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US005204241A
United States Patent Patent Number: 5,204,241
Pero Date of Patent: Apr. 20, 1993
GLUTATHIONE-S-TRANSFERASE MU AS A MEASURE OF DRUG RESISTANCE
Inventor: Ronald W. Pero, New York, N.Y.
Assignee: Oxi-Gene Inc., New York, N.Y.
Appl. No.: 601,266
Filed: Oct. 22, 1990
Int. Cl. C12Q 1/48; C12N 9/00
U.S. Cl. 435/15; 435/183
Field of Search 435/15, 183
References Cited
PUBLICATIONS
Leyland-Jones B.R. Antineoplastic Drug Sensitivity ... Chem. Abst. 114: 94779h Mar. 18, 1991.
Warholm M. Purification of a New GST Mu ... Biochem & Biophys Rsch Com 98 2: 512-519 Jan. 30, 1981.
Yusa K. Comparison of GST Activity Between ... Chem Abstracts, vol. 109, #13 109:104335y 1988.
Singh S. V. GST and Glutathione Peroxidases ... Chem. Abstracts 112:327t Jan. 1, 1990.
Morrow C.S. GST and Drug Resistance ... Chem. Abst. 112: 171572y May 7, 1990.
Begleiter A. Activity of Quinone Alkylating ... Chem. Abst. 113:70708n Aug. 27, 1990.
Seidegard, Janeric, Characterization of Soluble GST Activity ... Biochem. Pharm. 33 19: 3053-3058 1984.
Seidegard J. Hereditary Differences in the Expression ... Proc. Natl. Acad. Sci. USA 85:7293-7297 1988.
Peters W.H.M. Immunodetection with a Monoclonal Antibody ... Biochem. Pharm. 39 3:591-597 Jan. 1, 1990.
Johnston et al, J. Nat. Can. Inst., vol. 82, No. 9: 776-779 (1990).
Seidegard et al, Carcinogenesis, vol. 6, No. 8: 1211-1216 (1985).
Seidegard et al, J. Biochem. 246: 783-785 (1987).
Primary Examiner--John W. Rollins
Assistant Examiner--Ralph Gitomer
Attorney, Agent, or Firm--Cooper & Dunham
ABSTRACT
It has been discovered that by determining or measuring a person's
glutathione-s-transferase (GST) mu activity one can determine or measure the
individual's resistance to drugs, particularly to chemotherapeutic drugs.
Approximately 50% of the human population exhibit substantially no GST mu
activity, with the remaining 50% showing GST mu activity. This remaining 50% of
the population accordingly, when treated with drugs, such as a chemotherapeutic
drug for cancer therapy, show less effective response to the drug therapy than
the other 50% of the population which have substantially no GST mu activity,
since GST mu tends to deactivate drugs. Accordingly, a person having GST mu
activity would exhibit drug resistance and would not benefit as much by or be
as good a candidate for cancer chemotherapy as a person who has no GST mu
activity.
13 Claims, 1 Drawing Sheet
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GLUTATHIONE-S-TRANSFERASE MU AS A MEASURE OF DRUG RESISTANCE
BACKGROUND OF THE INVENTION
Glutathione-s-transferases (GSTs) are a group of multi-functional proteins
which play an important role in the biotransformation of many different
biologically active compounds, including agents which damage DNA, such as
chemotherapeutic drugs, see Mannervik, B., Adv. Enzymol. Relat. Areas Mol.
Biol. 57: 357-417 (1985). Indeed, it is known that GSTs are usually associated
with the detoxification by conjugation of genotoxic and cytotoxic xenobiotic
electrophiles derived from drugs, carcinogens and environmental pollutants, see
Glutathione transferases; H. Sies and B. Ketterer (eds.), Glutathione
Conjugation. Academic Press, New York, pp. 74-135 (1988).
On the basis of physical and immunological properties and substrate
specificities and protein structure, the human GSTs have been divided into
three distinct classes, named alpha, mu and pi, see Mannervik B., et al, Proc.
Natl. Acad. Sci. U.S.A. 82: 7202-7206 (1985).
It is an object of this invention to employ glutathione-s-transferase
activity as a measure of drug resistance.
How this and other objects of this invention are achieved will become
apparent in the light of the accompanying disclosure, including the drawing
which graphically illustrates the subject invention. In at least one embodiment
of the practices of this invention at least one of the objects of this
invention will be achieved.
Cellular reduced glutathione, i.e. the co-substrate for GSTs, and GST
activity in general, i.e. total activity estimated using 1-chloro-3,
4-dinitrobenzene (CDNB) as a substrate, has been shown to be involved in the
mechanism of chemotherapeutic drug resistance, see Johnston et al, J. Natl.
Can. Inst. 82: 776-779 (1990) and Lai G-M, et al, J. Natl. Can. Inst. 81:
535-539 (1989).
Chemotherapeutic agents, such as chlorambucil cisplatin, nitrosoureas and
other chemotherapeutic drugs that can damage DNA or other cellular
macromolecules, such as RNA or protein, are electrophiles which can be
conjugated with glutathione directly or indirectly via GST activity. Hence,
high levels of glutathione and/or GST activity provide a mechanism of drug
resistance because cells having high levels have increased opportunities to
remove the drugs before the drugs can cause genotoxicity or cytotoxicity or
other adverse effects. Heretofore, however, it has not been known whether any
one of the known GST isozymes, either the alpha, pi or the mu class, is more
specifically involved in conjugating chemotherapeutic drugs with glutathione.
BRIEF DESCRIPTION OF THE INVENTION
It has now been discovered, and it is the basis of this invention, that
GST mu isozymes are specifically and preferentially involved in the metabolism
of chemotherapeutic drugs. In accordance with this invention it has now been
discovered that by measuring GST mu activity, one can estimate and/or measure
an individual's resistance to chemotherapeutic drugs.
The mu class of GSTs are distinguished by having a high substrate
specificity towards trans-stilbene oxide, see Seidegard, J.-E. et al, Biochem.
J. 246: 783-785 (1987). About 50% of the human population lack GST mu because
of a gene deletion, see Seidegard, J.-E. and Pero, R.W., Genet. 69: 66-68
(1985) and Seidegard et al Proc. Natl. Acad. Sci. U.S.A. 85: 7293-7295 (1988).
Individuals can be easily phenotyped for the presence (+) or absence (--) of
GST Mu activity. Because GST mu activity represents at least 60% of the total
GST activity in liver, see Warholm, M. et al, Biochemistry 22: 3610-3617
(1983), and since the liver is the main source of metabolism of xenobiotic
substances, including chemotherapeutic drugs, and since GST mu has been shown
to have high substrate specificity toward toxic agents, such as trans-stilbene
oxide, benzopyrene 4,5-oxide and ethylene oxide, but little substrate
specificity for other GST substrates, such as cis-stilbene oxide
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and 1-chloro-2,4-dinitrobenzene, see Seidergard, J.-E. et al, Carcinogenesis 6:
1121-1216) 1985, GST mu activity may be employed to estimate a genetic based
sensitivity of individuals to metabolize chemotherapeutic drugs.
BRIEF DESCRIPTION OF THE DRAWING
The single FIGURE is a graph in which glutathione transferase activity
toward tauromustine is plotted against glutathione transferase activity toward
trans-stilbene oxide.
DETAILED DESCRIPTION OF THE INVENTION
The following example is illustrative of the practices of this invention.
In the example tauromustine, a nitrosourea, a class of chemotherapeutic drugs,
was metabolized to a much greater extent by human liver cytosols having GST mu
activity than by human liver cytosols lacking GST mu activity. The data
illustrated in the accompanying drawing teach that the presence (+) or absence
(--) of GST mu can predict individual sensitivity to chemotherapeutic drugs,
such as nitrosoureas, which damage DNA.
EXAMPLE
The importance of the GST-tSBO phenotype in influencing drug metabolism is
indicated by the following. Tauromustine is a drug representative of the class
of chemotherapeutic agents known as the nitrosoureas and has the structural
formula:
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Human liver biopsies from ? 3 individuals were homogenized in 5 vol. of
0.25 M sucrose, centrifuged at 10,000 g for 15 minutes and then the resulting
supernatant was re-centrifuged at 105,000 g for one hour. The 105,000 g
supernatants were analyzed for glutathione transferase activity using
trans-stilbene oxide (tSBO) and tauromustine as substrates. Metabolism of these
substrates to glutathione conjugates was monitored by radiometric procedures
involving differential organic solvent extraction, see Gill, S., Ota, J. and
Hammock, B., D. Anal. Biochem. 131: 273-282 (1983), and high pressure liquid
chromatography (HPLC).
The results are graphically presented in the accompanying drawing. When
GST-tSBO activity in liver cytosols was 0-10 nmo/min/mg protein, the level of
glutathione transferase activity toward tauromustine was also very low ranging
from 0-2 nmol/min/mg protein. However, when there was easily detectable
GST-tSBO activity, i.e. 25-65 nmol/min/mg proteins, there was also substantial
metabolism of tauromustine (i.e. 4-19 nmol/min/mg protein).
As mentioned hereinabove, there are at least three different classes of
human glutathione transferases, the alpha, mu and pi classes. Each class is
composed of several isozymes and GST-tSBO has been determined to be a distinct
isozyme of the mu class. Hence, these data teach that the metabolism of
nitrosoureas, such as are represented by tauromustine, is mainly carried out by
GST-tSBO, identical to GST-mu, and not by the other isozymes of glutathione
transferase. It follows then, since GST-tSBO activity has been shown to be
under genetic control and to be absent in about 50% of the population with a
higher degree of resistance to chemotherapy, such as to chemotherapeutic drugs
represented by the nitorsoureas.
The embodiment of this invention recognizes the prior knowledge that
glutathione and total GST activity, usually measured with CDNB as substrate,
can contribute to drug resistance. However, CDNB can serve as a substrate for
all the GST ioszyme sub-groups (i.e. alpha, pi and mu classes), and it was not
obvious or recognized that any single GST isozyme was contributing more than
any other to drug metabolism. Moreover, the pi class of GST isozymes had been
the only GST to be more directly implicated in chemotherapeutic drug
resistance, see Moscow, J.A. and Cowan, K.H., J. Natl. Can. Inst. 80: 14-20
(1988)
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and even then, only in relation to reduced glutathione levels. In other words,
the selective metabolism of a chemotherapeutic drug by GST pi isozymes, or any
other GST other than GST mu, shown in Example 1, has not been demonstrated.
This has implied that the substrate specificity of GSTs is very broad and the
various classes of GSTs can metabolize drugs in a reasonably equal manner.
Therefore, it was unexpected that GST mu isozymes could contribute so
dramatically to chemotherapeutic drug resistance in individuals expressing GST
Mu activity compared to individuals lacking GST mu activity, even though both
GST mu (+) and (--) individuals have other classes of GST activity present.
Although the expression of the GSTs is organ specific, the expression of
GST mu is known to be controlled by genetic factors where about 50% of the
population has no GST mu activity. None of the other human GSTs have been shown
to be lacking in such a large portion of the population, nor have they been
shown to have a high degree of substrate specificity controlled by genetic
factors. The combination of these GST mu characteristics, together with the
demonstrated selective metabolism of nitrosoureas by GST mu show that the GST
mu phenotype can be predictive of chemotherapeutic drug resistance via
metabolism characterized by a selective conjugation with glutathione catalyzed
or brought about by GST mu.
In the practices of this invention testing of a person's GST mu activity
can be carried out by obtaining and testing the blood samples of the human to
be tested as well as tissue or organ samples, such as the liver, colon, breast
and adrenal glands. Particularly useful, from the point of view of convenience
in carrying out the tests and determinations in accordance with this invention,
would be to carry out the tests on the person's mononuclear leukocytes. All the
tests would be carried out employing trans-stilbene oxide as the substrate for
the glutathione transferase since trans-stilbene oxide is a specific substrate
for glutathione transferase GST mu. The level of glutathione transferase
activity towards trans-stilbene oxide would be measured as nmol/mn/mg protein
and level of four (4), especially a level higher than eight (8), would be
indicative that the person so tested for glutathione transferase mu activity
would posses substantial drug resistance and would not be a good candidate for
drug therapy or cancer chemotherapy and the like, even when the person so
tested might evidence glutathione transferase activity of the GST alpha and pi
classes.
Instead of using tSBO as a substrate to phenotype individuals for GST mu
affinity and thus drug resistance, other ways of determining GST mu activity
may be employed. For example the (+) or (--) GST mu activity can also be
determined by using antibodies derived from purified GST mu or a DNA probe
derived from or based on the GST mu gene.
All the above-cited publication references are herein incorporated and
made part of this disclosure.
As will be apparent to those skilled in the art in the light of the
foregoing disclosure, many modifications, alterations and substitutions are
possible in the practices of this invention without departing from the spirit
or scope thereof.
What is claimed is:
1. A method of determining the resistance of a human individual to a
nitrosourea which comprises determining the GST mu activity of the individual,
to establish a measured value of the individual's GST mu activity, and
comparing said measured value with a predetermined value, the presence of a GST
mu activity above said predetermined value indicting that the individual is
resistant to the nitrosourea.
2. A method according to claim 1, wherein the determining step is
performed using tSGO to
measure the individual's GST mu activity.
3. A method according to claim 1, wherein the determining step is
performed using an antibody to GST mu to measure the individual's GST mu
activity.
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4. A method according to claim 1, wherein the determining step is
performed using a labeled DNA probe for the GST mu gene to measure the
individual's GST mu activity.
5. A method according to claim 1, wherein the determining step is
performed by testing liver tissue of the individual.
6. A method according to claim 1, wherein the determining step is
performed by testing colon tissue of the individual.
7. A method according to claim 1, wherein the determining step is
performed by testing breast tissue of the individual.
8. A method according to claim 1, wherein the determining step is
performed by testing mononuclear leukocytes of the individual.
9. A method according to claim 2, wherein the determining step is
performed by testing liver tissue of the individual.
10. A method according to claim 2 wherein the determining step is
performed by testing mononuclear leukocytes of the individual.
11. A method according to claim 1 wherein to said measured value of GST mu
activity is measured towards tSBO, nmol/min/mg protein, and said predetermined
value is 4 or above.
12. A method according to claim 1 wherein said nitrosourea is
tauromustine.
13. A method of determining the resistance of a human individual to
tauromustine, comprising determining GST mu activity of liver tissue of the
individual using tSBO, to establish a measured value of said activity expressed
in nmol/min/mg protein and comparing said measured value with a value of 4, a
measured value above 4 indicating that the individual is resistant to
tauromustine.
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