KR101818296B1 - Method of diagnosis for bisphosphonate-related osteonecrosis of the jaw - Google Patents

Abstract

The present invention relates to a bisphosphonate-associated jaw necrosis diagnosis and a prognostic prediction method.
By using the biomarker of the present invention, bisphosphonate-related jaw necrosis is diagnosed early, and treatment and prevention methods based on the pathogenic mechanism are used to enhance the therapeutic effect of bisphosphonate-related jaw necrosis and to minimize side effects of treatment.

Description

METHOD OF DIAGNOSIS FOR BISPHOSPHONATE-RELATED OSTEONECROSIS OF THE JAW BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a bisphosphonate-

The present invention relates to a method for diagnosing bisphosphonate-associated jaw necrosis.

The incidence of bisphosphonate-associated jaw necrosis is also expected to increase explosively due to the use of bisphosphonates worldwide and the full-scale entry into the aging society.

Since the introduction of bisphosphonates for the first time in 2003, many efforts have been made to identify the etiology and pathogenesis of this disease. However, the mechanism of development is still unclear, and the disease is now understood to be a disease caused by impaired healing due to inhibition and inhibition of osteogenesis by inhibition of osteoclast action, which is the main action of bisphosphonates.

Based on the fact that bisphosphonate-related osteonecrosis of jaw (BRONJ) is a bone metabolic disorder, risk prediction through measurement of C-terminal telopeptide of type I collagen (CTx) is proposed for diagnosis of BRONJ However, many clinical studies have shown that there is no relationship between serum CTx and BRONJ. In addition, clinical studies using several bone metabolism biomarkers have been performed, but what has been identified as a marker for bisphosphonate-related jaw necrosis disease There is no bar.

In addition, most of the related studies have been conducted with retrospective patient reviews, but this is insufficient to elicit statistical significance due to the possibility of selection bias and the limitation of the number of patients, and studies to date have generally been based on osteoporosis The patients who had undergone the biomarker test on a regular basis were set as the control group. Therefore, the patients who did not have BRONJ in their paired groups, There is something difficult to study. In addition, biomarkers should be tested before the onset of BRONJ as a risk taker, but most studies have problems in conclusions from the results of BRONJ test.

Based on this background, it is urgent to introduce a new biomarker for bisphosphonate-associated jaw necrosis, to identify the mechanism of its development, and to diagnose and prognose diseases based on the detected biomarkers.

KR 10-1474053, 2014.12.11 KR 10-1463505, 2014.11.13

The present invention relates to a bisphosphonate-associated osteonecrosis agent comprising an agent for measuring an expression level of at least one selected from among Tartrate-resistant acid phosphatase isoform 5b, OPG (osteoprotegerin), or RANKL (Receptor activator of nuclear factor- To provide diagnostic compositions.

The present invention also provides a kit for the diagnosis of bisphosphonate-associated jaw necrosis comprising the composition.

The present invention relates to a method for measuring the level of TRACP 5b or OPG / RANKL (Receptor activator of nuclear factor-kappa beta ligand) in a sample isolated from a patient, And comparing the level of the TRACP 5b (Tartrate-resistant acid phosphatase isoform 5b) or the ratio of OPG (Osteoprotegerin) / RANKL (Receptor activator of nuclear factor-κβ ligand) to a baseline level for diagnosis of bisphosphonate-associated jaw necrosis We want to provide information providing method.

The present inventors have made intensive efforts to discover biomarkers for the diagnosis of bisphosphonate-associated jaw osteonecrosis. As a result, TRACP 5b (Tartrate-resistant acid phosphatase isoform 5b), OPG (Osteoprotegerin) or RANKL (Receptor activator of nuclear factor- The present inventors confirmed that it is effective as a biomarker for diagnosis of jaw necrosis and completed the present invention.

The present invention provides a biomarker for the diagnosis of at least one bisphosphonate-associated jaw necrosis selected from TRACP 5b (Tartrate-resistant acid phosphatase isoform 5b), OPG (Osteoprotegerin) or RANKL (Receptor activator of nuclear factor-κβ ligand).

The term "diagnosis" as used herein means to identify the presence or characteristic of a pathological condition. For the purposes of the present invention, the diagnosis is to ascertain the disease status, including the incidence of bisphosphonate-associated jaw necrosis and prediction of prognosis.

The term "bisphosphonate-related jaw necrosis " used in the present invention refers to all abnormal abnormal osteomyelitis symptoms, cytotoxicity and the like which are generated in the jaw by treatment with a bisphosphonate compound (for example, zoledronate, pamidronate, etc.) Abnormal clinical symptoms such as pain in the affected part due to the treatment, tooth fluctuation, mucosal edema, ulceration, and changes in the sensation of the affected part.

In the present invention, a normal control (non-diseased) means a normal subject not receiving bisphosphonate administration.

In the present invention, bisphosphonate-associated jaw-related necrosis is an entity in which nasal bone necrosis occurs after bisphosphonate administration and surgical intervention, and ulcerative lesions and / or osteolysis are present in exposed necrotic bones and are accompanied by inflammatory cell infiltration Refers to a group having symptoms of pseudoepitheliomatous-like hyperplasia in the epithelial tissue and the presence of abdomen and bacterial colonies.

In the present invention, non-bisphosphonate-associated jaw necrosis refers to an individual who normally heals without occurrence of jaw necrosis after administration of bisphosphonate and surgical intervention.

As used herein, the term "diagnostic biomarker" refers to a significant increase or decrease in the level of gene expression or protein expression in a bisphosphonate-associated jaw necrosis subject relative to a normal control (non-diseased subject) or non-bisphosphonate- And organic biomolecules such as polypeptides or nucleic acids (e.g., mRNA) showing lipophilic characteristics, lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides and the like), and the like.

The diagnostic biomarker of the present invention may preferably be TRACP 5b or RANKL (Rectal activator of nuclear factor-kappa beta ligand) / OPG (Osteoprotegerin) ratio.

The present invention relates to a bisphosphonate-associated osteonecrosis agent comprising an agent for measuring an expression level of at least one selected from among Tartrate-resistant acid phosphatase isoform 5b, OPG (osteoprotegerin), or RANKL (Receptor activator of nuclear factor- To provide diagnostic compositions.

The gene information of the three proteins of the present invention may be TRACP 5b (GeneBank Accession No. NP_001104506.1), OPG (GeneBank Accession No. AAB53709.1), or RANKL (GeneBank Accession No. AAB86811.1).

The TRACP 5b and RANKL specifically showed a decrease in the gene expression level or the expression level of the protein in the bisphosphonate-associated jaw necrosis individuals compared with the non-bisphosphonate-related jaw necrosis subjects, and OPG was decreased in comparison with the non-bisphosphonate- The level of gene expression or the expression level of the protein is specifically increased in bisphosphonate-associated jaw necrosis individuals.

Preferably, the diagnostic composition comprises an agent that measures TRACP 5b or RANKL (Receptor activator of nuclear factor-kappa beta ligand) / OPG (Osteoprotegerin) ratio.

Expression level measurement of the present invention is an mRNA expression level measurement or protein level measurement, preferably a protein level measurement.

The term " measurement of mRNA expression level "used in the present invention is used to determine the presence and expression level of mRNA in genes for the diagnosis of bisphosphonate-associated jaw necrosis in a biological sample to diagnose bisphosphonate-associated jaw necrosis. RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection (RPA), and reverse transcriptase-polymerase chain reaction assay, Northern blotting, DNA chip, and the like.

The agent for measuring the mRNA expression level of the gene is preferably a primer pair or a probe. Since the nucleic acid information of the genes is known by GeneBank et al., A person skilled in the art can use a primer or a probe specifically amplifying a specific region of these genes Can be designed.

The term " measurement of protein expression level "used in the present invention is a process for confirming the presence and expression level of a protein in a biological sample in order to diagnose bisphosphonate-related jaw necrosis. An amount of the protein can be confirmed by using an antibody, an interacting protein, a ligand, a nanoparticle or an aptamer that specifically binds to the protein or peptide fragment. However, the affinity specific to the protein or peptide fragment and may preferably comprise all of the detection means with affinity, and preferably the level of protein expression itself, without the use of antibodies, interacting proteins, ligands, nanoparticles or aptamers.

As a method for measuring or comparing the protein expression level, protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Desorption / Ionization Time of Flight Mass Spectrometry) analysis, SELDI- of Flight Mass Spectrometry analysis, radioimmunoassay, radial immunodiffusion, Oucheroton immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation, two-dimensional electrophoresis analysis, liquid chromatography (LC-MS), liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS), Western blotting, and enzyme linked immunosorbent assay (ELISA).

Preferably, the agent for measuring the protein level is selected from one or more proteins or peptide fragments selected from TRACP 5b (Tartrate-resistant acid phosphatase isoform 5b), OPG (Osteoprotegerin) or RANKL (Receptor activator of nuclear factor- Antibodies, interacting proteins, ligands, nanoparticles, or aptamers that bind to the antigen.

The term "antibody" as used herein refers to a specific protein molecule directed against an antigenic site. For the purpose of the present invention, the antibody specifically binds to at least one protein selected from TRACP 5b (Tartrate-resistant acid phosphatase isoform 5b), OPG (Osteoprotegerin) or RANKL (Receptor activator of nuclear factor- Refers to an antibody, and includes both a polyclonal antibody, a monoclonal antibody, and a recombinant antibody. The production of the antibody can be easily carried out using techniques well known in the art.

The antibodies of the present invention also include functional fragments of antibody molecules as well as complete forms with two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least an antigen binding function, and includes Fab, F (ab ') 2, F (ab') 2 and Fv.

As used herein, the term "aptamer" is a biopolymer substance that inhibits protein interaction through three-dimensional binding with a specific target protein in the form of single or double stranded DNA or RNA. As shown in Fig. Typically, the aptamer may be a small nucleic acid of 15-50 bases in length that folds into defined secondary and tertiary structures, such as a stem-loop structure. It is preferred that the aptamer binds to the target high-expression or low-expression protein at a kd of less than 10 ^-6 , 10 ^-8 , 10 ^-10 , or 10 ^-12 . The aptamer can bind a highly expressed protein or a low expression protein with a very high specificity and the aptamer can be composed of a mixture of multiple ribonucleotide units, deoxyribonucleotide units, or two types of nucleotide residues. The aptamers of the present invention may further comprise one or more modified base, sugar or phosphate backbone units.

In another aspect, the present invention provides a kit for the diagnosis of bisphosphonate-associated jaw necrosis comprising the composition for diagnosing bisphosphonate-associated jaw necrosis. Preferably, the kit may be an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a rapid kit, or a multiple reaction monitoring (MRM) kit. Preferably, the kit for diagnosing bisphosphonate-associated jaw necrosis further comprises one or more other component compositions, solutions or devices suitable for the assay method.

More preferably, it may be a diagnostic kit characterized by comprising essential elements necessary for performing ELISA. ELISA kits include antibodies, interacting proteins, ligands, nanoparticles or aptamers that specifically bind to the protein or peptide fragments. Antibodies are monoclonal antibodies, polyclonal antibodies or recombinant antibodies with high specificity and affinity for each marker protein and little cross reactivity to other proteins. Other ELISA kits can be used to detect antibodies that can bind a reagent capable of detecting the bound antibody, such as a labeled secondary antibody, chromophores, an enzyme (e. G., Conjugated to an antibody) Other materials, and the like.

The present invention relates to a method for measuring the level of TRACP 5b or OPG / RANKL (Receptor activator of nuclear factor-kappa beta ligand) in a sample isolated from a patient, And comparing the level of the TRACP 5b (Tartrate-resistant acid phosphatase isoform 5b) or the ratio of OPG (Osteoprotegerin) / RANKL (Receptor activator of nuclear factor-κβ ligand) to a baseline level for diagnosis of bisphosphonate-associated jaw necrosis Provide information providing method.

As used herein, the term "sample separated from a patient" refers to a sample such as a tissue, cell, whole blood, serum, plasma, saliva, cerebrospinal fluid or urine which shows a difference in gene expression level or protein expression level by the onset of bisphosphonate- But are not limited thereto.

In the present invention, the "reference level" means the same level as the level of expression of a gene or protein capable of exhibiting high diagnostic predictability as detected and determined from reference samples by the methods described herein. The reference level may be relative to the number or value obtained from a population study with the same disease, a subject of the same or similar age range, a population study in which the racial group includes the same or similar subjects, ≪ / RTI > This reference level can be derived from statistical analysis of the population and / or risk prediction data, obtained from mathematical algorithms and computed exponents. The reference specific coefficients may also be constructed and used by using statistical and structural classification algorithms and other methods.

In some embodiments, the term "reference level" is used herein to refer to a pre-measured value, for example, those skilled in the art will recognize that the reference level may be pre-measured and set to meet the requirements in terms of specificity, sensitivity and / have. For example, the sensitivity or specificity may be set to a certain limit, e.g., 60%, 70%, 80%, 90% or 95%, respectively, and these requirements may be defined in terms of positive or negative predictive value .

In the present invention, statistical analysis methods such as average value calculation or ROC curve analysis can be used to determine the reference level of the expression level.

The ROC curve analysis according to an embodiment of the present invention is a curve showing the performance of diagnosis, and it is composed of sensitivity, specificity, and accuracy. According to one embodiment of the present invention, the sensitivity is a fraction of a test in a diseased population that indicates that the disease is a disease, and the specificity is a percentage of a test group And the accuracy represents the hit ratio of the test results among all cases. In this case, when the specificity and the sensitivity are both high, the accuracy of the test becomes high. Therefore, in the ROC curve, the x-axis becomes 1-specific and the y-axis becomes sensitive, and the area under curve (AUC) It means the area below.

According to one embodiment of the present invention, the "reference level" is a cutoff value showing a high predictive effect on the onset of bisphosphonate-associated jaw necrosis. The ratio of TRACP 5b or OPG / RANKL (TRACP 5b) or RANKL (ratio of reactant to nuclear factor-κβ ligand) , And predicts a higher incidence rate for samples below the reference level. Preferably, the TRACP 5b may be 3.09 U / L or less and the RANKL / OPG ratio may be 0.88 or less.

In the present invention, the term " above "or" excess "means a level above the reference level, or 1%, 2%, 5%, 10% %, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% . In the present invention, the terms "below" or "below" means a level below the reference level, or 1%, 2%, 5%, 10% %, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% .

The ratio of TRACP 5b or RANKL / RANKL / OPG (Osteoprotegerin) is higher than that of non-bisphosphonate-associated jaw necrosis in the bisphosphonate-associated jaw necrosis , The expression level of the gene or the expression level of the protein is decreased. Accordingly, when the level is decreased, the bisphosphonate-associated jaw necrosis entity can be diagnosed and information can be provided.

Preferably, the protein expression level of the present invention can be measured and compared using antibodies, interacting proteins, ligands, nanoparticles or aptamers that specifically bind to the protein or peptide fragment. The antibody and the protein in the biological sample are allowed to form an antigen-antibody complex, and a method of detecting the antibody-antibody complex is used. As used herein, the term "antigen-antibody complex" refers to a combination of a protein antigen and an antibody recognizing the presence or absence of the gene in the biological sample. The detection of the antigen-antibody complex can be detected using methods such as those known in the art, for example, spectroscopic, photochemical, biochemical, immunochemical, electrical, absorbance, chemical and other methods.

More preferably, in the present invention, the measurement and comparison of the protein expression level is characterized by measuring and comparing the protein expression level itself without using the antibody. For the purpose of the present invention, protein level analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Desorption / Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF Enhanced Laser Desorption / Ionization Time of Flight Mass Spectrometry analysis, radioimmunoassay, radial immunodiffusion, Oucheronian immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation analysis, two-dimensional electrophoresis analysis, But are not limited to, liquid chromatography-mass spectrometry (LCMS), liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS), Western blotting and enzyme linked immunosorbent assay (ELISA).

By using the biomarker of the present invention, bisphosphonate-related jaw necrosis is diagnosed early, and treatment and prevention methods based on the pathogenic mechanism are used to enhance the therapeutic effect of bisphosphonate-related jaw necrosis and to minimize side effects of treatment.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a diagram illustrating an experimental design for identifying a bisphosphonate-associated jaw necrosis biomarker of the present invention.
FIG. 2 is a graph showing the clinical appearance of jaws of non-bisphosphonate-associated jaw-necrosis group and bisphosphonate-associated jaw-necrosis group.
FIG. 3 is a diagram showing a tissue analysis result of a necrotizing rat jaw having an ulcerative lesion and a necrotic bone.
FIG. 4 is a graph showing the time-dependent biomarker CTx (C-terminal crosslinked telopeptide of type I collagen), TRACP 5b (Tartrate-resistant acid phosphatase isoform 5b), OPG (Osteoprotegerin), Glu- OC (Undercarboxylated osteocalcin), RANKL activator of nuclear factor-kappa beta ligand) and RANKL / OPG ratio.
FIG. 5 shows ROC curve analysis results of serum TRACP 5b and RANKL / OPG ratio. FIG.
Figure 6 is a graph showing individual percentage changes in the ratio of serum TRACP 5b and RANKL / OPG.

Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described in detail below. The present invention may, however, be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Is provided to fully convey the scope of the invention to those skilled in the art, and the invention is only defined by the scope of the claims.

≪ Example 1 > Animal Model and Experimental Design

A female rat (Sprague Dawley, SD) (n = 48), an average weight of 220 g (range 190-247 g) of 8 weeks old purchased from Ewah Medical Research, Seoul, Korea, . Animals were housed three per cage, and were then numbered using an ear punch. The cage was placed in a room of purified air at a temperature of 22 ± 2 ° C and a relative humidity of 50% ± 10%. The 12 hour name / cancer cycle was maintained. The animals fed an average rodent meal and water ad libitum. The animals were acclimated one week before the start of the experiment. This study was approved by the Institutional animal research ethics committee and carried out in accordance with its guidelines.

The rats were randomly divided into two groups. The bisphosphonate group (n = 36) received 200 μg / kg of zoledronic acid and the control group (n = 12) received saline once per week. Considering that the pharmacokinetics of bisphosphonates were not known in rodents, dosages were determined according to the following factors: (1) a suitable zoledronate dose of human (67 μg / kg / 4 weeks) in oncology; (2) the relatively fast bone metabolism of rodents; (3) administration route of bisphosphonates (lower plasma concentration when subcutaneous administration was used versus intravenous administration); And (4) maximum drug exposure over a relatively short period of time.

After 6 weeks of injections, extraction was performed with surgery to induce necrotizing necrosis. Animals were treated with ketamine (100 mg / kg, Yuhan pharmacy, Seoul, Korea), xylazine (10 mg / kg Rompun, Germany Leverkusen, Bayer AG) and meloxicam (1.5 mg / , Boehringer Ingelheim). Appropriate dislocation was performed until the fluidity of the teeth was sufficiently obtained after the ligamentisation using a pointed dental probe. In this way, using the dental probe, the first to third molars of the left lower right were sequentially removed. The gauze was compressed to control the bleeding, and the wound was left open without any sutures. One injection per week lasted for 8 weeks and the period was selected as the onset of bisphosphonate-associated jaw necrosis progressed. The animals were then sacrificed for further histological evaluation.

The experimental design contents are shown in Fig.

≪ Example 2 >

The presence of ONJ was determined based on histological analysis according to the following criteria: (1) presence of ulcerative lesions and / or osteolysis in exposed and necrotic bones, (2) presence of inflammatory cell infiltration in epithelial tissues The presence of pseudoepitheliomatous-like hyperplasia, and (3) the presence of pelvic and bacterial colonies.

A photograph of the sacrum of the sacrificed rat is shown in Fig. FIG. 2 (a) is a jaw of a non-jaw osteoarthritis group showing a normal mucosa 8 weeks after surgery, and FIG. 2 (b) is a jaw of a jaw osteomalacia group. As shown in FIG. 2, it was confirmed that the cell necrosis occurred in the group of onset of jaw necrosis.

The samples were also fixed with 4% phosphate-buffered saline formalin for 2 weeks and decalcified for 28 days at 4 ° C with 10% EDTA (pH 7.2). The sample was dehydrated in several steps with ethanol, washed with xylene and dipped in paraffin. The samples were then embedded in paraffin and cut into 4-μm-thick sections. Paraffins were removed from the sections and rehydrated and stained with hematoxylin and eosin (H-E). The results are shown in Fig. As shown in Fig. 3, it was confirmed that necrosis bone and ulcerative lesions were seen in the section taken from the jaw necrosis group.

≪ Example 3 > Biomarkers and statistical analysis

In the biomarker survey, blood samples were collected after fasting for 12 hours at T ₀ (reference point), T ₁ (6 weeks; surgery), and T ₂ (14 weeks; Blood samples were collected via jugular vein (T ₀ , T ₁ ) and cardiac puncture (T ₂ ) and then allowed to solidify for 20 minutes. The collected serum samples were separated by centrifugation (1600 xg, 4 캜, 15 min), aliquoted and stored at -70 캜 until analysis. (RatTRAP, IDS, Boldon, UK), serum undercarboxylated osteocalcin (Glu-OC; Rat Glu-Osteocalcin high sensitive EIA, Takara Bio, Shiga, Japan), serum TRXP 5b , Tyne & Wear), serum receptor activator of nuclear factor-κβ ligand (RANKL; Rat sRANKL ELISA, Cusabio, Wuhan, Hubei, China) and serum osteoprotegerin (OPG; Rat sOPG ELISA, Cusabio) .

을 기준으로 결정되었으며, signal to ratio는 T ₂ (signal)에서 비스포스포네이트 집단의 각 바이오마커 변화를 바이오마커의 변동성(noise;

)으로 나누어서 결정하였다. 0.05 미만의 P 값이 통계적으로 유의하다고 여겨지며, 통계적 분석은 SPSS 20.0(IBM Corp., Armonk, NY, USA) 및 MedCalc 12.7.0(Medc software, Ostend, Belgium)을 사용하여 수행되었다.According to the histological analysis of 2. above, rats of the bisphosphonate group (n = 36) were further classified as non-jaw necrosis group and jaw necrosis group. The normal distribution of the biomarkers tested (CTx, Glu-OC, TRACP 5b, and RANKL / OPG ratio) and the identity of variance were checked before further statistical analysis. The overall group differences of biomarkers over time were compared via repeated measures analysis of variance (RM-ANOVA). An independent t-test was used to test for significant differences between the non-jaw necrosis group and the jaw necrosis group, and between the bisphosphonate group and the control group. The receiver operating characteristic (ROC) curve analysis at T ₁ and T ₂ was used to evaluate the biomarkers. The area under curve (AUC) and the sensitivity and specificity of the biomarker, the cutoff value, were calculated. The intra-assay coefficient of variability (CVa) of each biomarker was determined twice and measured as an average value. The inter-assay coefficient of variability (CVi) was determined as the mean change observed in T ₂ versus T ₀ in the control group. The least significant change LSC at P < 0.05 is given by the equation: LSC =

And the signal to ratio is the change in biomarker of the bisphosphonate population at T ₂ (signal) to the biomarker variability (noise).

). P values less than 0.05 were considered statistically significant and statistical analyzes were performed using SPSS 20.0 (IBM Corp., Armonk, NY, USA) and MedCalc 12.7.0 (Medc software, Ostend, Belgium).

The results of the analysis are shown in Table 1 and Figs. 4 to 6.

In the 36 rats of the bisphosphonate group, 27 rats were classified histologically as the ONG group, and 9 rats were classified as the non-ONJ group. The values of all tested markers are shown in Table 1.

[Table 1] Biomarker analysis results

As seen in Table 1 above, CTx, TRACP 5b, and RANKL were significantly reduced in the bisphosphonate group compared to the control group in both T ₁ and T ₂ .

In order to analyze the potential biomarkers of jaw necrosis, biomarker concentrations were analyzed according to the time course between non - jaw necrosis group and jaw necrosis group. The TRACP 5b and RANKL / OPG ratios are shown in Fig. In the RM-ANOVA, the ONJ group showed a significantly lower ratio of TRACP 5b and RANKL / OPG over time than the non-ONJ group ( P <0.05). These results were also consistent with the RANKL / OPG ratio of T ₁ and the TRACP 5b and RANKL / OPG ratios of T ₂ .

In order to evaluate the clinical performance as a biomarker of TRACP 5b and RANKL / OPG ratio in jaw necrosis, the ROC curve analysis results at T ₁ and T ₂ are shown in FIG. As shown in Figure 5, the AUC of TRACP 5b at T ₂ was 0.807 (95% confidence interval [CI], 0.641-0.919; P < 0.001) and cutoff value was 3.09 U / L at both maximum sensitivity and specificity Respectively. The sensitivity and specificity of TRACP 5b for diagnosis in this cutoff value were 88.9% and 66.7%, respectively. Also, the AUC of the RANKL / OPG ratio at T ₁ was 0.776 (95% CI, 0.606-0.897; P <0.001). The AUC at T ₂ was 0.765 (95% CI, 0.595-0.890; P = 0.002) and the cutoff value was less than 0.88 (sensitivity, 77.8%, specificity, 62.9%).

The percent change (T ₂ -T ₀ ) of the TRACP 5b and RANKL / OPG ratio, respectively, is shown in FIG. TRACP 5b was associated with low intra-individual CV (6.32%) and inter-individual CV (11.20%) compared to the RANKL / OPG ratios (intra-individual CV, 8.14%; inter-individual CV, 14.76%; LSC, 39.27% And LSC (29.6%).

Based on the above results, it was confirmed that the ratio of TRACP 5b and RANKL / OPG has a good function as a biomarker for jaw necrosis.

Claims (9)

delete delete delete delete delete Measuring the level of TRACP 5b (Tartrate-resistant acid phosphatase isoform 5b) in the sample isolated from the patient;
Comparing the level of TRACP 5b (Tartrate-resistant acid phosphatase isoform 5b) with a reference level; And
Comparing the measured TRACP 5b level with the reference level to select samples below a reference level. 7. The method of claim 6, wherein the level measurement is for the diagnosis of bisphosphonate-associated jaw necrosis, which is an antibody, interacting protein, ligand, nanoparticles or an aptamer that specifically binds to a protein or peptide fragment. Information delivery method. 7. The method of claim 6, wherein the level measurement and comparison method comprises at least one of protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Desorption / Ionization Time of Flight Mass Spectrometry) analysis, SELDI- Ionization Time of Flight Mass Spectrometry Analysis, Radiation Immunoassay, Radiation Immunodiffusion, Ouchteroni Immunodiffusion, Rocket Immunoelectrophoresis, Tissue Immunostaining, Complement Fixation Analysis, 2D Electrophoresis, Liquid Chromatography-Mass Spectrometry for the diagnosis of bisphosphonate-associated jaw necrosis selected from the group consisting of liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS), western blot, and enzyme linked immunosorbent assay Information delivery method. delete

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