WO2010132459A2 - Biomarqueurs pour l'évaluation d'une dégénérescence maculaire liée à l'âge - Google Patents

Biomarqueurs pour l'évaluation d'une dégénérescence maculaire liée à l'âge Download PDF

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WO2010132459A2
WO2010132459A2 PCT/US2010/034398 US2010034398W WO2010132459A2 WO 2010132459 A2 WO2010132459 A2 WO 2010132459A2 US 2010034398 W US2010034398 W US 2010034398W WO 2010132459 A2 WO2010132459 A2 WO 2010132459A2
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level
measured
cep
cml
pentosidine
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PCT/US2010/034398
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WO2010132459A3 (fr
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John W. Crabb
Ram H. Nagaraj
Jiayin Gu
Jiaqian Ni
Xianglin Yuan
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Cleveland Clinic Foundation
Case Western Reserve University
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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
    • G01N33/6893—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
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/16—Ophthalmology
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/16—Ophthalmology
    • G01N2800/164—Retinal disorders, e.g. retinopathy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease

Definitions

  • Age-related macular degeneration is a progressive, multifactorial disease and the leading cause of severe vision loss in the elderly in industrialized countries (1).
  • Deposition of debris (drusen) in the macular region of Bruch's membrane, the extracellular matrix separating the choriocapillaris from the retinal pigment epithelium (RPE), is an early, hallmark risk factor.
  • the disease can progress to advanced dry AMD (geographic atrophy), which is characterized by regional degeneration of photoreceptor and retinal pigment epithelial (RPE) cells, or to advanced wet AMD (choroidal neovascularization, or CNV), which is characterized by abnormal blood vessels growing from the choriocapillaris through Bruch's membrane beneath the retina.
  • CNV choroidal neovascularization
  • Oxidative stress appears to be involved as smoking significantly increases the risk of AMD (2), antioxidant vitamins can selectively slow AMD progression (3), and a host of oxidative protein and DNA modifications have been detected at elevated levels in AMD Bruch's membrane, drusen, retina, RPE and plasma (4-11).
  • Oxidative protein modifications like carboxyethylpyrrole (CEP) and ⁇ -carboxymethyllysine (CML), both elevated in AMD Bruch's membrane, stimulate neovascularization in vivo (12, 13), suggesting possible roles in CNV.
  • CEP carboxyethylpyrrole
  • CML ⁇ -carboxymethyllysine
  • AGEs advanced glycation endproducts
  • AMD pathology 4, 5, 7, 15, 16
  • AGEs are a heterogeneous group of mostly oxidative modifications resulting from the nonenzymatic Maillard glycosylation reaction that appear to contribute to age- related diseases and diabetic complications (17, 18).
  • CML was the first AGE to be associated with AMD Bruch's membrane and drusen (4).
  • Other AGEs have since been detected in AMD ocular tissues by multiple investigators (5, 7, 15), and in Bruch's membrane, drusen, RPE, and choridal extracellular matrix from healthy eyes (6, 19).
  • CML and pentosidine a fluorescent crosslinking AGE
  • Receptors for AGEs RAGE and AGE-Rl
  • RAGE and AGE-Rl also appear elevated on RPE and photoreceptor cells in early and advanced dry AMD (7), especially in RPE overlying drusen- like deposits on Bruch's membrane (16).
  • RAGE is a pattern-recognition receptor related to other receptors known to impact AMD pathology, namely complement (21-27).
  • AGE-R3 is elevated in advanced dry AMD Bruch's membrane (46).
  • biomarkers that can be used to assess the risk that a subject will develop age-related macular degeneration (AMD) and/ or to assess the severity of AMD in the subject.
  • the biomarkers are also useful in clinical trials to monitor the effect of a treatment and in preclinical trials to screen candidate therapies.
  • the biomarkers can include carboxyethylpyrrole (CEP) adducts, carboxymethyllysine (CML), and pentosidine and be measured individually and in various combinations. In many cases in which CEP is measured, one can also measure autoantibodies directed against CEP.
  • Described herein is a method for characterizing the risk that a subject will develop age-related macular degeneration (AMD), the method comprising:
  • Described herein is a method for characterizing the risk that a subject will develop age-related macular degeneration (AMD), the method comprising:
  • Described herein is a method for characterizing the risk that a subject will develop age-related macular degeneration (AMD), the method comprising:
  • Described herein is a method for assessing the risk that a subject will develop age-related macular degeneration (AMD), the method comprising:
  • Described herein is a method for characterizing the risk that a subject will develop age-related macular degeneration (AMD), the method comprising:
  • Described herein is a method for characterizing the risk that a subject will develop age-related macular degeneration (AMD), the method comprising:
  • CML measured is greater than the CML control value
  • the level of CEP adducts or anti-CEP antibodies measured is greater than the CEP control value
  • the level of pentosidine measured is greater than the pentosidine control value or characterizing the subject as at lesser risk of developing AMD if the level of CML measured is not greater than the CML control value, the level of CEP adducts or anti-CEP antibodies measured is not greater than the CEP control value, and the level of pentosidine measured is not greater than the pentosidine control value.
  • the step of measuring CML in a bodily fluid comprising obtaining a biological sample from the subject comprising a bodily fluid.
  • the step of measuring pentosidine in a bodily fluid comprising obtaining a biological sample from the subject comprising a bodily fluid.
  • the step of measuring CEP adducts or anti-CEP antibodies in a bodily fluid comprising obtaining a biological sample from the subject comprising a bodily fluid.
  • the bodily fluid is selected from serum and plasma.
  • the subject is not suffering from diabetes.
  • the method includes characterizing the subject as at lesser risk of developing AMD if the level of CML measured is greater than the CML control value and the level of CEP adducts or anti-CEP antibodies measured is not greater than the CEP control value.
  • the method includes characterizing the subject as at lesser risk of developing AMD if the level of pentosidine measured is greater than the pentosidine control value and the level of CEP adducts or anti-CEP antibodies measured is not greater than the CEP control value. In various cases, the method includes characterizing the subject as at lesser risk of developing AMD if the level of CML measured is greater than the CML control value, the level of CEP adducts or anti-CEP antibodies measured is not greater than the CEP control value, and the level of pentosidine measured is greater than the pentosidine control value.
  • the AMD is advanced AMD and the control value is a control value for mild to moderate AMD. In various cases, the AMD is mild to moderate AMD and the control value is a control value for non-AMD. In various cases, the AMD is dry AMD. In various cases, the AMD is wet AMD.
  • the method can further include determining whether the subject carries a genetic marker associated with increased risk for developing AMD wherein the genetic marker is present in a gene selected from the group consisting of complement factor; complement C3; ARMS2; and MTND2.
  • the characterizing step comprises characterizing the subject as having a less than 60% chance of developing AMD. In various cases, the characterizing step comprises characterizing the subject as having a greater than 60% chance of developing AMD.
  • Described herein is a method for identifying a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD), the method comprising:
  • test compound (d) identifying the test compound as a candidate compound for treating or reducing the risk of developing AMD if the increase in the level of pentosidine measured is less than the pentosidine control value increase.
  • Described herein is a method for identifying a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD), the method comprising:
  • test compound identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if one or more of: (i) the increase in the level of CML measured is less than the CML control value increase and (ii) the increase in the level of CEP adducts or anti-CEP antibodies measured is less than the CEP control value increase.
  • AMD age-related macular degeneration
  • step (e) comprises identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if (i) the increase in the level of CML measured is less than the CML control value increase and (ii) the increase in the level of CEP adducts or anti-CEP antibodies measured is less than the CEP control value increase.
  • AMD age-related macular degeneration
  • Described herein is a method for identifying a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD), the method comprising:
  • test compound identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if one or more of: (i) the increase in the level of CML measured is less than the CML control value increase and (ii) the increase in the level of pentosidine measured is less than the pentosidine control value increase.
  • AMD age-related macular degeneration
  • step (e) comprises identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if (i) the increase in the level of CML measured is less than the CML control value increase and (ii) the increase in the level of pentosidine measured is less than the pentosidine control value increase.
  • AMD age-related macular degeneration
  • Described herein is a method for identifying a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD), the method comprising:
  • test compound identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if one or more of: (i) the increase in the level of pentosidine measured is less than the pentosidine control value increase and (ii) the increase in the level of CEP adducts or anti-CEP antibodies measured is less than the CEP control value increase.
  • ALD age-related macular degeneration
  • step (e) comprises identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if (i) the increase in the level of pentosidine measured is less than the pentodine control value increase and (ii) the increase in the level of CEP adducts or anti-CEP antibodies measured is less than the CEP control value increase.
  • AMD age-related macular degeneration
  • Described herein is a method for identifying a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD), the method comprising:
  • test compound identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if one or more of: (i) the increase in the level of CML measured is less than the CML control value increase; (ii) the increase in the level of CEP adducts or anti-CEP antibodies measured is less than the CEP control value increase; and (iii) the increase in the level of pentosidine measured is less than the pentosidine control value increase
  • step (g) comprises identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if two or more of: (i) the increase in the level of CML measured is less than the CML control value increase; (ii) the increase in the level of CEP adducts or anti-CEP antibodies measured is less than the CEP control value increase; and (iii) the increase in the level of pentosidine measured is less than the pentosidine control value increase.
  • AMD age-related macular degeneration
  • step (g) comprises identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if :(i) the increase in the level of CML measured is less than the CML control value increase; (ii) the increase in the level of CEP adducts or anti-CEP antibodies measured is less than the CEP control value increase; and (iii) the increase in the level of pentosidine measured is less than the pentosidine control value increase.
  • AMD age-related macular degeneration
  • Described herein is a method for identifying a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD), the method comprising:
  • Described herein is a method for identifying a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD), the method comprising:
  • test compound (b) identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if the level of pentosidine measured after administering the test compound is less than the level measured before administering the test compound.
  • AMD age-related macular degeneration
  • Described herein is a method for identifying a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD), the method comprising:
  • test compound identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if one or more of: (i) the level of CEP adducts or anti-CEP antibodies measured after administering the test compound is less than the level measured before administering the test compoundand (ii) the level of CML measured after administering the test compound is less than the level measured before administering the test compound.
  • AMD age-related macular degeneration
  • step (c) comprises identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if both: (i) the level of CEP or anti-CEP antibodies measured after administering the test compound is less than the level measured before administering the test compound and (ii) the level of CML measured after administering the test compound is less than the level measured before administering the test compound.
  • AMD age-related macular degeneration
  • Described herein is a method for identifying a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD), the method comprising:
  • test compound identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if one or more of: (i) the level of pentosidine measured after administering the test compound is less than the level measured before administering the test compound and (ii) the level of CML measured after administering the test compound is less than the level measured before administering the test compound.
  • AMD age-related macular degeneration
  • step (e) comprises identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if both: (i) the level of pentosidine measured after administering the test compound is less than the level measured before administering the test compound and (ii) the level of CML measured after administering the test compound is less than the level measured before administering the test compound.
  • AMD age-related macular degeneration
  • Described herein is a method for identifying a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD), the method comprising: (a) measuring the level of pentosidine in a bodily of a subject at a first time before administering the test compound and at a second time after administering the test compound;
  • test compound identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if one or more of: (i) the level of pentosidine measured after administering the test compound is less than the level measured before administering the test compound and (ii) the level of CEP adducts or anti-CEP antibodies measured after administering the test compound is less than the level measured before administering the test compound.
  • AMD age-related macular degeneration
  • step (c) comprises identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if both (i) the level of pentosidine measured after administering the test compound is less than the level measured before administering the test compound; and (ii) the level of CEP adducts or anti-CEP antibodies measured after administering the test compound is less than the level measured before administering the test compound.
  • AMD age-related macular degeneration
  • Described herein is a method for identifying a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD), the method comprising:
  • AMD age-related macular degeneration
  • step (d) comprises identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if two or more of: (i) the level of pentosidine measured after administering the test compound is less than the level measured before administering the test compound and (ii) the level of CML measured after administering the test compound is less than the level measured before administering the test compound; and (iii) the level of CEP adducts or anti-CEP antibodies measured after administering the test compound is less than the level measured before administering the test compound
  • step (d) comprises identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if (i) the level of pentosidine measured after administering the test compound is less than the level measured before administering the test compound and (ii) the level of CML measured after administering the test compound is less than the level measured before administering the test compound; and (iii) the level of CEP adducts or anti-CEP antibodies measured after administering the test compound is less than the level measured before administering the test compound.
  • AMD age-related macular degeneration
  • the step of measuring comprises the use of mass spectrometry; the bodily fluid is obtained from a biological sample obtained from the subject the subject is a human taking part in a clinical trial; the subject is a non-human mammal.
  • AMD age-related macular degeneration
  • Described herein is a method for characterizing the risk that a subject will develop age-related macular degeneration (AMD), the method comprising:
  • Described herein is a method for characterizing the risk that a subject will develop age-related macular degeneration (AMD), the method comprising:
  • Described herein is a method for assessing the risk that a subject will develop age-related macular degeneration (AMD), the method comprising:
  • Described herein is a method for characterizing the risk that a subject will develop age-related macular degeneration (AMD), the method comprising:
  • Described herein is a method for characterizing the risk that a subject will develop age-related macular degeneration (AMD), the method comprising:
  • the patient is diagnosed as not suffering from AMD if the level of CML measured is greater than the CML control value and the level of CEP adducts or anti-CEP antibodies measured is not greater than the CEP control value.
  • the patient is diagnosed as not suffering from AMD if the level of pentosidine measured is greater than the pentosidine control value and the level of CEP adducts or anti-CEP antibodies measured is not greater than the CEP control value.
  • the patient is diagnosed as not suffering from AMD if the level of CML measured is greater than the CML control value, the level of CEP adducts or anti-CEP antibodies measured is not greater than the CEP control value, and the level of pentosidine measured is greater than the pentosidine control value.
  • CML and Pentosidine are Elevated in AMD Plasma.
  • P-values two sided t-Test were determined from log-transformed concentrations.
  • FIG. 1 Correlation Between CML, Pentosidine and CEP Adducts in AMD Plasma.
  • P-values two sided t-Test) were determined from log-transformed concentrations.
  • Correlation between CML and CEP adduct concentrations (C) and between pentosidine and CEP adduct concentrations (D) are shown with horizontal and vertical dashed lines indicating median control values. Odds ratios for AMD risk and 95% confidence intervals were determined by logistic regression based on two markers elevated relative to the median control values; p values were determined using the Fischer exact Test.
  • Plasma CML and Pentosidine Concentrations Stratified by Demographic and Health Factors. Plasma protein CML and pentosidine levels in the AMD and control cohorts are plotted based on donor status with regard to gender, status of smoking, hypertension, hyperlipidemia, diabetes and cardiovascular diseases. Sample size per group is indicated and asterisks reflect p- values from a two sample t-Test of log-transformed marker concentrations (*** p ⁇ 0.001, ** p ⁇ 0.01, * p ⁇ 0.05). F, female, M, male; S, smokers; NS, non-smoking; w, with; w/o, without.
  • FIG. 1 Furosine in AMD and Control Plasma. Furosine concentrations quantified by amino acid analysis are shown with median (o) ⁇ first and third quartiles (Ql, Q3) and mean ( ⁇ ) ⁇ SD. Non-diabetic AMD and control plasma donors exhibited -20% lower mean furosine levels than diabetic AMD donors. P-values (two sided t-Test) were determined from log-transformed concentrations.
  • Figure 6. A table of CML and pentosidine markers in control and AMD plasma.
  • Figure 7 A table of C-statistics for CML and pentosidine.
  • Figure 8 A table of sensitivity and specificity of CML, pentosidine and CEP adducts.
  • Figure 9 A table of the characteristics of the study population.
  • MS/MS spectra used to identify high intensity product ions for MRM monitoring of CML, pentosidine, argpyrimidine and the internal standard pyridylethyl-Cysteine (PEC). MS/MS conditions were adjusted to optimize the intensity of the parent ion and predominant product ion.
  • FIG. 11 Representative Chromatography from LC System 1 of CML, argpyrimidine and pentosidine standards and internal standard PEC in the presence of plasma hydro lysate. Chromatography details are as described in experimental procedures.
  • FIG. 12 Representative Chromatography of pentosidine and argyrimidine using LC system 2 and ammonium acetate/acetonitrile solvents with fluorescence detection at 335 nm excitation and 385 nm emission. Chromatography details are as described in Experimental Procedures.
  • Figure 13 Representative Chromatography of CML using LC system 2 and acetic acid/acetonitrile solvents and MRM at 205.1 / 84.1 m/z.
  • Figure 14 Representative Chromatography of PEC using LC system 2, ammonium acetate/acetonitrile solvents and MRM at 227.3 / 106.1 m/z.
  • Figure 15 Representative standard calibration curves using LC system 2 and MRM for CML and PEC and fluorescence monitoring for pentosidine. Each standard was analyzed at the indicated amounts in triplicate each day of analysis. Means + SD for each amount analyzed are shown.
  • Figurel 6 Representative chromatography for quantification of furosine using AccQ Tag TM amino acid analysis. Furosine peak 3, the putative di-derivatized form of the amino acid, was used for quantification of protein bound furosine. * Derivatization byproducts.
  • Age-related macular degeneration causes severe vision loss in the elderly and early identification of AMD susceptibility could help slow or prevent disease progression.
  • AMD Age-related macular degeneration
  • CML and pentosidine are advanced glycation endproducts and abundant in Bruch's membrane, the extracellular matrix separating the retinal pigment epithelium from the blood-bearing choriocapillaris.
  • Carboxyethylpyrrole (CEP) adducts an oxidative modifications generated from docosahexaenoate-containing lipids and also abundant in AMD Bruch's membrane, were elevated - 2-fold in these AMD plasma but autoantibody titers to CEP, CML and pentodsidine were not significantly increased. Compelling higher mean levels of CML and pentosidine were found in AMD plasma over a broad age range. Receiver operating curves indicate that CML, CEP adducts, and pentosidine alone discriminated between AMD and control plasma donors with 80%, 81%, and 92% accuracy, respectively, while CML in combination with pentosidine provided -92% accuracy and CEP plus pentosidine provided - 95% accuracy.
  • Plasma from a total of 30 control subjects and 60 AMD patients were analyzed, including 30 early-stage dry AMD and 30 advanced-stage AMD patients. Protein was quantified by both PTC and AccQ-Tag amino acid analysis, yielding well defined protein concentrations with excellent agreement between the amino acid analysis methods ( ⁇ 4% average difference). Amounts of CML and pentosidine in each HCl hydrolysate were corrected based on the percent recovery of the PEC internal standard which averaged 70.1 ⁇ 12.4 (mean ⁇ SD, n 90). Overall, AMD patients exhibited -55% higher CML and -74% higher pentosidine concentrations relative to control plasma (Fig IAB and Figure 6 (Table I)). Comparison of log-transformed values confirmed the results with p ⁇ 0.0001 for both AGEs.
  • CEP plasma biomarkers offer potential utility is assessing AMD susceptibility (11), therefore it was of interest to compare CEP biomarkers with CML and pentosidine.
  • CEP autoantibody titers were higher than CML and pentosidine autoantibody titers in the AMD cohort but none were significantly elevated relative to the control cohort.
  • c-statistic The area under the ROC curve (c-statistic) is a measure of the overall discriminating accuracy of the markers and comparison of c-statistics suggested no significant difference in discriminatory accuracy between CEP (-81%) and CML (-80%) alone (p - 0.81) and between pentosidine and CEP alone (p - 0.10), however pentosidine exhibited significantly higher discrimination accuracy (-92%) than CML (p -0.03).
  • C-statistics for the joint effect of combined markers were verified by bootstrap resampling and by 10-fold cross-validation.
  • FIG. 5 A comparison of plasma protein CML and pentosidine concentrations by gender and health history is shown in Fig 5, including smoking, hypertension, hyperlipidemia, diabetes and cardiovascular disease.
  • CML and pentosidine concentrations were observed between AMD and control donors.
  • the detection of differences within the AMD and control cohorts was limited by sample size, particularly for the control cohorts, and no significant differences in CML concentrations were detected within any of the cohorts. Small but significant differences were detected in pentosidine levels within two of the AMD cohorts. Specifically, mean pentosidine levels were higher in AMD pateints with hypertension and cardiovascular disease.
  • CML is a lysine modification and pentosidine is a fluorescent lysine-arginine crosslink, and both are formed through the ubiquitous Maillard reaction, which combines sugar carbonyls with primary amino groups to form glycated residues called Amadori products.
  • Amadori products undergo subsequent reactions including intramolecular rearrangements and oxidative fragmentations to produces heterogeneous modifications collectively known as AGEs.
  • Extracellular matrix proteins like collagen are particularly susceptible to AGE modification because of slow turnover rates and tissue and circulating AGE levels are higher in smokers and in those on a high AGE diet (18). AGE formation can lead to a myriad of effects, including altered protein function and activation of intracellular signaling pathways.
  • AGEs in the pathogenesis of AMD (4, 5, 7, 15, 16) and as well as other age- associated diseases, including atherosclerosis, arthritis, Alzheimer's disease, and diabetic complications (18, 35-41).
  • AGEs formation and diabetic retinopathy have been reduced or prevented in a rodent model by treatment with pyrioximine (42), a derivative of vitamin B 6 , and the risk of AMD in women (43) has been reduced by treatment with pyridoxine (vitamine B 6 ) in combination with folic acid and cyanocobalamin (vitamin Bi 2 ).
  • furosine concentrations within 1 SD of the mean diabetic level might be suspect diabetics and 5 donor plasma in our study group fit this criteria.
  • CML and pentosidine concentrations were ⁇ 1 SD from mean control or AMD level, respectively and medical records provided no indication that they were diabetics.
  • These 5 plasma also did not exhibit significantly altered CEP adduct levels, consistent with our previous finding (11) that CEP adducts are not elevated in AMD diabetic plasma (determined from the analysis of 796 diabetic and 130 non-diabetic AMD plasma).
  • C-statistics for the joint effect of markers were increased relative to those for the single markers but significant differences were only associated with pentosidine plus CEP, a combination that appeared to be a better discriminator of AMD than CEP alone (p -0.02), and possibly with CML plus pentosidine (-92%), a combination which also may be a better discriminator than CEP alone (p ⁇ 0.06).
  • Pentosidine and CML are the most well studied AGEs, partly because of the availability of a variety of assays for their detection and quantification. Nevertheless, comparison of quantitative results across multiple studies remains complicated by variations in sample preparation, specimen storage, AGEs assay methods, protein quantification methods and by the array of formats used to report quantitative AGEs data (eg, pmol/ml, pmol/mg protein, fmol/nmol Lys, mmol/mol hydroxyproline, among others).
  • AMD category 2 patients exhibited early- stage disease with multiple small drusen, single or nonextensive intermediate drusen (63- 124 ⁇ m), RPE pigmentary anormalities, or any combination of these, in one or both eyes and visual acuity of 20/30 or better in both eyes.
  • AMD category 4 patients exhibited advanced AMD with substantial CNV or geographic atrophy involving the macula in one or both eyes. Control donors lacked macular drusen and exhibited no clinical evidence of any retinal disorder. Human Plasma Preparation.
  • Nonfasting blood specimens were collected in BD Vacutainer ® K 2 EDTA tubes and plasma was prepared within 6 hours and aliquotted to vials containing the antioxidant butylated hydroxytoluene (BHT; 1 mg/ml plasma) and a protease inhibitor cocktail (Sigma product number P 8340; lO ⁇ l/ml plasma) (11).
  • BHT antioxidant butylated hydroxytoluene
  • P 8340 protease inhibitor cocktail
  • the plasma was flushed with argon, quench-frozen in liquid nitrogen immediately and stored at - 80 0 C until analysis.
  • storage time at - 80 0 C ranged from 4-34 months and averaged 13 months. All samples were frozen and thawed only once.
  • Plasma (-200 ⁇ l, —10 mg) was transferred to 6 x 50 mm glass hydrolysis tubes and protein was precipitated with 2 volumes of cold acetone. After incubation at 4 0 C for 10-20 min, the preparation was centrifuged briefly on a microfuge, the supernatant discarded and the pellet washed once with 67% acetone (400 ⁇ l) and vacuum dried (32). Plasma protein was prepared for hydrolysis by adding 60 ⁇ l of 6 N HCl to each dried pellet then the hydrolysis tubes were placed in a 40 ml screw-cap vial containing -300 ⁇ l of 6N HCl with a few small crystals of phenol.
  • the 40 ml vial was capped with a mininert slide valve, the valve was connected to a vacuum pump and argon source via a three-way stopcock and the vial alternately evacuated and flushed with argon 3x then sealed under vacuum (32).
  • Protein was hydro lyzed at HO 0 C for 16 h, then vacuumed dried, flushed with argon and stored at -2O 0 C until analysis.
  • Protein was quantified by PTC amino acid analysis (-80 ⁇ g derivatized and -3 ⁇ g analyzed) using an Agilent 1100 HPLC system, a Haisil PTC C18 column (220 x 2.1 mm, Applied Biosytems), and a Gilson model 116 UV detector (32).
  • Protein was also quantified by AccQ-TagTM amino acid analysis (-3.5 ⁇ g derivatized and -35 ng analyzed) using an Acquity Ultra Performance LC system (Waters) and AccQ-Tag Ultra column (100 x 2.1 mm) according to the vendor (33).
  • Bovine serum albumin from the National Bureau of Standard was used as a protein standard and hydrolysis control. Amino acid calibration standards were obtained from Pierce and Thermo Scientific.
  • Furosine Quantification Furosine [ -N-(2-furoylmethyl) lysine] was quantified by duplicate AccQ'TagTM amino acid analyses (-15 ⁇ g derivatized and -2.7 ⁇ g analyzed) using the Acquity LC system (Waters) described above. Furosine contains a primary and a secondary amino group and both are derivatized by the AccQ'TagTM reagent, yielding two different mono-derivatized forms and a di-derivatized species. The apparent di-derivatized species was well separated from other amino acids, exhibited a constant response factor up to ⁇ 55 pmol derivatized and was used for quantification of furosine in protein hydrolysates. Furosine standard was purchased from NeoMPS, Inc.
  • Plasma protein HCl hydrolysates ( ⁇ 8 mg in 40 ⁇ l H 2 O) were spiked with a PEC internal standard (15 pmol) and fractionated on LC system 1 composed of an Agilent 1100 HPLC, a HypercarbTM porous graphite carbon column (5 ⁇ m particles, 50 x 10 mm, Thermo Scientific) maintained at 30 0 C with an Applied Biosytems 112A column oven, aqueous trifluoroacetic acid/acetonitrile solvents, and using gradient elution (0-100% acetonitrile over 13 min) and a flow rate of 1 ml/min.
  • LC system 1 composed of an Agilent 1100 HPLC, a HypercarbTM porous graphite carbon column (5 ⁇ m particles, 50 x 10 mm, Thermo Scientific) maintained at 30 0 C with an Applied Biosytems 112A column oven, aqueous trifluoroacetic acid/acetonitrile solvents, and using
  • the eluent was monitored for fluorescence ( 335 nm excitation, 385 nm emission) with a WatersTM 474 scanning fluorimeter and initially split with 20% directed to an API 3000 triple quadrupole electrospray mass spectrometer (Applied Biosystem) and 80% to a fraction collector. After determining reproducible elution times using control plasma spiked with standard AGEs and PEC, 100% of the eluant was directed to the fraction collector and three fractions were collected, one each for CML, PEC, and the co-eluting pentosidine plus argpyrimidine.
  • fractions were vacuum dried and re-fractionated on LC system 2 composed of the same HPLC equipment but with an aqueous normal phase Cogent Diamond HydrideTM Column (4.2 ⁇ m particles, 150 x 2.1 mm) used at room temperature. Fractions containing CML were re-chromatographed using aqueous acetic acid/acetonitrile solvents and gradient elution (95%-0% acetonitrile in 13.6 min) at a flow rate of 400 ⁇ l/min.
  • Fractions containing PEC, argpyrimidine and pentosidine were re-chromatographed using 10 mM ammonium acetate, pH 6.0/acetonitrile solvents, gradient elution (100%-0% acetonitrile in 15 min) at a flow rate of 400 ⁇ l/min.
  • Aqueous normal phase chromatography was monitored by fluorescence detection followed by 100% of the eluant directed to the mass spectrometer.
  • CML and PEC were quantified by multiple reaction monitoring (MRM) and pentosidine and argpyrimidine were measured by fluorescence; final CML and pentosidine amounts were adjusted based on the recovery of the PEC internal standard.
  • MRM multiple reaction monitoring
  • Plasma protein argpyrimidine concentrations were below reliable detection limits in this analytical system and not reported. Calibration curves were developed in triplicate each day of analysis using LC system 2 and external standards.
  • CML standard was purchased from NeoMPS, Inc.
  • pentosidine was obtained from the International Maillard Reaction Society (Case Western Reserve University, Cleveland, OH)
  • argpyrimidine was prepared in our laboratories by RN
  • s- ⁇ (4-pyridylethyl)-L-cysteine (PEC) was purchased from Sigma.
  • Mass Spectrometry The mass spectrometer was operated with Analyst 1.3.1 software (Applied Biosytems) and MS/MS spectra were generated on singly charged precursor ions for CML, pentosidine, argpyrimidine and PEC and specific transition ions for each modified amino acid were analyzed by MRM. The declustering potential, focusing potential, collision energy, and exit potential were optimized for each ion to ⁇ 0.1 Da and ⁇ 1 volts. Ion spray voltage was set at 5300 V and source temperature at 425°C in LC system 1 and at 490 0 C in LC system 2.
  • CML was quantified by MRM using precursor ion 205.1 and product ion 84.1; PEC was quantified using precursor ion 227.3 and product ion 106.1; pentosidine was monitored by MRM using precursor ion 379.2 and product ion 187.2; and argpyrimidine was monitored using precursor ion 255.2 and product ion 237.3. Peak areas and external standard calibration curves were used for quantification of CML and PEC by MRM and pentosidine by fluorescence.
  • Pentosidine and CML Autoantibody Assays were measured by direct ELISA using pentosidine modified bovine serum albumin (pentosidine-BSA) and unmodified BSA as coating antigen ( ⁇ 8 ⁇ g/well).
  • CML autoantibody titers were measured with the same methodology using CML-BSA and BSA as coating agent ( ⁇ 15 ⁇ g/well). Plasma (100 ⁇ l of a 1 :10 dilution) was applied to coating agents and the ELISA developed as previously described for the CEP autoantibody assay (9, 11).
  • Anti-CML R&D Systems, product MAB3247
  • anti-pentosidine monoclonal antibodies Trans Genie Inc, Japan, product KHO 12
  • Titer was defined as the ratio of plasma binding to antigen (A) versus binding to BSA (A 0 ) (9).
  • Pentosidine-BSA was prepared by mixing pentosidine (1 mg, NeoMPS, product SC1535) in dimethylformamide (100 ⁇ l) with l-ethyl-3-(3- dimethylaminopropyl) carbodiimide HCl (5 mg, Pierce) and N-hydroxysuccinimide (6 mg, Pierce) at room temperature for 2 hours followed by the addition of BSA (2.5 mg in 0.5 ml PBS, Sigma product A6003) and continued incubation at 37 0 C for 48 h.
  • CML-BSA was prepared by mixing BSA in PBS with 25 mM glyoxylic acid and 50 mM NaCNBH 3 for 48 h at room temperature followed by dialysis against PBS for 24 h with two changes (34). Protein was quantified by the BCA assay (Pierce) and pentosidine and CML modifications confirmed by Western analysis before and after the modification reactions using anti-CML monoclonal antibody (R&D Systems, product MAB3247) or anti-pentosidine monoclonal antibody (Trans Genie Inc, Japan, product KHO 12).
  • CEP Adduct and CEP Autoantibody Assays The CEP adduct and autoantibody concentrations of plasma used in this study were previously reported among 1404 plasma (11). Briefly, CEP adducts were quantified with a competitive ELISA using rabbit anti-CEP polyclonal antibody, CEP modified bovine serum albumin as coating agent and known amounts of CEP modified human serum albumin as reference protein. CEP autoantibody titers were measured by direct ELISA using CEP-BSA as coating antigen. These methods are well documented (9, 11).
  • C-statistics measured the model's ability to discriminate between AMD and controls, and odds ratios (ORs) showed the change in risk of AMD based on the predictors.
  • ORs odds ratios
  • c-statistics and p-values were determined based on log-transformed marker concentrations.
  • Validation of c-statistics was performed using 2000 bootstrap (random) resamplings to calculate empirical 95% confidence intervals (CI) and by performing 10-fold cross-validation. Sensitivity and specificity were calculated to maximize the sum of the two values using receiver operating characteristic (ROC) curves constructed with SAS 9.1 from the output of logistic regression analysis fit with either CML, pentosidine or CEP adducts alone or in combination.
  • ROC receiver operating characteristic
  • AGEs advanced glycation end-products
  • AMD age-related macular degeneration
  • AccQ 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate
  • AREDS age-related eye disease study
  • CEP 2-( ⁇ -carboxyethyl)pyrrole
  • CI confidence interval
  • CML N ⁇ - (carboxymethyl)lysine
  • CNV choroidal neovascularization
  • OR odds ratio
  • PEC s- ⁇ (4- pyridylethyl)-L-cysteine
  • PTC phenylthiocarbamyl
  • ROC receiver operating characteristic
  • RPE retinal pigment epithelium
  • RSD relative standard deviation
  • SD standard deviation.
  • Age-related Eye Disease Study Group (2001) A Randomized, Placebo-Controlled, Clinical Trial of High-Dose Supplementation with Vitamin C and E, Beta Carotene, and Zinc for Age-Related Macular Degeneration and Vision Loss. Arch Ophthalmol 119, 1417-1436.
  • N epsilon-(carboxymethyl)lysine is a dominant advanced glycation end product (AGE) antigen in tissue proteins. Biochemistry 34, 10872-10878.

Abstract

L'invention porte sur des biomarqueurs qui peuvent être utilisés pour évaluer le risque pour un sujet de développer une dégénérescence maculaire liée à l'âge (AMD) et/ou pour évaluer la gravité d'une AMD chez le sujet. Les biomarqueurs sont également utiles pour des essais cliniques pour surveiller l'effet d'un traitement et pour des essais précliniques pour cribler des thérapies candidates. Les biomarqueurs peuvent comprendre des produits d'addition de carboxyéthylpyrrole (CEP), la carboxyméthyllysine (CML) et la pentosidine et peuvent être mesurés individuellement et dans diverses combinaisons.
PCT/US2010/034398 2009-05-11 2010-05-11 Biomarqueurs pour l'évaluation d'une dégénérescence maculaire liée à l'âge WO2010132459A2 (fr)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015023596A1 (fr) * 2013-08-12 2015-02-19 Genentech, Inc. Compositions et méthode pour le traitement de troubles associés au complément
US10179821B2 (en) 2014-05-01 2019-01-15 Genentech, Inc. Anti-factor D antibodies
US10407510B2 (en) 2015-10-30 2019-09-10 Genentech, Inc. Anti-factor D antibodies and conjugates
US10654932B2 (en) 2015-10-30 2020-05-19 Genentech, Inc. Anti-factor D antibody variant conjugates and uses thereof
US11267803B2 (en) 2016-06-21 2022-03-08 Orion Ophthalmology LLC Carbocyclic prolinamide derivatives
US11377439B2 (en) 2016-06-21 2022-07-05 Orion Ophthalmology LLC Heterocyclic prolinamide derivatives
KR20220140431A (ko) * 2021-04-09 2022-10-18 가천대학교 산학협력단 글리코톡신, 글리코톡신에 결합된 특정 단백질 또는 글리코톡신-특정 단백질 복합체를 포함하는 신경퇴행성 질환용 바이오마커
JP7426026B2 (ja) 2019-08-26 2024-02-01 学校法人同志社 試料前処理方法及び分析方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004041160A2 (fr) * 2002-10-30 2004-05-21 University Of Kentucky Research Foundation Procedes et cobaye animal d'analyse de la degenerescence maculaire liee a l'age
WO2007127151A2 (fr) * 2006-04-24 2007-11-08 The Cleveland Clinic Foundation Modèle non humain de maladie auto-immune
WO2008013797A2 (fr) * 2006-07-24 2008-01-31 The Cleveland Clinic Foundation Compositions et procédés pour le traitement d'une dégénérescence maculaire
US20080160505A1 (en) * 2001-04-30 2008-07-03 Hollyfield Joe G Diagnostic methods for age related macular degeneration

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080160505A1 (en) * 2001-04-30 2008-07-03 Hollyfield Joe G Diagnostic methods for age related macular degeneration
WO2004041160A2 (fr) * 2002-10-30 2004-05-21 University Of Kentucky Research Foundation Procedes et cobaye animal d'analyse de la degenerescence maculaire liee a l'age
WO2007127151A2 (fr) * 2006-04-24 2007-11-08 The Cleveland Clinic Foundation Modèle non humain de maladie auto-immune
WO2008013797A2 (fr) * 2006-07-24 2008-01-31 The Cleveland Clinic Foundation Compositions et procédés pour le traitement d'une dégénérescence maculaire

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
NI, J. ET AL.: 'Plasma Protein Pentosidine and Carboxymethyllysine, Biomarker s for Age-related Macular Degeneration' MOLECULAR & CELLULAR PROTEOMICS 8.8 11 May 2009, *

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EP3033436A1 (fr) * 2013-08-12 2016-06-22 Genentech, Inc. Compositions et méthode pour le traitement de troubles associés au complément
US10093978B2 (en) 2013-08-12 2018-10-09 Genentech, Inc. Compositions for detecting complement factor H (CFH) and complement factor I (CFI) polymorphisms
TWI656217B (zh) * 2013-08-12 2019-04-11 建南德克公司 用於治療補體相關病況之組合物及方法
US10947591B2 (en) 2013-08-12 2021-03-16 Genentech, Inc. Compositions and method for treating complement-associated conditions
US10179821B2 (en) 2014-05-01 2019-01-15 Genentech, Inc. Anti-factor D antibodies
US10654932B2 (en) 2015-10-30 2020-05-19 Genentech, Inc. Anti-factor D antibody variant conjugates and uses thereof
US10407510B2 (en) 2015-10-30 2019-09-10 Genentech, Inc. Anti-factor D antibodies and conjugates
US10961313B2 (en) 2015-10-30 2021-03-30 Genentech, Inc. Anti-factor D antibody variant conjugates and uses thereof
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US11866422B2 (en) 2016-06-21 2024-01-09 Orion Ophthalmology LLC Carbocyclic prolinamide derivatives
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