CIRCULATING TOTAL-NT-PROBNP (GLYCOSYLATED AND UNGLYCOSYLATED NT-PROBNP) AND ITS RATIO WITH NT-PROBNP (UNGLYCOSYLATED NT-PROBNP) IN THE ASSESSMENT OF ATRIAL FIBRILLATION

Abstract

The present invention relates to a method for diagnosing atrial fibrillation in a subject, said method comprising the steps of a) determining the amount of total NT-proBNP in sample from the subject, b) determining the amount of unglycosylated NT-proBNP in a sample from the subject, c) calculating a score of the amounts determined in steps a) and b), d) comparing the calculated score with a reference score, and e) diagnosing atrial fibrillation in a subject.

Claims

1. A method for diagnosing atrial fibrillation in a subject, said method comprising determining an amount of total NT-proBNP in a sample from the subject, determining an amount of unglycosylated NT-proBNP in the sample from the subject, calculating a score of the amounts of the total NT-proBNP and the unglycosylated NT-proBNP, comparing the calculated score with a reference score, and diagnosing atrial fibrillation in the subject based on the comparison of the calculated score with the reference score.

2. The method of claim 1, wherein the sample is a blood, a serum, or a plasma sample, and/or wherein the subject is a human subject.

3. The method of claim 1, wherein the subject is suspected to suffer from atrial fibrillation.

4. The method of claim 1, wherein unglycosylated NT-proBNP is unglycosylated at one or more positions selected from T36, S37, S44, T48, S53, T58, and/or T71 of human NT-proBNP.

5. The method of claim 4, wherein unglycosylated NT-proBNP is unglycosylated at least at position S44.

6. The method of claim 1, wherein the determination of the amount of unglycosylated NT-proBNP comprises contacting the sample with an antibody, or antigen-binding fragment thereof, which specifically detects unglycosylated NT-proBNP.

7. The method of claim 6, wherein the epitope of the antibody, or antigen-binding fragment thereof, comprises amino acid residues 42 to 46 of human NT-proBNP.

8. The method of claim 1, wherein the amount of total NT-proBNP is the amount of glycosylated and unglycosylated NT-proBNP.

9. The method of claim 8, wherein the antibody, or antigen-binding fragment thereof, which specifically detects total NT-proBNP, binds to a region of human NT-proBNP which does not carry an O-glycosylation site.

10. The method of claim 9, wherein the antibody, or antigen-binding fragment thereof, binds to an epitope present within the first 35 amino acids of NT-proBNP, and wherein the epitope of the antibody, or antigen-binding fragment thereof, comprises amino acid residues 13 to 16 of human NT-proBNP.

11. The method of claim 1, wherein the calculated score is a ratio, and wherein the ratio is the ratio of the amount of total NT-proBNP to the amount of unglycosylated NT-proBNP, wherein a ratio which is lower than the reference ratio is indicative for a subject who suffers from atrial fibrillation, or wherein the ratio is the ratio of the amount of unglycosylated NT-proBNP to the amount of total NT-proBNP, wherein a ratio which is larger than the reference ratio is indicative for a subject who suffers from atrial fibrillation.

12. The method of claim 1, wherein atrial fibrillation is paroxysmal or persistent atrial fibrillation.

13. A computer-implemented method for diagnosing atrial fibrillation in a subject, comprising receiving at a processing unit a value for an amount of total NT-proBNP in a sample from the subject, and a value for an amount of unglycosylated NT-proBNP in the sample from the subject: processing the values for total NT-proBNP and unglycosylated NT-proBNP with the processing unit, wherein said processing comprises calculating a score of the values for total NT-proBNP and unglycosylated NT-proBNP, and comparing the calculated score with a reference score; and providing the diagnosis via an output device, wherein said diagnosis is based on processing the values for total NT-proBNP and unglycosylated NT-proBNP.

14. (canceled)

15. A kit comprising at least one agent which specifically binds to unglycosylated NT-proBNP and at least one agent which specifically binds to total NT-proBNP.

16. The method of claim 8, wherein the determination of the amount of total NT-proBNP comprises contacting the sample with an antibody, or antigen-binding fragment thereof, which specifically detects total NT-proBNP.

17. The method of claim 6, wherein the antibody is the monoclonal antibody MAB 1.21.3, or an antigen-binding fragment thereof.

18. The method of claim 16, wherein the epitope of the antibody, or antigen-binding fragment thereof, comprises amino acid residues 13 to 16 of human NT-proBNP.

19. The method of claim 16, wherein the antibody is the monoclonal antibody MAB 17.3.1, or an antigen-binding fragment thereof.

20. The method of claim 6, wherein the antibody, or antigen-binding fragment thereof, is used in a sandwich assay as a capture antibody in combination with at least one other antibody that binds to an epitope of NT-proBNP which is not glycosylated.

21. The method of claim 3, wherein the subject who is suspected to suffer from atrial fibrillation has a history of atrial fibrillation.

Description

[0150] The Figures show:

[0151] FIG. 1 Differential expression of NPPB in right atrial appendage tissue: A) persistent Aftb; B) paroxysmal Afib.

[0152] FIG. 2 Overview on O-glycosylation sites in proBNP epitopes. The sequence of human NT-proBNP is underlined twice. In the studies underlying the present invention, two antibodies were used: one antibody binding to epitope aa13-16 of NT-proBNP (for total NT-proBNP) and one antibody binding to epitope aa42-46 of NT-proBNP for NT-proBNP, i.e. unglycosylated NT-proBNP Other antibodies binding to other epitopes can be used as well, for example, the antibodies from Hytest which bind to epitopes as shown in the Figure. The antibodies are also disclosed in US20090163415A1.

[0153] FIG. 3: Differentiation between patients with ongoing atrial fibrillation and patients in sinus rhythm based on total-NT-proBNP

[0154] FIG. 4: Differentiation between patients with ongoing atrial fibrillation and patients in sinus rhythm based on unglycosylated NT-proBNP

[0155] FIG. 5: Differentiation between patients with ongoing atrial fibrillation and patients in sinus rhythm based on ratio [unglycosylated NT-proBNP) / [total-NT-proBNP].

EXAMPLES

[0156] The invention will be merely illustrated by the following Examples. The said Examples shall, whatsoever, not be construed in a manner limiting the scope of the invention.

Example 1: Differential Expression of Human NPPB in Right Atrial Appendage Tissue (Mapping Study)

[0157] Right atrial appendage tissue was sampled during open chest surgery because of CABG or valve surgery. Evidence of AF or SR (controls, sinus rhythm) was generated during surgery with simultaneous Endo-Epicardial High Density Activation Mapping. Tissue samples were taken during surgery. Patients with AF and controls were matched with regard to gender, age and comorbidities.

[0158] Atrial tissue samples were prepared for [0159] paroxysmal AF patients; n=14 patients persistent AF patients; n=8 patients [0160] control patients in SR; n=27 patients.

[0161] Differential expression of NPPB was determined in RNAseq analyses applying the algorithms RSEM and DESEQ2. The results are shown in FIG. 1 [A) persistent Afib B) paroxysmal Afib].

[0162] As shown in FIG. 1, NPPB expression was found to be upregulated in the analyzed right atrial appendage tissues of the patients with paroxysmal and persistent AF versus the control patients in sinus rhythms. The highest levels were observed in patients with persistent AF. These data support the hypothesis of atrial appendage dependency of NT-proBNP and its increase with disease severity.

Example 2. Detection of NT-proBNP

[0163] The amounts of total-NT-proBNP and NT-proBNP were measured by sandwich assays. Total NT-proBNP was determined by using an antibody against amino acids 13 to 16 of NT-proBNP as capture antibody (monoclonal antibody 17.3.1). NT-proBNP was determined by the using an antibody against amino acids 42 to 46 of NT-proBNP as capture antibody (monoclonal antibody 1.21.03) using the Roche Elecays® proBNP 11 NT-proBNP assay following the manufacturer’s instructions. This antibody detects NT-proBNP which is not O-glycosylated position S44.

[0164] For both assays, a detection antibody was used which binds to amino acids 27 to 31 (monoclonal antibody 18.4.34, see FIG. 2).

[0165] The epitopes are shown in FIG. 2.

[0166] Higher values generated with non-glycosylated total-NT-proBNP assay due to elimination of effects caused by O-Glycosylation blocking detection at position S44. In clinical setting this invention increases the sensitivity, where detection of different entities causes improvement in specificity and the sum in sensitivity.

[0167] A ratio between total NT-proBNP and unglycosylated NT-proBNP can be formed which allows normalization.

Example 3: Detection of Atrial Fibrillation in the Biomarker Sub-Study of the GISSI-AF Trial

[0168] In the biomarker sub-study of the GISSI-AF trial, blood samples were collected at study entry, and after 6 and 12 months of follow-up. For more details on the GISSI-AF trial, see the main publication: GISSI-AF Investigators, New Engl J Meet 2009;360:1606-17. For more details on the biomarker substudy, see: Latini R et al., J Intern Meet 2011;269-160-71

[0169] For 382 patients total-NT-proBNP and NT-proBNP values from plasma samples were obtained at baseline. After 24 weeks, out of 360 patients 38 developed paroxysmal atrial fibrillation. After 52 weeks, 48 out of 357 have developed atrial fibrillation.

[0170] NT-proBNP and total NT-proBNP was determined in the atrial fibrillation sub-cohort selected from the GISSI AF study. Elevated circulating NT-proBNP and total NT-proBNP levels were observed in samples from subjects with on-going atrial fibrillation versus controls.

TABLE-US-00002 Association with Prevalent Atrial Fibrillation: Baseline 24 weeks 12 months Sinus Rhythm 382 (100%) 322 309 (86.6%) Atrial Fibrillation 0 38 48 (13.4%)

[0171] The sample size considered is composed of 382 patients with measurements of NTproBNP at baseline. The variable SR (sinus rhythm) vs AF (atrial fibrillation) was recorded from variable “ritmo all′ECG″” in the CRF.

[0172] Conclusion: Measurement of total-NT-proBNP and NT-proBNP showed upon analysis that combination of both parameters results in far better diagnostic value than each single marker.

TABLE-US-00003 Biomarker concentrations and biomarker ratio and atrial fibrillation/sinus rhythm recorded by ECG during scheduled visits at 6 and 12 months Biomark er Time N Median AUC 95 %Cl P total NT-proBNP pg/mL 6 months Sinus Rhythm 280 865 0.78 (0.68-0.87) < 0.0001 Atrial Fibrillation 34 2532 12 months Sinus Rhythm 268 878 0.77 (0.70-0.84) < 0.0001 Atrial Fibrillation 45 2241 NT-proBNP pg/mL 6 months Sinus Rhythm 284 128.0 0.87 (0.78-0.95) < 0.0001 Atrial Fibrillation 34 724.5 12 months Sinus Rhythm 276 116.5 0.86 (0.81-0.92) < 0.0001 Atrial Fibrillation 45 580.0 Ratio NT-proBNP / total-NTpro BNP 6 months Sinus Rhythm 280 0.14 0.93 (0.88-0.97) < 0.0001 Atrial Fibrillation 34 0.3 12 months Sinus Rhythm 268 0.13 0.91 (0.85-0.96) < 0.0001 Atrial Fibrillation 45 0.28

[0173] To assess the strength of the association between studied biomarkers and ongoing AF (i.e. biomarkers assayed while an AF rhythm was recorded), ROC analyses were done. At 6 and 12 months for the ratio NT-proBNP/ total NT-proBNP the observed AUC was higher compared with the AUCs for each biomarker alone : ratio NTproBNP/ total NTproBNP (AUC6m=0.93 and AUC12m=0.91), total NTproBNP (AUC6M=0.78 and AUC12M=0.77), NT-proBNP (AUC6M=0.87 and AUC12M--0.86).

[0174] It can be not in the table 2 and in the FIGS. 3, 4 and 5, that the ratio of NT-proBNP/ total NT-proBNP proved to be better in the prediction of prevalent / ongoing AF than either one of the natriuretic peptides alone.

[0175] It was further observed in the GISSI AF study that the ratio NT-proBNP/total NT-proBNP was associated with incident / recurrent AF (p= 0.058).

[0176] In conclusion in patients with ongoing AF a lower degree of glycosylation of NT-proBNP has been found versus patients in sinus rhythm. Determination of the ratio NTproBNP/total NTproBNP is suitable to further improve the diagnostic accuracy versus each of the natriuretic peptides alone.

TABLE-US-00004 Biomarker concentrations and biomarker ratio and atrial fibrillation/sinus rhythm recorded by ECG during scheduled visits at 6 and 12 months in patients divided by gender Biomarker Gender Time N AUC 95%Cl P Ratio NT-proBNP /total-NTpro BNP Females 6 months Sinus Rhythm 100 0.94 (0.87-0.99) < 0.0001 Atrial Fibrillation 11 12 months Sinus Rhythm 96 0.95 (0.90-0.99) < 0.0001 Atrial Fibrillation 11 Ratio NT-proBNP /total-NTpro BNP Males 6 months Sinus Rhythm 180 0.92 (0.86-0.97) < 0.0001 Atrial Fibrillation 23 12 months Sinus Rhythm 172 0.92 (0.86-0.97) < 0.0001

[0177] To assess the strength of the association between studied biomarkers and ongoing AF in female patients versus male patients (i.e. biomarkers assayed while an AF rhythm was recorded), ROC analyses were done separately for male and female study participants. At 6 and 12 months for the ratio NT-proBNP/ total NT-proBNP the observed AUC was higher in female patients as compared with male patients the ratio NTproBNP/ total NTproBNP in female patients (AUC6m-0.94 and AUC12m-0.95), the ratio NTyroBNP/total NTproBNP in male patients (AUC6M=0.92 and AUC12M=0.92).

[0178] As shown in the table 3 the ratio of NTproBNP/total NT-proBNP was found to be particularly well performing in the detection of ongoing AF in the subgroup of female participants of the study. While the Ratio NT-proBNP / total-NTpro BNP was ∼0.15 in Afib (Table 2), but higher in reports of heart failure (0.19-0.31; Vodovar N et al European Heart Journal (2014) 35, 3434-3441), the present invention of using the ratio of NT-proBNP / total-NTpro BNP in AFib allows to better differentiate AFib versus HF as source of NT-proBNP elevation, and thus to better differentiate AFib vs heart failure.

[0179] In conclusion for the detection of AF in females the ratio NTproBNP/total NTpro-BNP may be very useful

[0180] The GISSI AF study comprises patients with moderate severe comorbidities: clinically diagnosed HF or LVEF<40% was low (11%), history of stroke (4%), diabetes (13 %), history of hypertension (84 %), documented CAD (11 %). The incidence of AF was not significantly different in patients with any of the observed comorbidities versus patients without respective comorbidities.

[0181] In conclusion, patients with incident AF or with a history of AF were found to have a lower extent of glycosylation of NTproBNP as compared to patients in sinus rhythm.

[0182] Overall it was observed that the ratio NTproBNP/total NTproBNP allows for an improved diagnosis of prevalent AF in patients with a history of AF or other risk factors for incident AF recurrence.