U.S. patent application number 12/942280 was filed with the patent office on 2011-05-12 for methods to evaluate cytochrome p450 2c19 isoenzyme activity using a breath test.
This patent application is currently assigned to Cambridge Isotope Laboratories, Inc.. Invention is credited to Zeruesenay Desta, David A. Flockhart, Yasuhisa Kurogi, Anil S. Modak.
Application Number | 20110111510 12/942280 |
Document ID | / |
Family ID | 39136939 |
Filed Date | 2011-05-12 |
United States Patent
Application |
20110111510 |
Kind Code |
A1 |
Flockhart; David A. ; et
al. |
May 12, 2011 |
METHODS TO EVALUATE CYTOCHROME P450 2C19 ISOENZYME ACTIVITY USING A
BREATH TEST
Abstract
The present invention relates, generally to a method of
determining and assessing cytochrome P450 2C19-related (CYP2C19)
metabolic capacity in an individual mammalian subject via a breath
assay, by determining the relative amount of .sup.13CO.sub.2
exhaled by the subject upon intravenous or oral administration of a
.sup.13C-labeled CYP2C19 substrate compound. The present invention
is useful as anon-invasive, in vivo assay for evaluating CYP2C19
enzyme activity in a subject using the metabolite .sup.13CO.sub.2
in expired breath, to phenotype individual subjects and to
determine the selection, optimal dosage and timing of drug
administration.
Inventors: |
Flockhart; David A.;
(Indianapolis, IN) ; Desta; Zeruesenay; (Carmel,
IN) ; Modak; Anil S.; (Haverhill, MA) ;
Kurogi; Yasuhisa; (North Andover, MA) |
Assignee: |
Cambridge Isotope Laboratories,
Inc.
|
Family ID: |
39136939 |
Appl. No.: |
12/942280 |
Filed: |
November 9, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11848750 |
Aug 31, 2007 |
7833744 |
|
|
12942280 |
|
|
|
|
60841999 |
Sep 1, 2006 |
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Current U.S.
Class: |
436/56 ;
546/273.7 |
Current CPC
Class: |
Y10T 436/204998
20150115; A61K 51/0455 20130101; G01N 33/497 20130101; Y10T 436/13
20150115; A61K 49/00 20130101 |
Class at
Publication: |
436/56 ;
546/273.7 |
International
Class: |
G01N 37/00 20060101
G01N037/00; C07D 401/12 20060101 C07D401/12 |
Claims
1-29. (canceled)
30. A method of selecting a mammalian subject for inclusion in a
clinical trial for determining the efficacy of a compound to
prevent or treat a medical condition, comprising the steps of: (a)
administering a .sup.13C-labeled cytochrome P450 2C19 isoenzyme
substrate compound to the subject; (b) measuring a metabolite
excretion pattern of an isotope-labeled metabolite excreted from
the body of the subject; (c) comparing the obtained metabolite
excretion pattern in the subject to a reference standard excretion
pattern; (d) classifying the subject according to a metabolic
phenotype selected from the group consisting of: poor metabolizer,
intermediate metabolizer, extensive metabolizer, and ultrarapid
metabolizer, based on the obtained metabolite excretion pattern;
and (e) selecting the subject classified as extensive metabolizer
in step (d) for inclusion in the clinical trial.
31. The method according to claim 30, wherein the isotope-labeled
metabolite excreted from the body of the subject is isotope-labeled
CO.sub.2 in the expired air.
32. A kit comprising a .sup.13C-labeled cytochrome P450 2C19
isoenzyme substrate compound and instructions provided with the
substrate that describe how to determine .sup.13C-labeled
cytochrome P450 2C19 isoenzyme substrate compound metabolism in a
subject.
33. The kit according to claim 32, further comprising at least
three breath collection bags.
34. The kit of according to claim 32, further comprising a
cytochrome P450 2C19 modulating agent.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 11/848,750, filed Aug. 31, 2007, which claims the benefit
of provisional application Ser. No. 60/841,999, filed Sep. 1, 2006,
the contents of which are incorporated herewith by reference in
their entireties.
BACKGROUND OF THE INVENTION
[0002] The CYP2C19 gene is mapped to chromosome 10 (10q24.1-q24.3)
and contains nine exons that code for a microsomal protein
consisting of 490 amino acids. CYP219 is expressed primarily in
human liver, and to a smaller extent in several extra-hepatic
tissues (e.g., gut-wall). Hepatic cytochrome P450 (CYP) 2C19 is an
important enzyme in the metabolism of widely used drugs such as the
proton pump inhibitors (omeprazole, esomeprazole, lansoprazole and
pantoprazole), diazepam, phenyloin, proguanil, clopidogrel,
voriconazole, nelfinavir and cyclophosphamide (Desta, Z. et al.,
Clin. Pharmacokinet., 41:913-58, 2002). As a result of genetic
polymorphisms in the CYP2C19 gene and nongenetic factors (e.g.,
drug interactions), wide interindividual variability is seen in the
in vitro as well as in vivo activity of CYP2C19. This variability
accounts for a significant part of the substantial differences in
the clearance and response to the large number of drugs metabolized
by this enzyme. Therefore, identification of the mechanisms and
causes for interindividual variability in CYP2C19 activity and
developing means to prospectively predict them is important to
optimize therapy with its substrates.
[0003] Conventional medical approaches to diagnosis and treatment
of disease is based on clinical data alone, or made in conjunction
with a diagnostic test. These traditional practices often lead to
therapeutic choices that are not optimal for the efficacy of the
prescribed drug therapy or to minimize the likelihood of side
effects for an individual subject. Therapy-specific diagnostics
(a.k.a., theranostics) is an emerging medical technology field that
provides tests useful to diagnose a disease, choose the correct
treatment regime, and monitor a subject's response. That is,
theranostics are useful to predict and assess drug response in
individual subjects, i.e., individualized medicine. Theranostic
tests are useful to select subjects for treatments that are
particularly likely to benefit from the treatment or to provide an
early and objective indication of treatment efficacy in individual
subjects, so that the treatment can be altered with a minimum of
delay. Theranostic tests may be developed in any suitable
diagnostic testing format, which include, but is not limited to,
e.g., non-invasive breath tests, immunohistochemical tests,
clinical chemistry, immunoassay, cell-based technologies, and
nucleic acid tests.
[0004] There is a need in the art for a reliable theranostic test
to define a subject's phenotype or the drug metabolizing capacity
to enable physicians to individualize therapy thereby avoiding
potential drug related toxicity in poor metabolizers and increasing
efficacy. Accordingly, there is a need in the art to develop new
diagnostic assays useful to assess the metabolic activity of drug
metabolizing enzymes such as the cytochrome P450 enzymes (CYPs) in
order to determine individual optimized drug selection and
dosages.
SUMMARY OF THE INVENTION
[0005] The present invention relates to a diagnostic, noninvasive,
in vivo phenotype test to evaluate CYP2C19 activity using a CYP2C19
substrate compound labeled with a detectable isotope incorporated
at least at one specific position. The present invention utilizes
the CYP2C19 enzyme-substrate interaction such that there is release
of, for example, stable isotope-labeled CO.sub.2 (e.g.,
.sup.13CO.sub.2) in the expired breath of a mammalian subject. The
subsequent quantification of stable isotope-labeled CO.sub.2 allows
for the indirect determination of pharmacokinetics of the substrate
and the evaluation of CYP2C19 enzyme activity (i.e.,
CYP2C19-related metabolic capacity).
[0006] In one aspect, the invention is directed to a preparation
for determining CYP2C19-related metabolic capacity, comprising as
an active ingredient a CYP2C19 substrate compound in which at least
one of the carbon or oxygen atoms is labeled with an isotope,
wherein the preparation is capable of producing isotope-labeled
CO.sub.2 after administration to a mammalian subject. In one
embodiment of the preparation, the isotope is at least one isotope
selected from the group consisting of: .sup.13C; .sup.14C; and
.sup.18O.
[0007] In another aspect, the invention is directed to a method for
determining cytochrome P450 2C19 isoenzyme-related metabolic
capacity, comprising administering to a mammalian subject a
preparation comprising as an active ingredient a cytochrome P450
2C19 isoenzyme substrate compound in which at least one of the
carbon or oxygen atoms is labeled with an isotope, wherein the
preparation is capable of producing isotope-labeled CO.sub.2 after
administration to a mammalian subject, and measuring the excretion
pattern of an isotope-labeled metabolite excreted from the body of
the subject. In a particular embodiment, the isotope-labeled
metabolite is excreted from the body as isotope-labeled CO.sub.2 in
the expired air.
[0008] In another embodiment, the invention is directed to a method
for determining cytochrome P450 2C19 isoenzyme-related metabolic
capacity in a mammalian subject, comprising administering to the
mammalian subject a preparation comprising as an active ingredient
a cytochrome P450 2C19 isoenzyme substrate compound in which at
least one of the carbon or oxygen atoms is labeled with an isotope,
wherein the preparation is capable of producing isotope-labeled
CO.sub.2 after administration to a mammalian subject, measuring the
excretion pattern of an isotope-labeled metabolite excreted from
the body of the subject, and assessing the obtained excretion
pattern in the subject. In a particular embodiment, the method
further comprises comparing the obtained excretion pattern in the
subject or a pharmacokinetic parameter obtained therefrom with the
corresponding excretion pattern or parameter in a healthy subject
with a normal cytochrome P450 2C19 isoenzyme-related metabolic
capacity.
[0009] In another embodiment, the invention is directed to a method
for determining the existence, nonexistence or degree of cytochrome
P450 2C19 isoenzyme-related metabolic disorder in a mammalian
subject, comprising administering to the mammalian subject a
preparation comprising as an active ingredient a cytochrome P450
2C19 isoenzyme substrate compound in which at least one of the
carbon or oxygen atoms is labeled with an isotope, wherein the
preparation is capable of producing isotope-labeled CO.sub.2 after
administration to a mammalian subject, measuring the excretion
pattern of an isotope-labeled metabolite excreted from the body of
the subject, and assessing the obtained excretion pattern in the
subject.
[0010] In yet another embodiment, the invention is directed to a
method for selecting a prophylactic or therapeutic treatment for a
subject, comprising: (a) determining the phenotype of the subject;
(b) assigning the subject to a subject class based on the phenotype
of the subject; and (c) selecting a prophylactic or therapeutic
treatment based on the subject class, wherein the subject class
comprises two or more individuals who display a level of cytochrome
P450 2C19 isoenzyme-related metabolic capacity that is at least
about 10% lower than a reference standard level of cytochrome P450
2C19 isoenzyme-related metabolic capacity. In a particular
embodiment, the subject class comprises two or more individuals who
display a level of cytochrome P450 2C19 isoenzyme-related metabolic
capacity that is at least about 10% higher than a reference
standard level of cytochrome P450 2C19 isoenzyme-related metabolic
capacity. In another embodiment, the subject class comprises two or
more individuals who display a level of cytochrome P450 2C19
isoenzyme-related metabolic capacity within at least about 10% of a
reference standard level of cytochrome P450 2C19 isoenzyme-related
metabolic capacity. In one embodiment, the treatment is selected
from the group consisting of: administering a drug, selecting a
drug dosage and selecting the timing of a drug administration.
[0011] In another embodiment, the invention is directed to a method
for evaluating cytochrome P450 2C19 isoenzyme-related metabolic
capacity, comprising administering a .sup.13C-labeled cytochrome
P450 2C19 isoenzyme substrate compound to a mammalian subject,
measuring .sup.13CO.sub.2 exhaled by the subject, and determining
cytochrome P450 2C19 isoenzyme-related metabolic capacity from the
measured .sup.13CO.sub.2. In one embodiment, the .sup.13C-labeled
cytochrome P450 2C19 isoenzyme substrate compound is a
.sup.13C-labeled pantoprazole or .sup.13C-labeled omeprazole. In
one embodiment, the .sup.13C-labeled cytochrome P450 2C19 isoenzyme
substrate compound is administered non-invasively, intravenously or
orally. In one embodiment, the exhaled .sup.13CO.sub.2 is measured
spectroscopically, e.g., by infrared spectroscopy, or with a mass
analyzer. In a particular embodiment, the exhaled .sup.13CO.sub.2
is measured over at least three time periods to generate a dose
response curve, and the cytochrome 2C19 isoenzyme-related metabolic
activity is determined from the area under the curve. In another
embodiment, the exhaled .sup.13CO.sub.2 is measured over at least
three time periods to calculate a delta over baseline (DOB), and
the cytochrome 2C19 isoenzyme-related metabolic activity is
determined from the DOB. In another embodiment, the exhaled
.sup.13CO.sub.2 is measured over at least three time periods to
calculate a percentage dose recovery (PDR), and the cytochrome 2C19
isoenzyme-related metabolic activity is determined from the PDR. In
one embodiment, the exhaled .sup.13CO.sub.2 is measured over at
least two different dosages of the .sup.13C-labeled cytochrome P450
2C19 isoenzyme substrate compound. In another embodiment, the
exhaled .sup.13CO.sub.2 is measured during at least the following
time points: t.sub.0, a time prior to ingesting the
.sup.13C-labeled cytochrome P450 2C19 isoenzyme substrate compound;
t.sub.1, a time after the .sup.13C-labeled cytochrome P450 2C19
isoenzyme substrate compound has been absorbed in the bloodstream
of the subject; and t.sub.2, a time during the first elimination
phase. In one embodiment, the cytochrome P450 2C19
isoenzyme-related metabolic capacity is determined from as the a
slope of .delta..sup.13CO.sub.2 at time points t.sub.1 and t.sub.2
calculated according to the following equation:
slope=[(.delta..sup.13CO.sub.2).sub.2-(.delta..sup.13CO.sub.2).sub.1]/(t.-
sub.2-t.sub.1) wherein .delta..sup.13CO.sub.2 is the amount of
exhaled .sup.13CO.sub.2. In a particular embodiment, at least one
cytochrome P450 2C19 isoenzyme modulating agent, e.g., a cytochrome
P450 2C19 inhibitor or a cytochrome P450 2C19 inducer, is
administered to the subject before administrating a
.sup.13C-labeled cytochrome P450 2D6 isoenzyme substrate
compound.
[0012] In another embodiment, the invention is directed to a method
of selecting a mammalian subject for inclusion in a clinical trial
for determining the efficacy of a compound to prevent or treat a
medical condition, comprising: (a) administering a .sup.13C-labeled
cytochrome P450 2C19 isoenzyme substrate compound to the subject;
(b) measuring a metabolite excretion pattern of an isotope-labeled
metabolite excreted from the body of the subject; (c) comparing the
obtained metabolite excretion pattern in the subject to a reference
standard excretion pattern; (d) classifying the subject according
to a metabolic phenotype selected from the group consisting of:
poor metabolizer, intermediate metabolizer, extensive metabolizer,
and ultrarapid metabolizer, based on the obtained metabolite
excretion pattern; and (e) selecting the subject classified as
extensive metabolizer in step (d) for inclusion in the clinical
trial. In a particular embodiment, the isotope-labeled metabolite
excreted from the body of the subject is isotope-labeled CO.sub.2
in the expired air.
[0013] In another embodiment, the invention is directed to a kit
comprising a .sup.13C-labeled cytochrome P450 2C19 isoenzyme
substrate compound and instructions provided with the substrate
that describe how to determine .sup.13C-labeled cytochrome P450
2C19 isoenzyme substrate compound metabolism in a subject. In a
particular embodiment, the kit further comprises at least three
breath collection bags. In another embodiment, the kit further
comprises a cytochrome P450 2C19 modulating agent.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 shows breath curves (delta over baseline (DOB) versus
time (left panel) and percentage dose recovery (PDR) curves versus
time (right panel) for eight volunteers for the Ptz
O.sup.13CH.sub.3 breath test.
[0015] FIG. 2 shows breath curves for ten subjects who were *1*1
(EM), nine subjects who were either *1*2 or *1*3 (IM) and one
subject who was *2*2 (PM).
DETAILED DESCRIPTION OF THE INVENTION
[0016] The discovery of the first poor metabolizer (PM) of
mephenyloin in 1979 and the subsequent demonstration of the genetic
basis for this phenotype have spurred extensive research into the
genetic basis for the interindividual variations of (S)-mephenyloin
4-hydroxylation (CYP2C19) and the role that this enzyme plays in
human drug metabolism. On the basis of the ability to metabolize
(S)-mephenyloin (or other CYP2C19 substrates), individuals can be
classified as extensive metabolizers (EMs) or poor metabolisers
(PMs) of CYP2C19. It is now established that the high
interindividual and interethnic variability in the pharmacokinetics
of CYP2C19 substrates is mainly due to polymorphisms in the CYP2C19
gene (Goldstein, J., Br. J. Clin. Pharmacol., 52:349-55, 2001).
Over 25 alleles and allelic variants of CYP2C19 have been
described. Most of these variants are null alleles (e.g.,
CYP2C19*2, *3, *4, *5, *6, *7 and *8) that result in complete
absence of enzyme function (De Morais, S. et al., Mol. Pharmacol.,
46:594-8, 1994; De Morais, S. et al., J. Biol. Chem., 1994;
269:15419-22; Ibeanu, G. et al., J. Pharmacol. Exp. Ther.,
286:1490-5, 1998; Ibeanu, G. et al., Pharmacogenetics, 8:129-35,
1998; Ibeanu, G. et al., J. Pharmacol. Exp. Ther., 290:635-40,
1999; Ferguson, R. et al., J. Pharmacol. Exp. Ther., 284:356-61,
1998) while others result in either reduced (e.g., *9, *11, *13) or
increased activity (CYP2C19*17) (Sim, S. et al., Clin. Pharmacol.
Ther., 79:103-13, 2006). Of these variants, the most frequent and
functionally relevant alleles are CYP2C19*2 (CYP2C19 ml) and
CYP2C19*3 (CYP2C19 m2). In both variants, a premature stop codon
resulting in truncated and inactive enzyme or a truncated protein
that is unable to bind to the haem moiety is produced. CYP2C19*2 is
a single base pair G681A mutation in exon 5 of CYP2C19. CYP2C19*3
is a single base pair G636A mutation in exon 4 of CYP2C19 that
results in a premature stop codon. Several studies indicate that
the distribution of these common alleles is different among
different populations. For example, the frequency of CYP2C19*2 has
been reported to be .about.17%, 30% and .about.15% in
African-Americans, Chinese and Caucasians, respectively, and the
CYP2C19*3 allele was shown to be more frequent in Asians
(.about.5%) than in Caucasians (0.04%) and Blacks (0.4%). These two
alleles account for almost all PMs in Asian and Black African
populations. CYP2C19*2 accounts for approximately 75 to 85% of
variant alleles responsible for PMs in Asians and Caucasians. While
CYP2C19*3 is extremely rare in Caucasian populations, it accounts
for almost all the remaining defective alleles in Asians.
Accordingly, there is a considerable interethnic heterogeneity in
the frequency of PMs. Population distribution of CYP2C19 poor
metabolizers (determined by phenotype and genotype) show that the
Asian population had a higher incidence of CYP2C19 poor
metabolizers (up to 25% in some studies), while in other
populations except Vanuatu islands in eastern Melanesia (38-70%) it
is below 8% (Desta, Z. et al., Clin. Pharmacokinet., 41:913-58,
2002).
[0017] In addition to genetic polymorphisms, other factors
contribute to the interindividual variability in activity of
CYP2C19. These factors include exposure to inhibitor or inducer
drugs or other chemicals influence CYP2C19 activity. For example,
drugs such as omeprazole, meclobemide, ticlopidine, clopidogrel,
fluvoxamine and oral contraceptives are known to significantly
impair CYP2C19 activity, while rifampin, efavirenz, ritonavir and
St. John's wort enhance the clearance of its substrates. In
addition, age, pregnancy, cancer, liver and inflammatory diseases
appear to reduce the activity of CYP2C19 activity in vivo in
humans.
[0018] Many therapeutic compounds are effective in about 30-60% of
patients with the same disease. Further, a subset of these patients
may suffer severe side effects, which are among the leading cause
of death in the United States and have an estimated $100 billion
annual economic impact (Lazarou, J. et al., J. Amer. Med. Assoc.,
279:1200-1205, 1998). Many studies have shown that patients differ
in their pharmacological and toxicological reactions to drugs due,
at least in part, to genetic polymorphisms that contribute to the
relatively high degree of uncertainty inherent in the treatment of
individuals with a drug. Single nucleotide polymorphisms
(SNPs)--variations in DNA at a single base that are found in at
least 1% of the population--are the most frequent polymorphisms in
the human genome. Such subtle changes in the primary nucleotide
sequence of a gene encoding a pharmaceutically-important protein
may be manifested as significant variation in expression, structure
and/or function of the protein.
[0019] CYP 2C19 is involved in the biotransformation of close to 30
therapeutic drugs including proton pump inhibitors as summarized in
Table 1. Isotopic labeling of the CYP2C19 substrates of Table 1
such that administration of the isotope-labeled substrate to a
subject results in the release of stable isotopically-labeled
.sup.13CO.sub.2 yields compounds useful in the methods of the
present invention.
TABLE-US-00001 TABLE 1 2C19 substrates 2C19 inhibitors 2C19
inducers amitriptyline cimetidine carbamazepine chloramphenicol
felbamate noretbindrone citalopram fluoxetine prednisone
clomipramine fluvoxamine rifampin cyclophosphamide indomethacin
diazepam ketoconazole E-3810 lansoprazole escitalopram modafinil
hexobarbital omeprazole imipramine probenicid indomethacin
ticlopidine lansoprazole topiramate S-mephenytoin R-mephobarbital
moclobemide nelfinavir nilutamide omeprazole oxcarbazepine
pantoprazole phenytoin phenobarbitone primidone progesterone
proguanil propranolol rabeprazole r-warfarin teniposide
[0020] Select agents can induce or inhibit CYP2C19 activity (i.e.,
CYP 2C19 modulating agents). CYP modulating agents are important in
the present invention as they can induce (increase) or inhibit
(decrease) CYP 2C19 enzyme activity. Compounds known to inhibit and
induce CYP 2C19 are summarized in Table 1.
[0021] It is to be appreciated that certain aspects, modes,
embodiments, variations and features of the invention are described
below in various levels of detail to provide a substantial
understanding of the present invention. The present invention
relates to a diagnostic, noninvasive, in vivo phenotype test to
evaluate CYP2C19 activity, using a CYP2C19 substrate compound
labeled with isotope incorporated at least at one specific
position. The present invention utilizes the CYP2C19
enzyme-substrate interaction such that there is release of stable
isotope-labeled CO.sub.2 (e.g., .sup.13CO.sub.2) in the expired
breath of a mammalian subject. The subsequent quantification of
stable isotope-labeled CO.sub.2 allows for the indirect
determination of pharmacokinetics of the substrate and the
evaluation of CYP2C19 enzyme activity (i.e., CYP2C19-related
metabolic capacity based on the oral or i.v. administration of a
stable isotope .sup.13C-labeled CYP2C19 substrate compound and
measurement of the .sup.13CO.sub.2/.sup.12CO.sub.2 ratio in expired
breath using commercially available instrumentation, e.g., mass or
infrared (IR) spectrometers.
[0022] The clinical relevance of CYP2C19 genetic polymorphisms has
been recently demonstrated. It is well established that in EMs
approximately 80% of doses of the proton pump inhibitors (PPIs)
such as omeprazole, lansoprazole and pantoprazole are cleared by
CYP2C19; about six-fold higher exposure to these drugs is observed
in PMs than in EMs of CYP2C19 and as a result PMs achieve greater
acid suppression (Furuta, T. et al., Clin. Pharmacol. Ther.,
65:552-561, 1999). The first report that CYP2C19 genotyping might
influence clinical response to PPIs came from Furuta et al., who
showed that treatment with omeprazole 20 mg/day plus amoxicillin
brought about eradication of H. pylori infection in 28.6, 60 and
100% of homozygous EMs, heterozygous EMs and PMs of CYP2C19,
respectively, and that this cure rate was parallel with the healing
rates for both duodenal and gastric ulcers in the respective three
groups (Furuta, T. et al., Ann. Intern. Med., 129:1027-30, 1998).
Several subsequent studies have established that patients who carry
CYP2C19 genetic variants respond better than those who carry the
wild-type allele (Furuta, T. et al., Drug Metab. Pharmacokinet.,
20:153-67, 2005). Therefore, genotyping for the common alleles of
CYP2C19 before initiating PPIs for the treatment of reflux disease
and H. pylori infection has been suggested to be a cost effective
tool to select appropriate duration of treatment and dosage
regimens.
[0023] Other drugs that are significantly influenced by CYP2C19
genotype include the anti-platelet drug clopidogrel and the
anticancer drug cyclophosphamide. Clopidogrel is a widely used drug
in the prevention and treatment of thrombotic complications
following stroke, unstable angina, myocardial infarction and
coronary stent placement. Clopidogrel is a prodrug that requires
conversion to active metabolite by the CYP system before it exerts
its anti-platelet effects. The lack or diminished anti-platelet
response in a substantial number of patients receiving clopidogrel
is a major problem during its clinical use.
[0024] A recent report indicate that clopidogrel had no
anti-platelet effect in healthy volunteers who carry CYP2C19*2
compared to those who carry CYP2C19*1*1 genotype (Hulot, J. et al.,
Blood, 108:2244-7, 2006). This observation implies clopidogrel
nonresponsive platelets are at risk for thrombotic events, with
devastating outcome to the patient. Similarly, cyclophosphamide is
a prodrug that requires metabolic activation by cytochrome P450
(CYP) enzymes to 4-hydroxycyclophosphamide. Multiple CYPs have been
implicated in this activation (Roy, P. et al., Drug Metab. Dispos.,
27:655-66, 1997; Rae, J. et al., Drug Metab. Dispos., 30:525-30,
2002), including CYP2A6, 2B6, 2C19, 2C9, 3A4, and 3A5, but CYP2C19
appears to be a key enzyme particularly at low cyclophosphamide
concentrations. For lupus nephritis patients taking
cyclophosphamide, it was found that CYP2C19*2 is a predictor of
premature ovarian failure and progression to end-stage renal
failure (Takada, K. et al., Arthritis Rheum., 50:2202-10,
2004).
[0025] Given the clinical relevance of this enzyme, it would be
important to develop a diagnostic test that identifies and predicts
a subgroup of patients who are at high-risk for failure of therapy
during initiation of CYP2C19 substrates such as clopidogrel,
cyclophosphamide and proton-pump inhibitors. Reliable genotype
tests are now commercially available to assay CYP2C19 variants
(e.g., Roche AmpliChip), but they do not capture differences in
CYP2C19 activity fully, as non-genetic factors have important
effects on CYP2C19 activity in vivo in humans. In vivo phenotypic
tests that can capture both genetic and non-genetics causes of
variable CYP2C19 function have been instrumental to better
understand the role of genetic and nongenetic factors on CYP2C19
function (e.g., S-mephenyloin 4-hydroxylation or omeprazole
5-hydroxylation) (Desta, Z. et al., Clin. Pharmacokinet.,
41:913-58, 2002). Their use for dose adjustment or selection of
appropriate drugs, however, is very limited in part because they
are often expensive, time and resource intensive and invasive
(requires pharmacokinetic sampling). Therefore, a quick, simple and
noninvasive method that can assess CYP2C19 phenotype is needed
prior to prescribing of CYP2C19 substrate drugs.
[0026] The mammalian liver plays a primary role in the metabolism
of steroids, the detoxification of drugs and xenobiotics, and the
activation of procarcinogens. The liver contains enzyme systems,
e.g., the CYP system, which converts a variety of chemicals to more
soluble products. The CYPs are among the major constituent proteins
of the liver mixed function monooxygenases. There are a number of
classes of CYPs that include the hepatic isoenzymes, e.g., CYP3As
(40-60% hepatic P-450 isoenzymes); CYP2D6 (2-5% hepatic P-450
isoenzymes), CYP1A2, CYP2Cs. The action of CYPs facilitates the
elimination of drugs and toxins from the body. Indeed, CYP action
is often the rate-limiting step in pharmaceutical elimination. CYPs
also play a role in the conversion of prodrugs to their
biologically active metabolites.
[0027] The CYPs are quantitatively the most important phase I drug
biotransformation enzymes and genetic variation of several members
of this gene superfamily has been examined. In phase I metabolism
of drugs and environmental pollutants, CYPs often modify substrate
with one or more water-soluble groups (such as hydroxyl), thereby
rendering it vulnerable to attack by the phase II conjugating
enzymes. The increased water-solubility of phase I and especially
phase II products permits ready excretion. Consequently, factors
that lessen the activity of CYPs usually prolong the effects of
pharmaceuticals, whereas factors that increase CYP activity have
the opposite effect.
[0028] Since the use of the naturally occurring .sup.13C compound
.sup.13C-glucose for a metabolic study in humans was described in
1973 (Lacroix, M. et al., Science, 181:445-6, 1973), stable isotope
.sup.13C-labeled compounds have been widely used as diagnostic
probes in research laboratories for over 30 years (Modak, Breath
Analysis For Clinical Diagnosis and Therapeutic Monitoring. A.
Amann, D Smith (ed). pp 457-478. World Scientific, Singapore
(2005). 2005). Subsequent to the wide availability of low cost
.sup.13C-substrates and the development of non-dispersive isotope
selective IR spectrometry (NDIRS) as opposed to previous
quantitation methods (e.g., mass spectrometry detection or other
complicated analytical assays), diagnostic .sup.13C stable isotope
probes are increasingly used to provide precise evaluations of the
presence or absence of etiologically, significant changes in
metabolism due to a specific disease or the lack of a specific
enzyme but also in assessing the metabolic status of drug
metabolizing enzymes.
Select Clinical Applications of the Method of the Invention
[0029] 1. Correlating a Subject to a Standard Reference
Population
[0030] One aspect of the invention relates to diagnostic assays for
determining CYP2C19-related metabolic capacity, in the context of a
biological sample (e.g., expired air) to thereby determine whether
an individual is afflicted with a disease or disorder, or is at
risk of developing a disorder, associated with aberrant CYP2C19
expression or activity. To deduce a correlation between clinical
response to a treatment and a gene expression pattern or phenotype,
it is necessary to obtain data on the clinical responses exhibited
by a population of individuals who received the treatment, i.e., a
clinical population. This clinical data can be obtained by
retrospective analysis of the results of a clinical trial.
Alternatively, the clinical data can be obtained by designing and
carrying out one or more new clinical trials. The analysis of
clinical population data is useful to define a standard reference
population, which, in turn, are useful to classify subjects for
clinical trial enrollment or for selection of therapeutic
treatment. It is preferred that the subjects included in the
clinical population have been graded for the existence of the
medical condition of interest, e.g., CYP2C19 PM phenotype, CYP2C19
IM phenotype, CYP2C19 EM phenotype, or CYP2C19 UM phenotype.
Grading of potential subjects can include, e.g., a standard
physical exam or one or more tests such as the breath test of the
present invention. Alternatively, grading of subjects can include
use of a gene expression pattern, e.g., CYP2C19 allelic variants.
For example, gene expression pattern is useful as grading criteria
where there is a strong correlation between gene expression pattern
and phenotype or disease susceptibility or severity.
[0031] ANOVA is used to test hypotheses about whether a response
variable is caused by, or correlates with, one or more traits or
variables that can be measured. Such standard reference population
comprising subjects sharing gene expression pattern profile and/or
phenotype characteristics, are useful in the methods of the present
invention to compare with the measured level of CYP2C19-related
metabolic capacity or CYP2C19 metabolite excretion pattern in a
given subject. In one embodiment, a subject is classified or
assigned to a particular genotype group or phenotype class based on
similarity between the measured expression pattern of CYP2C19
metabolite and the expression pattern of CYP2C19 metabolite
observed in a reference standard population. The method of the
present invention is useful as a diagnostic method to identify an
association between a clinical response and a genotype or haplotype
(or haplotype pair) for the CYP2C19 gene or a CYP2C19 phenotype.
Further, the method of the present invention is useful to determine
those individuals who will or will not respond to a treatment, or
alternatively, who will respond at a lower level and thus may
require more treatment, i.e., a greater dose of a drug.
[0032] 2. Monitoring Clinical Efficacy
[0033] The method of the present invention is useful to monitoring
the influence of agents (e.g., drugs, compounds) on the expression
or activity of CYP2C19-related metabolic capability and can be
applied in basic drug screening and in clinical trials. For
example, the effectiveness of an agent determined by a CYP2C19
phenotype assay of the invention to increase CYP2C19-related
metabolic activity can be monitored in clinical trials of subjects
exhibiting decreased CYP2C19-related metabolic capability.
Alternatively, the effectiveness of an agent determined by a
CYP2C19 phenotype assay of the invention to CYP2C19-related
metabolic activity can be monitored in clinical trials of subjects
exhibiting increased CYP2C19-related metabolic capacity.
[0034] Alternatively, the effect of an agent on CYP2C19-related
metabolic capability during a clinical trial can be measured using
the CYP2C19 phenotype assay of the present invention. In this way,
the CYP2C19 metabolite expression pattern measured using the method
of the present invention can serve as a benchmark, indicative of
the physiological response of the subject to the agent.
Accordingly, this response state of a subject can be determined
before, and at various points during treatment of the individual
with the agent.
[0035] The following Examples are presented in order to more fully
illustrate the preferred embodiments of the invention. These
Examples should in no way be construed as limiting the scope of the
invention, as defined by the appended claims.
EXAMPLES
Example 1
Classification of Human Subject by Pantoprazole (Ptz) Metabolic
Capacity Using the .sup.13CO.sub.2 Breath Test Method of the
Invention
[0036] The semisynthetic proton pump inhibitor pantoprazole (Ptz)
is useful to suppress acid production in the stomach. Ptz
metabolism is genetically polymorphous. CYP2C19 mediates the
O-demethylation of Ptz-O--.sup.13CH.sub.3 as detailed below.
##STR00001##
[0037] In addition to genetic factors, the apparent phenotype of an
individual subject and overall significance of CYP2C19 in the
biotransformation of a given substrate is influenced by the
quantitative importance of alternative metabolic routes. For
example, agents that are preferentially metabolized by CYP2C19,
pharmacologic inhibitors can modify enzyme activity such that the
magnitude of change in substrate metabolism can mimic that of
genetically determined poor metabolizers (i.e., an apparent change
in phenotype from an extensive metabolizer to a poor metabolizer).
With inhibitors of CYP2C19, the metabolism of coadministered
CYP2C19 substrates can be significantly altered in extensive
metabolizers. Such interactions can decrease the efficacy of a
prodrug requiring metabolic conversion to its active moiety or,
alternately, can result in toxicity for CYP2C19 substrates that
have a narrow therapeutic index. Non-invasive
diagnostic/theranostic tests, e.g., breath tests, are useful to
assess the CYP2C19 metabolic status of an individual subject.
[0038] The present studies employed the .sup.13CO.sub.2 breath test
method of the present invention to classify individual human
subjects (i.e., Volunteers 1-8) by their ability to metabolize
Ptz-O--.sup.13CH.sub.3. Briefly, following an 4-8 h fast normal
human subjects ingested 20-200 mg of Ptz-O--.sup.13CH.sub.3 and 2.1
g of sodium bicarbonate to protect the Ptz-O--.sup.13CH.sub.3 from
acid degradation in the stomach. A breath sample was collected
prior to drug ingestion and then at 5 min time points up to 30 min,
at 10 min intervals to 90 min, after ingestion of
Ptz-O--.sup.13CH.sub.3. The breath curves with SD (DOB versus Time
(Panel A) and PDR curves with SD versus Time (Panel B)) for eight
volunteers for the Ptz-O--.sup.13CH.sub.3 breath test are depicted
in FIG. 1.
[0039] Four volunteers were 2C19 extensive metabolizers (EM), two
were 2C19 intermediate metabolizers (IM) and two were poor
metabolizers (PM). The present studies demonstrate that either DOB
or PDR values at a specific time point are useful to differentiate
EM from IM and from PM.
[0040] The Ptz-O--.sup.13CH.sub.3 phenotyping procedure with a
.sup.13CO.sub.2 breath test has several potential advantages over
existing phenotyping methods, as mass spectrometry detection can be
replaced by infrared spectrometry. In addition to the safety and
demonstrated utility of Ptz as a probe for CYP2C19 activity, the
breath test affords phenotype determinations within a shorter time
frame (1 h or less after Ptz administration) and directly in
physicians' offices or other healthcare settings using relatively
cheap instrumentation (UBiT-IR.sub.300 IR spectrophotometer;
Meretek).
Example 2
Clinical Study Results Using the .sup.13CO.sub.2 Breath Test Method
of the Invention
[0041] Twenty subjects were admitted to the GCRC at Indiana School
of medicine at about 7 AM following an 8-hour overnight fasting.
Baseline breath samples were collected in 1.2-liter aluminum-lined
bags (Otsuka Pharmaceuticals, Tokushima, Japan). Subjects were then
administered 100 mg pantoprazole-.sup.13C (98%; Cambridge Isotope
Laboratories Inc., Andover, Mass.) with 2.1 g sodium bicarbonate to
prevent gastric acid degradation of the drug and water. Twelve
breath samples were collected over 120 minutes (obtained at 2.5, 5,
10, 15, 20, 25, 30, 40, 50, 60, 90, and 120 minutes). All subjects
were genotyped. Ten subjects were *1*1 (EM), nine subjects were
either *1*2 or *1*3 (IM) and one subject was *2*2 (PM). The breath
curves are depicted in FIG. 2.
[0042] In one embodiment of the breath test procedure of the
invention, .sup.13C-labeled CYP2C19 substrate compound (0.1 mg-500
mg) is ingested by a subject after overnight fasting (4-12 h), over
a time period of approximately 10-15 seconds. Breath samples are
collected prior to ingestion of .sup.13C-labeled CYP2C19 substrate
compound and then at 5 min intervals to 30 min, at 10 minute
intervals to 90 min, and at 30 min intervals thereafter to 90 min
after isotope-labeled substrate ingestion. The breath samples are
collected by having the subject momentarily hold their breath for 3
seconds prior to exhaling into a sample collection bag. The breath
samples are analyzed on a UBiT IR-300 spectrophotometer (Meretek,
Denver, Colo.) to determine the .sup.13CO.sub.2/.sup.12CO.sub.2
ratio in expired breath, or sent to a reference lab.
EQUIVALENTS
[0043] The present invention is not to be limited in terms of the
particular embodiments described in this application, which are
intended as single illustrations of individual aspects of the
invention. Many modifications and variations of this invention can
be made without departing from its spirit and scope, as will be
apparent to those skilled in the art. Functionally equivalent
methods and apparatuses within the scope of the invention, in
addition to those enumerated herein, will be apparent to those
skilled in the art from the foregoing descriptions. Such
modifications and variations are intended to fall within the scope
of the appended claims. The present invention is to be limited only
by the terms of the appended claims, along with the full scope of
equivalents to which such claims are entitled.
* * * * *