U.S. patent application number 17/101433 was filed with the patent office on 2021-03-11 for use of 1,25-dihydroxyvitamin d values in ratio with pth as a prognostic biomarker.
This patent application is currently assigned to DiaSorin S.p.A.. The applicant listed for this patent is DiaSorin S.p.A.. Invention is credited to Frank Blocki, Fabrizio Bonelli, Francesco Colotta, Claudia Zierold.
Application Number | 20210072264 17/101433 |
Document ID | / |
Family ID | 1000005234921 |
Filed Date | 2021-03-11 |
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United States Patent
Application |
20210072264 |
Kind Code |
A1 |
Colotta; Francesco ; et
al. |
March 11, 2021 |
Use of 1,25-Dihydroxyvitamin D Values in Ratio with PTH as a
Prognostic Biomarker
Abstract
The present invention relates to the use of
1,25-dihydroxyvitamin D values in ratio with PTH as a prognostic
biomarker. More particularly, the present invention relates to a
method for predicting or stratifying the risk of worsening renal
function (WRF) in a patient at risk of renal injury or affected by
renal injury. Levels of 1,25-dihydroxyvitamin D (1,25(OH).sub.2D)
are measured in a biological sample and taken together with
parathyroid hormone (PTH) levels to provide a ratio indicative of
the risk of worsening renal function.
Inventors: |
Colotta; Francesco; (Segrate
(Milano), IT) ; Bonelli; Fabrizio; (Casale Monferrato
(Alessandria), IT) ; Blocki; Frank; (Hayfield,
MN) ; Zierold; Claudia; (St. Paul, MN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DiaSorin S.p.A. |
Saluggia (Vercelli) |
|
IT |
|
|
Assignee: |
DiaSorin S.p.A.
Saluggia (Vercelli)
IT
|
Family ID: |
1000005234921 |
Appl. No.: |
17/101433 |
Filed: |
November 23, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15651517 |
Jul 17, 2017 |
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17101433 |
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PCT/IB2016/050230 |
Jan 18, 2016 |
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15651517 |
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15606284 |
May 26, 2017 |
10501548 |
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15651517 |
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14763264 |
Jul 24, 2015 |
10196449 |
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PCT/EP2014/051482 |
Jan 27, 2014 |
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15606284 |
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16681241 |
Nov 12, 2019 |
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14763264 |
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15606284 |
May 26, 2017 |
10501548 |
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16681241 |
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14763264 |
Jul 24, 2015 |
10196449 |
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PCT/EP2014/051482 |
Jan 27, 2014 |
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15606284 |
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62104802 |
Jan 18, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2800/347 20130101;
G01N 2333/635 20130101; G01N 33/6893 20130101; G01N 33/5005
20130101; G01N 33/74 20130101; G01N 33/78 20130101; G01N 33/5091
20130101; G01N 33/82 20130101; G01N 2800/60 20130101 |
International
Class: |
G01N 33/82 20060101
G01N033/82; G01N 33/50 20060101 G01N033/50; G01N 33/68 20060101
G01N033/68; G01N 33/78 20060101 G01N033/78; G01N 33/74 20060101
G01N033/74 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 28, 2013 |
EP |
13152851.5 |
Claims
1. A method for predicting or stratifying the risk of worsening
renal function (WRF) in a patient at risk of renal injury or
affected by renal injury, the method comprising: (a) incubating a
sample of serum or plasma or blood from said patient in a reaction
container in the presence of a receptor protein comprising the
Ligand Binding Domain of Vitamin D Receptor (VDR-LBD), and a
capture moiety that specifically binds to a conformational epitope
of VDR-LBD when said VDR-LBD is complexed with
1,25-dihydroxyvitamin D; wherein said complexing of VDR-LBD and
1,25-dihydroxyvitamin D does not naturally occur in serum, plasma
or blood, and said capture moiety does not specifically bind to
VDR-LBD or to 1,25-dihydroxyvitamin D that are not complexed with
one another; and wherein said incubation is under conditions
sufficient to permit the determination of the concentration of
1,25-dihydroxyvitamin D in said serum, plasma or blood; and (b)
assaying the concentration of parathyroid hormone (PTH) in said
serum, plasma or blood; wherein: when the [1,25-dihydroxyvitamin
D]/[parathyroid hormone] ratio is above a predetermined threshold,
the patient is predicted or stratified not to have an increased
risk of worsening renal function, and when the
[1,25-dihydroxyvitamin D]/[parathyroid hormone] ratio is below a
predetermined threshold, the patient is predicted or stratified to
have an increased risk of worsening renal function.
2. The method according to claim 1, wherein said capture moiety is
an antibody or an epitope-binding fragment thereof.
3. The method according to claim 2, wherein said antibody is a
monoclonal antibody.
4. The method according to claim 1, wherein the predetermined
threshold is comprised within the range of from 0.92 to 1.8.
5. The method according to claim 4, wherein the predetermined
threshold is 0.92, 0.98 or 1.68.
6. The method according to claim 1, wherein 1,25-dihydroxyvitamin D
and/or parathyroid hormone are detected and quantified using an
immunoassay.
7. The method according to claim 6, wherein the immunoassay is a
chemiluminescent assay.
8. The method according to claim 6, wherein 1,25-dihydroxyvitamin D
is detected and quantified using an immunoassay which comprises:
(i) contacting 1,25-dihydroxyvitamin D in said sample with a
receptor protein comprising the Ligand Binding Domain of Vitamin D
Receptor (VDR-LBD), thereby obtaining a first complex; (ii)
contacting said first complex with a capture moiety that
specifically binds to a conformational epitope on said first
complex, thereby obtaining a second complex; wherein said capture
moiety does not specifically bind to either 1,25-dihydroxyvitamin D
or VDR-LBD that is not bound in said first complex; and (iii)
detecting and quantifying said second complex as an indication of
the amount of 1,25-dihydroxyvitamin D in the sample.
9. The method according to claim 8, wherein said capture moiety is
an antibody or an epitope-binding fragment thereof.
10. The method according to claim 9, wherein said antibody is a
monoclonal antibody.
11. The method according to claim 8, wherein said capture moiety is
immobilized on or to a solid support.
12. The method according to claim 8, wherein said
1,25-dihydroxyvitamin D is detected and quantified using a sandwich
immunoassay.
13. The method according to claim 12, wherein in said step (ii)
said capture moiety is detectably-labeled, and said step (iii)
comprises detecting and quantifying detectably-labeled second
complex.
14. The method according to claim 8, wherein the predetermined
threshold is comprised within the range of from 0.92 to 1.8.
15. The method according to claim 14, wherein the predetermined
threshold is 0.92, 0.98 or 1.68.
16. The method according to claim 8, wherein parathyroid hormone is
detected and quantified using an immunoassay.
17. A method for determining the ratio of the concentration of
1,25-dihydroxyvitamin D to parathyroid hormone (PTH) in the serum,
plasma or blood of a patient, wherein said method comprises: (a)
incubating a sample of serum or plasma or blood from said patient
in a reaction container in the presence of a receptor protein
comprising the Ligand Binding Domain of Vitamin D Receptor
(VDR-LBD), and a capture moiety that specifically binds to a
conformational epitope of VDR-LBD when said VDR-LBD is complexed
with 1,25-dihydroxyvitamin D; wherein said complexing of VDR-LBD
and 1,25-dihydroxyvitamin D does not naturally occur in serum,
plasma or blood, and said capture moiety does not specifically bind
to VDR-LBD or to 1,25-dihydroxyvitamin D that are not complexed
with one another; and wherein said incubation is under conditions
sufficient to permit the determination of the concentration of
1,25-dihydroxyvitamin D in said serum, plasma or blood; (b)
assaying the concentration of parathyroid hormone (PTH) in said
serum, plasma or blood; and (c) determining the ratio of said
determined concentration of 1,25-dihydroxyvitamin D to said assayed
concentration of parathyroid hormone.
18. The method according to claim 17, wherein said capture moiety
is detectably-labeled.
19. The method according to claim 17, wherein said capture moiety
is an antibody or an epitope-binding fragment thereof.
20. The method according to claim 19, wherein said antibody is a
monoclonal antibody.
21. The method according to claim 17, wherein 1,25-dihydroxyvitamin
D and/or parathyroid hormone are detected and quantified using an
immunoassay.
22. The method according to claim 21, wherein said
1,25-dihydroxyvitamin D is detected and quantified using a sandwich
immunoassay.
23. The method according to claim 21, wherein the immunoassay is a
chemiluminescent assay.
24. The method according to claim 17, wherein said capture moiety
is immobilized on or to a solid support.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is: [0002] (A) a divisional
application of U.S. patent application Ser. No. 15/651,517 (filed
on Jul. 17, 2017; pending), which application is: [0003] (1) a
continuation-in-part of U.S. Patent Appln. Serial No.
PCT/IB2016/050230 (filed on Jan. 18, 2016; expired), which claims
priority to U.S. Patent Appln. Ser. No. 62/104,802 (filed on Jan.
18, 2015, expired); and [0004] (2) a continuation-in-part of U.S.
patent application Ser. No. 15/606,284 (filed on May 26, 2017;
issued Dec. 10, 2019 as U.S. Pat. No. 10,501,548), which
application is a division of U.S. patent application Ser. No.
14/763,264 (filed Jul. 24, 2015; issued Feb. 5, 2019 as U.S. Pat.
No. 10,196,449), which application is a .sctn. 371 Application of
PCT/EP2014/051482 (filed on Jan. 27, 2014; now lapsed), which
claims priority to European Patent Application EP 13152851.5 (filed
on Jan. 28, 2013); and [0005] (B) a continuation-in-part of U.S.
patent application Ser. No. 16/681,241 (filed on Nov. 12, 2019;
pending); which application is a continuation-in-part of U.S.
patent application Ser. No. 15/606,284 (filed on May 26, 2017;
issued Dec. 10, 2019 as U.S. Pat. No. 10,501,548), which
application is a division of U.S. patent application Ser. No.
14/763,264 (filed Jul. 24, 2015; issued Feb. 5, 2019 as U.S. Pat.
No. 10,196,449), which application is a .sctn. 371 Application of
PCT/EP2014/051482 (filed on Jan. 27, 2014; now lapsed), which
claims priority to European Patent Application EP 13152851.5 (filed
on Jan. 28, 2013); [0006] each of these patent applications is
hereby incorporated by reference in its entirety.
REFERENCE TO SEQUENCE LISTING
[0007] This application includes one or more Sequence Listings
pursuant to 37 C.F.R. 1.821 et seq., which are disclosed in
computer-readable media and were filed (file name:
0400_0007C_ST25_1.txt, created on Jul. 14, 2017, and having a size
of 4,356 bytes), which file is herein incorporated by reference in
its entirety.
FIELD OF THE INVENTION
[0008] The present invention relates to a method for predicting or
stratifying the risk of worsening renal function (WRF) in a patient
at risk of renal injury or in a patient affected by renal injury.
Levels of 1,25-dihydroxyvitamin D (1,25(OH).sub.2D) are measured in
a biological sample and taken together with parathyroid hormone
(PTH) levels to provide a ratio indicative of the risk of worsening
renal function.
BACKGROUND OF THE INVENTION
[0009] Progressive deterioration of renal function is common in
patients with different diseases such as chronic heart failure
(HF), chronic kidney disease (CKD) and metabolic syndrome, and is
associated with unfavorable outcomes, which can be improved by
timely interventions.
[0010] The cross talk between the diseased heart and kidney is a
growing burden for health care systems as the incidence of HF and
chronic kidney disease (CKD) has been steadily increasing and will
further increase due to ageing of the general population and better
treatment of acute cardiac and renal diseases. It has also been
realized that the progressive development of worsening renal
function (WRF) over time carries an increased risk of death and
hospitalizations.
[0011] Identifying patients at risk of worsening renal function
(WRF) is important for their clinical management, and might lead to
less frequent hospitalizations and to the prevention of adverse
outcomes. Early prediction and identification of patients at risk
for future WRF may also be useful to optimize therapies and to
improve outcomes. Circulating biomarkers may therefore provide a
simple and objective means to predict deterioration in renal
function in patients with chronic HF or other diseases such as
chronic kidney disease (CKD) or IgA nephropathy. For instance, a
common marker of renal injury is serum creatinine, which however is
slowly affected by changes in renal function and is also dependent
on a plurality of different factors such as muscle mass, sex, race
and age.
[0012] Disturbances of mineral metabolism, and in particular to the
parathyroid hormone (PTH)/vitamin D axis, are characteristic of
decreased renal function (Evenepoel, P. et al. (2014) "Laboratory
Abnormalities in CKD-MBD: Markers, Predictors, or Mediators of
Disease?" Semin. Nephrol. 34(2):151-163). Recent studies indicate
that vitamin D-deficiency may promote or accelerate the progression
of CKD. Cross-sectional studies have shown higher circulating
levels of PTH and lower levels of vitamin D metabolites as CKD
progresses (lower estimated glomerular filtration rate, eGFR). To
the inventors' knowledge, there are, however, scant reports on the
ability of circulating markers of bone mineral metabolism to
predict deterioration of renal function over time.
[0013] Therefore, the inventors examined the relation between two
markers of the vitamin D/PTH axis and worsening of renal function
(WRF) in a large cohort of patients with HF.
[0014] Lack of reliable automated testing of 1,25-dihydroxyvitamin
D (1,25(OH).sub.2D), the biologically active metabolite of vitamin
D, has in the past precluded evaluation of the prognostic value of
this measurement.
[0015] Therefore, there is a need to identify biomarkers for
predicting or stratifying the risk of worsening renal function
(WRF) in a patient at risk of renal injury or in a patient affected
by renal injury.
SUMMARY OF THE INVENTION
[0016] The present invention provides a method for predicting or
stratifying the risk of worsening renal function (WRF) in a patient
at risk of renal injury or in a patient affected by renal injury,
using the level of 1,25(OH).sub.2D in conjunction with the level of
parathyroid hormone (PTH) to determine the ratio of 1,25(OH).sub.2D
to PTH. The ratio value allows for risk prediction or
stratification of worsening renal function in the patient.
[0017] The term "PTH" as used in the present description preferably
refers to parathyroid hormone 1-84 (PTH 1-84), which is the
biologically active hormone produced by the parathyroid glands and
secreted into the systemic circulation.
[0018] Therefore, the invention provides a method for predicting or
stratifying the risk of worsening renal function (WRF) in a patient
at risk of renal injury or in a patient affected by renal injury,
the method comprising: (a) detecting and quantifying
1,25(OH).sub.2D in a sample from the patient; (b) detecting and
quantifying PTH in a sample from the patient; and (c) calculating
the 1,25(OH).sub.2D to PTH ratio, wherein when the ratio is above a
predetermined threshold, the patient is predicted or stratified not
to have an increased risk of worsening renal function, and when the
ratio is below a predetermined threshold, the patient is predicted
or stratified to have an increased risk of worsening renal
function.
[0019] The measurement of the ratio of 1,25(OH).sub.2D to PTH in
patients at risk of renal injury or in patients affected by renal
injury offers several advantages, such as improvement of the area
under the receiver operating curve, translating added clinical
value, integration of more than one physiopathologicaly
interrelated biomarker and modulation of two different hormones to
increase the significance of the ratio.
[0020] In various embodiments, 1,25(OH).sub.2D and/or PTH are
determined from blood, plasma or serum samples from the patient. In
these and other embodiments, 1,25(OH).sub.2D and/or PTH are
determined using an immunoassay. In particular embodiments, the
immunoassay is a chemiluminescent assay.
[0021] In one embodiment, when the ratio of 1,25(OH).sub.2D to PTH
is below the predetermined threshold, the patient is predicted or
stratified as having a high risk of worsening of renal
function.
[0022] In another embodiment, when the 1,25(OH).sub.2D to PTH ratio
is above the predetermined threshold, the patient is predicted or
stratified as having a low risk of worsening renal function.
[0023] The present invention is particularly directed to a method
for predicting or stratifying the risk of worsening renal function
(WRF) in a patient at risk of renal injury or affected by renal
injury, the method comprising: [0024] (a) incubating a sample of
serum or plasma or blood from the patient in a reaction container
in the presence of a receptor protein comprising the Ligand Binding
Domain of Vitamin D Receptor (VDR-LBD), and a capture moiety that
specifically binds to a conformational epitope of VDR-LBD when the
VDR-LBD is complexed with 1,25-dihydroxyvitamin D; [0025] wherein
the complexing of VDR-LBD and 1,25-dihydroxyvitamin D does not
naturally occur in serum, plasma or blood, and the capture moiety
does not specifically bind to VDR-LBD or to 1,25-dihydroxyvitamin D
that are not complexed with one another; and wherein the incubation
is under conditions sufficient to permit the determination of the
concentration of 1,25-dihydroxyvitamin D in the serum, plasma or
blood; and [0026] (b) assaying the concentration of parathyroid
hormone (PTH) in the serum, plasma or blood; [0027] wherein: [0028]
when the [1,25-dihydroxyvitamin D]/[parathyroid hormone] ratio is
above a predetermined threshold, the patient is predicted or
stratified not to have an increased risk of worsening renal
function, and when the [1,25-dihydroxyvitamin D]/[parathyroid
hormone] ratio is below a predetermined threshold, the patient is
predicted or stratified to have an increased risk of worsening
renal function.
[0029] Preferably, the predetermined threshold in these embodiments
is comprised within the range of from 0.92 to 1.8, more preferably
the predetermined threshold is 0.92, 0.98 or 1.68.
[0030] In yet another embodiment, 1,25(OH).sub.2D is determined
using an immunoassay which comprises (i) contacting the
1,25(OH).sub.2D in the sample from the patient with a receptor
protein comprising the Ligand Binding Domain of Vitamin D Receptor
(VDR-LBD), thereby obtaining a first complex; (ii) contacting the
first complex with a capture moiety that specifically binds to a
conformational epitope on the first complex, but does not bind to
either 1,25(OH).sub.2D or VDR-LBD that is not bound in the first
complex, thereby obtaining a second complex; and (iii) detecting
and quantifying the second complex as an indication of the amount
of 1,25(OH).sub.2D in the sample.
[0031] The invention particularly includes a method wherein
1,25-dihydroxyvitamin D is detected and quantified using an
immunoassay which comprises: [0032] (i) contacting
1,25-dihydroxyvitamin D in the sample with a receptor protein
comprising the Ligand Binding Domain of Vitamin D Receptor
(VDR-LBD), thereby obtaining a first complex; [0033] (ii)
contacting the first complex with a capture moiety that
specifically binds to a conformational epitope on the first
complex, thereby obtaining a second complex; wherein the capture
moiety does not specifically bind to either 1,25-dihydroxyvitamin D
or VDR-LBD that is not bound in the first complex; and [0034] (iii)
detecting and quantifying the second complex as an indication of
the amount of 1,25-dihydroxyvitamin D in the sample.
[0035] In a preferred embodiment, the capture moiety is a
monoclonal antibody.
[0036] Preferably, the capture moiety is immobilized on or to a
solid support.
[0037] In another preferred embodiment, the immunoassay of the
claimed method is a sandwich immunoassay.
[0038] In a more preferred embodiment, step (iii) of detecting and
quantifying the second complex is carried out by means of a labeled
anti-VDR-LBD detector antibody.
[0039] Further, while the present 1,25(OH).sub.2D data was obtained
using a new immunoassay that provides rapid, sensitive and
reproducible data using significantly smaller volumes of samples
than other available assays, those skilled in the art will
appreciate that any method of collecting reliable (sufficiently
sensitive and accurate) values for 1,25(OH).sub.2D and PTH is
contemplated. For example, such methods may include GC-MS, LC-MS/MS
and the like.
[0040] The invention also provides a kit, being an article of
manufacture, that comprises: [0041] (A) a first container that
contains Ligand Binding Domain of Vitamin D Receptor (VDR-LBD) and
an antibody, or epitope-binding fragment thereof, that specifically
binds to a conformational epitope of VDR-LBD when the VDR-LBD is
complexed with 1,25-dihydroxyvitamin D in serum or plasma or blood,
and does not specifically bind to VDR-LBD or to
1,25-dihydroxyvitamin D that are not complexed with one another;
and [0042] (B) a second container that contains a
detectably-labelled reagent capable of binding to parathyroid
hormone (PTH).
[0043] The invention particularly pertains to the embodiment of
such a kit wherein the antibody, or epitope-binding fragment
thereof, that specifically binds to the conformational epitope of
VDR-LBD is immobilized to a solid support (and especially a
magnetic bead) in the first container.
[0044] The invention also pertains to the embodiment of such kits
wherein the antibody that specifically binds to the conformational
epitope of VDR-LBD is a monoclonal antibody.
[0045] The invention particularly pertains to the embodiment of
such kits wherein the kit is a Reagent Integral that is a
multi-chambered cartridge specially adapted for use in an automated
analyzing device, wherein the first and second containers of the
kit are chambers of the Reagent Integral.
[0046] These and other features and advantages of the present
invention will be set forth or will become more fully apparent in
the description that follows and in the appended claims. The
features and advantages may be realized and obtained e.g. by means
of the instruments and combinations particularly pointed out in the
appended claims. Furthermore, the features and advantages of the
invention may be learned by the practice of the invention or will
be apparent from the description, as set forth hereinafter.
BRIEF DESCRIPTION OF THE FIGURES
[0047] Various exemplary embodiments of the compositions and
methods according to the invention will be described in detail,
with reference to the following figures wherein:
[0048] FIG. 1 Illustrates one-site, non-competitive immunoassays
according to the invention, wherein the complex formed via the
binding of 1,25(OH).sub.2D to the labeled receptor protein which
comprises the Ligand Binding Domain of Vitamin D Receptor (VDR-LBD)
is captured by the conformation-specific capture antibody of the
invention (designated as "Monoclonal Anti-Bound LBD") immobilized
on or to a solid support (a paramagnetic particle (PMP)) and the
label is Amino-Butyl-Ethyl-Isoluminol (ABEI). The step of adjusting
the pH of the biological fluid sample with the assay buffer and the
step of adding the receptor protein comprising the VDR-LBD to the
sample, are performed simultaneously.
[0049] FIG. 2 Illustrates one-site, non-competitive immunoassays
according to the invention, wherein the complex formed via the
binding of 1,25(OH).sub.2D to the labeled receptor protein which
comprises the Ligand Binding Domain of Vitamin D Receptor (VDR-LBD)
is captured by the conformation-specific capture antibody of the
invention (designated as "Monoclonal Anti-Bound LBD") immobilized
on or to a solid support (a paramagnetic particle (PMP)) and the
label is Amino-Butyl-Ethyl-Isoluminol (ABEI). The step of adjusting
the pH of the biological fluid sample with the assay buffer and the
step of adding the receptor protein comprising the VDR-LBD to the
sample, are carried our sequentially.
[0050] FIG. 3 Illustrates a sandwich immunoassay which involves the
binding of the VDR-LBD/1,25(OH).sub.2D complex to the
conformation-specific capture antibody (designated as "Monoclonal
Anti-Bound LBD") immobilized on or to a solid support (e.g. a
paramagnetic particle, PMP) and the use of a labeled detector
antibody as the second part of the sandwich. The detector antibody
is either directly labeled or it is recognized by a conjugate
consisting of a labeled anti-immunoglobulin antibody (in the
specific example of FIG. 3 the detector antibody is directly
labeled with ABEI). The amount of labeled antibody directly or
indirectly bound to the VDR-LBD/1,25(OH).sub.2D complex is then
measured by suitable means
[0051] FIG. 4: Illustrates an exemplary "Reagent Integral," which
is a multi-chambered cartridge that is specially adapted for use in
an automated analyzing device, such as the Liaison.RTM.
Analyzer.
[0052] FIG. 5: Receiver operator curve (ROC) and area under the
curve (AUC) for the 1,25(OH).sub.2D/PTH ratio with WRF
[0053] FIG. 6: Kaplan-Meier curve for the first occurrence of WRF
stratified by the [1,25(OH).sub.2D]/[PTH] ratio
DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENTS
[0054] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
reference unless the context clearly dictates otherwise. As well,
the terms "a" (or "an"), "one or more" and "at least one" can be
used interchangeably herein. It is also to be noted that the terms
"comprising", "including", "characterized by" and "having" can be
used interchangeably.
[0055] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
ordinary skill in the art to which this invention belongs. All
publications and patents specifically mentioned herein are
incorporated by reference for all purposes including describing and
disclosing the chemicals, instruments, statistical analyses and
methodologies which are reported in the publications which might be
used in connection with the invention. All references cited in this
specification are to be taken as indicative of the level of skill
in the art. Nothing herein is to be construed as an admission that
the invention is not entitled to antedate such disclosure by virtue
of prior invention.
[0056] Abbreviations used throughout this text are as follows: HF,
Heart Failure; NYHA, New York Heart Association; PPV, Positive
Predictive Value; NPV, Negative Predictive Value; LVEF, left
ventricular ejection fraction; GFR, glomerular filtration rate;
eGFR, estimated glomerular filtration rate; CV, cardiovascular;
1,25(OH).sub.2D, 1,25-dihydroxyvitamin D; PTH, parathyroid hormone;
ROC, receiver operating characteristics; AUC, area under the curve;
CART, Classification and regression trees.
[0057] The term "antibody" as used in the present description
encompasses a whole antibody molecule (including polyclonal,
monoclonal, chimeric, humanized, or human versions having full
length heavy and light chains) as well as an antigen binding
antibody fragment. An "antibody fragment" includes any
immunoglobulin fragment having the same binding specificity as the
corresponding whole antibody. Such fragments are produced according
to standard methods; cf. for example Harlow and Lane, "Antibodies,
A Laboratory Manual", CSH Press, Cold Spring Harbor, USA, 1988.
Non-limiting examples of antibody fragments include F(ab), Fab',
F(ab')2, F(v), single chain antibodies (scFv), F(c), F(d).
[0058] The term "vitamin D" as used in the present description
refers both to vitamin D.sub.3 (cholecalciferol) and vitamin
D.sub.2 (ergocalciferol), and the term "1,25(OH).sub.2D" refers to
both 1,25(OH)D.sub.3 and 1,25(OH)D.sub.2. Analogues of
1,25(OH).sub.2D include modified versions and structural analogues
thereof, such as for example, 19-nor-1.alpha.-25-dihydroxyvitamin
D.sub.2 (e.g. Zemplar or paricalcitol from Abbott),
1.alpha.-hydroxyvitamin D.sub.2 or 1.alpha.-hydroxyergocalciferol
(e.g. Hectorol or doxercalciferol from Genzyme), and
2-methylene-19-nor-(20S)-1.alpha.,25-(OH).sub.2D.sub.3 (e.g. 2MD
from Deltanoid Pharmaceuticals).
[0059] Vitamin D is a steroid hormone which plays a fundamental
role in skeletal metabolism and calcium homeostasis. In humans and
animals, the major forms of vitamin D are vitamin D.sub.3
(cholecalciferol) and vitamin D.sub.2 (ergocalciferol). Vitamin
D.sub.3 is primarily synthesized in the skin from
7-dehydrocholesterol in response to exposure to solar ultraviolet-B
(UVB), but vitamin intake can also occur from dietary sources such
as oily fish, i.e. salmon and mackerel. Vitamin D.sub.2 is
primarily acquired in the diet from fungal and vegetable sources as
well as from supplementation (e.g. Drisdol.TM. or Sterogyl 15
"A").
[0060] Irrespective of the source, the conversion of vitamins
D.sub.2 and D.sub.3 into a bioactive compound requires two separate
hydroxylation steps. In the liver, the enzyme 25-hydroxylase
converts vitamin D to 25-hydroxyvitamin D (hereinafter designated
as "25(OH)D"). This intermediary metabolite is the major
circulating form of the hormone and serves as a reservoir for
further hydroxylation to the biologically active metabolite
1,25-dihydroxyvitamin D (hereinafter designated as
"1,25(OH).sub.2D").
[0061] The latter step takes place primarily in the renal tubular
cells and is catalyzed by the enzyme 1-alpha-hydroxylase. The
plasma concentrations of 1,25(OH).sub.2D are highly regulated by a
variety of factors, including the serum parathyroid hormone (PTH),
and they are normally about 1000-fold lower than the precursor
compound 25(OH)D.
[0062] Because of their lipophilic nature, the majority of vitamin
D and metabolites thereof circulate in the blood-stream bound to
the vitamin D binding protein (DBP) (80-90%), also known as
Gc-Globulin, and albumin (10-20%). DBP has high affinity for
vitamin D metabolites (Ka=5.times.10.sup.8M.sup.-1 for 25(OH)D and
24,25(OH).sub.2D, 4.times.10.sup.7M.sup.-1 for 1,25(OH).sub.2D and
vitamin D), such that under normal circumstances only approximately
0.03% 25(OH)D and 24,25(OH).sub.2D and approximately 0.4%
1,25(OH).sub.2D are in a free form.
[0063] The biological effects of 1,25(OH).sub.2D are mediated
primarily by the binding of this bioactive hormone to a specific
intracellular Vitamin D Receptor (VDR), which acts primarily by
regulating the expression of genes whose promoters contain specific
DNA sequences known as Vitamin D Response Elements (VDREs).
[0064] The Vitamin D Receptor (VDR) is a ligand-dependent
transcriptional regulator belonging to the superfamily of nuclear
receptors (NRs). Like the other members of this receptor family,
the VDR possesses a modular structure which comprises an
amino-terminal A/B domain, a highly conserved DNA-Binding Domain
(DBD), a flexible linker region and a C-terminal Ligand-Binding
Domain (LBD) which is more variable (Mangelsdorf D J et al., 1995,
Cell 83(6):835-9). The C-terminal LBD is a globular multifunctional
domain, responsible for hormone binding, dimerization with Retinoid
X Receptor (RXR) and interaction with co-repressors and
co-activators, which all together are critical for the regulation
of transcriptional activities (Haussler M R, et al. 1998, J Bone
Miner Res. 13(3): 325-49).
[0065] The Ligand Binding Domain (LBD) of VDR has been crystallized
and its structure solved (Rochel N, Wurtz J M, Mitschler A, Klaholz
B, Moras D., "The Crystal Structure Of The Nuclear Receptor For
Vitamin D Bound To Its Natural Ligand," Mol Cell 2000;
5:173-179).
[0066] The binding of the ligand to the VDR induces a
conformational change at the Ligand Binding Domain of the receptor,
which in turn increases heterodimerization of VDR with a cofactor,
the Retinoid X Receptor (RXR), on a Vitamin D-Responsive Element
(VDRE) in the promoter region of the target genes. This in turn
leads to opening of the promoter to the transcriptional machinery
(Glenville J. et al., 1998 Physiological Reviews 78(4):
1193-1231).
[0067] Nuclear receptor Ligand Binding Domains (LBDs) are known to
have a high content of alpha-helix, which may undergo a large
conformational change in response to ligand binding, forming up a
hydrophobic pocket. Recently, differences in the conformation of
the Rattus norvegicus Ligand-Binding Domain (r-VDR-LBD) when bound
to diverse ligands were solved by NMR spectroscopy (Kiran K.
Singarapu et al. 2011 Biochemistry 50 (51): 11015-24).
[0068] Vitamin D is currently recognized as a pro-hormone which has
multiple roles in maintaining optimal health in human beings. It
has long been established that marked vitamin D deficiency results
in histologically evident bone diseases such as osteomalacia in
adults and rickets in children, while vitamin D insufficiency may
cause alterations in the parathyroid hormone concentration which,
if persisting over time, may contribute to bone loss and fracture.
However, although initially identified as a classic regulator of
calcium homeostasis, vitamin D is now known to have a broader
spectrum of actions, driven by the wide expression and distribution
in human tissues of the vitamin D receptor (VDR).
[0069] In the last decades, clinical and epidemiological data have
provided several evidences that impaired levels of 25(OH)D are
associated with an increasing risk of various chronic diseases
including cardiovascular diseases, hypertension, myocardial
infarction, diabetes, cancer, reduced neuromuscular function,
infectious and autoimmune diseases. Even complications of pregnancy
such as pre-eclampsia, gestational diabetes, cesarean section, and
premature birth might be the tragic sequela of gestational vitamin
D deficiencies (Holick M F; 2007 N Engl J Med. 357(3):266-81,
Holick M F and Chen T C. 2008 Am J Clin Nutr.; 87(4):1080S-6S).
[0070] Impaired levels of 25-Hydroxyvitamin D (25(OH)D) (calcidiol)
have previously been shown to be associated with an increased risk
of various diseases including cardiovascular disease, hypertension,
myocardial infarction, diabetes, cancer, reduced neuromuscular
function, infectious and autoimmune disease. However, under
conditions of vitamin D (cholecalciferol) deficiency, this
biomarker may not be as predictive as its biologically active
metabolite 1,25-dihydroxyvitamin D (1,25(OH).sub.2D) or calcitriol.
1,25(OH).sub.2D and PTH control calcium and phosphate homeostasis.
The potential value of 1,25(OH).sub.2D testing as a significant
predictor of renal injury or of worsening renal function in a
cohort of patients, more particularly HF patients, was pursued in a
patient model population.
[0071] However, very few studies have been carried out to associate
risks of chronic disease to 1,25(OH).sub.2D levels, due to both
complexity and lack of reliability of the measurement methods which
are available today.
[0072] Therefore, the determination of circulating 1,25(OH).sub.2D,
which is the active form of vitamin D, is becoming of increasing
relevance in many different clinical applications, either as a
diagnostic marker and/or as a therapy monitoring indicator. For
instance, the determination of serum 1,25(OH).sub.2D and
parathyroid hormone (PTH) levels and a possible correlation thereof
may represent an important measure for aiding in the diagnosis of
parathyroid diseases as well as for the detection of the onset of
secondary hyperparathyroidism in the course of renal failure or the
development of vitamin D-resistant rickets (VDRR).
[0073] Currently, both in routine clinical and research use there
is a wide range of methodologies available for measuring the
circulating levels of total 25(OH)D (i.e.,
25(OH)D.sub.3+25(OH)D.sub.2). Commercial, fast, automated
chemiluminescence-based immunoassay methods are supplied by Abbott
Diagnostics (Abbott Park, Ill., USA, ARCHITECT 25-OH vitamin D
assay), DiaSorin Inc. (Stillwater, Minn., USA, LIAISON.RTM. 25 OH
Vitamin D Total Assay), Immunodiagnostic Systems (Boldon, England,
IDS-iSYS 25-Hydroxy Vitamin D (250HD)), Roche Diagnostics
(Mannheim, Germany, Modular Analytics E170 Elecsys.RTM. Vitamin D
Total assay), and Siemens Healthcare Diagnostics (Tarrytown, N.Y.,
USA, ADVIA Centaur.RTM. Vitamin D Total assay). Besides these assay
platforms, there has recently been a steady increase in the use of
physical methods based on chromatographic separation followed by
non-immunological direct detection (semi-automated liquid
chromatography-tandem mass spectrometry, LC-MS/MS), which have been
principally developed in specialist laboratories in the United
States (e.g. Esoterix Inc. in Calabasas Hills, Calif., Mayo Clinic
in Rochester, Minn., ARUP Laboratories in Salt Lake City, Utah and
Quest Diagnostics in Lyndhurst, N.J.), Europe (e.g. Ghent
University in Ghent, Belgium, and CHU de Liege in Liege, Belgium)
and Australia (e.g. Pathology Queensland in Herston Queensland, and
Douglass Hanly Moir Pathology in Macquarie Park NSW).
[0074] Despite the wide selection of assay platforms for measuring
25(OH)D, there are no automated assay methods currently available
for the quantitative determination of the active form of vitamin D
in clinical samples. The systemic circulating levels of
1,25(OH).sub.2D are extremely low, in the pg/ml range, and
therefore represent a significant bioanalytical challenge for
clinical monitoring. Quantitation of 1,25(OH).sub.2D in plasma has
been traditionally carried-out by radioimmunoassay (MA). In order
to avoid problems related to handling of radioactivity and the
limited shelf-life of radioactive labels, new vitamin D testing
methods have recently emerged which mainly rely upon the employment
of the LC-MS/MS methodology. However, the reported LC-MS/MS
bioanalytical assays for 1,25(OH).sub.2D suffer from the extensive
sample preparation procedures or derivatization protocols which
need to be carried out in order to achieve the requisite
sensitivity and selectivity. At present, the main methods available
for the detection of 1,25(OH).sub.2D require performing a number of
sample pre-treatment or pre-analytical steps which are usually
carried-out manually and may therefore be very time consuming,
labor-intensive, and expensive.
[0075] EP 0 583 945 A discloses an assay for 1,25(OH).sub.2D which
involves extracting blood serum using an organic solvent such as
ethyl acetate, separating out potentially interfering other vitamin
D metabolites using a silica column, and then adding pig receptor
protein, radiolabeled 1,25(OH).sub.2D, biotinylated antibody
capable of binding to the receptor, and a facilitator protein such
as BSA as part of an immunoprecipitation competitive binding
assay.
[0076] WO 89/01631 discloses a competitive binding assay for
1,25(OH).sub.2D which involves adding pig receptor protein,
radiolabeled 1,25(OH).sub.2D and biotinylated antibody capable of
binding to the receptor to untreated blood serum. The competitive
binding assay requires the use of vitamin D transport protein which
acts as a screen to minimize interference from related
metabolites.
[0077] Swami, S. et al., Bone, Vol. 28, No. 3, March 2001:319-326
discloses an antibody which binds to the hinge portion of the
vitamin D receptor (VDR) and which is used in a method for the
measurement of VDR. However, such antibody is not able to
distinguish between ligand-occupied and -unoccupied VDR and is
therefore not useful for the detection of 1,25(OH).sub.2D.
[0078] The DiaSorin RIA (Part No. 65100E/100 Tubes;
1,25-Dihydroxyvitamin D) involves the use of organic solvents,
extraction instrumentation, and C18-OH columns to separate out
potentially interfering vitamin D metabolites such as
24,25(OH).sub.2D, 25,26(OH).sub.2D and 25(OH)D in order to isolate
1,25(OH).sub.2D from the test sample prior to metabolite
measurement.
[0079] Even the recently commercialized automated assay supplied by
Immunodiagnostics for the determination of 1,25(OH).sub.2D (Part
No. IS-2400; IDS-iSYS 1,25-Dihydroxyvitamin D) requires a
time-consuming and labor-intensive sample pre-treatment step which
makes use of the IDS proprietary Immunocapsules.
[0080] Furthermore, the prior art methods often suffer from
limitations in term of assay specificity since cross-reactivity
events with other vitamin D metabolites not completely removed from
the test specimens during the pre-analytical or sample
pre-treatment steps may lead to the measurement of erroneous higher
concentrations of 1,25(OH).sub.2D. For example, most immunoassay
antibodies significantly cross-react with 25(OH)D,
24,25(OH).sub.2D, and 25,26(OH).sub.2D which may be present in
blood at levels 1000-fold greater than 1,25(OH).sub.2D.
[0081] There is therefore a strong need to develop an assay method
for detecting total 1,25(OH).sub.2D
(1,25(OH).sub.2D.sub.2+1,25(OH).sub.2D.sub.3) which does not suffer
from the drawbacks and limitations of the prior art.
[0082] In particular, there is a need for an assay method which
would enable precise, sensitive and accurate detection of total
1,25(OH).sub.2D (1,25(OH).sub.2D.sub.2+1,25(OH).sub.2D.sub.3)
without requiring time-consuming and labor-intensive sample
pre-treatment steps and which may possibly be provided in an
automated format.
[0083] There is also a need for a 1,25(OH).sub.2D assay method
which substantially does not cross-react with other vitamin D
metabolites which may be present in the test sample.
[0084] These and other needs are met by the method, and the related
kit and antibodies, disclosed in PCT Patent Publication WO
2014/114780 and in US Patent Publication No. 2015-0361178, which
form an integral part of the present description.
[0085] In particular, PCT Patent Publication WO 2014/114780 and US
Patent Publication No. 2015-0361178 disclose the finding that the
pH of the medium in which the assay is performed significantly
influence the binding affinity of vitamin D binding protein (DBP)
and of the Ligand Binding Domain of Vitamin D Receptor (VDR-LBD) to
1,25(OH).sub.2D.
[0086] The results of experiments presented in PCT Patent
Publication WO 2014/114780 and in US Patent Publication No.
2015-0361178 clearly showed that a shift in the pH value of the
test sample above 6, preferably above 7, surprisingly induces an
increase of about 200-fold in the affinity of VDR-LBD for
1,25(OH).sub.2D over 25(OH)D, while at the same pH value DBP
exhibits about 1000-fold greater affinity for 25(OH)D over
1,25(OH).sub.2D. The exploitation of such an advantageous effect of
the pH on the equilibrium between 1,25(OH).sub.2D bound to DBP and
1,25(OH).sub.2D bound to VDR-LBD represents therefore a unique tool
in terms of both ease and effectiveness for selectively capturing
circulating 1,25(OH).sub.2D from natural DBP in the presence of a
molar excess of VDR-LBD, while leaving at the same time the
majority of 25(OH)D in a sequestered form bound to DBP. Such an
approach is particularly advantageous over the prior art methods,
which require time-consuming and labor-intensive sample
pre-treatment steps to allow the determination of 1,25(OH).sub.2D
in clinical samples.
[0087] Since the binding of 1,25(OH).sub.2D to VDR-LBD is known to
induce a conformational change in the VDR-LBD molecule, the present
inventors have conducted extensive experimentation to develop a
capture moiety, such as an antibody, capable of specifically
recognizing and binding to VDR-LBD bound to 1,25(OH).sub.2D without
cross-reacting with uncomplexed VDR-LBD, in order to selectively
discriminate the VDR-LBD/1,25(OH).sub.2D complex from unbound
VDR-LBD in various biological matrices. Such conformation-specific
capture moiety is particularly useful, since it represents an
invaluable tool for the rapid and reliable detection of the
circulating active form of vitamin D.
[0088] As mentioned above, a characterizing feature of the
detection method disclosed in PCT Patent Publication WO 2014/114780
and in US Patent Publication No. 2015-0361178 is that the pH of the
biological fluid sample under examination is adjusted to a value
above 6, i.e. comprised between 6 and 9. Preferred pH values are
comprised between 7 and 8.6, such as 7.2, 7.3, 7.4, 7.5, 7.6, 7.7.,
7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5 or 8.6. Buffering agents and
buffer solutions suitable for adjusting the pH of a biological
fluid sample to the above-mentioned values are well known to those
skilled in the art.
[0089] In the context of the present invention, the biological
fluid sample is preferably selected from the group consisting of
whole blood, serum, plasma, and urine. The biological fluid sample
may optionally include further components, such as for example:
diluents, preservatives, stabilizing agents and/or buffers. If
needed, dilutions of the biological fluid sample are prepared using
any suitable diluent buffer known in the art.
[0090] The detection method disclosed in PCT Patent Publication WO
2014/114780 and in US Patent Publication No. 2015-0361178 is
further characterized in that a receptor protein comprising the
Ligand Binding Domain of Vitamin D Receptor (VDR-LBD) is employed
in order to bind 1,25(OH).sub.2 D or analog thereof.
[0091] The term "receptor protein comprising the Ligand Binding
Domain of Vitamin D Receptor (VDR-LBD)" as used in the present
description encompasses both the whole Vitamin D Receptor protein
(VDR), which includes the C-terminal Ligand Binding Domain, and the
Ligand Binding Domain (LBD) of Vitamin D Receptor in an isolated or
engineered form.
[0092] For example, the whole Vitamin D Receptor protein or the
Ligand Binding Domain thereof is a recombinant protein generated by
DNA technologies. Nucleotide sequences encoding Vitamin D Receptor
from various animal species are available and characterized.
[0093] Thus, the whole Vitamin D Receptor protein or the Ligand
Binding Domain thereof used in PCT Patent Publication WO
2014/114780 and in US Patent Publication No. 2015-0361178 as the
receptor protein is, for example but without limitation, of
mammalian origin (e.g., a human, mouse or rat protein), or of avian
origin, or of amphibian origin; alternatively, it is a mutated
variant of any of such proteins.
[0094] Optionally, the whole Vitamin D Receptor protein or the
Ligand Binding Domain thereof used as the receptor protein in the
present invention further comprises or is coupled to an affinity
tag, in order to substantially improve purification and/or
detection procedures. Among the most common affinity tags,
polyhistidine tags ("His-tag") attached at the C-terminal or
N-terminal of the protein of interest are routinely employed in
protein sciences and their use within the context of the present
invention is therefore well within the knowledge of the person
skilled in the art. Expressed His-tagged proteins are easily
purified e.g. on matrices containing transitional metal ions, and
the use of anti-His-tag antibodies represents a useful and known
tool in localization and immunoprecipitation studies.
[0095] In order to produce recombinant VDR-LBD proteins to be used
as suitable reagents for the methods and kits of the invention, the
plasmid-based expression vector described in PCT Patent Publication
WO 2014/114780 and in US Patent Publication No. 2015-0361178 is
preferably employed. Briefly, DNA coding for the ligand binding
domain of the vitamin D receptor from Rattus norvegicus residues
116-423 with deletion of a 47 amino acid internal loop (165-211)
(rVDR-LBD) was cloned into the pET-29b plasmid (Novagen) by using
the Nde I/Bgl II restriction site combination. To facilitate the
detection and purification of recombinant VDR-LBD protein, a
polyhistidine tag can be added at the C-terminus of the protein of
interest by cloning a His tag coding sequence downstream of the
VDR-LBD coding sequence, followed by a stop codon.
[0096] Therefore, in a preferred embodiment of the method disclosed
in PCT Patent Publication WO 2014/114780 and in US Patent
Publication No. 2015-0361178, the whole Vitamin D Receptor protein
or the Ligand Binding Domain thereof used as the receptor protein
is a recombinant His-tagged fusion protein. However, other affinity
tags such as, for example, Argy, Strep-tag II, FLAG, fluorescein
(FITC), Poly(A), Poly(dT) and biotin may be employed. Techniques
for the production of epitope-tagged recombinant proteins are
generally known in the art. In another preferred embodiment, the
whole Vitamin D Receptor protein or the Ligand Binding Domain
thereof used as the receptor protein is coupled to a chaperone
protein or in general to any other protein which has a
chaperone-like function, in order to help protein folding and/or
improve stability. A receptor protein (i.e. the whole Vitamin D
Receptor protein or the Ligand Binding Domain thereof, possibly
coupled to an affinity tag or a chaperone or chaperone-like
protein) bearing an amino acid sequence mutation aimed at improving
stability may also be employed within the context of the
invention.
[0097] As mentioned above, the detection method disclosed in PCT
Patent Publication WO 2014/114780 and in US Patent Publication No.
2015-0361178 involves the use of a capture moiety capable of
binding the VDR-LBD/1,25(OH).sub.2D complex by specifically
recognizing the conformationally modified VDR-LBD bound to
1,25(OH).sub.2D or analog thereof, without cross-reacting with
uncomplexed VDR-LBD.
[0098] Preferably, the antibody of the invention is a monoclonal
antibody, and more preferably, the monoclonal antibody disclosed in
PCT Patent Publication WO 2014/114780 and in US Patent Publication
No. 2015-0361178. As disclosed therein, a number of hybridoma
clones producing monoclonal antibodies which are able to
specifically recognize and bind to the conformationally modified
VDR-LBD bound to 1,25(OH).sub.2D without substantially
cross-reacting with uncomplexed VDR-LBD, were produced. Methods for
producing such antibodies are disclosed in PCT Patent Publication
WO 2014/114780 and in US Patent Publication No. 2015-0361178. In
brief, individual use aliquots of the immunogen (i.e., the binding
domain of VDR-LBD bound to 1,25(OH).sub.2D), formulated with the
appropriate adjuvant are injected into BALB/c mice. Following 4-,
6- and 8-weeks, lymphocytes from mice spleens are fused with SP2/0
mouse myeloma cells using polyethylene glycol (PEG) as fusion
agent. The hybrid cells are plated over 384 wells in a high
through-put 96 well culture plate format.
[0099] The hybridoma clone, designated as 11B4H11H10, was isolated
in this manner, and found to produce a monoclonal antibody that was
fully characterized by sequencing in order to identify the nucleic
acid and amino acid sequences of its heavy and light chain variable
domains (see, PCT Patent Publication WO 2014/114780 and US Patent
Publication No. 2015-0361178). The CDRs (CDR1, CDR2 and CDR3) of
both the heavy and light chain variable domains were also
identified.
[0100] Such nucleic and amino acid sequences are illustrated in the
Sequence Listing, which forms an integral part of the description;
in the Sequence Listing, the amino acid and nucleic acid sequences
of the heavy chain variable domain of 11B4H11H10 are designated as
SEQ ID NO:7 and SEQ ID NO:8, respectively; the amino acid and
nucleic acid sequences of the light chain variable domain of
11B4H11H10 are designated as SEQ ID NO:9 and SEQ ID NO:10,
respectively; the CDRs of the heavy chain variable domain of
11B4H11H10 are designated as SEQ ID NOs:1, 2 and 3 and the CDRs of
the light chain variable domain of 11B4H11H10 are designated as SEQ
ID NOs:4, 5 and 6.
[0101] Therefore, according to a preferred embodiment, the antibody
of the invention is a monoclonal antibody comprising a heavy chain
variable domain and a light chain variable domain, wherein the
heavy chain variable domain comprises at least one CDR selected
from the group consisting of SEQ ID NOs:1, 2 and 3 and/or the light
chain variable domain comprises at least one CDR selected from the
group consisting of SEQ ID NOs:4, 5 and 6.
[0102] In a more preferred embodiment, the heavy chain variable
domain comprises one, two or all three of the CDRs SEQ ID NOs:1, 2
and 3 and/or the light chain variable domain comprises one, two or
all three of the CDRs SEQ ID NOs:4, 5 and 6.
[0103] In a particular embodiment, the heavy chain variable domain
comprises the amino acid sequence SEQ ID NO:7 or is encoded by a
nucleic acid comprising the sequence SEQ ID NO:8 and/or the light
chain variable domain comprises the amino acid sequence SEQ ID NO:9
or is encoded by a nucleic acid comprising the sequence SEQ ID
NO:10.
[0104] The present study was designed using a new, fully-automated
1,25(OH).sub.2D assay with improved analytical performance,
sensitivity, and reliability. The inventors tested the hypothesis
that levels of 1,25(OH).sub.2D and its ratio to PTH are biomarkers
that allow for risk prediction or stratification of worsening renal
function (WRF) in patients at risk of renal injury or in patients
affected by renal injury.
[0105] As discussed above, prior determinations for Vitamin D
sufficiency have relied upon determining levels of circulating
25-Hydroxyvitamin D (25(OH)D) (calcifediol), which is produced in
the liver by hydroxylation of vitamin D (cholecalciferol) but which
is biologically inactive. 25(OH)D is used for such determinations
as bone weakness, bone malformation, or abnormal metabolism of
calcium (reflected by abnormal calcium, phosphorus, PTH) occurring
as a result of a deficiency or excess of vitamin D. However,
circulating 25(OH)D is transported to the kidneys where it is
converted to its active form 1,25(OH).sub.2D (calcitriol).
1,25(OH).sub.2D acts on the gastrointestinal tract to promote the
absorption of dietary calcium, acts upon the kidney to increase
renal tubular reabsorption of calcium, and on the bone to mobilize
calcium. 1,25(OH).sub.2D circulates in the blood bound to the
vitamin D binding protein, and enters target cells where the
1,25(OH).sub.2D is made available to bind to the vitamin D receptor
(VDR). This ligand/receptor complex readily translocates across the
nuclear membrane to act as a transcription factor. 1,25(OH).sub.2D
is now known to have a broader spectrum of action, and has been
associated with increased risks for various chronic infectious and
autoimmune conditions, diabetes, cancer, cardiovascular ailments,
hypertension, obesity and overweight and complications during
pregnancy. Therefore, the inventors hypothesized that
1,25(OH).sub.2D levels may be more indicative of homeostatic health
and aberrations therefrom as manifested in cardiac, intestinal,
immune, bone, neuronal degenerative, cancerous and diabetic
conditions. While serum 1,25(OH).sub.2D values are not generally
taken, it is considered that normal circulating levels of
1,25(OH).sub.2D in the United States are in the range of 19.9-79.3
pg/ml with a median of about 47.8 pg/ml (LIAISON.RTM. XL 1,25
Dihydroxyvitamin D Assay (REF 310980, REF 310981) IFU (Instructions
for Use)).
[0106] Since the binding of 1,25(OH).sub.2D to the VDR-LBD is known
to induce a conformational change, immunoassay methods for
detecting total 1,25(OH).sub.2D may involve the use of a
conformation-specific capture moiety, such as an antibody, capable
of specifically recognizing and binding to VDR-LBD bound to
1,25(OH).sub.2D, in order to selectively discriminate the
VDR-LBD/1,25(OH).sub.2D complex from either unbound 1,25(OH).sub.2D
or unbound VDR-LBD, as described in WO 2014/114780. Preferably, the
capture moiety of the method of the invention is a monoclonal
antibody specific to the VDR-LBD-1,25(OH).sub.2D complex
conformation.
[0107] Furthermore, in such detection methods, the detection of the
captured VDR-LBD/1,25(OH).sub.2D complex may be accomplished
through a detectable signal, which is generated directly, for
example, by employing a labeled receptor protein or indirectly, for
example, via a labeled detector molecule which is capable of
specifically binding the VDR-LBD/1,25(OH).sub.2D complex captured
by the capture moiety. Typically, the detector molecule is an
antibody directed to an epitope on the VDR-LBD/1,25(OH).sub.2D
complex which is different from the epitope recognized by the
capture moiety.
[0108] According to a preferred embodiment, the 1,25(OH).sub.2D
immunoassay of the method of the invention is a sandwich
immunoassay, more preferably a chemiluminescence immunoassay.
Depending on the format of the immunoassay, the capture antibody
may be immobilized on or to a solid support. Non-limiting examples
of suitable solid supports are the wells of a microtitre plate, the
surface of a microparticle such as a latex, polystyrene, silica,
chelating sepharose or magnetic beads, membranes, strips or
chips.
[0109] The antibody of the present invention is preferably produced
by animal immunization. Briefly, monoclonal antibodies are
generated by injecting animals, for example rats, hamsters, rabbits
or mice, with an immunogen comprising the conformationally modified
VDR-LBD bound to 1,25-(OH).sub.2 vitamin D or analog thereof,
according to methods known per se (Costagliola et al., J Immunol
1998; 160:1458-65). The presence of specific antibody production is
monitored after the initial injection and/or after a booster
injection by performing an immunodetection assay on a serum sample
obtained from the injected animals. From the animals which are
found to produce the specific antibody(ies) of interest, spleen
cells are removed and subsequently fused with a myeloma cell fusion
partner to generate hybridoma cell lines which are then screened
for their ability to secrete the antibody(ies) of interest, i.e.,
antibodies which specifically bind to the VDR-LBD of the complex
formed between VDR-LBD and 1,25(OH).sub.2D or analog thereof.
[0110] In the detection method of the present invention, the
detection of the captured VDR-LBD/1,25(OH).sub.2D complex may be
accomplished through a wide range of techniques. For example, a
detectable signal may be generated directly by employing a labeled
receptor protein or indirectly via a labeled detector molecule
which is capable of binding the VDR-LBD/1,25(OH).sub.2D complex
captured by the capture moiety. Typically, the detector molecule is
another antibody directed to an epitope on the
VDR-LBD/1,25(OH).sub.2D complex which is different from the epitope
recognized by the capture moiety of the invention (i.e., an
anti-VDR-LBD detector antibody).
[0111] The detectable label may be any substance capable of
producing a signal that is detectable by visual or instrumental
means. Suitable labels for use in the present invention include for
example fluorescent compounds, chemiluminescent compounds,
radioactive compounds, enzymes and enzyme substrates, molecules
suitable for colorimetric detection, binding proteins, epitopes,
enzymes or substrates. In practice, any signal molecule or label
known in the art may be incorporated in embodiments of the method
and kit of the present invention.
[0112] Any assay format which enables contact between the
biological fluid sample and the receptor protein comprising the
Ligand Binding Domain of Vitamin D Receptor(VDR-LBD) is suitable
for carrying out the detection method of the invention.
[0113] According to a preferred embodiment, the detection method of
the invention is an in vitro immunoassay performed on a biological
fluid sample of a subject or patient. Immunoassays include both
homogeneous and heterogeneous assays, as well as competitive and
non-competitive sandwich assays.
[0114] By way of example, one-site, non-competitive immunoassays
may be conducted as described in PCT Patent Publication WO
2014/114780 and in US Patent Publication No. 2015-0361178 (FIG. 1
and FIG. 2), wherein the complex formed via the binding of
1,25(OH).sub.2D to the labeled receptor protein which comprises the
Ligand Binding Domain of Vitamin D Receptor (VDR-LBD) is captured
by the conformation-specific capture antibody of the invention
(which in FIG. 1 and FIG. 2 is designated a "Monoclonal Anti-Bound
LBD") immobilized on or to a solid support. In the examples of FIG.
1 and FIG. 2, the solid support is a paramagnetic particle (PMP)
and the label is Amino-Butyl-Ethyl-Isoluminol (ABEI).
[0115] In a specific embodiment described in PCT Patent Publication
WO 2014/114780 and in US Patent Publication No. 2015-0361178 (FIG.
1) the step of adjusting the pH of the biological fluid sample with
the assay buffer and the step of adding the receptor protein
comprising the VDR-LBD to the sample, are performed simultaneously.
In the specific embodiment of FIG. 2, such steps are carried out
sequentially.
[0116] PCT Patent Publication WO 2014/114780 and in US Patent
Publication No. 2015-0361178 illustrates (FIG. 3), by way of
example, a suitable sandwich immunoassay. The general features and
procedures of sandwich immunoassays are well-established and known
to the person skilled in the art. A sandwich immunoassay is a
particularly preferred embodiment of the method of the present
invention.
[0117] The sandwich immunoassay disclosed in PCT Patent Publication
WO 2014/114780 and in US Patent Publication No. 2015-0361178 (FIG.
3) involves the binding of the VDR-LBD/1,25(OH).sub.2D complex to
the conformation-specific capture antibody (designated as
"Monoclonal Anti-Bound LBD") immobilized on or to a solid support
(e.g. a paramagnetic particle, PMP) and the use of a labeled
detector antibody as the second part of the sandwich. The detector
antibody is either directly labeled or it is recognized by a
conjugate consisting of a labeled anti-immunoglobulin antibody (in
the specific example of FIG. 3, the detector antibody is directly
labeled with ABEI). The amount of labeled antibody directly or
indirectly bound to the VDR-LBD/1,25(OH).sub.2D complex is then
measured by suitable means.
[0118] A sandwich immunoassay suitable for use in the present
invention may involve the use of a tagged receptor protein
comprising VDR-LBD in combination with an anti-tag detector
antibody. In this embodiment, the detection of the
VDR-LBD/1,25(OH).sub.2D complex captured by the
conformational-specific capture antibody is achieved by the
specific binding of the detector antibody to the tag which is
present on the complex. Preferably, the tag is a polyhistidine tag.
In a more specific embodiment, the tag is a chaperone protein.
[0119] The immunoassays falling within the scope of the invention
may, however, be in any suitable format, such as, for example,
radioimmunoassays (MA), chemiluminescence- or
fluorescence-immunoassays, Enzyme-linked immunoassays (ELISA),
Luminex-based bead arrays, protein microarray assays, or rapid test
formats such as, for instance, immunochromatographic strip
tests.
[0120] Depending on the format of the immunoassay, the capture
antibody and/or the detector antibody may be immobilized on or to a
solid support. Non-limiting examples of suitable solid supports are
the wells of a microtitre plate, the surface of a microparticle
such as a latex, polystyrene, silica, chelating sepharose or
magnetic beads, membranes, strips or chips.
[0121] Parathyroid hormone ("PTH") is secreted by the parathyroid
glands and acts to increase the concentration of calcium in the
blood by binding to the parathyroid receptor (expressed at high
levels in the bone and kidney) or to parathyroid hormone 2 receptor
(expressed in the CNS, pancreas, testis and placenta). Further, PTH
increases the activity of renal 1-.alpha.-hydroxylase, which
converts 25-hydroxyvitamin D to 1,25(OH).sub.2D to support
endocrine function. 1-.alpha.-hydroxylase is also expressed in
various other tissues, whose cells may convert 25(OH)D for
autocrine and paracrine functions. Normal values for PTH are
considered to be 5.72 to 45.4 pg/mL and 5.68 to 47.8 pg/mL in EDTA
plasma and serum, respectively (LIAISON.RTM. 1-84 PTH Assay (REF
310630, REF 310631) IFU (Instructions for Use). The median for the
ratio of 1,25(OH).sub.2D to PTH in normal individuals is
approximately 2.7 (range of 1.2-9.1).
[0122] The ratio of 1,25(OH).sub.2D to PTH is identified herein as
being a biomarker for predicting or stratifying the risk of
worsening renal function (WRF) in patients at risk of renal injury
or in patients affected by renal injury.
[0123] As mentioned above, a further aspect of the present
invention is a kit for detecting and quantifying (i.e., determining
the amount or concentration) of 1,25(OH).sub.2D or analog thereof
in a biological fluid sample, and for additionally detecting PTH in
an aliquot of such sample, or in another biological fluid sample of
a subject patient. Preferably, the kit will comprise the Vitamin D
receptor protein and the capture moiety as defined above in
connection with the method, as well as a binding buffer which has a
pH comprised between 6 and 9. Preferred pH values are comprised
between 7 and 8.6, such as 7.2, 7.3, 7.4, 7.5, 7.6, 7.7., 7.8, 7.9,
8.0, 8.1, 8.2, 8.3, 8.4, 8.5 or 8.6. Preferred but not limiting
examples of the binding buffer for adjusting the pH of the test
sample include 50 mM Tris buffer (pH 7.4), Hepes (6.5-7.5), PBS.
The kit will also contain reagents for detecting and quantifying
the amount or concentration of PTH.
[0124] The kit of the invention may further comprise a solid
support such as, without limitation, beads, microparticles,
nanoparticles, super paramagnetic particles, a microtitre plate, a
cuvette, a lateral flow device, a flow cell, or any surface to
which a protein or peptide can be passively or covalently bound.
Either the receptor protein or the capture moiety of the kit of the
invention may be immobilized on or to the solid support.
[0125] Further, the kit of the invention may contain detection
means as described above in connection with the detection
method.
[0126] A "Reagent Integral" is a multi-chambered cartridge that is
specially adapted for use in an automated analyzing device, such as
the Liaison.RTM. Analyzer. FIG. 4 shows an exemplary Reagent
Integral. A Research Integral will comprise one or more "Reagent
Chambers," one or more optional "Calibrator Chambers," which will
be sealed to prevent contamination and spillage prior to use, a
chamber for mixing reagents, which may have an agitator, such as a
stirring wheel. The Research Integral may comprise a handle to
facilitate transport, and a nose to facilitate movement of the
Research Integral within the analyzer. The respective "Reagent
Chambers" hold reagents needed to conduct a desired assay, and are
constructed of a non-reactive material (e.g., glass, plastic,
resin, etc.). The Reagent Integral is preferably accompanied by
software, barcodes, etc. that is used by the automated analyzing
device to analyze the obtained data. The Reagent Integrals may
additionally comprise one or more "Calibrator Chambers" that are
specific to each Research Integral, and that are assayed to create
a "Working Curve," in conjunction with the Research Integral's
software (which provides a "Master Curve").
[0127] In some embodiments, the Research Integral will be specially
adapted to analyze a reagent by conducting an assay for that
reagent in accordance with a particular assay format, such as a
format in which one or more reactants is immobilized on or to a
solid support. Preferably, this is accomplished by immobilizing
such reagent directly or indirectly to a magnetic bead present in a
chamber or container of the Reagent Integral, and thus the Reagent
Integral may comprise a chamber or container that contains such
beads.
[0128] Thus, such Reagent Integral kits are adapted to be used to
analyze the concentrations or amounts of one or more target
analytes in a biological sample (e.g., serum, plasma, blood, etc.)
(and particularly in aliquots of the same biological sample) of a
patient.
[0129] In a preferred embodiment, a kit of the present invention
will be a "Reagent Integral" that is specially adapted for
determining the concentrations or amounts of 1,25-dihydroxyvitamin
D and PTH. In a more preferred embodiment, such a Reagent Integral
kit of the present invention is a single multi-chambered cartridge
specially adapted for determining the concentrations or amounts of
both 1,25-dihydroxyvitamin D and PTH. Thus, in a preferred
embodiment, a Reagent Integral kit of the present invention will
comprise: [0130] (A) a first container (e.g., a Reagent Chamber)
that contains Ligand Binding Domain of Vitamin D Receptor (VDR-LBD)
and an antibody, or epitope-binding fragment thereof, that
specifically binds to a conformational epitope of VDR-LBD when the
VDR-LBD is complexed with 1,25-dihydroxyvitamin D in serum or
plasma or blood, and does not specifically bind to VDR-LBD or to
1,25-dihydroxyvitamin D that are not complexed with one another;
and [0131] (B) a second container (e.g., a Reagent Chamber) that
contains a detectably-labelled reagent capable of binding to
parathyroid hormone (PTH).
[0132] In a preferred embodiment, the antibody, or epitope-binding
fragment thereof, that specifically binds to the conformational
epitope of VDR-LBD is immobilized on or to a solid support in the
first container. In a further embodiment, the antibody, or
epitope-binding fragment thereof, of the first container is a
monoclonal antibody (or epitope-binding fragment thereof). In a
further embodiment, the antibody, or epitope-binding fragment
thereof, of the first container may be detectably labeled.
[0133] Various exemplary embodiments of devices and compounds as
generally described above and methods according to this invention
will be understood more readily by reference to the following
examples, which are provided by way of illustration and are not
intended to be limiting of the invention in any fashion.
EXPERIMENTAL SECTION
Example 1: 1,25(OH).sub.2D assay
[0134] One of the preferred embodiment of the assay of the
invention is described in in PCT Patent Publication WO 2014/114780
and in US Patent Publication No. 2015-0361178. In brief,
paramagnetic microparticles (PMPs) (Dynal, Norway) were coated with
a conformation-specific monoclonal antibody capable of recognizing
the VDR-LBD/1,25(OH).sub.2D complex antibody following the supplier
instructions. The recombinant VDR-LBD that was used in the assay
was prepared as described in Example 1, and was coupled to an
affinity tag (designated in the following as "TAG"). The 11B4H11H10
monoclonal antibody, described above, was used for this purpose. A
mouse monoclonal anti-TAG antibody was conjugated with cyclic
AminoButhylEthylisoluminol (cABEI) in PBS buffer pH 7.4. The
calculated cABEI incorporation was from 2-3 molecules per antibody
molecule. Calibrators were prepared by adding different
concentrations of an ethanolic solution of 1,25(OH).sub.2D into a
steroid-free, charcoal-stripped human serum. The assay buffer
formulation consisted of TRIS 50 mM pH 7.4, CHAPS 0.02%, EDTA 1 mM,
heparin at 8 mg/ml and 1% mouse serum to mitigate heterophilic
human anti mouse (HAMA) interferences.
[0135] A major challenge of an automated assay not using any
off-line pre-analytical/sample pre-treatment steps is the ability
of the assay to specifically capture and detect the whole amount of
1,25(OH).sub.2D, or analogues of the active form of vitamin D, in a
biological matrix (e.g. serum or plasma) without interference by
other vitamin D metabolites such as 25(OH)D, 24,25(OH).sub.2D and
25,26(OH).sub.2D which can be present at levels 1000-fold higher
than 1,25(OH).sub.2D. This challenge is further complicated by the
presence of Vitamin D binding protein (DBP) and albumin, which are
abundant in circulation and serve as the major binding proteins for
25(OH)D, 1,25(OH).sub.2D, and other metabolites of vitamin D,
whereby 85% to 90% is bound to DBP and 10 to 15% is bound to
albumin. Furthermore, DBP levels increases up to 2-5 fold in
high-estrogen states, such as pregnancy.
[0136] The assay schematically illustrated in FIG. 3 was carried
out on the DiaSorin LIAISON.RTM. analyzer (Saluggia, Italy). First,
50 .mu.l of human serum sample was incubated with 100 .mu.l of
assay buffer and 50 .mu.l of VDR-LBD-TAG for 30 minutes. Next, 20
.mu.l of PMPs coated with 11B4H11H10 monoclonal antibody were added
and the reaction mixture was incubated for an additional 30
minutes. After washing the reaction mixture, 40 .mu.l of
cABEI-conjugated anti TAG monoclonal antibody was added and the
reaction mixture incubated for an additional 30 min. After a second
wash, trigger solutions were added and the reaction mixture was
read as Relative Lights Units (RLUs) in the analyzer reading
chamber.
Example 2: The 1,25(OH).sub.2D/PTH Ratio as Biomarker of Renal
Injury
[0137] Materials and Methods
[0138] The cohort studied consisted of 1083 patients enrolled in a
biomarker substudy of a randomized, double-blind, placebo
controlled, multicenter study that enrolled 6975 patients with
clinical evidence of chronic and stable HF (NYHA II-IV),
irrespective of the cause and the level of left ventricular
ejection fraction (LVEF). In relation to renal function, 180
patients of the cohort were normal (i.e., eGFR (mL/min/1.73
m.sup.2).gtoreq.90) and the remainder had eGFR below 90. Venous
blood samples were drawn on EDTA at randomization and after three
months of follow-up. Patients rested supine for at least 15 min
before blood sampling. Blood was centrifuged at 4.degree. C. within
10 minutes of draw and plasma aliquots were shipped on dry ice to a
central laboratory. Samples were stored at -70.degree. C. until
assayed. In the analysis, worsening of renal function (WRF) was
employed as the endpoint.
[0139] The plasma concentrations of 1,25-dihydroxyvitamin D
(1,25(OH).sub.2D) and PTH were assayed in a central laboratory in a
blinded fashion and in a single batch. 1,25(OH).sub.2D was
determined with a new fully automated and sensitive immunoassay
that uses a recombinant fusion construct of the vitamin D receptor
ligand binding domain for specific capture of 1,25(OH).sub.2D
(DiaSorin, Saluggia, Italy) and a conformation-specific monoclonal
antibody capable of recognizing the VDR-LBD/1,25(OH).sub.2D complex
antibody, such as the 11B4H11H10 (also referred to as "11B4")
monoclonal antibody. The limit of quantitation for this
1,25(OH).sub.2D assay is 5 pg/mL and the reference interval
determined in healthy volunteers ranged between 19.9 and 79.3 pg/mL
with a median of 47.8 pg/mL. PTH levels were determined using a
sensitive immune assay for the determination of PTH in blood, serum
or plasma (Liaison 1-84 PTH, DiaSorin, Saluggia, Italy, #310630)
with a measurement range of between 4 and 1800 pg/ml, with the
limit of detection being 1.7 pg/ml and the limit of quantitation
being 4 pg/ml. The reference interval determined for healthy
25(OH)D sufficient, volunteers ranged from 5.5 to 48 (LIAISON.RTM.
1-84 PTH Assay (REF 310630, REF 310631) IFU (Instructions for Use))
with a median of 15.3 pg/ml.
[0140] Serum creatinine was measured in local laboratories as part
of a national quality control surveillance, at randomization and
during follow-up after 1, 3, 6, 12, 24, 36, 48 and 60 months.
Glomerular filtration rate (eGFR, mL/min/1.73 m.sup.2) was
estimated using the simplified modification of diet in renal
disease (MDRD) formula. WRF was defined as the first increase in
serum creatinine concentration .gtoreq.0.3 mg/dL and .gtoreq.25% at
two consecutive measurements at any time during the study (Damman,
K. et al. (2014) "Terminology and Definition of Changes to Renal
Function in Heart Failure," Eur. Heart J. 35(48):3413-3416).
[0141] Statistical methods: Continuous variables were expressed as
mean.+-.SD if normally distributed or median [Q1-Q3], as
appropriate; categorical variables were reported as absolute
numbers and percentages.
[0142] Linear multilevel analysis was used to assess the
association of baseline patient characteristics with decreasing
baseline levels of the 1,25(OH).sub.2D to PTH ratio, which was
transformed on a natural logarithmic scale. The model allowed
consideration of variable patient characteristics as fixed effects
whereas multiple clinical centers introduced random effects.
[0143] A Cox proportional hazards model aiming to assess the
independent prognostic value of the 1,25(OH).sub.2D to PTH ratio on
the occurrence of WRF was built, adjusting for the covariates that
were statistically significant in the univariate analysis
(P<0.05). Similarly, multivariable Cox models were adopted for
the secondary outcomes of the present analysis.
[0144] For all of the categorical variables, the proportionality of
risk required by the Cox model was assessed using Schoenfeld
residuals. The ratio was initially fitted as a single continuous
measurement. Because there was clear evidence of non-linearity of
risk detected by the restricted cubic splines technique (RCS), it
was transformed with natural logarithms, hence satisfying the
linearity assumption imposed by the Cox model.
[0145] To establish the incremental prognostic value of the
1,25(OH).sub.2D to PTH ratio on the occurrence of WRF, in addition
to the conventional risk factors that emerged as statistically
significant in the multivariable model, the category-free Net
Reclassification Index (cfNRI) was calculated (Pencina, M. J. et
al. (2011) "Extensions of Net Reclassification Improvement
Calculations to Measure Usefulness of New Biomarkers," Stat. Med.
30:11-21).
[0146] A 2-sided P value of <0.05 was considered statistically
significant. Statistical analyses were performed with SAS software,
version 9.3 (SAS Institute, Inc., Cary, N.C.) and with the R
program and the rms package
(http://CRAN.R-project.org/package=rms).
Results
[0147] Study Population--Baseline Characteristics:
[0148] The distribution of baseline characteristics and laboratory
values across the entire cohort according to 25(OH)D and
1,25(OH).sub.2D levels are displayed in Table 1.
[0149] Univariate and Multivariable Cox Proportional Hazard
Models
[0150] COX proportional hazard (CPH) analyses were carried out by
entering biomarker concentration values into the models as
loge-transformed variables. Table 2 shows the results of the Cox
proportional hazard analysis for the association of WRF with
baseline 1,25(OH).sub.2D alone, PTH alone and their ratio. WRF
occurred at 189 [82-735] days after randomization (equivalent to
6.2 [2.7-24.1] months) (median [Q1-Q3]).
TABLE-US-00001 TABLE 1 All No WRF WRF Variable N = 1130 N = 795
(70.4%) N = 335 (29.6%) P Age (years) 66.8 .+-. 10.8 66.2 .+-. 11.1
68.1 .+-. 9.8 0.004 Sex (% males) 917 (81.2) 638 (80.3) 279 (83.3)
0.23 BMI (kg/m.sup.2) 26.8 .+-. 4.3 26.7 .+-. 4.4 26.9 .+-. 4.2
0.49 NYHA III-IV (%) 290 (25.7) 191 (24.0) 99 (29.6) 0.052 Ischemic
HF (%) 579 (51.2) 400 (50.3) 179 (53.4) 0.34 HR (bpm) 71.5 .+-.
13.6 70.9 .+-. 13.0 72.5 .+-. 14.7 0.07 SBP (mmHg) 124.9 .+-. 18.8
125.0 .+-. 18.8 125 .+-. 19.0 0.64 DBP (mmHg) 76.4 .+-. 10.4 76.7
.+-. 10.4 75.7 .+-. 10.5 0.14 LVEF (%) 33.1 .+-. 9.4 33.4 .+-. 9.3
32.5 .+-. 9.5 0.18 CVP > 6 cm H.sub.2O (%) 89 (7.9) 61 (7.7) 28
(8.4) 0.70 Medical History Previous MI (%) 495 (43.8) 338 (42.5)
157 (46.9) 0.18 Previous stroke (%) 53 (4.7) 36 (4.5) 17 (5.1) 0.69
History of hypertension (%) 619 (54.8) 427 (53.7) 192 (57.3) 0.27
History of diabetes (%) 295 (26.1) 201 (25.3) 94 (28.1) 0.33
History of atrial fibrillation (%) 208 (18.4) 137 (17.2) 71 (21.2)
0.12 History of COPD (%) 210 (18.6) 138 (17.4) 72 (21.5) 0.10
Laboratory parameters Serum creatinine (mg/dL) 1.20 .+-. 0.42 1.16
.+-. 0.37 1.29 .+-. 0.45 <0.0001 eGFR (mL/min/1.73 m.sup.2) 68.6
.+-. 23.5 71.0 .+-. 23 63.7 .+-. 22.9 <0.0001 Serum bilirubin
(mg/dL) 0.84 .+-. 0.55 0.86 .+-. 0.61 0.80 .+-. 0.36 0.07 Serum
fibrinogen (mg/dL) 375 .+-. 108 372 .+-. 104 379 .+-. 118 0.36
Serum cholesterol (mg/dL) 190 .+-. 41 193 .+-. 42 184 .+-. 40 0.002
Serum LDL-cholesterol 115 .+-. 36 118 .+-. 36 111 .+-. 35 0.006
(mg/dL) Serum HDL-cholesterol 47 .+-. 15 48 .+-. 15 44 .+-. 15
0.0004 (mg/dL) Serum triglycerides 123 [91-174] 123 [90-176] 124
[93-168] 0.90 (mg/dL) Medical Therapy ACEi (%) 926 (82.0) 645
(81.1) 281 (83.9) 0.27 ARB (%) 196 (17.4) 134 (16.9) 62 (18.5) 0.50
Diuretics (%) 1045 (92.5) 719 (90.4) 326 (97.3) <0.0001
Beta-blockers (%) 771 (68.2) 549 (69.1) 222 (66.3) 0.36
Spironolactone (%) 479 (42.4) 313 (39.4) 166 (49.6) 0.002 Digitalis
(%) 387 (34.3) 262 (33.0) 125 (37.3) 0.16 ASA (%) 569 (50.4) 421
(53.0) 148 (44.2) 0.007 Nitrates (%) 358 (31.7) 249 (31.3) 109
(32.5) 0.69 Amiodarone (%) 230 (20.4) 141 (17.7) 89 (26.6) 0.0008
Randomization to n-3 569 (50.4) 407 (51.2) 162 (48.4) 0.38 PUFA (%)
Randomization to 334 (50.7) 235 (50.5) 99 (51.0) 0.91 rosuvastatin
(%) Biomarkers 1,25(OH)2 vitamin D (pg/mL) 31.3 [23.0-42.2] 32.2
[24.1-43.1] 28.7 [21.4-38.6] 0.0001 PTH (1-84) (pg/mL) 33.7
[24.3-49.3] 32.5 [23.6-46.4] 36.3 [26.2-53.4] 0.0008 1,25(OH).sub.2
vitamin D/PTH 0.89 [0.55-1.45] 0.98 [0.60-1.53] 0.73 [0.46-1.26]
<0.0001 Blood samples drawn in 328 (29.0) 235 (29.6) 93 (27.8)
0.26 winter (No., %)
TABLE-US-00002 TABLE 2A Univariate No. HR [95% CI] P
1,25(OH).sub.2D 1098 0.61 [0.49-0.75] <0.0001 PTH (1-84) 1099
1.57 [1.28-1.93] <0.0001 1,25(OH).sub.2D/PTH 1067 0.60
[0.52-0.70] <0.0001
[0151] When multivariable COX analyses were performed, the
following variables were entered into the Cox multivariate models:
1,25(OH).sub.2D/PTH (loge-transformed), age, eGFR (MDRD equation),
NYHA class, serum concentrations of total cholesterol and
bilirubin, prescriptions of diuretics, spironolactone, aspirin or
amiodarone.
TABLE-US-00003 TABLE 2B Multivariate No. HR [95% CI] P
1,25(OH).sub.2D 1043 0.76 [0.59-0.97] 0.03 PTH (1-84) 1040 1.20
[0.95-1.50] 0.12 1,25(OH).sub.2D/PTH 1012 0.75 [0.62-0.90]
0.003
[0152] Variables significantly associated with WRF in the
multivariable models: With 1,25(OH).sub.2D: serum cholesterol
(p=0.0003), amiodarone (p=0.0005), aspirin (p=0.01), diuretics
(p=0.02), serum bilirubin (p=0.02), spironolactone (p=0.04).
[0153] With PTH: amiodarone (p=0.0003), serum cholesterol
(p=0.0004), aspirin (p=0.02), diuretics (p=0.03), spironolactone
(p=0.03), serum bilirubin (p=0.04).
[0154] With 1,25(OH).sub.2D/PTH: serum cholesterol (p=0.0001),
amiodarone (p=0.0008), aspirin (p=0.01), diuretics (p=0.02),
spironolactone (p=0.03), serum bilirubin (p=0.04), eGFR (0.05).
Prognostic Accuracy of Baseline 1,25(OH).sub.2D/PTH for WRF
ROC Curves
[0155] a) ROC Curves of Baseline 1,25(OH).sub.2D/PTH with WRF.
[0156] In ROC analysis, the area under the curve (AUC) criterion
was applied to WRF at the end of the 3.9 year follow-up (FIG. 5).
The receiver operator curve depicted in FIG. 5 for WRF at 3.9 years
follow up to baseline randomization discloses an AUC of 60%, with
specificity of 54% and sensitivity of 64% using the optimal
1,25(OH).sub.2D/1-84 PTH ratio threshold of 0.92.
TABLE-US-00004 TABLE 3 AUROC .+-. SD P Specificity Sensitivity
Optimal cut-off 1,25(OH).sub.2D/PTH 0.60 .+-. 0.02 <0.0001 0.54
0.64 0.92
[0157] Kaplan-Meier curves for the first occurrence of WRF
according to the 1,25(OH).sub.2D to PTH ratio are presented in FIG.
6. The Kaplan Meier curves in FIG. 6 depict the ability of the
1,25(OH).sub.2D/1-84PTH ratio to discriminate amongst HF subjects
those which have a significantly greater likelihood to both develop
WRF and whose WRF will progress more quickly. With advancing time
post randomization, those HF subjects whose baseline
1,25(OH).sub.2D/1-84PTH ratio were below the 0.92 threshold
developed WRF at a faster rate than those whose ratio were
>0.92.
b) Maximal Negative Predictive Value
[0158] The maximal negative predictive value (NPV=0.82) is observed
at a ratio of 1,25(OH).sub.2D to PTH of 1.68, with a sensitivity of
0.90, a specificity of 0.21 and a positive predictive value
PPV=0.32. A total of 187 patients (17.5%) have a ratio above the
level of 1.68. At this cut-off, the contingency table for the
occurrence of WRF is as follows:
TABLE-US-00005 WRF N Yes (positive) No (negative) 1,25(OH).sub.2
D/PTH <1.68 (positive) 285 595 .gtoreq.1.68 (negative) 33
154
True positive: 285/1067=26.7% False positive: 595/1067=55.8% False
negative: 33/1067=3.1% True negative: 154/1067=14.4% Test
performance at the cutoff of the ratio 1.68 (maximal NPV):
PPV=TP/(TP+FP)=A/(A+B)=285/(285+595)=0.3239
NPV=TN/(TN+FN)=D/(D+C)=154/(154+33)=0.8235
Sensitivity=TP/(TP+FN)=A/(A+C)=285/(285+33)=0.8962
Specificity=TN/(TN+FP)=D/(D+B)=154/(154+595)=0.2056
TABLE-US-00006 [0159] Statistic Value (%) Sensitivity 89.6
Specificity 20.6 Negative Predictive Value (NPV) 82.4 Positive
Predictive Value (PPV) 32.4
c) Best Cut-Off Value
[0160] A CART approach was then used to define the best cut-off
value of the 1,25(OH).sub.2D to PTH ratio to predict WRF.
Classification and regression trees (CART) is a model-free approach
used to find the best splitting criterion. This method, a form of
recursive partitioning, developed on 0.9 of the data allows to
validate best on the remaining 0.1 of the data. At the optimal
cut-off of 0.98, the contingency table for the occurrence of WRF is
as follows:
TABLE-US-00007 WRF N Yes (positive) No (negative 1,25(OH).sub.2
D/PTH <0.98 (positive) 217 372 .gtoreq.0.98 (negative) 101
377
True positive: 217/1067=20.3% False positive: 372/1067=34.9% False
negative: 101/1067=9.5% True negative: 377/1067=35.3% Test
performance at the cutoff of the ratio >0.98 (best cut-off
identified with CART method)
PPV=TP/(TP+FP)=A/(A+B)=217/(217+372)=0.3684
NPV=TN/(TN+FN)=D/(D+C)=377/(377+101)=0.7887
Sensitivity=TP/(TP+FN)=A/(A+C)=217/(217+101)=0.6823
Specificity=TN/(TN+FP)=D/(D+B)=377/(377+372)=0.5033
TABLE-US-00008 [0161] Statistic Value (%) Sensitivity 68.2
Specificity 50.3 Negative Predictive Value (NPV) 78.9 Positive
Predictive Value (PPV) 36.8
[0162] While this invention has been described in conjunction with
the various exemplary embodiments outlined above, various
alternatives, modifications, variations, improvements and/or
substantial equivalents, whether known or that are or may be
presently unforeseen, may become apparent to those having at least
ordinary skill in the art. Accordingly, the exemplary embodiments
according to this invention, as set forth above, are intended to be
illustrative not limiting. Various changes may be made without
departing from the spirit and scope of the invention. Therefore,
the invention is intended to embrace all known or later-developed
alternatives, modifications, variations, improvements and/or
substantial equivalents of these exemplary embodiments.
DISCUSSION
[0163] The present inventors found that the ratio of circulating
1,25(OH).sub.2D and PTH, two hormones involved in bone-mineral
metabolism, can surprisingly predict deterioration of renal
function better than either marker alone. These results were
obtained in a large, representative cohort of patients with HF,
some of which were affected by renal injury, enrolled in a
controlled, multicenter clinical trial.
[0164] Early prediction and identification of patients at risk for
WRF may be useful to optimize therapies, and to improve outcomes.
The search for new markers of changes in renal function is
currently very active. They should be more sensitive and specific
to early changes in renal function than serum creatinine, which is
slowly affected and can be confounded by muscle mass and
anthropometric factors.
[0165] In the present study, the inventors showed that a low
circulating ratio of 1,25(OH).sub.2D to PTH predicted future
episodes of WRF in patients. Although this ratio was strongly
associated with concomitant serum creatinine levels, its prognostic
value was independent of eGFR estimated from creatinine levels,
suggesting a net contribution of vitamin D metabolism to the
prediction of WRF.
[0166] In conclusion, the ratio of 1,25(OH).sub.2D to PTH is a new,
powerful indicator of future risk of deterioration of renal
function in patients at risk of renal injury and affected by renal
injury.
Sequence CWU 1
1
10110PRTMus musculus 1Gly Phe Thr Phe Ser Asn Phe Gly Met Gln1 5
10210PRTMus musculus 2Tyr Ile Ser Ser Gly Ser Ser Thr Ile Tyr1 5
1039PRTMus musculus 3Ser Gly Leu Ile Asp Gly Phe Ala Tyr1
5411PRTMus musculus 4His Ala Ser Gln Gly Ile Ser Ser Asn Ile Gly1 5
1057PRTMus musculus 5His Gly Thr Asn Leu Glu Asp1 569PRTMus
musculus 6Val Gln Tyr Ala Gln Phe Pro Phe Thr1 57110PRTMus musculus
7Gly Gly Leu Val Gln Pro Gly Gly Ser Arg Lys Leu Ser Cys Ala Ala1 5
10 15Ser Gly Phe Thr Phe Ser Asn Phe Gly Met Gln Trp Val Arg Gln
Ala 20 25 30Pro Glu Lys Gly Leu Glu Trp Val Ala Tyr Ile Ser Ser Gly
Ser Ser 35 40 45Thr Ile Tyr Tyr Ala Asp Thr Val Lys Gly Arg Phe Thr
Ile Ser Arg 50 55 60Asp Asn Pro Lys Asn Thr Leu Phe Leu Gln Met Thr
Ser Leu Arg Ser65 70 75 80Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg
Ser Gly Leu Ile Asp Gly 85 90 95Phe Ala Tyr Trp Gly Gln Gly Thr Thr
Val Thr Val Ser Ser 100 105 1108330DNAMus musculus 8ggaggcttag
tgcagcctgg agggtcccgg aaactctcct gtgcagcctc tggattcact 60ttcagtaact
ttggaatgca gtgggttcgt caggctccag agaaggggct agagtgggtc
120gcatacatca gtagtggcag tagtaccatc tactatgcag acacagtgaa
gggccgattc 180accatatcca gagacaatcc caagaatacc ctgttcctgc
aaatgaccag tctaaggtct 240gaggacacgg ccatgtatta ctgtgcaaga
tcgggtttaa tcgacgggtt tgcttactgg 300ggccaaggga ccacggtcac
cgtctcctca 330995PRTMus musculus 9Gln Ser Pro Ser Ser Met Ser Val
Ser Leu Gly Asp Thr Val Ser Ile1 5 10 15Thr Cys His Ala Ser Gln Gly
Ile Ser Ser Asn Ile Gly Trp Leu Gln 20 25 30Gln Lys Pro Gly Lys Ser
Phe Lys Gly Leu Ile Tyr His Gly Thr Asn 35 40 45Leu Glu Asp Gly Val
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Ala 50 55 60Asp Tyr Ser Leu
Thr Ile Ser Ser Leu Glu Ser Glu Asp Phe Ala Asp65 70 75 80Tyr Tyr
Cys Val Gln Tyr Ala Gln Phe Pro Phe Thr Phe Gly Ser 85 90
9510287DNAMus musculus 10cagtctccat cctccatgtc tgtatctctg
ggagacacag tcagcatcac ttgccatgca 60agtcagggca ttagcagtaa tatagggtgg
ttgcagcaga aaccagggaa atcatttaag 120ggcctgatct atcatggaac
caacttggaa gatggagttc catcaaggtt cagtggcagt 180ggatctggag
cagattattc tctcaccatc agcagcctgg aatctgaaga ttttgcagac
240tattactgtg tacagtatgc tcagtttcca ttcacgttcg gctcggg 287
* * * * *
References