MARKERS FOR ALTERNATED CONSOLIDATION OF BONE FRACTURES

专利摘要:
The present application particularly describes interleukin-8 (IL-8) as a novel biomarker for the prediction, diagnosis, prognosis and / or monitoring of altered consolidation of bone fractures; and associated methods, uses and kits.
公开号:BE1021759B1
申请号:E2012/0389
申请日:2012-06-11
公开日:2016-01-15
发明作者:Valérie Gangji；Jean-Philippe Hauzeur；Seny Dominique De；Myrielle Mathieu；Aude Ingels；Sabrina Rigutto；Delphine SPRUYT；Enrico Bastianelli；Valentina Albarani；Xavier Pesesse；Michel Malaise
申请人:Universite Libre De Bruxelles；Bone Therapeutics Sa；Universite De Liege；Centre Hospitalier Universitaire De Liege；
IPC主号:

专利说明:

BACKGROUND OF THE INVENTION The invention relates to biomarkers and parameters useful for the diagnosis, prediction, prognosis and / or monitoring of diseases and conditions in subjects, particularly the consolidation impaired bone fractures, such as but not limited to fractures with no consolidation, fractures with vicious consolidation or delayed-consolidation fractures; and methods, uses, kits and associated devices.
BACKGROUND OF THE INVENTION
There is a continuing need for other, and preferably improved, ways of obtaining accurate prediction, diagnosis, prognosis, and / or monitoring of diseases and conditions in subjects to inform and guide treatment choices.
Altered fracture consolidation encompasses all abnormalities and defects of bone fracture healing such as inadequate, delayed or absent bone fracture consolidation, including but not limited to, vicious consolidations, delayed consolidation and lack of consolidation. Fractures with no consolidation, also known as absence of consolidation (NU), including fractures with tight and unstable consolidation failures (non-union) are characterized by failure of the fracture repair process, without hope of spontaneous consolidation. The reported rate of fractures with no consolidation varies between 2% and 10% of all fractures, according to the authors (Gaston et al., J. Bone Joint Surg Br., 2007, vol 89 (12), 1553-1560 Tzioupis and Giannoudis, Injury, 2007, vol 38 Suppl 2, S3-S9). Fractures with no consolidation may be classified as hypertrophic or oligotrophic if bone fragment sites are vascular. Fractures with no hypertrophic consolidation are usually explained by instability of the fracture site. Fractures with no oligotrophic consolidation typically occur after significant displacement of fracture sites and show an inadequate consolidation response as indicated by the absence of callus. For fractures with no consolidation such as atrophic, the bone fragments are avascular, adynamic and incapable of biological reaction (Frolke et al Injury, 2007, vol 38 Suppl 2, S19-S22).
Vicious consolidations are characterized by imperfect consolidation of the previously fragmented bone. Delayed consolidation can be defined as a break for which consolidation has not occurred at the right time and the outcome remains uncertain.
In the normal consolidation process, a bone fracture engages a suite of inflammation, repair and. remodeling that can restore the bone to its original state. In humans, the inflammatory phase lasts about 5 to 7 days and begins with the development of a hematoma followed by the invasion of inflammatory cells. These cells, in combination with local cells, secrete cytokines, chemokines and growth factors to encourage the recruitment of osteogenic progenitor cells and endothelial progenitor cells, essential for the initiation of the repair process (Einhom Clin Orthop Report Res., 1998, vol 355 Suppl: S7-21). Progenitor cell recruitment is divided into four phases: mobilization, migration, invasion and cell transplantation at the site of the fracture. In particular, alteration of one or more of the above processes may result in altered consolidation of bone fractures.
The diagnosis is currently radiological and is performed after 3 to 4 months without consolidation for delayed consolidations and after 6 to 9 months without consolidation for fractures with no consolidation including nonunion, depending on the site and the type of fracture (Frolke et al. Supra). Clinically useful biomarker-based screening tests for impaired bone fracture consolidation are, to our knowledge, not available. In fact, the use of biomolecules as biomarkers for impaired bone fracture consolidation has never been investigated in patients with non-union fractures (NU). In pathophysiological studies, authors have demonstrated in situ that the levels of TGF-β, PDGF, FGF, and BMP 2/4 were the same in NU subjects and healthy 1 week after trauma, but that they decreased 8 weeks later in NU patients (Brownlow et al Injury, 2001, vol 32 (7), 519-524). Others have demonstrated that serum levels of TGF-β, PDGF-AB and FGF were lower at 2 and 4 weeks post-injury in NU patients. TGF-β 1 and PDGF-AB were still low in NU patients at 12 weeks post-injury (Weiss et al., Arch Orthop.Trauma Surg, 2009, 129, 989-997, Zimmermann et al. , 2005, 36 (5), 779-785).
Since knowledge, indication or warning that a fracture in a subject shows or has an increased possibility of showing an impaired consolidation, so that the progression goes towards a lack of consolidation, may help to practice therapeutic interventions on the subject, the provision of alternative methods and means, alternative and preferably improved methods and means for the diagnosis, prediction, prognosis and / or monitoring of altered bone fracture consolidation continues to be of the utmost importance.
SUMMARY OF THE INVENTION
Having conducted extensive experiments and tests, the inventors have identified biological molecules whose levels are closely predictive and / or indicative of the impaired consolidation of bone fractures and which thus constitute useful and promising biomarkers for altered fracture consolidation. The synonyms "altered bone fracture consolidation" or "altered fracture consolidation" as used herein encompass all abnormalities, abnormalities, and bone fracture consolidation defects, such as inadequate, delayed, or absent fracture healing. The sentences should specifically include and preferably indicate vicious consolidations, delayed consolidations and absence of consolidation, indicate even more preferably the absence of consolidation, including including tight and unstable consolidation absences (non-union).
According to the invention, other methods and means and methods and methods significantly improved for the diagnosis, prediction, prognosis and / or monitoring of altered fracture consolidation are achieved by the provision of one or several stromal-derived factor-1 (SDF-1 or CXCL12), platelet-derived growth factor BB (PDGF-BB), interleukin-8 (IL-8 or CXCL8) and interleukin-6 (IL-6) as biomarker (s) ) for impaired consolidation of bone fractures.
Thus, the present invention relates to the use of one or more of SDF-1, PDGF-BB, IL-8 and IL-6 as a biomarker, more particularly as a biomarker for altered fracture consolidation, even more particularly as a biomarker for diagnosis, prediction, prognosis and / or monitoring of altered fracture consolidation. Preferably, the present invention relates to the use of IL-8 as a biomarker, more particularly as a biomarker for the altered consolidation of fractures, and more particularly as a biomarker for the diagnosis, prediction, prognosis and / or monitoring of the consolidation. impaired fractures. Also, the present invention relates to the use of one or more SDF-1, PDGF-BB, IL-8 and IL-6 for the diagnosis, prediction, prognosis and / or monitoring of altered fracture consolidation. Preferably, the present invention relates to the use of IL-8 for the diagnosis, prediction, prognosis and / or monitoring of altered fracture consolidation.
In preferred embodiments of the present uses, impaired bone fracture consolidation may be selected from the group consisting of vicious consolidation fracture, delayed consolidation fracture, and non-healing fracture.
The present invention also relates to a method for the diagnosis, prediction, prognosis and / or monitoring of altered fracture consolidation in a subject, comprising measuring the level of one or more SDF-1s, PDGF-BB , IL-8 and IL-6 in a sample from said subject. Preferably, the present invention relates to a method for the diagnosis, prediction, prognosis and / or monitoring of altered fracture consolidation in a subject, comprising measuring the level of IL-8 in a sample from said subject. In order to measure the level of one or more biomarkers, the methods present and particularly the examination phase of such methods in which the data are collected from and / or about the subject, typically include the measurement or determination of the level. (i.e., the amount, volume) of said one or more biomarker (s) in a sample from the subject.
A method for the diagnosis, prediction, prognosis and / or monitoring of altered fracture consolidation in a subject as taught herein in preferred embodiments may include the steps of: (i) measuring the amount of one or more of SDF-1, PDGF-BB, IL-8 and IL-6 in a sample from the subject; (ii) comparing the amount as measured in (i) with a reference value representing a known diagnosis, prediction and / or prognosis of impaired fracture consolidation; (iii) finding a deviation or deviation of the amount as measured in (i) from the reference value; and (iv) attributing said deviation finding or no deviation for a specific diagnosis, prediction and / or prognosis of impaired fracture consolidation in the subject. Preferably, a method as taught herein for the diagnosis, prediction and / or prognosis of altered fracture consolidation in a subject may include the steps of: (i) measuring the amount of IL-8 in a sample from the subject; (ii) comparing the amount as measured in (i) with a reference value representing a known diagnosis, prediction and / or prognosis of impaired fracture consolidation; (iii) finding a deviation or deviation of the amount as measured in (i) from the reference value; and (iv) attributing said deviation finding or no deviation for a specific diagnosis, prediction and / or prognosis of impaired fracture consolidation in the subject. The method may also be performed for a subject at two or more successive time points and the respective results at said successive time points may be compared, according to which the presence or absence of a change between diagnosis, prediction and / or the prognosis of altered consolidation of fractures at said successive time points is determined. When so applied, the method may control a change in the diagnosis, prediction and / or prognosis of impaired fracture consolidation in the subject over time.
For example, a difference in the amount of one or more of the SDF-1, PDGF-BB, IL-8 and IL-6, preferably the amount of IL-8, in a sample from a subject, compared to a reference value representing the prediction or diagnosis of any altered fracture consolidation or a good prognosis for impaired fracture consolidation may indicate respectively that the subject has impaired fracture consolidation or is at risk of fracture consolidation. have impaired fracture consolidation or may indicate poor prognosis for impaired fracture consolidation in the subject. In another example, the absence of such a deviation from the reference value representing the prediction or diagnosis of any impaired fracture consolidation or a good prognosis for altered fracture consolidation may indicate that the subject does not show impaired fracture consolidation or may indicate a good prognosis for impaired fracture consolidation in the subject. In yet another example, the absence of such a deviation from the reference value representing the prediction or diagnosis of any impaired fracture consolidation or poor prognosis for altered fracture consolidation may indicate respectively that the subject has impaired fracture consolidation or the risk of impaired fracture consolidation or may indicate a poor prognosis for impaired fracture consolidation in the subject. A reference value representing the prediction or diagnosis of any impaired fracture consolidation may, for example, represent a healthy state, i.e., a state without fracture, or may represent a state with a fracture that is not impaired fracture consolidation. A reference value representing the prediction or diagnosis of altered fracture consolidation may for example represent a diseased state, that is, a state with impaired fracture consolidation.
Preferably, but not limited to, the inventors realized that a reduced amount of SDF-1, a reduced amount of PDGF-BB, a high amount of IL-8 and / or a high amount of IL-6 in a sample from of the subject, particularly in serum or plasma from the subject, compared to the respective reference value (s) representing the prediction or diagnosis of any impaired fracture consolidation, more preferably representing a healthy state or a normally consolidating fracture, or a good prognosis for impaired fracture consolidation, may indicate that the subject has impaired fracture consolidation or is at risk for impaired fracture consolidation or may indicate poor fracture consolidation. prognosis for fracture consolidation in the subject.
Preferably, but without limitation, the inventors realized that a reduced amount of SDF-1 and / or a reduced amount of IL-6 in a sample from the subject, particularly in the supernatant of cultured osteoblast cells or mesenchymal stem cells obtained in the subject, compared to the respective reference value (s) representing the prediction or diagnosis of any impaired fracture consolidation, more preferably representing a healthy state or a normally consolidating fracture, or representing a good prognosis for impaired fracture consolidation may indicate that the subject has impaired fracture consolidation or the risk of impaired fracture consolidation or may indicate a poor prognosis for fracture consolidation in the subject.
A method for monitoring altered fracture consolidation or for monitoring the probability of development of altered fracture consolidation in a subject as taught herein may in preferred embodiments include the steps of: (i) measuring the amount of one or more of SDF-1, PDGF-BB, IL-8 and IL-6 in a sample from the subject at two or more successive time points; (ii) comparing the amount as measured in (i) between said two or more successive time points; (iii) finding a deviation or deviation of the amount as measured in (i) between said two or more successive time points; (iv) attributing said gap finding or no deviation to a change in the altered fracture consolidation or to a change in the likelihood of developing impaired fracture consolidation in the subject between said two or more successive time points . Preferably, a method as taught here for monitoring the impaired consolidation of bone fractures or for monitoring the risk of developing impaired bone fracture consolidation in a subject may include the steps of (i) measuring the amount of IL-1. 8 in a sample from the subject at two or more successive time points; (ii) comparing the amount as measured in (i) between said two or more successive time points; (iii) finding a deviation or deviation of the amount as measured in (i) between said two or more successive time points; (iv) attributing said finding of deviation or any deviation to a change in the impaired consolidation of bone fractures or to a change in the likelihood of developing impaired bone fracture consolidation in the subject between said two or more points successive temporals.
Preferably, but not limited to, the inventors realized that a reduced amount of SDF-1, a reduced amount of PDGF-BB, a high amount of IL-8 and / or a high amount of IL-6 in a sample from of the subject, particularly in serum or plasma from the subject, in a last of said two or more successive time points compared to the respective amount in a first of said two or more successive time points, may indicate that the risk of development of impaired fracture consolidation in the subject has increased.
Preferably, but without limitation, the inventors realized that a reduced amount of SDF-1 and / or a high amount of IL-6 in a sample from the subject, particularly in the supernatant of cultured osteoblast cells or mesenchymal stem cells obtained in the subject, in one of the said two or more successive temporal points compared to the respective quantity in a first of said two or more successive temporal points, may indicate that the risk of developing an altered fracture consolidation in the subject has increases.
The present invention is a method for determining whether a subject is or is not (such as, for example, is still, or no longer) in need of a therapeutic or prophylactic (preventive) treatment for altered consolidation of fractures comprising measuring the level of one or more of SDF-1, PDGF-BB, IL-8 and IL-6 in a sample from said subject. In preferred embodiments, the method may comprise the steps of: (i) measuring the amount of one or more of SDF-1, PDGF-BB, IL-8 and IL-6 in a sample from the subject ; (ii) comparing the amount as measured in (i) with a reference value representing a known diagnosis, prediction and / or prognosis of impaired fracture consolidation; (iii) finding a deviation or deviation of the amount as measured in (i) from the reference value; and (iv) inferring from said finding of presence or absence of a need for therapeutic or prophylactic treatment for altered fracture consolidation. Preferably, a method for determining whether a subject is or is not in need of therapeutic or prophylactic treatment for altered bone fracture consolidation may include measuring the level of IL-8 in a sample from said subject, may preferably comprise the steps of: (i) measuring the amount of IL-8 in a sample from the subject; (ii) comparing the amount as measured in (i) with a reference value representing a known diagnosis, prediction and / or prognosis of impaired fracture consolidation; (iii) finding a deviation or deviation of the amount as measured in (i) from the reference value; and (iv) inferring from said finding of presence or absence of a need for therapeutic or prophylactic treatment for impaired bone fracture consolidation. Treatment may be particularly appropriate where the method permits a conclusion that the subject has impaired fracture consolidation or is at risk of impaired fracture consolidation or has a poor diagnosis of impaired fracture consolidation.
In this context, the present invention further describes a method for determining the outcome of therapeutic or prophylactic treatment of altered fracture consolidation in a subject, including measuring the level of one or more of the SDF-1s, PDGF. -BB, IL-8 and IL-6, preferably the level of IL-8, in a sample from said subject.
In preferred embodiments of the present methods, impaired bone fracture consolidation may be selected from the group consisting of vicious consolidation fracture, delayed consolidation fracture, and non-healing fracture.
Treatments for altered fracture consolidation include, but are not limited to, non-invasive treatments such as electrical stimulation, ultrasound or specialized orthopedic devices, and invasive procedures such as, for example, surgical removal of dead tissue, insertion of internal orthopedic device (eg stem, plate or screw), bone graft insertion, bone morphogenetic protein (BMP) injection or multiple or amputation to avoid greater injury.
Preferably, but not limited to, the inventors realized that a reduced amount of SDF-1, a reduced amount of PDGF-BB, a high amount of IL-8 and / or a high amount of IL-6 in a sample from of the subject, particularly in serum or plasma from the subject, compared to the respective reference value (s) representing the prediction or diagnosis of any impaired fracture consolidation, more preferably representing a healthy state , or representing a good prognosis for altered fracture consolidation, may indicate that the subject needs therapeutic or prophylactic treatment for altered fracture consolidation.
Preferably, but without limitation, the inventors realized that a reduced amount of SDF-1 and / or a reduced amount of IL-6 in a sample from the subject, particularly in the supernatant of cultured osteoblast cells or mesenchymal stem cells obtained in the subject, compared to the respective reference value (s) representing the prediction or diagnosis of any impaired fracture consolidation, more preferably representing a healthy state, or representing a good prognosis for impaired consolidation fractures, may indicate that the subject needs therapeutic or prophylactic treatment for altered fracture consolidation. One or more of SDF-1, PDGF-BB, IL-8, and / or IL-6 may reveal its diagnosis, prediction, prognosis, and / or value monitoring for impaired bone fracture, pre-fracture, and bone fracture consolidation. / or postfracture. In non-limiting embodiments, one or more of SDF-1, PDGF-BB, IL-8 and / or IL-6 may be particularly evaluated in subjects between the time of fracture and about 40 months post fracture, such as at approximately 1, 2, 3 and / or 4 weeks post-fracture, and / or at about 1 to 4 months, about 5 to 8 months, about 9 to 12 months, about 13 to 16 months, about 17 to 20 months months, approximately 21 to 24 months, approximately 25 to 28 months, approximately 29 to 32 months, approximately 33 to 36 months and / or approximately 37 to 40 months post-fracture. Any one of the biomarkers, uses and methods present may be preferably combined with or may supplement, or may be scheduled at the same time as, one or more diagnostic measures for altered fracture consolidation, such as without limitation , conventional imaging and radiological techniques including x-rays, positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI), ultrasound imaging. Any one or more of the biomarkers, uses, and methods described herein may be particularly useful in known or expected subjects as the risk of developing impaired bone fracture consolidation, for example, having one or more risk for impaired consolidation of bone fractures. Without limitation the risk factors associated with impaired bone fracture consolidation include lifestyle and health factors that may interfere with bone healing, such as smoking, alcohol abuse, poor condition nutritional, general poor health, impairments of physical state and diabetes; factors that may contribute to the loss of bone strength, such as the use of non-steroidal anti-inflammatory drugs (NSAIDs), the use of immunosuppressive drugs, other medications such as anticonvulsants, and replacement of thyroid hormone, thyroxine; the type and site of fracture such as fracture in a weakly vascular site, instability at the site of the fracture, high energy trauma and poor state of soft tissue around the bone; ancestry such as persons with ancestry from Europe or Asia who are at increased risk of osteoporosis; age such as those who are at increased risk of bone breakdown; women with early menopause, late menarche or loss of ovaries, and women at increased risk of bone weakness; etc.
In embodiments, any one or more of the biomarkers, use, and methods present may be supplemented or associated with the determination of the presence or absence and / or level of the one or more risk factors for the impaired consolidation of bone fractures in the subject. Any one or more of the biomarkers, use and methods present may preferably allow sensitivity and / or specificity (preferably, sensitivity and specificity) of at least 50%, at least 60%, at least 70% or at least 80%, for example> 85% or> 90% or> 95%, for example between approximately 80% and 100% or between approximately 85% and 95%. Any one or more of the biomarkers, use and methods present may be applied to subjects who have not yet been diagnosed with impaired bone fracture consolidation (eg, preventive screening). , or who have been subject to a fracture, or who have been diagnosed with impaired bone fracture consolidation, or who are thought to have impaired bone fracture consolidation (eg, presentation of one or several characteristic signs and / or symptoms), or who are at risk of developing impaired bone fracture consolidation (eg, genetic predisposition, presence of one or more developmental, environmental or behavioral risk factors). Any one or more of the biomarkers, use and methods present may be used to detect various stages of progression or severity of altered bone fracture consolidation; to detect the response of fracture consolidation to prophylactic or therapeutic treatments or other interventions; to assist the physician in making a decision on worsening, statuquo, partial recovery or total recovery of the subject of impaired bone fracture consolidation, resulting in either another treatment or observation or the authorization of exit from the subject of a medical care center. Similarly, any one or more of the biomarkers, use and methods present may be used for population screening such as, for example, screening of a general population or stratified population on the basis of one or more criteria, for example, age, ancestry, use, presence or absence of risk factors for altered fracture consolidation, etc. Any one or more of the biomarkers, use and methods present may also benefit from being supplemented or associated with the evaluation of one or more other biomarkers, signs, symptoms and / or parameters. clinically relevant for the impaired consolidation of bone fractures. By way of examples and without limitation, potentially useful biomarkers in the assessment of altered fracture consolidation, the amount of which can be measured advantageously in present uses and methods, include any one or more of the beta-factors. transformants (TGF-β), other platelet-derived growth factor (PDGF) isoforms (other isoforms), fibroblast growth factors (FGF) and bone morphogenetic proteins (BMPs), levels of which have been reported as decreased in patients with impaired fracture consolidation (Brownlow et al 2001, Weiss S et al 2009, Zimmermann et al 2005, garlic supra).
Thus, certain embodiments relate to uses and methods as defined herein, further comprising measuring the level of any one or more TGF-β, PDGF, FGF and BMP in a sample from the subject.
Also, certain embodiments relate to uses and methods as defined herein, further comprising measuring the level of any one or more of SDF-1, PDGF-BB, IL-6, TGF-β, PDGF, FGF and BMP in a sample from the subject.
The respective amount of any one or more of the biomarkers present may be evaluated separately and individually, i.e., each amount compared to its corresponding reference value. Otherwise, the amounts of any two or more biomarkers present may be used to establish a biomarker profile, which may be suitably compared to a corresponding multiparameter reference value. Alternatively, the amounts of any two or more biomarkers may each be modulated by an appropriate weighting factor and added to achieve a single value which can then be suitably compared to a corresponding corresponding reference value. It will be appreciated that such weighting factors may be dependent on the methodology used to quantify the biomarkers, and for each specific experimental setting may be determined and included in a model appropriate to the diagnosis, prediction and / or prognosis of the biomarker. impaired consolidation of fractures.
Reference values as used herein can be established according to known procedures previously used for biomarkers. Reference values can be established either in (that is, constituting a step of) or external to (that is, not constituting a step of) the uses and methods taught herein. Accordingly, any of the uses and methods taught herein may include a step of establishing a required reference value.
Thus, the present invention also relates to a method for establishing a reference value for any one or more biomarkers as taught herein, said value representing: (a) a prediction or diagnosis of lack of consolidation altered fractures or a good prognosis for impaired fracture consolidation, or (b) a prediction or diagnosis of impaired fracture consolidation or poor prognosis for impaired fracture consolidation, including: (i) measurement of the amount of fracture; any one or more biomarkers in a sample from: (iia) one or more subjects with no altered fracture consolidation or who do not have the risk of impaired fracture consolidation or impaired good prognosis fractures, or (ib) one or more subjects with impaired fracture consolidation or current risk have impaired fracture consolidation or poor prognosis of impaired fracture consolidation, and (ii) establishment from the quantity of any one or more biomarkers as measured in (ia), the value of reference representing the prediction or diagnosis of the lack of impaired fracture consolidation or the good prognosis for impaired fracture consolidation, or (ii) the establishment from the quantity of any one or more biomarkers as as measured in (ib), the reference value representing the prediction or diagnosis of altered fracture consolidation or representing poor prognosis for impaired fracture consolidation.
Preferably, the present invention is also a method for establishing a reference value for IL-8, said value representing: (a) a prediction or diagnosis of lack of impaired fracture consolidation or a good prognosis for the impaired consolidation of bone fractures, or (b) a prediction or diagnosis of altered fracture consolidation or poor prognosis for altered bone fracture consolidation, including: (i) measuring the amount of IL-8 in a sample from: (iia) one or more subjects who do not have impaired bone fracture consolidation or do not run the risk of impaired bone fracture consolidation or have a good, altered healing prognosis of bone fractures, or (ib) one or more subjects with impaired bone fracture consolidation or the risk of impaired bone consolidation bone actures or having a poor prognosis of impaired consolidation of bone fractures, and (ii a) establishment from the amount of IL-8 as measured in (ia), the reference value representing the prediction or diagnosis the lack of impaired consolidation of bone fractures or the good prognosis for impaired consolidation of bone fractures, or (ii) the establishment from the amount of IL-8 as measured in (ib), the a reference value representing the prediction or diagnosis of impaired bone fracture consolidation or poor prognosis for impaired bone fracture consolidation.
In preferred embodiments, the present uses and methods can measure the systemic amount of one or more biomarkers as taught herein. Preferably, the present uses and methods may include measuring the systemic amount of IL-8. The systemic amount of any one or more biomarkers, preferably IL-8, may be suitably evaluated in samples which comprise, consist essentially of or consist of whole blood or a minute component thereof, such as preferably plasma or serum.
In other embodiments, the present uses and methods may measure the local amount of one or more biomarkers as taught herein at the site of the impaired consolidation fracture. Preferably, the present uses and methods can measure the local amount of IL-8 at the site of the impaired consolidation fracture. The local amount of one or more biomarkers, preferably IL-8, may be suitably determined in samples that comprise, consisting essentially of tissue taken from the site of the altered consolidation fracture, such as tissue obtained by biopsy or other tissue recovery techniques.
In other embodiments, the present uses and methods may measure the local amount of bone marrow of one or more biomarkers as taught herein at a different bone marrow harvesting site. Preferably, the present uses and methods can measure the amount of IL-8 bone marrow at different bone marrow harvest sites. The bone marrow can be obtained from the bone presenting the fracture with impaired consolidation in the subject, or from a site distant from said fracture with impaired consolidation.
In yet other embodiments, the following uses and methods can measure the amount of one or more biomarkers as taught herein in the cells or in the supernatant of the cells obtained in the subject and then cultured in vitro. Preferably, the following uses and method can measure the amount of IL-8 in the cells or in the supernatant of the cells obtained in the subject and then cultured in vitro. Preferably, the cells may be bone or progenitor cells, more preferably osteoblast cells (OB) or mesenchymal stem cells (MSCs). Preferably, the biomarkers can be measured in primary cell cultures and / or other cultures (eg, secondary, tertiary, etc.). The cells may be obtained from the site of the altered consolidation fracture in the subject, or from a site remote from said altered consolidation fracture.
The present invention further describes in a kit, particularly a kit for the diagnosis, prediction, prognosis and / or monitoring of altered fracture consolidation in a subject, the kit comprising (i) a means for measuring the amount of of any one or more of SDF-1, PDGF-BB, IL-8 and / or IL-6, particularly in a sample from the subject, and (ii) optionally and preferably one or more reference values or means for establishing said one or more reference values, wherein said one or more reference values represents a known diagnosis, prediction and / or prognosis of impaired fracture consolidation.
The means for measuring the amount of a biomarker may include one or more linkers capable of specifically binding to said biomarker. Exemplary binding agents may include hybridization evidence (oligonucleotides), amplification primers, antibodies, aptamers, photoaptamers, proteins, peptides, peptidomimetics, or small molecules. The binding agents may advantageously be immobilized on a solid phase or a solid support.
The present invention thus also describes in a kit, particularly a kit for the diagnosis, prediction, prognosis and / or monitoring of altered fracture consolidation in a subject, the kit comprising (i) one or more capable binding agents specifically bind to any one or more of SDF-1, PDGF-BB, IL-8 and / or IL-6, particularly in a sample from the subject, (ii) preferably, a known amount or concentration of said one or several SDF-1, PDGF-BB, IL-8 and / or IL-6, such as for use as controls, standards and / or calibrators, (iii) optionally and preferably, one or more reference values or means of establishing one or more reference values, wherein said one or more reference values represents a known diagnosis, prediction and / or prognosis of impaired fracture consolidation. The said components under (i) and / or (ii) may be appropriately labeled as taught in another part of these specifications.
In preferred embodiments, the kits may be configured as portable devices, such as, for example, individual devices, for home use or in clinical facilities.
It may be appreciated that the means or tools for collecting a sample from a subject, such as, for example, a conventional blood collection tube comprising an anticoagulation agent, may be provided separately from the kit or included in the kits described here.
The present invention further describes the use of a kit as described herein for the diagnosis, prediction, prognosis and / or monitoring of altered fracture consolidation.
Preferably, the present invention consists in the use of a kit for the diagnosis, prediction, prognosis and / or monitoring of the impaired consolidation of bone fractures in a subject, the kit comprising (i) a measurement means of the amount of IL-8, particularly in a sample from the subject, and (ii) optionally and preferably one or more reference values or means for establishing said one or more reference values, wherein said one or more reference values represents a known diagnosis, prediction and / or prognosis of impaired bone fracture consolidation. The present invention further comprises the use as taught herein wherein the means for collecting a sample from a subject is provided separately from the kit or is included in the kit.
The present invention also describes reagents and tools useful for measuring any one or more biomarkers as taught herein. The present invention therefore describes a nucleic acid chip or biochip or a protein, polypeptide, or peptide chip or biochip comprising any one, preferably two, preferably three, more preferably the four SDF-1s, PDGF-BB, IL-1. 8 and / or IL-6. The present invention also discloses a linker chip or biochip comprising one or more linkers capable of binding specifically to any one, preferably two, preferably three, more preferably the four SDF-1s, PDGF-BB, IL. And / or IL-6, preferably comprising a known amount or concentration of said one or more linkers.
The present invention further discloses the use of a microarray as described herein for the diagnosis, prediction, prognosis and / or monitoring of altered fracture consolidation.
Preferably, the present invention describes the use of a nucleic acid chip or biochip or a protein, polypeptide or peptide chip or biochip for the diagnosis, prediction, prognosis and / or monitoring of altered fracture consolidation. bones, said chip or biochip comprising a nucleic acid encoding IL-8 or comprising IL-8. Preferably, the present invention also describes the use of a linker chip or biochip for the diagnosis, prediction, prognosis and / or monitoring of altered bone fracture consolidation, said chip or biochip comprising one or more a plurality of binding agents capable of binding specifically to IL-8, preferably comprising a known amount or concentration of said one or more linkers.
The above aspects and other aspects and preferred embodiments of the invention are described in the following sections and in the appended claims. The subject of the appended claims is here specifically incorporated in these specifications.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 illustrates the plasma levels of SDF-1 in an experiment comparing a group of patients with nonunion (NU) fractures with healthy controls (HV), (A) all samples (HV, n = 49; NU, n = 15), (B) samples where plasma is collected in heparin tubes (HV, n = 26, NU, n = 11), (C) samples where plasma is collected in EDTA tubes (HV n = 40, NU, n = 5).
Figure 2 illustrates serum levels of PDGF-BB in an experiment comparing a group of patients with non-union fractures (NU, n = 9) with healthy controls (HV, n = 20).
Figure 3 illustrates serum levels of IL-8 in an experiment comparing a group of patients with non-union fractures (NU, n = 4) with healthy controls (HV, n = 18).
Figure 4 illustrates serum levels of IL-6 in an experiment comparing a group of patients with non-union fractures (NU, η = 13) with healthy controls (HV, n = 29).
Figure 5A illustrates serum levels of SDF-1 in the supernatant of osteoblastic cell culture (OB) comparing a group of patients with non-union fractures (NU, n = 6) with healthy controls (HV, n = 9). ).
Figure 5B illustrates levels of SDF-1 in the supernatant of mesenchymal cell culture (MSC) comparing a group of patients with non-union fractures (NU, n = 6) with healthy controls (HV, n = 9) .
Figure 6 illustrates IL-6 levels in the supernatant of osteoblastic cell culture (OB) comparing a group of patients with non-union fractures (NU, n = 6) with healthy controls (HV, n = 10). ).
DETAILED DESCRIPTION OF THE INVENTION
As used herein, the singular forms "one", "one" and "the" include both singular and plural referents unless the context clearly specifies otherwise.
The terms "comprising", "includes" and "understood" as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open and do not exclude any steps additional, unexposed members, elements or methods. The term also encompasses "consisting of" and "consisting essentially of".
The recitation of the digital ranges by turn point includes all the numbers and fractions subsumed in the respective ranges, as well as the exposed turn points.
The term "about" as used herein when referring to a measurable value such as a parameter, amount, time duration and the like involves encompassing variations of or from the specified value, within particular variations of +/- 10% or less, preferably + 1-5% or less, more preferably +/- 1% or less and still more preferably +/- 0.1% or less of and from the specified value, in the extent to which such variations are suitable for carrying out the disclosed invention. It should be understood that the value to which the "about" modifier refers is itself also specifically and preferably described.
While the term "one or more", such as one or more members of a group of members is clear in itself, by means of another exemplification, the term includes in particular a reference to any one of such members, such as, for example, one of the> 3,> 4,> 5,> 6 or> 7 of the said members and to all such members .
All documents cited in the present specifications are here incorporated by reference in their entirety.
Unless otherwise indicated, all the terms used in the description of the invention, including technical and scientific terms, have the meaning most commonly understood by one of the ordinary rules of the art to which the invention belongs. By further guidance, term definitions may be included to better appreciate the teaching of the present invention.
The inventors have identified a stromal derived factor-1 (SDF-1), platelet derived growth factor BB (PDGF-BB), interleukin-8 (IL-8 or CXCL8) and interleukin-6 (IL-6) as a biomarker (s) new and useful for the diagnosis, prediction, prognosis and / or monitoring of impaired bone fracture consolidation. In particular, the inventors have identified IL-8 as a novel and useful biomarker for the diagnosis, prediction, prognosis and / or monitoring of impaired bone fracture consolidation.
The term "biomarker" is broad in the art and can broadly indicate a biological molecule and / or a detectable portion thereof whose qualitative and / or quantitative evaluation in a subject is alone or associated with other data. , predictive and / or informative (eg, prediction, diagnosis and / or prognosis) with respect to one or more aspects of the phenotype and / or genotype of the subject such as, for example, with respect to the subject's condition with respect to a disease or condition. In particular, biomarkers are intended herein to be RNA-based (particularly mRNA-based) or preferably can be protein-based, polypeptide- or peptide-based.
The terms "fracture with no consolidation", "non-fracture consolidation", "no consolidation" or "NU" interchangeably concern a fracture that, due to various factors, fails in its consolidation over a period of normal time. NU includes tight and unstable consolidation absences or nonunion. The terms "vicious consolidation fracture", "vicious fracture consolidation" or "vicious consolidation" interchangeably relate to the imperfect consolidation of the previously fragmented bone. The terms "delayed consolidation fracture" or "delayed consolidation" refer interchangeably to a fracture in which consolidation has not occurred in a timely manner and the outcome remains uncertain. Fractures with no consolidation, vicious consolidation or delayed consolidation are included here under the term "impaired bone fracture consolidation" or "altered fracture consolidation". Altered fracture consolidation therefore requires some form of intervention to stimulate consolidation.
The period of time at which the impaired consolidation of fractures actually ends varies according to the specific fracture, but it is generally agreed that an unbound fracture within 6 months after injury will not consolidate without intervention. It has also been suggested to conclude that impaired fracture consolidation will occur if a fracture shows no sign of progression towards consolidation within 3 months after the injury or simply if a fracture has not consolidated within the time frame in which a surgeon specialized in fractures would have planned his consolidation.
The reference in these specifications to diseases or conditions encompasses all those diseases or conditions as described herein to the extent that they are consistent with the context of a particular recitation, more specifically encompass impaired fracture consolidation.
The terms "predicting" or "prediction", "diagnosing" or "diagnosis" and "prognostic" or "prognosis" are commonplace and are well understood in medical and clinical practice. It should be understood that the phrase "a method for the diagnosis, prediction and / or prognosis" of a given disease or condition may also be interchanged with phrases such as "a method for diagnosing, predicting and / or prognosing" said disease or condition or "a method for realizing (or determining or establishing) the diagnosis, prediction and / or prognosis" of said disease or condition or the like.
By another explanation is without limitation, "prediction" or "prediction" generally refers to a statement, indication or announcement of a disease or condition in a subject who has not (yet) said disease or condition. For example, a prediction of a disease or condition in a subject may indicate a likelihood, a chance, or a risk that the subject will develop said disease or condition, for example, within a certain period of time or at a certain age. Said probability, chance or risk may be indicated in particular as a value, range or absolute statistics or may be indicated in relation to an appropriate control of the subject or population of the subject (such as, for example, with respect to a subject or a general, normal, or healthy population of subjects The likelihood, chance, or risk that a subject develops an illness or condition may therefore be advantageously indicated as increased or decreased, or multiplied or divided against a subject or population of appropriate control subjects As used herein, the term "prediction" of states or diseases as taught here in a subject may also mean that the subject has a "positive" prediction. Of these, that is to say that the subject runs a risk of having them (for example, the risk is significantly increased vis-à-vis a subject or a population of subjects The term "predefined Noting diseases or conditions as taught here as described herein in a subject may particularly mean that the subject has a "negative" prediction thereof, that is, the risk that the subject these are not significantly increased in relation to a subject or population of control subject.
The terms "diagnose" or "diagnosis" generally refer to the process or act of recognizing, deciding or concluding a disease or condition in a subject based on the symptoms or signs and / or from the results of the various diagnostic procedures. (such as, for example, from knowledge of the presence, absence, and / or amount of one or more characteristics of the biomarkers of the diagnosed disease or condition.) As used herein "diagnosis of" Diseases or conditions as taught here in a subject may particularly mean that the subject presents them and therefore has been diagnosed with presenting them. "Diagnosis of steps" of diseases or conditions as taught here in a subject can particularly mean that the subject does not present them and therefore was the subject of the diagnosis not to present them.A subject may be diagnosed not to present x-ci despite the presence of one or more conventional symptoms or signs suggestive of them.
The terms "prognostic" or "prognosis" generally refer to an anticipation of the progression of a disease or condition and the perspective (for example, likelihood, duration and / or extent) of recovery. A good prognosis of the diseases or conditions taught herein can generally include anticipating a satisfactory partial or complete recovery from diseases or conditions, preferably within an acceptable period of time. A good prognosis of these can more generally include an anticipation of non-aggravation or other aggravation thereof, preferably in a given period of time. A poor prognosis for diseases or conditions as taught herein can generally include anticipating sub-standard recovery and / or slow or poor recovery, or substantially no recovery, or even worsening thereof.
Thus, the prediction or prognosis of a disease or condition may, inter alia, permit prediction or prognosis of the occurrence of the disease or condition, or predict or establish a prognosis of progression, worsening, the attenuation or recurrence of the disease or condition or response to treatment or other external or internal factors, situations or causes of stress, etc.
In addition, surveillance of a disease or condition may, inter alia, predict the occurrence of the disease or condition, or monitor the progression, aggravation, attenuation or recurrence of the disease or condition. status, or response to treatment or other external or internal factors, situations or causes of stress, etc. Advantageously, the monitoring may be applied during a medical treatment of a subject, preferably a medical treatment for the purpose of attenuation of the disease or condition thus monitored. Such monitoring can be understood, for example, in decision-making for discharging a patient, for the purpose of modifying a treatment or the need for another hospitalization. As provided herein, a reference for monitoring an illness or condition also specifically includes monitoring the likelihood, risk or chance of a subject developing the disease or condition, i.e. ie, monitoring the change (s) of said probability, risk or chance over time.
The term "subject" or "patient" as used herein typically and preferably refers to humans, but may also include reference to non-human animals, preferably warm-blooded animals, more preferably vertebrates, still more preferably humans. mammals, such as, for example, non-human primates, rodents, canines, felines, horses, sheep, pigs and others. Particularly targeted are subjects known as or suspected to have suffered a bone fracture, more particularly in whom the bone fracture has not been consolidated (as established, for example, by radiological research). Relevant topics may include those presenting a doctor with symptoms and signs indicative of unconsolidated bone fracture or impaired bone fracture consolidation.
The terms "sample" or "biological sample" as used herein include any biological specimen obtained from a subject. Exemplary biological specimens include, but are not limited to, whole blood, plasma, serum, erythrocytes, leukocytes (e.g., peripheral blood mononuclear cells), total bone marrow, stem and serum cells derived from bone marrow, saliva, urine , stool (ie feces), tears, sweat, sebum, nipple aspirate, canal wash, tumor exudate, synovial fluid, cerebrospinal fluid, lymph, needle fluid, amniotic fluid, other body fluids , nail clippings, cell lysate, cell secretion products, inflammatory fluid, vaginal secretions, biopsies, bone biopsies, bone tissue homogenates, etc. Preferred samples may include those having one or more biomarkers as taught herein in detectable amounts. More preferred samples, particularly for the determination of systemic levels of biomarkers, include whole blood or a minute component thereof, such as particularly preferably plasma or serum. In some embodiments, a sample may comprise or may be represented by cell or cell supernatant, said cells (preferably MSC or OB) having been obtained from the subject and subsequently grown in vitro. Preferably, the cells can be evaluated during primary and / or secondary culture. Preferably a sample is readily available by minimally invasive methods for removing or isolating said sample from the subject.
A molecule or analyte such as a metabolite, nucleic acid, RNA, DNA or cDNA, protein, polypeptide or peptide is "measured" in a sample when the presence or absence and / or amount of said molecule or substance analyzing said group of molecules or analytes is detected or determined in the sample, preferably substantially for the exclusion of other molecules and analytes. For example, a biomarker may be measured by measuring the mRNA encoding it or by measuring the encoded protein or polypeptide or a peptide thereof.
The terms "quantity", "volume" and "level" are synonymous and generally well understood in the art. With respect to the molecules or substances to be analyzed, the terms may particularly refer to an absolute quantification of the molecule or analyte in a sample, or to a relative quantification of the molecule or analyte in the sample that is, relating to another value such as a reference value as taught herein, or to a range of values indicating a base expression of a biomarker. These values or ranges can be obtained from a single patient or from a group of patients.
An absolute amount of a molecule or analyte in a sample can be advantageously expressed in weight or molar volume, or more commonly in concentration, eg weight per volume or molecular weight per volume.
A relative amount of a molecule or analyte in a sample can be advantageously expressed as an increase or decrease or as a multiplication or a decrease related to said other value, such as relative to a reference value such as taught here. Performing a relative comparison between the first variable and the second variable (e.g., first and second quantity) may but need not determine the absolute values of said first and second variables. For example, a measurement method may produce quantifiable measurements (such as, for example, signal intensities) for said first and second variables, wherein said measurements are a function of the value of said variables and wherein said measurements may be directly compared to produce a relative value for the first variable in relation to the second variable, without the current need to first convert the measurements to absolute values of the respective variables.
As used herein, the reference to any of the biomarkers, nucleic acid, protein, polypeptide or peptide corresponds to the biomarker, nucleic acid, protein, polypeptide or peptide commonly known as the respective names in art. The terms encompass such biomarkers, nucleic acids, proteins, polypeptides or peptides of any organism where they have been found, and particularly animals, preferably warm-blooded animals, more preferably vertebrates, most preferably mammals, including human and non-human mammals, still more preferably humans. The terms particularly include such biomarkers, nucleic acids, proteins, polypeptides or peptides with a native sequence, i.e. those whose primary sequence is the same as that of the biomarkers, nucleic acids, proteins, polypeptides or peptides found. in or from nature. An experienced person understands that native sequences may differ between different species due to genetic divergence between such species. In addition, native sequences may differ between or among different individuals of the same species due to the normal genetic diversity (variation) within given species. Similarly, native sequences may differ between or even within different individuals of the same species due to post-transcriptional or post-translational modifications. These variants or isoforms of biomarkers, nucleic acids, proteins, polypeptides or peptides are described herein. As a result, all sequences of biomarkers, nucleic acids, proteins, polypeptides or peptides found in or from nature are considered "native". The terms include biomarkers, nucleic acids, proteins, polypeptides or peptides when they are part of a living organism, organ, tissue or cell, when they form part of a biological sample, as well as, when they are at least partially isolated from such sources. The terms also include biomarkers, nucleic acids, proteins, polypeptides or peptides when produced by recombinant or synthetic means.
Human exemplary biomarkers, nucleic acids, proteins, polypeptides, or peptides as taught herein may be annotated with the accession numbers NCBI Genbank (http://www.ncbi.nlm.nih.gov/) given below. An experienced person may also be aware that in some cases said sequences may be precursors (eg preproteins) of biomarkers, nucleic acids, proteins, polypeptides or peptides as taught herein and may include portions that are treated away from biomarkers. , nucleic acids, proteins, polypeptides or mature peptides. An experienced person may further be aware that although only one or more isoforms may be listed below, all isoforms are searched for. Unless otherwise specified, the entries below are presented as: Name (Code, Genbank accession number for one or more representative mRNA sequences (eg, isoforms), followed by a period and version of the Genbank sequence; Genbank accession number for a corresponding representative amino acid sequence (eg isoforms), followed by a period and version of the Genbank sequence):
Stromal derived factor-1 and isoforms α, β, γ, φ and ε (SDF-1 or CXCL12; NM_199168.3, NM 000609.5, NM_001033886.2, NM_001178134.1; NP_954637.1, NP_000600.1, NP_001029058.1, NP_001171605.1)
Platelet-derived growth factor BB, i.e. a PDGFB beta polypeptide homodimer (PDGFB; NM_002608.2, NM_033016.2; NP_002599.1, NP_148937.1)
Interleukin-8 (IL-8, CXCL8, GCP-1, LECT, LUCT, LYNAP, MDNCF, MONAP, NAF, NAP-1 or NAP1; NM 000584.2; NP_000575.1)
Interleukin-6 (IL-6, HSF, HGF, CDF, BSF2 or IFNB2, NM 000600.3, NP_000591.1)
Preferably, when the sample is whole blood or a fractional component thereof such as plasma or serum, any biomarker, nucleic acid, protein, polypeptide or peptide may be a circulating form (i.e. -cellular or membrane-bound).
Unless otherwise apparent in context, the reference here to any biomarker, nucleic acid, protein, polypeptide or peptide thereof can generally also include modified forms of said biomarkers, nucleic acids, proteins, polypeptides or peptides such as modifications of post-expression influence including, for example, phosphorylation, glycosylation, lipidation, methylation, cysteinylation, sulfonation, glutathionylation, acetylation, oxidation of methionine to methionine sulfoxide or methionine sulfone, and the like.
In one embodiment, any biomarker, nucleic acid, protein, polypeptide or peptide may be human, i.e., their primary sequence may be the same as a corresponding primary sequence of or present in a biomarker, acidic nucleic acid, protein, polypeptide or human peptide occurring naturally. Thus the term "human" in this connection refers to the primary sequence of the respective biomarker, nucleic acid, protein, polypeptide or peptide, rather than to its origin or source. For example, such biomarkers, nucleic acids, proteins, polypeptides or peptides may be present in or isolated from samples of human subjects or may be obtained by other means (e.g., recombinant expression, acellular translation or non-peptide synthesis). -organic).
Preferably, the biomarkers as described herein are base protein, polypeptide or peptide.
Reference here to any biomarker, nucleic acid, protein, polypeptide or peptide may also include fragments thereof. Thus, the reference here for measuring (or measuring the amount of) any biomarker, nucleic acid, protein, polypeptide or peptide may include measuring biomarkers, nucleic acids, proteins, polypeptides or peptides and / or measuring one or more fragments of these. For example, any biomarker, nucleic acid, protein, polypeptide or peptide and / or one or more fragments thereof can be measured collectively, so that the amount measured corresponds to the added volumes of the species measured collectively. In another example, any biomarker, nucleic acid, protein, polypeptide or peptide and / or one or more fragments thereof can be individually measured individually.
The term "fragment" of a nucleic acid generally refers to deleted or truncated 5'- and / or 3'-terminal forms of said nucleic acid. The term "fragment" of a protein, polypeptide or peptide generally refers to deleted or truncated N- and / or C-terminal forms of said protein, polypeptide or peptide. Without limitation, a fragment of a nucleic acid, a protein, a polypeptide or a peptide may represent at least about 5%, or at least about 10%, for example,> 20%,> 30% or> 40%, preferably> 50%, for example,> 60%,> 70% or> 80%, or more preferably> 90% or> 95% of the nucleotide sequence of said nucleic acid or sequence sequence amino acid of said protein, polypeptide or peptide.
In some embodiments, the biomarkers or other reagents described herein may include a detectable tracer. The term "tracer" refers to any atom, molecule, moiety or biomolecule that can be used to provide a detectable and preferably quantifiable measure or property, and that may be related to or be part of an entity of interest, such as a peptide or a polypeptide or a specific binding agent. The tracers may be suitably detectable by mass spectrometric, spectroscopic, optical, colorimetric, magnetic, photochemical, biochemical, immunochemical or chemical means. Tracers include without limitation dyes; radio-tracers such as' P, P, S, I, I; electron-dense reagents; enzymes (eg, horseradish peroxidase or alkaline phosphatase as commonly used in immunoassays); characteristic binding groups such as biotin, maltose; haptens such as digoxigenin, his-tag, myc-tag; luminogenic, phosphorescent or fluorogenic characteristic groups; mass tags; and fluorescent dyes alone or in combination with moieties that can suppress or displace the fluorescence resonance energy transfer (FRET) emission spectra. Examples of combinations that may be used in the tracer: Binding partner disposition may include, for example, biotinistreptavidin, his-tag: metal ion (e.g., Ni2 +), maltose maltose binding protein. Also contemplated herein is the use of any biomarker, nucleic acid, protein, polypeptide or peptide as taught herein, optionally including a detectable tracer, as (positive) controls, standard or calibrators in qualitative or quantitative detection assays (methods measurement) of said biomarkers, nucleic acids, proteins, polypeptides or peptides, and particularly in these methods for the diagnosis, prediction, prognosis and / or monitoring of diseases or conditions as taught here in subjects. The biomarkers, nucleic acids, proteins, polypeptides or peptides may be delivered in any form, in particular as precipitates, dried under vacuum, lyophilizates, in liquid or frozen solutions or covalently or non-covalently immobilized in solid phase, such as by for example, on a solid chromatographic matrix or on glass or plastic or any other suitable surface (for example, as part of the chips and peptide biochips). Biomarkers, nucleic acids, proteins, polypeptides or peptides can be easily prepared, for example, isolated from natural sources or prepared recombinantly or synthetically.
The present invention further discloses linkers capable of specifically binding to biomarkers, nucleic acids, proteins, polypeptides or peptides as taught herein. Binding agents as provided in all these specifications may include hybridization evidence, amplification primers, antibodies, aptamer, photoaptamer, protein, peptide, peptidomimetics or a small molecule. Bonding agents may be suitably labeled.
The term "specifically bind" as used in these specifications means that an agent (hereinafter also referred to as "binding-specific") binds to one or more desired molecules or analytes to the exclusion of others. molecules that are random or unrelated, and optionally substantially excluding other molecules that are structurally related. The term "specifically bind" does not necessarily require an agent to bind exclusively to his or her intended target (s). For example, an agent may be considered to bind specifically to the target (s) of interest if its affinity for such target (s) under the binding states is at least 2-fold larger, preferably at least about 5 times larger, more preferably at least about 10 times larger, still more preferably at least about 25 times larger, still more preferably at least about 50 times larger, and even more preferably at least about about 100 times or greater, than its affinity for a non-target molecule. For hybridization evidence, specific binding can be assayed under high rigor hybridization states as known in the art. For amplification primers, specific binding can be evidenced by selective amplification of the desired target.
Preferably, the agent can bind to its target (s) with affinity constant (KA) of such a binding KA> lxlO6 M'1, more preferably KA> lxlO7 M'1, still more preferably KA > lxlO8 M'1, even more preferably KA> lxlO9 M'1, and still more preferably KA> lxlO10 M1 or KA> lxlO11 M "1, where KA = [SBA_T] / [SBA] [T], SBA indicates the Specific binding agent, T indicates the desired target The determination of KA can be performed by methods known in the art, such as, for example, the use of equilibrium dialysis and Scatchard chart analysis.
As used herein, the term "antibody" is used in a broad sense and generally refers to any immunological binding agent. The term specifically encompasses intact monoclonal antibodies, polyclonal antibodies, multivalent antibodies (eg, 2-, 3- or more-valent) and / or multispecific antibodies (e.g., bi- or more specific antibodies) formed from at least two intact antibodies and antibody fragments to the extent that they exhibit the desired biological activity (particularly, the ability to bind specifically to an antigen of interest), as well as multivalent composites and / or multispecific of these fragments. The term "antibody" is not only inclusive of antibodies generated by methods comprising immunization, but also includes any polypeptide, for example a recombinantly expressed polypeptide, which is made to encompass at least one region of complementarity determining ( CDR) capable of binding specifically to an epitope or antigen of interest. The term therefore applies to these molecules regardless of whether they are produced in vitro or in vivo.
An antibody can be one of the IgA, IgD, IgE, IgG and IgM categories and preferably an IgG class antibody. An antibody may be a polyclonal antibody, for example an antiserum or immunoglobulins purified therefrom (e.g., purified affinity). An antibody may be a monoclonal antibody or a mixture of monoclonal antibodies. Monoclonal antibodies can target a particular antigen or epitope in an antigen with greater selectivity and reproducibility. By way of example and without limitation, monoclonal antibodies can be made by the hybridoma method first described by Kohler et al. 1975 (Nature 256: 495), or can be made by DNA methods (e.g., as in US 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using techniques as described by Clackson et al. 1991 (Nature 352: 624-628) and Marks et al. 1991 (J Mol Biol 222: 581-597), for example. Other binding agents may be antibody fragments. "Antibody fragments" include a portion of an intact antibody, including the antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab ', F (ab') 2, Fv and scFv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multivalent and / or multispecific antibodies formed from antibody fragment (s), for example, dibodies, tribodies, and multibodies. The above designations Fab, Fab ', F (ab') 2, Fv, scFv etc. are considered to have their meaning as established by art.
The term antibody includes antibodies from or comprising one or more portions derived from any animal species, preferably vertebrate species, including for example birds and mammals. Without limitation, the antibodies may be chicken, turkey, goose, duck, guinea fowl, quail or pheasant. Also without limitation, antibodies can be human, murine (eg mouse, rat, etc.), monkey, rabbit, goat, sheep, guinea pig, camel (eg Camelus bactrianus and Camelus dromaderius), lama (eg , Lama paccos, Lama glama or Lama vicugna) or horse.
An experienced person will understand that an antibody may include one or more deletions, additions and / or amino acid substitutions (eg, conservative substitutions), as long as such alterations preserve its binding of the respective antigen. An antibody may also include one or more native or artificial modifications of its constituent amino acid residues (eg, glycosylation, etc.)
Methods of producing polyclonal and monoclonal antibodies as well as fragments thereof are well known in the art, as are methods for producing recombinant antibodies or fragments thereof (see for example Harlow and Lane, "Antibodies: A Laboratory Manual," Cold Spring Harbor Laboratory, New York, 1988, "Harlow and Lane," Using Antibodies: A Laboratory Manual, "Cold Spring Harbor Laboratory, New York, 1999, ISBN 0879695447;" Monoclonal Antibodies A Manual of Techniques, "by Zola, ed., CRC Press 1987, ISBN 0849364760;" Monoclonal Antibodies: A Practical Approach, "by Dean & Shepherd, eds., Oxford University Press 2000, ISBN 0199637229; Methods in Molecular Biology , 248: "Antibody Engineering: Methods and Protocols," Lo, ed., Humana Press 2004, ISBN 1588290921).
The term "aptamer" refers to a single-stranded or double-stranded oligo-DNA, oligo-RNA or oligo DNA / RNA or any analog thereof, which can specifically bind to a target molecule such as a peptide. Advantageously, aptamers can display high specificity and affinity (for example KA in the order of 1x10 9 M'1) for their targets. The aptamer production is described in particular in US 5,270,163; Ellington & Szostak 1990 (Nature 346: 818-822); Tuerk & Gold 1990 (Science 249: 505-510); or "The Aptamer Handbook: Functional Oligonucleotides and Their Applications", by Klussmann, ed., Wiley-VCH 2006, ISBN 3527310592, incorporated herein by reference. The term "photoaptamer" refers to an aptamer that contains one or more photoreactive functional groups that can bind covalently or cross-link with a target molecule. The term "peptidomimetic" refers to a non-peptide agent that is a topological analog of a corresponding peptide. Methods for rational designation of peptide peptidomimetics are known in the art. For example, the rational design of the three peptidomimetics based on the 8-mer sulfated peptide CCK26-33, and two peptidomimetics based on the 11-mer Substance P peptide, and related to the design principles of peptidomimetics, is described in Horwell 1995 (Trends Biotechnol 13: 132-134).
The term "small molecule" refers to compounds, preferably organic compounds, with a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (eg, proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da, for example up to about 4000, preferably up to 3000 Da, more preferably up to 2000 Da, even more preferably up to about 1000 Da, for example up to about 900, 800, 700, 600 or up to about 500 Da.
All available, existing or conventional separation, detection and quantification methods can be used here to measure the presence or absence (eg, present-to-absent measurement, or detectable volume to non-detectable volume) and / or the amount (e.g., being an absolute or relative amount, such as, for example, absolute or relative concentration) of biomarkers, nucleic acids, proteins, polypeptides or peptides thereof in samples (any molecules or substances to be analyzed of interest thus measured in samples, including any one or more biomarkers, nucleic acids, proteins, polypeptides or peptides as taught herein, can here be mentioned collectively as biomarkers).
For example, such methods may include biochemical test methods, immunoassay methods, mass spectrometry analysis methods or chromatography methods, or combinations thereof.
The term "immunoassay" generally refers to known methods such as those for detecting one or more molecules or analytes of interest in a sample, where the specificity of an immunoassay for the molecule (s) or the the analyte (s) of interest is conferred by a specific binding between a specific binding agent, commonly an antibody and the molecule (s) or analyte (s) of interest . Immunoassay technologies include, without limitation, direct ELISA (enzyme-linked immunoadsorbent assay), indirect ELISA, sandwich ELISA, competitive ELISA, multiplex ELISA, radioimmunoassay (RIA), ELISPOT technologies, and other similar techniques known in the art. art. The principles of these immunoassay methods are known in the art, for example, John R. Crowther, "The ELISA Guidebook," ed., Humana Press 2000, ISBN 0896037282.
By way of another explanation and without limitation, direct ELISA uses a labeled primary antibody to bind to and thus quantify the target antigen in a sample immobilized on a solid medium such as a Microwell plate. Indirect ELISA uses an unlabeled primary antibody that binds to the target antigen and a secondary labeled antibody that recognizes and quantifies the primary antibody bound to the antigen. In sandwich ELISA, the target antigen is captured from a sample using a "capture" antibody that binds to an antigen site in the antigen, and following the removal of unrelated analytes, Antigen thus captured is detected using a "detection" antibody that binds to another antigenic site in said antigen, wherein the detection antibody may be directly labeled or indirectly detectable as above. Competitive ELISA uses a labeled "competitor" that can be either the primary antibody or the target antigen. In one example, the unlabeled primary antibody is incubated with a sample, this reaction is allowed to reach equilibrium, and then the labeled target antigen is added. The latter will be bound to the primary antibody wherever its binding sites will not yet be occupied by an unlabeled target antigen from the sample. Thus, the detected volume of labeled bound antigen is inversely correlated with the volume of unlabeled antigen in the sample. Multiplexed ELISA allows the simultaneous detection of two or more analytes in a single compartment (eg Microwell plate) usually in a plurality of sample addresses (see, for example, Nielsen & Geierstanger 2004. J Immunol Methods 290: 10720 and Ling et al., 2007 Expert Rev Mol Diagn 7: 87-98 for other tips). As considered, labeling in ELISA technologies is usually done by enzyme conjugation (such as, for example, horseradish peroxidase) and the turning point is typically colorimetric, chemiluminescent or fluorescent, magnetic, piezoelectric, pyroelectric and the like.
Radioimmunoassay (RIA) is a competitive technique and involves mixing known amounts of radioactively labeled (e.g., 125I- or 131I labeled) labeled antigen with antibodies to said antigen and then adding unlabeled or "cold" antigen. from a sample and measuring the volume of the tagged antigen displaced (see, for example, "An Introduction to Radioimmunoassay and Related Techniques", by Chard T, ed., Elsevier Science 1995, ISBN 0444821198 for guidance). Generally, any mass spectrometry (MS) technique that can obtain precise information on the mass of the peptides, and preferably also on fragmentation and / or (partial) amino acid sequence of the selected peptides (for example in spectrometry tandem mass, MS / MS, or in post-source fragmentation, TOF MS FTI), is useful here. Suitable MS and MS / MS techniques and systems are well known per se (see, for example, Methods in Molecular Biology, 146: "Mass Spectrometry of Proteins and Peptides", by Chapman, ed., Humana Press 2000, ISBN Biemann 1990. Methods Enzymol 193: 455-79 or Methods in Enzymology, Vol 402: "Biological Mass Spectrometry", by Burlingame, eds., Academie Press 2005, ISBN 9780121828073) and can be used here. The peptide ion fragmentation into MS (MS / MS) arrangements in tandem can be achieved using art-established ways, such as, for example, collision induced dissociation (CID). Detection and quantification of biomarkers by mass spectrometry may involve multiple reaction monitoring (MRM) as described by others by Kuhn et al. 2004 (Proteomics 4: 1175-86). The MS peptide analysis methods can be advantageously associated with downstream protein or peptide fractionation or fractionation methods, such as, for example, chromatographic methods.
Chromatography can also be used to measure biomarkers. As used herein, the term "chromatography" encompasses methods for separating chemicals, referred to as such and widely available in the art. In a preferred approach, chromatography refers to a process in which a mixture of chemicals (analytes) carried by a moving liquid or gas stream ("mobile phase") is separated into components as a result of the differential distribution. substances to be analyzed, since they flow around or on a stationary liquid or a solid phase ("stationary phase") between said mobile phase and said stationary phase. The stationary phase may be usually a finely divided solid, a sheet of filter material or a thin film of liquid on the surface of a solid, or the like. Chromatography is also widely applicable for the separation of chemical compounds of biological origin, such as, for example, amino acids, proteins, protein fragments or peptides, etc.
Chromatography as used herein may be preferably columnar (i.e. where the stationary phase is deposited or packed in a column), preferably liquid chromatography and most preferably HPLC. While the specifics of chromatography are well known in the art, for other tips, see for example Meyer M., 1998, ISBN: 047198373X, and "Practical HPLC Methodology and Applications", Bidlingmeyer, BA, John Wiley &; Sons Inc., 1993. Exemplary types of chromatography include, but are not limited to, high performance liquid chromatography (HPLC), normal phase HPLC (NP-HPLC), reverse phase HPLC (RP-HPLC), chromatography. ion exchange (IEC), such as cation exchange or anion chromatography, hydrophilic interaction chromatography (HILIC), hydrophobic interaction chromatography (HIC), exclusion-diffusion chromatography (SEC), including gel filtration chromatography or permeate gel chromatography, chromatofocusing, affinity chromatography such as immunoaffinity, immobilized transmetallic affinity chromatography and the like. Other methods of separating, identifying or quantifying peptides or polypeptides may be used, optionally in conjunction with one of the methods of analysis described above, for measuring the biomarkers of the present invention. Such methods include, but are not limited to, chemical extraction fractionation, isoelectric focusing (IEF) including capillary isoelectric focusing (CIEF), capillary isotachophoresis (CITP), capillary electrochromatography (CEC), and the like. one-dimensional polyacrylamide gel electrophoresis (PAGE), two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), capillary gel electrophoresis (CGE), zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC) ), continuous liquid electrophoresis (FFE), etc.
The level of biomarkers at the RNA level can be detected using the standard quantitative RNA measurement tools known in the art. Examples without limitation include hybridization-based analysis, biochip expression analysis, digital gene expression (DGE), in situ RNA hybridization (RISH), Northern blotting, and others. ; PCR, RT-PCR, RT-qPCR, PCR turning point, digital PCR or others; supported oligonucleotide detection, pyrosequencing, synthetic polony cyclic sequencing, simultaneous two-way sequencing, single-molecule sequencing, single-molecule real-time sequencing, single-molecule true sequencing, hybridization assisted nanopore sequencing , and synthetic sequencing.
The various aspects and embodiments taught herein may further depend on comparing the amount of biomarkers measured in samples from patients with reference values, where said reference values represent known predictions, diagnoses and / or prognoses of diseases and conditions as taught here.
For example, separate reference values may represent the prediction of a risk (eg, a high risk of abnormality) of having a given disease or condition as taught here in relation to the prediction of no risk or risk normal to have said disease or condition. In another example, separate reference values may represent predictions of different degrees of risk of having such a disease or condition.
In another example, separate reference values may represent the diagnosis of a given disease or condition as taught here in the face of the diagnosis of any disease or condition of this kind (such as, for example, the diagnosis of health or recovering said disease or condition, etc.). In another example, distinct reference values may represent the diagnosis of such a disease or of such a state of variable severity.
In yet another example, separate reference values may represent a good prognosis for a given disease or condition as taught here in view of a poor prognosis for said disease or condition. In another example, separate reference values may represent favorable or unfavorable prognoses for such a disease or condition.
Such a comparison may generally include any means of determining the presence or absence of at least one difference and optionally the size of this difference between compared values. A comparison may include visual inspection, arithmetic or statistical comparison of measurements. Such statistical comparisons include, but are not limited to, the application of a rule.
Reference values can be established according to known procedures previously used for other biomarkers and parameters. For example, a reference value can be established in an individual or population of individuals characterized by a particular diagnosis, prediction and / or prognosis of said disease or condition (ie, for whom said diagnosis, said prediction and / or said prognosis of the disease or condition remains true). Such a population may include without limitation> 2,> 10,> 100, or even several hundred or more individuals.
A "deviation" of a first value from a second value may generally encompass any direction (e.g., increase: first value> second value; or decrease: first value <second value) and any extent of corruption.
For example, a deviation may include a decrease in a first value, without limitation, of at least about 10% (about 0.9-times or less), or at least about 20% (about 0.8-times or less), or at least about 30% (about 0.7-fold or less), or at least about 40% (about 0.6-fold or less), or at least about 50% (about 0.5-fold or less) , or at least about 60% (about 0.4-fold or less), or at least about 70% (about 0.3-fold or less), or at least about 80% (about 0.2-fold or less) ), or at least about 90% (about 0.1-times or less), relating to a second value with which a comparison is made.
For example, a deviation may include an increase in a first value, without limitation, of at least about 10% (about 1.1-fold or more), or at least about 20% (about 1.2-fold or more) , or at least about 30% (about 1.3-fold or more), or at least about 40% (about 1.4-fold or more), or at least about 50% (about 1.5-fold or more) ), or at least about 60% (about 1.6-fold or more), or at least about 70% (about 1.7-fold or more), or at least about 80% (about 1.8-fold or more) more), or at least about 90% (about 1.9-fold or more), or at least about 100% (about 2-fold or more), or at least about 150% (about 2.5-fold) or more), or at least about 200% (about 3- or more times), or at least about 500% (about 6-fold or more, or at least about 700% (about 8-fold) or more), or other relating to a second value with which a comparison is made.
Preferably, a deviation can refer to a statistically significant observed alteration. For example, a deviation may refer to an observed impairment that falls outside the error margins of the reference values in a given population (as expressed, for example, by a standard deviation or a standard error, or by a multiple predetermined, for example, ± 1xSD or ± 2xSD, or ± 1xSE or ± 2xSE). The deviation can also refer to a value falling outside a reference range defined by values in a given population (for example, outside a range that includes> 40%,> 50%,> 60%,> 70%,> 75% or> 80% or> 85% or> 90% or> 95% or even> 100% of values in said populations).
In another embodiment, a discrepancy can be concluded if an observed alteration is beyond a given threshold or cutoff. Such a threshold or such a cleavage can be selected as generally known in the art to provide a selected sensitivity and / or specificity of the methods of diagnosis, prediction and / or prognosis, for example the sensitivity and / or specificity of at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%.
It is therefore obvious that the biomarkers, uses and method that provide substantial benefits in the diagnosis, prediction, prognosis and / or monitoring of altered fracture consolidation have been provided according to the invention. While the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art in light of the above description. Accordingly, it is intended to encompass all of these alternatives, modifications and variations as follows in the spirit and scope of the appended claims.
The aspects and embodiments of the invention are further supported by the following non-limiting examples.
EXAMPLE
Example 1: Measurement of Biomarker Levels in Serum / Plasma
Two groups of subjects were included in the study: (1) healthy volunteers (HV), (2) patients with impaired fracture consolidation, particularly with non-union fractures (NU). The patient population was distributed as follows:
The average age in both groups ranged between thirty and forty years. Patients with fracture with no consolidation were older (P = 0.012) and were predominantly male. However, the results remained unchanged regardless of sex and age. The bone sites were found in the long bones (radius, humerus, fibula, tibia and ulna) except for 2 metatarsal fractures and 2 calcaneal fractures. The delay between the fracture and the sample collected varied from approximately 25 months with a standard deviation of 15 months.
To identify systemic biomarkers of fractures with no consolidation, the sera were collected in dry tubes and plasma was collected in heparin or EDTA tubes, centrifuged, aliquoted and frozen at -20 ° C until use. They were used to determine the level of growth factors and proteins using enzyme-linked immunoadsorbent assays (ELISAs).
Stromal derived factor 1 was measured in plasma (SDF-1 / CXCL12, Duoset, R & D Systems, Abingdon, UK). The following biomarkers were measured in serum: platelet-derived BB growth factor (PDGF-BB, Quantikine ™ R & D Systems, Abingdon, UK), interleukin-8 (IL8 / CXCL8, Quantikine ™, R &D; Systems, Abingdon, UK) and interleukin 6 (IL-6, Quantikine ™, R & D Systems, Abingdon, UK).
All continuous values are expressed as mean ± standard error of mean (SEM), all reported P values are one-sided, and statistical significance is evaluated on the 10% level. The normality of the distribution was tested with a Kolmogorov-Smimov test. When the Kolmogorov-Smimov test failed, the differences between the groups were analyzed by a Mann-Whitney test.
Compared with HV, in NU patients plasma levels of SDF-1 decreased (Fig IA). The decrease was more pronounced in plasma collected in heparin tubes compared to plasma collected in EDTA tubes (Fig. IB and IC). Thus, heparin may be a preferred blood collection agent, specifically, an anti-coagulation agent, for collecting blood or plasma samples for the measurement of SDF-1. Based on the reading of this particular example, and by way of illustration only and without limitation to the invention as broadly described herein, an amount of SDF-1 in the plasma of a human subject collected using heparin, the amount of which is less than about 200 μg / ml, preferably less than about 150 μg / ml, more preferably less than about 100 μg / ml, such as between about 20 and about 150 μg / ml or between about 50 and about 100 μg / ml may indicate that the subject has impaired fracture consolidation or is at risk of impaired fracture consolidation or may indicate a poor prognosis for impaired fracture consolidation in the subject , and may indicate that the subject needs therapeutic or prophylactic treatment for impaired fracture consolidation; and an amount of SDF-1 in the plasma of a human subject collected using heparin, the amount of which is more than about 200 μg / ml may indicate that the subject has no impaired fracture consolidation or that there is no risk of impaired fracture consolidation or may indicate a good prognosis for impaired fracture consolidation in the subject, and may indicate that the subject does not require therapeutic or prophylactic treatment for consolidation impaired fractures.
The serum PDGF-BB level decreased in NU patients compared to healthy controls (HV) (Fig. 2). Based on the reading of this particular example and by way of illustration only and without limitation to the invention as broadly described herein, an amount of PDGF-BB in the serum of a human subject that is less than about 2.50 ng / ml, preferably less than about 2.25 ng / ml, more preferably about 2.00 ng / ml or less, such as between about 1.5 and about 2.5 ng / ml or between about 1.75 and about 2.25 ng / ml. / ml may indicate that the subject has impaired fracture consolidation or is at risk of impaired fracture consolidation or may indicate a poor prognosis for impaired fracture consolidation in the subject, and may indicate that the subject needs therapeutic or prophylactic treatment for altered fracture consolidation; and an amount of PDGF-BB in the serum of a human subject which is more than about 2.50 ng / ml, preferably more than about 2.70 ng / ml, such as between about 2.50 and about 3.00 ng / ml may indicate that the subject has no altered fracture consolidation or is at risk of impaired fracture consolidation or may indicate a good prognosis for impaired fracture consolidation in the subject, and may indicate that the subject does not need therapeutic or prophylactic treatment for altered fracture consolidation.
Compared with healthy volunteers (HV), the serum level of IL-8 increased in NU patients (Fig. 3). Based on the reading of this particular example and by way of illustration only and without limitation to the invention as broadly described herein, an amount of IL-8 in the serum of a human subject that is more than about 15 μg / ml, preferably more than about 20 μg / ml, more preferably more than about 25 μg / ml, more preferably more than about 30 μg / ml, such as between about 15 and about 45 μg / ml. pg / ml or between about 20 and about 40 μg / ml or between about 30 and about 35 μg / ml may indicate that the subject has impaired fracture consolidation or is at risk for impaired fracture consolidation or may indicate a poor prognosis for impaired fracture consolidation in the subject, and may indicate that the subject requires therapeutic or prophylactic treatment for impaired fracture consolidation; and an amount of IL-8 in the serum of a human subject that is less than about 15 μg / ml, preferably less than about 12.5 μg / ml, such as between about 5 and about 15 μg / ml or between about 7.5 and about 12.5 μg / ml or about 10 μg / ml may indicate that the subject has no altered fracture consolidation or is at risk of impaired fracture consolidation or may indicate a good prognosis for impaired fracture consolidation in the subject, and may indicate that the subject does not require therapeutic or prophylactic treatment for impaired fracture consolidation.
In comparison with healthy volunteers (HV), the serum level of IL-6 has tended to increase in NU patients (Fig.4A and 4B). Based on the reading of this particular example and by way of illustration only and without limitation to the invention as broadly described herein, an amount of IL-6 in the serum of a human subject that is more than about 1.0 μg / ml, preferably more than about 1.2 μg / ml, more preferably more than about 1.5 μg / ml, more preferably more than about 2 μg / ml, such as between about 1.3 and about 2.5 pg / ml or between about 1.5 and about 2.5 μg / ml may indicate that the subject has impaired fracture consolidation or is at risk of impaired fracture consolidation or may indicate a poor prognosis for impaired consolidation of fractures. fractures in the subject, and may indicate that the subject is in need of therapeutic or prophylactic treatment for impaired fracture consolidation; and an amount of IL-6 in the serum of a human subject that is less than about 1.0 μg / ml may indicate that the subject has no altered fracture consolidation or is at risk of have impaired fracture consolidation or may indicate a good prognosis for impaired fracture consolidation in the subject, and may indicate that the subject does not require therapeutic or prophylactic treatment for impaired fracture consolidation.
Example 2: Culture of cells from subjects
As noted, the amount of biomarkers can also be measured in cells or in the supernatant of cells obtained from subjects and cultured in vitro, preferably from osteoblastic cells (OB) or mesenchymal stem cells (MSCs). ). The following provides the appropriate protocols for isolation, differentiation and stem cell culture.
Twenty to sixty ml of heparinized bone marrow (BM) was obtained from the iliac crest distant from the site of the fracture. BM was mixed with phosphate buffered saline (PBS: BM ratio (v: v): 2) and layered on a Ficoll solution of density gradients. After centrifugation, mononuclear cells were cultured from the interface and washed twice in PBS. The cells were cultured in flasks of 1.43 x 10 cells / 25 cm 2 in two different media; (1) a mesenchymal medium composed of DNEM, 10% fetal bovine serum, 1% L-glutamine, 1% penicillin and 1% streptomycin; (2) an osteogenic medium. The cells were stored in a humidified atmosphere at 37 ° C containing 5% CO2. Changes of medium were made every 2 to 3 days. When they were confluent, cells from the first culture were detached and re-cultured for secondary culture. Supernatants from these 2 culture passages were collected and frozen until use.
The ELISA reagent protocols used for the blood samples were applied to the cell supernatant with routine adaptation.
Example 3: Autocrine / paracrine activity of osteoblastic cells and mesenchymal stem cells
To study the autocrine / paracrine activity of osteoprogenitor cells in altered fracture consolidation, the level of secreted growth factors in supernatant osteoblastic cell (OB) or mesenchymal cell (MSC) culture was assessed by ELISA. The following growth factors were measured; stromal derivative factor 1 (SDF-1 / CXCL12, Duoset, R & D Systems, Abingdon, UK), and interleukin-6 (IL-6, Duoset, R & D Systems, Abingdon, UK). Values were expressed as μg / ml supernatant.
Compared with healthy volunteers (HV), SDF-1 was less secreted in the supernatant of the OB and MSC culture of patients with no fracture consolidation (NU) at the end of the primary cell culture (Figs. 5A and 5B). .
In addition, IL-6 was less secreted in the OB culture supernatant of NU patients at the end of primary and secondary cell cultures compared with HV (Fig 6).

权利要求:
Claims (17)
[1]
claims
1. Use of interleukin-8 (IL-8) as a biomarker for altered bone fracture consolidation.
[2]
2. Use of IL-8 for the diagnosis, prediction, prognosis and / or monitoring of impaired bone fracture consolidation.
[3]
Use according to any one of claims 1 or 2, wherein the impaired consolidation of bone fractures is selected from the group consisting of vicious consolidation fracture, delayed consolidation fracture, and non-healing fracture.
[4]
A method for the diagnosis, prediction, prognosis and / or monitoring of altered bone fracture consolidation in a subject comprising measuring the level of IL-8 in a sample from said subject.
[5]
A method for the diagnosis, prediction, prognosis and / or monitoring of altered bone fracture consolidation in a subject according to claim 4 comprising the steps of: (i) measuring the amount of IL-8 in a sample from the subject; (ii) comparing the amount as measured in (i) with a reference value representing a known diagnosis, prediction and / or prognosis of impaired fracture consolidation; (iii) finding a deviation or deviation of the amount as measured in (i) from the reference value; and (iv) attributing said deviation finding or no deviation for a specific diagnosis, prediction and / or prognosis of impaired fracture consolidation in the subject.
[6]
A method for monitoring the impaired consolidation of bone fractures or for monitoring the risk of development of altered bone fracture consolidation in a subject according to claim 4 comprising the steps of: (i) measuring the amount of IL-8 in a sample from the subject at two or more successive time points; (ii) comparing the amount as measured in (i) between said two or more successive time points; (iii) finding a deviation or deviation of the amount as measured in (i) between said two or more successive time points; (iv) attributing said finding of deviation or deviation to a change in the impaired consolidation of bone fractures or to a change in the likelihood of developing impaired bone fracture consolidation in the subject between said two or more points successive temporals.
[7]
A method of determining whether a subject is or is not in need of therapeutic or prophylactic treatment for altered bone fracture consolidation comprising measuring the level of IL-8 in a sample from said subject, preferably comprising the steps of: (i) measuring the amount of IL-8 in a sample from the subject; (ii) comparing the amount as measured in (i) with a reference value representing a known diagnosis, prediction and / or prognosis of impaired bone fracture consolidation; (iii) finding a deviation or deviation of the amount as measured in (i) from the reference value; and (iv) inferring from said finding of presence or absence of a need for therapeutic or prophylactic treatment for impaired bone fracture consolidation.
[8]
The method according to any one of claims 4 to 7, wherein the altered consolidation of bone fractures is selected from the group consisting of vicious consolidation fracture, delayed consolidation fracture, and non-healing fracture.
[9]
Use according to any one of claims 1 to 3 or method according to any one of claims 4 to 8, further comprising measuring the level of any one or more SDF-1s, PDGF-BB, EL -6, TGF-β, PDGF, FGF and BMP in a sample from the subject.
[10]
10. Method for establishing a reference value for IL-8, said reference value representing: (a) a prediction or diagnosis of lack of impaired consolidation of bone fractures or a good prognosis for impaired bone fracture consolidation, or (b) a prediction or diagnosis of impaired bone fracture consolidation or poor prognosis for impaired bone fracture consolidation, comprising: (i) measuring the amount of IL-8 in a sample from: (i) one or more subjects who do not have impaired consolidation of bone fractures or who do not run the risk of having an impaired consolidation of bone fractures or who have a good prognosis of impaired bone fracture consolidation, or (ib) one or more subjects with impaired consolidation of bone fractures or running the risk of impaired bone fracture consolidation or impaired consolidation prognosis of bone fractures, and (ii a) establishment from the amount of IL-8 as measured in (ia), the reference value representing the prediction or diagnosis of the lack of consolidation altered bone fractures or representing the good prognosis for impaired bone fracture consolidation, or (ii) the establishment from the amount of IL-8 as measured in (ib), the reference value representing the prediction or the diagnosis of impaired consolidation of bone fractures or poor prognosis for impaired consolidation of bone fractures.
[11]
Use according to any one of claims 1 to 3 or 9 or method according to any one of claims 4 to 10, comprising measuring the systemic amount of IL-8, preferably in which the sample comprises, consists essentially or consists of whole blood or a fractional component thereof, more preferably plasma or serum.
[12]
Use according to any one of claims 1 to 3 or 9 or method according to any one of claims 4 to 10, comprising measuring the amount of IL-8 in the cells or in the supernatant of the cells obtained at from the subject and subsequently cultured in vitro.
[13]
Use or method according to claim 12, wherein the cells are osteoblasts (OB) or mesenchymal stem cells (MSC).
[14]
14. Use of a kit for the diagnosis, prediction, prognosis and / or monitoring of altered bone fracture consolidation in a subject, the kit comprising (i) one or more linkers capable of binding specifically to IL -8, particularly in a sample from the subject, and (ii) optionally and preferably one or more reference values or means for establishing said one or more reference values, wherein said one or more reference values represent a diagnosis, a prediction and / or a known prognosis of impaired consolidation of bone fractures.
[15]
The use of claim 14, wherein the means for collecting a sample from a subject is provided separately from the kit or is included in the kit.
[16]
16. Use of a nucleic acid chip or biochip or a protein, polypeptide or peptide chip or biochip for the diagnosis, prediction, prognosis and / or monitoring of altered bone fracture consolidation, said microarray or biochip comprising a nucleic acid encoding IL-8 or comprising IL-8.
[17]
17. Use of a linker chip or biochip for the diagnosis, prediction, prognosis and / or monitoring of impaired bone fracture consolidation, said microarray or biochip comprising one or more linkers capable of specifically bind IL-8, preferably comprising a known amount or concentration of said one or more linkers.

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法律状态:
2018-06-04| FG| Patent granted|Effective date: 20160115 |

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2018-06-04| MM| Lapsed because of non-payment of the annual fee|Effective date: 20170630 |

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