U.S. patent application number 10/655407 was filed with the patent office on 2004-04-22 for treatment of tuberous sclerosis associated neoplasms.
Invention is credited to Arbiser, Jack.
Application Number | 20040077661 10/655407 |
Document ID | / |
Family ID | 32069686 |
Filed Date | 2004-04-22 |
United States Patent
Application |
20040077661 |
Kind Code |
A1 |
Arbiser, Jack |
April 22, 2004 |
Treatment of tuberous sclerosis associated neoplasms
Abstract
The present invention relates to the use of PDGF receptor
tyrosine kinase or bcr-abl tyrosine kinase inhibitors, especially
of N-phenyl-2-pyrimidine-amine derivatives of formula I, 1 in which
the symbols and substituents have the meaning as defined herein in
free form or in pharmaceutically acceptable salt form, in the
manufacture of a pharmaceutical composition for the treatment of
tuberous sclerosis associated neoplasms; to a method of treatment
of warm-blooded animals, including humans, suffering from a
tuberous sclerosis associated neoplasms.
Inventors: |
Arbiser, Jack; (Atlanta,
GA) |
Correspondence
Address: |
THOMAS HOXIE
NOVARTIS, CORPORATE INTELLECTUAL PROPERTY
ONE HEALTH PLAZA 430/2
EAST HANOVER
NJ
07936-1080
US
|
Family ID: |
32069686 |
Appl. No.: |
10/655407 |
Filed: |
September 4, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60408550 |
Sep 5, 2002 |
|
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Current U.S.
Class: |
514/255.05 ;
514/275 |
Current CPC
Class: |
A61P 35/00 20180101;
A61K 31/505 20130101; A61K 31/506 20130101 |
Class at
Publication: |
514/255.05 ;
514/275 |
International
Class: |
A61K 031/506; A61K
031/505 |
Claims
What is claimed is:
1. A method of treating tuberous sclerosis associated neoplasms,
which comprises administering to a subject in need thereof an
effective amount of a PDGF receptor tyrosine kinase inhibitor or a
bcr-abl tyrosine kinase inhibitor.
2. A method according to claim 1 which comprises administering an
effective amount of an N-phenyl-2-pyrimidine-amine derivative of
the formula I 3wherein R.sub.1 is 4-pyrazinyl;
1-methyl-1H-pyrrolyl; amino- or amino-lower alkyl-substituted
phenyl, wherein the amino group in each case is free, alkylated or
acylated; 1H-indolyl or 1H-imidazolyl bonded at a five-membered
ring carbon atom; or unsubstituted or lower alkyl-substituted
pyridyl bonded at a ring carbon atom and unsubstituted or
substituted at the nitrogen atom by oxygen; R.sub.2 and R.sub.3 are
each independently of the other hydrogen or lower alkyl; one or two
of the radicals R.sub.4, R.sub.5, R.sub.6, R.sub.7 and R.sub.8 are
each nitro, fluoro-substituted lower alkoxy or a radical of formula
II --N(R.sub.9)--C(.dbd.X)--(Y).sub.n--R.sub.10 (II), wherein
R.sub.9 is hydrogen or lower alkyl, X is oxo, thio, imino, N-lower
alkyl-imino, hydroximino or O-lower alkyl-hydroximino, Y is oxygen
or the group NH, n is 0 or 1 and R.sub.10 is an aliphatic radical
having at least 5 carbon atoms, or an aromatic, aromatic-aliphatic,
cycloaliphatic, cycloaliphatic-aliphatic, heterocyclic or
heterocyclic-aliphatic radical, and the remaining radicals R.sub.4,
R.sub.5, R.sub.6, R.sub.7 and R.sub.8 are each independently of the
others hydrogen, lower alkyl that is unsubstituted or substituted
by free or alkylated amino, piperazinyl, piperidinyl, pyrrolidinyl
or by morpholinyl, or lower alkanoyl, trifluoromethyl, free,
etherified or esterifed hydroxy, free, alkylated or acylated amino
or free or esterified carboxy, or a pharmaceutically acceptable
salt thereof.
3. A method according to claim 2 wherein one or two of the radicals
R.sub.4, R.sub.5, R.sub.6, R.sub.7 and R.sub.8 are each nitro or a
radical of formula II wherein R.sub.9 is hydrogen or lower alkyl, X
is oxo, thio, imino, N-lower alkyl-imino, hydroximino or O-lower
alkyl-hydroximino, Y is oxygen or the group NH, n is 0 or 1 and
R.sub.10 is an aliphatic radical having at least 5 carbon atoms or
an aromatic, aromatic-aliphatic, cycloaliphatic,
cycloaliphatic-aliphatic, heterocyclic or heterocyclic-aliphatic
radical, and the remaining radicals R.sub.4, R.sub.5, R.sub.6,
R.sub.7 and R.sub.8 are each independently of the others hydrogen,
lower alkyl that is unsubstituted or substituted by free or
alkylated amino, piperazinyl, piperidinyl, pyrrolidinyl or by
morpholinyl, or lower alkanoyl, trifluoromethyl, free, etherified
or esterifed hydroxy, free, alkylated or acylated amino or free or
esterified carboxy, or a pharmaceutically acceptable salt
thereof.
4. A method according to claim 3 wherein R.sub.1 is pyridyl or
N-oxido-pyridyl each of which is bonded at a carbon atom, R.sub.2
and R.sub.3 are each hydrogen, R.sub.4 is hydrogen or lower alkyl,
R.sub.5 is hydrogen, lower alkyl or trifluoromethyl, R.sub.6 is
hydrogen, R.sub.7 is nitro, fluoro-substituted lower alkoxy or a
radical of formula II wherein R.sub.9 is hydrogen, X is oxo, n is 0
and R.sub.10 is pyridyl bonded at a carbon atom, phenyl that is
unsubstituted or substituted by halogen, cyano, lower alkoxy,
carboxy, lower alkyl or by 4-methyl-piperazinyl-meth- yl, or
C.sub.5-C.sub.7alkyl, thienyl, 2-naphthyl or cyclohexyl, and
R.sub.8 is hydrogen, or a pharmaceutically acceptable salt
thereof.
5. A method according to claim 4 wherein at least one of the
radicals R.sub.4 and R.sub.8 is lower alkyl, and the remaining
substituents are as defined in the respective generic claim, or a
pharmaceutically acceptable salt thereof.
6. A method according to claim 5, wherein R.sub.1 is pyridyl bonded
at a carbon atom, R.sub.2, R.sub.3, R.sub.5, R.sub.6 and R.sub.8
are each hydrogen, R.sub.4 is lower alkyl, R.sub.7 a radical of
formula II wherein R.sub.9 is hydrogen, X is oxo, n is 0 and
R.sub.10 is 4-methyl-piperazinyl-methyl, or a pharmaceutically
acceptable salt thereof.
7. A method according to claim 1, wherein
N-{5-[4-(4-methyl-piperazino-met-
hyl)-benzoylamido]-2-methylphenyl}-4-(3pyridinyl)-2-pyrimidine-amine,
or a pharmaceutically acceptable salt thereof, is administered.
8. A method according to claim 7, wherein
N-{5-[4-(4-methyl-piperazino-met-
hyl)-benzoylamido]-2-methylphenyl}-4-(3pyridinyl)-2-pyrimidine-amine,
monomesylate salt is administered.
9. A method of claim 1 wherein the tuberous sclerosis associated
neoplasm is selected from angiomyolipomas, rhabdomyomas,
lymphangioleiomyomatosis, subependymal giant cell astrocytomas,
angiofibromas and periungual fibromas.
10. A method of claim 1 wherein the monomethanesulfonate salt of
N-{5-[4-(4-methyl-piperazino-methyl)-benzoylamido]-2-methylphenyl}-4-(3-p-
yridinyl)-2-pyrimidine-amine is administered at a daily dose
corresponding to 100 to 1000 mg of
N-{5-[4-(4-methyl-piperazino-methyl)-benzoylamido]-2-
-methylphenyl}-4-(3-pyridinyl)-2-pyrimidine-amine free base.
11. A method of inhibiting the formation of neoplasms in a patient
suffering from tuberous sclerosis, which comprises administering to
the patient an effective amount of a PDGF receptor tyrosine kinase
inhibitor or a bcr-abl tyrosine kinase inhibitor.
12. A method according to claim 11 comprises administering an
effective amount of an N-phenyl-2-pyrimidine-amine derivative of
the formula I 4wherein R.sub.1 is 4-pyrazinyl;
1-methyl-1H-pyrrolyl; amino- or amino-lower alkyl-substituted
phenyl, wherein the amino group in each case is free, alkylated or
acylated; 1H-indolyl or 1H-imidazolyl bonded at a five-membered
ring carbon atom; or unsubstituted or lower alkyl--substituted
pyridyl bonded at a ring carbon atom and unsubstituted or
substituted at the nitrogen atom by oxygen; R.sub.2 and R.sub.3 are
each independently of the other hydrogen or lower alkyl; one or two
of the radicals R.sub.4, R.sub.5, R.sub.6, R.sub.7 and R.sub.8 are
each nitro, fluoro-substituted lower alkoxy or a radical of formula
II --N(R.sub.9)--C(.dbd.X)--(Y).sub.n--R.sub.10 (II), wherein
R.sub.9 is hydrogen or lower alkyl, X is oxo, thio, imino, N-lower
alkyl-imino, hydroximino or O-lower alkyl-hydroximino, Y is oxygen
or the group NH, n is 0 or 1 and R.sub.10 is an aliphatic radical
having at least 5 carbon atoms, or an aromatic, aromatic-aliphatic,
cycloaliphatic, cycloaliphatic-aliphatic, heterocyclic or
heterocyclic-aliphatic radical, and the remaining radicals R.sub.4,
R.sub.5, R.sub.6, R.sub.7 and R.sub.8 are each independently of the
others hydrogen, lower alkyl that is unsubstituted or substituted
by free or alkylated amino, piperazinyl, piperidinyl, pyrrolidinyl
or by morpholinyl, or lower alkanoyl, trifluoromethyl, free,
etherified or esterifed hydroxy, free, alkylated or acylated amino
or free or esterified carboxy, or a pharmaceutically acceptable
salt thereof.
13. A method according to claim 12 wherein one or two of the
radicals R.sub.4, R.sub.5, R.sub.6, R.sub.7 and R.sub.8 are each
nitro or a radical of formula II wherein R.sub.9 is hydrogen or
lower alkyl, X is oxo, thio, imino, N-lower alkyl-imino,
hydroximino or O-lower alkyl-hydroximino, Y is oxygen or the group
NH, n is 0 or 1 and R.sub.10 is an aliphatic radical having at
least 5 carbon atoms or an aromatic, aromatic-aliphatic,
cycloaliphatic, cycloaliphatic-aliphatic, heterocyclic or
heterocyclic-aliphatic radical, and the remaining radicals R.sub.4,
R.sub.5, R.sub.6, R.sub.7 and R.sub.8 are each independently of the
others hydrogen, lower alkyl that is unsubstituted or substituted
by free or alkylated amino, piperazinyl, piperidinyl, pyrrolidinyl
or by morpholinyl, or lower alkanoyl, trifluoromethyl, free,
etherified or esterifed hydroxy, free, alkylated or acylated amino
or free or esterified carboxy, or a pharmaceutically acceptable
salt thereof.
14. A method according to claim 13 wherein R.sub.1 is pyridyl or
N-oxido-pyridyl each of which is bonded at a carbon atom, R.sub.2
and R.sub.3 are each hydrogen, R.sub.4 is hydrogen or lower alkyl,
R.sub.5 is hydrogen, lower alkyl or trifluoromethyl, R.sub.6 is
hydrogen, R.sub.7 is nitro, fluoro-substituted lower alkoxy or a
radical of formula II wherein R.sub.9 is hydrogen, X is oxo, n is 0
and R.sub.10 is pyridyl bonded at a carbon atom, phenyl that is
unsubstituted or substituted by halogen, cyano, lower alkoxy,
carboxy, lower alkyl or by 4-methyl-piperazinyl-meth- yl, or
C.sub.5-C.sub.7alkyl, thienyl, 2-naphthyl or cyclohexyl, and
R.sub.8 is hydrogen, or a pharmaceutically acceptable salt
thereof.
15. A method according to claim 14 wherein at least one of the
radicals R.sub.4 and R.sub.8 is lower alkyl, and the remaining
substituents are as defined in the respective generic claim, or a
pharmaceutically acceptable salt thereof.
16. A method according to claim 15, wherein R.sub.1 is pyridyl
bonded at a carbon atom, R.sub.2, R.sub.3, R.sub.5, R.sub.6 and
R.sub.8 are each hydrogen, R.sub.4 is lower alkyl, R.sub.7 a
radical of formula II wherein R.sub.9 is hydrogen, X is oxo, n is 0
and R.sub.10 is 4-methyl-piperazinyl-methyl, or a pharmaceutically
acceptable salt thereof.
17. A method according to claim 11, wherein
N-{5-[4-(4-methyl-piperazino-m-
ethyl)-benzoylamido]-2-methylphenyl}-4-(3-pyridinyl)-3-pyrimidine-amine
or a pharmaceutically acceptable salt thereof, is administered.
18. A method according to claim 17, wherein
N-{5-[4-(4-methyl-piperazino-m-
ethyl)-benzoylamido]-2-methylphenyl}-4-(3-pyridinyl)-3-pyrimidine-amine
monomesylate salt is administered.
19. A method according to claim 11 wherein the monomethanesulfonate
salt of
N-{5-[4-(4-methyl-piperazino-methyl)-benzoylamido]-2-methylphenyl}-4-(-
3-pyridinyl)-3-pyrimidine-amine is administered at a daily dose
corresponding to 100 to 1000 mg of
N-{5-[4-(4-methyl-piperazino-methyl)-b-
enzoylamido]-2-methylphenyl}-4-(3-pyridinyl)-3-pyrimidine-amine
free base.
Description
[0001] The present invention relates to a new use of PDGF receptor
tyrosine kinase inhibitors or bcr-abl receptor tyrosine kinase
inhibitors, especially of N-phenyl-2-pyrimidine-amine derivatives
of formula I (hereinafter: "COMPOUNDS of formula I") in which the
symbols and substituents have the meaning as given hereinafter in
free form or in pharmaceutically acceptable salt form, in the
manufacture of a pharmaceutical composition for the treatment of
tuberous sclerosis associated neoplasms, and to a method of
treatment of warm-blooded animals, including humans, in which a
therapeutically effective dose of a PDGF receptor tyrosine kinase
or bcr-abl receptor tyrosine kinase inhibitor is administered to a
warm-blooded animal suffering from tuberous sclerosis associated
neoplasms.
[0002] Tuberous sclerosis (TSC) is an autosomal dominant disorder,
with variable penetrance and a frequency in the general population
of 1 in 6,000 to 1 in 10,000. The disease is characterized by the
development of hamartomas and focal dysplasias in multiple organs
including the brain (cortical and subcortical tubers, subependymal
nodules, and giant cell astrocytomas, cerebral atrophy, cerebral
infarct, cerebral aneurysm, arachnoid cyst), kidney
(angiomyolipomas, cysts, carcinomas), skin (hypomelanotic macules,
shagreen patches, facial angiofibromas, periungual fibromas), eye
(retinal hamartomas), heart (rhabdomyomas), lungs
(lymphangioleiomyomatosis), musculoskeletal manifestations
(Sclerotic lesions, bone lucencies) and, to a lesser extent, other
organs (K. S. Caldemeyer, et al; Tuberous sclerosis--Part I and
Part II; J. Am. Acad. Dermatol., (2001) Vol. 45, No. 3, p
448-451).
[0003] Tuberous sclerosis is a common autosomal dominant disorder,
which leads to neoplasms of the kidney, brain, skin, heart and lung
in children and adults. It has been demonstrated that benign
neoplasms of tuberous sclerosis are highly angiogenic.
TSC-associated brain lesions typically include cortical tubers,
subependymal nodules and subependymal giant cell astrocytomas. In
the brain, a distinct neoplasm arises in a periventricular area and
is termed a subependymal giant cell astrocytoma (SEGA). This
neoplasm is cytologically bland, but may grow to large sizes and
become surgically unresectable. Radiation therapy of SEGA may
result in transformation to a high-grade neoplasm. In the kidney
and the heart, the most common TSC-associated lesions are
angiomyolipomas (a benign neoplasm of adipose tissue (lipoma) in
which muscle cells and vascular structures are fairly conspicuous)
and rhabdomyomas, respectively, although oncocytomas and renal cell
carcinomas are observed with an increased frequency. Finally,
tuberous sclerosis manifests with cutaneous neoplasms including
angiofibromas and periungual fibromas. Although not
life-threatening as are other visceral tumors, these lesions can
cause significant disfigurement and can be difficult to treat.
Tumors involving the heart and kidneys can severely affect organ
function, while brain lesions can result in seizures, mental
retardation, hydrocephalus and autism. Cardiac rhabdomyomas (benign
neoplasms derived from striated muscle, occurring in the heart in
children, probably as a hamartomatous process) occur around birth
and usually resolve spontaneously. Angiomyolipomas are uncommon
renal tumors, which are a distinct neoplasm of perivascular
epithelioid cells, and exhibit phenotypic markers of smooth muscle,
fat, and melanocytes. They are found spontaneously in 1 of 1000
humans, and are the most common renal tumor in tuberous sclerosis
complex. Multifocal renal lesions are the major cause of morbidity
and mortality in adults with tuberous sclerosis, resulting in
hemorrhage, renal failure, and occasional malignant transformation.
A malignant version of angiomyolipomas occur sporadically in
pancreas, omentum, and female genitourinary tissues, and these
lesions may demonstrate loss of tuberin. In addition, similar
lesions occur in the lungs of young women and are known as
lymphangiomyomatosis. Lymphangiomyomatosis is a hormone sensitive
neoplasm, and can sometimes be ameliorated with hormonal therapy.
However, the primary modality for treatment of TSC is surgical, and
effective medical therapies are lacking for TSC neoplasms.
Currently, no medical therapy exists for the treatment of
angiomyolipomas of the kidney or SEGAs of the brain, and lesions
are treated by either resection or, in the case of smaller
angiomyolipomas, by embolization.
[0004] Pursuing research in this field, the applicant has
surprisingly discovered that platelet-derived growth factor
receptor (PDGFR) tyrosine kinase inhibitor especially
N-phenyl-2-pyrimidineamine derivatives of formula I such as
COUMPOUND I, inhibit the growth of human SV7tert angiomyolipoma
cells and murine tsc2ang1 cells in vitro. The applicant has further
demonstrated that PDGFRP is functionally active in the above cell
lines, and that functional PDGFR, is present in authentic human
angiomyolipoma tissue.
[0005] The instant invention is a response to the need for an
alternative therapy in the treatment of tuberous sclerosis
associated neoplasms.
[0006] It has now surprisingly been demonstrated that tuberous
sclerosis associated neoplasms can be successfully treated with
PDGF receptor tyrosine kinase or bcr-abl tyrosine kinase
inhibitors, especially N-phenyl-2-pyrimidine-amine derivatives of
formula I, or pharmaceutically acceptable salt thereof.
[0007] The present invention thus concerns the use of PDGF receptor
tyrosine kinase or bcr-abl tyrosine kinase inhibitors or a
pharmaceutically acceptable salt thereof, for the manufacture of a
medicament for treating tuberous sclerosis associated
neoplasms.
[0008] The present invention furthermore concerns the use of PDGF
receptor tyrosine kinase or bcr-abl tyrosine kinase inhibitors or a
pharmaceutically acceptable salt thereof, for the manufacture of a
medicament for the prevention of neoplasms in patients suffering
from tuberous sclerosis.
[0009] In particular, the present invention relates to a new use of
N-phenyl-2-pyrimidine-amine derivatives of formula I (hereinafter:
"COMPOUNDS of formula I"), 2
[0010] wherein
[0011] R.sub.1 is 4-pyrazinyl; 1-methyl-1H-pyrrolyl; amino- or
amino-lower alkyl-substituted phenyl, wherein the amino group in
each case is free, alkylated or acylated; 1H-indolyl or
1H-imidazolyl bonded at a five-membered ring carbon atom; or
unsubstituted or lower alkyl-substituted pyridyl bonded at a ring
carbon atom and unsubstituted or substituted at the nitrogen atom
by oxygen;
[0012] R.sub.2 and R.sub.3 are each independently of the other
hydrogen or lower alkyl;
[0013] one or two of the radicals R.sub.4, R.sub.5, R.sub.6,
R.sub.7 and R.sub.8 are each nitro, fluoro-substituted lower alkoxy
or a radical of formula II
--N(R.sub.9)--C(.dbd.X)--(Y).sub.n--R.sub.10 (II),
[0014] wherein
[0015] R.sub.9 is hydrogen or lower alkyl,
[0016] X is oxo, thio, imino, N-lower alkyl-imino, hydroximino or
O-lower alkyl-hydroximino,
[0017] Y is oxygen or the group NH,
[0018] n is 0 or 1 and
[0019] R.sub.10 is an aliphatic radical having at least 5 carbon
atoms, or an aromatic, aromatic-aliphatic, cycloaliphatic,
cycloaliphatic-aliphatic- , heterocyclic or heterocyclic-aliphatic
radical,
[0020] and the remaining radicals R.sub.4, R.sub.5, R.sub.6,
R.sub.7 and R.sub.8 are each independently of the others hydrogen,
lower alkyl that is unsubstituted or substituted by free or
alkylated amino, piperazinyl, piperidinyl, pyrrolidinyl or by
morpholinyl, or lower alkanoyl, trifluoromethyl, free, etherified
or esterifed hydroxy, free, alkylated or acylated amino or free or
esterified carboxy,
[0021] or of a salt of such a compound having at least one
salt-forming group,
[0022] for the manufacture of a medicament for treating tuberous
sclerosis associated neoplasms or for the manufacture of a
medicament for the prevention of neoplasms in patients suffering
from tuberous sclerosis.
[0023] 1-Methyl-1H-pyrrolyl is preferably 1-methyl-1H-pyrrol-2-yl
or 1-methyl-1H-pyrrol-3-yl.
[0024] Amino- or amino-lower alkyl-substituted phenyl R.sub.1
wherein the amino group in each case is free, alkylated or acylated
is phenyl substituted in any desired position (ortho, meta or para)
wherein an alkylated amino group is preferably mono- or di-lower
alkylamino, for example dimethylamino, and the lower alkyl moiety
of amino-lower alkyl is preferably linear C.sub.1-C.sub.3alkyl,
such as especially methyl or ethyl.
[0025] 1H-Indolyl bonded at a carbon atom of the five-membered ring
is 1H-indol-2-yl or 1H-indol-3-yl.
[0026] Unsubstituted or lower alkyl-substituted pyridyl bonded at a
ring carbon atom is lower alkyl-substituted or preferably
unsubstituted 2-, 4- or preferably 3-pyridyl, for example
3-pyridyl, 2-methyl-3-pyridyl or 4-methyl-3-pyridyl. Pyridyl
substituted at the nitrogen atom by oxygen is a radical derived
from pyridine N-oxide, i.e. N-oxido-pyridyl.
[0027] Fluoro-substituted lower alkoxy is lower alkoxy carrying at
least one, but preferably several, fluoro substituents, especially
trifluoromethoxy or 1,1,2,2-tetrafluoro-ethoxy.
[0028] When X is oxo, thio, imino, N-lower alkyl-imino, hydroximino
or O-lower alkyl-hydroximino, the group C.dbd.X is, in the above
order, a radical C.dbd.O, C.dbd.S, C.dbd.N--H, C.dbd.N-lower alkyl,
C.dbd.N--OH or C.dbd.N--O-lower alkyl, respectively. X is
preferably oxo.
[0029] n is preferably 0, i.e. the group Y is not present.
[0030] Y, if present, is preferably the group NH.
[0031] The term "lower" within the scope of this text denotes
radicals having up to and including 7, preferably up to and
including 4 carbon atoms.
[0032] Lower alkyl R.sub.1, R.sub.2, R.sub.3 and R.sub.9 is
preferably methyl or ethyl.
[0033] An aliphatic radical R.sub.10 having at least 5 carbon atoms
preferably has not more than 22 carbon atoms, generally not more
than 10 carbon atoms, and is such a substituted or preferably
unsubstituted aliphatic hydrocarbon radical, that is to say such a
substituted or preferably unsubstituted alkynyl, alkenyl or
preferably alkyl radical, such as C.sub.5-C.sub.7alkyl, for example
n-pentyl. An aromatic radical R.sub.10 has up to 20 carbon atoms
and is unsubstituted or substituted, for example in each case
unsubstituted or substituted naphthyl, such as especially
2-naphthyl, or preferably phenyl, the substituents preferably being
selected from cyano, unsubstituted or hydroxy-, amino- or
4-methyl-piperazinyl-substituted lower alkyl, such as especially
methyl, trifluoromethyl, free, etherified or esterified hydroxy,
free, alkylated or acylated amino and free or esterified carboxy.
In an aromatic-aliphatic radical R.sub.10 the aromatic moiety is as
defined above and the aliphatic moiety is preferably lower alkyl,
such as especially C.sub.1-C.sub.2alkyl, which is substituted or
preferably unsubstituted, for example benzyl. A cycloaliphatic
radical R.sub.10 has especially up to 30, more especially up to 20,
and most especially up to 10 carbon atoms, is mono- or poly-cyclic
and is substituted or preferably unsubstituted, for example such a
cycloalkyl radical, especially such a 5- or 6-membered cycloalkyl
radical, such as preferably cyclohexyl. In a
cycloaliphatic-aliphatic radical R.sub.10 the cycloaliphatic moiety
is as defined above and the aliphatic moiety is preferably lower
alkyl, such as especially C.sub.1-C.sub.2alkyl, which is
substituted or preferably unsubstituted. A heterocyclic radical
R.sub.10 contains especially up to 20 carbon atoms and is
preferably a saturated or unsaturated monocyclic radical having 5
or 6 ring members and 1-3 hetero atoms which are preferably
selected from nitrogen, oxygen and sulfur, especially, for example,
thienyl or 2-, 3- or 4-pyridyl, or a bi- or tri-cyclic radical
wherein, for example, one or two benzene radicals are annellated
(fused) to the mentioned monocyclic radical. In a
heterocyclic-aliphatic radical R.sub.10 the heterocyclic moiety is
as defined above and the aliphatic moiety is preferably lower
alkyl, such as especially C.sub.1-C.sub.2alkyl, which is
substituted or preferably unsubstituted.
[0034] Etherified hydroxy is preferably lower alkoxy. Esterified
hydroxy is preferably hydroxy esterified by an organic carboxylic
acid, such as a lower alkanoic acid, or a mineral acid, such as a
hydrohalic acid, for example lower alkanoyloxy or especially
halogen, such as iodine, bromine or especially fluorine or
chlorine.
[0035] Alkylated amino is, for example, lower alkylamino, such as
methylamino, or di-lower alkylamino, such as dimethylamino.
Acylated amino is, for example, lower alkanoylamino or
benzoylamino.
[0036] Esterified carboxy is, for example, lower alkoxycarbonyl,
such as methoxycarbonyl.
[0037] A substituted phenyl radical may carry up to 5 substituents,
such as fluorine, but especially in the case of relatively large
substituents is generally substituted by only from 1 to 3
substituents. Examples of substituted phenyl that may be given
special mention are 4-chloro-phenyl, pentafluoro-phenyl,
2-carboxy-phenyl, 2-methoxy-phenyl, 4-fluoro-phenyl, 4-cyano-phenyl
and 4-methyl-phenyl.
[0038] Salt-forming groups in a compound of formula I are groups or
radicals having basic or acidic properties. Compounds having at
least one basic group or at least one basic radical, for example a
free amino group, a pyrazinyl radical or a pyridyl radical, may
form acid addition salts, for example with inorganic acids, such as
hydrochloric acid, sulfuric acid or a phosphoric acid, or with
suitable organic carboxylic or sulfonic acids, for example
aliphatic mono- or di-carboxylic acids, such as trifluoroacetic
acid, acetic acid, propionic acid, glycolic acid, succinic acid,
maleic acid, fumaric acid, hydroxymaleic acid, malic acid, tartaric
acid, citric acid or oxalic acid, or amino acids such as arginine
or lysine, aromatic carboxylic acids, such as benzoic acid,
2-phenoxy-benzoic acid, 2-acetoxy-benzoic acid, salicylic acid,
4-aminosalicylic acid, aromatic-aliphatic carboxylic acids, such as
mandelic acid or cinnamic acid, heteroaromatic carboxylic acids,
such as nicotinic acid or isonicotinic acid, aliphatic sulfonic
acids, such as methane-, ethane- or 2-hydroxyethane-sulfonic acid,
or aromatic sulfonic acids, for example benzene-, p-toluene- or
naphthalene-2-sulfonic acid. When several basic groups are present
mono- or poly-acid addition salts may be formed.
[0039] COMPOUNDS of formula I having acidic groups, for example a
free carboxy group in the radical R.sub.10, may form metal or
ammonium salts, such as alkali metal or alkaline earth metal salts,
for example sodium, potassium, magnesium or calcium salts, or
ammonium salts with ammonia or suitable organic amines, such as
tertiary monoamines, for example triethylamine or
tri-(2-hydroxyethyl)-amine, or heterocyclic bases, for example
N-ethyl-piperidine or N,N'-dimethyl-piperazine.
[0040] COMPOUNDS of formula I having both acidic and basic groups
can form internal salts.
[0041] Preference is given to COMPOUNDS of formula I wherein one or
two of the radicals R.sub.4, R.sub.5, R.sub.6, R.sub.7 and R.sub.8
are each nitro or a radical of formula II wherein
[0042] R.sub.9 is hydrogen or lower alkyl,
[0043] X is oxo, thio, imino, N-lower alkyl-imino, hydroximino or
O-lower alkyl-hydroximino,
[0044] Y is oxygen or the group NH,
[0045] n is 0 or 1 and
[0046] R.sub.10 is an aliphatic radical having at least 5 carbon
atoms or an aromatic, aromatic-aliphatic, cycloaliphatic,
cycloaliphatic-aliphatic- , heterocyclic or heterocyclic-aliphatic
radical,
[0047] and the remaining radicals R.sub.4, R.sub.5, R.sub.6,
R.sub.7 and R.sub.8 are each independently of the others hydrogen,
lower alkyl that is unsubstituted or substituted by free or
alkylated amino, piperazinyl, piperidinyl, pyrrolidinyl or by
morpholinyl, or lower alkanoyl, trifluoromethyl, free, etherified
or esterifed hydroxy, free, alkylated or acylated amino or free or
esterified carboxy,
[0048] and the remaining substituents are as defined above.
[0049] Preference is given especially to COMPOUNDS of formula I
wherein
[0050] R.sub.1 is pyridyl or N-oxido-pyridyl each of which is
bonded at a carbon atom,
[0051] R.sub.2 and R.sub.3 are each hydrogen,
[0052] R.sub.4 is hydrogen or lower alkyl,
[0053] R.sub.5 is hydrogen, lower alkyl or trifluoromethyl,
[0054] R.sub.6 is hydrogen,
[0055] R.sub.7 is nitro, fluoro-substituted lower alkoxy or a
radical of formula II wherein
[0056] R.sub.9 is hydrogen,
[0057] X is oxo,
[0058] n is 0 and
[0059] R.sub.10 is pyridyl bonded at a carbon atom, phenyl that is
unsubstituted or substituted by halogen, cyano, lower alkoxy,
carboxy, lower alkyl or by 4-methyl-piperazinyl-methyl, or
C.sub.5-C.sub.7alkyl, thienyl, 2-naphthyl or cyclohexyl, and
[0060] R.sub.8 is hydrogen.
[0061] Special preference is given to COMPOUNDS of formula I
wherein at least one of the radicals R.sub.4 and R.sub.8 is lower
alkyl, and the remaining substituents are as defined above.
[0062] Preference is given above all to COMPOUNDS of formula I
wherein
[0063] R.sub.1 is pyridyl bonded at a carbon atom,
[0064] R.sub.2, R.sub.3, R.sub.5, R.sub.6 and R.sub.8 are each
hydrogen,
[0065] R.sub.4 is lower alkyl,
[0066] R.sub.7 a radical of formula II wherein
[0067] R.sub.9 is hydrogen,
[0068] X is oxo,
[0069] n is 0 and
[0070] R.sub.10 is 4-methyl-piperazinyl-methyl.
[0071] Preference is given above all especially to the COMPOUND of
formula I which is CGP 57148B
{N-{5-[4-(4-methyl-piperazino-methyl)-benzoylamido]-
-2-methylphenyl}-4-(3-pyridyl)-2-pyrimidine-amine} or a
pharmaceutically acceptable salt thereof.
N-{5-[4-(4-methyl-piperazino-methyl)-benzoylamid-
o]-2-methylphenyl}-4-(3-pyridyl)-2-pyrimidine-amine (hereinafter:
"COMPOUND I" also known as "imatinib" [International
Non-proprietary Name]) and the use thereof, are described in
Example 21 of European patent application EP-A-0 564 409, which was
published on 6 Oct. 1993, and in equivalent applications and
patents in numerous other countries, e.g. in U.S. Pat. No.
5,521,184 and in Japanese patent 2706682. Another preference is
given to the .beta.-crystal form of 4-(4-methylpiperazin-1--
ylmethyl)-N-[4-4-methyl-3-(4-pyridin-3-yl)pyrimidin-2-ylamino)phenyl]-benz-
amide monomethanesulfonate as described in the European patent
application No. 998 473 published on May 10, 2000.
N-{5-[4-(4-methyl-piperazino-methy-
l)-benzoylamido]-2-methylphenyl}-4-(3-pyridyl)-2-pyrimidine-amino
amine or a pharmaceutically acceptable salt will be referred herein
as COMPOUND l; the .beta.-crystal form of the mesylate salt of
COMPOUND I will be referred as COMPOUND I mesylate.
[0072] The COMPOUNDS of formula I are generically and specifically
disclosed in the patent applications EP 0 564 409 A1 and WO
99/03854, in particular in the compound claims and the final
products of the working examples, the subject-matter of the final
products, the pharmaceutical preparations and the claims are hereby
incorporated into the present application by reference to these
publications. Comprised are likewise the corresponding
stereoisomers as well as the corresponding polymorphs, e.g. crystal
modifications, which are disclosed therein. In EP 0 564 409 A1 the
COMPOUNDS of formula I are described as PDGF receptor tyrosine
kinase inhibitors useful for the treating cancer, thrombosis,
psoriasis, fibrosis, dermatosclerosis and atherosclerosis.
[0073] Pharmaceutically acceptable salts of COMPOUNDS of formula I
are pharmaceutically acceptable acid addition salts, like for
example with inorganic acids, such as hydrochloric acid, sulfuric
acid or a phosphoric acid, or with suitable organic carboxylic or
sulfonic acids, for example aliphatic mono- or di-carboxylic acids,
such as trifluoroacetic acid, acetic acid, propionic acid, glycolic
acid, succinic acid, maleic acid, fumaric acid, hydroxymaleic acid,
malic acid, tartaric acid, citric acid or oxalic acid, or amino
acids such as arginine or lysine, aromatic carboxylic acids, such
as benzoic acid, 2-phenoxy-benzoic acid, 2-acetoxy-benzoic acid,
salicylic acid, 4-aminosalicylic acid, aromatic-aliphatic
carboxylic acids, such as mandelic acid or cinnamic acid,
heteroaromatic carboxylic acids, such as nicotinic acid or
isonicotinic acid, aliphatic sulfonic acids, such as methane-,
ethane- or 2-hydroxyethane-sulfonic acid, or aromatic sulfonic
acids, for example benzene-, p-toluene- or naphthalene-2-sulfonic
acid.
[0074] The monomethanesulfonic acid addition salt of COMPOUND I
(hereinafter "COMPOUND I mesylate") and crystal forms thereof, e.g.
a preferred beta (.beta.) crystal form thereof are described in PCT
patent application WO99/03854 published on Jan. 28, 1999.
[0075] Very preferably a N-phenyl-2-pyrimidine-amine derivative of
formula I is used in the form of its monomesylate salt.
[0076] For the purposes of isolation or purification, as well as in
the case of compounds that are used further as intermediates, it is
also possible to use pharmaceutically unacceptable salts. Only
pharmaceutically acceptable, non-toxic salts are used for
therapeutic purposes and those salts are therefore preferred.
[0077] Examples of suitable PDGF receptor tyrosine kinase
inhibitors are COMPOUND I, RPR 101511A, SU 102, AG1296, AG1295,
AG17, CT52923, RP-1776, KY11784, GFB-111,
pyrrolo[3,4-c]-beta-carboline-diones, etc.
[0078] CT52923
(4-(6,7-dimethoxy-4-quinazolinyl)-N-(3,4-methylenedioxybenz-
yl)-1-piperazinethiocarboxamide) was described by Matsuno K, et al.
(Synthesis and structure activity relationships of PDGF receptor
phosphorylation inhibitor-1., in 18th Symposium on Medicinal
Chemistry; Nov. 25-27, 1998; Kyoto, Japan. The Pharmaceutical
Society of Japan, Division of Medicinal Chemistry, Tokyo, Japan.
Abstract 2-P-05).
[0079] KY11784 and RP-1776 novel cyclic peptides, were described by
Toki S, Agatsuma T, et al (J Antibiot (Tokyo) May
2001;54(5):405-14).
[0080] GFB-111 was described by Blaskovich M A, et al. (Nat
Biotechnol October 2000;18(10):1065-70).
[0081] AG1296 and AG1295 were described by Kovalenko M, et al.
(Cancer Res Dec. 1, 1994;54(23):6106-14).
[0082] RPR 101511A was described by Bilder G, et al. (Circulation
Jun. 29, 1999;99(25):3292-9)
[0083] Pyrrolo[3,4-c]-beta-carboline-diones was described by Teller
S, (Eur J Med Chem April 2000;35(4):413-27).
[0084] AG17 (3,5-di-tert-butyl-4-hydroxybenzylidene-malononitrile
or NSC 242557) was described by Gazit A et al.(Synthesis and
biological activity of protein tyrosine kinase inhibitors. J Med
Chem 1989;32:2344).
[0085] The present invention particularly concerns the use of PDGF
receptor tyrosine kinase or bcr-abl tyrosine kinase inhibitors
selected from COMPOUNDS of formula I, for the manufacture of a
medicament for treating tuberous sclerosis associated
neoplasms.
[0086] The present invention particularly concerns the use of PDGF
receptor tyrosine kinase or bcr-abl tyrosine kinase inhibitors
selected from COMPOUNDS of formula I, for the manufacture of a
medicament for the prevention of neoplasms in patients suffering
from tuberous sclerosis.
[0087] The present invention particularly concerns the use of
COMPOUND I, which is a PDGF receptor tyrosine kinase inhibitor and
a bcr-abl tyrosine kinase inhibitor.
[0088] By the term "tuberous sclerosis" the applicant means an
autosomal dominant disorder characterized by the development of
hamartomas and focal dysplasias in multiple organs including the
brain (i.e. cortical and subcortical tubers, subependymal nodules,
and giant cell astrocytomas, cerebral atrophy, cerebral infarct,
cerebral aneurysm, arachnoid cyst), kidney (i.e. angiomyolipomas,
cysts, carcinomas), skin (i.e. hypomelanotic macules, shagreen
patches, facial angiofibromas, periungual fibromas), eye (i.e.
retinal hamartomas), heart (i.e. rhabdomyomas), lungs (i.e.
lymphangioleiomyomatosis), musculoskeletal manifestations (i.e.
Sclerotic lesions, bone lucencies) and, to a lesser extent, other
organs.
[0089] By the term "tuberous sclerosis associated neoplasms" the
applicant means a tuberous sclerosis associated with abnormal
tissues that grow by cellular proliferation more rapidly than
normal and continue to grow after the stimuli that initiated the
new growth cease. Neoplasms show partial or complete lack of
structural organisation and functional coordination with the normal
tissue, and usually form a distinct mass of tissue that may be
either benign (benign tumor) or malignant (cancer). Preferred
examples of tuberous sclerosis associated neoplasms are
angiomyolipomas, e.g. renal angiomyolipomas, rhabdomyomas,
lymphangioleiomyomatosis, subependymal giant cell astrocytomas or
cutaneous neoplasms including fibrous plaques on the forehead and
scalp, angiofibromas, e.g. facial angiofribromas, and periungual
fibromas.
[0090] The term "treatment" as used herein means curative treatment
and prophylactic treatment.
[0091] The term "curative" as used herein means efficacy in
treating ongoing episodes of tuberous sclerosis associated
neoplasms.
[0092] The term "prophylactic" means the prevention of the onset or
recurrence of tuberous sclerosis associated neoplasms in patients
suffering from tuberous sclerosis.
[0093] In accordance with the particular findings of the invention,
the present invention also provides a method of treatment of a
subject or patient that is a warm-blooded animal, including
particularly a human, in which a therapeutically effective dose of
a PDGF receptor tyrosine kinase or bcr-abl tyrosine kinase
inhibitor or a pharmaceutically acceptable salt thereof is
administered to such a warm-blooded animal, preferably a human,
suffering from tuberous sclerosis associated neoplasms.
[0094] The invention relates also to a method for prophylactic
treatment of neoplasms or prevention of neoplasms in a subject or
patient that is a warm-blooded animal, including particularly a
human, in which a therapeutically effective dose of a PDGF receptor
tyrosine kinase or bcr-abl tyrosine kinase inhibitor or a
pharmaceutically acceptable salt thereof is administered to such a
warm-blooded animal, preferably a human, suffering from tuberous
sclerosis.
[0095] Depending on species, age, individual condition, mode of
administration, and the clinical picture in question, effective
doses of COMPOUND I, e.g. COMPOUND I mesylate, for example daily
doses of about 100-1000 mg, preferably 200-800 mg, e.g. 200 to 600
mg, or 400 mg, are administered to warm-blooded animals of about 70
kg bodyweight. For adult patients with unresectable tuberous
sclerosis neoplasms, a starting daily dose corresponding to 400 mg
of COMPOUND I free base can be used. For patients with an
inadequate response after an assessment of response to therapy with
400 mg daily, dose escalation can be safely considered and patients
may be treated as long as they benefit from treatment and in the
absence of limiting toxicities.
[0096] Preferably COMPOUND I, e.g. COMPOUND I mesylate, is
administered once daily for a period exceeding 3 months. The
invention relates especially to such methods wherein a daily dose
mesylate is administered corresponding to 100 to 1000 mg,
especially 200-800 mg, preferably 200-600 mg or 400 mg of COMPOUND
I free base.
[0097] It can be shown by established test models and especially
those test model described herein that the PDGF receptor tyrosine
kinase inhibitors, preferably COMPOUNDS of formula I, e.g. COMPOUND
I, or a pharmaceutically acceptable salt thereof, e.g. COMPOUND I
mesylate, result in a more effective prevention of neoplasms and in
an effective treatment of tuberous sclerosis associated neoplasms.
The person skilled in the pertinent art is fully enabled to select
a relevant test model to prove the hereinbefore and hereinafter
indicated therapeutic indications and beneficial effects. The
pharmacological activity may, for example, be demonstrated in a
clinical study or in the test procedure as essentially described
hereinafter.
[0098] Tumors often exhibit activation of specific tyrosine
kinases, which may allow targeting of therapy through inhibition of
tyrosine kinase signaling. It was hypothesized that tuberous
sclerosis neoplasms may also show activation of a specific tyrosine
kinase receptor, explaining in part the benign tissue specific
neoplasms observed in tuberous sclerosis. Tuberous sclerosis
associated cell lines were submitted to a battery of small
molecular weight tyrosine kinase inhibitors. Tuberous sclerosis
(TSC) model cells derived from tuberin heterozygous mice and from a
human renal angiomyolipoma are highly sensitive to PDGF receptor
antagonists and these cells express PDGFR.beta.. Expression of
phosphorylated (active) PDGFR.beta. in vivo was detected using
phospho-specific antibodies, as well as high level expression of
the PDGFR.beta. signaling partner grb7 in human angiomyolipomas.
Grb7 is highly expressed in angiomyolipomas, the most common tumor
seen in TSC, but rarely in renal cell carcinoma. SV7tert human
angiomyolipoma cells are particularly sensitive to COMPOUND I, and
that COMPOUND I down regulates signaling molecules in human
angiomyolipoma, including phosphorylation of Akt. In particular,
the sensitivity to COMPOUND I occurred in a dose-dependent fashion.
In addition, COMPOUND I inhibits PDGFR.beta. and bcr-abl tyrosine
kinases. Thus, our results suggest that COMPOUND I might be useful
in the treatment of neoplasms commonly seen in patients with
TSC.
[0099] The following Examples illustrate the invention described
above, but are not, however, intended to limit the scope of the
invention in any way.
Methods
Derivation of Cell Lines
[0100] SV7tert (ATCC CRL 2461) is a cell line derived from a human
angiomyolipoma through the sequential introduction of SV40 large T
antigen and telomerase into primary human angiomyolipoma cells
(Arbiser, J. L. et al. The generation and characterization of a
cell line derived from a sporadic renal angiomyolipoma: use of
telomerase to obtain stable populations of cells from benign
neoplasms. Am.J.Pathol. 159, 483-491 (2001)). Angiomyolipomas are
benign tumors of the kidney derived from putative perivascular
epithelioid cells, that may undergo differentiation into cells with
features of melanocytes, smooth muscle, and fat. Tsc2ang1 (ATCC CRL
2620) is a murine cell line derived from a cutaneous sarcoma
arising in the extremity of a mouse heterozygous for tsc2. The
sarcoma tissue was digested with collagenase and processed as
described for SV7tert cells (Arbiser, J. L. et al.). Mice
heterozygous for tsc2 develop cutaneous sarcomas at a frequency of
approximately 10-15% (Onda, H., Lueck, A., Marks, P. W., Warren, H.
B., & Kwiatkowski, D. J. Tsc2(.+-.) mice develop tumors in
multiple sites that express gelsolin and are influenced by genetic
background. J.Clin Invest 104, 687-695 (1999)).
Tyrosine Kinase Inhibitor Studies
[0101] The following tyrosine kinase inhibitors were reconstituted
as stock solutions in dimethyl sulfoxide (DMSO) immediately prior
to use; AG9, AG17, AG18, AG30, AG82, AG99, AG112, AG370, AG490,
AG879, AG957, AG1295, AG1296, AG1433, 2'thioadenosine, ST638,
COMPOUND I, lavendustin C, oxindole 1, JAK3 inhibitors 1,2, and 3,
as well as JAK3 inhibitor negative control. They can be obtained
from Calbiochem (San Diego, Calif.). 10,000 cells per well in a 24
well dish were plated on day 1, and were treated with inhibitors in
doses ranging from 0-2 .mu.g/ml (LaMontagne Jr K R Jr et al.
Inhibition of MAP kinase kinase causes morphological reversion and
dissociation between soft agar growth and in vivo tumorigenesis in
angiosarcoma cells; Am.J.Pathol. 157,1937-1945 (2000)). Cells were
counted 72 hours after treatment with inhibitors using a Coulter
Counter (Coulter, Hialeah,Fla.).
[0102] Demonstration of PDGFR.beta. signal transduction in SV7tert
and tsc2ang1 cells Subconfluent cells in 6-hole wells were
serum-starved over night and stimulated for 8 min with 50 ng/ml
PDGF-BB (Peprotech EC, Ltd). The cells were lysed and used for
immunoprecipitation with anti-PDGFR.beta. antibodies (Santa Cruz
Biotechnologies, Santa Cruz, Calif.). Immunoprecipitates were
immobilized on protein A-Sepharose beads which were washed and
boiled in SDS-sample buffer. The eluted material was separated on
10% SDS-polyacrylamide gels, transferred to filters and
immunoblotted using anti-phosphotyrosine antibodies (4G10,
Transduction Laboratories) or anti-PDGFR.beta. antibodies.
Immunoreactive proteins were detected by enhanced chemoluminescence
(ECL; Amersham Pharmacia Biotech). Western blot analysis of SV7tert
and tsc2ang1 cells was also performed with a grb7 polyclonal Ab
(Santa Cruz Biotechnologies, Santa Cruz, Calif.). Western blot
analysis of SV7tert and tsc2ang1 cells was also performed with a
grb7 polyclonal Ab (Santa Curx Biothechnologies, Santa Cruz,Calif.)
and a polyclonal phosphoPLCgamma1 Ab (Biosource International,
Camarillo, Calif.), following the instructions from the
manufacturers.
Immunohistochemistry of Human Angiomyolipomas for Phosphorylated
PDGFR.beta. and grb7
[0103] Sections of formalin-fixed, paraffin-embedded tissue (5
.mu.m) were tested for the presence of immunohistochemically
detectable antigen (phosphorylated PDGFR.beta.) with steam
heat-induced antigen retrieval, polyclonal anti-phosphorylated
PDGFR.beta. specific for phosphotyrosine 857 residue of PDGFR.beta.
(Bernard, A. & Kazlauskas, A. Phosphospecific antibodies reveal
temporal regulation of platelet-derived growth factor beta receptor
(PCGFR.beta.) signaling. Exp. Cell Res. 253, 704-712 (1999))
(1/600), an avidin-biotinylated enzyme complex kit (LSAB, DAKO,
Carpinteria, Calif.), and DAKO Autostainer. Sections were
deparaffinized and rehydrated, then steamed in citrate buffer
(pH=6) for 20 min and cooled for 5 min before immunostaining. All
tissues were then exposed to 3% hydrogen peroxide for 5 min,
primary antibody for 25 min, avidin-biotinylated enzyme complex for
25 min, diaminobenzidine as chromogen for 5 min and hematoxylin as
counterstain for 1 min. These incubations were performed at room
temperature; between incubations, sections were washed with buffer.
For the two negative controls, primary antibody was replaced by
buffer. Five-micron thick paraffin embedded tissue sections were
deparaffinized and rehydrated. Antigen retrieval was performed in
citrate buffer (pH6) using an electric pressure cooker at
120.degree. C. for 5 min. The tissue was then exposed to 3%
hydrogen peroxide for 5 min to quench endogenous tissue peroxidase.
The tissue sections were incubated for 25 min with rabbit
polyclonal anti-GRB 7 (1:40 dilution, Santa Cruz Biotechnology,
Santa Cruz, Calif., USA) for 25 min. After washing unbound primary
antibody, sections were treated with commercial biotinylated
secondary anti-immunoglobulin followed by avidin coupled to
biotinylated horseradish peroxidase, according to manufacturer's
instructions (LSAB2 kit for mouse and rabbit primary antibodies,
DAKO Corporation). Diaminobenzidine was used as the chromogenic
peroxidase substrate for 5 min, and sections were counterstained
with hematoxylin for 1 min after immunohistochemistry. These
incubations were performed using automated immunostainer (DAKO,
Carpinteria, Calif.). Specificity of the procedure was verified by
negative control reactions with primary antibody replaced with
buffer. Ten angiomyolipomas and ten renal cell carcinomas were
examined.
Results
[0104] In order to determine whether human angiomyolipoma and
tumors arising in mice heterozygous for tsc2 showed
hypersensitivity to tyrosine kinase inhibitors, relevant human and
mouse cell lines were exposed to a battery of tyrosine kinase
inhibitors. These are tyrosine inhibitors which inhibit specific
kinases, including JAK2 kinase, JAK3 kinase, platelet derived
growth factor (PDGFR), epidermal growth factor receptor (EGFR),
ERK1/2 inhibitor, p140 c-trk, p210 bcr-abl, p60 c-src, ErbB2, and
vascular endothelial growth factor receptor 2 (VEGFR2/flk-1). Human
SV7tert cells showed highest sensitivity to AG17 (Table 1), an
inhibitor of PDGFR tyrosine kinase, and AG957, an inhibitor of p210
bcr-abl. Murine tsc2ang1 cells demonstrated hypersensitivity to
AG17. Inhibitory compound AG17 is a substituted aromatic
malononitriles. AG957 caused approximately 90% inhibition at 0.5
.mu.g/ml (data not shown). AG1478 (Levitzki & Gazit. 1995.
Science 267: 1782-1788; European Patent Application 0520722) and
ST638 (Exp Cell Res August 1989;183(2):335-42) are selective
epidermal growth factor (EGF) receptor tyrosine kinase inhibitors
and AG30 is a c-ErbB tyrosine kinase inhibitor. The c-ErbB tyrosine
kinase receptor specific tyrphostin AG30 specifically blocked STAT5
activation (Cell Growth Differ May 1997;8(5):481-93).
1TABLE 1 Inhibition of proliferation of SV7tert and tsc2ang1 cells
by tyrosine kinase inhibitors (CC: concentration, N number of
living cells after 72 h treatment). Human SV7tert cells Murine
tsc2ang1 cells Compound DMSO AG17 ST638 AG30 AG1478 DMSO AG17 CC in
.mu.g/ml 10 20 10 20 10 20 10 20 10 20 10 20 10 20 N(X100) 252 248
23 22 250 205 250 270 251 210 185 185 23 23
[0105] AG17, a PDGFR kinase inhibitor, strongly inhibited both
murine and human cell lines. In addition, COMPOUND I inhibits
PDGFRP tyrosine kinase in addition to bcr-abl and c-kit tyrosine
kinase. The expression of functional PDGFRP on both human SV7tert
cells as well as on the murine tsc2ang1 cells was demonstrated by
immunoblotting. Cells were treated with PDGF-BB or vehicle, and
PDGFRP was immunoprecipitated, followed by immunoblotting with
anti-phosphotyrosine antibodies. We found that the expression level
of PDGFRP was slightly higher in tsc2ang1 cells compared with the
SV7tert cells. In both cases, PDGF-BB treatment resulted in
increased levels of phosphotyrosine-containing PDGFR.beta..
Furthermore, PDGF-BB treatment of tsc2ang1 cells resulted in
activation of phospholipase C gamma (PLC.gamma.), as an increase in
phosphorylated PLC.gamma. was noted after stimulation with
PDGF-BB.
[0106] In order to determine whether our findings of PDGFR.beta.
activation was truly associated with human angiomyolipomas in vivo,
we stained authentic human angiomyolipoma tissue with an antibody
specific for phosphorylated PDGFR.beta.. We observed strong
staining for activated PDGFR.beta. in the tumor sections,
indicating that PDGF mediated signal transduction events occur in
human angiomyolipomas in vivo. In addition, we examined human
angiomyolipomas for the presence of grb7, a signaling partner of
SV7tert, and found it to be expressed in 10/10 human
angiomyolipomas, but in only {fraction (1/10)} renal cell
carcinomas. COMPOUND I was tested for its ability to inhibit the
growth of SV7tert and tsc2ang1 in vitro. Human SV7tert
angiomyolipoma cells demonstrated increased sensitivity to COMPOUND
I compared to murine tsc2ang1, and this sensitivity occurred in a
dose-dependent fashion. Treatment of SV7tert cells with COMPOUND I
resulted in downregulation of Akt phosphorylation after 24 hours of
treatment, suggesting that COMPOUND I may inhibit PDGFRP
stimulation of P13 kinase-signaling (seeTable 2).
2 TABLE 2 Inhibition of SV7tert cells by COMPOUND I. concentration
of COMPOUND I Control 2 .mu.g/ml 5 .mu.g/ml 10 .mu.g/ml No. of
Cells after 72 hours 22450 16500 10000 6500
[0107] Tuberous sclerosis model cells (associated with neoplasms)
derived from tuberin heterozygous mice and from a human renal
angiomyolipoma are highly sensitive to PDGF receptor antagonists
and these cells express PDGFR.beta..
EXAMPLE 2
Capsules with
4-[(4-methyl-1-piperazin-1-ylmethyl)-N-[4-methyl-3-[[4-(3-py-
ridinyl)-2-pyrimidinyl]amino]phenyl]benzamide methanesulfonate, or
its .beta.-crystal form
[0108] Capsules containing 119.5 mg of the compound named in the
title (=COMPOUND I mesylate) corresponding to 100 mg of COMPOUND I
(free base) as active substance are prepared in the following
composition:
3 COMPOUND I mesylate 119.5 mg Cellulose MK GR 92 mg Crospovidone
XL 15 mg Aerosil 200 2 mg Magnesium stearate 1.5 mg 230 mg
[0109] The capsules are prepared by mixing the components and
filling the mixture into hard gelatin capsules, size 1.
EXAMPLE 3
Capsules with
4-[(4-methyl-1-piperazin-1-ylmethyl)-N-[4-methyl-3-[4-(3-pyr-
idinyl)-2-pyrimidinyl]amino]phenyl]benzamide methanesulfonate,
b-crystal form
[0110] Capsules containing 119.5 mg of SALT I corresponding to 100
mg of COMPOUND I (free base) as active substance are prepared in
the following composition:
4 Active substance 119.5 mg Avicel 200 mg PVPPXL 15 mg Aerosil 2 mg
Magnesium stearate 1.5 mg 338.0 mg
[0111] The capsules are prepared by mixing the components and
filling the mixture into hard gelatin capsules, size 1.
* * * * *