EP2997383A1 - Fgf23 utilisé en tant que biomarqueur en vue de prédire le risque de mortalité dû à une maladie du foie de stade final - Google Patents

Fgf23 utilisé en tant que biomarqueur en vue de prédire le risque de mortalité dû à une maladie du foie de stade final

Info

Publication number
EP2997383A1
EP2997383A1 EP14725123.5A EP14725123A EP2997383A1 EP 2997383 A1 EP2997383 A1 EP 2997383A1 EP 14725123 A EP14725123 A EP 14725123A EP 2997383 A1 EP2997383 A1 EP 2997383A1
Authority
EP
European Patent Office
Prior art keywords
fgf23
concentration
patient
risk
mortality
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP14725123.5A
Other languages
German (de)
English (en)
Inventor
Gérard FRIEDLANDER
Dominique PRIE
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Assistance Publique Hopitaux de Paris APHP
Institut National de la Sante et de la Recherche Medicale INSERM
Universite Paris Descartes
Original Assignee
Assistance Publique Hopitaux de Paris APHP
Institut National de la Sante et de la Recherche Medicale INSERM
Universite Paris Descartes
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Assistance Publique Hopitaux de Paris APHP, Institut National de la Sante et de la Recherche Medicale INSERM, Universite Paris Descartes filed Critical Assistance Publique Hopitaux de Paris APHP
Priority to EP14725123.5A priority Critical patent/EP2997383A1/fr
Publication of EP2997383A1 publication Critical patent/EP2997383A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G01N2333/50—Fibroblast growth factors [FGF]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/08—Hepato-biliairy disorders other than hepatitis
    • G01N2800/085—Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease

Definitions

  • FGF23 AS A BIOMARKER FOR PREDICTING THE RISK OF MORTALITY DUE TO END STAGE LIVER DISEASE
  • the invention relates to a method for predicting the risk of mortality of patients suffering from end stage liver disease (ESLD). More particularly, the method comprises a step of measuring FGF23 concentration in a blood sample obtained from said patients.
  • ESLD end stage liver disease
  • MELD Model for End-Stage Liver Disease
  • Fibroblast growth factor 23 is a circulating hormone whose concentration has been associated with mortality in chronic kidney disease [6, 7].
  • international patent application WO 2008/089936 relates to a method for the determination or prediction of the progression of CKD in a subject suspected to suffer from CKD, said method comprising the step of determining the expression levels of at least one marker selected from (a) FGF23; and (b) adiponectin in a biological sample.
  • International patent application WO 2009/091556 discloses that FGF23 is capable of predicting the risk of mortality due to CKD in an asymptomatic patient. This hormone inhibits renal phosphate reabsorption and calcitriol synthesis [8].
  • FGF23 can be cleaved between amino acids 176-179 into two smaller peptides.
  • the enzyme responsible for FGF23 cleavage and its location remains to be identified.
  • FGF23 mR A is mainly expressed in bone cells but is also present at lower levels in the liver [9].
  • Physiological triggers of FGF23 synthesis are high blood phosphate and calcitriol concentrations [10-15].
  • FGF23 concentration also increases early with the decline of renal function [16, 17]. Plasma FGF23 levels predict the risk of progression of chronic kidney disease: the higher FGF23 concentrations, the higher risk of decrease in renal function [18].
  • High FGF23 plasma levels are also associated with an increased risk of death in patients with end stage renal disease in the first year following the initiation of the hemodialysis procedure [7] and in patients with earlier stage of chronic kidney diseases [19, 20]. Furthermore FGF23 concentration was found associated to mortality in the absence of alteration of renal function [21] suggesting that FGF23 could also predict the risk of death in non renal disorders (i.e. in coronary artery diseases).
  • liver failure remains associated with high morbidity and mortality and there still exits an urge need for an easily measurable biomarker predicting the risk of death of patients suffering from ESLD.
  • a biomarker would be thus useful for determining amongst these patients which have priority for liver transplantation.
  • FGF23 concentration might be related with risk of mortality due to ESLD.
  • the invention relates to a method for predicting the risk of mortality of a patient suffering from end stage liver disease (ESLD), said method comprising the steps of:
  • step (ii) comparing the concentration obtained at step (i) to a predetermined value, wherein a difference between said concentration and said predetermined value is indicative of a risk of mortality for said patient.
  • the invention also relates to a method for determining priority for liver transplantation in a patient suffering from ESLD, comprising the steps of:
  • the invention further relates to a kit for performing a method as described above wherein the kit comprises means for measuring the concentration of FGF23 polypeptide in a biological sample obtained from said patient.
  • the invention relates to the use of FGF23 polypeptide as a biomarker for predicting the risk of mortality of a patient affected with ESLD.
  • the invention relates to a method for predicting or determining the severity of the liver disease in a patient, comprising the steps of:
  • the invention relates to the use of FGF23 polypeptide as a biomarker for determining the severity of a liver disease in patient.
  • the invention further relates to a FGF23 antagonist for use in the improvement of the survival time of a patient suffering from ESLD and having a risk of mortality as provided by a method for the prediction of mortality according to the invention.
  • the inventors have demonstrated that an increase in blood sample of FGF23 concentration is related with high risk of morality in patients suffering from end stage liver disease (ESLD). Therefore, measuring blood FGF23 concentration in a patient may represent a classifying test for patients at risk for of morality due to ESLD and thus a valuable mean for determining which patients have the priority for liver transplantation among patients on a liver-transplant waiting list. They have also shown that FGF23 concentration is associated with the severity of the liver disease (or of the severity of liver dysfunction).
  • ESLD end stage liver disease
  • ESLD refers to an irreversible condition that leads to the imminent complete failure of the liver and subsequently to the death.
  • ESLD may be the final stage of many liver diseases.
  • ESLD may be provoked by a disease selected in the group consisting of cirrhosis, viral hepatitis, genetic disorders, liver cancer (e.g. hepatocellular carcinoma (HCC), autoimmune disease, obesity and intoxication may be the factors that cause end stage liver disease.
  • HCC hepatocellular carcinoma
  • FGF23 fibroblast growth factor 23
  • FGF23 fibroblast growth factor 23
  • FGF23 is a member of the fibroblast growth factor (FGF) family which is responsible for phosphate metabolism.
  • FGF23 gene encodes a protein consisting of 251 amino acids. For instance, the naturally occurring human protein has an aminoacid sequence shown in Genbank Accession number NP 065689.
  • FGF23 polypeptide includes naturally occurring FGF23 polypeptide as well as variants, fragments and modified forms thereof.
  • FGF23 polypeptide thus refers to the FGF23 protein or a fragment thereof (e.g. the C-terminal fragment).
  • polypeptide means herein a polymer of amino acids having no specific length. Thus, peptides, oligopeptides and proteins are included in the definition of “polypeptide” and these terms are used interchangeably throughout the specification, as well as in the claims.
  • polypeptide does not exclude post-translational modifications that include but are not limited to phosphorylation, acetylation, glycosylation and the like.
  • blood sample refers to a blood sample (e.g. whole blood sample, serum sample, or plasma sample) obtained for the purpose of in vitro evaluation.
  • the term "patient” denotes a mammal, such as a rodent, a feline, a canine, and a primate.
  • a patient is a human.
  • the patient to be tested in the context of the invention shall suffer from an end stage liver disease and is registered on a liver transplant waiting list.
  • the invention in a first aspect, relates to a method for the predicting risk of mortality of a patient suffering from end stage liver disease (ESLD) comprising the steps of: i. measuring the concentration of FGF23 polypeptide in a blood sample obtained from said patient, and ii. comparing the concentration obtained at step (i) to a predetermined value, wherein a difference between said concentration and said predetermined value is indicative of a risk of mortality for said patient.
  • ESLD end stage liver disease
  • the risk of mortality of a patient shall be predicted.
  • the term "predicting the risk”, as used herein, refers to assessing the probability according to which the patient as referred to herein will die. More preferably, the risk/probability of mortality within a certain time window is predicted.
  • the predictive window preferably, is an interval between 2 and 800 days. Preferably, said predictive window is calculated from the time point at which the sample to be tested has been obtained.
  • the p-values are, preferably, 0.1, 0.05, 0.01, 0.005, or 0.0001.
  • the probability envisaged by the invention allows that the prediction of an increased risk will be correct for at least 60%, at least 70%>, at least 80%>), or at least 90%> of the patients of a given cohort or population.
  • the term preferably, relates to predicting whether or not there is an increased risk of mortality compared to the average risk of mortality in a population of patients rather than giving a precise probability for the said risk.
  • predicting the risk of mortality means that the patient to be analyzed by the method of the invention is allocated either into the group of patients being at risk of mortality, or into a group of patients being not at risk of mortality. Having a risk of mortality as referred to in accordance with the invention, preferably, means that the risk of mortality is increased (preferably, within the predictive window). Preferably, said risk is elevated as compared to the average risk in a cohort of patients suffering from ESLD.
  • the method for predicting the risk of mortality of the invention involves comparing the concentration of FGF23 polypeptide to a predetermined value.
  • the "predetermined value" according to the invention can be a single value such as a reference value derived from the concentration of FGF23 in blood samples from patients who are at particular stages of liver diseases, preferably patients with ESLD, or a control value derived from the concentration of FGF23 in blood samples from healthy patients.
  • the concentration of FGF23 has been assessed for 100 blood samples of 100 patients.
  • the 100 samples are ranked according to the concentration of FGF23.
  • Sample 1 has the highest level and sample 100 has the lowest level.
  • a first grouping provides two subsets: on one side sample Nr 1 and on the other side the 99 other samples.
  • the next grouping provides on one side samples 1 and 2 and on the other side the 98 remaining samples etc., until the last grouping: on one side samples 1 to 99 and on the other side sample Nr 100.
  • estimates of survival were calculated using the Kaplan Meier method (or any other survival analysis method) and tested with the log-rank test (or any other suitable test).
  • the predetermined reference value is then selected such as the discrimination based on the criterion of the minimum p value is the strongest.
  • concentration of FGF23 corresponding to the boundary between both subsets for which the p value is minimum is considered as the predetermined reference value. It should be noted that the predetermined reference value is not necessarily the median value of concentration of FGF23.
  • a single "cut-off value thus allows discrimination between a poor and a good prognosis with respect to the overall survival (OS) for a patient.
  • high statistical significance values e.g. low P values
  • a range of values is provided instead of using a definite predetermined reference value. Therefore, a minimal statistical significance value (minimal threshold of significance, e.g. maximal threshold P value) is arbitrarily set and a range of a plurality of arbitrary quantification values for which the statistical significance value calculated at step g) is higher (more significant, e.g.
  • This range of quantification values includes a "cut-off (for which the p value is the lowest) value as described above. For example, on a hypothetical scale of 1 to 10, if the ideal cut-off value (the value with the highest statistical significance) is 5, a suitable (exemplary) range may be from 4-6. Therefore, a patient may be assessed by comparing values obtained by measuring the concentration of FGF23, where values greater than 5 reveal a poor prognosis and values less than 5 reveal a good prognosis.
  • a patient may be assessed by comparing values obtained by measuring the concentration of FGF23 and comparing the values on a scale, where values above the range of 4-6 indicate a poor prognosis and values below the range of 4-6 indicate a good prognosis, with values falling within the range of 4-6 indicating an intermediate prognosis.
  • a concentration of FGF23 higher than the predetermined value is indicative of an increased risk of mortality (i.e. a major or extreme risk of mortality) for said patient.
  • the concentration of FGF23 polypeptide in the blood sample of a healthy patient is less than 120 RU/ml (control value).
  • the concentration of FGF23 polypeptide in the blood sample of a patient having a risk of mortality due to ESLD is more than 241 RU/ml.
  • the predetermined reference value may be 241 RU/ml as described in the section "EXAMPLE".
  • the patient is a patient suffering from ESLD.
  • the patient is affected by disease leading to ESLD selected in the group consisting of cirrhosis, viral hepatitis, genetic disorders, liver cancer (e.g. hepatocellular carcinoma (HCC), autoimmune disease, obesity and intoxication.
  • ESLD disease leading to ESLD selected in the group consisting of cirrhosis, viral hepatitis, genetic disorders, liver cancer (e.g. hepatocellular carcinoma (HCC), autoimmune disease, obesity and intoxication.
  • HCC hepatocellular carcinoma
  • the patient has already received a liver transplant.
  • the concentration of FGF23 polypeptide may be measured by any known method in the art.
  • Measuring in a blood sample the concentration of FGF23 polypeptide may be performed by any known method in the art.
  • the concentration may be measured by using standard immunodiagnostic techniques, including immunoassays such as competition, direct reaction, array chips, or sandwich type assays.
  • immunoassays include, but are not limited to, Western blots; agglutination tests; enzyme-labeled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; Immunoelectrophoresis; immunoprecipitation, gas chromatography, high performance liquid chromatography (HPLC), size exclusion chromatography, solid-phase affinity, etc.
  • the methods according to the invention comprise contacting the blood sample with a binding partner capable of selectively interacting with FGF23 polypeptide in said blood sample.
  • the binding partner may be generally an antibody that may be polyclonal or monoclonal, preferably monoclonal.
  • Polyclonal antibodies directed against FGF23 can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others.
  • a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others.
  • Various adjuvants known in the art can be used to enhance antibody production.
  • Monoclonal antibodies of the invention can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975); the human B-cell hybridoma technique (Cote et ah, 1983); and the EBV-hybridoma technique (Cole et ah, 1985). Alternatively, techniques described for the production of single chain antibodies (see e.g. U.S. Pat. No. 4,946,778) can be adapted to produce single chain antibodies.
  • Antibodies useful in practicing the present invention also include fragments including but not limited to F(ab') 2 fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab') 2 fragments.
  • Fab and/or scFv expression libraries can be constructed to allow rapid identification of fragments having the desired specificity.
  • phage display of antibodies may be used.
  • single-chain Fv (scFv) or Fab fragments are expressed on the surface of a suitable bacteriophage, e. g., M13. Briefly, spleen cells of a suitable host, e.
  • the binding partner may be an aptamer.
  • Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition.
  • Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity.
  • Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990.
  • the random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence.
  • Peptide aptamers consist of conformationally constrained antibody variable regions displayed by a platform protein, such as E. coli Thioredoxin A, that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996).
  • the aforementioned assays may involve the binding of the binding partner (ie. antibody or aptamer) to a solid support.
  • Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e. g., in membrane or microtiter well form); polyvinylchloride (e. g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like.
  • binding partners of the invention such as antibodies or aptamers, may be labelled with a detectable molecule or substance, such as a fluorescent molecule, a radioactive molecule or any others labels known in the art.
  • a detectable molecule or substance such as a fluorescent molecule, a radioactive molecule or any others labels known in the art.
  • Labels are known in the art that generally provide (either directly or indirectly) a signal.
  • the term "labeled”, with regard to the antibody or aptamer, is intended to encompass direct labeling of the antibody or aptamer by coupling (i.e., physically linking) a detectable substance, such as a radioactive agent or a fluorophore (e.g. fluorescein isothiocyanate (FITC), phycoerythrin (PE) or Indocyanine (Cy5)) to the antibody or aptamer, as well as indirect labeling of the antibody or aptamer by reactivity with a detectable substance.
  • FITC fluorescein isothiocyanate
  • PE phycoerythrin
  • Cy5 Indocyanine
  • the antibody or aptamer may also be labeled with a radioactive molecule by any method known in the art.
  • radioactive molecules include but are not limited radioactive atom for scintigraphic studies such as 1123, 1124, Inl 11, Rel86, Rel88.
  • an ELISA method can be used, wherein the wells of a microtiter plate are coated with a set of antibodies against FGF23. A blood sample containing or suspected of containing FGF23 is then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody-antigen complexes, the plate(s) can be washed to remove unbound moieties and a detectably labeled secondary binding molecule added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate washed and the presence of the secondary binding molecule detected using methods well known in the art.
  • Examples of said ELISA include, but are not limited to, the Immutopics C-terminal ELISA kits (Human FGF23 C-terminal ELISA kit, Immutopics International, San Clemente California USA and the Kainos intact FGF23 Elisa kit (Kainos Laboratories Japan) as described in the Example Section below.
  • Immutopics C-terminal ELISA kits Human FGF23 C-terminal ELISA kit, Immutopics International, San Clemente California USA and the Kainos intact FGF23 Elisa kit (Kainos Laboratories Japan) as described in the Example Section below.
  • measuring the concentration of FGF23 may also include separation of the proteins: centrifugation based on the protein's molecular weight; electrophoresis based on mass and charge; HPLC based on hydrophobicity; size exclusion chromatography based on size; and solid-phase affinity based on the protein's affinity for the particular solid-phase that is use.
  • FGF23 may be identified based on the known "separation profile" e. g., retention time, for that protein and measured using standard techniques.
  • the separated proteins may be detected and measured by, for example, a mass spectrometer.
  • methods of the invention comprise measuring the concentration of at least one further bio marker.
  • biomarker refers generally to a molecule, the expression of which in a blood sample from a patient can be detected by standard methods in the art (as well as those disclosed herein), and is predictive or denotes a condition of the patient from which it was obtained.
  • the other biomarker may be the serum sodium concentration.
  • methods of the invention comprise measuring at least one further physiological parameter.
  • physiological parameter refers generally to any parameter that may be monitored to determine one or more quantitative physiological levels and/or activities associated with the patient.
  • such physiological parameter may be selected from the group consisting of Model for End-Stage Liver Disease (MELD) and MELD-Na scores and glomerular filtration rate (GFR) value.
  • MELD Model for End-Stage Liver Disease
  • GFR glomerular filtration rate
  • kits for performing a method of the invention comprising means for measuring the concentration of FGF23 polypeptide in a blood sample obtained from a patient.
  • the kit may include an antibody, or a set of antibodies as above described. In a particular embodiment, the antibody or set of antibodies are labelled as above described.
  • the kit may also contain other suitably packaged reagents and materials needed for the particular detection protocol, including solid-phase matrices, if applicable, and standards.
  • the kit may also contain one or more means for the detection of a further biomarker and/or physiological parameter.
  • a further aspect of the invention relates to the use of FGF23 polypeptide a biomarker for predicting the risk of mortality of a patient affected with ESLD.
  • the invention also relates to the use of a kit of the invention for predicting the risk of mortality of a patient affected with ESLD.
  • the invention relates to the use of a kit comprising means for measuring the concentration of FGF23 polypeptide in a biological sample obtained from a patient affected with ESLD for performing a method for predicting the risk of mortality of said patient.
  • the invention further provides methods for determining which patients have the priority for liver transplantation among patients on a liver-transplant waiting list. Information gained by way of the methods described above can be used to determine which patient should undergo a liver transplantation
  • the invention relates to a method for determining priority for liver transplantation in a patient suffering from ESLD, comprising the steps of: i. performing the method for determining the risk of mortality of the invention, and ii. determining whether said patient has the priority for liver transplantation.
  • the patient is affected by disease leading to ESLD selected in the group consisting of cirrhosis, viral hepatitis, genetic disorders, liver cancer (e.g. hepatocellular carcinoma (HCC), autoimmune disease, obesity and intoxication.
  • ESLD disease leading to ESLD selected in the group consisting of cirrhosis, viral hepatitis, genetic disorders, liver cancer (e.g. hepatocellular carcinoma (HCC), autoimmune disease, obesity and intoxication.
  • HCC hepatocellular carcinoma
  • the patient has already received a liver transplant.
  • the invention also relates to the use of a kit comprising means for measuring the concentration of FGF23 polypeptide in a biological sample obtained from a patient suffering from ESLD for performing a method for determining priority for liver transplantation in said patient.
  • Another aspect of the invention relates to a method for predicting or determining the severity of the liver disease in a patient, comprising the steps of: i. measuring the concentration of FGF23 polypeptide in a blood sample obtained from said patient, and ii. comparing the concentration obtained at step (i) to a predetermined value, wherein a difference between said concentration and said predetermined value is indicative of a severe liver disease (or of the severity of liver dysfunction).
  • a concentration of FGF23 higher than the predetermined value is indicative of a severe liver disease (or of the severity of liver dysfunction).
  • the patient is a patient suffering from ESLD.
  • the patient is affected by disease leading to ESLD selected in the group consisting of cirrhosis, viral hepatitis, genetic disorders, liver cancer (e.g. hepatocellular carcinoma (HCC), autoimmune disease, obesity and intoxication.
  • ESLD disease leading to ESLD selected in the group consisting of cirrhosis, viral hepatitis, genetic disorders, liver cancer (e.g. hepatocellular carcinoma (HCC), autoimmune disease, obesity and intoxication.
  • HCC hepatocellular carcinoma
  • the patient has already received a liver transplant.
  • concentration of FGF23 polypeptide may be measured by any known method in the art as previously disclosed.
  • the invention also relates to the use of FGF23 polypeptide for predicting or determining the severity of the liver disease in a patient.
  • the invention further relates to the use of a kit of the invention for predicting or determining the severity of the liver disease in a patient.
  • the invention relates to the use of a kit comprising means for measuring the concentration of FGF23 polypeptide in a biological sample obtained from a patient for performing a method for predicting or determining the severity of the liver disease in a patient.
  • Another aspect of the invention relates to a method for screening an asymptomatic patient at risk of mortality due a liver disease, said method comprising measuring the concentration of FGF23 in a blood sample obtained from said patient.
  • the invention also provides methods and compositions (such as pharmaceutical compositions) for improving the survival time of a patient suffering from ESLD and having a risk of mortality.
  • the invention also relates to a FGF23 antagonist for use in the improvement of the survival time of a patient suffering from ESLD and having a risk of mortality as provided by a method for the prediction of mortality according to the invention.
  • the invention also relates to a FGF23 antagonist for use in the improvement of the survival time of a patient suffering from ESLD.
  • the patient has an increased risk of mortality (i.e. a major or extreme risk of mortality).
  • FGF23 antagonist refers to any FGF23 antagonist that is currently known in the art or that will be identified in the future, and includes any chemical entity that, upon administration to a patient, results in inhibition of a biological activity associated with activation of the FGFR (and more precisely FGFR type 1, 3 or 4) and/or Klotho by FGF23 in the patient, including any of the downstream biological effects otherwise resulting from the binding to FGFR and/or Klotho with FGF23.
  • Such an antagonist can act by binding directly to the intracellular domain of the receptor and inhibiting its kinase activity.
  • such an antagonist can act by occupying the ligand binding site or a portion thereof of the FGFR and/or Klotho, thereby making the receptor inaccessible to its natural ligand so that its normal biological activity is prevented or reduced.
  • such an antagonist can also act by binding directly to the FGF23, thereby preventing the binding of FGF23 to FGFR (i.e. FGFR- 1, FGFR-2 or FGFR-4) and/or Klotho.
  • the FGF23 antagonist is an inhibitor of the interaction between
  • FGF23 and FGFR or between FGF23 and Klotho.
  • blocking the interaction means preventing or reducing the direct or indirect association of one or more molecules, peptides, proteins, enzymes or receptors; or preventing or reducing the normal activity of one or more molecules, peptides, proteins, enzymes, or receptors.
  • the term “inhibitor of the interaction between FGF23 and FGFR” refers to a molecule which can prevent the interaction between FGFR23 and FGFR by competition or by fixing to one of the molecules.
  • the term “inhibitor of the interaction between FGF23 and Klotho” refers to a molecule which can prevent the interaction between FGFR23 and Klotho by competition or by fixing to one of the molecules.
  • the FGF23 antagonist may be a molecule which binds to FGF23, FGFR or Klotho selected from the group consisting of antibodies, aptamers, polypeptides and small organic molecules.
  • the FGF23 antagonist is an antibody (the term including antibody fragment or portion) that can block the interaction of FGFR with FGF23 or the interaction of Klotho with FGF23.
  • the FGF23 antagonist may consist in an antibody directed against the FGF23, FGFR or Klotho, in such a way that said antibody impairs the binding of a FGF23 to FGFR or Klotho ("neutralizing antibody").
  • the antibody is a monoclonal antibody.
  • the antibody is a polyclonal antibody.
  • the antibody is a humanized antibody.
  • the antibody is a chimeric antibody.
  • the portion of the antibody comprises a light chain of the antibody.
  • the portion of the antibody comprises a heavy chain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fab portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a F(ab')2 portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fc portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fv portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a variable domain of the antibody.
  • the portion of the antibody comprises one or more CDR domains of the antibody.
  • antibody includes both naturally occurring and non-naturally occurring antibodies. Specifically, “antibody” includes polyclonal and monoclonal antibodies, and monovalent and divalent fragments thereof. Furthermore, “antibody” includes chimeric antibodies, wholly synthetic antibodies, single chain antibodies, and fragments thereof. The antibody may be a human or nonhuman antibody. A nonhuman antibody may be humanized by recombinant methods to reduce its immunogenicity in man.
  • an anti-FGF23 antibody is a monoclonal antibody produced by hybridoma CIO (Accession No. FERM BP- 10772) as described in the patent application US2009148461, which is hereby incorporated by reference in its entirety.
  • anti-FGF23 antibodies are described in the patent US7981419 which is hereby incorporated by reference in its entirety.
  • the anti-FGF23 antibody is KRN23 (Kyowa Hakko Kirin Pharma, Inc.)
  • Antibodies are prepared according to conventional methodology. Monoclonal antibodies may be generated using the method of Kohler and Milstein (Nature, 256:495, 1975). To prepare monoclonal antibodies useful in the invention, a mouse or other appropriate host animal is immunized at suitable intervals (e.g., twice-weekly, weekly, twice-monthly or monthly) with antigenic forms of FG23. The animal may be administered a final "boost" of antigen within one week of sacrifice. It is often desirable to use an immunologic adjuvant during immunization.
  • Suitable immunologic adjuvants include Freund's complete adjuvant, Freund's incomplete adjuvant, alum, Ribi adjuvant, Hunter's Titermax, saponin adjuvants such as QS21 or Quil A, or CpG-containing immunostimulatory oligonucleotides.
  • Other suitable adjuvants are well-known in the field.
  • the animals may be immunized by subcutaneous, intraperitoneal, intramuscular, intravenous, intranasal or other routes. A given animal may be immunized with multiple forms of the antigen by multiple routes.
  • the recombinant FG23 may be provided by expression with recombinant cell lines.
  • Recombinant form of FG23 may be provided using any previously described method.
  • lymphocytes are isolated from the spleen, lymph node or other organ of the animal and fused with a suitable myeloma cell line using an agent such as polyethylene glycol to form a hydridoma.
  • cells are placed in media permissive for growth of hybridomas but not the fusion partners using standard methods, as described (Coding, Monoclonal Antibodies: Principles and Practice: Production and Application of Monoclonal Antibodies in Cell Biology, Biochemistry and Immunology, 3rd edition, Academic Press, New York, 1996).
  • cell supernatants are analyzed for the presence of antibodies of the desired specificity, i.e., that selectively bind the antigen.
  • Suitable analytical techniques include ELISA, flow cytometry, immunoprecipitation, and western blotting. Other screening techniques are well-known in the field. Preferred techniques are those that confirm binding of antibodies to conformationally intact, natively folded antigen, such as non-denaturing ELISA, flow cytometry, and immunoprecipitation.
  • the Fc' and Fc regions are effectors of the complement cascade but are not involved in antigen binding.
  • an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region designated an Fab fragment
  • Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd.
  • the Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation.
  • CDRs complementarity determining regions
  • FRs framework regions
  • CDR1 through CDRS complementarity determining regions
  • compositions and methods that include humanized forms of antibodies.
  • humanized describes antibodies wherein some, most or all of the amino acids outside the CDR regions are replaced with corresponding amino acids derived from human immunoglobulin molecules.
  • Methods of humanization include, but are not limited to, those described in U.S. Pat. Nos. 4,816,567,5,225,539,5,585,089, 5,693,761, 5,693,762 and 5,859,205, which are hereby incorporated by reference.
  • the above U.S. Pat. Nos. 5,585,089 and 5,693,761, and WO 90/07861 also propose four possible criteria which may used in designing the humanized antibodies.
  • the first proposal was that for an acceptor, use a framework from a particular human immunoglobulin that is unusually homologous to the donor immunoglobulin to be humanized, or use a consensus framework from many human antibodies.
  • the second proposal was that if an amino acid in the framework of the human immunoglobulin is unusual and the donor amino acid at that position is typical for human sequences, then the donor amino acid rather than the acceptor may be selected.
  • the third proposal was that in the positions immediately adjacent to the 3 CDRs in the humanized immunoglobulin chain, the donor amino acid rather than the acceptor amino acid may be selected.
  • the fourth proposal was to use the donor amino acid reside at the framework positions at which the amino acid is predicted to have a side chain atom within 3 A of the CDRs in a three dimensional model of the antibody and is predicted to be capable of interacting with the CDRs.
  • the above methods are merely illustrative of some of the methods that one skilled in the art could employ to make humanized antibodies.
  • One of ordinary skill in the art will be familiar with other methods for antibody humanization.
  • humanized forms of the antibodies some, most or all of the amino acids outside the CDR regions have been replaced with amino acids from human immunoglobulin molecules but where some, most or all amino acids within one or more CDR regions are unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they would not abrogate the ability of the antibody to bind a given antigen.
  • Suitable human immunoglobulin molecules would include IgGl, IgG2, IgG3, IgG4, IgA and IgM molecules.
  • a "humanized" antibody retains a similar antigenic specificity as the original antibody.
  • the affinity and/or specificity of binding of the antibody may be increased using methods of "directed evolution", as described by Wu et al, /. Mol. Biol. 294: 151, 1999, the contents of which are incorporated herein by reference.
  • Fully human monoclonal antibodies also can be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. See, e.g., U.S. Pat. Nos. 5,591,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584, and references cited therein, the contents of which are incorporated herein by reference. These animals have been genetically modified such that there is a functional deletion in the production of endogenous (e.g., murine) antibodies. The animals are further modified to contain all or a portion of the human germ-line immunoglobulin gene locus such that immunization of these animals will result in the production of fully human antibodies to the antigen of interest.
  • monoclonal antibodies can be prepared according to standard hybridoma technology. These monoclonal antibodies will have human immunoglobulin amino acid sequences and therefore will not provoke human anti-mouse antibody (KAMA) responses when administered to humans.
  • KAMA human anti-mouse antibody
  • the present invention also provides for F(ab') 2 Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric F(ab')2 fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; and chimeric Fd fragment antibodies in which the FR and/or CDR1 and/or CDR2 regions have been replaced by homologous human or non-human sequences.
  • the present invention also includes so-called single chain antibodies.
  • the various antibody molecules and fragments may derive from any of the commonly known immunoglobulin classes, including but not limited to IgA, secretory IgA, IgE, IgG and IgM.
  • IgG subclasses are also well known to those in the art and include but are not limited to human IgGl, IgG2, IgG3 and IgG4.
  • the antibody according to the invention is a single domain antibody.
  • the term "single domain antibody” (sdAb) or “VHH” refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called “nanobody®”. According to the invention, sdAb can particularly be llama sdAb.
  • the FGF23 antagonist is an antibody directed against FGFR or Klotho.
  • monoclonal antibodies which are capable of blocking the function of FGFRs e.g., FGFR4
  • FGFR4 FGFR4
  • neutralizing antibodies of FGF23 are selected.
  • the antibody which binds to FGF23 is a neutralizing anti-FGF23 antibody (i.e. an antibody which blocks the activity of FGF23).
  • the FGF23 antagonist is an aptamer directed against FGF23.
  • Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition.
  • Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity.
  • Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990.
  • the random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence.
  • Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al, 1996). Then, neutralizing aptamers of FGF23 are selected as above described.
  • the FGF23 antagonist is an aptamer directed against FGFR or Klotho.
  • the FGF23 antagonist is a Klotho polypeptide.
  • Klotho polypeptide refers to a polypeptide that specifically bind to FGF23 can be used as a FGF23 antagonist that bind to and sequester the FGF23 protein, thereby preventing it from signaling.
  • the Klotho polypeptide is soluble.
  • a soluble Klotho receptor polypeptide exerts an inhibitory effect on the biological activity of the FGF23 protein by binding to the protein, thereby preventing it from binding to Klotho present on the surface of target cells. It is undesirable for a Klotho polypeptide not to become associated with the cell membrane.
  • the soluble Klotho polypeptide lacks any amino acid sequences corresponding to the transmembrane and intracellular domains from the Klotho from which it is derived.
  • said polypeptide is a soluble Klotho (sKlotho) polypeptide or a functional equivalent thereof.
  • soluble Klotho or "sKlotho”, as used herein, refer to a polypeptide comprising or consisting of the extracellular region of the Klotho or a fragment thereof.
  • sKlotho may include all the extracellular domain of human Klotho.
  • a “functional equivalent of sKlotho” is a molecule which is capable of binding to FGF23, preferably which is capable of specifically binding to FGF23.
  • the term “functional equivalent” includes fragments and variants of sKlotho as above described.
  • binding specifically means that the biologically active fragment has high affinity for FGF23 but not for control proteins. Specific binding may be measured by a number of techniques such as ELISA, flow cytometry, western blotting, or immunoprecipitation.
  • the functionally equivalent specifically binds to FGF2 at nanomolar or picomolar levels.
  • the FGF23 antagonist is a FGFR polypeptide.
  • the FGF23 antagonist is a small organic molecule.
  • small organic molecule refers to a molecule of size comparable to those organic molecules generally sued in pharmaceuticals. The term excludes biological macro molecules (e.g.; proteins, nucleic acids, etc.); preferred small organic molecules range in size up to 2000 Da, and most preferably up to about 1000 Da.
  • the FGF23 antagonist useful in invention is a tyrosine kinase inhibitor (TKI) that inhibits FGFR activity.
  • TKI tyrosine kinase inhibitor
  • A' KI that inhibits FGFR activity means an inhibitor of receptor tyrosine kinase activity that selectively or non-selectively reduces the tyrosine kinase activity of a FGFR receptor.
  • Such an inhibitor generally reduces FGFR tyrosine kinase activity without significantly effecting the expression of FGFR and without effecting other FGFR activities such as ligand-binding capacity.
  • TKI that inhibits FGFR activity
  • PD 173074 and other pyrido[2,3-d] pyrimidine compounds described in the patent US5733913, which is hereby incorporated by reference in its entirety.
  • Other small molecule inhibitors of FGFR which may be used include SU6668 and SU5402.
  • the FGF23 antagonist is an inhibitor of FGF23 gene expression.
  • An “inhibitor of expression” refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene. Therefore, an “inhibitor of FGF23 gene expression” denotes a natural or synthetic compound that has a biological effect to inhibit the expression of FGF23 gene.
  • said inhibitor of FGF23 gene expression is a siR A, an antisense oligonucleotide or a ribozyme.
  • Inhibitors of FGF23 gene expression for use in the present invention may be based on antisense oligonucleotide constructs.
  • Anti-sense oligonucleotides including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of FGF23 mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of FGF23, and thus activity, in a cell.
  • antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding FGF23 can be synthesized, e.g., by conventional phosphodiester techniques and administered by e.g., intravenous injection or infusion.
  • RNA interference small double stranded RNA
  • Ribozymes can also function as inhibitors of FGF23 gene expression for use in the present invention.
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleo lytic cleavage.
  • Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of FGF23 mRNA sequences are thereby useful within the scope of the present invention.
  • ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.
  • Antisense oligonucleotides, siRNAs and ribozymes useful as inhibitors of FGF23 gene expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life.
  • Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-0-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.
  • Antisense oligonucleotides, siRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector.
  • a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA or ribozyme nucleic acid to the cells and preferably cells expressing FGF23.
  • the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector.
  • the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siR A or ribozyme nucleic acid sequences.
  • Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and R A virus such as a retrovirus.
  • retrovirus such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus
  • retrovirus such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus
  • adenovirus adeno
  • Non-cytopathic viral vectors are based on non-cytopathic eukaryotic viruses in which non- essential genes have been replaced with the gene of interest.
  • Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA.
  • Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle).
  • retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo.
  • adeno-viruses and adeno-associated viruses are double-stranded DNA viruses that have already been approved for human use in gene therapy.
  • the adeno-associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions.
  • the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection.
  • adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event.
  • the adeno- associated virus can also function in an extrachromosomal fashion.
  • Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g., SA BROOK et al, "Molecular Cloning: A Laboratory Manual," Second Edition, Cold Spring Harbor Laboratory Press, 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigen-encoding genes to cells in vivo.
  • Plasmids may be delivered by a variety of parenteral, mucosal and topical routes.
  • the DNA plasmid can be injected by intramuscular, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally. It may also be administered into the epidermis or a mucosal surface using a gene-gun.
  • the plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and microencapsulation.
  • the FGF23 antagonist is an inhibitor of FGFR or Klotho gene expression.
  • said antagonist is administered in a therapeutically effective amount.
  • a “therapeutically effective amount” is meant a sufficient amount of the FGF23 antagonist improve survival time of a patient suffering from ESLD and having a risk of mortality at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidential with the specific polypeptide employed; and like factors well known in the medical arts.
  • the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day.
  • the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated.
  • a medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient.
  • An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.
  • compositions of the invention are provided.
  • the FGF23 antagonist as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a FGF23 antagonist according to the invention and a pharmaceutically acceptable carrier for use in the improvement of the survival time of a patient suffering from ESLD and having a risk of mortality as provided by a method for the prediction of mortality according to the invention.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a FGF23 antagonist according to the invention and a pharmaceutically acceptable carrier for use in the improvement of the survival time of a patient suffering from ESLD.
  • “Pharmaceutically” or “pharmaceutically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate.
  • a pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • the antagonist contained in the pharmaceutical composition can be administered in several dosages or as a single dose until a desired response has been achieved.
  • the treatment is typically monitored and repeated dosages can be administered as necessary.
  • Compounds of the invention may be administered according to dosage regimens established whenever inactivation of FGF23 is required.
  • the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day.
  • the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated.
  • a medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient.
  • An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 10 mg/kg of body weight per day.
  • the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability, and length of action of that compound, the age, the body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy.
  • the active principle in the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings.
  • Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.
  • the appropriate unit forms of administration include forms for oral administration, such as tablets, gelatine capsules, powders, granules and solutions or suspensions to be taken orally, forms for sublingual and buccal administration, aerosols, implants, forms for subcutaneous, intramuscular, intravenous, intranasal or intraocular administration and forms for rectal administration.
  • the active principle is generally formulated as dosage units containing from 0.5 to 1000 mg, preferably from 1 to 500 mg, more preferably from 2 to 200 mg of said active principle per dosage unit for daily administrations.
  • a wetting agent such as sodium laurylsulfate can be added to the active principle optionally micronized, which is then mixed with a pharmaceutical vehicle such as silica, gelatine, starch, lactose, magnesium stearate, talc, gum arabic or the like.
  • a pharmaceutical vehicle such as silica, gelatine, starch, lactose, magnesium stearate, talc, gum arabic or the like.
  • the tablets can be coated with sucrose, with various polymers or other appropriate substances or else they can be treated so as to have a prolonged or delayed activity and so as to release a predetermined amount of active principle continuously.
  • a preparation in the form of gelatin capsules is obtained by mixing the active principle with a diluent such as a glycol or a glycerol ester and pouring the mixture obtained into soft or hard gelatine capsules.
  • a diluent such as a glycol or a glycerol ester
  • a preparation in the form of a syrup or elixir can contain the active principle together with a sweetener, which is preferably calorie-free, methyl-paraben and propylparaben as an antiseptic, a flavoring and an appropriate color.
  • a sweetener which is preferably calorie-free, methyl-paraben and propylparaben as an antiseptic, a flavoring and an appropriate color.
  • the water-dispersible powders or granules can contain the active principle mixed with dispersants or wetting agents, or suspending agents such as polyvinyl-pyrrolidone, and also with sweeteners or taste correctors.
  • Rectal administration is effected using suppositories prepared with binders which melt at the rectal temperature, for example cacao butter or polyethylene glycols.
  • Parenteral, intranasal or intraocular administration is effected using aqueous suspensions, isotonic saline solutions or sterile and injectable solutions which contain pharmacologically compatible dispersants and/or wetting agents, for example propylene glycol, butylene glycol, or polyethylene glycol.
  • pharmacologically compatible dispersants and/or wetting agents for example propylene glycol, butylene glycol, or polyethylene glycol.
  • a cosolvent for example an alcohol such as ethanol or a glycol such as polyethylene glycol or propylene glycol, and a hydrophilic surfactant such as Tween (R) 80, can be used to prepare an aqueous solution injectable by intravenous route.
  • the active principle can be solubilized by a triglyceride or a glycerol ester to prepare an oily solution injectable by intramuscular route.
  • Transdermal administration is effected using multilaminated patches or reservoirs into which the active principle is in the form of an alcoholic solution.
  • Administration by inhalation is effected using an aerosol containing for example sorbitan trioleate or oleic acid together with trichlorofluoromethane, dichlorotetrafluoroethane or any other biologically compatible propellant gas.
  • the active principle can also be formulated as microcapsules or microspheres, optionally with one or more carriers or additives.
  • implants can be used. These can be prepared in the form of an oily suspension or in the form of a suspension of microspheres in an isotonic medium.
  • the active principle can also be presented in the form of a complex with a cyclodextrin, for example .alpha.-, .beta.- or .gamma.-cyclodextrin, 2-hydroxypropyl-.beta.- cyclodextrin or methyl- .beta, -cyclodextrin.
  • a cyclodextrin for example .alpha.-, .beta.- or .gamma.-cyclodextrin, 2-hydroxypropyl-.beta.- cyclodextrin or methyl- .beta, -cyclodextrin.
  • FIGURES are a diagrammatic representation of FIGURES.
  • FIG. 1 FGF23 concentration according to glomerular filtration rate (GFR) in patients with end-stage liver disease (ESLD). Dashed lines indicate upper and normal values of FGF23 and GFR respectively. FGF23 concentration was above normal values in most of subjects with ESLD and normal renal function (GFR>70ml/min).
  • FIG. 2 Comparison of FGF23 concentration between patients with end-stage liver disease (ESLD) or normal liver function (control).
  • GFR Glomerular filtration rate
  • B FGF23 concentration was significantly higher in patients with ESLD than in control subjects (p ⁇ 0.0001). Results are mean ⁇ SD.
  • Figure 3 Patient survival according to plasma FGF23 concentration at the time of registration on the transplant waiting list.
  • A Kaplan-Meier survival-plot for patients on the liver-transplant waiting list.
  • B Patient survival in relation to plasma FGF23 concentration measured at the time of the registration on the transplant waiting list. Dashed line represents FGF23 concentration > 241 RU /ml and plain line FGF23 concentration ⁇ 241 RU/ml.
  • FIG 4 FGF23 plasma concentration and FGF23 mRNA level quantification in control and DEN- treated mice.
  • GFR glomerular filtration rate
  • FGF23 plasma concentration was measured using Immutopics c-terminal Elisa kits (Human FGF23 c- terminal Elisa kit, Immutopics International, San Clemente California USA).
  • the inventors also used Kainos intact FGF23 Elisa kit (Kainos Laboratories Japan) to measure plasma intact FGF23 in a subgroup of patients. As reported by other groups, they found a good correlation between the FGF23 values obtained with these two methods ( Figure 6). Consequently only the c-terminal kit (Immutopics Inc San Clemente CA), which measures both intact FGF23 and its carboxyl terminal by-product, was used to measure FGF23 concentration in all patients.
  • For FGF23 plasma determination 5 ml of blood were drawn on EDTA and immediately centrifuged at 4°C. The supernatant was stored at -80°C and used for measurement within two weeks.
  • Plasma FGF23 concentration was also determined in patients without acute or chronic liver disease ( ⁇ -glutamyl transpeptidase, aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase, INR within the normal ranges) who were referred to the Department Explorations Fonctionnelles at Necker-Enfants Malades hospital for the control of renal function from January 2005 to October 2008. These patients had either renal lithiasis, or renal insufficiency or were eligible in the absence of altered renal function for a treatment by cyclosporine for psoriasis.
  • Plasma FGF23 concentration and glomerular filtration rate were measured with the same procedures in all patients. Glomerular filtration rate was measured by two methods of reference: inulin and iohexol clearance. The patients were injected with inulin (Inutest 25% Serb Laboratoires, France) or iohexol (Omnipaque 300 GE Healthcare). Urine and blood samples were collected every hour for 5 hours for the measurements of inulin and iohexol. Inulin was used when an allergy to iohexol was suspected on the base of the patient's medical record.
  • the MELD score was calculated with the use of the standard formula [38].
  • MELD score ranges from 6 to 40 with higher values indicating more severe disease.
  • Plasma intact FGF23 concentrations were assessed using a commercial ELISA according to the manufacturer's protocol (Kainos Laboratories Inc.) Statistical analysis: As the variable FGF23 concentration was not normally distributed, we used its log transformation in the whole analysis. Quantitative variables were described using mean ⁇ SD or median (range).
  • Survival time was defined as the time from the date of FGF23 measurement to death on waiting list, transplantation or last follow-up.
  • FGF23 plasma concentration in the patients on the liver-transplant waiting list The inventors measured plasma FGF23 concentration in 200 patients with end stage liver disease at the time when they were considered as eligible for liver transplantation. The main characteristics of these patients are presented in Table 1. At registration on the waiting list the median MELD score was 13.5 (range 6 to 40) and the median serum sodium concentration was 137 mmol/L (range 122 to 146). Twenty six percent of the patients (51 patients) had hyponatremia (serum sodium concentration ⁇ 135 mmol/L). Forty two percent (84 patients) had hepatocellular carcinoma. The median FGF23 concentration was 241 RU/ml (range 5 to 17620).
  • Plasma FGF23 concentration was above normal value (120 RU/ml) in 63% of patients (126 patients). Since on physiological condition FGF23 production is stimulated by serum phosphate or calcitriol concentration we measured these two parameters at the same time that FGF23 concentration.
  • the median phosphate serum concentration was 0.95 mmol/L (range 0.53 to 1.86) at the time of FGF23 concentration measurement and hyperphosphatemia (serum phosphate concentration above 1.40 mmol/L) was present in only 3 patients.
  • Median plasma calcitriol concentration was 23 pg/ml (range 5 to 117) (Table 1) and was below the upper normal value (50 pg/ml) in 185 (92.5%>) of the patients.
  • Plasma FGF23 concentration was not correlated with phosphate or ionized calcium serum concentration or fractional excretion of phosphate in urine (Table 2) but was inversely correlated with plasma calcitriol concentration.
  • FGF23 concentration correlated with MELD score and sodium serum concentration (Table 2) suggesting that it may be associated with the severity of the liver disease.
  • FGF23 plasma concentration and mortality Since the increase in FGF23 plasma concentration was significantly associated with two known prognostic markers of survival (MELD score and hyponatremia) in patients with liver diseases, we examined the association between FGF23 plasma levels and the risk of death in the patients on the transplant waiting list.
  • liver FGF23 mRNA expression significantly increased between month 3 and month 9 ( Figure 4B).
  • Table 1 Characteristics of the patients on the liver-transplant waiting list:
  • Plasma calcitriol concentration pg/ml (median, min-max)
  • Table 3 Univariate analysis of prognostic factors on the waiting list.
  • GFR glomerular filtration rate
  • Table 4 Multivariate analysis of prognostic factors on the waiting list.
  • GFR glomerular filtration rate
  • the inventors report for the first time that plasma FGF23 concentration is increased in patients with end stage liver disease and they show that FGF23 plasma levels predict the risk of death in patients on the liver-transplant waiting list. Multivariate analysis indicates that FGF23 concentration was the best predictor of the risk of mortality. Although FGF23 concentration is correlated to GFR, this is a predictor of mortality independent of renal function in patients on the liver-transplant waiting list.
  • the sample on the liver-transplant waiting list did not differ by its characteristic from those reported in other studies, regarding the distributions of age, MELD and MELD-Na score, serum sodium concentration, sex ratio, the causes of cirrhosis or the number of deaths during the 44 months of the study [44,45,46,47,48]. As reported in other studies they observed that the MELD and MELD-Na scores and serum sodium concentration were associated with an increased risk of death in our patients.
  • FGF23 Only cells that co-express a FGFR and Klotho are sensitive to FGF23 signaling. However when FGF23 plasma levels increase above physiological values FGF23 can exhibit off-target effects. It can stimulate FGFR in the absence of Klotho and trigger new signaling pathway in particular on cardiomyocytes [24]]. This mechanism participates to the left ventricular hypertrophy and the increased risk of mortality associated with high FGF23 levels. FGF23 may similarly increase the risk of death in patients with end stage liver disease. However it is likely that FGF23 "off-target" effects are not restricted to the heart. FGF23 may also increase the sensitivity to infection.
  • FGF23 inhibits calcitriol synthesis and enhances the expression of CYP24A1, the enzyme that degrades calcitriol and 250H vitamin D.
  • Calcitriol induces innate antimicrobial response, suppresses pro -inflammatory cytokine response via endocrine, paracrine and autocrine activity.
  • Low calcitriol and 25 OH vitamin D concentrations have been associated with an increase risk of infection and an increased risk of death [53,54]. The increase in FGF23 concentration was not triggered by the physiological stimuli of
  • FGF23 production by bone cells Indeed plasma calcitriol concentrations were below the upper normal range in most of the patients elevated FGF23. Similarly a decrease in renal function cannot account for the increase in FGF23 concentration since first most subjects with ESLD and elevated FGF23 concentration had normal renal function and second when matched for GFR with control subjects, ESLD patients had higher FGF23 concentrations.
  • the main source of FGF23 is the osteocyte [3].
  • low levels of FGF23 mRNA have been detected in normal liver [3,12,55,56]. We found that FGF23 mRNA was present in the liver of mouse fetuses but not in adults.
  • Pathol 163 1653-1662.
  • Circulating FGF-23 is regulated by lalpha,25-dihydroxyvitamin D3 and phosphorus in vivo. J Biol Chem 280: 2543-2549.
  • FGF-23 is a potent regulator of vitamin D metabolism and phosphate homeostasis. J Bone Miner Res 19: 429-435.
  • Dihydroxyvitamin D3 upregulates FGF23 gene expression in bone: the final link in a renal-gastrointestinal- skeletal axis that controls phosphate transport. Am J Physiol
  • FGF23 is a strong predictor of survival in systolic heart failure. Peptides 37: 258-262.
  • Fibroblast growth factor 23 (FGF23) predicts progression of chronic kidney disease: the Mild to
  • MLD Liver Disease
  • MELD model including serum sodium and age improves the prediction of early mortality in patients with cirrhosis.
  • Growth Factor 23 is a Risk Factor for Kidney Transplant Loss and Mortality. Journal of the American Society of Nephrology 22: 956-966.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Microbiology (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Endocrinology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne un procédé destiné à prédire le risque de mortalité d'un patient souffrant d'une maladie du foie de stade final (ESLD), ce procédé comprenant une étape de mesure de la concentration de FGF23 dans un échantillon de sang prélevé chez ce patient.
EP14725123.5A 2013-05-16 2014-05-16 Fgf23 utilisé en tant que biomarqueur en vue de prédire le risque de mortalité dû à une maladie du foie de stade final Withdrawn EP2997383A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP14725123.5A EP2997383A1 (fr) 2013-05-16 2014-05-16 Fgf23 utilisé en tant que biomarqueur en vue de prédire le risque de mortalité dû à une maladie du foie de stade final

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
EP13305627 2013-05-16
PCT/EP2014/060052 WO2014184334A1 (fr) 2013-05-16 2014-05-16 Fgf23 utilisé en tant que biomarqueur en vue de prédire le risque de mortalité dû à une maladie du foie de stade final
EP14725123.5A EP2997383A1 (fr) 2013-05-16 2014-05-16 Fgf23 utilisé en tant que biomarqueur en vue de prédire le risque de mortalité dû à une maladie du foie de stade final

Publications (1)

Publication Number Publication Date
EP2997383A1 true EP2997383A1 (fr) 2016-03-23

Family

ID=48470875

Family Applications (1)

Application Number Title Priority Date Filing Date
EP14725123.5A Withdrawn EP2997383A1 (fr) 2013-05-16 2014-05-16 Fgf23 utilisé en tant que biomarqueur en vue de prédire le risque de mortalité dû à une maladie du foie de stade final

Country Status (2)

Country Link
EP (1) EP2997383A1 (fr)
WO (1) WO2014184334A1 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3432796A4 (fr) * 2016-03-23 2019-09-18 The Methodist Hospital System Estimation de clonalité de tcr avant transplantation pour prédire la survie après transplantation hépatique
CN114317756B (zh) * 2022-01-07 2024-05-07 深圳市龙华区人民医院 标志物的应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008089936A1 (fr) * 2007-01-22 2008-07-31 Medizinische Universität Innsbruck Nouveaux marqueurs pour la néphropathie chronique

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
None *
See also references of WO2014184334A1 *

Also Published As

Publication number Publication date
WO2014184334A1 (fr) 2014-11-20

Similar Documents

Publication Publication Date Title
JP6803230B2 (ja) アルツハイマー病に関するバイオマーカー及び方法
US20160313349A1 (en) Method of diagnosis and treatment
US20140200184A1 (en) Chitinase-3-Like Protein 1 as a Biomarker of Recovery from Kidney Injury
US12140594B2 (en) Early and non invasive method for assessing a subject's risk of having pancreatic ductal adenocarcinoma and methods of treatment of such disease
EP1902145A2 (fr) Méthodes de diagnostic et de traitement d'une réponse inflammatoire
CN107667294A (zh) 用于预测慢性肾病发生风险的方法
WO2008079877A2 (fr) Compositions et procédés destinés à diagnostiquer et à traiter des troubles associés au fer
US20080318241A1 (en) Methods and Systems for Detecting Antiangiogenesis
JP7271442B2 (ja) 腎機能を診断またはモニターする方法、または腎機能障害を診断することを補助する方法
WO2014184334A1 (fr) Fgf23 utilisé en tant que biomarqueur en vue de prédire le risque de mortalité dû à une maladie du foie de stade final
US20200088732A1 (en) Methods for the diagnosis and treatment of pancreatic ductal adenocarcinoma
EP3283889A2 (fr) Procédé et dosage pour le diagnostic rapide de la glomérulonéphrite évolutive chez un sujet
US20140248284A1 (en) Methods for the detection and the treatment of cardiac remodeling
US20230305023A1 (en) Methods of treatment and diagnostic of pathological conditions associated with intense stress
US8748493B2 (en) Inhibitors of cathepsin S for prevention or treatment of obesity-associated disorders
Qutaina et al. An angiopoietin-2 vaccine improves arteriovenous malformation pathology in hereditary hemorrhagic telangiectasia mice
JP2022037510A (ja) 抗骨髄腫剤および抗骨髄腫剤のスクリーニング方法
US20250258183A1 (en) Enzyme-linked immunosorbent assay (elisa) for anti-bsep autoantibody
WO2020144366A1 (fr) Procédé in vitro pour déterminer la probabilité d'apparition d'un rejet microvasculaire aigu contre une allogreffe rénale chez un individu
WO2019121872A1 (fr) Procédés de diagnostic et de traitement du cancer du foie
AU2012271293A1 (en) Biomarkers for epithelial cancer diagnosis and treatment
US20150377888A1 (en) Methods for Predicting and Preventing Metastasis in Triple Negative Breast Cancers
WO2016186173A1 (fr) Procédé permettant de diagnostiquer une insuffisance hépatique fulminante, et agent de prévention ou agent de traitement
WO2019234099A1 (fr) Procédés de diagnostic, de prédiction du résultat et de traitement d'un patient souffrant d'insuffisance cardiaque à fraction d'éjection préservée
JP2015520373A (ja) 巣状分節性糸球体硬化症を診断及び処置するための方法

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20151216

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

AX Request for extension of the european patent

Extension state: BA ME

DAX Request for extension of the european patent (deleted)
17Q First examination report despatched

Effective date: 20170207

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20170818