rapid, point-of-care diagnostic assay that the wound care professional can use to gain biochemical information that will help guide him or her in the best treatment option at that time.
  • Ten minutes is an optimal time point that is neither too short for adequate confidence in the result nor too long so as to preclude the capture of that piece of diagnostic information within the time frame of a typical patient consultation.
  • the peptide substrate may contain a quencher on the one side (e.g. the amino-terminal side) of the scissile bond and a fluorophore on the other side (e.g. the carboxyl-terminal side) of the scissile bond such that when the scissile bond is cleaved by HNE, the fluorophore signal is no longer quenched by the quencher and a fluorescent signal will occur in the sample.
  • quenchers that could be used include: DABCYL; QXS-520; FITC; Dnp; BHQ-I (see also TABLE 2).
  • fluorophores that could be used include: EDANS; 5-FAM; Rhodamine; Mca (see also TABLE 1).
  • the range of wavelengths used to achieve a fluorescent signal would depend upon the FRET pair chosen.
  • 5-FAM is used as the fluorophore and Dabsyl as the quencher.
  • the excitation optics would put out light at 745 ⁇ 15 nm (e.g. from a blue LED such as is contained in the Turner Biosystems Picofluor fluorometer.
  • the emission optics e.g. a photodiode detector within the Turner Biosystems Picofluor fluorometer
  • Rhodamine is used as the fluorophore
  • an excitation wavelength of 525 ⁇ 20 nm e.g. using the green LED light source within the Turner Biosystems Picofluor fluorometer
  • the emission optics would be set at >570 nm, again using the Turner Biosystems Picofluor fluorometer.
  • the Picofluor fluorometer (see htlp ://w ww . turnerbiosy s tern s . com/i n slrumen ts/PicoFluor-han dheld-tTuorometer-f luori meler- DNA:ENArPlote[ ⁇ i.php_) is commercially available from Turner Biosystems, Inc.
  • the Picofluor has two dedicated optical channels that allow for the measurement of two different fluorophores in the same experiment, if required. It has an internal data logging package with interfacing software to Microsoft and Excel through a Serial Interface cable connection to a computer.
  • the Picofluor can log up to 1,000 data points. It is CE marked for immediate use under European regulatory guidelines.
  • the fluorescence can be read and qualified using this portable hand-held fluorometer and recorded in patient medical records.
  • the sequential HNE activity levels in any single wound can be followed over time to guide the physician in management of the wound.
  • a second generation fluorometer that may be used will contain similar features with expanded firmware and software options that could automatically manage patient data over time.
  • the fluorescence can be read and quantified in a portable hand-held fluorometer and recorded in patient medical records. The level can be followed over time to guide the physician in management of the wound.
  • the HNE diagnostic test will be a rapid, Point-of-care indicator of the level of Human Neutrophil Elastase activity in wound fluid. It is anticipated that the test will have utility both the first time a wound care professional measures HNE activity in a patient, and then with subsequent regular (e.g. weekly) measurements of the HNE activity over time. Both measurement types (the initial and the sequential) would add to other diagnostic parameters (including subjective sensory perception of wound size, depth, color, swelling, level of exudates, visual presence of slough, and smell) to aid the physician in their treatment strategy (the initial measurement to help choose a treatment at that time; and the sequential measurements to indicate if that treatment strategy is working or whether it needs to be changed/modified).
  • diagnostic parameters including subjective sensory perception of wound size, depth, color, swelling, level of exudates, visual presence of slough, and smell
  • the test provides the first objective biochemical measurement of a parameter in wound fluid that is associated with the chronicity of wounds.
  • a high HNE activity is known to be associated with chronic, non-healing wounds (Yager et al., 1997, Ability of chronic wound fluids to degrade peptide growth factors is associated with increased levels of elastase activity and diminished levels of protease inhibitors.
  • the present invention teaches that a high activity level of this enzyme would indicate the wound was infected and that this infection was directly contributing to the failure of the wound to heal.
  • PHI polyhydrated ionogens, available from Greystone Pharmaceuticals, Inc.
  • Other wound care preparations may be administered to impede the re-establishment of biofilm after sharp debridement. See U.S. patent numbers 6,149,947 and 7,148,170, incorporated herein by reference in their entirety.
  • PHI-based compound used in this regard includes PHI and a hydrogel compound that also impedes the re-establishment of biofilm after debridement.
  • the HNE indicator would guide wound care professionals in what treatment options to apply to the wound. If they had previously embarked on one treatment option, the indicator would show if that strategy was being successful. The caregiver might well change the therapeutic strategy being applied to any given wound based upon the results of the test. Treatment would not stop until full closure of the wound had occurred.
  • the test would have a "cut-off or baseline level of HNE activity, above which it would indicate the presence of active infection, possibly involving biofilm, and below which there was no such infection.
  • the cut-off level is in the range of 0.25 mU/mg protein - 0.5 mU/mg protein.
  • HNE-specific amino acid sequences used with the present invention can be derived in several ways: first from reproducing a portion of the natural protein substrate sequence of HNE such as those in several of the collagens, elastin, fibronectin, cartilage proteoglycans, E.Coli OmpA, and virulence factors of several bacteria such as Shigella, Salmonella, and Yersinia.
  • the different sequences within the various natural substrates HNE cleaves could be used with the invention.
  • GPLGIA GITGARGLAGP SEQ ID NO: 3
  • oci-protease inhibitor oci-antitrypsin
  • Example sequences coming from this method include, but are not limited to, the inhibitory loop sequences of various serpins including oci -proteinase inhibitor, (LEAIPMSIPPEVKFNKPF (SEQ ID NO: 4) and truncated versions and derivatives); monocyte/neutrophil elastase inhibitor (GIATFCMLMPEENFTAD (SEQ ID NO: 5) and truncated versions and derivatives); plasminogen activator inhibitor- 1 (VIVSARMAPEEIIMDRP (SEQ ID NO: 6) and truncated versions and derivatives); proteinase inhibitor-9 (CFVV AECCMES GPRFCA (SEQ ID NO: 7) and truncated versions and derivatives); proteinase inhibitor-6 (AIMMMRCARFVPRFCAD (SEQ ID NO: 8) and truncated versions and derivatives); cowpox virus serpin CrmA (CALVADCASTVTNEFCA (SEQ ID NO: 9) and truncated versions
  • a third way of designing amino acid substrate sequences for HNE is from the list of synthetic inhibitors that are commercially available. This latter class of sequences include AAPV (SEQ ID NO: 14), AAPA (SEQ ID NO: 15) , and AAAA (SEQ ID NO: 16).
  • AAPV SEQ ID NO: 14
  • AAPA SEQ ID NO: 15
  • AAAA SEQ ID NO: 16
  • cleavage rates and specificity will be analyzed.
  • a sequence used with the present invention will preferably cleave rapidly at "room temperature” (see below) and be as specific for HNE as possible.
  • room temperature see below
  • One example substrate with a suitable sequence is described as follows:
  • the substrate is formulated as a "FRET peptide", that is, a peptide containing a fluorophore and a quencher spaced between 10-100 A apart, and can be represented by diagrams such as are shown in Figures 4 (a) and (b):
  • the APEEIMRRQ (SEQ ID NO: 10), AAPV (SEQ ID NO: 14) or other sequences would be contained within the construct as a specific HNE cleavage site.
  • the fluorophore is released from the distance constraint of the quencher and fluorescence occurs (see explanation of the principles of this process below).
  • flanking sequences There may or may not be flanking sequences around the HNE recognition/cleavage sequences (Fig. 4 shows flanking sequences).
  • a fluorophore has the ability to absorb energy from light, transfer this energy internally, and emit this energy as light of a characteristic wavelength. Fluorescence is generated when a substance absorbs light energy at a short (higher energy) wavelength, and then emits light energy at a longer (lower energy) wavelength. Thus, for any fluorescent molecule, the wavelength of emission is always longer than the wavelength of absorption (Stokes' Law).
  • Peak absorbance and peak emission wavelengths for some of the common fluorophores used in molecular applications, and which we might use in the present invention, are shown in TABLE 1 :
  • fluorescence detection techniques are based on quenching of fluorescence by energy transfer from one fluorophore to another fluorophore, or to a non-fluorophore.
  • the acceptor (quencher) moiety can be either fluorescent or non-fluorescent.
  • quenchers have been other fluorophores that accept a photon from energetically excited donor fluorophores. As a result, the energy level of the donor fluorophore returns to the ground state, without emitting fluorescence.
  • a requirement is that the fluorescence emission spectrum of the donor must overlap the absorption spectrum of the acceptor. If the acceptor is a fluorophore, the transferred energy can be emitted as fluorescence from the acceptor, but at a different wavelength that is not being measured by the fluorometer and so won't be "seen” as fluorescence. If the acceptor is not fluorescent, the absorbed energy is lost as heat.
  • Peak absorbance wavelength for FAM is 490 nm with a peak emission wavelength of 518 nm. If FAM and TAMRA are tethered at the amino-terminal and carboxyl-terminal of a peptide, respectively, and this construct is excited at 490 nm, so long as the peptide remains intact, emission will be at 580 nm (TABLE 1) and not at 518 nm (the wavelength being measured in the fluorometer) due to FAM transferring its energy to TAMRA. The fluorescence of the donor is thereby quenched through resonance energy transfer. Enzyme hydrolysis of the peptide results in spatial separation of the donor and acceptor, which leads to the recovery of the fluorescence of the donor, in this case at 518 nm, which wavelength the fluorometer has been set to measure by the practitioner of the experiment.
  • Fluorophores with an emission maximum between 500 and 550 nm such as 5-FAM, TET, and HEX (TABLE 1), are best quenched by quenchers with absorption maxima between 450 and 550 nm, such as Dabcyl and BHQ-I (TABLE 2).
  • Fluorophores with an emission maximum above 550 nm such as Rhodamines (including Rhodamine Red, TAMRA, ROX and Texas Red) and Cy dyes (including Cy3 and Cy5), are best quenched by quenchers with absorption maxima above 550 nm (including BHQ-2 [TABLE 2]).
  • Example 1 Use as a diagnostic of active infection in chronic dermal wounds.
  • the wound care provider would remove the wound dressing from the patient's wound and take a sample of wound fluid before applying any treatment to the wound. In this way the HNE activity in the wound can be assessed at the time of consultation and would represent the wound environment over the past 24 hours or longer.
  • a soft sterile Dacron swab designed to hold 80-100 ⁇ L of fluid, will be inserted into the top of the device.
  • the wound care professional would then swab the patient's wound by translating the swab gently over the entire wound surface so as to collect sample from as much of the wound surface area as possible, while simultaneously gently rotating the swab so as to expose all surfaces of the swab to the wound.
  • the practitioner will insert the swab into the elongated hollow tube of the device and replace the top on the tube [Figure 3 (a)].
  • the FRET peptide could consist of any sequence cleaved by HNE, as given by example herein, for example, APEEIMRRQ (SEQ ID NO: 10) flanked on the amino-terminal side by any quencher in TABLE 2, for example DABCYL and flanked on the carboxyl-terminal end by any fluorophore in TABLE 1, for example 5-FAM.
  • the amino-terminal and carboxy- terminal ends of the substrate can be modified to accept the fluorophore and quencher, i.e., DABCYL-GABA-APEEIMRRQK(5-FAM).
  • the quencher and fluorophore could be attached at opposite ends of the peptide to those mentioned above, so long as they straddle the scissile bond of the peptide and are within 10-100 A of one another in the FRET peptide.
  • the HNE peptide substrate sequence will be optimized for enzyme kinetics, HNE specificity, and to minimize promiscuity by other proteases. If necessary, non-specific proteolytic cleavage will be eliminated by the inclusion of protease inhibitors that are specific for the interfering substances.
  • the FRET peptide buffer solution would perfuse the swab to elute and dilute the wound fluid sample and bathe the swab in the reaction solution for a period of time, say 10 minutes [ Figure 3 (c)].
  • This solution also dissolves a cocktail of one or more protease inhibitors to prevent non-specific cleavage of the HNE substrate peptide during the reaction phase of the test.
  • HNE cleaves a peptide substrate to release a fluorescent signal. The more HNE activity in the wound fluid sample, the more fluorescence would be released.
  • the wound care provider pushes down on the top of the device [depicted by the arrow in Figure 3 (d)] to cause the tip of the swab to perforate the thin foil membrane and allow the reaction mixture to flow to the bottom of the tube [Figure 3 (d)].
  • an HNE neutralizing antibody which may be located in the bottom of the tube and/or another specific HNE inhibitor dissolves in the reaction buffer and inhibits further HNE proteolysis of the peptide substrate such that no more fluorescence is generated.
  • the bottom of the device is then inserted in the reading chamber of the fluorometer [Figure 2 (c)] which has been pre-calibrated using a solid fluorescent standard.
  • the readout result on the fluorometer is directly proportional to the HNE activity in the patient wound fluid and will be entered into the patient's records.
  • the fluorometric readout will be correlated with the presence of bacteria associated with biofilms, for example, Pseudomonas aeruginosa, Enterococcus faecalis, and Staphylococcus aureus and others (Sun et al,. 2008; In vitro multispecies Lubbock chronic wound biofilm model. Wound Rep 16(6): 805-813) and whether those biofilms or planktonic bacteria are causing active infection leading to a chronic inflammatory wound. These studies will result in fluorometric levels of HNE activity that correlate with active wound infection.
  • Example 2 Use as a diagnostic of active infection in periodontal disease.
  • the device would be used in the same way as for chronic dermal wounds except the sample collected would be gingival crevicular fluid.
  • Example 3 Use as a diagnostic of active infection in chronic lung disease.
  • the device would be used in the same way as for chronic dermal wounds except the sample collected would be sputum.
  • the present invention can also be formulated as kits.
  • the kit could be comprised of different components that could be packaged together or separately.
  • the kit could be comprised of a pack of devices as shown in Figure 2 A, a corresponding number of sterile swabs [Figure 2B], a fluorometer [Figure 2C], and a number of devices depicted in Figure 2 A containing a swab that had been impregnated with known amounts of active HNE as a standard.
  • the standard could be used to either calibrate the system with respect to HNE activity or they could be for the purpose of demonstrating that the reagents in that particular manufactured lot or device were still viable at the time of use by the wound care provider and that the wound care provider could competently operate the diagnostic test system.
  • the components could be packaged separately such that a fluorometer could be obtained, or a number of test devices and swabs could be obtained, or a number of standards could be obtained.

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Abstract

L'invention concerne une mesure du niveau d'activité de HNE dans un site de plaie, permettant de diagnostiquer si une plaie chronique a une infection active. Un substrat peptidique de FRET contenant une séquence d'acides aminés spécifique à HNE est incubé avec un fluide de plaie et mesuré pour un rayonnement fluorescent afin de déterminer le niveau d'activité HNE. L’analyse de diagnostic de HNE est un indicateur rapide, sur le lieu d’intervention, du niveau de l'activité HNE dans un fluide de plaie en tant qu'indicateur d'une infection "active".
PCT/US2009/054532 2008-08-20 2009-08-20 Procédés d'utilisation d'élastase neutrophile humaine en tant qu'indicateur d'infection de plaie active WO2010022281A1 (fr)

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Cited By (7)

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US20120135443A1 (en) * 2009-06-26 2012-05-31 Schultz Gregory S Rapid Bed-Side Measurement of Neutrophil Elastase Activity in Biological Fluids
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
US20120129186A1 (en) * 2010-11-23 2012-05-24 Kci Licensing, Inc. Devices and methods for the diagnosis and treatment of wounds using biomarkers
US9932622B2 (en) 2011-01-31 2018-04-03 Woundchek Laboratories B.V. Wound prognosis
GB2494934A (en) * 2011-09-23 2013-03-27 Systagenix Wound Man Ip Co Bv Wound prognosis
WO2014144572A3 (fr) * 2013-03-15 2014-11-06 Bayer Healthcare Llc Profilage de substrat de protéases dans des pièges extracellulaires neutrophiles
CN105339780A (zh) * 2013-03-15 2016-02-17 拜耳医药保健有限公司 蛋白酶在中性粒细胞胞外诱捕网中的底物性质
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US11104933B1 (en) 2016-03-22 2021-08-31 Cleu Diagnostics, Llc Compositions and methods for determining the presence of active leukocyte cells using an electrochemical assay

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