Methods of diagnosing tissue fibrosis
View Patent ↗The present invention provides a method of diagnosing the presence or severity of tissue fibrosis in an individual by detecting α2-macroglobulin (α2-MG) in a sample from the individual; detecting hyaluronic acid (HA) in a sample from the individual; detecting tissue inhibitor of metalloproteinases-1 (TIMP-1) in a sample from the individual; and diagnosing the presence or severity of tissue fibrosis in the individual based on the presence or level of α2-MG, HA and TIMP-1.
1. A method for diagnosing the presence or severity of liver fibrosis in an individual, the method comprising:
(a) determining a level of α2-macroglobulin (α2-MG) in a sample from said individual by contacting said sample with an α2-MG-specific binding agent;
(b) determining a level of hyaluronic acid (HA) in a sample from said individual by contacting said sample with an HA-specific binding agent;
(c) determining a level of tissue inhibitor of metalloproteinases-1 (TIMP-1) in a sample from said individual by contacting said sample with a TIMP-1-specific binding agent;
(d) calculating an index value for the individual using a regression algorithm based upon said level of α2-MG, HA, and TIMP-1, wherein said regression algorithm has a formula:
Index Value=Exp( b 0 +b 1 *x 1 +b 2 *x 2 b 3 *x 3 )/(1+Exp( b 0 +b 1 *x 1 +b 2 *x 2 +b 3 *x 3 )),
wherein
b 0 is an intercept value, wherein said intercept value is based upon the levels of α2-MG, HA, and TIMP-1 in a population of liver fibrosis patients;
b 1 is a regression coefficient value for HA, wherein said regression coefficient value is based upon the level of HA in said population of liver fibrosis patients;
x 1 is the concentration level of HA;
b 2 is a regression coefficient value for TIMP-1, wherein said regression coefficient value is based upon the level of TIMP-1 in said population of liver fibrosis patients;
x 2 is the concentration level of TIMP-1;
b 3 is a regression coefficient value for α2-MG, wherein said regression coefficient value is based upon the level of α2-MG in said population of liver fibrosis patients;
x 3 is the concentration level of α2-MG; and wherein said formula is:
Index
Value
=
Exp
[
-
4.3633
+
(
0.0108
*
HA
ng
/
ml
)
+
(
0.0015
*
TIMP
-
1
ng
/
ml
)
+
(
0.5357
*
α2
-
MG
mg
/
ml
)
]
1
+
Exp
[
-
4.3633
+
(
0.0108
*
HA
ng
/
ml
)
+
(
0.0015
*
TIMP
-
1
ng
/
ml
)
+
(
0.5357
*
α2
-
MG
mg
/
ml
)
]
;
(e) comparing the index value calculated for the individual to an index cutoff value, wherein said index cutoff value is between 0.2 and 0.7; and
(f) determining the presence or severity of liver fibrosis in said individual based upon whether the index value is above or below the index cutoff value.
2. The method of claim 1 , wherein the index cutoff value is between 0.3 and 0.6.
3. The method of claim 1 , wherein an index value greater than 0.42 correlates with moderate or severe liver fibrosis.
4. The method of claim 1 , wherein said individual has viral hepatitis.
5. The method of claim 4 , wherein said individual is infected with hepatitis C virus.
6. The method of claim 4 , wherein said individual is infected with hepatitis B virus.
7. The method of claim 4 , wherein said individual is infected with hepatitis A virus.
8. The method of claim 4 , wherein said individual is co-infected with at least two viruses.
9. The method of claim 8 , wherein one of the viruses is selected from the group consisting of hepatitis A, hepatitis B, and hepatitis C.
10. The method of claim 1 , wherein the level of α2-MG protein is determined using an anti-α2-MG antibody.
11. The method of claim 1 , wherein step (a) comprises determining a level of α2-MG activity.
12. The method of claim 1 , wherein the level of HA is determined using an HA-binding protein.
13. The method of claim 12 , wherein the level of HA is determined using an anti-HA antibody.
14. The method of claim 1 , wherein the level of TIMP-1 protein is determined using an anti-TIMP-1 antibody.
15. The method of claim 1 , wherein step (c) comprises determining a level of TIMP-1 activity.
16. The method of claim 1 , wherein the level of α2-MG protein, HA and TIMP-1 protein each is determined using an enzyme-linked assay.
17. The method of claim 1 , wherein a single sample is obtained from said individual.
18. The method of claim 17 , wherein said sample is selected from the group consisting of blood, serum, plasma, urine, saliva, and liver tissue.
19. The method of claim 18 , wherein said sample is a serum sample.
20. The method of claim 1 , comprising differentiating no or mild liver fibrosis from moderate to severe liver fibrosis.
21. A method for monitoring the efficacy of anti-fibrotic therapy in a patient, the method comprising:
(a) determining a level of α2-macroglobulin (α2-MG) in a sample from a patient administered an anti-fibrotic therapy by contacting said sample with an α2-MG-specific binding agent;
(b) determining a level of hyaluronic acid (HA) in a sample from said patient by contacting said sample with an HA-specific binding agent;
(c) determining a level of tissue inhibitor of metalloproteinases-1 (TIMP-1) in a sample from said patient by contacting said sample with a TIMP-1-specific binding agent;
(d) calculating an index value for said patient using a regression algorithm based upon said level of α2-MG, HA, and TIMP-1, wherein said regression algorithm has the a formula:
Index Value=Exp( b 0 +b 1 *x 1 +b 2 *x 2 +b 3 *x 3 )/(1Exp( b 0 +b 1 *x 1 +b 2 *x 2 +b 3 *x 3 )),
wherein
b 0 is an intercept value, wherein said intercept value is based upon the levels of α2-MG, HA, and TIMP-1 in a population of liver fibrosis patients;
b 1 is a regression coefficient value for HA, wherein said regression coefficient value is based upon the level of HA in said population of liver fibrosis patients;
x 1 is the concentration level of HA;
b 2 is a regression coefficient value for TIMP-1, wherein said regression coefficient value is based upon the level of TEMP-1 in said population of liver fibrosis patients;
x 2 is the concentration level of TIMP-1;
b 3 is a regression coefficient value for α2-MG, wherein said regression coefficient value is based upon the level of α2-MG in said population of liver fibrosis patients;
x 3 is the concentration level of α2-MG ,wherein said formula is:
Index
Value
=
Exp
[
-
4.3633
+
(
0.0108
*
HA
ng
/
ml
)
+
(
0.0015
*
TIMP
-
1
ng
/
ml
)
+
(
0.5357
*
α2
-
MG
mg
/
ml
)
]
1
+
Exp
[
-
4.3633
+
(
0.0108
*
HA
ng
/
ml
)
+
(
0.0015
*
TIMP
-
1
ng
/
ml
)
+
(
0.5357
*
α2
-
MG
mg
/
ml
)
]
;
(e) comparing the index value calculated for the individual to an index cutoff value, wherein said index cutoff value is between 0.2 and 0.7; and
(f) determining the presence or severity of liver fibrosis in said patient based upon whether the index value is above or below the index cutoff value, thereby monitoring the efficacy of anti-fibrotic therapy.
22. The method of claim 21 , further comprising comparing the presence or severity of liver fibrosis determined in step (e) to the presence or severity of liver fibrosis in said patient at an earlier time.
23. The method of claim 21 , wherein the level of α2-MG protein is determined using an anti-α2-MG antibody.
24. The method of claim 21 , wherein the level of TIMP-1 protein is determined using an anti-TIMP-1 antibody.
25. A method of differentiating no or mild liver fibrosis from moderate/severe liver fibrosis in an individual, the method comprising:
(a) determining a level of α2-macroglobulin (α2-MG) in a sample from said individual by contacting said sample with an α2-MG-specific binding agent;
(b) determining a level of hyaluronic acid (HA) in a sample from said individual by contacting said sample with an HA-specific binding agent;
(c) determining a level of tissue inhibitor of metalloproteinases-1 (TIMP-1) in a sample from said individual by contacting said sample with a TIMP--specific binding agent;
(d) calculating an index value for the individual using a regression algorithm based upon said level of α2-MG, HA, and TIMP-1, wherein said regression algorithm has following a formula:
Index Value=Exp( b 0 +b 1 *x 1 +b 2 *x 2 +b 3 *x 3 )/(1+Exp( b 0 +b 1 *x 1 +b 2 *x 2 +b 3 *x 3 ))
wherein
b 0 is an intercept value, wherein said intercept value is based upon the levels of α2-MG, HA, and TIMP-1 in a population of liver fibrosis patients;
b 1 is a regression coefficient value for HA, wherein said regression coefficient value is based upon the level of HA in said population of liver fibrosis patients;
x 1 is the concentration level of HA;
b 2 is a regression coefficient value for TIMP-1, wherein said regression coefficient value is based upon the level of TIMP-1 in said population of liver fibrosis patients;
x 2 is the concentration level of TIMP-1;
b 3 is a regression coefficient value for α2-MG, wherein said regression coefficient value is based upon the level of α2-MG in said population of liver fibrosis patients;
x 3 is the concentration level of α2-MG, wherein said formula is:
Index
Value
=
Exp
[
-
4.3633
+
(
0.0108
*
HA
ng
/
ml
)
+
(
0.0015
*
TIMP
-
1
ng
/
ml
)
+
(
0.5357
*
α2
-
MG
mg
/
ml
)
]
1
+
Exp
[
-
4.3633
+
(
0.0108
*
HA
ng
/
ml
)
+
(
0.0015
*
TIMP
-
1
ng
/
ml
)
+
(
0.5357
*
α2
-
MG
mg
/
ml
)
]
;
and
(e) diagnosing said individual as having no or mild liver fibrosis when the index value is below an index cutoff value of 0.42 and diagnosing said individual as having moderate or severe liver fibrosis when the index value is above the index cutoff value of 0.42.
26. The method of claim 25 , wherein said individual has a disorder selected from the group consisting of viral hepatitis, autoimmune liver disease, alcoholic liver disease, fatty liver disease and drug-induced liver disease.
27. The method of claim 26 , wherein said individual is infected with a virus selected from hepatitis A, hepatitis B, and hepatitis C virus.
28. The method of claim 25 , wherein said samples are independently selected from the group consisting of blood, serum, plasma, urine, saliva and liver tissue.
29. The method of claim 28 , wherein said level of α2-MG, HA and TIMP-1 each is determined in a serum sample.
30. A method for monitoring the progression of liver fibrosis in an individual, the method comprising:
(a) determining a level of α2-macroglobulin (α2-MG) in a sample from said individual by contacting said sample with an α2-MG-specific binding agent;
(b) determining a level of hyaluronic acid (HA) in a sample from said individual by contacting said sample with an HA-specific binding agent;
(c) determining a level of tissue inhibitor of metalloproteinases- 1 (TIMP-1) in a sample from said individual by contacting said sample with a TIMP-1-specific binding agent;
(d) calculating an index value for the individual using a regression algorithm based upon said level of α2-MG, HA, and TIMP-1, wherein said regression algorithm has a formula:
Index Value=Exp( b 0 +b 1 *x 1 +b 2 *x 2 +b 3 *x 3 )/(1Exp( b 0 +b 1 *x 1 +b 2 *x 2 +b 3 *x 3 )),
wherein
b 0 is an intercept value, wherein said intercept value is based upon the levels of α2-MG, HA, and TIMP-1 in a population of liver fibrosis patients;
b 1 is a regression coefficient value for HA, wherein said regression coefficient value is based upon the level of HA in said population of liver fibrosis patients;
x 1 is the concentration level of HA;
b 2 is a regression coefficient value for TIMP-1, wherein said regression coefficient value is based upon the level of TIMP-1 in said population of liver fibrosis patients;
x 2 is the concentration level of TIMP-1;
b 3 is a regression coefficient value for α2-MG, wherein said regression coefficient value is based upon the level of α2-MG in said population of liver fibrosis patients;
x 3 is the concentration level of α2-MG, wherein said formula is:
Index
Value
=
Exp
[
-
4.3633
+
(
0.0108
*
HA
ng
/
ml
)
+
(
0.0015
*
TIMP
-
1
ng
/
ml
)
+
(
0.5357
*
α2
-
MG
mg
/
ml
)
]
1
+
Exp
[
-
4.3633
+
(
0.0108
*
HA
ng
/
ml
)
+
(
0.0015
*
TIMP
-
1
ng
/
ml
)
+
(
0.5357
*
α2
-
MG
mg
/
ml
)
]
;
(e) comparing the index value calculated for the individual to an index cutoff value, wherein said index cutoff value is between 0.2 and 0.7; and
(f) determining the presence or severity of liver fibrosis in said individual based upon whether the index value is above or below the index cutoff value to monitor progression.
31. The method of claim 30 , further comprising comparing the presence or severity of liver fibrosis determined in step (e) to the presence or severity of liver fibrosis in said individual at an earlier time.
32. The method of claim 30 , comprising determining the levels of at most three markers of fibrosis.
33. The method of claim 30 , comprising determining the levels of at least three markers of fibrosis.
34. The method of claim 30 , wherein the level of α2-MG protein is determined using an anti-α2-MG antibody.
35. The method of claim 30 , wherein the level of TIMP-1 protein is determined using an anti-TIMP-1 antibody.
36. A method of monitoring a response to a therapeutic agent in an individual in need of such agent, the method comprising:
(a) determining a level of α2-macroglobulin (α2-MG) in a sample from said individual by contacting said sample with an α2-MG-specific binding agent;
(b) determining a level of hyaluronic acid (HA) in a sample from said individual by contacting said sample with an HA-specific binding agent;
(c) determining a level of tissue inhibitor of metalloproteinases-1 (TIMP-1) in a sample from said individual by contacting said sample with a TIMP-1-specific binding agent;
(d) calculating an index value for the individual using a regression algorithm based upon said level of α2-MG, HA, and TIMP-1, wherein said regression algorithm has following a formula:
Index Value=Exp( b 0 +b 1 *x 1 +b 2 *x 2 +b 3 *x 3 )/(1Exp( b 0 +b 1 *x 1 +b 2 *x 2 +b 3 *x 3 )),
wherein
b 0 is an intercept value, wherein said intercept value is based upon the levels of α2-MG, HA, and TIMP-1 in a population of liver fibrosis patients;
b 1 is a regression coefficient value for HA, wherein said regression coefficient value is based upon the level of HA in said population of liver fibrosis patients;
x 1 is the concentration level of HA;
b 2 is a regression coefficient value for TIMP-1, wherein said regression coefficient value is based upon the level of TIMP-1 in said population of liver fibrosis patients;
x 2 is the concentration level of TIMP-1;
b 3 is a regression coefficient value for α2-MG, wherein said regression coefficient value is based upon the level of α2-MG in said population of liver fibrosis patients;
x 3 is the concentration level of α2-MG, wherein said formula is:
Index
Value
=
Exp
[
-
4.3633
+
(
0.0108
*
HA
ng
/
ml
)
+
(
0.0015
*
TIMP
-
1
ng
/
ml
)
+
(
0.5357
*
α2
-
MG
mg
/
ml
)
]
1
+
Exp
[
-
4.3633
+
(
0.0108
*
HA
ng
/
ml
)
+
(
0.0015
*
TIMP
-
1
ng
/
ml
)
+
(
0.5357
*
α2
-
MG
mg
/
ml
)
]
;
(e) comparing the index value calculated for the individual to an index cutoff value, wherein said index cutoff value is between 0.2 and 0.7; and (f) determining the presence or severity of liver fibrosis in said individual based upon whether the index value is above or below the index cutoff value to monitor response.
37. The method of claim 36 , further comprising comparing the presence or severity of liver fibrosis determined in step (e) to the presence or severity of liver fibrosis in said individual at an earlier time.
38. The method of claim 36 , wherein the level of α2-MG protein is determined using an anti-α2-MG antibody.
39. The method of claim 36 , wherein the level of TIMP-1 protein is determined using an anti-TIMP-1 antibody.
40. The method of claim 36 , wherein the individual has a viral disease.
41. The method of claim 40 , wherein the viral disease is selected from the group consisting of hepatitis A, hepatitis B, and hepatitis C.
42. The method of claim 36 , wherein the therapeutic agent is an anti-fibrotic agent.
43. The method of claim 36 , wherein the therapeutic agent is an anti-viral agent.
44. The method of claim 1 , wherein said individual is diagnosed as having no or mild liver fibrosis when the index value is less than the index cut-off value.
45. The method of claim 1 , wherein said individual is diagnosed as having moderate or severe liver fibrosis when the index value is greater than the index cut-off value.
46. The method of claim 1 , wherein said regression algorithm uses logistic or linear regression.