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WO2025096715A1 - Compositions et procédés pour la détection et le traitement de la pancréatite aiguë - Google Patents

Compositions et procédés pour la détection et le traitement de la pancréatite aiguë Download PDF

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Publication number
WO2025096715A1
WO2025096715A1 PCT/US2024/053805 US2024053805W WO2025096715A1 WO 2025096715 A1 WO2025096715 A1 WO 2025096715A1 US 2024053805 W US2024053805 W US 2024053805W WO 2025096715 A1 WO2025096715 A1 WO 2025096715A1
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WIPO (PCT)
Prior art keywords
elastase
level
individual
detection
sample
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PCT/US2024/053805
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English (en)
Inventor
Maisam ABU-EL-HAIJA
Brian C. SEARLE
Venkata AKSHINTALA
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Cincinnati Childrens Hospital Medical Center
Johns Hopkins University
Ohio State Innovation Foundation
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Cincinnati Childrens Hospital Medical Center
Johns Hopkins University
Ohio State Innovation Foundation
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Publication of WO2025096715A1 publication Critical patent/WO2025096715A1/fr
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    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • 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
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/06—Gastro-intestinal diseases
    • G01N2800/067—Pancreatitis or colitis

Definitions

  • Acute pancreatitis is an inflammatory pancreatic disorder, with recent studies reporting increasing global incidence over time in pediatric and adult populations. Despite the burden and implications of inflammatory pancreatic disorders, the diagnosis of AP remains challenging. AP in children is an emerging problem, with few published data regarding diagnostic methods, and should be studied separately from adults. Serum lipase, a biomarker identified in the early 1900s, has limited specificity, yet it remains the standard molecular diagnostic for AP.3 Misdiagnosis of AP has major consequences, requiring patients to undergo invasive endoscopic interventions or surgeries that may not be warranted. Another unmet need in clinical practice is the absence of a reliable point-of-care test for AP that can be used in real time to facilitate timely management and tailored interventions for AP. BRIEF SUMMARY
  • the methods comprise detecting Elastase 2A and/or 2B in a sample obtained from the individual, for example via detection of Elastase 2A and/or 2B protein and/or an mRNA encoding Elastase 2A and/or 2B. Further disclosed are kits for detecting Elastase 2A and/or 2B.
  • FIG. 1A-1E Two experiment cohorts were selected to separate patients with AP from a variety of control individuals, including patients with CP, FRCs, and HCs.
  • FIG. IB A data-independent acquisition mass spectrometry strategy to capture the urine proteome that separates protein detection (of a sample pool) from protein quantification (of individual samples). Using this approach, more than 2000 proteins were detected from a detailed 6-injection proteomics analysis of a sample pool, forming a library for this experiment. With the library, quantified 1694 proteins were quantified with measured signals in every sample of at least one group.
  • FIG. 1A Two experiment cohorts were selected to separate patients with AP from a variety of control individuals, including patients with CP, FRCs, and HCs.
  • FIG. IB A data-independent acquisition mass spectrometry strategy to capture the urine proteome that separates protein detection (of a sample pool) from protein quantification (of individual samples). Using this approach, more than 2000 proteins were detected from a detailed 6-injection proteomics analysis of
  • FIG. ID Box plots representing fold changes for key AP-specific candidate protein biomarkers. Bold lines show median log2 intensities, and boxes indicate the interquartile range. Whiskers show 5% and 95% points, and dots indicate each individual measurement. Fold changes are indicated relative to the HC median.
  • FIG. 2A-2B Volcano plot showing pairwise significant proteins (FDR ⁇ 0.05, pink) in the discovery cohort when comparing AP patients relative to CP patients, HCs, and FRCs.
  • FIG. 2B Validation cohort results demonstrating differentiation between AP and control groups for elastase 2A, different amylase paralogs, and CRP protein expression. Bold lines show median log2 intensities, and boxes indicate the interquartile range. Whiskers show 5% and 95% points, and dots indicate each individual measurement. Fold changes are indicated relative to the HC median.
  • “about” may mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value.
  • the term may mean within an order of magnitude, preferably within 5-fold, and more preferably within 2- fold, of a value.
  • the term “effective amount” means the amount of one or more active components that is sufficient to show a desired effect. This includes both therapeutic and prophylactic effects. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.
  • the terms “individual,” “host,” “subject,” and “patient” are used interchangeably to refer to an animal that is the object of treatment, observation and/or experiment. Generally, the term refers to a human patient, but the methods and compositions may be equally applicable to non-human subjects such as other mammals. In some aspects, the terms refer to humans. In further aspects, the terms may refer to children.
  • Acute pancreatitis is a common inflammatory disease of the exocrine pancreas.
  • AP causes severe abdominal pain and multiple organ dysfunction and may lead to pancreatic necrosis and persistent organ failure.
  • Overall global incidence of AP is 30-40 cases per 100,000 population per year. The global incidence is rising, although studies suggest rates are currently more stable in Asia.
  • AP leads to significant short- and long-term morbidity, which in a significant minority causes prolonged debility, recurrent disease, and pancreatic exocrine and/or endocrine insufficiency. Quality of life can be significantly impaired as a result of chronic pain and the socio- economic consequences of prolonged hospitalization.
  • pancreatitis Presentations of pancreatitis include epigastric or diffuse abdominal pain (80-95%), nausea and vomiting (40-80%), abdominal distension, fever, breathlessness, irritability, and impaired consciousness, with pyrexia, low oxygen saturation, tachypnoea, tachycardia, hypotension, abdominal guarding, ileus and/or oliguria.
  • a diagnosis of acute pancreatitis requires two out of three criteria: (1) abdominal pain consistent with pancreatitis, (2) a serum amylase or lipase three or more times the upper limit of normal, and (3) findings consistent with pancreatitis on cross-sectional abdominal imaging. See, e.g., Szatmary, Peter et al. “Acute Pancreatitis: Diagnosis and Treatment.” Drugs vol. 82,12 (2022): 1251-1276. doi:10.1007/s40265-022-01766-4.
  • kits for the detection of one or both of Elastase 2 A and Elastase 2B are disclosed, the kits being useful for the diagnosis and/or treatment of AP in an individual in need thereof.
  • the individual is a pediatric patient.
  • the individual is an adult patient.
  • the individual is an individual presenting with one symptom of pancreatitis, for example at least one of a family history of acute pancreatitis, chronic pancreatitis, vomiting, abdominal pain, and fever.
  • the individual is an individual presenting with two symptoms of pancreatitis, for example at least one of a family history of acute pancreatitis, chronic pancreatitis, vomiting, abdominal pain, and fever.
  • the individual is suspected of having acute pancreatitis.
  • the individual is an individual presenting with three symptoms of pancreatitis, for example at least one of a family history of acute pancreatitis, chronic pancreatitis, vomiting, abdominal pain, and fever.
  • the biological sample for detection of the biomarker may be selected from blood, urine, saliva, cerebrospinal fluid, tissue biopsies, swabs (nasal, throat, or buccal), plasma, serum, and combinations thereof.
  • the biological sample is urine.
  • the biological sample is previously frozen urine.
  • the biological sample is freshly obtained (e.g., never frozen, and/or obtained within an hour of testing) at the time of subjecting the sample to the disclosed methods.
  • Methods of preparing biological samples for detection of a biomarker protein and/or nucleic acid are known, and the sample obtained from the individual can be subjected to one or more processes prior to detection of the biomarker.
  • Exemplary methods include, for example: centrifugation to separate plasma or scrum from blood; filtration or centrifugation to remove particulates from urine; collection and possible dilution or stabilization of saliva samples using specialized devices; filtration of cerebrospinal fluid collected via lumbar puncture; homogenization or lysis of tissue biopsies to release cellular contents; elution of swabs in buffer solutions; and treatments such as dilution, filtration, or enzymatic digestion for other bodily fluids.
  • the sample is a urine sample, and is not subjected to any processes prior to detection of the biomarker.
  • Elastase 2A and 2B are a serine proteases secreted by the pancreas. Enzymatically, serine proteases digest other proteins, where Elastase 2A and 2B have chymotrypsin-like activity, cleaving proteins at hydrophobic amino acids. The proteins arc often referenced by their full names: chymotrypsin- like elastase family member 2A and 2B, or their gene names: CELA2A and CELA2B. From a sequence perspective, Elastase 2A and 2B are nearly identical and diverge by only a few amino acids (88.1% identity).
  • the signal peptide for CELA2A is MIRTLLLSTLVAGALS (SEQ ID NO: 2) (l-16aa) and it has a propeptide CGDPTYPPYVTR (SEQ ID NO: 3) (17-28aa). The complete function of the propeptide is not clear'.
  • the active protein is 29-269aa.
  • the canonical protein sequence for CELA2B before the signal peptide is removed is:
  • the method comprises detecting Elastase 2A and/or 2B in a sample obtained from the individual.
  • the detecting comprises detecting at least a portion of Elastase 2A.
  • the detecting comprises detecting at least a portion of Elastase 2B.
  • the detecting comprises detecting a conserved region of Elastase 2A.
  • the detecting comprises detecting a conserved region of Elastase 2B.
  • the detecting comprises detecting a region of SEQ ID NO: 1.
  • the detecting comprises detecting a region of SEQ ID NO: 4.
  • the detecting comprises detecting a region of SEQ ID NO: 5. In aspects, the detecting comprises detecting a region of SEQ ID NO: 6. In aspects, the detection of SEQ ID NO: 1 comprises detection of a region that is not the signal sequence. In aspects, the detection of SEQ ID NO: 4 comprises detection of a region that is not the signal sequence.
  • the disclosed methods comprise the step of detecting one or both of Elastase 2A and Elastase 2B in a sample.
  • the method comprises detecting one or both of Elastase 2A and Elastase 2B protein.
  • the method comprises detecting one or both of Elastase 2A and Elastase 2B expression levels, comprising detecting an mRNA level of one or both of Elastase 2A and Elastase 2B.
  • Various methods are known in the art for detection of protein and/or mRNA, as described below.
  • Enzyme-Linked Immunosorbent Assay ELISA
  • the disclosed biomarker may be detected via Enzyme-Linked Immunosorbent Assay (ELISA).
  • ELISA methods are generally known in the art.
  • an antibody is used to bind the biomarker of interest.
  • a biological sample such as blood, urine, cell lysate, or serum, is introduced to a surface coated with a capture antibody specific to the target biomarker.
  • a secondary antibody conjugated to an enzyme such as, for example, horseradish peroxidase (HRP), is added.
  • HRP horseradish peroxidase
  • the enzyme Upon substrate introduction, the enzyme catalyzes a reaction that produces a detectable signal, typically colorimetric or chemiluminescent, which correlates with the biomarker's concentration in the sample.
  • a detectable signal typically colorimetric or chemiluminescent
  • the ELISA is used for quantitative detection.
  • the ELISA is used for qualitative detection.
  • Western Blot In aspects, the disclosed biomarker may be detected via Western blot. Western blot methods arc generally known in the art. In brief, proteins in a biological sample, such as blood or tissue lysate, are separated based on molecular weight using gel electrophoresis and then transferred onto a membrane, such as nitrocellulose or PVDF. The membrane is subsequently probed with a primary antibody specific to the target biomarker.
  • a secondary antibody conjugated to an enzyme such as, for example, horseradish peroxidase (HRP)
  • HRP horseradish peroxidase
  • the enzyme catalyzes a reaction that produces a detectable signal, typically chemiluminescent, which correlates with the presence of the biomarker on the membrane.
  • Western blot is used for both qualitative detection and semi-quantitative analysis of the biomarker.
  • IHC Immunohistochemistry
  • the disclosed biomarker may be detected via Immunohistochemistry (IHC).
  • IHC methods are generally known in the art. In brief, IHC involves the detection of a biomarker directly within tissue sections or cell samples. The sample is first fixed and then exposed to a primary antibody specific to the target biomarker. Following this, a secondary antibody conjugated to an enzyme, such as, for example, horseradish peroxidase (HRP), or a fluorescent tag is introduced. Upon addition of a suitable substrate, the enzyme catalyzes a reaction that generates a colorimetric signal, or the fluorescence is detected, indicating the presence and localization of the biomarker within the sample.
  • IHC provides both qualitative detection and spatial localization of the biomarker in tissues.
  • MS Mass Spectrometry
  • proteins from a biological sample such as blood or tissue lysate, are first digested into smaller peptides using an enzyme like trypsin.
  • the resulting peptides are then ionized and introduced into a mass spectrometer, where they are separated based on their mass-to-charge (m/z) ratio.
  • the mass spectrometer detects these ionized peptides, generating a mass spectrum that allows for the identification and quantification of the biomarker based on its unique peptide profile.
  • MS provides highly sensitive and accurate quantitative detection of the biomarker, as well as detailed structural information about the protein.
  • SPR Surface Plasmon Resonance
  • the disclosed biomarker may be detected via Surface Plasmon Resonance (SPR).
  • SPR methods arc generally known in the art.
  • SPR detects biomolecular interactions in real time without the need for labeling.
  • a biological sample containing the biomarker of interest is passed over a sensor surface coated with a capture molecule, such as an antibody specific to the biomarker. Binding of the biomarker to the capture molecule causes a change in the refractive index at the sensor surface, which is detected as a shift in the resonance angle of the surface plasmon wave. This shift is directly proportional to the amount of biomarker bound, allowing for both qualitative and quantitative analysis.
  • SPR provides sensitive and real-time detection of the biomarker, enabling the measurement of binding kinetics and concentration.
  • Flow Cytometry the disclosed biomarker may be detected via Flow Cytometry.
  • Flow cytometry methods are generally known in the art. In brief, cells from a biological sample, such as blood or tissue, are first labeled with a fluorescently tagged antibody specific to the target biomarker. The labeled cells are then passed through a laser beam within a flow cytometer, where individual cells are analyzed based on their fluorescence intensity, which corresponds to the amount of biomarker present on or within each cell. The flow cytometer collects data on various parameters, including cell size, complexity, and fluorescence, allowing for both qualitative detection and quantitative analysis of biomarker expression across a large number of cells. In aspects, flow cytometry provides high-throughput and multiparametric detection of the biomarker in a heterogeneous cell population.
  • Luminex Assay mRNA Detection Assays
  • RT-qPCR Reverse Transcription Quantitative PCR
  • the disclosed biomarker may be detected via Reverse Transcription Quantitative PCR (RT-qPCR).
  • RT-qPCR methods are generally known in the art.
  • RNA from a biological sample such as blood or tissue
  • cDNA complementary DNA
  • reverse transcriptase reverse transcriptase
  • the resulting cDNA is then amplified using quantitative PCR, where specific primers targeting the biomarker sequence are used.
  • fluorescent dyes or probes such as SYBR Green or TaqMan probes, bind to the DNA, and the fluorescence intensity is measured in real time. The amount of fluorescence correlates with the amount of the biomarker's mRNA originally present in the sample.
  • RT-qPCR provides sensitive, specific, and quantitative detection of biomarkcr mRNA expression.
  • the disclosed biomarker may be detected via Northern Blotting.
  • Northern Blotting methods are generally known in the art.
  • RNA is extracted from a biological sample and separated by size using gel electrophoresis.
  • the RNA is then transferred onto a membrane, such as nitrocellulose or nylon, where it is hybridized with a labeled probe that is complementary to the target biomarker’s mRNA.
  • Detection of the labeled probe typically via radioactive or chemiluminescent methods, allows for the visualization and quantification of the biomarker’s mRNA.
  • Northern blotting provides both qualitative and semi-quantitative detection of specific mRNA transcripts.
  • ISH In Situ Hybridization
  • the disclosed biomarker may be detected via In Situ Hybridization (ISH).
  • ISH In Situ Hybridization
  • ISH methods are generally known in the art.
  • ISH involves the use of a labeled probe that binds to the target biomarker’s mRNA directly within a fixed tissue or cell sample.
  • the labeled probe which is complementary to the specific mRNA of interest, hybridizes to the target within the tissue, allowing for spatial visualization of biomarker expression.
  • Detection is achieved through either fluorescent (FISH) or colorimetric signals.
  • FISH fluorescent
  • ISH colorimetric signals.
  • ISH provides both qualitative detection and precise localization of mRNA expression within tissue samples.
  • RNA-Seq Next-Generation Sequencing
  • the disclosed biomarker may be detected via RNA-Seq (Next-Generation Sequencing).
  • RNA-Seq methods are generally known in the art. In brief, RNA is isolated from a biological sample and converted into complementary DNA (cDNA) through reverse transcription. The cDNA is then subjected to high-throughput sequencing, where millions of short reads are generated and aligned to a reference genome. This allows for the quantification of the biomarker’s mRNA expression levels across the entire transcriptome.
  • RNA-Seq provides quantitative detection of mRNA with high sensitivity and can also reveal alternative splicing, novel transcripts, and other RNA modifications.
  • Microarray the disclosed biomarker may be detected via Microarray analysis.
  • Microarray methods are generally known in the art.
  • RNA from a biological sample is converted into labeled complementary DNA (cDNA), which is then hybridized to a microarray chip containing thousands of probes corresponding to specific genes, including the target biomarkcr.
  • the level of hybridization at each probe site generates a signal that can be quantified to determine the relative expression levels of the biomarker’s mRNA.
  • microarray technology allows for the simultaneous detection and quantification of the biomarker along with other mRNAs, providing high-throughput analysis of gene expression.
  • NanoString nCounter the disclosed biomarker may be detected via the NanoString nCounter system.
  • NanoString nCounter methods are generally known in the art.
  • RNA from a biological sample is hybridized with specific color-coded probes complementary to the target biomarker’s mRNA. These probes are directly counted using a digital detection system without the need for amplification. The resulting counts reflect the absolute quantity of the biomarker’s mRNA in the sample.
  • the NanoString nCounter system provides highly multiplexed, quantitative detection of the biomarker and other targets in a single assay, offering a balance of sensitivity and simplicity.
  • ddPCR Digital Droplet PCR
  • the disclosed biomarker may be detected via Digital Droplet PCR (ddPCR).
  • ddPCR methods are generally known in the art. In brief, RNA is first reverse-transcribed into complementary DNA (cDNA), which is then partitioned into thousands of droplets. Each droplet undergoes PCR amplification individually, with some droplets containing the target biomarker’s DNA and others not. Fluorescent probes are used to detect amplification within the droplets, and the number of positive droplets is counted to determine the absolute quantity of the biomarker’s mRNA. In aspects, ddPCR provides highly sensitive, precise, and absolute quantification of mRNA, even in low-abundance samples.
  • Branched DNA (bDNA) Assay the disclosed biomarker may be detected via the Branched DNA (bDNA) Assay.
  • bDNA methods are generally known in the art. In brief, RNA from a biological sample is hybridized with specific probes that bind to the target biomarker’s mRNA. Signal amplification is achieved through the addition of secondary "branched" DNA probes that further bind to the primary probes, enhancing the signal without the need for RNA amplification. The signal is typically detected using chemiluminescence, with the intensity correlating to the biomarker’s mRNA concentration.
  • the bDNA assay allows for quantitative detection of mRNA with high sensitivity and minimal background noise. Kits
  • kits for the detection of the disclosed biomarker may comprise any of the aforementioned detection methods, such as ELISA and/or a lateral flow assay.
  • the kit is a home-based assay.
  • the kit is for self-administration.
  • the kit for detection of the biomarker comprises a sample receiving region and a capture surface comprising an immobilized detection agent specific for the biomarker.
  • detection agents include an antibody or binding fragment thereof specific for the biomarker (as in the case of a protein), or a nucleotide sequence specific to the biomarker (as in the case of mRNA).
  • the detection agent can be an antibody, aptamer, or other molecules with high affinity and specificity for the biomarker.
  • the sample receiving region is designed to accommodate and direct a biological sample towards the capture surface, he sample receiving region may also be configured to handle various types of biological samples, such as that of blood, urine, saliva, tissue extracts, and combinations thereof. This region may include features such as wells, channels, or pads to ensure efficient application and distribution of the sample.
  • the sample Upon application of the sample to the sample receiving region, the sample is directed to the capture surface where the biomarker, if present, binds to the immobilized detection agent. This binding event generates a detectable signal, which can be measured using various detection methods. For example, a colorimetric assay may produce a visible color change, while other assays may use fluorescence or electrochemical signals to indicate the presence of the biomarker.
  • the kit provides a rapid result, the detection of the biomarker occurring in less than 1 hour, or less than 30 minutes, or less than 15 minutes, or less than 10 minutes, or less than 5 minutes.
  • the kit further comprises one or more components selected from buffers, reagents, control samples, and combinations thereof.
  • the kit may be used to carry out one or more of the disclosed methods.
  • the method comprises detecting one or both of Elastase Al and Elastase A2, in a biological sample obtained from an individual.
  • the biological sample is a urine sample that is contacted with a receiving region of the kit.
  • the kit may be configured for home use.
  • the kit may be configured for use in a physician’s office or hospital.
  • the kit may contain a solution or buffer for stabilizing and preserving the biological sample upon collection, such as a saline solution or phosphate- buffered solution.
  • Collection tools such as swabs, lancets, or needles, may be included for obtaining different types of samples, including saliva, blood, or tissue.
  • the kit may further comprise one or more collection receptacles, such as sterile tubes, vials, or microtiter plates.
  • the collection receptacle may comprise labels.
  • the kits may further comprises a collection receptacle designed to be securely sealed for transport.
  • absorbent pads or wipes may be provided for cleaning a collection site prior to sample retrieval.
  • the kit may also include detailed written instructions or visual guides to ensure proper sample collection and handling, which can be in the form of printed materials or accessible through a QR code linking to instructional videos.
  • the kit may further include reagents necessary for biomarker detection, such as antibodies, primers, or enzyme-based solutions for quantitative assays.
  • a small portable reader or device may be included for processing or analyzing the sample, providing immediate feedback on biomarker levels or preliminary classifications.
  • the kit may also include pre-labeled shipping materials for sending the collected sample to a laboratory for further analysis, including tamper-proof seals to ensure the integrity of the sample during transport. Additional components may include gloves, alcohol wipes, and biohazard bags for safe handling and disposal of materials.
  • software access or a mobile application may be provided, allowing users or healthcare professionals to input the results and receive an algorithm-generated classification for guiding further treatment decisions.
  • an individual may be diagnosed with acute pancreatitis when a level of Elastase 2A and/or 2B is at least two times that of a normal control (e.g. an individual who does not have acute pancreatitis).
  • an individual is diagnosed with acute pancreatitis when a level of Elastase 2A and/or 2B that is at least 5 times that of a normal control.
  • an individual is diagnosed with acute pancreatitis when a level of Elastase 2A and/or 2B that is at least 10 times that of a normal control.
  • an individual is diagnosed with acute pancreatitis when a level of Elastase 2A and/or 2B that is at least 20 times that of a normal control. In aspects, an individual is diagnosed with acute pancreatitis when a level of Elastase 2A and/or 2B that is at least 30 times that of a normal control. In aspects, an individual is diagnosed with acute pancreatitis when a level of Elastase 2A and/or 2B that is at least 40 times that of a normal control. In aspects, an individual is diagnosed with acute pancreatitis when a level of Elastase 2A and/or 2B that is at least 50 times that of a normal control. In aspects, an individual is diagnosed with acute pancreatitis when a level of Elastase 2A and/or 2B that is at least 60 times that of a normal control.
  • values can be optionally normalized to a control value (such as a baseline or reference standard).
  • a control value such as a baseline or reference standard.
  • an algorithm is applied to the normalized levels, wherein the algorithm compares the levels to a reference baseline associated with a predefined population or condition.
  • the algorithm may be used to provide a score, such as a a numerical or categorical value, which is indicative of the individual's classification as having AP, likely to have AP, or not having AP, or unlikely to have AP. This classification can be used to determine the subsequent therapeutic regimen for administration to the individual.
  • Additional steps may include preprocessing of the biological sample to remove contaminants or interfering substances, amplification or enrichment of the biomarker signals to enhance detection sensitivity, and validation of the results using a secondary assay or independent biomarkers to confirm the reliability of the classification.
  • the method may involve adjusting the algorithm based on demographic factors, such as age, gender, or disease severity, to improve the accuracy of the classification and the corresponding treatment recommendation.
  • the method may be used to, in whole or in pail, exclude a diagnosis of one or more of chronic pancreatis, appendicitis, gastrointestinal infection, and combinations thereof.
  • Administering a Therapy may be used to, in whole or in pail, exclude a diagnosis of one or more of chronic pancreatis, appendicitis, gastrointestinal infection, and combinations thereof.
  • the individual is diagnosed as having, or likely to have, acute pancreatitis and is treated via one or more of the following: fluid resuscitation, pain control, nutritional support, antibiotic use, insulin/plasmapheresis (for hypertriglyceridemia induced AP), low-molecular weight heparan (LMWH), protease inhibitors, and endoscopic retrograde cholangiopancreatography (ERCP).
  • fluid resuscitation for hypertriglyceridemia induced AP
  • LMWH low-molecular weight heparan
  • protease inhibitors for endoscopic retrograde cholangiopancreatography (ERCP).
  • the individual is diagnosed as having, or likely to have, acute pancreatitis and is administered additional monitoring for development of acute pancreatitis.
  • the individual is diagnosed as having, or likely to have, acute pancreatitis and is administered fluids, for example IV fluid and/or lactated Ringer’s is administered until electrolyte and fluid balance is restored.
  • the individual may be administered a moderate fluid resuscitation (10 ml/kg bolus in case of hypovolemia, followed by 1.5 ml/kg/h) using Ringers lactate.
  • the individual is diagnosed as having, or likely to have, acute pancreatitis and is administered electrolytes.
  • the individual is diagnosed as having, or likely to have, acute pancreatitis and is administered a pre-biotic and/or a probiotic.
  • the individual is administered Bifilac and/or Bacillus subtilis and Enterococcus faecium.
  • the individual is diagnosed as having, or likely to have, acute pancreatitis and is administered micro-encapsulated tributyrin (a butyrate prodrug).
  • the individual is diagnosed as having, or likely to have, acute pancreatitis and is administered an analgesic.
  • the analgesic is selected form an opioid, a nonsteroidal anti-inflammatory drug (NSAID), acetaminophen, an epidural, and combinations thereof.
  • NSAID nonsteroidal anti-inflammatory drug
  • acetaminophen an epidural
  • a combination of acetaminophen, metamizole and opiate is administered.
  • the individual is diagnosed as having, or likely to have, acute pancreatitis and is administered a feeding tube.
  • the individual is diagnosed as having, or likely to have, acute pancreatitis and is administered an antibiotic.
  • the antibiotic is carbapcmcns.
  • the individual is diagnosed as having, or likely to have, acute pancreatitis and is administered a surgery.
  • the surgery is a cholecystectomy.
  • the individual is diagnosed as having, or likely to have, acute pancreatitis and is administered insulin/plasmapheresis (for hypertriglyceridemia induced AP).
  • the individual is diagnosed as having, or likely to have, acute pancreatitis and is administered low-molecular weight heparan (LMWH).
  • LMWH low-molecular weight heparan
  • the individual is diagnosed as having, or likely to have, acute pancreatitis and is administered a protease inhibitor.
  • the individual is diagnosed as having, or likely to have, acute pancreatitis and is administered the peripherally acting p-opioid receptor antagonist methylnaltrexone, which counteracts inhibitory effects of opiates on gut function and immune responses, without affecting analgesia.
  • the peripherally acting p-opioid receptor antagonist methylnaltrexone which counteracts inhibitory effects of opiates on gut function and immune responses, without affecting analgesia.
  • the individual is diagnosed as having, or likely to have, acute pancreatitis and is administered an endoscopic cholangiopancreatography (ERCP).
  • ERCP combines upper gastrointestinal endoscopy and x-rays to treat narrowing or blockage of a bile or pancreatic duct.
  • a prospective cohort consisting of children (aged 0-21 years) presenting with Acute Pancreatitis (AP) compared to control individuals was used.
  • a summary of the overall study design and steps is shown in FIG. 1A.
  • a total of 130 patients were included in the discovery cohort and 29 in the validation cohort.
  • Discovery urine samples were obtained from 28 patients with AP, 50 with chronic pancreatitis (CP), 21 healthy control individuals (HCs), and 31 fracture pain control individuals (FRCs).
  • the validation cohort included 12 patients with AP, 7 patients with CP, and 10 HCs. All AP patients had lipase or amylase elevation of >3 times the upper limit of normal and either presented with abdominal pain or met criteria for imaging findings.
  • the severity of AP was classified using the North American Society for Pediatric Gastroenterology, Hepatology & Nutrition and the International Study Group of Pediatric Pancreatitis: In Search for a Cure criteria. Of the 40 patients with AP, 33 had a mild AP episode, 16 had abdominal imaging performed at the time of presentation, and 17 had imaging later in the course. The most common etiology for AP was idiopathic (36.8%), followed by gallstones (26.3%). CP diagnosis was made using the International Study Group of Pediatric Pancreatitis: In Search for a Cure criteria.
  • Proteomic analysis showed altered levels of urine proteins in AP patients compared to CP patients and control individuals. Briefly, global quantitative proteomics was performed on the discovery cohort urine specimens (FIG. IB). From these, 2137 proteins were identified in a urine chromatogram library and 1694 proteins were quantified across all samples. 105 differential proteins were significantly associated with AP (false discovery rate [FDR], ⁇ 0.05) with both global analysis of variance testing (FIG. 1C) and pairwise AP vs individual control t tests (patients with CP, HCs, FRCs) (FIG. 2A).
  • FDR false discovery rate
  • CELA2A/B Although many consistently significant proteins were downregulated in AP, five proteins showed promise as potential biomarkers: CELA2A/B, AMY2A, REGIA, SBSN, and GDF15 (FIG. ID).
  • AMY2A a specific isoform of amylase (2A)
  • Serum lipase was not observed in any urine sample subgroup.
  • CELA2A elastase 2A
  • Elastase 2A and 2B are highly similar gene paralogs, and the mass spectrometry experiments used here cannot differentiate between these putative proteins.
  • elastase 2A is referred to as the putative biomarker.
  • Using machine learning to combine elastase 2 A with the conventional biomarker amylase 2A dramatically increased the accuracy of AP diagnosis (FIG. IE) with an area under the curve of 0.91 ( ⁇ 0.03) compared to amylase 2A alone (0.82 ⁇ 0.04).
  • Other combinations including CRP, REGIA, SBSN, and GDF15 did not improve diagnostic accuracy.
  • elastase 2A continued to outperform amylase 2A and CRP (62-fold increase relative to control groups) (Supplementary FIG. IB). In this analysis, signals from the amylase 2A paralog from amylase IB and 2B could be separated; it was observed that only elevated amylase 2A and 2B indicate AP.
  • the AP biomarkers serum amylase and lipase without imaging have poor specificity, with limited application in children. Obtaining imaging as pail of the diagnosis for every AP episode is not always feasible or timely and contributes to high health care costs.
  • this study is the first to investigate urine proteomics in children with AP to identify novel biomarkers that improve diagnosis accuracy.
  • urine elastase 2A was identified as a promising diagnostic marker that can potentially be used as an accurate AP point-of-care test. The study shows a promising role for urinary markers in the diagnosis of AP in children through urinary noninvasive testing for timely and accurate diagnosis.
  • Voided urine samples were collected in a sterile urine collection cup (Coviden Precision), aliquoted in 1-1.5-mL amounts and saved in a -80°C freezer until ready for analysis. Urine samples were thawed, and a 400-mL aliquot was used for acetone protein precipitation. For this, 1600 mF of -20°C acetone was added, following by vortexing and an overnight incubation at -20°C. Samples were then centrifuged at 20,000g for 20 minutes to pellet proteins, after which the acetone was carefully removed. The pellet was washed twice with 500 mF of -20°C acetone following a second centrifugation at 20,000g for 20 minutes for each wash.
  • Pellets were air dried for at least 15 minutes and then resuspended in 50-200 mF of 5% sodium dodecyl sulfate and 50 mmol/E triethylammonium bicarbonate buffer.
  • Proteins were then reduced with 20 mmol/L dithiothreitol for 15 minutes at 65°C and alkylated with 40 mmol/L iodoacctamidc in the dark for 30 minutes at room temperature.
  • Reduced and alkylated proteins were acidified with 1.2% phosphoric acid and mixed with 7x S-trap binding buffer made of 90% methanol and 100 mmol/L triethylammonium bicarbonate.
  • Proteins were loaded onto a ProtiFi Mini S-Trap, washed with 3x 200-mL binding buffer, and then digested overnight at 37°C with a 1:50 ratio of trypsin to substrate. Peptides were eluted according to the manufacturer’s instructions using a mixture of acetonitrile, water, and formic acid. Finally, peptides were dried down using a speed vacuum concentrator.
  • Peptides were separated with a Thermo Scientific Ulti-Mate 3000 UHPLC using a 2- cm PepMap C18 trap column followed by a 25-cm PepMap EASY-Spray C18 analytical column and emitted into a Thermo Scientific Fusion tandem mass spectrometer.
  • Solvent A was 0.1% formic acid in water
  • solvent B was 0.1% formic acid in 80% acetonitrile.
  • approximately 1 mg was loaded and eluted using a 90-minute gradient from 2% to 35% solvent B, followed by a 30-minute washing gradient.
  • Thermo Scientific Fusion was configured to acquire mass spectrometry data using data-independent acquisition (DIA) using 38 xl6-m/z-wide windows in a staggered window pattern.
  • DIA windows were placed from 392.43 to 1008.70 m/z using EncyclopeDIA (version 1.12.31 ) with forbidden-zone-optimized window placements and acquired as targeted fragment mass spectra (MS2) using 17,500 resolution and an automatic gain control (AGC) target of 4e5.
  • Selected ion monitoring precursor spectra were placed every 38 scans (1 per cycle) from 390 to 1010 m/z using 60,000 resolution and an AGC target of 4e5.
  • a pool of representative peptides from each sample group was made from sample subaliquots and used for library generation with gas-phase fractionated (GPF) DIA.
  • GPF gas-phase fractionated
  • 6 injections of the pooled peptide sample were performed focused on different m/z regions (ie, 396.43-502.48, 496.48-602.52, 596.52-702.57, 696.57-802.61, 796.61-902.66, and 896.6-1002.70 m/z). Measurements were performed as for normal DIA as described, except MS2 spectra were configured using only 4-m/z-wide staggered windows.
  • peptides in a downloaded Homo sapiens Uniprot FASTA database were required to have either +2H or +3H charge states and fall within 396.43 and 1002.70 precursor m/z, considering up to 1 missed tryptic cleavage.
  • Peptides that were detected in the 6 GPF-DIA injections at a 1% peptide level FDR as judged by Percolator (version 3.01) were compiled into the chromatogram library using instrument-specific fragmentation and retention time values. Searches of the quantitative DIA injections were performed using this chromatogram library, again filtered to a 1% peptide-level FDR by Percolator.
  • Quantitative data were normalized by the precursor total ion current in each injection. Peak areas for each peptide were generated by summing peak areas from up to 6 total MS2 transitions. Protein peak areas were similarly generated by summing peptides uniquely assigned to that protein (e.g., not matching anywhere else in the canonical human genome). For each protein, 1-way analysis of variance and 2-sample, 2-tailed t tests were performed to assess the significance of quantitative changes. P values from these tests were FDR-corrected using the Benjamini- Hochberg approach. Proteins were marked as changing quantitatively if they passed a ⁇ 0.05 FDR threshold. Protein quantitative changes were additionally filtered using t tests ( ⁇ 0.05 FDR) to determine the direction and consistency of the change.
  • LDA linear discriminant analysis
  • the validation cohort protein samples were measured using the same DIA proteomics approach as the global cohort. However, rather than analyze the entire global proteome, specific peptides for targeted quantitative interpretation were selected using Skyline (version 22.2.0.527). Again, a predicted spectrum library was generated by Prosit to aid in the analysis of all potential +2H and +3H peptides from AMY1A, AMY1B, AMY2A, AMY2B, CRP, and CELA2A. Peak areas were calculated and compared using analysis of variance.
  • Example 1 The following examples relate to various non-exhaustive ways in which the teachings herein may be combined or applied. The following examples are not intended to restrict the coverage of any claims that may be presented at any time in this application or in subsequent filings of this application. No disclaimer is intended. The following examples are being provided for nothing more than merely illustrative purposes. It is contemplated that the various teachings herein may be arranged and applied in numerous other ways. It is also contemplated that some variations may omit certain features referred to in the below examples. Therefore, none of the aspects or features referred to below should be deemed critical unless otherwise explicitly indicated as such at a later date by the inventors or by a successor in interest to the inventors. If any claims are presented in this application or in subsequent filings related to this application that include additional features beyond those referred to below, those additional features shall not be presumed to have been added for any reason relating to patentability. [0098] Example 1
  • a method for identifying and/or treating an individual having, or likely to have, acute pancreatitis comprising detecting Elastase 2A and/or 2B in a sample obtained from the individual.
  • the detecting comprising detecting an Elastase 2A and/or 2B protein.
  • the detecting comprising detecting an mRNA encoding Elastase 2 A and/or 2B.
  • the detection is carried out via a test comprising a region for receiving sample, the sample being a urine sample, an antibody that binds Elastase 2A and/or 2B, a surface that allows the sample to come into contact with the antibody; and a signal that is visible when Elastase 2A and/or 2B is present in the sample and binds to the antibody.
  • Example 20 [0137] The method of any preceding example, further comprising detection of AMY2A.
  • a method of detecting and/or treating a pancreatitis flare in an individual comprising detecting a level of Elastase 2A and/or 2B in a sample obtained from the individual.
  • any of examples 27-31 further comprising diagnosing an individual with acute pancreatitis when a level of Elastase 2A and/or 2B that is at least 5 times that of a Elastase 2A and/or 2B level of a normal control, or at level of Elastase 2A and/or 2B that is at least 10 times that of a Elastase 2 A and/or 2B level of a normal control, or a level of Elastase 2A and/or 2B that is at least 20 times that of a Elastase 2A and/or 2B level of a normal control, or a level of Elastase 2A and/or 2B that is at least 30 times that of a Elastase 2A and/or 2B level of a normal control, or a level of Elastase 2A and/or 2B that is at least 40 times that of a Elastase 2A and/or 2B level of a normal control, or a level of Elastase 2A and/or 2
  • any of examples 27-40 wherein the detection is carried out via a test comprising a region for receiving sample, the sample being a urine sample, an antibody that binds Elastase 2A and/or 2B, a surface that allows the sample to come into contact with the antibody; and a signal that is visible when Elastase 2A and/or 2B is present in the sample and binds to the antibody.
  • Example 47 A kit for detecting Elastase 2A and/or 2B, comprising a sample receiving region and a surface comprising a test region comprising an immobilized detection agent specific for Elastase 2A and/or 2B.
  • a method comprising contacting the sample receiving region of the kit of example 47 with a sample from an individual.

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Abstract

L'invention concerne des procédés d'identification et/ou de traitement d'un individu ayant, ou susceptible de présenter, une pancréatite aiguë. Selon certains aspects, les procédés comprennent la détection de l'élastase 2A et/ou 2B dans un échantillon obtenu auprès de l'individu, par exemple par détection de la protéine élastase 2A et/ou 2B et/ou d'un ARNm codant pour l'élastase 2A et/ou 2B. L'invention concerne en outre des kits de détection d'élastase 2A et/ou 2B.
PCT/US2024/053805 2023-11-02 2024-10-31 Compositions et procédés pour la détection et le traitement de la pancréatite aiguë Pending WO2025096715A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013134860A1 (fr) * 2012-03-16 2013-09-19 University Health Network Biomarqueurs du cancer et leurs procédés d'utilisation

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013134860A1 (fr) * 2012-03-16 2013-09-19 University Health Network Biomarqueurs du cancer et leurs procédés d'utilisation

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
AKSHINTALA VENKATA S ET AL: "959 URINE PROTEOMICS PROFILING IDENTIFY NOVEL ACUTE AND CHRONIC PANCREATITIS DIAGNOSTIC BIOMARKERS IN CHILDREN", GASTROENTEROLOGY, W.B. SAUNDERS, vol. 164, no. 6, 1 May 2023 (2023-05-01), XP087352727, ISSN: 0016-5085, [retrieved on 20230706], DOI: 10.1016/S0016-5085(23)01467-1 *
SATHIYASEKARAN MALATHI ET AL: "Pancreatitis in Children", INDIAN JOURNAL OF PEDIATRICS, SPRINGER INDIA, NEW DELHI, vol. 83, no. 12, 6 June 2016 (2016-06-06), pages 1459 - 1472, XP036108868, ISSN: 0019-5456, [retrieved on 20160606], DOI: 10.1007/S12098-016-2115-1 *
SZATMARY, PETER ET AL.: "Acute Pancreatitis: Diagnosis and Treatment", DRUGS, vol. 82, no. 12, 2022, pages 1251 - 1276

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