WO2024056233A1 - Procédé pour déterminer un paramètre de diagnostic pour caractériser l'état d'inflammation, de préférence dans l'environnement d'un implant dentaire, et système d'analyse et corps de test rapide pour la mise en oeuvre du procédé - Google Patents

Procédé pour déterminer un paramètre de diagnostic pour caractériser l'état d'inflammation, de préférence dans l'environnement d'un implant dentaire, et système d'analyse et corps de test rapide pour la mise en oeuvre du procédé Download PDF

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Publication number
WO2024056233A1
WO2024056233A1 PCT/EP2023/068786 EP2023068786W WO2024056233A1 WO 2024056233 A1 WO2024056233 A1 WO 2024056233A1 EP 2023068786 W EP2023068786 W EP 2023068786W WO 2024056233 A1 WO2024056233 A1 WO 2024056233A1
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Prior art keywords
inflammation
detection
inflammatory
analysis system
molecules
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PCT/EP2023/068786
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German (de)
English (en)
Inventor
Holger Zipprich
Oliver Seitz
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Holger Zipprich
Oliver Seitz
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Publication of WO2024056233A1 publication Critical patent/WO2024056233A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6869—Interleukin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/18—Dental and oral disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/70—Mechanisms involved in disease identification
    • G01N2800/7095—Inflammation

Definitions

  • the invention relates to a method for determining a diagnostic parameter for characterizing an inflammatory condition, in particular in the vicinity of a tooth or a dental implant. It further relates to an analysis system for carrying out a test to detect an inflammatory condition and to a rapid test body, in particular for carrying out a rapid test to detect the inflammatory condition in the area of a dental implant.
  • inflammatory processes can be triggered in the human body and inflammation can become established.
  • Such inflammatory situations can occur, especially in the mouth or throat area, especially in the dental area, which can sometimes lead to serious problems and consequential damage for those affected.
  • periodontitis is an inflammation in the dental area that can lead to impaired dental health and even tooth loss.
  • such inflammatory processes can also occur in the area of inserted dental implants.
  • a biofilm can form, particularly on the usually micro-rough implant surface, which is colonized by bacteria, which can ultimately lead to chronic and recurring infections.
  • This clinical picture is known as peri-implantitis.
  • Periodontitis is a combination of neglected oral hygiene, adhesion of biofilm to the usually micro-rough surface of the inserted implant and other factors that cause the full picture of peri-implantitis, which is characterized by increasing stress and destruction of the hard and soft tissue.
  • the areas of the dental implants where the hard and/or soft tissue retreats are usually covered with a biofilm.
  • the invention is therefore based on the object of specifying a method for determining a diagnostic parameter for the early characterization of an inflammatory condition, in particular in the vicinity of a dental implant. Furthermore, an analysis system suitable for carrying out such a test, preferably a suitable rapid test, should be specified.
  • this object is achieved according to the invention by taking body fluid or secretion from an inflammatory area of a patient, for example from or on a natural tooth, mouth, nose or throat, an anal area, a vaginal area or from the penis, blood or other body fluids.
  • body fluid or secretion from an inflammatory area of a patient, for example from or on a natural tooth, mouth, nose or throat, an anal area, a vaginal area or from the penis, blood or other body fluids.
  • sulcus fluid in particular from the patient's peri-implant tissue
  • the invention is based on the idea that inflammatory processes in particular, particularly in the area of the tooth pockets, of a patient should be specifically monitored and analyzed.
  • Selected analytical or inflammatory markers should be monitored and evaluated that are particularly meaningful and suitable for the desired analysis of the inflammatory processes in the intended area of application, especially in implantology.
  • account should be taken of the fact that, especially in the area of dental applications, for example when sulcus fluid is taken from the area around a tooth or dental implant, it is only possible to take a quantitatively determinable amount of sample only rarely and under difficult conditions.
  • quantitative determination is usually necessary, as only determining the concentration of an inflammatory marker in the respective sample allows conclusions to be drawn about the status of the inflammatory process.
  • Inflammatory markers can generally be viewed as messenger substances with which the immune system is stimulated to react depending on the type and extent of the inflammation present. Accordingly, there are a large number of such messenger substances or inflammatory markers, the occurrence of which usually depends heavily on the degree of inflammation in question; In particular, the occurrence of an inflammatory marker is usually dependent on the degree of inflammation and has a local or global maximum at a certain degree of inflammation. I.e. There are inflammatory markers whose maximum occurrence occurs with relatively mild inflammation. dung exists and which are therefore the first to be detected in the event of progressive inflammation. In contrast, there are other inflammatory markers whose maximum occurrence only occurs with moderate inflammation or even with severe inflammation.
  • the diagnostic parameter is determined by comparing number key figures for at least two of interleukin-23, interleukin-17 or . Lipocalin-2.
  • the method can be carried out in any manner that allows the quantitative determination of number characteristics of inflammatory markers.
  • a PCR test can be provided for this purpose.
  • a suitable analysis system can also be provided for carrying out the method.
  • the stated task is solved with a detection body which is provided with a number of, preferably at least three, detection fields, each of which is equipped with a detector molecule assigned to a target molecule.
  • a detection body which is provided with a number of, preferably at least three, detection fields, each of which is equipped with a detector molecule assigned to a target molecule.
  • at least two, in particular at least three, detection fields with detector molecules assigned to different target molecules are provided.
  • one of the detection fields is each equipped with detector molecules for detecting at least two of interleukin-23, interleukin-17 or lipocalin-2 as target molecules.
  • the analysis system is designed as a rapid test that allows easy handling in the doctor's or dentist's office or, in particular, that the test can be carried out by the patient himself.
  • a rapid test body is provided, which is designed as an analysis system of the type described above and which has a test strip designed as a detection body with the detection fields mentioned.
  • FIG. 1 a quick test body in a perspective view
  • FIG. 2 a section of the test strip of the rapid test body.
  • FIG. 1 in plan view
  • FIG. 3 schematically shows the test tire of the quick test body.
  • FIG. 1 with detection molecules for target molecules.
  • a rapid test body 1 for the short-term detection of pathogen material, the basic structure of which has been in use in large numbers since it was widely used in the corona pandemic at the latest. It is pointed out that The functionality of the test method will be explained below based on the design of such a rapid test body 1. According to aspects of the invention, however, the operation of the method described below can also be implemented in another way, for example by means of the PCR method.
  • the rapid test body 1 has a carrier body 2 in which a test strip 6, for example made of a nitrocellulose membrane, is arranged within a window 4 accessible from the outside. Furthermore, an application field 8 is provided in the carrier body 2.
  • a sample 10 taken from the person to be examined and in liquid form, for example a blood sample, a saliva sample or a sample obtained from a mouth, throat or nose swab and kept in a solvent, can be applied to the application field 8. After being applied to the application field, the sample is drawn along the path shown in FIG. 1 due to the capillary forces in the test strip 6. 1 transported in the flow direction shown as arrow 12 in the test strip 6.
  • the test strip 6 is, as particularly shown in the enlarged detail in FIG. 2 can be removed, with a plurality of, in the exemplary embodiment three, detection fields 14 equipped.
  • Each detection field 14 is included, as shown in the schematic representation in FIG. 3 shown, equipped with specifically selected detection molecules 16, each of which specifically binds a quorum sensing target molecule 18 and/or a quorum sensing-associated target molecule 20.
  • the sample 10 first passes through the three detection fields 14. As soon as the sample 10 comes into contact with the detection molecules 16 in the respective detection field 14, the corresponding target molecule 18, 20, if present in the sample 10, binds specifically to it at. The sample 10 then reaches a control field 22 provided below, in which proof is provided that the sample 10 has completely passed through the test strip 6.
  • the respective target molecule 18, 20 is present in the sample 10 and comes into contact with the detection molecules 16 present there when the sample 10 passes through the respective detection field 14, it binds specifically to the respective detection molecule 16.
  • the detection molecules 16 are in turn directly on Test strip 6 attached and immobilized on it so that the ones bound to the detection molecule 16 Target molecules 18, 20 are retained in the corresponding detection field 14 when the sample 10 has passed the respective detection field 14 along its flow direction.
  • the control field 22, on the other hand, may have a means (not shown) which indicates that the sample 10, starting from the application field 8, has passed all detection fields 14 in the flow direction and flowed into the control field 22.
  • control field 22 depending on the type of sample 10, an antibody directed against an antibody that is already present there, for example in the case of a blood sample against a component that is always present in the blood, for example a gluboline or albumin, or, in the case of a sample of sulcus fluid, a corresponding antibody , be immobilized.
  • the control field 22 can be designed in such a way that it only indicates the wetting with sample liquid in an externally recognizable manner by outputting a measurable binding signal 30.
  • the sample 10 taken can first be placed in a suitable solvent after it has been taken before it is placed in the detection field 14 together with the solvent.
  • the sample 10 taken which can be in the form of a soaked paper or non-woven fabric tip in the case of a swab, is placed in this solvent and usually shaken or allowed to take effect.
  • the solvent for later evaluation, even small amounts of sample contained in the solvent can be sufficient, which can serve as evidence that the respective detection field 14 has been completely passed through.
  • a further antibody is added as a detection reagent to generate a binding signal 30 that can be evaluated at least qualitatively.
  • the additional antibody used as a detection reagent is provided with a marker and is able to specifically bind the target molecule 18, 20 bound to the detection molecule 16.
  • the binding of the detection reagent to the target molecule 18, 20 then indicates a binding signal 30 generated by the marker of the detection reagent.
  • the binding Signal 30 can be, for example, a color signal if the marker is a fluorescent compound or an enzyme that catalyzes a color reaction.
  • an analyte is added as a detection reagent, which competes with the target molecule 18, 20 for the binding site on the detection molecule 16.
  • the analyte forming the detection reagent is also provided with a marker which is designed to output a binding signal 30.
  • the competitive assay has the advantage that it can not only be qualitatively proven that the target molecule 18, 20 is contained in the sample 10, but also a quantitative determination of the amount of target molecule 18, 20 in the sample 10 can be carried out particularly reliably . The more target molecules 18, 20 are contained in the sample 10, the more target molecules 18, 20 remain bound to the detection molecule 16 even after the detection reagent has been added.
  • Another option for checking the binding of a target molecule 18, 20 to a detection molecule 16 is the so-called sandwich assay, which is also used in conventional ELISA (enzyme-linked immunosorbent assays) methods.
  • sandwich assay In a first detection step, a secondary antibody is first added, which is able to bind specifically to the target molecule 18, 20 bound to the detection molecule 16. Then, in a second step, the actual detection reagent is added, which is an additional antibody provided with a marker that specifically binds the secondary antibody. This results in a sandwich structure in which the marker of the detection reagent emits a measurable binding signal 30.
  • the respective reagents that generate the measurable binding signal 30 can be present in the test strip 6 directly within the detection fields 14; Alternatively, they can also be placed in front of the river in the direction of flow respective detection field 14 can be placed in the carrier material in such a way that the sample 10 takes the reagents with it and transports them into the detection fields 14.
  • An optical binding signal 30, for example a color stripe or a change in brightness in a black-and-white display concept, can be generated as a binding signal 30 if the respective target molecule 18, 20 specifically binds the corresponding detection molecule 16 in the respective detection field 14 or the applied sample 10 binds the control field 22 achieved, which then also becomes visible as a colored stripe.
  • the binding signal 30 can also be an electrical signal, for example a measured electrical resistance between two electrodes suitably placed in the area of the respective detection field 14, which changes accordingly in the presence of binding of the respective target molecules 18, 20 to the assigned detection molecules 16.
  • the binding of the target molecule 18, 20 to the respective detector molecule 16 can also leave behind an electrical signal, for example, if the binding partner is an electroactive substance and is activated by e.g. B. Increase in mass due to binding changes its conductivity.
  • the signal output can also be carried out potentiometrically, because charge carriers are released by the enzymatic turnover and these output the change in current flow as a measured value.
  • the binding can be detected or detected colometrically in that the binding changes the light reflection Z refraction properties, which light particle sensors can detect.
  • a quantitative detection of the target molecules 18, 20 bound in the detection fields 14 is provided, so that, on the one hand, the detection of a time course of the number of bound target molecules 18, 20 and, on the other hand, another Processing of the measurement results obtained in a quantitative manner is possible.
  • the temporal development of the amount of bound target molecules 18, 20 should be traceable
  • storage should also be possible, for example, in a central database, which is a Evaluation of the test results based on a sequence of several samplings, which should even be possible over a period of several months or years, for example.
  • a quantitatively evaluable binding signal 30 is generated, for example using one of the methods mentioned above, and, if necessary, converted into a characteristic value for the number of target molecules 18, 20 bound in the detection field 14 (hereinafter referred to as “number - Characteristic value”) converted or converted.
  • the rapid test and accordingly the rapid test body 1 provided for carrying out the rapid test, is designed for a determination and, if necessary, evaluation of a relative diagnostic statement, in which the values determined for the individual detection fields 14 are calculated for the number of In each case, the target molecules 18, 20 bound in the detection field 14 are set in relation to each other and the respective diagnosis is derived from this relation.
  • the mathematical ratio of two number parameters could be determined and further used, in particular by dividing the respective number parameters, or a quantitative larger/smaller comparison of two number parameters could be carried out, or the temporal progressions of two numbers could be carried out -Characteristics are compared with each other.
  • the number characteristics from three detection fields 14 are used in combination with one another and in relation to one another for the evaluation and determination of a diagnostic statement.
  • different detection molecules 16 are provided in the detection fields 14, so that when the sample 10 passes through, different target molecules are present in each detection field 14 18, 20 can be tied.
  • Such a relative diagnostic concept is viewed as particularly advantageous since the result and reliability of the test does not depend or almost does not depend on the absolute amount of the sample taken or the target molecules contained therein. Usually, especially when taking, for example, a swab with a paper tip or similar, it is not at all apparent to the user what volume or amount of material was taken and in what concentration the target molecule is actually present. Nevertheless, with such a relative diagnosis, the statements remain unchanged by comparing the results for the different target molecules, since the same volume, quantity or concentration ratios are present for all target molecules (at least assuming homogeneous sampling as such).
  • the ratios of several, preferably three, inflammatory markers to one another are used to assess the severity of the inflammation.
  • the volume of the sample taken has little or no influence on the results and therefore not on the diagnosis or therapy.
  • a combination of (at least two) markers or target molecules can be provided for evaluation, at least one of which increases in value as the extent of inflammation increases, and at least one further of which increases as the extent of inflammation increases Inflammation decreases in value.
  • this is based on the idea that the immune system communicates internally via the special markers.
  • the overall response of these markers provides information about the inflammatory picture, the degree of inflammation and the necessary defensive measures. It is a highly complex structure and highly complex communication that has not yet been fully deciphered. In principle, not all markers can be used to read or interpret the degree of inflammation. Due to the complexity of this communication system, it is hardly possible to determine the degree of inflammation based on the presence of a single marker. Even if one could determine the quantity of a marker in low, medium and high or even more finely as a concentration in a defined amount of substance, which, as described, is hardly possible from the ulcer / tooth or implant pocket, a direct conclusion can be drawn Degree of inflammation extremely vague and imprecise.
  • the rapid test can therefore, with suitable selection and specification of the detection molecules, be intended and designed for a large number of applications in the sense of the invention, for example to determine information about a possible inflammation using a sample or a swab from the mouth, nose or throat, from the rectal or anal area, from the vaginal or penile area, from a blood sample or from other body fluids.
  • the rapid test described is intended for use in the dental or dental field, i.e. in diagnosis or prevention in the field of dental or oral care.
  • the rapid test in particular using the rapid test body 1, is used according to one aspect of the invention to determine and/or monitor and/or diagnose whether there is an inflammation in the patient's oral cavity, what type it is, and if necessary whether and if necessary, how this progresses.
  • the rapid test is intended for the analysis of periodontitis.
  • the rapid test is intended and designed as a robust, prognostic rapid test for analyzing inflammatory processes in implantation surgery.
  • the rapid test according to one aspect of the invention is specifically designed for the detection and later evaluation, also in relation to one another, for specifically selected analysis or inflammatory markers, which have surprisingly been found to be for the desired and according to one aspect of the Analysis of the inflammatory processes in implantology provided by the invention is particularly meaningful and suitable.
  • the test described below and the evaluation described could be used, for example, in the Elisa or, if necessary, in a PCR method, as a test for periodontitis, for chronic genital diseases and/or for other chronic inflammations, possibly also for self-testing for patients.
  • in particular inflammatory processes in the area of a patient's tooth pockets are monitored and analyzed.
  • some sulcus fluid from healthy tooth pockets corresponds to the healthy condition
  • some sulcus fluid from peri-implant tissue can be taken as sample 10.
  • the proinflammatory cytokine interleukin is used as analysis markers and thus as target molecules 18, 20 in the sense mentioned above 23, IL-23 for short, proinflammatory cytokine interleukin 17, IL-17 for short, and the protein lipocalin-2.
  • these target molecules are essentially assigned the following properties, which are then converted into a diagnostic statement when the results are evaluated:
  • IL-23 if a comparatively high value is determined, it is concluded that there is a physiological and therefore harmless inflammation, but if a low or no value is determined, it is concluded that there is unphysiological inflammation and thus a need for action is determined
  • IL-17 if a comparatively low value or no value at all is determined, it is concluded that there is a physiological and therefore harmless inflammation, but if a high value is determined, it is concluded that there is unphysiological inflammation and thus a need for action is determined
  • Lipocalin-2 if a comparatively low value or no value at all is determined, a physiological and therefore harmless inflammation is concluded, but if a high value is determined, an unphysiological, possibly pathological or pathogenic inflammation is concluded and a need for action is therefore determined
  • these trendy individual pieces of information can be converted into a rough diagnostic grid by suitable combination with each other, for example cross-comparisons and/or trend comparisons, from which conclusions can be drawn as to the condition or progression of an inflammation and a recommendation for action for the dentist can be derived from this can.
  • This could, for example, be implemented in a kind of traffic light system for the further treatment of a patient, for example as part of preventive treatment or subsequent therapy.
  • a predominantly physiological (and therefore viewed as “harmless”) inflammation is detected, no need for action is diagnosed (“traffic light green”, in particular in accordance with the determination that only or at least predominantly IL-23 is present), whereas in one predominantly non-physiological (and therefore considered serious) inflammation is concluded from the need for immediate action (“traffic light red”, in particular according to the finding that lipocalin is present).
  • traffic light red in particular according to the finding that lipocalin is present.
  • Intermediate values can then indicate further preventive or care measures or even shorter monitoring intervals or the like (“yellow traffic light”, in particular according to the determination that a combination of IL-23 and IL-17 is present).
  • diagnosis can be designed according to the following criteria:
  • IL-23 secrete interleukin (IL)-23.
  • the analysis and evaluation of IL-23 can therefore be viewed as a good basis for a pro-inflammatory detection system.
  • IL-23 is, so to speak, at the 'base' of the resulting inflammation.
  • IL-23 is an amplifier of M(
  • Low IL-23 expression means controlled, normal inflammation and a good prognosis.
  • a lot of IL-23 initiates the next step in the inflammatory cascade and thus means a further increase in inflammation: poor prognosis.
  • IL-23 leads to the amplification of Th17 cells in the inflammatory area. Th17 cells secrete IL-17. IL-17 is therefore further upstream in the expanding inflammatory cascade. If IL-17 is detected, it indicates a further worsening of the prognosis: IL-17 detection means an even worse prognosis/transition to implant loss. IL-17 itself induces the production of inflammatory proteins in the tissue: IL-1, IL-6, TNFalpha and cyclooxygenase. At this point the inflammation now has the potential to become tissue destructive. IL-17 itself recruits neutrophils, whose presence confirms the problematic inflammatory situation and marks an end state. Neutrophils can be detected in the analysis using the specific marker “human neutrophil lipocalin” (HNL).
  • HNL human neutrophil lipocalin
  • the detection fields 14 are arranged at a distance from one another on the test strip 6, so that in a suitable evaluation unit, for example optically, the binding signals 30 of the detection fields 14 are recorded separately and independently of one another and evaluated can be supplied.
  • the binding signals 30 are then evaluated quantitatively as described above, so that the corresponding number characteristics can be suitably compared with one another and the diagnostic statement can be formed.
  • a color change can also be generated in the respective detection field 14 as a binding signal 30.
  • the detector and detection molecules are selected in such a way that the color change and thus the color coupling takes place according to the RGB system.
  • the detection fields 14 in the test strip 6 can be arranged in the same or immediately adjacent spatial area, so that the color change appears to overlap optically for the viewer.
  • the selection is made in such a way that the color change of all three components in the same spatial area results in an overlapping color signal, which, depending on the relationship of the components to one another, overall corresponds to the red-green-blue scheme Result generated.
  • the detection fields 14 can also be arranged in groups, each with three detection fields 14 designed according to the aforementioned RGB system and positioned together.
  • the diagnostic parameters obtained in the manner described above are stored and stored centrally, in particular in a database in the manner of a digital patient file, so that the diagnosis can also be tracked over a longer period of time and the therapy can be followed up if necessary.
  • Particular preference is given to integrating the historical data into the digital patient file, so that each tooth and implant or quadrant or jaw can be assigned a corresponding historical development. This makes it possible to monitor the inflammation and, in combination with therapy, to record/prove the effect of the therapy.

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Abstract

Selon l'invention, un corps de test rapide (1), en particulier pour effectuer un test rapide pour détecter l'état d'inflammation dans l'environnement d'un implant dentaire, lequel corps de test rapide présente une bandelette réactive (6), est pourvu d'un certain nombre de champs de détection (14) qui sont équipés de molécules de détection (16) pour détecter, dans un champ de détection respectif, l'interleukine 23, l'interleukine 17 et la lipocaline 2 en tant que molécule cible (18, 20).
PCT/EP2023/068786 2022-09-16 2023-07-06 Procédé pour déterminer un paramètre de diagnostic pour caractériser l'état d'inflammation, de préférence dans l'environnement d'un implant dentaire, et système d'analyse et corps de test rapide pour la mise en oeuvre du procédé WO2024056233A1 (fr)

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DE102022209790.4A DE102022209790A1 (de) 2022-09-16 2022-09-16 Verfahren zur Ermittlung eines Diagnoseparameters zur Charakterisierung des Entzündungszustands in der Umgebung eines Dentalimplantats und Schnelltestköper zur Durchführung des Verfahrens
DE102022209790.4 2022-09-16

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