U.S. patent application number 11/332406 was filed with the patent office on 2006-05-25 for combinations comprising n- {5-[4- [4-methy l-piperazino-methyl) -benzoylamido] -2-methylphenyl) -4- (3-pyridyl) -2-pyrimidine-amine and at least one telomerase inhibitor.
Invention is credited to Tetsuzo Tauchi.
Application Number | 20060111365 11/332406 |
Document ID | / |
Family ID | 27806713 |
Filed Date | 2006-05-25 |
United States Patent
Application |
20060111365 |
Kind Code |
A1 |
Tauchi; Tetsuzo |
May 25, 2006 |
Combinations comprising N- {5-[4- [4-methy L-piperazino-methyl)
-benzoylamido] -2-methylphenyl) -4- (3-pyridyl) -2-pyrimidine-amine
and at least one telomerase inhibitor
Abstract
The invention relates to a method of treating a warm-blooded
animal, especially a human, having a proliferative disease
comprising administering to the animal a combination which
comprises (a)
N-{5-[4-(4-methyl-piperazino-methyl)-benzoylamido]-2-methylphenyl}-4-(3-p-
yridyl)-2-pyrimidine-amine and (b) at least one telomerase
inhibitor, to a combination comprising (a) and (b) as defined above
and optionally at least one pharmaceutically acceptable carrier for
simultaneous, separate or sequential use, in particular for the
delay of progression or treatment of a proliferative disease and
finally to the use of at least one telomerase inhibitor for the
preparation of a medicament for the delay of progression or
treatment of Imatinib-resistant leukemia.
Inventors: |
Tauchi; Tetsuzo;
(Kawasaki-city, JP) |
Correspondence
Address: |
NOVARTIS;CORPORATE INTELLECTUAL PROPERTY
ONE HEALTH PLAZA 104/3
EAST HANOVER
NJ
07936-1080
US
|
Family ID: |
27806713 |
Appl. No.: |
11/332406 |
Filed: |
January 13, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10272837 |
Oct 17, 2002 |
|
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11332406 |
Jan 13, 2006 |
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Current U.S.
Class: |
514/252.18 |
Current CPC
Class: |
A61K 45/06 20130101;
A61K 31/00 20130101; A61K 31/426 20130101 |
Class at
Publication: |
514/252.18 |
International
Class: |
A61K 31/506 20060101
A61K031/506 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 18, 2001 |
GB |
0125105.7 |
Jul 4, 2002 |
GB |
0215583.6 |
Claims
1. Method of treating a warm-blooded animal having a proliferative
disease comprising administering to the animal a combination which
comprises (a)
N-{5-[4-(4-methyl-piperazino-methyl)-benzoylamido]-2-methylphenyl}-4-(3-p-
yridyl)-2-pyrimidine-amine and (b) at least one telomerase
inhibitor, in a quantity which is jointly therapeutically effective
against a proliferative disease and in which the compounds can also
be present in the form of their pharmaceutically acceptable salts,
wherein the telomerase inhibitor is a glitazone compound.
2. A Method according to claim 1, wherein the proliferative disease
is leukemia or Imatinib-resistant leukemia.
3. Method of treating a warm-blooded animal having a
Imatinib-resistant leukemia comprising administering to the animal
at least one telomerase inhibitor, in a quantity which is
therapeutically effective against leukemia and in which the
compounds can also be present in the form of their pharmaceutically
acceptable salts wherein the telomerase inhibitor is a glitazone
compound.
4. A combination which comprises (a)
N-{5-[4-(4-methyl-piperazino-methyl)-benzoylamido]-2-methylphenyl}-4-(3-p-
yridyl)-2-pyrimidine-amine and (b) at least one telomerase
inhibitor, wherein the active ingredients are present in each case
in free form or in the form of a pharmaceutically acceptable salt,
and optionally at least one pharmaceutically acceptable carrier;
for simultaneous, separate or sequential use and wherein the
telomerase inhibitor is a glitazone compound.
5. Combination according to claim 4 wherein the compound (a) is
used in the form of its monomethanesulfonate salt.
6. Combination according to claim 4, which is a combined
preparation or a pharmaceutical composition.
7. A pharmaceutical composition comprising a quantity which is
jointly therapeutically effective against a proliferative disease
of a combination according to claim 4 and at least one
pharmaceutically acceptable carrier.
8. Use of a combination according to claim 4 for the delay of
progression or treatment of a proliferative disease.
9. Use of a combination according to claim 4 for the preparation of
a medicament for the delay of progression or treatment of a
proliferative disease.
10. Use of a combination according to claim 8, wherein the
proliferative disease is leukemia or Imatinib-resistant
leukemia.
11. Use of at least one telomerase inhibitor for the preparation of
a medicament for the delay of progression or treatment of
Imatinib-resistant leukemia.
12. Use of at least one telomerase inhibitor for the delay of
progression or treatment of Imatinib-resistant leukemia.
13. A method according to claim 1, in which the combination
partners (a) and (b) are administered in synergistically effective
amounts.
14. A commercial package comprising a combination according to
claim 4, together with instructions for simultaneous, separate or
sequential use thereof in the delay of progression or treatment of
a proliferative disease.
15. A method according to claim 1, in wherein the glitazone
compound is selected from troglitazone, pioglitazone,
rosiglitazone, englitazone, ciglitazone or a combination thereof.
Description
[0001] The invention relates to a method of treating a warm-blooded
animal, especially a human, having a proliferative disease
comprising administering to the animal a combination which
comprises (a)
N-{5-[4-(4-methyl-piperazino-methyl)-benzoylamido]-2-methylphenyl}-4-(3-p-
yridyl)-2-pyrimidine-amine and (b) at least one telomerase
inhibitor, especially as defined herein; a combination comprising
(a) and (b) as defined above and optionally at least one
pharmaceutically acceptable carrier for simultaneous, separate or
sequential use, in particular for the delay of progression or
treatment of a proliferative disease, especially a tumor disease
and leukemia; a pharmaceutical composition comprising such a
combination; the use of such a combination for the preparation of a
medicament for the delay of progression or treatment of a
proliferative disease, and finally to the use of at least one
telomerase inhibitor for the preparation of a medicament for the
delay of progression or treatment of an Imatinib-resistant
leukemia; and to a commercial package or product comprising such a
combination.
[0002] The preparation of
N-{5-[4-(4-methyl-piperazino-methyl)-benzoylamido]-2-methylphenyl}-4-(3-p-
yridyl)-2-pyrimidine-amine and the use thereof, especially as an
antiproliferative agent, are described in EP-A-0 564 409, which was
published on 6 Oct. 1993, and in equivalent applications in
numerous other countries. This compound is also known and
hereinafter referred to as Imatinib [International Non-proprietary
Name].
[0003] Telomerase is a DNA polymerase with an endogenous RNA
template, on which the nascent telomeric repeats are synthesized.
It is known that approximately 85-90% of all human cancers are
positive for telomerase, both in cultured tumor cells and primary
tumor tissue, whereas most somatic cells appear to lack detectable
levels of telomerase. This finding has been extended to a wide
range of human tumors (see, for example, Hiyama et al.,
"Correlating telomerase activity levels with human neuroblastoma
outcomes," Nature Medicine, 1:249-255, 1995a.). Therefore, Human
telomerase is now considered as a novel and potentially highly
selective target for antitumor drug design, and many new promising
telomerase inhibitors have been discovered (Anne E. Pitts and David
R. Corey.sup.A, "The telomerase challenge--an unusual problem in
drug discovery"; Drug Discovery Today 1999, 4:155-161).
[0004] Surprisingly, it has been found that the effect in treating
a proliferative disease of a combination which comprises (a)
Imatinib or pharmaceutically acceptable salts thereof, and (b) at
least one telomerase inhibitor is greater than the effects that can
be achieved with either type of combination partner alone, i.e. a
supra-additive or synergistic effect.
[0005] This combination shows especially good results for treating
leukemia or Imatinib-resistant leukemia.
[0006] Furthermore, it was surprisingly found that telomerase
inhibitors are particularly useful for treating leukemia resistant
to Imatinib or pharmaceutically acceptable salts thereof and
resulted in unexpected strong inhibition of telomerase activity and
reduction of telomere length.
[0007] Hence, in a first embodiment, the present invention relates
to a method of treating a warm-blooded animal having
Imatinib-resistant leukemia comprising administering to the animal
at least one telomerase inhibitor in a quantity which is
therapeutically effective against leukemia, in which method said
compounds can also be present in the form of their pharmaceutically
acceptable salts.
[0008] In a second embodiment, the present invention relates to the
use of at least one telomerase inhibitor for the manufacture of a
drug useful for treating a warm-blooded animal having I
Imatinib-resistant leukemia.
[0009] In a third aspect embodiment, the present invention relates
to a method of treating a warm-blooded animal having
Imatinib-resistant leukemia comprising administering to the animal
at least one telomerase inhibitor in a quantity which is
therapeutically effective against leukaemia, in which method said
compounds can also be present in the form of their pharmaceutically
acceptable salts.
[0010] Furthermore, the present invention relates to a combination,
such as a combined preparation or a pharmaceutical composition,
which comprises (a)
N-{5-[4-(4-methyl-piperazino-methyl)-benzoylamido]-2-methylphenyl}-4-(3-p-
yridyl)-2-pyrimidine-amine and (b) at least one telomerase
inhibitor, wherein the active ingredients are present in each case
in free form or in the form of a pharmaceutically acceptable salt,
and optionally at least one pharmaceutically acceptable carrier;
for simultaneous, separate or sequential use.
[0011] The present invention also concerns a method of treating a
warm-blooded animal having a proliferative disease comprising
administering to the animal a combination which comprises (a)
N-{5-[4-(4-methyl-piperazino-methyl)-benzoylamido]-2-methylphenyl}-4-(3-p-
yridyl)-2-pyrimidine-amine and (b) at least one telomerase
inhibitor, in a quantity which is jointly therapeutically effective
against a proliferative disease and in which the compounds can also
be present in the form of their pharmaceutically acceptable
salts.
[0012] Furthermore, the present invention pertains to a
pharmaceutical composition comprising a quantity, which is jointly
therapeutically effective against a proliferative disease of a
combination as defined herein and at least one pharmaceutically
acceptable carrier.
[0013] In the herein disclosed methods, combinations, compositions
or uses, the combination partners (a) and (b) are preferably
administered in synergistically effective amounts.
[0014] The term "proliferative disease" includes malignant and
non-malignant proliferative diseases, e.g. atherosclerosis,
carcinomas and leukemia, tumors, thrombosis, psoriasis, restenosis,
sclerodermitis and fibrosis.
[0015] The term "tumor" as used herein includes, but is not limited
to breast cancer, melanoma, epidermoid cancer, cancer of the colon
and generally the GI tract, lung cancer, in particular small-cell
lung cancer, and non-small-cell lung cancer, head and neck cancer,
genitourinary cancer, e.g. cervical, uterine, ovarian, testicles,
prostate or bladder cancer; Hodgkin's disease or Kaposi's sarcoma.
The combinations of the present invention inhibit the growth of
liquid tumors and, in particular, solid tumors. Furthermore,
depending on the tumor type and the particular combination used a
decrease of the tumor volume can be obtained. The combinations
disclosed herein are also suited to prevent the metastatic spread
of tumors and the growth or development of micrometastases. The
combinations disclosed herein are in particular suitable for the
treatment of poor prognosis patients, e.g. such poor prognosis
patients having non-small-cell lung cancer.
[0016] The term "leukemia" as used herein includes, but is not
limited to, chronic myelogenous leukemia (CML) and acute lymphocyte
leukemia (ALL), especially Philadelphia-chromosome positive acute
lymphocyte leukemia (Ph+ ALL) as well as Imatinib-resistant
leukemia. Preferably, the variant of leukemia to be treated by the
methods disclosed herein is CML.
[0017] The term "Imatinib-resistant leukemia" as used herein
defines especially a leukemia in which Imatinib shows a reduction
of its therapeutic effectiveness or the relive of its therapeutic
activity for the treatment of leukemia.
[0018] The term "a combined preparation", as used herein defines
especially a "kit of parts" in the sense that the combination
partners (a) and (b) as defined above can be dosed independently or
by use of different fixed combinations with distinguished amounts
of the combination partners (a) and (b), i.e., simultaneously or at
different time points. The parts of the kit of parts can then,
e.g., be administered simultaneously or chronologically staggered,
that is at different time points and with equal or different time
intervals for any part of the kit of parts. Very preferably, the
time intervals are chosen such that the effect on the treated
disease in the combined use of the parts is larger than the effect
which would be obtained by use of only any one of the combination
partners (a) and (b). The ratio of the total amounts of the
combination partner (a) to the combination partner (b) to be
administered in the combined preparation can be varied, e.g. in
order to cope with the needs of a patient sub-population to be
treated or the needs of the single patient which different needs
can be due to the particular disease, age, sex, body weight, etc.
of the patients. Preferably, there is at least one beneficial
effect, e.g., a mutual enhancing of the effect of the combination
partners (a) and (b), in particular a synergism, e.g. a more than
additive effect, additional advantageous effects, less side
effects, a combined therapeutical effect in a non-effective dosage
of one or both of the combination partners (a) and (b), and very
preferably a strong synergism of the combination partners (a) and
(b).
[0019] The term "delay of progression" as used herein means
administration of the combination to patients being in a pre-stage
or in an early phase of the disease to be treated, in which
patients for example a pre-form of the corresponding disease is
diagnosed or which patients are in a condition, e.g. during a
medical treatment or a condition resulting from an accident, under
which it is likely that a corresponding disease will develop.
[0020] It will be understood that references to the combination
partners (a) and (b) are meant to also include the pharmaceutically
acceptable salts. If this combination partners (a) and (b) have,
for example, at least one basic center, they can form acid addition
salts. Corresponding acid addition salts can also be formed having,
if desired, an additionally present basic center. The combination
partners (a) and (b) having an acid group (for example COOH) can
also form salts with bases. The combination partner (a) or (b) or a
pharmaceutically acceptable salt thereof may also be used in form
of a hydrate or include other solvents used for crystallization.
N-{5-[4-(4-methyl-piperazino-methyl)-benzoylamido]-2-methylphenyl}-4-(3-p-
yridyl)-2-pyrimidine-amine, i.e. combination partner (a), is
preferably used in the present invention in the form of its
monomesylate salt. Depending on the chemical structure of the
telomerase inhibitor, a salt form thereof may not exist.
[0021] The combination partner (a) can be prepared and administered
as described in WO 99/03854, especially the monomesylate salt of
N-{5-[4-(4-methyl-piperazino-methyl)-benzoylamido]-2-methylphenyl}-4-(3-p-
yridyl)-2-pyrimidine-amine can be formulated as described in
Examples 4 and 6 of WO 99/03854.
[0022] The term "telomerase inhibitor" is simply meant a reagent,
drug or chemical which is able to decrease the activity of the
telomerase enzyme in vitro or in vivo. Such inhibitors can be
readily identified using standard screening protocols in which a
cellular extract or other preparation having telomerase activity is
placed in contact with a potential inhibitor, and the level of
telomerase activity measured in the presence or absence of the
inhibitor, or in the presence of varying amounts of inhibitor. In
this way, not only can useful inhibitors be identified, but the
optimum level of such an inhibitor can be determined in vitro for
further testing in vivo. Examples of inhibitors are Telomestatin
(J. Am. Chm. Soc. 2001, 123, 1262-1263), Dimethyl sulfoxide,
protein kinase C inhibitors (bisindolylmaleimide I and H-7),
cisplatin, antisense c-myc oligonucleotides (Kohtaro Fujimoto,
Morinobu Takahashi, Biochem Biophys Res Commun "Telomerase Activity
in Human Leukemic Cell Lines Is inhibited by Antisense
Pentadecadeoxynucleotides Targeted against c-myc mRNA", 1997
December, 241:775-81), G-quadruplex structures described in WO
01/402377, selective DNA triplex interactive compounds (Fox et al.,
"A molecular anchor for stabilizing triple-helical DNA," Proc; Haq
et al., "Molecular anchoring of duplex and triplex DNA by
disubstituted anthracene-9/1 0-diones", J. Am. Chem. Soc.,
118:10693-10701, 1996), 2,6-diamido-anthraquinones reported as
DNA-interactive agents (Collier and Neidle, "Synthesis, molecular
modeling, DNA binding, and antitumor properties of some substituted
amidoanthraquinones," Med. Chem., 31:847-857, 1988; Agbandje et
al., "Anthracene-9,10-diones as potential anticancer agents.
Synthesis, DNA binding, and biological studies on a series of
2,6-disubstituted derivatives," Med. Chem., 35:1418-1429, 1992.),
compounds as described in WO 99/65845 or carbocyanine dye,
3,3'-diethyloxadicarbocyanine (DODC,), reported to bind dimeric
hairpin G-quadruplex structures (Chen et al., "Spectroscopic
recognition of guanine dimeric hairpin quadruplexes by a
carbocyanine dye," Proc. Natl. Acad. Sci. USA, 93:2635-2639, 1996.)
or thiazolidinedione compounds. Representative known
thiazolidinedione compounds include the glitazones, such as, for
example, troglitazone (also known as CS-045 (Sankyo) and CI-991
(Park-Davis)), pioglitazone (also known as AD-4833 and U-72107E),
rosiglitazone (also known as BRL49653), englitazone (also known as
CP-68,722), and ciglitazone or compounds described in WO 01/02377.
Anne E. Pitts and David R. Corey reported other inhibitors of human
telomerase activity such as Phosphorothioate DNA, Ribozyme,
Oligonucleotide with 2'-5' A linkage, Tea catechins, Nucleoside
derivatives, Perylenetetracarboxylic diimide, Cationic porphyrin,
Anthraquinone derivatives, Phosphodiester DNA oligos, Peptide
nucleic acid, antisense RNA (Anne E. Pitts and David R. Corey, "The
telomerase challenge--an unusual problem in drug discovery"; Drug
Discovery Today 1999, 4:155-161).
[0023] The structure of the active agents identified by code nos.,
generic or trade names may be taken from the actual edition of the
standard compendium "The Merck Index" or from databases, e.g.
Patents International (e.g. IMS World Publications). The
corresponding content thereof is hereby incorporated by
reference.
[0024] A combination which comprises (a)
N-{5-[4-(4-methyl-piperazino-methyl)-benzoylamido]-2-methylphenyl}-4-(3-p-
yridyl)-2-pyrimidine-amine and (b) at least one telomerase
inhibitor, in which the active ingredients are present in each case
in free form or in the form of a pharmaceutically acceptable salt
and optionally at least one pharmaceutically acceptable carrier,
will be referred to hereinafter as a COMBINATION OF THE INVENTION.
Depending on the structure of the telomerase inhibitor, a salt form
may impossible.
[0025] The nature of proliferative diseases like solid tumor
diseases is multifactorial. Under certain circumstances, drugs with
different mechanisms of action may be combined. However, just
considering any combination of drugs having different mode of
action does not necessarily lead to combinations with advantageous
effects.
[0026] The utility of the invention for the treatment of
proliferative diseases such as leukemia is demonstrated, by the
ability of the COMBINATION OF THE INVENTION or a telomerase
inhibitor taken alone to inhibit the transformation of BCR-ABL
positive OM9;22 cells or Imatinib-resistant BCR-ABL positive
OM9;22R cells.
[0027] In the present study, the clinical candidate telomerase
inhibitor, telomestatin (J. Am. Chem. Soc. 123; 1262, 2001), was
characterized for its ability to inhibit BCR-ABL transformation.
When tested against BCR-ABL positive OM9;22 cells or
Imatinib-resistant BCR-ABL positive OM9;22R cells, a human leukemia
cell line derived from Ph positive acute lymphoblastic leukemia
patient, treatment of telomestatin resulted in unexpected strong
inhibition of telomerase activity and reduction of telomere length.
Treatment of telomestatin potently inhibited soft agar colony
formation, slowed proliferation within 2 weeks. Cell cycle analysis
of OM9;22 or OM9;22R cells treated with telomestatin revealed G1/S
blockage. Telomestatin induced phosphorylation of p95/NBS protein,
essential for the cellular response to DNA damage, and increased
the expression of p21CIP1 and p27KIP1 in OM9;22R cells. In
addition, we examined the impact of telomestatin on human normal
hematopoietic progenitor cells by a clonogenic assay, and we
observed significantly less sensitive at the concentrations 10
times higher than those that completely inhibited colonies from
OM9;22R cells. These results demonstrate that disruption of
telomere maintenance by telomestatin alters the chemotherapeutic
profile of Imatinib-resistant BCR-ABL transformed cells, and point
the combined use of Imatinib and telomestatin as an effective
therapeutic approach of Ph positive leukemias.
[0028] The combination of telomestatin with Imatinib or
daunorubicine showed supra-additive or synergistic effects in soft
agar colony formation, whereas, the combination of telomestatin
with cytosine arabinoside or stoposide did not show any synergistic
effect and even no additive effect.
[0029] All the more surprising is the experimental finding that the
administration of a COMBINATION OF THE INVENTION, especially
comprising telomestatine as combination partner (b), results not
only in a beneficial effect, especially a supra-additive or
synergistic therapeutic effect, e.g. with regard to slowing down
the formation of soft agar colonies, but also in further beneficial
effects, e.g. less side-effects, an improved quality of life and a
decreased mortality and morbidity, compared to a monotherapy
applying only one of the pharmaceutically active ingredients used
in the COMBINATION OF THE INVENTION, in particular in the treatment
of proliferative diseases refractory to other chemotherapeutics
known as anti-cancer agents. In particular, an unexpected increased
up-take of the combination partner (b) in tumor tissue, tumor cells
or human leukemia cells (i.e. BCR-ABL) is supposed, when applied in
combination with combination partner (a).
[0030] A further benefit is that lower doses of the active
ingredients of the COMBINATION OF THE INVENTION can be used, for
example, that the dosages need not only often be smaller but are
also applied less frequently, or can be used in order to diminish
the incidence of side-effects. This is in accordance with the
desires and requirements of the patients to be treated. This
supra-additive interaction is not associated with a similar
increase in adverse effects potential.
[0031] It can be shown by established test models and in particular
those test models described herein that a COMBINATION OF THE
INVENTION results in a more effective delay of progression or
treatment of a proliferative disease compared to the effects
observed with the single combination partners. The person skilled
in the pertinent art is fully enabled to select a relevant test
model to prove the hereinbefore and hereinafter mentioned
therapeutic indications and beneficial effects. The pharmacological
activity of a COMBINATION OF THE INVENTION may, for example, be
demonstrated in a clinical study or in a test procedure as
essentially described hereinafter.
[0032] Suitable clinical studies are, for example, open label
non-randomized, dose escalation studies in patients with advanced
proliferative diseases. Such studies can in particular prove the
synergism of the active ingredients of the COMBINATIONS OF THE
INVENTION. The beneficial effects on proliferative diseases can be
determined directly through the results of these studies or by
changes in the study design which are known as such to a person
skilled in the art. Such studies are, in particular, suitable to
compare the effects of a monotherapy using the active ingredients
and a COMBINATION OF THE INVENTION. Preferably, the combination
partner (a) is administered with a fixed dose and the dose of the
combination partner (b) is escalated until the Maximum Tolerated
Dosage is reached. In a preferred embodiment of the study, each
patient receives daily doses of the combination partner (a).
[0033] The efficacy of the treatment can be determined in such
studies, e.g., after 18 or 24 weeks by radiologic evaluation of the
tumors every 6 weeks.
[0034] Alternatively, a placebo-controlled, double blind study can
be used in order to prove the benefits of the COMBINATION OF THE
INVENTION mentioned herein.
[0035] The COMBINATION OF THE INVENTION can also be applied in
combination with surgical intervention, mild prolonged whole body
hyperthermia and/or irradiation therapy.
[0036] In a preferred embodiment of the invention the telomerase
inhibitor is telomestatin as described in the European patent
application No. 1 123 937 filed in Oct. 20, 1999, or by K. Shin-ya
et al. (J. Am. Chem. Soc. 2001, 123, 1262-1263).
[0037] The COMBINATION OF THE INVENTION can be a combined
preparation or a pharmaceutical composition.
[0038] It is one objective of this invention to provide a
pharmaceutical composition comprising a quantity, which is jointly
therapeutically effective against a proliferative disease
comprising the COMBINATION OF THE INVENTION. In this composition,
the combination partners (a) and (b) can be administered together,
one after the other or separately in one combined unit dosage form
or in two separate unit dosage forms. The unit dosage form may also
be a fixed combination.
[0039] The pharmaceutical compositions for separate administration
of the combination partners (a) and (b) and for the administration
in a fixed combination, i.e. single galenical compositions
comprising at least two combination partners (a) and (b), according
to the invention can be prepared in a manner known per se and are
those suitable for enteral, such as oral or rectal, and parenteral
administration to mammals (warm-blooded animals), including man,
comprising a therapeutically effective amount of at least one
pharmacologically active combination partner alone or in
combination with one or more pharmaceutically acceptable carries,
especially suitable for enteral or parenteral application.
[0040] Novel pharmaceutical composition contain, for example, from
about 10% to about 100%, preferably from about 20% to about 60%, of
the active ingredients. Pharmaceutical preparations for the
combination therapy for enteral or parenteral administration are,
for example, those in unit dosage forms, such as sugar-coated
tablets, tablets, capsules or suppositories, and furthermore
ampoules. If not indicated otherwise, these are prepared in a
manner known per se, for example by means of conventional mixing,
granulating, sugar-coating, dissolving or lyophilizing processes.
It will be appreciated that the unit content of a combination
partner contained in an individual dose of each dosage form need
not in itself constitute an effective amount since the necessary
effective amount can be reached by administration of a plurality of
dosage units.
[0041] In particular, a therapeutically effective amount of each of
the combination partner of the COMBINATION OF THE INVENTION may be
administered simultaneously or sequentially and in any order, and
the components may be administered separately or as a fixed
combination. For example, the method of delay of progression or
treatment of a proliferative disease according to the invention may
comprise (i) administration of the combination partner (a) in free
or pharmaceutically acceptable salt form and (ii) administration of
a combination partner (b) in free or pharmaceutically acceptable
salt form, simultaneously or sequentially in any order, in jointly
therapeutically effective amounts, preferably in synergistically
effective amounts, e.g. in daily dosages corresponding to the
amounts described herein. The individual combination partners of
the COMBINATION OF THE INVENTION can be administered separately at
different times during the course of therapy or concurrently in
divided or single combination forms. Furthermore, the term
administering also encompasses the use of a pro-drug of a
combination partner that convert in vivo to the combination partner
as such. The instant invention is therefore to be understood as
embracing all such regimes of simultaneous or alternating treatment
and the term "administering" is to be interpreted accordingly.
[0042] An example of sequential administration could be a first
administration of
N-{5-[4-(4-methyl-piperazino-methyl)-benzoylamido]-2-methylphenyl}-4-(3-p-
yridyl)-2-pyrimidine-amine until a resistance to the therapy is
observed, followed by the administration of a telomerase inhibitor
taken alone or in combination with Imatinib.
[0043] The effective dosage of each of the combination partners
employed in the COMBINATION OF THE INVENTION may vary depending on
the particular compound or pharmaceutical composition employed, the
mode of administration, the condition being treated, the severity
of the condition being treated. Thus, the dosage regimen the
COMBINATION OF THE INVENTION is selected in accordance with a
variety of factors including the route of administration and the
renal and hepatic function of the patient. A physician, clinician
or veterinarian of ordinary skill can readily determine and
prescribe the effective amount of the single active ingredients
required to prevent, counter or arrest the progress of the
condition. Optimal precision in achieving concentration of the
active ingredients within the range that yields efficacy without
toxicity requires a regimen based on the kinetics of the active
ingredients' availability to target sites.
[0044]
N-{5-[4-(4-methyl-piperazino-methyl)-benzoylamido]-2-methylphenyl}-
-4-(3-pyridyl)-2-pyrimidine-amine monomesylate, is preferably
administered to a human in a dosage in the range of about 2.5 to
850 mg/day, more preferably 5 to 600 mg/day and most preferably 20
to 300 mg/day. Unless stated otherwise herein, the compound is
preferably administered from one to four times per day, more
preferably once daily.
[0045] Furthermore, the present invention pertains to the use of a
COMBINATION OF THE INVENTION for the delay of progression or
treatment of a proliferative disease and to the use of a
COMBINATION OF THE INVENTION for the preparation of a medicament
for the delay of progression or treatment of a proliferative
disease.
[0046] Preferably, the proliferative disease is leukemia,
Imatinib-resistant leukemia and tumors.
[0047] Moreover, the present invention provides a commercial
package comprising a COMBINATION OF THE INVENTION, together with
instructions for simultaneous, separate or sequential use thereof
in the delay of progression or treatment of a proliferative
disease.
[0048] The following Example illustrates the invention described
above, but is not, however, intended to limit the scope of the
invention in any way. The beneficial effects of the COMBINATION OF
THE INVENTION (i.e. good therapeutic margin, less side effects,
synergistic therapeutic effect and other advantages mentioned
herein), can also be determined by other test models known as such
to the person skilled in the pertinent art. The synergistic
therapeutic effect, may for example, be demonstrated in a clinical
study or in the test procedure as essentially described
hereinafter.
MATERIALS AND METHODS
[0049] Antibodies and Reagents: Anti-ATM Ab (K-19), anti-Chk2 Ab
(H-300), anti-NBS1 mAb (N-19), anti-phospho-NBS1 Ab (Ser343),
anti-p21.sup.CIP1 Ab (F-15), anti-p27.sup.KIP1 Ab (F-8),
anti-p15.sup.INK4B Ab (K-18), and anti-p16.sup.INK4A Ab (H-156)
were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz,
Calif.). Imatinib was kindly provided by Novartis Inc. (Basel,
Switzerland). Daunorubicin (DNR), cytosine arabinoside (Ara C), and
etoposide (VP-16) were obtained from Sigma (St Louis, Mo.).
Telomestatin was purified as previously described (Shin-ya et al.,
2001).
[0050] Cells and cell culture: The Ph-positive acute lymphoblastic
cell line OM9;22 has been described previously (Ohyashiki et al.,
1993). K562 cells were obtained from the American Type Culture
Collection (Rockville, Md.). These cell lines were cultured in
McCoy's 5A modified medium (Life Technology, Inc.) supplemented
with 10% fetal calf serum (Hyclone Laboratories, Logan, Utah).
[0051] Generation of stable clones expressing DN-hTERT mutants.
pBABE-DN-hTERT was a gift from Dr. Robert Weinberg (Massachusetts
Institute of Technology). OM9;22 cells were transfected with the
expression vector pBABE-puro-DN-hTERT by electroporation. Beginning
48 hours after electroporation, cells were selected with 2 .mu.g/ml
of puromycin and cloned by limiting dilution. PD 0 was defined as
the time at which cultures reached confluence in 10-cm culture
dishes.
[0052] Telomerase assay and measurement of TRF. Telomerase activity
was examined by a telomere repeat amplification protocol (TRAP)
assay using a TRAP.sub.EzE telomerase detection kit (Oncor,
Gaithersburg, Md.). The PCR products were subjected to 12%
acrylamide denaturing electrophoresis in an automated laser
fluorescence DNA sequencer II (Pharmacia LKB Biotechnology, AB) and
analyzed by the Fragment Manager program (Pharmacia LKB
Biotechnology, AB). Activity in the extract-based PCR TRAP assay
was detected as a periodic 6-bp peak of telomerase products and, in
each sample, relative telomerase activity was calculated
semiquantitatively in comparison with a 36-bp internal standard. To
measure TRF, genomic DNA was digested with the restriction enzymes
HinfI and RsaI, fractionated on 0.7% agarose gels and transferred
onto nylon membranes. Hybridization was performed by using the Telo
TTAGGG telomere length assay kit (Roche Molecular Biochemicals,
Mannheim, Germany).
[0053] Apoptosis assay. The incidence of apoptosis was determined
by flow cytometric analysis with the FITC-conjugated APO2.7
monoclonal antibody (clone 2.7), which was raised against the 38
kDa mitochondrial membrane protein (7A6 antigen) and is expressed
by cells undergoing apoptosis (Nakajima et al., 2001).
[0054] Fluorescence in situ hybridization and quantitative image
analysis. Individual telomere length was analysed by quantitative
fluorescence in situ hybridization (Q-FISH) as described previously
(Martens et al., 1998). Digital images of metaphase spreads were
recorded with a digital camera (Sensys, Photometrics) on a Zeiss
Axioplan II fluorescence microscope using the Vysis workstation
QUIPS. Telomere profiles were analysed by use of the TFL-TELO
software (Poon et al., 1999). Telomere fluorescence intensity
values were expressed in arbitrary units.
[0055] Immunoblotting and Immunoprecipitation. Immunoblotting and
immunoprecipitation were performed as described previously (Tauchi
et al., 1994). For immune complex kinase assays, immunoprecipitated
proteins were incubated with 30 .mu.l of kinase buffer (50 mM
HEPES, pH 8.0, 10 mM MnCl.sub.2, 2.5 mM EDTA, 1 mM dithiothreitol,
10 mM ATP and 30 mCi of [.gamma.-.sup.32P]ATP at 30.degree. C. for
30 min. The reaction products were separated by SDS-PAGE, and
transferred to the membranes for autoradiography.
[0056] Statistical analysis. Comparisons between groups were
analyzed by the Mann-Whitney U test. Values of P<0.05 were
considered to indicate statistical significance. The statistical
tests were performed using the Microsoft Word Excel (Brain Power
Inc, Calabashes, Calif.) software package for the Macintosh
personal computer.
[0057] The utility of the invention for the treatment of
proliferative disease such as leukemia can also be demonstrated,
e.g., in the proliferation test using bcr-Abl transfected 32D cells
as follows:
[0058] Bcr-Abl-transfected 32D cells (32D pGD p210 Bcr-Abl;
Bazzoni, G.; et al. J. Clin. Invest. (1996), 98(2), 521-528) are
cultured in RPMI 1640 (BioConcept, Allschwil, Switzerland; cat.
No.: 1-41F01), 10% fetal calf serum, 2 mM glutamine. 10000 cells in
50 .mu.L per well are seeded into flat bottom 96 well tissue
culture plates. Complete medium alone (for controls) or serial
threefold dilutions of compounds are added in triplicates to a
final volume of 100 .mu.L and the cells are incubated at 37.degree.
C., 5% CO.sub.2 for 65 to 72 h. The cell proliferation reagent
WST-1 (Roche Diagnostics GmbH; cat. no.: 1 664 807) is added at 10
.mu.L per well followed by 2 h incubation at 37.degree. C. Colour
development, depending on the amount of living cells, is measured
at 440 nm. The effect for each compound or combination is
calculated as percent inhibition of the value (OD.sub.440) obtained
for the control cells (100%) and plotted against the compound
concentrations. The IC.sub.50s are calculated from the dose
response curves by graphic extrapolation.
[0059] Compounds inhibiting the growth of 32D-Bcr-Abl cells can be
further tested on IL-3 dependent 32D wt cells to prove the
specificity of the compounds for the bcr-Abl kinase and to exclude
compound toxicity.
RESULTS
[0060] Effects of telomestatin on cell Proliferation. We
characterize the growth properties of telomestatin-treated cells.
The growth kinetics of telomestatin-treated cells initially did not
differ from those of untreated control cells, regardless of the
cell line used. K562 cell cultures in the absence or presence of 2
.mu.M of telomestatin exhibit no or only minor differences in
proliferation during 20 days of treatment. However, after 30 days,
telomestatin-treated K562 cells show an almost complete inhibition
of proliferation. Telomestatin-treated OM9;22 or OM9;22R cells also
cease to proliferate after 15 days. Telomestatin-treated cells show
distinctive morphological features associated with apoptosis. In a
further study, we can characterize the ability of telomestatin to
inhibit BCR-ABL transformation. When tested against
Imatinib-resistant BCR-ABL positive OM9;22R cells, a human leukemia
cell line derived from Ph positive acute lymphoblastic leukemia
patient, treatment of telomestatin results in unexpected strong
inhibition of telomerase activity and reduction of telomere length.
Treatment of telomestatin potently inhibite soft agar colony
formation and slows proliferation within 2 weeks.
[0061] Cell cycle analysis of OM9;22R cells treated with
telomestatin reveal G1/S blockage. Telomestatin induces
phosphorylation of p95/NBS protein, essential for the cellular
response to DNA damage, and increases the expression of p21CIP1 and
p27KIP1 in OM9;22R cells. In addition, we can examine the impact of
telomestatin on human normal hematopoietic progenitor cells by a
clonogenic assay, and we observe significantly less sensitive at
the concentrations 10 times higher than those that completely
inhibit colonies from OM9;22R cells. These results demonstrate that
disruption of telomere maintenance by telomestatin alters the
chemotherapeutic profile of Imatinib-resistant BCR-ABL transformed
cells, and point the combined use of Imatinib and telomestatin as
an effective therapeutic approach of Ph positive leukemias.
[0062] Enhancement of apoptosis in telomestatin-treated K562 cells
by chemotherapeutic agents. Since early passaged
telomestatin-treated K562 cells did not show induction of
apoptosis, we decided to examine the impact of telomerase
inhibition on chemotherapeutic responses (Table A). Mechanistically
distinct classes of reagents are selected for analysis, including
imatinib, daunorubicin (DNR), mitoxantrone (MIT), and vincristine
(VCR). To assess the effects of telomerase inhibition in modulating
responses to these reagents, experiments focused on early passaged
telomestatin-treated K562 cells (PD10). In this series of
experiments, K562 cells are cultured with telomestatin for 10 days,
subsequently the telomestatin-treated K562 cells are incubated with
the agents for 48 hours, and the incidence of apoptosis is
determined by flow cytometric analysis with APO2.7 mAb (Table A).
Apo2.7 is the apoptosis index. Higher score means that apoptosis is
induced more powerfully. TABLE-US-00001 TABLE A APO2.7 (%) 0 nM 10
nM 50 nM 100 nM 500 nM 1000 nM Imatinib 8 9 12 28 (control)
Imatinib + 8 22 62 90 Telom 2 .mu.M DNR (control) 10 11 23 36 53
DNR + 10 32 50 70 85 Telomest 2 .mu.M MIT (control) 9 10 12 17 29
38 MIT + 9 10 11 33 47 62 Telomest 2 .mu.M VCR (control) 5 12 18 24
29 34 VCR + 5 18 32 44 57 74 Telomest 2 .mu.M
The telomestatin-treated K562 cells show enhanced induction of
apoptosis compared with control cells after exposure to imatinib,
DNR, MIT and VCR (Table A), whereas significant chemosensitivity is
not observed in cells exposed to etoposide (VP-16),
6-mercaptopurine (6-MP), methotrexate (MTX), cytosine arabimoside
and stoposide (data not shown). These results, demonstrating
enhanced sensitivity to some classes of chemotherapeutic agents,
imply cytotoxic synergy between telomere dysfunction and these
agents. Thus synergistic effects of combinations comprising
telomestatin with imatinib, DNR, MIT and VCR have been shown.
[0063] We conclude that telomerase inhibitors combined with use of
imatinib and other chemotherapeutic agents may be very useful for
the treatment of BCR-ABL-positive leukemia.
* * * * *