U.S. patent application number 12/484148 was filed with the patent office on 2010-06-24 for methods of quantifying methotrexate metabolites.
This patent application is currently assigned to CYPRESS BIOSCIENCE, INC.. Invention is credited to Thierry DERVIEUX, Russell RICHERSON.
Application Number | 20100159493 12/484148 |
Document ID | / |
Family ID | 31977076 |
Filed Date | 2010-06-24 |
United States Patent
Application |
20100159493 |
Kind Code |
A1 |
DERVIEUX; Thierry ; et
al. |
June 24, 2010 |
METHODS OF QUANTIFYING METHOTREXATE METABOLITES
Abstract
The present invention provides a method for efficiently
converting methotrexate polyglutamates to methotrexate in a
cellular extract by contacting the cellular extract with gamma
glutamyl hydrolase under conditions suitable for efficient
conversion of methotrexate polyglutamates to methotrexate.
Inventors: |
DERVIEUX; Thierry; (San
Diego, CA) ; RICHERSON; Russell; (Tucson,
AZ) |
Correspondence
Address: |
COOLEY GODWARD KRONISH LLP;ATTN: Patent Group
Suite 1100, 777 - 6th Street, NW
WASHINGTON
DC
20001
US
|
Assignee: |
CYPRESS BIOSCIENCE, INC.
San Diego
CA
|
Family ID: |
31977076 |
Appl. No.: |
12/484148 |
Filed: |
June 12, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10232760 |
Aug 30, 2002 |
7563590 |
|
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12484148 |
|
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Current U.S.
Class: |
435/18 |
Current CPC
Class: |
C12Q 1/34 20130101; G01N
33/5014 20130101; A61K 35/18 20130101; C12Q 1/37 20130101 |
Class at
Publication: |
435/18 |
International
Class: |
C12Q 1/34 20060101
C12Q001/34 |
Claims
1. A method for predicting therapeutic efficacy or reducing
toxicity associated with methotrexate (MTX) therapy administered to
an individual, comprising the steps of: (a) converting methotrexate
polyglutamates (MTXPGs) to MTX in a cellular extract from said
individual under conditions suitable for efficient conversion of
MTXPGs to MTX; (b) determining a level of MTX in said cellular
extract, said level of MTX correlating with a level of MTXPGs,
thereby determining a level of MTXPGs in said cellular extract,
wherein a drug or dosage subsequently administered to said
individual is selected based on said level of MTXPGs.
Description
BACKGROUND OF THE INVENTION
[0001] This invention relates generally to methods for monitoring
drug therapy and, more specifically, to methods for monitoring
efficacy and toxicity of anti-folate drug therapy.
[0002] Folate (folic acid) is a vitamin that is essential for the
life-sustaining processes of DNA synthesis, replication and repair.
Folate is also important for protein biosynthesis, another process
that is central to cell viability. The pteridine compound,
methotrexate (MTX), is structurally similar to folate and as a
result can bind to the active sites of a number of enzymes that
normally use folate as a coenzyme for the biosynthesis of the
purine and pyrimidine nucleotide precursors of DNA and for the
interconversion of amino acids during protein biosynthesis. Despite
its structural similarity to folic acid, MTX cannot be used as a
cofactor by enzymes that require folate, and instead competes with
the folate cofactor for enzyme binding sites, thereby inhibiting
protein and DNA biosynthesis and, hence, cell division.
[0003] The ability of methotrexate to inhibit cell division has
been exploited in the treatment of a number of diseases and
conditions that are characterized by rapid or aberrant cell growth.
As an example, autoimmune diseases are characterized by an
inappropriate immune response directed against normal autologous
(self) tissues and are mediated by rapidly replicating T-cells or
B-cells. Autoimmune diseases that have been treated with MTX
include, for example, multiple sclerosis, rheumatoid arthritis,
psoriasis, the autoimmune stage of diabetes mellitus
(juvenile-onset or Type 1 diabetes), autoimmune uveoretinitis,
myasthenia gravis, autoimmune thyroiditis, and systemic lupus
erythematosus.
[0004] Because many malignant cells proliferate more rapidly than
normal cells, MTX can also be used to selectively impair cancerous
cell growth. As a consequence, methotrexate is a widely used
anticancer agent, employed, for example, in the treatment of acute
lymphocytic leukemia, breast cancer, epidermoid cancers of the head
and neck, advanced mycosis fungoides, lung cancer, non-Hodgkins
lymphomas, gestational choriocarcinoma, chorioadenoma destruens,
and hydatidiform moles.
[0005] Despite its therapeutic efficacy for a wide variety of
diseases and conditions, treatment with methotrexate can present a
risk to the patient. In particular, because MTX interferes with
processes required for replication and division of normal as well
as diseased cells, inappropriately high levels of the drug can lead
to destruction of actively proliferating non-target tissues such as
bone marrow and intestinal mucosa. MTX consequently is associated
with renal and hepatic toxicity when administered in the "high-dose
regimen" that is required for some conditions. In addition,
low-dose MTX therapy can lead to toxicity and unwanted side-effects
in some patients, where the dosage is not appropriate due to
individual variability in pharmacokinetic parameters influencing,
for example, drug uptake, targeting and clearance. This situation
is especially problematic in the treatment of chronic conditions
such as rheumatoid arthritis, where methotrexate can be
administered over a period of many years.
[0006] Because individual differences in pharmacokinetic parameters
can be difficult to predict, safe and effective methotrexate
treatment strategies require that methotrexate or methotrexate
metabolite levels be monitored in patients being treated. A variety
of methods have been developed for monitoring MTX drug
concentrations in plasma including bioassays, immunological
detection and chromatographic assays. Such plasma detection methods
have been useful for monitoring high dose MTX therapy in some
clinical applications. However, these plasma detection methods have
not been useful in monitoring low-dose methotrexate therapy.
[0007] Methotrexate is metabolized upon uptake by mammalian cells,
such that one or more glutamyl moieties are added to MTX to yield a
mixture of methotrexate polyglutamates (MTXPGs). The number of
glutamyl moieties that can be added to MTX generally varies from
two to seven. MTXPGs do not readily efflux from cells and thus are
able to exert their cytotoxic effects over long periods of time.
Levels of intracellular MTXPGs have been shown to be higher in
patients that responded to MTX therapy as compared the
intracellular levels in patients that did not respond. Currently
available methods for measuring cellular MTXPG levels are based on
a dihydrofolate reductase enzyme assay in which MTXPG levels are
calculated based on inhibition of the dihydrofolate reductase
enzyme. However, the extent of enzyme inhibition in these assays is
dependent upon the number of glutamyl residues attached to MTX,
rendering an accurate determination of intracellular MTXPGs levels
impossible by this method. The variability of dihydrofolate
reductase based assays can be further exacerbated in some
situations because folates, which are present in different amounts
depending upon an individual's response to MTX therapy and the
amount of folate contributed by diet, also influence the results of
the assay.
[0008] Thus, there exists a need for new methods for determining
intracellular levels of methotrexate polyglutamates and for
monitoring the efficacy and toxicity of methotrexate therapy such
as low-dose methotrexate therapy. The present invention satisfies
this need and provides related advantages as well.
SUMMARY OF THE INVENTION
[0009] The present invention provides a method for efficiently
converting methotrexate polyglutamates (MTXPGs) to methotrexate
(MTX) in a cellular extract by contacting the cellular extract with
gamma glutamyl hydrolase under conditions suitable for efficient
conversion of MTXPGs to MTX. The method is useful for quantitating
the level of MTXPGs in a cellular extract.
[0010] The invention further provides a method for determining a
level of methotrexate polyglutamates in a cellular extract. The
method includes the steps of: (a) converting the MTXPGs to
methotrexate in a cellular extract under conditions suitable for
efficient conversion of MTXPGs to MTX, and (b) determining a level
of the MTX, where the level of MTX can be correlated with the level
of MTXPGs in the cellular extract.
[0011] The invention also provides a method for determining a level
of methotrexate in a cellular extract by (a) irradiating the
cellular extract, thereby producing a fluorescent MTX photolytic
product; and (b) determining a level of the fluorescent MTX
photolytic product, wherein the level of the fluorescent MTX
photolytic product can be correlated with the level of MTX. By
indirectly determining the intracellular level of methotrexate
polyglutamates, a method of the invention can be useful, for
example, for monitoring the efficacy or toxicity of MTX
therapy.
[0012] The invention provides a method of optimizing therapeutic
efficacy or reducing toxicity associated with methotrexate therapy
administered to an individual. The method includes the steps of:
(a) converting methotrexate polyglutamates to MTX in a cellular
extract from an individual under conditions suitable for efficient
conversion of MTXPGs to MTX; and (b) determining a level of MTX in
the cellular extract, the level of MTX correlating with a level of
MTXPGs, thereby determining a level of MTXPGs in the cellular
extract, wherein a drug or dosage subsequently administered to the
individual is selected based on the level of MTXPGs.
[0013] The invention further provides an isolated deglutamated
composition which is characterized as (a) present in a red blood
cell extract following deglutamation; (b) having a retention time
of about 12 minutes in a mobile phase consisting of a 15-minute
linear gradient from 2% acetonitrile/98% mobile phase A to 25%
acetonitrile/75% mobile phase A at a flow rate of 1 ml/min through
a Terra MS 018 reverse phase column, where the reverse phase column
has dimensions of 25 cm.times.4.6 mm and a particle size of 5
micrometers and where the mobile phase A contains 10 mM ammonium
acetate, pH 6.5, and 0.06% hydrogen peroxide in water; and (c)
having a fluorescence excitation spectrum with peaks at 299 nm and
402 nm in water and a fluorescence emission spectra with a peak at
460 nm in water.
[0014] The deglutamated endogenous composition of the invention can
be produced by: (a) converting polyglutamated compounds in a red
blood cellular extract to produce deglutamated RBC compounds; (b)
fractionating the deglutamated RBC compounds under conditions
including passage of a mobile phase consisting of a 15-minute
linear gradient from 2% acetonitrile/98% mobile phase A to 25%
acetonitrile/75% mobile phase A at a flow rate of 1 ml/min through
a Terra MS 018 reverse phase column, wherein the reverse phase
column has dimensions of 25 cm.times.4.6 mm and a particle size of
5 micrometers and wherein the mobile phase A includes 10 mM
ammonium acetate, pH 6.5 and 0.06% hydrogen peroxide in water; and
(c) isolating a compound that elutes in a peak at 12 minutes, where
the peak is detectable by emission at 464 nm upon excitation with
radiation at 400 nm following photolysis of the compound by
irradiation at 254 nm for 3 seconds in 80% mobile phase A/20%
acetonitrile.
[0015] The deglutamated endogenous composition of the invention can
combined, if desired, with a pharmaceutically acceptable carrier to
produce a pharmaceutical composition. Such a pharmaceutical
composition can be useful, for example, in a therapeutic method and
can optionally include methotrexate. According to the invention,
the deglutamated endogenous composition of the invention is also
provided as a kit for use in a therapeutic or diagnostic
method.
[0016] A deglutamated endogenous composition of the invention can
be useful for reducing toxicity associated with methotrexate
therapy. A deglutamated endogenous composition of the invention
also can be useful in diagnostic methods such as methods for
determining an intracellular level of methotrexate or a method of
optimizing therapeutic efficacy or reducing toxicity associated
with methotrexate therapy.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 shows the structures of methotrexate and methotrexate
polyglutamates. (A) The chemical structure of methotrexate. (B) The
chemical structure for methotrexate polyglutamate, where n refers
to the number of glutamates attached to methotrexate.
[0018] FIG. 2 shows the effect of mobile phase composition on
formation of fluorescent MTX photolytic products. (A) A plot of
peak area detected at different pH values for MTX photolytic
product A or product B; (B) A plot of peak area detected at
different acetonitrile concentrations for MTX photolytic product A
or product B; (C) A chromatogram of the photolytic products of MTX;
and (D) Fluorescence excitation and emission spectra for photolytic
product A of the compound eluting at 12.42 min from two separate
chromatography runs.
[0019] FIG. 3 shows typical chromatograms of red blood cell
extracts. (A) A typical chromatogram of a deglutamated red blood
cell extract following photolysis; (B) A typical chromatogram of a
deglutamated, MTXPG.sub.2-7-spiked red blood cell extract following
photolysis; and (C) Fluorescence excitation spectrum of photolytic
product A produced from the peak eluting at 12.5 minutes in the
chromatogram of part B (indicated as "RBC") overlayed with
excitation spectrum for photolytic product A produced from a
methotrexate standard in water (indicated as "water").
[0020] FIG. 4 shows the conversion results obtained for various
methotrexate polyglutamates. (A) Timecourse of conversion of
MTXPG.sub.2 to MTX. (B) Timecourse of conversion of MTXPG.sub.2-7
to MTX.
[0021] FIG. 5 shows a plot of detected MTX concentration versus the
concentration of MTXPG.sub.2 or MTXPG.sub.2-7 added to a red blood
cell sample prior to deglutamation. MTX from samples with
MTXPG.sub.2 is shown as squares. MTX from samples with
MTXPG.sub.2-7 is shown as triangles.
[0022] FIG. 6 shows characterization of a red blood cell patient
sample. (A) Chromatogram of a deglutamated RBC extract from a
patient having received low-dose MTX therapy. (B) Fluorescence
excitation spectrum of photolytic product A produced from the peak
eluting at 12.42 minutes in the chromatogram of part A (indicated
as "RBC") overlayed with excitation spectrum for photolytic product
A produced from a methotrexate standard in water (indicated as
"water").
[0023] FIG. 7 shows a fluorescence excitation spectrum obtained for
the eluate at 12.02 minutes in the chromatograph of FIG. 6A.
DETAILED DESCRIPTION OF THE INVENTION
[0024] As disclosed herein, intracellular levels of methotrexate
(MTX) were quantitated by fluorimetric detection of MTX photolytic
products. As disclosed in Example I, a cell extract containing MTX
was chromatographically separated and photolysed to yield MTX
photolytic products which were of fluorimetrically detected. The
method was demonstrated to detect MTX at low concentrations such as
5 nmol/L.
[0025] As further disclosed herein, methotrexate polyglutamates
(MTXPGs) in a cellular extract were efficiently converted to MTX by
contacting the extract with plasma as a source of gamma glutamyl
hydrolase. Efficient conversion of intracellular MTXPGs to MTX,
combined with fluorimetric detection of MTX photolytic products,
allowed accurate quantitation of cellular MTXPG levels as
demonstrated in Example II. As further disclosed in Example III, a
method of the invention for quantitating cellular MTXPG levels was
useful in monitoring intracellular MTXPG levels in patients
undergoing low-dose MTX therapy. Thus, as disclosed herein, the
methods of the invention can be useful, for example, for
determining the percentage of MTX that has been metabolized to
MTXPGs in a cellular extract. The methods of the invention also can
be useful for quantitating cellular MTXPG levels in order to
determine the efficacy or toxicity of methotrexate therapy.
[0026] The present invention further is directed to the discovery
of an endogenous compound that is present in deglutamated red blood
cell extracts. As demonstrated in Example IV, the endogenous
compound can be chromatographically separated from unrelated red
blood cell components. The endogenous compound is characterized by
(a) being present in red blood cells following deglutamation; (b)
having a retention time of about 12 minutes in a mobile phase
consisting of a 15-minute linear gradient from 2% acetonitrile/98%
mobile phase A to 25% acetonitrile/75% mobile phase A at a flow
rate of 1 ml/min through a Terra MS C18 reverse phase column, which
has dimensions of 25 cm.times.4.6 mm and a particle size of 5
micrometers, where mobile phase A contains 10 mM ammonium acetate,
pH 6.5, and 0.06% hydrogen peroxide in water; and (c) having a
fluorescence excitation spectrum with peaks at about 299 nm and 402
nm in water and fluorescence emission at about 464 nm in water. The
endogenous compound can be, for example, photostable to UV light
for at least 3 seconds and further can be selectively present in a
deglutamated RBC extract but absent from an untreated RBC
extract.
[0027] As further disclosed herein, detection or monitoring of the
levels of the endogenous compound can be used to optimize
therapeutic efficacy or reduce toxicity associated with
methotrexate therapy. In particular, the levels of MTX and the
endogenous deglutamated compound can be monitored and compared to
optimize therapeutic efficacy or reduce toxicity associated with
methotrexate or other anti-folate therapy. The endogenous compound
also can function as an analog of folic acid and can be a useful
adjunct to methotrexate therapy. As an example, the isolated
endogenous compound can be administered to an individual undergoing
methotrexate therapy in order to reduce attendant toxicity.
[0028] Based on these findings, the present invention provides a
method for efficiently converting MTXPGs to MTX in a cellular
extract by contacting the cellular extract with gamma glutamyl
hydrolase under conditions suitable for efficient conversion of
MTXPGs to MTX. A method of the invention can be useful, for
example, for removing MTXPGs from a cellular extract, for isolating
MTX from a cellular extract or for quantitating the level of MTXPGs
in a cellular extract as described further below.
[0029] As used herein, the term "methotrexate" is synonymous with
"MTX" and means a molecule having the structure shown in FIG. 1A.
MTX includes, in part, a 2,4-diamino substituted pterine ring
moiety linked at the 6 position to the amino group of a
p-aminobenzoyl moiety, the p-aminobenzoyl moiety having a
methylated amino group and being amide bonded to a glutamic acid
moiety. Methotrexate functions as an inhibitor of dihydrofolate
reductase (DHFR), decreasing the production of tetrahydrofolate
(THF) from dihydrofolate (DHF). As a consequence, MTX indirectly
inhibits purine and thymidine synthesis and amino acid
interconversion. MTX also exhibits anti-proliferative activity
through inhibition of thymidylate synthesis, which is required to
synthesize DNA (Calvert, Semin. Oncol. 26:3-10 (1999)). MTX and its
synthesis and properties are described in further detail in U.S.
Pat. Nos. 2,512,572; 3,892,801; 3,989,703; 4,057,548; 4,067,867;
4,079,056; 4,080,325; 4,136,101; 4,224,446; 4,306,064; 4,374,987;
4,421,913; and 4,767,859. Methods of using MTX to treat cancer are
described, for example, in U.S. Pat. Nos. 4,106,488, 4,558,690, and
4,662,359.
[0030] Methotrexate, which is useful in the treatment of a variety
of autoimmune diseases and cancers, is typically administered by
the oral or parenteral route. MTX is readily distributed to body
tissues, where it is transported into cells by a specific carrier
system which includes components such as the reduced folate
carrier, RCF1, and the folate receptor. Due to its high polarity at
physiological pH, MTX does not readily pass through the cell
membrane, and the majority of MTX enters cells via specific
carriers. Once inside the cell, MTX is converted to MTXPGs by
specific enzymes such as folylpoly-gamma-glutamate synthetase,
which add one or more glutamic acid moieties, linked by
iso-peptidic bonds to the y-carboxyl of MTX as described, for
example, in Kamen, Semin. Oncol. S18:30-39 (1997).
[0031] As used herein, the term "methotrexate polyglutamate" is
synonymous with "MTXPG" and means a derivative of MTX having two or
more glutamates which are amide bonded to the p-aminobenzoyl moiety
of MTX as shown in the generalized structure of FIG. 1B. The number
of glutamates in a methotrexate polyglutamate varies from two to
seven or more; the number of glutamate moieties can be denoted by
"n" using the nomenclature MTXPG.sub.n such that, for example,
MTXPG.sub.2 is MTXPG having two glutamates, MTXPG.sub.3 is MTXPG
having three glutamates, MTXPG.sub.4 is MTXPG having four
glutamates, MTXPG.sub.5 is MTXPG having five glutamates,
MTXPG.sub.6 is MTXPG having six glutamates, MTXPG.sub.7 is MTXPG
having seven glutamates, and MTXPG.sub.2-7 is a mixture containing
MTXPG.sub.2, MTXPG.sub.3, MTXPG.sub.4, MTXPG.sub.5, MTXPG.sub.5 and
MTXPG.sub.7 with the ratio of the individual polyglutamated forms
in the mixture not defined. It is understood that, consistent with
this nomenclature, MTXPG.sub.1 is synonymous with MTX.
[0032] An extract from cells that have taken up MTX will typically
contain a mixture of methotrexate polyglutamated species that
differ in the number of attached glutamate moieties. Such mixtures
of MTXPGs can be efficiently converted to MTX by a method of the
invention as set forth below and demonstrated in Examples II and
III. A method of the invention can also be used to efficiently
convert MTXPGs that are homogeneous with respect to the number of
attached glutamate moieties as demonstrated by the efficient
conversion of MTXPG.sub.2 to MTX in Example II. In particular
embodiments, the methods of the invention are used to efficiently
convert an MTXPG.sub.n to MTX, where n is any integer greater than
1.
[0033] In a method of the invention, MTXPGs can be converted to MTX
by gamma glutamyl hydrolase. As used herein, the term "gamma
glutamyl hydrolase" is synonymous with "GGH" and means a molecule
that catalyzes cleavage of a gamma glutamyl bond to remove a
glutamate moiety from a polyglutamated substrate. In one embodiment
of the invention, a GGH catalyzes cleavage of a gamma glutamyl bond
to remove a glutamate moiety from a polyglutamated substrate and
lacks activity in cleaving the glutamate moiety from a
monoglutamated substrate. GGHs useful in the invention including,
but are not limited to, enzymes such as EC 3.4.19.9, or a
functional equivalent thereof including, for example, gamma-glu-X
carboxypeptidase, folate conjugase, pteroyl-poly-alpha-glutamate
hydrolase, carboxypeptidase G and lysosomal gamma-glutamyl
carboxypeptidase. Examples of substrates of a gamma glutamyl
hydrolase are MTXPGs and other polyglutamated antifolates. It is
understood that a GGH can catalyze removal of any number of
glutamate residues that are linked to the gamma carboxyl of a
monoglutamated moiety of a polyglutamated substrate, and under
certain conditions can yield a monoglutamated product. As an
example, MTXPG.sub.n includes n-1 glutamates that can be
catalytically removed by GGH; MTXPG.sub.2 includes a single
glutamate that can be catalytically removed by GGH; and MTXPG.sub.7
includes 6 glutamates that can be catalytically removed by GGH. As
discussed further below, it is understood that the actual number of
glutamates hydrolyzed, if any, depends on the conditions and length
of time the enzyme is in contact with the polyglutamated
substrate.
[0034] In one embodiment of the invention, a GGH is in contact with
a cellular extract under conditions suitable for cleavage of a
gamma glutamyl bond such that all but one glutamate moiety are
cleaved from a polyglutamated substrate. As an example,
carboxypeptidase G is a GGH that under particular conditions can
convert MTXPG to MTX and, additionally, can remove the glutamate
moiety from MTX to yield 4-amino-4-deoxy-N.sup.10-methylpteroic
acid (DAMPA). Carboxypeptidase G can be used to convert MTXPG to
MTX in a method of the invention under conditions that reduce or
prevent the latter activity, such as in the presence of an
inhibitor, thereby removing all but one glutamate moiety from
MTXPGs and converting the MTXPGs to MTX.
[0035] Native gamma-glutamyl hydrolase is a glycoprotein typically
having a molecular mass of 80 to 120 kDa (Yao et al., Proc. Natl.
Acad. Sci. USA 93:10134-10138 (1996)). The protein portions of
human and rat GGH are about 35 kDa with multiple potential
asparagine-linked glycosylation sites, rat GGH having seven and
human GGH having four based on sequence homology (Yao et al.,
supra, 1996). Depending upon the tissue, GGH is found in lysosomal
compartments or secreted (Yao et al., J. Biol. Chem. 271:8525-8528
(1996)). GGH typically has an acidic pH optimum and is often
sulfhydryl- and Zn.sup.2+-dependent (Yao et al., J. Biol. Chem.
271:8525-8528 (1996)).
[0036] A variety of GGHs are useful in the invention, including
those with naturally occurring and non-naturally occurring
sequences, glycosylated and non-glycosylated forms, species
homologs, or in crude, partially purified or purified forms. GGHs
useful in the invention include, without limitation, biochemically
purified native proteins as well as recombinant proteins and
fragments or isoforms thereof that retain gamma glutamyl hydrolase
activity. A variety of GGHs can be useful in the invention
including, but not limited to, mammalian, primate, human, non-human
primate, bovine, porcine, rat, and murine GGHs. Furthermore, GGHs
with improved or diminished enzymatic activity, stability or other
function as compared to naturally occurring (wild type) GGH can be
useful in the invention, as can point mutants, deletion or
insertion mutants and fusion proteins including GGH or an active
fragment thereof. In particular embodiments, the invention is
practiced with a source containing a GGH with at least 40% amino
acid identity to the human GGH provided in Genbank accession
NM.sub.--003878, or with at least 50%, 60%, 70%, 80%, 90% or more
amino acid identity as compared to the human GGH provided in
Genbank accession NM.sub.--003878.
[0037] A GGH useful in the invention can be provided, without
limitation, as plasma or a fraction thereof; or as an extract from
a cell expressing GGH or a fraction thereof; or in any purified or
partially purified form. Under optimal conditions, a GGH can have,
for example, an activity of at least 30% conversion of MTXPG.sub.2
to MTX per hour, for example, at least 25% conversion of
MTXPG.sub.2 to MTX per hour, or at least 10% conversion of
MTXPG.sub.2 to MTX per hour. A GGH can be a GGH of any species
origin, for example, a human GGH having NCBI Accession
NM.sub.--003878 (see, also, Rhee et al., Mol. Pharmacol. 53:
1040-1046 (1998), and Yao et al., Proc. Natl. Acad. Sci. USA
93:10134-10138 (1996)); the rat GGH having NCBI Accession
NM.sub.--012960 (see, also, Yao et al., J. Biol. Chem. 271:
8525-8528 (1996)); or the mouse GGH having NCBI Accession
NM.sub.--010281 (see, also, Esaki et al., Gene 234:93-100
(1999)).
[0038] Plasma containing GGH can be used to efficiently convert
MTXPGs to MTX as demonstrated in Examples II and III. As used
herein, the term "plasma" means the acellular fluid portion of
blood. Plasma useful in a method of the invention can be obtained
from any of a variety of animals. As non-limiting examples, plasma
useful in the invention can be human plasma, primate plasma,
non-human primate plasma, rat plasma, mouse plasma or plasma from
any other mammal. Plasma can be used in the invention as fresh
plasma or, alternatively, following storage under conditions that
maintain GGH activity. Plasma can be obtained using methods well
known in the art such as centrifugation of blood at low speed and
isolation of the supernatant fraction. Plasma can also be obtained
from a commercial source, such as Sigma (St Louis, USA), which
provides lyophilized plasma (Cat. No. P9523). Exemplary conditions
that can be used to store plasma useful in the invention include,
without limitation, lyophilization, refrigeration, freezing or a
combination thereof.
[0039] A cell expressing GGH also can be a source of GGH useful in
a method of the invention. Such a cell can be any cell that
expresses endogenous or recombinant GGH or both. Many cancer cells,
including, for example, human breast cancer cells, hepatoma cell
lines and leukemia cell lines are known to express high levels of
GGH and can therefore serve as sources of GGH for use in the
invention. See, for example, Rhee et al., Cell Pharmacol. 2:289-292
(1995), and Pizzorno et al., Cancer Res. 55:566-573 (1995).
[0040] It further is understood that GGH can be obtained from a
recombinant cell that expresses exogenous GGH. Nucleic acid
molecules encoding GGH are known in the art, as set forth above, or
can be obtained by routine cloning methods including, for example,
isolation of a GGH-encoding nucleic acid molecule from a cDNA
library or genomic library with a natural or artificially designed
gene-specific nucleic acid probe, or PCR amplification using a
sequence specific primer. The GGH-encoding nucleic acid molecule
can be cloned into an expression vector and used to transform cells
for recombinant expression of GGH by routine methods, as described
for human and rat GHH, for example, in Rhee et al., supra, 1998,
and Yao et al., supra, 1996. Those skilled in the art will be able
to obtain recombinant cells expressing any of a variety of GGHs
using these and other routine cloning methods that are well known
in the art as described, for example, in Sambrook et al., Molecular
Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press,
Plainview, N.Y. (2001); and Ausubel et al. (Current Protocols in
Molecular Biology (Supplement 47), John Wiley & Sons, New York
(1999).
[0041] Purified or partially purified GGH also can be useful in a
method of the invention. As non-limiting examples, GGH can be
obtained from a native source such as plasma or a tumor cell or a
recombinant source such as a bacterial, yeast or insect cell. GGH
can be purified or partially purified using known methods as
described, for example, in Scopes, Protein Purification: Principles
and Practice, 3.sup.rd Ed., Springer-Verlag, New York (1994) and
Coligan et al., Current Protocols in Protein Science, John Wiley
and Sons, Baltimore, Md. (2000). Isolation of GGH by these and
other methods can be monitored using an antibody to GGH or an assay
for GGH activity as described, for example, in Yao et al., supra,
1996, or Rhee et al., supra, 1998. As a non-limiting illustration,
an E. coli cell lysate containing recombinant human gamma glutamyl
hydrolase can obtained by sonicating E. coli cells that express the
recombinant enzyme as described, for example, in Yao et al., supra,
1996. As a further illustration, recombinant human gamma glutamyl
hydrolase can be expressed in intracellular or secreted form using
recombinant insect cells and purified with cation exchange
chromatography or immunoaffinity chromatography as described, for
example, in Rhee et al., supra, 1998.
[0042] The methods of the invention involve contacting a cellular
extract with gamma glutamyl hydrolase under conditions suitable for
efficient conversion of MTXPGs to MTX. As used herein, the term
"efficient conversion" means the production at least a 60% molar
equivalent of MTX product compared to MTXPG substrate. Thus, the
phrase "under conditions suitable for efficient conversion of
MTXPGs to MTX" means an environment in which at least a 60% molar
equivalent of MTX is produced from MTXPGs. In particular
embodiments, the methods of the invention for efficiently
converting MTXPGs to MTX produce at least 65%, 70%, 75%, 80%, 85%,
90%, 95%, 98% or 99% molar equivalent of MTX product compared to
MTXPG substrate. In addition embodiments, the methods of the
invention produce at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,
98% or 99% molar equivalent of MTX product from MTXPG.sub.2
substrate. Efficient conversion of MTXPG.sub.2 to MTX can occur
within a time period of, for example, 2 hours, 4 hours, 6 hours, 8
hours, 10 hours, 12 hours or longer. Thus, at least 65%, 70%, 75%,
80%, 85%, 90%, 95%, 98% or 99% molar equivalent of MTX product
compared to MTXPG substrate can be produced, for example, within 2
hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours or longer.
[0043] In one embodiment, a method of the invention is practiced by
converting MTXPGs to MTX under conditions that include a pH of at
least 4. The methods of the invention can be practiced, for
example, under conditions that include a pH in the range of about 6
to 7. It is understood that the desired pH can be maintained using
a buffer. Those skilled in the art can determine an appropriate
buffer to maintain a particular pH based on the pKa of the sample
being used and known pKa values of buffers as described, for
example, in Segel, Biochemical Calculations, 2nd Ed., John Wiley
and Sons, New York (1976). As illustrated in Example II, because
plasma and RBCs are strongly buffered at physiological pH, addition
of 100 mM phosphate buffer pH 4.5 to a mixture of plasma and RBC
extract at a volume/volume ratio of 2 parts buffer:1 part RBC
extract:2 parts plasma yields a final pH of about 6.5. Those
skilled in the art will recognize that buffers having a similar pKa
can be substituted for each other in order to maintain a desired pH
in a method of the invention. The concentration of phosphate in the
conditions of the invention can be, for example, below about 400 mM
such as below 350 mM or below 300 mM.
[0044] By using an appropriate buffer, a method of the invention
can be practiced under conditions that include, for example, a pH
of about 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0 or 8.5.
In particular embodiments, a method of the invention is practiced
under conditions that include a pH of at least 3.5, at least 4.0,
at least 4.5, at least 5.0, at least 5.5, at least 6.0, at least
6.5 or at least 7.0. In other embodiments, a method of the
invention is practiced with a pH in the range of 4.0 to 8.0, 4.5 to
8.0, 5.0 to 8.0, 5.5 to 8.0, 6.0 to 8.0, 6.5 to 8.0, 7.0 to 8.0,
4.0 to 7.5, 4.0 to 7.0, 4.0 to 6.5, 4.0 to 6.0, 5.0 to 7.5, 5.0 to
7.0, 5.0 to 6.5, 5.0 to 6.0, 6.0 to 7.0, or 6.5 to 7.0.
[0045] Conditions suitable for efficient conversion of MTXPGs to
MTX can include, for example, a reducing agent as demonstrated in
Example II, where 2-mercaptoethanol was included. The concentration
of 2-mercaptoethanol in a method of the invention can be, for
example, about 50 mM or more. Other reducing agents can be useful
in the invention including, without limitation, dithiothreitol,
dithionite, cysteamine or glutathione. It is understood that the
concentration of the reducing agent can vary from those exemplified
herein, so long as the conditions lead to efficient conversion of
MTXPGs to MTX.
[0046] Efficient conversion of at least 60% MTXPG.sub.2-7 to MTX
typically occurs within about 3 hours or less using plasma as a
source of GGH. As an example, FIG. 4A shows that GGH provided in a
plasma fraction converted greater than 60% MTXPG.sub.2 to MTX in
about 3 hours and greater than 90% MTXPG.sub.2 to MTX in about 5
hours. As a further example, FIG. 4B shows that GGH provided in a
plasma fraction converted greater than 60% MTXPG.sub.2-7 to MTX in
about 2 hours and greater than 90% MTXPG.sub.2-7 to MTX in about 5
hours. Those skilled in the art will understand that the time for
the conversion reaction can be altered from the times used for
plasma GGH as exemplified herein depending, for example, on the use
of a more concentrated or purified fraction of GGH, recombinant GGH
or another molecule having GGH activity. As non-limiting examples,
a method of the invention can be practiced by contacting a cellular
extract containing MTXPGs with plasma or another source of with
gamma glutamyl hydrolase for a time that is within the range of 2
to 15 hours, 2 to 10 hours, 2 to 8 hours, 2 to 5 hours, 3 to 15
hours, 3 to 10 hours, 3 to 8 hours, 3 to 5 hours, 5 to 15 hours, 5
to 10 hours, 5 to 8 hours, 6 to 24 hours, 9 to 24 hours, 6 to 15
hours or 9 to 15 hours.
[0047] Plasma used as a source of GGH in a method of the invention
can be provided, for example, in a volume excess of about 2 fold or
more compared to the volume of a RBC extract, provided at a
concentration of about 0.5.times.10.sup.9 red blood cells per 100
microliters. As exemplified in Example II, MTXPGs were efficiently
converted to MTX in a RBC extract using a 2 fold excess of plasma
that had been reconstituted from lyophilized form according to the
manufacturer's instructions. Although more concentrated conditions
can be used in a method of the invention, precipitation of some of
the MTXPGs by hemoglobin in the mixture can reduce the amount that
is converted to MTX. Higher volume excess of plasma to RBC extract
can also be used such as 2.5 fold, 3 fold, 3.5 fold, 4 fold or
higher volume excess of plasma compared to RBC extract, so long as
MTXPGs are efficiently converted to MTX. Those skilled in the art
will recognize that dilution of RBC's in a method of the invention
can be achieved in other ways including, without limitation,
increasing the volume of buffer present or decreasing the number of
RBC's present. For example, when purified GGH or GGH from
recombinant cells or enriched plasma fractions is used in place of
plasma in a method of the invention, the concentration of the RBC's
can be diluted, if desired, using buffer.
[0048] In a method of the invention MTXPGs are efficiently
converted to MTX in a cellular extract. As used herein, the term
"cellular extract" means a mixture containing a heterogenous
plurality of cellular components. A cellular extract useful in the
invention can contain, for example, a heterogeneous plurality of
soluble cellular compounds, proteins or metabolites. Heterogeneity
of a cellular extract can be characterized by various criteria.
According to one criteria, a cellular extract useful in the
invention can be heterogeneous with respect to the variety of
cellular components present in the extract. In this regard, a
cellular extract can contain, for example, at least 100, 1000,
1.times.10.sup.4 or 1.times.10.sup.5 or more different cellular
components, for example, at least 100, 1000, 1.times.10.sup.4 or
1.times.10.sup.5 or more different proteins. Heterogeneity can also
be expressed as the percent of the total number of different
components of the cell from which the extract is derived. For
example, a cellular extract can contain cellular components
representing at least 5%, 10%, 15%, 20%, 25%, 50% or 75% of the
variety of components present in the cell from which the extract
was derived. Heterogeneity can also be determined based on the
percentage of any one cellular component in a cellular extract
compared to the total amount of other components in the cellular
extract. A cellular extract useful in the invention can have, for
example, a cellular component that represents at most about 90%,
80%, 70%, 60%, 50%, 25%, or 10% of the total amount of other
cellular components in the mixture. A cellular extract used in a
method of the invention can contain mixtures of components such as
proteins, components that are larger than 100 Da or components that
absorb radiation between about 303 nm and 313 nm or at about 370
nm.
[0049] A cellular extract that is useful in a method of the
invention can be any cell extract that contains one or more MTXPGs.
Additional exogenous MTXPGs can be added to a cellular extract as
set forth in Example II. The addition of one or more exogenous
MTXPGs into a cellular extract can be useful for determining a
standard curve for quantitating MTX or for otherwise testing or
optimizing MTX detection conditions. A cellular extract containing
MTXPGs can be obtained by adding MTX to a cellular extract and
allowing polyglutamation to occur in vitro. Thus, a method of the
invention can be used to monitor or determine polyglutamation
activity of a cell or a component thereof, such as
folylpoly-gamma-glutamate synthetase. A cellular extract can also
be prepared from a cell isolated from an individual that has been
administered methotrexate by any route. Efficient conversion of
MTXPGs to MTX in a cellular extract obtained from an individual
undergoing low-dose MTX therapy is disclosed in Example III. The
method of the invention for efficiently converting MTXPGs to MTX in
a cellular extract from an individual treated with methotrexate can
be useful for optimizing therapeutic efficacy or reducing toxicity
associated with MTX therapy in the individual, as set forth in
greater detail below.
[0050] The methods of the invention are well suited to efficiently
converting MTXPGs to MTX in a red blood cellular extract as
demonstrated in Examples II and III. The conditions exemplified
herein can also be readily applied to other types of cellular
extracts in order to efficiently convert MTXPGs to MTX. The
cellular extract can be from a cell that is a target for MTX
therapy or otherwise is a cell indicative of efficacy or toxicity
of MTX therapy. Non-limiting examples of cellular extracts that are
useful in the invention include extracts prepared from tissue
biopsies, erythrocyte extracts, neutrophil extracts and leukocyte
extracts. Additional cellular extracts useful in the invention
include neoplastic or cancer cell extracts such as those obtained
from any of the specific cancers set forth below. Cellular extracts
useful in a method of the invention further include, without
limitation, mammalian cellular extracts, primate cellular extracts,
human cellular extracts, non-human primate cellular extracts, rat
cellular extracts, mouse cellular extracts, cat cellular extracts,
dog cellular extracts, bird cellular extracts and horse cellular
extracts.
[0051] A cellular extract useful in the invention can be obtained
from any individual treated with methotrexate therapy, including
low-dose and high-dose therapy. In one embodiment, a cellular
extract useful in a method of the invention is from an individual
having an autoimmune disease. As used herein, the term "autoimmune
disease" means a disease resulting from an immune response against
a self tissue or tissue component and can include a self antibody
response or cell-mediated response. The term autoimmune disease, as
used herein, encompasses organ-specific autoimmune diseases, in
which an autoimmune response is directed against a single tissue,
such as Crohn's disease and ulcerative colitis, Type I diabetes
mellitus, myasthenia gravis, vitiligo, Graves' disease, Hashimoto's
disease, Addison's disease and autoimmune gastritis; and autoimmune
hepatitis. The term autoimmune disease also encompasses non-organ
specific autoimmune diseases, in which an autoimmune response is
directed against a component present in several or many organs
throughout the body. Such autoimmune diseases include, for example,
rheumatoid disease, systemic lupus erythematosus, progressive
systemic sclerosis and variants, polymyositis and dermatomyositis.
Additional autoimmune diseases include pernicious anemia,
autoimmune gastritis, primary biliary cirrhosis, autoimmune
thrombocytopenia, Sjogren's syndrome, multiple sclerosis and
psoriasis. One skilled in the art will know that a number of the
autoimmune diseases set forth above have been treated with MTX
therapy or can be treated with MTX therapy. One skilled in the art
recognizes that the methods of the invention can be used with a
cellular extract obtained from an individual having any of the
above or another autoimmune disease.
[0052] In another embodiment a cellular extract useful in a method
of the invention is obtained from an individual having arthritis.
As used herein, the term "arthritis" means an inflammatory
condition that affects joints. Arthritis can be infective,
autoimmune or traumatic in origin; the term arthritis includes, but
is not limited to, acute arthritis, acute gouty arthritis,
bacterial arthritis, chronic inflammatory arthritis, degenerative
arthritis (osteoarthritis), infectious arthritis, juvenile
arthritis, mycotic arthritis, neuropathic arthritis, polyarthritis,
proliferative arthritis, psoriatic arthritis, juvenile rheumatoid
arthritis, venereal arthritis, and viral arthritis.
[0053] In a further embodiment a cellular extract useful in a
method of the invention is obtained from an individual having
rheumatoid arthritis. Rheumatoid arthritis is a chronic systemic
disease primarily of the joints, usually polyarticular, marked by
inflammatory changes in the synovial membranes and articular
structures and by muscle atrophy and rarefaction of the bones.
Methotrexate is widely used in the treatment of rheumatoid
arthritis, and one skilled in the art recognizes that the methods
of the invention can be practiced with a cellular extract from an
individual having rheumatoid arthritis or another form of
arthritis.
[0054] In another embodiment, a method of the invention is
practiced with a cellular extract from an individual having cancer.
As used herein, the term "cancer" is intended to mean any member of
a class of diseases characterized by the uncontrolled growth of
aberrant cells. The term includes all known cancers and neoplastic
conditions, whether characterized as malignant, benign, soft tissue
or solid tumor and cancers of all stages and grades including pre-
and post-metastatic cancers. The term cancer encompasses, without
limitation, leukemias such as acute lymphocytic leukemia and acute
myelocytic leukemia; lymphomas; choriocarcinomas; head and neck
cancers; and osteogenic sarcomas, each of which are widely treated
with methotrexate. The term cancer further includes, but is not
limited to, digestive and gastrointestinal cancers such as anal
cancer, bile duct cancer, gastrointestinal carcinoid tumors and
colon cancer; esophageal cancer, gallbladder cancer, liver cancer,
pancreatic cancer, rectal cancer, appendix cancer, small intestine
cancer and stomach (gastric) cancer; breast cancer; ovarian cancer;
lung cancer; renal cancer; cancer of the central nervous system;
and skin cancer. In a particular embodiment, a method of the
invention is practiced with a cellular extract obtained from an
individual having leukemia.
[0055] Rheumatoid arthritis and a variety of other autoimmune
disorders such as psoriasis, systemic lupus erythematosus, and
graft-versus-host disease are typically treated with low-dose MTX
therapy, which is also used in some cancer treatment regimens. In
one embodiment, a method of the invention is practiced with a
cellular extract from an individual undergoing low-dose MTX
therapy. As used herein, the term "low-dose MTX therapy" means
administration of MTX to an individual at a dose that is less than
40 mg/m.sup.2 of body surface per week. Typically, low-dose MTX
therapy is administered at a dose in the range of 2.5 to 40
mg/m.sup.2 of body surface per week depending upon the condition
being treated. Low-dose MTX therapy is typically administered
orally because relatively low doses of MTX are absorbed by the
gastrointestinal tract.
[0056] The methods of the invention can also be used with a
cellular extract from an individual undergoing high-dose MTX
therapy, for example, for treatment of cancer. As used herein, the
term "high-dose MTX therapy" means administration of MTX to an
individual at a dose that is at least 40 mg/m.sup.2 of body surface
per day, for example, at least 100, 500, 1000, 1500, 3000
mg/m.sup.2 or 5000 mg/m.sup.2 of body surface per day. One skilled
in the art understands that high-dose MTX therapy can be
administered at doses up to 5 g/m.sup.2 of body surface per day or
higher depending upon the condition or disease being treated. One
skilled in the art recognizes that the doses of MTX typically used
in high-dose MTX therapy can be administered, for example,
intravenously or orally and that such high-dose MTX therapy
generally requires a period of recovery, which can include
leucovorin rescue or other forms of folate replacement.
[0057] It will be understood that the dosage ranges of MTX set
forth above in the definitions of high- and low-dose MTX therapy
are generalized with respect to treatment of a variety of diseases
and that the range of MTX dose that is administered for one disease
can differ from the range administered for another. Accordingly, a
dose of 40 mg/m.sup.2 of body surface per day, although generally
considered high-dose MTX therapy, may be considered by those
skilled in the art of cancer therapy as a relatively low dose for
treating cancer. Similarly, a dose of 30 mg/m.sup.2 of body surface
per day, although generally considered as low-dose MTX therapy, may
be considered by those skilled in the art of rheumatology as a
relatively high-dose for treating rheumatoid arthritis.
[0058] Cellular extracts useful in the invention can be prepared
from a cell or tissue using methods well known in the art. Those
skilled in the art will know or be able to determine an appropriate
method for obtaining source cells based on their location and
characteristics. As an example, red blood cells and other blood
cells can be obtained by harvesting through intravenous routes.
Cells can also be removed from tissues, such as cancer tissues,
using known biopsy methods including, for example, those utilizing
an open surgical incision, biopsy needle or endoscope. Cells will
be lysed by any of a variety of means depending, in part, on the
properties of the cell. As non-limiting examples, cells can be
lysed by mechanical disruption with glass beads, a Dounce
homogenizer, french press, or sonication; enzymatic disruption with
lysozyme or other enzyme that degrades the cell wall; osmotic
disruption or a combination of these methods.
[0059] A cellular extract useful in a method of the invention can
be a fractionated extract, which can be enriched in MTX or MTXPGs
as compared to the total extract. As an example, an extract can be
fractionated by centrifugation to remove insoluble material such as
membranes and large cellular structures. Fractionation to separate
MTX, MTXPGs, or analogs thereof, from other cellular components can
include, without limitation, centrifugation, protein precipitation,
liquid-liquid extraction, solid-phase extraction, or chromatography
such as reverse phase chromatography, ion pairing chromatography or
ion exchange chromatography, as described, for example, in Rubino,
J. Chromatog. 764:217-254 (2001). Additional methods that can be
used to obtain and fractionate cellular extracts are well known in
the art, as described, for example, in Scopes, supra, 1994, and
Coligan et al., supra, 2000.
[0060] The methods described above for efficiently converting
MTXPGs to MTX also can be used to efficiently deglutamate other
glutamated and polyglutamated substrates, including polyglutamated
antifolates. A number of antifolates useful in treating diseases
and conditions characterized by aberrant cell proliferation can be
polyglutamated in cells. Such antifolates are advantageous for
therapeutic use because intracellular derivatization of an
antifolate with polyglutamate prevents the derivatized antifolate
from being extruded from the cell, in contrast to monoglutamated
antifolates, which are generally extruded. Thus, a polyglutamated
antifolate is maintained in a cell for long periods of time and,
consequently, has prolonged cytotoxic activity without requiring
high levels of administration.
[0061] Thus, the invention provides a method for efficiently
deglutamating a polyglutamated substrate in a cellular extract by
contacting the cellular extract with gamma glutamyl hydrolase under
conditions suitable for efficiently converting the polyglutamated
substrate to a monoglutamated substrate or an unglutamated
substrate. The invention also provides a method for efficiently
deglutamating a polyglutamated antifolate in a cellular extract by
contacting the cellular extract with gamma glutamyl hydrolase under
conditions suitable for efficient conversion of the polyglutamated
antifolate to an antifolate. Such conditions allow gamma glutamyl
hydrolase to remove glutamates from polyglutamated antifolates by
cleavage of one or more iso-peptide bonds.
[0062] As used herein, the term "antifolate" means a molecule
having structural similarity to folate and activity as a folate
antagonist against one or more folate-dependent enzymes.
Polyglutamylatable antifolates are antifolates that can be
polyglutamated in a cell by an enzyme such as
folylpoly-gamma-glutamate synthetase. Examples of
polyglutamylatable antifolates include, without limitation,
aminopterin, raltitrexed, lometrexol, multitargeted antifolate
(MTA), AQA, MTX and analogs thereof. Aminopterin, for example,
possesses a hydrogen instead of a methyl group at position N-10
compared to the structure of methotrexate. Raltitrexed is a
selective inhibitor of thymidylate synthase as described, for
example, in Kamen, Semin. Oncol. S18:30-39 (1997). Lometrexol
selectively inhibits glycinamide ribonucleotide formyltransferase,
the first enzyme involved in the pathway of de novo purine
synthesis as described, for example, in Calvert, supra, 1999. MTA
is an inhibitor of multiple folate-dependent enzymes, such as
dihydrofolate reductase, thymidylate synthase, and glycinamide
ribonucleotide formyltransferase (see, for example, Calvert, supra,
1999).
[0063] One type of antifolate useful in the invention is a
methotrexate analog. As used herein, the term "methotrexate analog"
means a molecule having structural and functional similarity to
methotrexate. Methotrexate analogs are functionally characterized,
in part, by their inhibitory activity against dihydrofolate
reductase. A methotrexate analog useful in the invention acts as a
substrate for polyglutamation in a cell by an enzyme such as
folylpoly-gamma-glutamate synthetase. MTX analogs include, but are
not limited to, 4-amino derivatives with halogen substitution on
the para-aminobenzoic moiety, such as dichloromethotrexate (see,
for example, Frei et al., Clin. Pharmacol. Therap. 6:160-71
(1965)); 7-methyl substituted MTX (see, for example, Rosowsky and
Chen, J. Med. Chem. 17:1308-11 (1974)); 3',5'-difluoro MTX, (see,
for example, Tomcuf, J. Organic Chem, 26:3351 (1961)); 2' and 3'
monofluorinated derivatives of aminopterin (see, for example,
Henkin and Washtien, J. Med. Chem. 26:1193-1196 (1983)); and
7,8-dihydro-8-methyl-MTX (see, for example, Chaykovsky, J. Org.
Chem. 40:145-146 (1975)). The skilled person understands that the
methods of the invention can be used to optimize or monitor
efficacy or toxicity associated with methotrexate analogs or other
antifolates in the same manner as disclosed herein for monitoring
methotrexate therapy.
[0064] The invention further provides a method for determining a
level of methotrexate polyglutamates in a cellular extract. The
method includes the steps of: (a) converting the MTXPGs to
methotrexate in a cellular extract under conditions suitable for
efficient conversion of MTXPGs to MTX, and (b) determining a level
of the MTX, where the level of MTX can be correlated with the level
of MTXPGs in the cellular extract. Step (a) of the method can
further include, for example, contacting the cellular extract with
gamma glutamyl hydrolase under conditions suitable for efficient
conversion of MTXPGs to MTX, for example, any of the conditions set
forth above. The method is useful for monitoring the efficacy or
toxicity of MTX therapy, as set forth in further detail below.
[0065] As discussed above, the invention provides conditions
suitable for efficient conversion of MTXPGs to MTX. Thus, the level
of MTX in a "converted" cellular extract can be correlated with the
level of MTXPGs in the cellular extract after correction for any
baseline level of methotrexate. The term "level," when used in
reference to a particular molecule in a sample, means an amount or
concentration of the particular molecule in the sample. As an
example, a level of MTX within a cellular extract is the amount or
concentration of MTX in the cellular extract; a level of MTXPGs in
a cellular extract is the amount or concentration of MTXPGs in the
cellular extract. In view of the above, it is understood that a
level can be an absolute level such as a molar concentration or
weight or a relative level such as a percent or fraction compared
to one or more other molecules in a sample. Specifically excluded
from the definition of "level," as used herein, are estimates or
measurements based on enzymatic activity, such as inhibition of
DHFR or other enzyme activity.
[0066] Subsequent to efficient conversion of methotrexate
polyglutamates to methotrexate, the methods of the invention can
include a step of determining the level of methotrexate.
Quantitative and accurate detection of MTX at low levels in
cellular extracts can be achieved using the methods set forth
below.
[0067] The invention also provides a method for determining a level
of methotrexate in a cellular extract by (a) irradiating the
cellular extract, thereby producing a fluorescent MTX photolytic
product; and (b) determining a level of the fluorescent MTX
photolytic product, wherein the level of the fluorescent MTX
photolytic product can be correlated with the level of MTX. By
indirectly determining the intracellular level of methotrexate
polyglutamates, a method of the invention can be useful for
monitoring the efficacy or toxicity of MTX therapy.
[0068] In a method of the invention, MTX can be irradiated to
produce a photolytic product. As used herein, the term "photolytic
product" means a molecule that is produced by cleavage of bonds in
a substrate that is electronically excited by radiation. The
process of producing a photolytic product is referred to as
photolysis. Photolysis of MTX to produce a "MTX photolytic product"
can be performed, for example, with UV light, which is a term
understood in the art to include any wavelength in the range of
about 200 to 400 nm. It further is understood that any light source
which produces UV light can be useful in a method of the invention
including, for example, a lamp such as an arc lamp or quartz
halogen lamp, or a laser. As demonstrated in Example I, a
fluorescent MTX photolytic product was produced by irradiating MTX
with a low pressure mercury UV lamp which emits radiation in the
range of 225 to 275 nm and has a peak output at 254 nm. It is
understood that MTX can be selectively irradiated with a particular
wavelength in the UV range by using an appropriate light source,
optical filter or combination of these components in accordance
with their known optical characteristics.
[0069] A sample, such as a cellular extract, that contains MTX, can
be irradiated according to a method of the invention for the
appropriate period of time to yield a fluorescent MTX photolytic
product, where the level of the fluorescent MTX photolytic product
can be correlated with the level of MTX in the sample. In
particular embodiments, a method of the invention is practiced by
irradiating a cellular extract containing MTX for 0.5 to 60
seconds. Shorter periods of irradiation within this range can also
be useful in a method of the invention including, for example,
irradiation for 0.5 to 15 seconds. In further embodiments, the
cellular extract is irradiated for 0.1 to 100 seconds, 0.2 to 60
seconds, 0.5 to 60 seconds, 0.5 to 45 seconds, 0.5 to 30 seconds,
0.5 to 20 seconds, 0.5 to 15 seconds, 0.5 to 10 seconds, 1 to 20
seconds, 1 to 10 seconds, 2 to 20 seconds or 2 to 10 seconds. In
additional embodiments, the cellular extract is irradiated for 0.5
to 6, 0.5 to 5, 0.5 to 4, 1 to 6, 1 to 5, 1 to 4, or 2 to 4
seconds.
[0070] As demonstrated in Example I, irradiation for 3 seconds
yielded primarily fluorescent MTX photolytic product A. As further
demonstrated in Example I, irradiation for 24 seconds yielded
primarily fluorescent MTX photolytic product B. In view of the
above, it is understood that the time of irradiation can be varied
to produce the desired fluorescent MTX photolytic product having
characteristic properties as desired for a particular application.
One skilled in the art further understands that the methods of the
invention can be practiced by detecting a single fluorescent MTX
photolytic product such as MTX photolytic product A or MTX
photolytic product B. The MTX photolytic products A and B have
different properties including, for example, characteristic
fluorescence excitation and emission peak maxima, and
characteristic fluorescence intensity levels depending upon the pH
and amount of acetonitrile present during detection (see Example
I).
[0071] Photolysis of MTX can be carried out in the presence of
hydrogen peroxide (H.sub.2O.sub.2) or another peroxide. As a
non-limiting example, when hydrogen peroxide is added, the final
concentration can be 0.03% or higher. In particular embodiments,
the final concentration of hydrogen peroxide is in the range of
0.05% to 1%, 0.2% to 1% or 0.3% to 0.6%.
[0072] A level of MTX in a cellular extract can be determined based
on the level of a fluorescent MTX photolytic product. The term
level, when used in reference to a fluorescent MTX photolytic
product, means an amount or concentration of the fluorescent MTX
photolytic product. The amount or concentration of a fluorescent
MTX photolytic product can be determined, for example, based on the
intensity of fluorescence from the photolytic product.
[0073] As used herein, the term "fluorescence" means an emission of
photons in the ultraviolet (UV), visible (VIS) or infrared (IR)
region of the spectrum in response to electronic excitation by
radiation. The term "fluorescent," when used in reference to a MTX
photolytic product, means a photolytic product that emits photons
in the UV, VIS or IR region of the spectrum in response to
electronic excitation by radiation. Thus, a fluorescent MTX
photolytic product is a photolytic product derived from
methotrexate that emits photons in the UV, VIS or IR region of the
spectrum in response to electronic excitation by radiation. A
fluorescent MTX photolytic product can be characterized, for
example, as emitting photons at a quantum yield of at least 0.01
when excited by radiation in solution. In particular embodiments, a
fluorescent MTX photolytic product is characterized by a quantum
yield of fluorescence that is at least 0.1, 0.2, 0.3, 0.4, 0.5,
0.6, 0.7, 0.8, 0.9 or higher when excited by radiation in
solution.
[0074] A fluorescent molecule, such as a fluorescent MTX photolytic
product, also can be characterized with respect to maximum emission
wavelength or maximum excitation wavelength. In particular
embodiments, a method of the invention involves determining the
level of a fluorescent MTX photolytic product having a maximum
excitation wavelength in the IR, red, orange, yellow, green, blue,
violet or UV region of the spectrum. In additional embodiments, a
method of the invention is practiced by determining the level of a
fluorescent MTX photolytic product having a maximum emission
wavelength in the IR, red, orange, yellow, green, blue, violet or
UV region of the spectrum.
[0075] Fluorescence can be detected in a method of the invention
using any of a variety of excitation sources and emission
detectors. Excitation of a fluorescent MTX photolytic product can
be achieved with an excitation source such as a lamp or laser
including, without limitation, any of those described above in
regard to photolysis. Excitation at a particular wavelength or in a
particular wavelength range can be achieved in a method of the
invention using, for example, a laser that is tuned to the desired
wavelength or a lamp having an output that includes the wavelength
range. An appropriate optical filter can be placed between the
excitation source and fluorescent MTX photolytic product to further
limit the range of wavelengths contacting the fluorescent MTX
photolytic product if desired. As shown in FIG. 2D and set forth in
Example I, fluorescent MTX photolytic product A has two excitation
peaks in the range of 240 nm to 420 nm, including the peak from
about 240 nm to 300 nm and the peak from about 360 nm to 410 nm. In
particular embodiments of the invention, a fluorescent MTX
photolytic product can be detected by excitation at a wavelength in
the range of about 240 nm to 420 nm, about 240 nm to 300 nm or
about 360 nm to 410 nm. If desired, the methods of the invention
can include excitation at or near the peak of 275 nm or in a range
near this peak including, for example, excitation at a wavelength
in the range of 250 nm to 290 nm or 260 nm to 280 nm. An excitation
at or near the peak of 385 nm or in a range near this peak can also
be used including, for example, excitation at a wavelength in the
range of 250 nm to 290 nm or 260 nm to 280 nm.
[0076] Emission can be detected from a fluorescent MTX photolytic
product using a detector such as, without limitation, a
photomultiplier tube, diode, diode array or charge coupled device
camera. A detector that detects light at a particular wavelength or
in a particular wavelength range can be used in a method of the
invention. If desired, an optical filter can be placed between the
fluorescent MTX photolytic product and the detector to limit the
range of wavelengths detected. As shown in FIG. 2D and set forth in
Example I, fluorescent MTX photolytic product A emits from about
320 nm to 550 nm and has a primary peak from about 440 nm to 520
nm. In particular embodiments of the invention, emission from a
fluorescent MTX photolytic product can be detected at a wavelength
in the range of about 320 nm to 550 or about 440 nm to 520 nm. If
desired, the methods of the invention can include detection of
emission at or near the peak of 464 nm or in a range near this peak
including, for example, emission at a wavelength in the range of
450 nm to 510 nm or 460 nm to 490 nm.
[0077] A level of MTX also can be determined using, for example,
chromatography or mass spectrometric analysis. MTX can be
chromatographically separated from other cellular components using,
for example, reverse phase chromatography as set forth in Examples
I through III and subsequently quantitated, for example, by
comparison to known reference standards. As demonstrated herein,
MTX was accurately detected using reverse phase chromatography on a
high performance liquid chromatography system. As a non-limiting
example, chromatographic analysis can include passing an MTX
containing sample through a C18 reverse phase column in a mobile
phase consisting of a linear gradient from 2% acetonitrile/98%
mobile phase A to 25% acetonitrile/75% mobile phase A, wherein
mobile phase A is 10 mM ammonium acetate, pH 6.5.
[0078] The content of a solution that is used to detect MTX, or a
photolytic product thereof, can be varied, for example, with
respect to pH or acetonitrile content. The pH at which MTX, or a
photolytic product thereof, is detected in a method of the
invention can be in the range of, for example, about pH 2 to 8 or
in the range of about pH 4 to 7. In particular embodiments, MTX, or
a photolytic product thereof, can be detected, for example, at pH
4, 4.5, 5, 5.5, 6, 6.5 or 7. The amount of acetonitrile present
during detection of MTX, or a photolytic product thereof, can be in
the range of, for example, about 0% to 20% or about 10% to 20%. In
particular embodiments the amount of acetonitrile present can be,
for example, 5%, 10%, 15% or 20%.
[0079] A reverse phase column useful for separating MTX from other
cellular components can have, for example, dimensions of 25
cm.times.4.6 mm, as exemplified herein. Columns having larger or
smaller diameters, lengths or both can also be used, for example,
to accommodate larger or smaller sample size. Flow rates can vary,
for example, from 0.2 to 2.5 ml/min. As demonstrated herein, the
flow rate for the mobile phase can be, for example, 1 ml/min.
However, the flow rate of the mobile phase can be altered as
desired. A slower flow rate, such as 0.8 ml/min, 0.5 ml/min or 0.2
ml/min, can be used, for example, with a smaller column or to
increase MTX retention times. Alternatively a faster flow rate,
such as 1.5 ml/min or 2.0 ml/min, can be used, for example, with a
larger column or to decrease MTX retention times.
[0080] A level of MTX in a sample also can be detected in a method
of the invention based on other properties of MTX including, for
example, ultraviolet or visible light absorption properties,
fluorescence, electrochemical properties, or mass. Methods for
detection of MTX based on these properties are known in the art as
described, for example, in Rubino, supra, 2001. As non-limiting
examples, a level of MTXPG in a sample can be determined by
efficiently converting MTXPGs to MTX under conditions suitable for
efficient conversion and detecting the resulting MTX with UV/Vis
absorption spectroscopy, fluorimetry, electrochemical detection, or
mass spectrometry. Those skilled in the art will know or be able to
determine an appropriate means for detecting methotrexate based on
the accuracy and sensitivity desired and the presence of
potentially interfering substances in the particular sample being
analyzed.
[0081] The invention provides a method of optimizing therapeutic
efficacy or reducing toxicity associated with methotrexate therapy
administered to an individual. The method includes the steps of:
(a) converting methotrexate polyglutamates to MTX in a cellular
extract from an individual under conditions suitable for efficient
conversion of MTXPGs to MTX; and (b) determining a level of MTX in
the cellular extract, the level of MTX correlating with a level of
MTXPGs, thereby determining a level of MTXPGs in the cellular
extract, wherein a drug or dosage subsequently administered to the
individual is selected based on the level of MTXPGs.
[0082] Intracellular levels of methotrexate polyglutamates can be
associated with efficacy, with higher levels associated with
toxicity. By determining the intracellular level of methotrexate
polyglutamates, the methods of the invention can be useful for
adjusting the amount or frequency of methotrexate therapy in order
that the methotrexate polyglutamates are within the therapeutic
range and do not exceed this range, thereby producing undesirable
toxic side effects. Any of a variety of types of cellular extracts
can be useful in a method of the invention for optimizing
therapeutic efficacy or reducing toxicity associated with
methotrexate therapy. Cell extracts can be prepared from any cell
or tissue that is indicative of the efficacy or toxicity of
methotrexate therapy such as a diseased cell or tissue, a target
cell for MTX therapy or a cell that is representative of the amount
of drug in diseased cells. As used herein, the term "target cell
for MTX therapy" means a cell for which uptake of MTX is desired to
treat a disease or condition. As non-limiting examples, cell
extracts can be prepared from red blood cells, leukocytes,
neutrophils, cancer cells, and tissue biopsies.
[0083] A method of the invention for optimizing therapeutic
efficacy or reducing toxicity associated with methotrexate therapy
can further include altering the methotrexate dosage subsequently
administered to the individual based on the determined level of
methotrexate polyglutamates. Where the determined level of MTXPGs
is below a therapeutic range, the dose or frequency of methotrexate
can be increased. Where the determined level of MTXPGs is above a
therapeutic range, the dose or frequency of methotrexate can be
reduced to avoid toxicity. A therapeutic range can be determined
from dose-response information for a particular disease or
condition and, if desired, for the age, gender and medical
condition of the individual being treated. As an example, a level
of MTXPG determined in a red blood cellular extract can be compared
to dose-response information correlating red blood cell
methotrexate polyglutamate levels with reduction of an arthritis
symptoms. Dose-response information also can be obtained using well
known clinical procedures relevant to the particular pathological
condition.
[0084] In a particular embodiment of the invention, a cellular
extract useful in a method of the invention for optimizing
therapeutic efficacy or reducing toxicity associated with MTX
therapy can be obtained from a non-target cell for MTX therapy. As
used herein, the term "non-target cell for MTX therapy" means a
cell for which uptake of MTX is not desired. Such a non-target cell
can be a normal cell that is sensitive to levels of MTX or that is
disposed to take up MTX that is administered by a particular route.
Determining a MTXPG level in a cellular extract from a non-target
cell also can be useful to determine toxicity of MTX therapy.
[0085] Methotrexate therapy can cause a variety of unwanted adverse
effects that mimic folate deficiency including, for example,
gastrointestinal intolerance, stomatitis, alopecia and cytopenia.
Many adverse effects of methotrexate therapy are dose dependent and
can be alleviated by administration of compensatory doses of
folate. Accordingly, a method of the invention for optimizing
therapeutic efficacy or reducing toxicity associated with
methotrexate therapy can further include, if desired, altering the
dose of folate, or a derivative thereof, subsequently administered
to the individual based on the determined level of intracellular
methotrexate polyglutamates.
[0086] The invention further provides an isolated deglutamated
composition which is characterized as (a) present in a red blood
cell extract following deglutamation; (b) having a retention time
of about 12 minutes in a mobile phase consisting of a 15-minute
linear gradient from 2% acetonitrile/98% mobile phase A to 25%
acetonitrile/75% mobile phase A at a flow rate of 1 ml/min through
a Terra MS 018 reverse phase column, which has dimensions of 25
cm.times.4.6 mm and a particle size of 5 micrometers, and where the
mobile phase A contains 10 mM ammonium acetate, pH 6.5, and 0.06%
hydrogen peroxide in water; and (c) having a fluorescence
excitation spectrum with peaks at 299 nm and 402 nm in water and a
fluorescence emission spectra with a peak at 460 nm in water. The
endogenous compound can be further characterized as being
photostable to irradiation with a 254 nm low pressure mercury
ultraviolet lamp for 3 seconds, wherein following irradiation with
254 nm for 3 seconds the fluorescence excitation spectrum of the
isolated deglutamated composition has peaks at 299 nm and 402 nm in
water and the isolated deglutamated composition emits fluorescence
at 464 nm in water. The endogenous compound can further be
characterized, for example, as having a fluorescence emission peak
at 460 nm in water.
[0087] The present invention further provides an isolated
composition produced by: (a) converting polyglutamated compounds in
a red blood cellular extract to produce deglutamated RBC compounds;
(b) fractionating the deglutamated RBC compounds under conditions
comprising passage of a mobile phase consisting of a 15-minute
linear gradient from 2% acetonitrile/98% mobile phase A to 25%
acetonitrile/75% mobile phase A at a flow rate of 1 ml/min through
a Terra MS 018 reverse phase column, which has dimensions of 25
cm.times.4.6 mm and a particle size of 5 micrometers, and where the
mobile phase A includes 10 mM ammonium acetate, pH 6.5 and 0.06%
hydrogen peroxide in water; and (c) isolating a compound that
elutes in a peak at 12 minutes, where the peak is detectable by
emission at 464 nm upon excitation with radiation at 400 nm.
[0088] The invention also provides an isolated deglutamated
composition, such as the endogenous compound described in Example
IV, together with a pharmaceutically acceptable carrier. An
isolated deglutamated composition of the invention can be, for
example, a folate derivative; in this case, a pharmaceutical
composition containing the derivative can be useful as a surrogate
for folate in any of a variety of applications. Such a
pharmaceutical composition can be useful, for example, in a method
of reducing toxicity associated with methotrexate therapy. Thus,
the present invention also provides a method of reducing toxicity
associated with methotrexate therapy by administering to an
individual treated with methotrexate therapy an effective dose of a
pharmaceutical composition of the invention.
[0089] Any of a variety of pharmaceutical compositions are
encompassed by the invention. As one example, a deglutamated
composition can be administered as a solution or suspension
together with a pharmaceutically acceptable carrier. Such a
pharmaceutically acceptable carrier can be, for example, water,
sodium phosphate buffer, phosphate buffered saline, normal saline
or Ringer's solution or other physiologically buffered saline, or
other solvent or vehicle such as a glycol, glycerol, an oil such as
olive oil or an injectable organic ester. Pharmaceutically
acceptable carriers can be sterile or substantially free from
contaminating particles and organisms. A composition of the
invention that includes a pharmaceutically acceptable carrier can
be of sufficient purity and quality for use in humans. A
pharmaceutical composition of the invention containing a
deglutamated compound can optionally include methotrexate.
[0090] A pharmaceutical composition of the invention can be
formulated for parenteral administration such as subcutaneous,
intraperitoneal, intramuscular, intravenous, intradermal,
intracranial, intratracheal, and epidural administration. A
pharmaceutical composition of the invention also can be formulated
for oral, topical (including buccal and sublingual), rectal,
ophthalmic, nasal, intrauterine or vaginal administration. A
pharmaceutical composition of the invention can be presented in
unit dosage form and can be prepared by pharmaceutical techniques
well known to those skilled in the art. Such techniques include the
step of bringing into association the active ingredient and a
pharmaceutical carrier or excipient.
[0091] Formulations suitable for parenteral administration include
aqueous and non-aqueous sterile injectable solutions including any
of the pharmaceutically acceptable carriers described above. The
solutions can additionally contain, for example, one or more
anti-oxidants, buffers, bacteriostats or solutes which render the
formulation isotonic with the blood of the intended recipient.
Other formulations include, for example, aqueous and non-aqueous
sterile suspensions which can include suspending agents and
thickening agents. The formulations can be presented in unit-dose
or multi-dose containers, for example, sealed ampules and vials,
and can be stored in a lyophilized condition requiring, for
example, the addition of the sterile liquid carrier, immediately
prior to use. Extemporaneous injection solutions and suspensions
can be prepared from sterile powders, granules and tablets of the
kind previously described.
[0092] A pharmaceutically acceptable carrier can additionally
contain any of a variety of physiologically acceptable compounds
that act, for example, to stabilize the deglutamated compound. Such
physiologically acceptable compounds include, for example,
carbohydrates such as glucose, sucrose or dextrans; antioxidants
such as ascorbic acid or glutathione; chelating agents such as
EDTA, which disrupts microbial membranes; divalent metal ions such
as calcium or magnesium; low molecular weight proteins; lipids or
liposomes; and other stabilizers or excipients.
[0093] A variety of routes of administration can be useful for
delivering a pharmaceutical composition of the invention. Such
routes include those described previously and will depend, in part,
on the particular therapeutic need and formulation. One skilled in
the art understands that an effective amount of the deglutamated
compound is included in the pharmaceutical composition and depends
on the pathological condition to be treated and the specific
physical characteristics of the individual affected with the
disease. Those skilled in the art will know or can determine a
specific regime of administration which is effective for a
particular application using the teachings and guidance provided
herein together with diagnostic and clinical criteria known within
the field of art of the particular pathological condition.
[0094] The invention further provides a kit including the
endogenous deglutamated compound described in Example IV. In one
embodiment, the kit further includes methotrexate. Those skilled in
the art can readily incorporate a composition of the invention into
kit form in combination with appropriate buffers and solutions for
the practice of one or more of the methods disclosed herein.
[0095] A kit can include packaging material that houses the
contents of the kit. The packaging material is constructed by well
known methods, preferably to provide a sterile, contaminant-free
environment. The packaging materials employed herein in relation to
a kit or diagnostic system can be any acceptable material that is
customarily utilized in small molecule-based therapeutic or
diagnostic systems. A package can be any solid matrix or material
such as glass, plastic, paper, foil, and the like, capable of
holding within fixed limits a compound of the present invention.
Thus, for example, a package can be a glass vial or microtiter
plate used to contain microgram or milligram quantities of the
endogenous deglutamated compound disclosed herein in Example
IV.
[0096] The packaging material can include a label which indicates
that the compositions can be used for reducing toxicity associated
with methotrexate therapy, for determining an intracellular level
of methotrexate, or for optimizing methotrexate therapy. In
addition, the packaging material can include instructions
indicating how the materials within the kit are employed. Such
instructions will be in accordance with the description set forth
herein for performing the methods. The instructions will typically
include a tangible expression describing the concentration of the
compound or at least one therapeutic or diagnostic method parameter
including, without limitation, the relative amounts of the compound
and methotrexate or some other component to be admixed, maintenance
time periods for components of the kit, recommended temperature for
storage or use, buffer conditions, and the like.
[0097] As further disclosed herein, intracellular levels of a
deglutamated composition of the invention can be determined and
used to calculated the intracellular level of methotrexate. Thus,
the present invention provides a method for determining an
intracellular level of methotrexate, by deglutamating a cellular
extract to produce a deglutamated composition of the invention; and
determining the level of deglutamated composition, where the level
of the deglutamated composition can be correlated with the
intracellular level of methotrexate. In one embodiment, the
cellular extract is a red blood cellular extract.
[0098] The present invention further provides a method of
optimizing therapeutic efficacy or reducing toxicity associated
with methotrexate therapy administered to an individual by
deglutamating compounds in a cellular extract from the individual;
and determining a level of a deglutamated composition of the
invention in the cellular extract, where a drug or dosage
subsequently administered to the individual is selected based on
the level of the deglutamated composition. The level of
methotrexate can optionally also be determined in the cellular
extract; in this case, a drug or dosage subsequently administered
to the individual is selected based on the ratio of the level of
methotrexate to the level of the deglutamated composition.
[0099] The following examples are intended to illustrate but not
limit the present invention.
Example I
[0100] Quantification of Methotrexate by Fluorimetric Detection of
Methotrexate-Photolytic Products
[0101] This example describes quantification of methotrexate using
high performance liquid chromatography (HPLC) and post-column
photolysis followed by fluorometric detection of photolytic
products.
[0102] The MTX detection system was based on an HPLC system
(Agilent 1100 HPLC chemstation system) equipped with a C18 reversed
phase column (25 cm.times.4.6 mm X Terra MS C18, 5 micrometer
particle size; Waters, Milford, Mass.), a post-column photochemical
reactor unit (Aura Industries; New York, N.Y.) and a post-reactor
unit fluorometer detector. The system also included a C18
pre-column that was changed every 200 injections.
[0103] Samples containing 20 to 1000 nmol/L MTX (Sigma, St. Louis,
USA) in RBC extracts were run in the HPLC system at a flow rate of
1 ml/min, with a 15-minute linear gradient from 2% acetonitrile/98%
mobile phase A (10 mM ammonium acetate, pH 6.5, unless otherwise
indicated and 0.2% hydrogen peroxide in water) to 25%
acetonitrile/75% mobile phase A, unless otherwise indicated. After
15 minutes, mobile phase was returned to 100% mobile phase A and
the column was re-equilibrated for 5 minutes. The samples were
maintained at 4.degree. C. and injected every 20 minutes with an
autoinjector.
[0104] Eluate from the column passed through the photochemical
reactor unit in TEFLON.TM. tubing ( 1/16 inch outer diameter, 0.25
mm inner diameter, 8 meter length) that was connected on-line after
the column and before the fluorometer detector and assembled as a
knitted coil. The knitted coil was extended lengthwise through the
photochemical reactor unit which also housed an elongated 254 nm
low pressure mercury ultraviolet lamp (Aura Industries; New York,
N.Y.). The time of irradiation of MTX in the photochemical reactor
was 24 seconds. Irradiation for less than 24 seconds was achieved
by masking all but a portion of the elongated lamp with foil such
that only a portion of the knitted coil was irradiated. In
particular, the lamp was masked such that only 1 meter of the coil
was irradiated with the lamp which at a flow rate of 1 ml/min
corresponded to 3 seconds irradiation. Methotrexate photolytic
products were measured at an excitation wavelength set at 400 nm
and an emission wavelength at 470 nm, unless otherwise indicated.
Chromatograms were acquired and analyzed on a Hewlett-Packard
Vector XP computer. The retention times described herein for the
above-described HPLC system were measured from the time of
injection to time of detection at the post-reactor unit fluorometer
detector.
[0105] As shown in FIG. 2C, HPLC chromatography with in line
photolysis under conditions that formed methotrexate photolytic
product A, and fluorescence detection at an excitation wavelength
of 400 nm and emission wavelength at 464 nm produced a single peak
having a retention time of about 12.4 minutes. The limit of
detection, defined as three times the signal-to-noise ratio, was 5
nmol/L for MTX. The analytical column demonstrated no deterioration
of its performance after up to 1000 injections.
[0106] Two methotrexate photolytic products were obtained from the
post-column derivatization. Product A was formed from the
equivalent of a 3-second irradiation and exhibited an excitation
spectrum with a wavelength maximum at 274 nm and a smaller peak
with a maximum at 380 nm and an emission spectrum with wavelength
maximum at 464 nm. FIG. 2D shows the fluorescence excitation and
emission spectra for product A of the peak eluting at 12.42 minutes
in two separate HPLC separations under conditions similar to those
described for the chromatogram of FIG. 2C. Product B was formed
following irradiation corresponding to 24 seconds using an 8 meter
knitted reactor coil. Product B had maximum excitation at 370 nm
and maximum emission at 417 nm.
[0107] The effect of pH on MTX detection was analyzed using the MTX
detection system described above except that the mobile phase was
isocratic and consisted of 10 mM ammonium acetate, 0.2% hydrogen
peroxide in water, 10% acetonitrile and a different pH from 4.0 to
7.0. As shown in FIG. 2A, when detection was based on conversion to
photolytic product A, increasing pH resulted in an increase in the
peak area for MTX. However, the peak area was relatively unchanged
at different pH values in this range, when detection was based on
conversion to photolytic product B. At pH greater than about 4.2,
product A exhibited higher fluorescence intensity compared to
product B.
[0108] The effect of acetonitrile on MTX detection was analyzed
using the MTX detection system described above except that the
mobile phase was isocratic and consisted of 10 mM ammonium acetate,
pH 6.0, 0.2% hydrogen peroxide in water and different percentages
of acetonitrile varying from 10 to 20%. As shown in FIG. 2B, when
detection was based on conversion to photolytic product A,
increasing percentages of acetonitrile resulted in a decrease in
the peak area for MTX. However, the peak area was relatively
unchanged at different acetonitrile percentages in this range, when
detection was based on conversion to photolytic product B. In the
range of acetonitrile tested, product A exhibited higher
fluorescence intensity compared to product B.
[0109] These results indicate that pH and percentage acetonitrile
of the mobile phase and the time of irradiation can be changed to
modify the sensitivity of MTX detection. In particular, these
results indicate that the sensitivity of detection of MTX
photolytic product A can be changed by altering pH and acetonitrile
concentration.
Example II
Conversion of Methotrexate Polyglutamates to Methotrexate
[0110] This example describes efficient conversion of methotrexate
polyglutamates (MTXPGs) to methotrexate (MTX) and further
demonstrates quantitation of methotrexate polyglutamates in a
cellular extract.
[0111] MTXPGs were enzymatically converted to MTX by incubation
with RBC extract and plasma as follows. RBCs were isolated from
healthy donors (Blood bank; San Diego, Calif.). Hemolysates were
prepared having 0.88.times.10.sup.9 RBC per 100 .mu.l. For spiked
samples, MTXPG (Schirks Laboratories; Jona, Switzerland) was added
to RBC extracts at a final concentration of 1 .mu.M (from stock
solutions containing 100 .mu.M MTXPG in 0.1 N potassium hydroxide).
Reconstituted plasma was prepared from lyophilized plasma (Sigma,
St. Louis, USA; Cat. No. P9523) and 100 .mu.l was added to 50 .mu.l
of RBC extract, mixed for 30 seconds, and 100 .mu.l of buffer
containing 100 mM potassium phosphate pH 4.5 and 150 mM
mercaptoethanol was added. Because the reconstituted plasma and RBC
extract are strongly buffered at physiological pH, the mixture had
a final pH of about 6.5. Samples were incubated for 12-14 hours,
unless otherwise indicated, in the dark at 37.degree. C. After
incubation 30 .mu.l of 70% perchloric acid was added to the
mixture, vortexed for 15 seconds and centrifuged for 10 minutes. A
total volume of 80 .mu.l was injected onto the HPLC system for
analysis using the MTX detection system described in Example I.
[0112] FIG. 3 shows typical chromatograms of an RBC extract blank
(Panel A) and an RBC extract spiked with MTXPG.sub.2-7 (Panel B,
100 nmol/L RBC) under the above-described conditions for enzymatic
conversion to MTX. Although the maximum excitation wavelength for
the methotrexate photolytic product was 274 nm, excitation
wavelength was set at 400 nm to avoid interfering signals observed
in the RBC extract blank. As shown in FIG. 3C, the excitation
spectrum for the MTX photolytic product from the RBC extract that
eluted at 12.5 minutes in the chromatogram shown in FIG. 3B was
similar to the excitation spectrum for the photolytic product
obtained from an MTX standard, measured in water. The overlap in
the excitation spectra confirm that the 12.5 minute peak is
MTX.
[0113] Standard curves demonstrated a linear relationship between
peak area and concentration with correlation coefficients >0.995
for MTX, and for MTX formed after enzymatic conversion of
MTXPG.sub.2 (MTXPG having 2 glutamates) or MTXPG.sub.2-7 (equimolar
mixture of MTXPG.sub.2, MTXPG.sub.3, MTXPG.sub.4, MTXPG.sub.5,
MTXPG.sub.6, and MTXPG.sub.7), Spiked standards were assayed in
duplicate. Standard curves were fit by linear regression using peak
areas versus concentrations. Equations describing the standard
curves were: MTX, y=0.401x+0.281; MTX after enzymatic conversion of
MTXPG.sub.2, y=0.383.times.+0.115; and MTX after enzymatic
conversion of MTXPG.sub.2-7, y=0.391x-0.122 where y=peak area and
x=spiked concentration.
[0114] As shown in FIG. 4, MTXPG.sub.2 (Panel A) and MTXPG.sub.2-7
(Panel B) were completely converted to MTX after an 8 hour
incubation at 37.degree. C., in the dark, at concentration of 1000
nmol/L RBC and the conversion was largely complete by 4 hours. The
percent conversion of MTXPG to MTX was determined by comparing the
peak area of MTX formed during the enzymatic conversion at
different times with the peak area of MTX spiked in the red blood
cells at the same concentration.
[0115] The intra-day accuracy and inter-day accuracy for
quantitation of MTX and MTXPGs are shown in Table I. RBC extracts
were incubated with 20, 100 or 500 nmol/L of MTX, MTXPG.sub.2 or
MTXPG.sub.2-7 (Target concentrations; see Table I), and the
observed concentration of MTX was determined by the HPLC method
described in Example I. For the intra-day accuracy assessment, the
mean observed MTX concentration was determined for 10 spiked
replicates at each target concentration (see Table I). The mean
observed MTX concentrations for inter-day accuracy assessment were
based on three replicates from five different days (Table 1).
Accuracy was calculated as the percentage error of the mean
observed MTX concentration from the target concentration value for
each set of spiked samples (mean observed concentration/target
concentration.times.100%). Precision was determined by the relative
standard deviation (RSD).
TABLE-US-00001 TABLE I Intra-day (n = 10) Inter-day (n = 5) Mean
Mean Mean Mean Target observed accuracy observed accuracy conc.
MTXPG.sub.1 RSD of target MTXPG.sub.1 RSD of target Standard nmol/L
conc. nmol/L % value (%) conc. nmol/L % value (%) MTXPG.sub.1 50.0
45.0 4.8 90.0 48.9 8.4 97.9 250.0 250.2 3.8 100.1 260.8 3.8 104.3
500.0 504.2 4.1 100.8 517.3 2.3 103.5 MTXPG.sub.2 50.0 48.2 1.8
96.4 48.8 6.8 97.7 250.0 231.1 4.2 92.5 238.5 2.7 95.4 500.0 473.2
3.6 94.6 490.8 4.2 98.2 MTXPG.sub.2-7 50.0 45.4 4.7 90.7 47.0 4.3
94.1 250.0 244.5 3.3 97.8 245.6 4.1 98.2 500.0 473.2 2.3 94.6 492.8
6.1 98.6
[0116] Highly efficient conversion of MTXPG.sub.2 and
MTXPG.sub.2-7, respectively, to MTX was demonstrated by the
accuracies of greater than 95% and a coefficient of variation less
than 6% for all target concentrations as shown in Table I.
Furthermore, as shown in FIG. 5, the plots of observed MTX
concentration versus the concentration of spiked MTXPG.sub.2 or
MTXPG.sub.2-7 had slopes of 0.98 and 1.0 respectively,
demonstrating that the method was accurate and converted greater
than 95% methotrexate polyglutamates to methotrexate. The lower
limit of quantification of MTXPG by conversion to MTX and analysis
on the MTX detection system was 10 nmol/L MTXPG.
[0117] These results demonstrate that MTXPG can be efficiently
converted to MTX in RBC extract samples using plasma as a source of
gamma glutamyl hydrolase. The results further demonstrate that
methotrexate polyglutamates can be accurately and quantitatively
detected in RBC extracts by converting the MTXPGs to MTX and
quantitating the amount of MTX by fluorescent detection of a MTX
photolytic product.
Example III
Monitoring Methotrexate Polyglutamate Levels in Patients Receiving
Low-Dose Methotrexate Therapy
[0118] Methotrexate requires intracellular activation to
methotrexate polyglutamate to exert anti-arthritic effects. This
example describes a method for quantitating the levels of
intracellular methotrexate polyglutamates in the RBCs of a patient
following administration of low-dose MTX therapy.
[0119] Blood samples (5 ml) from polyarthritis patients receiving
low-dose MTX were collected after written informed consent. The
patients had received a median of 15 mg MTX (range 2.5 mg to 40 mg
per week) for at least three months. Blood samples were centrifuged
for 10 minutes to separate plasma and buffy coat from RBCs. After
washing the RBC pellet with 2 volumes of saline, RBC count was
determined with a Beckman Coulter Onyx Counter; RBCs were
subsequently stored at -70.degree. C. until analysis. Results were
normalized to 10 red blood cells. Patient results were expressed as
average .+-.SEM (range).
[0120] MTXPG concentrations were determined using the methods
described in Example II except that RBC extracts were not spiked
with MTXPG. MTXPG concentrations were calculated by subtracting MTX
concentrations measured after enzymatic treatment with MTX
concentrations measured in a corresponding sample not subject to
enzymatic treatment. To quantify MTX concentrations without
enzymatic treatment, a second 100 .mu.l aliquot of patient RBC
extract was obtained but not treated with plasma or buffer. Rather,
200 .mu.l water was added to the RBC extract prior to immediate
deproteinization with 30 .mu.l 70% perchloric acid as described
above.
[0121] An exemplary determination of MTXPG concentration in the red
blood cells of a patient on 2.5 mg per week MTX therapy follows. A
total of 0.88.times.10.sup.9 RBC cells were obtained from the
patient and analyzed and resulted in the chromatogram shown in FIG.
6A. The fluorescence excitation spectrum of photolytic product A
produced from the peak eluting at 12.42 minutes in the chromatogram
of FIG. 6A was similar to the excitation spectrum for
methotrexate's photolytic product in water as shown in FIG. 6B. The
overlap in the excitation spectra confirm that the 12.42 minute
peak is MTX.
[0122] The concentration of MTXPG in the sample was determined to
be 7.9 pmol MTXPG/10.sup.9 cells by subtracting the MTX
concentration prior to enzymatic conversion, which was 16.5 pmol
MTXPG/10.sup.9 cells, from MTX after enzymatic conversion, which
was 28.6 pmol MTXPG/10.sup.9 cells. Based on these results, it was
determined that 27% of the total MTX in the patient's RBCs was
present as a polyglutaminated derivative.
[0123] In 12 patients, total MTXPG ranged from 3.4 to 37.0 pmol
MTXPG/10.sup.9 cells with a median of 16.5 pmol MTXPG/10.sup.9
cells. Prior to conversion, MTX concentration ranged from 1.9 to
30.0 pmol/10.sup.9 cells. The percentage of MTXPG in the patients'
RBCs ranged from 35% to 100% of total MTX with a median of 45%.
[0124] These results demonstrate an assay that is capable of
detecting the amount of RBC intracellular MTX in a patient on low
dose MTX therapy as well as the amount of this MTX that has been
converted to the active MTXPG form.
Example IV
A Compound Isolated from a Deglutamated Red Blood Cellular
Extract
[0125] This example describes isolation and characterization of a
compound isolated from red blood cells following deglutamation.
[0126] RBCs were isolated, hemolysates prepared and the hemolysates
deglutamated by incubation with reconstituted plasma as described
in Example II. Deglutamated samples were injected onto the HPLC
system for analysis using the MTX detection system described in
Example I.
[0127] As shown in FIG. 3A, an endogenous peak having a retention
time of 11.90 minutes was detected in the deglutamated RBC extract.
The endogenous peak had a retention time of 11.91 minutes in a
deglutamated RBC extract that had been spiked with MXTPG and was
resolved from the MTX peak, which had a retention time of 12.5
minutes (FIG. 3B). In a deglutamated RBC extract from a patient
undergoing methotrexate therapy, the endogenous peak had a
retention time of 12.02 minutes and was again resolved from the MTX
peak, which had a retention time of 12.42 minutes (FIG. 6). In both
the MTXPG spiked sample and patient sample, the resolution was
sufficient to allow the endogenous peak to be integrated for
quantitative comparison between samples. The endogenous peak had
retention times that were about 0.6 to 0.4 minutes less than the
retention times for MTX.
[0128] A fluorescence excitation spectrum was obtained for the
material eluting with the endogenous peak by detecting emission at
464 nm for the HPLC column eluate having a retention time of 12.02
minutes. As shown in FIG. 7, the excitation spectrum had peak
maxima at about 299 nm and 402 nm and minima at about 259 nm, 336
nm and 429 nm.
[0129] The excitation spectrum of the endogenous peak shown in FIG.
7 was obtained following 3 second photolysis under the conditions
set forth in Example I. Also shown in FIG. 7 is the excitation
spectrum obtained for the endogenous peak under similar HPLC
conditions except that the sample was not irradiated prior to
detection. The spectra for the endogenous peak with and without
photolysis were similar, indicating that the endogenous compound
isolated from deglutamated red blood cellular extracts is
photostable to UV irradiation for at least 3 seconds in the
presence of peroxide.
[0130] The endogenous compound that is present in the endogenous
peak is obtained by isolating from the column eluate the fraction
corresponding in retention time to the endogenous peak. The
presence of the compound in the isolated fraction is confirmed by
analysis of a portion of the fraction. This portion is analyzed by
reinjection onto the HPLC system using the post-column photolysis
and fluorescence detection system described in Example I and the
presence of the compound identified by elution at the appropriate
time.
[0131] The endogenous compound is characterized by co-injection
with known folates in the HPLC system or other analytical
chromatography system. A known folate that coelutes with the
endogenous compound is identified as potentially having similar
structure. Further characterization of the endogenous compound is
based on the rate at which it is produced by GGH compared to the
rate at which a known polyglutamated folate is converted to a
monoglutamated folate or the photostability of the endogenous
compound compared to photostability of a known folate. Similarity
in the reaction rates, photostability or both also identifies the
endogenous compound as having a structure that is similar to the
known folate.
[0132] The molecular weight of the endogenous compound is
determined by mass spectrometry using methods known in the art.
Further structural characterization is carried out by nuclear
magnetic resonance spectroscopy using methods known in the art as
applied to analysis of methotrexate and other folates.
[0133] Throughout this application various patent and non-patent
publications have been referenced. The disclosures of these
publications in their entireties are hereby incorporated by
reference in this application in order to more fully describe the
state of the art to which this invention pertains.
[0134] The term "comprising" is intended herein to be open-ended,
including not only the recited elements, but further encompassing
any additional elements.
[0135] Although the invention has been described with reference to
the examples provided above, it should be understood that various
modifications can be made without departing from the spirit of the
invention. Accordingly, the invention is limited only by the
claims.
* * * * *