U.S. patent application number 15/053189 was filed with the patent office on 2016-06-16 for means and methods for assessing increased peroxisomal proliferation.
This patent application is currently assigned to BASF SE. The applicant listed for this patent is Georgia Coelho Palermo Cunha, Eric Fabian, Michael Herold, Hennicke Kamp, Edgar Leibold, Ralf Looser, Werner Mellert, Alexander Prokudin, Volker Strasse, Bennard van Ravenzwaay, Tilmann Walk, Jan C Wiemer. Invention is credited to Georgia Coelho Palermo Cunha, Eric Fabian, Michael Herold, Hennicke Kamp, Edgar Leibold, Ralf Looser, Werner Mellert, Alexander Prokudin, Volker Strasse, Bennard van Ravenzwaay, Tilmann Walk, Jan C Wiemer.
Application Number | 20160169863 15/053189 |
Document ID | / |
Family ID | 41213166 |
Filed Date | 2016-06-16 |
United States Patent
Application |
20160169863 |
Kind Code |
A1 |
van Ravenzwaay; Bennard ; et
al. |
June 16, 2016 |
Means and Methods for Assessing Increased Peroxisomal
Proliferation
Abstract
The present invention pertains to the field of toxicological
assessments for risk stratification of chemical compounds.
Specifically, it relates to a method for diagnosing increased
peroxisomal proliferation. It also relates to a method of
determining whether a compound is capable of inducing such
peroxisomal proliferation in a subject and to a method of
identifying a drug for treating increased peroxisomal
proliferation. Furthermore, the present invention relates to a data
collection comprising characteristic values of at least five
metabolites, a data storage medium comprising said data collection,
and a system and a device for diagnosing increased peroxisomal
proliferation. Finally, the present invention pertains to the use
of a group of metabolites or means for the determination thereof
for the manufacture of a diagnostic device or composition for
diagnosing increased peroxisomal proliferation in a subject.
Inventors: |
van Ravenzwaay; Bennard;
(Altrip, DE) ; Mellert; Werner; (Hassloch, DE)
; Fabian; Eric; (Speyer, DE) ; Strasse;
Volker; (Bad Duerkheim, DE) ; Walk; Tilmann;
(Kleinmachnow, DE) ; Looser; Ralf; (Berlin,
DE) ; Leibold; Edgar; (Carlsberg, DE) ; Kamp;
Hennicke; (Bischheim, DE) ; Coelho Palermo Cunha;
Georgia; (Sao Paulo, BR) ; Herold; Michael;
(Berlin, DE) ; Wiemer; Jan C; (Berlin, DE)
; Prokudin; Alexander; (Berlin, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
van Ravenzwaay; Bennard
Mellert; Werner
Fabian; Eric
Strasse; Volker
Walk; Tilmann
Looser; Ralf
Leibold; Edgar
Kamp; Hennicke
Coelho Palermo Cunha; Georgia
Herold; Michael
Wiemer; Jan C
Prokudin; Alexander |
Altrip
Hassloch
Speyer
Bad Duerkheim
Kleinmachnow
Berlin
Carlsberg
Bischheim
Sao Paulo
Berlin
Berlin
Berlin |
|
DE
DE
DE
DE
DE
DE
DE
DE
BR
DE
DE
DE |
|
|
Assignee: |
BASF SE
Ludwigshafen
DE
|
Family ID: |
41213166 |
Appl. No.: |
15/053189 |
Filed: |
February 25, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
14152411 |
Jan 10, 2014 |
9304136 |
|
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15053189 |
|
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12994705 |
Feb 11, 2011 |
8658427 |
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PCT/EP2009/056386 |
May 26, 2009 |
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14152411 |
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Current U.S.
Class: |
506/12 |
Current CPC
Class: |
G01N 2405/00 20130101;
G01N 2800/046 20130101; G01N 33/92 20130101; G01N 2500/00 20130101;
G01N 2800/24 20130101; Y10T 436/24 20150115; G01N 2800/60 20130101;
G01N 33/487 20130101; G01N 33/68 20130101; Y10T 436/200833
20150115; G01N 33/82 20130101; G01N 33/6812 20130101; H01J 49/0036
20130101; G01N 2800/10 20130101; G01N 33/50 20130101 |
International
Class: |
G01N 33/487 20060101
G01N033/487; H01J 49/00 20060101 H01J049/00 |
Foreign Application Data
Date |
Code |
Application Number |
May 28, 2008 |
EP |
08157118.4 |
Jan 28, 2009 |
EP |
09151549.4 |
Claims
1. A method for diagnosing a disorder associated with increased
peroxisomal proliferation comprising: (a) selecting a male or
female subject suspected to suffer from increased peroxisomal
proliferation; (b) obtaining a body fluid test sample from the male
or female subject; (c) determining the amount of at least five of
the following analytes Coenzyme Q10, 16-Methylheptadecanoic acid,
17-Methyloctadecanoic acid, Eicosatrienoic acid (C20:3), Threonine,
Proline, Tyrosine, trans-4-Hydroxyproline from the body fluid test
sample from the male subject, or the amount of at least five of the
following analytes Pantothenic acid, Coenzyme Q9, Glycerol,
Palmitic acid (C16:0), Linoleic acid (C18:cis[9,12]2),
14-Methylhexadecanoic acid, gamma-Linolenic acid
(C18:cis[6,9,12]3), 16-Methylheptadecanoic acid, Threonic acid,
Cytosine, Phosphatidylcholine (C18:0/C22:6) from the body fluid
test sample from the female subject; (d) comparing the amounts
determined in step (c) to corresponding reference results, wherein
the reference results comprise (i) reference results obtained from
one or more samples derived from one or more subjects which has
been brought into contact with Benzylbutyl Phthalate, Fenofibrate,
Clofibrate, Fenoforbrate, Diethylhexylphthalate or Wy 14643, (ii)
reference results obtained from one or more samples derived from
one or more subjects which has not been brought into contact with
Benzylbutyl Phthalate, Fenofibrate, Clofibrate, Fenoforbrate,
Diethylhexylphthalate or Wy 14643, (iii) reference results obtained
from one or more samples derived from one or more subjects which
suffers from increased peroxisomal proliferation, or (iv) reference
results obtained from one or more samples derived from one or more
subjects known to not suffer from increased peroxisomal
proliferation; and (e) based on the comparison of step (d),
diagnose the disorder by monitoring, confirmation, or
classification of the disorder or its symptoms in the male or
female subject.
2. The method of claim 1, wherein said male or female subject has
been brought into contact with a compound suspected to be capable
of inducing increased peroxisomal proliferation.
3. The method of claim 2, wherein said compound is at least one
compound selected from the group consisting of: Benzylbutyl
Phthalate, Fenofibrate, Clofibrate, Fenoforbrate,
Diethylhexylphthalate, and Wy 14643.
4. The method of claim 1, wherein said reference result is obtained
from one or more samples derived from (i) one or more subjects
which suffers from increased peroxisomal proliferation; or (ii) one
or more subjects which has been brought into contact with at least
one compound selected from the group consisting of: Benzylbutyl
Phthalate, Fenofibrate, Clofibrate, Fenoforbrate,
Diethylhexylphthalate, and Wy 14643.
5. The method of claim 4, wherein identical amounts for the
analytes in the body fluid test sample and the reference result are
indicative for increased peroxisomal proliferation.
6. The method of claim 1, wherein said reference result is obtained
from one or more samples derived from (i) one or more subjects
known to not suffer from increased peroxisomal proliferation; or
(ii) one or more subjects which has not been brought into contact
with at least one compound selected from the group consisting of:
Benzylbutyl Phthalate, Fenofibrate, Clofibrate, Fenoforbrate,
Diethylhexylphthalate, and Wy 14643.
7. The method of claim 6, wherein amounts for the analytes which
differ in the body fluid test sample in comparison to the reference
result are indicative for increased peroxisomal proliferation.
8. The method of claim 1, wherein indicative for increased
peroxisomal proliferation are amounts of the analytes in comparison
to the reference result of one or more subjects known to not suffer
from increased peroxisomal proliferation which differ as follows:
(i) in a body fluid sample of a male: Coenzyme Q10 decreased,
16-Methylheptadecanoic acid decreased, 17-Methyloctadecanoic acid
decreased, Eicosatrienoic acid (C20:3) increased, Threonine
decreased, Proline decreased, Tyrosine decreased,
trans-4-Hydroxyproline decreased; and (ii) in a body fluid sample
of a female subject: Pantothenic acid increased, Coenzyme Q9
increased, Glycerol increased, Palmitic acid (C16:0) increased,
Linoleic acid (C18:cis[9,12]2) increased, 14-Methylhexadecanoic
acid increased, gamma-Linolenic acid (C18:cis[6,9,12]3) decreased,
16-Methylheptadecanoic acid decreased, Threonic acid increased,
Cytosine decreased, Phosphatidylcholine (C18:0/C22:6)
decreased.
9. The method of claim 1, wherein said increased peroxisomal
proliferation results in a predisposition for at least one disorder
or disease selected from the group consisting of: cancer, thyroid
disorders, reproductive dysfunction, skeletal and cardiac
myopathies, and dysfunction of the immune system.
10. The method of claim 1, wherein said determining the amount of
analytes comprises mass spectrometry (MS).
11. The method of claim 10, wherein said mass spectrometry is
liquid chromatography (LC)-MS or gas chromatography (GC)-MS.
12. The method of claim 1, wherein said subject is a mammal.
13. The method of claim 11, wherein the method is automated.
14. The method of claim 13, wherein the method comprises computer
assisted data processing.
15. A method for diagnosing a disorder associated with increased
peroxisomal proliferation comprising: (a) selecting a male or
female subject suspected to suffer from increased peroxisomal
proliferation; (b) obtaining a body fluid test sample from the male
or female subject; (c) determining the amount of at least five of
the following analytes Coenzyme Q10, 16-Methylheptadecanoic acid,
17-Methyloctadecanoic acid, Eicosatrienoic acid (C20:3), Threonine,
Proline, Tyrosine, trans-4-Hydroxyproline from the body fluid test
sample from the male subject, or the amount of at least five of the
following analytes Pantothenic acid, Coenzyme Q9, Glycerol,
Palmitic acid (C16:0), Linoleic acid (C18:cis[9,12]2),
14-Methylhexadecanoic acid, gamma-Linolenic acid
(C18:cis[6,9,12]3), 16-Methylheptadecanoic acid, Threonic acid,
Cytosine, Phosphatidylcholine (C18:0/C22:6) from the body fluid
test sample from the female subject.
16. The method of claim 15, wherein said male or female subject has
been brought into contact with a compound suspected to be capable
of inducing increased peroxisomal proliferation.
17. The method of claim 16, wherein said compound is at least one
compound selected from the group consisting of: Benzylbutyl
Phthalate, Fenofibrate, Clofibrate, Fenoforbrate,
Diethylhexylphthalate, and Wy 14643.
18. The method of claim 15, wherein reference results are obtained
from one or more samples derived from (i) one or more subjects
which suffers from increased peroxisomal proliferation; or (ii) one
or more subjects which has been brought into contact with at least
one compound selected from the group consisting of: Benzylbutyl
Phthalate, Fenofibrate, Clofibrate, Fenoforbrate,
Diethylhexylphthalate, and Wy 14643.
19. The method of claim 18, wherein identical amounts for the
analytes in the body fluid test sample and corresponding reference
results are indicative for increased peroxisomal proliferation.
20. The method of claim 15, wherein reference results are obtained
from one or more samples derived from (i) one or more subjects
known to not suffer from increased peroxisomal proliferation; or
(ii) one or more subjects which has not been brought into contact
with at least one compound selected from the group consisting of:
Benzylbutyl Phthalate, Fenofibrate, Clofibrate, Fenoforbrate,
Diethylhexylphthalate, and Wy 14643.
21. The method of claim 20, wherein amounts for the analytes which
differ in the body fluid test sample relative to corresponding
reference results are indicative for increased peroxisomal
proliferation.
22. The method of claim 20, wherein indicative for increased
peroxisomal proliferation are amounts of the analytes relative to
corresponding reference results of one or more subjects known to
not suffer from increased peroxisomal proliferation which differ as
follows: (i) in a body fluid sample of a male: Coenzyme Q10
decreased, 16-Methylheptadecanoic acid decreased,
17-Methyloctadecanoic acid decreased, Eicosatrienoic acid (C20:3)
increased, Threonine decreased, Proline decreased, Tyrosine
decreased, trans-4-Hydroxyproline decreased; and (ii) in a body
fluid sample of a female subject: Pantothenic acid increased,
Coenzyme Q9 increased, Glycerol increased, Palmitic acid (C16:0)
increased, Linoleic acid (C18:cis[9,12]2) increased,
14-Methylhexadecanoic acid increased, gamma-Linolenic acid
(C18:cis[6,9,12]3) decreased, 16-Methylheptadecanoic acid
decreased, Threonic acid increased, Cytosine decreased,
Phosphatidylcholine (C18:0/C22:6) decreased.
23. The method of claim 15, wherein said increased peroxisomal
proliferation results in a predisposition for at least one disorder
or disease selected from the group consisting of: cancer, thyroid
disorders, reproductive dysfunction, skeletal and cardiac
myopathies, and dysfunction of the immune system.
24. The method of claim 15, wherein said determining the amount of
analytes comprises mass spectrometry (MS).
25. The method of claim 24, wherein said mass spectrometry is
liquid chromatography (LC)-MS or gas chromatography (GC)-MS.
26. The method of claim 15, wherein said subject is a mammal.
27. The method of claim 25, wherein the method is automated.
28. The method of claim 27, wherein the method comprises computer
assisted data processing.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 14/152,411, filed Jan. 10, 2014, which is a continuation
of U.S. patent application Ser. No. 12/994,705, filed Feb. 11,
2011, now U.S. Pat. No. 8,658,427, issued Feb. 25, 2014, which is a
national stage application (under 35 U.S.C. .sctn.371) of
PCT/EP2009/056386, filed May 26, 2009, which claims benefit of
European Application Number 08157118.4, filed May 28, 2008, and
European Application Number 09151549.4, filed Jan. 28, 2009.
[0002] The present invention pertains to the field of toxicological
assessments for risk stratification of chemical compounds.
Specifically, it relates to a method for diagnosing peroxisomal
proliferation. It also relates to a method of determining whether a
compound is capable of inducing such peroxisomal proliferation in a
subject and to a method of identifying a drug for treating
peroxisomal proliferation. Furthermore, the present invention
relates to a data collection comprising characteristic values of at
least five metabolites, a data storage medium comprising said data
collection, and a system and a device for diagnosing peroxisomal
proliferation. Finally, the present invention pertains to the use
of a group of metabolites or means for the determination thereof
for the manufacture of a diagnostic device or composition for
diagnosing peroxisomal proliferation in a subject.
[0003] Peroxisomes are intracellular organelles of eukaryotic
cells. Their enzymatic content varies across species. However,
peroxisomes, in principle, contain enzymes for oxidative reactions,
like the beta-oxidation of very-long-chain fatty acids. In this
process, the fatty acids are broken down into two carbon units at a
time, converted to Acetyl-CoA, which is then transported back to
the cytosol for further use. In animal cells, beta-oxidation can
also occur in the mitochondria while in yeast and plant cells, this
process occurs exclusively in the peroxisomes. Moreover, in
animals, the first steps in the formation of plasmalogen occur in
peroxisomes. Plasmalogen is the most abundant phospholipid in
myelin. Deficiency of plasmalogens causes profound abnormalities in
the myelination of nerve cells and, thus, leads to diseases of the
nervous system. Peroxisomes also play a role in the synthesis of
bile acids and proteins. Impaired peroxisomal function leads to
peroxisomal disorders, a class of medical conditions that lead to
lipid metabolism diseases.
[0004] An increase in peroxisome numbers will lead to an increased
formation of radical oxidative molecular species, such as reactive
oxygen species (ROS), and, thus, to an increased oxidative stress
for the cell. Accordingly, increased peroxisomal activity will
elicit disorders caused by increased oxidative stress, such as
damages on proteins, lipids or nucleic acids. Therefore,
peroxisomal proliferation will increase the somatic mutagenesis
rate as well as induce cell death by apoptosis or even necrosis.
Additionally, a liver tumor promoting effect of peroxisome
proliferators is discussed (Reddy, J. K. and Lalwani, N.D. (1983)
Carcinogenesis by hepatic peroxisome proliferators: evaluation of
the risk of hypolipidemic drugs and industrial plasticizers to
humans. Crit Rev. Toxicol. 12, 1-58; Moody, D. E., Reddy, J. K.,
Lake, B. G., Popp, J. A. and Reese, D. H. (1991) Peroxisome
proliferation and nongenotoxic carcinogenesis. Commentary on a
symposium. Fundam. Appl. Toxicol. 16, 232-248).
[0005] Chemical compounds, such as phthalates, fenofibrate, or
clofibrate, have been shown to induce peroxisomal proliferation.
Such compounds are also called "peroxisomal proliferators".
Moreover, due to their capability of inducing peroxisomal
proliferation, and subsequent events (radical oxygen species
formation and cell proliferation) these compounds also increase the
risk for somatic mutations resulting in disease such as cancer or
impaired metabolism (e.g. thyroid disorders, reproductive
dysfunction, skeletal and cardiac myopathies, dysfunction of the
immune system; Youssef, J. and Badr, M. (1998) Extraperoxisomal
Targets of Peroxisome Proliferators: Mitochondrial, Microsomal, and
Cytosolic Effects. Implications for Health and Disease. Crit. Rev.
Toxicol. 28: 1-33).
[0006] Further, chemical compounds which are used in any kind of
industry in the European Community, e.g., will now need to comply
with REACH (Registration, Evaluation, and Authorisation of
Chemicals). It will be understood that the potential of a chemical
compound to induce peroxisomal proliferation will be deemed as a
high risk for the compound and, consequently, the compound will be
available only for limited applications and when obeying high
security standards.
[0007] Sensitive and specific methods for assessing the capability
of a compound to induce peroxisomal proliferation in an efficient
and reliable manner, and to differentiate between said mode of
action and other hepatotoxic effects are not yet available but
would, nevertheless, be highly appreciated.
[0008] Thus, the technical problem underlying the present invention
could be seen as the provision of means and methods for complying
with the aforementioned needs. The technical problem is solved by
the embodiments characterized in the claims and described herein
below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIGS. 1 and 1a show a fragmentation pattern of
Phosphatidylcholine (C18:0/C22:6).
DETAILED DESCRIPTION
[0010] Accordingly, the present invention relates to a method for
diagnosing increased peroxisomal proliferation comprising: [0011]
(a) determining the amount of at least one, preferably, at least
five of the following analytes Coenzyme Q10, 16-Methylheptadecanoic
acid, 17-Methyloctadecanoic acid, Eicosatrienoic acid (C20:3),
Threonine, Proline, Tyrosine, trans-4-Hydroxyproline in a test
sample of a male subject suspected to suffer from increased
peroxisomal proliferation or at least one, preferably, at least
five of the following analytes Pantothenic acid, Coenzyme Q9,
Glycerol, Palmitic acid (C16:0), Linoleic acid (C18:cis[9,12]2),
14-Methylhexadecanoic acid, gamma-Linolenic acid
(C18:cis[6,9,12]3), 16-Methylheptadecanoic acid, Threonic acid,
Cytosine, Phosphatidylcholine (C18:0/C22:6) in a test sample of a
female subject suspected to suffer from increased peroxisomal
proliferation, and [0012] (b) comparing the amounts determined in
step (a) to a reference, whereby increased peroxisomal
proliferation is to be diagnosed.
[0013] The term "Phosphatidylcholine (C18:0/C22:6)" as used herein
refers to molecular species, preferably, characterized by the sum
parameter of glycerophosphorylcholines containing the combination
of a C18:0 fatty acid unit and a C22:6 fatty acid unit. The
mass-to-charge ratio (m/z) of the ionised spezies is 834.6 Da
(+/-0.3 Da). A preferred fragmentation pattern is shown in FIG. 1
below.
[0014] The expression "method for diagnosing" as referred to in
accordance with the present invention means that the method either
essentially consists of the aforementioned steps or may include
further steps. However, it is to be understood that the method, in
a preferred embodiment, is a method carried out ex vivo, i.e. not
practised on the human or animal body. Diagnosing as used herein
refers to assessing the probability according to which a subject is
suffering from a disorder. As will be understood by those skilled
in the art, such an assessment, although preferred to be, may
usually not be correct for 100% of the subjects to be diagnosed.
The term, however, requires that a statistically significant
portion of subjects can be identified as suffering from the
disorder or as having a predisposition therefor. Whether a portion
is statistically significant can be determined without further ado
by the person skilled in the art using various well known statistic
evaluation tools, e.g., determination of confidence intervals,
p-value determination, WELCH test, Mann-Whitney test, etc. Details
are found in Dowdy and Wearden, Statistics for Research, John Wiley
& Sons, New York 1983. Preferred confidence intervals are at
least 50%, at least 60%, at least 70%, at least 80%, at least 90%,
at least 95%. The p-values are, preferably, 0.2, 0.1, 0.05.
[0015] Diagnosing according to the present invention includes
monitoring, confirmation, and classification of the relevant
disorder or its symptoms. Monitoring relates to keeping track of an
already diagnosed disorder, e.g. to analyze the progression of the
disorder, the influence of a particular treatment on the
progression of the disorder. Confirmation relates to the
strengthening or substantiating a diagnosis already performed using
other indicators or markers.
[0016] Classification relates to allocating the diagnosis according
to the strength or kind of symptoms into different classes. Some of
the conditions which appear in connection with increased
peroxisomal proliferation may be accompanied by further metabolic
changes.
[0017] The term "peroxisomal proliferation" or "increased
peroxisomal proliferation" as used herein relates to a
significantly increased number of peroxisomes which are formed in a
cell or to a significantly increased peroxisomal activity found in
a cell or both. Increased peroxisomal proliferation results in an
impaired oxidative stress in the cell and, thus, in an increased
predisposition for mutagenesis or cell death as well as other
cellular disorders associated with an imbalanced ratio of radical
oxidative molecular species and antioxidants. Preferably, induced
peroxisomal proliferation as used herein results in a
predisposition for cancer and for diseases selected from thyroid
disorders, reproductive dysfunction, skeletal and cardiac
myopathies, or dysfunction of the immune system. The symptoms and
clinical signs of the aforementioned manifestations of increased
peroxisomal proliferation are well known to the person skilled in
the art and are described in detail in H. Marquardt, S. G. Schafer,
R. O. McClellan, F. Welsch (eds.), "Toxicology", Chapter 13: The
Liver, 1999, Academic Press, London.
[0018] Each of the analytes to be determined in the method of the
present invention is also suitable for diagnosing the diseases or
disorders referred to herein when analysed alone. However, it was
found in accordance with the present invention that a combination
of at least five different analytes further strengthen the
diagnosis since each of the analytes is an apparently statistically
independent predictor of equal value for the diagnosis. Moreover,
the specificity for liver toxicity is also significantly increased
since influences from other tissues on the marker abundance are
counterbalanced. Preferred marker combinations for specific liver
enzyme inducing compound classes are those found in Table 1 and 2,
below.
[0019] It is to be understood that in addition to a group
consisting of at least five of the aforementioned analytes,
additional analytes are, preferably, determined in the method of
the present invention. The additional analytes are, preferably,
also selected from the aforementioned group. In other words,
preferably, at least six, at least seven, at least eight, at least
nine, at least ten or all of the analytes of the aforementioned
group are determined in the method of the present invention. The
additional determination of these analytes even further strengthen
the result obtained by the method of this invention. Furthermore,
other analytes or metabolites (i.e. metabolites not specifically
recited in the aforementioned group) or biomarkers, such as
enzymes, may still be determined in addition.
[0020] In a particular preferred embodiment, the at least one
analyte in the male sample is selected from the group consisting
of: Coenzyme Q10, 16-Methylheptadecanoic acid,
17-Methyloctadecanoic acid, Eicosatrienoic acid (C20:3), and
trans-4-Hydroxyproline. In an even more preferred embodiments, all
of the aforementioned analytes are determined.
[0021] However, it is also preferably envisaged that in a group of
at least five analytes to be determined in accordance with the
present invention, one, two, three, or four analytes are from the
aforementioned group of preferred analytes while the remaining
analytes are analytes for male samples as specified elsewhere
herein.
[0022] Thus, if the first analyte of a preferred group of five
analytes to be determined in accordance with the present invention
is Coenzyme Q10, the remaining four analytes are selected from a
group consisting of the following analytes: 16-Methylheptadecanoic
acid, 17-Methyloctadecanoic acid, Eicosatrienoic acid (C20:3),
Threonine, Proline, Tyrosine, and trans-4-Hydroxyproline.
[0023] If the first and second analyte are Coenzym Q10 and
16-Methylheptadecanoic acid, the remaining three analytes are
selected from a group consisting of: 17-Methyloctadecanoic acid,
Eicosatrienoic acid (C20:3), Threonine, Proline, Tyrosine, and
trans-4-Hydroxyproline.
[0024] If the first, second and third analytes are Coenzyme Q10,
16-Methylheptadecanoic acid, 17-Methyloctadecanoic acid, the
remaining two analytes are selected from a group consisting of:
Eicosatrienoic acid (C20:3), Threonine, Proline, Tyrosine, and
trans-4-Hydroxyproline.
[0025] If the first, second, third and fourth analytes are Coenzyme
Q10, 16-Methylheptadecanoic acid, 17-Methyloctadecanoic acid,
Eicosatrienoic acid (C20:3), the remaining analyte is selected from
a group consisting of: Threonine, Proline, Tyrosine, and
trans-4-Hydroxyproline.
[0026] In a particular preferred embodiment, the at least one
analyte in the female is selected from the group consisting of:
Pantothenic acid, Glycerol, Linoleic acid (C18:cis[9,12]2),
16-Methylheptadecanoic acid, Cytosine, and Phosphatidylcholine
(C18:0/C22:6). In an even more preferred embodiments, all six of
the aforementioned analytes are determined. However, it is
preferably envisaged that in a group of at least five analytes to
be determined in accordance with the present invention, one, two,
three, or four analytes are from the aforementioned group of
preferred analytes while the remaining analytes are analytes for
female samples as specified elsewhere herein.
[0027] However, it is also preferably envisaged that in a group of
at least five analytes to be determined in accordance with the
present invention, one, two, three, or four analytes are from the
aforementioned group of preferred analytes while the remaining
analytes are analytes for male samples as specified elsewhere
herein.
[0028] Thus, if the first analyte of a preferred group of five
analytes to be determined in accordance with the present invention
is Pantothenic acid, the remaining four analytes are selected from
a group consisting of the following analytes: Coenzyme Q9,
Glycerol, Palmitic acid (C16:0), Linoleic acid (C18:cis[9,12]2),
14-Methylhexadecanoic acid, gamma-Linolenic acid
(C18:cis[6,9,12]3), 16-Methylheptadecanoic acid, Threonic acid,
Cytosine, Phosphatidylcholine (C18:0/C22:6).
[0029] If the first and second analyte are Pantothenic acid and
Glycerol, the remaining three analytes are selected from a group
consisting of: Coenzyme Q9, Palmitic acid (C16:0), Linoleic acid
(C18:cis[9,12]2), 14-Methylhexadecanoic acid, gamma-Linolenic acid
(C18:cis[6,9,12]3), 16-Methylheptadecanoic acid, Threonic acid,
Cytosine, Phosphatidylcholine (C18:0/C22:6).
[0030] If the first, second and third analytes are Pantothenic
acid, Glycerol, and Linoleic acid (C18:cis[9,12]2), the remaining
two analytes are selected from a group consisting of: Coenzyme Q9,
Palmitic acid (C16:0), 14-Methylhexadecanoic acid, gamma-Linolenic
acid (C18:cis[6,9,12]3), 16-Methylheptadecanoic acid, Threonic
acid, Cytosine, Phosphatidylcholine (C18:0/C22:6).
[0031] If the first, second, third and fourth analytes are
Pantothenic acid, Glycerol, Linoleic acid (C18:cis[9,12]2),
16-Methylheptadecanoic acid, the remaining analyte is selected from
a group consisting of: Coenzyme Q9, Palmitic acid (C16:0),
14-Methylhexadecanoic acid, gamma-Linolenic acid
(C18:cis[6,9,12]3), Threonic acid, Cytosine, Phosphatidylcholine
(C18:0/C22:6).
[0032] Analyte as used herein refers to at least one molecule of a
specific analyte up to a plurality of molecules of the said
specific analyte. It is to be understood further that a group of
analytes means a plurality of chemically different molecules
wherein for each analyte at least one molecule up to a plurality of
molecules may be present. An analyte in accordance with the present
invention encompasses all classes of organic or inorganic chemical
compounds including those being comprised by biological material
such as organisms. Preferably, the analyte in accordance with the
present invention is a small molecule compound. More preferably, in
case a plurality of analytes is envisaged, it will be understood
that each analyte represents a metabolite and that the plurality of
metabolites represent a metabolome. The metabolome is the
collection of metabolites being comprised by an organism, an organ,
a tissue or a cell at a specific time and under specific
conditions. The analyte may differ from the metabolite which is
represented thereby due to chemical modifications arising as
arising as a result of purification steps, GC derivatives or other
modifications required for determination. However, the person
skilled in the art will readily be able to allocate the analyte to
a metabolite or class of metabolites.
[0033] Metabolites are small molecule compounds, such as substrates
for enzymes of metabolic pathways, intermediates of such pathways
or the products obtained by a metabolic pathway. Metabolic pathways
are well known in the art and may vary between species. Preferably,
said pathways include at least citric acid cycle, respiratory
chain, glycolysis, gluconeogenesis, hexose monophosphate pathway,
oxidative pentose phosphate pathway, production and
.beta.-oxidation of fatty acids, urea cycle, amino acid
biosynthesis pathways, protein degradation pathways such as
proteasomal degradation, amino acid degrading pathways,
biosynthesis or degradation of: lipids, polyketides (including e.g.
flavonoids and isoflavonoids), isoprenoids (including eg. terpenes,
sterols, steroids, carotenoids, xanthophylls), carbohydrates,
phenylpropanoids and derivatives, alcaloids, benzenoids, indoles,
indole-sulfur compounds, porphyrines, hormones, vitamins, cofactors
such as prosthetic groups or electron carriers, glucosinolates,
purines, pyrimidines, nucleosides, nucleotides and related
molecules such as tRNAs, microRNAs (miRNA) or mRNAs. Accordingly,
small molecule compound metabolites are preferably composed of the
following classes of compounds: alcohols, alkanes, alkenes,
alkines, aromatic compounds, ketones, aldehydes, carboxylic acids,
esters, amines, imines, amides, cyanides, amino acids, peptides,
thiols, thioesters, phosphate esters, sulfate esters, thioethers,
sulfoxides, ethers, or combinations or derivatives of the
aforementioned compounds. The small molecules among the metabolites
may be primary metabolites which are required for normal cellular
function, organ function or animal growth, development or health.
Moreover, small molecule metabolites further comprise secondary
metabolites having essential ecological function, e.g. metabolites
which allow an organism to adapt to its environment. Furthermore,
metabolites are not limited to said primary and secondary
metabolites and further encompass artificial small molecule
compounds. Said artificial small molecule compounds are derived
from exogenously provided small molecules which are administered or
taken up by an organism but are not primary or secondary
metabolites as defined above. For instance, artificial small
molecule compounds may be metabolic products obtained from drugs by
metabolic pathways of the animal. Moreover, metabolites further
include peptides, oligopeptides, polypeptides, oligonucleotides and
polynucleotides, such as RNA or DNA. More preferably, a metabolite
has a molecular weight of 50 Da (Dalton) to 30,000 Da, most
preferably less than 30,000 Da, less than 20,000 Da, less than
15,000 Da, less than 10,000 Da, less than 8,000 Da, less than 7,000
Da, less than 6,000 Da, less than 5,000 Da, less than 4,000 Da,
less than 3,000 Da, less than 2,000 Da, less than 1,000 Da, less
than 500 Da, less than 300 Da, less than 200 Da, less than 100 Da.
Preferably, a metabolite has, however, a molecular weight of at
least 50 Da. Most preferably, a metabolite in accordance with the
present invention has a molecular weight of 50 Da up to 1,500
Da.
[0034] Analytes as referred to in accordance with this invention
are molecular species which are derived from the naturally
occurring metabolite due to the purification and/or determination
process. In some cases, the analyte will be identical. In other
cases, however, it will be a chemical derivative thereof.
Nevertheless, it is to be understood that the appearing of the
analyte inevitably allows drawing conclusions on the occurrence of
the metabolite.
[0035] The term "test sample" as used herein refers to samples to
be used for the diagnosis of increased peroxisomal proliferation by
the method of the present invention. Said test sample is a
biological sample. Samples from biological sources (i.e. biological
samples) usually comprise a plurality of metabolites. Preferred
biological samples to be used in the method of the present
invention are samples from body fluids, preferably, blood, plasma,
serum, saliva, bile, urine or cerebrospinal fluid, or samples
derived, e.g., by biopsy, from cells, tissues or organs, preferably
from the liver. More preferably, the sample is a blood, plasma or
serum sample, most preferably, a plasma sample. Biological samples
are derived from a subject as specified elsewhere herein.
Techniques for obtaining the aforementioned different types of
biological samples are well known in the art. For example, blood
samples may be obtained by blood taking while tissue or organ
samples are to be obtained, e.g., by biopsy.
[0036] The aforementioned samples are, preferably, pre-treated
before they are used for the method of the present invention. As
described in more detail below, said pre-treatment may include
treatments required to release or separate the compounds or to
remove excessive material or waste. Suitable techniques comprise
centrifugation, extraction, fractioning, ultrafiltration, protein
precipitation followed by filtration and purification and/or
enrichment of compounds. Moreover, other pre-treatments are carried
out in order to provide the compounds in a form or concentration
suitable for compound analysis. For example, if gas-chromatography
coupled mass spectrometry is used in the method of the present
invention, it will be required to derivatize the compounds prior to
the said gas chromatography. Suitable and necessary pre-treatments
depend on the means used for carrying out the method of the
invention and are well known to the person skilled in the art.
Pre-treated samples as described before are also comprised by the
term "sample" as used in accordance with the present invention.
[0037] The term "subject" as used herein relates to animals,
preferably to mammals such as mice, rats, guinea pigs, rabbits,
hamsters, pigs, sheep, dogs, cats, horses, monkeys, or cows and,
also preferably, to humans. More preferably, the subject is a
rodent and, most preferably, a rat. Other animals which may be
diagnosed applying the method of the present invention are fishes,
birds or reptiles. Preferably, said subject was in or has been
brought into contact with a compound suspected to be capable of
inducing increased peroxisomal proliferation. A subject which has
been brought into contact with a compound suspected to induce
increased peroxisomal proliferation may, e.g., be a laboratory
animal such as a rat which is used in a screening assay for, e.g.,
toxicity of compounds. A subject suspected to have been in contact
with a compound capable of inducing increased peroxisomal
proliferation may be also a subject to be diagnosed for selecting a
suitable therapy. Preferably, a compound capable of inducing
increased peroxisomal proliferation as used herein is Benzylbutyl
Phthalate, Fenofibrate, Clofibrate, Fenoforbrate,
Diethylhexylphthalate or Wy 14643.
[0038] The term "determining the amount" as used herein refers to
determining at least one characteristic feature of each analyte of
the said at least five analytes. Characteristic features in
accordance with the present invention are features which
characterize the physical and/or chemical properties including
biochemical properties of a analyte. Such properties include, e.g.,
molecular weight, viscosity, density, electrical charge, spin,
optical activity, colour, fluorescence, chemoluminescence,
elementary composition, chemical structure, capability to react
with other compounds, capability to elicit a response in a
biological read out system (e.g., induction of a reporter gene) and
the like. Values for said properties may serve as characteristic
features and can be determined by techniques well known in the art.
Moreover, the characteristic feature may be any feature which is
derived from the values of the physical and/or chemical properties
of a analyte by standard operations, e.g., mathematical
calculations such as multiplication, division or logarithmic
calculus. Most preferably, the at least one characteristic feature
allows the determination and/or chemical identification of the
analyte and its amount. Accordingly, the characteristic value,
preferably, also comprises information relating to the abundance of
the metabolite from which the characteristic value is derived. For
example, a characteristic value of a metabolite may be a peak in a
mass spectrum. Such a peak contains characteristic information of
the metabolite, i.e. the m/z (mass to charge ratio) information, as
well as an intensity value being related to the abundance of the
said analyte (i.e. its amount) in the sample.
[0039] As discussed before, each analyte of the group of analytes
to be determined in accordance with the method of the present
invention may be, preferably, determined quantitatively or
semi-quantitatively. For quantitative determination, either the
absolute or precise amount of the metabolite will be determined or
the relative amount of the analyte will be determined based on the
value determined for the characteristic feature(s) referred to
herein above. The relative amount may be determined in a case were
the precise amount of a analyte can or shall not be determined.
[0040] In said case, it can be determined whether the amount in
which the analyte is present is enlarged or diminished with respect
to a second sample comprising said metabolite in a second amount.
Quantitatively analysing a analyte, thus, also includes what is
sometimes referred to as semiquantitative analysis of a
analyte.
[0041] Moreover, determining as used in the method of the present
invention, preferably, includes using a compound separation step
prior to the analysis step referred to before. Preferably, said
compound separation step yields a time resolved separation of the
analytes comprised by the sample. Suitable techniques for
separation to be used preferably in accordance with the present
invention, therefore, include all chromatographic separation
techniques such as liquid chromatography (LC), high performance
liquid chromatography (HPLC), gas chromatography (GC), thin layer
chromatography, size exclusion or affinity chromatography. These
techniques are well known in the art and can be applied by the
person skilled in the art without further ado. Most preferably, LC
and/or GC are chromatographic techniques to be envisaged by the
method of the present invention. Suitable devices for such
determination of metabolites are well known in the art. Preferably,
mass spectrometry is used in particular gas chromatography mass
spectrometry (GC-MS), liquid chromatography mass spectrometry
(LC-MS), direct infusion mass spectrometry or Fourier transform
ion-cyclotrone-resonance mass spectrometry (FT-ICR-MS), capillary
electrophoresis mass spectrometry (CE-MS), high-performance liquid
chromatography coupled mass spectrometry (HPLC-MS), quadrupole mass
spectrometry, any sequentially coupled mass spectrometry, such as
MS-MS or MS-MS-MS, inductively coupled plasma mass spectrometry
(ICP-MS), pyrolysis mass spectrometry (Py-MS), ion mobility mass
spectrometry or time of flight mass spectrometry (TOF). Most
preferably, LC-MS and/or GC-MS are used as described in detail
below. Said techniques are disclosed in, e.g., Nissen, Journal of
Chromatography A, 703, 1995: 37-57, U.S. Pat. No. 4,540,884 or U.S.
Pat. No. 5,397,894, the disclosure content of which is hereby
incorporated by reference. As an alternative or in addition to mass
spectrometry techniques, the following techniques may be used for
compound determination: nuclear magnetic resonance (NMR), magnetic
resonance imaging (MRI), Fourier transform infrared analysis
(FT-IR), ultraviolet (UV) spectroscopy, refraction index (RI),
fluorescent detection, radiochemical detection, electrochemical
detection, light scattering (LS), dispersive Raman spectroscopy or
flame ionisation detection (FID). These techniques are well known
to the person skilled in the art and can be applied without further
ado. The method of the present invention shall be, preferably,
assisted by automation. For example, sample processing or
pre-treatment can be automated by robotics. Data processing and
comparison is, preferably, assisted by suitable computer programs
and databases. Automation as described herein before allows using
the method of the present invention in high-throughput
approaches.
[0042] Moreover, the analyte can also be determined by a specific
chemical or biological assay. Said assay shall comprise means which
allow for specifically detecting the analyte in the sample.
Preferably, said means are capable of specifically recognizing the
chemical structure of the analyte or are capable of specifically
identifying the analyte based on its capability to react with other
compounds or its capability to elicit a response in a biological
read out system (e.g., induction of a reporter gene). Means which
are capable of specifically recognizing the chemical structure of a
metabolite are, preferably, antibodies or other proteins which
specifically interact with chemical structures, such as receptors
or enzymes. Specific antibodies, for instance, may be obtained
using the metabolite as antigen by methods well known in the art.
Antibodies as referred to herein include both polyclonal and
monoclonal antibodies, as well as fragments thereof, such as Fv,
Fab and F(ab).sub.2 fragments that are capable of binding the
antigen or hapten. The present invention also includes humanized
hybrid antibodies wherein amino acid sequences of a nonhuman donor
antibody exhibiting a desired antigen-specificity are combined with
sequences of a human acceptor antibody. Moreover, encompassed are
single chain antibodies. The donor sequences will usually include
at least the antigen-binding amino acid residues of the donor but
may comprise other structurally and/or functionally relevant amino
acid residues of the donor antibody as well. Such hybrids can be
prepared by several methods well known in the art. Suitable
proteins which are capable of specifically recognizing the
metabolite are, preferably, enzymes which are involved in the
metabolic conversion of the said metabolite. Said enzymes may
either use the metabolite as a substrate or may convert a substrate
into the metabolite. Moreover, said antibodies may be used as a
basis to generate oligopeptides which specifically recognize the
metabolite. These oligopeptides shall, for example, comprise the
enzyme's binding domains or pockets for the said metabolite.
Suitable antibody and/or enzyme based assays may be RIA
(radioimmunoassay), ELISA (enzyme-linked immunosorbent assay),
sandwich enzyme immune tests, electrochemiluminescence sandwich
immunoassays (ECLIA), dissociation-enhanced lanthanide fluoro
immuno assay (DELFIA) or solid phase immune tests. Moreover, the
metabolite may also be identified based on its capability to react
with other compounds, i.e. by a specific chemical reaction.
Further, the metabolite may be determined in a sample due to its
capability to elicit a response in a biological read out system.
The biological response shall be detected as read out indicating
the presence and/or the amount of the metabolite comprised by the
sample. The biological response may be, e.g., the induction of gene
expression or a phenotypic response of a cell or an organism.
[0043] The term "reference" refers to values of characteristic
features of each of the analytes of the group of analytes which can
be correlated to increased peroxisomal proliferation. Such
reference results are, preferably, obtained from a sample derived
from a subject which has been brought into contact with Benzylbutyl
Phthalate, Fenofibrate, Clofibrate, Fenoforbrate,
Diethylhexylphthalate or Wy 14643. A subject may be brought into
contact with the said compounds by each topic or systemic
administration mode as long as the compounds are bioavailable. The
reference results may be determined as described hereinabove for
the amounts of the analytes. Alternatively, but nevertheless also
preferred, the reference results may be obtained from sample
derived from a subject which has not been brought into contact with
Benzylbutyl Phthalate, Fenofibrate, Clofibrate, Fenoforbrate,
Diethylhexylphthalate or Wy 14643 or a healthy subject with respect
to increased peroxisomal proliferation and, more preferably, other
diseases as well. Moreover, the reference, also preferably, could
be a calculated reference, most preferably the average or median,
for the relative or absolute amount for each of the analytes of the
group of analytes derived from a population of individuals
comprising the subject to be investigated. However, it is to be
understood that the population of subjects to be investigated for
determining a calculated reference, preferably, either consist of
apparently healthy subjects (e.g. untreated) or comprise a number
of apparently healthy subjects which is large enough to be
statistically resistant against significant average or median
changes due to the presence of the test subject(s) in the said
population. The absolute or relative amounts of the analytes of
said individuals of the population can be determined as specified
elsewhere herein. How to calculate a suitable reference value,
preferably, the average or median, is well known in the art. The
population of subjects referred to before shall comprise a
plurality of subjects, preferably, at least 5, 10, 50, 100, 1,000
or 10,000 subjects. It is to be understood that the subject to be
diagnosed by the method of the present invention and the subjects
of the said plurality of subjects are of the same species.
[0044] More preferably, the reference results, i.e. values for at
least one characteristic features of the analyte, will be stored in
a suitable data storage medium such as a database and are, thus,
also available for future diagnoses. This also allows efficiently
diagnosing predisposition for a disease because suitable reference
results can be identified in the database once it has been
confirmed (in the future) that the subject from which the
corresponding reference sample was obtained (indeed) showed
increased peroxisomal proliferation.
[0045] The term "comparing" refers to assessing whether the results
of the determination described hereinabove in detail, i.e. the
results of the qualitative or quantitative determination of a
analyte, are identical or similar to reference results or differ
therefrom.
[0046] In case the reference results are obtained from a sample
derived from a subject which has been brought into contact with
Benzylbutyl Phthalate, Fenofibrate, Clofibrate, Fenoforbrate,
Diethylhexylphthalate or Wy 14643, increased peroxisomal
proliferation can be diagnosed based on the degree of identity or
similarity between the test results obtained from the test sample
and the aforementioned reference results, i.e. based on an
identical or similar qualitative or quantitative composition with
respect to the aforementioned analytes. The results of the test
sample and the reference results are identical, if the values for
the characteristic features and, in the case of quantitative
determination, the intensity values are identical. Said results are
similar, if the values of the characteristic features are identical
but the intensity values are different. Such a difference is,
preferably, not significant and shall be characterized in that the
values for the intensity are within at least the interval between
1.sup.st and 99.sup.th percentile, 5.sup.th and 95.sup.th
percentile, 10.sup.th and 90.sup.th percentile, 20.sup.th and
80.sup.th percentile, 30.sup.th and 70.sup.th percentile, 40.sup.th
and 60.sup.th percentile of the reference value the 50.sup.th,
60.sup.th, 70.sup.th, 80.sup.th, 90.sup.th or 95.sup.th percentile
of the reference value.
[0047] In case the reference results are obtained a subject which
has not been brought into contact with Benzylbutyl Phthalate,
Fenofibrate, Clofibrate, Fenoforbrate, Diethylhexylphthalate or Wy
14643, increased peroxisomal proliferation can be diagnosed based
on the differences between the test results obtained from the test
sample and the aforementioned reference results, i.e. differences
in the qualitative or quantitative composition with respect to the
aforementioned analytes. The same applies if a calculated reference
as specified above is used. The difference may be an increase in
the absolute or relative amount of an analyte (sometimes referred
to as up-regulation of the metabolite; see also Examples) or a
decrease in either of said amounts or the absence of a detectable
amount of the analyte (sometimes referred to as up-regulation of
the metabolite; see also Examples). Preferably, the difference in
the relative or absolute amount is significant, i.e. outside of the
interval between 45.sup.th and 55.sup.th percentile, 40.sup.th and
60.sup.th percentile, 30.sup.th and 70.sup.th percentile, 20.sup.th
and 80.sup.th percentile, 10.sup.th and 90.sup.th percentile,
5.sup.th and 95.sup.th percentile, 1.sup.st and 99.sup.th
percentile of the reference value.
[0048] Preferably, the amounts of the analytes in comparison to the
reference differ as follows: (i) in a sample of a male: Coenzyme
Q10 decreased, 16-Methylheptadecanoic acid decreased,
17-Methyloctadecanoic acid decreased, Eicosatrienoic acid (C20:3)
increased, Threonine decreased, Proline decreased, Tyrosine
decreased, trans-4-Hydroxyproline decreased; and
(ii) in a sample of a female subject: Pantothenic acid increased,
Coenzyme Q9 increased, Glycerol increased, Palmitic acid (C16:0)
increased, Linoleic acid (C18:cis[9,12]2) increased,
14-Methylhexadecanoic acid increased, gamma-Linolenic acid
(C18:cis[6,9,12]3) decreased, 16-Methylheptadecanoic acid
decreased, Threonic acid increased, Cytosine decreased,
Phosphatidylcholine (C18:0/C22:6) decreased. For the specific
analytes referred to in this specification, preferred values for
the changes in the relative amounts (i.e. "fold"--changes) or the
kind of change (i.e. "up"- or "down"-regulation resulting in a
higher or lower relative and/or absolute amount) are indicated in
the Examples below.
[0049] The comparison is, preferably, assisted by automation. For
example, a suitable computer program comprising algorithm for the
comparison of two different data sets (e.g., data sets comprising
the values of the characteristic feature(s)) may be used. Such
computer programs and algorithm are well known in the art.
Notwithstanding the above, a comparison can also be carried out
manually.
[0050] The aforementioned methods for the determination of the
analytes can be implemented into a device. A device as used herein
shall comprise at least the aforementioned means. Moreover, the
device, preferably, further comprises means for comparison and
evaluation of the detected characteristic feature(s) of the
analytes and, also preferably, the determined signal intensity. The
means of the device are, preferably, operatively linked to each
other. How to link the means in an operating manner will depend on
the type of means included into the device. For example, where
means for automatically qualitatively or quantitatively determining
the metabolite are applied, the data obtained by said automatically
operating means can be processed by, e.g., a computer program in
order to facilitate the diagnosis. Preferably, the means are
comprised by a single device in such a case. Said device may
accordingly include an analyzing unit for the analytes and a
computer unit for processing the resulting data for the diagnosis.
Alternatively, where means such as test stripes are used for
determining the analytes, the means for diagnosing may comprise
control stripes or tables allocating the determined result data to
result data known to be accompanied with increased peroxisomal
proliferation or those being indicative for a healthy subject as
discussed above. Preferred devices are those which can be applied
without the particular knowledge of a specialized clinician, e.g.,
test stripes or electronic devices which merely require loading
with a sample.
[0051] Alternatively, the methods for the determination of the
analytes can be implemented into a system comprising several
devices which are, preferably, operatively linked to each other.
Specifically, the means must be linked in a manner as to allow
carrying out the method of the present invention as described in
detail above. Therefore, operatively linked, as used herein,
preferably, means functionally linked. Depending on the means to be
used for the system of the present invention, said means may be
functionally linked by connecting each mean with the other by means
which allow data transport in between said means, e.g., glass fiber
cables, and other cables for high throughput data transport.
Nevertheless, wireless data transfer between the means is also
envisaged by the present invention, e.g., via LAN (Wireless LAN,
W-LAN). A preferred system comprises means for determining
analytes. Means for determining analytes as used herein encompass
means for separating analytes, such as chromatographic devices, and
means for analyte determination, such as mass spectrometry devices.
Suitable devices have been described in detail above. Preferred
means for compound separation to be used in the system of the
present invention include chromatographic devices, more preferably
devices for liquid chromatography, HPLC, and/or gas chromatography.
Preferred devices for compound determination comprise mass
spectrometry devices, more preferably, GC-MS, LC-MS, direct
infusion mass spectrometry, FT-ICR-MS, CE-MS, HPLC-MS, quadrupole
mass spectrometry, sequentially coupled mass spectrometry
(including MS-MS or MS-MS-MS), ICP-MS, Py-MS or TOF. The separation
and determination means are, preferably, coupled to each other.
Most preferably, LC-MS and/or GCMS is used in the system of the
present invention as described in detail elsewhere in the
specification. Further comprised shall be means for comparing
and/or analyzing the results obtained from the means for
determination of analytes. The means for comparing and/or analyzing
the results may comprise at least one databases and an implemented
computer program for comparison of the results. Preferred
embodiments of the aforementioned systems and devices are also
described in detail below.
[0052] Advantageously, it has been found in the study underlying
the present invention that the amounts of a group of at least five
of the aforementioned analytes serve as biomarkers for increased
peroxisomal proliferation, in particular those peroxisomal
proliferation induced by the peroxisomal proliferators Benzylbutyl
Phthalate, Fenofibrate, Clofibrate, Fenoforbrate,
Diethylhexylphthalate or Wy 14643. The specificity and accuracy of
the method will be even more improved by determining all of the
aformetioned analytes. A change in the quantitative and/or
qualitative composition of the metabolome with respect to these
specific analytes is indicative for increased peroxisomal
proliferation. The morphological, physiological as well as
biochemical parameters which are currently used for diagnosing
peroxisomal proliferation are less specific and less sensitive in
comparison to the biomarker determination provided by the present
invention. Thanks to the present invention, the capability of a
compound to induce peroxisomal proliferation, i.e. to be a so
called "peroxisomal proliferators", can be more efficiently and
reliably assessed. Moreover, based on the aforementioned findings,
screening assays for drugs which ameliorate or inhibit increased
peroxisomal proliferation are feasible. Furthermore, chemical
compounds which, amongst other effects, induce peroxisomal
proliferation and have lipid lowering properties are compounds such
as MCPA (methyl-chloro-phenoxy-aceticacid), Dichlorprop
(dichloro-phenoxyproionicacid) and Mecoprop
(methyl-chloro-phenoxy-aceticacid). They can also be identified by
the method of the present invention.
[0053] The present invention, in principle, relates to the use of
at least one, preferably, at least five of the following analytes
Coenzyme Q10, 16-Methylheptadecanoic acid, 17-Methyloctadecanoic
acid, Eicosatrienoic acid (C20:3), Threonine, Proline, Tyrosine,
trans-4-Hydroxyproline or means for the detection thereof for the
manufacture of a diagnostic device or composition for diagnosing
increased peroxisomal proliferation in a male subject or at least
one, preferably, at least five of the following analytes
Pantothenic acid, Coenzyme Q9, Glycerol, Palmitic acid (C16:0),
Linoleic acid (C18:cis[9,12]2), 14-Methylhexadecanoic acid,
gamma-Linolenic acid (C18:cis[6,9,12]3), 16-Methylheptadecanoic
acid, Threonic acid, Cytosine, Phosphatidylcholine (C18:0/C22:6) or
means for the detection thereof for the manufacture of a diagnostic
device or composition for diagnosing increased peroxisomal
proliferation in a female subject.
[0054] All definitions and explanations of the terms made above
apply mutatis mutandis for the aforementioned methods and all other
embodiments described further below except stated otherwise in the
following.
[0055] It follows from the above that the present invention also
contemplates a method of determining whether a compound is capable
of inducing increased peroxisomal proliferation in a subject
comprising: [0056] (a) determining in a sample of a male subject
which has been brought into contact with a compound suspected to be
capable of inducing increased peroxisomal proliferation the amount
of at least one, preferably, at least five of the following
analytes Coenzyme Q10, 16-Methylheptadecanoic acid,
17-Methyloctadecanoic acid, Eicosatrienoic acid (C20:3), Threonine,
Proline, Tyrosine, trans-4-Hydroxyproline or in a test sample of a
female which has been brought into contact with a compound
suspected to be capable of inducing increased peroxisomal
proliferation at least one, preferably, at least five of the
following analytes Pantothenic acid, Coenzyme Q9, Glycerol,
Palmitic acid (C16:0), Linoleic acid (C18:cis[9,12]2),
14-Methylhexadecanoic acid, gamma-Linolenic acid
(C18:cis[6,9,12]3), 16-Methylheptadecanoic acid, Threonic acid,
Cytosine, Phosphatidylcholine (C18:0/C22:6); and [0057] (b)
comparing the amounts determined in step (a) to a reference,
whereby the capability of the compound to induce increased
peroxisomal proliferation is determined.
[0058] In a preferred embodiment of said method, said compound is
at least one compound selected from the group consisting of:
Benzylbutyl Phthalate, Fenofibrate, Clofibrate, Fenoforbrate,
Diethylhexylphthalate or Wy 14643.
[0059] Preferably, said reference is derived from a subject which
suffers from increased peroxisomal proliferation or from a subject
which has been brought into contact with at least one compound
selected from the group consisting of: Benzylbutyl Phthalate,
Fenofibrate, Clofibrate, Fenoforbrate, Diethylhexylphthalate or Wy
14643. More preferably, essentially identical amounts for the
analytes in the test sample and the reference are indicative for
increased peroxisomal proliferation.
[0060] Also preferably, said reference is derived from a subject
known to not suffer from increased peroxisomal proliferation or
from a subject which has not been brought into contact with at
least one compound selected from the group consisting of:
Benzylbutyl Phthalate, Fenofibrate, Clofibrate, Fenoforbrate,
Diethylhexylphthalate or Wy 14643. Alternatively, but also
preferred, the said reference is a calculated reference for the
analytes for a population of subjects. More preferably, amounts for
the analytes which differ in the test sample in comparison to the
reference are indicative for increased peroxisomal
proliferation.
[0061] Preferred indicative amounts for increased peroxisomal
proliferation are disclosed elsewhere in this specification.
[0062] The present invention also relates to a method of
identifying a substance for treating increased peroxisomal
proliferation comprising the steps of: [0063] (a) determining in a
sample of a male subject suffering from increased peroxisomal
proliferation which has been brought into contact with a candidate
substance suspected to be capable of treating increased peroxisomal
proliferation the amount of at least one, preferably, at least five
of the following analytes Coenzyme Q10, 16-Methylheptadecanoic
acid, 17-Methyloctadecanoic acid, Eicosatrienoic acid (C20:3),
Threonine, Proline, Tyrosine, trans-4-Hydroxyproline or in a sample
of a female subject suffering from increased peroxisomal
proliferation which has been brought into contact with a candidate
substance suspected to be capable of treating increased peroxisomal
proliferation the amount of at least one, preferably, at least five
of the following analytes Pantothenic acid, Coenzyme Q9, Glycerol,
Palmitic acid (C16:0), Linoleic acid (C18:cis[9,12]2),
14-Methylhexadecanoic acid, gamma-Linolenic acid
(C18:cis[6,9,12]3), 16-Methylheptadecanoic acid, Threonic acid,
Cytosine, Phosphatidylcholine (C18:0/C22:6); and [0064] (b)
comparing the amounts determined in step (a) to a reference,
whereby a substance capable of treating increased peroxisomal
proliferation is to be identified.
[0065] Specifically, in case of the method of identifying a
substance useful for treating increased peroxisomal proliferation,
said reference is, preferably, derived from a subject which has
been brought into contact with at least one compound selected from
the group consisting of: Benzylbutyl Phthalate, Fenofibrate,
Clofibrate, Fenoforbrate, Diethylhexylphthalate or Wy 14643 or a
subject which suffers from increased peroxisomal proliferation.
More preferably, amounts for the metabolites which differ in the
test sample and the reference are indicative for a substance useful
for treating increased peroxisomal proliferation.
[0066] Specifically, indicative for a substance capable of treating
increased peroxisomal proliferation are amounts of the analytes in
comparison to the reference which differ as follows: (i) in a
sample of a male: Coenzyme Q10 increased, 16-Methylheptadecanoic
acid increased, 17-Methyloctadecanoic acid increased,
Eicosatrienoic acid (C20:3) decreased, Threonine decreased, Proline
increased, Tyrosine increased, trans-4-Hydroxyproline increased and
(ii) in a sample of a female subject: Pantothenic acid decreased,
Coenzyme Q9 decreased, Glycerol decreased, Palmitic acid (C16:0)
decreased, Linoleic acid (C18:cis[9,12]2) decreased,
14-Methylhexadecanoic acid decreased, gamma-Linolenic acid
(C18:cis[6,9,12]3) increased, 16-Methylheptadecanoic acid
increased, Threonic acid decreased, Cytosine increased,
Phosphatidylcholine (C18:0/C22:6) increased.
[0067] Alternatively, the said reference may be, preferably, be
derived from a subject which has not been brought into contact with
Benzylbutyl Phthalate, Fenofibrate, Clofibrate, Fenoforbrate,
Diethylhexylphthalate or Wy 14643 or a subject known to not suffer
from increased peroxisomal proliferation or may be a calculated
reference for the analytes in a population of subjects. If such a
reference is used, identical or similar amounts for the metabolites
in the test sample and the reference are indicative for a substance
useful for treating increased peroxisomal proliferation.
[0068] The term "substance for treating increased peroxisomal
proliferation" refers to compounds which may directly interfere
with the biological mechanisms inducing increased peroxisomal
proliferation referred to elsewhere in this specification.
Substances to be identified by the method of the present invention
may be organic and inorganic chemicals, such as small molecules,
polynucleotides, oligonucleotides, peptides, polypeptides including
antibodies or other artificial or biological polymers. Preferably,
the substances are suitable as drugs, pro-drugs or lead substances
for the development of drugs or pro-drugs.
[0069] It is to be understood that if the methods of the present
invention are to be used for identifying drugs for the therapy of
increased peroxisomal proliferation or for toxicological
assessments of compounds (i.e. determining whether a compound is
capable of inducing increased peroxisomal proliferation), test
samples of a plurality of subjects may be investigated for
statistical reasons. Preferably, the metabolome within such a
cohort of test subjects shall be as similar as possible in order to
avoid differences which are caused, e.g., by factors other than the
compound to be investigated. Subjects to be used for the said
methods are, preferably, laboratory animals such as rodents and
more preferably rats. It is to be understood further that the said
laboratory animals shall be, preferably, sacrificed after
completion of the method of the present invention. All subjects of
a cohort test and reference animals shall be kept under identical
conditions to avoid any differential environmental influences.
Suitable conditions and methods of providing such animals are
described in detail in WO2007/014825. Said conditions are hereby
incorporated by reference.
[0070] The present invention also relates to a data collection
comprising characteristic values for the following analytes
Coenzyme Q10, 16-Methylheptadecanoic acid, 17-Methyloctadecanoic
acid, Eicosatrienoic acid (C20:3), Threonine, Proline, Tyrosine,
trans-4-Hydroxyproline and/or at least the following analytes
Pantothenic acid, Coenzyme Q9, Glycerol, Palmitic acid (C16:0),
Linoleic acid (C18:cis[9,12]2), 14-Methylhexadecanoic acid,
gamma-Linolenic acid (C18:cis[6,9,12]3), 16-Methylheptadecanoic
acid, Threonic acid, Cytosine, Phosphatidylcholine
(C18:0/C22:6).
[0071] The term "data collection" refers to a collection of data
which may be physically and/or logically grouped together.
Accordingly, the data collection may be implemented in a single
data storage medium or in physically separated data storage media
being operatively linked to each other. Preferably, the data
collection is implemented by means of a database. Thus, a database
as used herein comprises the data collection on a suitable storage
medium. Moreover, the database, preferably, further comprises a
database management system. The database management system is,
preferably, a network-based, hierarchical or object-oriented
database management system. Furthermore, the database may be a
federal or integrated database. More preferably, the database will
be implemented as a distributed (federal) system, e.g. as a
Client-Server-System. More preferably, the database is structured
as to allow a search algorithm to compare a test data set with the
data sets comprised by the data collection. Specifically, by using
such an algorithm, the database can be searched for similar or
identical data sets being indicative for increased peroxisomal
proliferation (e.g. a query search). Thus, if an identical or
similar data set can be identified in the data collection, the test
data set will be associated with increased peroxisomal
proliferation. Consequently, the information obtained from the data
collection can be used to diagnose increased peroxisomal
proliferation based on a test data set obtained from a subject.
[0072] Moreover, the present invention pertains to a data storage
medium comprising the said data collection.
[0073] The term "data storage medium" as used herein encompasses
data storage media which are based on single physical entities such
as a CD, a CD-ROM, a hard disk, optical storage media, or a
diskette. Moreover, the term further includes data storage media
consisting of physically separated entities which are operatively
linked to each other in a manner as to provide the aforementioned
data collection, preferably, in a suitable way for a query
search.
[0074] The present invention also relates to a system comprising
[0075] (a) means for comparing characteristic values of metabolites
of a sample operatively linked to [0076] (b) the data storage
medium of the present invention.
[0077] The term "system" as used herein relates to different means
which are operatively linked to each other. Said means may be
implemented in a single device or may be implemented in physically
separated devices which are operatively linked to each other. The
means for comparing characteristic values of analytes operate,
preferably, based on an algorithm for comparison as mentioned
before. The data storage medium, preferably, comprises the
aforementioned data collection or database, wherein each of the
stored data sets being indicative for increased peroxisomal
proliferation. Thus, the system of the present invention allows
identifying whether a test data set is comprised by the data
collection stored in the data storage medium. Consequently, the
system of the present invention may be applied as a diagnostic
means in diagnosing increased peroxisomal proliferation.
[0078] In a preferred embodiment of the system, means for
determining characteristic values of metabolites of a sample are
comprised.
[0079] The term "means for determining characteristic values of
metabolites" preferably relates to the aforementioned devices for
the determination of analytes such as mass spectrometry devices,
NMR devices or devices for carrying out chemical or biological
assays for the analytes.
[0080] The present invention also encompasses a diagnostic
composition comprising at least one, preferably, at least five of
the following analytes Coenzyme Q10, 16-Methylheptadecanoic acid,
17-Methyloctadecanoic acid, Eicosatrienoic acid (C20:3), Threonine,
Proline, Tyrosine, trans-4-Hydroxyproline and/or at least one,
preferably, at least five of the following analytes Pantothenic
acid, Coenzyme Q9, Glycerol, Palmitic acid (C16:0), Linoleic acid
(C18:cis[9,12]2), 14-Methylhexadecanoic acid, gamma-Linolenic acid
(C18:cis[6,9,12]3), 16-Methylheptadecanoic acid, Threonic acid,
Cytosine, Phosphatidylcholine (C18:0/C22:6) or means for the
determination thereof.
[0081] Further encompassed is a diagnostic device comprising [0082]
(a) means for determining characteristic values of at least one,
preferably, at least five of the following analytes Coenzyme Q10,
16-Methylheptadecanoic acid, 17-Methyloctadecanoic acid,
Eicosatrienoic acid (C20:3), Threonine, Proline, Tyrosine,
trans-4-Hydroxyproline and/or at least one, preferably, at least
five of the following analytes Pantothenic acid, Coenzyme Q9,
Glycerol, Palmitic acid (C16:0), Linoleic acid (C18:cis[9,12]2),
14-Methylhexadecanoic acid, gamma-Linolenic acid
(C18:cis[6,9,12]3), 16-Methylheptadecanoic acid, Threonic acid,
Cytosine, Phosphatidylcholine (C18:0/C22:6); and [0083] (b) means
for diagnosing liver toxicicty based on the characteristic values
determined by the means of (a).
[0084] The term "diagnostic means", preferably, relates to a
diagnostic device, system or biological or chemical assay as
specified elsewhere in the description in detail.
[0085] The expression "means for determining characteristic values
of a group of metabolites" refers to devices or agents which are
capable of specifically recognizing the metabolite(s). Suitable
devices may be spectrometric devices such as mass spectrometry, NMR
devices or devices for carrying out chemical or biological assays
for the metabolites. Suitable agents may be compounds which
specifically detect the metabolites. Detection as used herein may
be a two-step process, i.e. the compound may first bind
specifically to the metabolite to be detected and subsequently
generate a detectable signal, e.g., fluorescent signals,
chemiluminescent signals, radioactive signals and the like. For the
generation of the detectable signal, further compounds may be
required which are all comprised by the term "means for determining
characteristic values of a group of metabolites". Compounds which
specifically bind to the metabolite are described elsewhere in the
specification in detail and include, preferably, enzymes,
antibodies, ligands, receptors or other biological molecules or
chemicals which specifically bind to the metabolites.
[0086] All references referred to above are herewith incorporated
by reference with respect to their entire disclosure content as
well as their specific disclosure content explicitly referred to in
the above description.
[0087] The FIGURE shows a fragmentation pattern of
Phosphatidylcholine (C18:0/C22:6).
[0088] The following Examples are merely for the purposes of
illustrating the present invention. They shall not be construed,
whatsoever, to limit the scope of the invention in any respect.
Example
Biomarkers Associated with Increased Peroxisomal Proliferation
[0089] A group of each 5 male and female rats was dosed once daily
with the indicated compounds (see tables below) at 10 and 100 mg/kg
body weight per gavage over 28 days. Additional groups of each 5
male and female animals served as controls. Before starting the
treatment period, animals, which were 62-64 days old when supplied,
were acclimatized to the housing and environmental conditions for 7
days. All animals of the animal population were kept under the same
constant temperature (20-24.+-.3.degree. C.) and the same constant
humidity (30-70%). Each animal of the animal population was kept in
a separate cage. The animals of the animal population are fed ad
libitum. The food to be used was be essentially free of chemical or
microbial contaminants. Drinking water was also offered ad libitum.
Accordingly, the water was be free of chemical and microbial
contaminants as laid down in the European Drinking Water Directive
98/83/EG. The illumination period was 12 hours light followed by 12
hours darkness (12 hours light, from 6:00 to 18:00, and 12 hours
darkness, from 18:00 to 6:00).
[0090] In the morning of day 7, 14, and 28, blood was taken from
the retroorbital venous plexus from fasted anaesthetized animals.
From each animal, 1 ml of blood was collected with EDTA as
anticoagulant. The samples were be centrifuged for generation of
plasma. All plasma samples were covered with a N2 atmosphere and
then stored at -80.degree. C. until analysis.
[0091] For mass spectrometry-based metabolite profiling analyses
plasma samples were extracted and a polar and a non-polar fraction
was obtained. For GC-MS analysis, the non-polar fraction was tested
with methanol under acidic conditions to yield the fatty acid
methyl esters. Both fractions were further derivatised with
O-methyl-hydroxyamine hydrochloride and pyridine to convert
Oxo-groups to O-metyloximes and subsequently with a silyating agent
before hydrolysis. In LCMS analysis, both fractions were
reconstituted in appropriate solvent mixtures. HPLC was performed
by gradient elution on reversed phase separation columns. For mass
spectrometric detection metanomics proprietary technology was
applied which allows target and high sensitivity MRM (Multiple
Reaction Monitoring) profiling in parallel to a full screen
analysis.
[0092] Following comprehensive analytical validation steps, the
data for each analyte were normalized against data from pool
samples. These samples were run in parallel through the whole
process to account for process variability. The significance of
treatment group values specific for sex, dose group and metabolite
was determined by comparing means of the treated groups to the
means of the respective untreated control groups using Student's
t-test. Normalized treatment group values and their significance
were fed into a database for further statistics and data mining
processes.
[0093] The changes of the group of plasma metabolites being
indicative for increased peroxisomal proliferation after treatment
of the rats are shown in the following tables:
TABLE-US-00001 TABLE 1 Markers for peroxisomal proliferators in
male rats Benzylbutyl Phthalate Clofibrate (MOA6) (MOA50)
Metabolite Direction mh7 mh14 mh28 mh7 mh14 mh28 Coenzyme Q10 down
0.51 0.34 0.27 0.45 0.60 0.47 16-Methylheptadecanoic acid down 0.24
0.31 0.20 0.24 0.21 0.28 17-Methyloctadecanoic acid down 0.37 0.28
0.30 0.35 0.27 0.37 Eicosatrienoic acid (C20:3) up 2.56 3.12 4.12
1.65 1.72 2.54 Threonine down 0.55 0.69 0.81 0.68 1.02 1.06 Proline
down 0.74 0.85 0.84 0.78 0.80 0.87 Tyrosine down 0.74 0.83 0.97
0.87 0.95 1.02 trans-4-Hydroxyproline down 0.69 0.65 0.83 0.77 0.64
0.57
TABLE-US-00002 TABLE 2 Markers for peroxisomal proliferators in
female rats Benzylbutyl Phthalate Clofibrate (MOA6) (MOA50)
Metabolite Direction fh7 fh14 fh28 fh7 fh14 fh28 Pantothenic acid
up 1.82 2.06 2.42 1.07 1.75 1.22 Coenzyme Q9 up 1.44 1.50 1.78 1.86
1.64 2.55 Glycerol, lipid fraction up 1.15 1.48 2.43 1.39 1.64 4.99
Palmitic acid (C16:0) up 1.48 1.79 1.94 1.05 1.38 2.31
Gamma-Linolenic acid up 1.98 1.64 2.08 2.04 1.88 7.00
(C18:cis[6,9,12]3) 16-Methylheptadecanoic acid down 0.55 0.66 0.85
0.55 0.75 0.75 17-Methyloctadecanoic acid down 0.78 0.64 0.69 0.48
0.57 0.77 Threonic acid up 1.20 1.53 1.74 1.23 1.30 1.30 Cytosine
down 0.77 0.63 0.79 0.87 0.86 1.00 Phosphatidylcholine
(C18:0/C22:6) down 0.59 0.86 0.81 0.88 0.71 0.53
* * * * *