WO2026070830A1 - Method for determining presence or absence of fibrotic disease - Google Patents

Abstract

The present invention addresses the problem of providing a method for detecting fibrosis, the method having low invasiveness and high versatility, and finding a marker, of a fibrotic disease, having high discrimination accuracy and applicable to a relatively wide range of organs.　Provided are: a combination marker for identifying a fibrotic disease, the marker containing LOX-1 protein or a fragment thereof and LAB or a part thereof; and a method for determining the presence or absence of a fibrotic disease or the presence or absence of risk of onset thereof in a subject by using the marker.

Description

Method for determining whether or not one has fibrotic disease

The present invention relates to markers, methods, kits, and devices for determining the presence or absence of fibrotic disease or the risk of developing it.

Fibrotic diseases are conditions in which tissues and organs become dysfunctional as a result of incomplete tissue reconstruction by stromal cells and extracellular matrix when subjected to stress. Traditionally, such tissue fibrosis has been understood as a symptom resulting from the advanced stages of various disorders. However, recent reports (Non-Patent Literature 1) suggest that tissue plasticity may remain even in cases of advanced fibrosis, indicating the possibility of healthy tissue reorganization through therapeutic intervention.

Typically, a definitive diagnosis of fibrosis is made by examining a pathological biopsy taken from the patient to identify the fibrotic site. However, pathological biopsy is highly invasive and not suitable for patients with relatively mild symptoms. In particular, liver biopsies are reported to cause serious complications in approximately 0.5% of patients, making it impractical to perform them on all patients. On the other hand, in order to provide therapeutic intervention for tissue fibrosis, it is necessary to detect fibrosis before it progresses and severe symptoms appear.

Given this background, there is a need to develop a less invasive method for detecting fibrosis that can be applied to patients with relatively mild symptoms or those without clearly defined pathological conditions.

Terai, S. and Tsuchiya, J. Gastroenterol 52, 129-140 (2017)

Given the background described above, research has been conducted to find markers applicable to various fibrotic diseases. For example, markers based on autotaxin (a protein capable of binding to receptors expressed on sinusoidal endothelial cells in the liver's sinusoids) and markers based on the blood concentrations of liver enzymes such as AST and ALT have been established as markers for hepatic fibrotic diseases.

However, all of these methods can only detect fibrosis of a certain degree of severity. In addition, since established markers are all based on proteins specific to particular organs, their applicability is limited depending on the type of organ affected by fibrosis. Furthermore, the progression of fibrosis varies greatly depending on the target organ; as mentioned above, markers for lung and liver fibrosis are being actively searched for, but the development of markers for fibrosis in other organs is lagging behind.

On the other hand, extracellular matrix-based markers are known as markers that can detect fibrosis in a relatively wide range of organs. However, their discriminative accuracy is not high, and no established marker has sufficient discriminative accuracy to be used in actual diagnosis.

Given this background, despite its high invasiveness, pathological biopsy is currently used as the standard diagnostic method.

Therefore, the objective of this invention is to identify a marker for fibrotic disease that has high discriminative accuracy and is applicable to a relatively wide range of organs, and to provide a low-invasiveness, highly versatile method for detecting fibrosis.

The inventors noted that damage to vascular endothelial cells occurs in the early stages of many fibrotic disorders. To address this issue, the inventors conducted intensive research and discovered that by combining the amount of LOX-1 protein, which is widely expressed in vascular endothelial cells, and the amount of LAB, an oxidized form of LDL present in the blood throughout the body, in a specific manner, it is possible to differentiate fibrotic diseases with greater accuracy than previously established fibrosis markers. This invention is based on these novel findings and provides the following:
[1-1] A method for determining whether a subject has a fibrotic disease accompanied by vascular endothelial damage or is at risk of developing such a disease, comprising the steps of: measuring the amount of lectin-like oxidized low-density lipoprotein receptor 1 protein (LOX-1 protein) and apolipoprotein B-containing LOX-1 ligand (LAB) in a sample derived from the subject; and calculating the ratio of the amount of LOX-1 protein to the amount of LAB, wherein the ratio indicates that the subject has a fibrotic disease accompanied by vascular endothelial damage or is at risk of developing such a disease.
[1-2] A method for determining whether a subject has a fibrotic disease or is at risk of developing one, comprising the step of calculating a determination value based on the amount of lectin-like oxidized low-density lipoprotein receptor 1 protein (LOX-1 protein) and/or the amount of apolipoprotein B-containing LOX-1 ligand (LAB) in a sample derived from the subject, wherein the determination value indicates that the subject has a fibrotic disease or is at risk of developing one.
[1-3] The method according to [1-2], wherein the fibrotic disease is a fibrotic disease accompanied by vascular endothelial damage.
[1-4] The method according to [1-2] or [1-3], wherein the determination value is the ratio of the amount of LOX-1 protein to the amount of LAB.
[1-5] A method for determining whether a subject has a fibrotic disease accompanied by vascular endothelial damage or is at risk of developing such a disease, comprising the step of calculating the ratio of the amount of lectin-like oxidized low-density lipoprotein receptor 1 protein (LOX-1 protein) to the amount of apolipoprotein B-containing LOX-1 ligand (LAB) in a sample derived from the subject, wherein the ratio indicates that the subject has a fibrotic disease accompanied by vascular endothelial damage or is at risk of developing such a disease.
[1-6] The method according to any one of [1-2] to [1-5], further comprising the step of measuring the amount of LOX-1 protein and/or LAB in a sample derived from the subject.
[2] The method according to any one of [1-1] to [1-6], wherein the sample is a bodily fluid sample.
[3] The method according to [2], wherein the bodily fluid sample is a blood sample.
[4] The method according to any one of [1-1] to [3], wherein the fibrotic disease is further accompanied by liver damage and/or an autoimmune disease.
[5] The method according to [4], wherein the liver disorder includes cirrhosis and/or portal hypertension.
[6] The method according to [4] or [5], wherein the autoimmune disease includes an antinuclear antibody-positive disorder.
[7] The method according to [6], wherein the antinuclear antibody-positive disorder includes anticentromere antibody-positive disorder and/or Sjögren's syndrome.
[8-1] The method according to any one of [1-1] to [7], wherein the subject is shown to have a fibrotic disease accompanied by vascular endothelial damage or to be at risk of developing such a disease, based on a comparison of the ratio with a predetermined cutoff value or a control value similarly calculated for a sample derived from a control individual.
[8-2] A method according to any one of [1-1] to [7], wherein the subject is found to have a fibrotic disease or is at risk of developing one, based on a comparison of the determination value with a predetermined cutoff value or a control value similarly calculated for a sample derived from a control individual.
[9] A composition comprising a LOX-1 protein-binding molecule for use in any of the methods described in [1-1] to [8-2].
[10] A composition comprising a LAB-binding molecule for use in any of the methods described in [1-1] to [8-2].
[11] A kit for use in any of the methods described in [1-1] to [8-2], comprising a LOX-1 protein binding molecule and a LAB binding molecule.
[12] A device for use in any of the methods described in [1-1] to [8-2], comprising a LOX-1 protein binding molecule and a LAB binding molecule.
[13-1] A combination marker for the differential diagnosis of fibrotic diseases with vascular endothelial damage, comprising LOX-1 protein or a fragment thereof, and LAB or a part thereof.
[13-2] A combination marker for the differential diagnosis of fibrotic diseases, comprising LOX-1 protein or a fragment thereof, and LAB or a part thereof.
This specification includes the disclosures of Japanese Patent Application No. 2024-166159, which forms the basis of the priority claim of this application.

The markers, methods, kits, and devices of the present invention can detect the presence or absence of fibrotic diseases accompanied by vascular endothelial damage.

This figure shows the LOX-1 levels in individuals that are positive for and negative for anti-centromere antibodies (ACA). In the figure, the dots represent the data points, the shape of the ACA-positive dots indicates which individual the data was obtained from, and * indicates p<0.05. This figure shows the logarithm of LOX-1 protein levels in ACA-positive and ACA-negative individuals. In the figure, the dots represent the points for each data point, the shape of the ACA-positive dots indicates which individual the data was obtained from, and * indicates p<0.05. This figure shows the amount of LAB in ACA-positive and ACA-negative individuals. In the figure, the dots represent the points for each data point, the shape of the ACA-positive dots indicates which individual the data was obtained from, and *** indicates p<0.001. This figure shows the LOX-index ^(R) values for ACA-positive and ACA-negative individuals. In the figure, the dots represent the data points, the shape of the ACA-positive dots indicates which individual the data was obtained from, and "ns" indicates no significant difference. This figure shows the values of common liver fibrosis markers in ACA-positive and ACA-negative individuals. Figure 5A shows hyaluronic acid levels, and Figure 5B shows type IV collagen levels. In the figure, the dots represent the points for each data point, the shape of the dots for ACA-positive individuals indicates which individual the data was obtained from, and "n.s." indicates no significant difference. This figure shows the Fib4-index values, a common liver fibrosis marker, in ACA-positive and ACA-negative individuals. In the figure, the dots represent the data points, the shape of the ACA-positive dots indicates which individual the data was obtained from, and ** indicates p<0.01. In the figure, the dashed line represents the level at which the Fib4-index value is 2.67. This figure shows the ratio of LOX-1 to LAB levels in ACA-positive and ACA-negative individuals. In the figure, dots represent data points, the shape of the ACA-positive dots indicates which individual the data was obtained from, and ** indicates p<0.01.

1. Combination Marker for Fibrotic Disease Diagnosis 1-1. Overview The first aspect of the present invention is a combination marker for the differential diagnosis of fibrotic diseases. The combination marker of the present invention uses LOX-1 protein or a fragment thereof, and LAB or a part thereof as biomarkers. By measuring the amount of the marker contained in the subject's biological sample using the determination method described in the second aspect, it is possible to determine whether or not the subject has a fibrotic disease or whether or not there is a risk of developing it.

1-2. Definitions The terms frequently used in this specification are defined below.

"Fibrotic disease" refers to a disease characterized by the loss or dysfunction of parenchymal cells in tissues, followed by excessive migration and proliferation of fibroblasts to compensate, and subsequent rigidity due to the deposition of extracellular matrix. The causes of parenchymal cell loss or dysfunction, and the sites of onset, are not particularly limited. Specific causes include, for example, chemical stimuli such as drugs (e.g., antitumor agents, antibiotics, antibacterial agents, antiarrhythmic agents, anti-inflammatory agents, anti-rheumatic agents), physical stimuli such as excessive pressure, physiological stimuli such as inflammatory responses, and abnormalities in the body such as diseases or disorders (e.g., nephritis, myocarditis, enteritis, pneumonia), or combinations thereof. Sites of onset include, for example, the respiratory system such as the trachea and lungs, the digestive system such as the esophagus, stomach and intestines, the urinary system such as the kidneys, the circulatory system such as blood vessels, heart, and liver, or combinations thereof. Fibrotic disease is usually definitively diagnosed by confirming tissue fibrosis through histopathological findings. For example, in the liver, liver biopsy can be used to evaluate the condition using various methods, such as the New Inuyama Classification or the New European Classification by Desmet et al. Alternatively, morphological evaluation using CT or MRI, measurement of liver stiffness by MR elastography, evaluation of liver fibrosis by abdominal ultrasound elastography, and blood test data such as the ELF (Enhanced Liver Fibrosis) test, type IV collagen 7S, M2BPGi, autotaxin, and hyaluronic acid, or scoring systems such as the FIB-4 index, Child-Pugh classification, MELD score, or a combination of these can be used for diagnosis. For example, in FIB-4 index evaluation, a low value of less than 1.3 indicates low risk, an intermediate value of 1.3 to less than 2.67 indicates moderate fibrosis, and a high value of 2.67 or higher indicates severe fibrosis.

The severity of fibrotic disease is not particularly limited. In this specification, the severity of fibrotic disease may be defined as, for example, a stage of F1 or higher and a grade of A0 or higher according to the New Inuyama classification, or a stage of 2 or higher and a grade of 1 or higher according to the New European classification. In this specification, the severity of fibrosis may be defined as a value greater than 1.3 according to the FIB-4 Index. In this specification, fibrotic disease preferably involves vascular endothelial dysfunction.

"Endothelial dysfunction" refers to functional and/or structural impairment of vascular endothelial cells. Examples of endothelial cell functions include regulating vasodilation and vascular smooth muscle proliferation via vasoactive substances such as nitric oxide (NO) and endothelin, thereby regulating vasotonic tone (tonus), regulating intravascular blood coagulation, regulating vascular permeability, and other biological defense mechanisms, regulating inflammation, and regulating oxidation. In this specification, endothelial dysfunction refers to a decline in one or more of these functions. For example, in this specification, endothelial dysfunction refers to a state where flow-mediated dilation (FMD) is below a certain level. While the FMD value is not particularly limited, for example, in this specification, endothelial dysfunction refers to a state where FMD is less than 7%, less than 6%, less than 5%, or less than 4%.

In this specification, "fibrotic disease with vascular endothelial dysfunction" refers to fibrotic diseases that exhibit vascular endothelial dysfunction. For example, this includes fibrotic diseases accompanied by decreased FMD. Fibrotic diseases with vascular endothelial dysfunction in this specification may also be accompanied by liver dysfunction and/or autoimmune diseases (such as antinuclear antibody-positive disorders).

In this specification, "liver impairment" refers to a condition in which liver function is impaired. The specific type of liver impairment is not limited. Examples include cirrhosis, portal hypertension, hepatitis, fatty liver, liver atrophy, or associated systemic diseases (such as disorders of the heart, lungs, kidneys, eyes, skin, etc.).

In this specification, "autoimmune disease" refers to a disease characterized by an overactive immune system targeting the body's own cells or tissues. While not limited to specific diseases, autoimmune diseases include, for example, antinuclear antibody-positive disorders, Sjögren's syndrome, scleroderma, rheumatism, arthritis, asthma, psoriasis, dermatitis, urticaria, eczema, systemic lupus erythematosus, Addison's disease, polymyositis, and polyglomerulonephritis.

In this specification, "antinuclear antibody-positive disorder" refers to a disorder characterized by positive results for autoantibodies against factors constituting the nucleus of one's own cells, and is a type of autoimmune disease. Examples of such antinuclear antibodies include anti-centromere antibodies (ACAs), anti-RNA polymerase III antibodies, anti-nucleolar antibodies, and anti-topoisomerase I antibodies. Examples of antinuclear antibody-positive disorders in this specification include anti-centromere antibody-positive disorders and Sjögren's syndrome.

"Anti-centromere antibodies" refer to autoantibodies against the centromere. They are positive in many patients with systemic sclerosis. In this specification, "anti-centromere antibody-positive disorder" refers to a disorder characterized by a positive test for anti-centromere antibodies. The severity is not specifically limited, but examples include relatively mild localized cutaneous systemic sclerosis according to the LeRoy and Medsger classification. It should be noted that ACA positivity does not directly indicate the development of a specific pathological condition, and even if a specific condition is present, its progression is generally slow, so pathogenicity is not considered a clinical problem.

Sjögren's syndrome is an autoimmune disease characterized primarily by chronic sialadenitis and keratoconjunctivitis sicca, accompanied by the appearance of various autoantibodies and hypergammaglobulinemia. While dryness is the main symptom, not only the salivary and lacrimal glands but also exocrine glands throughout the body are systematically affected. Sjögren's syndrome is broadly classified into primary Sjögren's syndrome, which does not involve other collagen diseases, and secondary Sjögren's syndrome, which is associated with collagen diseases such as rheumatoid arthritis or systemic lupus erythematosus, but both are included in this specification. Furthermore, primary Sjögren's syndrome is broadly classified into glandular type, where lesions are limited to the lacrimal and salivary glands, and extraglandular type, where lesions extend to various organs throughout the body, but both are included in this specification. In addition to polyclonal hypergammaglobulinemia, autoantibodies such as antinuclear antibodies, rheumatoid factor, anti-SS-A antibodies, and anti-SS-B antibodies appear. Sjögren's syndrome can be diagnosed, for example, based on the revised diagnostic criteria for Sjögren's syndrome (SjS) (Ministry of Health, Labour and Welfare Research Group, 1999). This diagnosis is made based on a comprehensive assessment of biopsy histopathological examination, oral examination, ophthalmological examination, and blood test results.

LOX-1 (lectin-like oxidized low-density lipoprotein receptor-1) protein (often referred to as "LOX-1 protein" in this specification) is a single-pass transmembrane receptor membrane protein with its N-terminus exposed in the cytoplasm and its C-terminus exposed extracellularly. It forms homodimers via disulfide bonds and is expressed in vascular endothelial cells, smooth muscle, and macrophages, functioning as a scavenger receptor for oxidized LDL (described later). Recently, however, it has also attracted attention as a factor promoting arteriosclerosis. LOX-1 protein expression is known to be induced in platelets, endothelial cells, vascular smooth muscle, neurons, and macrophages by ischemia-reperfusion injury, reactive oxygen species, and inflammatory cytokines.

LDL (low-density lipoprotein) is a lipoprotein composed of a protein component (apo-protein B: apoB) and a lipid component (cholesterol, triglycerides, and phospholipids), and is characterized by its low specific gravity. LDL plays a role in transporting cholesterol produced in the liver throughout the body via the bloodstream. Because increased blood levels of LDL are a risk factor for arteriosclerosis, LDL is commonly referred to as "bad cholesterol."

"Oxidized LDL (ox-LDL)" refers to LDL in which the lipid or protein components have been oxidatively modified or damaged by free radicals such as reactive oxygen species. Oxidized LDL is also called denatured LDL or LAB (LOX-1 ligand containing apolipoprotein B). In this specification, unless otherwise specified, oxidized LDL or denatured LDL will be referred to as "LAB".

1-3. Composition The marker of this embodiment includes the LOX-1 protein or a fragment thereof, and LAB or a part thereof. Each component is described below.

(1) LOX-1 protein or fragment thereof In this specification, LOX-1 protein refers to LOX-1 protein derived from the target animal, preferably human LOX-1 protein. LOX-1 protein includes wild-type and mutant types. Specifically, wild-type human LOX-1 protein refers to human LOX-1 protein consisting of the amino acid sequence shown in SEQ ID NO: 2. Furthermore, "mutant LOX-1 protein" refers to a polypeptide in which a mutation has occurred in a part of the wild-type LOX-1 protein. The specific amino acid sequence of mutant LOX-1 protein is not particularly limited, but examples include an amino acid sequence in which one or more amino acids are added, deleted, or substituted in the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more amino acid identity with respect to the amino acid sequence shown in SEQ ID NO: 2. For example, examples include splicing variants and mutants based on SNPs, etc. For example, a mutant LOX-1 protein that retains its ability to bind to LAB can be suitably used as a marker in this embodiment.

In this specification, "multiple" refers to, for example, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3. Furthermore, "amino acid identity" refers to the percentage (%) of identical amino acid residues in the total number of amino acid residues when the amino acid sequences of two polypeptides being compared are aligned by inserting gaps as needed to maximize the number of identical amino acid residues in one or both sequences. Alignment of two amino acid sequences for calculating amino acid identity can be performed using known programs such as Blast, FASTA, or ClustalW.

In this specification, "(amino acid) substitution" refers to substitutions within the conserved amino acid group, which consists of 20 amino acids that make up natural proteins and have similar properties such as charge, side chain, polarity, and aromaticity. Examples include substitutions within the uncharged polar amino acid group with low-polarity side chains (Gly, Asn, Gln, Ser, Thr, Cys, Tyr), the branched-chain amino acid group (Leu, Val, Ile), the neutral amino acid group (Gly, Ile, Val, Leu, Ala, Met, Pro), the neutral amino acid group with hydrophilic side chains (Asn, Gln, Thr, Ser, Tyr, Cys), the acidic amino acid group (Asp, Glu), the basic amino acid group (Arg, Lys, His), and the aromatic amino acid group (Phe, Tyr, Trp). Amino acid substitutions within these groups are preferred because they are known to cause minimal changes in the properties of polypeptides.

A fragment of the LOX-1 protein may be used as a marker in this embodiment. In that case, the specific composition of the fragment is not particularly limited, as long as it can be distinguished as the LOX-1 protein from other biomolecules. For example, a fragment that retains LAB binding ability can be suitably used. A specific example of a fragment is soluble LOX-1 protein.

"Soluble form of LOX-1 protein" (often referred to as "sLOX-1 protein" in this specification) refers to a peptide fragment composed of the extracellular domain of the LOX-1 protein. The LOX-1 protein has a highly protease-sensitive site in its neck domain, which connects the extracellular domain at the C-terminus to the transmembrane domain at the N-terminus. When cleaved at this site, the extracellular domain becomes free and is released extracellularly. Since LOX-1 protein is expressed in vascular endothelial cells, sLOX-1 protein is known to exist in a free state in the blood. Because sLOX-1 protein possesses a LAB-binding domain, it retains the same LAB-binding ability as the full-length LOX-1 protein.

Specific examples of sLOX-1 proteins include, for example, a polypeptide or fragment thereof consisting of 213 amino acids corresponding to the amino acid region from positions 61 to 273 in the amino acid sequence of the human LOX-1 protein shown in SEQ ID NO: 2, and having the amino acid sequence shown in SEQ ID NO: 3. Examples of such fragments include a polypeptide consisting of 183 amino acids corresponding to positions 91 to 273 in the amino acid sequence shown in SEQ ID NO: 2, and having the amino acid sequence shown in SEQ ID NO: 4, or a polypeptide consisting of 180 amino acids corresponding to positions 94 to 273 in the amino acid sequence shown in SEQ ID NO: 2, and having the amino acid sequence shown in SEQ ID NO: 5. Furthermore, any peptide fragment composed of the extracellular region of the mutant LOX-1 protein may be used. For example, a polypeptide in which one or more amino acids are added, deleted, and/or substituted in the amino acid sequence shown in any of SEQ ID NOs: 3-5, or a polypeptide having 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more amino acid identity with respect to the amino acid sequence shown in any of SEQ ID NOs: 3-5.

(2) LAB or part thereof As described above, LAB is an apolipoprotein also called oxidized LDL (denatured LDL), and includes apolipoprotein B (apoB) as a protein component. In this specification, apoB is apoB derived from the target animal, preferably human apoB. In this specification, apoB includes both wild-type and mutant forms of any isoform, including apoB-48 and apoB-100. A specific example of wild-type human apoB is the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 6. In this specification, mutant apoB refers to a polypeptide in which a mutation has occurred in a part of wild-type apoB. The amino acid sequence of mutant apoB is not particularly limited, but examples include an amino acid sequence in which one or more amino acids are added, deleted, or substituted in the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence having 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more amino acid identity with the amino acid sequence shown in SEQ ID NO: 6. While not limited, specific examples include splicing variants and mutants based on SNPs, etc. For example, mutant apoB that retains the ability to bind to the LOX-1 protein can be suitably used as a marker in this embodiment.

The specific composition of a LAB is not particularly limited, as long as it can be distinguished as a LAB from other biomolecules. This includes apolipoproteins containing apoB that has the ability to bind to LOX-1, such as malondialdehyde-modified LDL (MDA-LDL) and AGE-LDL. The LAB used as a marker in this embodiment may be either a portion that has the ability to bind to the LOX-1 protein or a portion that does not; both are included in the LAB.

1-4. Applications The marker of this embodiment is used in methods for diagnosing fibrotic diseases (such as methods for diagnosing fibrotic diseases accompanied by vascular endothelial damage). Detailed information on the diagnosing method is described in the second embodiment.

The diseases to be differentiated are as described in the second embodiment and are not particularly limited. Depending on the type of disease to be differentiated, the markers in this embodiment may be, for example, markers for differentiating fibrotic diseases (fibrotic diseases accompanied by vascular endothelial damage, etc.), markers for differentiating scleroderma, markers for differentiating hepatic fibrosis, markers for differentiating liver damage, markers for differentiating portal hypertension, etc.

2. Method for Determining Fibrotic Disease 2-1. Overview The second aspect of the present invention is a method for determining whether or not a person has a fibrotic disease or is at risk of developing one. The method of this aspect includes a step of measuring the marker described in the first aspect (measurement step), a step of calculating a determination value (ratio, etc.) (calculation step), and a step of making a determination based on the determination value (ratio, etc.) (determination step). According to the determination method of the present invention, the presence or absence of a fibrotic disease and/or risk of developing one can be determined very simply.

2-2. Process The determination method of the present invention includes a measurement step, a calculation step, and a determination step. Each step will be described in detail below.

2-2-1. Measurement Process The "measurement process" is a process of measuring the amount of lectin-like oxidized low-density lipoprotein receptor 1 protein (LOX-1 protein) and/or apolipoprotein B-containing LOX-1 ligand (LAB) in a sample derived from the subject.

In this specification, "subject" refers to an animal individual used in the determination method of this embodiment. The specific animal species is not particularly limited, but examples include mammals such as humans, dogs, cats, horses, cattle, sheep, goats, camels, rabbits, ferrets, hamsters, and mice. Humans are preferred. The disease status of the subject is not particularly limited. For example, it may be an individual suspected of having a specific disease (such as fibrotic disease), an individual judged to have a low probability of having a specific disease, or an individual whose presence or absence of a specific disease is unknown.

In this specification, "an individual suspected of having a specific disease" refers to an individual exhibiting symptoms seen in patients with that specific disease based on clinical findings, etc., and who has, in principle, been diagnosed as suspected of having the disease by a physician or other healthcare professional. Diagnosis is primarily made by combining a medical history, clinical course, physical examination findings, and histopathological findings. Similarly, an individual diagnosed by a physician or other healthcare professional as having a low probability of having the disease is referred to as "an individual judged to have a low probability of having the specific disease," and an individual for whom a judgment has not been made by a physician or other healthcare professional is referred to as "an individual whose presence or absence of the specific disease is unknown."

The specific diseases referred to herein may be diseases related to fibrotic diseases (such as fibrotic diseases accompanied by vascular endothelial damage) that are subject to determination by the method of this embodiment, or diseases that are unrelated or whose relationship is unclear; they are not particularly limited. Preferred specific diseases include, for example, diseases related to or associated with fibrotic diseases (such as fibrotic diseases accompanied by vascular endothelial damage).

Examples of fibrotic diseases accompanied by vascular endothelial damage include fibrotic diseases accompanied by liver damage and/or autoimmune diseases (antinuclear antibody-positive disorders), particularly fibrotic diseases accompanied by cirrhosis and/or portal hypertension.

Diseases associated with fibrotic diseases involving vascular endothelial damage include, for example, fibrotic diseases themselves, and diseases that have been suggested to be associated with an increased risk of developing fibrotic diseases.

Specific fibrotic diseases include, in addition to the diseases listed below as those to be assessed, liver fibrosis (cirrhosis, (idiopathic) portal hypertension, viral hepatitis, etc.), renal fibrosis, scleroderma renal crisis, tubulointerstitial nephritis, interstitial pneumonia (pulmonary fibrosis, etc.), chronic obstructive pulmonary disease, viral hepatitis, chronic pancreatitis, scirrhous gastric cancer, idiopathic pulmonary arterial hypertension (IPAH), and connective tissue disease-associated pulmonary hypertension (CTD-PAH). Examples include hypertension, hypothyroidism, slowly progressive insulin-dependent diabetes mellitus (SPIDDM), type 1 diabetes mellitus, keratoconjunctivitis sicca, scleritis, uveitis, myocardial fibrosis (myocardial infarction, etc.), sclerosing peritonitis, systemic lupus erythematosus, scleroderma (diffuse cutaneous systemic scleroderma, localized cutaneous systemic scleroderma), Sjögren's syndrome, dermatomyositis, rheumatoid arthritis, scarring (burn scars, keloids, hypertrophic scars, etc.), uterine fibroids (uterine leiomyomas, etc.), benign prostatic hyperplasia, sclerotic peritonitis (retroperitoneal fibrosis, etc.), and myelofibrosis.

Diseases that have been suggested to be associated with an increased risk of developing fibrotic diseases include, for example, diseases known to be caused by vascular endothelial damage. Specific examples of such diseases include cardiovascular diseases such as arteriosclerosis, peripheral circulatory disorders, hypertension, coronary and cerebral vasospasm, periodontal disease, type 2 diabetes, and hyperlipidemia.

Furthermore, the individuals referred to here may also be individuals with risk factors for developing vascular endothelial dysfunction. While the specific types of risk factors are not limited, examples include hypertension, hyperlipidemia, diabetes, obesity, lifestyle-related diseases, lack of exercise, smoking, excessive salt intake, and menopause.

In this specification, "lifestyle-related diseases" refers to diseases in which lifestyle habits such as eating habits, exercise habits, rest, smoking, or drinking are involved in the onset and progression of the disease. Examples include hypertension, hyperlipidemia, hyperuricemia, obesity, malnutrition, anorexia nervosa, gout, arteriosclerosis, kidney stones, kidney disease, osteoporosis, periodontitis, emphysema, periodontal disease, aneurysms, and insomnia. Hypertension refers to a condition in which the systolic blood pressure is 140 mmHg or higher, or the diastolic blood pressure is 90 mmHg or higher. Hyperlipidemia refers to a condition in which, in fasting serum, LDL cholesterol is 140 mg/dL or higher, triglycerides are 150 mg/dL or higher, or HDL cholesterol is less than 40 mg/dL. In this specification, hyperlipidemia is used synonymously with hypercholesterolemia and dyslipidemia. Hyperuricemia refers to a condition in which the serum uric acid level exceeds 7.0 mg/dL. Furthermore, a high BMI (Body Mass Index) refers to a condition where the BMI is 30 or higher. While this is not a disease in itself, it has been linked to many lifestyle-related diseases.

In this specification, "sample" refers to any biological sample taken from a subject or control individual. Examples include tissue samples and bodily fluid samples.

In this specification, "body fluid sample" refers to a liquid sample collected from a subject and a control individual. Examples include cerebrospinal fluid (CSF), blood (including serum, plasma, and interstitial fluid), urine, lymph, digestive fluid, ascites, pleural fluid, periradicular fluid, and extracts of various tissues or cells. A preferred body fluid sample is a blood sample.

In this specification, "blood sample" refers to whole blood, serum, plasma, or interstitial fluid. The origin of the blood sample is not particularly limited. For example, venous blood, arterial blood, or a combination thereof may be used.

The amount of sample used in the method of this embodiment is not particularly limited, as long as it is sufficient to measure the amount of the marker. For example, when whole blood is used as the blood sample, the amount of sample can be 100 μL or more, 200 μL or more, etc. Also, for example, when serum or plasma is used as the blood sample, the amount of sample can be 50 μL or more, 100 μL or more, etc.

The method of collecting samples is widely known in this technical field and can be appropriately selected based on the type of sample, etc. For example, in the case of body fluid samples, especially blood samples, any blood collection method can be used and is not particularly limited. Specifically, for example, peripheral blood can be collected by injecting into a peripheral vein.

The sample may be used directly in the method of this embodiment after collection, or it may be subjected to additional processing. Specific processing is not particularly limited, but examples include storage at low temperatures, concentration, dilution with physiological saline, addition of anticoagulants such as heparin, removal of unwanted components, or combinations thereof.

In this specification, "measured value" refers to a value indicating the amount of marker measured in this process. This amount may be a relative quantity expressed as fluorescence intensity, luminescence intensity, turbidity, absorbance, radiation dose, ionic intensity, or concentration, or it may be an absolute quantity such as the weight or volume of marker contained in the sample.

In this step, to correct the measured values, a known protein that is expected to have no quantitative difference in the sample may be measured as an endogenous control. For example, albumin can be used as such an endogenous control. However, in this embodiment, if a ratio or the like is calculated as a judgment value in the subsequent calculation step, correction using an endogenous control is not necessary.

The measurement methods used in this process are described below.
In this step, the amount of the marker (LOX-1 protein and/or LAB) described in the first embodiment contained in the sample derived from the subject is measured. If this step is not performed, or if one of the markers is not measured in this step, the already measured value can be used in the next calculation step.

LOX-1 protein is a protein, and LAB is a lipoprotein. Therefore, its measurement can be performed using known quantitative methods for proteins or lipoproteins, and is not particularly limited. Examples include immunological detection methods, receptor-ligand binding analysis, aptamer analysis, gel filtration HPLC, mass spectrometry, or combinations thereof.

(1) Immunological Detection Method The "immunological detection method" is the most common method for detecting and quantifying a target molecule by forming an immune complex with the target molecule using an antibody or a fragment thereof that specifically binds to the target molecule as an antigen. In the present invention, since the target molecule is the LOX-1 protein or LAB, the measurement can be performed using an anti-LOX-1 antibody and/or an anti-LAB antibody. Note that the antibody here includes an antigen-binding fragment. The antibody is as described in the third embodiment.

Specific immunological detection methods include, for example, enzyme immunoassay, fluorescence immunoassay, luminescence immunoassay, surface plasmon resonance (SPR), quartz crystal microbalance (QCM), radioimmunoassay (RIA), immunoturbidimetry, latex agglutination immunoassay, latex turbidimetry, particle agglutination reaction, gold colloid method, capillary electrophoresis, Western blotting, or immunohistochemistry (immunostaining). All of these methods are well-known and, in principle, should be carried out in accordance with the usual methods in the relevant field. For example, Current protocols in Protein Sciences, 1995, John Wiley & Sons Inc.; Current protocols in Immunology, 2001, John Wiley & Sons Inc.; Green & Sambrook, Molecular Cloning, 2012, Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Japanese Society of Clinical Pathology (ed.), Special Issue No. 53 of Clinical Pathology, Immunology for Clinical Testing. The following publications describe the methods used: "Radioimmunoassay - Techniques and Applications," Clinical Pathology Publication Association, 1983; "Enzyme Immunoassay," 3rd edition, edited by Eiji Ishikawa et al., Igaku-Shoin, 1987; "Protein, Nucleic Acid, and Enzyme Supplement No. 31: Enzyme Immunoassay," edited by Tsunehiro Kitagawa et al., Kyoritsu Shuppan, 1987; "Radioimmunoassay," edited by Minoru Irie, Kodansha Scientific, 1974; "Radioimmunoassay, Continued," edited by Minoru Irie, Kodansha Scientific, 1979; "Real-Time Analysis Experimental Methods for Biomolecular Interactions," edited by Kazuhiro Nagata and Hiroshi Handa, Springer-Verlag Tokyo, 1988; and "Sensor Engineering," Toyosaka Moriizumi and Takamichi Nakamoto, Shokodo, 1997.

Enzyme-mediated immunoassay is a method that detects a primary antibody bound to a target molecule via a labeled secondary antibody, and quantifies the target molecule based on the color intensity or fluorescence intensity generated by the labeling. For example, one method involves capturing the anti-LAB antibody of a primary antibody bound to LAB with a labeled secondary antibody that binds to the primary antibody, and indirectly measuring LAB based on the signal intensity from the label. ELISA and sandwich ELISA methods are also included in this method.

The "Surface Plasmon Resonance (SPR) method" utilizes the surface plasmon resonance phenomenon, where the reflected light intensity is significantly attenuated at a specific incident angle (resonance angle) when the incident angle of laser light irradiated onto a metal thin film is changed. This method allows for highly sensitive detection and quantification of adsorbents on the surface of a metal thin film. In this invention, for example, an anti-LOX-1 antibody is immobilized on the surface of the metal thin film, and other parts of the metal thin film surface are blocked. Then, by flowing a sample across the metal thin film surface, the LOX-1 protein can be detected and quantified from the difference in measurement values before and after sample flow. Detection and quantification using the surface plasmon resonance method can be performed, for example, using an SPR sensor commercially available from Biacore.

The "Quartz Crystal Microbalance (QCM) method" is a mass measurement method that utilizes the phenomenon where the resonant frequency of a quartz crystal decreases in proportion to the mass of a substance adsorbed onto the electrode surface attached to a quartz crystal. It quantitatively detects minute amounts of adsorbed substances by measuring the change in resonant frequency. Similar to the SPR method, detection and quantification using this method can be performed using commercially available QCM sensors to detect target molecules. In this invention, for example, a marker can be quantified by an antigen-antibody reaction between an anti-LOX-1 antibody or anti-LAB antibody immobilized on the electrode surface and a marker in the sample.

(2) Receptor-Ligand Binding Analysis Method The "receptor-ligand binding analysis method" is a method applicable when the target molecule is a ligand or a receptor, and it utilizes receptor-ligand activity to capture the other present in the sample using one and measure its amount. Since the LOX-1 protein is a specific receptor for LAB, one of the target molecules of the present invention, LAB can be detected using the LOX-1 protein. Similarly, SR-A, CD36, etc. may be used. As a specific method, for example, the LOX-1 protein or a fragment thereof having LAB-binding ability can be immobilized on a substrate, and the amount of protein complex (receptor-ligand complex) formed by binding to LAB in the sample can be measured using the immunological detection methods described above. For example, when the measurement is performed by enzyme immunoassay, it can be measured indirectly by a modified sandwich ELISA method in which the LAB-LOX-1 protein complex on the substrate is detected with a labeled anti-LAB antibody. Alternatively, with the SPR method or QCM method, the LAB-LOX-1 protein complex formed on the surface of a metal thin film or on the electrode surface can be measured directly.

Furthermore, the amount of LOX-1 protein may be measured using a similar method, conversely, by using a portion of LAB, apoB, or a substance with LOX-1 protein binding ability. In this case, the apoB or LOX-1 protein used for detection may be recombinant protein.

(3) Aptamer Analysis Method The "aptamer analysis method" is a method for quantifying a target molecule using nucleic acid aptamers or peptide aptamers. Basically, it can be performed by replacing the antibody in the immunological detection method described above with an aptamer that can specifically bind to the target molecule. In the present invention, LAB-binding aptamers and/or LOX-1 protein-binding aptamers can be used in the same way as antibodies in the immunological detection method to detect and measure LAB in the sample.

(4) Mass Spectrometry Method Mass spectrometry is a method of analyzing substances in a sample by ionizing the sample under high vacuum and separating the ions electromagnetically. When the target molecule to be detected in the sample is known, the target molecule can be detected and quantified by comparing the mass spectrum of the sample with the mass spectrum of the target molecule used as a standard. In this invention, LOX-1 protein and LAB are the target molecules.

The specific mass spectrometry methods used are not limited, but examples include high-performance liquid chromatography-mass spectrometry (LC-MS), high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS), gas chromatography-mass spectrometry (GC-MS), gas chromatography-tandem mass spectrometry (GC-MS/MS), capillary electrophoresis-mass spectrometry (CE-MS), and intercellular plasma chromatography (ICP-MS).

2-2-2. Calculation Process The "calculation process" is the process of calculating a determination value based on the amount of LOX-1 protein and/or LAB. This process can be performed simultaneously with or after the measurement process.

This step calculates a determination value (such as the ratio of LOX-1 protein to LAB) for the amount of LOX-1 protein and/or LAB. If the aforementioned measurement step is performed, this step calculates the determination value (such as the ratio of LOX-1 protein to LAB) based on the values obtained in the measurement step. If the measurement step is not performed, or if one of the markers is not measured in the measurement step, the already measured value can be used in this step.

The content of the judgment value is not particularly limited, as long as it reflects the amount of LOX-1 protein and/or LAB. For example, the judgment value can be obtained by performing calculations on the amount of LOX-1 protein and/or LAB.

The calculations performed in this case are not particularly limited. Any calculations are possible, such as arithmetic operations, logarithms, exponents, roots, or combinations thereof. Typically, the ratio of the amount of LOX-1 protein to the amount of LAB can be used as the determination value. In this case, additional calculations may be performed on the ratio.

The ratio calculated in this case is the relative value of the amount of LAB to the amount of LOX-1 protein, or its reciprocal (the relative value of the amount of LOX-1 protein to the relative value of the amount of LAB).
A "ratio" refers to the quotient value between the values of multiple indicators.

The "relative value" of the first value relative to the second value refers to the value obtained by comparing the first value with the second value, and usually refers to the value obtained by dividing the first value by the second value.

The calculation of the ratio in this process may involve additional arithmetic operations. The specific content of these additional operations is not particularly limited. For example, in addition to arithmetic operations using any number (including the values of other markers), the operation may involve exponents of this ratio, logarithms of this ratio, powers, roots, or combinations thereof. The specific content of each operation is not particularly limited. For example, the ratio may be multiplied by 10^6, the natural logarithm or common logarithm of the ratio may be taken, or a combination thereof may be used.

The means for calculating relative values in this process are not particularly limited. For example, it may be done by manual calculation or by using a calculation device.

The judgment value (ratio, etc.) calculated in this process indicates that the subject is suffering from or at risk of developing a fibrotic disease (such as a fibrotic disease accompanied by vascular endothelial damage). The details of this judgment should be followed according to the information provided in the judgment process.

2-2-3. Judgment Process The "judgment process" is a process in which, based on the judgment values (ratios, etc.) obtained in the calculation process, the presence or absence of a fibrotic disease (fibrotic disease accompanied by vascular endothelial damage, etc.) in the subject and/or the presence or absence of a risk of developing it.

"Based on the judgment value (ratio, etc.) obtained in the calculation process" means "according to the value of the judgment value (ratio, etc.) which is the result of the calculation process." While the judgment method is not particularly limited, a specific method might involve determining whether the subject suffers from or is at risk of developing a fibrotic disease (such as a fibrotic disease accompanied by vascular endothelial damage) based on a pre-set cutoff value or a comparison with a control value similarly calculated for a sample derived from a control individual.

The diseases to be assessed in this process are fibrotic diseases, such as fibrotic diseases accompanied by vascular endothelial damage. While there are no specific limitations on the diseases to be assessed, examples include: hepatic fibrosis (cirrhosis, (idiopathic) portal hypertension, viral hepatitis, etc.), renal fibrosis, scleroderma renal crisis, tubulointerstitial nephritis, interstitial pneumonia (pulmonary fibrosis, etc.), chronic obstructive pulmonary disease, viral hepatitis, chronic pancreatitis, scirrhous gastric cancer, idiopathic pulmonary arterial hypertension, pulmonary hypertension associated with connective tissue disease, hypothyroidism, slowly progressive diabetes mellitus, type 1 diabetes mellitus, etc. Examples include keratoconjunctivitis sicca, scleritis, uveitis, myocardial fibrosis (myocardial infarction, etc.), sclerosing peritonitis, systemic lupus erythematosus, scleroderma (diffuse cutaneous systemic scleroderma, localized cutaneous systemic scleroderma), Sjögren's syndrome, dermatomyositis, rheumatoid arthritis, scarring (burn scars, keloids, hypertrophic scars, etc.), uterine fibroids (uterine leiomyomas, etc.), benign prostatic hyperplasia, sclerotic peritonitis (retroperitoneal fibrosis, etc.), and myelofibrosis.

The diseases to be assessed may also be accompanied by liver dysfunction and/or autoimmune diseases (such as antinuclear antibody-positive disorders). The specific types of liver dysfunction in this case are not particularly limited, but include, for example, one or more disorders selected from the group consisting of cirrhosis, portal hypertension, hepatitis, fatty liver, and liver atrophy, such as cirrhosis and/or portal hypertension, or systemic diseases associated therewith (such as heart, lung, kidney, eye, and skin disorders). Furthermore, the specific types of autoimmune diseases are not particularly limited, but include, for example, antinuclear antibody-positive disorders, more specifically, diseases exhibiting positivity for one or more antinuclear antibodies selected from the group consisting of anti-centromere antibodies, anti-RNA polymerase III antibodies, anti-nucleolar antibodies, and anti-topoisomerase I antibodies, such as anti-centromere antibody-positive disorders and Sjögren's syndrome.

The following details each diagnostic method, but in all cases, the diagnosis may be made based on a combination of other diagnostic criteria as needed. These other diagnostic criteria are not particularly limited. For example, they may include the medical history, clinical course, physical examination findings, FMD measurement, imaging findings, histopathological findings, biomarkers (e.g., autotaxin or Fib-4 index), or combinations thereof, commonly used in the diagnosis of the target fibrotic disease (such as fibrotic disease with vascular endothelial damage).

(1) Judgment method based on cutoff value The "judgment method based on cutoff value" is a method of making a judgment based on a judgment value (ratio, etc.) calculated from the subject and a predetermined cutoff value.

In this specification, "cutoff value" refers to a value that allows for the determination of the presence or absence of disease incidence and/or the risk of developing a disease, based on that value. Preferably, the cutoff value exhibits sufficiently high sensitivity and specificity. Generally, it is derived using known methods from ROC curves drawn based on a direct comparison between the control group and the disease group, but is not limited to this. For example, the cutoff value may be set without using an ROC curve. Any known method can be used to determine the cutoff value using an ROC curve. Specific methods include, for example, the Youden Index (sensitivity - (1 - specificity)) method, and the method using the straight-line distance from the upper left corner (the point where both sensitivity and specificity are 100%).

A "Receiver Operating Characteristic (ROC) curve" is created by plotting the true position fraction (TPF), i.e., sensitivity, on the vertical axis and the false position fraction (FPF), i.e., (1 - specificity), on the horizontal axis, while varying the cutoff point, the threshold value at which a test result is judged as positive, as a mediating variable. Here, specificity is the rate at which negative individuals are accurately judged as negative.

AUC (Area Under the Curve) refers to the area under the ROC curve and represents the discriminative ability of the index associated with that ROC curve. Generally, an AUC closer to 1 indicates high discriminative ability, while an AUC closer to 0.5 indicates low discriminative ability.

In this invention, it is preferable to use cutoff values that exhibit sufficiently high sensitivity and specificity, for example, sensitivity and/or specificity of 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, or 90% or higher. Furthermore, it is preferable that the AUC, which indicates the discriminative ability of the biomarker, is 0.75 or higher, 0.8 or higher, 0.85 or higher, or 0.9 or higher. Additionally, it is preferable to use cutoff values where the sensitivity-(1-specificity) value is 0.4 or higher or 0.45 or higher.

Typically, a positive result indicates that the individual has the target disease or is at risk of developing it, while a negative result indicates that the individual does not have the target disease or is not at risk of developing it. Typically, when a value above a cutoff value is considered positive and a value below the cutoff value is considered negative, if the subject's test value (ratio, etc.) is higher than the cutoff value, the subject is judged positive, meaning they have the target disease or a high risk of developing it. Conversely, if the test value is below the cutoff value, the subject is judged negative, meaning they do not have the target disease or a low risk of developing it.

Multiple cutoff values can be used in the determination process. In this case, the criteria for setting each cutoff value are not particularly limited. For example, cutoff values may be set according to the target disease, measurement method, sample type, sample size, etc., and separate cutoff values may be set for determining the presence or absence of the disease and for determining the presence or absence of the risk of developing the disease.

For example, if cutoff values are set for determining the presence or absence of a disease and for determining the presence or absence of a risk of developing the disease, if the subject's judgment value (ratio, etc.) exceeds the cutoff value for determining the presence or absence of the disease, the subject is determined to have the disease. If the value is below the cutoff value for determining the presence or absence of the disease, and above the cutoff value for determining the presence or absence of a risk of developing the disease, the subject is determined to have a risk of developing the disease. If the value is below the cutoff value for determining the presence or absence of a risk of developing the disease, the subject is determined not to have a risk of developing the disease.

(2) Determination method based on statistical significance In the determination method based on statistical significance, it is determined that the subject has a fibrotic disease (such as a fibrotic disease accompanied by vascular endothelial damage) or is at risk of developing one, based on a comparison with a control value similarly calculated for a sample derived from a control individual.

In this specification, "statistically significant" means that when the difference between the measured value of the subject and the control value is statistically analyzed, there is a significant difference between the two. For example, when the significance level of the obtained value is small, specifically less than 5% (p<0.05), less than 1% (p<0.01), or less than 0.1% (p<0.001). Here, "p (value)" represents the probability that the test statistic will coincidentally reach that value within the distribution based on the null hypothesis in a statistical test. Therefore, the smaller "p" is, the lower the probability that the test statistic will reach that value, and the more likely the null hypothesis is to be rejected. The statistical testing method used is not particularly limited; any known test method capable of determining significance can be used as appropriate. For example, Student's t-test, paired Student's t-test, Welch's t-test, Wilcoxon rank-sum test, analysis of variance, Tukey post-hoc test, etc., can be used, but are not particularly limited.

In this specification, when a determination is made based on statistical significance, the determination is made based on a comparison with a control value similarly calculated for a sample derived from a control individual.

In this case, the control individuals are those that have been found to have the target disease (affected individuals) or those that have been found not to have the target disease (healthy individuals).

When using a diseased individual as a control, if there is no statistically significant difference between the judgment value (ratio, etc.) calculated from the sample derived from the subject and the positive control value similarly calculated from the sample derived from the diseased individual (or diseased population), it can be determined that the subject has the target disease or is at risk of developing it. Conversely, if there is a statistically significant difference between the judgment value (ratio, etc.) calculated from the sample derived from the subject and the positive control value similarly calculated from the sample derived from the diseased individual (or diseased population), it can be determined that the subject does not have the target disease or is not at risk of developing it.

Furthermore, when using healthy individuals as control subjects, if there is a statistically significant difference between the judgment value (ratio, etc.) calculated from the sample derived from the subject and the negative control value similarly calculated from the sample derived from a healthy individual (or healthy population), it can be determined that the subject has the target disease or is at risk of developing it. Conversely, if there is no statistically significant difference between the judgment value (ratio, etc.) calculated from the sample derived from the subject and the negative control value similarly calculated from the sample derived from a healthy individual (or healthy population), it can be determined that the subject does not have the target disease or is not at risk of developing it.

In this specification, "affected individual" refers to an individual that is clearly diagnosed with the target disease, and, in principle, an individual of the same animal species as the subject, diagnosed with the target disease by a physician or other medical professional. Preferably, it is an individual that is diagnosed with the target disease.

In this specification, "healthy individual" refers to an individual that is clearly not suffering from the target disease, and, in principle, an individual of the same animal species as the subject, diagnosed by a physician or other medical professional as not suffering from the target disease. Preferably, it is an individual that is not suffering from any of the diseases.

In this specification, "control population" refers to a group consisting of multiple control individuals of the same animal species. The number of individuals is not particularly limited as long as it is two or more, but preferably five or more, more preferably ten or more, and even more preferably fifteen or more. Each individual constituting this population is preferably of the same animal species as the subject, of the same sex, and has the same or similar physical conditions as the subject, such as age, height, and weight.

The "control value" is a judgment value (ratio, etc.) calculated from the amounts of markers, namely LOX-1 protein and LAB, contained in samples taken from each individual constituting the control population. This judgment value (ratio, etc.) is, in principle, calculated using the same method as for the subject, based on measurements obtained using the same sample and measurement method as those used in the measurement process for the subject.

The measurement and calculation of control values may be performed simultaneously with or separately from the measurement and calculation of the subject. For example, the determination may be made using pre-measured and calculated values provided in a database or similar format; the determination may be made based on a comparison with values usable for determining statistical significance, such as confidence intervals; or the determination may be made based on a comparison with judgment values (proportions, etc.) calculated based on results measured at the same facility and/or at the same time as the measurement of the subject.

In the determination process, multiple significance levels and/or statistical tests may be used. In this case, the criteria for setting each significance level and statistical test are not particularly limited. For example, significance levels may be set according to the target disease, measurement method, sample type, sample size, etc., and separate significance levels may be set for determining the presence or absence of the disease and the presence or absence of the risk of developing the disease. Furthermore, for example, a significant trend and a statistically significant result may provide different determination results.

For example, if significance levels are set for determining the presence or absence of disease and for determining the presence or absence of risk of developing the disease, then, for instance, if the difference between the subject's judgment value (ratio, etc.) and the judgment value (ratio, etc.) of a healthy individual is significant based on the significance level for determining the presence or absence of disease, then the subject is determined to have the target disease. If the difference is not significant based on the significance level for determining the presence or absence of disease, but is significant based on the significance level for determining the presence or absence of risk of developing the disease, then the subject is determined to have a risk of developing the target disease. If the difference is not significant even based on the significance level for determining the presence or absence of risk of developing the disease, then the subject is determined to not have a risk of developing the target disease.

2-3. Effects When vascular endothelial cells, which make up the inner lining of blood vessels throughout the body, become dysfunctional, the risk of arteriosclerosis and thrombus formation increases. Not only do these pathological conditions increase the risk of tissue fibrosis, but the alteration of the properties of vascular endothelial cells themselves also increases the risk of tissue fibrosis. For example, various mechanisms have been pointed out, such as the induction of an inflammatory response at the site of damage due to damage to vascular endothelial cells, the alteration of vascular endothelial cells to fibroblast-like properties due to inflammatory cytokines, and the migration of cells that would not normally flow into the bloodstream to other tissues via the bloodstream due to dysfunction of vascular endothelial cells, which are responsible for the barrier function of blood vessels, and the induction of an excessive inflammatory response due to the presence of ectopic cells at the destination.

The method of the present invention is based on markers that reflect damage to vascular endothelial cells that occurs prior to fibrosis, and allows for early detection of fibrosis.

Furthermore, the LOX-1 protein used in the method of the present invention is expressed in vascular endothelial cells throughout the body, and LAB is present in the blood throughout the body. Therefore, according to the method of the present invention, fibrosis can be comprehensively detected in any organ or tissue, regardless of the site of fibrosis.

Furthermore, unlike the LOX-index ^(R) , which focuses on the accumulation of LAB and the resulting increase in LOX-1 protein, the marker of the present invention can detect the onset of fibrotic diseases by capturing pathological conditions in which LOX-1 protein expression is induced by mechanisms other than LAB stimulation. This makes it possible to detect other pathological conditions that could not be detected by LOX-index ^(R) , even while using the same molecular abundances as those used to measure LOX-index ^(R) .

3. Fibrotic Disease Diagnosis Kit and Device 3-1. Overview A third aspect of the present invention is a fibrotic disease diagnosis kit and device. The kit and device of this aspect include a LOX-1 protein-binding molecule and a LAB-binding molecule as essential components. The kit and device of this aspect can be used in the method described in the second aspect to determine whether or not a person has a fibrotic disease (such as a fibrotic disease accompanied by vascular endothelial damage) or whether or not they are at risk of developing it.

3-2. Composition The LOX-1 protein-binding molecule and the LAB-binding molecule, which are components of the kit of the present invention, will be described in detail below.

"LOX-1 protein-binding molecule" refers to a drug that has specific binding ability to LAB, and "LAB-binding molecule" refers to a drug that has specific binding ability to LAB.

The two binding molecules differ only in whether the target molecule is the LOX-1 protein or LAB; their basic structural requirements are the same.

3-2-1. Binding molecule The binding molecule may consist of a peptide, nucleic acid, small molecule compound, or a combination thereof. The binding molecule in this embodiment may also include additional elements, such as a labeling substance, as needed.

In this specification, "labeled substance" refers to a substance that emits a signal that can be detected. Examples of labeled substances include fluorescent molecules, luminescent labeling substances that emit light under specific conditions such as chemiluminescent substances, sound-emitting labeling substances that emit sound waves such as photoacoustic effect probes, and radioactive labeling substances. Examples of fluorescent molecules, though not limited to them, include fluorescent proteins, fluorescein and its derivatives, pyrene and its derivatives, and quantum dots. Examples of chemiluminescent substances include enzymes such as peroxidase (HRP) and alkaline phosphatase (ALP). Examples of radioactive labeling substances include reagents containing ^14C , ^3H , ^125I , etc. The photoacoustic effect is a phenomenon in which thermoelastic waves are generated by adiabatic expansion accompanying light absorption, and these thermoelastic waves can be detected as acoustic waves. Examples of photoacoustic effect probes include indocyanine green or its derivatives, curcumin derivatives, or choline derivatives. If the absorbance properties of the binding molecule and labeling substance used are known, it is not always necessary to use a labeling substance for photoacoustic effects; for example, a luminescent labeling substance may be detected based on its photoacoustic effect.

(1) Peptide binding molecules: When the binding molecule is composed of a peptide, the specific peptide used is not particularly limited. Examples include antibodies, peptide aptamers, and receptor or ligand proteins.

(i) Antibodies that can be used as binding molecules refer to antibodies that can immunologically and specifically bind to LOX-1 protein or LAB as an antigen, and in this specification also include fragments thereof that have antigen-binding ability.

The species from which the antibodies originate is not particularly limited. They can be derived from animals, including mammals and birds. Examples of such animals include mice, rats, guinea pigs, rabbits, goats, donkeys, sheep, camels, horses, chickens, or humans.

Any type of antibody may be used, including polyclonal antibodies, monoclonal antibodies, recombinant antibodies, synthetic antibodies, and combinations thereof.

A "polyclonal antibody" refers to a group of multiple immunoglobulins that recognize and bind to different epitopes of the same antigen. Polyclonal antibodies can be obtained from the serum of animals after immunizing them with a target molecule (in this case, LOX-1 protein or LAB) as an antigen. Polyclonal antibodies obtained using LOX-1 protein as an antigen are referred to as "anti-LOX-1 polyclonal antibodies" in this specification, and polyclonal antibodies obtained using LAB as an antigen are referred to as "anti-LAB polyclonal antibodies" in this specification.

A "monoclonal antibody" refers to a group of antibodies consisting of clones of a single immunoglobulin. Each immunoglobulin constituting a monoclonal antibody contains a common framework region (FR) and a common complementarity-determining region (CDR), enabling them to recognize and bind to the same epitope of the same antigen. Monoclonal antibodies can be obtained from hybridomas derived from single cells. Monoclonal antibodies obtained using the LOX-1 protein as an antigen are referred to as "anti-LOX-1 monoclonal antibodies" in this specification, and monoclonal antibodies obtained using LAB as an antigen are referred to as "anti-LAB monoclonal antibodies" in this specification.

A typical immunoglobulin molecule is composed of a tetramer in which two sets of polypeptide chains, called a heavy chain and a light chain, are interconnected by disulfide bonds. The heavy chain consists of a variable heavy chain region at the N-terminus (V region of the heavy chain; hereinafter referred to as "VH") and a constant heavy chain region at the C-terminus (C region of the heavy chain; hereinafter referred to as "CH"), while the light chain consists of a variable light chain region at the N-terminus (V region of the light chain; hereinafter referred to as "VL") and a constant light chain region at the C-terminus (C region of the light chain; hereinafter referred to as "CL"). Of these, VH and VL are particularly important in that they are involved in the binding specificity of antibodies. Both VH and VL consist of approximately 110 amino acid residues and contain three CDRs (CDR1, CDR2, CDR3) that are directly involved in antigen binding specificity, and four FRs (FR1, FR2, FR3, FR4) that function as the backbone structure of the variable region, in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the N-terminus. CDRs are known to form a complementary three-dimensional structure with the antigen molecule and determine the specificity of the antibody (E. A. Kabat et al, 1991, Sequences of proteins of immunological interest, Vol. 1, eds. 5, NIH publication). In the variable region, the CDRs and FRs are arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the N-terminus to the C-terminus. Within immunoglobulin molecules, VL and VH form antigen-binding sites by dimerizing relative to each other.

When the antibody is a polyclonal or monoclonal antibody, immunoglobulin molecules are known to belong to the classes IgG, IgM, IgA, IgE, and IgD; however, the antibody of the present invention may belong to any of these classes. For example, IgG can be suitably used.

The specific method for producing a hybridoma that recognizes and binds to the marker of the present invention is not particularly limited and can be produced in accordance with antibody production methods known in the art.

In this specification, "recombinant antibody" includes chimeric antibodies, humanized antibodies, or multispecific antibodies. A "chimeric antibody" is an antibody produced by combining the amino acid sequences of antibodies derived from different animals, where the V region of one antibody is replaced with the V region of another antibody. For example, an antibody in which the V region of a mouse-derived anti-human LAB monoclonal antibody that specifically binds to human LABs is replaced with the V region of a human antibody, resulting in an antibody where the V region is mouse-derived and the C region is human-derived. A "humanized antibody" refers to a graft antibody in which the CDRs (CDR1, CDR2, CDR3) in the V region of an antibody derived from a mammal other than a human, such as a mouse, are replaced with CDRs from a human antibody. A "multispecific antibody" is a polyvalent antibody, i.e., an antibody having multiple antigen-binding sites within a single molecule, where each antigen-binding site binds to a different epitope. For example, in the case of an antibody with two antigen-binding sites, such as IgG, a bispecific antibody is an example where each antigen-binding site specifically binds to the same or different markers described in the first embodiment.

In this specification, "synthetic antibody" refers to an antibody synthesized by chemical methods or recombinant DNA methods. For example, this includes a monomeric polypeptide molecule or a polymer polypeptide thereof, obtained by artificially linking one or more VLs and one or more VHs of a specific antibody via a linker peptide of appropriate length and sequence. Specific examples of such polypeptides include single-chain Fv (scFv: single-chain Fragment of variable region) (see Pierce Catalog and Handbook, 1994-1995, Pierce Chemical Co., Rockford, IL), scFv-Fc, sc(Fv) ₂ , Fv, diabody, triabody, or tetrabody.

In immunoglobulin molecules, the VL and VH regions are usually located on separate polypeptide chains (L and H chains). A "single-stranded Fv" is a synthetic antibody fragment that has a structure in which the V regions on these two polypeptide chains are linked by a sufficiently long flexible linker and incorporated into a single polypeptide chain. Within the single-stranded Fv, both V regions can self-assemble to form a single functional antigen-binding site. Single-stranded Fv can be obtained by incorporating the recombinant DNA encoding it into a phage genome using known techniques and then expressing it.

A "diabody" is a molecule based on the dimeric structure of a single-stranded Fv molecule, possessing two functional antigen-binding sites (Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-6448). For example, if the length of the linker is shorter than 12 amino acid residues, the two variable sites within the single-stranded Fv molecule cannot structurally self-assemble. However, by forming a diabody and causing two single-stranded Fv molecules to interact with each other, the VL of one Fv chain can assemble with the VH of the other Fv chain, thereby forming two functional antigen-binding sites (Marvin et al., 2005, Acta Pharmacol. Sin. 26:649-658). Furthermore, by adding a cysteine residue to the C-terminus of a single-chain Fv, disulfide bonds can be formed between two Fv chains, creating a stable diabody (Olafsen et al., 2004, Prot. Engr. Des. Sel. 17:21-27). "Triabodies" and "tetrabodies," like diabodies, have trimer and tetramer structures based on a single-chain Fv structure. They are trivalent and tetravalent antibody fragments, respectively, and may also be multispecific antibodies.

In the case of diabodies and other multivalent antibodies, each antigen-binding site may bind to the same epitope, or it may possess multispecificity, recognizing and specifically binding to different epitopes.

In this specification, "the active fragment" refers to a partial fragment of the polyclonal or monoclonal antibody described above, which is a polypeptide chain or complex thereof having substantially equivalent activity to the antigen-specific binding activity of the antibody. For example, this includes an antibody portion containing at least one antigen-binding site, i.e., a polypeptide chain having at least one pair of VL and VH, or a complex thereof. Specific examples include numerous well-characterized antibody fragments produced by cleaving immunoglobulins with various peptidases. More specific examples include Fab, F(ab') ₂ , Fab', etc. Fab is a fragment produced by papain cleaving an IgG molecule at a position adjacent to the N-terminus of the disulfide bond in its hinge region, and consists of a polypeptide comprising CH1 adjacent to VH among the three domains (CH1, CH2, CH3) that constitute VH and CH, and a light chain. F(ab') ₂ is a dimer of Fab' produced by pepsin cleaving an IgG molecule at a position adjacent to the C-terminus of the disulfide bond in its hinge region. Fab' has a slightly longer H chain due to the inclusion of a hinge region, but is essentially structurally equivalent to Fab (Fundamental Immunology, Paul ed., 3d ed., 1993). Fab' can be obtained by reducing F(ab') ₂ under mild conditions and cleaving the disulfide linkage in the hinge region. All of these antibody fragments contain an antigen-binding site and have the ability to specifically bind to the target molecule, which is the antigen.

(ii) Peptide Aptamers An "aptamer" is a ligand molecule that has the ability to bind strongly and specifically to a target substance due to its three-dimensional structure. Aptamers can be broadly classified into nucleic acid aptamers and peptide aptamers depending on the type of molecule that makes up the aptamer.

A "peptide aptamer" is an aptamer composed of amino acids, similar to an antibody, that recognizes the surface structure of a target molecule and specifically binds to the target substance based on its three-dimensional structure. It is a peptide molecule with a diameter of 1 kD to 6 kD. The production method for peptide aptamers can be based on methods known in the relevant field. For example, one can refer to Whaley, S.R., et al., 2000, Nature, 405, 665-668, which specifically describes production methods using phage display or cell surface display.

(iii) Receptor or ligand protein Examples of "LAB receptor protein" include the LOX-1 protein or a fragment thereof having LAB binding ability, as described in relation to the measurement step of the second embodiment. The LOX-1 protein was described in detail in the first embodiment, so its description is omitted here. The LOX-1 protein here may be a natural protein or a recombinant protein.

A "recombinant protein" is a protein obtained by expressing a gene encoding a protein, obtained through gene cloning technology, in a gene expression system using host cells. Recombinant proteins may be prepared by expressing the gene encoding the target protein in host cells according to standard methods in this field, or commercially available recombinant proteins may be used.

In this specification, the LOX-1 gene used is preferably the LOX-1 gene derived from the animal to which the kit and device of this embodiment are applied. For example, if the target is human, the human LOX-1 gene is preferably used. The LOX-1 gene in this specification includes both wild-type and mutant forms. The wild-type human LOX-1 gene is the gene that encodes the human LOX-1 protein consisting of the amino acid sequence shown in SEQ ID NO: 2. Specifically, examples include polynucleotides consisting of the base sequence shown in SEQ ID NO: 1.

Examples of "LOX-1 ligands" include LAB, apoB, or a subset of those having LOX-1 protein binding ability, as described in relation to the measurement step of the second embodiment. Here, LAB may be a natural lipoprotein or a lipoprotein containing recombinant protein.

In this specification, the apoB gene preferably used is the apoB gene derived from the animal to which the kit and device of this embodiment are applied. For example, if the target is a human, then the human apoB gene is used. In this specification, the apoB gene includes both wild-type and mutant forms. The wild-type human apoB gene is a gene encoding human apoB or a part thereof, consisting of the amino acid sequence shown in SEQ ID NO: 6 or 8. Specifically, examples include polynucleotides consisting of the base sequence shown in SEQ ID NO: 9, polynucleotides consisting of the base sequence shown in SEQ ID NO: 7, and so on.

(2) When the nucleic acid binding molecule is composed of nucleic acids, specific examples, though not limited to them, include nucleic acid aptamers.

A "nucleic acid aptamer" refers to an aptamer composed of nucleic acids. The nucleic acids constituting a nucleic acid aptamer may be DNA, RNA, or a combination thereof. If necessary, chemically modified nucleic acids such as PNA, LNA/BNA, methylphosphonate DNA, phosphorothioate DNA, and 2'-O-methyl RNA may also be included.

Nucleic acid aptamers can be prepared based on the target molecule using methods known in the field. For example, RNA aptamers can be prepared by in vitro selection using the SELEX (systematic evolution of ligands by exponential enrichment) method. The SELEX method involves selecting RNA molecules bound to the target molecule from an RNA pool consisting of numerous RNA molecules with random sequence regions and primer-binding regions at both ends, recovering them, amplifying them by RT-PCR, transcribing the resulting cDNA molecule using it as a template, and then using this as the RNA pool for the next round. This cycle is repeated for several to tens of rounds to select RNA with stronger binding affinity to the target molecule. In this case, there are no particular limitations on the base sequence lengths of the random sequence region and the primer-binding region. Generally, the random sequence region is in the range of 20 to 80 bases, and the primer-binding region is in the range of 15 to 40 bases. The RNA molecules finally obtained by the above method are used as LAB-binding RNA aptamers. The specific method can be followed, for example, in accordance with Pan et al. (Proc. Natl. Acad. Sci. 1995, U.S.A., 92:11509-11513).

3-2-2. Other Components The kit and device of the present invention may further include means for collecting a sample, a container for recovering the sample, a reagent for purifying the sample, means for calculating a determination value (ratio, etc.), a dilution or reaction buffer containing components necessary for measurement, a washing solution, a colorimetric reagent, a reaction vessel, etc.

The kit and device of the present invention may include a container. The material of the container is not limited as long as it does not contaminate the contents or is not contaminated by the contents. Examples include plastics such as polypropylene and polystyrene, glass, or paper with a special surface coating.

When detection equipment is included, its type is not particularly limited. The detection method can be appropriately selected according to the label and reagents used, and the properties of the sample, and the equipment necessary for that detection method can be used. For example, when a fluorescent dye or luminescent substance is used, detection can be performed by visual inspection, using a microscope (e.g., a fluorescence microscope), using a detector (e.g., a photometer, absorbance spectrophotometer), or a combination thereof. When a non-colored low-molecular-weight substance that acts as an enzyme substrate or antigen is used as a label, detection can be performed, for example, after processing such as enzyme treatment, using the same detection method as when a fluorescent dye or luminescent substance is used. When a radioisotope is used as a label, detection can be performed by autoradiography, scintillation counter, positron emission tomography (PET), or a combination thereof.

The kit and device of the present invention may, as needed, include instructions for use, as well as application methods such as syringes, droppers, and micropipettes, and storage methods.

The kits and devices of the present invention are used in the method described in the second embodiment. Therefore, the kits and devices of the present invention can be configured as kits for any purpose described in the second embodiment (e.g., any target disease and type of sample).

In the kit and device of the present invention, the above-mentioned binding molecules may be immobilized on a substrate. "Substrate" refers to a solid-phase support for immobilizing the binding molecules.

The substrate material should be one that allows for the direct or indirect solidification of binding molecules onto its surface. While not limited, it is preferable that the material be water-insoluble. Examples include plastics, glass, metals, ceramics, natural resins (e.g., natural rubber or lacquer), natural or chemical fibers or aggregates thereof (e.g., paper, nonwoven fabrics, filters), polysaccharide polymers (e.g., agar), gelling proteins (e.g., gelatin, collagen), or mixtures thereof.

The material used should be appropriately selected depending on the measurement method used. For example, when measuring using enzyme immunoassay methods such as ELISA, fluorescence, or colorimetric methods, plastic or glass is preferred for reasons of cost, processing, and operation, although there are no limitations. Furthermore, while the appearance of the substrate is not particularly limited, transparent materials are preferably used in this case. Specifically, if using plastic, examples include polyvinyl chloride, polyvinylidene chloride, polystyrene, polyurethane, polysulfone, polycarbonate, polyarylate, polyamide, and polyvinyl alcohol. Also, when measuring with SPR or QCM sensors, metals such as gold (Au), platinum (Pt), silver (Ag), and copper (Cu) that constitute the sensor chip are preferred.

The shape of the substrate can be appropriately determined depending on the application of the kit and device of the present invention. Examples of shapes include plates (including rectangular plates such as 96-well microtiter plates), dishes, tubes, sticks, beads, plates, or test pieces. When solidifying the substrate on a bead surface, the substrate can be a sphere with a diameter of approximately 1 μm to approximately 1 cm. Furthermore, for example, if the kit and device of the present invention are used as a sensor chip for an SPR measurement sensor, the substrate should be shaped to match the SPR measurement sensor being used.

The substrate can also be a multilayer structure composed of two or more materials. For example, a substrate in which a thin gold film is laminated on a glass surface falls into this category. When the substrate has such a multilayer structure, at least the layers constituting the substrate surface are composed of materials capable of solidifying binding molecules.

The "substrate surface" refers to the portion of the substrate that can come into direct contact with the sample taken from the subject. Therefore, the substrate surface varies depending on the shape of the substrate used. For example, if the substrate is plate-shaped, such as a microscope slide, the substrate surface includes both the front and back surfaces, as well as the sides. If the substrate is tube-shaped, the substrate surface includes the outer surface, inner surface, and cross-section. Furthermore, if the substrate is spherical, the substrate surface is generally the outer surface of the sphere; however, if the substrate has an internal space that is partially open to the outside, the internal surface is also included. Examples include hollow beads and porous materials.

"Immobilization on the substrate surface" refers to fixing binding molecules to the substrate surface. The method of immobilization is not particularly limited. Examples include chemical adsorption, physical adsorption, affinity, or a combination thereof. Chemical adsorption includes chemical bonds such as covalent or ionic bonds. Physical adsorption includes van der Waals forces.

The kit and device of this embodiment may further include additional components used for disease determination, as needed. The specific additional components are not particularly limited, but may include, for example, multiple types of the aforementioned binding molecules, means for detecting other markers as exemplified in the second embodiment, elements used in other diagnostic criteria (e.g., questionnaires), or combinations thereof.

3-3. Other Aspects The present invention further relates to a composition for use in the method of the second aspect, comprising a LOX-1 protein-binding molecule and/or a LAB-binding molecule.
The compositions of this embodiment may optionally include a carrier and/or a solvent.

The carriers and media used in the compositions of this embodiment are not particularly limited, as long as they facilitate the use of the composition, the detection and measurement of the marker, maintain the binding ability of the active ingredient binding molecule, and/or control the rate of action. For example, solutions or additives commonly used in the measurement method used may be used as carriers or solvents.

Specific examples of carriers usable in this invention include protective agents, antioxidants, UV absorbers, emulsifiers, chelating agents, and pH buffers. These carriers may be added beforehand or immediately before use.

Protective agents are expected to have effects such as reducing damage caused by ultraviolet rays. Examples include skim milk, casein, and gelatin.

Examples of antioxidants include phenolic antioxidants, sulfur-based antioxidants, and phosphorus-based antioxidants.

Examples of UV absorbers include benzotriazole-based UV absorbers, benzophenone-based UV absorbers, other organic UV absorbers (e.g., para-aminobenzoic acid-based, oxybenzone-based, cinnamic acid-based, urocanic acid-based, anti-fading agents, etc.), and inorganic UV absorbers (e.g., titanium dioxide, zinc oxide, silica, talc, and kaolin, etc.).

Examples of emulsifiers include sorbitan fatty acid esters, glycerin fatty acid esters, sucrose fatty acid esters, and propylene glycol fatty acid esters.

Examples of chelating agents include EDTA, EGTA, citric acid, and salicylates.

Examples of pH adjusters include alkaline agents such as sodium hydroxide and potassium hydroxide, and acidic agents such as citric acid, sodium citrate, glycolic acid, and ascorbic acid.

A pharmaceutically acceptable carrier can be used as the carrier. "Pharmaceutically acceptable carrier" refers to additives commonly used in the field of pharmaceutical formulation technology. Examples include, in addition to those mentioned above, excipients, binders, disintegrants, fillers, flow additive modifiers, lubricants, and human serum albumin.

Excipients include, for example, sugars such as monosaccharides, disaccharides, cyclodextrins, and polysaccharides, metal salts, citric acid, tartaric acid, glycine, polyethylene glycol, ^Pluronic® , kaolin, silicic acid, or combinations thereof.

Examples of binders include starch paste made from plant starch, pectin, xanthan gum, simple syrup, glucose solution, gelatin, tragacanth, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, shellac, paraffin, polyvinylpyrrolidone, or combinations thereof.

Examples of disintegrants include the aforementioned starch, lactose, carboxymethyl starch, cross-linked polyvinylpyrrolidone, agar, laminaran powder, sodium bicarbonate, calcium carbonate, alginic acid or sodium alginate, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, monoglyceride stearate, or salts thereof.

Examples of fillers include petrolatum, the aforementioned sugars, and/or calcium phosphate.

Examples of fluidity modifiers and lubricants include silicates, talc, stearates, or polyethylene glycol.

In addition to the above, if necessary, the composition may also contain solubilizers, suspending agents, diluents, dispersants, surfactants, stabilizers, bulking agents, humectants, moisturizers, wetting agents, adsorbents, deodorizers, disintegration inhibitors, coating agents, colorants, preservatives, antiseptics, buffering agents, isotonic agents, etc., which are commonly used in pharmaceutical compositions.

The solvent used in the composition of the present invention is not particularly limited as long as it can maintain the binding ability of the active ingredient (binding molecule) and maintain the detection ability of the marker.

The solvents usable in this invention may include not only solvents commonly used for measuring biomolecules, but also pharmaceutically acceptable solvents. "Pharmaceutically acceptable solvents" refers to solvents commonly used in the pharmaceutical technology field. Examples include water or aqueous solutions, or organic solvents. Aqueous solutions include, for example, physiological saline, isotonic solutions containing glucose or other adjuvants, phosphate buffers, phosphate-buffered saline, sodium acetate buffer, glycol, or ethanol solutions. Adjuvants include, for example, D-sorbitol, D-mannose, D-mannitol, sodium chloride, and other low concentrations of nonionic surfactants, polyoxyethylene sorbitan fatty acid esters, etc. An example of an organic solvent is ethanol.

The compositions of this embodiment, like those of the kit and device, may further include additional components used for disease determination, as necessary. The specific details of these additional components are as described above for the kit and device.

3-4. Applications The kit, device, and composition of this embodiment are used in methods for diagnosing fibrotic diseases (such as methods for diagnosing fibrotic diseases accompanied by vascular endothelial damage). The details of the diagnosing method are as described in the second embodiment.

The diseases to be differentiated are as described in the second embodiment, and are not particularly limited. Depending on the type of disease to be differentiated, the kits, devices, and compositions of this embodiment can be, for example, kits, devices, and compositions for differentiating fibrotic diseases (fibrotic diseases accompanied by vascular endothelial damage, etc.), scleroderma, hepatic fibrosis, liver damage, systemic diseases accompanied by liver damage (disorders of the heart, lungs, kidneys, eyes, skin, etc.), autoimmune diseases, antinuclear antibody-positive disorders, anticentromere antibody-positive disorders, Sjögren's syndrome, portal hypertension, etc.

<Example 1. Measurement of Markers>
(the purpose)
To explore markers that change depending on the presence or absence of anti-centromere antibody (ACA) positive disorder, which is a disorder accompanied by fibrosis, the amounts of various markers will be measured in patient samples.

(method)
1. Subjects In this example, anti-centromere antibody (ACA) positive disorder was used as the fibrotic disease.
Six patients who were ACA-positive were selected from those with a history of outpatient visits to the Department of General Medicine at Tokyo Medical and Dental University, and whose ACA concentration detected by quantitative anti-centromere antibody testing (SRL Co., Ltd.) on serum samples was 10.0 U/mL or higher. These patients were later confirmed to be ACA-positive through the measurements described below.

As ACA-negative patients, we used 19 patients who had a history of outpatient visits to the Department of General Medicine at Tokyo Medical and Dental University and who had one or more of the following conditions: diabetes, dyslipidemia (LDL ≥ 120 or taking medication), or obesity (fatty liver). These patients were confirmed to be ACA-negative in the measurements described below.

Blood samples were collected from each patient after obtaining informed consent. Blood was collected in dedicated test tubes for measuring LOX-1 protein and LAB levels.

2. Measurements For each patient, routine blood tests (conducted at Tokyo Medical and Dental University Hospital) were performed during outpatient consultations to measure platelet count, AST, ALT, hyaluronic acid, and type IV collagen.

The concentration of ACA was measured using an anti-centromere antibody quantitative test (SRL Co., Ltd.). A concentration of 10.0 U/mL or higher was considered positive, and a concentration below 10.0 U/mL was considered negative.
LOX-1 protein and LAB levels were measured at Primedica.

(1) Measurement of LAB amount The LAB amount was measured in accordance with Japanese Patent Publication No. 2021-042986. The measurement method is roughly as follows.

Plasma was obtained by collecting the supernatant after centrifugation.
Plasma from each patient was dispensed in 100 μL portions into each well of an sLOX-1 immobilized plate and incubated at room temperature for 2 hours. The sLOX-1 immobilized plate used was a 96-well microplate in which sLOX-1 protein was immobilized by adding 100 μL of human recombinant soluble LOX-1 (sLOX-1) protein solution (5 μL/mL) consisting of the amino acid sequence shown in SEQ ID NO: 3 to each well, and then blocked with a blocking solution (diluted with distilled water to make a 3% Block Ace, 2% sucrose).

HRP-labeled anti-Apo antibody solution was dispensed into each well of a plasma-removed sLOX-1 immobilized plate in 100 μL increments. The mixture was stirred at 1000 rpm for 1 minute and then incubated at room temperature for 1 hour. The HRP-labeled anti-Apo antibody solution used was a PBS solution (concentration: 1 μg/mL) of HRP-labeled chicken anti-human apolipoprotein monoclonal antibody (HUC20: Creative Biolabs: conjugates to both apoB-48 and apoB-100), diluted 420-fold with dilution PBS solution (containing 0.4% Block Ace).

The HRP-based signals were detected by dispensing 100 μL of luminescent solution into each well and using a plate reader (Infinite ^(R) 200 PRO: Tecan Japan Co., Ltd.), and quantified based on the light intensity. As the luminescent solution, a 1:1 mixture of Peroxide Solution and Luminol/Enhancer Solution included with SuperSignal ^™ ELISA Pico Chemiluminescent Substrate (Thermo Fisher Scientific) was used.

(2) Measurement of LOX-1 protein amount The quantitative value of sLOX-1 protein was used as the LOX-1 protein amount. The measurement of sLOX-1 protein amount was performed according to the procedure described in Inoue et al, 2010, Clinical Chemistry 56(4) (doi:10.1373/clinchem.2009.140707). The general procedure is the same as for the measurement of LAB amount, except that anti-human LOX-1 antibody was used instead of sLOX-1 and anti-human LOX-1 antibody was used instead of HRP-labeled anti-Apo antibody.

3. Calculation The value of each marker was calculated using the following method:
LOX-index ^(R) : LOX-1 protein amount (ng/L) × LAB amount (μg/L);
LOX/LAB: LOX-1 protein amount (ng/L)/LAB amount (μg/L);
Fib4-index: {age (years) x AST (IU/L)} / {platelet count (10^9/L) x √ALT (IU/L)}.

<Example 2. Evaluation of the marker of the present invention>
(the purpose)
The differential performance of the markers of the present invention is evaluated based on the measurement results of each marker obtained from patient samples and their comparison with the pathological condition of each patient.

(method)
1. Examination of the pathological condition: Flow-dependent vasodilation (FMD) was tested using UNEXEF18VG (UNEX Corporation).

The severity of fibrosis was determined according to the New Inuyama classification based on the observation of fibrotic liver tissue in pathological biopsy. However, in cases where liver biopsy was not possible, morphological evaluation using CT or MRI, and measurement of liver stiffness by MR elastography were used as alternative methods. Furthermore, the severity was assessed through evaluation of liver fibrosis using abdominal ultrasound elastography and a comprehensive evaluation combining blood test data such as ELF test, type IV collagen 7S, M2BPGi, autotaxin, and hyaluronic acid.
Other findings were obtained based on the medical history, physical examination of the patient, and blood pressure measurement.

2. Statistical Analysis: The Wilcoxon two-sample test was used to compare the two groups.
To analyze the correlation between the two markers, we used a test of no correlation based on Spearman's rank correlation coefficient.

For the analysis of unit odds ratios related to LOX/LAB and FIB-4 index, logistic regression analysis was performed with ACA positive/negative as the dependent variable and the LOX-1/LAB ratio and FIB-4 index as independent variables.
All statistical analyses were performed using JMP (version 18, JMP Statistical Discovery).

(result)
The results are shown in Table 1 and Figures 1-7.
As shown in Figure 1, LOX-1 protein levels were significantly higher in the ACA-positive group compared to the ACA-negative group. However, there was a large overlap in the distribution ranges of the values in both groups, making it difficult to clearly distinguish between the two groups based solely on these values. Furthermore, the distribution remained unchanged even when the natural logarithm of the LOX-1 protein levels was taken (Figure 2).

On the other hand, as shown in Figure 3, LAB levels were significantly lower in the ACA-positive group compared to the ACA-negative group. However, low LAB levels, i.e., low oxidized LDL levels, alone are not sufficient to distinguish between healthy individuals with a low risk of lifestyle-related diseases and those with low LAB levels.

Therefore, when we compared the LOX-index ^(R) values, which are the product of LAB levels and LOX-1 protein levels, none of the patients examined were classified as high-risk for stroke or myocardial infarction, and there was no significant difference between the two groups (Figure 4). In addition, individuals in the ACA-positive group had low LAB levels, and all of them were classified as low-risk or medium-high-risk for stroke or myocardial infarction based on the LOX-index ^(R) .

Furthermore, when we examined hyaluronic acid (Figure 5A) and type IV collagen (Figure 5B), which are extracellular matrix-related factors and are considered common liver fibrosis markers, we found no significant difference between the ACA-positive and ACA-negative groups for either marker. This result is consistent with the general understanding that these markers do not have high specificity for discriminating fibrosis.

The FIB-4 index is a well-established marker for liver fibrosis. Therefore, a comparison using the FIB-4 index (Figure 6) showed that the ACA-positive group had significantly higher FIB-4 scores compared to the ACA-negative group. However, only 2 out of 6 ACA-positive individuals had an FIB-4 index of 2.67 or higher (above the dashed line in Figure 6), which is considered high.

On the other hand, in the case of LOX/LAB, the marker of the present invention (Figure 7), the values were significantly higher in the ACA-positive group compared to the ACA-negative group, and the distribution ranges of the values in both groups were clearly distinguishable. Furthermore, LOX/LAB showed a significant correlation with the FIB-4 index (p = 0.0326), demonstrating that this marker is a highly reliable indicator.

To further clarify the accuracy of the FIB-4 index and LOX/LAB, the unit odds ratios for each marker were calculated using ACA positivity as the dependent variable and the FIB-4 index value and the natural logarithm of LOX/LAB as independent variables. The results are shown in Table 1.

As shown in Table 1, the unit odds ratio for log(LOX/LAB) was larger than that of the Fib4-index. The unit odds ratio directly reflects the coefficients of the explanatory variables in a logistic regression equation where the dependent variable is expressed as a polynomial of the explanatory variables. This indicates that log(LOX/LAB) has a higher contribution to ACA positivity compared to the Fib4-index. Furthermore, since the confidence interval for the unit odds ratio of log(LOX/LAB) does not cross 1, it is shown that log(LOX/LAB) significantly contributes to ACA positivity compared to the Fib4-index.

Furthermore, the degree of vascular endothelial damage, the severity of fibrosis, and other findings in ACA-positive individuals are shown in Table 2 below.

As shown in Table 2, the individual with the highest Fib4-index among the ACA-positive group (indicated by a black triangle in the figure) had a low FMD value of 3.8%, indicating vascular endothelial dysfunction. Furthermore, this individual presented with idiopathic portal hypertension and localized cutaneous systemic scleroderma, both associated with hepatic fibrosis. Thus, when fibrosis progresses in the liver and cirrhosis occurs, portal pressure often increases, and as the disease progresses further, blood reflux from veins into arteries leads to elevated arterial blood pressure in other parts of the body. Pulmonary hypertension was also observed in this individual.

Furthermore, among the two individuals with high LOX/LAB values, the individual indicated by the black square in the figure had an even lower FMD value and exhibited scleroderma. The individual indicated by the black circle in the figure, similar to the aforementioned individual, showed Sjögren's syndrome, suggesting the progression of fibrotic disease.

Since these two individuals had FIB-4 index levels below 2.67, detailed pathological examinations were not performed on their specimens. However, based on the findings of these individuals, it was suggested that the marker of the present invention can capture a wider range of fibrotic conditions than conventional markers.

Furthermore, LOX-1 protein is expressed in vascular endothelial cells throughout the body, and LAB is a lipoprotein present in the blood regardless of its location. Therefore, the markers of this invention suggest that fibrosis can be broadly detected throughout the body, regardless of the specific body part where it occurs.

From the above, it is suggested that the marker of the present invention is highly reliable and, compared to conventional established markers, is superior in both versatility and discrimination accuracy.
All publications, patents, and patent applications cited herein shall be incorporated herein by direct reference.

Claims (16)

A method for determining whether a subject has a fibrotic disease or is at risk of developing one,
The process includes a step of calculating a determination value based on the amount of lectin-like oxidized low-density lipoprotein receptor 1 protein (LOX-1 protein) and/or the amount of apolipoprotein B-containing LOX-1 ligand (LAB) in a sample derived from the subject,
The aforementioned determination value indicates that the subject is suffering from or at risk of developing a fibrotic disease.
The aforementioned method. The method according to claim 1, wherein the fibrotic disease is a fibrotic disease accompanied by vascular endothelial damage. The method according to claim 1, wherein the determination value is the ratio of the amount of LOX-1 protein to the amount of LAB. The method according to claim 1, further comprising the step of measuring the amount of the LOX-1 protein and/or the LAB in a sample derived from the subject. The method according to claim 4, wherein the sample is a bodily fluid sample. The method according to claim 5, wherein the bodily fluid sample is a blood sample. The method according to claim 1, wherein the fibrotic disease is further accompanied by liver damage and/or an autoimmune disease. The method according to claim 7, wherein the liver disorder includes cirrhosis and/or portal hypertension. The method according to claim 7, wherein the autoimmune disease includes an antinuclear antibody-positive disorder. The method according to claim 9, wherein the antinuclear antibody-positive disorder includes anticentromere antibody-positive disorder and/or Sjögren's syndrome. The method according to claim 1, wherein, based on a comparison of the determination value with a predetermined cutoff value or a control value similarly calculated for a sample derived from a control individual, it is indicated that the subject is suffering from or at risk of developing a fibrotic disease. A composition for use in the method according to any one of claims 1 to 11, comprising a LOX-1 protein-binding molecule. A composition comprising a LAB-binding molecule, for use in the method according to any one of claims 1 to 11. A kit for use in the method according to any one of claims 1 to 11, comprising a LOX-1 protein-binding molecule and a LAB-binding molecule. A device for use in the method according to any one of claims 1 to 11, comprising a LOX-1 protein-binding molecule and a LAB-binding molecule. A combination marker for the differential diagnosis of fibrotic diseases, comprising LOX-1 protein or a fragment thereof, and LAB or a part thereof.