MONITORING INFLAMMATION STATUS

FIELD OF THE INVENTION

The present invention relates to the identification of markers that predict or identify an inflammatory event. In particular, the invention relates to prediction and identification of exacerbation events, more specifically pulmonary exacerbations, based upon measuring urinary markers. The invention permits monitoring of inflammatory status by an individual by providing a personalised home use test.

BACKGROUND TO THE INVENTION

There are a number of different disorders of the respiratory tract, many of which have an inflammatory component. Examples included chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF).

The chronic infection and inflammation of lung disease can cause a progressive decline of lung function resulting in daily symptoms such as cough and sputum production.

There are intermittent episodes of acute worsening of symptoms, more commonly referred to as pulmonary exacerbations. Pulmonary exacerbations (PEx) are a major cause of morbidity, mortality and hospital admission.
DESCRIPTION OF THE INVENTION
It would be useful to be able to accurately monitor subjects to predict PEx events before they happen, or identify the early onset of a PEx. This would allow early interventions to minimize the inflammatory damage caused by the PEx. Ideally, this can be achieved in a home self-testing approach. The inventors have discovered that urine samples are a useful source of markers which can predict PEx events, enabling home testing for predicting PExs. The markers may be indicative of neutrophil activation and thus may be termed "neutrophil activation markers".
Accordingly, in a first aspect, the invention prvides a method for monitoring
inflammation status of a subject, the method comprising determining levels of at least one (neutrophil activation) marker in urine samples taken from the subject at multiple time points, wherein increased levels of the at least one neutrophil activation marker in a urine sample are indicative of or predictive of an exacerbation of inflammation.

The method is preferably implemented in a system or kit for home use monitoring.
Accordingly, the invention also provides a system or test kit for monitoring inflammation status in a subject, comprising:
a. One or more testing devices for determining levels of at least one
(neutrophil activation) marker in a urine sample
b. A processor; and
c. A storage medium comprising a computer application that, when executed by the processor, is configured to:

i. Access and/or calculate the determined levels of the at least one neutrophil activation marker in the urine sample on the one or more testing devices

ii. Calculate whether there is an increased or decreased level (or whether the level remains the same) of the at least one neutrophil activation marker in the urine sample; and
iii. Output from the processor the current inflammation status of the subject, wherein increased levels of the at least one neutrophil activation marker in a urine sample are indicative of or predictive of an exacerbation of inflammation.
The invention also relates to a corresponding computer application for use in the system or test kit.

The inventors have determined that specific and sensitive results may be achieved by combining a plurality of urinary markers in order to monitor or predict PEx events.

Accordingly, the invention also provides a method for monitoring inflammation status of a subject, the method comprising determining levels of at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more markers in urine samples taken from the subject at multiple time points, wherein increased levels of at least one of the markers in a urine sample indicates or predicts an exacerbation of inflammation.

Similarly, the invention also provides a system or test kit for monitoring inflammation status in a subject, comprising:

a. One or more testing devices for determining levels of at least 2, 3, 4, 5, 6,

7, 8, 9, 10 or more markers in a urine sample

b. A processor; and

c. A storage medium comprising a computer application that, when executed by the processor, is configured to:

i. Access and/or calculate the determined levels of each marker in the urine sample on the one or more testing devices

ii. Calculate whether there is an increased or decreased level (or whether the level remains the same) of at least one of the markers in the urine sample; and

iii. Output from the processor the current inflammation status of the subject, wherein increased levels of at least one of the markers in a urine sample are indicative of or predictive of an exacerbation of inflammation.

The invention also relates to a corresponding computer application for use in the system or test kit.

According to all aspects of the invention, the markers are useful for monitoring of inflammation. Thus as well as proving useful for identifying or predicting an inflammatory event such as a PEx, the markers may also be useful for indicating a recovery from, or successful treatment of, the inflammatory event. Accordingly, in some embodiments decreased levels of the at least one marker in a urine sample following an increase are indicative or predictive of recovery from, or successful treatment of, an exacerbation of inflammation. The decrease may be down to pre-exacerbation levels and then may be measured on an on-going basis to ensure they are maintained thereafter. The invention may thus be used in conjunction with and to guide treatment of individual subjects. The invention may be used to monitor on-going treatment and to assist with determination of whether treatments should be altered (e.g. the dosage adjusted, level of intervention altered), stopped or replaced with an alternative. Similarly, stable levels of markers in urine of subjects may indicate the condition is being managed and the inflammatory status is stable.

The invention may be applicable to identifying bacterial or viral causes of an

exacerbation in some embodiments.

In specific embodiments according to all aspects of the invention the monitored inflammation status is lung inflammation status. In further embodiments, the

exacerbation of inflammation that is indicated and/or predicted is a pulmonary

exacerbation.

The subject is a mammalian subject, typically a human. In certain embodiments, the subject is suffering from a respiratory disorder. More specifically, the respiratory disorder may be chronic obstructive pulmonary disease (COPD) or cystic fibrosis (CF). The inventors have accumulated data showing the effectiveness of this approach in these specific disease conditions. COPD represents a collection of lung diseases including chronic bronchitis, emphysema and chronic obstructive airways disease and thus any of these lung diseases may be monitored according to the invention. The invention may also be applicable to monitoring of asthma and interstitial lung disease (ILD). The invention may also be applied to bronchiectasis.

It should be noted that the invention is performed in vitro based upon isolated urine samples. The urine sample may be a mid-stream urine sample in some embodiments. The methods of the invention may include steps of obtaining a urine sample for testing in some embodiments. Similarly, in some embodiments, the systems and test kits include suitable vessels for receiving a urine sample. Those vessels may be specifically adapted for urine collection and may be different depending upon the gender of the subject. Commercially available examples include the Peezy MSU Urine Collection Device (Williams Medical). The container may be coloured to protect any light sensitive analytes.

By "marker" is meant a molecule indicative of inflammation, typically indicative of neutrophil activation. In some embodiments, the at least one marker is selected from a signalling molecule or an effector/effector inhibitor molecule. It should be noted that throughout the specification the term "the at least one marker" includes "at least one of the markers" where levels of a plurality of markers are being detected. Signalling molecules may be responsible for recruitment of the molecules that cause inflammatory damage. The effector molecules cause inflammatory damage. They may be enzymes. The effector inhibitor molecules inhibit the activity of the effector molecules. They may be enzyme inhibitors.

In some embodiments the effector molecule is selected from a protease activity, Neutrophil gelatinase-associated lipocalin (NGAL), calprotectin or myeloperoxidase (MPO). The effector molecule, in particular NGAL activity, may be either free or in complex. In further embodiments, the protease activity is selected from matrix metalloproteinase (MMP) activity, human neutrophil elastase (HNE) activity and cathepsin G activity. There are various MMPs which may usefully be detected, as discussed in further detail herein. In specific embodiments, MMP activity comprises MMP9 and/or MMP8 activity.

According to the invention, levels of effector inhibitor molecules may additionally or alternatively be determined. In specific embodiments, the effector inhibitor molecule comprises, consists essentially of or consists of (i.e. is) a protease inhibitor molecule. In some embodiments, the protease inhibitor molecule is selected from Tissue Inhibitor of metalloproteinase (TIMP), cystatin c, secreted leukocyte protease inhibitor (SLPI) and alpha-1 antitrypsin (A1 AT).

According to the invention, levels of signalling molecules may additionally or alternatively be determined. In specific embodiments, the signalling molecule is selected from ICAM- 1 , IL-6, IL-1 β, IL-8, N-formyl-Met-Leu-Phe (fMLP), IL-6 induced fibrinogen, fragments of complement proteins and cytokine induced beta-2-microglobulin (B2M).

In certain embodiments, the at least one marker comprises or further comprises a molecule produced as a consequence of inflammation. In specific embodiments, the molecule produced as a consequence of inflammation comprises a degradation product of protease activity, such as an extracellular matrix breakdown product and/or a product of oxidative damage such as chlorinated peptides and/or metabolites such as lactic acid and free fatty acid. Examples of extracellular matrix breakdown products include collagen breakdown products such as Ac-PGP, elastin fragments/peptides, desmosine.

In specific embodiments, however, the levels of desmosine are not measured. In some embodiments, levels of large elastin fragments (LEF) may be measured.

Specific markers useful in the method include TIMP1 , a tissue inhibitor of

metalloproteinases, and cystatin c, a lysosomal proteinase inhibitor. Other useful markers which may be employed in combination include MMP level or activity and A1AT level or activity. The markers may be selected from those listed in Figure 1 in some embodiments. In certain embodiments, the marker or markers is/are selected from TIMP (in particular TIMP2), NGAL, A1AT, IL-6, FMLP, creatinine, cystatin c, HSA, RBP4 and beta 2 microglobulin. IL-8 may also be a useful marker. Desmosine may be a useful marker when measured by ELISA. Each of these markers has been shown to be individually useful in indicating an inflammatory exacerbation (see Table 1 and Example 2 herein). Other specific marker combinations which may be useful in the invention include:

A method for monitoring inflammation status of a subject, the method comprising determining levels of at least one neutrophil activation marker in urine samples taken from the subject at multiple time points, wherein increased levels of the at least one neutrophil activation marker in a urine sample are indicative of or predictive of an exacerbation of inflammation and/or wherein decreased levels of the at least one neutrophil activation marker in a urine sample following an increase are indicative or predictive of recovery from, or successful treatment of, an exacerbation of

inflammation.

A method for monitoring inflammation status of a subject, the method comprising determining levels of at least three markers in urine samples taken from the subject at multiple time points, wherein increased levels of at least one of the markers in a urine sample indicates or predicts an exacerbation of inflammation and/or wherein decreased levels of at least one of the markers in a urine sample following an increase indicate or predict recovery from, or successful treatment of, an

exacerbation of inflammation.

The method of claim 1 or 2 wherein at least one of the markers is selected from CRP, CC16 and large elastin fragments (LEF).

A method for monitoring inflammation status of a subject, the method comprising determining levels of C-reactive protein (CRP) in urine samples taken from the subject at multiple time points, wherein increased levels CRP in a urine sample are indicative of or predictive of an exacerbation of inflammation and/or wherein decreased levels of CRP in a urine sample following an increase are indicative or predictive of recovery from, or successful treatment of, an exacerbation of

inflammation.

The method of claim 3 further comprising determining levels of at least one further marker in the urine samples.

The method according to claim 1 wherein the at least one neutrophil activation marker is selected from a signalling molecule or an effector/effector inhibitor molecule, or according to claim 2 wherein at least one of the markers is selected from a signalling molecule or an effector/effector inhibitor molecule, or according to claim 5 wherein at least one further marker is selected from a signalling molecule or an effector/effector inhibitor molecule.

The method according to claim 6 wherein the effector molecule is selected from a protease activity, Neutrophil gelatinase-associated lipocalin (NGAL) (either free or in complex), calprotectin or myeloperoxidase (MPO).

The method according to claim 7 wherein the protease activity is selected from matrix metalloproteinase (MMP) activity, HNE activity and cathepsin G activity, optionally wherein MMP activity comprises MMP9 and/or MMP8 activity.

The method according to claim 7 or 8 wherein protease activity is determined by measuring cleavage of a peptide substrate.

The method according to any one of claims 7 to 9 wherein protease activity is determined by a method comprising:

a. bringing an indicator molecule into contact with the test sample, said indicator molecule comprising

i. a cleavage region comprising at least one cleavage site, which can be cleaved by said protease if present; and

ii. a capture site;

wherein cleavage of the at least one cleavage site produces a novel binding site;

b. adding to the test sample binding molecules capable of binding to the novel binding site, wherein the binding molecules are incapable of binding to the indicator molecule unless and until cleavage has occurred;

c. capturing the part of the indicator molecule containing the novel binding site at a capture zone through binding of capture molecules in the capture zone to the capture site; and

d. detecting cleavage of the at least one cleavage site by determining binding of the binding molecules to the novel binding site of the indicator molecule captured in the capture zone.

The method according to any one of claims 6 to 8 wherein the effector inhibitor molecule is a protease inhibitor molecule, optionally wherein the protease inhibitor molecule is selected from Tissue Inhibitor of metalloproteinase (TIMP), cystatin C and alpha-1 antitrypsin (A1 AT).

12. The method according to claim 6 wherein the signalling molecule is selected from ICAM-1 , IL-6, IL-1 β, IL-8, N-formyl-Met-Leu-Phe (fMLP), IL-6 induced fibrinogen and cytokine induced beta-2-microglobulin (B2M).

13. The method according to any preceding claim wherein the at least one neutrophil activation marker or at least one of the markers or at least one further marker comprises or further comprises a molecule produced as a consequence of inflammation, optionally wherein the molecule produced as a consequence of inflammation comprises a degradation product of protease activity, such as an extracellular matrix breakdown product (e.g. Ac-PGP, elastin fragments/peptides, desmosine) and/or a product of oxidative damage such as chlorinated peptides and/or metabolites such as lactic acid and free fatty acid.

14. The method according to any preceding claim wherein the inflammation status is lung inflammation status.

1 5. The method according to any preceding claim wherein the exacerbation of

inflammation is a pulmonary exacerbation.

16. The method according to any preceding claim wherein the subject is suffering from a respiratory disorder, optionally wherein the respiratory disorder is chronic obstructive pulmonary disease (COPD) or cystic fibrosis (CF).

17. The method according to any preceding claim wherein increased or decreased levels of the at least one neutrophil activation marker or at least one of the markers or at least one further marker are calculated with reference to a threshold level of the marker that is adapted to the subject.

18. The method according to claim 17 wherein the threshold level of the marker is set by determining the levels of the marker in urine samples taken from the subject at earlier time points, optionally wherein the earlier time points comprise at least two earlier measurements immediately preceding the determination of the level of the marker in the current urine sample.
19. The method according to claim 17 or 18 wherein the threshold level of the marker is set by determining the levels of the marker in urine samples taken from the subject at earlier time points at which the subject was not suffering from an exacerbation of inflammation and an increase above threshold predicts or identifies an exacerbation or wherein the threshold level of the marker is set by determining the levels of the marker in urine samples taken from the subject at earlier time points at which the subject was suffering from an exacerbation of inflammation and a decrease below threshold predicts or identifies recovery from, or successful treatment of, an exacerbation of inflammation.
20. The method according to any preceding claim wherein marker levels are determined at least twice a week.
21 . The method according to any preceding claim wherein the frequency of determining the marker levels in urine samples taken from the subject is increased if an increase in the marker levels is detected, optionally wherein the frequency of determining the marker levels in urine samples taken from the subject is maintained until a decrease in the marker levels is detected.
22. The method according to any preceding claim comprising determining levels of at least two or three neutrophil activation markers in urine samples taken from the subject at multiple time points.
23. The method according to any preceding claim (in which levels of a plurality of
markers is measured) wherein increased levels of at least one of the markers in a urine sample are indicative of or predictive of an exacerbation of inflammation and/or wherein decreased levels of at least one of the markers in a urine sample following an increase are indicative or predictive of recovery from, or successful treatment of, an exacerbation of inflammation and/or wherein the determined levels of the at least two or three markers are analysed in a pre-determined sequence to monitor the inflammation status of the subject and/or wherein the determined levels of the at least two or three markers are weighted.
24. The method according to any preceding claim wherein levels of at least one marker are determined by normalising against the levels of a reference marker, optionally wherein the reference marker comprises urinary creatinine or fibrinogen.
25. A system or test kit for monitoring inflammation status in a subject, comprising:
a. One or more testing devices for determining levels of at least one neutrophil activation marker in a urine sample
b A processor; and
c. A storage medium comprising a computer application that, when executed by the processor, is configured to:
i. Access and/or calculate the determined levels of the at least one neutrophil activation marker in the urine sample on the one or more testing devices
i. Calculate whether there is an increased or decreased level of the at least one neutrophil activation marker in the urine sample; and iii. Output from the processor the current inflammation status of the
subject, wherein increased levels of the at least one neutrophil activation marker in a urine sample are indicative of or predictive of an exacerbation of inflammation and/or wherein decreased levels of the at least one neutrophil activation marker in a urine sample following an increase are indicative or predictive of recovery from, or successful treatment of, an exacerbation of inflammation.
26. A system or test kit for monitoring inflammation status in a subject, comprising:
a. One or more testing devices for determining levels of at least three markers in a urine sample
b. A processor; and
c. A storage medium comprising a computer application that, when executed by the processor, is configured to:
i. Access and/or calculate the determined levels of each marker in the urine sample on the one or more testing devices
ii. Calculate whether there is an increased or decreased level of at least one of the markers in the urine sample; and
iii. Output from the processor the current inflammation status of the
subject, wherein increased levels of at least one of the markers in a urine sample are indicative of or predictive of an exacerbation of inflammation and/or wherein decreased levels of at least one of the
markers in a urine sample following an increase are indicative or predictive of recovery from, or successful treatment of, an
exacerbation of inflammation.
27. The system or test kit of claim 25 or 26 wherein at least one of the markers is
selected from CRP, CC1 6 and large elastin fragments (LEF).
28. A system or test kit for monitoring inflammation status in a subject, comprising :
a. One or more testing devices for determining levels of C-reactive protein
(CRP) in a urine sample
b. A processor; and
c. A storage medium comprising a computer application that, when executed by the processor, is configured to:
i. Access and/or calculate the determined levels of CRP in the urine sample on the one or more testing devices
ii. Calculate whether there is an increased or decreased level of CRP in the urine sample; and
iii. Output from the processor the current inflammation status of the
subject, wherein increased levels of CRP are indicative of or predictive of an exacerbation of inflammation and/or wherein decreased levels of CRP in a urine sample following an increase are indicative or predictive of recovery from, or successful treatment of, an
exacerbation of inflammation.
29. The system or test kit of claim 28 which further determines levels of at least one further marker in the urine samples.

30. The system or test kit of any one of claims 25 to 29 further comprising a display for the output from the processor and/or wherein the one or more testing devices are disposable single use devices and/or wherein the one or more testing devices comprise lateral flow test strips, optionally comprising a lateral flow test strip for each marker that is determined.
31 . The system or test kit of any one of claims 25 to 30 wherein at least one marker is selected from a signalling molecule or an effector/effector inhibitor molecule, optionally wherein the effector molecule is selected from a protease activity,

Neutrophil gelatinase-associated lipocalin (NGAL) (either free or in complex),

calprotectin or myeloperoxidase (MPO), optionally wherein the protease activity is selected from matrix metalloproteinase (MMP) activity, HNE activity and cathepsin G activity, optionally wherein MMP activity comprises MMP9 and/or MMP8 activity, optionally wherein the one or more testing devices comprises a testing device for measuring cleavage of a peptide substrate as an indicator of protease activity, optionally wherein the testing device comprises:
a. an indicator molecule for adding to the urine sample, said indicator molecule comprising
i. a cleavage region comprising at least one cleavage site, which can be cleaved by said protease activity if present; and
ii. a capture site;
wherein cleavage of the at least one cleavage site produces a novel binding site; b. a capture zone to receive the urine sample, wherein the capture zone

comprises capture molecules capable of binding to the capture site of the indicator molecule in order to immobilise the indicator molecule including the novel binding site; and

c. binding molecules capable of binding to the novel binding site, wherein the binding molecules are incapable of binding to the indicator molecule unless and until cleavage has occurred.
32. The system or test kit of claim 31 wherein the effector inhibitor molecule is a protease inhibitor molecule, optionally wherein the protease inhibitor molecule is selected from Tissue Inhibitor of metalloproteinase (TIMP), cystatin C and alpha-1 antitrypsin (A1 AT), or wherein the signalling molecule is selected from ICAM-1 , IL-6, IL-1 β, IL-8, N-formyl-Met-Leu-Phe (fMLP), IL-6 induced fibrinogen and cytokine induced beta-2- microglobulin (B2M).

33. The system or test kit of any one of claims 25 to 32 wherein the marker or markers comprises or further comprises a molecule produced as a consequence of inflammation, optionally wherein the molecule produced as a consequence of inflammation comprises a degradation product of protease activity, such as an extracellular matrix breakdown product (e.g. Ac-PGP, elastin fragments/peptides, desmosine) and/or a product of oxidative damage such as chlorinated peptides and/or metabolites such as lactic acid and free fatty acid.
34. The system or test kit of any one of claims 25 to 33 wherein the inflammation status is lung inflammation status and/or wherein the exacerbation of inflammation is a pulmonary exacerbation and/or wherein the subject is suffering from a respiratory disorder, optionally wherein the respiratory disorder is chronic obstructive pulmonary disease (COPD) or cystic fibrosis (CF).
35. The system or test kit of any one of claims 25 to 34 wherein the computer application causes the processor to calculate levels of the marker or markers with reference to a threshold level of the marker that is adapted to the subject, optionally wherein the threshold level of the marker is set based upon determined levels of the marker in urine samples taken from the subject at earlier time points, optionally wherein the earlier time points comprise at least two earlier measurements immediately preceding the determination of the level of the marker in the current urine sample.

36. The system or test kit of claim 35 wherein the threshold level of the marker is set based upon determined levels of the marker in urine samples taken from the subject at earlier time points at which the subject was not suffering from an exacerbation of inflammation and an increase above threshold predicts or identifies an exacerbation and/or wherein the threshold level of the marker is set based upon determined levels of the marker in urine samples taken from the subject at earlier time points at which the subject was suffering from an exacerbation of inflammation and a decrease below threshold predicts or identifies recovery from, or successful treatment of, an exacerbation of inflammation.

37. The system or test kit of any one of claims 25 to 36 wherein the computer application causes the processor to indicate to the subject the requirement to determine the levels of at the marker or markers and/or wherein the computer application is further configured to output from the processor a requirement to increase the frequency of determining the levels of the marker or markers in urine samples taken from the subject where an increase in the levels of the at least one marker is calculated, optionally wherein the computer application is further configured to output from the processor a requirement to maintain the increased frequency of determining the levels of the at least one neutrophil activation marker until a decrease in the levels of the at least one marker is calculated.

38. The system or test kit of any one of claims 25 to 37 comprising one or more testing devices for determining levels of at least two or three neutrophil activation markers in urine samples taken from the subject at multiple time points.

39. The system or test kit of any one of claims 25 to 38 wherein the computer application is configured to calculate increased levels of at least one of the markers and provide an output from the processor that a calculated increase in levels of at least one of the markers is indicative of or predictive of an exacerbation of inflammation
and/orwherein the computer application is configured to calculate decreased levels of at least one of the neutrophil activation markers and provide an output from the processor that a calculated decrease in levels of at least one of the markers following an increase are indicative or predictive of recovery from, or successful treatment of, an exacerbation of inflammation and/or wherein the computer application is configured to analyse the calculated levels of the at least two or three markers in a pre-determined sequence to monitor the inflammation status of the subject and/or wherein the computer application is configured to apply a weighting to the

determined levels of the at least two or three markers.
40. The system or test kit of any one of claims 25 to 39 wherein the computer application is configured to calculate levels of at least one neutrophil activation marker by normalising against the levels of a reference marker, optionally wherein the reference marker comprises urinary creatinine or fibrinogen and/or wherein the computer application is further configured to incorporate inputs from other indicators of exacerbation of inflammation into the calculation of the current inflammation status of the subject, optionally wherein the other indicators of exacerbation of inflammation comprise shortness of breath, increased wheeze, increased pulse rate, dyspnoea, increased sputum purulence, increased sputum colour, sore throat, increased cough, cold and fever.
41 . A computer application as defined in any one of claims 25 to 40.