U.S. patent application number 13/357972 was filed with the patent office on 2012-06-21 for inhibitors of the mutant form of kit.
Invention is credited to Elisabeth Buchdunger, Doriano Fabbro.
Application Number | 20120157441 13/357972 |
Document ID | / |
Family ID | 34619508 |
Filed Date | 2012-06-21 |
United States Patent
Application |
20120157441 |
Kind Code |
A1 |
Buchdunger; Elisabeth ; et
al. |
June 21, 2012 |
INHIBITORS OF THE MUTANT FORM OF KIT
Abstract
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 form midostaurin, vatalanib and compound A is
administered. ##STR00001##
Inventors: |
Buchdunger; Elisabeth;
(Neuenburg, DE) ; Fabbro; Doriano; (Arlesheim,
CH) |
Family ID: |
34619508 |
Appl. No.: |
13/357972 |
Filed: |
January 25, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12700106 |
Feb 4, 2010 |
8124611 |
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13357972 |
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10579586 |
Jan 17, 2007 |
8017621 |
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PCT/EP2004/013045 |
Nov 17, 2004 |
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12700106 |
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60520714 |
Nov 18, 2003 |
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Current U.S.
Class: |
514/211.08 ;
514/248 |
Current CPC
Class: |
A61K 31/506 20130101;
G01N 2800/44 20130101; A61P 35/02 20180101; A61K 31/553 20130101;
A61P 3/04 20180101; A61P 35/00 20180101; A61K 31/502 20130101; A61P
43/00 20180101 |
Class at
Publication: |
514/211.08 ;
514/248 |
International
Class: |
A61K 31/553 20060101
A61K031/553; A61P 35/02 20060101 A61P035/02; A61P 35/00 20060101
A61P035/00; A61K 31/502 20060101 A61K031/502 |
Claims
1. A method of treating a KIT dependent disease in a patient, which
comprises: (a) identifying a mutant form of KIT associated with the
KIT dependent disease; and (b) administering to said patient an
effective mutant KIT inhibiting amount of an inhibitor selected
from the group consisting of midostaurin and vatalanib.
2. A method of claim 1, wherein the mutant form of KIT is selected
from 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 T6701.
3. A method of claim 2, wherein the mutant form of KIT is selected
from D816F, D816H, D816N, D816Y, D816V, K642E, Y823D, Del 550-558,
Del 557-561, N822K, V654A, N822H, Del 550-558+V654A, Del
557-561+V654A.
4. A method of claim 1, wherein the KIT dependent disease is
resistant to treatment with imatinib.
5. A method of claim 3, wherein the mutant form of KIT is D816F and
the inhibitor is selected from the group consisting of
midostaurin.
6. A method of claim 3, wherein the mutant form of KIT is D816H and
the inhibitor is selected from the group consisting of
midostaurin.
7. A method of claim 3, wherein the mutant form of KIT is D816N and
the inhibitor is selected from the group consisting of
midostaurin.
8. A method of claim 3, wherein the mutant form of KIT is D816Y and
the inhibitor is selected from the group consisting of
midostaurin.
9. A method of claim 3, wherein the mutant form of KIT is D816V and
the inhibitor is selected from the group consisting of
midostaurin.
10. A method of claim 3, wherein the mutant form of KIT is K642E
and the inhibitor is selected from the group consisting of
midostaurin and vatalanib.
11. A method of claim 3, wherein the mutant form of KIT is Y823D
and the inhibitor is selected from the group consisting of
midostaurin and vatalanib.
12. A method of claim 3, wherein the mutant form of KIT is Del
550-558 and the inhibitor is selected from the group consisting of
midostaurin and vatalanib.
13. A method of claim 3, wherein the mutant form of KIT is Del
557-561 and the inhibitor is selected from the group consisting of
midostaurin and vatalanib.
14. A method of claim 3, wherein the mutant form of KIT is N822K
and the inhibitor is selected from the group consisting of
midostaurin and vatalanib.
15. A method of claim 3, wherein the mutant form of KIT is V654A
and the inhibitor is selected from the group consisting of
midostaurin.
16. A method of claim 3, wherein the mutant form of KIT is N822H
and the inhibitor is selected from the group consisting of
midostaurin and vatalanib.
17. A method of claim 3, wherein the mutant form of KIT is Del
550-558 V654A and the inhibitor is selected from the group
consisting of midostaurin and vatalanib.
18. A method of claim 3, wherein the mutant form of KIT is Del
557-561+V654A and the inhibitor is selected from the group
consisting of midostaurin.
19. A method of claim 2, wherein the mutant form of KIT is selected
from the group consisting of D816H, D816F, D816N and D816Y and the
inhibitor is midostaurin.
20. A method of claim 2, wherein the mutant form of KIT is selected
from the group consisting of D816V, K642E, Y823D, De1550-558, Del
557-561, N822K, V654A, N822H, Del 550-558+V654A, and Del
557-561+V654A and the inhibitor is midostaurin.
21. A method of claim 2, wherein the mutant form of KIT is selected
from the group consisting of K642E, Y823D, Del 550-558, Del
557-561, N822K and N822H and the inhibitor is vatalanib.
22. A method according to claim 1, wherein the KIT dependent
disease is selected from mast cell diseases, acute myelogenous
leukemia, gastrointestinal stromal tumors, seminomas and
dysgerminomas.
23. A method of claim 4, wherein the inhibitor is midostaurin.
24. A method of claim 4, wherein the inhibitor is vatalanib.
Description
[0001] 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.
[0002] 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.
[0003] Imatinib, 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.
[0004] Thus, the present invention relates to a method of treating
a KIT dependent disease in a patient, which comprises [0005] (a)
identifying the mutant form of KIT associated with the KIT
dependent disease; and [0006] (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.
[0007] 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.
[0008] 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, T801I, C809G,
D820Y, N822K, N822H, Y823D, Y823C and T670I.
[0009] 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 IC.sub.50 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.
[0010] 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.
[0011] The KIT inhibitors utilized in accordance with the present
method include midostaurin, vatalanib and compound A. Midostaurin
(U.S. Pat. No. 5,093,330) and vatalanib (WO 98/35958) are known in
the art. Compound A is a compound of the formula
##STR00002##
[0012] And may be produced according to WO 04/005281.
[0013] Appropriate dosages of midostaurin, vatanalib and compound A
are determined by routine methods.
[0014] 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.
[0015] 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.
[0016] 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.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 illustrates mutant insertion points in the Bac-to-Bac
donor vector pFB-GST-01.
EXAMPLES
[0018] 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 H1 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 inserted into the Bac-to-Bac donor vector pFB-GST-01 using the
restriction enzymes BamH1-EcoR1 for each mutant shown in FIG. 1.
Automated sequencing confirmed the correct sequence to be present
for each mutant plasmid.
[0019] Bacmid 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 anti-GST antibodies for
immonudetection.
TABLE-US-00001 Vatalanib Compound A Kit Mutation IC.sub.50 (.mu.M)
(avg) IC.sub.50 (.mu.M) (avg) D816F >10 >10 D816H >10
>10 D816N >10 <10 D816Y >10 >10 D816V >10 >10
K642E <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 average N.degree. of IC50 .mu.M SEM
values HIS preparation HT-KIT-TA23 wt 1.7 0.15 2 HT-KIT TA23 -
D820G 0.084 0.05 2 HT-KIT TA23 - T670I 0.89 0.21 2 GST preparation
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-KIT 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 D816V 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: 1 .mu.M ATP, 5 .mu.g/ml Poly-EY, 10 min
incubation at ambient temperature
[0020] 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 cm.sup.2 round tissue culture plates were seeded
with 5.times.10.sup.7 cells/plate and infected with 1 mL of
virus-containing media (approximately 5 MOIs). After 3 days, the
cells were scraped off the plate and centrifuged at 500 rpm for 5
minutes. Cell pellets from 10-20, 100 cm.sup.2 plates, were
re-suspended in 50 mL of ice-cold lysis buffer (25 mM Tris-HCl, 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.
[0021] The centrifuged cell lysate was loaded onto a 2 mL
glutathione-sepharose column (Pharmacia) and washed 3.times. with
10 mL of 25 mM Tris-HCl, pH 7.5, 2 mM EDTA, 1 mM DTT, 200 mM NaCl.
The GST-tagged proteins were then eluted by 10 applications (1 mL
each) of 25 mM Tris-HCl, pH 7.5, 10 mM reduced-glutathione, 100 mM
NaCl, 1 mM DTT, 10% glycerol and stored at -70.degree. C.
[0022] The protein kinase activities of the various Kit mutants
200-500 ng were assayed in the presence or absence of inhibitors,
20 mM Tris-HCl, pH 7.6, 3 mM MnCl.sub.2, 3 mM MgCl.sub.2, 1 mM DTT,
10 .mu.M Na.sub.3VO.sub.4, 3 .mu.g/mL poly(Glu,Tyr) 4:1, 1% DMSO,
1.5 .mu.M ATP (.gamma.-[.sup.33P]-ATP 0.1 .mu.Ci). The assay (30
.mu.L) was carried out in 96-well plates at ambient temperature for
30 minutes and the reaction terminated by the addition of 20 .mu.L
of 125 mM EDTA. Subsequently, 30 .mu.l of the reaction mixture were
transferred onto Immobilon-PVDF membrane (Millipore, Bedford,
Mass., USA) previously soaked for 5 minutes with methanol, rinsed
with water, then soaked for 5 minutes with 0.5% H.sub.3PO.sub.4 and
mounted on vacuum manifold with disconnected vacuum source. After
spotting all samples, vacuum was connected and each well rinsed
with 200 .mu.L 0.5% H.sub.3PO.sub.4. Membranes were removed and
washed 4.times. on a shaker with 1.0% H.sub.3PO.sub.4, once with
ethanol. Membranes were counted after drying at ambient
temperature, mounting in Packard TopCount 96-well frame, and
addition of 10 .mu.L/well of Microscint (Packard). IC.sub.50 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 .mu.M). One unit of protein kinase
activity is defined as 1 nmole of .sup.33P transferred from
[.gamma..sup.33P]ATP to the substrate protein/minute/mg of protein
at RT.
* * * * *