In vitro method to identify whether a subject treated with bone-modifying agents may develop osteonecrosis of the jaws

Abstract

The present invention belongs to the medical field. Specifically, it relates to a method based on the detection of biomarkers to identify whether a subject treated with bone-modifying agents (BMAs) may develop medication-related osteonecrosis of the jaws (MRONJ), to assess whether it is advisable to carry out an invasive dental procedure in subjects treated with BMAs, and / or to select subjects treated with BMAs based on their risk of developing MRONJ.

Description

[0001] IN VITRO METHOD TO IDENTIFY WHETHER A SUBJECT TREATED WITH BONE-MODIFYING AGENTS MAY DEVELOP OSTEONECROSIS OF THE JAWS

[0002] FIELD OF THE INVENTION

[0003] The present invention belongs to the medical field. Specifically, it relates to a method based on the detection of biomarkers to identify whether a subject treated with bone-modifying agents (BMAs) may develop medication-related osteonecrosis of the jaws (MRONJ), to assess whether it is advisable to carry out an invasive dental procedure in subjects treated with BMAs, and / or to select subjects treated with BMAs based on their risk of developing MRONJ.

[0004] PRIOR ART

[0005] Bone-modifying agents (BMAs) are drugs aimed at improving the bone mineral density and microstructure. They are primarily used in the treatment of osteoporosis, as anti-tumour treatment, and in control of metastatic bone disease. Medication-related osteonecrosis of the jaws (MRONJ) is a potentially serious complication commonly observed in patients treated with BMAs. According to the definition by the American Association of Oral and Maxillofacial Surgeons (AAOMS), MRONJ is defined as exposed bone, or bone that can be probed with an intraoral or extraoral fistula, that i) does not heal after 8 weeks, ii) occurs in a patient who has been treated with BMAs or angiogenic inhibitors, and iii) who has no history of head and neck radiation. This treatment sequela appears mainly in patients treated with three types of BMAs (bisphosphonates, antiangiogenics and monoclonal antibodies such as denosumab) who have undergone an invasive dental procedure (tooth extraction, dental implant, apicoectomy, etc.). MRONJ is clinically similar to osteoradionecrosis caused by the direct and indirect action of radiotherapy in the head and neck region but has specific clinical and prognostic characteristics. Treatment of patients with MRONJ varies according to the severity of the necrosis, ranging from antibiotics and antiseptics in the early stages to bone resections that affect the facial bone structure and impair the quality of life of affected patients, limiting their masticatory and phonatory function, social environment and quality of life.

[0006] In this context, there is a pressing need to improve the determination of reliable predictive biomarkers which allow assessing the risk of developing MRONJ in patients taking BMAs, especially in order to avoid possible invasive dental procedures that may favour the development of MRONJ. The present invention aims to solve this problem and proposes a method to identify whether a subject treated with BMAs may develop MRONJ, to assess whether it is advisable to carry out an invasive dental procedure in subjects treated with BMAs, and / or to select subjects treated with BMAs based on their risk of developing MRONJ.

[0007] DESCRIPTION OF THE INVENTION

[0008] Brief description of the invention

[0009] The present invention relates to a method based on the detection of biomarkers to identify whether a subject treated with BMAs may develop MRONJ, to assess whether it is advisable to carry out an invasive dental procedure in subjects treated with BMAs, and / or to select subjects treated with BMAs based on their risk of developing MRONJ.

[0010] By means of comparing the salivary proteome of patients with MRONJ to two control groups (one of them taking BMAs without MRONJ and the other group being healthy patients who are not taking BMAs), investigators have identified that the combination of markers (MMP9, metalloproteinase 9; AACT, Alpha- 1 -anti chymotrypsin; and HBD, haemoglobin subunit delta), with at least one marker selected from MMP8 (metalloproteinase 8), A1BG (Alpha-IB- glycoprotein) and / or HBB (haemoglobin subunit beta), allow identification of the presence of MRONJ with both a sensitivity and a specificity above 85% (Table 1). Namely, combinations i) MMP9, AACT, HBD and MMP8, ii) MMP9, AACT, HBD, MMP8 and A1BG, and iii) MMP9, AACT, HBD, MMP8, A1BG and HBB resulted in a sensitivity in the 85-93% range and a specificity of 89.5%.

[0011] Table 1: Sensitivity and specificity provided by biomarkers MMP9, AACT and HBD, combined with at least one protein selected from MMP8, A1BG and / or HBB.

[0012] Particularly, it is unexpected that the AUC value provided by combining the proteins MMP9, AACT and HBD (0.879) would be substantially improved to values higher than 0.9, when the three biomarkers MMP9, AACT and HBD are further combined with at least one, two or the three following proteins: MMP8, A1BG and / or HBB. According to Table 1, when the proteins MMP9, AACT and HBD were further combined with MMP8 the AUC increased to 0.9237, when the proteins MMP9, AACT and HBD were further combined with MMP8 and A1BG the AUC increased to 0.9342 and when the proteins MMP9, AACT and HBD were further combined with MMP8, A1BG and HBB the AUC increased to 0.9342.

[0013] So, in summary, a precise selection of proteins to be combined with MMP9, AACT and HBD (namely MMP8, A1BG and / or HBB) was made in the present invention to obtain higher- ranked signatures.

[0014] On the other hand, the combination of markers MMP8, MMP9 and A1BG discriminated the presence of MRONJ with the sensitivity and specificity shown in Table 2. Furthermore, the predictive capability was improved by combining these three proteins with at least one of the proteins selected from AACT, HBB and / or HBD. (Table 2). Namely, combinations i) MMP8, MMP9, A1BG and AACT, ii) MMP8, MMP9, A1BG, AACT and HBB and iii) MMP8, MMP9, A1BG, AACT, HBB and HBD provided a sensitivity of 89-93% and a specificity of 78.9-92.4%. It should be noted that the specificity provided by the combination of MMP8, MMP9, A1BG, AACT, HBB and HBD shown in Table 2 (92.5%) is slightly higher than that shown in Table 1 (89.5%) due to slight differences in the statistical fit applied to each model.

[0015] Table 2: Sensitivity and specificity provided by biomarkers MMP8, MMP9 and A1BG, combined with at least one protein selected from AACT, HBB and / or HBD. Therefore, based on the results shown, the first aspect of the invention relates to an in vitro method to identify whether a subject treated with one or more BMAs may develop MRONJ, to assess whether it is advisable to carry out an invasive dental procedure in subjects treated with one or more BMAs, and / or to select subjects treated with one or more BMAs based on their risk of developing MRONJ, which comprises: a) determining the level of expression of the combination of the following three proteins: MMP9, AACT and HBD combined with at least one protein selected from the group consisting of: MMP8, A1BG and / or HBB, or the genes encoding said proteins, in a biological sample obtained from a subject, b) wherein a high level of expression, in comparison with a pre-established threshold value, is indicative that the subject may develop MRONJ, that it is not advisable to carry out an invasive dental procedure, and / or that the subject has the risk of developing MRONJ.

[0016] In a preferred embodiment of the invention, step a) comprises determining the level of expression of the combination of the following three proteins: MMP9, AACT and HBD combined with at least two proteins selected from the group consisting of: MMP8, A1BG and / or HBB, or the genes encoding said proteins.

[0017] In a preferred embodiment of the invention, step a) comprises determining the level of expression of the combination of the MMP9, AACT, HBD and MMP8; MMP9, AACT, HBD, MMP8 and A1BG; and / or MMP9, AACT, HBD, MMP8, A1BG and HBB proteins, or the genes encoding said proteins.

[0018] In a preferred embodiment of the invention, the BMAs are selected from the group comprising: immunotherapy, monoclonal antibodies, bisphosphonates, anti angiogenic drugs and / or antiresorptive drugs.

[0019] In a preferred embodiment of the invention, the BMAs are selected from the group of immunotherapy, monoclonal antibodies, bisphosphonates and / or anti angiogenic drugs comprising: etidronate, clodronate, alendronate, risedronate, tiludronate, pamidronate, ibandronate, zoledronate, neridronate, olpadronate, bevacizumab, aflibercept, pazopanib, cabozantinib, sunitinib, axitinib, dasatinib, imatinib, erlotinib, sorafenib, infliximab, adalimumab, rituximab, bevacizumab, denosumab, methotrexate, temsirolimus and / or everolimus. In a preferred embodiment of the invention, the invasive dental procedure is selected from the group comprising: simple tooth extraction, complex tooth extraction, periapical surgery, resective periodontal surgery, regenerative periodontal surgery, dental implant, any oral surgery technique involving the potential exposure of alveolar bone, and / or basic periodontal treatment.

[0020] In a preferred embodiment of the invention, the biological sample is selected from: saliva and / or a bone biopsy, preferably saliva.

[0021] The second aspect of the invention relates to the in vitro use of the following three proteins: MMP9, AACT and HBD combined with at least one protein selected from the group consisting of: MMP8, A1BG and / or HBB, or transcripts encoding said proteins, or a kit comprising reagents to measure the level of expression of the following three proteins: MMP9, AACT and HBD combined with at least one protein selected from the group consisting of: MMP8, A1BG and / or HBB, or the genes encoding said proteins, to identify whether a subject treated with one or more BMAs may develop MRONJ, to assess whether it is advisable to carry out an invasive dental procedure in subjects treated with one or more BMAs, and / or to select subjects treated with one or more BMAs based on their risk of developing MRONJ.

[0022] A preferred embodiment of the invention encompasses the in vitro use of the following three proteins: MMP9, AACT and HBD combined with at least two proteins selected from the group consisting of: MMP8, A1BG and / or HBB, or the transcripts encoding said proteins; or a kit comprising reagents to measure the level of expression of the following three proteins: MMP9, AACT and HBD combined with at least two proteins selected from the group consisting of: MMP8, A1BG and / or HBB, or the genes encoding said proteins.

[0023] A preferred embodiment of the invention encompasses the in vitro use of the combination the MMP9, AACT, HBD and MMP8; MMP9, AACT, HBD, MMP8 and A1BG; and / or MMP9, AACT, HBD, MMP8, A1BG and HBB proteins, or the transcripts encoding said proteins; or a kit comprising reagents to measure the level of expression of the combination of the MMP9, AACT, HBD and MMP8; MMP9, AACT, HBD, MMP8 and A1BG; and / or MMP9, AACT, HBD, MMP8, A1BG and HBB proteins, or the genes encoding said proteins.

[0024] In a preferred embodiment of the invention, the BMAs are selected from the group comprising: immunotherapy, monoclonal antibodies, bisphosphonates, anti angiogenic and / or antiresorptive drugs. In a preferred embodiment of the invention, the BMAs are selected from the group of immunotherapy, monoclonal antibodies, bisphosphonates and / or anti angiogenic drugs comprising: etidronate, clodronate, alendronate, risedronate, tiludronate, pamidronate, ibandronate, zoledronate, neridronate, olpadronate, bevacizumab, aflibercept, pazopanib, cabozantinib, sunitinib, axitinib, dasatinib, imatinib, erlotinib, sorafenib, infliximab, adalimumab, rituximab, bevacizumab, denosumab, methotrexate, temsirolimus and / or everolimus.

[0025] In a preferred embodiment of the invention, the invasive dental procedure is selected from the group comprising: simple tooth extraction, complex tooth extraction, periapical surgery, resective periodontal surgery, regenerative periodontal surgery, dental implant, any oral surgery technique involving the potential exposure of alveolar bone, and / or basic periodontal treatment.

[0026] The third aspect of the present invention is a kit comprising: a) reagents to measure the level of expression of the following three proteins: MMP9, AACT and HBD combined with at least one protein selected from the group consisting of: MMP8, A1BG and / or HBB, or the genes encoding said proteins; or b) reagents to measure the level of expression of the following three proteins: MMP9, AACT and HBD combined with at least two proteins selected from the group consisting of: MMP8, A1BG and / or HBB, or the genes encoding said proteins; or c) reagents to measure the level of expression of the combination of the MMP9, AACT, HBD and MMP8; MMP9, AACT, HBD, MMP8 and A1BG; and / or MMP9, AACT, HBD, MMP8, A1BG and HBB proteins, or the genes encoding said proteins.

[0027] Alternatively, the present invention relates to an invasive dental treatment methodology, such as surgery, for example, in a patient treated with one or more BMAs which comprises assessing whether it is advisable to carry out an invasive dental procedure using any of the methods described above.

[0028] Furthermore, the present invention relates to a method to detect the biomarkers indicated above in a sample from a patient treated with BMAs with the risk of developing MRONJ, wherein the method comprises: (a) contacting the biological sample with a reagent capable of binding specifically to the biomarkers of the invention, for example through an immunoassay, and (b) measuring the level of the biomarkers in the biological sample. In a preferred embodiment, the present invention is a computer-implemented invention, wherein a processing unit (hardware) and a software are configured to: i) receive the values of the level of the biomarkers or signatures mentioned above, ii) process the values of the level of concentration received to find substantial variations or deviations, and iii) provide a response through a terminal about the variation or deviation of the level, wherein the variation or deviation of the level indicates that the patient treated with BMAs has the risk of developing MRONJ.

[0029] For a better interpretation of the scope of the present invention, the following definitions are provided:

[0030] • The term “comprising” means that it includes, but is not limited to, whatever follows the work “comprising”. In that sense, use of the term “comprising” indicates that the listed elements are necessary or mandatory, but that other elements are optional and may or may not be present.

[0031] • The term “consisting of’ means that it includes, and is limited to, whatever follows the expression “consisting of’. Therefore, the expression “consisting of’ indicates that the listed elements are necessary or mandatory, and that no other elements can be present.

[0032] • The term “pre-established threshold value”, in the context of the present invention, refers to a value determined in subjects without active oral pathologies (including mucosal or periodontal pathologies) and without systemic diseases that compromise immunity (including cancer and immunodeficiencies), or immunosuppressive treatments. This pre-established threshold value can be determined experimentally, empirically or theoretically. In relation to the present invention, for example, a subject would be more likely to develop MRONJ if he or she exhibited levels of expression of the aforementioned markers higher than the pre-established threshold value. This value can be selected arbitrarily based on experimental or clinical data, as would be obvious to one skilled in the art. This value must be set taking into account the desired sensitivity and specificity, which do not have to be the same in every context. Typically, values that provide optimal sensitivity and specificity are determined using ROC (receiver operating characteristic) curves based on experimental data.

[0033] Description of the figures

[0034] Figure 1. Comparative analysis of samples of osteonecrosis by ELISA. Left side (new samples for validation in new population). Right side (samples of the initial study). Detailed description of the invention

[0035] Embodiments of the invention are shown below for the purpose of illustrating said invention and without intending to limit the degree of protection.

[0036] Example 1. Materials and methods

[0037] The patient at risk of MRONJ is selected prior to the dental procedure. The patient takes BMAs by any route of administration, from 1 month for intravenous route and 3 months for oral route.

[0038] The saliva sample is preferably taken in the morning to avoid the influence of the circadian rhythm and on an empty stomach. Brushing is avoided two hours prior so as to avoid possible blood contamination. Two ml of unstimulated saliva collected by expectoration or other described and scientifically accepted method are required. The saliva is stored at -20°C for later analysis or applied directly to the medical device, as appropriate.

[0039] The analysis of the proposed biomarker panels is performed using qTRAP technology, lateral flow ELISA devices for each protein, or using electrochemical biosensors.

[0040] Example 2. Results

[0041] The inventors of the present invention performed a study of cases (N=18) and controls (N=20) for the purpose of studying the salivary proteome in patients with MRONJ compared to two control groups, one of them taking BMAs without MRONJ and the other group being healthy patients who are not taking BMAs. Unstimulated global saliva samples were obtained from Hospital Clinico Universitario in Valencia (the main MRONJ reference centre in Spain), following previously established protocols [Lorenzo-Pouso, Alejandro I et al. “Protein-Based Salivary Profiles as Novel Biomarkers for Oral Diseases.” Disease marker 5 vol. 2018 6141845. 7 Nov. 2018, doi : 10.1155 / 2018 / 6141845], A proteomic study was performed by means of SWATH-MS associated with an in-depth bioinformatics analysis. Out of the total number of proteins identified (586), 175 showed differences between MRONJ and controls. These proteins had different functional roles, but an integrative mathematical analysis (subsequently verified by artificial intelligence) allowed the identification of several combinations of biomarkers that could discriminate between subjects with and without MRONJ.

[0042] On one hand, the combination of markers MMP9, AACT and HBD with at least one marker selected from MMP8, A1BG and / or HBB, allow identification of the presence of MRONJ with both a sensitivity and a specificity above 85% (Table 1). Namely, combinations i) MMP9, AACT, HBD and MMP8, ii) MMP9, AACT, HBD, MMP8 and A1BG, and iii) MMP9, AACT, HBD, MMP8, A1BG and HBB resulted in a sensitivity in the 85-93% range and a specificity of 89.5%.

[0043] On the other hand, the combination of markers MMP8, MMP9 and A1BG discriminated the presence of MRONJ with the sensitivity and specificity shown in Table 2. Furthermore, the predictive capability was improved by combining these three proteins with at least one of the proteins selected from AACT, HBB and / or HBD (Table 2). Namely, combinations i) MMP8, MMP9, A1BG and AACT, ii) MMP8, MMP9, A1BG, AACT and HBB and iii) MMP8, MMP9, A1BG, AACT, HBB and HBD provided a sensitivity of 89-93% and a specificity of 78.9-92.4%.

[0044] Moreover, additional experiments have been carried out to validate the results previously obtained in new osteonecrosis samples with new patients in the control group. For this purpose, the ELISA method for proteins has been used. The summarized results can be seen in Figure 1. The overexpression of the proteins in the MRONJ group is verified by comparing it with the healthy control patients. Moreover, it is also established, with these experiments, the validated units of measurement for the design of the commercial device. The specific data for the validated proteins in all samples are as follows.

[0045] Table 3

[0046] B-, OWl- and S- are internal coding for different patients / samples

[0047] The three proteins were carefully selected for validation of the diagnostic kit based on their clinical relevance and association with the disease or condition under investigation. We have chosen to initially validate three proteins to ensure the feasibility of the assay and guarantee the quality of the results obtained. This allows us to focus our resources and efforts on the optimization and proper validation of a manageable subset of biomarkers. Validating a limited number of proteins initially allows us to maximize the efficiency of available resources and minimize the costs associated with diagnostic kit development. Validation of three proteins provides a solid basis for initial evaluation of kit performance, allowing us to make informed decisions about the inclusion of additional proteins at later stages of development. Our three- protein validation approach initially aligns with an iterative approach to diagnostic kit development. Once the initial validation is complete, we will evaluate the results obtained and use this information to guide the selection and validation of additional proteins in later iterations of the kit development process.

Claims

CLAIMS1. An in vitro method to identify whether a subject treated with one or more bone-modifying agents (BMAs) may develop medication-related osteonecrosis of the jaws (MRONJ), to assess whether it is advisable to carry out an invasive dental procedure in subjects treated with one or more BMAs, and / or to select subjects treated with one or more BMAs based on their risk of developing MRONJ, which comprises: a) determining the level of expression of the combination of the following three proteins: MMP9, AACT and HBD combined with at least one protein selected from the group consisting of: MMP8, A1BG and / or HBB, or the genes encoding said proteins, in a biological sample obtained from a subject, b) wherein a high level of expression, in comparison with a pre-established threshold value, is indicative that the subject may develop MRONJ, that it is not advisable to carry out an invasive dental procedure, and / or that the subject has the risk of developing MRONJ.

2. The in vitro method according to claim 1, characterised in that step a) comprises determining the level of expression of the combination of proteins: MMP9, AACT, HBD and MMP8; MMP9, AACT, HBD, MMP8 and A1BG; and / or MMP9, AACT, HBD, MMP8, A1BG and HBB, or the genes encoding said proteins.

3. The in vitro method according to any of claims 1 or 2, wherein the BMAs are selected from the group comprising: immunotherapy, monoclonal antibodies, bisphosphonates, antiangiogenic drugs and / or antiresorptive drugs.

4. The in vitro method according to any of claims 1 to 3, wherein the BMAs are selected from the group of immunotherapy, monoclonal antibodies, bisphosphonates and / or antiangiogenic drugs comprising: etidronate, clodronate, alendronate, risedronate, tiludronate, pamidronate, ibandronate, zoledronate, neridronate, olpadronate, bevacizumab, aflibercept, pazopanib, cabozantinib, sunitinib, axitinib, dasatinib, imatinib, erlotinib, sorafenib, infliximab, adalimumab, rituximab, bevacizumab, denosumab, methotrexate, temsirolimus and / or everolimus.

5. The in vitro method according to any of claims 1 to 4, wherein the invasive dental procedure is selected from the group comprising: simple tooth extraction, complex tooth extraction,periapical surgery, resective periodontal surgery, regenerative periodontal surgery, dental implant, any oral surgery technique involving the potential exposure of alveolar bone, and / or basic periodontal treatment.

6. The in vitro method according to any of claims 1 to 5, wherein the biological sample is selected from: saliva and / or a bone biopsy, preferably saliva.

7. In vitro use of the following three proteins: MMP9, AACT and HBD combined with at least one protein selected from the group consisting of: MMP8, A1BG and / or HBB, or transcripts encoding said proteins, or a kit comprising reagents to measure the level of expression of the following three proteins: MMP9, AACT and HBD combined with at least one protein selected from the group consisting of: MMP8, A1BG and / or HBB, or the genes encoding said proteins, to identify whether a subject treated with one or more BMAs may develop MRONJ, to assess whether it is advisable to carry out an invasive dental procedure in subjects treated with one or more BMAs, and / or to select subjects treated with one or more BMAs based on their risk of developing MRONJ.

8. In vitro use, according to claim 7, of the combination of proteins MMP9, AACT, HBD and MMP8; MMP9, AACT, HBD, MMP8 and A1BG; and / or MMP9, AACT, HBD, MMP8, A1BG and HBB proteins, or the transcripts encoding said proteins; or of a kit comprising reagents to measure the level of expression of the combination of proteins MMP9, AACT, HBD and MMP8; MMP9, AACT, HBD, MMP8 and A1BG; and / or MMP9, AACT, HBD, MMP8, A1BG and HBB proteins, or the genes encoding said proteins.

9. In vitro use, according to any of claims 7 or 8, wherein the BMAs are selected from the group comprising: immunotherapy, monoclonal antibodies, bisphosphonates, antiangiogenic and / or antiresorptive drugs.

10. In vitro use, according to any of claims 7 to 9, wherein the BMAs are selected from the group of immunotherapy, monoclonal antibodies, bisphosphonates and / or antiangiogenic drugs comprising: etidronate, clodronate, alendronate, risedronate, tiludronate, pamidronate, ibandronate, zoledronate, neridronate, olpadronate, bevacizumab, aflibercept, pazopanib, cabozantinib, sunitinib, axitinib, dasatinib, imatinib, erlotinib, sorafenib,infliximab, adalimumab, rituximab, bevacizumab, denosumab, methotrexate, temsirolimus and / or everolimus.

11. In vitro use according to any of claims 7 to 10, wherein the invasive dental procedure is selected from the group comprising: simple tooth extraction, complex tooth extraction, periapical surgery, resective periodontal surgery, regenerative periodontal surgery, dental implant, any oral surgery technique involving the potential exposure of alveolar bone, and / or basic periodontal treatment.

12. A kit, suitable for performing the method of any of the claims 1 to 6, comprising: a) reagents to measure the level of expression of the following three proteins: MMP9, AACT and HBD combined with at least one protein selected from the group consisting of: MMP8, A1BG and / or HBB, or the genes encoding said proteins; or b) reagents to measure the level of expression of the following three proteins: MMP9, AACT and HBD combined with at least two proteins selected from the group consisting of: MMP8, A1BG and / or HBB, or the genes encoding said proteins; or c) reagents to measure the level of expression of the combination of the MMP9, AACT, HBD and MMP8; MMP9, AACT, HBD, MMP8 and A1BG; and / or MMP9, AACT, HBD, MMP8, A1BG and HBB proteins, or the genes encoding said proteins.

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