WO2022221264A1 - Procédés et compositions pour l'analyse d'une lésion rénale aiguë - Google Patents

Procédés et compositions pour l'analyse d'une lésion rénale aiguë Download PDF

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WO2022221264A1
WO2022221264A1 PCT/US2022/024395 US2022024395W WO2022221264A1 WO 2022221264 A1 WO2022221264 A1 WO 2022221264A1 US 2022024395 W US2022024395 W US 2022024395W WO 2022221264 A1 WO2022221264 A1 WO 2022221264A1
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aki
levels
patient
suspected
renal
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PCT/US2022/024395
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English (en)
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Michael Ryan OTT
Clyde W. Shearman
Stephen L. Carrithers
S. Aaron L. CARRITHERS
Brennan M. CARRITHERS
Shawn Mark O'hara
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Ethos Biosciences, Inc.
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Publication of WO2022221264A1 publication Critical patent/WO2022221264A1/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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • 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
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/34—Genitourinary disorders
    • G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy

Definitions

  • the present disclosure relates to the field of medical diagnostic assays and subsequent treatment and, more specifically, relates to immunoassay detection of the novel biomarker, interleukin 18 (IL-18) alone, or in combination with other biomarkers, in biological fluid samples and methods and kits thereof for early identification or stratification or management of acute kidney injury in a patient.
  • IL-18 interleukin 18
  • the kidney is responsible for water and solute excretion from the body. Its functions include maintenance of acid-base balance, regulation of electrolyte concentrations, control of blood volume, and regulation of blood pressure. As such, loss of kidney function through injury and/or disease results in substantial morbidity and mortality. A detailed discussion of renal injuries is provided in Harrison's Principles of Internal Medicine, 17th Ed., McGraw Hill, New York, pages 1741-1830, which is hereby incorporated by reference in its entirety. Renal disease and/or injury may be acute or chronic.
  • Acute disease is described as follows, from Current Medical Diagnosis & Treatment 2008, 47th Ed, McGraw Hill, New York, pages 785-815, which is hereby incorporated by reference in its entirety: "Acute renal failure is worsening of renal function over hours to days, resulting in the retention of nitrogenous wastes (such as urea nitrogen) and creatinine in the blood. Retention of these substances is called azotemia.”
  • Acute kidney injury (AKI) (also known as acute renal failure) is an abrupt (typically detected within about 48 hours to 1 week) reduction in glomerular filtration. This loss of filtration capacity results in retention of nitrogenous (urea and creatinine) and non-nitrogenous waste products that are normally excreted by the kidney, a reduction in urine output, or both. It is reported that AKI complicates about 5% of hospital admissions, 4-15% of cardiopulmonary bypass surgeries, and up to 30% of intensive care admissions. AKI may be categorized as prerenal, intrinsic renal, or postrenal in causation. Intrinsic renal disease can be further divided into glomerular, tubular, interstitial, and vascular abnormalities.
  • a commonly reported criteria for defining and detecting AKI is an abrupt (typically within about 2-7 days, or within a period of hospitalization) elevation of serum creatinine.
  • serum creatinine elevation to define and detect AKI is well established, the magnitude of the serum creatinine elevation and the time over which it is measured to define AKI varies considerably among publications.
  • relatively large increases in serum creatinine such as 100%, 200%, an increase of at least 100% to a value over 2 mg/dL and other definitions were used to define AKI.
  • the recent trend has been towards using smaller serum creatinine rises to define AKI.
  • “Failure” serum creatinine increased 3.0 fold from baseline OR creatinine >355 umol/L (with a rise of >44) or urine output below 0.3 ml/kg/hr for 24 h or anuria for at least 12 hours;
  • “Loss” persistent need for renal replacement therapy for more than four weeks.
  • “ESRD” End stage renal disease - the need for dialysis for more than 3 months.
  • RIFLE criteria which provide a useful clinical tool to classify renal status.
  • the RIFLE criteria provide a uniform definition of AKI which has been validated in numerous studies.
  • Standardization I increase in serum creatinine of more than or equal to 0.3 mg/dL (> 26.4 mihoI/L) or increase to more than or equal to 150% (1.5-fold) from baseline OR urine output less than 0.5 mL/kg per hour for more than 6 hours;
  • Stage II increase in serum creatinine to more than 200% (> 2-fold) from baseline OR urine output less than 0.5 mL/kg per hour for more than 12 hours;
  • Standardization III increase in serum creatinine to more than 300% (> 3-fold) from baseline OR serum creatinine > 354 pmol/L accompanied by an acute increase of at least 44 pmol/L OR urine output less than 0.3 mL/kg per hour for 24 hours or anuria for 12 hours.
  • small changes in serum creatinine such as 0.3 mg/dL or 25%, are sufficient to detect AKI (worsening renal function) and that the magnitude of the serum creatinine change is an indicator of the severity of the AKI and mortality risk. It is important to note the overall clinical utility of these definitions and staging systems thereof has not been validated in clinical assessment and management of patients with AKI.
  • IL-18 is a proinflammatory cytokine that is produced by epithelial cells in the proximal convoluted tubule (PCT) of the nephron (first portion of kidney cells), activated by caspase-1 in response to ischemic injury and directly excreted into tubular fluid, thus readily presenting in the urine immediately following hypoxia-mediated AKI. Elevated urinary IL-18 levels have been found to be an early marker for AKI, preceding even small changes in serum creatinine by up to 48 hours, which correlate with upregulated IL-18 messenger ribonucleic acid (mRNA) levels in the PCT, particularly following hypoxic-ischemia-mediated, as well as proinflammatory insults.
  • mRNA messenger ribonucleic acid
  • This proinflammatory cytosolic cascade has not only shown to be initiated by hypoxic-ischemic renal injury, but also through antigen binding and activation of molecular specific membrane-bound pattern recognition receptors (PRRs), specifically toll-like receptors (TLRs), which recognize endogenous molecules released during significant host tissue injury and/or death referred to as damage associated molecular patterns (DAMPs).
  • PRRs molecular specific membrane-bound pattern recognition receptors
  • TLRs toll-like receptors
  • Renal-associated TLR-2 and TLR-4 are constitutively expressed on tubular epithelial ceils (TECs) and play a vital, yet detrimental role in the induction of inflammation and injury following ischemic renal injury.
  • Mice studies have demonstrated that RNA expression of TLRs-2/-4 were notably enhanced in proximal TECs following ischemic-hypoxic kidney injury, while genetic knockout mice lacking TLR-2 and/or TLR-4 had significant reductions in local cytokine and chemokine levels, renal leukocyte infiltration, neutrophil influx, and tubular apoptosis compared to controls.
  • TLRs-2/-4 antisense treatment prior to ischemia demonstrated protection from renal injury and dysfunction demonstrated by histological evaluation and renal functional parameters compared with nonsense treatment.
  • DAMPs and TLRs are among the most important pathophysiological systemic components in cardiorenal syndromes, specifically type-1 (CRS-1), characterized by an acute cardiac disorder leading to AKI.
  • CRS-1 type-1
  • AKI is comprised of several subtypes and multiple disease states, highlighting the need to improve phenotyping.
  • the proinflammatory response facilitated by IL-18 can be detrimental to surrounding tissue and further perpetuate severity of the insult.
  • Neutralization of IL-18 prior to ischemic renal injury were functionally and histologically protected against kidney injury as determined by blood urea nitrogen (BUN), serum creatinine, and acute tubular necrosis (ATN) score.
  • BUN blood urea nitrogen
  • ATN acute tubular necrosis
  • the present disclosure is directed to methods for the diagnosis and treatment of renal dysfunction. More specifically, the disclosure provides a urine or other bodily fluid test with high sensitivity and specificity for IL-18 or fragments thereof alone or in combination with other biomarkers which provides the means for an early diagnosis and staging of acute kidney injury (AKI), and treatment or management thereof.
  • AKI acute kidney injury
  • the present disclosure is directed to a method of diagnosing and treating or managing AKI, comprising: (a) the measurement of IL-18 alone or in combination with other biomarkers in urine, blood, saliva or other similar matrix, (b) the comparison of that measurement to a calibration standards or a reference controls, (c) the determination that the measurement of IL-18 alone or in combination with other biomarkers in that matrix is above a normal range of values for people without kidney injury, and if so, (d) treatment or management thereof. Methods are also provided for evaluating the efficacy of treatments using the described reagents, as well.
  • the present disclosure is also directed as an immunoassay system/kit for diagnosing an early stage of acute kidney injury, one that may not yet be detectable by conventional testing methods (i.e. , serum creatinine), encompassing one or more containers comprising: (a) a labeled antibody specific for a modified or native form or region of the protein(s), or using human expressed IL-18 binding protein (IL-18BP) as a capture mechanism in place of an IL-18 specific labeled-antibody; (b) reagents for developing labeled-antibody reaction; and (c) instructions on how to use components (a) and (b) to carry out the diagnosis. Also provided are compositions comprising the described labeled antibody.
  • epithelial cells of the proximal convoluted tubule (located in the first portion of each nephron) are particularly prone to renal insult due to high oxygen demands, and in turn readily activate inflammatory mediators such as caspase-1 before other nephron segments are affected.
  • Caspase-1 activates interleukin-18 (IL-18) in response to early hypoxic injury, which is then directly secreted into the tubular fluid and excreted in the urine.
  • IL-18 interleukin-18
  • the present disclosure relates to methods for identifying or staging AKI in a patient deficient in systemic blood flow due to cardiac dysfunction or failure which compromises systemic organ function due to hypoxia or ischemia.
  • These analytical methods comprise: Calculating a staging and or a risk score derived from the IL-18 levels alone or in combination with other biomarkers measured from a biological fluid sample (urine, blood, saliva, etc.) and comparing this value to predetermined threshold values or a well-characterized calibration standard.
  • the urinary concentration of IL-18 alone or in combination with other biomarkers which are measured using analytical instruments (e.g., any spectrometry, including but not limited to mass spectrometry, electrophoresis, chromatography, FTIR, NMR, SPR, and variations thereof) that receives the urine sample, performs a specific analytical detection or binding assays, and reports the assay result(s) in a form readable by the operator.
  • analytical instruments e.g., any spectrometry, including but not limited to mass spectrometry, electrophoresis, chromatography, FTIR, NMR, SPR, and variations thereof
  • the concentrations may be obtained by introducing the urine sample obtained from the patient into an analytical or immunoassay instrument; wherein the analytical instrument measures a characteristic identifying physical or chemical property, or an immunoassay instrument comprises a solid phase, an IL-18 antibody alone or in combination with other biomarkers or binding protein immobilized at a location on the solid phase; wherein the instrument causes the urine sample to contact that location; wherein the instrument measures the amount of IL-18 which binds to the IL-18 antibody or IL-18BP immobilized at the designated location and determines therefrom the concentration of IL-18 in the urine sample; wherein the instrument/software mathematically translates the concentration of IL-18 alone or in combination with other biomarkers into a given value that is used to derive an AKI risk score; and wherein the instrument reports the stage and or risk score in a human readable form.
  • the analytical instrument measures a characteristic identifying physical or chemical property
  • an immunoassay instrument comprises a solid phase, an IL-18 antibody alone or in combination with other biomarkers or binding protein
  • the urine sample obtained from the patient may be further contacted first by a capture antibody then with a second IL-18 alone or in combination with other biomarkers antibody(s) conjugated to detectable label; wherein sandwich complexes are formed between the IL-18 antibody or IL-18BP, IL-18 present in the urine sample, and the second IL-18 antibody; and wherein the amount of IL-18 which binds to the IL-18 antibody or IL-18- BP is determined by the instrument detecting the detectable label bound at the designated location.
  • the methods of the present disclosure may also use methods for determining the level of IL-18 known in the art, such as enzymatic and/or chemical protein determination or immunological assay-based methods.
  • Immunological assays for determining the level of IL-18 or creatinine can be performed in a variety of formats including sandwich assays (i.e. , enzyme linked immunosorbent assay; ELISA), competition assays (i.e., competitive radioimmunoassay; RIA), lateral flow assays, and non-competitive assay formats, to generate a signal that is related to the presence or amount of the biomarker of interest. Suitable assay formats also include chromatographic, mass spectrographic, and protein "blotting" methods. Additionally, certain methods and devices, such as biosensors and optical immunoassays, may be employed to determine the presence or level of analytes without the need for a labeled molecule.
  • CAD coronary artery disease
  • urine or blood samples at different times from a subject with CAD may be obtained and analyzed for IL-18 presence or concentration and this information may be used to assess the condition, or state of renal disease, of the subject.
  • these samples may be samples that do not have an increase of serum creatinine detected in the subject’s blood.
  • AMI acute myocardial infarction
  • a sample from a subject with AMI is obtained and IL-18 alone or in combination with other biomarkers are analyzed.
  • urine or blood samples at different times from a subject with AMI may be obtained and analyzed for IL-18 presence or concentration and this information may be used to assess the condition of the subject.
  • these samples may be samples that do not have an increase of serum creatinine detected in the subject’s blood. Patients of this nature require AKI risk analysis upon initial assessment in the emergency department.
  • the disclosed methods allow the rapid assessment of organ health of a patient such as the renal health of a patient predisposed to deficiencies in systemic or renal blood flow.
  • at least one sample from a subject admitted to the emergency department or intensive care unit may be obtained and analyzed for IL-I8 alone or in combination with other biomarkers and the subject evaluated based on the information obtained on IL-I8.
  • urine or blood samples at different times from a subject admitted to the emergency department or intensive care unit may be obtained and analyzed for the presence or absence of IL-18.
  • multiple parameters of an admitted subject such as age and gender, may be examined in combination with the IL-18 factor to assess the survivability of a subject.
  • the criteria may be used to assess the efficacy of treatment of the subject with at least one therapeutic agent.
  • additional clinical indicia may be used in conjunction with IL-18 alone or in combination with other biomarkers assay results wherein a combination of biomarker levels relevant to kidney health or dysfunction are measured which include but are not limited to neutrophil gelatinase (NGAL), kidney injury molecule-1 (KIM-1), or L-fatty acid binding protein (L-FABP).
  • NGAL neutrophil gelatinase
  • KIM-1 kidney injury molecule-1
  • L-FABP L-fatty acid binding protein
  • biomarkers include but are not limited to Cystatin-C, urea, IGFBP-7 (insulin-like growth factor-binding protein-7), TIMP-2 (tissue inhibitor of metalloproteinases-2), Calprotectin, IP-10 (interferon gamma-induced protein-10), MCP-1 (monocyte chemoattractant protein-1), P53 (tumor protein 53), Ins2 (insulin-2), LepR (leptin receptor), Beta trace protein (lipocalin prostaglandin D2 synthase), ADMA (asymmetric dimethylarginine), SDMA, Uromodulin (Tamm-Horsfall protein), NAG (N-acetyl-glucosaminidase), GST (glutathione-S-transferase), GGT (gamma-glutamyl transpeptidase), AAP (alanine aminopeptidase), LDH (Lactate Dehydrogenase), Gd-lgA1 (galactose-deficient lg
  • KDIGO Clinical Practice Guideline for the Evaluation and Management of AKI analyzed management strategies aimed at addressing prevention of AKI progression.
  • an elevated risk of AKI is identified by clinical measures and laboratory parameters, managing the patient as high risk for future AKI may be performed as described in the KDIGO Clinical Practice Guideline for Acute Kidney Injury, Kidney Inti. 2 (Suppl. 1), March 2012, pp 1-138.
  • management can comprise one or more of discontinuing or avoiding one or more nephrotoxic agents, maintaining kidney oxygen perfusion during the surgical procedure, maintaining a protein intake of at least 0.8 g/kg/day, maintaining plasma glucose within the range of 110-149 mg/dL, and performing intravenous administration of isotonic solutions to maintain hemodynamic status.
  • the methods described herein may be used prophylactically in advance of, or as a treatment following, various treatments or conditions that are known to be injurious to the kidney.
  • the present methods are based on the use of a biomarker alone or in combination with other biomarkers, and/or fragments thereof, associated with normal kidney function that advances the diagnostic efficiency of identifying AKI.
  • Urinary IL-18 levels alone or in combination with other biomarkers also appear to be complementary to filtration-dependent markers, yet a distinct mechanism may help define pathophysiologic events in the initiation of renal disease.
  • a diagnostic test that is simple and convenient to administer, stratifies patients by level or risk, and provides treatment based on the determined level or risk of AKI, is provided.
  • FIG. 1 is a schematic diagram of an IL-18 assay sandwich immunoassay detection system in accordance with some embodiments of the present disclosure.
  • FIG. 2 is a flow chart illustrating a non-limiting method in accordance with some embodiments of the present disclosure.
  • the range of “from about 2 to about 10” also discloses the range “from 2 to 10.”
  • the term “about” may refer to plus or minus 10% of the indicated number.
  • “about 10%” may indicate a range of 9% to 11 %, and “about 1” may mean from 0.9-1.1.
  • the present disclosure provides a method for the detection of renal dysfunction by determining the level of lL-18 alone or in combination with other biomarkers in biological fluids (including but not limited to: urine, plasma, serum, saliva, cerebrospinal fluid). Further, based on the detection of renal dysfunction, the subject is treated, as described herein.
  • biological fluids including but not limited to: urine, plasma, serum, saliva, cerebrospinal fluid.
  • IL-18 In healthy individuals, the levels of IL-18 are present at a low maintenance level supporting its role in inflammation. In people with renal injury, IL-18 can rise significantly above normal levels as the kidney damage is occurring.
  • PCT proximal convoluted tubule
  • caspase-1 activates interleukin-18 (IL-18) in response to early hypoxic injury, which is then directly secreted into the tubular fluid and excreted in the urine.
  • IL-18 levels increase significantly, relative to the healthy patient, in parallel with ischemic renal insult thereof.
  • normoxia normoxia
  • Exogenous recombinant IL-18 (1 pg per 6 ml of tubule suspension) exacerbated sublethal (12 minutes) hypoxic proximal tubular injury.
  • LDH release was 10% ⁇ 1% in normoxic tubules, 13% ⁇ 1 % in hypoxic tubules preincubated with vehicle (saline) (NS vs.
  • IL-18 plays a role in renal hypoxic injury and is a marker thereof.
  • IL-18BP human-expressed IL-18 binding protein
  • the antibody can be a monoclonal antibody (mAb), polyclonal antibody (pAb), a single chain antibody, and Fab fragment, or a recombinant antibody.
  • the IL-18BP can be a fragment of the protein thereof that can bind to IL-18.
  • clinical indicia of health status, and particularly of renal sufficiency may be combined with the IL-18 measurement such as the ratios of other biomarkers in the methods described herein.
  • Such clinical indicia may include one or more of: a baseline urine output value for the patient, a baseline change in serum creatinine for the patient, demographic information (e.g., weight, sex, age, race), medical history (e.g., family history, type of surgery, pre-existing disease such as aneurism, congestive heart failure, preeclampsia, eclampsia, diabetes mellitus, hypertension, coronary artery disease, proteinuria, renal insufficiency, or sepsis, type of toxin exposure such as NSAIDs, cyclosporines, tacrolimus, aminoglycosides, foscarnet, ethylene glycol, hemoglobin, myoglobin, ifosfamide, heavy metals, methotrexate, radiopaque contrast
  • a glomerular filtration rate an estimated glomerular filtration rate (eGFR)
  • eGFR estimated glomerular filtration rate
  • a urine production rate a serum or plasma creatinine concentration, a urine creatinine concentration, a fractional excretion of sodium, a urine sodium concentration, a urine creatinine to serum or plasma creatinine ratio, a urine specific gravity, a urine osmolality, a urine urea nitrogen to plasma urea nitrogen ratio (BUN), a plasma BUN to creatinine ratio, a renal failure index calculated as urine sodium / (urine creatinine/plasma creatinine), L-fatty acid binding protein (L-FABP) concentration, kidney injury molecule-1 (KIM-1) concentration, a serum or plasma neutrophil gelatinase (NGAL) concentration, a urine NGAL concentration, a serum or plasma cystatin C concentration, a serum or plasma cardiac troponin concentration, a serum
  • Types of immunoassays may include both competitive and noncompetitive assay systems, including, but not limited to the ELISA, “sandwich” immunoassay such as the sandwich ELISA, counting immunoassay (CIA), chemiluminescence immunoassay (CLIA), radioimmunoassay (RIA), immunoradiometric assay (IRMA) and Western blot.
  • ELISA electrospray immunoassay
  • CIA counting immunoassay
  • CLIA chemiluminescence immunoassay
  • RIA radioimmunoassay
  • IRMA immunoradiometric assay
  • FIG. 1 The following method was used for a paired-antibody (binding protein) sandwich ELISA (FIG. 1).
  • Each well 1 of a microtiter/multi-well plate i.e. , 96-well, 384- well
  • mAb, pAb proprietary biomarker-specific primary “capture” monoclonal or polyclonal antibodies
  • IL-18 specific binding protein or fragments thereof 2
  • Sample Buffer i.e., 1 part clinical specimen with 9 parts Sample Buffer (Sample Buffer: 10 mM MOPS, protein-free, pH 6.3-7.3).
  • Sample Buffer 10 mM MOPS, protein-free, pH 6.3-7.3.
  • the diluted biological sample is then added to one of the wells of the Capture Plate.
  • the sample is incubated in the well for specified amount of time. In one embodiment, the sample is incubated for approximately one hour (55-65 minutes) in the microtiter well at room temperature (20-25°C). The samples are aspirated out of the well, and the well is washed.
  • the well is washed and aspirated three times with 350 uL of Plate-Wash Buffer (10-25 mM Tris, 150 mM NaCI, 0.05-0.5% Tween-20, pH 7.8).
  • the analyte, or IL-18 3 or fragments thereof, within the biological sample is captured by the capture mAb.
  • the proprietary “detection” mAb or pAb 4 is added, which is conjugated to a detectable marker.
  • the detectable or conjugated marker is an enzyme such as horseradish peroxidase (HRP) 5.
  • the detection mAb or pAb 2 may be conjugated but not limited to other enzymes such as alkaline phosphatase or a fluorophore.
  • the detection mAb/enzyme complex is incubated in the microtiter well 1. In one embodiment, the detection complex is incubated in the microtiter well for one hour at room temperature (as described above). Following incubation, the detection mAb solution is aspirated out of the microtiter well, and again, the well is washed with Plate-Wash Buffer. In one embodiment, the well is washed and aspirated three times with 350 uL of Plate-Wash Buffer. In an embodiment using an enzyme as the detectable marker, a solution of enzyme substrate, i.e.
  • TMB tetramethylbenzidine
  • the incubation is for twenty minutes at room temperature (20-25°C).
  • the TMB reaction is quenched with the addition of Acid/(0.12N HCL, 0.16M h SC ySTOP-Solution (in the case where TMB is used, the color of the wells will turn from blue to yellow), and the wells / plate is read at an O.D. 450 nm and 650 nm (absorbance readings at A630-A650 act as a reference absorbance only).
  • Proprietary capture 2 and detection 4 monoclonal or polyclonal antibodies are developed by Ethos Biosciences, using an immunization technique for antibodies against IL-18.
  • the calibrator for the biomarker human IL-18 was also developed by Ethos Biosciences.
  • the calibrator protein was independently quantitated by tryptic digestion followed by LC/MS methods and Western blot.
  • FIG. 2 illustrates a non-limiting example of a method 100 in accordance with some embodiments of the present disclosure.
  • the method 100 includes identifying a patient with a suspected kidney injury 110, detecting and/or measuring IL-18 levels in the patient 120, analyzing the IL-18 levels to determine if the patient has a kidney injury 130, and treating the patient for the kidney injury (if applicable) 140.
  • the analysis may include comparing one or more measured/detected IL-18 levels to a predetermined threshold value.
  • the one or more levels may refer to bound IL-18, free IL-18, or a ratio of free IL-18 to bound IL-18.
  • IL-18 binding protein is naturally secreted in humans and has an exceptionally high affinity for IL-18.
  • Aptamers are oligonucleic acid or peptide molecules that bind to a specific target molecule. Aptamers are created by selection from large random sequence pools, but natural aptamers also exist. High-affinity aptamers containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions and may include amino acid side chain functionalities.
  • An IL-18 curve may be generated whereby several parameters of the sample are obtained, relative to that of a simultaneously run standard curve using recombinant human IL-18. Specific measurements may include the IL-I8 level itself or the IL-I8 level corrected for the amount of serum creatinine.
  • the IL-I8 assay is reproducible and analytically sensitive to the IL-I8 component in the renal system, as well as to physiologic alterations in progression to renal failure.
  • the measurement of these parameters may be applied to assess subjects with known and as yet undefined organ conditions.
  • IL-I8 assay results may be analyzed in an individual suffering from renal conditions.
  • renal conditions include but are not limited to AKI, prerenal azotemia, intrinsic kidney injury, postrenal azotemia, glomerular diseases, and interstitial nephritis.
  • IL-I8 assay results may be analyzed in an individual suffering from coronary artery disease including stable angina, unstable angina, non- ST-elevated myocardial infarction, and ST-elevated myocardial infarction.
  • a non limiting example includes acute myocardial infarction (AMI).
  • the IL-18 assay results may be analyzed to aid in selection or exclusion of a patient as a candidate for a specific therapeutic drug, as such, serving as a companion diagnostic device.
  • the IL-18 biomarker assay results would indicate immediate treat with an I L-18-specific neutralizing agent to mitigate the further renal dysfunction initiated by the presence of the proinflammatory cytokine.
  • the IL-18 assay would be used to assess the renal dysfunction status and clinical response to the aforementioned therapeutic agent, which would not be limited to IL-18 neutralizing therapies.
  • AKI acute kidney injury
  • identifying a patient with suspected acute myocardial infarction (AMI) event AMI
  • detecting and or measuring Interleukin 18 (IL-18) levels in the patient within about 24 hours of suspected AMI event AMI event
  • IL-18 Interleukin 18
  • a compatible treatment regimen comprising one or more of initiating renal replacement therapy, administering a compound that decreases inflammatory response, administering a compound that lowers renal inflammation, administering a compound that decreases IL-18 levels, withdrawing delivery of compounds that are known to be damaging to the kidney, delaying procedures that are known to be damaging to the kidney, modifying diuretic administration, and consulting a nephrologist up to
  • treating the patient for AKI can involve various treatment regimens.
  • renal replacement therapy includes hemodialysis, peritoneal dialysis, hemofiltration and/or renal transplantation.
  • steps can be taken such as pharmacological inhibition (such as eritoran, TAK242, pirfenidone, etc.) or neutralization of any step within the pathway that leads to substantially lower levels of renal inflammation or of free IL-18.
  • the detecting or measuring the IL- 18 levels consists of detecting or measuring free IL-18 levels (i.e. IL-18 not bound to IL- 18BP).
  • a binding molecule that specifically binds Interleukin 18 (IL-18), wherein the binding molecule does not bind the IL-18 binding protein (IL-18BP) complex and wherein the binding molecule is not IL- 18BP.
  • the binding molecule can be an isolated fully human, humanized or chimeric antibody or a fragment thereof.
  • the binding molecule or isolated antibody or fragment thereof as described herein are capable of only detecting free IL-18, not bound to IL-18BP.
  • free IL-18 is the biologically active molecule, it becomes immediately apparent that a binding molecule capable of recognizing only such entity may allow differentiation of free IL-18 from IL-18 bound to IL-18BP, which is inactive.
  • the binding molecules of this aspect of the disclosure do not encompass the IL-18BP, either isolated or naturally occurring, they lend themselves not only as therapeutic agents but also as tools in many other applications such as in diagnosis or in a diagnostic kit.
  • the disclosure includes a diagnostic kit comprising the binding molecule or the isolated antibody or fragment thereof which binds free IL-18 and does not bind IL-18/IL-18 binding protein (IL-18BP) complex and wherein the binding molecule is not IL-18BP and/or the complex comprising IL-18.
  • the kit optionally contains a first control compound.
  • the first control compound is IL-18.
  • the kit can also optionally include a second control compound.
  • a medical or diagnostic device which includes the binding molecule or isolated antibody or a fragment thereof which binds IL-18 and does not bind IL-18/IL-18 binding protein (IL- 18BP) complex and wherein the binding molecule is not IL-18BP and/or the complex comprising IL-18.
  • IL- 18BP IL-18/IL-18 binding protein

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Abstract

Procédé de traitement d'une lésion rénale aiguë (AKI) comprenant les étapes suivantes : identification d'un patient présentant une suspicion d'AKI ; détection et ou mesure des niveaux d'interleukine 18 (IL-18) chez le patient présentant une suspicion d'AKI ; analyse des niveaux d'IL-18 pour déterminer la présence d'une AKI chez le patient ; et le traitement du patient pour une AKI avec un régime thérapeutique compatible comprenant un ou plusieurs des éléments suivants : initiation d'une thérapie de remplacement rénal, administration d'un composé réduisant la réponse inflammatoire, administration d'un composé réduisant l'inflammation rénale, administration d'un composé réduisant les niveaux d'IL-18, retrait de l'administration de composés connus pour être délétères pour les reins, report des procédures identifiées comme étant délétères pour les reins, modification de l'administration de diurétiques et consultation d'un néphrologue.
PCT/US2022/024395 2021-04-12 2022-04-12 Procédés et compositions pour l'analyse d'une lésion rénale aiguë WO2022221264A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7141382B1 (en) * 2004-10-12 2006-11-28 Parikh Chirag R Methods for detection of IL-18 as an early marker for diagnosis of acute renal failure and predictor of mortality
US20190085068A1 (en) * 2012-09-07 2019-03-21 Novartis Ag Il-18 binding molecules

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7141382B1 (en) * 2004-10-12 2006-11-28 Parikh Chirag R Methods for detection of IL-18 as an early marker for diagnosis of acute renal failure and predictor of mortality
US20190085068A1 (en) * 2012-09-07 2019-03-21 Novartis Ag Il-18 binding molecules

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHEN TIEN-HSING, CHANG CHIH-HSIANG, LIN CHAN-YU, JENQ CHANG-CHYI, CHANG MING-YANG, TIAN YA-CHUNG, HUNG CHENG-CHIEH, FANG JI-TSENG,: "Acute Kidney Injury Biomarkers for Patients in a Coronary Care Unit: A Prospective Cohort Study", PLOS ONE, PUBLIC LIBRARY OF SCIENCE (PLOS), vol. 7, no. 2, 1 February 2012 (2012-02-01), pages e32328, XP055983281, DOI: 10.1371/journal.pone.0032328 *
MOHAMMAD ET AL.: "Reno-protective effects of TAK-242 on acute Kidney Injury in a Rat Model", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 503, no. 1, 3 September 2018 (2018-09-03), pages 304 - 308, XP085965395, DOI: 10.1016/j.bbrc.2018.06.020 *

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