WO2007051259A1

WO2007051259A1 - Sleep apnea diagnosis or monitoring based on detecting levels of marker proteins in blood

Info

Publication number: WO2007051259A1
Application number: PCT/AU2006/001652
Authority: WO
Inventors: Glenn Richards; Adam Vivian Benjafield
Original assignee: Resmed Ltd
Priority date: 2005-11-04
Filing date: 2006-11-03
Publication date: 2007-05-10
Also published as: CN101356440A

Abstract

The invention is the use of a blood-based assay to aid the diagnosis and treatment of patients. A further aspect of the invention is to utilize a blood-based assay as an indication for the presence of sleep disordered breathing (SDB). In addition, the progress of the disease in a patient is monitored using a blood-based assay as well as the effectiveness, and compliance by the patient, of a treatment regime.

Description

SLEEP APNEA DIAGNOSIS OR MONITORING BASED ON DETECTING LEVELS OF MARKER PROTEINS IN BLOOD

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of the following application: 1 ) Australian Provisional Serial No. 2005906112, filed November 4, 2005. The application is hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

[0002] The invention relates to methods and apparatus to aid the diagnosis and management of Sleep Disordered Breathing.

BACKGROUND OF THE INVENTION

[0003] Sleep disordered breathing (SDB), in particular obstructive sleep apnea (OSA) syndrome is a common health problem affecting as much as 4 to 9% of the adult population. It has been associated with increased morbidity and mortality from cardiovascular and cerebrovascular conditions. OSA is also associated with excessive daytime somnolence leading to intellectual deterioration, mood changes and increased motor vehicle accidents.

[0004] SDB, namely OSA, is currently diagnosed and assessed by full polysomnography (for example Puritan Bennett's Sandman or Medcare's Embla) and by other portable recording devices such as the Embletta (Medcare) and ApneaLink (ResMed). These methods can be time consuming, labour intensive and expensive. [0005] As well as this, resources are limited for the mass screening of populations because the devices record overnight sleep variables. For example full polysomnography can require that a patient spend two nights in a sleep clinic. In addition, in some hospital clinics a patient may need to wait for a year before a suitable bed is available.

[0006] The present invention is directed towards a simple, cheap and quick assay for the presence or level of a sleep disorder such as OSA. SUMMARY OF THE INVENTION

[0007] A first aspect of the invention is to use a blood-based assay to aid the diagnosis and treatment of patients. In one form a blood-based assay is used as an indication of the presence of sleep disordered breathing. In another form, the progress of disease in a patient is monitored using a blood-based assay. In another form, patient compliance with a treatment regime is monitored using a blood-based assay. In another form, the effectiveness of a form of treatment is monitored using a blood-based assay. [0008] In another form, the blood-based assay is used in conjunction with a non- blood assay, for example an Epworth Sleepiness test.

[0009] In one form of the invention an ELISA is used to detect a number of biochemical markers indicative of sleep disordered breathing.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] A number of biochemical markers found in blood are significantly altered in people with SDB compared with the levels of non-SDB sufferers. The present invention contemplates using one or more of these markers to monitor and diagnose SDB. By combining these markers into a panel a significant improvement is made in the accuracy of the test.

[0011] In one form the panel of markers is tested by enzyme-linked immunosorbent assay (ELISA), and by standard biochemical blood testing for C- reactive protein (CRP) and creatine phosphokinase (CK). ELISA is a rapid immunochemical test that involves an enzyme (a protein that catalyzes a biochemical reaction), and also involves an antibody or antigen (immunologic molecules).

[0012] In another form the blood-based test is used to quantify changes in SDB patients after treatment, for example, continuous positive airway pressure (CPAP).

[0013] In one form, the blood-based assay tests for a lowering of the occurrence of hypoxia/reoxygenation. In aother form the blood based assay tests for sympathetic nervous system activation.

[0014] In one form improvement in these markers is used to monitor compliance with CPAP.

[0015] The procedure for performing this assay begins with a qualified professional drawing a blood sample from the patient under standard blood collecting procedures.

The blood sample is prepared and tested by a pathology organization. An aliquot of whole blood is used for measuring CRP and CK. The remaining markers are measured from serum obtained by using standard laboratory techniques for obtaining serum from whole blood.

[0016] All of the ELISA based assays are performed in microtitre plates suitable for this method with each of the markers, listed below, individually allocated to a specific well of a microtitre plate. Standard laboratory procedures for performing an ELISA, as well as measuring CRP and CK are to be performed to quantify the level of these markers.

[0017] In one form the following markers are tested:

Tumor necrosis factor-α (TN F-α) lnterleukin-6 (IL-6) lnterleukin-8 (IL-8) lnterleukin-18 (IL-18)

C-reactive protein (CRP)

Serum amyloid A (SAA)

Matrix metalloproteinase-9 (MMP-9)

Creatine phosphokinase (CK) .

8-isoprostane

Vascular endothelial growth factor (VEGF)

Intercellular adhesion molecule-1 (ICAM-1)

Granulocyte chemotactic proteiri-2 (GCP-2)

[0018] Each of these markers, and their relevance to SDB, are described later. [0019] The greater the number of markers that are elevated the greater the risk of SDB. Elevation of all of the tested markers indicates a high risk of SDB. Sensitivity and specificity of the assay are improved further by incorporating the results of body habitus (for example, neck circumference, BMI and snoring), sleep related questionnaires (for example, Epworth Sleepiness Scale), blood pressure and related medical conditions (for example, stroke, coronary artery disease, atherosclerosis, congestive heart disease and type Il diabetes) with the results for the blood-based test(s). When this additional information is suggestive of the characteristics of a typical sufferer of SDB, fewer number of blood-based marker elevations are required to indicate the risk of SDB. MARKERS

Tumor necrosis factor- α

[0020] A proinflammatory cytokine that is involved in the pathogenesis of many diseases. Proinflammatory cytokines have a direct effect on glucose and lipid metabolism. TNF-α is involved in atherosclerosis by inducing/stimulating ICAM-T, vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1 by endothelial cells. Research shows serum levels of TNF-α are determined mainly by the production from adipocytes and monocytes due to hypoxia. TNF-α as well as IL-6 play a significant role in mediating sleepiness and fatigue in disorders of excessive daytime sleepiness. Levels of TNF-α in OSA patients are elevated. A measurement of TNF-α >

2.0 pg/ml indicates possible OSA.

lnterleukin-6

[0021] Hormonally regulated cytokine and production is suppressed by glucocorticoids and stimulated by catecholamines through β-adrenergic receptors. The increased peripheral sympathetic nervous activity in OSA elevates IL-6 levels. Hypoxia induces expression of this proinflammatory cytokine. Increased proinflammatory cytokine production in OSA patients has important consequences to the patient in regards to increased risk for developing cardiovascular and cerebrovascular diseases. IL-6 is significantly raised in OSA patients. A measurement of IL-6 > 1.1 pg/ml indicates possible OSA.

lnterleukin-8

[0022] Hypoxia induces the synthesis and expression of IL-8 via activation of NF- KB. OSAinduced hypoxic stress increases circulating inflammatory mediators, leading to cardiovascular lesions. IL-8 is produced and secreted by adipose tissue, and plays an important role in the development of atherosclerosis. It also increases the numbers and expression of adhesion molecules such as L-selectin. Levels of this cytokine are elevated in OSA patients.

lnterleukin-18

[0023] A potent proinflammatory cytokine that promotes atherosclerosis and increased levels of IL-18 correlate with cardiovascular events. Expression of IL-18 can be induced by other cytokines such as TNF-α and IL-6. Body mass index and plasma levels of IL-18 are positively correlated in obese subjects. Elevated levels of IL-18 are found in OSA patients. A measurement of IL-18 > 225.0 pg/ml indicates possible OSA.

C-reactive protein

[0024] CRP is a nonspecific marker for inflammation. It is a prooxidant that induces production of monocyte chemoattractant protein-1 and expression of adhesion molecules such as ICAM-1 and vascular cell adhesion molecule-1. Hypoxia increases IL-6 production through activation of NF-κB and thus increases CRP levels by the liver. Elevated levels of CRP are found in OSA patients.

Serum amyloid A

[0025] SAA is one of the major acute-phase proteins in humans that is upregulated by inflammatory cytokines, including IL-1 and IL-6. It is synthesized predominantly by the liver. Elevated SAA is associated with increased risk of coronary heart disease. Hypoxia stimulates the genes of acute-phase proteins, as well as cytokines known to induce these proteins. OSA patients have elevated SAA levels. A measurement of SAA

> 15.0 μg/ml indicates possible OSA.

Matrix metalloproteinase-9

[0026] Expression of MMPs in increased in the remodelling processes of atherosclerosis and myocardial infarction. They regulate the degradation of the extracellular matrix and play an important role in cardiac and vascular remodeling. MMP-9 is stimulated by hypoxia and by several cytokines, such as IL-6 and TNF-α. Levels of this endoproteinase in OSA patients are elevated. A measurement of MMP-9

> 140.0 ng/ml indicates possible OSA.

Creatine phosphokinase

[0027] Intermittent hypoxia leads to possible oxygenation reperfusion injury, which produces large amounts of free radicals. These free radicals damage the mitochondria leading to a change in lipid oxidation and inducing hyperlipidemia and atherosclerosis. Reduced mitochondrial function and free radicals affect endothelial function (blood pressure), cause accumulation of lipids in tissue and induce insulin resistance/type 2 diabetes. There is a deficiency in total creatine in OSA patients and this limits their ability to buffer these repetitive hypoxic episodes. Elevation of CK is a marker for these events. Serum levels of CK are elevated in OSA patients. A measurement of CK > 150.0 U/L indicates possible OSA.

8-isoprostane

[0028] lsoprostanes are formed by the effect of oxidative stress on arachidonic acid, which is generated from membrane phospholipids by phospholipase A2. Their stability, specificity for lipid peroxidation and relative abundance in biological fluids make isprostanes very reliable biomarkers of lipid peroxidation and oxidative stress. 8- isoprostane, as a marker of oxidative stress, has been widely investigated in pulmonary disease. It provides a quantitative measure of oxidant stress due to hypoxia/reoxygenation in OSA, as levels are elevated in OSA patients. A measurement of 8-isoprostane > 8.5 pg/ml indicates possible OSA.

Vascular endothelial growth factor

[0029] VEGF is a glycoprotein that stimulates normal and abnormal vessel growth and has a well established role in the pathophysiology of cardiovascular disease. Expression of the VEGF gene is mainly stimulated by hypoxia and pulsatile stretch caused by apnea-related blood pressure oscillations. These stimulate VEGF secretion in OSA. Angtiotensiή Il (Ang II) also stimulates VEGF production and elevated levels of Ang Il are observed in OSA. Levels of VEGF are elevated in OSA patients. A measurement of VEGF > 380.0 pg/ml indicates possible OSA.

Intercellular adhesion molecule- 1

[0030] Synthesis and expression of ICAM-1 , via activation of NF-κB, is induced by hypoxia. Leukocyte migration to inflamed tissue requires the leukocytes to adhere to the microvascular endothelium. Potential mediators responsible for this attachment include ICAM-1. ICAM-1 plays a role in ischemic heart disease, and levels are elevated in OSA patients. A measurement of ICAM-1 > 300.0 ng/ml indicates possible OSA.

Granulocyte chemotactic protein-2

[0031] Chemotactic cytokines (chemokines) such as GCP-2 and IL-8 play a major role in inflammation. GCP-2 is considered a backup chemokine of IL-8. This potent neutrophil chemokine plays a role in the existence of systemic inflammation in OSA patients. Levels of GCP-2 are shown to be elevated in OSA patients. A measurement of GCP-2 > 300.0 pg/ml indicates possible OSA.

COMBINATIONS OF MARKERS

[0032] Combining the results in a panel increases sensitivity and specificity of the bloodbased assay. That is, it enables the assay to distinguish OSA from other medical conditions. For example elevated IL-6.could indicate both OSA and inflammation from an infection (e.g. the cold virus ). By combining a test for IL-6 with a test for VEGF - which is not generally elevated in infection - the assay is more specific for OSA.

ADVANTAGES OF THE INVENTION

[0033] A blood-based test(s) offers a more practical and cheaper alternative to screen the population of possible SDB sufferers. Patients with a positive result from the bloodbased test(s) can then be referred for having a full polysomnography to confirm . the blood based test(s) results. This reduces demand on polysomnography equipment in the community, and allows this expensive test to be performed on those at a confirmed high risk for having SDB.

OTHER FORMS OF THE INVENTION

[0034] While the preferred form of the invention has been described, other forms are contemplated as being within the spirit and scope of the invention.

Claims

1. A method of detecting the presence a sleep disorder in a person comprising the step of: conducting a blood-based assay; and determining that the person has sleep disordered breathing when the blood based assay is indicative of the presence of the sleep disorder.

2. The method of claim 1 wherein the sleep disorder is obstructive sleep apnea.

3. The method of claim 1 wherein the sleep disorder is central sleep apnea.

4. The method of claim 1 wherein the blood-based assay includes a panel of tests.

5. The method of claim 1 wherein the blood-based assay detects biochemical markers of hypoxia.

6. The method of claim 1 wherein the blood-based assay detects activation of the sympathetic nervous system.

7. The method of claim 1 wherein the blood-based assay detects biochemical markers of endothelial dysfunction.

8. The method of claim 1 wherein the blood-based assay includes an enzyme- linked immunosorbent assay (ELISA).

9. The method of claim 1 wherein the blood-based assay includes a biochemical assay for C-reactive protein (CRP).

10. The method of claim 9 whereby it is determined that the person has a sleep disorder when C-reactive protein is elevated.

11. The method of claim 1 wherein the blood-based assay includes a biochemical assay for creatine phosphokinase (CK).

12. The method of claim 11 whereby it is determined that the person has a sleep disorder when creatine phosphokinase is elevated.

13. The method of claim 12 whereby it is determined that the person has a sleep disorder creatine phosphokinase is greater than 150.0 U/L

14. The method of claim 1 wherein the blood-based assay includes an assay for the presence of tumor necrosis factor-α (TN F-α).

15. The method of claim 14 whereby it is determined that the person has a sleep disorder when tumor necrosis factor-α is elevated.

16. The method of claim 15 whereby it is determined that the person has a sleep disorder when tumor necrosis factor-α is greater than 2.0 pg/ml.

17. The method of claim 1 wherein the blood-based assay includes an assay for the presence of interleukin-6 (IL-6).

18. The method of claim 17 whereby it is determined that the person has a sleep disorder when interleukin-6 is elevated.

19. The method of claim 18 whereby it is determined that the person has a sleep disorder when interleukin-6 is greater than 1.1 pg/ml.

20. The method of claim 1 wherein the blood-based assay includes an assay for the presence of interleukin-8 (IL-8)..

21. The method of claim 20 whereby it is determined that the person has a sleep disorder when interleukin-8 is elevated.

22. The method of claim 1 wherein the blood-based assay includes an assay for the presence of interleukin-18 (IL-18).

23. The method of claim 22 whereby it is determined that the person has a sleep disorder when interleukin-18 is elevated.

24. The method of claim 23 whereby it is determined that the person has a sleep disorder when interleukin-18 is greater than 225.0 pg/ml.

25. The method of claim 1 wherein the blood-based assay includes an assay for the presence of serum amyloid A (SAA).

26. The method of claim 25 whereby it is determined that the person has a sleep disorder when serum amyloid A is elevated.

27. The method of claim 26 whereby it is determined that the person has a sleep disorder when serum amyloid A is greater than 15.0 μg/ml.

28. The method of claim 1 wherein the blood-based assay includes an assay for the presence of matrix metalloproteinase-9 (MMP-9).

29. The method of claim 28 whereby it is determined that the person has a sleep disorder when matrix metalloproteinase-9 is elevated.

30. The method of claim 29 whereby it is determined that the person has a sleep disorder when matrix metailoproteinase-9 is greater than 140.0 ng/ml.

31. The method of claim 1 wherein the blood-based assay includes an assay for the presence of 8-isoprostane.

32. The method of claim 31 whereby it is determined that the person has a sleep disorder when 8-isoprostane is elevated.

33. The method of claim 32 whereby it is determined that the person has a sleep disorder when 8-isόprostane is greater than 8.5 pg/ml. 50

34. The method of claim 1 wherein the blood-based assay includes an assay for the presence of vascular endothelial growth factor (VEGF).

35. The method of claim 34 whereby it is determined that the person has a sleep disorder when vascular endothelial growth factor is elevated.

36. The method of claim 35 whereby it is determined that the person has a sleep disorder when vascular endothelial growth factor is greater than 380.0 pg/ml.

37. The method of claim 1 wherein the blood-based assay includes an assay for the presence of intercellular adhesion molecule-1 (ICAM-1).

38. The method of claim 37 whereby it is determined that the person has a sleep disorder when intercellular adhesion molecule-1 is elevated.

39. The method of claim 38 whereby it is determined that the person has a sleep disorder when intercellular adhesion molecule-1 is greater than 300.0 ng/ml.

40. The method of claim 1 wherein the blood-based assay includes an assay for the presence of granulocyte chemotactic protein-2 (GCP-2).

41. The method of claim 40 whereby it is determined that the person has a sleep disorder when granulocyte chemotactic protein-2 is elevated.

42. The method of claim 41 whereby it is determined that the person has a sleep disorder when granulocyte chemotactic protein-2 is greater than 300.0 pg/ml.

43. A method of determining the effectiveness of a treatment of sleep disordered breathing comprising the step of: conducting a blood-based assay; and determining that treatment is effective when the blood-based assay indicates that treatment is effective.

44. An ELISA kit to assist in the diagnosis of sleep disordered breathing wherein the ELISA kit includes reagents for the detection of:

Tumor necrosis factor-α (TNF-α); lnterleukin-6 (IL-6); lnterleukin-8 (IL-8); lnterleukin-18 (IL-18);

Serum amyloid A (SAA);

Matrix metalloproteinase-9 (MMP-9);

8-isoprostane;

Vascular endothelial growth factor (VEGF);

Intercellular adhesion molecule-1 (ICAM-1); and

Granulocyte chemotactic protein-2 (GCP-2).