COMBINATION THERAPY FOR TREATING CANCER AND DIAGNOSTIC
ASSAYS FOR USE THEREIN
This application is a continuation-in-part of U.S. Serial No. 12/120,914, filed on
May 15, 2008, which is a divisional of U.S. Serial No. 11/432,937, filed on May 12,
2006, now U.S. Patent 7,390,799, which claims priority to U.S. Provisional Application
Ser. No. 60/718,618, September 20, 2005 and U.S. Provisional Application Ser. No
60/680,107, May 12, 2005.
FIELD
The present disclosure relates to a combination of therapeutic agents for use in
treating a patient a subject suffering from cancer. The combination comprises (1) at least
one polyploidy inducing agent; and (b) at least one Bcl-2 family protein inhibitor. In
addition, the present disclosure also relates to diagnostic assays useful in classification of
patients for treatment with a polyploidy inducing agent, a Bcl-2 family protein inhibitor
or a combination of a polyploidy inducing agent and a Bcl-2 family protein inhibitor. In
particular, the present disclosure relates to identifying the presence or absence of at least
one mutation in a BAX gene, a BAK gene or a NOXA gene and then classifying patients
as eligible for receiving treatment with a polyploidy inducing agent, a Bcl-2 family
protein inhibitor or a combination of a polyploidy inducing agent and a Bcl-2 family
protein inhibitor.
BACKGROUND
Anti-apoptotic Bcl-2 family protein members are associated with a number of
diseases and thus are under investigation as potential therapeutic drug targets. These
important targets for interventional therapy include, for example, the Bcl-2 family of
proteins Bcl-2, B C1-XL and Bcl-w. Recently inhibitors of Bcl-2 family members have
been reported in the literature, see, for example, WO 2005/049594, Oltersdorf, et. al,
Nature, 435:677-681 (2005), US 6,720,338 and US 7,030,115. While this art teaches
inhibitors having high binding to the target protein, this is only one of many parameters
that must be considered as a compound is investigated for further or continued drug
development. As part of this development, it is highly desirable to produce compounds
that are efficacious in animal models of cancer after oral administration. To achieve this
oral efficacy, it is well known in the art that a compound must not only display potent
activity against a tumor type or cell line under investigation, but must also achieve
acceptable levels of systemic exposure after oral administration. A typical measure of a
compound's cellular activity is the concentration eliciting 50% cellular effect (EC50). A
typical measure of systemic exposure is the area under the curve resulting from graphing
the plasma compound concentration after oral administration vs. time (AUC). The ratio
between these parameters (AUC/EC50) is well known in the art to constitute a useful
pharmacodynamic parameter to predict oral efficacy.
In one aspect, this disclosure is directed to a series of haloalkylsulfonylaryl
analogs that demonstrate enhanced and unexpected properties with respect to cellular
efficacy and systemic exposure after oral administration in animals. Specifically,
compounds of this disclosure maintain potent cellular efficacy while exhibiting suitable
systemic exposure after oral administration to animals. This results in AUC/EC50 ratios
significantly higher than that of the compounds taught in the art. Other aspects of the
disclosure are disclosed and apparent herein.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows comparative antitumorigenesis of EXAMPLE 1, etoposide and
combinations thereof on B-cell lymphoma.
FIG. 2 shows comparative antitumorigenesis of EXAMPLE 1, vincristine and
combinations thereof on B-cell lymphoma.
FIG. 3 shows comparative antitumorigenesis of EXAMPLE 1, CHOP and
combinations thereof on B-cell lymphoma.
FIG. 4 shows comparative antitumorigenesis of EXAMPLE 1, rituximab and
combinations thereof on B-cell lymphoma.
FIG. 5 shows comparative antitumorigenesis of EXAMPLE 1, rapamycin and
combinations thereof on B-cell lymphoma.
FIG. 6 shows comparative antitumorigenesis of EXAMPLE 1, R-CHOP and
combinations thereof on mantle cell lymphoma.
FIG. 7 shows comparative antitumorigenesis of EXAMPLE 1, bortezomib and
combinations thereof on mantle cell lymphoma.
FIG. 8 shows that the BH3 mimetic ABT-263 and the pan-Aurora inhibitor VX-
680 are synergistically lethal. FIG. 8A shows that VX-680 was screened at a single, fixed
dose ( 1 mM) in combination with 19 other chemotherapeutics in 6-pt dose-response in a
panel of 7 cell lines derived from various solid malignancies. Cell viability was
determined 3 days after exposure to the combination regimens. Synergy or antagonism
of drug combinations was determined using the Bliss additivism model of drug-drug
interactions (See, Berenbaum, M.C., Cancer Res 35, 269-335 (1981)). The value in
excess of Bliss additivism for each dose was determined for all combinations and defined
as follows: >15 = synergistic, 0-15 = no interaction, <-15 = antagonistic. These values
are expressed in a heatmap using SPOTFIRE® (TIBCO®) data analysis software where
synergy is indicated in red, no interaction in gray, and antagonism in blue. Each dose
response included a point for VX-680 alone (0 mM combination compound). These
values are not applicable for Bliss analysis and were excluded from the heatmap.
Compounds tested in combination are shown at the right. The targets/mechanisms of
action of each of these agents and the corresponding dose responses for the combination
screen is summarized in Table A in EXAMPLE 40. FIG. 8B shows Bliss analysis of cell
viability determinations for VX-680 in combination with ABT-263 in dose-response in
D54MG, HCT1 16, SW620, A549, PC3, and EJ1 cells. FIG. 8C shows cell viability
determinations analyzed in FIG. 8B.
FIG. 9 shows that inhibition of Aurora B and B C1-X Lis sufficient for synergistic
cytotoxicity. FIG. 9A shows HCT1 16 colon carcinoma cells were either not transfected
or transfected with the luciferase, Aurora A, or Aurora B siR As respectively. 24 hours
post-transfection, cells were treated with ABT-263 in dose response, and cell viability
was determined 72 hours after the addition of ABT-263. FIG. 8B shows immunoblots of
HCT1 16 lysates demonstrated knockdown of target proteins 72 hours post-transfection
with siRNAs used in FIG. 9A. MLN8054 (FIG. 9C) or AZD1 152 (FIG. 9D) were tested
in combination with DMSO or 1 mM ABT-263 in HCT1 16 cells. Cell viability was
determined 72 hours after treatment. FIG. 8E shows immunoblots of HCT1 16 lysates
demonstrated knockdown of Bcl-2, B C1-XL, and Mcl-1 72 hours post-transfection. EJ1
(FIG. 9F) or HCTl 16 (FIG. 9G) cells were transfected with a luciferase siRNA or the
same set of siR As against Bcl-2, B C1-X L, or Mcl-1 siRNAs as in FIG. 9E. 24 hours
post-transfection, cells were treated with different amounts of AZDl 152. Cell viability
was determined 72 hours after exposure to AZDl 152. FIG. 9H shows HCTl 16 cells
were transfected with different amount of Aurora B or B C1-X LsiRNAs individually or in
combination. Cell viability was determined 72 hours post-transfection. In all figures,
cell viability was expressed as the percentage of untransfected, DMSO-treated control
cells. The red viability curve represents a synergistic combination.
FIG. 10 shows that polyp loidization renders cells B C1-XLdependent. FIG. 10A
shows that HCT 116 cells were transfected with INCENP or Bcl-XL siRNAs in doseresponse
individually or in combination. Cell viability was determined 72 hours posttransfection.
The red curve represents a synergistic combination. FIG. 10B shows
immunoblots of HCTl 16 lysates demonstrated knockdown of FNCENP 72 hours posttransfection
with the same siRNA as in FIG. 10A. HCTl 16 cells (FIG. 10C-FIG. 10F)
were treated for 24 hours with DMSO or 200 nM AZD 1152. AZD 1152 was then either
left in the culture medium (AZDl 152) or washed out by replacing the medium with drugfree
growth medium (AZDl 152 washout). Cells were cultured for an additional 72 hours
before being subjected to the following treatment/analysis: Western analysis (FIG. IOC)
to determine the amounts of p53, phosphorylated (pSer 10) and unphosphorylated histone
H3, or Phase contrast imaging (FIG. 10D) to show morphological signs of polyploidy,
such as dramatically increased cell size and multinucleation, in AZDl 152 treated cells
regardless of washout, further treatment of 1 mM ABT-263 for 4 hours (FIG. 10E) and
assaying for Caspase-3 activity, and further treatment with 1 mM ABT-263 (FIG. 10F)
for 24 hours and assaying for cell viability.
FIG. 11 shows that modulation of the Bcl-2 network during polyploidization.
HCTl 16 cells (FIG. 11A) were treated with DMSO or 200 nM AZDl 152 for 72 hours
followed by 2 hours with 1 mM ABT-263. Conformationally active BAK or BAX was
immunoprecipitated and detected by immunoblotting. D54MG and HCTl 16 cells (FIG.
1IB) were treated DMSO or 200 nM AZDl 152 for 72 hours prior to the addition of 1
mM ABT-263 for 4 hours. Conformationally active BAK or BAX was
immunoprecipitated and detected by immunoblotting (top and middle panel). Cell lysates
were also fractionated and Cytochrome C release into the cytosolic fraction was detected
by immunoblotting. HCTl 16, SW620, and D54MG cells (FIG. 11C) were treated with
DMSO (untreated) or 200 nM AZDl 152 (AZDl 152) for 72 hours and the expression of
Bcl-2 network components were detected by immunoblotting (FIG. 1ID- FIG 1IE)
HCTl 16 cells were treated as in FIG. 1IB. Endogenous B C1-XL and Mcl-l were
immunoprecipitated, and the Bcl-2 network components in the immunecomplexes were
detected by immunoblotting.
FIG. 12 shows that elements of Bcl-2 network are required for B C1-XL "addiction"
in polyploid cells and for polyploidization-mediated apoptosis. FIG. 12A shows a
focused library of siR As targeting Bcl-2 network components was transfected as
individual oligos into HCTl 16 cells. EJ1 and DLD1 cells (FIG. 12B) were transfected
with individual NOXA siRNAs or BAX and BA siRNAs in combination. In both FIG.
12A and FIG. 12B, 24 hours post-transfection, cells were treated with 200 nM AZDl 152
to induce polyploidy. 72 hours after the addition of AZDl 152, cells were treated with 1
mM ABT-263 and cell viability was determined 24 hours after the addition of ABT-263.
HCTl 16 cells (FIG. 12C and FIG. 12D) were transfected with a control, nontargeting
siRNA or a NOXA siRNA. 24 hours post-transfection, cells were treated with 200 nM
AZDl 152 for 72 hours. 1 mM ABT-263 was then added for 2 hours prior to cell lysis
and immunoprecipitation of B C1-XL or Mcl-l. HCTl 16 (FIG. 12E) cells were transfected
with the focused siRNA library as in FIG. 12A. 24 hours post-transfection, cells were
treated with 200 nM AZDl 152 to induce polyploidy. AZDl 152 was washed out by
replacing the medium with drug-free growth medium 3 and 7 days after the addition of
AZDl 152. Cell viability was determined 10 days after the addition of AZDl 152 to
assess polyploidization-induced lethality.
FIG. 13 shows that ABT-263 and AZDl 152 produce synergistic tumor growth
inhibition in vivo . FIG. 13A shows the growth curve of HCT 11 tumors in mice when
administered vehicle, ABT-263, AZDl 152, or the combination of AZDl 152 and ABT-
263 as indicated in Example 40. CT1 16 tumor-bearing mice (FIG. 13B) were treated
with vehicle or AZDl 152 (100 mg/kg/day (hereinafter "mkd", IP, QD) for 3 consecutive
days prior to the administration of vehicle or ABT-263 (75 mkd, PO). 4 hours and 8
hours after the administration of ABT-263, tumors were collected, and tumor lysates
were analyzed for the abundance of p53 and the conformationally active BAX (IP with
6A7, an antibody specifically detect active BAX, followed by BAX immunob lotting).
FIG. 13C shows a model for B C1-XL "addiction" and sensitivity to ABT-263 in polyploid
cells. Polyp loidization functionally neutralizes Mcl-1 by downregulating Mcl-1 protein,
inducing NOXA, increasing the interaction of NOXA with Mcl-1 , or some combination
of these effects. Additionally, B C1-XL becomes burdened by BH3-only proteins, such as
tBID and BIK. This renders cell survival increasingly dependent upon B C1-XL. This
dependence or "addiction" represents a molecular Achilles' heel of polyploidization, as
ABT-263, which disables the anti-apoptotic function of B C1-XL, elicits rapid and robust
apoptosis in polyploid tumor cells.
FIG. 14 BA and BAX activation in tumor cell lines. D54MG, SW620, and EJ1
cells were treated as in FIG. 17B. Conformationally active BAK and BAX was
immunoprecipitated and detected by immunoblotting.
FIG. 15 shows gene expression changes in the Bcl-2 network. D54MG, SW620,
and HCTl 16 cells were treated for 72 hours with 200 nM AZDl 152, 250 nM MLN8054,
or 1 mM VX-680. These treatments result in the following cellular Aurora selectivities:
Aurora B, Aurora A, or pan- Aurora, respectively. Gene expression changes in the Bcl-2
network were detected by microarray. These data were hierarchically clustered using a
correlation similarity measure using SPOTFIRE® (TIBCO®) data analysis software.
FIG. 16 shows the identification of B C1-XL and Mcl-1 interactomes. FIG. 17A
shows a schematic of the workflow to identify B C1-XL and Mcl-1 interactors is shown.
HCTl 16 cells are either untreated or treated with 200 nM AZDl 152, 1 mM ABT-263 or
the combination. Endogenous B C1-XL and Mcl-1 is immunoprecipitated, and
immunoprecipitated proteins are subjected to in-gel tryptic digestion and LC-MS/MS.
The protein identified from all treatment conditions were consolidated into a single list as
B C1-XL and Mcl-1 interactomes and applied to a manually constructed Bcl-2 network
map. Interactions are indicated in green. Interactome components were subsequently
confirmed by co-immunoprecipitation and immunoblotting (FIG. 17B and FIG. 17C)
BCI-XL and Mcl-1 interactomes.
FIG. 18 shows that siRNA-mediated knockdown of Bcl-2 network components.
HCTl 16 cells were transfected with siRNAs as indicated. 24 hours post-transfection,
cells were treated with 200 nM AZD1 152, and lysates were prepared 72 hours later and
analyzed by immunoblotting. Ral-A serves as a loading control and siNT, non-targeted,
control siR A.
FIG. 19 shows that time-course of polyploidization-mediated apoptosis. HCT1 16
cells were treated with 200 nM AZD 1152, and cell viability was determined over
indicated time course.
FIG. 20 shows that Mcl-1 is epistatic to NOXA in the sensitization of polyploid
cells to ABT-263. HCT1 16 cells (FIG. 20A) were transfected with siRNAs as indicated.
24 hours post-transfection, cells were treated with 200 nM AZD 1152 for a further 72
hours. Cells were then treated for 4 hours with 1 mM ABT-263, and Caspase-3 activity
was determined. HCT1 16 cells (FIG. 20B) were transfected with siRNAs as indicated.
24 hours post-transfection, cells were treated with 200 nM AZD 1152 for a further 72
hours. Cells were then treated for 24 hours with 1 mM ABT-263, and cell viability was
determined.
SUMMARY
In one aspect, the present disclosure comprises compounds having formula (II)
(P) ,
and therapeutically acceptable salts, prodrugs, salts of prodrugs and metabolites thereof,
wherein X is CI or F;
X4 is azepan-l-yl, morpholin-l-yl, 1,4-oxazepan-4-yl, pyrrolidin-l-yl, N(CH )2,
N(CH3)(CH(CH3) ), 7-azabicyclo[2.2.1]heptan-l-yl or 2-oxa-5-azabicyclo[2.2.1]hept-5-
l, and ° is
, wherein
X5 is CH2, C(CH ) or CH2CH2;
X and X are both hydrogen or are both methyl; and
X8 is F, CI, Br or I; or
X is azepan-l-yl, morpholin-l-yl, pyrrolidin-l-yl, N(CH3)(CH(CH3)2) or
7-azabicyclo[2.2.1]heptan-l-yl, and R is
X is C¾ )2or morpholin-l-yl, and R is
Another aspect comprises compounds having formula (II), and therapeutically
3
acceptable salts, prodrugs, salts of prodrugs and metabolites thereof, wherein X is CI or
F;
X is azepan-l-yl, morpholin-l-yl, 1,4-oxazepan-4-yl, pyrrolidin-l-yl, N(CH )2
N(CH3)(CH(CH3) ), 7-azabicyclo[2.2.1]heptan-l-yl or 2-oxa-5-azabicyclo[2.2.1]hept-5-
l, and R° is
wherein
X5 is CH2, C(CH3)2 or CH2CH2;
X6 and X7 are both hydrogen or are both methyl; and
X8 is F, CI, Br or I; or
4
X is azepan- 1-yl, morpholin- 1-yl, pyrrolidin- 1-yl, N(CH3)(CH(CH3)2) or
7-azabicyclo[2.2.1]heptan-l-yl, and R° is
X is C¾ )2 or morpholin-l-yl, and R is
Still another aspect comprises compounds having formula (II), and therapeutically
3
acceptable salts, prodrugs, salts of prodrugs and metabolites thereof, wherein X is CI or
X is azepan-l-yl, morpholin-l-yl, 1,4-oxazepan-4-yl, pyrrolidin-l-yl, N(CH )2,
N(CH3)(CH(CH3) ), 7-azabicyclo[2.2.1]heptan-l-yl or 2-oxa-5-azabicyclo[2.2.1]hept-5-
l, and R° is
wherein X5 is CH2, C(CH )2 or CH2CH2, and X6 and X7 are both
hydrogen or are both methyl; and
X8 is F, CI, Br or I .
Still another aspect comprises compounds having formula (II), and therapeutically
acceptable salts, prodrugs, salts of prodrugs and metabolites thereof, wherein X is CI or
X is azepan-l-yl, morpholin-l-yl, pyrrolidin-l-yl, (CH3)(CH(CH )2) or
7-azabicyclo [2.2.1 Jheptan- 1-yl;
wherein X and X are both hydrogen or are both methyl; and
Still another aspect comprises compounds having formula (II), and therapeutically
acceptable salts, prodrugs, salts of prodrugs and metabolites thereof, wherein X is CI or
X is (CI¾) or morpholin-l-yl;
R is
X° is F, CI, Br or I .
Still another aspect comprises a compound having formula (II), and
therapeutically acceptable salts, prodrugs, salts of prodrugs and metabolites thereof,
wherein X3 is F; X4 is morpholin-l-yl;
, wherein X5 is C(CH3)2; X6 and X7 are both methyl; and
X° is Cl.
Still another aspect comprises
N-(4-(4-((2-(4-chlorophenyl)-5 ,5-dimethyl- 1-cyclohex- 1-en- 1-
yl)methyl)piperazin- 1-yl)benzoyl)-4-((( 1R)-3-(morpholin-4-yl)- 1-
((phenylsulfanyl)methyl)propyl)amino)-3-((trifluoromethyl)sulfonyl)benzenesulfonam
(alternatively referred to herein as ABT-263),
3-((chloro(difiuoro)methyl)sulfonyl)-N-(4-(4-((2-(4-chlorophenyl)-4,4-
dimethylcyclohex-1-en- 1-yl)methyl)piperazin-1-yl)benzoyl)-4-(((1R)-3-(morpholin-4-
yl)-1-((phenylsulfanyl)methyl)propyl)arnino)benzenesulfonamide,
3-((chloro(difluoro)methyl)sulfonyl)-N-(4-(4-((2-(4-chlorophenyl)-1-cyclohex-1-
en- 1-yl)methyl)piperazin- 1-yl)benzoyl)-4-(((1R)-3-(morpholin-4-yl)- 1-
((phenylsulfanyl)methyl)propyl)amino)benzenesulfonamide,
N-(4-(4-((2-(4-chlorophenyl)-5 ,5-dimethylcyclohex-1-en-1-yl)methyl)piperazin-
1-yl)benzoyl)-4-(((1R)-3-(isopropyl(methyl)amino)-1-
((phenylsulfanyl)methyl)propyl)amino)-3-
((trifluoromethyl)sulfonyl)benzenesulfonamide,
N-(4-(4-((2-(4-chlorophenyl)-l -cyclohepten- 1-yl)methyl)piperazin- 1-yl)benzoyl)-
4-(((lR)-3-(isopropyl(methyl)amino)-l-((phenylsulfanyl)methyl)propyl)amino)-3-
((trifluoromethyl)sulfonyl)benzenesulfonamide,
3-((chloro(difiuoro)methyl)sulfonyl)-N-(4-(4-((2-(4-chlorophenyl)cyclohex-l-enl-
yl)methyl)piperazin-l-yl)benzoyl)-4-(((lR)-3-((lS,4S)-2-oxa-5-azabicyclo[2.2.1]hept-
5-yl)-l-((phenylsulfanyl)methyl)propyl)amino)benzenesulfonamide,
N-(4-(4-((2-(4-chlorophenyl)cyclohex- 1-en- 1-yl)methyl)piperazin-1-yl)benzoyl)-
4-(((1R)-3-(morpholin-4-yl)- 1-((phenylsulfanyl)methyl)propyl)amino)-3-
((trifluoromethyl)sulfonyl)benzenesulfonamide,
4-(((lR)-3-(7-azabicyclo[2.2.1]hept-7-yl)-l-
((phenylsulfanyl)methyl)propyl)amino)-N-(4-(4-((2-(4-chlorophenyl)cyclohex-1-en-1-
yl)methyl)piperazin-l-yl)benzoyl)-3-((trifluoromethyl)sulfonyl)benzenesulfonamide,
N-(4-(4-((2-(4-chlorophenyl)cyclohex- 1-en- 1-yl)methyl)piperazin-1-yl)benzoyl)-
4-(((lR)-3-(2-oxa-5-azabicyclo[2.2.1]hept-5-yl)-l-
((phenylsulfanyl)methyl)propyl)amino)-3-
((trifluoromethyl)sulfonyl)benzenesulfonamide,
N-(4-(4-((2-(4-chlorophenyl)-5 ,5-dimethylcyclohex-1-en-1-yl)methyl)piperazinl-
yl)benzoyl)-4-(((lR)-3-(2-oxa-5-azabicyclo[2.2.1]hept-5-yl)-l((
phenylsulfanyl)methyl)propyl)amino)-3-
((trifluoromethyl)sulfonyl)benzenesulfonamide,
3-((chloro(difluoro)methyl)sulfonyl)-N-(4-(4-((2-(4-chlorophenyl)-5,5-
dimethylcyclohex- 1-en- 1-yl)methyl)piperazin- 1-yl)benzoyl)-4-((( 1R)-3-(2-oxa-5 -
azabicyclo [2.2.1 ]hept-5 -yl)- 1-
((phenylsulfanyl)methyl)propyl)amino)benzenesulfonamide,
N-(4-(4-((2-(4-chlorophenyl)cyclohept- 1-en- 1-yl)methyl)piperazin- 1-yl)benzoyl)-
4-(((lR)-3-(2-oxa-5-azabicyclo[2.2.1]hept-5-yl)-l-
((phenylsulfanyl)methyl)propyl)amino)-3-
((trifluoromethyl)sulfonyl)benzenesulfonamide,
3-((chloro(difluoro)methyl)sulfonyl)-N-(4-(4-((2-(4-chlorophenyl)cyclohept-len-
1-yl)methyl)piperazin- 1-yl)benzoyl)-4-((( 1R)-3 -(2-oxa-5-azabicyclo[2.2. 1]hept-5-yl)-
l-((phenylsulfanyl)methyl)propyl)amino)benzenesulfonamide,
N-(4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex- 1-en- 1-yl)methyl)piperazin-
1-yl)benzoyl)-4-((( 1R)-3-(isopropyl(methyl)amino)- 1-
((phenylsulfanyl)methyl)propyl)amino)-3-
((trifluoromethyl)sulfonyl)benzenesulfonamide,
N-(4-(4-((2-(4-chlorophenyl)cyclohex- 1-en- 1-yl)methyl)piperazin- 1-yl)benzoyl)-
4-(((lR)-3-(l,4-oxazepan-4-yl)-l-((phenylsulfanyl)methyl)propyl)amino)-3-
((trifluoromethyl)sulfonyl)benzenesulfonamide,
4-(((lR)-3-(azepan-l-yl)-l-((phenylsulfanyl)methyl)propyl)amino)-N-(4-(4-((2-
(4-chlorophenyl)- 1-cyclohex- 1-en- 1-yl)methyl)piperazin- 1-yl)benzoyl)-3-
((trifluoromethyl)sulfonyl)benzenesulfonamide,
N-(4-(4-((2-(4-chlorophenyl)cyclohept- 1-en- 1-yl)methyl)piperazin- 1-yl)benzoyl)-
4-((( 1R)-3-(dimethylamino)- 1-((phenylsulfanyl)methyl)propyl)amino)-3 -
((trifluoromethyl)sulfonyl)benzenesulfonamide,
3-((chloro(difluoro)methyl)sulfonyl)-N-(4-(4-((2-(4-chlorophenyl)cyclohex-l-en-
1-yl)methyl)piperazin- 1-yl)benzoyl)-4-((( 1R)-3-(dimethylamino)- 1-
((phenylsulfanyl)methyl)propyl)amino)benzenesulfonamide,
3-((chloro(difluoro)methyl)sulfonyl)-N-(4-(4-((2-(4-chlorophenyl)-5,5-
dimethylcyclohex- 1-en- 1-yl)methyl)piperazin- 1-yl)benzoyl)-4-((( 1R)-3-(morpholin-4-
yl)- 1-((^henylsulfanyl)methyl)propyl)amino)benzenesulfonamide,
N-(4-(4-((2-(4-chlorophenyl)-5 ,5-dimethyl- 1-cyclohex- 1-en- 1-
yl)methyl)piperazin- 1-yl)benzoyl)-4-((( 1R)-3-(dimethylamino)- 1-
((phenylsulfanyl)methyl)propyl)amino)-3-
((trifluoromethyl)sulfonyl)benzenesulfonamide,
N-(4-(4-((4-(4-chlorophenyl)-5 ,6-dihydro-2H-pyran-3 -yl)methyl)piperazin- 1-
((trifluoromethyl)sulfonyl)benzenesulfonamide,
3-((chloro(difluoro)methyl)sulfonyl)-N-(4-(4-((2-(4-chlorophenyl)-4,4-
dimethylcyclohex- 1-en- 1-yl)methyl)piperazin- 1-yl)benzoyl)-4-((( 1R)-3-
(isopropyl(methyl)amino)-l-((phenylsulfanyl)methyl)propyl)amino)benzenesulfonamide,
3-((chloro(difluoro)methyl)sulfonyl)-N-(4-(4-((2-(4-chlorophenyl)-5,5-
dimethylcyclohex- 1-en- 1-yl)methyl)piperazin- 1-yl)benzoyl)-4-((( 1R)-3-
(isopropyl(methyl)amino)-l-((phenylsulfanyl)methyl)propyl)amino)benzenesulfonamide,
N-(4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex- 1-en- 1-yl)methyl)piperazin-
1-yl)benzoyl)-4-((( 1R)-3-(morpholin-4-yl)- 1-((phenylsulfanyl)methyl)propyl)amino)-3 -
((trifluoromethyl)sulfonyl)benzenesulfonamide,
3-((chloro(difluoro)methyl)sulfonyl)-N-(4-(4-((2-(4-chlorophenyl)-4,4-
dimethylcyclohex- 1-en- 1-yl)methyl)piperazin- 1-yl)benzoyl)-4-((( 1R)-3-(( 1S,4S)-2-oxa-
5-azabicyclo [2.2.1 ]hept-5 -y1)- 1-
((phenylsulfanyl)methyl)propyl)amino)benzenesulfonamide,
N-(4-(4-((2-(4-chlorophenyl)cyclohex- 1-en- 1-yl)methyl)piperazin- 1-yl)benzoyl)-
4-((( 1R)-3-(dimethylamino)- 1-((phenylsulfanyl)methyl)propyl)amino)-3 -
((trifluoromethyl)sulfonyl)benzenesulfonamide,
N-(4-(4-((2-(4-chlorophenyl)cyclohex- 1-en- 1-yl)methyl)piperazin- 1-yl)benzoyl)-
4-((( 1R)- 1-((phenylsulfanyl)methyl)-3 -(pyrrolidin- 1-yl)propyl)amino)-3 -
((trifluoromethyl)sulfonyl)benzenesulfonamide,
3-((chloro(difluoro)methyl)sulfonyl)-N-(4-(4-((2-(4-chlorophenyl)-4,4-
dimethylcyclohex-1-en- 1-yl)methyl)piperazin-1-yl)benzoyl)-4-((( 1R)-3-
(dimethylamino)-l-((phenylsulfanyl)methyl)propyl)amino)benzenesulfonamide,
N-(4-(4-((2-(4-chlorophenyl)cyclohept- 1-en- 1-yl)methyl)piperazin- 1-yl)benzoyl)-
4-((( 1 )-1-((phenylsulfanyl)methyl)-3-(pyrrolidin-1-yl)propyl)amino)-3-
((trifluoromethyl)sulfonyl)benzenesulfonamide,
3-((chloro(difluoro)methyl)sulfonyl)-N-(4-(4-((2-(4-chlorophenyl)-5,5-
dimethylcyclohex-1-en- 1-yl)methyl)piperazin-1-yl)benzoyl)-4-((( 1R)-1-
((phenylsulfanyl)methyl)-3-(pyrrolidin- 1-yl)propyl)amino)benzenesulfonamide,
3-((chloro(difluoro)methyl)sulfonyl)-N-(4-(4-((2-(4-chlorophenyl)-4,4-
dimethylcyclohex-1-en- 1-yl)methyl)piperazin-1-yl)benzoyl)-4-((( 1R)-1-
((phenylsulfanyl)methyl)-3-(pyrrolidin- 1-yl)propyl)amino)benzenesulfonamide,
3-((chloro(difluoro)methyl)sulfonyl)-N-(4-(4-((2-(4-chlorophenyl)cyclohept-len-
1-yl)methyl)piperazin- 1-yl)benzoyl)-4-((( 1R)-1-((phenylsulfanyl)methyl)-3-
(pyrrolidin- 1-yl)propyl)amino)benzenesulfonamide,
N-(4-(4-((2-(4-chlorophenyl)cyclohex- 1-en- 1-yl)methyl)piperazin-1-yl)benzoyl)-
4-(((lR)-3-(isopropyl(methyl)amino)-l-((phenylsulfanyl)methyl)propyl)amino)-3-
((trifluoromethyl)sulfonyl)benzenesulfonamide,
N-(4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex- 1-en-1-yl)methyl)piperazin-
1-yl)benzoyl)-4-((( 1R)- 1-((phenylsulfanyl)methyl)-3-(pyrrolidin-1-yl)propyl)amino)-3-
((trifluoromethyl)sulfonyl)benzenesulfonamide,
3-((chloro(difluoro)methyl)sulfonyl)-N-(4-(4-((2-(4-chlorophenyl)cyclohex-l-en-
1-yl)methyl)piperazin- 1-yl)benzoyl)-4-(((1R)- 1-((phenylsulfanyl)methyl)-3-(pyrrolidinl-
yl)propyl)amino)benzenesulfonamide,
N-(4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex- 1-en-1-yl)methyl)piperazin-
1-yl)benzoyl)-4-((( 1R)-3-(dimethylamino)-1-((phenylsulfanyl)methyl)propyl)amino)-3-
((trifluoromethyl)sulfonyl)benzenesulfonamide,
N-(4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex- 1-en-1-yl)methyl)piperazinl-
yl)benzoyl)-4-(((lR)-3-((lS,4S)-2-oxa-5-azabicyclo[2.2.1]hept-5-yl)-l-
((phenylsulfanyl)methyl)propyl)amino)-3-
((trifluoromethyl)sulfonyl)benzenesulfonamide,
N -(4-(4-((4'-chloro(l ,r-biphenyl)-2-yl)methyl)-l-piperazinyl)benzoyl)-4-(((lR)-
3-(dimethylamino)-l-((phenylsulfanyl)methyl)propyl)amino)-3-
((trifluoromethyl)sulfonyl)benzenesulfonamide, and
N -(4-(4-((4'-chloro(l ,r-biphenyl)-2-yl)methyl)-l-piperazinyl)benzoyl)-4-(((lR)-
3-(4-morpholinyl)- 1-((phenylsulfanyl)methyl)propyl)amino)-3-
((trifluoromethyl)sulfonyl)benzenesulfonamide,
and therapeutically acceptable salts, prodrugs, salts of prodrugs and metabolites thereof.
Still another aspect comprises compositions for treating diseases during which are
expressed one or more than one o f antiapoptotic B C1-XL protein, antiapoptotic Bcl-2
protein or antiapoptotic Bcl-w protein, said compositions comprising an excipient and a
therapeutically effective amount of the compound having formula (II).
Still another aspect comprises methods o f treating diseases in a patient during
which are expressed one or more than one of antiapoptotic B C1-X protein, antiapoptotic
Bcl-2 protein or antiapoptotic Bcl-w protein, said methods comprising administering to
the patient a therapeutically effective amount of a compound having formula (II).
Still another aspect comprises compositions comprising an excipient and a
therapeutically effective amount of the compound having formula (II) for treating
diseases o f abnormal cell growth and/or dysregulated apoptosis, such as cancer,
including, for example, mesothioloma, bladder cancer, pancreatic cancer, skin cancer,
cancer o f the head or neck, cutaneous or intraocular melanoma, ovarian cancer, breast
cancer, uterine cancer, carcinoma o f the fallopian tubes, carcinoma of the endometrium,
carcinoma of the cervix, carcinoma o f the vagina, carcinoma o f the vulva, bone cancer,
ovarian cancer, cervical cancer, colon cancer, rectal cancer, cancer of the anal region,
stomach cancer, gastrointestinal (gastric, colorectal, and duodenal), chronic lymphocytic
leukemia, esophageal cancer, cancer of the small intestine, cancer of the endocrine
system, cancer of the thyroid gland, cancer o f the parathyroid gland, cancer o f the adrenal
gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, testicular cancer,
hepatocellular cancer (hepatic and billiary duct), primary or secondary central nervous
system tumor, primary or secondary brain tumor, Hodgkin's disease, chronic or acute
leukemia, chronic myeloid leukemia, lymphocytic lymphomas, lymphoblastic leukemia,
follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma,
multiple myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, prostate
cancer, small cell lung cancer, cancer of the kidney and ureter, renal cell carcinoma,
carcinoma of the renal pelvis, neoplasms of the central nervous system, primary central
nervous system lymphoma, non Hodgkin's lymphoma, spinal axis tumors, brain stem
glioma, pituitary adenoma, adrenocortical cancer, gall bladder cancer, cancer of the
spleen, cholangiocarcinoma, fibrosarcoma, neuroblastoma, retinoblastoma, or a
combination thereof.
Still another aspect comprises methods of treating mesothioloma, bladder cancer,
pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular
melanoma, ovarian cancer, breast cancer, uterine cancer, carcinoma of the fallopian
tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina,
carcinoma of the vulva, bone cancer, ovarian cancer, cervical cancer, colon cancer, rectal
cancer, cancer of the anal region, stomach cancer, gastrointestinal (gastric, colorectal, and
duodenal), chronic lymphocytic leukemia, esophageal cancer, cancer of the small
intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the
parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the
urethra, cancer of the penis, testicular cancer, hepatocellular cancer (hepatic and billiary
duct), primary or secondary central nervous system tumor, primary or secondary brain
tumor, Hodgkin's disease, chronic or acute leukemia, chronic myeloid leukemia,
lymphocytic lymphomas, lymphoblastic leukemia, follicular lymphoma, lymphoid
malignancies of T-cell or B-cell origin, melanoma, multiple myeloma, oral cancer,
ovarian cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, cancer
of the kidney and ureter, renal cell carcinoma, carcinoma of the renal pelvis, neoplasms
of the central nervous system, primary central nervous system lymphoma, non Hodgkin's
lymphoma, spinal axis tumors, brain stem glioma, pituitary adenoma, adrenocortical
cancer, gall bladder cancer, cancer of the spleen, cholangiocarcinoma, fibrosarcoma,
neuroblastoma, retinoblastoma, or a combination of one or more of the above cancers in a
patient, said methods comprising administering thereto a therapeutically effective amount
of a compound having formula (II).
Still another aspect comprises compositions for treating bladder cancer, brain
cancer, breast cancer, bone marrow cancer, cervical cancer, chronic lymphocytic
leukemia, colorectal cancer, esophageal cancer, hepatocellular cancer, lymphoblastic
leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin,
melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small cell
lung cancer, prostate cancer, small cell lung cancer and spleen cancer, said compositions
comprising an excipient and a therapeutically effective amount of the compound having
formula (II).
Still another aspect comprises methods of treating bladder cancer, brain cancer,
breast cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia,
colorectal cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia,
follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma,
myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung
cancer, prostate cancer, small cell lung cancer and spleen cancer in a patient, said
methods comprising administering to the patient a therapeutically effective amount of a
compound having formula (II).
Still another aspect comprises compositions for treating diseases in a patient
during which are expressed one or more than one of antiapoptotic BC1-XL protein,
antiapoptotic Bcl-2 protein or antiapoptotic Bcl-w protein, said compositions comprising
an excipient and a therapeutically effective amount of the compound having formula (II)
and a therapeutically effective amount of one additional therapeutic agent or more than
one additional therapeutic agent.
Still another aspect comprises methods of treating diseases in a patient during
which is expressed one or more than one of antiapoptotic BC1-XL protein, antiapoptotic
Bcl-2 protein or antiapoptotic Bcl-w protein, said methods comprising administering to
the patient a therapeutically effective amount of a compound having formula (II) and a
therapeutically effective amount of one additional therapeutic agent or more than one
additional therapeutic agent.
Still another aspect comprises compositions for treating mesothioloma, bladder
cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or
intraocular melanoma, ovarian cancer, breast cancer, uterine cancer, carcinoma of the
fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the
vagina, carcinoma of the vulva, bone cancer, ovarian cancer, cervical cancer, colon
cancer, rectal cancer, cancer of the anal region, stomach cancer, gastrointestinal (gastric,
colorectal, and duodenal), chronic lymphocytic leukemia, esophageal cancer, cancer of
the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of
the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the
urethra, cancer of the penis, testicular cancer, hepatocellular cancer (hepatic and billiary
duct), primary or secondary central nervous system tumor, primary or secondary brain
tumor, Hodgkin's disease, chronic or acute leukemia, chronic myeloid leukemia,
lymphocytic lymphomas, lymphoblastic leukemia, follicular lymphoma, lymphoid
malignancies of T-cell or B-cell origin, melanoma, multiple myeloma, oral cancer,
ovarian cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, cancer
of the kidney and ureter, renal cell carcinoma, carcinoma of the renal pelvis, neoplasms
of the central nervous system, primary central nervous system lymphoma, non Hodgkin's
lymphoma, spinal axis tumors, brain stem glioma, pituitary adenoma, adrenocortical
cancer, gall bladder cancer, cancer of the spleen, cholangiocarcinoma, fibrosarcoma,
neuroblastoma, retinoblastoma, or a combination of one or more of the above cancers,
said compositions comprising an excipient and therapeutically effective amount of a
compound having formula (II) and one additional therapeutic agent or more than one
additional therapeutic agent.
Still another aspect comprises methods of treating mesothioloma, bladder cancer,
pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular
melanoma, ovarian cancer, breast cancer, uterine cancer, carcinoma of the fallopian
tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina,
carcinoma of the vulva, bone cancer, ovarian cancer, cervical cancer, colon cancer, rectal
cancer, cancer of the anal region, stomach cancer, gastrointestinal (gastric, colorectal, and
duodenal), chronic lymphocytic leukemia, esophageal cancer, cancer of the small
intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the
parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the
urethra, cancer of the penis, testicular cancer, hepatocellular cancer (hepatic and billiary
duct), primary or secondary central nervous system tumor, primary or secondary brain
tumor, Hodgkin's disease, chronic or acute leukemia, chronic myeloid leukemia,
lymphocytic lymphomas, lymphoblastic leukemia, follicular lymphoma, lymphoid
malignancies of T-cell or B-cell origin, melanoma, multiple myeloma, oral cancer,
ovarian cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, cancer
of the kidney and ureter, renal cell carcinoma, carcinoma of the renal pelvis, neoplasms
of the central nervous system, primary central nervous system lymphoma, non Hodgkin's
lymphoma, spinal axis tumors, brain stem glioma, pituitary adenoma, adrenocortical
cancer, gall bladder cancer, cancer of the spleen, cholangiocarcinoma, fibrosarcoma,
neuroblastoma, retinoblastoma, or a combination of one or more of the above cancers in a
patient, said methods comprising administering thereto therapeutically effective amounts
of a compound having formula (II) and one additional therapeutic agent or more than one
additional therapeutic agent.
Still another aspect comprises methods of treating mesothioloma, bladder cancer,
pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular
melanoma, ovarian cancer, breast cancer, uterine cancer, carcinoma of the fallopian
tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina,
carcinoma of the vulva, bone cancer, ovarian cancer, cervical cancer, colon cancer, rectal
cancer, cancer of the anal region, stomach cancer, gastrointestinal (gastric, colorectal, and
duodenal), chronic lymphocytic leukemia, esophageal cancer, cancer of the small
intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the
parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the
urethra, cancer of the penis, testicular cancer, hepatocellular cancer (hepatic and billiary
duct), primary or secondary central nervous system tumor, primary or secondary brain
tumor, Hodgkin's disease, chronic or acute leukemia, chronic myeloid leukemia,
lymphocytic lymphomas, lymphoblastic leukemia, follicular lymphoma, lymphoid
malignancies of T-cell or B-cell origin, melanoma, multiple myeloma, oral cancer,
ovarian cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, cancer
of the kidney and ureter, renal cell carcinoma, carcinoma of the renal pelvis, neoplasms
of the central nervous system, primary central nervous system lymphoma, non Hodgkin's
lymphoma, spinal axis tumors, brain stem glioma, pituitary adenoma, adrenocortical
cancer, gall bladder cancer, cancer of the spleen, cholangiocarcinoma, fibrosarcoma,
neuroblastoma, retinoblastoma, or a combination of one or more of the above cancers in
a patient, said methods comprising administering thereto therapeutically effective
amounts of a compound having formula (II) and one or more than one of etoposide,
vincristine, CHOP, rituximab, rapamycin, R-CHOP, bortezomib, MLN8237, tozasertib
(also known as VX-680 or MK-0457), PHA-739358, AT9283, AZDl 152, BI811283,
ENMD-2076, CYC1 16, AMG900, PF-03814735, R763, SNS-314 or TAK-901.
Still another aspect comprises methods of treating B-cell lymphoma in a patient
comprising administering thereto a therapeutically effective amount of a compound
having formula (II) and etoposide.
Still another aspect comprises methods of treating B-cell lymphoma in a patient
comprising administering thereto therapeutically effective amounts of a compound
having formula (II) and vincristine.
Still another aspect comprises methods of treating B-cell lymphoma in a patient
comprising administering thereto therapeutically effective amounts of a compound
having formula (II) and CHOP.
Still another aspect comprises methods of treating B-cell lymphoma in a patient
comprising administering thereto therapeutically effective amounts of a compound
having formula (II) and rituximab.
Still another aspect comprises methods of treating B-cell lymphoma in a patient
comprising administering thereto therapeutically acceptaible amounts of a compound
having formula (II) and rapamycin.
Still another aspect comprises methods of treating mantle cell lymphoma in a
patient comprising administering thereto therapeutically acceptable amouns of a
compound having formula (II) and R-CHOP.
Still another aspect comprises methods of treating mantle cell lymphoma in a
patient comprising administering thereto therapeutically effective amounts of a
compound having formula (II) and bortezomib.
In still yet another aspect, the present disclosure relates to a method of treating a
patient suffering from cancer. The method comprises the steps of:
a) administering to a patient suffering from cancer a therapeutically effective
amount of at least one polyploidy inducing agent; and
b) administering to the patient a therapeutically effective amount of at least
one Bcl-2 family protein inhibitor.
In the above method, the polyploidy inducing agent can be an Aurora Kinase
inhibitor. The Aurora Kinase inhibitor can be an Aurora Kinase A inhibitor, an Aurora
Kinase B inhibitor, an Aurora Kinase C inhibitor or combinations thereof. More
specifically, the Aurora Kinase inhibitor is an Aurora Kinase B inhibitor. For example,
the Aurora Kinase B inhibitor is AZD 1152, ZM447439, VX-680/MK0457 or
Hersperadin.
In the above method, the Bcl-2 family protein inhibitor can be ABT-263, ABT-
737, a B C1-XL selective inhibitor or combinations thereof.
In another aspect, the present disclosure relates to a combination of therapeutic
agents for use in treating a patient suffering from cancer. The combination comprises:
a) at least one polyploidy inducing agent for use in inducing polyploidization in
one or more cancer cells in the patient; and
b) at least one Bcl-2 family protein inhibitor.
In the above combination, the polyploidy inducing agent can be an Aurora Kinase
inhibitor. The Aurora Kinase inhibitor can be an Aurora Kinase A inhibitor, an Aurora
Kinase B inhibitor, an Aurora Kinase C inhibitor or combinations thereof. More
specifically, the Aurora Kinase inhibitor is an Aurora Kinase B inhibitor. For example,
the Aurora Kinase B inhibitor is AZD 1152, ZM447439, VX-680/MK0457 or
Hersperadin.
In the above combination, the Bcl-2 family protein inhibitor can be ABT-263,
ABT-737, a B C1-XL selective inhibitor or combinations thereof.
In yet another aspect, the present disclosure relates to method of classifying a
patient for eligibility for treatment with a polyploidy inducing agent, a Bcl-2 family
protein inhibitor or a combination of a polyploidy inducing agent and a Bcl-2 family
protein inhibitor. The method comprising the steps of:
a) providing a test sample from a patient;
b) determining the presence or absence of at least one mutation in at least one
gene selected from the group consisting of: a BAX gene, a BAK gene or a NOXA gene
in the test sample; and
c) classifying the patient as being eligible for receiving treatment with a
polyploidy inducing agent, a Bcl-2 family protein inhibitor or a combination of a
polyploidy inducing agent and a Bcl-2 family protein inhibitor based on the presence or
absence of at least one mutation as determined in step b).
In the above method, the polyploidy inducing agent can be an Aurora Kinase
inhibitor. The Aurora Kinase inhibitor can be an Aurora Kinase A inhibitor, an Aurora
Kinase B inhibitor, an Aurora Kinase C inhibitor or combinations thereof. More
specifically, the Aurora Kinase inhibitor is an Aurora Kinase B inhibitor. For example,
the Aurora Kinase B inhibitor is AZD1 152, ZM447439, VX -680/MK0457 or
Hersperadin.
In the above method, the Bcl-2 family protein inhibitor can be ABT-263, ABT-
737, a B C1-XL selective inhibitor or combinations thereof.
In the above method, the test sample can comprise a tissue sample. For example,
the tissue sample comprises a peripheral blood sample, a tumor tissue or a suspected
tumor tissue, a thin layer cytological sample, a fine needle aspirate sample, a bone
marrow sample, a lymph node sample, a urine sample, an ascites sample, a lavage
sample, an esophageal brushing sample, a bladder or lung wash sample, a spinal fluid
sample, a brain fluid sample, a ductal aspirate sample, a nipple discharge sample, a
pleural effusion sample, a fresh frozen tissue sample, a paraffin embedded tissue sample
or an extract or processed sample produced from any of a peripheral blood sample, a
tumor tissue or a suspected tumor tissue, a thin layer cytological sample, a fine needle
aspirate sample, a bone marrow sample, a urine sample, an ascites sample, a lavage
sample, an esophageal brushing sample, a bladder or lung wash sample, a spinal fluid
sample, a brain fluid sample, a ductal aspirate sample, a nipple discharge sample, a
pleural effusion sample, a fresh frozen tissue sample or a paraffin embedded tissue
sample.
Moreover, in the above method, the determination step (b) can be performed by in
situ hybridization. For example, the in situ hybridization can be performed with a nucleic
acid probe that is fluorescently labeled. Alternatively, the in situ hybridization is
performed with at least two nucleic acid probes. In still yet another alternative, the in
situ hybridization is performed with a peptide nucleic acid probe.
Alternatively, in the above method, the determination step (b) can be performed
by polymerase chain reaction.
In the above method, the patient may also, contemporaneously therewith, be
receiving treatment with chemotherapy, radiation or combinations thereof.
In another aspect, the present disclosure relates to a method of monitoring a
patient suffering from cancer and being treated with a polyploidy inducing agent, a Bcl-2
family protein inhibitor or a combination of a polyploidy inducing agent and a Bcl-2
family protein inhibitor. The method comprises the steps of:
a) providing a test sample from a patient suffering from cancer and currently
being treated with at least one polyploidy inducing agent, a Bcl-2 family protein inhibitor
or a combination of a polyploidy inducing agent and a Bcl-2 family protein inhibitor;
b) determining the presence or absence of at least one mutation in at least one
gene selected from the group consisting of: a BAX gene, a BAK gene or a NOXA gene
in the test sample; and
c) determining whether the patient should continue to be treated with a
polyploidy inducing agent, a Bcl-2 family protein inhibitor or a combination of a
polyploidy inducing agent and a Bcl-2 family protein inhibitor based on the presence or
absence of at least one mutation as determined in step b).
In the above method, the polyploidy inducing agent can be an Aurora Kinase
inhibitor. The Aurora Kinase inhibitor can be an Aurora Kinase A inhibitor, an Aurora
Kinase B inhibitor, an Aurora Kinase C inhibitor or combinations thereof. More
specifically, the Aurora Kinase inhibitor is an Aurora Kinase B inhibitor. For example,
the Aurora Kinase B inhibitor is AZD1 152, ZM447439, VX-680/MK0457 or
Hersperadin.
In the above method, the Bcl-2 family protein inhibitor can be ABT-263, ABT-
737, a B C1-XL selective inhibitor or combinations thereof.
In the above method, the test sample can comprise a tissue sample. For example,
the tissue sample comprises a peripheral blood sample, a tumor tissue or a suspected
tumor tissue, a thin layer cytological sample, a fine needle aspirate sample, a bone
marrow sample, a lymph node sample, a urine sample, an ascites sample, a lavage
sample, an esophageal brushing sample, a bladder or lung wash sample, a spinal fluid
sample, a brain fluid sample, a ductal aspirate sample, a nipple discharge sample, a
pleural effusion sample, a fresh frozen tissue sample, a paraffin embedded tissue sample
or an extract or processed sample produced from any of a peripheral blood sample, a
tumor tissue or a suspected tumor tissue, a thin layer cytological sample, a fine needle
aspirate sample, a bone marrow sample, a urine sample, an ascites sample, a lavage
sample, an esophageal brushing sample, a bladder or lung wash sample, a spinal fluid
sample, a brain fluid sample, a ductal aspirate sample, a nipple discharge sample, a
pleural effusion sample, a fresh frozen tissue sample or a paraffin embedded tissue
sample.
Moreover, in the above method, the determination step (b) can be performed by in
situ hybridization. For example, the in situ hybridization can be performed with a nucleic
acid probe that is fluorescently labeled. Alternatively, the in situ hybridization is
performed with at least two nucleic acid probes. In still yet another alternative, the in
situ hybridization is performed with a peptide nucleic acid probe.
Alternatively, in the above method, the determination step (b) can be performed
by polymerase chain reaction.
In the above method, the patient may also, contemporaneously therewith, be
receiving treatment with chemotherapy, radiation or combinations thereof.
In still yet another aspect, the present disclosure relates to a method of classifying
a patient as having a cancer that is resistant to treatment with a polyploidy inducing
agent, a Bcl-2 family protein inhibitor or a combination of a polyploidy inducing agent
and a Bcl-2 family protein inhibitor, the method comprising the steps of:
a) providing a test sample from a patient suffering from cancer;
b) determining the presence or absence of at least one mutation in at least one
gene selected from the group consisting of: a BAX gene, a BAK gene or aNOXA gene in
the test sample; and
c) classifying the patient as having a cancer that is resistant to treatment with a
polyploidy inducing agent, a Bcl-2 family protein inhibitor or a combination of a
polyploidy inducing agent and a Bcl-2 family protein inhibitor based on the presence or
absence of at least one mutation determined in step b).
In the above method, the polyploidy inducing agent can be an Aurora Kinase
inhibitor. The Aurora Kinase inhibitor can be an Aurora Kinase A inhibitor, an Aurora
Kinase B inhibitor, an Aurora Kinase C inhibitor or combinations thereof. More
specifically, the Aurora Kinase inhibitor is an Aurora Kinase B inhibitor. For example,
the Aurora Kinase B inhibitor is AZD1 152, ZM447439, VX-680/MK0457 or
Hersperadin.
In the above method, the Bcl-2 family protein inhibitor can be ABT-263, ABT-
737, a B C1-XLselective inhibitor or combinations thereof. In the above method, the test
sample can comprise a tissue sample. For example, the tissue sample comprises a
peripheral blood sample, a tumor tissue or a suspected tumor tissue, a thin layer
cytological sample, a fine needle aspirate sample, a bone marrow sample, a lymph node
sample, a urine sample, an ascites sample, a lavage sample, an esophageal brushing
sample, a bladder or lung wash sample, a spinal fluid sample, a brain fluid sample, a
ductal aspirate sample, a nipple discharge sample, a pleural effusion sample, a fresh
frozen tissue sample, a paraffin embedded tissue sample or an extract or processed
sample produced from any of a peripheral blood sample, a tumor tissue or a suspected
tumor tissue, a thin layer cytological sample, a fine needle aspirate sample, a bone
marrow sample, a urine sample, an ascites sample, a lavage sample, an esophageal
brushing sample, a bladder or lung wash sample, a spinal fluid sample, a brain fluid
sample, a ductal aspirate sample, a nipple discharge sample, a pleural effusion sample, a
fresh frozen tissue sample or a paraffin embedded tissue sample.
Moreover, in the above method, the determination step (b) can be performed by in
situ hybridization. For example, the in situ hybridization can be performed with a nucleic
acid probe that is fluorescently labeled. Alternatively, the in situ hybridization is
performed with at least two nucleic acid probes. In still yet another alternative, the in
situ hybridization is performed with a peptide nucleic acid probe.
Alternatively, in the above method, the determination step (b) can be performed
by polymerase chain reaction.
In the above method, the patient may also, contemporaneously therewith, be
receiving treatment with chemotherapy, radiation or combinations thereof.
DETAILED DESCRIPTION
In one aspect, the present disclosure relates to the discovery of a synergistic effect
that occurs in tumor and cancer cells when these cells are administered a therapeutically
effective amount of at least one polyploidy inducing agent and a therapeutically effective
amount of at least one Bcl-2 family protein inhibitor. Specifically, the inventors of the
present disclosure discovered that the induction of polyploidization in certain tumor and
cancer cells makes or renders the survival of these resulting polyploidy cells dependent
on the anti-apoptotic activity of BC1-XL. More specifically, inducing polyploidy in these
tumor and cancer cells sensitizes these cells to BC1-XL inhibition. Thus, the inventors
have discovered a polyploidy-mediated method for inducing apoptosis or cell death in
these cells. This method involves administering to a patient suffering from cancer a
combination of therapeutic agents. Specifically, the patient is administered at least one
polyploidy inducing agent. The purpose of the polyploidy inducing agent is to induce
polyploidy in one or more cancer cells. As mentioned previously herein, after induction
of polyploidy, the survival of these polyploid tumor and cancer cells is dependent on Bcl-
XL. Apoptosis or death of these polyploidy tumor and cancer cells can be obtained or
achieved by administering to the patient at least one Bcl-2 family protein inhibitor. The
order in which the at least one polyploidy inducing agent and the at least one Bcl-2
family protein inhibitor are administered is not critical. However, because tumor and
cancer cells do not become sensitized to Bcl-2 family protein inhibitors until after the
induction of polyploidy, it is preferred to administer the at least one polyploidy inducing
agent to the patient prior to or along with the at least one Bcl-2 family protein inhibitor.
In connection with the above discovery, the inventors of the present disclosure also
discovered that tumor and cancer cells (regardless of whether or not these cells are
induced to be polyploid or not) which contain at least one mutation in (i) a human Bcl-2
pro-apoptotic encoding gene (such as a human BAX gene and a human BAK gene), (ii) a
human NOXA gene, or (iii) a human Bcl-2 pro-apoptotic encoding gene (such as a
human BAX gene and a human BAK gene) and a human NOXA gene are immune or
exhibit resistance to treatment with a Bcl-2 family protein inhibitor after induction of
polyploidy (such as with at least one polyploidy inducing agent). In other words,
polyploid tumor and cancer cells which contain at least one mutation in (i) a human Bcl-2
pro-apoptotic encoding gene (such as a human BAX gene and a human BAK gene), (ii) a
human NOXA gene, or (iii) a human Bcl-2 pro-apoptotic encoding gene (such as a
human BAX gene and a human BAK gene) and human NOXA gene do not exhibit or
experience apoptosis or cell death when simultaneously or subsequently treated with at
least one Bcl-2 family protein inhibitor.
Thus, in view of this further discovery, in another aspect, the present disclosure
also provides methods and compositions for monitoring cancer and tumor cells for
resistance to polyploidy inducing agent (such as an Aurora Kinase inhibitor) therapy,
Bcl-2 family protein inhibitor therapy or a combination of polyploidy inducing agent and
Bcl-2 family protein inhibitor therapy. The disclosure provides diagnostic assays for
identifying, classifying and monitoring cancer patients which comprises assessing a test
sample for the presence or absence of at least one mutation in (i) a human Bcl-2 proapoptotic
encoding gene (such as a human BAX gene and a human BAK gene), (ii) a
human NOXA gene, or (iii) a human Bcl-2 pro-apoptotic encoding gene (such as a
human BAX gene and a human BAK gene) and a human NOXA gene. The inventive
assays include assay methods for identifying patients eligible to receive a polyploidy
mducmg agent, Bcl-2 family protein inhibitor therapy or a polyploidy inducing agent and
Bcl-2 family protein inhibitor therapy (as a combination therapy together or together with
yet another therapy (e.g., such as with chemotherapy, radiation or combinations thereof)
and for monitoring patient response to such therapy. The disclosure comprises, for
example, determining by fluorescent in situ hybridization the presence or absence of at
least one mutation in (i) a human Bcl-2 pro-apoptotic encoding gene (such as a human
BAX gene and a human BAK gene), (ii) a human NOXA gene, or (iii) a human Bcl-2
pro-apoptotic encoding gene (such as a human BAX gene and a human BAK gene) and a
human NOXA gene. The methods herein can be performed prior to the induction of
polyploidy or after the induction of polyploidy. Patients classified as having one or
mutations or an increase in one or more mutations over a baseline (or predetermined)
level in (i) a human Bcl-2 pro-apoptotic encoding gene (such as a human BAX gene and
a human BAK gene), (ii) a human NOXA gene, or (iii) a human Bcl-2 pro-apoptotic
encoding gene (such as a human BAX gene and a human BAK gene) and a human
NOXA gene would be considered to be ineligible to receive at least one polyploidy
inducing agent, at least one Bcl-2 family protein inhibitor or the combination therapy of a
polyploidy inducing agent and Bcl-2 family protein inhibitor therapy because such
patients would be considered to be less likely to respond to each of these therapies.
Specifically, patients who are identified as having one or more mutations in (i) a human
Bcl-2 pro-apoptotic encoding gene (such as a human BAX gene and a human BAK
gene), (ii) a human NOXA gene, or (iii) a human Bcl-2 pro-apoptotic encoding gene
(such as a human BAX gene and a human BAK gene) and a human NOXA gene are
believed to be immune, resistant or less sensitized to treatment with at least one Bcl-2
family protein inhibitor before treatment with or after treatment with at least one
polyploidy inducing agent (to induce polyploidization).
In one aspect, the disclosure comprises a method for identifying or classifying a
patient as eligible for treatment with a polyploidy inducing agent, a Bcl-2 family protein
inhibitor or with a polyploidy inducing agent and a Bcl-2 family protein inhibitor (either
together or in combination with a third therapy). The method comprising the steps of:
(a) providing a tissue sample from a patient;
(b) determining the presence or absence of at least one mutation in (i) a human
Bcl-2 pro-apoptotic encoding gene (such as a human BAX gene and a human BAK
gene), (ii) a human NOXA gene, or (iii) a human Bcl-2 pro-apoptotic encoding gene
(such as a human BAX gene and a human BAK gene) and a human NOXA gene; and
(c) classifying the patient as being eligible for treatment with a polyploidy
inducing agent, a Bcl-2 family protein inhibitor or both a polyploidy inducing agent and
Bcl-2 family protein inhibitor based on the presence or absence of at least one mutation
in a (i) a human Bcl-2 pro-apoptotic encoding gene (such as a human BAX gene and a
human BAK gene), (ii) a human NOXA gene, or (iii) a human Bcl-2 pro-apoptotic
encoding gene (such as a human BAX gene and a human BAK gene) and a human
NOXA gene. In the above method, a patient would be ineligible for treatment with a
polyploidy inducing agent, a Bcl-2 family protein inhibitor or both a polyploidy inducing
agent and a Bcl-2 family protein inhibitor based on the presence of at least one mutation
or an increase in the number of one or more mutations over a baseline (or predetermined)
level in a (i) a human Bcl-2 pro-apoptotic encoding gene (such as a human BAX gene
and a human BAK gene), (ii) a human NOXA gene, or (iii) a human Bcl-2 pro-apoptotic
encoding gene (such as a human BAX gene and a human BAK gene) and a human
NOXA gene. As with the methods mentioned previously herein, this method can be
performed before or after induction of polyploidy in the patient.
In this aspect, the cancer can be any type of cancer, such as colorectal carcinoma
or pancreatic cancer. Moreover, in this aspect, the gene amplification can be determined
by a multi-color fluorescent in situ hybridization (FISH) assay, for example, performed
on a lung cancer tumor biopsy sample. In other aspects, the quantitative polymerase
chain reaction (Q-PCR) method is used.
In yet another aspect, the disclosure comprises a method for identifying or
classifying a patient having a cancer that is resistant to therapy with a polyploidy
inducing agent, a Bcl-2 family protein inhibitor, or a combination of a polyploidy
inducing agent and a Bcl-2 family protein inhibitor, the method comprising the steps of:
(a) providing a test sample (e.g. , such as a tissue sample) from a patient;
(b) determining the presence or absence of at least one mutation in a (i) a human
Bcl-2 pro-apoptotic encoding gene (such as a human BAX gene and a human BAK
gene), (ii) a human NOXA gene, or (iii) a human Bcl-2 pro-apoptotic encoding gene
(such as a human BAX gene and a human BAK gene) and a human NOXA gene; and
(c) classifying the patient as having a cancer that is resistant to a polyploidy
inducing agent, a Bcl-2 family protein inhibitor or a combination of a polyploidy
inducing agent and a Bcl-2 family protein inhibitor based on the presence of at least one
mutation in a (i) a human Bcl-2 pro-apoptotic encoding gene (such as a human BAX gene
and a human BAK gene), (ii) a human NOXA gene, or (iii) a human Bcl-2 pro-apoptotic
encoding gene (such as a human BAX gene and a human BAK gene) and a human
NOXA gene. As with the methods mentioned previously herein, this method can be
performed before or after induction of polyploidy in the patient.
In this aspect, the cancer can be any type of cancer, such as colorectal carcinoma
or pancreatic cancer. Moreover, in this aspect, the gene amplification can be determined
by a multi-color fluorescent in situ hybridization (FISH) assay, for example, performed
on a lung cancer tumor biopsy sample. In other aspects, the polymerase chain reaction
(PCR) is used.
In still yet another aspect, the disclosure is directed to methods for monitoring a
patient being treated with a polyploidy inducing agent, a Bcl-2 family protein inhibitor or
a combination of a polyploidy inducing agent and a Bcl-2 family protein inhibitor, the
method comprising the steps of:
(a) providing a test sample from a cancer patient being treated with at least one
polyploidy inducing agent, a Bcl-2 family protein inhibitor or a combination of a
polyploidy inducing agent and a Bcl-2 family protein inhibitor (optionally, tumor or
cancer cells obtained from a tissue sample can be identified or extracted);
(b) determining in the test sample (for example, in the tumor or cancer cells) the
presence or absence of at least one mutation in (i) a human Bcl-2 pro-apoptotic encoding
gene (such as a human BAX gene and a human BAK gene), (ii) a human NOXA gene, or
(iii) a human Bcl-2 pro-apoptotic encoding gene (such as a human BAX gene and a
human BAK gene) and a human NOXA gene; and
(c) comparing the number of mutations in (i) a human Bcl-2 pro-apoptotic
encoding gene (such as a human BAX gene and a human BAK gene), (ii) a human
NOXA gene, or (iii) a human Bcl-2 pro-apoptotic encoding gene (such as a human BAX
gene and a human BAK gene) and a human NOXA gene from the test sample (such as in
the tumor or cancer cells) against a baseline level or a predetermined level; and
(d) determining whether the patient should continue to be treated with the
polyploidy inducing agent, Bcl-2 family protein inhibitor or a combination of a
polyploidy inducing agent and Bcl-2 family protein inhibitor based on the comparison in
step (c). As with the methods mentioned previously herein, this method can be
performed before or after induction of polyploidy in the patient.
The comparison (or informational analysis) of the number of one or more
mutations determined from the test sample with the baseline or predetermined level can
be done by an automated system, such as a software program or intelligence system that
is part of, or compatible with, the equipment (e.g., computer platform) on which the assay
is carried out. Alternatively, this comparison or informational analysis can be done by a
physician.
Specifically, if the test sample (e.g., the tumor or cancer cells) having at least one
mutation in (i) a human Bcl-2 pro-apoptotic encoding gene (such as a human BAX gene
and a human BAK gene), (ii) a human NOXA gene, or (iii) a human Bcl-2 pro-apoptotic
encoding gene (such as a human BAX gene and a human BAK gene) and a human
NOXA gene is the same as or higher then the baseline level or predetermined level, then
treatment with the polyploidy inducing agent, Bcl-2 family protein inhibitor or
combination of a polyploidy inducing agent and a Bcl-2 family protein inhibitor can be
discontinued, stopped or terminated. Alternatively, the treating physician may decide to
combine the polyploidy inducing agent with at least a second therapy (for example,
treatment with a second small molecule) as a combination therapy. Still further
alternatively, the treating physician may decide to combine the Bcl-2 family protein
inhibitor with at least a second therapy (for example, treatment with a second small
molecule) as a combination therapy. However, if at least one mutation in (i) a human
Bcl-2 pro-apoptotic encoding gene (such as a human BAX gene and a human BAK
gene), (ii) a human NOXA gene, or (iii) a human Bcl-2 pro-apoptotic encoding gene
(such as a human BAX gene and a human BAK gene) and a human NOXA gene is less
then the baseline level or the predetermined level or if no mutations are detected in (i) a
human Bcl-2 pro-apoptotic encoding gene (such as a human BAX gene and a human
BAK gene), (ii) a human NOXA gene, or (iii) a human Bcl-2 pro-apoptotic encoding
gene (such as a human BAX gene and a human BAK gene) and a human NOXA gene
then treatment with the polyploidy inducing agent, a Bcl-2 family protein inhibitor or a
combination of a polyploidy inducing agent and a Bcl-2 family protein inhibitor can be
continued. Again, depending on the results obtained with said treatment, the treating
physician may decide to combine the polyploidy inducing agent with at least a second
therapy (for example, treatment with a second small molecule) as a combination therapy.
Alternatively, depending on the results obtained with said treatment, the treating
physician may decide to combine the Bcl-2 family protein inhibitor therapy with at least
a second therapy (for example, treatment with a second small molecule).
Again, FISH and PCR methods can be used to detect the presence or absence of at
least one mutation in (i) a human Bcl-2 pro-apoptotic encoding gene (such as a human
BAX gene and a human BAK gene), (ii) a human NOXA gene, or (iii) a human Bcl-2
pro-apoptotic encoding gene (such as a human BAX gene and a human BAK gene) and a
human NOXA gene in a test sample obtained from a patient.
The disclosure is also directed to kits that package, for example, oligo- or
polynucleotides engineered to be used as PCR primers, FISH probes, etc.
The disclosure has significant capability to provide improved stratification of
patients for cancer therapy, and in particular for polyploidy inducing agent therapy, Bcl-2
family protein inhibitor therapy or a combination of polyploidy inducing agent therapy
and Bcl-2 family protein inhibitor therapy. The assessment of these biomarkers
according to the present disclosure also allows tracking of individual patient response to
the therapy.
A. Definitions
Section headings as used in this section and the entire disclosure herein are not
intended to be limiting.
As used herein, the singular forms "a," "an" and "the" include plural referents
unless the context clearly dictates otherwise. For the recitation of numeric ranges herein,
each intervening number there between with the same degree of precision is explicitly
contemplated. For example, for the range 6-9, the numbers 7 and 8 are contemplated in
addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6,
6.7, 6.8, 6.9 and 7.0 are explicitly contemplated.
a) Antitumorigenesis
As used herein, the term "antitumorigenesis," refers to a reduction of tumor
growth.
b) Aurora Kinase Inhibitor
An "Aurora Kinase inhibitor" refers to a therapeutic compound of any type (e.g.,
non-selective or selective), including small molecule, antibody, antisense, small
interfering R A, or microRNA-based compounds, that binds to at least one of Aurora
Kinase A, Aurora A, Aurora Kinase B, Aurora B, Aurora Kinase C or Aurora C, and
antagonizes the activity of at least one Aurora Kinase A, Aurora A, Aurora Kinase B,
Aurora B, Aurora Kinase C or Aurora C related nucleic acid or protein.
c) Aurora Kinase A Inhibitor
An "Aurora Kinase A inhibitor" refers to a therapeutic compound of any type
(e.g., non-selective or selective), including small molecule, antibody, antisense, small
interfering RNA, or microRNA-based compounds, that binds to at least one of Aurora
Kinase A or Aurora A, and antagonizes the activity of the Aurora Kinase A or Aurora A
related nucleic acid or protein. The methods of the present disclosure are useful with any
known or hereafter developed Aurora Kinase A inhibitor. Examples of an Aurora Kinase
A inhibitor are PHA-739358, MLN-8054, R-763, JNJ-7706621, MP-529 and MP-235.
) Aurora Kinase B Inhibitor
An "Aurora Kinase B inhibitor" refers to a therapeutic compound of any type
(e.g., non-selective or selective), including small molecule, antibody, antisense, small
interfering R A, or microRNA-based compounds, that binds to at least one of Aurora
Kinase B or Aurora B, and antagonizes the activity of the Aurora Kinase B or Aurora B
related nucleic acid or protein. For example, a number of Aurora Kinase B inhibitors are
known to inhibit at least one of histone H3 phosphorylation or cell division. In addition,
a number of Aurora Kinase B inhibitors are known to induce apoptosis in at least one cell
system (such as an acute myeloid leukemia cell line, a primary acute myeloid leukemia
culture, etc.). The methods of the present disclosure are useful with any known or
hereafter developed Aurora Kinase B inhibitor. Examples of an Aurora Kinase B
inhibitor are AZD 11 2, ZM447439, VX-680/MK0457 and Hesperadin.
AZDl 152, also known as, 2-[[3-({4-[(5-{2-[(3-Fluorophenyl)amino]-2-
oxoethyl}-l H-pyrazol-3-yl)amino]-quinazolin-7-yl}oxy)propyl](ethyl)amino]ethyl
dihydrogen phosphate, is a prodrug of a pyrazoloquinazoline Aurora Kinase inhibitor
(AZDl 152-hydroxyquinazo line pyrazol anilide (HQPA)) and is converted rapidly to the
active AZDl 152-HQPA in plasma (See, Mortlock, AA, et al, J. Med. Chem., 50:2213-24
(2007)). AZDl 152-HQPA is a highly potent and selective inhibitor of Aurora B.
ZM447439, also known as 4-(4-(N-benzoylamino)anilino)-6-methoxy-7-(3-(lmorpholino)
propoxy)quinazoline, is a quinazoline derivative, inhibits Aurora A and
Aurora B. The chemical structure of ZM447439 is provided in Ditchfield, C , et al, J.
Cell Bio., 161(2):267-280 (2003) and Montembault, E., et al., Drugs of the Future,
30(1): 1-9 (2005).
VX-680/MK0457 is a cyclopropane carboxylic acid of {4-[4-(4-methyl-piperazinl-
yl)-6-(5-methyl-2H-pyrazol-3-ylamino)-pyrimidin-2-ylsulphanyl]-phenyl}-amide and
inhibits Aurora A, Aurora B and Aurora C. The chemical structure of VX-680/MK0457
is provided in Montembault, E., et al, Drugs of the Future, 30(1): 1-9 (2005).
Hesperadin, an indolinone, inhibits Aurora B. The chemical structure of
Hesperadin is provided in Hauf, S., et al., J. Cell Bio., 161(2):28 1-294 (2003) and
Montembault, E., et al, Drugs of the Future, 30(1): 1-9 (2005).
e) Aurora Kinase C Inhibitor
An "Aurora Kinase C inhibitor" refers to a therapeutic compound of any type
(e.g., non-selective or selective), including small molecule, antibody, antisense, small
interfering R A, or microRNA-based compounds, that binds to at least one of Aurora
Kinase C or Aurora C, and antagonizes the activity of the Aurora Kinase C or Aurora C
related nucleic acid or protein. The methods of the present disclosure are useful with any
known or hereafter developed Aurora Kinase C inhibitor. Examples of an Aurora Kinase
C inhibitor are AZD1 1 2 and VX-680/MK-0457.
f) Consisting Essentially of a Polynucleotide Having a %Sequence Identity
"Consisting essentially of a polynucleotide having a % sequence identity" means
that the polynucleotide does not substantially differ in length, but may differ substantially
in sequence. Thus, a polynucleotide "A" consisting essentially of a polynucleotide
having at least 80% sequence identity to a known sequence "B" of 100 nucleotides means
that polynucleotide "A" is about 100 nucleotides (nts) long, but up to 20 nts can vary
from the "B" sequence. The polynucleotide sequence in question can be longer or
shorter due to modification of the termini, such as, for example, the addition of 1-15
nucleotides to produce specific types of probes, primers and other molecular tools, etc.,
such as the case of when substantially non-identical sequences are added to create
intended secondary structures. Such non-identical nucleotides are not considered in the
calculation of sequence identity when the sequence is modified by "consisting essentially
of."
g) Bcl-2
As used herein, the term "Bcl-2" refers to a family of pro- and anti-apoptotic
proteins that constitute a critical control point for apoptosis. Members of this family
include both pro- and anti-apoptotic proteins and share homology in up to four conserved
regions termed Bcl-2 homology (BH) 1-4 domains. The family can be divided into three
main sub-classes: anti-apoptotic proteins, pro-apoptotic proteins, "BH3-only" proteins.
The anti-apoptotic proteins, which include Bcl-2 and B C1-XL, are all
"multidomain," sharing homology throughout all four BH domains. However, the proapoptotic
proteins can be further subdivided and include multidomain proteins, such as
BAX and BAK, which possess sequence homology in BH1-3 domains. The more
distantly related "BH3-only" proteins are to date all pro-apoptotic and share sequence
homology within the amphipathic a-helical BH3 region, which is required for their
apoptotic function.
The human BAK gene encoding human BAK has Accession no. U23765, and is
described in Chittenden et al. Nature 374:733-736 (1995). The human BAK-2 gene has
Accession no. U16812, and is described in Kiefer et al., Nature 374:736-739 (1995). The
human BAX genes encoding human BAX have Accession nos. L22475, L22474 and
L22473, and are described in Oltvai et al, Cell 74:609-619 (1993).
As used herein, the phrase "human Bcl-2 pro-apoptotic encoding gene" refers to a
gene that encodes at least one human Bcl-2 pro-apoptotic protein or fragment thereof
such as, for example, human BAK or human BAX.
The "BH3-only proteins" constitute the third subset of the Bcl-2 family and
include, for example, BID, NOXA, PUMA, BIK, BIM and BAD. These proteins share
sequence homology only in the amphipathic -helical BH3 region which mutation analysis
indicated is required in pro-apoptotic members for their death activity.
The human BID gene encoding human BID has Accession no. NM l 97966,
NM_197967, NM_001 196 and is described in Genbank. The human NOXA gene
encoding human NOXA has Accession no. NM_021 127 and is described in Genbank.
The human PUMA gene encoding human PUMA has Accession no. NM_001 127242,
NM_001 127241, NM_001 127240, NM_014417 and is described in Genbank. The
human BIK gene encoding human BIK has Accession no. NM 001 197 and is described
in Genbank. The human BIM gene encoding human BIM has Accession no.
NMJ38621, NM_207002, NM_006538, BC033694, AY305714, AY305716,
AY423443, AY423442, AY423441 and is described in Genbank. The human BAD gene
encoding human BAD has Accession no. NM_032989, NM_004322, BC095431 and is
described in Genbank.
As used herein, the phrase, "human BH3 encoding gene" refers to a gene that
encodes at least one human Bcl-2 BH3-only protein or fragment thereof, such as, for
example, human BID, human NOXA, human PUMA, human BIK, human BIM and
human BAD.
h) Bcl-2 Family Protein Inhibitor, Bcl-2 Family Inhibitor or Inhibitor of a
Bcl-2 Family Protein
As used herein, the phrases "Bcl-2 Family Protein Inhibitor", "Bcl-2 Family
Inhibitor" or "Inhibitor of a Bcl-2 Family Protein" as used interchangeably herein, refer
to a therapeutic compound of any type (e.g., non-selective or selective), including small
molecule-, antibody-, antisense-, small interfering RNA, or microRNA-based
compounds, that binds and antagonizes or inhibits the activity of at least one gene or
protein of the Bcl-2 family that governs mitochondrial outer membrane permeabilization
(MOMP) and is anti-apoptotic (such as Bcl-2, Bcl-X L, Bcl-w, Bcl-B, BFL-1 and MCI-1).
Examples of Bcl-2 family protein inhibitors include the compounds described in Section
B herein (namely, ABT-263 (which is N-(4-(4-((2-(4-chlorophenyl)-5,5-dimethyl-lcyclohex-
1-en- 1-yl)methy l)piperazin- 1-yl)benzoyl)-4-((( 1R)-3 -(morpholin-4-yl)- 1-
((phenylsulfanyl)methyl)propyl)amino)-3-
((trifluoromethyl)sulfonyl)benzenesulfonamide)), ABT-737 (N-(4-(4-((4'-chloro(l ,1'-
biphenyl)-2-yl)methyl)piperazin- 1-yl)benzoyl)-4-((( 1R)-3 -(dimethylamino)- 1-
((phenylsulfanyl)methyl)propyl)amino)-3-nitrobenzenesulfonamide, described in
published US Patent Application Nos. 20050159427 and 20060128706, Bcl-X L inhibitors
\ and combinations thereof.
i) Bcl-XL selective inhibitor(s)
As used herein, the phrase "BC1-XL selective inhibitor(s)", refers to a B C1-XL
inhibitor that exhibits a selectivity for Bcl-XL Over Bcl-2. Methods for determining
whether a B C1-XL inhibitor is a B C1-XL selective inhibitor are well known in the art.
Specifically, such methods involve determining the inhibition constants (Ki) for the Bcl-
X L inhibitor compounds and the binding selectivity ratio (for example, B C1-XL K;:Bcl-2
Ki). The inhibition constant (Ki) is the dissociation constant of an enzyme-inhibitor
complex or a protein/small molecule complex, wherein the small molecule is inhibiting
binding of one protein to another protein or peptide. Inhibition constants can be
determined using Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET)
assay. Where the K for a compound is represented as ">" (greater than) a certain
numerical value, it is intended to mean that the binding affinity value (e.g., for B C1-XL) is
greater than that determined in the assay. Where the binding selectivity ratio for a
compound is represented as ">" (greater than) a certain numerical value, it is intended to
mean that the selectivity of a particular compound for Bcl-XL over Bcl-2 is at least as
great as the number indicated. Where the ¾ for a compound is represented as "<" (less
than) a certain numerical value, it is intended to mean that the binding affinity value (e.g.,
for Bcl-2) is lower than the limit of detection of the assay used. Inhibition constants were
determined using Wang's equation (Wang Zx,. An Exact Mathematical Expression For
Describing Competitive Binding Of Two Different Ligands To A Protein Molecule.
FEBSLett. 1995, 360:1 11-4).
j) Expression, Antisense Inhibition and Co-Suppression
"Expression" refers to the production of a functional end-product. Expression of
a gene involves transcription of the gene and translation of the mRNA into a precursor or
mature protein. "Antisense inhibition" refers to the production of antisense RNA
transcripts capable of suppressing the expression of the target protein. "Co-suppression"
refers to the production of sense RNA transcripts capable of suppressing the expression
of identical or substantially similar foreign or endogenous genes (U.S. Patent No.
5,231,020).
k) Isolated
As used herein, the term "isolated" in the context of nucleic acid molecules or
polynucleotides refers to a nucleic acid molecule or polynucleotide which is separated
from other nucleic acid molecules or polynucleotides which are present in the natural
source of the nucleic acid molecule or polynucleotide. Moreover, an "isolated" nucleic
acid molecule or polynucleotide, such as a cDNA molecule, can be substantially free of
other cellular material, or culture medium when produced by recombinant techniques, or
substantially free of chemical precursors or other chemicals when chemically
synthesized. In one aspect, nucleic acid molecules or polynucleotides are isolated.
1) Gene
"Gene" refers to a nucleic acid fragment that expresses a specific protein,
including regulatory sequences preceding (5' non-coding sequences) and following (3'
non-coding sequences) the coding sequence.
m) Inhibition Constant or Ki
As used herein, "inhibition constant" or "Ki" refers the dissociation constant of
an enzyme-inhibitor complex or a protein/small molecule complex, wherein the small
molecule is inhibiting binding of one protein to another protein. A large Ki value
indicates a low binding affinity, and a small Ki value indicates a high binding affinity.
Ki can be determined using any method known in the art, such as by using Wang's
equation (Wang Z X,. An Exact Mathematical Expression For Describing Competitive
Binding Of Two Different Ligands To A Protein Molecule. FEBSLett. 1995; 360:1 11-
4). A typical measure of binding affinity of an anti-apoptotic protein inhibitor is the
balance between the binding and dissociation processes between the protein and the
inhibitor (Ki).
n) Native Gene and Chimeric Construct
"Native gene" refers to a gene as found in nature with its own regulatory
sequences. In contrast, "chimeric construct" refers to a combination of nucleic acid
fragments that are not normally found together in nature. Accordingly, a chimeric
construct may comprise regulatory sequences and coding sequences that are derived from
different sources, or regulatory sequences and coding sequences derived from the same
source, but arranged in a manner different than that normally found in nature.
o) Percent (%) Nucleic Acid Sequence Identity
"Percent (%) nucleic acid sequence identity" with respect to nucleic acid
sequences is defined as the percentage of nucleotides in a candidate sequence that are
identical with the nucleotides in the sequence of interest, after aligning the sequences and
introducing gaps, if necessary, to achieve the maximum percent sequence identity.
Alignment for purposes of determining % nucleic acid sequence identity can be achieved
in various ways that are within the skill in the art, for instance, using publicly available
computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)
software. Those skilled in the art can determine appropriate parameters for measuring
alignment, including any algorithms needed to achieve maximal alignment over the full
length of the sequences being compared.
When nucleotide sequences are aligned, the % nucleic acid sequence identity of a
given nucleic acid sequence C to, with, or against a given nucleic acid sequence D
(which can alternatively be phrased as a given nucleic acid sequence C that has or
comprises a certain % nucleic acid sequence identity to, with, or against a given nucleic
acid sequence D) can be calculated as follows:
% nucleic acid sequence identity = W/Z* 100
where
W is the number of nucleotides scored as identical matches by the sequence
alignment program's or algorithm's alignment of C and D
and
Z is the total number of nucleotides in D.
When the length of nucleic acid sequence C is not equal to the length of nucleic
acid sequence D, the % nucleic acid sequence identity of C to D will not equal the %
nucleic acid sequence identity of D to C.
p) Polymerase Chain Reaction or PCR
"Polymerase Chain Reaction" or "PCR", a technique for the synthesis of large
quantities of specific DNA segments, consists of a series of repetitive cycles (Perkin
Elmer Cetus Instruments, Norwalk, CT). Typically, the double stranded DNA is heatdenatured,
the two primers complementary to the 3' boundaries of the target segment are
annealed at low temperature and then extended at an intermediate temperature. One set
of these three consecutive steps is referred to as a cycle.
PCR is a powerful technique used to amplify DNA millions of fold, by repeated
replication of a template, in a short period of time. ( (Mullis, K., et al, Cold Spring Harb
Symp Quant Biol. 5 1 Pt 1:263-73 (1986)); European Patent Application No. 50,424;
European Patent Application No. 84,796; European Patent Application No. 258,017,
European Patent Application No. 237,362; European Patent Application No. 201,184,
U.S. Patent No. 4,683,202; U.S. Patent No. 4,582,788; and U.S. Patent No. 4,683,194).
The process uses sets of specific in vitro synthesized oligonucleotides to prime DNA
synthesis. The design of the primers is dependent upon the sequences of DNA that are to
be analyzed. The technique is carried out through many cycles (usually 20-50) of
melting the template at high temperature, allowing the primers to anneal to
complementary sequences within the template and then replicating the template with
DNA polymerase.
The products of PCR reactions can be analyzed by separation in agarose gels
followed by ethidium bromide staining and visualization with UV transillumination.
Alternatively, radioactive dNTPs can be added to the PCR in order to incorporate label
into the products. In this case the products of PCR are visualized by exposure of the gel
to x-ray film. The added advantage of radiolabeling PCR products is that the levels of
individual amplification products can be quantitated.
q) Polynucleotide
A "polynucleotide" is a nucleic acid polymer of ribonucleic acid (RNA),
deoxyribonucleic acid (DNA), modified RNA or DNA, or RNA or DNA mimetics (such
as PNAs), and derivatives thereof, and homologues thereof. Thus, polynucleotides
include polymers composed of naturally occurring nucleic bases, sugars and covalent
inter-nucleoside (backbone) linkages as well as polymers having non-naturally-occurring
portions that function similarly. Such modified or substituted nucleic acid polymers are
well known in the art and are referred to as "analogues." Oligonucleotides are generally
short polynucleotides from about 10 to up to about 160 or 200 nucleotides.
Polynucleotides also comprise primers that specifically hybridize to target
sequences, including analogues and/or derivatives of the nucleic acid sequences, and
homologues thereof.
Polynucleotides can be prepared by conventional techniques, such as solid-phase
synthesis using commercially available equipment, such as that available from Applied
Biosystems USA Inc. (Foster City, CA; USA), DuPont, (Wilmington, DE; USA), or
Milligen (Bedford, MA; USA). Modified polynucleotides, such as phosphorothioates
and alkylated derivatives, can also be readily prepared by similar methods known in the
art (See, U.S. Patent Nos. 4,948,882, 5,464,746, and 5,424,414).
r) Polynucleotide Analogues
As used herein, the term "polynucleotide analogues" refers to polymers having
modified backbones or non-natural inter-nucleoside linkages. Modified backbones
include those retaining a phosphorus atom in the backbone, such as phosphorothioates,
chiral phosphorothioates, phosphorodithioates, phosphotriesters,
aminoalkylphosphotriesters, methyl and other alkyl phosphonates, as well as those no
longer having a phosphorus atom, such as backbones formed by short chain alkyl or
cycloalkyl inter-nucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside
linkages, or one or more short chain heteroatomic or heterocyclic internucleoside
linkages. Modified nucleic acid polymers (analogues) can contain one or
more modified sugar moieties.
Analogs that are RNA or DNA mimetics, in which both the sugar and the internucleoside
linkage of the nucleotide units are replaced with novel groups, are also useful.
In these mimetics, the base units are maintained for hybridization with the target
sequence. An example of such a mimetic, which has been shown to have excellent
hybridization properties, is a peptide nucleic acid (PNA) (See, Buchardt, 0., P. Nielsen,
and R. Berg. 1992. Peptide Nucleic Acids).
s) Polyploidy Inducing Agent
As used herein, the phrase "polyploidy inducing agent" refers to a therapeutic
compound of any type (e.g., non-selective or selective), including small molecule,
antibody, antisense, small interfering RNA, or microRNA-based compounds, that induce
polyploidy in one or more cells. Methods for determining the induction or evidence of
polyploidy in one or more cells can be obtained using routine techniques known in the
art. For example, evidence of polyploidy can be determined by detecting elevated
expression of p53. p53 is a surrogate for polyploidization in cells harboring wildtype p53
(See, Gizatullin, F. et al, "The Aurora Kinase inhibitor VX-680 induces
endoreduplication and apoptosis preferentially in cells with compromised p53-dependent
postmitotic checkpoint function," Cancer Res. 66, 7668-77. (2006)). Additionally, cells
in which polyploidy has been induced exhibit a gross morphological increase in cell size
and multinucleation, both of which can be detected using routine techniques known in the
art.
Examples of polyploidy inducing agents, include, but are not limited to, Aurora
Kinase inhibitors, microtubule inhibitors (such as, for example, Taxotere, vincristine,
nocodazole, paclitaxel or colcemid), pan-kinase inhibitors (such as, for example,
staurosporine), oncolytic viruses (such as, for example, ONYX-015), Acridine orange,
Dolastain-10, Noscapine, toposiomerase II inhibitors (such as, for example, ICRF-187 or
ICRF-193), 2-{4-[(7-chloro-2-quinoxalinyl)oxy]phenoxy}propionic acid, 2-{4-[(7-
bromo-2-quinolinyl)oxy]phenoxy}propionic acid, Platycodin D, microtubule poisons
(such as, for example, JG-03-14), actin polymerization inhibitors (such as, for example,
Cytochalasin B), Bistramide A or antitumor antibiotics (such as, for example,
Mithramycin SKI).
t) Predetermined Level
As used herein, the term "predetermined level" refers generally to an assay cut-off value
that is used to assess diagnostic results by comparing the assay results against the
predetermined level, and where the predetermined level already has been linked or
associated with various clinical parameters (e.g., assessing risk, severity of disease,
progression/non-progression/improvement, determining the age of a test sample,
determining whether a test sample (e.g., serum or plasma) has hemolyzed, etc.). An
example of a predetermined level that can be used is a baseline level obtained from one
or more subjects that may optionally be suffering from one or more diseases or
conditions. It is well known that cutoff values may vary dependent on the nature of the
assay. It further is well within the ordinary skill in the art to adapt the disclosure herein
for other assays to obtain assay-specific cut-off values for those other assays based on
this description.
u) Primer or Probe
A "probe" or "primer" as used herein is a polynucleotide that is at least 8
nucleotides in length and forms a hybrid structure with a target sequence, due to
complementarity of at least one sequence in the probe or primer with a sequence in the
target region. The polynucleotide regions of the probe can be composed of DNA and/or
RNA and/or synthetic nucleotide analogs. Preferably, the probe does not contain a
sequence that is complementary to the sequence or sequences used to prime for a target
sequence during the polymerase chain reaction.
v) Recombinant
"Recombinant" refers to an artificial combination of two otherwise separated
segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated
segments of nucleic acids by genetic engineering techniques.
w) Specifically hybridize
"Specifically hybridize" refers to the ability of a nucleic acid to bind detectably
and specifically to a second nucleic acid. Polynucleotides specifically hybridize with
target nucleic acid strands under hybridization and wash conditions that minimize
appreciable amounts of detectable binding by non-specific nucleic acids.
x) Stringency or Stringent Conditions
The specificity of single stranded DNA to hybridize complementary fragments is
determined by the stringency of the reaction conditions. Hybridization stringency
increases as the propensity to form DNA duplexes decreases. In nucleic acid
hybridization reactions, the stringency can be chosen to favor specific hybridizations
(high stringency). Less-specific hybridizations (low stringency) can be used to identify
related, but not exact, DNA molecules (homologous, but not identical) or segments.
DNA duplexes are stabilized by: (1) the number of complementary base pairs, (2)
the type of base pairs, (3) salt concentration (ionic strength) of the reaction mixture, (4)
the temperature of the reaction, and (5) the presence of certain organic solvents, such as
formamide, which decrease DNA duplex stability. A common approach is to vary the
temperature: higher relative temperatures result in more stringent reaction conditions
(See, Ausubel, F.M., R. Brent, R.E. Kingston, et al. 1987. Current Protocols in
Molecular Biology. John Wiley & Sons, New York) provide an excellent explanation of
stringency of hybridization reactions.
Hybridization under "stringent conditions" means hybridization protocols in
which nucleotide sequences at least 60% homologous to each other remain hybridized.
Polynucleotides can include other appended groups such as peptides (e.g., for targeting
host cell receptors in vivo), or agents facilitating transport across the cell membrane. In
addition, oligonucleotides can be modified with hybridization-triggered cleavage agents
(See, van der Krol et al., Biotechniques. 6:958-76 (1988) or intercalculating agents (Zon,
G., Pharm Res. 5:539-49 (1988)). The oligonucleotide can be conjugated to another
molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent,
a hybridization-triggered cleavage agent, and the like
y) Subject(s) or Patient(s)
As used herein, the terms "subject" and "patient" are used interchangeably
irrespective of whether the subject has or is currently undergoing any form of treatment.
As used herein, the terms "subject" and "subjects" refer to any vertebrate, including, but
not limited to, a mammal (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep,
hamsters, guinea pig, cat, dog, rat, and mouse, a non-human primate (for example, a
monkey, such as a cynomolgous monkey, chimpanzee, etc) and a human). Preferably,
the subject is a human. Subjects or patients can be living or expired
z) Target Sequence or Target Nucleic Acid Sequence
"Target sequence" or "target nucleic acid sequence" means a nucleic acid
sequence encompassing, for example, a gene, or complements or fragments thereof, that
is amplified, detected, or both using a polynucleotide primer or probe. Additionally,
while the term target sequence sometimes refers to a double stranded nucleic acid
sequence; a target sequence can also be single-stranded. In cases where the target is
double-stranded, polynucleotide primer sequences preferably amplify both strands of the
target sequence. A target sequence can be selected that is more or less specific for a
particular organism. For example, the target sequence can be specific to an entire genus,
to more than one genus, to a species or subspecies, serogroup, auxotype, serotype, strain,
isolate or other subset of organisms.
aa) Test sample
"Test sample" means a sample taken from a subject, or a biological fluid, wherein
the sample may contain a target sequence. A test sample can be taken from any source,
for example, tissue, blood, saliva, sputa, mucus, sweat, urine, urethral swabs, cervical
swabs, urogenital or anal swabs, conjunctival swabs, ocular lens fluid, cerebral spinal
fluid, etc. A test sample can be used (i) directly as obtained from the source; or (ii)
following a pre-treatment to modify the character of the sample. Thus, a test sample can
be pre-treated prior to use by, for example, preparing plasma or serum from blood,
disrupting cells or viral particles, preparing liquids from solid materials, diluting viscous
fluids, filtering liquids, adding reagents, purifying nucleic acids, etc.
bb) Therapeutically Effective Amount
The term "therapeutically effective amount" means an amount of drug, which is
effective for producing a desired therapeutic effect upon administration to a patient, for
example, to stem the growth, or result in the shrinkage, of a cancerous tumor or to
produce the death of a cancerous cell or tumor.
cc) "Time Resolved-Fluorescence Resonance Energy Transfer"
As used herein, the phrase "Time Resolved-Fluorescence Resonance Energy Transfer" or
"TR-FRET" refers to an assay that unites the principles of TRF (Time-Resolved
Fluorescence) and FRET (Fluorescence Resonance Energy Transfer). A number of TRFRET
assays are known and commercially available in the art. An example of a TRFRET
that can be used in the present invention is described below.
Probe Synthesis
All reagents described below for use can be obtained from the vendor unless
otherwise specified. Peptide synthesis reagents including diisopropylethylamine (DIEA),
dichloromethane (DCM), N-methylpyrrolidone (NMP), 2-(lH-benzotriazole-l-yl)-
1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), N-hydroxybenzotriazole
(HOBt) and piperidine can be obtained from Applied Biosystems, Inc. (ABI), Foster
City, CA or American Bioanalytical, Natick, MA. Preloaded 9-
Fluorenylmethyloxycarbonyl (Fmoc) amino acid cartridges (Fmoc-Ala-OH, Fmoc-
Cys(Trt)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Phe-OH, Fmoc-Gly-OH,
Fmoc-His(Trt)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Met-OH,
Fmoc-Asn(Trt)-OH, Fmoc-Pro-OH, Fmor-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-
Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Val-OH, Fmoc-Trp(Boc)-OH, Fmoc-Tyr(tBu)-
OH) can be obtained from ABI or Anaspec, San Jose, CA. The peptide synthesis resin
(Fmoc-Rink amide MBHA resin) and Fmoc-Lys(Mtt)-OH can be obtained from
Novabiochem, San Diego, CA. Single-isomer 6-carboxyfluorescein succinimidyl ester
(6-FAM-NHS) can be obtained from Anaspec. Trifluoroacetic acid (TFA) can be
obtained from Oakwood Products, West Columbia, SC. Thioanisole, phenol,
triisopropylsilane (TIS), 3,6-dioxa-l,8-octanedithiol (DODT) and isopropanol can be
obtained from Aldrich Chemical Co., Milwaukee, WI. Matrix-assisted laser desorption
ionization mass-spectra (MALDI-MS) can be recorded on an Applied Biosystems
Voyager DE-PRO MS). Electrospray mass-spectra (ESI-MS) can be recorded on
Finnigan SSQ7000 (Finnigan Corp., San Jose, CA) in both positive and negative ion
mode.
General Procedure For Solid-Phase Peptide Synthesis (SPPS)
Peptides can be synthesized with, at most, 250 mihoΐ preloaded Wang resin/vessel
on an ABI 433A peptide synthesizer using 250 m oΐ scale Fastmoc™ coupling cycles.
Preloaded cartridges containing 1mmol standard Fmoc-amino acids, except for the
position of attachment of the fluorophore, where 1mmol Fmoc-Lys(Mtt)-OH can be
placed in the cartridge, and used with conductivity feedback monitoring. N-terminal
acetylation can be accomplished by using 1 mmol acetic acid in a cartridge under
standard coupling conditions.
Removal of 4-Methyltrityl (Mtt) From Lysine
The resin from the synthesizer can be washed thrice with DCM and kept wet.
150 mL of 95:4:1 dichloromethane:triisopropylsilane:trifluoroacetic acid can be flowed
through the resin bed over 30 minutes. The mixture should turn deep yellow and then fad
to pale yellow. 100 mL of DMF can be flowed through the bed over 15 minutes. The
resin can then be washed thrice with DMF and filtered. Ninhydrin tests should show a
strong signal for primary amine.
Resin Labeling With 6-Carboxyfluorescein-NHS (6-FAM-NHS)
The resin can be treated with 2 equivalents 6-FAM-NHS in 1% DIEA/DMF and
stirred or shaken at ambient temperature overnight. When completed, the resin can be
drained, washed thrice with DMF, thrice with (1% DCM and 1% methanol) and dried to
provide an orange resin that was negative by ninhydrin test.
General Procedure For Cleavage And Deprotection Of Resin-Bound Peptide
Peptides can be cleaved from the resin by shaking for 3 hours at ambient
temperature in a cleavage cocktail consisting of 80% TFA, 5% water, 5% thioanisole, 5%
phenol, 2.5% TIS, and 2.5% EDT ( 1 mL/0.1 g resin). The resin can be removed by
filtration and rinsing twice with TFA. The TFA can be evaporated from the filtrates, and
product precipitated with ether (10 mL/0.1 g resin), recovered by centrifugation, washed
twice with ether (10 mL/0.1 g resin) and dried to give the crude peptide.
General Procedure For Purification Of Peptides
The crude peptides can be purified on a Gilson preparative HPLC system running
Unipoint® analysis software (Gilson, Inc., Middleton, WI) on a radial compression
column containing two 25 x 100 mm segments packed with Delta-Pak™ C18 15 mih
particles with 100 Apore size and eluted with one of the gradient methods listed below.
One to two milliliters of crude peptide solution (10 mg/mL in 90% DMSO/water) can be
purified per injection. The peaks containing the product(s) from each run can be pooled
and lyophilized. All preparative runs can be run at 20 mL/min with eluents as buffer A:
0.1% TFA-water and buffer B: acetonitrile.
General Procedure For Analytical HPLC
Analytical HPLC can be performed on a Hewlett-Packard 1200 series system with
a diode-array detector and a Hewlett-Packard 1046A fluorescence detector running
HPLC 3D ChemStation software version A.03 .04 (Hewlett-Packard. Palo Alto, CA) on a
4.6 x 250 mm YMC column packed with ODS-AQ 5 m h particles with a 120 Apore size
and eluted with one of the gradient methods listed below after preequilibrating at the
starting conditions for 7 minutes. Eluents can be buffer A: 0.1% TFA-water and buffer
B: acetonitrile. The flow rate for all gradients may be 1 mL/minute.
F-Bak: Peptide Probe: Acetyl-(SEQ ID NO: 1)GQVG QLAIIGDK(6-FAM)-(SEQ ID
NO: 2) INR-NH .
Fmoc-Pvink amide MBHA resin can be extended using the general peptide
synthesis procedure to provide the protected resin-bound peptide ( 1.020 g). The Mtt
group can be removed, labeled with 6-FAM-NHS and cleaved and deprotected as
described hereinabove to provide the crude product as an orange solid (0.37 g). This
product can be purified by RP-HPLC. Fractions across the main peak can be tested by
analytical RP-HPLC, and the pure fractions can be isolated and lyophilized, with the
major peak providing the title compound (0.0802 g) as a yellow solid; MALDI-MS m z =
2137.1 ((M+H)+) .
Alternative Synthesis of Peptide Probe F-Bak: Acetyl~(SEQ ID NO: 1)
GQVGRQLAIIGDK(6-FAM)- -(SEQ ID NO: 2) INR-NH2
The protected peptide can be assembled on 0.25 mmol Fmoc-Rink amide MBHA
resin (Novabiochem) on an Applied Biosystems 433A automated peptide synthesizer
running Fastmoc™ coupling cycles using pre-loaded 1mmol amino acid cartridges,
except for the fluorescein(6-FAM)-labeled lysine, where 1 mmol Fmoc-Lys(4-
methyltrityl) can be weighed into the cartridge. The N-terminal acetyl group can be
incorporated by putting 1mmol acetic acid in a cartridge and coupled as described
hereinabove. Selective removal of the 4-methyltrityl group can be accomplished with a
solution of 95:4: 1 DCM:TIS:TFA (v/v/v) flowing through the resin over 15 minutes,
followed by quenching with a flow of dimethylformamide. Single-isomer
6-carboxyfluorescein-NHS can be reacted with the lysine side-chain in 1% DIEA in
DMF and confirmed complete by ninhydrin testing. The peptide can be cleaved from the
resin and side-chains deprotected by treating with 80:5:5:5:2.5:2.5 TFA:water: phenol:
thioanisole:triisopropylsilane: 3,6-dioxa-l,8-octanedithiol (v/v/v/v/v/v), and the crude
peptide recovered by precipitation with diethyl ether. The crude peptide can be purified
by reverse-phase high-performance liquid chromatography, and its purity and identity
confirmed by analytical reverse-phase high-performance liquid chromatography and
matrix-assisted laser-desorption mass-spectrometry (m/z = 2137.1 ((M+H)+)).
Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay
Representative compounds can be serially diluted in dimethyl sulfoxide (DMSO)
starting at 50 mM (2 starting concentration; 10% DMSO) and 10 m transferred into a
384-well plate. Then 10 m of a protein/probe/antibody mix can be added to each well at
final concentrations listed in the below Table AA
TABLE AA. Protein, Probe And Antibody For Use in TR-FRET Assays
No.2)INR-amide)
F-Bak(SEQ. ID. No. 1) ]
(GQVGRQLAIIGDK(6- |
GST- 1 FAM) (SEQ ID Tb-anti-
B l-X 1 No.2)INR-amide) 1 100 GST 1
6-FAM = 6- carboxyfluorescein.; Tb = terbium; GST = glutathione S-transferase
The samples can then mixed on a shaker for 1 minute and incubated for an
additional 3 hours at room temperature. For each assay, the probe/antibody and
protein/probe/antibody can be included on each assay plate as negative and positive
controls, respectively. Fluorescence can be measured on the Envision (Perkin Elmer)
using a 340/35 nm excitation filter and 520/525 (F-Bak peptide) and 495/510 nm (Tblabeled
anti-Histidine antibody) emission filters.
) Treat, Treating or Treatment
The terms "treat", "treating" or "treatment" as used herein refer to administering
one or more active agents or compounds to a subject in an effort to (i) prevent a
pathologic condition from occurring (e.g. prophylaxis); (ii) inhibit the pathologic
condition or arrest its development; (iii) relieve a pathologic condition and/or prevent or
reduce the severity one or more symptoms associated with such a pathologic condition,
regardless of whether any of items (i) through (iii) are successful in a subject.
ee) Variant Polynucleotide or Variant Nucleic Acid Sequence
A "variant polynucleotide" or a "variant nucleic acid sequence" means a
polynucleotide having at least about 60% nucleic acid sequence identity, more preferably
at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,
74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% nucleic acid sequence identity
and yet more preferably at least about 99% nucleic acid sequence identity with a given
nucleic acid sequence. Variants do not encompass the native nucleotide sequence.
Ordinarily, variant polynucleotides are at least about 8 nucleotides in length, often
at least about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29 30, 35, 40, 45, 50, 55, 60 nucleotides in length, or even about 75-200 nucleotides in
length, or more.
The realm of nucleotides includes derivatives wherein the nucleic acid molecule
has been covalently modified by substitution, chemical, enzymatic, or other appropriate
means with a moiety other than a naturally occurring nucleotide.
B. Bcl-2 Family Protein Inhibitors
In one aspect, the present disclosure relates to certain Bcl-2 family protein
inhibitor compounds.
Variable moieties of compounds herein are represented by identifiers (capital
letters with numerical and/or alphabetical superscripts) and may be specifically
embodied.
It is meant to be understood that proper valences are maintained for all moieties
and combinations thereof and that monovalent moieties having more than one atom are
attached through their left ends.
It is also meant to be understood that a specific aspect of a variable moiety may
be the same or different as another specific aspect having the same identifier.
Compounds of this disclosure may contain asymmetrically substituted carbon
atoms in the R or S configuration, wherein the terms "R" and "S" are as defined in Pure
Appl. Chem. (1976) 45, 13-10. Compounds having asymmetrically substituted carbon
atoms with equal amounts of R and S configurations are racemic at those atoms. Atoms
having excess of one configuration over the other are assigned the configuration in
excess, preferably an excess of about 85%-90%, more preferably an excess of about
95%-99%, and still more preferably an excess greater than about 99%. Accordingly,
this disclosure is meant to embrace racemic mixtures and relative and absolute
diastereoisomers of the compounds thereof.
Compounds of this disclosure may also contain carbon-carbon double bonds or
carbon-nitrogen double bonds in the Z or E configuration, in which the term "Z"
represents the larger two substituents on the same side of a carbon-carbon or
carbon-nitrogen double bond and the term "E" represents the larger two substituents on
opposite sides of a carbon-carbon or carbon-nitrogen double bond. The compounds of
this disclosure may also exist as a mixture of "Z" and "E" isomers.
Compounds of this disclosure may also exist as tautomers or equilibrium
mixtures thereof wherein a proton of a compound shifts from one atom to another.
Examples of tautomers include, but are not limited to, keto-enol, phenol-keto, oximenitroso,
nitro-aci, imine-enamine and the like.
Compounds having formula (II) having NH, C(0)OH, OH or SH moieties may
have attached thereto prodrug-forming moieties. The prodrug-forming moieties are
removed by metabolic processes and release the compounds having the freed NH,
C(0)OH, OH or SH in vivo. Prodrugs are useful for adjusting such pharmacokinetic
properties of the compounds as solubility and/or hydrophobicity, absorption in the
gastrointestinal tract, bioavailability, tissue penetration, and rate of clearance.
Metabolites of compounds having formula (II), produced by in vitro or in vivo
metabolic processes, may also have utility for treating diseases associated with
expression of an anti-apoptotic family protein member such as of B C1-XL protein, Bcl-2
protein or Bcl-w protein.
Certain precursor compounds which may be metabolized in vitro or in vivo to
form compounds having formula (II) may also have utility for treating diseases
associated with expression of an anti-apoptotic family protein member such as of
B C1-XL protein, Bcl-2 protein or Bcl-w protein.
Compounds having formula (II) may exist as acid addition salts, basic addition
salts or zwitterions. Salts of compounds having formula (II) are prepared during their
isolation or following their purification. Acid addition salts are those derived from the
reaction of a compound having formula (II) with acid. Accordingly, salts including the
acetate, adipate, alginate, bicarbonate, citrate, aspartate, benzoate, benzenesulfonate
(besylate), bisulfate, butyrate, camphorate, camphorsufonate, digluconate, formate,
fumarate, glycerophosphate, glutamate, hemisulfate, heptanoate, hexanoate,
hydrochloride, hydrobromide, hydroiodide, lactobionate, lactate, maleate,
mesitylenesulfonate, methanesulfonate, naphthylenesulfonate, nicotinate, oxalate,
pamoate, pectinate, persulfate, phosphate, picrate, propionate, succinate, tartrate,
thiocyanate, trichloroacetic, trifluoroacetic, para-toluenesulfonate and undecanoate salts
of the compounds having formula (II) are meant to be embraced by this disclosure.
Basic addition salts of compounds are those derived from the reaction of the compounds
having formula (II) with the bicarbonate, carbonate, hydroxide or phosphate of cations
such as lithium, sodium, potassium, calcium and magnesium.
Compounds having formula (II) may be administered, for example, bucally,
ophthalmically, orally, osmotically, parenterally (intramuscularly, intraperintoneally
intrasternally, intravenously, subcutaneously), rectally, topically, transdermally,
vaginally and intraarterially as well as by intraarticular injection, infusion, and
placement in the body, such as, for example, the vasculature by means of, for example, a
stent.
Therapeutically effective amounts of a compound having formula (II) depend on
recipient of treatment, disease treated and severity thereof, composition comprising it,
time of administration, route of administration, duration of treatment, potency, rate of
clearance and whether or not another drug is co-administered. The amount of a
compound having formula (II) used to make a composition to be administered daily to a
patient in a single dose or in divided doses is from about 0.03 to about 200 mg/kg body
weight. Single dose compositions contain these amounts or a combination of
submultiples thereof.
Compounds having formula (II) may be administered with or without an
excipient. Excipients include, but are not limited to, encapsulators and additives such as
absorption accelerators, antioxidants, binders, buffers, coating agents, coloring agents,
diluents, disintegrating agents, emulsifiers, extenders, fillers, flavoring agents,
humectants, lubricants, perfumes, preservatives, propellants, releasing agents, sterilizing
agents, sweeteners, solubilizers, wetting agents, mixtures thereof and the like.
Excipients for preparation of compositions comprising a compound having
formula (II) to be administered orally include, but are not limited to, agar, alginic acid,
aluminum hydroxide, benzyl alcohol, benzyl benzoate, 1,3-butylene glycol, carbomers,
castor oil, cellulose, cellulose acetate, cocoa butter, corn starch, corn oil, cottonseed oil,
cross-povidone, diglycerides, ethanol, ethyl cellulose, ethyl laureate, ethyl oleate, fatty
acid esters, gelatin, germ oil, glucose, glycerol, groundnut oil, hydroxypropylmethyl
celluose, isopropanol, isotonic saline, lactose, magnesium hydroxide, magnesium
stearate, malt, mannitol, monoglycerides, olive oil, peanut oil, potassium phosphate salts,
potato starch, povidone, propylene glycol, Ringer's solution, safflower oil, sesame oil,
sodium carboxymethyl cellulose, sodium phosphate salts, sodium lauryl sulfate, sodium
sorbitol, soybean oil, stearic acids, stearyl fumarate, sucrose, surfactants, talc, tragacanth,
tetrahydrofurfuryl alcohol, triglycerides, water, mixtures thereof and the like. Excipients
for preparation of compositions comprising a compound having formula (II) to be
administered ophthalmically or orally include, but are not limited to, 1,3-butylene glycol,
castor oil, corn oil, cottonseed oil, ethanol, fatty acid esters of sorbitan, germ oil,
groundnut oil, glycerol, isopropanol, olive oil, polyethylene glycols, propylene glycol,
sesame oil, water, mixtures thereof and the like. Excipients for preparation of
compositions comprising a compound having formula (II) to be administered osmotically
include, but are not limited to, chlorofluorohydrocarbons, ethanol, water, mixtures
thereof and the like. Excipients for preparation of compositions comprising a compound
having formula (II) to be administered parenterally include, but are not limited to, 1,3-
butanediol, castor oil, corn oil, cottonseed oil, dextrose, germ oil, groundnut oil,
liposomes, oleic acid, olive oil, peanut oil, Ringer's solution, safflower oil, sesame oil,
soybean oil, U.S.P. or isotonic sodium chloride solution, water, mixtures thereof and the
like. Excipients for preparation of compositions comprising a compound having
formula (II) to be administered rectally or vaginally include, but are not limited to, cocoa
butter, polyethylene glycol, wax, mixtures thereof and the like.
This disclosure also comprises combination therapeutic methods of treating
disease conditions involving abnormal cell growth and/or dysregulated apoptosis, such as
cancer, in a patient comprising administering thereto a therapeutically effective amount
of a pharmaceutical composition comprising a compound having formula (II) and a
therapeutically effective amount of one or more than one additional therapeutic agents
and / or ionizing radiation.
The combination therapeutic methods include administering compositions of a
compound having formula (II) and one or more than one additional therapeutic agents or
ionizing radiation to a patient using any desired dosing and/or scheduling regimen.
Compounds having formula (II) are expected to be useful when used with
alkylating agents, angiogenesis inhibitors, antibodies, antimetabolites, antimitotics,
antiproliferatives, antivirals, aurora kinase inhibitors, other apoptosis promoters (for
example, Bcl-xL, Bcl-w and Bfl-1) inhibitors, activators of death receptor pathway, BcrAbl
kinase inhibitors, BiTE (Bi-Specific T cell Engager) antibodies, antibody drug
conjugates, biologic response modifiers, cyclin-dependent kinase inhibitors, cell cycle
inhibitors, cyclooxygenase-2 inhibitors, DVDs, leukemia viral oncogene homolog
(ErbB2) receptor inhibitors, growth factor inhibitors, heat shock protein (HSP)-90
inhibitors, histone deacetylase (HDAC) inhibitors, hormonal therapies, immunologicals,
inhibitors of inhibitors of apoptosis proteins (IAPs), intercalating antibiotics, kinase
inhibitors, kinesin inhibitors, Jak2 inhibitors, mammalian target of rapamycin inhibitors,
microR A' s, mitogen-activated extracellular signal-regulated kinase inhibitors,
multivalent binding proteins, non-steroidal anti-inflammatory drugs (NSAIDs), poly
ADP (adenosine diphosphate)-ribose polymerase (PARP) inhibitors, platinum
chemotherapeutics, polo-like kinase (Plk) inhibitors, phosphoinositide-3 kinase (PI3K)
inhibitors, proteosome inhibitors, purine analogs, pyrimidine analogs, receptor tyrosine
kinase inhibitors, etinoids/deltoids plant alkaloids, small inhibitory ribonucleic acids
(siRNAs), topoisomerase inhibitors, ubiquitin ligase inhibitors, and the like, and in
combination with one or more of these agents.
BiTE antibodies are bi-specific antibodies that direct T-cells to attack cancer
cells by simultaneously binding the two cells. The T-cell then attacks the target cancer
cell. Examples of BiTE antibodies include adecatumumab (Micromet MT201),
blinatumomab (Micromet MT103) and the like. Without being limited by theory, one of
the mechanisms by which T-cells elicit apoptosis of the target cancer cell is by exocytosis
of cytolytic granule components, which include perforin and granzyme B. In this regard,
Bcl-2 has been shown to attenuate the induction of apoptosis by both perforin and
granzyme B. These data suggest that inhibition of Bcl-2 could enhance the cytotoxic
effects elicited by T-cells when targeted to cancer cells (V.R. Sutton, D.L. Vaux and J.A.
Trapani (1997) J. of Immunology. 158 (12): 5783).
SiRNAs are molecules having endogenous RNA bases or chemically modified
nucleotides. The modifications do not abolish cellular activity, but rather impart
increased stability and/or increased cellular potency. Examples of chemical
modifications include phosphorothioate groups, 2'-deoxynucleotide, 2'-OCH3-containing
ribonucleotides, 2'-F-ribonucleotides, 2'-methoxyethyl ribonucleotides, combinations
thereof and the like. The siRNA can have varying lengths (e.g., 10-200 bps) and
structures (e.g., hairpins, single/double strands, bulges, nicks/gaps, mismatches) and are
processed in cells to provide active gene silencing. A double-stranded siRNA (dsRNA)
can have the same number of nucleotides on each strand (blunt ends) or asymmetric ends
(overhangs). The overhang of 1-2 nucleotides can be present on the sense and/or the
antisense strand, as well as present on the 5'- and/ or the 3'-ends of a given strand. For
example, siRNAs targeting Mcl-1 have been shown to enhance the activity of ABT-263,
(i.e. , N-(4-(4-((2-(4-chlorophenyl)-5 ,5-dimethyl- 1-cyclohex- 1-en- 1-yl)methyl)piperazinl-
yl)benzoyl)-4-(((lR)-3-(morpholin-4-yl)-l-((phenylsulfanyl)methyl)propyl)amino)-3-
((trifluoromethyl)sulfonyl)benzenesulfonamide) or ABT-737 (i.e., N-(4-(4-((4'-
chloro( 1,1'-biphenyl)-2-yl)methyl)piperazin- 1-yl)benzoyl)-4-((( 1R)-3-(dimethylamino)-
l-((phenylsulfanyl)methyl)propyl)amino)-3-nitrobenzenesulfonamide) in multiple tumor
cell lines (Tse et. al (2008) Cancer Research. 68(9): 3421 and references therein).
Multivalent binding proteins are binding proteins comprising two or more antigen
binding sites. Multivalent binding proteins are engineered to have the three or more
antigen binding sites and are generally not naturally occurring antibodies. The term
"multispecific binding protein" means a binding protein capable of binding two or more
related or unrelated targets. Dual variable domain (DVD) binding proteins are tetravalent
or multivalent binding proteins binding proteins comprising two or more antigen binding
sites. Such DVDs may be monospecific (i.e., capable of binding one antigen) or
multispecific (i.e., capable of binding two or more antigens). DVD binding proteins
comprising two heavy chain DVD polypeptides and two light chain DVD polypeptides
are referred to as DVD Ig's. Each half of a DVD Ig comprises a heavy chain DVD
polypeptide, a light chain DVD polypeptide, and two antigen binding sites. Each binding
site comprises a heavy chain variable domain and a light chain variable domain with a
total of 6 CDRs involved in antigen binding per antigen binding site.
Alkylating agents include altretamine, AMD-473, AP-5280, apaziquone,
bendamustine, brostallicin, busulfan, carboquone, carmustine (BCNU), chlorambucil,
CLORETAZINE® (laromustine, VNP 40101M), cyclophosphamide, decarbazine,
estramustine, fotemustine, glufosfamide, ifosfamide, KW-2170, lomustine (CCNU),
mafosfamide, melphalan, mitobronitol, mitolactol, nimustine, nitrogen mustard N-oxide,
ranimustine, temozolomide, thiotepa, TREANDA® (bendamustine), treosulfan,
rofosfamide and the like.
Angiogenesis inhibitors include endothelial-specific receptor tyrosine kinase (Tie-
2) inhibitors, epidermal growth factor receptor (EGFR) inhibitors, insulin growth factor-2
receptor (IGFR-2) inhibitors, matrix metalloproteinase-2 (MMP-2) inhibitors, matrix
metalloproteinase-9 (MMP-9) inhibitors, platelet-derived growth factor receptor
(PDGFR) inhibitors, thrombospondin analogs, vascular endothelial growth factor
receptor tyrosine kinase (VEGFR) inhibitors and the like.
Antimetabolites include ALIMTA® (pemetrexed disodium, LY231514, MTA), 5-
azacitidine, XELODA® (capecitabine), carmofur, LEUSTAT® (cladribine), clofarabme,
cytarabine, cytarabine ocfosfate, cytosine arabinoside, decitabine, deferoxamine,
doxifluridine, eflornithine, EICAR (5-ethynyl-l- -D-ribofuranosylimidazole-4-
carboxamide), enocitabine, ethnylcytidine, fludarabine, 5-fluorouracil alone or in
combination with leucovorin, GEMZAR® (gemcitabine), hydroxyurea,
ALKERAN®(melphalan), mercaptopurine, 6-mercaptopurine riboside, methotrexate,
mycophenolic acid, nelarabine, nolatrexed, ocfosfate, pelitrexol, pentostatin, raltitrexed,
Ribavirin, triapine, trimetrexate, S-1, tiazofurin, tegafur, TS-1, vidarabine, UFT and the
like.
Antivirals include ritonavir, hydroxychloroquine and the like.
Bcr-Abl kinase inhibitors include DASATINIB® (BMS-354825), GLEEVEC®
(imatinib) and the like.
CDK inhibitors include AZD-5438, BMI-1040, BMS-032, BMS-387, CVT-2584,
flavopyridol, GPC-286199, MCS-5A, PD0332991, PHA-690509, seliciclib (CYC-202,
R-roscovitine), ZK-304709 and the like.
COX-2 inhibitors include ABT-963, ARCOXIA® (etoricoxib), BEXTRA®
(valdecoxib), BMS347070, CELEBREX® (celecoxib), COX-189 (lumiracoxib), CT-3,
DERAMAXX® (deracoxib), JTE-522, 4-methyl-2-(3,4-dimethylphenyl)-l-(4-
sulfamoylphenyl-lH-pyrrole), MK-663 (etoricoxib), NS-398, parecoxib, RS-57067, SC-
58125, SD-8381, SVT-2016, S-2474, T-614, VIOXX® (rofecoxib) and the like.
EGFR inhibitors include ABX-EGF, anti-EGFR immunoliposomes, EGF-vaccine,
EMD-7200, ERBITUX® (cetuximab), HR3, IgA antibodies, IRESSA® (gefitinib),
TARCEVA® (erlotinib or OSI-774), TP-38, EGFR fusion protein, TYKERB®
(lapatinib) and the like.
ErbB2 receptor inhibitors include CP-724-714, CI- 1033 (canertinib),
HERCEPTIN® (trastuzumab), TYKERB® (lapatinib), OMNITARG® (2C4,
petuzumab), TAK-165, GW-572016 (ionafarnib), GW-282974, EKB-569, PI-166,
dHER2 (HER2 vaccine), APC-8024 (HER-2 vaccine), anti-HER^2neu bispecific
antibody, B7.her2IgG3, AS HER2 trifunctional bispecific antibodies, mAB AR-209,
mAB 2B-l and the like.
Histone deacetylase inhibitors include depsipeptide, LAQ-824, MS-275, trapoxin,
suberoylanilide hydroxamic acid (SAHA), TSA, valproic acid and the like.
HSP-90 inhibitors include 17-AAG-nab, 17-AAG, CNF- 101, CNF-1010, CNF-
2024, 17-DMAG, geldanamycin, IPI-504, KOS-953, MYCOGRAB® (human
recombinant antibody to HSP-90), NCS-683664, PU24FC1, PU-3, radicicol, SNX-2112,
STA-9090 VER49009 and the like.
Inhibitors of inhibitors of apoptosis proteins include HGS 1029, GDC-0 45, GDC-
0152, LCL-161, LBW-242 and the like.
Antibody drug conjugates include anti-CD22-MC-MMAF, anti-CD22-MCMMAE,
anti-CD22-MCC-DMl, CR-01 1-vcMMAE, PSMA-ADC, MEDI-547, SGN-
19Am SGN-35, SGN-75 and the like.
Activators of death receptor pathway include TRAIL, antibodies or other agents that
target TRAIL or death receptors (e.g., DR4 and DR5) such as Apomab, conatumumab,
ETR2-ST01, GDC0145, (lexatumumab), HGS-1029, LBY-135, PRO-1762 and
trastuzumab.
Kinesin inhibitors include Eg5 inhibitors such as AZD4877, ARRY-520; CENPE
inhibitors such as GSK923295A and the like.
JAK-2 inhibitors include CEP-701 (lesaurtinib), XL019 and INCBO 18424 and the
like.
MEK inhibitors include ARRY-142886, ARRY-438162 PD-325901, PD-98059
and the like.
mTOR inhibitors include AP-23573, CCI-779, everolimus, RAD-001, rapamycin,
temsirolimus, ATP-competitive TORC1/TORC2 inhibitors, including PI-103, PP242,
PP30, Torin 1 and the like.
Non-steroidal anti-inflammatory drugs include AMIGESIC® (salsalate),
DOLOBID® (diflunisal), MOTRIN® (ibuprofen), ORUDIS® (ketoprofen), RELAFEN®
(nabumetone), FELDENE® (piroxicam), ibuprofen cream, ALEVE® (naproxen) and
NAPROSYN® (naproxen), VOLTAREN® (diclofenac), INDOCIN® (indomethacin),
CLINORIL® (sulindac), TOLECTIN® (tolmetin), LODINE® (etodolac), TORADOL®
(ketorolac), DAYPRO® (oxaprozin) and the like.
PDGFR inhibitors include C-451, CP-673, CP-868596 and the like.
Platinum chemotherapeutics include cisplatin, ELOXATFN® (oxaliplatin)
eptaplatin, lobaplatin, nedaplatin, PARAPLATFN® (carboplatin), satraplatin, picoplatin
and the like.
Polo-like kinase inhibitors include BI-2536 and the like.
Phosphoinositide-3 kinase (PI3K) inhibitors include wortmannin, LY294002, XL-
147, CAL-120, ONC-21, AEZS-127, ETP-45658, PX-866, GDC-0941, BGT226,
BEZ235, XL765 and the like.
Thrombospondin analogs include ABT-510, ABT-567, ABT-898, TSP-1 and the
like.
VEGFR inhibitors include AVASTIN® (bevacizumab), ABT-869, AEE-788,
ANGIOZYME™ (a ribozyme that inhibits angiogenesis (Ribozyme Pharmaceuticals
(Boulder, CO.) and Chiron, (Emeryville, CA)) , axitinib (AG-13736), AZD-2171, CP-
547,632, IM-862, MACUGEN (pegaptamib), NEXAVAR® (sorafenib, BAY43-9006),
pazopanib (GW-786034), vatalanib (PTK-787, ZK-222584), SUTENT® (sunitinib, SU-
11248), VEGF trap, ZACTIMA™ (vandetanib, ZD-6474) and the like.
Antibiotics include intercalating antibiotics aclarubicin, actinomycin D,
amrubicin, annamycin, adriamycin, BLENOXANE® (bleomycin), daunorubicin,
CAELYX® or MYOCET® (liposomal doxorubicin), elsamitrucin, epirbucin, glarbuicin,
ZAVEDOS® (idarubicin), mitomycin C, nemorubicin, neocarzinostatin, peplomycin,
pirarubicin, rebeccamycin, stimalamer, streptozocin, VALSTAR® (valrubicin),
zinostatin and the like.
Topoisomerase inhibitors include aclarubicin, 9-aminocamptothecin, amonafide,
amsacrine, becatecarin, belotecan, BN-80915, CAMPTOSAR® (irinotecan
hydrochloride), camptothecin, CARDIOXANE® (dexrazoxine), diflomotecan,
edotecarin, ELLENCE® or PHARMORUBICIN® (epirubicin), etoposide, exatecan, 10-
hydroxycamptothecin, gimatecan, lurtotecan, mitoxantrone, orathecin, pirarbucin,
pixantrone, rubitecan, sobuzoxane, SN-38, tafluposide, topotecan and the like.
Antibodies include AVASTIN® (bevacizumab), CD40-specific antibodies,
chTNT-l/B, denosumab, ERBITUX® (cetuximab), HUMAX-CD4® (zanolimumab),
IGFlR-specific antibodies, lintuzumab, PANOREX® (edrecolomab), RENCAREX®
(WX G250), RITUXAN® (rituximab), ticilimumab, trastuzimab, CD20 antibodies types
I and II and the like.
Hormonal therapies include ARIMIDEX® (anastrozole), AROMASIN®
(exemestane), arzoxifene, CASODEX® (bicalutamide), CETROTIDE® (cetrorelix),
degarelix, deslorelin, DESOPAN® (trilostane), dexamethasone, DROGENIL®
(flutamide), EVISTA® (raloxifene), AFEMA™ (fadrozole), FARESTON®
(toremifene), FASLODEX® (fulvestrant), FEMARA® (letrozole), formestane,
glucocorticoids, HECTOROL® (doxercalciferol), RENAGEL® (sevelamer carbonate),
lasofoxifene, leuprolide acetate, MEGACE® (megesterol), MIFEPREX® (mifepristone),
NILANDRON™ (nilutamide), NOLVADEX® (tamoxifen citrate), PLENAXIS™
(abarelix), prednisone, PROPECIA® (finasteride), rilostane, SUPREFACT® (buserelin),
TRELSTAR® (luteinizing hormone releasing hormone (LHRH)), VANTAS® (Histrelin
implant), VETORYL® (trilostane or modrastane), ZOLADEX® (fosrelin, goserelin) and
the like.
Deltoids and retinoids include seocalcitol (EB1089, CB1093), lexacalcitrol
(KH1060), fenretinide, PANRETIN® (aliretinoin), ATRAGEN® (liposomal tretinoin),
TARGRETFN® (bexarotene), LGD-1550 and the like.
PARP inhibitors include ABT-888, olaparib, KU-59436, AZD-2281, AG-014699,
BSI-201, BGP-15, GNO-1001, ONO-2231 and the like.
Plant alkaloids include, but are not limited to, vincristine, vinblastine, vindesine,
vinorelbine and the like.
Proteasome inhibitors include VELCADE® (bortezomib), MG132, NPI-0052,
PR- 171 and the like.
Examples of immunologicals include interferons and other immune-enhancing
agents. Interferons include interferon alpha, interferon alpha-2a, interferon alpha-2b,
interferon beta, interferon gamma- la, ACTIMMUNE® (interferon gamma- lb) or
interferon gamma-nl, combinations thereof and the like. Other agents include
ALFAFERONE®, (IFN-), BAM-002 (oxidized glutathione), BEROMUN® (tasonermin),
BEXXAR® (tositumomab), CAMPATH® (alemtuzumab), CTLA4 (cytotoxic
lymphocyte antigen 4), decarbazine, denileukin, epratuzumab, GRANOCYTE®
(lenograstim), lentinan, leukocyte alpha interferon, imiquimod, MDX-010 (anti-CTLA-
4), melanoma vaccine, mitumomab, molgramostim, MYLOTARG™ (gemtuzumab
ozogamicin), NEUPOGEN® (filgrastim), OncoVAC-CL, OVAREX® (oregovomab),
pemtumomab (Y-muHMFGl), PROVENGE® (sipuleucel-T), sargaramostim, sizofilan,
teceleukin, THERACYS® (Bacillus Calmette-Guerin), ubenimex, VIRULIZIN®
(immunotherapeutic, Lorus Pharmaceuticals), Z-100 (Specific Substance of Maruyama
(SSM)), WF-10 (Tetrachlorodecaoxide (TCDO)), PROLEUKIN® (aldesleukin),
ZADAXIN® (thymalfasin), ZENAPAX® (daclizumab), ZEVALIN® (90Y-Ibritumomab
tiuxetan) and the like.
Biological response modifiers are agents that modify defense mechanisms of
living organisms or biological responses, such as survival, growth or differentiation of
tissue cells to direct them to have anti-tumor activity and include krestin, lentinan,
sizofiran, picibanil PF-35 12676 (CpG-8954), ubenimex and the like.
Pyrimidine analogs include cytarabine (ara C or Arabinoside C), cytosine
arabinoside, doxifluridine, FLUDARA® (fludarabine), 5-FU (5-fluorouracil),
fioxuridine, GEMZAR® (gemcitabine), TOMUDEX® (ratitrexed), TROXATYL™
(triacetyluridine troxacitabine) and the like.
Purine analogs include LANVIS® (thioguanine) and PURI-NETHOL®
(mercaptopurine).
Antimitotic agents include batabulin, epothilone D (KOS-862), N-(2-((4-
hydroxyphenyl)amino)pyridin-3-yl)-4-methoxybenzenesulfonamide, ixabepilone (BMS
247550), paclitaxel, TAXOTERE® (docetaxel), PNU100940 (109881), patupilone, XRP-
98 1 (larotaxel), vinflunine, ZK-EPO (synthetic epothilone) and the like.
Ubiquitin ligase inhibitors include MDM2 inhibitors, such as nutlins, NEDD8
inhibitors such as MLN4924 and the like.
Compounds of this disclosure can also be used as radiosensitizers that enhance
the efficacy of radiotherapy. Examples of radiotherapy include external beam
radiotherapy, teletherapy, brachytherapy and sealed, unsealed source radiotherapy and
the like.
Additionally, compounds having Formula (II) may be combined with other
chemotherapeutic agents such as ABRAXANE™ (ABI-007), ABT- 100 (farnesyl
transferase inhibitor), ADVEXIN® (Ad5CMV-p53 vaccine), ALTOCOR® or
MEVACOR® (lovastatin), AMPLIGEN® (poly I:poly C12U, a synthetic RNA),
APTOSYN® (exisulind), AREDIA® (pamidronic acid), arglabin, L-asparaginase,
atamestane (l-methyl-3,17-dione-androsta-l,4-diene), AVAGE® (tazarotene), AVE-
8062 (combreastatin derivative) BEC2 (mitumomab), cachectin or cachexin (tumor
necrosis factor), canvaxin (vaccine), CEAVAC® (cancer vaccine), CELEUK®
(celmoleukin), CEPLENE® (histamine dihydrochloride), CERVARIX® (human
papillomavirus vaccine), CHOP® (C: CYTOXAN® (cyclophosphamide); H:
ADRIAMYCIN® (hydroxydoxorubicm); O: Vincristine (ONCOVIN®); P: prednisone),
CYPAT™ (cyproterone acetate), combrestatin A4P, DAB(389)EGF (catalytic and
translocation domains of diphtheria toxin fused via a His-Ala linker to human epidermal
growth factor) or TransMID-107R™ (diphtheria toxins), dacarbazine, dactinomycin, 5,6-
dimethylxanthenone-4-acetic acid (DMXAA), eniluracil, EVIZON™ (squalamine
lactate), DIMERICINE® (T4N5 liposome lotion), discodermolide, DX-8951f (exatecan
mesylate), enzastaurin, EPO906 (epithilone B), GARDASIL® (quadrivalent human
papillomavirus (Types 6, 11, 16, 18) recombinant vaccine), GASTRIMMUNE®,
GENASENSE®, GMK (ganglioside conjugate vaccine), GVAX® (prostate cancer
vaccine), halofuginone, histerelin, hydroxycarbamide, ibandronic acid, IGN-101, IL-13-
PE38, IL-13-PE38QQR (cintredekin besudotox), IL-13-pseudomonas exotoxin,
interferon-a, interferon-g , JUNOVAN™ or MEPACT™ (mifamurtide), lonafamib, 5,10-
methylenetetrahydrofolate, miltefosine (hexadecylphosphocholine),
NEOVASTAT®(AE-941), NEUTREXIN® (trimetrexate glucuronate), NIPENT®
(pentostatin), ONCONASE® (a ribonuclease enzyme), ONCOPHAGE® (melanoma
vaccine treatment), ONCOVAX® (IL-2 Vaccine), ORATHECIN™ (rubitecan),
OSIDEM® (antibody-based cell drug), OVAREX® MAb (murine monoclonal antibody),
paclitaxel, PANDIMEX™ (aglycone saponins from ginseng comprising
20(S)protopanaxadiol (aPPD) and 20(S)protopanaxatriol (aPPT)), panitumumab,
PANVAC®-VF (investigational cancer vaccine), pegaspargase, PEG Interferon A,
phenoxodiol, procarbazine, rebimastat, REMOVAB® (catumaxomab), REVLIMID®
(lenalidomide), RSR13 (efaproxiral), SOMATULINE® LA (lanreotide), SORIATANE®
(acitretin), staurosporine (Streptomyces staurospores), talabostat (PT100),
TARGRETIN® (bexarotene), TAXOPREXIN® (DHA-paclitaxel), TELCYTA®
(canfosfamide, TLK286), temilifene, TEMODAR® (temozolomide), tesmilifene,
thalidomide, THERATOPE® (STn-KLH), thymitaq (2-amino-3,4-dihydro-6-methyl-4-
oxo-5-(4-pyridylthio)quinazoline dihydrochloride), TNFERADE™ (adenovector: DNA
carrier containing the gene for tumor necrosis factor-a), TRACLEER® or ZAVESCA®
(bosentan), tretinoin (Retin-A), tetrandrine, TRISENOX® (arsenic trioxide),
VIRULIZIN®, ukrain (derivative of alkaloids from the greater celandine plant), vitaxin
(anti-alphavbeta3 antibody), XCYTRIN® (motexafm gadolinium), XINLAY™
(atrasentan), XYOTAX™ (paclitaxel poliglumex), YONDELIS® (trabectedin), ZD-
6126, ZINECARD® (dexrazoxane), ZOMETA® (zolendronic acid), zorubicin and the
like.
BAX and BAD peptides are reported in Zhang, H. C , Nimmer, P., Rosenberg, S.
H., Ng, S. C., and Joseph, M. (2002). Development of a High-Throughput Fluorescence
Polarization Assay for Bcl-x(L). Analytical Biochemistry 307, 70-75.
Binding affinity of compounds having formula (II) to B C1-X protein is indicia of
their inhibition of the activity of this protein. To determine the binding affinity of
compounds having formula (II) to B C1-XLprotein, representative examples were diluted in
DMSO to concentrations between 100 mM and 1 pM and added to each well of a 96-well
microtiter plate. A mixture comprising 125 mE per well of assay buffer (20 mM phosphate
buffer (pH 7.4), 1mM EDTA, 50 mM NaCl, 0.05% PF-68), 6 nM of Bcl-XL protein
(prepared as described in Science 1997, 275, 983-986), 1 nM fluorescein-labeled BAD
peptide (prepared in-house) and the DMSO solution of the compound was shaken for 2
minutes and placed in a LJL Analyst (LJL Bio Systems, CA). A negative control (DMSO,
15 nM BAD peptide, assay buffer) and a positive control (DMSO, 1 nM BAD peptide, 6 nM
B C1-X , assay buffer) were used to determine the range of the assay. Polarization was
measured at room temperature using a continuous Fluorescein lamp (excitation 485 nm,
emission 530 nm). Percentage of inhibition was determined by (l-((mP value of wellnegative
control)/range)) x 100%. The results are shown in TABLE 1.
Binding affinity of compounds having formula (II) to Bcl-2 protein is indicia of their
inhibition of the activity of this protein. To determine the binding affinity of compounds
having formula (II) to Bcl-2, representative examples were diluted in DMSO to
concentrations between 10 mM and 10 pM and added to each well of a 96-well microtiter
plate. A mixture comprising 125 L per well of assay buffer (20 mM phosphate buffer (pH
7.4), 1mM EDTA, 50 mM NaCl, 0.05% PF-68), 10 nM of Bcl-2 protein (prepared according
to the procedure described in PNAS 2001, 98, 3012 - 3017), 1 nM fluorescein-labeled BAX
peptide (prepared in-house) and the DMSO solution of the representative EXAMPLE was
shaken for 2 minutes and placed in a LJL Analyst (LJL Bio Systems, CA. Polarization was
measured at room temperature using a continuous Fluorescein lamp (excitation 485 nm,
emission 530 nm). The results are also shown in TABLE 1.
These data demonstrate the utility of compounds having formula (II) as binders to
and inhibitors of anti-apopotic B C1-XLprotein and anti-apopotic Bcl-2.
It is expected that, because compounds having formula (II) bind to and inhibit the
activity of B C1-XL and Bcl-2, they would also have utility as inhibitors of anti-apopotic
family protein members having close structural homology to B C1-X L and Bcl-2 such as,
for example, anti-apopotic Bcl-w protein.
Accordingly, compounds having formula (II) are expected to have utility in
treatment of diseases during which anti-apopotic B C1-X Lprotein, anti-apopotic Bcl-2
protein, anti-apopotic Bcl-w protein or a combination thereof, are expressed.
Determination of Cellular Efficacy in Human Tumor Cell Line
NCI-H146 (ATCC, Manassas, VA.) human small cell lung carcinoma cells were
plated 50,000 cells per well in 96-well tissue culture plates in a total volume of 100 m
tissue culture medium supplemented with 10% human serum (Invitrogen, Carlsbad, CA.)
instead of fetal bovine serum and treated with a 2-fold serial dilution of the compounds of
interest from 10 mM to 0.020 mM. Each concentration was tested in duplicate at least 3
separate times. The number of viable cells following 48 hours of compound treatment
was determined using the CellTiter 96® AQueous non-radioactive cell proliferation MTS
assay according to manufacturer's recommendations (Promega Corp., Madison, WI).
The results are also shown in TABLE 1.
Pharmacokinetic Evaluation of Selected Compounds in Rat
The pharmacokinetic behavior of compounds of this disclosure was determined
following a single 2 mg/kg intravenous or 5 mg/kg oral dose in male Sprague-Dawley
derived rats (n=3 per group). The compounds were prepared as 2 mg/mL solution in a
10% DMSO in PEG-400 formulation for both oral and intravenous administration. The
1 mL/kg intravenous dose was administered as a slow bolus (about 1-2 minutes) in the
jugular vein of a rat under light ether anesthetic. The oral dose was administered by
gavage. Serial blood samples were obtained from a tail vein of each rat prior 0.1 (IV
only), 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8 and 24 hours after dosing. The heparinized samples
were thoroughly mixed and placed in an ice bath. Plasma was separated by
centrifugation and stored frozen prior to analysis. The results are also shown in TABLE
1.
The compounds of interest were separated from the plasma using protein
precipitation with acetonitrile. A plasma (100-200 ΐ , sample or spiked standard)
aliquot was combined with 50 of internal standard (structurally related analog
prepared in acetonitrile) and 1ml acetonitrile in a 96-well polypropylene deep well plate.
The plates were vortexed for 30 seconds followed by centrifugation (3500 rpm x 15
minutes, 4°C). In an automated manner, the supernatant was transferred to a clean 96-
well plate. The samples were evaporated to near dryness on a Micro-Vap™ under a
stream of dry nitrogen over low heat (~37°C). The samples were reconstituted vortexing
with 0.2 mL 5% DMSO in acetonitrile. A 0.1-0.2 ml aliquot of acetonitrile: 0.1 % >
trifluoroacetic acid (20:80, by volume) was added to each well, followed by an additional
30 second vortexing. The plates were centrifuged (3500 rpm x 15 minutes, 4°C) prior to
HPLC-MS/MS analysis. Samples were analyzed simultaneously with spiked plasma
standards. All samples from each study were analyzed as a single batch on the LCMS/
MS.
The compounds of interest and the internal standard were separated from each
other and co-extracted contaminants on a 50 x 3 mm Keystone Betasil CN 5 mhi column
with an acetonitrile: 0.1% trifluoroacetic acid mobile phase (50:50, by volume) at a flow
rate of 0.7 ml/min. Analysis was performed on a Sciex API 300™ Biomolecular Mass
Analyzer using a heated nebulizer interface. Peak areas of the title compounds and
internal standards were determined using the Sciex MacQuan™ software. Calibration
curves were derived from peak area ratio (parent drug/internal standard) of the spiked rat
plasma standards using least squares linear regression of the ratio versus the theoretical
concentration. The methods were generally linear over the range of the standard curve
(correlation coefficients > 0.99) with an estimated quantitation limit of 0.01 mg mL. The
plasma concentration data for each animal were submitted to multi-exponential curve
fitting using WinNonlin. The area under the plasma concentration-time curve from 0 to
t hours (time of the last measurable plasma concentration) after dosing (AUCo-t) was
calculated using the linear trapezoidal rule for the plasma concentration-time profiles.
The residual area extrapolated to infinity, determined as the final measured plasma
concentration (Ct) divided by the terminal elimination rate constant (b), was added to
AUCo-t to produce the total area under the curve (AUCo-¥) . The results are also shown in
TABLE 1. Ki values shown in the tables below were determined using Wang's equation
(Wang Z X,. An Exact Mathematical Expression For Describing Competitive Binding Of
Two Different Ligands To A Protein Molecule. FEBSLett. 1995; 360:11 1-4)).
TABLE 1
1 ! < 0.001 i < 0.001 mM 0.0891 mM \ 5.01 mM 56
2 < 0.001 i l < 0.001 mM 0.0291 mM 7.01 mM 241
3 ! < 0.001 i l < 0.001 mM 0.0288 mM ! 4.13 mM 144
4 < 0.001 i l < 0.001 mM 0.0587 mM 6.34 108
! 5 < 0.001 i l < 0.001 mM 0.0388 mM 2.22 mM 57
6 < 0.001 i l < 0.001 0.0010 0.91 M 9 1
7 < 0.001 i l < 0.001 mM 0.0589 mM 3.87 mM 66
8 j < 0.001 i l < 0.001 mM 0.0212 mM 1. 1 L V 52
! 9 \ < 0.001 i l < 0.001 mM 0.0137 mM \ 1.88 mM 137
i o < 0.001 i l < 0.001 mM 0.0342 mM 2.48 mM 73
11 < 0.002 i l < 0.003 uM 0.0206 mM 2.40 mM 117
12 i < 0.001 i l < 0.001 mM 0.0271 mM i 2.07 mM 76
I 13 \ < 0.001 i l < 0.001 mM 0.0190 mM \ 2.06 mM 108
14 < o.oo i i < 0.001 0.0309 m 1 3.42 mM J i
15 < 0.001 i l < 0.001 mM 0.0099 mM 1.25 mM 126
16 i < 0.002 i l < 0.002 uM 0.0374 mM i 2.37 mM 63
! 17 < 0.001 i l < 0.001 mM 0.0287 mM \ 0.88 mM 3 1
18 < 0.001 L V < 0.001 mM 0.0154 mM 0.61 mM 40
! 19 < 0.001 i l < 0.001 mM 0.0158 mM 7.12 mM 451
! 20 < 0.001 i l < 0.001 mM 0.0277 mM \ 3.1 1 mM 112
i 2 1 < 0.001 i l < 0.001 mM 0.0643 mM 1.81 mM 28
22 < 0.001 i l < 0.001 mn 0.0388 mM 4.08 mM 105
i 23 < 0.001 l < 0.001 mM 0.0528 mM ! 3.54 mM 67
24 < 0.001 LIV < 0.001 mM 0.0443 mM 8.04 mM 181
25 < 0.001 i l < 0.001 mM 0.0164 mM \ 1.67 mM 102
26 < 0.001 i l < 0.001 mM 0.0243 mM 0.80 mM 33
27 < 0.001 i l < 0.001 mM 0.0185 mM ! 2.08 mM 112
\ 28 < 0.001 i l < 0.001 mM 0.0242 mM 6.30 mM 260
29 < 0.001 l < 0.001 mM 0.0298 mM 1.74 mM 58
30 < 0.001 i < 0.001 M 0.0 17 m 3.39 m 107
! 3 1 < 0.001 i l < 0.001 mM 0.0130 mM 5.10 392
32 < 0.001 i l < 0.001 mM 0.0187 mM ! 1.38 mM 73.9
! 33 < 0.001 i l < 0.001 mM 0.0378 mM \ 3.01 mM 79.8
34 < 0.001 i l < 0.001 mM 0.0200 m 1 .07 mV 554
35 < 0.001 mM < 0.001 mM 0.0076 mM 1.26 mM 166
36 < 0.001 mM j < 0.001 L V \0.0242 mM 4.22 mM 174
37 < 0.001 mM i < 0.001 mM i 0.0175 mM 7.30 mM 417
38 < 0.001 mM < 0.001 mM 0.0394 mM 0.67 m 17
39 < 0.001 mM < 0.001 mM 0.0827 mM 1.66 mM 20
The compounds of the present disclosure were compared to compounds disclosed
in WO 2005/049594, identified herein as EXAMPLES A-L, by determining the ratio of
systemic exposure to cellular efficacy. This measure, sometimes reported as AUC/EC50,
is well known to those skilled in the art of pharmaceutical drug discovery and drug
development as a useful pharmacodynamic predictor of oral efficacy.
The examples of the present disclosure and compounds disclosed in
WO 2005/049594 were both tested in an HI46 cellular assay and evaluated for oral
pharmacokinetic properties in rat, both as previously described herein. The results are
shown in TABLES 2 and 3. As can be seen with reference to the data, the compounds of
the present disclosure have a more preferred pharmacodynamic profile (meaning that the
compounds of the present invention exhibit higher AUC/EC50values) as compared to the
compounds known in the art. From these results, a number of observations can be drawn.
It can be observed that the compounds having a O moiety at position W1 tend to have
good to excellent cellular potency (e.g., EC50 < 1 mM) . However, when the
pharmacokinetic properties of these same compounds are determined, it can be seen that
the systemic exposure after oral administration is poor, resulting in AUC/ EC50 ratios of
from 0.5 to 19.7. On the other hand, when compounds having a CF3 or CN moiety at
position W1 are tested in the cellular assay, these derivatives have relatively poor cellular
efficacy (e.g., EC50 > 1 mM) even though they have suitable sytstemic exposure after
oral administration. Again, this combination provides overall AUC/EC50 ratios from
about 2.8 to about < 7.4. Surprisingly, compounds of the present disclosure demonstrate
cellular efficacy on par with compounds having an O moiety while maintaining
suitable systemic exposure after oral administration. The resulting AUC/EC50 ratios for
the compounds of the disclosure are from about 20 to about 554.
TABLE 2
Pharmacokineti PK/PD
NCI- Pharmacokineti
c area under the ratio
H146 c peak plasma
plasma
Cellular concentration
concentration
Efficacy (rat)
curve (rat)
H146,
a AUC,
EXAMPLE w w2 w3
EC50, AUC/EC50
(mM) (mM)
(mM)
38 S0 2CF3 N(CH 3)2 H 0.039 0.072 0.665 16.9
A CF N(CH 3) H 1.599 0.371 4.412 2.8
B N0 2 N(CH 3) H 0.063 0.039 0.283 4.5
C CN N(CH 3)2 H 1.807 0.315 1.917 1.1
D CF N(CH 3)2 F 7.329 0.386 3.827 0.5
39 S0 2CF3 — N 0 H 0.083 0.290 1.657 20.0
E N0 2 — N 0 H 0.974 0.195 1.157 1.2
F CF — N 0 H > 1.00 0.592 7.365 <7.4
TABLE 3
Pharmacokinetic PK/PD
NCI-H146 Pharmacokinetic area under the ratio
Cellular peak plasma plasma
Efficacy concentration (rat) concentration
Curve (rat)
H146
r
m ax AUC,
EXAMPLE w 1 w 2 EC50, AUC/ECso
(mM) (mM)
(mM)
18 S0 2CF2C 1 N(CH 3) 0.015 0.059 0.609 39.5
26 S0 2CF3 N(CH 3) 0.024 0.097 0.803 33.0
G N0 2 N(CH 3) 0.026 0.057 0.507 19.7
H CF3 N(CH 3) 0.410 0.215 1.973 4.8
3 S0 2CF C 1 — N 0 0.029 0.385 4.131 143.6
7 S0 2CF3 — N 0 0.059 _ 0.518 3.867 65.6
I N0 — N 0 0.094 0.267 1.977 21.0
S0 2CF3 0.021 0.071 1.098 51.7
N0 2 0.049 0.077 0.368 7.5
N(z-
S0 2CF3 0.038 0.129 3.013 79.8
Pr)CH3
N(z-
N0 2 0.034 0.0367 0.615 18.2
Pr)CH3
-Pr means iso-propyl
As shown in Figures 1-7, studies pertaining to the oral efficacy of EXAMPLE 1
in combination with etoposide, vincristine, CHOP, rituximab, rituximab with CHOP,
rapamycin, and VELCADE demonstrated that EXAMPLE 1 synergistically enhanced
efficacy of these cytotoxic agents during combination therapy when administered orally.
Further, combinations comprising EXAMPLE 1 and vincristine resulted in 10%
complete tumor regression.
Still further, combinations comprising EXAMPLE 1 and rituximab resulted in
70% complete tumor regression whereas no tumor regressions were observed for
rituximab alone.
Still further, combinations comprising EXAMPLE 1 and rapamycin resulted in
70% complete tumor regression whereas 10%> tumor regressions were observed for
rapamycin alone.
Still further, combinations comprising EXAMPLE 1 and rituximab with CHOP
resulted in 90%> complete tumor regression whereas 10% tumor regressions were
observed for rituximab with CHOP only.
Still further, combinations comprising EXAMPLE 1 and bortexomib resulted in
10% complete tumor regression whereas no tumor regressions were observed for
bortexomib alone.
Diseases during which anti-apopotic B C1-X protein, anti-apopotic Bcl-2 protein,
anti-apopotic Bcl-w protein or a combination thereof, are expressed include, but are not
limited to, cancer and autoimmune disorders, wherein cancer includes, but is not limited
to, acoustic neuroma, acute leukemia, acute lymphocytic leukemia, acute myelocytic
leukemia (monocytic, myeloblasts, adenocarcinoma, angiosarcoma, astrocytoma,
myelomonocytic and promyelocyte), acute t-cell leukemia, basal cell carcinoma, bile
duct carcinoma, bladder cancer, brain cancer, breast cancer, bronchogenic carcinoma,
cervical cancer, chondrosarcoma, chordoma, choriocarcinoma, chronic leukemia, chronic
lymphocytic leukemia, chronic myelocytic (granulocytic) leukemia, colorectal cancer,
craniopharyngioma, cystadenocarcinoma, diffuse large B -cell lymphoma,
dysproliferative changes (dysplasias and metaplasias), embryonal carcinoma, endometrial
cancer, endotheliosarcoma, ependymoma, epithelial carcinoma, erythroleukemia,
esophageal cancer, estrogen-receptor positive breast cancer, essential thrombocythemia,
Ewing's tumor, fibrosarcoma, follicular lymphoma, germ cell testicular cancer, glioma,
heavy chain disease, hemangioblastoma, hepatoma, hepatocellular cancer, hormone
insensitive prostate cancer, leiomyosarcoma, liposarcoma, lung carcinoma,
lymphagioendotheliosarcoma, lymphangiosarcoma, lymphoblastic leukemia, lymphoma
(Hodgkin's andnon-Hodgkin's), malignancies and hyperproliferative disorders of the
bladder, breast, colon, lung, ovaries, pancreas, prostate, skin and uterus, lymphoid
malignancies of T-cell or B-cell origin, leukemia, lymphoma, medullary carcinoma,
medulloblastoma, melanoma, meningioma, mesothelioma, multiple myeloma,
myelogenous leukemia, myeloma, myxosarcoma, neuroblastoma, non-small cell lung
cancer, oligodendroglioma, oral cancer, osteogenic sarcoma, ovarian cancer, pancreatic
cancer, papillary adenocarcinomas, papillary carcinoma, pinealoma, polycythemia vera,
prostate cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma,
sebaceous gland carcinoma, seminoma, small cell lung carcinoma, solid tumors
(carcinomas and sarcomas), small cell lung cancer squamous cell carcinoma, synovioma,
sweat gland carcinoma, Waldenstrom's macroglobulinemia, testicular tumors, uterine
cancer and Wilms' tumor (Cancer Res., 2000, 60, 6101-10 and Medicine, 2d Ed., J.B.
Lippincott Co., Philadelphia (1985)); autoimmune disorders include, but are not limited
to, acquired immunodeficiency disease syndrome, autoimmune lymphoproliferative
syndrome, hemolytic anemia, inflammatory diseases, and thrombocytopenia (Current
Allergy and Asthma Reports 2003, 3:378-384; Br. J. Haematol. 2000 Sep; 110(3): 584-
90; Blood 2000 Feb 15;95(4): 1283-92; and New England Journal of Medicine 2004 Sep;
351(14): 1409-1418).
It is also expected that compounds having formula (II) would inhibit the growth
of cells derived from a cancer or neoplasm such as breast cancer (including estrogenreceptor
positive breast cancer), colorectal cancer, endometrial cancer, lung cancer
(including small cell lung cancer), lymphoma (including follicular or Diffuse Large
B-cell), lymphoma (including non-Hodgkin's lymphoma), neuroblastoma, ovarian
cancer, prostate cancer (including hormone-insensitive prostate cancer), testicular cancer
(including germ cell testicular cancer).
It is also expected that compounds having formula (II) would inhibit the growth
of cells derived from a pediatric cancer or neoplasm such as embryonal
rhabdomyosarcoma, pediatric acute lymphoblastic leukemia, pediatric acute
myelogenous leukemia, pediatric alveolar rhabdomyosarcoma, pediatric anaplastic
ependymoma, pediatric anaplastic large cell lymphoma, pediatric anaplastic
medulloblastoma, pediatric atypical teratoid/rhabdoid tumor of the central nervous
syatem, pediatric biphenotypic acute leukemia, pediatric Burkitts lymphoma, pediatric
cancers of Ewing's family of tumors such as primitive neuroectodermal rumors, pediatric
diffuse anaplastic Wilm's tumor, pediatric favorable histology Wilm's tumor, pediatric
glioblastoma, pediatric medulloblastoma, pediatric neuroblastoma, pediatric
neuroblastoma-derived myelocytomatosis, pediatric pre-B-cell cancers (such as
leukemia), pediatric psteosarcoma, pediatric rhabdoid kidney tumor, pediatric
rhabdomyosarcoma, and pediatric T-cell cancers such as lymphoma and skin cancer
(commonly-owned United States Application Ser No. 10/988,338), Cancer Res., 2000,
60, 6101-10); autoimmune disorders include, but are not limited to, acquired
immunodeficiency disease syndrome, autoimmune lymphoproliferative syndrome,
hemolytic anemia, inflammatory diseases, and thrombocytopenia (Current Allergy and
Asthma Reports 2003, 3:378-384; Br. J . Haematol. 2000 Sep; 110(3): 584-90; Blood
2000 Feb 15;95(4):1283-92; and New England Journal of Medicine 2004 Sep; 351(14):
1409-1418).
Compounds having formula (II) may be made by synthetic chemical processes,
examples of which are shown hereinbelow. It is meant to be understood that the order of
the steps in the processes may be varied, that reagents, solvents and reaction conditions
may be substituted for those specifically mentioned, and that vulnerable moieties may be
protected and deprotected, as necessary.
Protecting groups for C(0)OH moieties include, but are not limited to,
acetoxymethyl, allyl, benzoylmethyl, benzyl, benzyloxymethyl, tert-butyl,
tert-butyldiphenylsilyl, diphenylmethyl, cyclobutyl, cyclohexyl, cyclopentyl,
cyclopropyl, diphenylmethylsilyl, ethyl, para-methoxybenzyl, methoxymethyl,
methoxyethoxymethyl, methyl, methylthiomethyl, naphthyl, para-nitrobenzyl, phenyl, npropyl,
2,2,2-trichloroethyl, tnethylsilyl, 2-(trimethylsilyl)ethyl, 2-
(trimethylsilyl)ethoxymethyl, triphenylmethyl and the like.
Protecting groups for C(O) and C(0)H moieties include, but are not limited to,
1.3-dioxylketal, diethylketal, dimethylketal, 1,3-dithianylketal, O-methyloxime,
O-phenyloxime and the like.
Protecting groups for NH moieties include, but are not limited to, acetyl, alanyl,
benzoyl, benzyl (phenylmethyl), benzylidene, benzyloxycarbonyl (Cbz),
tert-butoxycarbonyl (Boc), 3,4-dimethoxybenzyloxycarbonyl, diphenylmethyl,
diphenylphosphoryl, formyl, methanesulfonyl, para-methoxybenzyloxycarbonyl,
phenylacetyl, phthaloyl, succinyl, trichloroethoxycarbonyl, triethylsilyl, trifluoroacetyl,
trimethylsilyl, triphenylmethyl, triphenylsilyl, para-toluenesulfonyl and the like.
Protecting groups for OH and SH moieties include, but are not limited to, acetyl,
allyl, allyloxycarbonyl, benzyloxycarbonyl (Cbz), benzoyl, benzyl, tert-butyl,
tert-butyldimethy lsilyl, tert-butyldiphenylsilyl, 3,4-dimethoxybenzyl,
3.4-dimethoxybenzyloxycarbonyl, l,l-dimethyl-2-propenyl, diphenylmethyl, formyl,
methanesulfonyl, methoxyacetyl, 4-methoxybenzyloxycarbonyl, para-methoxybenzyl,
methoxycarbonyl, methyl, para-toluenesulfonyl, 2,2,2-trichloroethoxycarbonyl,
2,2,2-trichloroethyl, triethylsilyl, trifluoroacetyl, 2-(trimethylsilyl)ethoxycarbonyl,
2-trimethylsilylethyl, triphenylmethyl, 2-(triphenylphosphonio)ethoxycarbonyl and the
like.
The following abbreviations have the meanings indicated.
ADDP means l,l'-(azodicarbonyl)dipiperidine; AD-mix- b means a mixture of
(DHQD) 2PHAL, 3Fe(CN) , K2C0 3 and K2S0 4); AIBN means 2,2'-azobis(2-
methylpropionitrile); 9-BBN means 9-borabicyclo[3.3.1]nonane; (DHQD)2PHAL means
hydroquinidine 1,4-phthalazinediyl diethyl ether; DBU means 1,8-
diazabicyclo[5.4.0]undec-7-ene; DIBAL means diisobutylaluminum hydride; DIEA
means diisopropylethylamine; DMAP means N,N-dimethylaminopyridine; DME means
1,2-dimethoxyethane; DMF means N,N-dimethylformamide; dmpe means 1,2-
bis(dimethylphosphino)ethane; DMSO means dimethylsulfoxide; dppb means 1,4-
bis(diphenylphosphino)butane; dppe means 1,2-bis(diphenylphosphino)ethane; dppf
means l,l'-bis(diphenylphosphino)ferrocene; dppm means 1,1-
bis(diphenylphosphino)methane; EDAC means l-(3-dimethylaminopropyl)-3-
ethylcarbodiimide; Fmoc means fluorenylmethoxycarbonyl; HATU means 0-(7-
azabenzotriazol-l-yl)-N,NTS['N'-tetramethyluronium hexafluorophosphate; HMPA means
hexamethylphosphoramide; IPA means isopropyl alcohol; LDA means lithium
diisopropylamide; LHMDS means lithium bis(hexamethyldisilylamide); MP-BH3 means
macroporus triethylammonium methylpolystyrene cyanoborohydride; LAH means

WHAT IS CLAIMED IS:
1. A method of treating a patient suffering from cancer, the method
comprising the steps of:
a) administering to a patient suffering from cancer a therapeutically effective
amount of at least one polyploidy inducing agent; and
b) administering to the patient a therapeutically effective amount of at least
one Bcl-2 family protein inhibitor.
2 . The method of claim 1, wherein the at least one polyploidy inducing agent
is an Aurora Kinase inhibitor.
3. The method of claim 2, wherein the Aurora Kinase inhibitor is an Aurora
Kinase B inhibitor.
4 . The method of claim 3, wherein the Aurora Kinase B inhibitor is VX-680,
AZD1 152, ZM44739 or Hersperadin.
5. The method of claim 1, wherein the Bcl-2 family protein inhibitor is ABT-
263, ABT-737, a BC1-XL selective inhibitor or combinations thereof.
6. A combination of therapeutic agents for use in treating a patient suffering
from cancer, wherein said combination comprises:
a) at least one polyploidy inducing agent for use in inducing polyploidization
in one or more cancer cells in the patient; and
b) at least one Bcl-2 family protein inhibitor.
7. The combination of claim 6, wherein the at least one polyploidy inducing
agent is an Aurora Kinase inhibitor.
8. The combination of claim 7, wherein the Aurora Kinase inhibitor is an
Aurora Kinase B inhibitor.
9. The combination of claim 8, wherein the Aurora Kinase B inhibitor is VX-
680, AZD1 152, ZM44739 or Hersperadin.
10. The combination of claim 6, wherein the Bcl-2 family protein inhibitor is
ABT-263, ABT-737, a BC1-XL selective inhibitor or combinations thereof.
11. A method of classifying a patient for eligibility for treatment with a
polyploidy inducing agent, a Bcl-2 family protein inhibitor or a combination of a
polyploidy inducing agent and a Bcl-2 family protein inhibitor, the method comprising the
steps of:
a) providing a test sample from a patient;
b) determining the presence or absence of at least one mutation in at least one
gene selected from the group consisting of: a BAX gene, a BAK gene or a NOXA gene in
the test sample; and
c) classifying the patient as being eligible for receiving treatment with a
polyploidy inducing agent, a Bcl-2 family protein inhibitor or a combination of a
polyploidy inducing agent and a Bcl-2 family protein inhibitor based on the presence or
absence of at least one mutation as determined in step b).
12. The method of claim 11, wherein the polyploidy inducing agent is an
Aurora Kinase inhibitor.
13. The method of claim 12, wherein the Aurora Kinase inhibitor is an Aurora
Kinase A inhibitor, an Aurora Kinase B inhibitor, an Aurora Kinase C inhibitor or
combinations thereof.
14. The method of claim 13, wherein the Aurora Kinase inhibitor is an Aurora
Kinase B inhibitor.
15. The method of claim 14, wherein the Aurora Kinase B inhibitor is
AZD1 152, ZM447439, VX-680/MK0457 or Hersperadin.
16. The method of claim 11, wherein the Bcl-2 family protein inhibitor is
ABT-263, ABT-737, a BC1-XL selective inhibitor or combinations thereof.
17. The method of claim 11, wherein the test sample comprises a tissue
sample.
18. The method of claim 17, wherein the tissue sample comprises a peripheral
blood sample, a tumor tissue or a suspected tumor tissue, a thin layer cytological sample, a
fine needle aspirate sample, a bone marrow sample, a lymph node sample, a urine sample,
an ascites sample, a lavage sample, an esophageal brushing sample, a bladder or lung
wash sample, a spinal fluid sample, a brain fluid sample, a ductal aspirate sample, a nipple
discharge sample, a pleural effusion sample, a fresh frozen tissue sample, a paraffin
embedded tissue sample or an extract or processed sample produced from any of a
peripheral blood sample, a tumor tissue or a suspected tumor tissue, a thin layer
cytological sample, a fine needle aspirate sample, a bone marrow sample, a urine sample,
an ascites sample, a lavage sample, an esophageal brushing sample, a bladder or lung
wash sample, a spinal fluid sample, a brain fluid sample, a ductal aspirate sample, a nipple
discharge sample, a pleural effusion sample, a fresh frozen tissue sample or a paraffin
embedded tissue sample.
19. The method of claim 11, wherein the determining step (b) is performed by
in situ hybridization.
20. The method of claim 19, wherein the in situ hybridization is performed
90 with a nucleic acid probe that is fluorescently labeled.
21. The method of claim 20, wherein the in situ hybridization is performed
with at least two nucleic acid probes.
95 22. The method of claim 20, wherein the in situ hybridization is performed
with a peptide nucleic acid probe.
23. The method of claim 11, wherein the determining step (b) is performed by
polymerase chain reaction.
100
24. The method of claim 11, wherein the patient is also being treated with
chemotherapy, radiation or combinations thereof.
25. A method of monitoring a patient suffering from cancer and being treated
105 with a polyploidy inducing agent, a Bcl-2 family protein inhibitor or a combination of a
polyploidy inducing agent and a Bcl-2 family protein inhibitor, the method comprising the
steps of:
a) providing a test sample from a patient suffering from cancer and currently
being treated with at least one polyploidy inducing agent, a Bcl-2 family protein inhibitor
110 or a combination of a polyploidy inducing agent and a Bcl-2 family protein inhibitor;
b) determining the presence or absence of at least one mutation in at least one
gene selected from the group consisting of: a BAX gene, a BAK gene or a NOXA gene in
the test sample; and
c) determining whether the patient should continue to be treated with a polyploidy
115 inducing agent, a Bcl-2 family protein inhibitor or a combination of a polyploidy inducing
agent and a Bcl-2 family protein inhibitor based on the presence or absence of at least one
mutation as determined in step b).
26. The method of claim 25, wherein the polyploidy inducing agent is an
120 Aurora Kinase inhibitor.
27. The method of claim 26, wherein the Aurora Kinase inhibitor is an Aurora
Kinase A inhibitor, an Aurora Kinase B inhibitor, an Aurora Kinase C inhibitor or
combinations thereof.
125
28. The method of claim 27, wherein the Aurora Kinase inhibitor is an Aurora
Kinase B inhibitor.
29. The method of claim 28, wherein the Aurora Kinase B inhibitor is
130 AZD1 152, ZM447439, VX -680/MK0457 or Hersperadin.
30. The method of claim 25, wherein the Bcl-2 family protein inhibitor is
ABT-263, ABT-737, a BC1-XL selective inhibitor or combinations thereof.
135 31. The method of claim 25, wherein the test sample comprises atissue
sample.
32. The method of claim 3 1, wherein the tissue sample comprises a peripheral
blood sample, a tumor tissue or a suspected tumor tissue, a thin layer cytological sample, a
140 fine needle aspirate sample, a bone marrow sample, a lymph node sample, a urine sample,
an ascites sample, a lavage sample, an esophageal brushing sample, a bladder or lung
wash sample, a spinal fluid sample, a brain fluid sample, a ductal aspirate sample, a nipple
discharge sample, a pleural effusion sample, a fresh frozen tissue sample, a paraffin
embedded tissue sample or an extract or processed sample produced from any of a
145 peripheral blood sample, a tumor tissue or a suspected tumor tissue, a thin layer
cytological sample, a fine needle aspirate sample, a bone marrow sample, a urine sample,
an ascites sample, a lavage sample, an esophageal brushing sample, a bladder or lung
wash sample, a spinal fluid sample, a brain fluid sample, a ductal aspirate sample, a nipple
discharge sample, a pleural effusion sample, a fresh frozen tissue sample or a paraffin
150 embedded tissue sample.
33. The method of claim 25, wherein the determining step (b) is performed by
in situ hybridization.
155 34. The method of claim 33, wherein the in situ hybridization is performed
with a nucleic acid probe that is fluorescently labeled.
35. The method of claim 33, wherein the in situ hybridization is performed
with at least two nucleic acid probes.
160
36. The method of claim 33, wherein the in situ hybridization is performed
with a peptide nucleic acid probe.
37. The method of claim 25, wherein the determining step (b) is performed by
165 polymerase chain reaction.
38. The method of claim 25, wherein the patient is also being treated with
chemotherapy, radiation or combinations thereof.
170 39. A method of classifying a patient as having a cancer that is resistant to
treatment with a polyploidy inducing agent, a Bcl-2 family protein inhibitor or a
combination of a polyploidy inducing agent and a Bcl-2 family protein inhibitor, the
method comprising the steps of:
a) providing a test sample from a patient suffering from cancer;
175 b) determining the presence or absence of at least one mutation in at least one
gene selected from the group consisting of: a BAX gene, a BAK gene or a NOXA gene in
the test sample; and
c) classifying the patient as having a cancer that is resistant to treatment with a
polyploidy inducing agent, a Bcl-2 family protein inhibitor or a combination of a
180 polyploidy inducing agent and a Bcl-2 family protein inhibitor based on the presence or
absence of at least one mutation determined in step b).
40. The method of claim 39, wherein the polyploidy inducing agent is an
Aurora Kinase inhibitor.
185
4 1. The method of claim 40, wherein the Aurora Kinase inhibitor is an Aurora
Kinase A inhibitor, an Aurora Kinase B inhibitor, an Aurora Kinase C inhibitor or
combinations thereof.
190 4 . The method of claim 4 1, wherein the Aurora Kinase inhibitor is an Aurora
Kinase B inhibitor.
43. The method of claim 42, wherein the Aurora Kinase B inhibitor is
AZD1 152, ZM447439, VX-680/MK0457 or Hersperadin.
195
44. The method of claim 39, wherein the Bcl-2 family protein inhibitor is
ABT-263, ABT-737, a BC1-XL selective inhibitor or combinations thereof.
45. The method of claim 39, wherein the test sample comprises a tissue
sample.
46. The method of claim 45, wherein the tissue sample comprises a peripheral
blood sample, a tumor tissue or a suspected tumor tissue, a thin layer cytological sample, a
fine needle aspirate sample, a bone marrow sample, a lymph node sample, a urine sample,
205 an ascites sample, a lavage sample, an esophageal brushing sample, a bladder or lung
wash sample, a spinal fluid sample, a brain fluid sample, a ductal aspirate sample, a nipple
discharge sample, a pleural effusion sample, a fresh frozen tissue sample, a paraffin
embedded tissue sample or an extract or processed sample produced from any of a
peripheral blood sample, a tumor tissue or a suspected tumor tissue, a thin layer
210 cytological sample, a fine needle aspirate sample, a bone marrow sample, a urine sample,
an ascites sample, a lavage sample, an esophageal brushing sample, a bladder or lung
wash sample, a spinal fluid sample, a brain fluid sample, a ductal aspirate sample, a nipple
discharge sample, a pleural effusion sample, a fresh frozen tissue sample or a paraffin
embedded tissue sample.
215
47. The method of claim 39, wherein the determining step (b) is performed by
in situ hybridization.
48. The method of claim 47, wherein the in situ hybridization is performed
220 with a nucleic acid probe that is fluorescently labeled.
49. The method of claim 47, wherein the in situ hybridization is performed
with at least two nucleic acid probes.
225 50. The method of claim 47, wherein the in situ hybridization is performed
with a peptide nucleic acid probe.
51. The method of claim 39, wherein the determining step (b) is performed by
polymerase chain reaction.
230
52. The method of claim 39, wherein the patient is also being treated with
chemotherapy, radiation or combinations thereof.