U.S. patent application number 10/487932 was filed with the patent office on 2004-09-09 for method for predicting the sensitivity to chemotherapy.
Invention is credited to Fowst, Camilla, Franzanne, Vreeland, Margaret, Tursi Jennifer, Rosa, Geroni Maria Cristina.
Application Number | 20040175774 10/487932 |
Document ID | / |
Family ID | 25506133 |
Filed Date | 2004-09-09 |
United States Patent
Application |
20040175774 |
Kind Code |
A1 |
Fowst, Camilla ; et
al. |
September 9, 2004 |
Method for predicting the sensitivity to chemotherapy
Abstract
Herewith described is a novel method for predicting the
sensitivity towards chemotherapy, of a patient in need thereof,
which comprises obtaining a blood sample from the patient and
detecting the levels of blood glutathione (GSH) as a surrogate
marker for glutathione-S-transfera- se (GST) activity in tumor
tissues.
Inventors: |
Fowst, Camilla; (Milan,
IT) ; Rosa, Geroni Maria Cristina; (Milan, IT)
; Margaret, Tursi Jennifer; (Milan, IT) ;
Franzanne, Vreeland; (Groton, MI) |
Correspondence
Address: |
ARENT FOX KINTNER PLOTKIN & KAHN
1050 CONNECTICUT AVENUE, N.W.
SUITE 400
WASHINGTON
DC
20036
US
|
Family ID: |
25506133 |
Appl. No.: |
10/487932 |
Filed: |
March 2, 2004 |
PCT Filed: |
September 19, 2002 |
PCT NO: |
PCT/EP02/10647 |
Current U.S.
Class: |
435/7.23 |
Current CPC
Class: |
G01N 2800/52 20130101;
G01N 2333/91171 20130101; G01N 33/57488 20130101; A61P 35/00
20180101; G01N 33/574 20130101 |
Class at
Publication: |
435/007.23 |
International
Class: |
G01N 033/574 |
Claims
1. A method for predicting the sensitivity towards chemotherapy, of
a patient in need thereof, which comprises obtaining a blood sample
from the patient and detecting the presence of blood glutathione
(GSH) as a surrogate marker for glutathione-S-transferase (GST)
activity in tumor tissues.
2. The method of claim 1 which allows predicting whether the given
tumor is associated with GST over expression.
3. The method of claim 2 wherein the tumor is selected from the
group consisting of gastrointestinal tumors, uterine and ovarian
cancers, head and neck cancer, lung carcinomas, sarcomas, liver
tumors, pancreatic cancer, breast cancer, prostate cancer, melanoma
and haematological tumors.
4. The method of claim 3 wherein the tumor is selected from head,
neck and lung cancer.
5. A method for selecting the proper chemotherapeutic treatment for
a patient in need thereof, which first comprises predicting his
sensitivity towards chemotherapy by obtaining a blood sample from
the patient, detecting the levels of blood glutathione (GSH) as a
surrogate marker for glutathione-S-transferase (GST) activity in
tumor tissues, determining whether the blood GSH levels fall within
a range indicative of a potential for the patient to exhibit de
novo or later progression to resistance to anticancer
chemotherapeutic agents, and selecting a suitable and effective
chemotherapeutic treatment based on the above GSH levels.
6. The method of claim 5 for selecting the proper chemotherapeutic
treatment for a patient suffering of a tumor selected from the
group consisting of gastrointestinal tumors, uterine and ovarian
cancers, head and neck cancer, lung carcinomas, sarcomas, liver
tumors, pancreatic cancer, breast cancer, prostate cancer, melanoma
and haematological tumors
7. The method of claim 6 wherein the tumor is selected from head,
neck and lung cancer.
8. The method of claim 5 which, based on the blood GSH levels,
allows to select the proper chemotherapeutic treatment which may
comprise the administration to the patient in need thereof of the
compound N-(5-{[(5-{[(5-{[(2-{[amino(imino)methyl]amino}
ethyl)amino]carbonyl}-1-m- ethyl-1H-pyrrol-3-yl)amino]
carbonyl}-1-methyl-1H-pyrrol-3-yl)amino]carbon-
yl}-1-methyl-1H-pyrrol-3-yl)-4-[(2-bromoacryloyl)amino]-1-methyl-1H-pyrrol-
e-2-carboxamide (internal code PNU 166196) or a pharmaceutically
acceptable salt thereof or, alternatively, of a conventional
antitumor agent.
9. The method of claim 8 wherein the conventional antitumor agent
is selected from the group consisting of alkylating agents,
anthracyclines and platinum derivatives.
10. A method for treating a patient suffering from a tumor
over-expressing the GSH/GST system, which first comprises
predicting his sensitivity towards chemotherapy by obtaining a
blood sample from the patient, detecting the levels of blood GSH as
a surrogate marker for GST activity in tumor tissues, determining
whether the GSH levels fall within a range indicative of a
potential for the patient to exhibit de novo or later progression
to resistance to chemotherapeutic agents, and selecting a suitable
and effective chemotherapeutic treatment based on the above GSH/GST
levels which treatment may comprise the administration of an
effective amount of N-(5-{[(5-{[(5-{[(2-{[amino(imino)methyl]amino}
ethyl)amino]carbonyl}-1-methyl-1H-pyrrol-3-yl)amino]carbonyl}-1-methyl-1H-
-pyrrol-3-yl)amino]carbonyl}-1-methyl-1H-pyrrol-3-yl)-4-[(2-bromoacryloyl)-
amino]-1-methyl-1H-pyrrole-2-carboxamide (internal code PNU
166196), or of a pharmaceutically acceptable salt thereof.
11. The method of claim 10 wherein the tumor is selected from the
group consisting of gastrointestinal tumors, uterine and ovarian
cancers, head and neck cancer, lung carcinomas, sarcomas, liver
tumors, pancreatic cancer, breast cancer, prostate cancer, melanoma
and haematological tumors.
12. The method of claim 11 wherein the tumor is selected from head,
neck and lung cancer.
13. Use of a kit for determining blood GSH levels as a surrogate
marker for GST activity in tumor tissues.
Description
[0001] The present invention relates to the field of cancer
treatment and, more particularly, it relates to a method for
predicting the sensitivity towards chemotherapy of a patient, by
measuring glutathione (GSH) or GSH-related enzyme
glutathione-S-transferase (GST) blood levels of the said patient
undergoing chemotherapeutic treatment.
[0002] The levels of glutathione (GSH) or (GST) are known in the
art to be correlated with the response to cytotoxic antitumor
treatments since high levels of GSH or GST confer resistance to
several antitumor drugs such as, for instance, alkylating agents
(e.g. melphalan, chlorambucil, cyclophosphamide, ifosfamide
mustards, BCNU), platinum complexes (e.g. cisplatin, carboplatin
and oxaliplatin) and anthracyclines (e.g. doxorubicin, epirubicin,
idarubicin and daunorubicin) [Biochem. Pharmacol 35: 3405-3409
(1986)].
[0003] Both GSH and GST are ubiquitously present in several human
tissues such as, for instance, blood cells, plasma, serum
circulating blasts and pathologic (tumor) tissues.
[0004] See, for general references to GSH and GST, Cancer Res. 54:
4313-4320 (1994); Brit. J. Cancer 72(2): 324-326 (1995); Drug
Discovery Today 3:113-121 (1998).
[0005] GST, and most prominently GST-.pi., are present at high
levels in a preponderance of tumor types. Increased levels of GSH
and activity of GST in comparison to normal tissues has been found
in several tumor types comprising, for instance, gastrointestinal
tumors, uterine and ovarian cancers, head and neck cancer, lung
carcinomas, sarcomas, liver tumors and haematological tumors
[Cancer Res. 49:5225-5229 (1989); Clinical Reviews in Biochemistry
and Molecular Biology 27(4.5):337-386 (1992)].
[0006] GSH plays a crucial protective role against cellular injury
produced by a number of toxic insults. Preclinical and clinical
studies have established a correlation between GSH/GST over
expression and cancer or cancer response to chemotherapy.
[0007] Alterations of the GSH-based detoxification system
(consisting of GSH and GSH related enzymes, GSTs) have been also
associated with varying responsiveness to several antineoplastic
agents.
[0008] So far, because of the low rate of responsiveness to
conventional chemotherapy in those tumors over expressing GSH/GST,
the identification of new markers predicting sensitivity to therapy
is of utmost importance.
[0009] Of additional importance was the requirement to identify
these new predictive markers from a relatively non-invasive source,
for instance blood or blood component, to allow these predictive
markers to be readily analyzed for the evaluation of chemotherapy
sensitivity.
[0010] We have now found that GST activity in tumor tissues is
strongly correlated with blood GSH levels, hence indicating blood
GSH levels as a possible surrogate marker for GST activity in tumor
tissues.
[0011] FIG. 1: correlation between GST activity in tumor tissue and
GSH levels in matched whole blood specimens from lung cancer
patients.
[0012] FIG. 2: correlation between GST activity in tumor tissue and
GSH levels in matched whole blood specimens from head and neck
cancer patients.
[0013] Therefore, it is a first object of the present invention a
method for predicting the sensitivity towards chemotherapy of a
patient in need thereof, which comprises obtaining a blood sample
from the patient and detecting the presence of blood glutathione
(GSH) as a surrogate marker for glutathione-S-transferase (GST)
activity in tumor tissues.
[0014] According to the method of the invention, it is thus
possible to identify whether a given tumor is associated with
GSH/GST over expression, hence allowing the selection of the most
suitable antitumor therapy.
[0015] It is therefore a further object of the invention a method
for selecting the proper chemotherapeutic treatment for a patient
in need thereof, which first comprises predicting his sensitivity
towards chemotherapy by obtaining a blood sample from the patient,
detecting the presence of blood glutathione (GSH) as a surrogate
marker for glutathione-S-transferase (GST) activity in tumor
tissues, determining whether the blood GSH levels fall within a
range indicative of a potential for the patient to exhibit de novo
or later progression to resistance to chemotherapeutic agents, and
selecting a suitable and effective chemotherapeutic treatment.
[0016] In other words, once the blood levels of GSH being thus
detected are so high to indicate, for the patient, the possibility
of exhibiting resistance to conventional chemotherapeutic agents,
for instance alkylating agents, anthracyclines or platinum
complexes, a suitable and effective chemotherapeutic treatment,
based on the above GSH levels, might comprise the administration of
an antitumor agent which is effective in the treatment of those
tumors over expressing GSH/GST.
[0017] In this respect, the compound
N-(5-{[(5-{[(5-{[(2-{[amino(imino)met- hyl]amino}ethyl)
amino]carbonyl}-1-methyl-1H-pyrrol-3-yl)amino]carbonyl}-1-
-methyl-1H-pyrrol-3-yl)
amino]carbonyl}-1-methyl-1H-pyrrol-3-yl)-4-[(2-bro-
moacryloyl)amino]-1-methyl-1H-pyrrole-2-carboxamide (internal code
PNU 166196), and pharmaceutically acceptable salts thereof,
recently appeared to be effective in the treatment of a tumor known
to be poorly responsive or resistant to conventional antitumor
therapies and described in the literature as potentially
over-expressing GSH/GST.
[0018] For a general reference to the above compound of formula
1
[0019] and to its effectiveness against tumors over expressing
GSH/GST system, see the international patent application WO
98/04524 and WO 01/85144 (filed on Apr. 19, 2001 and claiming
priority from UK patent application No. 0011059.3, filed on May 8,
2000), both in the name of the Applicant itself and herewith
incorporated by reference.
[0020] Preferably, a suitable therapy could thus comprise the
administration to a patient in need thereof, of the proper amounts
of the compound PNU 166196, for instance according to the
administration schedule reported in the international patent
application WO 02/28389 (claiming priority from U.S. Ser. No.
09/676,770, filed on Oct. 2, 2000) in the name of the Applicant
itself and herewith incorporated by reference.
[0021] According to a preferred embodiment of the invention, the
above method for predicting the sensitivity towards chemotherapy
could be advantageously used in several tumor forms including, for
instance, gastrointestinal tumors, uterine and ovarian cancers,
head and neck cancer, lung carcinomas, sarcomas, liver tumors,
pancreatic cancer, breast cancer, prostate cancer, melanoma and
haematological tumors.
[0022] Even more preferably, the said tumor is selected from lung,
head and neck cancer.
[0023] In addition, the above method may also be applied to select
the proper antitumor therapy as a second line therapy, for instance
once a previous chemotherapy treatment, for example a first-line
chemotherapy treatment with conventional antitumor agents, e.g.
alkylating agents, platinum derivatives or anthracyclines, failed
to give the expected results because of the occurrence, among other
effects, of the aforementioned resistance effects.
[0024] Several methods are known in the art for the assay of GSH
and related kits are commercially available.
[0025] According to the present invention, therefore, any
commercially available kit for detecting GSH levels in blood
samples may be conveniently employed.
[0026] In this respect, it is a further object of the invention the
use of a kit for determining blood GSH levels as a surrogate marker
for GST activity in tumor tissues.
[0027] With the aim of illustrating the present invention, without
posing any limitation to it, the following experimental part is now
given.
Experimental Part
[0028] The following experimental part was used to demonstrate the
strong correlation existing between the GSH levels in blood versus
the GST activity in tumor tissues, so as to render GSH detection in
blood as a surrogate marker for GST levels in tumor tissues.
[0029] As formerly indicated, FIGS. 1 and 2 clearly show the above
correlations between GSH levels in blood of lung cancer patients
and head and neck cancer patients, with the GST activities in tumor
tissues of the said patients.
[0030] Tissue and blood samples from 29 patients with lung cancer
(NSCLC) and 23 patients with head and neck cancer (SCC) were
enrolled, as per the following table I.
1TABLE I Patient series Principal characteristics Head and neck
cancer Lung cancer No. 23 29 Age 56 (29-72) 67 (28-80) Sex 16 m - 7
f 24 m - 5 f Tumor type SCC 26 (NSCLC) 2 (lung adenocarcinoma) 1
(spino cell.)
Sampling Modalities
[0031] Tissue from primary or relapsed tumor. A sample (E 200 mg)
of tumor tissue adjacent to the sample submitted for histological
examination was collected from each patient. Tissue samples were
put immediately in crushed ice. Samples were frozen in liquid
nitrogen within 30 minutes (max 1 hour) from the excision.
[0032] Blood (before treatment of the primary tumor or at time of
failure). Blood (15 ml) was collected in a pre-chilled syringe and
processed as follows.
[0033] 3 ml were dispensed in K.sub.3EDTA (or ACD-solution A) tubes
and stored at -20.degree. C. (whole blood).
Analytical Methods
[0034] GSH quantity GSH level in cytosol and whole blood samples
was measured by a commercially available GSH assay kit (Cayman, Ann
Arbor, Mich., USA). This kit utilizes an enzymatic recycling method
based on the reaction between GSH and DTNB that produces a yellow
coloured compound (TNB). The rate of TNB production is directly
proportional to the concentration of GSH in the sample. Measurement
of the absorbance of TNB at 405 nm provides an accurate estimation
of GSH in the sample.
[0035] Before assaying, samples were deproteinated with 10%
metaphosphoric acid (MPA) to avoid interferences due to sulfhydryl
groups on the proteins in the assay. 50 .mu.l of the deproteinated
sample (whole or diluted 1:3 with kit Wash Buffer) were assayed in
duplicate according to manufacturer's instructions. GSH
concentration was measured by comparison with a standard curve
obtained by plotting the absorbance at 25 min vs. GSH concentration
(nmol/ml). Cytosol GSH levels were normalised for protein content
(nmol/mg).
[0036] GST activity. 10 .mu.l of cytosol was analysed by a
commercially available assay kit (Novagen, Darmstadt, Germany)
according to manufacture's instructions. The kit is designed in
order to perform a colorimetric-enzymatic assay of glutathione
S-transferase (GST). The sample is combined with
1-chloro-2,4-dinitrobenzene (CDNB) substrate in the supplied
reaction buffer and the absorbance of the reaction is monitored at
.lambda.=340 nm. The rate of change in A.sub.340 is proportional to
the amount of GST activity in the sample. The absorbance at 340 nm
was monitored every 30 sec. over a period of 5 min for cytosol
samples.
[0037] GST activity of all samples was compared with a standard
(cytosol of human placenta) and was measured as U*/mg prot for
cytosol sample.
[0038] *U=(dA/min of 10 .mu.l placenta)/mg prot of placenta
Assay Validation
[0039] The validation of the methods was planned taking into
account: sensitivity, specificity, precision (intra-assay,
inter-assay, inter-batch), calibration range, reagent stability,
and analyte stability in different storage conditions.
[0040] GSH
[0041] The analytical sensitivity (evaluated as the mean+3 SD of 8
replicates of the zero standard) was 0.33 nmol/ml.
[0042] Functional sensitivity was evaluated by plotting the
imprecision profile of the method.
[0043] The minimum concentration with a coefficient of variation
(C.V.) less then 10% was 0.4 nmol/ml.
[0044] Assay kit is based on a reaction between GST-reductase and
DTNB that reacts with all groups--SH contained in the sample. A
high specificity is expected since:--all thiol protein groups are
removed by deproteination;--GST-reductase is a specific enzyme for
GSH substrate;--the reaction is monitored at .lambda.=405 that is
specific for GSH. No further confirmation experiments were thus
performed.
[0045] Precision was evaluated by analysing, for 5 consecutive
runs, a duplicate of whole blood. We obtained an inter-assay C.V.
below 12% while the intra-assay C.V. was below 5% of variability
(tables 5 and 6).
[0046] The calibration curve ranges between 0.6-40 nmol/ml.
[0047] All reagents must be stored at +4.degree. C. until
expiration date indicated by manufacturers.
[0048] After opening, reagents are stable for 2 weeks at +4.degree.
C.
[0049] Samples and deproteinated samples are stable up to 6 months
if stored at -80.degree. C. and -20.degree. C., respectively.
[0050] GST Activity
[0051] Analytical sensitivity was evaluated by 8 replicates of the
zero standard and resulted 0.0055 U/ml.
[0052] Functional sensitivity was evaluated on 8 replicates of low
activity sample. Since C.V. of replicates was less than 10% (9.2%)
the corresponding mean activity level (0.008 U of activity) was
considered as functional sensitivity.
[0053] Activity assay kit is based on an enzymatic reaction between
GST and CDNB, that is a specific substrate of the enzyme.
Accordingly the specificity of the method used is largely
demonstrated in literature (Habig W. H., 1974; Smith D. B., 1988).
We therefore did not perform further confirmatory experiments.
[0054] Accuracy was evaluated with dilution test of a cytosol
sample. Recovery was between 112% and 133%.
[0055] Precision was evaluated on 2 cytosol samples with two
different activity levels. Four replicates of the samples were
assayed on 5 different runs. Inter and intra-assay C.V. were
respectively under 9% of variability in high activity level sample
and under 14% of variability in low activity level sample.
[0056] The calibration curve ranges between 0.01-0.4 dA/min
[0057] All reagents must be stored at -20.degree. C. until
expiration date indicated by manufacturers. Samples are stable up
to 6 months if stored at -80.degree. C.
Results
[0058] GSH levels were measured in whole blood from 29 patients
with lung cancer and 23 with head and neck cancer. Mean level in
blood is 516 nmol/ml (S.D.=117) in lung cancer and 428 nmol/ml
(S.D.=97) in head and neck cancer.
2TABLE II GSH levels Summary Statistics Whole blood (nmol/ml)
Overall mean 477 median 458 10.degree.-90.degree.% 350-620 n 52
paired Wilcoxon test <0.0001 (0.0001) Lung cancer mean 516
median 494 10.degree.-90.degree.% 383-681 n 29 paired Wilcoxon test
0.0004 (0.0001) Head and neck cancer mean 428 median 426
10.degree.-90.degree.% 317-566 n 23 paired Wilcoxon test 0.03
(0.0532)
GST Activity
[0059] Total GST activity was measured in cytosol but not in plasma
sample, because of low levels of the GST enzymes in this matrix. In
fact we tested 21 plasma samples of 29 available lung cancer
patients and 15 plasma samples of 23 available head and neck cancer
patients. GST activity was close to sensibility threshold of the
method being not detectable in 11/21 lung and 3/15 head and neck
samples.
[0060] GST activity was measured in 29 tissue samples of lung
cancer and in 22 of head and neck cancer. Mean activity is 1.72
U/Mg (S.D.=0.89) in lung cancer tissue. In head and neck, mean
activity is 2.61 U/mg (S.D.=1.74).
3TABLE III GST activity Summary Statistics Cancer tissue U/mg
Overall mean 2.1 median 1.72 10.degree.-90.degree.% 1.06-3.31 n 51
paired Wilcoxon test <0.0001 (0.0001) Lung cancer mean 1.72
median 1.37 10.degree.-90.degree.% 0.87-2.97 n 29 paired Wilcoxon
test 0.0002 (0.0001) Head and neck cancer mean 2.61 median 2.49
10.degree.-90.degree.% 1.11-3.42 n 22 paired Wilcoxon test 0.02
(0.0789)
CONCLUSIONS
[0061] The evaluated methods are reliable and robust for routine
use in tissue extracts (GST activity) and in whole blood (GSH
level).
[0062] A highly significant positive correlation was found between
whole blood GSH and tissue GST activity.
[0063] In particular, the GST activity in cancer tissue vs. GSH
level in whole blood resulted to be correlated in lung cancer
(r0.53, p=0.003, FIG. 1) and in head and neck cancer (r=0.89,
p<0.0001; FIG. 2).
4TABLE IV GST activity in cancer tissue vs. whole blood GSH levels
Tumor Spearman Correlation p value Lung 0.53 0.004 Head and neck
0.89 <0.0001
[0064] The above results clearly provide evidence that the GSH
levels in blood samples of a cancer patient can be used as a
surrogate marker for GST activities in tumor tissues, thus allowing
to predict whether the patient responsiveness to chemotherapy is
associated with GSH/GST system over expression.
* * * * *