U.S. patent application number 15/363376 was filed with the patent office on 2017-03-23 for biomarkers for sensitive detection of statin-induced muscle toxicity.
The applicant listed for this patent is Zora Biosciences OY. Invention is credited to Kim Ekroos, Reini Hurme, Minna Janis, Riikka Katainen, Reijo Laaksonen, Kirill Tarasov.
Application Number | 20170082644 15/363376 |
Document ID | / |
Family ID | 46966286 |
Filed Date | 2017-03-23 |
United States Patent
Application |
20170082644 |
Kind Code |
A1 |
Laaksonen; Reijo ; et
al. |
March 23, 2017 |
BIOMARKERS FOR SENSITIVE DETECTION OF STATIN-INDUCED MUSCLE
TOXICITY
Abstract
The present invention inter alia provides a method, and uses
thereof, of predicting statin-induced muscle toxicity or its
complications, such as myalgia, myopathy and rhabdomyolysis, by
detecting the lipid concentrations or lipid-lipid concentration
ratios of a biological sample and comparing them to a control. This
method has identified lipid markers that are more specific and
sensitive in detecting these statin-induced muscle toxicity than
the currently utilized clinical markers. Also provided is an
antibody towards said lipids, and the use thereof for predicting,
diagnosing, statin-induced muscle toxicity. The invention
additionally relates to kits comprising lipids and/or an antibody
thereto, for use in the prediction and/or diagnosis of
statin-induced muscle toxicity.
Inventors: |
Laaksonen; Reijo; (Lempaala,
FI) ; Ekroos; Kim; (Helsinki, FI) ; Hurme;
Reini; (Espoo, FI) ; Katainen; Riikka;
(Helsinki, FI) ; Janis; Minna; (Espoo, FI)
; Tarasov; Kirill; (Espoo, FI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Zora Biosciences OY |
Espoo |
|
FI |
|
|
Family ID: |
46966286 |
Appl. No.: |
15/363376 |
Filed: |
November 29, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13332773 |
Dec 21, 2011 |
9541565 |
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15363376 |
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PCT/EP2011/055569 |
Apr 8, 2011 |
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13332773 |
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61545603 |
Oct 11, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2800/325 20130101;
G01N 33/92 20130101; G01N 2800/10 20130101; G01N 2800/2871
20130101; G01N 2570/00 20130101 |
International
Class: |
G01N 33/92 20060101
G01N033/92 |
Claims
1.-31. (canceled)
32. A method for determining whether a subject is at risk to
develop, or is suffering from muscle toxicity induced by a lipid
lowering drug and/or one or more of its complications, comprising
(a) determining in a sample from said subject the concentration(s)
of one or more lipid(s), wherein (an) increased or decreased
concentration(s) in said sample, when compared to a control, is
(are) indicative of said subject suffering from said muscle
toxicity induced by the lipid lowering drug and/or said
complication(s), wherein the one or more lipid(s) whose increase in
concentration is (are) compared to the control is (are) selected
from: LacCer(d18:1/22:0), Gb3(d18:1/24:1), Gb3(d18:1/22:0),
Gb3(d18:1/24:0), LacCer(d18:1/24:0), Total eicosanoids, Total Gb3,
Cer(d18:1/20:0), Gb3(d18:1/20:0), LacCer(d18:1/24:1),
LacCer(d18:1/20:0), PGE2, 12-OXOETE, 17-HDoHE, PGD2, TXB3, PS
18:0/18:1 and SM (d18:1/24:2); and wherein the one or more lipid(s)
whose decrease in concentration is (are) compared to the control is
(are) selected from: Gb3(d18:1/16:0) and Gb3(d18:1/18:0); or (b)
determining in a sample from said subject one or more lipid-lipid
concentration ratio(s), wherein (an) increased or decreased
lipid-lipid concentration ratio(s) in said sample, when compared to
a control, is (are) indicative of said subject suffering from said
muscle toxicity induced by the lipid lowering drug and/or said
complication(s), wherein the one or more lipid-lipid concentration
ratio(s) whose increase is (are) compared to the control is (are)
selected from: 12-HETE/15-HETrE, 12-HETE/14_15-DHET,
12-HETE/Gb3(d18:1/16:0), 12-HETE/Glc/GalCer(d18:1/24:1),
12-HETE/Glc/GalCer(d18:1/18:0), Gb3(d18:1/24:1)/LacCer(d18:1/16:0),
LacCer(d18:1/22:0)/LacCer(d18:1/24:1),
Cer(d18:1/20:0)/Glc/GalCer(d18:1/24:1); and wherein the one or more
lipid-lipid concentration ratio(s) whose decrease is (are) compared
to the control is (are) selected from:
LacCer(d18:1/16:0)/LacCer(d18:1/22:0),
Gb3(d18:1/16:0)/Gb3(d18:1/24:1) and 11-HETE/12-HETE, and (c)
adjusting a treatment of the subject who is determined to be at
risk to develop, or is suffering from muscle toxicity induced by a
lipid lowering drug and/or one or more of its complications,
wherein adjusting the treatment comprises (i) reducing a lipid
lowering drug dose; (ii) stopping a lipid lowering drug treatment;
or (iii) changing to a different lipid lowering drug.
33. A method for determining whether the treatment with a lipid
lowering drug of a subject needs adjustment, comprising (a)
determining in a sample from said subject the concentration(s) of
one or more lipid(s), wherein (an) increased or decreased
concentration(s) in said sample, when compared to a control, is
(are) indicative of said treatment requiring adjustment, wherein
the one or more lipid(s) whose increase in concentration is (are)
compared to the control is (are) selected from: LacCer(d18:1/22:0),
Gb3(d18:1/24:1), Gb3(d18:1/22:0), Gb3(d18:1/24:0),
LacCer(d18:1/24:0), Total eicosanoids, Total Gb3, Cer(d18:1/20:0),
Gb3(d18:1/20:0), LacCer(d18:1/24:1), LacCer(d18:1/20:0), PGE2,
12-OXOETE, 17-HDoHE, PGD2, TXB3, PS 18:0/18:1 and SM (d18:1/24:2);
and wherein the one or more lipid(s) whose decrease in
concentration is (are) compared to the control is (are) selected
from: Gb3(d18:1/16:0) and Gb3(d18:1/18:0); or (b) determining in a
sample from said subject one or more lipid-lipid concentration
ratio(s), wherein (an) increased or decreased lipid-lipid
concentration ratio(s) in said sample, when compared to a control,
is (are) indicative of said treatment requiring adjustment, wherein
the one or more lipid-lipid concentration ratio(s) whose increase
is (are) compared to the control is (are) selected from:
12-HETE/15-HETrE, 12-HETE/14_15-DHET, 12-HETE/Gb3 (d18:1/16:0),
12-HETE/Glc/GalCer(d18:1/24:1), 12-HETE/G1c/GalCer(d18:1/18:0),
Gb3(d18:1/24:1)/LacCer(d18:1/16:0),
LacCer(d18:1/22:0)/LacCer(d18:1/24:1),
Cer(d18:1/20:0)/Glc/GalCer(d18:1/24:1); and wherein the one or more
lipid-lipid concentration ratio(s) whose decrease is (are) compared
to the control is (are) selected from:
LacCer(d18:1/16:0)/LacCer(d18:1/22:0),
Gb3(d18:1/16:0)/Gb3(d18:1/24:1) and 11-HETE/12-HETE, and (c)
adjusting the treatment with the lipid lowering drug in the subject
who is determined to need adjustment, wherein adjusting the
treatment comprises (i) reducing a lipid lowering drug dose; (ii)
stopping a lipid lowering drug treatment; (iii) re-commencing a
lipid lowering drug treatment; or (iv) changing to a different
lipid lowering drug.
34. The method of claim 32, wherein the lipid whose increase in
concentration is compared to the control is selected from:
LacCer(d18:1/22:0) and Gb3(d18:1/24:1).
35. The method of claim 33, wherein the lipid whose increase in
concentration is compared to the control is selected from:
LacCer(d18:1/22:0) and Gb3(d18:1/24:1).
36. The method of claim 33, wherein the adjustment of said
treatment with a lipid lowering drug comprises (a) a reduction of a
statin dose; (b) a cessation of a statin treatment; (c) a
re-commencement of a statin treatment; (d) a change to a different
statin drug; (e) a change to a different lipid lowering drug; or
(f) a cessation of another drug treatment which led to muscle
toxicity due to its interaction with one or more statins.
37. The method of claim 32, wherein the method is for evaluating
the degree of muscle toxicity induced by a novel statin or a novel
lipid lowering medication in a subject undergoing treatment with
said statin or lipid lowering medication.
38. The method of claim 32, wherein the method is for (a)
determining early warning signs of muscle toxicity in said subject
and/or (b) determining whether the symptoms of muscle toxicity
found in a subject are due to statin-induced muscle toxicity.
39. The method of claim 32, comprising determining at least 2, at
least 3, at least 4, or at least 5, of said lipid concentrations or
lipid-lipid concentration ratios.
40. The method of claim 33, comprising determining at least 2, at
least 3, at least 4, or at least 5, of said lipid concentrations or
lipid-lipid concentration ratios.
41. The method of claim 32, wherein said lipid lowering drug is a
statin and wherein (a) said subject is being treated with one or
more statins; (b) said subject had undergone statin treatment, but
discontinued said treatment due to onset of muscle pain; or (c)
said subject has not yet been treated with statins.
42. The method of claim 33, wherein said lipid lowering drug is a
statin and wherein (a) said subject is being treated with one or
more statins; (b) said subject had undergone statin treatment, but
discontinued said treatment due to onset of muscle pain; or (c)
said subject has not yet been treated with statins.
43. The method of claim 32, wherein the subject is at a high risk
for developing statin-induced muscle toxicity and/or one or more of
its complications.
44. The method of claim 33, wherein the subject is at a high risk
for developing statin-induced muscle toxicity and/or one or more of
its complications.
45. The method claim 32, wherein said control to which comparison
is made is: (a) a control sample from the same subject undergoing
statin treatment prior to the onset of muscle toxicity; (b) a
control sample from the same subject prior to statin treatment or
during discontinuation of statin treatment; (c) a control sample
from a subject with no signs or history of statin-induced muscle
toxicity; (d) a control sample from a population of subjects with
no signs or history of statin-induced muscle toxicity; (e) a
control value established from one or more subject(s) not on statin
treatment and with no signs or history of muscle toxicity; or (f) a
control value established from one or more subject(s) on statin
treatment and with no signs or history of muscle toxicity.
46. The method claim 33, wherein said control to which comparison
is made is: (a) a control sample from the same subject undergoing
statin treatment prior to the onset of muscle toxicity; (b) a
control sample from the same subject prior to statin treatment or
during discontinuation of statin treatment; (c) a control sample
from a subject with no signs or history of statin-induced muscle
toxicity; (d) a control sample from a population of subjects with
no signs or history of statin-induced muscle toxicity; (e) a
control value established from one or more subject(s) not on statin
treatment and with no signs or history of muscle toxicity; or (f) a
control value established from one or more subject(s) on statin
treatment and with no signs or history of muscle toxicity.
47. The method of claim 32, further comprising determining or
evaluating the level of creatine kinase in said subject or in a
sample from said subject.
48. The method of claim 47, wherein the subject has or does not
have elevated creatine kinase levels.
49. The method of claim 33, further comprising determining or
evaluating the level of creatine kinase in said subject or in a
sample from said subject.
50. The method of claim 49, wherein the subject has or does not
have elevated creatine kinase levels.
51. The method of claim 32, wherein (a) the sample is blood,
plasma, serum, a lipoprotein fraction of blood, urine or muscle
biopsy tissue; and/or (b) the lipid concentration(s) or lipid-lipid
concentration ratio(s) is (are) determined by using mass
spectrometry, nuclear magnetic resonance spectroscopy, fluorescence
spectroscopy or dual polarisation interferometry, a high
performance separation method such as HPLC or UPLC, an immunoassay
such as an ELISA and/or with a binding moiety capable of
specifically binding the analyte.
52. The method of claim 33, wherein (a) the sample is blood,
plasma, serum, a lipoprotein fraction of blood, urine or muscle
biopsy tissue; and/or (b) the lipid concentration(s) or lipid-lipid
concentration ratio(s) is (are) determined by using mass
spectrometry, nuclear magnetic resonance spectroscopy, fluorescence
spectroscopy or dual polarisation interferometry, a high
performance separation method such as HPLC or UPLC, an immunoassay
such as an ELISA and/or with a binding moiety capable of
specifically binding the analyte.
53. A kit for predicting statin-induced muscle toxicity and/or one
or more of its complications, wherein the kit comprises: (a) lipid
standard(s) chosen from the lipids defined in claim 1, and
optionally one or more further reference compounds selected from:
(b) one or more control markers; (c) positive and/or negative
controls; (d) internal and/or external standards; (e) calibration
line controls; and (f) an agent, optionally an antibody, capable of
binding any one of the lipids defined in claim 1.
54. Use of a kit as defined in claim 53 for predicting
statin-induced muscle toxicity and/or one or more of its
complications, wherein the lipid concentration(s) or lipid-lipid
concentration ratio(s) in a sample from a subject is (are)
optionally determined by using mass spectrometry.
55. The method of claim 32, wherein: (a) said statin is selected
from the group consisting of atorvastatin, cerivastatin,
fluvastatin, fluvastatin XL, lovastatin, pitavastatin, pravastatin,
rosuvastatin and/or simvastatin; (b) said lipid lowering drug may
be selected from any of the following: a HMG-CoA reductase
inhibitor, niacin (nicotinic acid), a cholesterol absorption
inhibitor, a cholesteryl ester transfer protein (CETP) inhibitor, a
bile acid sequestrant; a fibrate or a phytosterol; wherein
optionally said cholesterol absorption inhibitor is ezetimibe or
SCH-48461; said cholesteryl ester transfer protein (CETP) inhibitor
is torcetrapib, anacetrapib or dalcetrapib; said bile acid
sequestrant is colesevelam, cholestyramine or colestipol; and said
fibrate is fenofibrate, gemfibrozil, clofibrate, or bezafibrate;
(c) the muscle toxicity is associated with a muscle disease, for
example, a muscle dystrophy; and/or (d) the one or more
complications of statin-induced muscle toxicity are selected from
myalgia, myositis, myopathy and rhabdomyolysis.
56. The method of claim 33, wherein: (a) said statin is selected
from the group consisting of atorvastatin, cerivastatin,
fluvastatin, fluvastatin XL, lovastatin, pitavastatin, pravastatin,
rosuvastatin and/or simvastatin; (b) said lipid lowering drug may
be selected from any of the following: a HMG-CoA reductase
inhibitor, niacin (nicotinic acid), a cholesterol absorption
inhibitor, a cholesteryl ester transfer protein (CETP) inhibitor, a
bile acid sequestrant; a fibrate or a phytosterol; wherein
optionally said cholesterol absorption inhibitor is ezetimibe or
SCH-48461; said cholesteryl ester transfer protein (CETP) inhibitor
is torcetrapib, anacetrapib or dalcetrapib; said bile acid
sequestrant is colesevelam, cholestyramine or colestipol; and said
fibrate is fenofibrate, gemfibrozil, clofibrate, or bezafibrate;
(c) the muscle toxicity is associated with a muscle disease, for
example, a muscle dystrophy; and/or (d) the one or more
complications of statin-induced muscle toxicity are selected from
myalgia, myositis, myopathy and rhabdomyolysis.
57. The method of claim 32, wherein the lipid lowering drug is a
statin.
58. The method of claim 33, wherein the lipid lowering drug is a
statin.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of International
Application PCT/EP2011/055569, filed 8 Apr. 2011 (pending), and
claims the benefit of, and relies on the filing date of U.S.
provisional patent application No. 61/545,603, filed 11 Oct. 2011,
the entire disclosures of which are incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] This invention relates to methods and uses involving lipid
levels to predict or diagnose statin-induced muscle toxicity. The
invention is applicable, inter alia, to determining whether a
subject requires adjustment of statin treatment and to the
evaluation of muscle toxicity induced by new lipid lowering drugs.
The methods include analyzing lipid levels of a biological sample,
and comparing it to a control.
BACKGROUND OF THE INVENTION
[0003] Statins are currently the most widely used lipid-lowering
drugs because they reduce the incidence of hard cardiovascular
end-points (cardiovascular death, myocardial infarction and stroke)
by 25% to 35% in different patient populations. These populations
include those with stable or unstable coronary artery disease,
diabetics, and hypertensive patients with other risk factors. In
general, statins are well tolerated although muscular, liver and
gastrointestinal side effects can occur. Statins can be associated
with a wide range of muscular side effects, from non-specific or
atypical myalgias, to myopathy and the full-blown rhabdomyolysis
syndrome.
[0004] Myalgias are defined as muscle pain or complaints of sore
muscles that can either be generalized or localized. Such symptoms
occur in up to 10% of patients and can force physicians to reduce
dose, switch to another statin using a trial-and-error approach or
stop the medication completely. These muscular symptoms can also
contribute to the relatively high rate of patients stopping statin
therapy within the first two years of the treatment. Thus, even the
more benign muscular symptoms can have important consequences and
limit the large clinical and socio-economic benefits potentially
offered by these agents.
[0005] Myopathy, while less devastating than rhabdomyolysis, can
also occur after treatment with statins and is defined as muscle
pain and/or weakness with increased creatine kinase (CK) levels at
least 10 times the upper limit of normal. The incidence of myopathy
is approximately 1-5%. The known predisposing risk factors for
statin-related muscle toxicity include renal insufficiency,
hypothyroidism, hereditary or acquired muscle diseases, history of
muscle toxicity with another statin or a fibrate, concomitant use
of a fibric acid derivative, alcohol abuse, clinical settings where
increased plasma levels of statins could occur, as well as Asian
ancestry.
[0006] Rhabdomyolysis is a rare event (well below 0.1% of statin
users) but constitutes a life-threatening condition characterized
by severe muscle toxicity, large increase in plasma creatine kinase
(CK) levels (exceeding 10,000 U/L) and renal insufficiency
secondary to myoglobin toxicity. Rhabdomyolysis has caused several
patient deaths and has led to the withdrawal of one statin from the
market, cerivastatin, (Baycol, Bayer). The incidence of
rhabdomyolysis was also recently shown to be increased with another
HMG-CoA reductase inhibitor, simvastatin (Zocor, Merck & Co.),
when administered at a high dose (A to Z trial).
[0007] Currently plasma/serum Creatine kinase (CK) measurement is
used as a biomarker for statin-induced muscle toxicity. For the
vast majority of cases, CK measurement remains uninformative
despite the presence of symptoms. Plasma/serum CK is an unspecific
marker because it can be elevated for many other reasons, including
physical exercise. An even greater limitation is its poor
sensitivity, since it becomes indicative only after a substantial
damage to muscle cells involving CK leakage to plasma from tissues.
Thus, to this end it is well justified to develop new biomarkers
for diagnosis of statin-induced muscle toxicity. Earlier studies
(Phillips P S et al.: "Statin-associated myopathy with normal
creatine kinase levels." Ann Intern Med. 2002 Oct. 1; 137(7):581-5)
on muscle specimens obtained from patients during acute muscle pain
have demonstrated, e.g., an accumulation of inflammatory cells in
histopathological studies.
[0008] The number of lipid mediators in the human body is
overwhelming. Attempts have been made to facilitate their
identification and quantification by advances in mass spectrometry
and lipid biochemistry, which today enable the simultaneous high
throughput identification and quantification of hundreds of
molecular lipid species in several lipid classes (Ejsing C S, et
al: Global analysis of the yeast lipidome by quantitative shotgun
mass spectrometry. Proc Natl Acad Sci USA 2009, 106:2136-2141;
Stahlman M, et al: High-throughput shotgun lipidomics by quadrupole
time-of-flight mass spectrometry. J Chromatogr B Analyt Technol
Biomed Life Sci 2009 Hiukka A, et al: ApoCIII-enriched LDL in type
2 diabetes displays altered lipid composition, increased
susceptibility for sphingomyelinase, and increased binding to
biglycan. Diabetes 2009, 58:2018-2026; Linden D, et al:
Liver-directed overexpression of mitochondrial glycerol-3-phosphate
acyltransferase results in hepatic steatosis, increased
triacylglycerol secretion and reduced fatty acid oxidation. FASEB J
2006, 20:434-443.) collectively referred to as the lipidome.
Lipidomic studies have sought to identify lipid cellular
distribution and to describe their biochemical mechanisms,
interactions and dynamics. Lipidomics is capable in principle of
quantifying the exact chemical composition of lipidomes (Han X,
Gross R W: Global analyses of cellular lipidomes directly from
crude extracts of biological samples by ESI mass spectrometry: a
bridge to lipidomics. J Lipid Res 2003, 44:1071-1079).
[0009] The bulk of the lipid data in the art today presents lipids
in a sum composition format, i.e., phosphatidylcholine (PC) 34:1
(Brugger B, et al: Quantitative analysis of biological membrane
lipids at the low picomole level by nano-electrospray ionization
tandem mass spectrometry. Proc Natl Acad Sci USA 1997,
94:2339-2344) where the molecular lipid and the attached fatty acid
tails remain unidentified. The identification of molecular lipid
species, e.g., PC 16:0/18:1 (Ekroos K, et al: Charting molecular
composition of phosphatidylcholines by fatty acid scanning and ion
trap MS3 fragmentation. J Lipid Res 2003, 44:2181-2192) is the main
feature of advanced lipidomics, which delivers highly resolved
molecular lipid species rather than summed fatty acid information.
For example, the information of the type of fatty acids and their
positions of attachment to the glycerol backbone making up the
particular PC molecule is revealed. There are conventional
techniques such as thin-layer chromatography combined with gas
chromatography but they not only require considerably larger sample
amounts and laborious sample preparation, but they do not deliver
the molecular lipid species. Despite multiple mass spectrometry
techniques capable of characterizing lipid entities, most of them
are still unable to deliver reliable high-quality quantitative data
in terms of absolute or close-to absolute concentrations.
[0010] There is a need for specific and reliable methods for the
detection and diagnosis of statin-induced muscle toxicity, as well
as markers useful in this regard. There is also a need for
improvements of existing treatment regimes with statins or lipid
lowering drugs.
SUMMARY OF THE INVENTION
[0011] The present invention inter alia provides novel lipidomic
markers, also referred to herein as "biomarkers", for the detection
and diagnosis of statin-induced muscle toxicity, such as
statin-induced muscle toxicity associated with muscle disease,
muscle dystrophy, myalgia, myositis, myopathy or
rhabdomyolysis.
[0012] In one aspect of the present invention, methods, lipidomic
markers, agents such as antibodies and kits are inter alia
disclosed and/or claimed herein for detecting statin associated
muscular side effects, from non-specific or atypical myalgias to
myopathy and the full-blown rhabdomyolysis syndrome. Myalgias are
defined as muscle pain or complaints of sore muscles that can
either be generalized or localized. Myopathy, while less
catastrophic than rhabdomyolysis, can also occur after treatment
with statins and is defined as muscle pain and/or weakness with
increased CK levels at least 10 times the upper limit of
normal.
[0013] Methods according to the invention may, e.g., comprise the
steps of: a) providing a biological sample from a subject being
treated, to be treated, or having been treated with a statin; b)
determining the concentration(s) of one or more lipid(s) and/or
lipid-lipid concentration ratio(s) identified herein as useful
lipidomic markers in accordance with the invention in said sample;
and c) comparing said determined lipid concentration(s) and/or
lipid-lipid concentration ratio(s) to the corresponding lipid
concentration(s) and/or to (a) lipid ratio(s) in a control.
[0014] The lipidomic markers of the present invention allow for
sensitive detection of statin-induced muscle toxicity. It will be
appreciated that the same applies to the complications of
statin-induced muscle toxicity. This will facilitate improving
patient care, lessening symptom development and suffering, and
achieving decreased morbidity/mortality associated with statin
side-effects. Thus, the lipidomic markers described and claimed
herein allow for individual tailoring of drug intervention
regarding patients treated, or to be treated, with statins. Also,
the invention is applicable to animal experiments where statins and
statin-like compounds are tested. The invention will inter alia
allow a better safety assessment of novel lipid lowering
medications to be made.
[0015] The lipidomic markers may be eicosanoids, ceramides or
cerebrosides. Moreover, the lipidomic markers are selected from the
lipid classes of ceramides, lactosylceramides, galactosyl- or
glucosylceramides, globotriaosylceramides (Gb3),
hydroxyeicosatetraenoic acids (HETE), dihydroxyeicosatrienoic acids
(DHET), hydroxyeicosapentaenoic acids (HEPE) and/or prostaglandins.
The lipidomic marker may also be a metabolic product of
12/15-lipoxygenase or a metabolic product of cyclooxygenase-2.
[0016] Accordingly, a method is inter alia provided herein for
determining whether a subject is at risk to develop, or is
suffering from statin-induced muscle toxicity and/or one or more of
its complications, comprising determining in a sample from said
subject the concentration(s) of one or more lipid(s), wherein (an)
increased or decreased concentration(s) in said sample, when
compared to a control, is (are) indicative of said subject
suffering from said statin-induced muscle toxicity and/or said
complication(s), wherein the one or more lipid(s) whose increase in
concentration is (are) compared to the control is (are) selected
from: 12-HETE, LacCer(d18:1/22:0), 15-HETE, Gb3(d18:1/24:1),
Gb3(d18:1/22:0), Gb3(d18:1/24:0), LacCer(d18:1/24:0), AA, Total
eicosanoids, Total Gb3, Cer(d18:1/20:0), Gb3(d18:1/20:0), 12-HEPE,
PGE2, LacCer(d18:1/20:0), 12-OXOETE, 17-HDOoHE, PGD2, TXB3, PS
18:0/18:1, SM (d18:1/24:2) and LacCer(d18:1/24:1) (see Tables 3 and
4); and wherein the one or more lipid(s) whose decrease in
concentration is (are) compared to the control is (are) selected
from: Gb3(d18:1/16:0), Gb3(d18:1/18:0), 14_15-DHET, and 8_9-DHET
(see Table 3).
[0017] Alternatively, a method is provided herein for determining
whether a subject is at risk to develop, or is suffering from
statin-induced muscle toxicity and/or one or more of its
complications, comprising determining in a sample from said subject
one or more lipid-lipid concentration ratio(s), wherein (an)
increased or decreased lipid-lipid concentration ratio(s) in said
sample, when compared to a control, is (are) indicative of said
subject suffering from said statin-induced muscle toxicity and/or
said complication(s), wherein the one or more lipid-lipid
concentration ratio(s) whose increase is (are) compared to the
control is (are) selected from: 12-HETE/15-HETrE,
12-HETE/14_15-DHET, 12-HETE/Gb3 (d18:1/16:0),
12-HETE/Glc/GalCer(d18:1/24:1), 12-HETE/Glc/GalCer(d18:1/18:0),
Gb3(d18:1/24:1)/LacCer(d18:1/16:0),
LacCer(d18:1/22:0)/LacCer(d18:1/24:1),
Cer(d18:1/20:0)/Glc/GalCer(d18:1/24:1) (Table 6); and wherein the
one or more lipid-lipid concentration ratio(s) whose decrease is
(are) compared to the control is (are) selected from
LacCer(d18:1/16:0)/LacCer(d18:1/22:0),
Gb3(d18:1/16:0)/Gb3(d18:1/24:1) and 11-HETE/12-HETE (Table 6).
[0018] Another aspect of the invention, provided herein is a method
for determining whether a subject is at risk to develop, or is
suffering from statin-induced muscle toxicity and/or one or more of
its complications, wherein the subject is female, comprising
determining in a sample from said subject the concentration(s) of
one or more lipid(s), wherein (a) decreased concentration(s) in
said sample, when compared to a control, is (are) indicative of
said subject suffering from said statin-induced muscle toxicity
and/or said complication(s), wherein the one or more lipid(s) whose
decrease in concentration is (are) compared to the control is (are)
selected from: Total LacCer, LacCer(d18:1/16:0) and
Glc/GalCer(d18:1/24:1) (see Table 3).
[0019] In a preferred embodiment, the one or more lipid(s) whose
increase in concentration is (are) compared to the control is (are)
selected from: 12-HETE, LacCer(d18:1/22:0) and Gb3(d18:1/24:1) (see
Table 5).
[0020] In another preferred embodiment, wherein the subject is
female, the one or more lipid(s) whose decrease in concentration is
(are) compared to the control is (are) selected from:
LacCer(d18:1/16:0) and Glc/GalCer(d18:1/24:1) (see Table 5).
[0021] In another aspect, the present invention relates to a method
for determining whether the statin treatment and/or the treatment
with a lipid lowering drug of a subject needs adjustment,
comprising determining in a sample from said subject the
concentration(s) of one or more lipid(s), wherein (an) increased or
decreased concentration(s) in said sample, when compared to a
control, is (are) indicative of said treatment requiring
adjustment, wherein the one or more lipid(s) whose increase in
concentration is (are) compared to the control is (are) selected
from: 12-HETE, LacCer(d18:1/22:0), 15-HETE, Gb3(d18:1/24:1),
Gb3(d18:1/22:0), Gb3(d18:1/24:0), LacCer(d18:1/24:0), AA, Total
eicosanoids, Total Gb3, Cer(d18:1/20:0), Gb3(d18:1/20:0), 12-HEPE,
PGE2, LacCer(d18:1/20:0), 12-OXOETE, 17-HDOoHE, PGD2, TXB3, PS
18:0/18:1, SM (d18:1/24:2) and LacCer(d18:1/24:1) (see Tables 3 or
4); and wherein the one or more lipid(s) whose decrease in
concentration is (are) compared to the control is (are) selected
from: Gb3(d18:1/16:0), Gb3(d18:1/18:0), 14_15-DHET and 8_9-DHET
(see Table 3).
[0022] In another aspect, the present invention relates to a method
for determining whether the statin treatment and/or the treatment
with a lipid lowering drug of a subject needs adjustment,
comprising determining in a sample from said subject one or more
lipid-lipid concentration ratio(s), wherein (an) increased or
decreased lipid-lipid concentration ratio(s) in said sample, when
compared to a control, is (are) indicative of said treatment
requiring adjustment, wherein the one or more lipid-lipid
concentration ratio(s) whose increase is (are) compared to the
control is (are) selected from: 12-HETE/15-HETrE,
12-HETE/14_15-DHET, 12-HETE/Gb3(d18:1/16:0), 12-HETE/Glc/GalC
er(d18:1/24:1), 12-HETE/Glc/GalCer(d18:1/18:0),
Gb3(d18:1/24:1)/LacCer(d18:1/16:0),
LacCer(d18:1/22:0)/LacCer(d18:1/24:1),
Cer(d18:1/20:0)/Glc/GalCer(d18:1/24:1) (Table 6); and wherein the
one or more lipid-lipid concentration ratio(s) whose decrease is
(are) compared to the control is (are) selected from
LacCer(d18:1/16:0)/LacCer(d18:1/22:0),
Gb3(d18:1/16:0)/Gb3(d18:1/24:1) and 11-HETE/12-HETE (Table 6).
[0023] Yet in another aspect, the present invention relates to a
method for determining whether the statin treatment and/or the
treatment with a lipid lowering drug of a subject needs adjustment,
wherein the subject is female, comprising determining in a sample
from said subject the concentration(s) of one or more lipid(s),
wherein (a) decreased concentration(s) in said sample, when
compared to a control, is (are) indicative of said treatment
requiring adjustment, wherein the one or more lipid(s) whose
decrease in concentration is (are) compared to the control is (are)
selected from: Total LacCer, LacCer(d18:1/16:0) and
Glc/GalCer(d18:1/24:1) (see Table 3).
[0024] In a preferred embodiment, the one or more lipid(s) whose
increase in concentration is (are) compared to the control is (are)
selected from: 12-HETE, LacCer(d18:1/22:0) and Gb3(d18:1/24:1) (see
Table 5).
[0025] In another preferred embodiment, wherein the subject is
female, the one or more lipid(s) whose decrease in concentration is
(are) compared to the control is (are) selected from:
LacCer(d18:1/16:0) and Glc/GalCer(d18:1/24:1) (see Table 5).
[0026] For the purpose of the method for determining whether the
statin treatment or the treatment with a lipid lowering drug of a
subject needs adjustment, the adjustment of said statin treatment
may comprise (a) a reduction of statin dose; (b) a cessation of
statin treatment; (c) a re-commencement of statin treatment; (d) a
change to a different statin drug; (e) a change to a different
lipid lowering drug; or (f) a cessation of another drug treatment
which led to muscle toxicity due to its interaction with one or
more statins.
[0027] In a further embodiment, the methods of the invention may be
used for evaluating the degree of muscle toxicity induced by a
novel statin or a novel lipid lowering medication in a subject
undergoing treatment with said statin or lipid lowering
medication.
[0028] The methods of the invention may be used for determining
early warning signs of muscle toxicity in said subject.
[0029] In addition, or alternatively, the methods may be used for
determining whether the symptoms of muscle toxicity found in a
subject are due to statin-induced muscle toxicity.
[0030] For the purposes of the methods of the invention, at least
one lipid concentration from Tables 3 or 4 or lipid-lipid
concentration ratio from Table 6 may be determined to assess
whether a subject is at risk to develop, or is suffering from
statin-induced muscle toxicity and/or one or more of its
complications, or to determine whether the statin treatment or
treatment with a lipid lowering drug of a subject needs adjustment.
However, it is also possible, and may be advantageous, to determine
at least 2, at least 3, at least 4, at least 5, at least 6, at
least 7, or at least 8 lipid concentrations from Tables 3 or 4, or
at least 2, at least 3, at least 4, at least 5, at least 6, at
least 7, or at least 8 lipid-lipid concentration ratios from Table
6. Where more than one lipidomic marker is determined and used for
the assessment, it may be advantageous that a specific lipid
concentration or lipid-lipid concentration ratio is given greater
weight than others in the above-mentioned assessment.
[0031] The methods of the invention encompass the determination of
(a) lipid concentration(s) or (a) lipid-lipid concentration
ratio(s) in a sample from a subject that is being treated with one
or more statins.
[0032] Alternatively, the methods of the invention encompass the
determination of (a) lipid concentration(s) or (a) lipid-lipid
concentration ratio(s) in a sample from a subject that had
undergone statin treatment, but discontinued said treatment, e.g.,
due to onset of muscle pain.
[0033] In a further alternative, the methods of the invention
encompass the determination of (a) lipid concentration(s) or (a)
lipid-lipid concentration ratio(s) in a sample from a subject that
has not yet been treated with statins.
[0034] The methods of the invention may further encompass the
determination of (a) lipid concentration(s) or (a) lipid-lipid
concentration ratio in a sample from a subject that is at a high
risk for developing statin-induced muscle toxicity and/or one or
more of its complications.
[0035] For the purposes of the methods of the invention, a
comparison of the subject's sample is made in respect of a
control.
[0036] In a preferred embodiment, the control is, for example, a
control sample, preferably a control sample that corresponds to the
subject's sample.
[0037] In a preferred embodiment, the control sample is from the
same subject undergoing statin treatment, but prior to the onset of
muscle toxicity. The control may, however, also be a sample from
the same subject prior to statin treatment or during
discontinuation of statin treatment. In another preferred
embodiment, it may also be from another subject with no signs or
history of statin-induced muscle toxicity.
[0038] In another preferred embodiment, the control is a control
sample from a population of subjects with no signs or history of
statin-induced muscle toxicity.
[0039] In yet another preferred embodiment, however, the control is
not a sample but merely a control value established from one or
more subject(s) not on statin treatment and with no signs or
history of muscle toxicity. Alternatively, the control may
advantageously be a control value established from one or more
subject(s) on statin treatment and with no signs or history of
muscle toxicity.
[0040] In accordance with the present invention, the
concentration(s) of the individual lipid(s) and/or the lipid-lipid
concentration ratio(s) of lipids in the sample from the subject are
preferably compared to the concentration(s) of the corresponding
lipid(s) or to (a) lipid ratio(s) (e.g., the corresponding
lipid-lipid concentration ratio(s)) in the control, be it a control
sample or a control value, for determining whether a subject is at
risk to develop, or is suffering from statin-induced muscle
toxicity (and/or one or more of its complications), or to determine
whether the statin treatment or treatment with a lipid lowering
drug of a subject needs adjustment. Some illustrative examples of
the comparisons that can be made between a subject's sample and a
control are shown in the table below:
TABLE-US-00001 TABLE 1 Example on comparison pairs between a
subject's sample and a control. Comparison pairs Case Control
Readout 1 Subject on Statin with Subject(s) on Statin with Increase
or decrease in muscle toxicity or a high risk no muscle toxicity
concentration of lipid(s) in subject on statin with no Tables 3 or
4 muscle toxicity 2 Subject on Statin with Same subject on statin
Increase or decrease in muscle toxicity prior to muscle toxicity
concentration of lipid(s) in Tables 3 or 4 3 Subject on Statin with
Same subject prior to statin Change or lack of change in muscle
toxicity therapy concentration of lipid(s) in Tables 3 or 4 e.g.,
a) increased concentration of 12-HETE, b) decreased concentration
of "decreased" lipids, c) lack of change in remaining lipids 4
Subject on statin with muscle Same subject after statin Change or
lack of change in toxicity withdrawal concentration of lipid(s) in
Tables 3 or 4 e.g., a) increased concentration of 12-HETE, b)
decreased concentration of "decreased" lipids, c) lack of change in
remaining lipids
[0041] On the other hand, the comparison in accordance with the
present invention of the concentration(s) of the individual
lipid(s) or lipid-lipid concentration ratio(s) in the sample from
said subject may also be made to the concentration(s) or
concentration ratio(s) of (an)other individual molecule(s) in the
control, again either control sample or control value, for
determining whether a subject is at risk to develop, or is
suffering from statin-induced muscle toxicity (and/or one or more
of its complications), or to determine whether the statin treatment
or treatment with a lipid lowering drug of a subject needs
adjustment. Such other individual molecule(s) in the control is
(are) preferably molecule(s) where the concentration(s) or
concentration ratio(s) thereof is (are) similar, or essentially
similar, in all or at least a majority of subjects, so that the
concentration(s) or concentration ratio(s) is (are) suitable as
point of reference for determining whether there is an increase or
decrease in said sample in respect of the lipidomic markers
according to the invention. Preferred in this regard is/are, for
example, (an)other lipid(s). Also preferred in this regard is/are,
for example, (a) protein(s). Particularly preferred in this regard
is/are (a) molecule(s) in the control that is/are regularly
measured in a clinical setting. For example, preferred are
embodiments where the comparison is made to the concentration of
apoA, apoB, albumin or total PC in the control (again control
sample or control value), or combinations thereof.
[0042] In a further embodiment, the methods of the invention may
further comprise determining or evaluating the level of creatine
kinase (CK) in the subject or in a sample from the subject. In one
embodiment of the invention, the subject has elevated creatine
kinase levels. In another embodiment of the invention, the subject
does not have elevated creatine kinase levels.
[0043] In accordance with the methods of the invention, the sample
can be blood plasma, blood serum, or muscle biopsy tissue. The
sample may also be a fraction of blood, blood plasma or blood
serum, e.g., a lipoprotein fraction. A blood sample can be prepared
and plasma or serum, or fractions thereof, can be separated
therefrom with techniques well known to the person skilled in the
art. Alternatively, both the sample from the subject and the
control sample may also be a urine sample or a tissue sample, e.g.,
muscle biopsy tissue.
[0044] Collecting information on a lipidomic marker (i.e., a lipid
concentration or lipid-lipid concentration ratio, as described and
claimed herein) according to the methods of the present invention
from the subject's sample, and also from the control sample, can be
performed via various chemical and high resolution analytical
techniques. Particularly suitable analytical techniques include,
but are not limited to, mass spectrometry and nuclear magnetic
resonance spectroscopy. Indeed, any high resolution technique
capable of resolving individual lipids or lipid classes and
providing structural information of the same can be used to
determine the lipidomic markers according to the invention from the
subject's sample, and also from the control sample. For the
purposes of the methods of the present invention the lipid
concentration(s) or lipid-lipid concentration ratio(s) are thus
preferably determined by using mass spectrometry. However, nuclear
magnetic resonance spectroscopy, fluorescence spectroscopy or dual
polarisation interferometry, high performance separation methods
such as HPLC or UPLC, an immunoassay such as an ELISA and/or the
use of a binding moiety capable of specifically binding the lipid
analyte are also useful in this regard.
[0045] As indicated above, according to an alternative or further
embodiment of the methods of the invention, a lipid analyte in a
sample can be detected and/or quantified by combining the analyte
with a binding moiety capable of specifically binding the analyte.
The binding moiety can include, for example, a member of a
ligand-receptor pair, i.e., a pair of molecules capable of having a
specific binding interaction. The binding moiety can also include,
for example, a member of a specific binding pair, such as
antibody-antigen, enzyme-substrate, nucleic acid-based ligands,
other protein ligands, or other specific binding pairs known in the
art.
[0046] In a particularly preferred embodiment, the lipidomic
markers of the present invention are determined with mass
spectrometry (MS), wherein the MS instrument is optionally coupled
to direct infusion methods and/or high performance separation
methods such as HPLC or UPLC. The amount of the individual lipids
or lipid classes in the collected lipidomic markers is used when
comparing the collected lipid profile to a control.
[0047] In another aspect of the present invention, an agent capable
of binding to any one of the lipids in Tables 3, 4 or 6, is used
for predicting, diagnosing, preventing or treating statin-induced
muscle toxicity and/or one or more of its complications in a
subject. Similarly, the present invention relates to a method of
predicting, diagnosing, preventing or treating statin-induced
muscle toxicity and/or one or more of its complications in a
subject using, or administering a therapeutically effective amount
of an agent that binds to any one of the lipids in Tables 3, 4 or
6. In a preferred embodiment, the said agent is an antibody.
[0048] For example, since 12/15-lipoxygenase or cyclooxygenase-2
derived metabolites may be related to pain signaling (Mathie A. Ion
channels as novel therapeutic targets in the treatment of pain. J
Pharm Pharmacol. 2010 September; 62(9):1089-95; Ma W, Quirion R.
Does COX2-dependent PGE2 play a role in neuropathic pain? Neurosci
Lett. 2008 Jun. 6; 437(3):165-9; Xie C, Wang D H. Inhibition of
Renin Release by Arachidonic Acid Metabolites, 12(s)-HPETE and
12-HETE: Role of TRPV 1 Channels. Endocrinology. 2011 October;
152(10):3811-9), inhibition of one or both of these metabolic
pathways with an agent may offer (an) efficient mechanism(s) for
treating or preventing statin-induced muscle toxicity or muscle
pain caused by statin induced muscle toxicity.
[0049] Ceramides are known to activate cytosolic phospholipase A2
(cPLA2), which leads to increased 12-HETE levels through the
release of arachidonic acid from phospholipids (Pettus B J,
Bielawska A, Subramanian P, Wijesinghe D S, Maceyka M, Leslie C C,
Evans J H, Freiberg J, Roddy P, Hannun Y A, Chaliant C E. Ceramide
1-phosphate is a direct activator of cytosolic phospholipase A2. J
Biol Chem. 2004 Mar. 19; 279(12):11320-6. Epub 2003 Dec. 15;
Farooqui A A, Horrocks L A. Phospholipase A2-generated lipid
mediators in the brain: the good, the bad, and the ugly.
Neuroscientist. 2006 June; 12(3):245-60; Nanda B L, Nataraju A,
Rajesh R, Rangappa K S, Shekar M A, Vishwanath B S. PLA2 mediated
arachidonate free radicals: PLA2 inhibition and neutralization of
free radicals by anti-oxidants--a new role as anti-inflammatory
molecule. Curr Top Med Chem. 2007; 7(8):765-77). Since the present
invention shows that ceramides and 12-HETE are upregulated in
statin-induced muscle toxicity, inhibition of ceramide synthesis
and/or arachidonic acid production with an agent may also be useful
in treating or preventing statin-induced muscle toxicity or muscle
pain caused by statin induced muscle toxicity. For example, as
eicosanoids are involved in arachidonic acid metabolism (Zeldin, D
C. Epoxygenase Pathways of Arachidonic Acid Metabolism. JBC. 2001.
276 (39):36059-36062), they may be an appropriate target for an
agent to inhibit statin-induced muscle toxicity. In one preferred
embodiment of the invention, an agent capable of binding to any one
of the eicosanoids of the present invention invention (e.g.,
12-HETE, 15-HETE, AA, 14.sub.--15-DHET, 8.sub.--9-DHET (Table 3),
12-HEPE, PGE2, 12-OXOETE, 17-HDoHE, PGD2 or TXB3 (Table 4) is used
for predicting, diagnosing, preventing or treating statin-induced
muscle toxicity and/or one or more of its complications in a
subject. Similarly, the present invention relates to a method of
predicting, diagnosing, preventing or treating statin-induced
muscle toxicity and/or one or more of its complications in a
subject using, or administering a therapeutically effective amount
of an agent that binds to any one of the eicosanoids of the present
invention (e.g., 12-HETE, 15-HETE, AA, 14_15-DHET, 8_9-DHET (Table
3), 12-HEPE, PGE2, 12-OXOETE, 17-HDoHE, PGD2 or TXB3 (Table 4). In
a preferred embodiment, the said agent is an antibody.
[0050] Alternatively, the present invention relates to an agent for
use in preventing or treating statin-induced muscle toxicity and/or
one or more of its complications in a subject, wherein the agent
affects the activity, functionality or concentration of an enzyme,
wherein said enzyme catalyzes a reaction that produces or degrades
any one of the lipids in Tables 3, 4 or 6. Similarly, the present
invention relates to a method of preventing or treating
statin-induced muscle toxicity and/or one or more of its
complications in a subject using, or administering an agent,
wherein the agent affects the activity, functionality or
concentration of an enzyme, wherein said enzyme catalyzes a
reaction that produces or degrades any one of the lipids in Tables
3, 4 or 6.
[0051] Also encompassed by the present invention is a kit for
predicting statin-induced muscle toxicity and/or one or more of its
complications, or for performing the methods or uses described
and/or claimed herein, wherein the kit comprises reagents and
reference compounds. The reference compounds may be one or more of
the following, but are not limited to: (a) (a) lipid standard(s)
chosen from the lipids in Tables 3, 4 or 6, (b) one or more control
markers (for example, a lipid or lipids, preferably a lipid
corresponding to any of the lipidomic markers described and/or
claimed herein, or (an)other lipid(s), e.g., total PC, or another
molecule, e.g., a protein; c) positive and/or negative controls; d)
internal and/or external standards; e) calibration line controls;
(f) an antibody or other binding moiety capable of binding any one
of the lipids in Tables 3, 4 or 6. The reagents are solution(s),
solvent(s), and/or buffer(s) useful for performing said methods or
uses.
[0052] In one embodiment of the invention, a kit is provided for
predicting statin-induced muscle toxicitiy and/or one or more of
its complications, or for performing the methods of the invention,
wherein the kit comprises (a) (a) lipid standard(s) chosen from the
lipids in Tables 3, 4 or 6, and optionally one or more further
reference compound(s) selected from: (b) one or more control
markers (for example, a lipid or lipids, preferably a lipid
corresponding to any of the lipidomic markers described and/or
claimed herein, or another lipid(s), e.g., total PC, or another
molecule, e.g., a protein); c) positive and/or negative controls;
d) internal and/or external standards, which may or may not be
chemically modified, tagged or non-endogenous occurring molecules
in human; e) calibration line controls; and (f) an agent,
optionally an antibody, capable of binding any one of the lipids in
Tables 3, 4 or 6, and (g) (a) reagent(s) for performing said
methods or uses.
[0053] Preferred kits according to the invention comprise, for
example, the following combinations of the above listed
constituents: (a) and (b), and optionally (g); (a) and (c), and
optionally (g); (a) and (d), and optionally (g); (a) and (e), and
optionally (g); (a) and (f), and optionally (g); (a), (b) and (c),
and optionally (g); (a), (c) and (d), and optionally (g); (a), (d)
and (e), and optionally (g); or (a), (e) and (f), and optionally
(g).
[0054] In one preferred embodiment, the one or more control
marker(s) of the claimed kit is/are (a) molecule(s) that is/are
regularly measured in a clinical setting. For example, preferred
are embodiments wherein the one or more said control marker(s) is
CK.
[0055] In a preferred embodiment, the kit is used to predict
statin-induced muscle toxicity and/or one or more of its
complications, or to perform any of the methods encompassed by the
present invention, wherein the lipid concentration(s) or
lipid-lipid concentration ratio(s) in a sample from a subject is
(are) determined by using mass spectrometry. The sample may be
subjected to a purification and/or other sample pre-preparation
step(s) before mass spectrometry analysis. The purification step
may be, but is not limited to chromatography, for example, high
performance liquid chromatography (HPLC) and/or ultra high
performance liquid chromatography (UHPLC). The sample
pre-preparation step may be, but is not limited to solid-phase
extraction (SPE), derivatization and/or liquid-liquid extraction.
The said mass spectrometry determination may be done by tandem mass
spectrometry.
[0056] In another aspect, the present invention relates to a statin
or a lipid lowering drug for use in the treatment of a subject at
risk to develop or suffering from atherosclerosis or cardiovascular
disease (CVD) and/or one or more of their complications, wherein
said subject would be identified as being at risk to develop or as
suffering from statin-induced muscle toxicity when applying any of
the methods, agents, kits or uses described and/or claimed herein.
Similarly, the present invention relates to a method of treating a
subject at risk to develop or suffering from atherosclerosis or
cardiovascular disease (CVD) and/or one or more of their
complications with a statin or a lipid lowering drug, wherein said
subject would be identified as being at risk to develop or as
suffering from statin-induced muscle toxicity when applying any of
the methods, agents, kits or uses described and/or claimed
herein.
[0057] In a further embodiment, the present invention relates to a
statin or a lipid lowering drug for use in the treatment of a
subject at risk to develop or suffering from atherosclerosis or
cardiovascular disease (CVD) and/or one or more of their
complications, wherein said subject actually has been identified as
being at risk to develop or as suffering from statin-induced muscle
toxicity by any of the methods, agents, kits or uses described
and/or claimed herein. Similarly, the present invention relates to
a method of treating a subject at risk to develop or suffering from
atherosclerosis or cardiovascular disease (CVD) and/or one or more
of their complications with a statin or a lipid lowering drug,
wherein said subject actually has been identified as being at risk
to develop or as suffering from statin-induced muscle toxicity by
any of the methods, agents, kits or uses described and/or claimed
herein.
[0058] In yet another aspect, the present invention relates to a
statin or a lipid lowering drug for use in the treatment of a
subject at risk to develop or suffering from atherosclerosis or
cardiovascular disease (CVD) and/or one or more of their
complications, wherein said subject would be identified as not
being at risk to develop or as not suffering from statin-induced
muscle toxicity when applying any of the methods, agents, kits or
uses described and/or claimed herein. Similarly, the present
invention relates to a method of treating a subject at risk to
develop or suffering from atherosclerosis or cardiovascular disease
(CVD) and/or one or more of their complications with a statin or a
lipid lowering drug, wherein said subject would be identified as
not being at risk to develop or as not suffering from
statin-induced muscle toxicity when applying any of the methods,
agents, kits or uses described and/or claimed herein.
[0059] In a further embodiment, the present invention relates to a
statin or a lipid lowering drug for use in the treatment of a
subject at risk to develop or suffering from atherosclerosis or
cardiovascular disease (CVD) and/or one or more of their
complications, wherein said subject actually has been identified as
not being at risk to develop or as not suffering from
statin-induced muscle toxicity by any of the methods, agents, kits
or uses described and/or claimed herein. Similarly, the present
invention relates to a method of treating a subject at risk to
develop or suffering from atherosclerosis or cardiovascular disease
(CVD) and/or one or more of their complications with a statin or a
lipid lowering drug, wherein said subject actually has been
identified as not being at risk to develop or as not suffering from
statin-induced muscle toxicity by any of the methods, agents, kits
or uses described and/or claimed herein.
[0060] In yet another aspect, the present invention relates to a
statin or a lipid lowering drug for use in the treatment of a
subject at risk to develop or suffering from atherosclerosis or
cardiovascular disease (CVD) and/or one or more of their
complications, wherein said treatment is, or has been, assessed
using any of the methods for determining the need for treatment
adjustment described and/or claimed herein, and wherein said
treatment is, or has been, adjusted accordingly. Similarly, the
present invention relates to a method of treating a subject at risk
to develop or suffering from atherosclerosis or cardiovascular
disease (CVD) and/or one or more of their complications with a
statin or a lipid lowering drug, wherein said treatment is, or has
been, assessed using any of the methods for determining the need
for treatment adjustment described and/or claimed herein, and
wherein optionally, said treatment is, or has been, adjusted
accordingly. In connection with this aspect of the invention, such
adjustment may suitably comprise, but is not limited to: (a) a
reduction of statin dose; (b) a cessation of statin treatment; (c)
a re-commencement of statin treatment; (d) a change to a different
statin drug; (e) a change to a different lipid lowering drug; or
(f) a cessation of another drug treatment which led to muscle
toxicity due to its interaction with one or more statins.
[0061] In the context of all aspects and embodiments of the
invention described and claimed herein, a statin may be one
selected from, but not limited to, the group consisting of
atorvastatin, cerivastatin, fluvastatin, fluvastatin XL,
lovastatin, pitavastatin, pravastatin, rosuvastatin and/or
simvastatin.
[0062] In the context of all aspects and embodiments of the
invention described and claimed herein, the determination of the
lipid concentration(s) or lipid-lipid concentration ratio(s) is
typically performed using an assay.
[0063] In the context of all aspects and embodiments of the
invention described and claimed herein, muscle toxicity may be
associated with a muscle disease, for example, a muscle
dystrophy.
[0064] In the context of all aspects and embodiments of the
invention described and claimed herein, statin-induced muscle
toxicity complications include in particular those selected from
myalgia, myositis, myopathy and rhabdomyolysis. Statin myopathy or
myositis is typically characterized by muscle pain (myalgia),
muscle weakness, joint pain or rhabdomyolysis.
DETAILED DESCRIPTION OF THE INVENTION
[0065] The present invention is the result of applying lipidomics
to the identification of biomarkers indicative of statin-induced
muscle toxicity. It will facilitate the mission of making sure the
right individual receives the right statin or cholesterol lowering
drug at the right time and dose, thereby opening this therapeutic
area towards personalizing hitherto more generally applied
medicines and/or treatment regimes.
[0066] Due to both high sensitivity and specificity of lipidomics,
even the smallest sample amounts can be analyzed.
[0067] According to the present invention, the lipids may be
analyzed by a variety of techniques. In the context of the present
invention, electrospray ionization mass spectrometry-based
lipidomics is the preferred technology. The superior quality and
specificity of shotgun and targeted analysis methods will meet
stringent regulatory standards, such as good laboratory practice
guidelines (GLP) when set-up in the proper environment.
[0068] As used herein, muscle toxicity is an adverse change in
muscle cell(s) and/or muscle tissue induced by a drug.
[0069] As used herein, myopathy is a general term referring to any
disease of muscles; myopathies can be acquired or inherited and can
occur at birth or later in life (Source: NINDS Myopathy
Page-http://accessible.ninds.nih.gov/health_and_medical/disorders/myopath-
y.htm).
[0070] As used herein, myalgia is a term that describes muscle ache
or weakness without creatine kinase (CK) elevation.
[0071] As used herein, myositis is a term to describe muscle
symptoms with increased CK levels.
[0072] Rhabdomyolysis as used herein is characterized by muscle
symptoms with marked CK elevation (typically substantially greater
than 10 times the upper limit of normal [ULN]) and with creatinine
elevation (usually with brown urine and urinary myoglobin).
[0073] A muscle disease as used herein is any disease or disorder
that affects the muscle system.
[0074] A muscle dystrophy as used herein is a hereditary muscle
disease that weakens the muscles. Muscular dystrophies are
characterized by progressive skeletal muscle weakness, defects in
muscle proteins, and the death of muscle cells and tissue. Muscle
dystrophies may include Duchenne, Becker, limb girdle, congenital,
facioscapulohumeral, myotonic, oculopharyngeal, distal, and/or
Emery-Dreifuss diseases.
[0075] As used herein, a complication of atherosclerosis or CVD
includes in particular a complication selected from myocardial
infarction (MI), AMI, angina pectoris, transient ischemic attack
(TIA), stroke and death.
[0076] Some abbreviations used herein have the following meaning:
CK is creatine kinase, ADR is adverse drug reaction, MS is mass
spectrometry, HPLC is high performance liquid chromatography, and
UPLC is ultra high performance liquid chromatography, ROC is
receiving operating characteristics, and AUC is area under
curve.
[0077] Moderate to severe creatine kinase elevations are those
considered greater than 10 times ULN or greater than 10,000 IU/L.
Mild CK elevation is considered to be greater than the ULN but less
than 10 times the ULN (Jacobson T A, et al: Toward "Pain-Free"
Statin Prescribing: Clinical Algorithm for Diagnosis and Management
of Myalgia. Mayo Clin Proc. June 2008; 83(4687-700; Joy T R and
Hegele R A, Narrative review: statin-related myopathy. Ann Intern
Med. 2009 Jun. 16; 150(12): 858-68).
[0078] A statin and a statin treatment, respectively, in accordance
with the present invention will preferably be the following statins
and treatments therewith, respectively: cerivastin (0.4 mg/d,
Phillips P S et al: Statin-AssociatedMyopathy with Normal Creatine
Kinase Levels. Ann Intern Med. 2002; 137:581-585; Evans M and Rees
A: The myotoxicity of statins. Current Opinion in Lipidology. 2002,
13:415-420); fluvastatin (80 mg/d, Jacobson T A, et al: Toward
"Pain-Free" Statin Prescribing:Clinical Algorithm for Diagnosis and
Management of Myalgia. Mayo Clin Proc. June 2008; 83(4687-700);
fluvastatin XL (80 mg/d, Jacobson T A, et al: Toward "Pain-Free"
Statin Prescribing: Clinical Algorithm for Diagnosis and Management
of Myalgia. Mayo Clin Proc. June 2008; 83(4687-700); Iovastatin (40
mg/d, Phillips P S et al: Statin-Associated Myopathy with Normal
Creatine Kinase Levels. Ann Intern Med. 2002; 137:581-585);
pravastatin (40 mg/d, Phillips P S et al: Statin-Associated
Myopathy with Normal Creatine Kinase Levels. Ann Intern Med. 2002;
137:581-585; Jacobson T A, et al: Toward "Pain-Free" Stalin
Prescribing:Clinical Algorithm for Diagnosis and Management of
Myalgia. Mayo Clin Proc. June 2008; 83(6):687-700); rosuvastatin
(2.5 to 20 mg, 1 to 7 times weekly, with a preferred embodiment of
5 or 10 mg per day, Joy T R and Hegele R A, Narrative review:
statin-related myopathy. Ann Intern Med. 2009 Jun. 16;
150(12):858-68); Jacobson T A, et al: Toward "Pain-Free" Statin
Prescribing:Clinical Algorithm for Diagnosis and Management of
Myalgia. Mayo Clin Proc. June 2008; 83 (6): 687-700); atorvasatin
(10 or 20 mg/d, Phillips P S et al: Statin-AssociatedMyopathy with
Normal Creatine Kinase Levels. Ann Intern Med. 2002; 137:581-585);
40 mg/d (Laaksonen R, et al: A Systems Biology Strategy Reveals
Biological Pathways and Plasma Biomarker Candidates for Potentially
Toxic Statin-Induced Changes in Muscle. PLoS ONE. December 2006,
Issue 1, e97: 1-9); 40 or 80 mg/d (Jacobson T A, et al: Toward
"Pain-Free" Statin Prescribing:Clinical Algorithm for Diagnosis and
Management of Myalgia. Mayo Clin Proc. June 2008; 83(6):687-700);
and/or simvastatin (40 or 80 mg/d, Phillips P S et al:
Statin-Associated Myopathy with Normal Creatine Kinase Levels. Ann
Intern Med. 2002; 137:581-585); 80 mg/d (Laaksonen R, et al: A
Systems Biology Strategy Reveals Biological Pathways and Plasma
Biomarker Candidates for Potentially Toxic Statin-Induced Changes
in Muscle. PLoS ONE. December 2006, Issue 1, e97: 1-9; Jacobson T
A, et al: Toward "Pain-Free" Stalin Prescribing: Clinical Algorithm
for Diagnosis and Management of Myalgia. Mayo Clin Proc. June 2008;
83(6):687-700). Alternatively, fluvastatin, lovastatin,
pravastatin, rosuvastatin, atorvasatin and/or simvastatin may be
administered at 40 mg/d (Jacobson T A, et al: Toward "Pain-Free"
Stalin Prescribing:Clinical Algorithm for Diagnosis and Management
of Myalgia. Mayo Clin Proc. June 2008; 83(6):687-700). This
treatment may or may not also comprise the administration of a
fibrate or ezetimibe (10 mg/d, Jacobson T A, et al: Toward
"Pain-Free" Statin Prescribing: Clinical Algorithm for Diagnosis
and Management of Myalgia. Mayo Clin Proc. June 2008;
83(6):687-700). Colesevelam may be additionally administered with
ezetimibe at a dosage of 3.75 g/d (Joy T R and Hegele R A,
Narrative review: statin-related myopathy. Ann Intern Med. 2009
Jun. 16; 150(12):858-68).
[0079] For the purposes of the present invention, a lipid lowering
drug or medication is preferably an HMG-CoA reductase inhibitor,
niacin (nicotinic acid), a cholesterol absorption inhibitor, a
cholesteryl ester transfer protein (CETP) inhibitor, a bile acid
sequestrant; a fibrate or a phytosterol.
[0080] For the purposes of the present invention, a cholesterol
absorption inhibitor is preferably ezetimibe or SCH-48461; a
cholesteryl ester transfer protein (CETP) inhibitor is preferably
torcetrapib, anacetrapib or dalcetrapib; a bile acid sequestrant is
preferably colesevelam, cholestyramine or colestipol; and a fibrate
is preferably fenofibrate, gemfibrozil, clofibrate, or
bezafibrate.
[0081] As used herein, a subject includes all mammals, including
without limitation humans, but also non-human primates, dogs, cats,
horses, sheep, goats, cows, rabbits, pigs and rodents. A
particularly preferred subject in accordance with the present
invention is a human.
[0082] As used herein a high risk subject is typically a subject,
particularly a human, on high statin dose and/or on multiple
medications (causing a risk for drug interactions), having a known
muscle disease, or having a disease that may increase the risk of
adverse events (e.g., hypothyroidism, renal insufficiency or a
liver disease).
[0083] As used herein, a control may be a control sample or merely
a control value. In case it is a control value, it will be
appreciated that it may have already been determined, calculated or
extrapolated prior to initiating the methods of the invention.
Alternatively, the control value may be determined, calculated or
extrapolated after conducting the determination of the
concentration(s) of said one or more lipid(s), or lipid-lipid
concentration ratio(s), in accordance with the methods of the
present invention. Thus, it will be appreciated that a suitable
control value in accordance with the present invention may well be
one that is taken from the literature.
[0084] A sample as used herein is defined as any biological sample
obtained from a subject or a group or population of subjects. For
the purposes of the present invention, the biological sample may be
whole blood, blood serum, or blood plasma, with blood serum and
blood plasma being preferred. The sample may also be urine. Taking
a blood and/or urine sample of a patient is a part of normal
clinical practice. The blood sample can be taken in connection with
e.g. measuring the cholesterol levels in the patients. The
collected blood sample can be prepared and serum or plasma can be
separated with techniques well known to a person skilled in the
art. Venous blood samples can be collected from patients using a
needle and a BD Vacutainer.RTM. Plastic Tubes or Vacutainer.RTM.
Plus Plastic Tubes (BD Vacutainer.RTM. SST.TM. Tubes contain
spray-coated silia and a polymer gel for serum separation). Serum
can be separated by centrifugation at 1300 RCF for 10 min at room
temperature and stored in small plastic tubes at -80.degree. C. The
urine sample can be collected and prepared with techniques well
know to a person skilled in art. The sample may also be a fraction
of whole blood, blood plasma or blood serum, e.g., a lipoprotein
fraction. In another preferred embodiment, the sample may also be a
tissue sample, e.g., muscle biopsy tissue.
[0085] The lipids or other molecules in the control to which the
comparison is made in accordance with the present invention are
referred to herein also as control markers.
[0086] As used herein, the reference to a control sample from the
same subject or from a(nother) subject may mean that the control
sample has been directly obtained from said subject. Alternatively,
however, it may also mean that it has been obtained as the result
of a physical or chemical treatment of a sample directly obtained
or taken from said subject, such as centrifugation, fractionation,
enzymatic digestion, precipitation, and the like. The same applies
to any reference herein to a control sample from a group of
subjects or from a population of subjects.
[0087] The terms control sample from a group of subjects or control
sample from a population of subjects as used herein furthermore
preferably entail that the control sample is representative of said
group or population. In this context, representative shall mean
that the concentration(s) of the one or more lipids or the
lipid-lipid concentration(s) in said control sample to which a
comparison is made in the context of the present invention
corresponds to the average concentration(s) of said lipid(s) or
lipid-lipid concentration ratio(s) in corresponding individual
samples from the subjects of said group or population. Preferably,
the concentrations of all lipids and lipid-lipid concentration
ratios in said control sample correspond to the average
concentrations of said lipids and lipid-lipid concentration ratios
in corresponding individual samples from the subjects of said group
or population. Likewise, where a comparison is made in the context
of the present invention to one or more other molecules, e.g.,
other lipids or proteins, such as total PC, or apoA, apoB, or
albumin, respectively, a representative control sample is one where
the concentration(s) of this (these) molecule(s) correspond(s) to
the average concentration(s) of said molecule(s) in corresponding
individual samples from the subjects of said group or population.
In a preferred embodiment, a control sample from a group of
subjects or a control sample from a population of subjects in the
sense of the present invention is obtained by mixing equal amounts
of samples directly obtained or taken from the subjects of said
group or population, or by mixing equal amounts of fractions,
constituents or reaction products (e.g., enzymatic reaction
products or precipitates) thereof.
[0088] As used herein a control sample corresponds to the subject's
sample if it has been obtained from the same type of biological
tissue or source in the same, or essentially the same, manner. For
example, if the subject's sample is a whole blood, blood plasma,
blood serum sample or urine, or a fraction thereof, a corresponding
control sample will likewise be a whole blood, blood plasma, blood
serum sample, or urine, or a fraction thereof, respectively. It
will be appreciated that such corresponding control sample would
include whole blood, blood plasma, blood serum samples, or urine,
or a fraction thereof, obtained by mixing the whole blood, blood
plasma, blood serum, or urine samples, or certain fractions
thereof, from a group or population of subjects (see also the
further explanations herein and the claims regarding suitable
control samples in accordance with the invention). The same applies
mutatis mutandis to, e.g., tissue samples.
[0089] A lipid as used herein is defined as hydrophobic or
amphiphilic small molecule.
[0090] For the purposes of the present invention, lipids are
referred to according to the following nomenclature: CE is
cholesteryl ester, Cer is ceramide, DAG is diacylglycerol, PC O is
ether-linked PC, Gb is Globotriaosylceramide, GD is
disialogangliosides, Glc/GalCer is galactosyl- or
glucosylceramides, GM is monosialogangliosides, LacCer is
lactosylceramides, LPC is lysophosphatidylcholine, PC is
phosphatidylcholine, PE is phosphatidylethanolamine, PI is
phosphatidylinositol, SM is sphingomyelin, S1P is
sphingosine-1-phosphate, HETE is hydroxyeicosatetraenoic acid, HEPE
is hydroxyeicosapentaenoic acid, DHET is dihydroxyeicosatrienoic
acid, PGE is prostaglandin E and AA is arachidonic acid.
[0091] The nomenclature X:Y indicates, X number of total carbon
atoms in the fatty acid(s) portions of the molecule, and Y the
total number of double bonds in the fatty acid portion(s) of the
molecule.
[0092] The nomenclature A/B indicates, for a molecule of DAG and
PC, A and B types of fatty acid moieties attached to the glycerol
backbone of the molecule.
[0093] The nomenclature (dC/A) indicates, for a molecule of Cer,
Gb, GlcCer, LacCer and SM, C the type of long-chain base with an
amide-linked, A, fatty acid moiety.
[0094] 15-HETE is formally known as
(.+-.)15-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid (CAS:
73836-87-0) and is referred to in Lehmann, W. D., Metzger, K.,
Stephan, M., et al. Quantitative lipoxygenase product profiling by
gas chromatography negative-ion chemical ionization mass
spectrometry. Anal Biochem 224 227-234 (1995) and Zijlstra, F. J.,
van Dijk, A. P. M., Wilson, J. H. P., et al. 15-HETE is the main
eicosanoid formed by human colonic mucosa. Agents Actions C53-C59
(1992). 12-HETE is formally known as
(.+-.)12-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (CAS:
71030-37-0) and is referred to in Lehmann, W. D., Metzger, K.,
Stephan, M., et al. Quantitative lipoxygenase product profiling by
gas chromatography negative-ion chemical ionization mass
spectrometry. Anal Biochem 224 227-234 (1995). O'Flaherty, J. T.,
Thomas, M. J., Lees, C. J., et al. Neutrophil-aggregating activity
of monohydroxyeicosatetraenoic acids. Am J Pathol 104 55-62 (1981).
LacCer(d18:1/22:0) is formally known as
N-(docosanoyl)-1-b-lactosyl-sphing-4-enine and belongs to the same
family as LacCer(d18:1/24:0) (CAS number is 105087-85-2). Gb3 is
formally known as ceramide trihexoside (CAS: 71965-57-6) and is
referred in Groener J E, Poorthuis B J, Kuiper S, Helmond M T,
Hollak C E, Aerts J M. HPLC for simultaneous quantification of
total ceramide, glucosylceramide, and ceramide trihexoside
concentrations in plasma. Clin Chem. 2007 April; 53(4):742-7 and
Mills K., Johnson A., Winchester B. Synthesis of novel internal
standards for the quantitative determination of plasma ceramide
trihexoside in Fabry disease by tandem mass spectrometry. FEBS
Lett. 2002 Mar. 27; 515 (1-3):171-6. 12-HEPE is formally known as
(.+-.)-12-hydroxy-5Z,8Z,10E,14Z,17Z-eicosapentaenoic acid (CAS:
81187-21-5) and is referred in Karanian, J. W., Kim, H. Y., and
Salem, N. Inhibitory effects of n-6 and n-3 hydroxy fatty acids on
thromboxane (U46619)-induced smooth muscle contraction. J Pharmacol
Exp Ther 270 1105-1109 (1994) and Takenaga, M., Hirai, A., Terano,
T., et al. Comparison of the in vitro effect of eicosapentaenoic
acid (EPA)-derived lipoxygenase metabolites on human platelet
function with those of arachidonic acid. Thromb Res 37 373-384
(1986).
[0095] An agent capable of binding to any one of the lipidomic
markers of the invention may be a small molecule (i.e., a molecule
having a molecular weight of less than 5 kDa, and more typically
less than 1 kDa, which may be a protein or a peptide sequence, or a
member of any of a wide variety of organics, e.g., a carbohydrate,
a sugar, a drug, an alcohol, a carboxylic acid, an amine, an
aldehyde or a ketone, a thiol, a cyclic or an acyclic compound), a
nucleic acid (e.g., an aptamer), a carbohydrate, a protein or
peptide, or a proteoglycan. In a preferred embodiment, the agent is
an antibody.
[0096] As used herein, the term antibody includes monoclonal and
polyclonal antibodies, whole antibodies, antibody fragments, and
antibody sub-fragments that exhibit specific binding to a said
lipid. Thus, suitable antibodies can be whole immunoglobulins of
any class, e.g., IgG, IgM, IgA, IgD, IgE, chimeric antibodies or
hybrid antibodies with dual or multiple antigen or epitope
specificities, or fragments, e.g., F(ab').sub.2, Fab', Fab and the
like, including hybrid fragments, and additionally includes any
immunoglobulin or any natural, synthetic or genetically engineered
protein that acts like an antibody by binding to a specific antigen
to form a complex. The term antibody encompasses antigen-binding
fragments of antibodies (e.g., single chain antibodies, Fab
fragments, F(ab').sub.2, a Fd fragment, a Fv fragment and dAb
fragments) as well as complete antibodies. For example, Fab
molecules can be expressed and assembled in a genetically
transformed host like E. coli. A lambda vector system is available
thus to express a population of Fab's with a potential diversity
equal to or exceeding that of subject generating the predecessor
antibody. See Huse W D, et al., Science 1989, 246:1275-81. Such
Fab's are included in the definition of antibody. The ability of a
given molecule, including an antibody fragment or sub-fragment, to
act like an antibody and specifically bind to a specific antigen
can be determined by binding assays known in the art, for example,
using the antigen of interest as the binding partner.
[0097] Antibodies against lipids in accordance with the present
invention may be prepared by methods well known to those skilled in
the art. For example, mice may be immunized with a lipid with
adjuvant.
[0098] Splenocytes are harvested as a pool from the mice that were
administered 3 immunizations at 2-week intervals with test bleeds
performed on alternate weeks for serum antibody titers. Splenocytes
are prepared as 3 aliquots that are either used immediately in
fusion experiments or stored in liquid nitrogen for use in future
fusions.
[0099] Fusion experiments are then performed according to the
procedure of Stewart & Fuller, J. Immunol. Methods 1989,
123:45-53. Supernatants from wells with growing hybrids are
screened by enzyme-linked immunosorbent assay (ELISA) for
monoclonal antibody (MAb) secretors on 96-well ELISA plates coated
with the said lipid. ELISA positive cultures are cloned by limiting
dilutions, typically resulting in hybridomas established from
single colonies after 2 serial cloning experiments.
EXAMPLES
Example 1
[0100] Materials and Methods
[0101] For this study the subjects were selected from a cohort of
patients presenting clear muscular intolerance phenotypes
determined according to strict criteria.
[0102] The inclusion criteria for the subjects were the following:
[0103] Written informed consent to participate in the study; [0104]
Men or women aged 18 years or older; [0105] Documentation of
statin-related muscle toxicity manifested by either: [0106] muscle
pain that occurs during statin treatment and stops after withdrawal
or reduction in dosage; or [0107] muscle pain that starts after
initiation of statin treatment and persists while still being
treated in patients in whom it is considered not possible to stop
statin administration; or [0108] muscle pain that occurs while
patient is being treated with a statin and clearly appears to be
statin-related in the opinion of his/her physician; or [0109]
patient in whom lipid-lowering regimen is changed from a statin to
ezetimibe because of intolerance to statins due to muscle pain or
weakness, myopathy or rhabdomyolysis; or [0110] elevation in plasma
CK level more than 1.5 times the upper limit of normal while being
treated with a statin, in the absence of other causes to explain
the abnormality; or [0111] presence of myoglobinuria or
myoglobinemia while being treated with a statin, in the absence of
other causes to explain the abnormality; [0112] clinical diagnosis
of rhabdomyolysis while being treated with a statin, in the absence
of other responsible causes.
[0113] Exclusion Criteria for the subjects were the following:
[0114] patient in whom muscle pain is not clearly associated with
the use of a statin in the physician's judgment; [0115]
Hypothyroidism that is not controlled with a stable dose of
supplement for at least the last 3 months and that occurred during
muscle toxicity; [0116] Known hyperthyroidism in the last year and
that occurred during muscle toxicity; [0117] History of alcohol or
drug abuse in the last year and that occurred during muscle
toxicity; [0118] Known renal insufficiency (not secondary to
rhabdomyolysis) with serum creatinine level of 200 .mu.mol/L or
more at the time of muscle toxicity; [0119] Known severe liver
disease with cirrhosis, biliary obstruction, acute or chronic
infectious hepatitis at the time of the muscle toxicity; [0120]
Known hereditary or acquired muscle disease; [0121] Any medical or
psychiatric condition that may make the patient an unsuitable
candidate for the study in the physician's opinion. [0122]
Participation in any other investigational drug study within 30
days of recruitment.
[0123] Inclusion Criteria for the controls were the following:
[0124] Written informed consent to participate in the study [0125]
Men or women aged 18 years or older; [0126] Known dyslipidemia
treated with a stable dose of a statin for at least 3 months;
[0127] Absence of current or past statin-related side effects.
[0128] Exclusion Criteria for the controls were the following:
[0129] Hypothyroidism that is not controlled with a stable dose of
supplement for at least the last 3 months unless the absence of
muscle toxicity due to statins has been confirmed before condition;
[0130] Known hyperthyroidism in the last year unless the absence of
muscle toxicity due to statins has been confirmed before condition
; [0131] History of alcohol or drug abuse in the last year unless
the absence of muscle toxicity due to statins has been confirmed
before condition; [0132] Known renal insufficiency with serum
creatinine level of 200 .mu.mol/L or more at the time of
recruitment unless the absence of muscle toxicity due to statins
has been confirmed before condition; [0133] Known severe liver
disease with cirrhosis, biliary obstruction, acute or chronic
infectious hepatitis at the time of recruitment unless the absence
of muscle toxicity due to statins has been confirmed before
condition; [0134] Known hereditary or acquired muscle disease;
[0135] Any medical or psychiatric condition that may make the
patient an unsuitable candidate for the study in the physician's
opinion. [0136] Participation in any other investigational drug
study within 30 days of recruitment.
TABLE-US-00002 [0136] TABLE 2 Background characteristics for statin
myopathy patients analyzed with Lipidomics Atorvastatin equivalent
N = dose Age CK Controls male 92 35 63.5 96.3 Controls female 58 28
64.3 87.8 Cases male, CK <200 U/l 50 28 64.1 98.5 Cases male, CK
>200 U/l 42 37 60 271 Cases female, CK <200 U/l 50 25 62 76
Cases female, CK >200 U/l 8 26 64 267 Total number of subjects
300
[0137] Analytical Methods
[0138] Mass Spectrometry Driven Lipidomics
[0139] Direct infusion coupled to tandem mass spectrometry, i.e.
shotgun lipidomics, and two liquid chromatography tandem mass
spectrometry (LC-MS/MS) approaches, i.e. ceramide and cerebroside
lipidomics and eicosanoid lipidomics, were used to identify
statin-induced muscle toxicity by analyzing molecular lipid species
in human plasma. The applied methods were optimized especially for
quantification of molecular cholesteryl esters (CE),
phosphatidylcholines (PC), lysophosphatidylcholines (LPC) and other
lysophospholipids (LPL), ether-linked phosphatidylcholines (PC O)
and other ether-linked phospholipids (PL O), phosphatidylserines
(PS), phosphatidylethanolamines (PE), phosphatidylglycerols (PG),
phosphatidylinositols (PI), phosphatidic acids (PA),
diacylglycerols (DAG), ceramides (Cer), glucosylceramides (GlcCer),
lactosylceramides (LacCer), globotriaosylceramides (Gb), free fatty
acids (FFA) and eicosanoids.
[0140] The following materials were used according to the methods.
HPLC or LC-MS grade of chloroform, methanol, water, acetonitrile,
formic acid, methanol, isopropanol, ammonium acetate, acetic acid,
potassium chloride and butylated hydroxytoluene (BHT) were
purchased from Sigma-Aldrich (St. Louis, Mo., USA).
[0141] HPLC column (Acquity BEH C18, 2.1.times.50 mm id. 1.7 .mu.m)
was purchased from Waters (Milford, Mass., USA). HPLC pre-column
(Widepore C18 4.times.2.0 mm) was purchased from Phenomenex
(Torrance, Calif., USA). All labware used for the extraction were
resistant to chloroform. Aerosol resistant filter tips (Molecular
BioProducts) and Eppendorf 2 ml safe-lock tubes, 96-well twin.tec
PCR plates, and Pierce-it-lite thermo-sealing foils were purchased
from VWR International (West Chester, Pa., USA). CO-RE Filter Tips
and 96-well 2 ml Whatman Uniplates were purchased from Hamilton
Robotics (Bonaduz, Switzerland). Synthetic lipid standards were
purchased from Avanti Polar Lipids (Alabaster, Ala., USA), Matreya
(Pleasant Gap, Pa., USA), and Cayman Chemical (Ann Arbor, Mich.,
USA).
[0142] Lipids were extracted in chloroform:methanol according to
the following protocols. Samples were spiked with known amounts of
non-endogenous synthetic internal standards for data normalization
and endogenous lipid quantification. Post-extract spiked
non-endogenous synthetic external standards were used for quality
controlling. Stock solutions of standards were prepared by
dissolving appropriately weighed amounts of each standard in
chloroform:methanol (2:1, v/v) to achieve a final concentration of
500 .mu.M. An internal standard mixture containing each of the
standard stock was created and used in lipid extraction.
[0143] 5 .mu.l of plasma was used for shotgun lipidomics and 10
.mu.l of plasma for ceramide and cerebroside lipidomics Lipid
extractions were carried out in automated fashion using a Hamilton
MICROLAB STAR system (Hamilton Robotics, Switzerland). Well-mixed
samples were aliquoted into a 96-well 2 ml Whatman Uniplate
containing ice-cold methanol and 0.1% BHT. The samples were mixed
thoroughly after each step in the extraction protocol. The
extraction proceeded at room temperature by adding an appropriate
volume of internal standard mixture and chloroform and methanol. In
shotgun and ceramide and cerebroside lipidomics, the organic phase
separation was facilitated by adding 20 mM acetic acid and
centrifuging the plate for 5 min at 500.times.g. The organic phase
was transferred into a new 96-well 2 ml Whatman Uniplate. The
remaining water-containing phase was washed by adding appropriate
volume of chloroform followed by centrifugation. The two organic
phases were pooled and evaporated under N.sub.2 until dryness. The
lipid extracts were then re-dissolved in chloroform:methanol (1:2,
v/v) including the addition of the synthetic external standard. The
extracts were stored in 2 ml safe-lock Eppendorf tubes at
-20.degree. C. prior to MS analysis. Required volumes of lipid
extracts were aliquoted into an Eppendorf 96-well twin.tec PCR
plate and the plate was heat-sealed with aluminum foil to avoid
evaporation.
[0144] In shotgun lipidomics, lipid extracts were analyzed on a
hybrid triple quadrupole/linear ion trap mass spectrometer (QTRAP
5500, AB Sciex) equipped with a robotic nanoflow ion source
(NanoMate HD, Advion Biosciences). The instruments were operated in
positive and negative ion modes. In positive ion the spray voltage
was set to 1.0 to 1.4 kV and in negative ion mode to -1.0 to -1.4
kV. A gas pressure of 0.3-0.8 psi was used and the interface heater
was set at 60.degree. C. The collision energy (CE) and declustering
potential (DP) was optimized for each lipid class using synthetic
standards. The mass spectrometer was operated in unit resolution
mode using a scan speed of 200 Da/s. Molecular lipids were analyzed
in both positive and negative ion modes using multiple precursor
ion scanning (MPIS) and neutral loss scanning (NLS) as described by
Stahlman and colleagues (Stahlman M, et al: High-throughput shotgun
lipidomics by quadrupole time-of-flight mass spectrometry. J
Chromatogr B Analyt Technol Biomed Life Sci 2009).
[0145] In ceramide and cerebroside lipidomics, the high performance
liquid chromatography (HPLC) analyses were conducted in the
following way. Chromatographic apparatus consisted of a CTC HTC PAL
autosampler (CTC Analytics AG, Switzerland), a Rheos Allegro UHPLC
pump (Flux Instruments AG, Switzerland), an external column heater
set to 60.degree. C. for ceramide and cerebroside lipidomics and
the Acquity BEH C18 column with an in-line pre-column. The
extracted samples, 10 .mu.l of each, were injected into the
pre-column followed by the analytical column and delivered to the
mass spectrometer at a flow rate of 500 .mu.l/min. In ceramide and
cerebroside lipidomics, A gradient was used for lipid analyte
separation with solvent A comprising 10 mM ammonium acetate in HPLC
grade water containing 0.1% formic acid and solvent B of 10 mM
ammonium acetate in acetonitrile:isopropanol (4:3, v/v) containing
0.1% formic acid. The gradient was constructed in the following
way: 0 min-65% B; 2 min-65% B; 2.5 min-75% B; 17.5 min-100% B; 22.5
min-100% B; 22.6 min-65% B; 25 min-65% B.
[0146] The lipid extracts were analyzed by HPLC-MS/MS. The MS
analysis was performed on a hybrid triple quadrupole/linear ion
trap mass spectrometer equipped with the Turbo V.TM. Ion Source
(4000 QTRAP, AB Sciex). The instrument was operating in positive
ion mode. The ion source voltage was set to 5500V for ceramide and
cerebroside lipidomics and to -4500V for ganglioside lipidomics,
and source temperature at 400.degree. C. The collision energy (CE)
and declustering potential (DP) was optimized for each lipid class
using synthetic standards. A 20 sec dwell time was applied for each
scan. Multiple reaction monitoring (MRM) scan mode was applied and
based on the description by Sullards and colleagues (Sullards M C,
et al: Structure-specific, quantitative methods for analysis of
sphingolipids by liquid chromatography-tandem mass spectrometry:
"inside-out" sphingolipidomics. Methods Enzymol 2007).
[0147] Eicosanoids were extracted using solid phase extraction
(SPE). 150 .mu.l plasma was extracted with 10% methanol containing
0.1% of butylated hydroxytoluene (BHT). Samples were spiked with
known amounts of non-endogenous synthetic internal standards for
data normalization and endogenous lipid quantification. An internal
standard mixture containing each of the standard stock was created
and used in lipid extraction. Strata-X 33 um SPE cartridges were
conditioned with HPLC grade methanol followed by a conditioning
step with ultra pure water (UPW). Samples were loaded onto the SPE
followed by a wash step using 35% methanol. Eicosanoids were eluted
with acetonitrile and the sample elutes were dried down under
nitrogen. The final sample extracts were reconstituted in methanol
and directly analyzed by mass spectrometry.
[0148] In the analysis for Eicosanoids, high performance liquid
chromatography (HPLC) analyses were conducted in the following way:
Chromatographic apparatus consisted of a CTC HTC PAL autosampler
(CTC Analytics AG, Switzerland), a Rheos Allegro UHPLC pump (Flux
Instruments AG, Switzerland), an external column heater set to
45.degree. C. and switching valve (Valco Instruments Co. Inc. and
VICI AG, Huston, USA). Separation was carried out using a
Phenomenex Jupiter, 250.times.2.0 mm id. 5 .mu.m HPLC column
(Phenomenex, Inc, Torrance, Calif.). The extracted samples, 10
.mu.l of each, were injected into the analytical column and
delivered to the mass spectrometer at a flow rate of 300 .mu.l/min.
A gradient was used for lipid analyte separation with solvent A
comprising of acetonitrile:water (63:37 (v/v)) containing 0.1%
formic acid and solvent B of acetonitrile: isopropanol (50:50
(v/v)). The gradient was constructed in the following way: 0 min-0%
B; 6 min-20% B; 6.50 min-55% B; 10.0 min-55% B; 12.0 min-100% B;
14.0 min-100% B; 14.50 min-0% B; 18.0 min-0% B.
[0149] The lipid extracts were analyzed by HPLC-MS/MS. The MS
analysis was performed on a hybrid triple quadrupole/linear ion
trap mass spectrometer equipped with the Turbo V.TM. Ion Source
(4000 QTRAP, AB Sciex). The instrument was operating in negative
ion mode and the ion source voltage was set to -4500V. The
collision energy (CE) and declustering potential (DP) was optimized
for each lipid class using synthetic standards where available.
Multiple reaction monitoring (MRM) scan mode was applied and based
on the description by Deems and colleagues (Deems, R., et al:
Detection and quantitation of eicosanoids via high performance
liquid chromatography-electrospray ionization-mass spectrometry.
Methods Enzymol 2007).
[0150] The data processing was done in the following way: Initially
the retention time (in LC mode) and identification of each peak was
done using endogenous standards and by Information Dependent
Acquisition (IDA) experiments where applicable. The raw data were
processed according to peak detected and retention time (in LC
mode) in automated fashion. A stringent cutoff was applied for
separating background noise from actual lipid peaks. Each sample
was controlled and only accepted when fulfilling the stringent
acceptance criteria. Peak area counts (cps) of detected peaks were
converted into a list of corresponding lipid names. Lipids were
normalized to their respective internal standard and sample volume
to retrieve their concentrations.
[0151] The ratio of synthetic Internal Standards (IS) to
corresponding post-extract spiked External Standards (ES), and MS
analysis of extracted matrix and solvents served as quality
controls (QC) of the analysis. In addition, extracted reference
plasma samples were analyzed for monitoring the instruments'
performance, i.e., the intra- and inter-assay variation.
[0152] A calibration line using synthetic or isolated standards was
obtained prior to sample analysis. Synthetic standards were chosen
based on application and had similar properties to the endogenous
lipids or analyte(s) of interest. The calibration line consisted of
a minimum of five standards points covering the expected
quantification range. The calibration line was used to determine
the dynamic quantification range for each lipid class monitored,
e.g., the linear quantification limits. As the internal standards
used behave in the same way as endogenous lipids they were used for
quantifying endogenous lipid species. The calibration lines were
based on the same internal standards that were used for
quantification of the endogenous lipids.
[0153] For each platform, a stringent cutoff was applied for
separating background noise from actual lipid peaks. Each sample
was controlled and only accepted when fulfilling the acceptance
criteria. Masses and counts of detected peaks were converted into a
list of corresponding lipid names. Lipids were normalized to their
respective internal standard and sample volume to retrieve their
concentrations.
[0154] Statistical Analyses
[0155] Percentage changes in lipid concentrations between control
and case groups were calculated as follows:
100*(AVG[C] in case group-AVG[C] in control group)/AVG[C] in
control group.
[0156] Statistical significance was assigned based on two
independent samples t-test and Mann-Whitney U-test p-values.
[0157] In addition, ROC curves were used for finding lipid
molecules and concentration cutoffs that separate the best cases
from controls. Sensitivity is calculated as a number of correctly
identified cases divided by the total number of cases. Specificity
is calculated as a number of correctly identified controls divided
by the total number of controls. Sensitivity and specificity was
calculated for each lipid concentration. Significant biomarkers
were defined as those molecules that have a t-test based p-value of
0.05 or Sensitivity>=60% and Specificity=>40%. Gender groups
were also analyzed separately in order to avoid any unexpected
gender specific results as men are generally more muscular and
physically active.
[0158] Results
[0159] In the study sample group the creatine kinase levels were
practically identical in controls and cases, therefore this
traditionally used enzyme marker was not predictive or diagnostic
for statin-induced myopathy.
[0160] On the other hand, lipidomic biomarkers appeared as
significant biomarkers of the statin-induced myopathy. A total of
290 molecular lipids were quantified in this study as described
above. Out of those 20 molecular lipids were significant biomarkers
based on set statistical criteria. The significant biomarker
candidates based on molecular lipid concentrations are presented in
Table 3.
[0161] The preferred embodiments selected among the identified
biomarker candidates are listed in Table 5.
TABLE-US-00003 TABLE 3 Significant biomarkers based on individual
lipid or fatty acid measurement. Species names, p-values,
percentage change, auc-values; and specificity and sensitivity are
presented. Percentage Measurement Name Change p-value AUC
Sensitivity Specificity Increased 12-HETE 460.03 0.0001 0.77 71.43
62.79 LacCer(d18:1/22:0) 65.01 0.0002 0.72 69.39 60.42 15-HETE
49.64 0.0150 0.63 63.64 60.61 Gb3(d18:1/24:1) 43.93 4.18 .times.
10.sup.-6 0.78 85.71 61.90 Gb3(d18:1/22:0) 43.21 0.0378 0.78 87.50
62.50 Gb3(d18:1/24:0) 40.21 0.0295 0.80 87.50 62.50
LacCer(d18:1/24:0) 31.81 0.0110 0.61 AA 30.59 0.0005 0.72 77.55
60.00 Total eicosanoids 24.18 0.0023 0.70 73.47 60.00 Total Gb3
23.40 0.0006 0.69 64.29 71.43 Cer(d18:1/20:0) 20.96 0.0145 0.62
Gb3(d18:1/20:0) 20.22 0.0219 0.65 66.67 61.29 LacCer(d18:1/24:1)
14.52 0.0213 0.62 Decreased Gb3(d18:1/16:0) -7.73 0.0220 0.61 Total
LacCer* -10.10 0.0347 0.64 64.00 68.00 LacCer(d18:1/16:0)* -11.70
0.0168 0.66 66.00 60.00 Gb3(d18:1/18:0) -14.34 0.0145 0.65 67.35
60.42 14_15-DHET -16.24 0.0468 0.60 Glc/GalCer(d18:1/24:1)* -17.09
0.0050 0.66 70.00 68.00 8_9-DHET -22.67 0.0394 0.64 60.00 68.18
*The markers are specific for females. Other markers are not gender
specific.
[0162] Lipid measurements that were below a detection limit in at
least 25% of control or case samples were converted into
dichotomous variables where 0 indicates that a lipid is absent and
1 indicated that a lipid is present in a sample. Next, we counted
how many times a given lipid was present in controls and cases and
significance of the fact that lipids were missing/present in
controls comparing to cases was evaluated using Fisher exact test.
Table 4 shows the potential biomarkers generated with Fisher
test.
TABLE-US-00004 TABLE 4 Significant markers generated by Fisher test
Control Case Direction of LIPID_NAME absent present absent present
Fisher p-value change LacCer (d18:1/20:0) 32 18 20 30 0.02718
Increased 12-HEPE 36 14 13 37 7.70E-06 Increased PGE2 47 3 37 13
0.01222 Increased 12-OXOETE 28 12 12 28 0.000695 Increased 17-HDoHE
31 9 19 21 0.010492 Increased PGD2 39 1 29 11 0.003255 Increased
TXB3 38 2 19 21 0.000003 Increased PS 18:0/18:1 34 6 25 15 0.040609
Increased SM (d18:1/24:2) 30 10 20 20 0.036835 Increased
TABLE-US-00005 TABLE 5 Preferred embodiments of biomarkers
Percentage Spe- Measurement Name Change p-value AUC Sensitivity
cificity Increased 12-HETE 460.03 0.0001 0.77 71.43 62.79
LacCer(d18:1/22:0) 65.01 0.0002 0.72 69.39 60.42 Gb3(d18:1/24:1)
43.93 0.0000 0.78 85.71 61.90 Decreased LacCer(d18:1/16:0)* -11.70
0.0168 0.66 66.00 60.00 Glc/ -17.09 0.0050 0.66 70.00 68.00
GalCer(d18:1/24:1)* *The markers are specific for females. Other
markers are not gender specific.
[0163] Furthermore, the significance of lipid-lipid concentration
ratios for identifying the statin-induced myopathy was
investigated. The lipid-lipid concentration ratios which showed
improved AUC values over individual lipids are shown in Table 6.
Table 6 shows that, in addition to the individual lipid
measurements, the lipid-lipid concentration ratios can be used as
biomarkers to identify the statin-induced myopathy or for
determining whether the statin treatment or the treatment with a
lipid lowering drug of a subject needs adjustment.
TABLE-US-00006 TABLE 6 Example on the lipid-lipid concentration
ratios and their significance Percentage Measurement name AUC
Sensitivity Specificity p-value change Increased 12-HETE/15-HETrE
0.83 83.3 61.9 0.0000195733 635.0 12-HETE/14_15-DHET 0.79 73.5 62.8
0.0000278151 587.8 12- 0.80 70.8 71.4 0.0002342687 494.8
HETE/Gb3(d18:1/16:0) 12- 0.78 70.8 71.4 0.0003976485 484.5
HETE/Glc/GalCer(d18:1/ 24:1) 12- 0.78 70.8 76.2 0.0002044722 477.8
HETE/Glc/GalCer(d18:1/ 18:0) Gb3(d18:1/24:1)/LacCer 0.82 83.3 61.9
0.0000042133 54.4 (d18:1/16:0) LacCer(d18:1/22:0)/Lac 0.71 74.0
60.0 0.0004157530 26.1 Cer(d18:1/24:1) Cer(d18:1/20:0)/Glc/Gal 0.72
74.0 62.0 0.0006147547 25.8 Cer(d18:1/24:1) Decreased
LacCer(d18:1/16:0)/Lac 0.69 74.0 66.0 0.0009722258 -20.9
Cer(d18:1/22:0) Gb3(d18:1/16:0)/Gb3(d18:1/ 0.80 83.3 61.9
0.0000005500 -23.9 24:1) 11-HETE/12-HETE 0.85 87.8 65.1
0.0000000008 -59.1
[0164] In summary, this study provides novel lipid markers of
statin-induced muscle toxicity. Since the creatine kinase levels in
the study sample group were practically identical in controls and
cases (Table 2), the lipidomic biomarkers were more specific and
sensitive markers of the statin-induced muscle toxicity.
* * * * *
References