WO2023101447A1 - Biomarqueur pour le diagnostic de la pancréatite chez un chien de compagnie - Google Patents

Biomarqueur pour le diagnostic de la pancréatite chez un chien de compagnie Download PDF

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WO2023101447A1
WO2023101447A1 PCT/KR2022/019278 KR2022019278W WO2023101447A1 WO 2023101447 A1 WO2023101447 A1 WO 2023101447A1 KR 2022019278 W KR2022019278 W KR 2022019278W WO 2023101447 A1 WO2023101447 A1 WO 2023101447A1
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pancreatitis
protein
level
mrna
group
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PCT/KR2022/019278
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Korean (ko)
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김경곤
김민정
염정훈
안희성
유지영
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재단법인 아산사회복지재단
울산대학교 산학협력단
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Priority claimed from KR1020220164050A external-priority patent/KR20230082583A/ko
Publication of WO2023101447A1 publication Critical patent/WO2023101447A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • 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

Definitions

  • the present invention relates to a biomarker for diagnosing canine pancreatitis and a method for diagnosing canine pancreatitis using the same.
  • Pancreatitis is a disease in which pancreatic secretory glands are destroyed by pancreatic enzymes or the like, or inflammation occurs locally or entirely in the pancreas.
  • Pancreatitis is a disease commonly observed in humans as well as other animals including dogs.
  • Pancreatitis is divided into acute and chronic pancreatitis.
  • Acute pancreatitis occurs when the exocrine function of the pancreas is impaired, digestive enzymes are activated, and this attacks the pancreas and surrounding tissues, causing severe inflammation.
  • Dogs suffering from acute pancreatitis cause vomiting, diarrhea, abdominal pain, etc., and severe symptoms such as bloody stool can lead to a poor prognosis, but if detected early and actively treated, the survival rate can be increased.
  • Chronic pancreatitis is characterized by impairment of both exocrine and endocrine functions due to chronic inflammation. It is accompanied by mild vomiting and diarrhea, and the condition may improve or worsen repeatedly. Canine pancreatitis has a mortality rate of about 40%, which is very high, and can cause complications such as diabetes, so timely treatment is very important.
  • the present invention was made to solve the above problems, and was completed by discovering a biomarker capable of diagnosing pancreatitis in dogs with high accuracy and sensitivity through qualitative and quantitative analysis of canine plasma proteins.
  • an object of the present invention is to provide a composition for diagnosing canine pancreatitis, which contains, as an active ingredient, an agent for measuring protein or mRNA levels of the biomarkers.
  • Another object of the present invention is to provide a kit for diagnosing canine pancreatitis, including an agent for measuring protein or mRNA levels of the biomarkers.
  • Another object of the present invention relates to a method for diagnosing canine pancreatitis by measuring protein or mRNA levels of the biomarkers.
  • the present invention relates to a dog product comprising, as an active ingredient, an agent for measuring the level of at least one protein or mRNA selected from the group consisting of PDE4DIP (phosphodiesterase 4D interacting protein), SERPINA3 (Serpin family A member 3), and FGG (Fibrinogen gamma chain).
  • PDE4DIP phosphodiesterase 4D interacting protein
  • SERPINA3 Serotonin-1
  • FGG Fibrinogen gamma chain
  • the protein or mRNA level of SERPINA3 may be increased in pancreatitis subjects compared to normal subjects, but is not limited thereto.
  • the protein or mRNA level of the PDE4DIP or FGG may be reduced in pancreatitis subjects compared to normal subjects, but is not limited thereto.
  • the composition comprises Pentaxin, Angiotensinogen, Peptidase D, Transferrin (TF), Complement C7, Coagulation factor X, Kallikrein B1, Factor XI, Complement C8 alpha chain, Coagulation factor XIII B chain (F13B) , Complement C3 (LOC484960), and may further include an agent for measuring the level of at least one protein or mRNA selected from the group consisting of Complement factor I, but is not limited thereto.
  • the agent for measuring the protein level may be an antibody or an aptamer specific to the protein, but is not limited thereto.
  • the agent for measuring the mRNA expression level may be a primer set or a probe that specifically binds to the mRNA, but is not limited thereto.
  • the protein or mRNA level may be at least one level selected from the group consisting of blood, whole blood, plasma, serum, and lymph, but is not limited thereto.
  • the present invention provides a kit for diagnosing canine pancreatitis, comprising the agent or composition according to the present invention.
  • the present invention provides a method for diagnosing canine pancreatitis, comprising the following steps:
  • S1 measuring the level of at least one protein or mRNA selected from the group consisting of phosphodiesterase 4D interacting protein (PDE4DIP), Serpin family A member 3 (SERPINA3), and FGG in a biological sample isolated from the subject; and
  • step (S2) the protein or mRNA level of SERPINA3 measured in step (S1) is increased compared to the control group; Diagnosing pancreatitis or determining that there is a risk of developing pancreatitis when the protein or mRNA level of PDE4DIP or FGG is reduced compared to the control group.
  • the method comprises Pentaxin, Angiotensinogen, Peptidase D, Transferrin (TF), Complement C7, Coagulation factor X, Kallikrein B1, Factor XI, Complement C8 alpha chain, A step of measuring the level of at least one protein or mRNA selected from the group consisting of Coagulation factor XIII B chain (F13B), Complement C3 (LOC484960), and Complement factor I may be further included, but is not limited thereto.
  • the biological sample may be at least one selected from the group consisting of blood, whole blood, plasma, serum, and lymph, but is not limited thereto.
  • the protein level is measured by protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF (Sulface Enhanced Laser Desorption/Ionization Time of Flight Mass Spectrometry) assay, radioimmunoassay, radioimmunodiffusion method, Oukteroni immunodiffusion method, rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, two-dimensional electrophoretic assay, liquid chromatography-mass At least one method selected from the group consisting of liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-mass spectrometry/ mass spectrometry (LC-MS/MS), western blotting, enzyme linked immunosorbentassay (ELISA), and FACS It may be measured as, but is not limited thereto.
  • MALDI-TOF Microx Assisted Laser Desorption/Ionization Time
  • the mRNA level is measured from the group consisting of PCR, RNase protection assay, northern blotting, southern blotting, in situ hybridization, DNA chip, and RNA chip. It may be measured by one or more selected methods, but is not limited thereto.
  • the present invention is an agent for measuring the level of at least one protein or mRNA selected from the group consisting of PDE4DIP (phosphodiesterase 4D interacting protein), SERPINA3 (Serpin family A member 3), and FGG, for preparing an agent or kit for diagnosing canine pancreatitis.
  • PDE4DIP phosphodiesterase 4D interacting protein
  • SERPINA3 Sepin family A member 3
  • FGG FGG
  • the present invention provides the use of an agent for measuring the level of at least one protein or mRNA selected from the group consisting of PDE4DIP (phosphodiesterase 4D interacting protein), SERPINA3 (Serpin family A member 3), and FGG for the diagnosis of canine pancreatitis. .
  • the present invention relates to a composition for diagnosing canine pancreatitis, and was completed by discovering a biomarker capable of diagnosing pancreatitis with high accuracy and sensitivity through qualitative and quantitative analysis of canine plasma proteins.
  • the present inventors performed proteomic analysis using high-resolution mass spectrometry on plasma proteins of dogs suffering from pancreatitis, and selected biomarkers showing significant expression differences compared to normal controls. It was confirmed that the pancreatitis diagnostic model established using the selected biomarker can diagnose pancreatitis with high specificity, sensitivity, and accuracy.
  • the present invention is a non-invasive method using a small amount of blood, which enables prediction and early diagnosis of the risk of pancreatitis in dogs, thereby preserving the health of dogs, reducing the burden of guardians' medical expenses, and contributing to the improvement of the quality of veterinary consulting. It is expected that
  • 1a is a diagram illustrating the principle of removing the top 14 abundant plasma proteins using a MARS14 column.
  • Figure 1b shows the result of pre-processing the top 14 abundant plasma proteins and trace proteins and confirming the UV spectrum.
  • Figure 1c is a flow chart showing the process of preparing plasma proteins into peptides by the suspension trap method.
  • 1d is a diagram showing parameters used in SWATCH LC-MS analysis.
  • 3 is a graph showing the number of proteins quantified in canine plasma samples.
  • Figure 4 is a Volcano plot showing increased or decreased proteins in pancreatitis subjects compared to normal controls.
  • 5 is a table showing information on 16 proteins increased or decreased in pancreatitis subjects compared to normal controls.
  • FIG. 6a and 6b are diagrams showing box plots and ROC curves of SERPINA3 (FIG. 6a) and Angiotensinogen (FIG. 6b), which are proteins increased in pancreatitis subjects compared to normal controls.
  • 8 is a result of screening 23 proteins showing differential expression levels in a normal control group, a chronic pancreatitis group, and an acute pancreatitis group using the Kruskal Wallis test.
  • FIG. 9 is a box plot comparing the expression levels of Angiotensinogen, SERPINA3, and PDE4DIP in a normal control group, a chronic pancreatitis group, and an acute pancreatitis group (from the left, in order, expression in the acute pancreatitis group, chronic pancreatitis group, and normal group). represents).
  • the present invention relates to a composition for diagnosing canine pancreatitis, and was completed by discovering a biomarker capable of diagnosing pancreatitis in dogs with high accuracy and sensitivity through qualitative and quantitative analysis of canine plasma proteins. Specifically, the present inventors performed quantitative and qualitative analysis on plasma proteins of normal and diseased dogs through high-resolution mass spectrometry, and through this, biomarkers showing significant differences between individuals were selected. In addition, it was confirmed that the diagnostic model established by combining the above biomarkers can distinguish between diseased individuals and normal individuals with high specificity, sensitivity, and accuracy.
  • the diagnosis method according to the present invention is an alternative to conventional invasive diagnosis and examination methods, and has the advantage of being able to diagnose diseases through a relatively simple and economical method of blood sampling.
  • the main object of the present invention is to provide an agent for measuring the level of at least one protein or mRNA selected from the group consisting of PDE4DIP (phosphodiesterase 4D interacting protein), SERPINA3 (Serpin family A member 3), and FGG as an active ingredient, It is to provide a composition for diagnosing pancreatitis in an individual.
  • PDE4DIP phosphodiesterase 4D interacting protein
  • SERPINA3 Sepin family A member 3
  • FGG FGG
  • subject means a subject requiring risk prediction, diagnosis, prognosis prediction, or treatment of a disease, and more specifically, non-human primates, mice, rats, dogs , It may mean mammals such as cats, horses, and cows, but preferably means dogs (dogs; scientific name, Canis lupus familiaris).
  • pancreatitis is a disease in which pancreatic secretory glands are destroyed by pancreatic enzymes or the like, or inflammation occurs locally or entirely in the pancreas.
  • Pancreatitis according to the present invention includes both acute pancreatitis and chronic pancreatitis.
  • Acute pancreatitis occurs when the exocrine function of the pancreas is impaired, digestive enzymes are activated, and this attacks the pancreas and surrounding tissues, causing severe inflammation.
  • Dogs suffering from acute pancreatitis cause vomiting, diarrhea, abdominal pain, and severe symptoms such as bloody stools.
  • the prognosis may be poor, but early detection and active treatment can increase the survival rate, so early diagnosis is more important than anything else. do.
  • pancreatitis is characterized by impairment of both exocrine and endocrine functions due to chronic inflammation. It is accompanied by mild vomiting and diarrhea, and the condition may improve or worsen repeatedly. Since pancreatitis has no clearly identified cause, it is important to screen for the risk of developing it or diagnose it early.
  • diagnosis means confirming the presence or characteristics of a pathological state, and confirming the presence or absence of pancreatitis, whether pancreatitis has occurred, the possibility of developing pancreatitis (risk of occurrence), the prognosis of pancreatitis, etc. It can mean including.
  • diagnosis refers to determining the susceptibility of a subject to a specific disease or disorder, and determining whether a subject currently has a specific disease or disorder. , determining the prognosis of a subject suffering from a particular disease or condition (e.g., identifying tumor status, determining the staging of a tumor, or determining the responsiveness of a cancer to treatment), or therametrics (e.g., treatment monitoring the state of the object to provide information about efficacy).
  • a particular disease or condition e.g., identifying tumor status, determining the staging of a tumor, or determining the responsiveness of a cancer to treatment
  • therametrics e.g., treatment monitoring the state of the object to provide information about efficacy
  • PDE4DIP phosphodiesterase 4D interacting protein
  • PDE4DIP phosphodiesterase 4D interacting protein
  • abnormal PDE4DIP induces myeloproliferative disorders associated with eosinophilia and the like, and is known to be associated with adult pineoblastoma and adult pineal parenchymal tumor.
  • canine pancreatitis and PDE4DIP there have been no reports of canine pancreatitis and PDE4DIP to date.
  • the present inventors confirmed that the level of PDE4DIP was significantly reduced in the pancreatitis group compared to the normal group through the canine plasma proteomic analysis.
  • Specific information on PDE4DIP can be found in public databases such as Uniprot (Uniprot registration number, A0A8C0SUT4).
  • SERPINA3 (Serpin family A member 3) is a member of the serpin family, a protein group that inhibits serine proteases. Polymorphisms in SERPINA3 appear tissue-specific and may affect protease targeting. In humans, SERPINA3 abnormalities have been reported to cause Alzheimer's disease, liver disease, Parkinson's disease, etc., but the correlation between SERPINA3 expression level and pancreatitis in dogs has not been known to date. The present inventors confirmed that it was increased in the pancreatitis group compared to the normal group through the dog plasma proteomic analysis. Specific information on SERPINA3 can be found at Uniprot, etc. (Uniprot registration number: A0A8I3MJD4).
  • FGG Fibrinogen gamma chain or fibrinogen gamma gene
  • FGA Fibrinogen alpha
  • FGB Fibrinogen beta
  • FGG is one of the major components of blood clots and plays an important role in hemostasis. It functions in the early stages of wound repair to stabilize the lesion and regulate cell migration during re-epithelialization. The present inventors confirmed that it was reduced in the pancreatitis group compared to the normal group through the dog plasma proteomic analysis. Specific information on FGG can be found in Uniprot, etc. (Uniprot registration number: P12800).
  • the composition is Pentaxin, Angiotensinogen, Peptidase D, Transferrin (TF), Complement C7, Coagulation factor X, Kallikrein B1, Factor XI, Complement C8 alpha chain, Coagulation factor XIII B chain (F13B), It may further include an agent for measuring the level of at least one protein or mRNA selected from the group consisting of Complement C3 (LOC484960) and Complement factor I.
  • the above proteins (genes) are markers confirmed to be statistically significantly increased or decreased in dogs suffering from pancreatitis compared to normal individuals.
  • Pentaxin is a protein also referred to as "Pentraxin”, which includes a Pentraxin protein domain and is known to be involved in an acute immunological response.
  • Pentaxin is produced by tissue cells, macrophages, monocytes, and dendritic cells at the site of infection or inflammation, and plays a role in suppressing infections such as fungi, bacteria, and viruses.
  • canine pancreatitis and Pentaxin nothing is known about the relationship between canine pancreatitis and Pentaxin, but the present inventors confirmed that the level of Pentaxin was significantly increased in pancreatitis subjects compared to normal subjects through canine plasma protein analysis.
  • Specific information on Pentaxin can be found in public databases such as Uniprot (Uniprot registration number: A0A8C0T016).
  • Angiotensinogen is a component of the renin-angiotensin system (RAS), a hormone system that regulates blood pressure and body fluid balance.
  • RAS renin-angiotensin system
  • Angiotensinogen is also known as a renin substrate and is a non-inhibitory member of the serpin family.
  • the present inventors confirmed that the level of angiotensinogen was significantly increased in pancreatitis subjects compared to normal subjects through canine plasma proteomic analysis.
  • Specific information on angiotensinogen can be found in public databases such as Uniprot (Uniprot registration number: A0A8C0PY30).
  • the Pentaxin, SERPINA3, and Angiotensinogen may be increased in subjects suffering from pancreatitis compared to normal controls.
  • Peptidase D Transferrin (TF), Complement C7, Coagulation factor X, Kallikrein B1, Factor XI, Complement C8 alpha chain, Coagulation factor XIII B chain (F13B), Complement C3 (LOC484960), Complement factor I, or PDE4DIP It may be characterized as being reduced in individuals suffering from pancreatitis compared to normal controls.
  • the level is increased means that the undetected is detected, or the detected amount is relatively higher than the normal level.
  • a level “increased” means that the level in the experimental group is at least 1%, 2%, 3%, 4%, 5%, 10% or more, such as 5%, compared to that in the control group. , 10%, 20%, 30%, 40%, or 50%, 60%, 70%, 80%, 90% or higher, and/or 0.5x, 1.1x, 1.2x, 1.4x, 1.6x , 1.8 times higher or higher.
  • 5.5 times, 5.5 to 6 times, 6 to 6.5 times, 6.5 to 7 times, 7 to 7.5 times, 7.5 to 8 times, 8 to 8.5 times, 8.5 to 9 times, 9 to 9.5 times, 9.5 to 10 times, or 10 times It may mean an increase of more than double, but is not limited thereto.
  • the meaning of the opposite term can be understood as having the opposite meaning according to the above definition by those skilled in the art.
  • the term "measurement” refers to measuring and confirming the presence (expression) of a substance of interest (mRNA or protein of a pancreatitis biomarker gene in the case of the present invention), or the presence level (expression of a substance of interest) level) is meant to include both measuring and confirming changes. That is, measuring the expression level of the protein means measuring whether or not it is expressed (that is, measuring whether or not it is expressed) or measuring the level of qualitative or quantitative change of the protein. The measurement can be performed without limitation including both qualitative methods (assays) and quantitative methods. Types of qualitative and quantitative methods for measuring protein levels are well known in the art, and include the experimental methods described herein. Specific protein level comparison methods for each method are well known in the art. Therefore, detecting the target protein means detecting the presence or absence of the collagen protein, or confirming an increase (up-regulation) or decrease (down-regulation) of the protein level.
  • analysis may preferably mean “measurement”, and the qualitative analysis may mean measuring and confirming the presence or absence of a target substance, and the quantitative analysis may mean It may mean measuring and confirming changes in the presence level (expression level) or amount of the desired substance.
  • analysis or measurement may be performed without limitation including both qualitative and quantitative methods, and preferably, quantitative measurement may be performed.
  • the agent for measuring the level of mRNA may be a primer set or a probe that specifically binds to mRNA.
  • the composition of the present invention including the mRNA-specific primer set or probe of the biomarker genes according to the present invention may further include an agent required for a known RNA detection method.
  • the mRNA level of the biomarkers can be measured in a subject by using a known method for detecting RNA using the composition of the present invention without limitation.
  • the "primer” is a fragment that recognizes a target gene sequence, and includes a forward and reverse primer pair, preferably a primer pair that provides an analysis result having specificity and sensitivity. High specificity can be imparted when the nucleic acid sequence of the primer is a sequence that is inconsistent with the non-target sequence present in the sample, so that the primer amplifies only the target gene sequence containing a complementary primer binding site and does not cause non-specific amplification. .
  • the "probe” means a substance that can specifically bind to a target substance to be detected in a sample, and means a substance that can specifically confirm the presence of a target substance in a sample through the binding.
  • the type of probe is not limited as a material commonly used in the art, but preferably may be peptide nucleic acid (PNA), locked nucleic acid (LNA), peptide, polypeptide, protein, RNA or DNA, and most preferably Most likely it is PNA.
  • the probe is a biomaterial, including one derived from or similar to a living organism or manufactured in vitro, for example, enzymes, proteins, antibodies, microorganisms, animal and plant cells and organs, nerve cells, DNA, and It may be RNA, DNA includes cDNA, genomic DNA, and oligonucleotides, RNA includes genomic RNA, mRNA, and oligonucleotides, and examples of proteins may include antibodies, antigens, enzymes, peptides, and the like.
  • primer, probe, or antisense nucleotide that specifically binds to the gene encoding the protein based on this information (amino acid sequence, nucleic acid sequence, etc.) can be easily designed.
  • Primers or probes may be chemically synthesized using a phosphoramidite solid support synthesis method or other well-known methods, and include all functional equivalents.
  • primers or probes may be variously modified according to methods known in the art to the extent that hybridization with mRNA is not hindered.
  • modifications are methylation, capping, substitution of one or more homologs of the natural nucleotide, and modifications between nucleotides, such as uncharged linkages such as methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc. ) or charged linkages (eg, phosphorothioate, phosphorodithioate, etc.), and binding of fluorescent or enzymatic labeling materials.
  • the agent for measuring the level of the protein may be an antibody or an aptamer specific to the protein.
  • an antibody refers to a specific protein molecule directed against an antigenic site.
  • an antibody means an antibody that specifically binds to a marker protein, and includes both polyclonal antibodies, monoclonal antibodies and recombinant antibodies.
  • a part of the entire antibody is also included in the antibody of the present invention, and all types of immunoglobulin antibodies that specifically bind to the protein of the present invention are included.
  • antibodies of the present invention include special antibodies such as humanized antibodies and chimeric antibodies and recombinant antibodies as long as they can specifically bind to the protein of the present invention.
  • the term "aptamer” refers to a substance capable of specifically binding to an analyte to be detected in a sample, and a single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) having a stable tertiary structure in itself. nucleic acid), it is possible to specifically confirm the presence of a target protein in a sample.
  • the aptamer is synthesized by determining the sequence of an oligonucleotide that is selective and highly binding to the target protein to be identified according to the general aptamer preparation method, and then the 5' or 3' end of the oligonucleotide is applied. It may be made by modification with -SH, -COOH, -OH or NH 2 so that it can bind to the functional group of the timer chip, but is not limited thereto.
  • composition of the present invention may additionally include an agent required for the method of detecting the biomarker protein, and the subject (or biological obtained from the subject) may be used without limitation by using a known protein detection method using the present composition.
  • the expression level of the protein in the sample) can be measured.
  • the composition according to the present invention can be used for analysis of a biological sample isolated from a subject in need of diagnosis.
  • the "biological sample” may be included without limitation as long as it is collected from a subject to diagnose pancreatitis or predict the risk of developing it.
  • the biological sample may be selected from liquid, whole blood, plasma, urine, saliva, tissue, cell, organ, bone marrow, fine needle aspiration specimen, core needle biopsy specimen, and vacuum aspiration biopsy specimen.
  • the composition according to the present invention is for non-invasive diagnosis, and the biological sample may be at least one selected from the group consisting of blood, whole blood, plasma, serum, and lymph.
  • the biological sample may be pretreated before being used for detection or diagnosis.
  • it may include homogenization, filtration, distillation, extraction, concentration, inactivation of interfering components, addition of reagents, and the like.
  • the sample may be prepared to increase the detection sensitivity of a protein marker, for example, a sample obtained from a subject may be subjected to anion exchange chromatography, affinity chromatography, size exclusion chromatography, liquid chromatography , it may be pretreated using methods such as sequential extraction or gel electrophoresis.
  • the present invention can provide a kit for diagnosing canine pancreatitis, comprising the composition according to the present invention.
  • kit refers to a device or set for diagnosis that can diagnose pancreatitis by measuring the amount of mRNA or protein of the biomarkers according to the present invention, which are included in a sample.
  • the kit may be an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a Rapid kit, a mass spectrometry kit, a microarray kit, or a multiple reaction monitoring (MRM) kit, but is limited thereto.
  • the kit of the present invention may include an antibody recognizing a target protein as a marker, a primer set recognizing mRNA as a marker, or a probe, as well as one or more other component compositions, solutions, or devices suitable for an analysis method.
  • the substance for detecting the expression level of the gene, mRNA, or protein can be acted on one or more times without limitation, there is no limitation before and after applying each substance, and the application of each substance may be performed simultaneously or microscopically. may proceed to
  • kit according to the present invention may further include instructions describing the diagnostic method and the like according to the present invention.
  • Another object of the present invention is to provide a method for diagnosing pancreatitis in an individual or a method for providing information for diagnosing pancreatitis in a dog, including the following steps:
  • S1 measuring the level of at least one protein or mRNA selected from the group consisting of phosphodiesterase 4D interacting protein (PDE4DIP), Serpin family A member 3 (SERPINA3), and FGG in a biological sample isolated from the subject; and
  • step (S2) the protein or mRNA level of SERPINA3 measured in step (S1) is increased compared to the control group; If the protein or mRNA level of PDE4DIP or FGG is reduced compared to the control group, diagnosing pancreatitis or judging that there is a risk (high probability of developing) pancreatitis.
  • the method comprises Pentaxin, Angiotensinogen, Peptidase D, Transferrin (TF), Complement C7, Coagulation factor X, Kallikrein B1, Factor XI, Complement C8 alpha chain, A step of measuring the level of at least one protein or mRNA selected from the group consisting of Coagulation factor XIII B chain (F13B), Complement C3 (LOC484960), and Complement factor I may be further included, but is not limited thereto.
  • pancreatitis is diagnosed or pancreatitis is diagnosed as having a risk (high probability of developing) Further steps may be included.
  • the method Peptidase D, Transferrin (TF), Complement C7, Coagulation factor X, Kallikrein B1, Factor XI, Complement C8 alpha chain, Coagulation factor XIII B chain (F13B), Complement C3 (LOC484960) of the subject , Complement factor I, and / or the level of PDE4DIP is reduced compared to the control group, the step of diagnosing pancreatitis or determining that there is a risk of developing pancreatitis (high probability of developing) may be further included.
  • the area under curve when analyzing receiver operating characteristics, may be 0.7 or more, 0.75 or more, 0.8 or more, 0.85 or more, 0.9 or more, 0.93 or more, 0.95 or more, 0.96 or more, 0.97 or more, 0.98 or more, or 0.99 or more.
  • the diagnostic method according to the present invention has a specificity and / or sensitivity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 96% or more, 97% or more or more, 98% or more, or 99% or more.
  • the diagnostic method according to the present invention includes the step of measuring the protein or mRNA level of SERPINA3, the diagnostic specificity may be 70% or more or 75% or more, and the diagnostic sensitivity is 70% or more, 75% or more, Or it may be 80% or more, and the ROC AUC may be 0.75 or more, 0.8 or more, or 0.85 or more, but is not limited thereto.
  • the diagnostic method according to the present invention includes the step of measuring the protein or mRNA level of the angiotensinogen
  • the diagnostic specificity may be 75% or more, 80% or more, or 85% or more
  • the diagnostic sensitivity may be 70% or more or 75% or more.
  • % or more and the ROC AUC may be 0.75 or more or 0.8 or more, but is not limited thereto.
  • the diagnostic accuracy can be further increased.
  • the diagnostic method according to the present invention includes the step of measuring the protein or mRNA levels of PDE4DIP, FGG, and SERPINA3, the diagnostic sensitivity is 80% or more, 85% or more, 90% or more, 93% or more, or 95% or greater, diagnostic specificity may be 0.7 or greater, 0.75 or greater, or 0.8 or greater, and ROC AUC may be 80% or greater, 85% or greater, 90% or greater, 93% or greater, or 95% or greater. It is not going to be
  • the diagnostic method according to the present invention can be used to determine the type (chronic or acute) of pancreatitis in a subject diagnosed with pancreatitis.
  • the diagnostic method according to the present invention includes measuring the mRNA or protein level of SERPINA3, and at this time, if the mRNA or protein level of SERPINA3 in the subject is increased compared to the control group, the subject has acute pancreatitis can be diagnosed as
  • the control group may be a normal individual or an individual suffering from chronic pancreatitis.
  • the diagnostic method according to the present invention includes the step of measuring the protein or mRNA level of PDE4DIP, and at this time, if the protein or mRNA level of PDE4DIP in the subject is reduced compared to the control group, the subject is diagnosed with acute pancreatitis.
  • the control group may be a normal individual or an individual suffering from chronic pancreatitis.
  • the term "method for providing information” refers to a method for providing information on diagnosis of a disease by analyzing a biological sample of a subject or increasing or decreasing the level of biomarkers according to the present invention. It refers to a method of obtaining information on the onset or onset (risk) of a disease by checking For example, a method for measuring the level of a biomarker according to the present invention and comparing it with a control group to provide information on whether an individual is likely to develop pancreatitis, has a relatively high likelihood of developing pancreatitis, or has already developed pancreatitis As a result, the risk of developing pancreatitis, that is, the high-risk group, can also be predicted through the method, and it can also be used as a method of providing information on the prevention and treatment of pancreatitis.
  • a "control" may be a biological sample isolated from a normal subject or a subject suffering from pancreatitis.
  • the method for measuring the protein level is not particularly limited as long as it is by a protein measuring method known in the art, but protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF (Sulface Enhanced Laser Desorption/Ionization Time of Flight Mass Spectrometry) analysis, radioimmunoassay, radioimmunodiffusion method, Oukteroni immunodiffusion method, rocket immunoelectrophoresis, tissue immunostaining, Complement fixation method, two-dimensional electrophoretic analysis, liquid chromatography-Mass Spectrometry (LC-MS), LC-MS/MS (liquid chromatography-Mass Spectrometry/ Mass Spectrometry), Western blotting, ELISA ( enzyme linked immunosorbent assay), and FACS.
  • MALDI-TOF Microx Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry
  • the method for measuring the mRNA level is not particularly limited as long as it is by a method for measuring mRNA known in the art, but PCR, RNase protection assay, Northern blotting, Southern blotting, It can be measured by methods such as in situ hybridization, DNA chip, and/or RNA chip.
  • the present invention may provide a device for diagnosing pancreatitis in an individual (eg, dog).
  • the measuring unit of the diagnostic device of the present invention measures the expression level of at least one protein selected from the group consisting of a biomarker according to the present invention, for example, PDE4DIP, SERPINA3, and FGG, or a gene encoding the same, with respect to a biological sample obtained from a subject of interest.
  • the expression level of a protein or gene can be measured using a measurement agent.
  • Pancreatitis can be diagnosed or diagnosed as having a high risk of developing pancreatitis by checking the expression level of the protein or gene using the preparation in the measuring unit.
  • the diagnostic device of the present invention may further include a detection unit that predicts and outputs the presence, stage, or type of pancreatitis of an individual based on the expression level of the protein or gene obtained from the measurement unit.
  • the detection unit can diagnose pancreatitis by generating and classifying information on pancreatitis according to the category of the expression level of the protein or gene obtained by the measurement unit.
  • the present invention provides a method for preventing or treating pancreatitis in dogs, comprising the following steps:
  • S1 measuring the level of at least one protein or mRNA selected from the group consisting of phosphodiesterase 4D interacting protein (PDE4DIP), Serpin family A member 3 (SERPINA3), and FGG in a biological sample isolated from the subject;
  • PDE4DIP phosphodiesterase 4D interacting protein
  • SERPINA3 Serpin family A member 3
  • step (S2) the protein or mRNA level of SERPINA3 measured in step (S1) is increased compared to the control group; diagnosing the subject as suffering from pancreatitis when the protein or mRNA level of PDE4DIP or FGG is decreased compared to a control group; and
  • the present invention is a method for characterizing a respondent (i.e., dog) to a therapeutic agent for canine pancreatitis (i.e., companion diagnostic method), comprising the following steps:
  • step (b) the protein or mRNA level of SERPINA3 measured in step (a) is increased compared to the control group; If the protein or mRNA level of PDE4DIP or FGG is decreased compared to the control group, determining the subject as a responder to a treatment for canine pancreatitis;
  • the canine pancreatitis treatment agent is at least one selected from the group consisting of maropitant, ondansetron, Panoquell-CA1, and metoclopramide.
  • the "treatment” refers to all activities that improve or beneficially change the desired disease and metabolic abnormalities, and include methods such as chemotherapy, radiation therapy, surgical operation, biological therapy, or antibiotic administration. can be used.
  • the treatment may be performed through fluid treatment, drugs, plasma transfusion, and dietary control.
  • the chemotherapy refers to the act of using a chemical substance for the treatment of a specific disease and the entire drug used at that time.
  • the drug include antiemetics, gastroprotective drugs, antibiotics, and the like, and more specifically, maropitant, ondansetron, Panoquell-CA1, and metoclopramide.
  • the surgical operation includes any therapeutic or diagnostic procedure involving the methodical action of the hands or instruments in conjunction with the body of a subject to achieve a curative, therapeutic or diagnostic effect. .
  • the biological therapy refers to a treatment method that directly or indirectly uses the body's immune system using a biological agent containing a material derived from a living organism or a material produced using a living organism, and the biological agent is a physical , vaccines, allergens, antigens, hormones, cytokines, enzymes, blood and plasma, immune sera, monoclonal antibodies, fermented products, antitoxins, and laboratory diagnostics for which potency and safety cannot be assessed by chemical testing alone.
  • a biological agent containing a material derived from a living organism or a material produced using a living organism
  • the biological agent is a physical , vaccines, allergens, antigens, hormones, cytokines, enzymes, blood and plasma, immune sera, monoclonal antibodies, fermented products, antitoxins, and laboratory diagnostics for which potency and safety cannot be assessed by chemical testing alone.
  • SWATCH LC-MS analysis was performed on the peptides obtained through the above examples. To this end, a spectrum was obtained by performing LC-MS analysis using the parameters shown in FIG. 1d.
  • the iRT standard provided by the iRT-Kit was added to the prepared Peptide sample in 1/10 volume to the sample, following the supplier instructions (Reference: https://pubmed.ncbi.nlm Retention times were calibrated according to .nih.gov/22577012/).
  • the total amount of each sample was 40 ⁇ g and was dissolved in 40 ⁇ L of solution.
  • a 4 ⁇ L sample injected was analyzed using a SCIEX TripleTOF 5600+ system mass spectrometer with the following LC-MS/MS settings.
  • a nanoLC 425 (Eksigent, Dublin, CA, USA) was used as an analytical column with an Eksigent Micro Trap C18 column (ChromXP C18CL, 5 ⁇ m, 120 ⁇ ) and an Eksigent column (C18-CL, 0.3 ⁇ 150 mm, particle size 3). ⁇ m, pore size 120 ⁇ ), the column temperature was maintained at 40° C. and the sample was loaded onto the trap column using buffer A at a flow rate of 10 ⁇ L/min.
  • the peptide mixture was separated through a concentration gradient using buffer A and buffer B (0.1% formic acid in HPLC acetonitrile) at a flow rate of 5 ⁇ L/min by 57 minutes.
  • SWATH mass spectrometry run
  • the SWATH parameters were set as follows: Lower m/z limit 400; m/z upper limit 1250; Window overlap (Da) 1.0; The CES was 5 for the small window, 8 for the large window, and 10 for the largest window.
  • MS2 spectra were collected in the range 100–1500 m/z for 2.5 ms in high-sensitivity mode, with a total cycle time of 2.8 seconds.
  • MS parameters were set as follows: ion source gas 1 (GS1) 15 psi; ion source gas 2 (GS2) 20 psi; Curtain Gas (CUR) 30 psi; Temperature (TEM) 250 °C; Floating Ion Spray Voltage (ISVF) 5500V.
  • GS1 ion source gas 1
  • GS2 ion source gas 2
  • CUR Curtain Gas
  • TEM Temperature
  • ISVF Floating Ion Spray Voltage
  • SWATH LC-MS analysis was performed on each sample to obtain a spectrum result, and quantitative and quantitative analysis of proteins from the spectra was performed using DIA-NN software and Pan-human proteome library. Sexual information was extracted.
  • DIA-NN software and Pan-human proteome library.
  • Example 2 based on S-trap, 116 plasma samples were prepared as peptides with trypsin/LysC, and the amount of peptides was quantified using Nanodrop (FIG. 2). As a result, it was confirmed that a minimum of 214 ⁇ g of peptide was obtained at a maximum of 1,023 ⁇ g. Considering that only about 8 ⁇ g of peptide is usually analyzed during protein analysis, it was found that a sufficient amount of peptide was prepared for the study.
  • DEPs Differently expressed proteins
  • Fig. 4 The p value and fold change for 16 DEPs are shown in FIG. 5 .
  • Pentaxin is a C-Reactive protein, which the present inventors have identified as a biomarker that is equally increased in lymphoma.
  • CRP and SERPINA3 are also proteins identified in DEP in lymphoma.
  • 10 proteins, including PEPD were commonly reduced in disease groups.
  • SERPINA3 Sepin family A member 3
  • Angiotensinogen are both proteins that are increased in pancreatitis.
  • SEPINAA3 showed a specificity of 78.1% and a sensitivity of 81.8%, and ROC AUC was 0.857, indicating excellent discrimination power (FIG. 6a).
  • angiotensinogen had a sensitivity of 78.8% and a specificity of 87.5%, and the ROC AUC was 0.848, confirming that the disease discrimination power was similarly excellent (FIG. 6b).
  • pancreatitis group acute and chronic
  • normal control group have a probability that they can be distinguished from each other using quantitative characteristics of proteins (Fig. 7).
  • F1PAL5 which means angiotensinogen, is marked on the right-axis graph of FIG. 7 .
  • angiotensinogen was found to be increased in both chronic pancreatitis and acute pancreatitis, and SERPINA3 showed quantitative characteristics with a higher level in normal, chronic, and acute stages.
  • PDE4DIP was high in the normal group and decreased in the chronic and acute pancreatitis groups (FIG. 9). That is, it was confirmed that a panel for distinguishing chronic pancreatitis from acute pancreatitis could be developed using the quantitative characteristics of the proteins.
  • the present invention relates to a composition for diagnosing canine pancreatitis, and was completed by discovering a biomarker capable of diagnosing pancreatitis with high accuracy and sensitivity through qualitative and quantitative analysis of canine plasma proteins.
  • the present inventors performed proteomic analysis using high-resolution mass spectrometry on plasma proteins of dogs suffering from pancreatitis, and selected biomarkers showing significant expression differences compared to normal controls. It was confirmed that the pancreatitis diagnostic model established using the selected biomarker can diagnose pancreatitis with high specificity, sensitivity, and accuracy.
  • the present invention is a non-invasive method using a small amount of blood, which enables prediction and early diagnosis of the risk of pancreatitis in dogs, thereby preserving the health of dogs, reducing the burden of guardians' medical expenses, and contributing to the improvement of the quality of veterinary consulting. It is expected that

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Abstract

La présente invention concerne une composition pour le diagnostic de la pancréatite canine. À l'origine de la présente invention, une découverte a été faite concernant un biomarqueur permettant de diagnostiquer la pancréatite avec une précision et une sensibilité élevées par analyse qualitative et quantitative de protéines plasmatiques canines. Plus précisément, les inventeurs de la présente invention ont effectué une analyse protéomique par spectrométrie de masse haute résolution sur les protéines plasmatiques de chiens souffrant de pancréatite et ont choisi des biomarqueurs présentant des différences d'expression significatives par comparaison avec des témoins normaux. Il a été observé que le modèle de diagnostic de pancréatite établi à l'aide du biomarqueur sélectionné peut diagnostiquer la pancréatite avec une spécificité, une sensibilité et une précision élevées. Par conséquent, en tant que méthode non invasive à l'aide d'une petite quantité de sang, ce qui permet une prédiction et un diagnostic précoce du risque de pancréatite chez les chiens, la présente invention est censée préserver la santé des chiens, réduire la charge des dépenses médicales du propriétaire et contribuer à l'amélioration de la qualité de la consultation vétérinaire.
PCT/KR2022/019278 2021-12-01 2022-11-30 Biomarqueur pour le diagnostic de la pancréatite chez un chien de compagnie WO2023101447A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20150001287A (ko) * 2013-06-27 2015-01-06 인하대학교 산학협력단 췌장염 진단용 바이오 마커 조성물
KR20190065281A (ko) * 2016-09-26 2019-06-11 지에이 제네릭 에세이즈 게엠베하 당단백질 2 아이소폼 알파(GP2a)의 검출에 의한 급성 췌장염(AP) 진단 방법

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20150001287A (ko) * 2013-06-27 2015-01-06 인하대학교 산학협력단 췌장염 진단용 바이오 마커 조성물
KR20190065281A (ko) * 2016-09-26 2019-06-11 지에이 제네릭 에세이즈 게엠베하 당단백질 2 아이소폼 알파(GP2a)의 검출에 의한 급성 췌장염(AP) 진단 방법

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
FRANCO-MARTÍNEZ LORENA, GELEMANOVIĆ ANDREA, HORVATIĆ ANITA, CONTRERAS-AGUILAR MARÍA DOLORES, MRLJAK VLADIMIR, CERÓN JOSÉ JOAQUÍN, : "The Serum and Saliva Proteome of Dogs with Diabetes Mellitus", ANIMALS, vol. 10, no. 12, 1 December 2020 (2020-12-01), pages 2261, XP093069860, DOI: 10.3390/ani10122261 *
KIM HAKHYUN, KIM HYUNG-JIN, KANG JI-HOUN, KANG BYEONG-TECK, YANG MHAN-PYO: "Evaluation of serum C-reactive protein and high mobility group box 1 concentrations in 22 dogs with acute pancreatitis: a pilot study", VETERINARY QUARTERLY., KLUWER, DORDRECHT., NL, vol. 39, no. 1, 1 January 2019 (2019-01-01), NL , pages 122 - 130, XP093069856, ISSN: 0165-2176, DOI: 10.1080/01652176.2019.1655178 *
WALDRON RICHARD T., LUGEA AURELIA, GULLA AISTE, PANDOL STEPHEN J.: "Proteomic Identification of Novel Plasma Biomarkers and Pathobiologic Pathways in Alcoholic Acute Pancreatitis", FRONTIERS IN PHYSIOLOGY, vol. 9, 30 August 2018 (2018-08-30), pages 1215, XP093069851, DOI: 10.3389/fphys.2018.01215 *
YOON JI-SEON, SUHEE KIM, JIN-HEE KANG, JINHO PARK, DOHYEON YU: "Alterations in serum protein electrophoresis profiles during the acute phase response in dogs with acute pancreatitis", CANADIAN JOURNAL OF VETERINARY RESEARCH, vol. 84, no. 1, 1 January 2020 (2020-01-01), pages 74 - 78, XP093069853 *

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