INHIBITEURS DE LA FORME MUTANTE DU KIT

INHIBITORS OF THE MUTANT FORM OF KIT

Abrégé anglais

The present invention relates to the treatment of KIT dependent
diseases that are characterized by a mutant form of KIT whereby the mutant KIT
is
identified and an appropriate inhibitor of the mutant KIT selected from
midostaurin
and vatalanib is administered.

Revendications

CLAIMS:
1. Use of an inhibitor which is midostaurin or vatalanib for the
manufacture
of a medicament for treating a KIT dependent disease in a patent, wherein a
mutant
form of KIT is associated with the KIT dependent disease.
2. The use of claim 1, wherein the mutant form of KIT is D816F, D816H,
D816N, D816Y, D816V, K642E, Y823D, Del 550-558, Del 557-561, N822K, V654A,
N822H, Del 550-558 + V654A, Del 557-561 + V654A, lns503AY, V560G, 558NP,
Del 557-558, Del W559-560, F522C, Del 579, R634W, K642E, T801I, C809G,
D820Y, N822K, N822H, Y823D, Y823C or 1670I.
3. The use of claim 2, wherein the mutant form of KIT is D816F, D816H,
D816N, K642E, Y823D, Del 550-558, Del 557-561, N822K, V654A, N822H,
Del 550-558 + V654A or Del 557-561 + V654A.
4. The use of claim 1, wherein the KIT dependent disease is resistant to
treatment with imatinib.
5. The use according to claim 2, wherein the mutant form of KIT is D816V,
K642E, Y823D, Del 550-558, Del 557-561, N822K, V654A, N822H,
Del 550-558 + V654A or Del 557-561 + V654A and the inhibitor is midostaurin.
6. The use according to claim 2, wherein the mutant form of KIT is K642E,
Y823D, Del 550-558, Del 557-561, N822K or N822H and the inhibitor is
vatalanib.
7. The use according to any one of claims 1 to 6, wherein the KIT
dependent disease is a mast cell disease, acute myelogenous leukemia, a
gastrointestinal stromal tumor, a seminoma or a dysgerminoma.
8. The use according to claim 4, wherein the inhibitor is midostaurin.
9. The use according to claim 4, wherein the inhibitor is vatalanib.
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10. Use of an inhibitor which is midostaurin or vatalanib for treating a KIT
dependent disease in a patient, wherein a mutant form of KIT is associated
with the
KIT dependent disease.
11. The use of claim 10, wherein the mutant form of KIT is D816F, D816H,
D816N, D816Y, D816V, K642E, Y823D, Del 550-558, Del 557-561, N822K, V654A,
N822H, Del 550-558 + V654A, Del 557-561 + V654A, Ins503AY, V560G, 558NP,
Del 557-558, Del W559-560, F522C, Del 579, R634W, K642E, T8O1l, C809G,
D820Y, N822K, N822H, Y823D, Y823C or 1670I.
12. The use of claim 11, wherein the mutant form of KIT is D816F, D816H,
D816N, K642E, Y823D, Del 550-558, Del 557-561, N822K, V654A, N822H,
Del 550-558 + V654A or Del 557-561 + V654A.
13. The use of claim 10, wherein the KIT dependent disease is resistant to
treatment with imatinib.
14. The use according to claim 11, wherein the mutant form of KIT is
D816V, K642E, Y823D, Del 550-558, Del 557-561, N822K, V654A, N822H,
Del 550-558 + V654A or Del 557-561 + V654A and the inhibitor is midostaurin.
15. The use according to claim 11, wherein the mutant form of KIT is
K642E, Y823D, Del 550-558, Del 557-561, N822K or N822H and the inhibitor is
vatalanib.
16. The use according to any one of claims 10 to 15, wherein the KIT
dependent disease is a mast cell disease, acute myelogenous leukemia, a
gastrointestinal stromal tumor, a seminoma or a dysgerminoma.
17. The use according to claim 13, wherein the inhibitor is midostaurin.
18. The use according to claim 13, wherein the inhibitor is vatalanib.
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19. An inhibitor which is midostaurin or vatalanib for use in the treatment
of
a KIT dependent disease in a patient, wherein a mutant form of KIT is
associated with
the KIT dependent disease.
20. The inhibitor of claim 19, wherein the mutant form of KIT is D816F,
D816H, D816N, D816Y, D816V, K642E, Y823D, Del 550-558, Del 557-561, N822K,
V654A, N822H, Del 550-558 + V654A, Del 557-561 + V654A, Ins503AY, V560G,
558NP, Del 557-558, Del W559-560, F522C, Del 579, R634W, K642E, T8011,
C809G, D820Y, N822K, N822H, Y823D, Y823C or T670I.
21. The inhibitor of claim 20, wherein the mutant form of KIT is D816F,
D816H, D816N, K642E, Y823D, Del 550-558, Del 557-561, N822K, V654A, N822H,
Del 550-558 + V654A or Del 557-561 + V654A.
22. The inhibitor of claim 19, wherein the KIT dependent disease is
resistant to treatment with imatinib.
23. The inhibitor according to claim 20, wherein the mutant form of KIT is
D816V, K642E, Y823D, Del 550-558, Del 557-561, N822K, V654A, N822H,
Del 550-558 + V654A or Del 557-561 + V654A and the inhibitor is midostaurin.
24. The inhibitor according to claim 20, wherein the mutant form of KIT is
K642E, Y823D, Del 550-558, Del 557-561, N822K or N822H and the inhibitor is
vatalanib.
25. The inhibitor according to any one of claims 19 to 24, wherein the KIT
dependent disease is a mast cell disease, acute myelogenous leukemia, a
gastrointestinal stromal tumor, a seminoma or a dysgerminoma.
26. The inhibitor according to claim 22, which is midostaurin.
27. The inhibitor according to claim 22, which is vatalanib.
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Description

CA 02807475 2013-02-22
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inhibitors of the mutant form of KIT
This is a divisional application of Canadian Patent Application No. 2,546,189
filed on
November 17, 2004. It should be understood that the expression "present
invention", or the like,
encompasses the subject matters of both this divisional application and the
parent application.
The present invention relates to the treatment of KIT dependent diseases that
are
characterized by a mutant form of KIT whereby the mutant KIT Is identified and
an
appropriate inhibitor of the mutant KIT Is administered.
The c-kit gene encodes a receptor protein tyrosine kinase, which is herein
referred to
as KIT, but which is also known as mast/stem cell growth factor receptor. The
amino acid
sequence of KIT and the nucleotide sequence of the c-kit gene are known. See
Swiss
Prot.: P10721. Upon binding its ligand, stem cell factor, KIT forms a dimer
that is
autophosphorylated and activates signaling cascades that lead to cell growth.
Mutations
that lead to an activated form of KIT, especially forms that are activated
independently of its
ligand, are known and are believed to play a role in certain proliferative
diseases, such as
mast cell diseases, like mastocytosis, particularly systemic mastocytosis,
acute
myelogenous leukemia, gastrointestinal stromal tumors, sinonasal NK/T-cell
lymphoma,
seminomas and dysgerminomas.
lmatinib, which is marketed as its mesylate salt under the brandname GLIVEC or
GLEEVEC, is known to inhibit wild type KIT and certain KIT mutations e.g.
those in exons
commonly found in gastrointestinal stromal tumors (GIST). However, it is also
inactive or
significantly less active against certain other mutant forms of KIT, for
example the D816V
mutation commonly found in systemic mastocytosis. The present invention Is
based upon
research that correlates the treatment of a disease characterized by a mutant
form of KIT
with an appropriate alternative pharmaceutical therapy based on the
alternative's ability to
inhibit the mutant KIT.
Thus, the present invention relates to a method of treating a KIT dependent
disease
in a patient, which comprises
(a) identifying the mutant form of KIT associated with the KIT dependent
disease; =
and
(b) administering to the patient an effective mutant KIT-inhibiting 'amount of
an
inhibitor selected from the group consisting of midostaurin, vatalanib and
compound A. =
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KIT dependent diseases are generally proliferative diseases that are
characterized
by excessive KIT kinase activity due to an activating mutation in KIT. Such
activating
mutations are known in the art and are identified by techniques known in the
art.
KIT dependent diseases include diseases characterized by the following known
KIT
mutations: D816F, D816H, D816N, D816Y, D816V, K642E, Y823D, Del 550-558, Del
557-
561, N822K, V654A, N822H, Del 550-558 + V654A, Del 557-561 + V654A, Ins503AY,
V560G, 558NP, Del 557-558, Del W559-560, F522C, Del 579, R634W; K642E, T8011,
C809G, D820Y, N822K, N822H, Y823D, Y823C and T670I.
In an important embodiment of the present invention, the KIT dependent disease
is
resistant to treatment with imatinib. A KIT dependent disease that is
resistant to imatinib is
generally a KIT dependent disease as described above wherein imatinib,
administered at a
dose of 400-1000 mg/day, does not provide sufficient inhibition of the mutant
KIT to effect a
significant therapeutic benefit. Generally, mutant KIT that is resistant to
imatinib has an
in vitro IC50 of the mutant KIT greater than about 3 micromolar. Imatinib
resistant KIT
mutations include D816F, D816H, D816N, D816Y, D816V, T670I and mutant forms
that
include V654A.
The selection of a compound that inhibits the mutant form of KIT is based on
testing
the compound or a number of compounds for their ability to inhibit the mutant
KIT. Such
testing is carried out by standard inhibition assays that are known in the art
or within the skill
of the artisan.
The KIT inhibitors utilized in accordance with the present method include
midostaurin, vatalanib and compound A. Midostaurin (US5;093,330) and vatalanib
(WO
98/35958) are known in the art. Compound A is a compound of the formula
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Compound A
0
N NN 1110 N SF
And may be produced according to WO 04/005281.
Appropriate dosages of midostaurin, vatanalib and compound A are determined by
routine methods.
An appropriate dose of midostaurin is administered, e.g., once, twice or three
times a
day, for a total dose of 25 ¨ 300 preferably 50-300 more preferably 50 -100
most preferably
100-300 mg daily, e.g., two or three times a day, for a total dose of 150-250
mg, preferably
225 mg daily.
An appropriate daily dose of vatanalib is an amount In the range from 300-4000
mg,
e.g., in the range from 300-2000 mg/day or 300-1500 mg/day, in particular,
300, 500, 750,
1000, 1250, 1500 or 2000 mg/day, particularly 1250 mg/day.
The daily dose of compound A for a 70 kg/person is from approximately 0.05-5
g,
preferably from approximately 0.25-1.5 g.
EXAMPLES
The human KIT gene encoding aa 544-976 was cloned into the baculovirus donor
plasmid pFB-GST-01. This coding sequence was excised using restriction
endonucleases
Barn HI and EcoR1 and ligated to a Bac-to-Bac donor vector pFB-GEX-P1 with
compatible
ends. Subsequently the desired mutations were brought into the KIT gene by
methods know
to a person skilled in the art. Due to a frame shift within the original
plasmid that was used
to generate the mutant coding sequences, the mutated plasmid inserts were
excised and
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inserted into the Bac-to-Bac donor vector pFB-GST-01 using the restriction
enzymes
BamH1-EcoR1 for each mutant shown in Figure 1. Automated sequencing confirmed
the
correct sequence to be present for each mutant plasmid.
Bacrnid DNA was generated from 10 colonies each of DH10Bac cells transformed
with pFB-G01-KIT-mutant plasmid clones as described in materials and methods
and these
transfected into Sf9 cells. The transfected cells were pelleted and the
resultant recombinant
baculovirus present in the supernatant medium amplified. Western blotting was
applied to
the lysed cell pellets to confirm the expression of the GST-c-KIT fusion
protein by the viral
clones using anti-KIT and antl-GST antibodies for immonudetection.
=
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=
Vatalanib Compound A
Kit Mutation Wse (I1M) ICs, (01) (avg)
D816F >10 >10
D81611 >10 >10
D816N >10 <10
D816Y >10 >10
D816V >10 >10
K642B <1 <10
Y823D <1 <1
Del 550-558 <1 <2
Del 557-561 <1 <2
N822K <2 <10
V654A >10 >10 =
N822H <2 <10
Del 550-558 + V654A <10 <10
Del 557-561 + V654A >10 >10
Midostaurin
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average N of
HIS preparation IC50 pM SEM values
HT-KIT-TA23 wt 1.7 0.15 2
HT-KIT TA23 -D820G 0.084 0.05 2
HT-KIT TA23 -T6701 0.89 0.21 2
average N of
GST preparation IC50 pM SEM values
GST-KIT wt 1.8 0.26 10
GST-KIT Del 557-561 0.32 0.042 3
GST-KIT Del 550-558 0.53 0.057 3
GST-KIT Del 550-558+ V654A 0.27 0.079 5
GST-KIT Del 557-561 + V654A 0.34 0.11 5
GST-KIT V654A 0.46 0.16 5
GST-KIT K642E 0.64 0.036 4
GST-KIT R634W 0.33 0.13 2
GST-K1T T670I + Del 550-558 0.11 0.05 2
GST-KIT D816F 0.41 0.055 5
GST-KIT D816H 0.35 0.078 5
GST-KIT D816N 0.74 0.25 5
GST-KIT D816Y 0.29 0.11 9
GST-KIT 0816V 0.25 0.039 3
GST-KIT D816H + R634W 0.08 0.04 2
GST-KIT N822H 0.37 0.12 5
GST-KIT N822K 0.15 0.058 5
GST-KIT Y823D 0.13 0.0075 3
Assay conditions: I pm ATP, 5 pg / ml Poly-EY, 10 min incubation at ambient
temperature
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Virus containing media was collected from the transfected cell culture and
used for
infection to increase its titer. Virus containing media obtained after two
rounds of infection
was used for large-scale protein expression. For large-scale protein
expression 100 cm2
round tissue culture plates were seeded with 5 x 107 cells/plate and infected
with 1 mL of
virus-containing media (approximately 5 MOls). After 3 days, the cells were
scraped off the
plate and centrifuged at 500 rpm for 5 minutes. Cell pellets from 10-20, 100
cm2 plates,
were re-suspended in 50 mL of ice-cold lysis buffer (25 mM Tris-HCI, pH 7.5, 2
mM EDTA,
1% NP-40, 1 mM DTT, 1 mM PMSF). The cells were stirred on ice for 15 minutes
and then
centrifuged at 5000 rpm for 20 minutes.
The centrifuged cell lysate was loaded onto a 2 mL glutathione-sepharose
column
(Pharmacia) and washed 3 x with 10 mL of 25 mM Tris-HCI, pH 7.5, 2 mM EDTA, 1
mM
DTT, 200 mM NaCI. The GST-tagged proteins were then eluted by 10 applications
(1 mL
each) of 25 mM Tris-HCI, pH 7.5, 10 mM reduced-glutathione, 100 mM NaCI, 1 mM
DTT,
10% glycerol and stored at -70 C.
The protein kinase activities of the various Kit mutants 200-500 ng were
assayed in
the presence or absence of inhibitors, 20 mM Tris-HCI, pH 7.6, 3 mM MnCl2, 3
mM MgC12,
1 mM DTT, 10 pM Na3VO4, 3 pg/mL poly(Glu,Tyr) 4:1, 1% DMSO, 1.5 pM ATP (y-
13311-ATP
0.1 pCi). The assay (30 pL) was carried out in 96-well plates at ambient
temperature for
30 minutes and the reaction terminated by the addition of 20 pL of 125 mM
EDTA.
Subsequently, 30 pl of the reaction mixture were transferred onto Immobilon-
PVDF
membrane (Millipore, Bedford, MA, USA) previously soaked for 5 minutes with
methanol,
rinsed with water, then soaked for 5 minutes with 0.5% H3PO4 and mounted on
vacuum
manifold with disconnected vacuum source. After spotting all samples, vacuum
was
connected and each well rinsed with 200 pL 0.5% H3PO4. Membranes were removed
and
washed 4 x on a shaker with 1.0% H3PO4, once with ethanol. Membranes were
counted
after drying at ambient temperature, mounting in Packard TopCount 96-well
frame, and
addition of 10 pL/well of Microscint (Packard). IC50 values were calculated by
linear
regression analysis of the percentage inhibition of each compound in
duplicate, at
4 concentrations (usually 0.01, 0.1, 1 and 10 pM). One unit of protein kinase
activity is
defined as 1 nmole of 33P transferred from [733P]ATP to the substrate
protein/minute/mg of
protein at RT.
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