JP6964322B2 - Pharmaceutical compositions for treating or preventing lupus nephritis and biomarkers of lupus nephritis - Google Patents

Description

The present application relates to a pharmaceutical composition containing a RAGE aptamer for treating or preventing lupus nephritis, and a method for diagnosing lupus nephritis, a composition and a kit for that purpose.

Systemic lupus erythematosus (SLE) is an autoimmune disease. Approximately 60% of adult SLE patients develop lupus nephritis, and approximately 25% of lupus renal inflammation cases develop end-stage renal disease within 10 years of onset (Non-Patent Documents 1 and 2). Lupus nephritis is mainly treated with steroids, but steroid-resistant lupus nephritis is present, and long-term oral steroids have serious side effects. Thus, lupus nephritis is an important factor in determining the survival rate and quality of life (QOL) of SLE patients (Non-Patent Document 3). Immunosuppressants such as glucocorticoids and mycophenolate mofetil are also used for lupus nephritis. However, immunosuppressants cannot completely prevent the progression of lupus nephritis to end-stage renal disease and can cause opportunistic infections. In addition, most immunosuppressants are expensive and place a heavy financial burden on patients and society. In addition, glucocorticoids have side effects such as hyperglycemia, obesity, and acne, and these side effects are directly linked to a decrease in activities of daily living (ADL) of patients. Therefore, the development of new drugs is an urgent task.

The present inventors have been studying the pathophysiological role of advanced glycation endproducts (AGEs) and their receptor RAGE (Receptor for AGEs) in diabetic nephropathy. We have also started drug discovery targeting the AGEs-RAGE system. For example, it was reported that when a nucleic acid aptamer for glyceraldehyde-derived AGE, which has strong cytotoxicity, was prepared and continuously administered to spontaneously-onset type 2 diabetic mice KKAy / Ta mice, the progression of diabetic nephropathy was markedly suppressed. (Patent Document 1, Non-Patent Document 4). However, AGEs contain a wide variety of compounds, and even if one type of AGE can be blocked, other AGE-induced cell damage is induced. In order to solve this problem, the present inventors focused on RAGE to which most AGEs bind, and created a RAGE aptamer to which it binds to the surface of RAGE (Patent Document 2). The RAGE aptamer significantly suppressed the binding of RAGE to glyceraldehyde-derived AGE, Human Morbidity Group Box-1 (HMGB-1).

Overexpression of RAGE in the renal tissue of human lupus nephritis has been reported (Non-Patent Document 5), and the relationship between the progression of lupus nephritis and RAGE has attracted attention. It has been reported that a DNA-containing immune complex bound to HMGB-1 induces activation of plasmacytoid dendritic cells and B cells via RAGE and induces autoimmune abnormalities (Non-Patent Document 6). In addition, it is known that the RAGE gene polymorphism that is susceptible to SLE and lupus nephritis is strongly associated with the onset of lupus nephritis and the amount of urinary protein (Non-Patent Document 7). However, there is no consensus on the inhibitory effect of RAGE inhibitors on the onset of SLE and the development of lupus nephritis.

International Publication No. 2012/070621 Japanese Unexamined Patent Publication No. 2016-79184

Buhaescu et al. Semin Arthritis Rheum. 2007; 36 (4): 224-37 Maroz N et al. Am J Med Sci. 2013; 346 (4): 319-23 Houssiau FA, Arthritis Res Ther. 2012 31; 14 (1): 202 Kaida et al. Diabetes 2013 62 (9): 3241-50 Tanji et al. JASN 2000; 11: 1656-1666 Tian J et al. Nat Immunol. 2007; 8 (5): 487-96 Martens HA et al. Lupus. 2012; 21 (9): 959-68 J Exp Med. 2013 21; 210 (11): 2447-63

The present application is intended to contribute to the treatment, prevention or diagnosis of lupus nephritis.

The present inventors prevent the onset and progress of lupus nephritis by increasing RAGE expression in the kidney of a subject suffering from lupus nephritis and increasing as the pathological condition progresses, and by administering RAGE aptamer. It was found that it can suppress renal function and protect renal function.

Thus, in some embodiments, the present application provides a pharmaceutical composition comprising RAGE aptamer for treating or preventing lupus nephritis.
In another aspect, the present application provides a composition comprising a RAGE aptamer for administration to a patient with systemic lupus erythematosus.

In some embodiments, the present application provides a method of diagnosing lupus nephritis, comprising measuring RAGE concentration in urine collected from a subject.
In another aspect, the present application provides a composition for diagnosing lupus nephritis, comprising a reagent for detecting RAGE.
In a further aspect, the present application provides a kit for diagnosing lupus nephritis, which comprises the composition.
In a further aspect, the present application provides the use of RAGE as a biomarker for lupus nephritis.

The subject's lupus nephritis can be treated, prevented or diagnosed in accordance with the disclosure of the present application.

Periodic acid-siff reagent staining of glomeruli, proximal tubules and distal tubules of MRL / lpr mice is shown. The expression level of RAGE in the glomerulus of MRL / lpr mice is shown. The expression level of RAGE in the tubular interstitium of MRL / lpr mice is shown. The results of immunostaining of RAGE in the renal tubules of MRL / lpr mice are shown. It shows the intracellular localization of RAGE in the distal tubules of MRL / lpr mice. The localization of RAGE in the glomerulus of MRL / lpr mice is shown. The amount of urinary RAGE excreted and the amount of urinary protein in MRL / lpr mice are shown. The correlation between the urinary RAGE excretion amount and the urinary NAG excretion amount in MRL / lpr mice is shown. The effect of RAGE aptamer on lupus nephritis in MRL / lpr mice is shown. The effect of RAGE aptamer on RAGE expression in the kidney of MRL / lpr mice is shown. The effect of RAGE aptamer on HMGB1 deposition in the kidney of MRL / lpr mice is shown. The effect of RAGE aptamer on the expression of IL-6 in MRL / lpr mice is shown. The effect of RAGE aptamer on the expression of TNFα in MRL / lpr mice is shown. The effect of RAGE aptamer on the expression of MCP-1 in the kidney of MRL / lpr mice is shown. The effect of RAGE aptamer on the expression of Kim1 in MRL / lpr mice is shown. The effect of RAGE aptamer on the formation of crescents in the glomeruli of MRL / lpr mice is shown. The effect of RAGE aptamer on the formation of wire loop lesions in the glomerulus of MRL / lpr mice is shown. The effect of RAGE aptamer on macrophage infiltration in glomeruli of MRL / lpr mice is shown.

Unless otherwise specified, the terms used herein have the meaning generally understood by those skilled in the art in the fields of organic chemistry, medicine, pharmacy, molecular biology, microbiology and the like. The following are definitions of some terms used herein, but these definitions supersede the general understanding herein.

In the present specification, when a numerical value is accompanied by the term "about", it is intended to include a range of ± 10% of the value. The range of numbers includes all numbers between the endpoints and the numbers at the endpoints. The "about" for a range applies to both ends of the range. Therefore, for example, "about 20 to 30" includes "20 ± 10% to 30 ± 10%".

"RAGE" is a transmembrane protein that functions as a receptor for advanced glycation end products (AGEs). As the ligand of RAGE, various AGEs including Nεcarboxymethyllysine and AGE-2 derived from glyceraldehyde, as well as HMGB-1 and LPS are known.

An "aptamer" is a single-stranded DNA or RNA that has the ability to specifically bind to a target substance, has various three-dimensional structures, binds to proteins and compounds, and exerts an antibody-like function. As used herein, the term "RAGE aptamer" means a single-stranded DNA or RNA that binds to RAGE and inhibits the binding of RAGE to its ligand.

RAGE aptamers can be prepared by the SELEX (Systematic Evolution of Ligands by EXponential enrichment) method. The outline of the SELEX method using single-stranded DNA as a library source will be described below. First, a template DNA containing a random sequence of an appropriate length sandwiched between two arbitrary primer sequences is synthesized. The appropriate length of the random sequence is 35 to 120 bases. This template DNA is amplified by PCR (Polymerase Chain Reaction) to obtain a random DNA aptamer pool. The random DNA aptamer pool is then associated with the target material, the unbound DNA is removed, and the bound DNA aptamer is extracted. The obtained DNA aptamer is amplified by PCR using the above primer sequence. At this time, ^{PCR is performed in the presence of 5 to 8 mM Mg 2+} to reduce the accuracy of replication and facilitate the introduction of mutations, so that new DNA that does not exist in the DNA aptamer pool before association with the target substance is present. A DNA aptamer pool containing aptamers is obtained. New DNA aptamers may be more binding, i.e., DNA aptamers may evolve. By repeating the above series of operations for 5 to 15 rounds on this evolved DNA aptamer pool, a DNA aptamer that binds to the target substance can be obtained. After the final round, the resulting DNA aptamer pool is cloned and then sequenced by procedures commonly performed by those skilled in the art. In this SELEX method, operations such as template DNA synthesis and PCR, as well as cloning and sequencing are performed by methods usually used by those skilled in the art. RAGE aptamers can be chemically synthesized by methods commonly used by those skilled in the art based on the sequences thus determined.

The RAGE aptamer may be composed of modified nucleotides to increase its stability. For example, the RAGE aptamer may be a phosphorothioate (S-modified) oligonucleotide in which the oxygen atom of the phosphate group at the binding site between nucleotides is replaced with a sulfur atom.

The RAGE aptamer may be either single-stranded DNA or single-stranded RNA, but is preferably single-stranded DNA. The RAGE aptamer comprises at least 35 bases, preferably from about 35 bases to about 120 bases, more preferably from about 35 bases to about 70 bases, and even more preferably from about 35 bases to about 50 bases.

In some embodiments, the RAGE aptamer is a single-stranded DNA comprising any of the sequences of SEQ ID NOs: 1-10 below.

CCTGATATGGTGTCACCGCCGCCTTAGTATTGGTGTCTAC (SEQ ID NO: 1)
TCTGTTCAGGTTGGTACGGTGGAAGGTGTGATTCACGAGG (SEQ ID NO: 2)
CTTGGGTTGTTTATGTCGACCGCCAGTTTTGTGTTCAGCA (SEQ ID NO: 3)
CATTCTTAGATTTTTGTCTCACTTAGGTGTAGATGGTGAT (SEQ ID NO: 4)
TTGGTTCTCTTGCCCTCTTCTATTTTGTACGATGTTTCCT (SEQ ID NO: 5)
TTCCACTGAGTGCCGCGGACTGTTGTTGGGAGGTGGTGTG (SEQ ID NO: 6)
ATGGGGAGGAGGGGTGTCGTGGGGGGTGGGGGAGTGGGGA (SEQ ID NO: 7)
CTGGGGATGACTCGGATAGGATGTACGAGTTTGATGTGTA (SEQ ID NO: 8)
TTTGATCGAGCTGCGCCCTCCTCGCCGTAGAAGAGTGTGT (SEQ ID NO: 9)
TTTGGGGTGGAGTTAGGGGTGGATCAAGCGGGATTGTGTA (SEQ ID NO: 10)

In some embodiments, the RAGE aptamer is an oligonucleotide comprising any of the sequences of SEQ ID NOs: 1-10 above and comprising a phosphorothioate bond at the position shown in the table below.

The RAGE aptamer is about 70% or more, preferably about 80% or more, more preferably about 85% or more, even more preferably about 90% or more, most preferably about 95% with any of the sequences of SEQ ID NOs: 1-10. It may be a single-stranded DNA containing a sequence having the above sequence identity and binding to RAGE to inhibit the binding of RAGE to its ligand. In addition, the RAGE aptamer contains a sequence in which 1, 2, or 3 bases are deleted, substituted, or added in any of the sequences of SEQ ID NOs: 1 to 10, and binds to RAGE to form RAGE and its ligand. It may be a single-stranded DNA that inhibits the binding of the DNA.

"Sequence identity" means the degree of sequence similarity between two oligonucleotides, and the two are aligned to the optimum state (the state where the sequence match is maximized) over the region of the base sequence to be compared. Determined by comparing sequences. The sequence identity number (%) determines the number of matching sites by determining the same bases present in both sequences, then dividing the number of matching sites by the total number of bases in the sequence region to be compared. , It is calculated by multiplying the obtained numerical value by 100. Algorithms for obtaining optimal alignment and sequence identity include various algorithms commonly available to those skilled in the art (eg, BLAST algorithm, FASTA algorithm, etc.). The sequence identity of the base sequence is determined by using sequence analysis software such as BLAST or FASTA.

Whether or not the RAGE aptamer binds to RAGE and inhibits the binding of RAGE to its ligand can be examined by, for example, ELISA or QCM (quartz crystal microbalance) method. In the case of ELISA, the V domain (v-RAGE) of RAGE that binds to AGE and the RAGE aptamer are pre-reacted, and this reaction mixture is added to an ELISA plate on which a ligand of RAGE (for example, AGE or HMGB1) is immobilized. RAGE bound to the ligand in the plate is detected with an anti-RAGE antibody. Alternatively, v-RAGE may be immobilized on an ELISA plate, a RAGE aptamer and its ligand may be added, and the ligand bound to v-RAGE may be detected by an antibody. In the QCM method, v-RAGE may be immobilized on a QCM sensor plate, a ligand of RAGE may be added in the presence or absence of a RAGE aptamer, and the binding of the ligand to v-RAGE may be monitored.

In a preferred embodiment, the RAGE aptamer is a single-stranded DNA comprising any of the sequences of SEQ ID NOs: 1-10, and more preferably a single-stranded DNA comprising any of the sequences of SEQ ID NOs: 1-10. In another preferred embodiment, the RAGE aptamer is a single-stranded DNA comprising the sequence of CATTCTTAGATTTTTGTCTCACTTAGGTGTAGATGGTGAT (SEQ ID NO: 4), more preferably a single-stranded DNA comprising the sequence of SEQ ID NO: 4.

RAGE aptamers can treat or prevent lupus nephritis by inhibiting the binding of RAGE to its ligand. "Lupus nephritis" means renal damage associated with systemic lupus erythematosus (SLE). SLE is an autoimmune disease that can affect the entire body, resulting in the formation of immune complexes and tissue damage by complement. The location of tissue damage varies from case to case, and damage can occur to various organs such as the kidneys, skin, lungs, and brain. As diagnostic criteria for SLE, for example, Updating the American College of Rheumatology revised criteria (1997) and The systemic lupus international collaborating clinics classification criteria for systemic lupus erythematosus (2012) are known.

In lupus nephritis, glomerulonephritis mainly occurs, but vasculitis and interstitial nephritis can also occur. Lupus nephritis includes micromesangial lupus nephritis, mesangial proliferative lupus nephritis, focal lupus nephritis, diffuse lupus nephritis, membranous lupus nephritis and advanced sclerosing lupus nephritis, especially diffuse lupus nephritis. Symptoms of lupus nephritis include, for example, proteinuria and swelling. As lupus nephritis progresses, renal function declines, which may lead to renal failure. The diagnosis or classification of lupus nephritis may be based on, for example, the 2003 classification of lupus nephritis by the International Society of Nephrology (ISN) or the ACR (American College of Rheumatology) practice guidelines for lupus nephritis (2012).

"Treatment" of lupus nephritis includes amelioration and remission of lupus nephritis or its symptoms, as well as suppression of the progression or progression of lupus nephritis or its symptoms. In addition, "prevention" of lupus nephritis includes suppression and delay of the onset of lupus nephritis or its symptoms. The target for treatment or prevention of lupus nephritis can be patients with SLE. The subject of treatment for lupus nephritis can be a subject suffering from lupus nephritis, eg, a patient with SLE suffering from lupus nephritis. The target for prevention of lupus nephritis may be a subject at risk of developing lupus nephritis, for example, a patient with SLE who does not develop lupus nephritis, or a subject at risk of developing SLE. The subject can be a human or a non-human mammal.

Treatment or prevention of lupus nephritis includes suppression or protection of deterioration of renal function and protection of renal tissue. Non-limiting examples of indicators of renal function include urinary albumin excretion, urinary albumin / creatinine ratio, creatinine clearance, blood urea nitrogen level, and blood creatinine level. Treatment or prevention of lupus nephritis includes increased mesangial matrix, thickening of the glomerular basement membrane, suppression of glomerular fibrosis (hardening), and / or renal enlargement (increased kidney / body weight ratio), renal tubules. Includes suppression of interstitial fibrosis and / or atrophy, and suppression of oxidative stress. An index of oxidative stress includes urinary 8OHdG (8-Hydroxydeoxyguanosine) excretion.

The pharmaceutical composition is prepared by blending RAGE aptamer with a pharmaceutically acceptable carrier as a solid preparation (for example, tablets, capsules, granules, powders, etc.) or a liquid preparation (for example, syrup, injection, etc.). Can be manufactured. Pharmaceutically acceptable carriers include, for example, excipients (eg, lactose, sucrose, dextrin, hydroxypropyl cellulose, polyvinylpyrrolidone, etc.), disintegrants (eg, starch, carboxymethyl cellulose, etc.), lubricants (eg, starch, carboxymethyl cellulose, etc.). , Magnesium stearate, etc.), surfactants (eg, sodium lauryl sulfate, etc.), solvents (eg, water, saline, soybean oil, etc.), preservatives (eg, p-hydroxybenzoic acid ester, etc.), etc. However, it is not limited to these.

The administration method of the above-mentioned pharmaceutical composition is appropriately selected depending on the age, body weight, health condition and the like of the administration subject. For example, the above-mentioned pharmaceutical composition can be administered by oral administration, intravenous administration, or subcutaneous administration, but it is preferably administered subcutaneously or intravenously. The dose of the pharmaceutical composition is also appropriately selected depending on the administration method, the age, body weight, health condition and the like of the administration target. For example, 0.01 μg to 100 g, for example, 0.01 μg to 1 mg of RAGE aptamer can be administered per day for an adult, but is not limited thereto.

In some embodiments, the present application is a method of treating or preventing lupus nephritis, comprising administering RAGE aptamer to a patient in need thereof, use of RAGE aptamer to treat or prevent lupus nephritis, And provide the use of RAGE aptamers for the manufacture of pharmaceuticals to treat or prevent lupus nephritis.

In some embodiments, the present application provides a composition comprising a RAGE aptamer for administration to a patient with SLE. This composition can be prepared in the same manner as the pharmaceutical composition for treating or preventing lupus nephritis described above and administered to patients with SLE.

As described in the examples below, the present inventors have increased urinary RAGE excretion in SLE mice, and are strongly positive for urinary NAG excretion, which is a marker of tubular damage, and urinary RAGE excretion. It was found that there is a correlation between. Therefore, RAGE can be used as a biomarker for lupus nephritis to diagnose lupus nephritis.

For example, a method for diagnosing lupus nephritis is provided, which comprises measuring the RAGE concentration in urine collected from a subject. This method may be used as a method for assisting the diagnosis of lupus nephritis. For example, the diagnosis may be for the following purposes, but the purpose of the diagnosis is not limited to these.
(A) Determine if the subject has lupus nephritis.
(B) Determine the risk of developing lupus nephritis.
(C) Predict the effect of treatment or prevention of lupus nephritis in a subject with a RAGE aptamer.
(D) Monitor the subject's lupus nephritis.

The urine used in the above method may be collected according to a conventional method. The collected urine may be immediately used for measuring the RAGE concentration, or may be stored at a low temperature, for example, refrigerated or frozen, and stored at, for example, 4 ° C. or lower, 0 ° C. or lower, or −20 ° C. or lower until the measurement. .. Diluted or concentrated urine may be used.

The urinary RAGE concentration can be measured by an immunological method using an antibody that specifically binds to RAGE. Examples of immunological methods include radioimmunoassay immunoassay (RIA method), enzyme-linked immunosorbent assay (ELISA method), and Western blotting.

Antibodies used to measure urinary RAGE levels can be selected from the group consisting of antibodies that bind to RAGE (anti-RAGE antibodies), i.e., immunoglobulin backbone-based affinity ligands. Antibodies include monoclonal and polyclonal antibodies of any origin and chimeric antibodies, including mice, rats, rabbits, goats, humans and other antibodies, as well as sequences derived from multiple species, eg, partially humanized. Includes antibodies, such as partially humanized mouse antibodies. The antibody can be produced by an existing general production method using each marker protein or a partial peptide having the antigenicity of the protein as an immunogen. For example, polyclonal antibodies can be produced by immunizing animals with antigens, and monoclonal antibodies can be produced using hybridoma technology. Alternatively, a commercially available antibody may be used.

The anti-RAGE antibody may be a fragment and a derivative thereof as long as it can selectively interact with RAGE. Antibody fragments and derivatives include, for example, from the heavy chain first constant domain (CH1), light chain constant domain (CL), heavy chain variable domain (VH) and light chain variable domain (VL) of the complete immunoglobulin protein. Fab fragment; Fv fragment consisting of two variable antibody domains VH and VL; single chain Fv fragment consisting of two VH and VL domains linked by a mobile peptide linker (scFv); heavy chain two of the camel family. Includes a metric and a single variable region, and a single domain skeleton such as a novel antigen receptor for the shark, and a minibody based on the variable heavy chain domain.

RAGE aptamers can also be used to measure urinary RAGE levels, as well as anti-RAGE antibodies. For this, the RAGE aptamer described above can be used.

The anti-RAGE antibody or RAGE aptamer used to measure urinary RAGE concentration may be labeled. The sign can be appropriately selected by those skilled in the art. Examples of labels include fluorescent dyes or metals (eg, fluorescein, rhodamine, phycoerythrin, fluoresamine), chromophore dyes (eg, rhodopsin), chemiluminescent compounds (eg, luminal, imidazole) and bioluminescent proteins (eg, luciferin). , luciferase), haptens (e.g., biotin), an enzyme (e.g., peroxidase, alkaline phosphatase, beta-lactamase), radioisotopes ^{(e.g., 3 H, 14 C, 32} P, 35 S or ¹²⁵ I), particles (for example, Includes metal particles such as gold) and fluorescent semiconductor nanocrystals (quantum dots). The various labels can be attached to the desired antibody or aptamer using various chemical reactions well known to those of skill in the art. Alternatively, the antibody or aptamer used to measure urinary RAGE concentration may be unlabeled. In this case, a labeled secondary antibody or nucleic acid probe that recognizes the anti-RAGE antibody or RAGE aptamer may be further used.

Alternatively, the urinary RAGE concentration can be measured by measuring the amount of ligand that binds to RAGE. For example, the concentration of RAGE can be measured by a ligand binding assay using a labeled ligand such as a fluorescently labeled ligand or a radioligand. Any RAGE ligand can be used for this purpose. For example, as the RAGE ligand, various AGEs including Nεcarboxymethyllysine and AGE-2 derived from glyceraldehyde, HMGB-1 and LPS can be used.

The anti-RAGE antibody, RAGE aptamer or RAGE ligand may be attached to a suitable support. Supports commonly used in the art can be used. The support may be of any shape or material and may be, for example, a membrane such as a nylon membrane, a plate, a multi-well plate, beads, glass, plastic and metal.

Since the urinary RAGE concentration changes depending on the state of water metabolism at the time of urine collection, it is preferable to correct the measured urinary RAGE concentration with the value of urinary creatinine (creatinine correction). Methods of creatinine correction are well known to those of skill in the art.

Regarding (A) above, a method (method (A)) for determining whether or not a subject has lupus nephritis is provided, which comprises measuring the RAGE concentration in urine collected from the subject. In method (A), RAGE is used as a biomarker for lupus nephritis.

The subject in method (A) can be a human or a non-human mammal and may be in any health condition. For example, the subject may be a patient with SLE or a subject suspected of developing SLE or lupus nephritis. Subjects may or may not be treated for SLE or lupus nephritis.

In one embodiment, the measured urinary RAGE concentration is compared to an index value to determine if the subject has lupus nephritis. The index value may be a value obtained by measuring the urinary RAGE concentration in an animal group (control group) of the same species as the subject. The index value may be a value determined for the control group at the same time as the measurement of the test subject, or before or after, or may be a predetermined value for each test animal species.

In some embodiments, the index value can be the value obtained for a group of animals (control group) that suffers from SLE but not lupus nephritis. In this case, if the urinary RAGE concentration of the subject is higher than the index value, it can be determined that the subject has lupus nephritis, and if it is lower than the index value, the subject is not suffering from lupus nephritis. Can be judged. In another embodiment, the index value can be a value obtained for a group of animals (control group) suffering from lupus nephritis. In this case, if the urinary RAGE concentration of the subject is higher than the index value, it can be determined that the subject has lupus nephritis, and if it is lower than the index value, the subject is not suffering from lupus nephritis. Can be judged. In addition, the severity of lupus nephritis can be further known based on the degree of test value relative to the index value.

Further, the cutoff value of the urinary RAGE concentration may be set in advance, and the measured value and the cutoff value may be compared. For example, if the urinary RAGE concentration of a subject is higher than the cutoff value, it can be determined that the subject has lupus nephritis, and if it is lower than the cutoff value, the subject is not suffering from lupus nephritis. Can be judged.

The "cutoff value" is also referred to as a threshold value, and refers to a value at which lupus nephritis can be diagnosed based on the threshold value. Preferably, the cutoff value exhibits both high diagnostic sensitivity and high diagnostic specificity. Here, sensitivity means a true positive rate. In addition, specificity means a true negative rate. For example, a urinary RAGE concentration showing a high positive rate in patients with lupus nephritis and a high negative rate in subjects not suffering from lupus nephritis can be set as a cutoff value. You can also set multiple cutoff values.

The cutoff value can be set by a method known per se, using various statistical analysis methods. In general, the cutoff value can be set by statistically analyzing the urinary RAGE concentration of a patient with lupus nephritis and the urinary RAGE concentration of a subject not suffering from lupus nephritis.

For example, the cutoff value can be set by ROC analysis (receiver opera ting characteristic analysis), which is generally used as a method for examining the usefulness of a diagnostic test. In the ROC analysis, when the threshold values are changed, the sensitivity (Sensitivity) at each threshold value is plotted on the vertical axis, and the FPF (False Positive Fraction, false positive rate: 1-specificity) is plotted on the horizontal axis. The ROC curve is created. In the ROC curve, a test with no diagnostic ability becomes a straight line on the diagonal line, but as the diagnostic ability improves, the diagonal line becomes a curve that draws an arc to the upper left, and a test with 100% diagnostic ability is on the left side- It becomes a curve that passes over the upper side. As for the setting of the cutoff value, for example, due to the fact that the ROC curve of the independent variable having excellent sensitivity and specificity approaches the upper left corner, the point where the distance from the upper left corner is the minimum is set as the cutoff value. There is a way to do it. Further, a method of setting the cutoff value at the point farthest from the diagonal line where the area under the curve (abbreviated as AUC) in the ROC curve is 0.500, that is, (sensitivity + specificity -1). ) Can be calculated and the Youden index, which is the maximum value, can be set as the cutoff value.

Specifically, the RAGE concentration in urine collected from a group of patients with lupus nephritis (reaction cases) and a group of subjects not suffering from lupus nephritis (non-reaction cases) was measured, and the diagnostic sensitivity and diagnostic sensitivity at the measured values were measured. The diagnostic specificity is determined, and based on these values, a ROC curve is created using commercially available analysis software. Then, a value when the diagnostic sensitivity and the diagnostic specificity are as close to 100% as possible can be obtained, and that value can be used as the cutoff value. In addition, for example, the diagnostic efficiency at the detected value (that is, the ratio of the total number of cases in which a patient with lupus nephritis is correctly diagnosed to a case in which a subject without lupus nephritis is correctly diagnosed) with respect to the total number of cases. The value obtained and the value at which the highest diagnostic efficiency is calculated can be used as the cutoff value.

The cutoff value can be changed as appropriate. That is, the cutoff value can be set according to conditions such as a method for measuring and comparing the urinary RAGE concentration, a clinical purpose of use, a desired sensitivity and specificity.

In one embodiment, a method of determining whether a subject has lupus nephritis.
(1) The process of collecting urine from the subject and
(2) Step of measuring RAGE concentration in the urine,
Methods are provided that include.

This method also
(3) A step of comparing the measured urinary RAGE concentration with an index value or a cutoff value,
(4) When the measured urinary RAGE concentration is higher than the index value or cutoff value, it is determined that the subject has lupus nephritis, and the measured urinary RAGE concentration is the index value or cutoff value. The step of determining that the subject is not suffering from lupus nephritis, if lower than
May include.

In a further embodiment, if the subject is determined to have lupus nephritis, it is determined to administer the RAGE aptamer to the subject. In a further embodiment, the subject is administered a RAGE aptamer if the subject is determined to have lupus nephritis.

Regarding (B) above, a method (method (B)) for determining the risk of developing lupus nephritis is provided, which comprises measuring the RAGE concentration in urine collected from a subject. Method (B) uses RAGE as a biomarker for the risk of developing lupus nephritis.

The subject in method (B) can be a human or a non-human mammal and may be in any health condition. For example, the subject can be a healthy subject, a patient with SLE, or a subject suspected of developing SLE or lupus nephritis. Subjects may or may not be treated for SLE.

In one embodiment, the risk of developing lupus nephritis in a subject is determined by comparing the measured urinary RAGE concentration with an index value. The index value may be a value obtained by measuring the urinary RAGE concentration in an animal group (control group) of the same species as the subject. The index value may be a value determined for the control group at the same time as the measurement of the test subject, or before or after, or may be a predetermined value for each test animal species.

In some embodiments, the index value can be the value obtained for a group of animals (control group) not affected by SLE. In this case, if the urinary RAGE concentration of the subject is higher than the index value, it can be determined that the subject has a high risk of developing lupus nephritis, and if it is lower than the index value, the subject is at risk of developing lupus nephritis. It can be judged to be low. In another embodiment, the index value can be the value obtained for a group of animals suffering from SLE but not developing lupus nephritis. In this case, if the urinary RAGE concentration of the subject is higher than the index value, it can be determined that the subject has a high risk of developing lupus nephritis, and if it is lower than the index value, the subject is at risk of developing lupus nephritis. It can be judged to be low. In addition, the magnitude of the risk of developing lupus nephritis can be further known based on the degree of the test value relative to the index value.

Further, the cutoff value of the urinary RAGE concentration may be set in advance, and the measured value and the cutoff value may be compared. For example, if the urinary RAGE concentration is higher than the cutoff value, it can be determined that the subject is at high risk of developing lupus nephritis, and if it is lower than the cutoff value, the subject is determined to be at high risk of developing lupus nephritis. Can be. The cutoff value can be set according to the above method (A) based on the measured value of an appropriate control group.

In one embodiment, a method of determining the risk of developing lupus nephritis in a subject.
(1) The process of collecting urine from the subject and
(2) Step of measuring RAGE concentration in the urine,
Methods are provided that include.

This method also
(3) A step of comparing the measured urinary RAGE concentration with an index value or a cutoff value,
(4) When the measured urinary RAGE concentration is higher than the index value or cutoff value, it is judged that the subject is at high risk of developing lupus nephritis, and the measured urinary RAGE concentration is the index value or cutoff value. The step of determining that a subject has a low risk of developing lupus nephritis if it is below the value,
May include.

In a further embodiment, the subject is determined to be administered RAGE aptamer if the subject is determined to be at high risk of developing lupus nephritis. In a further embodiment, the subject is administered a RAGE aptamer if the subject is determined to be at high risk of developing lupus nephritis.

Regarding (C) above, a method (method (C)) for predicting the effect of treatment or prevention of lupus nephritis in a subject with a RAGE aptamer, which comprises measuring the urinary RAGE concentration in urine collected from the subject, is provided. NS. As mentioned above, RAGE aptamers can be used to treat or prevent lupus nephritis, and RAGE is excreted in the urine of SLE patients and increases with the development of lupus nephritis. From this, it can be predicted that treatment or prevention of lupus nephritis with RAGE aptamer is effective for subjects in which RAGE is detected in urine. Such a method is commonly referred to as companion diagnostics.

In method (C), when predicting the effect of treatment of a subject's lupus nephritis with a RAGE aptamer, it is determined whether or not the subject has lupus nephritis according to method (A), and the subject suffers from lupus nephritis. If it is diagnosed as having, it is predicted that treatment of lupus nephritis with RAGE aptamer will be effective.

In the method (C), when predicting the effect of prevention of lupus nephritis in the subject by the RAGE aptamer, the risk of the subject suffering from lupus nephritis is determined according to the method (B), and when the risk is determined to be high. , Predict that prevention of lupus nephritis by RAGE aptamer is effective.

Method (C) can be performed before, during, or after treatment or prevention of lupus nephritis with RAGE aptamers. In certain embodiments, method (C) is performed on subjects who have not been treated or prevented for lupus nephritis with RAGE aptamers. Subjects who have not been treated or prevented for lupus nephritis by RAGE aptamers are those who have not been treated or prevented, and those who have been treated or prevented, but perform method (C). Includes objects that have not been received at this time. In another embodiment, method (C) is performed on a subject undergoing treatment or prevention of lupus nephritis with a RAGE aptamer to determine whether to continue treatment or prevention.

In certain embodiments, the subject is determined to be administered the RAGE aptamer when treatment or prevention of the subject's lupus nephritis with the RAGE aptamer is predicted to be effective. In certain embodiments, a subject is administered a RAGE aptamer when treatment or prevention of lupus nephritis in the subject with a RAGE aptamer is predicted to be effective. In certain embodiments, it is determined not to administer the RAGE aptamer to the subject if treatment or prevention of the subject's lupus nephritis with the RAGE aptamer is predicted to be ineffective. In certain embodiments, the treatment or prevention is interrupted, postponed or discontinued in subjects undergoing treatment or prevention of lupus nephritis with RAGE aptamers if the treatment or prevention is predicted to be ineffective.

In one embodiment, a method of predicting the effect of treatment or prevention of lupus nephritis in a subject with a RAGE aptamer.
(1) The process of collecting urine from the subject and
(2) Step of measuring RAGE concentration in the urine,
Methods are provided that include.

This method also
(3) A step of comparing the measured urinary RAGE concentration with an index value or a cutoff value,
(4) When the measured urinary RAGE concentration is higher than the index value or the cutoff value, it is predicted that treatment or prevention of the subject lupus nephritis with the RAGE aptamer is effective, and the measured urinary RAGE concentration is The step of predicting that treatment or prevention of lupus nephritis in a subject with a RAGE aptamer is not effective if it is below the index or cutoff value.
May include.

Regarding (D) above, a method (method (D)) for monitoring lupus nephritis in a subject is provided, which comprises measuring and comparing RAGE concentrations in urine collected from the subject at at least two time points. Method (D) uses RAGE as a biomarker for the progression of lupus nephritis.

The subject in method (D) can be a human or a non-human mammal and may be in any health condition. For example, the subject can be a healthy subject, a patient with SLE or lupus nephritis, or a subject suspected of developing SLE or lupus nephritis. Also, at least at least two points in time, the condition of the subject's SLE or lupus nephritis may be the same or different. Subjects may or may not be treated for SLE or lupus nephritis.

In method (D), urine collected at two or more time points can be used. The intervals at the time of collection may be constant or different. For example, once a day, two days, three days, four days, five days, six days, seven days, eight days, ten days, one week, two weeks, three weeks, once every four weeks, January, Urine collected once in February, March, April, once every six months, once a year, etc. can be used.

In one embodiment, as a result of comparing the RAGE concentrations in the urine collected at at least two time points, it can be determined that lupus nephritis has progressed when the urinary RAGE concentration of the subject increases, and the urinary RAGE concentration of the subject can be determined. When is reduced, it can be determined that lupus nephritis has been improved. Therefore, by this method, for example, it is possible to observe the pathological progression of lupus nephritis or confirm the effect of treatment for lupus nephritis.

In one embodiment, a method of monitoring lupus nephritis in a subject, the method of monitoring.
(1) A step of collecting urine from a subject at at least two time points,
(2) A step of measuring the RAGE concentration in the urine, and
(3) Step of comparing the measured urinary RAGE concentration,
Methods are provided that include.

This method also
(4) A step of determining that lupus nephritis has progressed when the urinary RAGE concentration of the subject increases, and determining that lupus nephritis has improved when the urinary RAGE concentration of the subject decreases.
May include.

In some embodiments, the present application provides a composition for diagnosing lupus nephritis, comprising at least one reagent for detecting RAGE. The reagent for detecting RAGE can measure the urinary RAGE concentration, and can be, for example, the above-mentioned anti-RAGE antibody, RAGE aptamer or RAGE ligand, particularly anti-RAGE antibody. Reagents for detecting RAGE can be dissolved in water or a suitable buffer, such as phosphate buffered saline (PBS), or provided lyophilized. The composition can be used, for example, in any of the above methods (A) to (D).

In some embodiments, the present application provides a kit for diagnosing lupus nephritis, comprising at least one reagent for detecting RAGE or a composition for diagnosing lupus nephritis described above. The kit further contains other components and reagents necessary for measuring urinary RAGE concentration, such as labeled secondary antibody, labeled probe, chromogenic substrate, blocking solution, wash buffer, diluent, ELISA plate, blotting membrane, etc. obtain. The kit may include a RAGE positive or negative control sample or control reagent. In addition, it may include other components desirable from a commercial and user standpoint, such as package inserts containing instructions for use (eg, documents, tapes, CD-ROMs, etc.).

Each component included in the kit is dissolved or frozen in water or a suitable buffer, such as Phosphate Buffered Saline (PBS), either separately or, if possible, mixed. In a dry state, it may be contained and provided in a suitable container. Suitable containers include bottles, vials, test tubes, plates, multi-well plates, membranes and the like. The container may be made of a variety of materials such as glass, plastic and metal. The container may have a label. The kit can be used, for example, in any of the above methods (A) to (D).

In some embodiments, the present application provides the use of reagents for detecting RAGE to diagnose lupus nephritis in a subject. Alternatively, the present application provides the use of reagents for detecting RAGE to produce a medicament for diagnosing lupus nephritis of interest. Reagents for detecting RAGE can be the anti-RAGE antibodies, RAGE aptamers or RAGE ligands described above, in particular anti-RAGE antibodies.

The present application provides, for example, the following embodiments.
[1] A pharmaceutical composition for treating or preventing lupus nephritis, which comprises a RAGE aptamer.
[2] The composition according to item 1, wherein the RAGE aptamer is selected from the group consisting of the following:
(1) Single-stranded DNA containing any of the sequences of SEQ ID NOs: 1 to 10;
(2) A single-stranded DNA that contains a sequence having 90% or more sequence identity with any of SEQ ID NOs: 1 to 10 and binds to RAGE to inhibit the binding of RAGE to its ligand;
(3) In any of the sequences of SEQ ID NOs: 1 to 10, 1, 2, or 3 bases are deleted, substituted, or added to the sequence, and the sequence binds to RAGE to bind RAGE to its ligand. Single-stranded DNA that inhibits.
[3] The composition according to item 1 or 2, wherein the RAGE aptamer comprises any of the sequences of SEQ ID NOs: 1-10.
[4] The composition according to any one of Items 1 to 3, wherein the RAGE aptamer comprises any of the sequences of SEQ ID NOs: 1 to 10.
[5] The composition according to any one of paragraphs 1 to 4, for administration to a patient with systemic lupus erythematosus.
[6] The composition according to any one of items 1 to 5, for treating lupus nephritis.
[7] The composition according to any one of items 1 to 5, for preventing lupus nephritis.
[8] A pharmaceutical composition containing a RAGE aptamer for administration to a patient with systemic lupus erythematosus.
[9] A method for diagnosing lupus nephritis, which comprises measuring the RAGE concentration in urine collected from a subject.
[10] The method according to paragraph 9, for determining whether or not the subject has lupus nephritis.
[11] The method according to paragraph 9, for determining the risk of developing lupus nephritis.
[12] The method according to paragraph 9, for predicting the effect of treatment or prevention of lupus nephritis in a subject with a RAGE aptamer.
[13] The method according to paragraph 9, for monitoring the subject lupus nephritis.
[14] The method according to any one of paragraphs 9 to 13, wherein the subject is a patient with systemic lupus erythematosus.
[15] A composition for diagnosing lupus nephritis, which comprises a reagent for detecting RAGE.
[16] The composition according to item 15, wherein the reagent for detecting RAGE is an anti-RAGE antibody, RAGE aptamer or RAGE ligand.
[17] The composition according to claim 15 or 16, wherein the reagent for detecting RAGE is an anti-RAGE antibody.
[18] A kit for diagnosing lupus nephritis, which comprises the composition according to any one of paragraphs 15 to 17.
[19] Use of RAGE as a biomarker for lupus nephritis.

Hereinafter, the disclosure of the present application will be further described with reference to Examples, but the present invention is not limited thereto.

Model Animal A model of systemic lupus erythematosus using MRL / MpJ-Fas ^lpr / J (MRL / lpr) mice spontaneously developing lupus nephritis (purchased from The Jackson Laboratory). MRL / lpr mice usually develop glomerulonephritis due to immune complexes at about 16 weeks of age, and about 50% die of renal failure in about 20 weeks (Arthritis Rheum. 2006 Jan; 54 (1): 325). -35). As controls, C57BL6 / J mice or MRL / MPJ mice were used. The MRL / MPJ mouse is a mouse having the same background as the MRL / lpr mouse, and is widely used as a control for the MRL / lpr mouse.

Experiment 1: Kidney Periodic Acid Schiff Reagent Staining Kidney tissue was collected from 8, 12, 16 and 18 week old female MRL / lpr mice and glomeruli, proximal tubules and distal convoluted tubules were collected in the tissue. The polysaccharides were stained with the Periodic Acid Schiff Reagent (PAS). The results are shown in FIG.

Experiment 2: RAGE expression in glomerular and tubular interstitium Renal tissue was collected from female MRL / lpr mice and MRL / MPJ mice aged 8, 12, 16 and 18 weeks, and glomerular and tubular stroma were immunofluorescent. Stained by the method. An anti-RAGE antibody (provided by Dr. Yasuhiko Yamamoto, Kanazawa University) was used as the primary antibody, and an Alexa Fluor 488-labeled anti-mouse antibody was used as the secondary antibody. The fluorescence intensity was measured with a confocal microscope, the RAGE positive area was measured, and the relative value of each measured value was calculated with the measured value of the control as 1. The results are shown in FIG. RAGE expression increased in glomeruli in a time-dependent manner (Fig. 2A). RAGE expression increased early in the renal tubules and was maintained (Fig. 2B).

Experiment 3: Localization of RAGE in renal tubules MRL / lpr mouse In order to investigate in which cells of the renal tubules RAGE expression is increased, the renal tubules of 8-week-old female MRL / lpr mice and MRL / MPJ mice are suppressed. Immunostaining with RAGE antibody. The results are shown in FIG. In addition, RAGE and NCX (distal tubular marker) were double-stained by the fluorescent antibody method. Anti-RAGE antibody and anti-NCX antibody were used as the primary antibody, and Alexa Fluor 488 and Alexa Fluor-labeled antibody were used as the secondary antibody, respectively. RAGE and NCX were significantly co-stained. This indicates that RAGE expression is increased in the distal tubules early in lupus nephritis.

Experiment 4: Intracellular localization of RAGE in the distal tubule In the steady state, RAGE is mainly localized in the cell membrane, but in FIG. 3, RAGE in the distal tubule is localized in the intracellular granules. The intracellular localization of this RAGE was investigated. RAGE and various markers were co-stained by the fluorescent antibody method and observed with a confocal microscope. The results are shown in FIG. RAGE was co-stained with the late endocytosis membrane surface protein Rab7 (upper) and the lysosomal enzyme cathepsin D (middle). However, it was not co-stained with LC3, which is an autophagy-related protein (lower row). These results suggest that RAGE is not involved in autophagy but in endocytosis and lysosomal proteolysis.

Experiment 5: Localization of RAGE in glomeruli The site of RAGE overexpression in MRL / lpr mouse glomeruli was investigated. In the glomeruli of 18-week-old female MRL / lpr mice, RAGE and various markers were co-stained by the fluorescent antibody method and observed with a confocal microscope. The results are shown in FIG. RAEG was co-stained with the vascular endothelial cell marker CD34 (upper). It is a marker of podocytes (glomerular epithelial cells) and was not co-stained with WT1 localized in the cytoplasm, and RAGE was observed around it (lower row). Podocytes showed that RAGE was predominantly localized to the cell membrane (steady state).

Experiment 6: Urinary RAGE excretion of 8, 12, 14, 16 and 18-week-old female MRL / lpr mice (n = 5) and 9-week-old MRL / MPJ mice (n = 5) (control) were collected. , Urinary RAGE excretion was measured by ELISA. The urinary RAGE excretion amount of the control was set to 1, and the urinary RAGE excretion amount was relatively compared. The amount of urinary protein was measured by urinary creatinine correction. The results are shown in FIG. In MRL / lpr mice, urinary RAGE excretion increased from 8 weeks of age (Fig. 6, top). There was no significant difference in urinary protein content during the entire study period (Fig. 6, bottom). These results indicate that SLE mice have high urinary RAGE levels.

Experiment 7: Correlation between urinary RAGE excretion and urinary NAG excretion Using the same sample as in Experiment 6, urinary RAGE excretion and urinary NAG (N-acetyl-β-D-), which is a marker for tubular damage, Glucosaminidase) The relationship with excretion was investigated. The measurement of urinary NAG excretion was outsourced to SRL Co., Ltd. and carried out by the ELISA or EIA method. The results are shown in FIG. Urinary NAG excretion in MRL / lpr mice increased at 18 weeks of age (Fig. 7, top). For all week-old samples, plotting the urinary NAG excretion (NAG / Cre ratio) of MRL / lpr mice on the horizontal axis and the urinary RAGE excretion (RAGE / Cre ratio) on the vertical axis is strongly positive. A correlation was seen (Fig. 7, bottom). These results indicate that urinary RAGE excretion increases with the progression of nephritis.

８週齢雌ＭＲＬ／ｌｐｒマウスを２群に分け、ＲＡＧＥアプタマー（ｎ＝６）またはコントロールアプタマー（ｎ＝９）を、浸透圧ポンプを用いて皮下持続投与した（２.０×１０^−４μｇ／日）。８、１２、１６および１８週齢で尿と血液を採取した。１８週齢で屠殺し、腎臓、肝臓、脾臓および肺を採取した。以下の実験では、これらのサンプルを使用した。 Administration of RAGE aptamer and sampling The following RAGE aptamer (RAGE-apt) and control aptamer (Ctr-apt) were used.

Eight-week-old female MRL / lpr mice were divided into two groups, and RAGE aptamer (n = 6) or control aptamer (n = 9) was continuously administered subcutaneously using an osmotic pump (2.0 × 10 ^-4 μg). /Day). Urine and blood were collected at 8, 12, 16 and 18 weeks of age. It was sacrificed at 18 weeks of age and the kidneys, liver, spleen and lungs were harvested. These samples were used in the experiments below.

Experiment 8: Effect of RAGE aptamer on lupus nephritis Blood urea nitrogen (BUN) of an 18-week-old sample was measured by the UV method. Masson's trichrome staining of renal tissue (staining in which the deposition of immune complex was dyed red) was performed, and the area of positive staining in the glomerulus was measured. The anti-ds-DNA antibody in the blood of the 18-week-old sample was measured by the ELISA method (Levis). The blood IgG concentration of the 18-week-old sample was measured by CLEIA. The blood C3 concentration of the 18-week-old sample was measured by the immunoturbidimetry method. The blood Na concentration of the 18-week-old sample was measured by the electrode method. The urinary albumin excretion of the 18-week-old sample was measured by the immunoturbidimetry method. The results are shown in FIG. 8 and the table below.

データは平均±ＳＥＭである。
*は、P < 0.05 vs. MRL/MPJを示す。#は、P < 0.05 vs. MRL/lpr + Ctrl-aptを示す。
**は、P=0.051 vs. MRL/MPJを示す。##は、P=0.055 vs. MRL/lpr + Ctrl-aptを示す。
略号；
ＢＰ：血圧、ＨＲ：心拍数、ＫＷ：腎臓重量、ＢＷ：体重、ＢＵＮ：血中尿素窒素、ＵＡＥ：尿アルブミン排泄量

The data are mean ± SEM.
* Indicates P <0.05 vs. MRL / MPJ. # Indicates P <0.05 vs. MRL / lpr + Ctrl-apt.
** indicates P = 0.051 vs. MRL / MPJ. ## indicates P = 0.055 vs. MRL / lpr + Ctrl-apt.
Abbreviation;
BP: Blood pressure, HR: Heart rate, KW: Kidney weight, BW: Body weight, BUN: Blood urea nitrogen, UAE: Urinary albumin excretion

BUN was low in the RAGE aptamer-administered group. This indicates that the administration of RAGE aptamer suppressed the decrease in renal function. Masson's trichrome staining of renal tissue indicates that administration of RAGE aptamer suppressed the deposition of immune complexes in the glomerulus (Fig. 8, upper row). In addition, in the mice to which the RAGE aptamer was administered, the concentration of IgG, which is the most involved in autoimmune diseases among the immunoglobulins produced from B cells, was low. Furthermore, the blood C3 concentration was high in the RAGE aptamer-administered group. The complement component C3, which decreases during the lupus nephritis activity phase, is improved by administration of RAGE aptamer, suggesting that the reduction of inflammation suppressed the consumption of C3 and suppressed the activity of lupus nephritis. be.

Experiment 9: Effect of RAGE aptamer on RAGE expression The amount of RAGE present in renal tissue was analyzed by Western blotting. In addition, mRNA was extracted from renal tissue, and the RAGE expression level in renal tissue was measured by real-time RT-PCR. The results are shown in FIG. In the Western blotting method, no significant difference was observed in the amount of RAGE between the RAGE aptamer-administered group and the control group (upper row). On the other hand, the RAGE expression level measured by real-time RT-PCR was significantly lower in the RAGE aptamer-administered group (lower row). This result indicates that the expression of RAGE in the kidney was suppressed in the RAGE aptamer-administered group. In the Western blotting method, RAGE derived from blood flow was also included in the sample, so it is considered that no significant difference was observed.

Experiment 10: Inhibition of HMGB1 deposition by RAGE aptamers HMGB1 is representative of SLE-related RAGE ligands. The degree of HMGB1 deposition in renal tissue was analyzed by immunostaining. The HMGB1 content of the entire renal tissue was analyzed by Western blotting. The amount of HMGB1 in plasma was measured by ELISA. The results are shown in FIG. Although there was no significant difference in the HMGB1 content (upper, right) and plasma HMGB1 (lower) of the entire renal tissue (90% of the renal tubules) between the RAGE aptamer-administered group and the control group, HMGB1 in the glomerulus was not observed. Deposition was suppressed (upper, left). In other words, by inhibiting RAGE, which is thought to play a role in incorporating HMGB1 which is a RAGE ligand into cells, deposition in tissues is suppressed, but the amount of HMGB1 present in bloodshed or urine does not change. It is considered that the HMGB1 concentration in the kidney and blood did not change.

Experiment 11: Effect of RAGE aptamer on inflammatory cytokine expression MRNA was extracted from kidney, liver, spleen and lung tissues, and the expression levels of inflammatory cytokines IL-6 and TNFα were measured by real-time RT-PCR. In addition, the expression level of MCP-1, which is an inflammatory cytokine, was measured in the kidney. The results are shown in FIGS. 11-13. Interestingly, the amount of IL-6 and TNFa mRNA in the RAGE aptamer-administered group decreased to about 1/10 in the kidney alone, and the amount of MCP-1 mRNA was significantly decreased in the kidney. This result suggests that RAGE has a unique role in the kidney and that inhibition of RAGE suppresses inflammation and provides a renal protective effect.

Experiment 12: Effect of RAGE aptamer on the expression of Kim1 The expression of Kim1 which is a marker of acute kidney injury and is involved in apoptosis was analyzed by renal cortex immunostaining. The results are shown in FIG. In the RAGE aptamer-administered group, Kim1 expression was clearly lower than that in the control group. In general, kidney cells (vascular endothelial cells, tubular cells) induced to undergo apoptosis by the inflammatory process express Kim1 and are phagocytosed by T cells. Considering the decrease in inflammatory cytokine expression in renal tissue due to the administration of RAGE aptamer, it is considered that the inflammatory process was suppressed by RAGE aptamer and the expression of Kim1 was also suppressed.

Experiment 13: Effect of RAGE aptamer on glomerular lesions The effect of RAGE aptamers on glomerular lesions was investigated. The formation of crescents in the glomerulus was observed by PAS staining. The formation of wire loop lesions in the glomerulus was observed by Masson's trichrome staining. Infiltration of macrophages in glomeruli was observed by immunofluorescence using anti-CD68 antibody and anti-RAGE antibody, which are macrophage markers. The results are shown in FIGS. 15-17. In MRL / lpr mice, administration of RAGE aptamers suppressed crescent formation and glomerular endothelial cell swelling (Fig. 15) and reduced the formation of wire loop lesions (Fig. 16). In addition, macrophage infiltration was observed in the glomeruli of the mice to which the control aptamer was administered and co-localized with RAGE, but the glomeruli of the mice to which the RAGE aptamer was administered had significantly less macrophage infiltration and RAGE. No co-localization with was observed (Fig. 17). This suggests that the RAGE aptamer suppressed the infiltration of macrophages into the glomerulus.

Discussion It has been reported that about 50% of MRL / lpr mice, which are a model of spontaneous SLE, die from renal failure due to nephritis around 24 weeks after birth. From the above experiments, it was revealed that the expression of RAGE increases with age in the renal tissue of MRL / lpr mice. RAGE in urine was also significantly increased in MRL / lpr mice as compared with control mice, and a strong correlation was observed between the NAG / Cre ratio and the RAGE / Cre ratio, which are markers of renal tubular damage.

From the above results, it was found that RAGE expression increased from a relatively early stage in SLE mice, and increased as the pathological condition of lupus nephritis progressed. Therefore, urinary RAGE can be used as a biomarker for the progression of lupus nephritis.

In addition, administration of RAGE aptamer to MRL / lpr mice not only suppressed the exacerbation of renal function, but also suppressed crescent formation and glomerular endothelial cell swelling, and reduced the formation of wire loop lesions. Wire-loop lesions and crescent formation are phenomena that are especially seen in diffuse lupus nephritis. In addition, since the infiltration of macrophages into the glomerulus was suppressed, the cells in the glomerulus did not incorporate molecules that induce antigen-antibody reactions such as double-stranded DNA and RNA, and the activity of immune cells in the glomerulus. It is considered that the conversion is suppressed. In addition, since the levels of inflammatory cytokines IL-6, TNFα, and MCP-1 mRNA in renal tissue were suppressed to about 1/10, RAGE aptamer suppressed inflammation and immune inducers in the kidney, thereby maintaining renal function. It is thought that it was.

Aptamers are next-generation molecular-targeted drugs whose main component is nucleic acid, and generally have low immunogenicity. Since the above-mentioned RAGE aptamer has a high affinity for RAGE, it can exert its effect with a small dose. In addition, mass production is possible and there is little difference between lots, so the production cost is low. Therefore, the RAGE aptamer can be suitably manufactured and used as a pharmaceutical.

SEQ ID NO: 1: Aptamer SEQ ID NO: 2: Aptamer SEQ ID NO: 3: Aptamer SEQ ID NO: 4: Aptamer SEQ ID NO: 5: Aptamer SEQ ID NO: 6: Aptamer SEQ ID NO: 7: Aptamer SEQ ID NO: 8: Aptamer SEQ ID NO: 9: Aptamer SEQ ID NO: 10: Aptamer SEQ ID NO: 11: Aptamer

Claims (12)

A pharmaceutical composition for treating or preventing lupus nephritis, which comprises a RAGE aptamer selected from the group consisting of:
(1) Single-stranded DNA containing the sequence of SEQ ID NO: 4;
(2) A single-stranded DNA that contains a sequence having 90% or more sequence identity with the sequence of SEQ ID NO: 4 and binds to RAGE to inhibit the binding of RAGE to its ligand;
(3) One that contains a sequence in which 1, 2, or 3 bases are deleted, substituted, or added in the sequence of SEQ ID NO: 4 and binds to RAGE to inhibit the binding of RAGE to its ligand. Strand DNA. The composition of claim 1, wherein the RAGE aptamer comprises the sequence of SEQ ID NO: 4. The composition according to claim 1 or 2, wherein the RAGE aptamer comprises the sequence of SEQ ID NO: 4. The composition according to any one of claims 1 to 3, for administration to a patient with systemic lupus erythematosus. The composition according to any one of claims 1 to 4, for treating lupus nephritis. The composition according to any one of claims 1 to 4, for preventing lupus nephritis. A pharmaceutical composition comprising a RAGE aptamer selected from the following group for administration to patients with systemic lupus erythematosus:
(1) Single-stranded DNA containing the sequence of SEQ ID NO: 4;
(2) A single-stranded DNA that contains a sequence having 90% or more sequence identity with the sequence of SEQ ID NO: 4 and binds to RAGE to inhibit the binding of RAGE to its ligand;
(3) One that contains a sequence in which 1, 2, or 3 bases are deleted, substituted, or added in the sequence of SEQ ID NO: 4 and binds to RAGE to inhibit the binding of RAGE to its ligand. Strand DNA. If the measured urinary RAGE concentration is higher than the index value or cutoff value, it is judged that the subject is at high risk of developing lupus nephritis, and the measured urinary RAGE concentration is higher than the index value or cutoff value. A method of assisting the diagnosis of lupus nephritis, which comprises measuring the RAGE concentration in urine collected from the subject to determine that the subject is at low risk of developing lupus nephritis when low. The method of claim 8, wherein the subject is a patient with systemic lupus erythematosus. A composition comprising an anti-RAGE antibody for diagnosing the risk of developing lupus nephritis by measuring the RAGE concentration in urine. A kit for diagnosing the risk of developing lupus nephritis, which comprises the composition according to claim 10. If the urinary RAGE concentration is higher than the index value or cutoff value, the subject is judged to be at high risk of developing lupus nephritis, and if the urinary RAGE concentration is lower than the index value or cutoff value, the subject is determined to be at high risk. Use of urinary RAGE as a biomarker to aid in the diagnosis of lupus nephritis , used to determine a low risk of developing lupus nephritis.

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