BIOMARKER TEST AND METHOD FOR ASSESSING MUCOSAL HEALING IN RESPONSE TO TREATMENT OF ULCERATIVE COLITIS

• US 20190331679A1
• Filed: 11/30/2017
• Published: 10/31/2019
• Est. Priority Date: 12/01/2016
• Status: Active Grant

• Current Assignee: Katholieke Universiteit Leuven, Glycominds, Ltd. (Storage Drop Storage Technologies Ltd.)
• Original Assignee: Katholieke Universiteit Leuven, Kepler Diagnostics, Inc.
• Inventors: DUKLER, Avinoam, RINGOLD, Randy, VERMEIRE, Severine, DE BRUYN, Magali, OPDENAKKER, Ghislain
• Granted Patent: US 11,480,569 B2
• Time in Patent Office: Days
• CPC Class Codes: G01N 2333/4737 C-reactive protein
• CPC Class Codes: G01N 2800/065 Bowel diseases, e.g. Crohn, ulcerative colitis, IBS
• CPC Class Codes: G01N 2800/52 Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
• CPC Class Codes: G01N 33/53 Immunoassay; Biospecific binding assay; Materials therefor
• CPC Class Codes: G01N 33/564 for pre-existing immune complex or autoimmune disease , i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
• CPC Class Codes: G01N 33/56972 White blood cells
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: G01N 33/74 involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
• CPC Class Codes: G16H 50/30 for calculating health indices; for individual health risk assessment
• CPC Class Codes: Y02A 90/10 Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

Abstract

Mucosal healing is an indication to the disease activity level in patients affected by inflammatory bowel diseases, and it is thus far mainly monitored by endoscopy. Instead or in addition to endoscopy, the invention provides blood test using biomarkers and an index that allows a practitioner to assess the status of mucosal healing, to change or adapt dosage of treatment and to predict which patient will become responder versus non-responder to treatment as assessed by endoscopy. While none of neutrophils cell count, c-reactive protein (CRP), Human type of Cathelicidin (LL-37), or Chitinase 3-like 1 (CHI3L1) alone is able to provide an assessment means of mucosal healing, the invention provides a novel combination of the levels of these biomarkers to assess the level of mucosal healing in relation to endoscopic healing.

Claims

1. A method for non-invasively determining mucosal healing in human subjects undergoing treatment for inflammatory bowel disease using a set of biomarkers, the method comprising the steps of:

obtaining a body specimen from a human subject;

obtaining a concentration of c-reactive protein (CRP) in said body specimen, comprising obtaining a weighted value of CRP concentration;

obtaining a cell count of neutrophils in a blood sample from said human subject, comprising obtaining a weighted value of neutrophils cell count;

obtaining a concentration of Human type of Cathelicidin (LL-37) in said body specimen, comprising obtaining a weighted value of said LL-37 concentration;

obtaining a concentration of Chitinase 3-like 1 (CHI3L1) in said body specimen, comprising obtaining a weighted value of CHI3L1 concentration;

obtaining an index value by summing said weighted value of CRP concentration, said weighted value of neutrophils cell count, said weighted value of said LL-37 concentration and said weighted value of CHI3L1 concentration; and

determining that said human subject has a low mucosal healing if said index is above the index upper quartile, when said weighted value of CRP concentration, said weighted value of neutrophils cell count, said weighted value of said LL-37 concentration and said weighted value of CHI3L1 concentration are adequately selected such that said index is a numerical range.

2. A method as in claim 1 further comprising said Index numerical ranges is from zero to ten.

3. A method as in claim 1 further comprising said Index numerical ranges is from zero to one hundred.

4. A method as in claim 1 further comprises obtaining said body specimen from a human subject affected by Ulcerative Colitis.

5. A method as in claim 4 further comprises obtaining a blood sample.

6. A method as in claim 5 further comprising:

obtaining said concentration of c-reactive protein (CRP) from a serum portion of said blood sample;

obtaining said cell count of neutrophils in said blood sample;

obtaining said concentration of Human type of Cathelicidin (LL-37) in said serum portion; and

obtaining said concentration of Chitinase 3-like 1 (CHI3L1) in said serum portion.

7. A method as in claim 6 further comprising alternatively obtaining at least one of said concentrations of CRP, LL-37 and CHI3L1 in a blood plasma sample.

8. A method as in claim 6 further comprising:

obtaining a discretized CRP concentration value of said concentration of CRP, wherein said discretized value of CRP having a value zero (0) if said concentration of CRP is less than about 2.8 ng/ml, and a value one (1) if said concentration of CRP is greater than about 2.8 ng/ml, and obtaining said weighted value of CRP concentration by multiplying said discretized CRP concentration value by a CRP coefficient;

obtaining a discretized cell count value of said cell count of neutrophils, wherein said discretized value of said cell count having a value zero (0) if said cell count of neutrophils is less than about 35,000 per ml, and having the value one (1) if said cell count of neutrophils is greater than about 35,000 per ml, and obtaining said weighted value of said neutrophils cell count by multiplying said discretized cell count value by a cell count coefficient;

obtaining a discretized LL-37 concentration value, wherein said discretized LL-37 concentration value having a value zero (0) if said concentration of LL-37 is less than about 46 ng/ml, and a value one (1) if said concentration of LL-37 is greater than about 46 ng/ml, and obtaining said weighted value of said LL-37 concentration by multiplying said discretized LL-37 concentration value by a LL-37 coefficient; and

obtaining a discretized CHI3L1 concentration value, wherein said discretized CHI3L1 concentration value having value “

0”

if said concentration of CHI3L1 is less than about 22 ng/ml, and having a value one (1) if said concentration of CHI3L1 is greater than about 22 ng/ml, and obtaining said weighted value of said CHI3L1 concentration by multiplying said discretized CHI3L1 concentration value by a CHI3L1 coefficient.

9. A method as in claim 8, further comprising:

assigning a value 1.2 to said CRP coefficient;

assigning a value 1.6 to said cell count coefficient;

assigning a value 0.9 to said LL-37 coefficient; and

assigning a value 1.1 to said CHI3L1 coefficient.

10. A method as in claim 1 further comprises obtaining said body specimen from a human subject affected by Crohn'"'"'s Disease.

11. A method as in claim 1, wherein said obtaining said body specimen further comprises obtaining a stool sample.

12. A method as in claim 1, wherein said obtaining said body specimen further comprises obtaining a tissue sample.

13. A method as in claim 1 further comprising:

obtaining a concentration of NGAL-MMP-9 and obtaining a weighted value of said NGAL-MMP-9; and

obtaining said index by summing said weighted value of CRP concentration, said weighted value of neutrophils cell count, said weighted value of said LL-37 concentration, said weighted value of CHI3L1 concentration and said weighted value of said NGAL-MMP-9.

14. A method for treating a subject who has inflammatory bowel disease and suffers from no mucosal healing, the method comprising the steps of:

obtaining a blood sample from a human subject and a serum sample from said blood sample;

obtaining a concentration of c-reactive protein (CRP) in said serum sample, comprising obtaining a weighted value of CRP concentration, and obtaining a discretized CRP concentration value of said concentration of CRP, wherein said discretized value of CRP having a value zero (0) if said concentration of CRP is less than about 2.8 ng/ml, and a value one (1) if said concentration of CRP is greater than about 2.8 ng/ml, and obtaining said weighted value of CRP concentration by multiplying said discretized CRP concentration value by a CRP coefficient having a value of 1.12;

obtaining a cell count of neutrophils in said blood sample from said human subject, comprising obtaining a weighted value of neutrophils cell count;

obtaining a discretized cell count value of said cell count of neutrophils, wherein said discretized value of said cell count having a value zero (0) if said cell count of neutrophils is less than about 35,000 per ml, and having the value one (1) if said cell count of neutrophils is greater than about 35,000 per ml, and obtaining said weighted value of said neutrophils cell count by multiplying said discretized cell count value by a cell count coefficient having a value of 1.6;

obtaining a concentration of Human type of Cathelicidin (LL-37) in said serum sample, comprising obtaining a weighted value of said LL-37 concentration;

obtaining a discretized LL-37 concentration value, wherein said discretized LL-37 concentration value having a value zero (0) if said concentration of LL-37 is less than about 46 ng/ml, and a value one (1) if said concentration of LL-37 is greater than about 46 ng/ml, and obtaining said weighted value of said LL-37 concentration by multiplying said discretized LL-37 concentration value by a LL-37 coefficient having a value of 0.9; and

obtaining a concentration of Chitinase 3-like 1 (CHI3L1) in said serum sample, comprising obtaining a weighted value of CHI3L1 concentration;

obtaining a discretized CHI3L1 concentration value, wherein said discretized CHI3L1 concentration value having value “

0”

if said concentration of CHI3L1 is less than about 22 ng/ml, and having a value one (1) if said concentration of CHI3L1 is greater than about 22 ng/ml, and obtaining said weighted value of said CHI3L1 concentration by multiplying said discretized CHI3L1 concentration value by a CHI3L1 coefficient having a value of 1.1;

obtaining an index value by summing said weighted value of CRP concentration, said weighted value of neutrophils cell count, said weighted value of said LL-37 concentration and said weighted value of CHI3L1 concentration and determining that said human subject has a low mucosal healing if said index is above the index upper quartile; and

administering a pharmaceutical composition containing at least one of an immunosuppressant agent and an anti-inflammatory agent for treating inflammatory bowel disease.

15. A method as in claim 14 further comprising alternatively obtaining at least one of said concentrations of CRP, LL-37 and CHI3L1 in a blood plasma sample.

16. A method as in claim 14, wherein said obtaining said blood sample further comprises obtaining said blood sample from a human subject affected by Crohn'"'"'s disease.

17. A method as in claim 14, wherein said obtaining said blood sample further comprises obtaining said blood sample from a human subject affected by Ulcerative Colitis.

18. A method as in claim 14 further comprising administering an pharmaceutical composition containing an Anti-TNF(α

).

19. A method as in claim 14 further comprising administering an pharmaceutical composition containing an Anti-α

4β

7 integrin

20. A method as in claim 14 further comprising administering an pharmaceutical composition containing an IL-23 inhibitor.

21. A method as in claim 14 further comprising administering an pharmaceutical composition containing a Janus kinase (JAK) inhibitor.

22. A method as in claim 14 further comprising administering an pharmaceutical composition containing an S1P1-receptor and S1P5-receptor modulator.

23. A method as in claim 14 further comprising administering an pharmaceutical composition containing an aminosalicylates.

24. A method as in claim 14 further comprising administering an pharmaceutical composition containing Corticosteroids.

25. A method as in claim 14 further comprising monitoring mucosal healing level by repeatedly obtaining said index from said human subject and further comprising adjusting a dose of said pharmaceutical composition.

26. A method for assessing the status of mucosal healing in relation to endoscopic healing according to the Mayo endoscopic subscore in a human subject affected by inflammatory bowel disease and undergoing treatment, said method comprising the steps of:

obtaining a blood sample from a human subject and a serum sample from said blood sample;

obtaining a concentration of c-reactive protein (CRP) in said serum sample, comprising obtaining a weighted value of CRP concentration, and obtaining a discretized CRP concentration value of said concentration of CRP, wherein said discretized value of CRP having a value zero (0) if said concentration of CRP is less than about 2.8 ng/ml, and a value one (1) if said concentration of CRP is greater than about 2.8 ng/ml, and obtaining said weighted value of CRP concentration by multiplying said discretized CRP concentration value by a CRP coefficient having a value of 1.2;

obtaining a cell count of neutrophils in said blood sample from said human subject, comprising obtaining a weighted value of neutrophils cell count;

obtaining a discretized cell count value of said cell count of neutrophils, wherein said discretized value of said cell count having a value zero (0) if said cell count of neutrophils is less than about 35,000 per ml, and having the value one (1) if said cell count of neutrophils is greater than about 35,000 per ml, and obtaining said weighted value of said neutrophils cell count by multiplying said discretized cell count value by a cell count coefficient having a value of 1.6;

obtaining a concentration of Human type of Cathelicidin (LL-37) in said serum sample, comprising obtaining a weighted value of said LL-37 concentration;

obtaining a discretized LL-37 concentration value, wherein said discretized LL-37 concentration value having a value zero (0) if said concentration of LL-37 is less than about 46 ng/ml, and a value one (1) if said concentration of LL-37 is greater than about 46 ng/ml, and obtaining said weighted value of said LL-37 concentration by multiplying said discretized LL-37 concentration value by a LL-37 coefficient having a value of 0.9; and

obtaining a concentration of Chitinase 3-like 1 (CHI3L1) in said serum sample, comprising obtaining a weighted value of CHI3L1 concentration;

obtaining a discretized CHI3L1 concentration value, wherein said discretized CHI3L1 concentration value having value “

0”

if said concentration of CHI3L1 is less than about 22 ng/ml, and having a value one (1) if said concentration of CHI3L1 is greater than about 22 ng/ml, and obtaining said weighted value of said CHI3L1 concentration by multiplying said discretized CHI3L1 concentration value by a CHI3L1 coefficient having a value of 1.1;

obtaining an index value by summing said weighted value of CRP concentration, said weighted value of neutrophils cell count, said weighted value of said LL-37 concentration and said weighted value of CHI3L1 concentration; and

determining that said human subject is responding to a treatment when said index value is within the index lower quartile indicative of a decrease in Mayo endoscopic subscore to Mayo endoscopic subscore level of zero or one of said human subject.

27. A method as in claim 26 further comprising alternatively obtaining at least one of said concentrations of CRP, LL-37 and CHI3L1 in a blood plasma sample.

28. A method as in claim 26 further comprising an assessment of the status of mucosal healing in relation to endoscopic healing according to the Mayo endoscopic subscore in a human subject is not responding to a treatment when said index value is within the index upper quartile indicative an increase in Mayo endoscopic subscore to Mayo endoscopic subscore level of 2 or 3 of said human subject.

29. A system for non-invasively determining mucosal healing in human subjects undergoing treatment for inflammatory bowel disease using a set of biomarkers, comprising:

a body specimen collection means for obtaining a body specimen from a human subject;

an assay for measuring a concentration of c-reactive protein (CRP) in said body specimen;

a cell count system for producing a cell count of neutrophils in a blood sample from said human subject;

an assay for measuring a concentration of Human type of Cathelicidin (LL-37) in said body specimen;

an assay for measuring a concentration of Chitinase 3-like 1 (CHI3L1) in said body specimen; and

a computer system configured to obtain data pertaining to said concentration of CRP, said a cell count of neutrophils, said concentration of LL-37 and said concentration of CHI3L1, and having an interface to produce an output for use by a practitioner assessing mucosal healing in response to treatment, said computer system is configured with program code, when executed, is configured to cause said computer system to;

generate a weighted value of CRP concentration;

generate a weighted value of neutrophils cell count;

generate a weighted value of said LL-37 concentration;

generate a weighted value of CHI3L1 concentration;

generate an index value by summing said weighted value of CRP concentration, said weighted value of neutrophils cell count, said weighted value of said LL-37 concentration and said weighted value of CHI3L1 concentration; and

determine that said human subject has a low mucosal healing if said index is above the index upper quartile, when said weighted value of CRP concentration, said weighted value of neutrophils cell count, said weighted value of said LL-37 concentration and said weighted value of CHI3L1 concentration are adequately selected such that said index ranges.

30. A system as in claim 29 further comprising said Index is ranges from zero to ten

31. A system as in claim 29 further comprising said Index is ranges from zero to one hundred

32. A system as in claim 29, wherein said body specimen collection means further comprises a blood sample collection means from a human subject affected by Ulcerative Colitis, further comprising an assay for measuring a concentration of CRP in said blood sample, an assay for measuring a concentration of LL-37 in said blood sample, an assay for measuring a concentration of CHI3L1 in said blood sample, and wherein said computer system further comprises computer program code configured to cause said computer system to:

generate a discretized CRP concentration value of said concentration of CRP, wherein said discretized value of CRP having a value zero (0) if said concentration of CRP is less than about 2.8 ng/ml, and a value one (1) if said concentration of CRP is greater than about 2.8 ng/ml, and obtaining said weighted value of CRP concentration by multiplying said discretized CRP concentration value by a CRP coefficient;

generate a discretized cell count value of said cell count of neutrophils, wherein said discretized value of said cell count having a value zero (0) if said cell count of neutrophils is less than about 35,000 per ml, and having the value one (1) if said cell count of neutrophils is greater than about 35,000 per ml, and obtaining said weighted value of said neutrophils cell count by multiplying said discretized cell count value by a cell count coefficient;

generate a discretized LL-37 concentration value, wherein said discretized LL-37 concentration value having a value zero (0) if said concentration of LL-37 is less than about 46 ng/ml, and a value one (1) if said concentration of LL-37 is greater than about 46 ng/ml, and obtaining said weighted value of said LL-37 concentration by multiplying said discretized LL-37 concentration value by a LL-37 coefficient; and

generate a discretized CHI3L1 concentration value, wherein said discretized CHI3L1 concentration value having value “

0”

if said concentration of CHI3L1 is less than about 22 ng/ml, and having a value one (1) if said concentration of CHI3L1 is greater than about 22 ng/ml, and obtaining said weighted value of said CHI3L1 concentration by multiplying said discretized CHI3L1 concentration value by a CHI3L1 coefficient.

33. A system as in claim 32, wherein said computer system further comprising computer program code configured to cause said computer to:

assign a value 1.12 to said CRP coefficient;

assign a value 1.6 to said cell count coefficient;

assign a value 0.9 to said LL-37 coefficient; and

assign a value 1.1 to said CHI3L1 coefficient.

34. A kit for assessing mucosal healing in human subjects affected by inflammatory bowel disease, comprising:

a kit for collecting a blood sample from a human subject;

a cell count system for counting neutrophils in said blood sample and providing a weighted value of neutrophils cell count;

an immuno-assay kit for measuring a concentration of c-reactive protein (CRP) in said blood sample and provide a weighted value of said concentration of CRP;

an immuno-assay kit for measuring a concentration of Human type of Cathelicidin (LL-37) in said blood sample and provided a weighted value of said concentration of LL-37; and

an immuno-assay for measuring a concentration of Chitinase 3-like 1 (CHI3L1) in said blood sample and provide a weighted value of said concentration of CHI3L1.

35. A kit as in claim 34, wherein said an immuno-assay kit for measuring said concentration of CRP, Human type of Cathelicidin (LL-37), Chitinase 3-like 1 (CHI3L1) further comprising multiplex monoclonal antibodies including a monoclonal antibody specific to CRP, a monoclonal antibody specific to Human type of Cathelicidin (LL-37) and a monoclonal antibody specific to Chitinase 3-like 1 (CHI3L1).

36. A kit as in claim 35, wherein said an immuno-assay kit for measuring said concentration of LL-37 further comprising an enzyme-linked to said monoclonal antibody.

37. A kit as in claim 34, wherein said immuno-assay kit for measuring said concentration of LL-37, further comprises micro titer wells for recognizing and capturing human LL-37.

38. A kit as in claim 37, wherein said immuno-assay kit for measuring said concentration of LL-37, further comprises biotinylated tracer antibody that binds to captured human LL-37.

39. A kit as in claim 38, further comprises Streptavidin-peroxidase conjugate for binding to the biotinylated tracer antibody.

40. A kit as in claim 34, wherein said immuno-assay kit for measuring said concentration of CHI3L1, further comprises a monoclonal antibody specific for human CHI3L1 pre-coated onto a microplate.

41. A kit as in claim 40, further comprises an enzyme-linked polyclonal antibody specific for human CHI3L1.

42. A kit as in claim 34 further comprising a monoclonal antibody specific to NGAL-MMP-9.

43. A kit as in claim 35 further comprises a multiplex monoclonal antibodies including a monoclonal antibody specific for human NGAL-MMP-9.