U.S. patent application number 16/259499 was filed with the patent office on 2022-09-01 for methods for identifying risk of chemotherapy-induced cardiotoxicity and targeted medical intervention.
This patent application is currently assigned to Detroit R & D, Inc.. The applicant listed for this patent is Hyesook Kim. Invention is credited to Hyesook Kim.
Application Number | 20220276265 16/259499 |
Document ID | / |
Family ID | 1000006534392 |
Filed Date | 2022-09-01 |
United States Patent
Application |
20220276265 |
Kind Code |
A9 |
Kim; Hyesook |
September 1, 2022 |
Methods for Identifying Risk of Chemotherapy-Induced Cardiotoxicity
and Targeted Medical Intervention
Abstract
This invention discloses diagnosis of risk of
chemotherapy-induced cardiotoxicity by measurement of increased
expression of soluble epoxide hydrolase in vitro and in vivo in
cells, tissues or animals including measurement of increased levels
of soluble epoxide hydrolase metabolites, e.g., 14,15-DHET and
11,12-DHET, in biological fluids. This invention also includes
diagnosis of risk of chemotherapy-induced cardiotoxicity by
measuring increased levels of oxidative stress in cells, tissues or
animals including measurement of increased levels of oxidative
stress biomarkers, e.g., 8-isoprostane, in biological fluids. Fatty
acid and protein biomarkers to diagnose the risk of
chemotherapy-induced cardiotoxicity are detected using various
detection methods including mass spectrometry and immunoassay such
as ELISA, Western blot analysis or label-free microwell and
nanowell technologies. This invention discloses targeted medical
intervention for a subject who is at risk or with
chemotherapy-induced cardiotoxicity by treating with soluble
epoxide hydrolase inhibitor(s) with or without antioxidants to
prevent or ameliorate the chemotherapy-induced cardiotoxicity.
Inventors: |
Kim; Hyesook; (Bloomfield
Hills, MI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Kim; Hyesook |
Bloomfield Hills |
MI |
US |
|
|
Assignee: |
Detroit R & D, Inc.
Bloomfield Hills
MI
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20200241019 A1 |
July 30, 2020 |
|
|
Family ID: |
1000006534392 |
Appl. No.: |
16/259499 |
Filed: |
January 28, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62623775 |
Jan 30, 2018 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2560/00 20130101;
G01N 2800/50 20130101; G01N 2800/32 20130101; G01N 2800/42
20130101; G01N 33/573 20130101; G01N 33/6887 20130101; G01N 33/92
20130101; G01N 2800/52 20130101; G01N 2405/00 20130101 |
International
Class: |
G01N 33/92 20060101
G01N033/92; G01N 33/68 20060101 G01N033/68; G01N 33/573 20060101
G01N033/573 |
Goverment Interests
GOVERNMENT SUPPORT
[0001] Research in this application was supported, in part, by a
Phase I SBIR Contract from the National Heart, Lung, and Blood
Institute (NHLBI Contract HHSN261201600028C).
Claims
1. A method to identify risk of chemotherapy-induced cardiotoxicity
using biomarkers that predict cardiotoxicity by the steps of:
determining early cardiotoxicity biomarker levels in biological
samples including biological fluids and Identifying the increased
level of the biomarker compared to control sample.
2. The method of claim 1, wherein the chemotherapy-induced
cardiotoxicity is induced by an anthracycline, a DNA/RNA
intercalator, such as doxorubicin, daunorubicin, epirubicin and
idarubicin.
3. The method of claim 2, wherein early cardiotoxicity biomarker is
a metabolite of soluble epoxide hydrolase.
4. The method of claim 3, wherein the metabolite is 14,15-DHET or
11,12-DHET.
5. The method of claim 4, wherein the sEH metabolites are
determined by mass spectrometry or immunoassay including ELISA, dot
blot analysis or lateral flow (dipstick) immunoassay or label-free
microwell and nanowell technologies.
6. The method of claim 2, wherein early cardiotoxicity biomarker is
8-isoprostane.
7. The method of claim 2, wherein multiple early cardiotoxicity
biomarkers consisted of 14,15-DHET and 8-isoprostane.
8. The method of claim 7, wherein with multiple early
cardiotoxicity biomarkers consisted of increased levels of
14,15-DHET, 8-isoprostane, troponin I (TnI), myeloperoxidase (MPO)
and/or GDF-15 (NAG-1).
9. The method of claim 2, wherein early cardiotoxicity biomarker is
the increased brain natriuretic peptide (BNP) level.
10. A method to identify sEH-dependent cardiotoxicity after
chemotherapy using a metabolite of sEH by the steps of: determining
the 14,15-DHET level in biological fluids and Identifying the
increased 14,15-DHET level compared to control sample.
11. The method of claim 10, wherein the cardiotoxicity is induced
by an anthracycline, a DNA/RNA intercalator, such as doxorubicin,
daunorubicin, epirubicin and idarubicin.
12. The method of claim 11, wherein the chemotherapy-associated
cardiotoxicity is diagnosed with cardiovascular disease biomarkers
or a reduction of left ventricular ejection fraction (LVEF).
13. The method of claim 12, wherein the sEH metabolites are
determined by mass spectrometry or immunoassays including ELISA,
dot blot analysis and lateral flow (dipstick) immunoassay or
label-free microwell and nanowell technologies.
14. The method of claim 13, wherein with multi-cardiotoxicity
biomarkers consisted of 14,15-DHET, 8-isoprostane, troponin I
(TnI), myeloperoxidase (MPO) and/or GDF-15 (NAG-1).
15. The method of claim 11, wherein cardiotoxicity biomarker is the
decreased brain natriuretic peptide (BNP) level.
16. Treatment of natural or synthetic soluble epoxide hydrolase
inhibitor or a mixture of soluble epoxide hydrolase inhibitors with
or without oxidative stress inhibitors to prevent or ameliorate the
chemotherapy-induced cardiotoxicity.
17. The method of claim 16, wherein the inhibitor is honokiol or a
honokiol-containing dietary supplement.
18. A method to measure fatty acids and proteins in biological
samples including biological fluids using label-free microwell or
nanowell technology by the steps of: Measuring cyclic voltammetry
or impedance levels of various concentrations of fatty acid or
protein using electrodes coated with the antibody or IgG ranging
from 1 to 10 .mu.g/ml; Drawing a standard curve of concentrations
of fatty acid or protein vs. cyclic voltammetry or impedance levels
(-Z'', Kohm); Selecting antibody or IgG levels to coat electrode
surface; and Measuring levels of fatty acid or protein in a target
biological fluid sample and control sample.
19. The method of claim 18, wherein the fatty acids are 14,15-DHET,
20-HETE and 8-isoprostane and protein is troponin I (TnI).
20. The method of claim 19, wherein the limit of detection (LOD) is
.ltoreq.100 fg/ml.
Description
BACKGROUND OF THE INVENTION
1. Technical Field
[0002] This invention relates to methods to identify risk of
chemotherapy-induced cardiotoxicity using biomarkers. The method
includes diagnosis of risk of chemotherapy-induced cardiotoxicity
by measuring increased levels of expression of soluble epoxide
hydrolase in vitro and in vivo including cells, tissues or animals.
The method includes diagnosis of risk of chemotherapy-induced
cardiotoxicity by measuring increased levels of activity of soluble
epoxide hydrolase including 14,15-DHET or 11,12-DHET in biological
fluids. The method also includes diagnosis of risk of
chemotherapy-induced cardiotoxicity by measuring increased levels
of oxidative stress in vitro and in vivo including cells, tissues
or animals. The method also includes diagnosis of risk of
chemotherapy-induced cardiotoxicity by measuring increased levels
of oxidative stress using 8-isoprostane levels in biological
fluids. Fatty acid and protein biomarkers to diagnose risk of
chemotherapy-induced cardiotoxicity are determined using various
detection methods including mass spectrometry, Western blot
analysis, ELISA or label-free microwell and nanowell technologies.
This invention includes treatment using soluble epoxide hydrolase
inhibitor with or without antioxidants to prevent or ameliorate
chemotherapy-induced cardiotoxicity.
2. Background Art
[0003] Center for Disease Control and Prevention (CDC), the
National Center for Health Statistics (NCHS) reported that during
1999-2013, 1 of every 4 deaths in the United States (0.6 million
people/year) was a result of coronary heart disease (CHD) (1),
which is predicted to increase each year (2).
[0004] Chemotherapy treatment of cancer patients without surgery
and before and after surgery has become a routine treatment for
cancer. Many successful chemotherapy agents which inhibit cell
proliferation and/or metastasis have increased the 5-year survival
rate of cancer patients. However, the chemotherapeutic agents are
toxic to heart tissue and linked to heart failure. An anthracycline
a DNA/RNA intercalator, doxorubicin (Adriamycin), daunorubicin,
epirubicin or idarubicin in combination with paclitaxel (Taxol)
(microtubule binder) is used to treat many cancers including
breast, ovarian and lung cancers. Anthracycline chemotherapy
efficiently treats various cancers. However, it has cardiotoxic
effects which lead to heart failure (6%) and subclinical heart
disease (18%) (3).
[0005] Over 3.1 million women in the US in 2017 were subjected to
breast cancer chemotherapy (4). Treatment with DOX for 3 months
followed by one-year treatment with trastuzumab for HER2-positive
breast cancer patients is associated with cardiac dysfunction
including symptomatic heart failure (HF) (18%) (5).
[0006] Chemotherapy-induced cardiotoxicity leads to heart failure,
symptomatic left ventricular dysfunction (LVD) and reduced left
ventricular ejection fraction (LVEF). The 2D-echocardiography
widely used for diagnosis of CHD including heart failure and LVD is
not suitable for early detection of cardiotoxicity (6). A few
drawbacks of the 2D-echocardiography are high screening cost, need
for a skilled technician and detection of cardiotoxicity only when
the damage is occurred to the extent that recovery opportunity of
heart function, LVD or LVEF, is not available (6).
[0007] Anthracycline and/or trastuzumab-associated cardiotoxicity
is defined as either a cardiomyopathy with a reduction of LVEF
.gtoreq.5% to <55% with symptoms of heart failure or an
asymptomatic reduction of LVEF .gtoreq.10% to <55% by the
Cardiac Review and Evaluation Committee (CREC) (7).
[0008] Early diagnosis (prediction) of chemotherapy-induced
cardiotoxicity may offer a targeted (precision) drug treatment
opportunity for patients.
[0009] Epoxyeicosatrienoic acids (EETs) are primary
cardioprotective metabolites formed by cytochrome P450 (CYP) 2C/2J.
Soluble epoxide hydrolase (sEH) rapidly metabolizes EETs to
dihydroxyeicosatrienoic acids (DHETs) causing hypertension
(8,10).
[0010] Blood and urinary 14,15-DHET is a biomarker of sEH-induced
hypertension and cardiovascular disease in rats and humans (8-10).
The sEH biomarker in human was validated in a blind test using
human urine specimens obtained from hypertensive (preeclamptic) and
normotensive patients (10).
[0011] An electrochemical nano-biosensor detects a change in
electrical signal due to hybridization of a target molecule with a
capture molecule, i.e., a fatty acid, a protein, a DNA or an RNA
biomarker in serum, which binds to an antibody or a complementary
DNA probe coated on a gold-surfaced nanowell (11,12).
SUMMARY OF INVENTION
[0012] The present invention provides methods to identify risk of
chemotherapy-induced cardiotoxicity using biomarkers. This
invention includes diagnosis of risk of chemotherapy-induced
cardiotoxicity by measuring increased levels of expression of
soluble epoxide hydrolase in vitro and in vivo including cells,
tissues or animals. The method includes diagnosis of risk or
presence of chemotherapy-induced cardiotoxicity by measuring
increased levels of activity of soluble epoxide hydrolase including
14,15-DHET in biological fluids. This invention also includes
diagnosis of risk of chemotherapy-induced cardiotoxicity by
measuring increased levels of oxidative stress in vitro and in vivo
including cells, tissues or animals. The method also includes
diagnosis of risk of chemotherapy-induced cardiotoxicity by
measuring increased levels of oxidative stress using 8-isoprostane
levels in biological fluids. Fatty acid and protein biomarkers to
diagnosis risk of chemotherapy-induced cardiotoxicity are detected
using various methods including mass spectrometry, Western blot
analysis, ELISA or label-free microwell and nanowell technologies.
This invention discloses targeted medical intervention of the
subject who has risk or presence of chemotherapy-induced
cardiotoxicity by treating with soluble epoxide hydrolase
inhibitor(s) with or without antioxidants to prevent or ameliorate
the chemotherapy-induced cardiotoxicity.
DESCRIPTION OF THE DRAWINGS
[0013] Other advantages of the present invention will be readily
appreciated as the same becomes better understood by reference to
the following detailed description when considered in connection
with the accompanying drawings wherein:
[0014] FIGS. 1A and 1B show the effect of doxorubicin (DOX) on H9c2
rat cardiomyocytes. Cells were treated for 2 hr with media
containing 1 .mu.M DOX, washed and kept for 2, 6 and 24 hr in media
without DOX. BNP mRNA levels in H9c2 cells were assessed by qRT-PCR
(A) and cell media was collected and extracted with ethyl acetate
for 14,15-DHET analyses using ELISA kit (Detroit R&D) (B).
*p<0.05;
[0015] FIG. 2 shows the effect of doxorubicin (DOX) on H9c2 rat
cardiomyocyte surface area as a measurement of cellular
hypertrophy. Cells were treated for 2 hr with media containing 1
.mu.M DOX, washed and kept for 2, 6 and 24 hr in media without DOX.
Cell sizes (cell surface area) were measured using image J
software. Results expressed as mean.+-.SD (n=6-44);
[0016] FIGS. 3A through 3D show the effect of doxorubicin (DOX)
treatment on BNP mRNA and serum 14,15-DHET and 20-HETE levels in
rat. Rats were euthanized at 48 hr (no recovery) or after 2 weeks
(2 weeks recovery) following injection of the rats for 2 weeks with
saline (control) or DOX (3 mg/kg/week). BNP mRNA levels in heart
were assessed by qRT-PCR and results were normalized by
.beta.-actin mRNA (A). Levels of 14,15-DHET (B), 20-HETE (C) and
8-isoprostane (D) were assessed after extraction of fatty acids
with ethyl acetate using ELISA kit (Detroit R&D). Results
expressed as mean.+-.SD (n=4-6). *p<0.05 and **p<0.01;
[0017] FIG. 4 shows the effect of doxorubicin (DOX) treatment on
levels of a biologically active fatty acid in plasma samples
obtained from breast cancer patients before and after 3 months of
treatment with DOX. Plasma samples were extracted using ethyl
acetate and levels of 14,15-DHET were assessed in triplicate for
ELISA. Results expressed as mean.+-.SD. *p<0.05;
[0018] FIGS. 5A and 5B show the effect of doxorubicin (DOX) on
soluble epoxide hydrolase (sEH) expression in rat H9c2
cardiomyocytes cells (A) and NAG-1 (MIC-1, GDF-15), a plasma
cardiotoxicity biomarker (B). Panel A, cells harvested at 6 hr with
(Lane 2) and without (Lane 1) treatment of doxorubicin (DOX) (2 hr,
final concentration 1 .mu.M). Protein expression of sEH, GAPDH and
beta-actin expressed in the cells were assessed by Western blot
analysis. Expression levels of sEH were normalized by beta-actin
protein levels and Panel B, Western blot analysis carried out under
non-reducing/non-denaturing condition for NAG-1 protein in plasma
samples obtained from healthy subjects (pooled plasma sample,
control) and 3 breast cancer patients with cardiovascular disease
(cardiotoxicity, Innovative Research);
[0019] FIGS. 6A and 6B show quantitation of 14,15-DHET (A) and TnI
(B) by standard curves using 8-channel nanowell electrodes. Various
amounts of 14,15-DHET (A) or TnI (B) (100 fg/ml, 1 pg/ml and 10
pg/ml) were added to the channels coated with anti-14,15-DHET or
TnI (Detroit R&D) and impedance was measured after dipping the
electrode in ferro/ferricyanide (5 mM each), 100 mM KCl in PBS
using an 8-channel Ivium potentio-n-stat (Ivium Technology).
Impedance increased proportionally with increasing concentration of
14,15-DHET or TnI.
[0020] FIGS. 7A through 7C show changes in impedance after addition
of plasma samples from pooled plasma from cancer-free women and 3
cardiomyopathy breast cancer patients (Innovative Research)
measured by label-free electrochemical analyses using 90 nm
nanowell electrodes conjugated with 14,15-DHET IgG (2.5 .mu.g/ml).
Impedance was measured after dipping the electrode in
ferro/ferricyanide (5 mM each), 100 mM KCl in PBS using an
8-channel Ivium potentio-n-stat (Ivium Technology) (A). Formula for
calculating 14,15-DHET (B). Percent of increase from control
average was calculated and expressed as a mean.+-.standard
deviation (SD). *p<0.05 (C).
DETAILED DESCRIPTION OF THE INVENTION
[0021] Chemo and radiation therapy are the most common treatments
given to cancer patients. However, this treatment may induce
cardiac events such as hypertension, left ventricular dysfunction,
cardiomyopathy and heart failure.
[0022] Acute cardiotoxicity from anthracyclines, e.g., doxorubicin
(DOX), occurs as early as 6 months after completion of 3 months
anthracycline treatment and chronic cardiotoxicity from
anthracyclines occurs in .about.2-5% of patients .about.1 year
after completion of chemotherapy. Detection of early biomarkers,
which predict cardiac dysfunction before any damage occurs, offers
an opportunity to adjust an individual's dosage and protocol during
chemo and radiation therapy and provide targeted medical
intervention.
[0023] The present invention provides methods to identify risk
and/or presence of chemotherapy- and radiation therapy-induced
cardiotoxicity by measuring increased levels of expression or
activity of soluble epoxide hydrolase in vitro and in vivo
including cells, tissues or animals and related cell media and
biological fluids using various detection methods including Western
blot analysis, ELISA and label-free microwell and nanowell
technologies.
[0024] Soluble epoxide hydrolase (sEH) rapidly metabolizes EETs to
DHETs causing hypertension (8,10) and cardiovascular diseases (9).
To find whether anthracycline treatment, which induce
cardiovascular diseases, increases sEH activity in heart cells,
H9c2 rat cardiomyocytes were incubated for 2 hr with media with and
without 1 .mu.M DOX (FIG. 1 and FIG. 2).
[0025] Cells and media were collected after 2, 6 and 26 hr recovery
periods (FIG. 1 and FIG. 2, Recovery).
[0026] Cell viability was determined using the
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)
assay (13) for cells incubated with and without 1 .mu.M of DOX in
media for 2 hr followed by fresh media for another 24 hours. The
DOX treated cells maintained more than 90% cell viability compared
to untreated control cells.
[0027] Increased cell surface area (hypertrophy) was found at 6 hr
and 24 hr after DOX treatment (p<0.05) whereas no changes were
observed after 2 hr (.about.100 cells measured/group) (FIG. 2).
[0028] Brain natriuretic peptide (BNP) mRNA levels of the control
and treated cells were measured by reverse-transcription/real time
PCR (Sybr green) as previously described (13). Results were
normalized using .beta.-actin mRNA levels obtained by
reverse-transcription/real time PCR (Sybr green) (13). Normalized
BNP levels were decreased 6 hr and 24 hr after 2 hr of DOX
treatment (p<0.05) (FIG. 1A) when cardiomyocyte hypertrophy
occurred (FIG. 1A). This result demonstrated that decreased BNP
mRNA levels are a biomarker of anthracycline-induced
cardiotoxicity.
[0029] It was surprising that the effect of DOX treatment induced
an almost 4-fold increase of BNP mRNA at 2 hr of recovery of the
DOX treatment (FIG. 1A) when no change in cell size was detected
(FIG. 2). This result suggested that 2 hr recovery is the best time
point to find biomarkers that precede the incidence of
cardiomyocyte hypertrophy.
[0030] An approximately 4-fold increase of BNP mRNA levels in heart
tissues without cardiotoxicity was also detected in a female rat
study (FIG. 3A).
[0031] These results strongly suggested that increased BNP levels
after DOX treatment are an early biomarker for DOX-induced
cardiotoxicity.
[0032] H9c2 cardiomyocytes were treated for 2 hr with and without 1
.mu.M DOX. Levels of 14,15-DHET in cell media increased
.about.3-fold (p<0.05) after a 2 hr recovery period prior to
detection of cellular hypertrophy at 6 hr and 24 hr recovery
periods. Levels of 14,15-DHET, a primary metabolite of cytosolic
sEH remained elevated after 6 and 24 hr of recovery periods
(p<0.05) (FIG. 1B).
[0033] This result demonstrated that sEH expression increased at an
early stage when no cellular hypertrophy occurred (FIG. 1B). Thus,
measurement of the sEH activity predicts cardiomyocyte damage in
advance and any biomarkers of increased sEH activity, e.g.,
14,15-DHET and 11,12-DHET, are early biomarkers for
anthracycline-induced cardiotoxicity.
[0034] Expression of sEH enzyme in the cells was confirmed by
detection of increased 14,15-DHET formation activity after 1 .mu.M
EET (substrate) treatment.
[0035] Increased sEH protein levels after DOX treatment was
detected by Western blot analysis using sEH antibody (Detroit
R&D) (FIG. 5A).
[0036] Increased 14,15-DHET level in biological fluids, e.g., blood
and urine, is a biomarker for hypertension and cardiovascular
diseases. Our cell study demonstrated that metabolites of sEH,
e.g., 14,15-DHET, detected in biological fluids are early
biomarkers of cardiotoxicity and the sEH enzyme is a target enzyme
to prevent occurrence of cardiotoxicity by treating the patient
with an sEH inhibitor.
[0037] The cell study also demonstrated that metabolites of sEH,
e.g., 14,15-DHET, detected in biological fluids are biomarkers of
sEH-dependent cardiotoxicity for the patient whose heart tissues
are damaged or heart function is decreased after anthracycline
treatment and the sEH-dependent diseases can be treated with an sEH
inhibitor.
[0038] A study on serum biomarkers to detect risk of
chemotherapy-induced cardiotoxicity was carried out using female
rats treated with and without DOX (3 mg/kg/week, i.v. for 2 weeks)
(no recovery group: Control and DOX, n=6/group) and with a 2-week
recovery period (2 weeks recovery group: Control and DOX,
n=6/group).
[0039] Decreased BNP mRNA levels are indicative of heart cell
damage (FIG. 1 and FIG. 2). As proven by the increased BNP mRNA
levels in the rat heart tissues (FIG. 3A), histochemical analysis
of the heart tissues revealed no vacuolization or heart tissue
damage occurred after a 2-week recovery period.
[0040] BNP mRNA levels in heart tissues were measured by
reverse-transcription/real time PCR (Sybr green) and normalized
using .beta.-actin mRNA levels (13). After 2 weeks of recovery
following DOX treatment (no heart tissue damage), BNP mRNA levels
in heart tissue increased .about.4-fold compared to the levels in
control tissue (p<0.01) (FIG. 3A), verifying the cardiomyocytes
study which revealed .about.4-fold increase of BNP mRNA levels
after 2 hr recovery when no hypertrophy occurred (p<0.05) (FIG.
1A). When cellular hypertrophy occurred at 6 hr and 24 hr recovery,
the BNP mRNA levels were lower than control (p<0.05) (FIG. 1A).
Decreased BNP mRNA levels in cardiomyocytes is a biomarker of
cellular hypertrophy.
[0041] As in the rat cardiomyocyte study (FIG. 1 and FIG. 2), BNP
mRNA levels in rat heart tissues increased prior to cardiac
hypertrophy. These results demonstrated that the increased BNP
level is an early biomarker for anthracycline-induced
cardiotoxicity.
[0042] To verify that 14,15-DHET levels can be used as an early
biomarker for prediction of DOX-dependent cardiotoxicity as
previously found in the rat cardiomyocyte study (FIG. 1 and FIG.
2), levels of 14,15-DHET (sEH metabolite) in serum samples from
control and DOX-treated rats with and without 2-week recovery were
measured using a 14,15-DHET ELISA kit (Detroit R&D).
[0043] Levels of 20-HETE (CYP4A/4F-dependent hypertension
biomarker) and 8-isoprostane (non-enzymatic oxidative stress
biomarker) in rat serum samples were also measured using 20-HETE
and 8-isoprostane ELISA kits, respectively, from Detroit
R&D.
[0044] The 14,15-DHET, 20-HETE and 8-isoprostane ELISA results
showed that all three biologically active fatty acids did not
significantly change after two weeks (3 mg/kg body weight/week) of
DOX treatment (no recovery) (FIGS. 3. B, C and D,
respectively).
[0045] However, two weeks after the second DOX injection (2 weeks
recovery), when cardiotoxicity was not detected, levels of
14,15-DHET and 8-isoprostane were increased compared to the control
group (FIGS. 3. B and D, respectively). No changes in levels of
20-HETE in rat serum samples between the two groups were observed
after DOX treatment with and without 2-week recovery (FIG. 3C).
[0046] These results demonstrated that 14,15-DHET and 8-isoprostane
are early biomarkers which predict DOX-induced cardiotoxicity.
Inhibition of the sEH activity will ameliorate DOX-induced
cardiotoxicity.
[0047] The 14,15-DHET and 8-isoprostane biomarkers are detected in
various biological fluids including blood and urine and an elevated
level of 14,15-DHET is a blood and urinary biomarker of
hypertension and cardiovascular disease (CVD) (8-10).
[0048] Biomarkers which predict anthracycline-caused cardiotoxicity
in rat serum and heart tissue samples are summarized in Table
1.
[0049] A blind test for the early biomarker, 14,15-DHET (metabolite
of sEH), was carried out with plasma samples from 5 breast cancer
patients obtained before and after 3 months of DOX treatment (FIG.
4). No cardiotoxicity was detected in the patients at the time 3
months of DOX-treatment.
[0050] Fatty acids in the plasma samples (50 .mu.l/sample) were
extracted with ethyl acetate and levels of 14,15-DHET were assessed
in triplicate samples using an ELISA kit from Detroit R&D.
[0051] Chemotherapy-associated cardiotoxicity is defined as either
a cardiomyopathy with a reduction of left ventricular ejection
fraction (LVEF) .gtoreq.5% to <55% with symptoms of heart
failure or an asymptomatic reduction of LVEF .gtoreq.10% to <55%
by the Cardiac Review and Evaluation Committee (CREC) (7).
[0052] The 14,15-DHET ELISA results showed that, although no
cardiotoxicity was detected in any of the 5 patients after 3 months
of DOX-treatment was completed, levels of 14,15-DHET significantly
increased by .about.38% and .about.55% in plasma samples obtained
from Patients #3 and #4, respectively (p<0.05) (FIG. 4).
[0053] According to our finding (FIG. 1 through FIG. 3), only
Patients #3 and #4 were predicted to develop cardiotoxicity during
recovery.
[0054] Indeed, only Patients #3 and #4 had reduction of LVEF higher
than 20% (.about.28% and 22%, respectively) at 3 months after 3
months of DOX-treatment was completed (6 months including 3 months
of treatment). Reduction of LVEF of Patients #3 and #4 remained
around 20% at 6 months after 3 months of DOX-treatment was
completed (9 months including 3 months of treatment).
[0055] Increased troponin I (TnI) levels which were found to be an
early biomarker of anthracycline-dependent cardiotoxicity (5) were
increased and decreased in plasma samples obtained from Patients #3
and #4, respectively, at 3 months after 3 months of DOX-treatment.
TnI levels of Patients #1, #2 and #5 (no cardiotoxicity) were
increased at 3 months after 3 months of DOX-treatment, which
strongly suggested that additional early biomarkers are needed to
compensate specificity of the TnI as an early cardiotoxicity
biomarker.
[0056] Another shortcoming of the use of early cardiotoxicity
biomarkers of heart muscle injury such as TnI is that a molecular
inhibitor of the injury is difficult to produce. Contrary,
inhibitors of sEH, which prevent conversion of a substrate of the
enzyme to a metabolite, e.g., 14,15-EET to 14,15-DHET, are
molecules which can inhibit the enzymatic activity by directly
binding to the enzyme.
[0057] Honokiol (Sigma), a component of magnolia bark, and
12[[(tricycle[3.3.1.13,7]dec-1-ylamino)carbonyl]amino]-dodecanoic
acid (AUDA) (Cayman), a synthetic sEH inhibitor, inhibited EET (1
.mu.M)-dependent sEH activity in a reconstituted system with
recombinant sEH and human kidney cells (ACHN). Both AUDA and
honokiol were found to be potent inhibitors of the sEH
activity.
[0058] sEH metabolites including 14,15-DHET are early serum
biomarkers for prediction of cardiotoxicity. Early diagnosis of
patients to predict cardiac dysfunction is necessary to adjust the
anti-cancer drug treatment protocol. Moreover, inhibition of
activity of the target enzyme, sEH, will ameliorate the
chemotherapy-induced cardiotoxicity.
[0059] Expression of GDF-15 (NAG-1) was detected by Western blot
analysis under non-reducing/non-denaturing condition using a
Detroit R&D antibody to GDF-15. DGF-15 protein was up-regulated
in plasma samples from 3 breast cancer patients with cardiovascular
disease (cardiotoxicity) compared to pooled healthy subject plasma
sample (Innovative Research) (FIG. 5). The result demonstrated that
an increase in plasma GDF-15 level is a biomarker of
cancer-therapy-induced cardiotoxicity.
[0060] Serum or plasma cardiotoxicity biomarkers cam be detected by
ELISA, dot blot analysis and lateral flow (dipstick)
immunoassay.
[0061] A facile label-free method using electrochemical microwell
and nanowell biosensors which uses 1 .mu.l blood or serum/plasma
samples with or without dilution was developed to detect a change
in current or impedance due to hybridization of a target molecule
(biomarker) with a capture molecule (antibody).
[0062] Hybridization of target fatty acids and proteins to the
antibodies coated on microwell or nanowell surface was monitored
using an Ivium Stat potentiometer (Ivium Technology). The impedance
resulting from fatty acids and proteins binding to the nanogold
surface was determined for redox conversion using
ferri/ferrocyanide (5 mM each). Using Nyquist plots, the charge
transfer resistance (Rct) increased as concentrations of
biologically active fatty acids, 14,15-DHET (FIG. 6A), 20-HETE and
8-isoprostane, or protein, TnI (FIG. 6B), increased. Limit of
detection (LOD) for 14,15-DHET, 8-isoprostane, 20-HETE and TnI
using nanowell technology was 100 fg/ml, which was 100-fold more
sensitive compared with the LOD of STIP-1 target protein (10
.mu.g/ml) (12), when an 8-channel electrode (700 .mu.m.sup.2 gold
surface) containing 2,025.times.2,025 nanowells (Detroit R&D)
was used.
[0063] The 14,15-DHET levels in plasma samples from 3 breast cancer
patients with cardiotoxicity were detected by nanowell technology
and compared to a pooled plasma sample from non-cancer female
donors (Innovative Research) (FIG. 7). Compared to the pooled
control plasma sample, levels of 14,15-DHET increased in all 3
breast cancer patients with cardiotoxicity (FIG. 7A). The plasma
14,15-DHET level for each channel was calculated by subtracting the
impedance level of 14,15-DHET IgG alone from the impedance level
obtained with the sample added to the IgG-coated electrode (FIG.
7B). These results showed that levels of 14,15-DHET significantly
(p<0.04) increased by .about.90% in plasma samples obtained from
breast cancer patients with cardiotoxicity compared to the control
sample (FIG. 7C).
[0064] The plasma samples of Patient #4 (10-fold diluted) who
developed DOX-dependent cardiotoxicity 3 and 6 months after
completion of the 3 months DOX treatment were obtained before and
after 3 months DOX treatment (FIG. 4).
[0065] By 14,15-DHET nanowell label-free analysis, it was found
that the 14,15-DHET level increased .about.20-fold in the plasma
sample obtained from Patient #4 after 3 months treatment with DOX
compared to the plasma sample obtained before the treatment (n=4,
p<0.05). The electrochemical result indicated that DOX treatment
induced an increase in the level of 14,15-DHET which could be used
to predict cardiotoxicity (early cardiotoxocity biomarker).
[0066] In addition, the fatty acid cardiotoxicity biomarkers,
14,15-DHET, 11,12-DHET and 8-isoprostane, can be isolated by
affinity chromatography and gas or liquid chromatography to
identify the fatty acids by mass spectrometry (MS). The protein
cardiotoxicity biomarkers can be isolated by affinity
chromatography or electrophoresis to identify the proteins by mass
spectrometry (MS) or N-terminal sequencing or Western blot
analysis.
[0067] The fatty acid and protein cardiotoxicity biomarkers can
also be detected by other technologies including label-free
nanotechnologies and dot blot and lateral flow immunoassays.
[0068] Interference RNA of mRNA of cardiotoxicity biomarkers
including sEH, GDF-15, TnI and MPO can be used as
anti-cardiotoxicity molecules in cells or animal disease models.
They can also be used to treat patients.
[0069] Natural plant sEH inhibitors including honokiol-containing
plants can be used to treat patients who have risk of sEH-dependent
cardiotoxicity.
[0070] Some of the techniques used for screening of glycan,
glycoprotein, glycan-binding protein and anti-antibody biomarkers
in the present disclosure are practiced in the art, and most
practitioners are familiar with the standard resource materials,
which describe specific conditions and procedures. The methods used
with and the utility of the present invention can be shown by the
following non-limiting examples and accompanying figures.
EXAMPLES
Example 1
[0071] Biomarkers of Risk or Presence of Chemotherapy-Induced
Cardiotoxicity Identified Using Rat H9c2 Cardiomyocyte Cells.
[0072] Rat H9c2 cell line was purchased from American Type Culture
Collection (Manassas, Va.) and cultured on polystyrene culture
plates in a humidified incubator at 37.degree. C. in an atmosphere
of 5% CO.sub.2 and 95% air. The H9c2 cells were grown in high
glucose DMEM supplemented with 4.5 g/l glucose, 0.15% sodium
bicarbonate, 0.11% sodium pyruvate, 10% fetal bovine serum, 20
.mu.M L-glutamine, 100 IU/ml penicillin and 10 .mu.g/ml
streptomycin. When cells reached 80-90% confluency, media was
replaced with media containing 1 .mu.M DOX (Sigma) dissolved in
DMSO. Cells were treated for 2 hr and collected immediately (0 hr)
or washed and kept for 2, 6 and 24 hr in media without DOX.
[0073] The effect of DOX on cell viability was determined by
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)
assay (13). We found that the cells treated with 1 .mu.M of DOX for
2 hr with medium replaced by fresh medium for another 24 hr
maintained more than 90% cell viability.
[0074] For cell hypertrophy assay, rat H9c2 cells grown overnight
in a plate containing a glass cover slip were fixed at 2, 6 and 24
hr after 2 hr 1 .mu.M DOX treatment and stained with hematoxylin
and eosin and images were taken using a using a Zeiss Axiovert
microscope. ImageJ software was used to measure the surface area of
the cells. After measurement of more than 100 cells per group, the
results demonstrated increased cell surface area at 6 hr and 24 hr
after DOX treatment. Importantly, no changes were observed in 2 hr
(FIG. 2).
[0075] Our novel finding suggests that 2 hr after 2 hr of DOX
treatment is the suitable time to measure a biomarker that can
predict hypertrophy of cardiomyocytes at later time points, e.g., 6
hr or 24 hr, in vitro using H9c2 cells. Thus, based on this
parameter (cell surface area), analysis of cell media at 2 hr (no
damage) and 6 hr or 24 hr (heart damage) after 2 hr DOX treatment
is a cell model to predict cellular damage in human patients
occurring after completion of 3 months DOX treatment (no
damage).
[0076] BNP mRNA was assessed in rat H9c2 cells. RNA (1 .mu.g) was
converted to cDNA using High Capacity cDNA Reverse Transcript Kit
(Applied Biosystems). BNP were measured by real time PCR (Sybr
green) using rat specific primers (13). Results were normalized
using .beta.-actin mRNA levels using a rat specific primer (13).
Our results have shown that BNP levels were decreased 6 and 24 hr
after 2 hr of DOX treatment (FIG. 1A) when cell hypertrophy was
detected (FIG. 2).
[0077] The effect of DOX treatment induced an almost 4-fold
increase of BNP mRNA at 2 hr of recovery after 2 hr DOX treatment
(FIG. 1A) when changes in cell size was not observed (FIG. 2). This
result confirms that increased BNP mRNA level is an early biomarker
that precedes the incidence of cardiomyocyte hypertrophy.
[0078] sEH enzyme converts 14,15-EET to 14,15-DHET, so differential
levels of 14,15-DHET will reflect sEH activity. After addition of 1
.mu.M 14,15-EET to H9c2 cells, .about.250 ng/ml 14,15-DHET was
formed in cell media in 30 min and 4.5-fold higher level of
14,15-DHET was formed when the EET level was increased from 1 .mu.M
to 5 .mu.M, verifying EET-dose-dependent 14,15-DHET formation by
rat H9c2 cardiomyocyte cells.
[0079] Levels of 14,15-DHET in cardiomyocyte (H9c2 cell) media were
assessed by the Detroit R&D 14,15-DHET ELISA. Media, 2 ml, was
extracted with 2 ml acetyl acetate. and levels of 14,15-DHET
increased after 2 hr, 6 hr and 24 hr following 2 hr DOX treatment
(2 hr, 6 hr and 24 hr recovery) (FIG. 1B).
[0080] The effect of DOX treatment dramatically induced 14,15-DHET
levels at 2 hr of recovery after 2 hr DOX treatment (FIG. 1B) when
changes in cell size was not observed (FIG. 2). This result
confirms that increased 14,15-DHET level is an early biomarker that
precedes the incidence of cardiomyocyte hypertrophy.
Example 2
[0081] Identification of Biomarkers for Risk of
Chemotherapy-Induced Cardiotoxicity Using Female Rats.
[0082] Early diagnosis followed by medical intervention is critical
to prevent cardiotoxicity. Early DOX cardiotoxicity biomarkers are
identified using rat H9c2 cardiomyocyte cells with and without 6 or
24 hr recovery period after DOX treatment (Example 1, FIG. 1 and
FIG. 2). Heart tissue and serum samples from female rats after
treatment with DOX for 2 weeks with and without a 2-week recovery
period were obtained and increased levels of BNP mRNA in heart
tissues and increased levels of 14,15-DHET in sera were detected
after 2 week recovery, when cardiotoxicity was not detected,
supporting our finding that BNP mRNA and 14,15-DHET (sEH
metabolite) are early biomarkers of anthracycline-induced
cardioxicity.
[0083] (DOX administration in laboratory animals has been performed
to mimic human chemotherapy treatment and to analyze the secondary
effect of DOX. Female Sprague-Dawley rats (Charles River, Ashland,
Ohio) received two intravenous injections of either 3 mg/kg body
weight (once a week for 2 weeks) of DOX (DOX-treated, n=12) or a
similar volume of saline (Control, n=12). Because one of the
outcomes of DOX treatment in animals is a reduced body weight gain,
body weights for the rats were measured daily. After 2 weeks of
recovery, the body weight or the weight gain of the control group
(325.+-.17) was .about.20% higher than the DOX group (269.+-.15
g).
[0084] Heart tissues were dehydrated, imbedded in paraffin and
sliced. Slices were incubated in Mayer's hematoxylin reagent
(ScyTek) for 5 min and covered with bluing reagent (ScyTek) for 15
seconds and eosin Y (ScyTek) solution for 3 minutes. Images of the
rat cardiomyocyte were taken using a Zeiss Axiovert 200 microscope
with RT Insight camera and SPOT Advanced Imaging software in
40.times. magnification at the Microscopy and Imaging Facility at
Wayne State University.
[0085] Presence of vacuolization in heart tissue, a sign of
cardiotoxicity (14), was not observed at 40.times. magnification.
Thus, histological analyses suggest that the period of 2 weeks
after DOX-injections using our rat-protocol is a time-period before
the development of cardiotoxicity.
[0086] Rat specific primers for RT-PCR were purchase from IDT
Integrated DNA technology (Table 1). Heart total RNA was extracted
using the TRIzol method, cDNA was synthesized using High Capacity
cDNA Reverse Transcript Kit (Applied Biosystems). Levels of mRNA in
cardiomyocytes were measured by real time PCR (Sybr green) using
rat specific primers. After 2 weeks recovery, levels of mRNA of
both natriuretic peptides released from the atrium (ANP) (Table 1)
and the ventricle (BNP) (Table 1, FIG. 3A) increased .about.6- and
4-folds, respectively. Early increase of the BNP mRNA level without
cell hypertrophy were confirmed using DOX-treated H9c2 rat
cardiomyocytes (FIG. 1A). After 2 weeks recovery when no cardiac
hypertrophy was detected, mRNA levels of myeloperoxidase (MPO)
which plays a role in the inflammatory processes and s100
calcium-binding protein A9 (S100A9) increased and decreased,
respectively, compared to mRNA levels of control rats (Table
1).
[0087] Biologically active fatty acids were extracted with ethyl
acetate from 75 .mu.l of serum samples (n=4) from control and
DOX-treated animals with and without 2 weeks recovery. Levels of
14,15-DHET (sEH metabolite), 20-HETE (metabolite of cytochrome P450
4A/4F) and 8-isoprostane (non-enzymatic oxidative stress product)
were measured using ELISA kits from Detroit R&D. The results
demonstrated that all three biologically active fatty acids did not
significantly change after two weeks (3 mg/kg body weight/week) of
DOX treatment (no recovery). However, two weeks after the second
injection (2 weeks recovery), when cardiotoxicity was still not
detected, levels of 14,15-DHET (a metabolite of soluble epoxide
hydrolase) and 8-isoprostane (non-enzymatic oxidative stress
biomarker) were increased compared to the control group (FIG. 3B
and FIG. 3D). No changes in levels of 20-HETE in rat serum samples
between the two groups were observed (FIG. 3C). These results and
rat cardiomyocyte study results demonstrated that increased
14,15-DHET and 8-isoprostane are early biomarkers to predict
DOX-induced cardiotoxicity.
Example 3
[0088] Increased 14,15-DHET Level in Plasma Sample is an Early
Biomarker which Predicts Cardiotoxicity (Decreased Heart Function)
of Breast Cancer Patients.
[0089] Plasma samples from 5 breast cancer patients obtained before
(n=5) and after (n=5) 3 months of DOX treatment (60
mg/m.sup.2/injection for 4 injections). Among them, only the
Patients #3 and #4 had reduction of LVEF higher than 20% at 3 and 6
months after 3 months of DOX-treatment was completed whereas no
significant changes were observed with the other patients.
[0090] Biologically active fatty acids in the plasma samples (50
.mu.l/sample) were extracted with ethyl acetate, dried and
re-suspended with 30 .mu.l of DMF followed by dilution buffer to
900 .mu.l. Levels of 14,15-DHET were assessed in triplicate using
an ELISA kit from Detroit R&D (100 .mu.l/well). Results showed
that levels of 14,15-DHET significantly increased (p<0.05) in
plasma samples from Patients #3 and #4. In samples from Patients #2
and #5, the 14,15-DHET levels did not significantly increase (FIG.
4). By these results, Patients #3 and #4 are predicted to develop
cardiotoxicity.
[0091] The results showed that 14,15-DHET is an early biomarker to
predict a decrease in LVEF after DOX treatment. LVEF is a
functional measurement used to verify the presence of abnormalities
in the heart (cardiotoxicity) (1). Once the LVEF is decreased, it
might be too late to recover to the normal LVEF by treatment. A
challenge in this field is identification of an early biomarker
that predicts such a change in the heart. Thus, early biomarkers
which can predict chemotherapy-induced reduction of LVEF could
allow patients to receive a therapy before cardiotoxicity occurs to
prevent chemotherapy-induced-induced heart damage.
Example 4
[0092] Label-Free Microwell and Nanowell Technologies to Detect
Biomarkers for Risk or Presence of Chemotherapy-Induced
Cardiotoxicity.
[0093] The bare gold surface of the 8-channel 90 nm or 200 nm
nanowell electrode (Detroit R&D) was coated with a
self-assembly monolayer (SAM) by incubating the electrode with 10
mM of 11-mercaptoundecanoic acid (MUA) (Sigma) for 1 hr at room
temperature and activated using 50 mM EDC and 50 mM NHS. The
14,15-DHET IgG (Detroit R&D) (FIG. 6A) or TnI IgG (Detroit
R&D) (FIG. 6B) in PBS (2.5 .mu.g/ml) was added to the
EDC/NHS-activated electrode. The IgG level to obtain optimal
results was experimentally obtained by using electrodes coated with
various concentrations of IgG ranging 1 to 10 .mu.g/ml.
[0094] Label-free 8-isoprostane nanowell analysis with 5 .mu.g/ml
and 10 .mu.g/ml IgG 8-isoprostane IgG revealed that analysis with
the nanowell electrode coated with 5 .mu.g/ml was better than the
10 .mu.g/ml IgG-coated electrode for a standard curve production
(LOD, 100 fg/ml).
[0095] Various amounts of 14,15-DHET or TnI protein (100 fg/ml, 1
pg/ml and 10 pg/ml) were added to the channels. Cyclic voltammetry
(CV) was measured after dipping the electrode in ferro/ferricyanide
(5 mM each), 100 mM KCl in PBS using an Ivium potentio-n-stat
potentiometer (Ivium Technology). Impedance increased
proportionally with increasing concentration of 14,15-DHET or TnI
added to the electrode (FIG. 6). Low variance of impedance values
among the channels was observed when 2.5 .mu.g/ml of IgG was used.
The LOD values of both 14,15-DHET and TnI by the label-free
technology were 100 fg/ml (FIG. 6).
[0096] The 14,15-DHET 90 nm nanowell analysis was carried out using
pooled female control plasma sample and plasma samples from 3
breast cancer patients with cardiotoxicity (.about.1 .mu.l/channel)
(Innovative Research) (FIG. 7A). Changes in impedance after
addition of the pooled normal plasma and 3 cardiotoxicity plasma
samples were measured by label-free electrochemical analyses using
90 nm nanowell electrodes conjugated with 14,15-DHET IgG (2.5
.mu.g/ml). Impedance was measured after dipping the electrode in
ferro/ferricyanide (5 mM each), 100 mM KCl in PBS using an
8-channel Ivium potentio-n-stat (Ivium Technology) (FIG. 7A). The
formula for calculating 14,15-DHET is shown (FIG. 7B). Percent
increase over the everage control value was calculated and
expressed as a mean.+-.standard deviation (SD). *p<0.05 (FIG.
7C).
[0097] Compared to the pooled control sample, levels of 14,15-DHET
of all 3 breast cancer patients with cardiotoxicity increased (FIG.
7A). The plasma 14,15-DHET level for each channel was calculated by
subtracting the impedance level of IgG alone from the impedance
level obtained with the sample added to the IgG-coated electrode
(FIG. 7B). Percent increases from an average of controls showed a
significant difference between the control and cardiotoxicity
groups (FIG. 7C). These results suggest that 2.5 .mu.g/ml
14,15-DHET IgG is the ideal concentration for measurements of
plasma 14,15-DHET levels and that levels of 14,15-DHET
significantly (p<0.04) increased .about.90% in plasma samples
obtained from breast cancer patients with cardiotoxicity.
TABLE-US-00001 TABLE 1 Biomarker candidates to predict
cardiotoxicity in rats after DOX injection. Sample was collected at
~48 hr after 2 weeks DOX injection (3 mg/kg body weight/week) (no
recovery) and at 2 weeks after the last injection of DOX (2 weeks
recovery). Results were assessed by ELISA and by real-time
(RT)-PCR. Heart tissue biomarkers were normalized by the
.beta.-actin mRNA level. 2-Weeks after 2 weeks DOX 2 Weeks DOX
Treatment Treatment Biomarker Method (No Recovery) (2 Weeks
Recovery) Serum 14,15-DHET ELISA No change .uparw. 3.5-fold.sup.
Bio- 20-HETE ELISA No change No change marker 8-Iso- ELISA No
change .uparw. 3-fold prostane Heart ANP RT-PCR .dwnarw. 2-fold
.uparw. 5-fold Tissue BNP RT-PCR No change .uparw. 4-fold Bio- MPO
RT-PCR No change .uparw. 2-fold marker S100A9 RT-PCR No change
.dwnarw. 2.5-fold.sup.
[0098] The invention has been described in an illustrative manner,
and it is to be understood that the terminology, which has been
used is intended to be in the nature of words of description rather
than of limitation.
[0099] Obviously, many modifications and variations of the present
invention are possible in light of the above teachings. It is,
therefore, to be understood that within the scope of the appended
claims, the invention can be practiced otherwise than as
specifically described.
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