U.S. patent application number 12/618612 was filed with the patent office on 2010-08-12 for therapies for hematologic malignancies.
Invention is credited to W. Michael GALLATIN, Neill A. Giese, Roger G. Ulrich.
Application Number | 20100202963 12/618612 |
Document ID | / |
Family ID | 41604034 |
Filed Date | 2010-08-12 |
United States Patent
Application |
20100202963 |
Kind Code |
A1 |
GALLATIN; W. Michael ; et
al. |
August 12, 2010 |
THERAPIES FOR HEMATOLOGIC MALIGNANCIES
Abstract
The invention provides methods that relate to a novel
therapeutic strategy for the treatment of hematological
malignancies and inflammatory diseases. In particular, the method
comprises administration of a compound of formula A, ##STR00001##
wherein R is H, halo, or C1-C6 alkyl; R' is C1-C6 alkyl; or a
pharmaceutically acceptable salt thereof; and optionally a
pharmaceutically acceptable excipient.
Inventors: |
GALLATIN; W. Michael;
(Mercer Island, WA) ; Ulrich; Roger G.;
(Sammamish, WA) ; Giese; Neill A.; (Seattle,
WA) |
Correspondence
Address: |
MORRISON & FOERSTER LLP
12531 HIGH BLUFF DRIVE, SUITE 100
SAN DIEGO
CA
92130-2040
US
|
Family ID: |
41604034 |
Appl. No.: |
12/618612 |
Filed: |
November 13, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61114434 |
Nov 13, 2008 |
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61142845 |
Jan 6, 2009 |
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61155057 |
Feb 24, 2009 |
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61180768 |
May 22, 2009 |
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61231278 |
Aug 4, 2009 |
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61245196 |
Sep 23, 2009 |
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Current U.S.
Class: |
424/1.49 ;
424/133.1; 424/649; 514/1.1; 514/1.2; 514/110; 514/263.21; 514/27;
514/7.6 |
Current CPC
Class: |
A61K 31/519 20130101;
A61K 38/05 20130101; A61K 31/4965 20130101; A61P 11/00 20180101;
A61P 35/00 20180101; A61P 37/00 20180101; A61K 39/3955 20130101;
A61P 19/02 20180101; A61P 29/00 20180101; A61K 31/495 20130101;
A61K 31/517 20130101; A61K 31/69 20130101; A61P 35/02 20180101;
A61P 11/06 20180101; A61K 45/06 20130101; A61K 2039/505 20130101;
A61K 2300/00 20130101; A61K 31/75 20130101; C07D 473/34 20130101;
A61P 37/02 20180101; A61P 11/02 20180101; A61P 37/08 20180101; A61K
31/52 20130101; A61P 43/00 20180101; A61K 31/5377 20130101; A61K
31/513 20130101 |
Class at
Publication: |
424/1.49 ;
514/263.21; 514/19; 514/110; 424/133.1; 514/27; 424/649 |
International
Class: |
A61K 31/52 20060101
A61K031/52; A61P 35/00 20060101 A61P035/00; A61P 35/02 20060101
A61P035/02; A61K 38/05 20060101 A61K038/05; A61K 31/661 20060101
A61K031/661; A61K 39/395 20060101 A61K039/395; A61K 51/10 20060101
A61K051/10; A61K 31/7048 20060101 A61K031/7048; A61K 33/24 20060101
A61K033/24 |
Claims
1. A method to treat a condition, wherein the condition is cancer
or an autoimmune condition, comprising administering to a subject
in need of such treatment, an effective amount of a compound having
a formula A, ##STR00017## wherein R is H, halo, or C1-C6 alkyl; R'
is C1-C6 alkyl; or a pharmaceutically acceptable salt thereof; and
optionally a pharmaceutically acceptable excipient.
2. The method according to claim 1, wherein the compound is
predominantly the S-enantiomer.
3. The method according to claim 1, wherein R is fluoro (F) and is
attached to position 5 or 6 of the quinazolinyl ring.
4. The method according to claim 1, wherein R is H or F; and R' is
methyl, ethyl or propyl.
5. The method according to claim 1, wherein the compound is
##STR00018##
6. The method according to claim 1, wherein the compound is
##STR00019##
7. The method according to claim 1, wherein the autoimmune
condition is allergic rhinitis, asthma, COPD, or rheumatoid
arthritis.
8. The method according to claim 1, wherein the condition is
cancer.
9. The method according to claim 8, wherein the cancer is a
hematological malignancy.
10. The method according to claim 9, wherein said hematological
malignancy is leukemia.
11. The method according to claim 9, wherein said hematological
malignancy is lymphoma.
12. The method according to claim 9, wherein the hematological
malignancy is selected from the group consisting of acute
lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic
lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL),
multiple myeloma (MM), non-Hodgkin's lymphoma (NHL), mantle cell
lymphoma (MCL), follicular lymphoma, Waldenstrom's
macroglobulinemia (WM), B-cell lymphoma and diffuse large B-cell
lymphoma (DLBCL).
13. The method according to claim 8, wherein the cancer is acute
lymphocytic leukemia (ALL).
14. The method according to claim 8, wherein the cancer is acute
myeloid leukemia (AML).
15. The method according to claim 8, wherein the cancer is chronic
lymphocytic leukemia (CLL).
16. The method according to claim 8, wherein the cancer is multiple
myeloma (MM).
17. The method according to claim 8, wherein the cancer is B-cell
lymphoma.
18. The method according to claim 8, wherein the cancer is diffuse
large B-cell lymphoma (DLBCL).
19. The method according to claim 8, wherein the cancer is B-cell
or T-cell ALL.
20. The method according to claim 8, wherein the cancer is
Hodgkin's lymphoma.
21. The method according to claim 8, wherein the cancer is breast
or ovarian cancer.
22. The method according to claim 8, wherein the subject is
refractory to chemotherapy treatment, or in relapse after treatment
with chemotherapy.
23. The method according to claim 8, further comprising
administering at least one additional therapeutic agent.
24. The method according to claim 23, wherein said additional
therapeutic agent is a proteasome inhibitor.
25. The method according to claim 23, wherein the additional
therapeutic agent is combined with the compound of Formula A.
26. The method according to claim 23, wherein the additional
therapeutic agent is selected from the group consisting of
Bortezomib (Velcade.RTM.), Carfilzomib (PR-171), PR-047,
disulfiram, lactacystin, PS-519, eponemycin, epoxomycin,
aclacinomycin, CEP-1612, MG-132, CVT-63417, PS-341, vinyl sulfone
tripeptide inhibitors, ritonavir, PI-083, (+/-)-7-methylomuralide,
and (-)-7-methylomuralide.
27. The method according to claim 23, wherein the additional
therapeutic agent is bortezomib.
28. The method according to claim 23, wherein the compound is
administered in combination with at least a group of at least two
agents, wherein said group of agents is selected from the groups
consisting of a-q, a) CHOP (cyclophosphamide, doxorubicin,
vincristine, prednisone); b) R-CHOP (rituximab-CHOP); c) hyperCV AD
(hyperfractionated cyclophosphamide, vincristine, doxorubicin,
dexamethasone, methotrexate, cytarabine); d) R-hyperCV AD
(rituximab-hyperCV AD); e) FCM (fludarabine, cyclophosphamide,
mitoxantrone); f) R-FCM (rituximab, fludarabine, cyclophosphamide,
mitoxantrone); g) bortezomib and rituximab; h) temsirolimus and
rituximab; i) temsirolimus and Velcade.RTM.; j) Iodine-131
tositumomab (Bexxar.RTM.) and CHOP; k) CVP (cyclophosphamide,
vincristine, prednisone); l) R-CVP (rituximab-CVP); m) ICE
(iphosphamide, carboplatin, etoposide); n) R-ICE (rituximab-ICE);
o) FCR (fludarabine, cyclophosphamide, rituximab); p) FR
(fludarabine, rituximab); and q) D.T. PACE (Dexamethasone,
Thalidomide, Cisplatin, Adriamycin, Cyclophosphamide,
Etoposide).
29. The method according to claims 1, wherein the compound of
formula A is present in a pharmaceutical composition comprising the
compound of formula A and at least one pharmaceutically acceptable
excipient.
30. A method to treat a condition, wherein the condition is
selected from the group consisting of multiple myeloma, acute
lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic
lymphocytic leukemia (CLL), B-cell lymphoma, diffuse large B-cell
lymphoma (DLBCL), B cell ALL, T cell ALL, Hodgkin's lymphoma,
breast, and ovarian cancer, comprising administering to a subject
in need of such treatment, an effective amount of a compound having
a formula I'' or formula II'' or a pharmaceutically acceptable salt
thereof: ##STR00020##
31. The method according to claim 30, wherein the subject is
refractory to chemotherapy treatment or in relapse after treatment
with chemotherapy.
32. The method according to claim 30, wherein the subject has a
cancer that constitutively expresses Akt phosphorylation
activity.
33. The method according to claim 30, wherein the subject has a
cancer with high p110.delta. activity and low p110.alpha.
activity.
34. The method according to claims 30, wherein the compound is used
in combination with Bortezomib.
35. A method to treat a hematological cancer comprising
administering to a subject in need of such treatment, an effective
amount of a compound having formula I'' or formula II'' and
bortezomib or carfilzomib.
36. The method according to claim 8, wherein the compound maintains
an average blood concentration above the EC.sub.50 level for
PI3K.delta. activation and below the level for EC.sub.50
PI3K.gamma. activation in basophils over a period of at least 12
hours from compound administration.
37. The method according to claim 8, wherein the compound maintains
an average blood plasma concentration between 100 nM and 1100 nM
over a period of at least 12 hours from compound
administration.
38. The method according to claim 8, wherein the subject is
resistant to standard chemotherapeutic treatments.
39. The method according to claim 8, wherein the subject has at
least one enlarged lymph node.
40. The method according to claim 22, wherein the subject is
refractory to at least two standard or experimental chemotherapy
treatments.
41. The method according to claim 40, wherein each chemotherapy
treatment is selected from the group consisting of fludarabine,
alkylating agents, rituximab, alemtuzumab, and the treatments a-q
listed in claim 28.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from U.S. provisional
application Nos. 61/245,196 filed Sep. 23, 2009; 61/231,278 filed
Aug. 4, 2009; 61/180,768 filed May 22, 2009; 61/155,057 filed Feb.
24, 2009; 61/142,845 filed Jan. 6, 2009; and 61/114,434 filed Nov.
13, 2008. The contents of these documents are incorporated herein
by reference.
TECHNICAL FIELD
[0002] The invention is in the field of therapeutics and medicinal
chemistry. In particular, the invention concerns uses of certain
quinazoline derivatives for the treatment of hematologic
malignancies and certain other conditions.
BACKGROUND ART
[0003] Cell signaling via 3'-phosphorylated phosphoinositides has
been implicated in a variety of cellular processes, e.g., malignant
transformation, growth factor signaling, inflammation, and
immunity. The enzyme responsible for generating these
phosphorylated signaling products, phosphatidylinositol 3-kinase
(PI 3-kinase; PI3K), was originally identified as an activity
associated with viral oncoproteins and growth factor receptor
tyrosine kinases that phosphorylates phosphatidylinositol (PI) and
its phosphorylated derivatives at the 3'-hydroxyl of the inositol
ring.
[0004] PI 3-kinase activation, is believed to be involved in a
range of cellular responses including cell growth, differentiation,
and apoptosis.
[0005] The initial purification and molecular cloning of PI
3-kinase revealed that it was a heterodimer consisting of p85 and
p110 subunits. Four distinct Class I PI3Ks have been identified,
designated PI3K .alpha., .beta., .delta., and .gamma., each
consisting of a distinct 110 kDa catalytic subunit and a regulatory
subunit. More specifically, three of the catalytic subunits, i.e.,
p110.alpha., p110.beta. and p110.delta., each interact with the
same regulatory subunit, p85; whereas p110.gamma. interacts with a
distinct regulatory subunit, p101. The patterns of expression of
each of these PI3Ks in human cells and tissues are also
distinct.
[0006] Identification of the p110.delta. isoform of PI 3-kinase is
described in Chantry et al., J Biol Chem, 272:19236-41 (1997). It
was observed that the human p110.delta. isoform is expressed in a
tissue-restricted fashion. It is expressed at high levels in
lymphocytes and lymphoid tissues, suggesting that the protein might
play a role in PI 3-kinase-mediated signaling in the immune system.
The p110.beta. isoform of PI3K may also play a role in
PI3K-mediated signaling in certain cancers.
[0007] There is a need for a treatment relating to PI3K mediated
disorders relating to cancers, inflammatory diseases, and
autoimmune diseases.
SUMMARY
[0008] The present invention provides a class of quinazolinone type
compounds and a method to use these compounds in the treatment of
cancer, inflammatory, and autoimmune diseases. In particular,
cancers that are hematologic malignancies, such as leukemia and
lymphoma, are treated by the methods herein. Also provided are
methods of using the quinazolinone compounds in combination with
other therapeutic treatments in patients in need thereof.
[0009] In one aspect, the invention provides the use of a compound
for the manufacture of a medicament for the treatment of a
condition in a subject, wherein the condition is cancer or an
autoimmune condition; wherein the compound is of formula A,
##STR00002##
[0010] wherein R is H, halo, or C1-C6 alkyl; R' is C1-C6 alkyl; or
a pharmaceutically acceptable salt thereof; and optionally a
pharmaceutically acceptable excipient.
[0011] In one embodiment, the compound is predominantly the
S-enantiomer.
[0012] In some of the foregoing embodiments, R is fluoro (F) and is
attached to position 5 or 6 of the quinazolinyl ring.
[0013] In some of the foregoing embodiments, R is H or F; and R' is
methyl, ethyl or propyl.
[0014] In some embodiments, the compound is
##STR00003##
[0015] In some embodiments, compound is
##STR00004##
[0016] In some of the foregoing embodiments, the autoimmune disease
is allergic rhinitis, asthma, COPD, or rheumatoid arthritis.
[0017] In some of the foregoing embodiments, the condition is
cancer.
[0018] In some of the foregoing embodiments, the cancer is a
hematological malignancy.
[0019] In some of the foregoing embodiments, the hematological
malignancy is leukemia.
[0020] In some of the foregoing embodiments, the hematological
malignancy is lymphoma.
[0021] In some of the foregoing embodiments, the hematological
malignancy is selected from the group consisting of acute
lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic
lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL),
multiple myeloma (MM), non-Hodgkin's lymphoma (NHL), mantle cell
lymphoma (MCL), follicular lymphoma, Waldenstrom's
macroglobulinemia (WM), B-cell lymphoma and diffuse large B-cell
lymphoma (DLBCL).
[0022] In some of the foregoing embodiments, the cancer is acute
lymphocytic leukemia (ALL).
[0023] In some of the foregoing embodiments, the cancer is acute
myeloid leukemia (AML).
[0024] In some of the foregoing embodiments, the cancer is chronic
lymphocytic leukemia (CLL).
[0025] In some of the foregoing embodiments, the cancer is multiple
myeloma (MM).
[0026] In some of the foregoing embodiments, the cancer is B-cell
lymphoma.
[0027] In some of the foregoing embodiments, the cancer is diffuse
large B-cell lymphoma (DLBCL).
[0028] In some of the foregoing embodiments, the cancer is B-cell
or T-cell ALL.
[0029] In some of the foregoing embodiments, the cancer is
Hodgkin's lymphoma.
[0030] In some of the foregoing embodiments, the cancer is breast,
lung, colon, prostate or ovarian cancer.
[0031] In some of the foregoing embodiments, the subject is
refractory to chemotherapy treatment, or in relapse after treatment
with chemotherapy.
[0032] In some of the foregoing embodiments, the compound is
prepared for administration with at least one additional
therapeutic agent.
[0033] In some of the foregoing embodiments, the additional
therapeutic agent is a proteasome inhibitor.
[0034] In some of the foregoing embodiments, the additional
therapeutic agent is combined with the compound of Formula A.
[0035] In some of the foregoing embodiments, the additional
therapeutic agent is selected from the group consisting of
bortezomib (Velcade.RTM.), carfilzomib (PR-171), PR-047,
disulfiram, lactacystin, PS-519, eponemycin, epoxomycin,
aclacinomycin, CEP-1612, MG-132, CVT 63417, PS-341, vinyl sulfone
tripeptide inhibitors, ritonavir, PI-083, (+/-) 7 methylomuralide,
(-)-7-methylomuralide.
[0036] In some of the foregoing embodiments, the additional
therapeutic agent is bortezomib.
[0037] In some of the foregoing embodiments, the compound is
prepared for administration with at least a group of at least two
agents, wherein said group of agents is selected from the groups
consisting of a-q,
[0038] a) CHOP (cyclophosphamide, doxorubicin, vincristine,
prednisone);
[0039] b) R CHOP (rituximab CHOP);
[0040] c) hyperCV AD (hyperfractionated cyclophosphamide,
vincristine, doxorubicin, dexamethasone, methotrexate,
cytarabine);
[0041] d) R-hyperCV AD (rituximab-hyperCV AD);
[0042] e) FCM (fludarabine, cyclophosphamide, mitoxantrone);
[0043] f) R-FCM (rituximab, fludarabine, cyclophosphamide,
mitoxantrone);
[0044] g) bortezomib and rituximab;
[0045] h) temsirolimus and rituximab;
[0046] i) temsirolimus and Velcade.RTM.;
[0047] j) Iodine-131 tositumomab (Bexxar.RTM.) and CHOP;
[0048] k) CVP (cyclophosphamide, vincristine, prednisone);
[0049] l) R-CVP (rituximab-CVP);
[0050] m) ICE (iphosphamide, carboplatin, etoposide);
[0051] n) R-ICE (rituximab-ICE);
[0052] o) FCR (fludarabine, cyclophosphamide, rituximab);
[0053] p) FR (fludarabine, rituximab); and
[0054] q) D.T. PACE (dexamethasone, thalidomide, cisplatin,
Adriamycin.RTM., cyclophosphamide, etoposide).
[0055] In some of the foregoing embodiments, the compound of
formula A is present in a pharmaceutical composition comprising the
compound of formula A and at least one pharmaceutically acceptable
excipient.
[0056] In another aspect, the invention provides the use of a
compound for the manufacture of a medicament for the treatment of a
condition in a subject, wherein the condition is selected from the
group consisting of multiple myeloma, acute lymphocytic leukemia
(ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia
(CLL), B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), B
cell ALL, T cell ALL, Hodgkin's lymphoma, breast, and ovarian
cancer, wherein the compound is a compound of formula I'' or
formula II'':
##STR00005##
[0057] In some of the foregoing embodiments, the subject is
refractory to chemotherapy treatment or in relapse after treatment
with chemotherapy.
[0058] In some of the foregoing embodiments, the subject has a
cancer that constitutively expresses Akt phosphorylation
activity.
[0059] In some of the foregoing embodiments, the subject has a
cancer with high p110.delta. activity and low p110.alpha.
activity.
[0060] In some of the foregoing embodiments, the compound is used
in combination with bortezomib.
[0061] In another aspect, the invention provides the use of a
compound I'' or H'' in the manufacture of a medicament for treating
a hematological cancer, wherein the medicament is prepared for
administration with bortezomib or carfilzomib.
[0062] In some of the foregoing embodiments, the compound maintains
an average blood concentration above the EC50 level for PI3K.delta.
activation and below the level for EC50 PI3K.gamma. activation in
basophils over a period of at least 12 hours from compound
administration.
[0063] In some of the foregoing embodiments, the compound maintains
an average blood plasma concentration between 100 nM and 1100 nM
over a period of at least 12 hours from compound
administration.
[0064] In some of the foregoing embodiments, the subject is
resistant to standard chemotherapeutic treatments.
[0065] In some of the foregoing embodiments, the subject has at
least one enlarged lymph node.
[0066] In some of the foregoing embodiments, the subject is
refractory to at least two standard or experimental chemotherapy
treatments had at least two prior chemotherapy treatments.
[0067] In some of the foregoing embodiments, each chemotherapy
treatment is selected from the group consisting of fludarabine,
alkylating agents, rituximab, alemtuzumab, and the treatments a-q
listed above.
BRIEF DESCRIPTION OF THE DRAWINGS
[0068] FIG. 1 shows a graphical summary of multiple myeloma (MM)
cell growth as a function of varying concentrations of cytokines
IGF-1 and IL-6 in combination with compound I, using LB cells.
[0069] FIG. 2 shows a graphical summary of cell growth of multiple
myeloma (MM) cells as a function of varying concentrations of
compound I and the presence or absence of bone marrow stromal cells
(BMSC) after 48 hours.
[0070] FIG. 3 shows a graphical summary of apoptosis of Chronic
Lymphocytic Leukemia (CLL) cells as a function of varying
concentrations of compound of formula I.
[0071] FIG. 4 shows a summary chart of the effect of compound I on
cell viability, reduction in Akt (Ser473) phosphorylation, and
caspase 3 activation in several different Acute Lymphoblastic
Leukemia (ALL) cell lines.
[0072] FIG. 5 shows a summary of the effect of compound I on the
cell cycle of acute lymphoblastic leukemia (ALL) cell lines.
[0073] FIG. 6 shows a graphical summary of the effect of varying
concentration of compound I on cellular growth in breast cancer
T47D and HS-578T cell lines at 48 hrs and 72 hrs.
[0074] FIG. 7 shows a graphical summary of the effect of varying
concentrations of compound I on cellular growth of ovarian IGROV-1
and OVCAR-3 cell lines at 48 hrs and 72 hrs.
[0075] FIG. 8 shows a summary of the effect of compound I on Akt
phosphorylation in many leukemia and lymphoma cell lines.
[0076] FIG. 9 shows SDS-PAGE images and displays of Akt and pAkt in
various hematopoietic cancer cell lines as a function of the
presence or absence of compound I, showing compound I inhibits Akt
phosphorylation.
[0077] FIG. 10 shows graphical summaries of apoptotic and viable
cell populations in breast cancer cell lines as a function of
varying concentrations of compound formula I, demonstrating that
the compound induces apoptosis.
[0078] FIG. 11 shows the concentration of compound I in the blood
of a healthy human subject over 12 hours after oral administration
of 50, 100 and 200 mg doses of said compound.
[0079] FIG. 12 shows the comparison of lesion areas in a human
patient diagnosed with mantle cell lymphoma after 28 days (1 cycle)
of treatment with compound I and lesion areas prior to
treatment.
[0080] FIG. 13 shows the ALC (absolute lymphocyte count) in the
blood of a patient over a period of 4 weeks after 28 days (1 cycle)
of treatment with the compound of formula I.
[0081] FIG. 14 shows the concentration of compound I in the blood
of patients with and without mantle cell lymphoma (MCL) over 6
hours after administration (50 mg BID) at day 28, compared to the
concentration in the blood of a normal healthy volunteer at day 7
(D7) using the same dosing schedule or dosing with 100 mg BID of
Compound I.
[0082] FIG. 15 shows PI3K isoform expression in a panel of lymphoma
and leukemia cell lines.
[0083] FIG. 16A shows cell viability and apoptosis data in leukemia
cell lines exposed to Compound I. In FIG. 16B the Annexin staining
indicates an increase in apoptosis in the treated cells.
[0084] FIGS. 17A-D shows PAGE results of different PI3K isoform
expression in CLL patient cells.
[0085] FIG. 18 shows the induction of (A) caspase 3 and (B) PARP
cleavage in the presence of compound I.
[0086] FIG. 19 shows increased apoptosis of Chronic Lymphocytic
Leukemia (CLL) cells from poor prognosis patients caused by
exposure to compound I, demonstrating that compound I is effective
in drug resistant patients.
[0087] FIG. 20 shows increased apoptosis of Chronic Lymphocytic
Leukemia (CLL) cells from refractory/relapsed patients caused by
exposure to the compound of formula I.
[0088] FIG. 21 shows the results of Phospho-Akt production in the
absence or presence of 0.1, 1.0, 10 .mu.M of compound I.
[0089] FIG. 22 shows flow cytometry results relating to PI3K
signaling in basophils, demonstrating that (B) anti-FC.epsilon.R1
or (C) fMLP increases CD63 expression compared to no stimulation
(A).
[0090] FIG. 23 shows inhibition of PI3K inhibition by compound I in
basophils, and demonstrates that Compound I is especially effective
at inhibition of CD63 expression induced by a p110.delta. pathway,
but also effective at micromolar concentration to inhibit
expression induced by a p110.gamma. pathway.
[0091] FIG. 24 shows pharmacokinetic data of (A) single dose
administration of compound I at different dose amounts in healthy
volunteers, and (B) a pharmacokinetic profile that maintains an
effective dosage over a 12 hour period.
[0092] FIG. 25 shows the effects of various doses of compound I on
(A) glucose and (B) insulin levels, exhibiting little off-target
activity.
[0093] FIG. 26A shows the PI3K isoform expression in a panel of
DLBCL cell lines.
[0094] FIG. 26 B shows an SDS-PAGE image of pAkt in DLBCL cell
lines in the presence or absence of compound I.
[0095] FIG. 27 shows the effects of a 10 .mu.M concentration of
compound I on the phosphorylation of Akt and S6 in ALL cell lines
in SDS-PAGE.
[0096] FIG. 28 shows a dose dependent reduction of phosphorylation
of Akt, S6, and GSK-3.beta. after treatment with a series of
compound I dilutions.
[0097] FIG. 29 shows dose dependent effects of compound I on ALL
cell lines in the downregulation of cFLIP, cleavage of Caspase 3,
and cleavage of PARP.
[0098] FIG. 30 shows expression of p110.delta. in A) MM cell lines
and B) patient MM cells; and C) in MM.1S and LB cells.
[0099] FIG. 31A shows expression of p110.delta. from LB and INA-6
cells transfected with p110.delta. siRNA (Si) or control siRNA
(mock).
[0100] FIG. 31B shows a graph of INA-6 cell growth after
transfection with p110.delta. siRNA (Si) or control siRNA
(mock).
[0101] FIG. 31C shows the % of viable cells cultured with or
without compound I for 48 hours.
[0102] FIG. 31D shows the % of viable MM cells after being cultured
with compound I at concentrations from 0 to 20 .mu.M for 48
hours.
[0103] FIG. 31E shows the % of viable peripheral blood mononuclear
cells from healthy donors after being cultured with compound I at
various concentrations for 72 hours.
[0104] FIG. 31F shows immunoblotting results of lysates from INA-6
cells cultured with compound I (0-5 .mu.M) for 120 hours.
[0105] FIG. 32 shows immunoblot AKT and ERK expression profiles
after culturing of A) INA-6 cells with compound I or LY294002 for
12 hours; B) INA-6 and MM.1S cells with compound I at various
concentrations for 6 hours; C) LB and INA-6 cells with compound I
for 0-6 hours.
[0106] FIG. 33A shows fluorescent and transmission electron
microscopic images of INA-6 and LB MM cells treated with compound I
for 6 hours and LC3 accumulation; arrows indicate
autophagosomes.
[0107] FIG. 33B shows fluorescence microscopy images of INA-6 cells
treated with 5 .mu.M of compound I or serum starvation for 6
hours.
[0108] FIG. 33C shows immunoblots of LC3 and beclin-1 protein
levels from INA-6 cells treated with or without compound 1 and 3-MA
(3-methyl adenine, a known inhibitor of autophagy).
[0109] FIG. 33D shows % growth of p110.delta. positive LB cells
after treatment with up to 100 .mu.M of 3-MA for 24 hours.
[0110] FIG. 34 shows the levels of growth inhibition of A) LB or B)
INA-6 cells co-cultured with 0, 5, and 10 .mu.M of compound I in
the presence or absence of varying amounts of IL-6 or IGF-1;
Legend: control media (.box-solid.); compound I at 5.0 .mu.M
(.box-solid.) or 10 .mu.M (.quadrature.).
[0111] FIGS. 34C and 34D show MM cell growth inhibition in the
presence of BMSC. Legend for 34C only: control media
(.quadrature.), Compound I 2.5 .mu.M (.quadrature.), 5 .mu.M
(.box-solid.), and 10 .mu.M (.box-solid.).
[0112] FIG. 34 E shows immunoblots of IL-6 in culture supernatants
from BMSCs cultured with compound I or control media for 48
hours.
[0113] FIG. 34F shows immunoblots of AKT and ERK expression
profiles in INA-6 cells treated with compound I cultured with our
without BMSCs.
[0114] FIG. 34G shows % BMSC cell growth in two different patients
after culturing with compound I for 48 hours.
[0115] FIG. 35A shows microscopic images of HuVECs (human umbilical
vein endothelial cells) cultured with 0, 1 and 10 .mu.M of compound
I for 8 hours and microtubule formation assessed.
[0116] FIG. 35B shows a bar chart summarizing endothelial cell tube
formation in HuVEC cells treated with compound I.
[0117] FIG. 35 C shows a graph charting % cell growth of HuVECs as
a function of the increasing culture concentration of compound
I.
[0118] FIG. 35 D shows decreasing Akt and ERK expression of HuVEC
cell lysates after being cultured with compound I for 8 hours.
[0119] FIG. 36A charts the tumor volume in SCID mice with human MM
xenografts treated with 0, 10 mg/kg or 30 mg/kg of compound II as a
function of time, showing strong in vivo activity on the human
xenograft tumor
[0120] FIG. 36 B shows a photograph comparing the tumor from human
MM xenografts on a mouse treated with compound II for 12 days to a
control mouse.
[0121] FIG. 36C shows the survival rate of SCID mice with human MM
xenografts treated with 0, 10, and 30 mg/kg compound II over
time.
[0122] FIG. 36D shows images from immuno-histochemistric analysis
of tumors harvested from a mouse treated with compound II in
comparison to the control; wherein CD31 and P-AKT positive cells
are dark brown.
[0123] FIG. 36E shows immunoblots detecting PDK-1 and AKT levels
from tumor tissues harvested from mice treated with compound II in
comparison to a control.
[0124] FIG. 36F shows a chart of sIL6R levels measured in mice
treated with 0, 10 mg/kg or 30 mg/kg of compound II over a period
of 4 weeks of treatment as determined by ELISA.
[0125] FIG. 37A show the % of viable LB or INA-6 MM cells after
treatment with compound I with varying amounts of bortezomib (B);
Legend: medium (.box-solid.), compound I 1.25 .mu.M (.box-solid.),
2.5 .mu.M (.quadrature.), or 5.0 .mu.M (.quadrature.).
[0126] FIG. 37B shows immunoblots comparing levels of
phosphorylation of AKT in INA-6 cells treated for 6 hours with
compound I and/or bortezomib.
[0127] FIG. 38 shows (A) PI3K isoform expression in a panel of
follicular lymphoma cell lines; (B) reduction in the expression of
pAkt, Akt, pS6 and S6 after exposure to compound I; and (C)
Increase in PARP and caspase-3 cleavage after exposure to compound
I in a dose-dependent manner.
[0128] FIG. 39 shows (A) amounts of constitutive PI3K signaling in
primary MCL cells in various amounts of compound I; (B) reduction
in pAkt production in MCL cell lines containing a survival factor
and varying amounts of compound I.
[0129] FIG. 40 show a computer tomography axillary image of a bulky
lymphadenopathy in a patient with CLL (A) before treatment with
compound I and (B) after 1 cycle of treatment with compound I.
MODES OF CARRYING OUT THE INVENTION
[0130] Unless otherwise defined, all terms of art, notations and
other scientific terms or terminology used herein are intended to
have the meanings commonly understood by those of skill in the art
to which this invention pertains. In some cases, terms with
commonly understood meanings are defined herein for clarity and/or
for ready reference, and the inclusion of such definitions herein
should not necessarily be construed to represent a substantial
difference over what is generally understood in the art. Many of
the techniques and procedures described or referenced herein are
well understood and commonly employed using conventional
methodology by those skilled in the art. As appropriate, procedures
involving the use of commercially available kits and reagents are
generally carried out in accordance with manufacturer defined
protocols and/or parameters unless otherwise noted.
[0131] The discussion of the general methods given herein is
intended for illustrative purposes only. Other alternative methods
and embodiments will be apparent to those of skill in the art upon
review of this disclosure.
[0132] A group of items linked with the conjunction "or" should not
be read as requiring mutual exclusivity among that group, but
rather should also be read as "and/or" unless expressly stated
otherwise. Although items, elements, or components of the invention
may be described or claimed in the singular, the plural is
contemplated to be within the scope thereof unless limitation to
the singular is explicitly stated.
[0133] The invention provides methods that relate to a novel
therapeutic strategy for the treatment of cancer and inflammatory
diseases. In one aspect, the invention provides a method of
treating cancer or an autoimmune disease in a subject comprising
administering to said subject a compound of formula A
##STR00006##
[0134] wherein R is H, halo, or C1-C6 alkyl; R' is C1-C6 alkyl; or
a pharmaceutically acceptable salt thereof; and optionally a
pharmaceutically acceptable excipient.
[0135] In a particular embodiment, halo is F; and R' is methyl,
ethyl or propyl.
[0136] In a particular embodiment, R is attached to position 5 of
the quinazolinyl ring, having the structure
##STR00007##
[0137] In a particular embodiment, R is attached to position 6 of
the quinazolinyl ring, having the structure
##STR00008##
[0138] The term `compound` used herein, unless otherwise specified,
refers to a compound of formula A, such as compound I, compound II,
or an enantiomer, such as I'' or II'', or an enantiomeric
mixture.
[0139] The "compound of formula I" or "compound I" refers to the
chemical compound
5-fluoro-3-phenyl-2-[1-(9H-purin-6-ylamino)-propyl]-3H-quinazoli-
n-4-one, structure of formula I:
##STR00009##
[0140] The S-enantiomer of compound I is shown here, designated
I'':
##STR00010##
[0141] The "compound of formula II" or "compound II" refers to the
chemical compound
2-(1-(9H-purin-6-ylamino)ethyl)-6-fluoro-3-phenylquinazolin-4(3H)-one,
structure of formula II:
##STR00011##
[0142] The S-enantiomer of compound II is shown here, designated
II'':
##STR00012##
[0143] In one embodiment, the compound of formula A is a compound
of formula I. In another embodiment, the compound of formula A is a
compound of formula II. In certain embodiments, the compound is a
racemic mixture of R- and S-enantiomers. In certain embodiments,
the compound is used as a mixture of enantiomers, and is often
enriched with the S-enantiomer. In some embodiments, the compound
is predominantly the S-enantiomer. In some embodiments, the
compound of formula A, used in the methods described herein is at
least 80% S-enantiomer. In certain embodiments, the compound is
primarily composed of the S-enantiomer, wherein the compound
comprises at least 66-95%, or 85-99% of the S-enantiomer. In some
embodiments the compound has an enantiomeric excess (e.e.) of at
least 90% or at least 95% of S-enantiomer. In some embodiments the
compound has an S-enantiomeric excess (e.e.) of at least 98% or at
least 99%. In certain embodiments, the compound comprises at least
95% of the S-enantiomer. In the cellular and patient experiments
provided in the Example section, the sample of compound I used was
over 95% S-enantiomer.
[0144] In specific embodiments, the compound of formula I or II,
used in the methods described herein is at least 80% S-enantiomer.
In certain embodiments, the compound of formula I or II is
primarily composed of the S-enantiomer, wherein the compound
comprises at least 66-95%, or 85-99% of the S-enantiomer. In some
embodiments the compound of formula I or II has an enantiomeric
excess (e.e.) of at least 90% or at least 95% of S-enantiomer. In
some embodiments the compound of formula I or II has an
S-enantiomeric excess (e.e.) of at least 98% or at least 99%. In
certain embodiments, the compound of formula I or II comprises at
least 95% of the S-enantiomer. In the cellular and patient
experiments provided in the Example section, the sample of compound
I used was over 95% S-enantiomer.
[0145] In a particular embodiment, the compound selectively
inhibits PI3K p110.delta. compared to other PI3K isoforms.
[0146] In a particular embodiment, the autoimmune disease is
allergic rhinitis, asthma, COPD, or rheumatoid arthritis.
[0147] In a particular embodiment, the cancer is a hematological
malignancy and/or solid tumor. In another particular embodiment,
the hematological malignancy is leukemia or lymphoma.
[0148] In some embodiments, lymphoma is a mature (peripheral)
B-cell neoplasm. In specific embodiments, the mature B-cell
neoplasm is selected from the group consisting of B-cell chronic
lymphocytic leukemia/small lymphocytic lymphoma; B-cell
prolymphocytic leukemia; Lymphoplasmacytic lymphoma; Marginal zone
lymphoma, such as Splenic marginal zone B-cell lymphoma (+/-villous
lymphocytes), Nodal marginal zone lymphoma (+/-monocytoid B-cells),
and Extranodal marginal zone B-cell lymphoma of mucosa-associated
lymphoid tissue (MALT) type; Hairy cell leukemia; Plasma cell
myeloma/plasmacytoma; Follicular lymphoma, follicle center; Mantle
cell lymphoma; Diffuse large cell B-cell lymphoma (including
Mediastinal large B-cell lymphoma, Intravascular large B-cell
lymphoma, and Primary effusion lymphoma); and Burkitt's
lymphoma/Burkitt's cell leukemia.
[0149] In some embodiments, lymphoma is selected from the group
consisting of multiple myeloma (MM) and non-Hodgkin's lymphoma
(NHL), mantle cell lymphoma (MCL), follicular lymphoma,
Waldenstrom's macroglobulinemia (WM) or B-cell lymphoma and diffuse
large B-cell lymphoma (DLBCL).
[0150] In a further particular embodiment, leukemia is selected
from the group consisting of acute lymphocytic leukemia (ALL),
acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL),
and small lymphocytic lymphoma (SLL). Acute lymphocytic leukemia is
also known as acute lymphoblastic leukemia and may be used
interchangeably herein. Both terms describe a type of cancer that
starts from the white blood cells, lymphocytes, in the bone
marrow.
[0151] In some embodiments, Non-Hodgkin's Lymphoma (NHL) falls into
one of two categories, aggressive NHL or indolent NHL. Aggressive
NHL is fast growing and may lead to a patient's death relatively
quickly. Untreated survival may be measured in months or even
weeks. Examples of aggressive NHL includes B-cell neoplasms,
diffuse large B-cell lymphoma, T/NK cell neoplasms, anaplastic
large cell lymphoma, peripheral T-cell lymphomas, precursor
B-lymphoblastic leukemia/lymphoma, precursor T-lymphoblastic
leukemia/lymphoma, Burkitt's lymphoma, Adult T-cell
lymphoma/leukemia (HTLV1+), primary CNS lymphoma, mantle cell
lymphoma, polymorphic post-transplantation lymphoproliferative
disorder (PTLD), AIDS-related lymphoma, true histiocytic lymphoma,
and blastic NK-cell lymphoma. The most common type of aggressive
NHL is diffuse large cell lymphoma.
[0152] Indolent NHL is slow growing and does not display obvious
symptoms for most patients until the disease has progressed to an
advanced stage. Untreated survival of patients with indolent NHL
may be measured in years. Non-limiting examples include follicular
lymphoma, small lymphocytic lymphoma, marginal zone lymphoma (such
as extranodal marginal zone lymphoma (also called mucosa associated
lymphoid tissue--MALT lymphoma), nodal marginal zone B-cell
lymphoma (monocytoid B-cell lymphoma), splenic marginal zone
lymphoma), and lymphoplasmacytic lymphoma (Waldenstrom's
macroglobulinemia).
[0153] In some cases, histologic transformation may occur, e.g.,
indolent NHL in patients may convert to aggressive NHL.
[0154] In some embodiments, the invention provides methods of
treating a patient with aggressive NHL or indolent NHL.
[0155] In some embodiments, the invention provides methods of
treating a patient with a condition selected from the group
consisting of mantle cell lymphoma (MCL), diffuse large B cell
lymphoma (DLBCL), follicular lymphoma (FL), acute lymphocytic
leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic
leukemia (CLL), and small lymphocytic lymphoma (SLL), multiple
myeloma (MM), and marginal zone lymphoma.
[0156] In some embodiments, the methods of the invention are
administered to patients with relapsed or refractory
conditions.
[0157] In another embodiment, the cancer is breast, lung, colon or
prostate cancer.
[0158] In a particular embodiment, the cancer or autoimmune disease
is associated with abnormal PI3K activity compared to PI3K activity
in a subject without cancer or without an autoimmune disease.
[0159] In a particular embodiment, the preferred subject is
refractory to chemotherapy treatment, or in relapse after treatment
with chemotherapy. In an alternative embodiment, the subject is a
de novo patient.
[0160] In a particular embodiment, the method comprises reducing
the level of PI3K.delta. activity in said patient.
[0161] In a particular embodiment, the subject is a human
subject.
[0162] Subjects that undergo treatment with known therapeutic
agents may experience resistance to treatment. For example,
although bortezomib was FDA approved for relapsed/refractory,
relapsed, and newly diagnosed MM, some patients do not respond and
others acquire resistance to bortezomib. In some embodiments, the
quinazolinone compound described herein synergistically augments
efficacy of a known therapeutic agent. In some embodiments, the
compounds described herein can augment any of the therapeutic
agents described herein. In more specific embodiments, the
compounds described herein synergistically augment proteasome
inhibitors. In some of the foregoing embodiments, the subject is
resistant to chemotherapeutic treatment. In some of the foregoing
embodiments, the subject is resistant to proteasome inhibitors. In
some of the foregoing embodiments, the subject is resistant
bortezomib or carfilzomib. In one example, the compounds described
herein synergistically augment bortezomib-induced MM cytotoxicity.
Without being bound by theory, in some embodiments, the compounds
discussed herein inhibit bortezomib-induced phosphorylation of AKT.
In some embodiments, the methods described herein are used to
overcome resistance to proteasome inhibitor treatment. In some
embodiments, the invention provides a method to treat a subject
that is resistant or has developed a resistance to therapeutic
agents.
[0163] While not being bound by theory, the synergistic effects
between a compound of formula A and conventional therapies may be
attributed to the ability of the compound of the invention to
induce tumor cell mobilization into peripheral circulation.
Inducing the peripheral circulation of the tumor cells increases
the ability of conventional therapy to act upon and more
effectively neutralize the tumor. This synergy has been
demonstrated in CLL patients.
[0164] Accordingly, the method comprises administering in addition
to a compound of formula A to a patient, a therapeutically
effective amount of at least one additional therapeutic agent
and/or a therapeutic procedure selected to treat said cancer or
autoimmune disease in said patient. "Therapeutic agent" may refer
to one or more compounds, as used herein. The therapeutic agent may
be a standard or experimental chemotherapy drug. The therapeutic
agent may comprise a combination of more than one chemotherapy
drug. Typical chemotherapy drug combinations are listed a-q herein.
A particular therapeutic agent may be chosen depending on the type
of disease being treated. Non-limiting examples of conventional
chemotherapeutic treatments for particular hematologic disease are
described in later sections. In a particular embodiment, the
invention provides a method to treat a hematopoietic cancer
patient, e.g., a CLL patient, with bortezomib and a compound of
formula A (e.g., formula I, II, I'', or II''), wherein the
combination provides a synergistic effect.
[0165] In a particular embodiment, said therapeutic agent is
selected from the following group consisting of bortezomib
(Velcade.RTM.), carfilzomib (PR-171), PR-047, disulfiram,
lactacystin, PS-519, eponemycin, epoxomycin, aclacinomycin,
CEP-1612, MG-132, CVT-63417, PS-341, vinyl sulfone tripeptide
inhibitors, ritonavir, PI-083, (+/-)-7-methylomuralide,
(-)-7-methylomuralide, perifosine, rituximab, sildenafil citrate
(Viagra.RTM.), CC-5103, thalidomide, epratuzumab (hLL2-anti-CD22
humanized antibody), simvastatin, enzastaurin, Campath-1H.RTM.,
dexamethasone, DT PACE, oblimersen, antineoplaston A10,
antineoplaston AS2-1, alemtuzumab, beta alethine, cyclophosphamide,
doxorubicin hydrochloride, PEGylated liposomal doxorubicin
hydrochloride, prednisone, prednisolone, cladribine, vincristine
sulfate, fludarabine, filgrastim, melphalan, recombinant interferon
alfa, carmustine, cisplatin, cyclophosphamide, cytarabine,
etoposide, melphalan, dolastatin 10, indium In 111 monoclonal
antibody MN-14, yttrium Y 90 humanized epratuzumab, anti-thymocyte
globulin, busulfan, cyclosporine, methotrexate, mycophenolate
mofetil, therapeutic allogeneic lymphocytes, Yttrium Y 90
ibritumomab tiuxetan, sirolimus, tacrolimus, carboplatin, thiotepa,
paclitaxel, aldesleukin, recombinant interferon alfa, docetaxel,
ifosfamide, mesna, recombinant interleukin-12, recombinant
interleukin-11, Bcl-2 family protein inhibitor ABT-263, denileukin
diftitox, tanespimycin, everolimus, pegfilgrastim, vorinostat,
alvocidib, recombinant flt3 ligand, recombinant human
thrombopoietin, lymphokine-activated killer cells, amifostine
trihydrate, aminocamptothecin, irinotecan hydrochloride,
caspofungin acetate, clofarabine, epoetin alfa, nelarabine,
pentostatin, sargramostim, vinorelbine ditartrate, WT-1 analog
peptide vaccine, WT1 126-134 peptide vaccine, fenretinide,
ixabepilone, oxaliplatin, monoclonal antibody CD19, monoclonal
antibody CD20, omega-3 fatty acids, mitoxantrone hydrochloride,
octreotide acetate, tositumomab and iodine I 131 tositumomab,
motexafin gadolinium, arsenic trioxide, tipifarnib, autologous
human tumor-derived HSPPC-96, veltuzumab, bryostatin 1, anti-CD20
monoclonal antibodies, chlorambucil, pentostatin, lumiliximab,
apolizumab, Anti-CD40, and Ofatumumab, or a combination thereof.
Combination of therapeutic agents are used in current and
experimental therapies such as those combinations a-q listed
above.
[0166] In some embodiments, the therapeutic agent is preferably a
proteasome inhibitor. In some embodiments, the methods comprise
administering a compound with a proteasome inhibitor. Proteasome
inhibitors include natural and synthetic compounds. Non-limiting
examples of proteasome inhibitors include bortezomib,
([(1R)-3-methyl-1-({(2S)-3-phenyl-2-[(pyrazin-2-ylcarbonyl)amino]propanoy-
l}amino)butyl]boronic acid), which is marketed as `Velcade.RTM.` by
Millennium pharmaceuticals; carfilzomib (PR-171) and the oral
analog, PR-047, both of which are developed by Proteolix, Inc.
Other examples of proteasome inhibitors include disulfiram;
lactacystin; synthetic compounds such as PS-519, eponemycin,
epoxomycin, and aclacinomycin; calpain inhibitors, such as
CEP-1612, MG-132, CVT-63417, PS-341; vinyl sulfone tripeptide
inhibitors; ritonavir; PI-083; (+/-)-7-methylomuralide; and
(-)-7-methylomuralide. In particular embodiments, the compound of
formula A is administered in combination with bortezomib or
carfilzomib. In more particular embodiments, the compound of
formula I is administered in combination with bortezomib or
carfilzomib. In other particular embodiments, the compound of
formula II is administered in combination with bortezomib or
carfilzomib.
[0167] In one aspect, the invention provides a pharmaceutical
composition comprising a compound of Formula I:
##STR00013##
[0168] or a pharmaceutically acceptable salt thereof; and at least
one pharmaceutically acceptable excipient. In one embodiment, the
composition is enriched with the S-enantiomer.
[0169] In another aspect, the invention provides a pharmaceutical
composition comprising a compound of Formula II:
##STR00014##
[0170] or a pharmaceutically acceptable salt thereof; and at least
one pharmaceutically acceptable excipient. In one embodiment, the
composition is enriched with the S-enantiomer.
[0171] In one aspect, the invention provides a method of treating
multiple myeloma (MM) in a patient comprising administering a
combination of a compound of formula A and an additional
therapeutic agent. In some embodiments, formula A is compound I or
II. In specific embodiments, formula A is compound I''. In other
embodiments, formula A is compound II''. In some of the foregoing
embodiments the additional therapeutic agent is a proteasome
inhibitor. In specific embodiments the additional therapeutic agent
is bortezomib. In a specific embodiment, the method of treating
multiple myeloma in a patient comprises administering compound I''
with bortezomib. In a specific embodiment, the method of treating
multiple myeloma in a patient comprises administering compound II''
with bortezomib. In some of the foregoing embodiments, compound I''
or II'' has an enantiomeric excess of at least 60%. In some of the
foregoing embodiments, compound I'' or II'' has an enantiomeric
excess of at least 70%. In some of the foregoing embodiments,
compound I'' or II'' has an enantiomeric excess of at least 80%. In
some of the foregoing embodiments, compound I'' or II'' has an
enantiomeric excess of at least 90%. In some of the foregoing
embodiments, compound I'' or II'' has an enantiomeric excess of at
least 95%. In some of the foregoing embodiments, compound I'' or
II'' has an enantiomeric excess of at least 98%. In some of the
foregoing embodiments, compound I'' or II'' has an enantiomeric
excess of at least 99%.
[0172] In a particular embodiment, a combination of therapeutic
agents is administered with a compound of Formula A, wherein said
combination is selected from the group consisting of
[0173] a) CHOP (cyclophosphamide, doxorubicin, vincristine,
prednisone);
[0174] b) R-CHOP (rituximab-CHOP);
[0175] c) hyperCV AD (hyperfractionated cyclophosphamide,
vincristine, doxorubicin, dexamethasone, methotrexate,
cytarabine);
[0176] d) R-hyperCV AD (rituximab-hyperCV AD);
[0177] e) FCM (fludarabine, cyclophosphamide, mitoxantrone);
[0178] f) R-FCM (rituximab, fludarabine, cyclophosphamide,
mitoxantrone);
[0179] g) bortezomib and rituximab;
[0180] h) temsirolimus and rituximab;
[0181] i) temsirolimus and Velcade.RTM.;
[0182] j) Iodine-131 tositumomab (Bexxar.RTM.) and CHOP;
[0183] k) CVP (cyclophosphamide, vincristine, prednisone);
[0184] l) R-CVP (rituximab-CVP);
[0185] m) ICE (iphosphamide, carboplatin, etoposide);
[0186] n) R-ICE (rituximab-ICE);
[0187] o) FCR (fludarabine, cyclophosphamide, rituximab);
[0188] p) FR (fludarabine, rituximab); and
[0189] q) D.T. PACE (dexamethasone, thalidomide, cisplatin,
Adriamycin.RTM., cyclophosphamide, etoposide).
[0190] In alternative embodiments, the compound is used in
combination with a therapeutic procedure. In a particular
embodiment, the therapeutic procedure is selected from the group
consisting of peripheral blood stem cell transplantation,
autologous hematopoietic stem cell transplantation, autologous bone
marrow transplantation, antibody therapy, biological therapy,
enzyme inhibitor therapy, total body irradiation, infusion of stem
cells, bone marrow ablation with stem cell support, in
vitro-treated peripheral blood stem cell transplantation, umbilical
cord blood transplantation, immunoenzyme technique,
immunohistochemistry staining method, pharmacological study,
low-LET cobalt-60 gamma ray therapy, bleomycin, conventional
surgery, radiation therapy, high-dose chemotherapy and
nonmyeloablative allogeneic hematopoietic stem cell
transplantation.
[0191] In a particular embodiment, the method further comprises
obtaining a biological sample from said patient; and analyzing said
biological sample with an analytical procedure selected from the
group consisting of blood chemistry analysis, chromosomal
translocation analysis, needle biopsy, fluorescence in situ
hybridization, laboratory biomarker analysis, immunohistochemistry
staining method, flow cytometry or a combination thereof.
[0192] For nomenclature purposes, the quinazolinyl and purinyl
components of the compound are numbered accordingly:
##STR00015##
[0193] As used herein, the term "alkyl," includes straight-chain,
branched-chain and cyclic monovalent hydrocarbyl radicals, and
combinations of these, which contain only C and H when they are
unsubstituted. Examples include methyl, ethyl, isobutyl,
cyclohexyl, cyclopentylethyl, and the like. The total number of
carbon atoms in each such group is sometimes described herein,
e.g., when the group can contain up to ten carbon atoms it can be
represented as 1-10C or as C1-C10 or C1-10.
[0194] "Halo", as used herein, includes fluoro, chloro, bromo and
iodo. Fluoro and chloro are often preferred.
[0195] The term "selective PI3K.delta. inhibitor" or "selective
PI3K.beta. inhibitor", etc., as used herein, refers to a compound
that inhibits the PI3K.delta. or PI3.beta. isozyme, respectively,
more effectively than at least one other isozymes of the PI3K
family. The selective inhibitor may also be active against other
isozymes of PI3K, but requires higher concentrations to achieve the
same degree of inhibition of the other isozymes. "Selective" can
also be used to describe a compound that inhibits a particular
PI3-kinase more so than a comparable compound. A "selective
PI3K.delta. inhibitor" compound is understood to be more selective
for PI3K.delta. than compounds conventionally and generically
designated PI3K inhibitors, e.g., wortmannin or LY294002.
Concomitantly, wortmannin and LY294002 are deemed "nonselective
PI3K inhibitors." In certain embodiments, compounds of any type
that selectively negatively regulate PI3K.delta. expression or
activity can be used as selective PI3K.delta. inhibitors in the
methods of the invention. Moreover, compounds of any type that
selectively negatively regulate PI3K.delta. expression or activity
and that possess acceptable pharmacological properties can be used
as selective PI3K.delta. inhibitors in the therapeutic methods of
the invention. Without being bound by theory, targeting p110 delta
inhibition with a compound of the invention provides a novel
approach for the treatment of hematological malignancies because
this method inhibits constitutive signaling resulting in direct
destruction of the tumor cell. In addition, without being bound by
theory, p110 delta inhibition represses microenvironmental signals
which are crucial for tumor cell homing, survival and
proliferation.
[0196] In an alternative embodiment, compounds of any type that
selectively negatively regulate PI3K.beta. expression or activity
can be used as selective PI3K.beta. inhibitors in the methods of
the invention. Moreover, compounds of any type that selectively
negatively regulate PI3K.beta. expression or activity and that
possess acceptable pharmacological properties can be used as
selective PI3K.beta. inhibitors in the therapeutic methods of the
invention.
[0197] "Treating" as used herein refers to inhibiting a disorder,
i.e., arresting its development; relieving the disorder, i.e.,
causing its regression; or ameliorating the disorder, i.e.,
reducing the severity of at least one of the symptoms associated
with the disorder. In some embodiments, "treating" refers to
preventing a disorder from occurring in an animal that can be
predisposed to the disorder, but has not yet been diagnosed as
having it. "Disorder" is intended to encompass medical disorders,
diseases, conditions, syndromes, and the like, without
limitation.
[0198] "Autoimmune disease" as used herein refers to any group of
disorders in which tissue injury is associated with humoral or
cell-mediated responses to the body's own constituents.
[0199] In another aspect, the invention includes a method for
suppressing a function of basophils and/or mast cells, and thereby
enabling treatment of diseases or disorders characterized by
excessive or undesirable basophil and/or mast cell activity.
According to the method, a compound of the invention can be used
that selectively inhibits the expression or activity of
phosphatidylinositol 3-kinase delta (PI3K.delta.) in the basophils
and/or mast cells. Preferably, the method employs a PI3K.delta.
inhibitor in an amount sufficient to inhibit stimulated histamine
release by the basophils and/or mast cells. Accordingly, the use of
such compounds and other PI3K.delta. selective inhibitors can be of
value in treating diseases characterized by histamine release,
i.e., allergic disorders, including disorders such as chronic
obstructive pulmonary disease (COPD), asthma, ARDS, emphysema, and
related disorders.
[0200] The present invention enables methods of treating such
diseases as arthritic diseases, such as rheumatoid arthritis,
psoriatic arthritis, monoarticular arthritis, osteoarthritis, gouty
arthritis, spondylitis; Behcet disease; sepsis, septic shock,
endotoxic shock, gram negative sepsis, gram positive sepsis, and
toxic shock syndrome; multiple organ injury syndrome secondary to
septicemia, trauma, or hemorrhage; ophthalmic disorders such as
allergic conjunctivitis, vernal conjunctivitis, uveitis, and
thyroid-associated opthalmopathy; eosinophilic granuloma; pulmonary
or respiratory disorders such as asthma, chronic bronchitis,
allergic rhinitis, ARDS, chronic pulmonary inflammatory disease
(e.g., chronic obstructive pulmonary disease), silicosis, pulmonary
sarcoidosis, pleurisy, alveolitis, vasculitis, emphysema,
pneumonia, bronchiectasis, and pulmonary oxygen toxicity;
reperfusion injury of the myocardium, brain, or extremities;
fibrosis such as cystic fibrosis; keloid formation or scar tissue
formation; atherosclerosis; autoimmune diseases, such as systemic
lupus erythematosus (SLE), autoimmune thyroiditis, multiple
sclerosis, some forms of diabetes, and Reynaud's syndrome; and
transplant rejection disorders such as graft-versus-host disease
(GVHD) and allograft rejection; chronic glomerulonephritis;
inflammatory bowel diseases such as chronic inflammatory bowel
disease (CIBD), Crohn's disease, ulcerative colitis, and
necrotizing enterocolitis; inflammatory dermatoses such as contact
dermatitis, atopic dermatitis, psoriasis, or urticaria; fever and
myalgias due to infection; central or peripheral nervous system
inflammatory disorders such as meningitis, encephalitis, and brain
or spinal cord injury due to minor trauma; Sjogren's syndrome;
diseases involving leukocyte diapedesis; alcoholic hepatitis;
bacterial pneumonia; antigen-antibody complex mediated diseases;
hypovolemic shock; Type I diabetes mellitus; acute and delayed
hypersensitivity; disease states due to leukocyte dyscrasia and
metastasis; thermal injury; granulocyte transfusion-associated
syndromes; and cytokine-induced toxicity.
[0201] The method can have utility in treating subjects who are or
can be subject to reperfusion injury, i.e., injury resulting from
situations in which a tissue or organ experiences a period of
ischemia followed by reperfusion. The term "ischemia" refers to
localized tissue anemia due to obstruction of the inflow of
arterial blood. Transient ischemia followed by reperfusion
characteristically results in neutrophil activation and
transmigration through the endothelium of the blood vessels in the
affected area. Accumulation of activated neutrophils in turn
results in generation of reactive oxygen metabolites, which damage
components of the involved tissue or organ. This phenomenon of
"reperfusion injury" is commonly associated with conditions such as
vascular stroke (including global and focal ischemia), hemorrhagic
shock, myocardial ischemia or infarction, organ transplantation,
and cerebral vasospasm. To illustrate, reperfusion injury occurs at
the termination of cardiac bypass procedures or during cardiac
arrest when the heart, once prevented from receiving blood, begins
to reperfuse. It is expected that inhibition of PI3K.delta.
activity will result in reduced amounts of reperfusion injury in
such situations.
[0202] In certain embodiments, the invention provides methods to
treat a solid tumor. In specific embodiments, the cancer is breast,
lung, colon, or prostate cancer. In certain embodiments, the
invention provides methods to treat a solid tumor that is
associated with abnormal or undesirable cellular signaling activity
mediated by PI3K.beta.. In certain embodiments, a solid tumor is
selected from the group consisting of pancreatic cancer; bladder
cancer; colorectal cancer; breast cancer, including metastatic
breast cancer; prostate cancer, including androgen-dependent and
androgen-independent prostate cancer; renal cancer, including,
e.g., metastatic renal cell carcinoma; hepatocellular cancer; lung
cancer, including, e.g., non-small cell lung cancer (NSCLC),
bronchioloalveolar carcinoma (BAC), and adenocarcinoma of the lung;
ovarian cancer, including, e.g., progressive epithelial or primary
peritoneal cancer; cervical cancer; gastric cancer; esophageal
cancer; head and neck cancer, including, e.g., squamous cell
carcinoma of the head and neck; melanoma; neuroendocrine cancer,
including metastatic neuroendocrine tumors; brain tumors,
including, e.g., glioma, anaplastic oligodendroglioma, adult
glioblastoma multiforme, and adult anaplastic astrocytoma; bone
cancer; and soft tissue sarcoma.
[0203] Genetic ablation of p110.delta. has been found to result in
mild phenotype restricted to immune system. General observations
include organisms that are fertile with no gross anatomical or
behavioral abnormalities. A histological examination revealed major
organs to appear normal. The total class I PI3K activity was
reduced 30-50% in B and T cells. In addition, no increase in
susceptibility to infections was observed. Furthermore, the effect
on the hematopoietic system includes normal peripheral blood cell
counts, the occurrence of lymphoid hypoplasia and the lack of
germinal centers in spleen and lymph nodes, a reduced number of
B220+IgM+B cell progenitors in bone marrow, a reduced level of
serum immunoglobulin, and normal T cell development in the
thymus.
[0204] Genetic ablation of p110.delta. affects myeloid and B cell
signaling, which is important for oncogenesis. In particular,
tyrosine kinase signaling, development, proliferation and survival
are affected in myeloid cells. B cell function is most affected and
includes proliferation, differentiation, apoptosis, and response to
B cell survival factors (BCR, CD40, IL-4, chemokines). Thus, the
invention includes methods of treating disease states in which one
or more of these myeloid and B cell functions are abnormal or
undesirable.
[0205] A pan PI3K inhibitor that targets on a molecular level
p110.alpha., p110.beta., p110.delta., p110.gamma., (hvPS34, mTOR,
DNA-PK, and others), in turn targets all tissues. The potential
clinical indication includes cancer but clinical adverse events
include hyperinsulinemia in cancer patients. The advantage of a
p110.delta. selective inhibitor which targets cells mediating
inflammation and cancer cells, wherein potential clinical
indication include cancer, rheumatoid arthritis, asthma, allergies
and COPD, is that treatment is well tolerated, and side effects
like hyperinsulinemia are avoided. Thus in one aspect the invention
provides a method to treat patients having insulin resistance, or
type 2 diabetes, for cancer, rheumatoid arthritis, asthma,
allergies, COPD, or other conditions treatable with the compounds
of the invention. For patients needing such treatment who have
excessive insulin conditions or tendencies, the compounds of the
invention are particularly advantageous over pan-PI3K inhibitors.
In certain embodiments, a compound of formula I or I'' is preferred
because it provides therapeutic benefits to treating hematologic
malignancies without adversely affecting insulin signaling.
[0206] In one embodiment, the invention relates to methods of
inhibiting PI3K p110.delta.. In another embodiment, the invention
relates to methods of inhibiting PI3K p110.beta. or
p110.gamma..
[0207] In certain embodiments, the method described herein has
little or no off target activity. In particular, compound of
formula I used in the method show little activity against over 300
protein kinases including those summarized in Table 3 of Example
16. In certain embodiments, the method described herein has no or
minimal hyperinsulinemia effects in cancer patients compared to
methods comprising the administration of pan-PI3K inhibitors. In
certain embodiments, the method described herein is useful in
targeting cells mediating Akt phosphorylation, because the
compounds of Formula A inhibit Akt phosphorylation. Suitable
patients for treatment with the compounds of the invention can thus
be selected, in one embodiment, by selecting a patient exhibiting
elevated Akt phosphorylation associated with a hematopoietic cancer
such as lymphoma, leukemia or multiple myeloma.
[0208] The methods herein avoid off-target liabilities and are
characterized by negative results in receptor gram screens, having
no hERG inhibition and no significant P450 inhibition.
[0209] Another advantage of the inventive method is the absence of
adverse cardiovascular, respiratory, or central nervous system
effects as demonstrated in safety pharmacology studies. In
addition, a 28-day toxicity study in rats and dogs demonstrated a
high therapeutic index, e.g., a NOAEL (no observable adverse effect
level)>>10 .mu.M. This is the highest experimental dose of a
chemical at which there is no statistically or biologically
significant increase in frequency or severity of a toxicological
effect between an exposed group and its appropriate control.
Adverse effects are defined as any effects that result in
functional impairment and/or pathological lesions that may affect
the performance of the whole organism or that reduce an organism's
ability to respond to an additional challenge.
[0210] In another embodiment, the inventive methods are
non-genotoxic in a standard battery of tests.
[0211] Another advantage of the invention is that compound
selectivity for one or two PI3K isoforms results in an improved
safety profile over compounds having pan-PI3K inhibition. In yet
another advantage, compound I has a favorable pharmacokinetic
profile with good target coverage, and no adverse effects on
glucose or insulin levels, and is well tolerated at doses above
commonly used therapeutic doses by normal healthy volunteers.
Another advantage of the invention includes the ability to treat a
wide range of hematological malignancies as demonstrated by the
examples herein.
[0212] In certain embodiments, the methods of the invention are
directed towards treating a cancer or an autoimmune disease. In
certain embodiments, the cancer is a hematological malignancy. In
specific embodiments, the hematological malignancy is selected from
the group consisting of acute lymphocytic leukemia (ALL), acute
myeloid leukemia (AML), chronic lymphocytic leukemia (CLL),
multiple myeloma (MM), and non-Hodgkin lymphoma (NHL). In certain
embodiments, the non-Hodgkin lymphoma is selected from the group
consisting of large diffuse B-cell lymphoma (LDBCL), mantle cell
lymphoma (MCL), Waldenstrom's macroglobulinemia (WM) and
lymphoplasmacytic lymphoma.
[0213] PI3K is implicated in many hematological malignancies and
preclinical proof of concept relating to treatment with compound I
has been established. The table below summarizes particular
hematological malignancies and the method of action on the primary
patient cell or disease cell line.
TABLE-US-00001 Effects of compounds Indication of formula A Chronic
Lymphocytic Leukemia Primary patient cells (CLL) Induces apoptosis
Blocks survival factors Acute Myelogenous Leukemia (AML) Primary
patient cells Blocks PI3K signaling Inhibits proliferation Acute
Lymphocytic Leukemia (ALL) Cell Lines Blocks PI3K signaling Induces
apoptosis Non-Hodgkin's Lymphomas (NHL) Cell Lines (MCL, DLBCL, FL)
Blocks PI3K signaling Induces apoptosis Multiple Myeloma (MM)
Primary patient cells P110 .delta. overexpressed in 24/24 samples
Induces apoptosis
[0214] Data provided herein demonstrates that the compounds of the
invention are useful to treat lymphomas and leukemias. Lymphomas
and leukemias generally express the delta isoform of p110
selectively, e.g., FIG. 15 demonstrates that p110.delta. is
prevalent in most lymphoma cell lines, while p110.alpha. is not
generally observed. Moreover, data presented in FIG. 16A shows that
cell cultures from six different leukemia cell lines were sensitive
to Compound I, and were strongly affected by 5-10 micromolar
concentrations of this compound. FIGS. 8 and 9 support compound I
as reducing Akt(Ser473) production in several cell lines.
[0215] CLL, for example, produces mainly p110.delta. and to a
lesser extent p110.gamma. for signaling purposes, thus compounds
that inhibit p110.delta. and/or p110.gamma. are expected to exhibit
selective cytotoxicity towards these cells. In Example 3, for
example, shows dose-dependent cytotoxicity for compound I (FIG. 3),
in CLL cells, including cells taken from poor prognosis patients
(FIG. 19), and cells from patients shown to be resistant to other
CLL treatments (FIG. 20). In addition, Example 13 and FIG. 13
demonstrate that compound I administered to a CLL patient at a rate
of 50 mg BID for a 28-day cycle provides a significant therapeutic
effect. An ALC concentration percent decrease in lymphocytes is
observed. Thus in one aspect, the invention provides methods for
treating CLL patients with drug-resistant CLL using compounds of
Formula A. On the other hand, Example 17 suggests that a fibroblast
cell line relying mainly on p110.alpha. for signaling was not
sensitive to Compound I. Thus in one aspect, patient selection can
include excluding patients having a cancer that relies mainly on
p110.alpha. for signaling.
[0216] The compounds of Formula A are also useful to treat
lymphoma, including both B-cell and T-cell lymphomas. Data in FIG.
4 demonstrates that six different ALL cell lines were sensitive to
Compound I, which caused a significant reduction in cell viability
in all six cell lines.
[0217] FIG. 12 and Example 12 demonstrate that mantle cell lymphoma
patients treated with 50 mg BID of Compound I for 28 days
experienced on average a 44% decrease in tumor burden. Moreover,
FIG. 14 demonstrates that an MCL patient at the end of the 28 day
cycle experienced similar plasma levels of Compound I following
administration of a 50 mg dose to that observed in a normal healthy
volunteer (NHV); thus the compound does not build up excessively
over the course of a cycle of treatment, nor does the patient
become tolerant by increased metabolism over the course of a
treatment cycle.
[0218] In addition, the compounds of Formula A, or Formula I, are
useful to treat hematopoietic cancers that constitutively express
Akt phosphorylation activity. Example 8, and FIGS. 8 and 9 list
cancer cell lines that demonstrate constitutive Akt
phosphorylation, including B-cell lymphomas, T-cell lymphomas, ALL,
malignant histiocytosis, DLBCL and AML. Exposure of the cell to
compound I results in the reduction of Akt phosphorylation. See
also Example 19, which shows that constitutive Akt phosphorylation
was inhibited by Compound I in 13 of 13 cell lines.
[0219] In certain embodiments, the cancer is a solid tumor. In
specific embodiments, the cancer is breast, ovarian, lung, colon,
or prostate cancer. FIG. 6, for example, shows that Compound I
reduces cellular proliferation of two breast cancer cell lines, and
FIG. 10 illustrates cytotoxicity to three different breast cancer
cell lines. Similarly, FIG. 7 demonstrates that Compound I is
cytotoxic to two ovarian cancer cell lines.
[0220] For the treatment of a solid tumor, it is advantageous to
use a compound of Formula A that expresses good activity (e.g.,
IC.sub.50 less than about 1 .mu.M, and preferably less than about
250 nM--see Example 15) against p110.beta., since solid tumors
often utilize this isozyme rather than or more than p110.delta..
Thus a compound of formula A that has an IC.sub.50 less than about
250 nM is preferred for treatment of a solid tumor; compound I,
I'', II, or II'' is suitable for this use, as demonstrated
herein.
[0221] In some embodiments, the subject for treatments described
herein as one who has been diagnosed with at least one of the
conditions described herein as treatable by the use of a compound
of Formula A. In some embodiments, the subject has been diagnosed
with a cancer named herein, and has proven refractory to treatment
with at least one conventional chemotherapeutic agent. For
instance, patients who have failed to respond to treatments such as
proteasome inhibitors, autologous stem cell transplant, CHOP
regimens, rituximab, fludarabine, alemtuzumab, conventional
anticancer nucleoside analogues and alkylating agents frequently
respond to the methods of treatment described herein. Thus, in one
embodiment, the treatments of the invention are directed to
patients who have received one or more than one such treatment.
[0222] In certain embodiments, the autoimmune disease is allergic
rhinitis, asthma, chronic obstructive pulmonary disease (COPD), or
rheumatoid arthritis.
[0223] In certain embodiments, the methods of the invention are
directed to B-cell, or B lymphocyte, related diseases. B-cells play
a role in the pathogenesis of autoimmune diseases.
[0224] The compounds of Formula A (particularly Formulas I, I'', II
and II'') are suitable for treating a variety of subjects having
the conditions described herein, especially hematological cancers
in humans. In some embodiments, the subject selected for treatment
of a hematological malignancy that is a subject experiencing
relapse after other treatments or is refractory to other
treatments. In some embodiments, the subject is selected for
treatment of a hematological malignancy that is resistant to other
cancer drugs. In some embodiments, the subject is selected for
treatment of a hematological malignancy that exhibits a high level
of p110.delta. activity. In some embodiments, the subject is
selected for treatment of a hematological malignancy that exhibits
a relatively low level of p110.alpha. activity. In some
embodiments, the subject is selected for treatment of a
hematological malignancy that constitutively expresses Akt
phosphorylation activity.
[0225] In one embodiment, the method described herein comprises
administering to a subject a compound of formula A described
herein, in combination with a therapy used to treat cancer or an
autoimmune disease. "Therapy" or "treatment", as used herein, is a
treatment of cancer or an autoimmune disease by any well-known
conventional or experimental form of treatment used to treat cancer
or an autoimmune disease that does not include the use of a
compound of formula A. In certain embodiments, the combination of a
compound of formula A with a conventional or experimental therapy
used to treat cancer or an autoimmune disease provides beneficial
and/or desirable treatment results superior to results obtained by
treatment without the combination. In certain embodiments,
therapies used to treat cancer or an autoimmune disease are
well-known to a person having ordinary skill in the art and are
described in the literature. Therapies include, but are not limited
to, chemotherapy, combinations of chemotherapy, biological
therapies, immunotherapy, radioimmunotherapy, and the use of
monoclonal antibodies, and vaccines.
[0226] In some of the foregoing embodiments, the combination method
provides for a compound of formula A administered simultaneously
with or during the period of administration of the therapy. In some
of the foregoing embodiments the compound of formula A is
administered simultaneously with the other chemotherapeutic
treatment. In certain embodiments, the combination method provides
for a compound of formula A administered prior to or after the
administration of the therapy.
[0227] In some of the foregoing embodiments, the subject is
refractory to at least one standard or experimental chemotherapy.
In some of the foregoing embodiments, the subject is refractory to
at least two standard or experimental chemotherapies. In some of
the foregoing embodiments, the subject is refractory to at least
three standard or experimental chemotherapies. In some of the
foregoing embodiments, the subject is refractory to at least four
standard or experimental chemotherapies.
[0228] In some of the foregoing embodiments, the subject is
refractory to at least one standard or experimental chemotherapy
selected from the group consisting of fludarabine, rituximab,
alkylating agents, alemtuzumab and the chemotherapy treatments a-q
listed above.
[0229] In some of the foregoing embodiments, the subject is
refractory to at least two standard or experimental chemotherapies
selected from the group consisting of fludarabine, rituximab,
alkylating agents, alemtuzumab and the chemotherapy treatments a-q
listed above.
[0230] In some of the foregoing embodiments, the subject is
refractory to at least three standard or experimental
chemotherapies selected from the group consisting of fludarabine,
rituximab, alkylating agents, alemtuzumab and the chemotherapy
treatments a-q listed above.
[0231] In some of the foregoing embodiments, the subject is
refractory to at least four standard or experimental chemotherapies
selected from the group consisting of fludarabine, rituximab,
alkylating agents, alemtuzumab and the chemotherapy treatments a-q
listed above.
[0232] The exact details regarding the administration of the
combination may be determined experimentally. The refinement of
sequence and timing of administering a compound of formula A with a
selected therapy will be tailored to the individual subject, the
nature of the condition to be treated in the subject, and
generally, the judgment of the attending practitioner.
[0233] Non-limiting examples of experimental or standard therapy
are described below. In addition, treatment of certain lymphomas is
reviewed in Cheson, B. D., Leonard, J. P., "Monoclonal Antibody
Therapy for B-Cell Non-Hodgkin's Lymphoma" The New England Journal
of Medicine 2008, 359(6), p. 613-626; and Wierda, W. G., "Current
and Investigational Therapies for Patients with CLL" Hematology
2006, p. 285-294. Lymphoma incidence patterns in the United States
is profiled in Morton, L. M., et al. "Lymphoma Incidence Patterns
by WHO Subtype in the United States, 1992-2001" Blood 2006, 107(1),
p. 265-276.
[0234] Treatment of non-Hodgkin's lymphomas, especially of B cell
origin, include, but are not limited to use of monoclonal
antibodies, standard chemotherapy approaches (e.g., CHOP, CVP, FCM,
MCP, and the like), radioimmunotherapy, and combinations thereof,
especially integration of an antibody therapy with
chemotherapy.
[0235] Non-limiting examples of unconjugated monoclonal antibodies
for Non-Hodgkin's lymphoma/B-cell cancers include rituximab,
alemtuzumab, human or humanized anti-CD20 antibodies, lumiliximab,
anti-TRAIL, bevacizumab, galiximab, epratuzumab, SGN-40, and
anti-CD74. Non-limiting examples of experimental antibody agents
used in treatment of Non-Hodgkin's lymphoma/B-cell cancers include
ofatumumab, ha20, PRO131921, alemtuzumab, galiximab, SGN-40,
CHIR-12.12, epratuzumab, lumiliximab, apolizumab, milatuzumab, and
bevacizumab. Any of the monoclonal antibodies can be combined with
rituximab, fludarabine, or a chemotherapy agent/regimen.
[0236] Non-limiting examples of standard regimens of chemotherapy
for Non-Hodgkin's lymphoma/B-cell cancers include CHOP
(cyclophosphamide, doxorubicin, vincristine, prednisone), FCM
(fludarabine, cyclophosphamide, mitoxantrone), CVP
(cyclophosphamide, vincristine and prednisone), MCP (mitoxantrone,
chlorambucil, and prednisolone), R-CHOP (rituximab plus CHOP),
R-FCM (rituximab plus FCM), R-CVP (rituximab plus CVP), and R-MCP
(R-MCP).
[0237] Non-limiting examples of radioimmunotherapy for
Non-Hodgkin's lymphoma/B-cell cancers include yttrium-90-labeled
ibritumomab tiuxetan, and iodine-131-labeled tositumomab. These
therapeutic agents are approved for use in subjects with relapsed
or refractory follicular or low-grade lymphoma.
[0238] Therapeutic treatments for mantle cell lymphoma include
combination chemotherapies such as CHOP (cyclophosphamide,
doxorubicin, vincristine, prednisone), hyperCV AD
(hyperfractionated cyclophosphamide, vincristine, doxorubicin,
dexamethasone, methotrexate, cytarabine) and FCM (fludarabine,
cyclophosphamide, mitoxantrone). In addition, these regimens can be
supplemented with the monoclonal antibody rituximab (Rituxan) to
form combination therapies R-CHOP, hyperCV AD-R, and R-FCM. Other
approaches include combining any of the abovementioned therapies
with stem cell transplantation or treatment with ICE (iphosphamide,
carboplatin and etoposide).
[0239] Another approach to treating mantle cell lymphoma includes
immunotherapy such as using monoclonal antibodies like Rituximab
(Rituxan). Rituximab is also effective against other indolent
B-cell cancers, including marginal-zone lymphoma, WM, CLL and small
lymphocytic lymphoma. A combination of Rituximab and chemotherapy
agents is especially effective. A modified approach is
radioimmunotherapy, wherein a monoclonal antibody is combined with
a radioisotope particle, such as Iodine-131 tositumomab
(Bexxar.RTM.) and Yttrium-90 ibritumomab tiuxetan (Zevalin.RTM.).
In another example, Bexxar.RTM. is used in sequential treatment
with CHOP. Another immunotherapy example includes using cancer
vaccines, which is based upon the genetic makeup of an individual
patient's tumor. A lymphoma vaccine example is GTOP-99
(MyVax.RTM.).
[0240] Another approach to treating mantle cell lymphoma includes
autologous stem cell transplantation coupled with high-dose
chemotherapy.
[0241] Another approach to treating mantle cell lymphoma includes
administering proteasome inhibitors, such as Velcade.RTM.
(bortezomib or PS-341), or antiangiogenesis agents, such as
thalidomide, especially in combination with Rituxan. Another
treatment approach is administering drugs that lead to the
degradation of Bcl-2 protein and increase cancer cell sensitivity
to chemotherapy, such as oblimersen (Genasense) in combination with
other chemotherapeutic agents. Another treatment approach includes
administering mTOR inhibitors, which can lead to inhibition of cell
growth and even cell death; a non-limiting example is Temsirolimus
(CCI-779), and Temsirolimus in combination with Rituxan.RTM.,
Velcade.RTM. or other chemotherapeutic agents.
[0242] Other recent therapies for MCL have been disclosed (Nature
Reviews; Jares, P. 2007). Non-limiting examples include
Flavopiridol, PD0332991, R-roscovitine (Selicilib, CYC202), Styryl
sulphones, Obatoclax (GX15-070), TRAIL, Anti-TRAIL DR4 and DR5
antibodies, Temsirolimus (CCI-779), Everolimus (RAD001),
BMS-345541, Curcumin, Vorinostat (SAHA), Thalidomide, lenalidomide
(Revlimid.RTM., CC-5013), and Geldanamycin (17-AAG).
[0243] Non-limiting examples of other therapeutic agents used to
treat Waldenstrom's Macroglobulinemia include perifosine,
bortezomib (Velcade.RTM.), rituximab, sildenafil citrate
(Viagra.RTM.), CC-5103, thalidomide, epratuzumab (hLL2-anti-CD22
humanized antibody), simvastatin, enzastaurin, campath-1H,
dexamethasone, DT PACE, oblimersen, antineoplaston A10,
antineoplaston AS2-1, alemtuzumab, beta alethine, cyclophosphamide,
doxorubicin hydrochloride, prednisone, vincristine sulfate,
fludarabine, filgrastim, melphalan, recombinant interferon alfa,
carmustine, cisplatin, cyclophosphamide, cytarabine, etoposide,
melphalan, dolastatin 10, indium In 111 monoclonal antibody MN-14,
yttrium Y 90 humanized epratuzumab, anti-thymocyte globulin,
busulfan, cyclosporine, methotrexate, mycophenolate mofetil,
therapeutic allogeneic lymphocytes, Yttrium Y 90 ibritumomab
tiuxetan, sirolimus, tacrolimus, carboplatin, thiotepa, paclitaxel,
aldesleukin, recombinant interferon alfa, docetaxel, ifosfamide,
mesna, recombinant interleukin-12, recombinant interleukin-11,
Bcl-2 family protein inhibitor ABT-263, denileukin diftitox,
tanespimycin, everolimus, pegfilgrastim, vorinostat, alvocidib,
recombinant flt3 ligand, recombinant human thrombopoietin,
lymphokine-activated killer cells, amifostine trihydrate,
aminocamptothecin, irinotecan hydrochloride, caspofungin acetate,
clofarabine, epoetin alfa, nelarabine, pentostatin, sargramostim,
vinorelbine ditartrate, WT-1 analog peptide vaccine, WT1 126-134
peptide vaccine, fenretinide, ixabepilone, oxaliplatin, monoclonal
antibody CD19, monoclonal antibody CD20, omega-3 fatty acids,
mitoxantrone hydrochloride, octreotide acetate, tositumomab and
iodine I-131 tositumomab, motexafin gadolinium, arsenic trioxide,
tipifarnib, autologous human tumor-derived HSPPC-96, veltuzumab,
bryostatin 1, and PEGylated liposomal doxorubicin hydrochloride,
and any combination thereof.
[0244] Non-limiting examples of other therapeutic agents used to
treat diffuse large B-cell lymphoma (DLBCL) drug therapies (Blood
2005 Abramson, J.) include cyclophosphamide, doxorubicin,
vincristine, prednisone, anti-CD20 monoclonal antibodies,
etoposide, bleomycin, many of the agents listed for Waldenstrom's,
and any combination thereof, such as ICE and R-ICE.
[0245] Non-limiting examples of therapeutic procedures used to
treat Waldenstrom's Macroglobulinemia include peripheral blood stem
cell transplantation, autologous hematopoietic stem cell
transplantation, autologous bone marrow transplantation, antibody
therapy, biological therapy, enzyme inhibitor therapy, total body
irradiation, infusion of stem cells, bone marrow ablation with stem
cell support, in vitro-treated peripheral blood stem cell
transplantation, umbilical cord blood transplantation, immunoenzyme
technique, pharmacological study, low-LET cobalt-60 gamma ray
therapy, bleomycin, conventional surgery, radiation therapy, and
nonmyeloablative allogeneic hematopoietic stem cell
transplantation.
[0246] Non-limiting examples of other therapeutic agents used to
treat Chronic Lymphocytic Leukemia (Spectrum, 2006, Fernandes, D.)
include Chlorambucil (Leukeran), Cyclophosphamide (Cyloxan,
Endoxan, Endoxana, Cyclostin), Fludarabine (Fludara), Pentstatin
(Nipent), Cladribine (Leustarin), Doxorubicin (Adriamycin.RTM.,
Adriblastine), Vincristine (Oncovin), Prednisone, Prednisolone,
Alemtuzumab (Campath, MabCampath), many of the agents listed for
Waldenstrom's, and combination chemotherapy and chemoimmunotherapy,
including the common combination regimen: CVP (cyclophosphamide,
vincristine, prednisone); R-CVP (rituximab-CVP); ICE (iphosphamide,
carboplatin, etoposide); R-ICE (rituximab-ICE); FCR (fludarabine,
cyclophosphamide, rituximab); and FR (fludarabine, rituximab).
[0247] In certain embodiments, the method comprises administering
in addition to a compound of I or II to said patient, a
therapeutically effective amount of at least one therapeutic agent
and/or therapeutic procedure selected to treat said cancer or
autoimmune disease in said patient. In certain embodiments, the
method comprises administering in addition to a compound of I or II
to said patient, a therapeutically effective amount of a
combination of therapeutic agents selected from the group
consisting of a) CHOP (cyclophosphamide, doxorubicin, vincristine,
prednisone); b) R-CHOP (rituximab-CHOP); c) hyperCV AD
(hyperfractionated cyclophosphamide, vincristine, doxorubicin,
dexamethasone, methotrexate, cytarabine); d) R-hyperCV AD
(rituximab-hyperCV AD); e) FCM (fludarabine, cyclophosphamide,
mitoxantrone); 1) R-FCM (rituximab, fludarabine, cyclophosphamide,
mitoxantrone); g) bortezomib and rituximab; h) temsirolimus and
rituximab; i) temsirolimus and Velcade.RTM.; j) Iodine-131
tositumomab (Bexxar.RTM.) and CHOP; k) CVP (cyclophosphamide,
vincristine, prednisone); l) R-CVP (rituximab-CVP); m) ICE
(iphosphamide, carboplatin, etoposide); n) R-ICE (rituximab-ICE);
o) FCR (fludarabine, cyclophosphamide, rituximab); and p) FR
(fludarabine, rituximab).
[0248] The compounds of the invention may be formulated for
administration to animal subject using commonly understood
formulation techniques well known in the art. Formulations which
are suitable for particular modes of administration and for the
compounds of formula A may be found in Remington's Pharmaceutical
Sciences, latest edition, Mack Publishing Company, Easton, Pa.
[0249] The compounds of the invention may be prepared in the form
of prodrugs, i.e., protected forms which release the compounds of
the invention after administration to the subject. Typically, the
protecting groups are hydrolyzed in body fluids such as in the
bloodstream thus releasing the active compound or are oxidized or
reduced in vivo to release the active compound. A discussion of
prodrugs is found in Smith and Williams Introduction to the
Principles of Drug Design, Smith, H. J.; Wright, 2.sup.nd ed.,
London (1988).
[0250] A compound of the present invention can be administered as
the neat chemical, but it is typically preferable to administer the
compound in the form of a pharmaceutical composition or
formulation. Accordingly, the present invention also provides
pharmaceutical compositions that comprise a compound of formula A
and a biocompatible pharmaceutical carrier, adjuvant, or vehicle.
The composition can include the compound of Formula A as the only
active moiety or in combination with other agents, such as oligo-
or polynucleotides, oligo- or polypeptides, drugs, or hormones
mixed with excipient(s) or other pharmaceutically acceptable
carriers. Carriers and other ingredients can be deemed
pharmaceutically acceptable insofar as they are compatible with
other ingredients of the formulation and not deleterious to the
recipient thereof.
[0251] The pharmaceutical compositions are formulated to contain
suitable pharmaceutically acceptable carriers, and can optionally
comprise excipients and auxiliaries that facilitate processing of
the active compounds into preparations that can be used
pharmaceutically. The administration modality will generally
determine the nature of the carrier. For example, formulations for
parenteral administration can comprise aqueous solutions of the
active compounds in water-soluble form. Carriers suitable for
parenteral administration can be selected from among saline,
buffered saline, dextrose, water, and other physiologically
compatible solutions. Preferred carriers for parenteral
administration are physiologically compatible buffers such as
Hank's solution, Ringer's solution, or physiologically buffered
saline. For tissue or cellular administration, penetrants
appropriate to the particular barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art.
For preparations comprising proteins, the formulation can include
stabilizing materials, such as polyols (e.g., sucrose) and/or
surfactants (e.g., nonionic surfactants), and the like.
[0252] Alternatively, formulations for parenteral use can comprise
dispersions or suspensions of the active compounds prepared as
appropriate oily injection suspensions. Suitable lipophilic
solvents or vehicles include fatty oils, such as sesame oil, and
synthetic fatty acid esters, such as ethyl oleate or triglycerides,
or liposomes. Aqueous injection suspensions can contain substances
that increase the viscosity of the suspension, such as sodium
carboxy-methylcellulose, sorbitol, or dextran. Optionally, the
suspension also can contain suitable stabilizers or agents that
increase the solubility of the compounds to allow for the
preparation of highly concentrated solutions. Aqueous polymers that
provide pH-sensitive solubilization and/or sustained release of the
active agent also can be used as coatings or matrix structures,
e.g., methacrylic polymers, such as the Eudragit.RTM. series
available from Rohm America Inc. (Piscataway, N.J.). Emulsions,
e.g., oil-in-water and water-in-oil dispersions, also can be used,
optionally stabilized by an emulsifying agent or dispersant
(surface active materials; surfactants). Suspensions can contain
suspending agents such as ethoxylated isostearyl alcohols,
polyoxyethlyene sorbitol and sorbitan esters, microcrystalline
cellulose, aluminum metahydroxide, bentonite, agar-agar, gum
tragacanth, and mixtures thereof.
[0253] Liposomes containing the active compound of Formula A also
can be employed for parenteral administration. Liposomes generally
are derived from phospholipids or other lipid substances. The
compositions in liposome form also can contain other ingredients,
such as stabilizers, preservatives, excipients, and the like.
Preferred lipids include phospholipids and phosphatidyl cholines
(lecithins), both natural and synthetic. Methods of forming
liposomes are known in the art. See, e.g., Prescott (Ed.), Methods
in Cell Biology, Vol. XIV, p. 33, Academic Press, New York
(1976).
[0254] The pharmaceutical compositions comprising the compound of
Formula A in dosages suitable for oral administration can be
formulated using pharmaceutically acceptable carriers well known in
the art. The preparations formulated for oral administration can be
in the form of tablets, pills, capsules, cachets, dragees,
lozenges, liquids, gels, syrups, slurries, elixirs, suspensions, or
powders. To illustrate, pharmaceutical preparations for oral use
can be obtained by combining the active compounds with a solid
excipient, optionally grinding the resulting mixture, and
processing the mixture of granules, after adding suitable
auxiliaries if desired, to obtain tablets or dragee cores. Oral
formulations can employ liquid carriers similar in type to those
described for parenteral use, e.g., buffered aqueous solutions,
suspensions, and the like.
[0255] Preferred oral formulations include tablets, dragees, and
gelatin capsules. These preparations can contain one or excipients,
which include, without limitation:
[0256] a) diluents, such as sugars, including lactose, dextrose,
sucrose, mannitol, or sorbitol;
[0257] b) binders, such as magnesium aluminum silicate, starch from
corn, wheat, rice, potato, etc.;
[0258] c) cellulose materials, such as methylcellulose,
hydroxypropylmethyl cellulose, and sodium carboxymethylcellulose,
polyvinylpyrrolidone, gums, such as gum arabic and gum tragacanth,
and proteins, such as gelatin and collagen;
[0259] d) disintegrating or solubilizing agents such as
cross-linked polyvinyl pyrrolidone, starches, agar, alginic acid or
a salt thereof, such as sodium alginate, or effervescent
compositions;
[0260] e) lubricants, such as silica, talc, stearic acid or its
magnesium or calcium salt, and polyethylene glycol;
[0261] f) flavorants and sweeteners;
[0262] g) colorants or pigments, e.g., to identify the product or
to characterize the quantity (dosage) of active compound; and
[0263] h) other ingredients, such as preservatives, stabilizers,
swelling agents, emulsifying agents, solution promoters, salts for
regulating osmotic pressure, and buffers.
[0264] In some preferred oral formulations, the pharmaceutical
composition comprises at least one of the materials from group (a)
above, or at least one material from group (b) above, or at least
one material from group (c) above, or at least one material from
group (d) above, or at least one material from group (e) above.
Preferably, the composition comprises at least one material from
each of two groups selected from groups (a)-(e) above.
[0265] Gelatin capsules include push-fit capsules made of gelatin,
as well as soft, sealed capsules made of gelatin and a coating such
as glycerol or sorbitol. Push-fit capsules can contain the active
ingredient(s) mixed with fillers, binders, lubricants, and/or
stabilizers, etc. In soft capsules, the active compounds can be
dissolved or suspended in suitable fluids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycol with or without
stabilizers.
[0266] Dragee cores can be provided with suitable coatings such as
concentrated sugar solutions, which also can contain gum arabic,
talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol,
and/or titanium dioxide, lacquer solutions, and suitable organic
solvents or solvent mixtures.
[0267] The pharmaceutical composition can be provided as a salt of
the active compound. Salts tend to be more soluble in aqueous or
other protonic solvents than the corresponding free acid or base
forms. Pharmaceutically acceptable salts are well known in the art.
Compounds that contain acidic moieties can form pharmaceutically
acceptable salts with suitable cations. Suitable pharmaceutically
acceptable cations include, for example, alkali metal (e.g., sodium
or potassium) and alkaline earth (e.g., calcium or magnesium)
cations.
[0268] Compounds of structural formula (A) that contain basic
moieties can form pharmaceutically acceptable acid addition salts
with suitable acids. For example, Berge, et al., describe
pharmaceutically acceptable salts in detail in J Pharm Sci, 66:1
(1977). The salts can be prepared in situ during the final
isolation and purification of the compounds of the invention or
separately by reacting a free base function with a suitable
acid.
[0269] Representative acid addition salts include, but are not
limited to, acetate, adipate, alginate, citrate, aspartate,
benzoate, benzenesulfonate, bisulfate, butyrate, camphorate,
camphorolsulfonate, digluconate, glycerophosphate, hemisulfate,
heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide,
hydroiodide, 2-hydroxyethanesulfonate (isothionate), lactate,
maleate, methanesulfonate or sulfate, nicotinate,
2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate,
3-phenylpropionate, picrate, pivalate, propionate, succinate,
tartrate, thiocyanate, phosphate or hydrogen phosphate, glutamate,
bicarbonate, p-toluenesulfonate, and undecanoate. Examples of acids
that can be employed to form pharmaceutically acceptable acid
addition salts include, without limitation, such inorganic acids as
hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric
acid, and such organic acids as oxalic acid, maleic acid, succinic
acid, and citric acid.
[0270] Basic nitrogen-containing groups can be quaternized with
such agents as lower alkyl halides such as methyl, ethyl, propyl,
and butyl chlorides, bromides and iodides; dialkyl sulfates like
dimethyl, diethyl, dibutyl, and diamyl sulfates; long chain alkyl
halides such as decyl, lauryl, myristyl, and stearyl chlorides,
bromides, and iodides; arylalkyl halides such as benzyl and
phenethyl bromides; and others. Products having modified solubility
or dispersibility are thereby obtained.
[0271] Compositions comprising a compound of the invention
formulated in a pharmaceutical acceptable carrier can be prepared,
placed in an appropriate container, and labeled for treatment of an
indicated condition. Accordingly, there also is contemplated an
article of manufacture, such as a container comprising a dosage
form of a compound of the invention and a label containing
instructions for use of the compound. Kits are also contemplated
under the invention. For example, the kit can comprise a dosage
form of a pharmaceutical composition and a package insert
containing instructions for use of the composition in treatment of
a medical condition. In either case, conditions indicated on the
label can include treatment of inflammatory disorders, cancer,
etc.
[0272] Methods of Administration
[0273] Pharmaceutical compositions comprising a compound of formula
A can be administered to the subject by any conventional method,
including parenteral and enteral techniques. Parenteral
administration modalities include those in which the composition is
administered by a route other than through the gastrointestinal
tract, for example, intravenous, intraarterial, intraperitoneal,
intramedullarly, intramuscular, intraarticular, intrathecal, and
intraventricular injections. Enteral administration modalities
include, for example, oral (including buccal and sublingual) and
rectal administration. Transepithelial administration modalities
include, for example, transmucosal administration and transdermal
administration. Transmucosal administration includes, for example,
enteral administration as well as nasal, inhalation, and deep lung
administration; vaginal administration; and rectal administration.
Transdermal administration includes passive or active transdermal
or transcutaneous modalities, including, for example, patches and
iontophoresis devices, as well as topical application of pastes,
salves, or ointments. Parenteral administration also can be
accomplished using a high-pressure technique, e.g.,
POWDERJECT.TM..
[0274] Surgical techniques include implantation of depot
(reservoir) compositions, osmotic pumps, and the like. A preferred
route of administration for treatment of inflammation can be local
or topical delivery for localized disorders such as arthritis, or
systemic delivery for distributed disorders, e.g., intravenous
delivery for reperfusion injury or for systemic conditions such as
septicemia. For other diseases, including those involving the
respiratory tract, e.g., chronic obstructive pulmonary disease,
asthma, and emphysema, administration can be accomplished by
inhalation or deep lung administration of sprays, aerosols,
powders, and the like.
[0275] In some foregoing embodiments, the compound of formula A is
administered before, during, or after administration of
chemotherapy, radiotherapy, and/or surgery. The formulation and
route of administration chosen will be tailored to the individual
subject, the nature of the condition to be treated in the subject,
and generally, the judgment of the attending practitioner.
[0276] The therapeutic index of the compound of formula A can be
enhanced by modifying or derivatizing the compounds for targeted
delivery to cancer cells expressing a marker that identifies the
cells as such. For example, the compounds can be linked to an
antibody that recognizes a marker that is selective or specific for
cancer cells, so that the compounds are brought into the vicinity
of the cells to exert their effects locally, as previously
described (see for example, Pietersz, et al., Immunol Rev, 129:57
(1992); Trail, et al., Science, 261:212 (1993); and
Rowlinson-Busza, et al., Curr Opin Oncol, 4:1142 (1992)).
Tumor-directed delivery of these compounds enhances the therapeutic
benefit by, inter alia, minimizing potential nonspecific toxicities
that can result from radiation treatment or chemotherapy. In
another aspect, the compound of formula A and radioisotopes or
chemotherapeutic agents can be conjugated to the same anti-tumor
antibody.
[0277] The characteristics of the agent itself and the formulation
of the agent can influence the physical state, stability, rate of
in vivo release, and rate of in vivo clearance of the administered
agent. Such pharmacokinetic and pharmacodynamic information can be
collected through preclinical in vitro and in vivo studies, later
confirmed in humans during the course of clinical trials. Thus, for
any compound used in the method of the invention, a therapeutically
effective dose can be estimated initially from biochemical and/or
cell-based assays. Then, dosage can be formulated in animal models
to achieve a desirable circulating concentration range that
modulates expression or activity of a particular PI3K isoform or
combination of isoforms. As human studies are conducted, further
information will emerge regarding the appropriate dosage levels and
duration of treatment for various diseases and conditions.
[0278] Although compounds of the invention are well tolerated, an
example of a limit to the treatment dosage is elevated liver
function tests (LFT). LFT involve standard clinical biochemistry
tests on the patient's serum or plasma to provide information about
the state of a patient's liver. Levels, such as alanine
transaminase, aspartate transaminase, alkaline phosphatase,
bilirubin, and gamma glutamyl transpeptidase, that are outside the
normal range can signal possible liver toxicity. Dosing of the
therapeutic compound can be adjusted to avoid or reduce elevated
liver function test values and subsequent potential for liver
toxicity. For instance, a subject may be administered escalating
doses of a compound. At a certain dose amount, the subject begins
to develop elevated LFT levels outside a normal range, signaling
potential liver toxicity at that dosage. In response, the dosage
may be reduced to an amount such that LFT levels are reduced to an
acceptable range as judged by the treating physician, e.g. a level
that is in the range normal for the subject being treated, or
within about 25% to 50% of normal. Therefore, liver function tests
can be used to titrate the administration dosage of a compound.
[0279] Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD.sub.50 (the
dose lethal to 50% of the population) and the ED.sub.50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the "therapeutic index,"
which typically is expressed as the ratio LD50/ED50. Compounds that
exhibit large therapeutic indices, i.e., the toxic dose is
substantially higher than the effective dose, are preferred. The
data obtained from such cell culture assays and additional animal
studies can be used in formulating a range of dosage for human use.
The dosage of such compounds lies preferably within a range of
circulating concentrations that include the ED.sub.50 with little
or no toxicity.
[0280] Dosage may be limited by treatment-related toxicity
symptoms. Such symptoms besides elevated liver function tests
include anemia, vision blurring, diarrhea, vomiting, fatigue,
mucositis, peripheral edema, pyrexia, peripheral neuropathy,
pleural effusion, night sweats, and orthopnea, or a combination
thereof. At a certain dose amount, if the subject develops
intolerable levels of such symptoms, the dosage may be reduced such
that the adverse event is eliminated and no longer adverse or
reduced to an acceptable level as judged by a treating
physician.
[0281] Another consideration in determining the appropriate dose of
compound for a patient is the desired concentration circulating in
the blood plasma. In a particular embodiment, the concentration of
compound in the blood is between 40-3,000 ng/mL over a 12 hour
period from the time of administration. In another particular
embodiment, the concentration of compound in the blood is between
75-2,000 ng/mL over a 12 hour period from the time of
administration. In another particular embodiment, the concentration
of compound in the blood is between 500-2,000 ng/mL over a 12 hour
period from the time of administration. In a preferred embodiment,
the concentration of compound in the blood is between 40-3,000
ng/mL over a 12 hour period from the time of administration,
wherein the compound is a formula of I, I'', II, or II'' and is
orally administered in an amount of about 50 mg, 100 mg, or 200 mg.
In a preferred embodiment, the concentration of compound in the
blood is between 40-3,000 ng/mL over a 12 hour period from the time
of administration, wherein the compound is a formula of I and is
orally administered in an amount of about 50 mg, 100 mg, or 200 mg.
In a preferred embodiment, the concentration of compound in the
blood is between 40-3,000 ng/mL over a 12 hour period from the time
of administration, wherein the compound is a formula of II and is
orally administered in an amount of about 50 mg, 100 mg, or 200 mg.
In some of the foregoing embodiments, the maximum concentration in
the blood plasma is achieved within two hours of
administration.
[0282] In certain embodiments, the dosage of the compound of
Formula I or II is selected to produce a plasma concentration of
drug of about 10 nM or higher over a period of 8 to 12 hours, on
average, and to provide a peak plasma concentration of about 500 nM
or higher, preferably about 1000 nM or higher. In certain
embodiments, the dosage of the compound of Formula I or II is
selected to produce a plasma concentration of drug of about 100 nM
or higher over a period of 8 to 12 hours, on average, and to
provide a peak plasma concentration of about 500 nM or higher,
preferably about 1000 nM or higher. In certain embodiments, the
dosage of the compound of Formula I or II is selected to produce a
plasma concentration of drug of about 200 nM or higher over a
period of 8 to 12 hours, on average, and to provide a peak plasma
concentration of about 500 nM or higher, preferably about 1000 nM
or higher.
[0283] In certain embodiments, the dosage of the compound of
formula I or II is selected to produce a plasma concentration
wherein the trough concentration of the compound is in the range
where a therapeutic effect, such as apoptosis of cancer cells, is
observed. In certain embodiments, the dosage of the compound of
formula I or II is selected to produce a trough plasma
concentration at or higher than the EC.sub.50 PI3K.delta. isoform
activation in blood plasma. In certain embodiments, the dosage of
the compound of formula I or II is selected to produce an trough
blood concentration above the EC.sub.50 level for PI3K.delta.
activation and below the level for EC.sub.50 PI3K.gamma. activation
in a cell during a period of at least 12 hours from compound
administration. For instance, if the EC.sub.50 value for
PI3K.delta. basophil activation is 65 nM and the EC.sub.50 value
for PI3K.gamma. basophil activation is 1100 nM in whole blood
plasma, then the dosage of the compound selected provides a trough
plasma concentration of the compound between 60 nM and 1100 nM
during a period of 8-12 hours from compound administration.
Similarly, a dosage can be selected to produce an trough blood
concentration above the EC.sub.50 level for PI3K.delta. basophil
activation and below the EC.sub.50 level for PI3K-.alpha., -.beta.
or -.gamma. basophil activation. The EC.sub.50 values for the PI3K
isoform activation or inhibition in vivo can be determined by a
person having ordinary skill in the art. In alternative
embodiments, the upper range of the trough concentration of the
drug may exceed and is not limited by the EC.sub.50 value of the
PI3K-.gamma., -.alpha., or -.beta. isoform in blood plasma.
Moreover, the blood concentration range of the drug is at a level
which is therapeutically beneficial in treating the hematologic
malignancy, while minimizing undesirable side effects.
[0284] For instance, while being delta-selective, the compounds can
exhibit sufficient activity on p110.gamma. to be clinically useful,
i.e., to be effective on a cancer that relies upon p110.gamma. for
signaling, because a plasma level above the effective dosage for
inhibition of p110.gamma. can be achieved while still being
selective relative to other isoforms, particularly the alpha
isoform. Thus, in some embodiments, the dosage of the compound is
selected to produce a blood concentration effective for selectively
inhibiting p110.delta. and p110.gamma..
[0285] In some embodiments, the dosage of the compound provides a
trough blood plasma concentration between 65 nM and 1100 nM during
a period of 8 to 12 hours from compound administration. In some
foregoing embodiments, the period is at least 12 hours from
compound administration.
[0286] In a particular embodiment, the compound is administered in
a therapeutically effective amount.
[0287] In a particular embodiment, the compound is administered at
a dose of 20-500 mg/day. In a particular embodiment, the compound
is administered at a dose of 50-250 mg/day.
[0288] In a particular embodiment, the compound is administered at
a dose of 25 to 150 mg per dose, and two doses are administered per
day (e.g., BID dosing with 25 to 150 mg doses). In a preferred
embodiment, a subject is treated with 50 mg to 100 mg of a compound
of formula A twice per day.
[0289] In a particular embodiment, the method comprises
administering to said patient an initial daily dose of 20-500 mg of
the compound and increasing said dose by increments until clinical
efficacy is achieved. Increments of about 25, 50, or 100 mg can be
used to increase the dose. The dosage can be increased daily, every
other day, twice per week, or once per week.
[0290] In a particular embodiment, the method comprises continuing
to treat said patient by administering the same dose of the
compound at which clinical efficacy is achieved or reducing said
dose by increments to a level at which efficacy can be
maintained.
[0291] In a particular embodiment, the method comprises
administering to said patient an initial daily dose of 20-500 mg of
the compound and increasing said dose to a total dosage of 50-400
mg per day over at least 6 days. Optionally, the dosage can be
further increased to about 750 mg/day.
[0292] In a particular embodiment, the compound is administered at
least twice daily.
[0293] In a particular embodiment, the compound is administered
orally, intravenously or by inhalation. Preferably, the compound is
administered orally. In some embodiments, it is administered orally
at a dosage of about 50 mg BID or at a dosage of about 100 mg
BID.
[0294] For the methods of the invention, any effective
administration regimen regulating the timing and sequence of doses
can be used. Doses of the agent preferably include pharmaceutical
dosage units comprising an effective amount of the agent. As used
herein, "effective amount" refers to an amount sufficient to
modulate PI3K.delta. expression or activity and/or derive a
measurable change in a physiological parameter of the subject
through administration of one or more of the pharmaceutical dosage
units. "Effective amount" can also refer to the amount required to
ameliorate a disease or disorder in a subject.
[0295] Suitable dosage ranges for the compounds of formula A vary
according to these considerations, but in general, the compounds
are administered in the range of 10.0 .mu.g/kg-15 mg/kg of body
weight; 1.0 .mu.g/kg-10 mg/kg of body weight, or 0.5 mg/kg-5 mg/kg
of body weight. For a typical 70-kg human subject, thus, the dosage
range is from 700 .mu.g-1050 mg; 70 .mu.g-700 mg; or 35 mg-350 mg
per dose, and two or more doses may be administered per day.
Dosages may be higher when the compounds are administered orally or
transdermally as compared to, for example, i.v. administration. The
reduced toxicity of a compound of formula A, permits the
therapeutic administration of relatively high doses. In some of the
foregoing embodiments, oral administration of up to 750 mg/day of a
compound of the invention is suitable. In some of the foregoing
embodiments, a compound of formula A is administered at a dose of
50 mg BID. In some of the foregoing embodiments, a compound of
formula A is administered at a dose of 100 mg BID. In some of the
foregoing embodiments, a compound of formula A is administered at a
dose of 200 mg BID. In some of the foregoing embodiments, a
compound of formula A is administered at a dose of 350 mg BID. In
specific embodiments, for treatment of leukemias, lymphomas and
multiple myeloma, a dosage of about 50-350 mg per dose,
administered orally once or preferably twice per day, is often
suitable.
[0296] In some of the foregoing embodiments, oral administration of
up to 750 mg/day of compound I'' or II'' is suitable. In some of
the foregoing embodiments, a compound of formula I'' or II'' is
administered at a dose of 50 mg BID. In some of the foregoing
embodiments, a compound of formula I'' or II'' is administered at a
dose of 100 mg BID. In some of the foregoing embodiments, a
compound of formula I'' or II'' is administered at a dose of 200 mg
BID. I In some of the foregoing embodiments, a compound of formula
I'' or II'' is administered at a dose of 350 mg BID. In some of the
foregoing embodiments, for treatment of leukemias, lymphomas and
multiple myeloma, a dosage of about 50-350 mg per dose of a
compound of formula I'' or II'', administered orally once or
preferably twice per day, is often suitable.
[0297] The compounds may be administered as a single bolus dose, a
dose over time, as in i.v. or transdermal administration, or in
multiple dosages.
[0298] Dosing may be continued for at least seven days. In some
embodiments, daily dosing is continued for about 28 days. In some
embodiments, dosing is continued for about 28 days and is then
discontinued for at least 7 days. In some embodiments, a complete
cycle is continuous daily dosing for 28 days. Evaluation of a
clinical response in the patient can be measured after each cycle.
The clinical results can be used to make a decision to increase,
decrease, discontinue or maintain the dosage.
[0299] Depending on the route of administration, a suitable dose
can be calculated according to body weight, body surface area, or
organ size. The final dosage regimen will be determined by the
attending physician in view of good medical practice, considering
various factors that modify the action of drugs, e.g., the agent's
specific activity, the identity and severity of the disease state,
the responsiveness of the patient, the age, condition, body weight,
sex, and diet of the patient, and the severity of any infection.
Additional factors that can be taken into account include time and
frequency of administration, drug combinations, reaction
sensitivities, and tolerance/response to therapy. Further
refinement of the dosage appropriate for treatment involving any of
the formulations mentioned herein is done routinely by the skilled
practitioner without undue experimentation, especially in light of
the dosage information and assays disclosed, as well as the
pharmacokinetic data observed in human clinical trials. Appropriate
dosages can be ascertained through use of established assays for
determining concentration of the agent in a body fluid or other
sample together with dose response data.
[0300] The frequency of dosing will depend on the pharmacokinetic
parameters of the compound of Formula A and the route of
administration. Dosage and administration are adjusted to provide
sufficient levels of the active moiety or to maintain the desired
effect. Accordingly, the pharmaceutical compositions can be
administered in a single dose, multiple discrete doses, continuous
infusion, sustained release depots, or combinations thereof, as
required to maintain desired minimum level of the compound.
Short-acting pharmaceutical compositions (i.e., short half-life)
can be administered once a day or more than once a day (e.g., two,
three, or four times a day). Long acting pharmaceutical
compositions might be administered every 3 to 4 days, every week,
or once every two weeks. Pumps, such as subcutaneous,
intraperitoneal, or subdural pumps, can be preferred for continuous
infusion.
[0301] Subjects that will respond favorably to the method of the
invention include medical and veterinary subjects generally,
including human patients. Among other subjects for whom the methods
of the invention is useful are cats, dogs, large animals, avians
such as chickens, and the like. In general, any subject who would
benefit from a compound of formula A is appropriate for
administration of the invention method. In some foregoing
embodiments, the patient has a cytogenetic characteristic of
del(17p) or del(11q). In some foregoing, embodiments, the patient
has a lymphadenopathy. In some foregoing embodiments, the use of
compound I, I'', II, or II'' reduces the size of a lymphadenopathy
in a patient. In some foregoing embodiments, the use of compound I,
I'', II, or II'' reduces the size of a lymphadenopathy after one
cycle of treatment. In some foregoing embodiments, the use of
compound I, I'', II, or II'' reduces the size of a lymphadenopathy
by at least 10% after one cycle of treatment. In some foregoing
embodiments, the use of compound I, I'', II, or II'' reduces the
size of a lymphadenopathy by at least 25% after one cycle of
treatment. In some foregoing embodiments, the use of compound I,
I'', II, or II'' reduces the size of a lymphadenopathy by at least
30% after one cycle of treatment. In some foregoing embodiments,
the use of compound I, I'', II, or II'' reduces the size of a
lymphadenopathy by at least 40% after one cycle of treatment. In
some foregoing embodiments, the use of compound I, I'', II, or II''
reduces the size of a lymphadenopathy by at least 50% after one
cycle of treatment. In some foregoing embodiments, the use of
compound I, I'', II, or II'' reduces the size of a lymphadenopathy
by at least 75% after one cycle of treatment.
[0302] In one aspect, the invention provides a method of treating a
condition, comprising administering a compound of formula I, II or
a pharmaceutically acceptable salt thereof and one or more
therapeutic agents to a subject in need of such treatment, wherein
the condition is a cancer or an autoimmune condition. In preferred
embodiments, the therapeutic agent is a proteasome inhibitor. In
more specific embodiments, the therapeutic agent is bortezomib. In
some of the foregoing embodiments, the condition is a hematologic
malignancy. In preferred embodiments, the condition is selected
from the group consisting of multiple myeloma, acute lymphocytic
leukemia, acute myeloid leukemia, chronic lymphocytic leukemia,
B-cell lymphoma, diffuse large B-cell lymphoma, B-cell ALL, T-cell
ALL and Hodgkin's lymphoma. In preferred embodiments, the compound
is substantially comprised of the S-enantiomer. In specific
embodiments, the compound comprises at least 95% of the
S-enantiomer. In some of the foregoing embodiments, the
administration of said compound and therapeutic agent provides a
synergistic benefit superior to results obtained without the
combination of the compound and therapeutic agent.
[0303] The following examples are offered to illustrate but not to
limit the invention. In the examples below, references to the
`compound of formula I` or `compound I` refer to the S-enantiomer
shown here, and samples used for these Examples exhibited a 98.2%
ee as measured by chiral HPLC methods:
##STR00016##
[0304] In addition, an analysis of this compound reveals the
following characteristics of the material:
TABLE-US-00002 Test Test Result Appearance Slightly off-white
powder 1H-NMR Spectrum conforms to the reference HPLC Assay 98.1%
(Anhydrous, solvent-free basis) Chiral Purity 98.2% ee (HPLC)
TABLE-US-00003 Test Test Result Residual on Ignition 0.11% Infrared
Spectroscopy Spectrum in agreement (FTIR) with the reference
13C-NMR Spectrum conforms to the reference Particle Size Analysis
Median diameter: 11.3 .mu.m Water (Coulometric 0.56% Karl
Fischer)
TABLE-US-00004 Test Result Property or Test Expected Found
Elemental Analysis % C 63.3 63.5 % C, H, F, N % H 4.4 4.4 % N 23.5
23.1 % F 4.5 4.5
Example 1
Inhibition of Cell Growth in MM Cells
[0305] This example demonstrates the compound of formula I inhibits
the cellular growth stimulatory effects of cytokines (IGF-1 and
IL-6) in multiple myeloma (MM) cells. LB cells (Myelomonocytic
myeloma cell line) were cultured for 48 h with control media; with
the compound of formula I, in the presence or absence of either
IL-6 or IGF-1. The inhibitory effect of the compound of formula I
on MM cell growth was assessed by measuring
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide (MTT;
Chemicon International) dye absorbance. Cells were pulsed with 10
.mu.L of 5 mg/mL MTT to each well for the last 4 hours of 48-hour
cultures, followed by 100 .mu.L isopropanol containing 0.04 N HCl.
Absorbance was measured at 570/630 nm using a spectrophotometer
(Molecular Devices). A summary of the results is shown in FIG. 1.
Exposure of 0.625 .mu.M-2.5 .mu.M of Compound I inhibits MM cell
growth even in the presence of cell growth stimulatory
cytokines.
Example 2
Effect of BMSC on Cytotoxicity
[0306] This example demonstrates Bone Marrow Stromal Cells (BMSCs)
do not protect against compound I-induced LB cell cytotoxicity. LB
cells were cultured with control media, and with the compound of
formula I for 48 hours, in the presence or absence of BMSCs. Cell
proliferation was assessed using [.sup.3H]-thymidine uptake assay.
All data represent mean (.+-.SD) of triplicate experiment. A
summary of the results is shown in FIG. 2. LB cell growth is
reduced after exposure to 0.625 .mu.M-10 .mu.M of compound I even
in the presence of BMSC.
Example 3
Effect of Compound on Apoptosis of CLL Cells
[0307] This example demonstrates the compound of formula I induces
apoptosis in patient chronic lymphocytic leukemia (CLL) cells.
Peripheral blood was obtained from patients with B-CLL through the
CLL Research Consortium from Ohio State University. Primary
CD19-positive cells were isolated using Rosette-Sep (StemCell
Technologies). Cells were maintained in RPMI 1640 (Invitrogen)
supplemented with 10% heat-inactivated fetal bovine serum, 2 mmol/L
L-glutamine, and penicillin (100 units/mL)/streptomycin (100
.mu.g/mL; Invitrogen) at 37.degree. C., 5% CO.sub.2, and high
humidity. After incubation with the compound of formula I or medium
for 96 hours, 5.times.10.sup.5 cells were washed with PBS and then
resuspended in binding buffer (10 mmol/L HEPES/NaOH, pH 7.4, 150
mmol/L NaCl 5 mmol/L KCl, 1 mmol/L MgCl.sub.2, 1.8 mmol/L
CaCl.sub.2) containing 2 .mu.L of Annexin V-FITC stock
(BioWhittaker, Inc) and 10 .mu.L of 20 .mu.g/mL PI (Sigma). After
incubation for 10 minutes at room temperature in a light-protected
area, the specimens were quantified by flow cytometry on a
FACScan.TM. (Becton Dickinson).
[0308] Treatment of CLL patient cells with compound I results in
apoptosis and the result appears to be dose-dependent, as seen in
FIG. 3.
[0309] Compound I induced apoptosis was seen in CLL cells from poor
prognosis patients, as the data indicates in FIG. 19.
[0310] Compound I induced apoptosis was also seen to be effective
in CLL cells from refractory/relapsed patients as shown in FIG.
20.
Example 4
Effect of Compound in ALL Cell Lines
[0311] This example demonstrates the compound of formula I results
in a reduction of Akt phosphorylation and a decrease in cellular
proliferation accompanied by cell death in both T-ALL and B-ALL
(Acute Lymphoblastic Leukemia) leukemic cell lines. Viability
assays of cell lines were performed using the AlamarBlue assay
(Invitrogen). Cells (1.times.10.sup.6 per well) in a volume of 100
.mu.L were placed in a 96-well flat-bottom plate and the compound
of formula I (100 .mu.L per well at 2.times. final concentration)
or medium alone was added to the plates. All were performed in
quadruplicate. Cells were incubated for fixed times (48 hours).
After the incubation, 10 .mu.L AlamarBlue.RTM. was added to each
well. Cells were incubated for 4 hours and the optical density at
530-560 nm was obtained using a SpectraMax.RTM. M5 plate reader
2001. Cell viability was expressed as a percentage of absorption
between treated cells/control sample. These results are summarized
in the table shown in FIG. 4. Exposure to compound I result in
substantial reduction in cellular viability in a variety ALL cell
lines as well as reduction in Akt phosphorylation.
Example 5
Effect of Compound on ALL Cell Cycle
[0312] This example demonstrates treatment of the acute
lymphoblastic leukemia (ALL) cell line CCRF-SB with the compound of
formula I results in G0/G1 cell cycle arrest. Representative
fluorescence-activated cell sorting (FACS) analysis of propidium
iodide-stained CCRF-SB cells under normal growth conditions, and
growth in the presence of the compound of formula I. The average
percentage of cells in G.sub.0-G.sub.1, S, and G.sub.2-M phases is
calculated in the table below the histographs. Results are shown in
FIG. 5.
Example 6
Inhibition of Proliferation of Breast Cancer Cells
[0313] This example demonstrates the compound of formula I inhibits
proliferation of breast cancer cell lines. T47D and HS-578T cell
lines were grown in the presence of serum plus the indicated
concentrations of the compound of formula I. Proliferation was
measured in triplicate wells by AlamarBlue.RTM. assay (Invitrogen)
96-well plates. Results of proliferation assays are expressed as
the mean cellular percentage values and shown in FIG. 6.
Example 7
Inhibition of Proliferation of Ovarian Cancer Cell Lines
[0314] This example demonstrates the compound of formula I inhibits
proliferation of ovarian cancer cell lines. IGROV-1 and OVCAR-3
cell lines were grown in the presence of serum plus the indicated
concentrations of the compound of formula I. Proliferation was
measured in triplicate wells by AlamarBlue assay (Invitrogen)
96-well plates. Results of proliferation assays are expressed as
the mean cellular percentage values and are shown in FIG. 7.
Example 8
Reduction of Akt Phosphorylation
[0315] This example demonstrates the compound of formula I reduces
constitutive Akt phosphorylation in hematopoietic tumor cell lines
that exhibited constitutive Akt phosphorylation. A large panel of
leukemia and lymphoma cell lines was assessed for constitutive Akt
phosphorylation. These cell lines represent B-lymphoma, T-lymphoma,
ALL, Malignant histiocytosis, DLBCL and AML. Cell lines that
demonstrated serum independent Akt phosphorylation were treated
with the compound of formula I for 2 hours. Thereafter, cell were
lysed, size-fractioned and immunoblotted with antibodies directed
against phospho-Akt(Ser473). Results are shown in FIG. 8. Reduction
in Akt(Ser473) was achieved for all cell lines after exposure to
compound I.
Example 9
Compound I Effective in DLBCL
[0316] This example provides evidence that compound I blocks PI3K
signaling and induces apoptosis in diffuse large B-cell lymphoma
cells. P110.delta. is expressed in DLBCL cell lines as shown in
FIG. 26A. FIG. 26B shows that exposure to compound I reduces pAKT
levels in several DLBCL cell lines.
Example 10
Inducement of Apoptosis in Breast Cancer Cells
[0317] This example demonstrates the compound of formula I induces
apoptosis in breast cancer cell lines. HS-578T, T47D, and MCF7
cells were treated with the compound of formula I or corresponding
DMSO concentrations for 24 h. The percentage of apoptotic cells was
determined by Annexin V-FITC/7AAD staining. Bottom left, viable
cells (Annexin V-FITC/PI negative); bottom right, early apoptotic
cells (Annexin V-FITC positive only); top right, mid-late apoptotic
cells (Annexin V-FITC/7AAD double-positive); and top left, late
apoptotic/necrotic (7AAD positive only). Percentages of cells in
each quadrant are indicated except for the bottom left quadrant
(viable cells). One experiment representative of three different
experiments that gave similar results is shown in FIG. 10.
Example 11
Steady State Blood Levels on Day 7 in Healthy Volunteers
[0318] This example provides data relating to the concentration of
the compound of formula I in the blood of a healthy human subject
on day 7. The concentration was monitored over a period of 12
hours, after oral administration of 50, 100, or 200 mg BID of the
compound of formula I on day 7 of the study. FIG. 11 follows the
plasma concentration of the drug over a period of 12 hours from
administration. The maximum concentration of drug is achieved
within two hours for all doses. Administration of 50, 100 or 200 mg
BID of said compound results in a concentration level that exceeds
the PI3K.delta. EC.sub.50 concentration in basophil for at least 12
hours.
[0319] In addition, single dose studies wherein 17-400 mg of the
compound of formula I was administered in healthy volunteers was
carried out. Concentration of the compound in the blood was
measured over 24 hours from administration and results are shown in
FIG. 24A. At about 6 hours, the concentration of compound I in the
blood for all administered doses is at least about 100 nM. At about
12 hours, the concentration of compound I in the blood for doses 50
mg and higher is over 50 nM. The maximum concentration of compound
I in the blood is achieved within 2 hours of administration.
[0320] In another experiment, the mean compound I concentration was
measured on the 7th day of 50 mg BID dosing in healthy volunteers
(N=6). The mean trough concentration was higher than the EC50 for
PI3K.delta. and the mean peak concentration was lower than the EC50
for PI3K.gamma. as determined in the whole blood basophil
activation assay, FIG. 24B. This example demonstrates the
concentration range of compound I administered at 50 mg BID is at a
level that is above the ED.sub.50 PI3K.delta. basophil activation
level but lower than the minimum ED.sub.50 PI3K.gamma. basophil
level activation level in whole blood for at least 12 hours.
[0321] Table 1, below, provides an overview of the subjects in the
study, wherein either a singe dose (SD) or multiple dose (MD) of
the compound of formula I is administered to a subject at varying
amounts. The "n" values refer to the number of subjects in each
group.
TABLE-US-00005 TABLE 1 Cohort Regimen Compound I Placebo 1 (n = 8)
SD 17 mg (n = 6) Placebo (n = 2) 2 (n = 8) SD 50 mg (n = 6) Placebo
(n = 2) 3 (n = 8) SD 125 mg (n = 6) Placebo (n = 2) 4 (n = 8) SD
250 mg (n = 6) Placebo (n = 2) 5 (n = 8) SD 400 mg (n = 6) Placebo
(n = 2) 6 (n = 8) MD 50 mg BID .times. 7 d (n = 6) Placebo BID
.times. 7 d (n = 2) 7 (n = 8) MD 100 mg BID .times. 7 d (n = 6)
Placebo BID .times. 7 d (n = 2) 8 (n = 8) MD 200 mg BID .times. 7 d
(n = 6) Placebo BID .times. 7 d (n = 2)
Example 12
Effect on Lesions in a Patient with Mantle Cell Lymphoma
[0322] This example provides data relating to the area of lesions
of a patient with mantle cell lymphoma after 1 cycle of treatment
(28 days) with the compound of formula I. The area of 6 lesions was
measured prior to treatment and after a cycle of treatment. The
response to 28 days of oral administration of 50 mg BID of the
compound of formula I, results in a decrease of lesion area
compared to area prior to treatment and represents a 44% decrease
in tumor burden. The results are summarized in a bar graph found in
FIG. 12.
Example 13
Response of a Patient with CLL to Treatment
[0323] This example provides data relating to the concentration of
absolute lymphocyte count (ALC) in the blood of a patient with CLL
after 1 cycle (28 days) of treatment with oral administration of
the compound of formula I. The blood ALC concentration was measured
over a period of 4 weeks after completion of one cycle of
treatment. A 55% decrease in lymphocytosis and a 38% decrease in
lymphadenopathy as a result of treatment were observed. A marked
decrease in ALC concentration is observed between week 1 and week
2, FIG. 13.
Example 14
Comparison of Lymphoma Patient to Healthy Volunteer
[0324] This example provides data comparing the concentration of
the compound of formula I in a lymphoma patient to normal healthy
volunteers. On the 28.sup.th day of oral administration of 50 mg
BID of compound in a patient with mantle cell lymphoma, the
concentration of the compound in the blood was measured over a
period of 6 hours after administration. The concentration of 50 and
100 mg oral administration in normal healthy volunteers on day 7 of
administration was also observed. The results are summarized in
FIG. 14. Thus, the compound does not build up excessively over the
course of a cycle of treatment, nor does the patient become
tolerant by increased metabolism over the course of the treatment
cycle.
Example 15
Activity of Compound I in Various Kinases
[0325] This example shows the IC.sub.50 profile of compound I
across classes of kinases as summarized in Table 2. While
especially active on p110.delta., Compound I was also active on
p110.gamma. and even active enough to be therapeutically useful at
non-toxic doses against p110.beta., due to the demonstrated high
NOAEL level of the compound; while exhibiting little activity on
Class II-V phosphoinositide kinases. Thus while being
delta-selective, the compounds can exhibit sufficient activity on
p110.gamma. to be clinically useful, i.e., to be effective on a
cancer that relies upon p110.gamma. for signaling, because a plasma
level above the effective dosage for inhibition of p110.gamma. can
be achieved while still being selective relative to other isoforms,
particularly the alpha isoform.
TABLE-US-00006 TABLE 2 Other Class I PI3Ks, Class II PI3K, Class
III PI3K, Class IV PI3K, Phosphoinositide IC.sub.50 (nM)
IC.sub.50(nM) IC.sub.50(nM) IC.sub.50 (nM) kinases Compound
P110.alpha. p110.beta. p110.delta. p110.gamma. CIIbeta hVPS34
DNA-PK mTOR PIP5K.alpha. PIP5K.beta. I 435 128 1 14 >10.sup.3
978 6,729 >10.sup.3 >10.sup.3 >10.sup.3 NVP-BEZ-235 19 293
63 267 .sup. 3 6 1 .sup. 2 ND* ND Novartis InvitroGen Adapta assay
*ND = not determined
Example 16
No Off-Target Activity of Compound I in Kinome-Wide Protein Kinase
Screen
[0326] This example demonstrates that compound I has little or no
off target activity in a kinome-wide protein kinase screen. Using
Ambit KINOMEscan.TM. a genome wide screen of over 350 protein
kinases failed to detect any activity at 10 .mu.M. Examples of some
kinases in the screen are shown below in Table 3.
TABLE-US-00007 TABLE 3 Examples of Relevant Kinases in Screen ABL
AKT ALK BLK BRAF BTK CDK CSF1R EGFR EPH FGFR1 VEGFR1 FLT3 FRK FYN
HCK HER2 ICK IGF1-R ITK JAK1 JAK2 JNK1 KIT LCK LYN MAPK MEK MET MLK
P38MAPK PDGFR PIM PKA PKC PLK RAF RET ROCK ROS S6K SLK SRC SYK TAK
TIE TRK TYK YES ZAP70
Example 17
Selectivity of Compound I for p110.delta.
[0327] This example demonstrates that compound I is selective for
p110.delta. as measured in isoform specific cell-based assays.
[0328] Swiss-3T3 fibroblasts and RAW-264 were seeded on a 96-well
tissue culture plate and allowed to reach at least 90% confluency.
Cells were starved and treated with either vehicle or serial
dilutions of compound I for 2 hrs and stimulated with PDGF or C5a
respectively. Akt phosphorylation and total AKT was detected by
ELISA. Purified B-cells were treated with either vehicle or serial
dilutions of compound I for 30 minutes at room temperature before
the addition of purified goat anti-human IgM. Results are expressed
as relative [.sup.3H] thymidine incorporation induced by IgM
crosslinking.
TABLE-US-00008 TABLE 4 PI3K.alpha. PI3K.delta. PI3K.gamma.
EC.sub.50 (nM) EC.sub.50 (nM) EC.sub.50 (nM) Fibroblast Cell Line
Primary B Cell Monocyte Cell Line PDGF induced pAKT BCR mediated
C5a induced pAKT proliferates >20,000 6 3,894 (n = 12) (n = 6)
(n = 11)
Example 18
Expression of p110.delta. in Leukemia and Lymphoma Cell Lines
[0329] This example demonstrates that PI3K p110.delta. is highly
expressed in a broad range of leukemia and lymphoma cell lines.
[0330] PI3K p110.delta. promotes proliferation and survival in a
wide range of leukemia and lymphoma cell lines. Among the cell
types investigated are MCL, DLBCL, AML, ALL, and CML.
[0331] Expression of PI3K p110.alpha., .beta., .gamma. and .delta.
in a panel of lymphoma and leukemia cell lines is demonstrated in
FIG. 15. Proteins from 10.sup.6 cells were separated by SDS-PAGE
and analyzed by Western blot using antibodies specific for the
.alpha., .beta., .gamma. and .delta. isoforms. Purified recombinant
p110 proteins were used as controls. Anti-actin antibodies were
used to assess equal loading of the samples. p110.delta. was
consistently expressed at a high level while other p110 isoforms
were highly variable. PI3K p110.delta. is known to be uniformly
expressed in patient AML cells as discussed by Sujobert, et al.,
Blood 2005 106(3), 1063-1066.
Example 19
Inhibitory Effect of Compound I on p110.delta.
[0332] Example 19 shows compound I inhibition of p110.delta. blocks
PI3K signaling in leukemia and lymphoma cell lines with
constitutive pathway activation.
[0333] The PI3K pathway is frequently deregulated in leukemia and
lymphoma cell lines. 48% of cell lines, or 13 out of 27, were found
to have constitutive p-AKT. In addition, PI3K pathway activation is
dependent on p110.delta.. Compound I was found to inhibit
constitutive AKT phosphorylation in 13 out of 13 cell lines.
[0334] PAGE results of FIG. 9 demonstrates that constitutive AKT
phosphorylation was inhibited by the presence of compound I in each
of 11 cell lines, including B-cell and T-cell lymphomas. Cells were
incubated for 2 hrs with 10 .mu.M compound I. Cell lysates were run
on SDS-PAGE and transferred onto PDVF membrane and probed with
appropriate antibodies. Compound I was found to inhibit
constitutive AKT phosphorylation in 11 out of 11 cell lines.
Additional cell line data for T-ALL and B-ALL cell lines is shown
in FIG. 27. A decrease in Akt and S6 phosphorylation after exposure
to a range concentrations of compound I (0.1 .mu.M to 10 .mu.M),
was quantitated by densitometry, expressed as the percent change,
FIG. 28.
Example 20
Compound I Inhibits Proliferation and Apoptosis in Leukemia Cell
Lines
[0335] Example 20 demonstrates that compound I inhibits
proliferation and induces apoptosis in leukemia cell lines. FIGS.
16A-B show that treatment with compound I for 24 hours reduces
cellular viability in a dose dependent manner.
[0336] Proliferation assays (AlamarBlue.RTM.) on ALL cell lines
grown in the presence of 10% FBS serum and measurements were taken
at 24 hrs. Proliferation was measured in triplicate wells in
96-well plates. The inhibition of PI3K signaling by compound I
resulted in a block of cell cycle progression, and/or cell death.
In each of six leukemia cell lines, viability was reduced by 40-50%
with 10 micromolar concentrations of Compound I, FIG. 16A.
[0337] Induction of apoptosis by compound I. Cells were treated
with DMSO (vehicle), 1 .mu.M or 10 .mu.M compound 1 for 24 hrs. The
percentage of apoptotic cells was determined by Annexin V-FITC/7AAD
staining. One experiment representative of different experiments
that gave similar results is shown in FIG. 16B.
Example 21
Expression of p110.delta. in CLL Cells
[0338] This example demonstrates PI3K p110.delta. and p110.delta.
isoform expression in patient CLL cells.
[0339] PI3K mediated signaling pathways have been implicated in
CLL. These pathways have a role in cell proliferation, prevention
of apoptosis and cell migration. Efforts were made to determine
PI3K isoform expression in patient CLL cells.
[0340] CLL patient demographics are summarized below in Table
5.
TABLE-US-00009 TABLE 5 CLL Patient Demographics (Total (N = 24) I)
Cytogenetic abnormalities 13q14.3 58% 11q22.3 33% 17p13.1 20%
Trisomy 12 12% II) Treatment History Fludarabine refractory 29%
Unknown 54% II) IgVH Status Mutated 33% Unmutated 33% Unknown
33%
[0341] The PAGE images of FIG. 17A-D compare the expression of
p110.alpha., p110.delta., p110.beta., and p110.gamma. in CLL cells
of patients A-E. p110.delta. and p110.gamma. is expressed in each
patient compared to the other PI3K isoforms.
Example 22
Compound I Induces Cleavage of Caspase 3 and PARP
[0342] This example demonstrates that compound I induced the
cleavage of caspase 3 and PARP. FIGS. 18A-B show results of caspase
3 and PARP (Poly(ADP) Ribose Polymerase) cleavage in the presence
of 1, 10 .mu.M of compound I or 25 .mu.M of LY294002.
[0343] Further experiments provide evidence of compound I inducing
caspase 2 and PARP cleavage. Cells were cultured with compound I or
vehicle alone for 24 hrs. Thereafter, cells were lysed and
sized-fractionated and immunoblotted with antibody directed against
FLIP, FIG. 29. Additionally, whole cell lysates were added to MDS
(Meso Scale Diagnostics) multi-spot 96-well 4 spot plates coated
with Total caspase-3, cleaved caspase-3, cleaved PARP, and BSA.
Proteins were detected with antibodies labeled with SULFO-TAG
reagent and quantified. A dose dependent response in the cleavage
of caspase 3 and PARP was achieved upon exposure to 5 or 10 .mu.M
of compound I.
Example 23
Compound I Blocks PI3K Signaling
[0344] This example demonstrates that compound I blocks PI3K
signaling in patient AML cells. PI3K.delta. is implicated in
signaling in AML patient cells. FIG. 21 shows the results of
Phospho-Akt production in the absence or presence of 0.1, 1.0, 10
.mu.M of Compound I. This provides evidence that compound I reduces
phospho-Akt production in patient AML cells.
Example 24
Measurement of PI3K Signaling in Basophils Founding Whole-Blood
[0345] This example demonstrates a whole-blood assay for
measurement of PI3K signaling in basophils using flow cytometry by
the induction of CD63 surface expression.
[0346] Inhibition of PI3K signaling in basophils permits compound
Ito be a useful pharmacodynamic marker. PI3K signaling is monitored
by CD63 surface expression. In particular, p110.delta. mediates
FC.epsilon.R1 signaling and p110.gamma. mediates fMLP receptor
signaling. The flow cytometry analysis of PI3K mediated CD63
expression on basophils comprises the following sequential
steps:
[0347] 1. Collect peripheral blood
[0348] 2. Basophil stimulation (fMLP or Anti-FC.epsilon.R1 Mab)
[0349] 3. Label basophils (Anti-CCR3-FITC and Anti-CD63-PE)
[0350] 4. Lyse and fix cells
[0351] 5. Analysis by flow cytometry
[0352] FIG. 22A-C compares the results of A) no stimulation, B)
stimulation with Anti-FC.epsilon.R1, or C) stimulation with
fMLP.
[0353] FIG. 23 shows that Compound I is especially active where
p110.delta. mediated signaling is most important, but is also
relatively active where p110.gamma. is utilized: it achieved 50%
reduction in SD63 expression at <<1 .mu.M for the p110.delta.
test, and ca. 10 .mu.M for the p110.gamma. test. Basophil
activation was measured in human whole blood using the Flow2
CAST.RTM. kit. Whole blood samples were treated with either vehicle
or serial dilutions of compound I prior to activation of basophils
either with anti-Fc.epsilon.RI mAb or fMLP. Cells were stained with
the combination of anti-human CD63-FITC and anti-human CCR3-PE
mAbs. The percent CD63 positive cells within the gated basophil
population were determined in different treatment groups and
normalized to the vehicle control.
Example 25
Compound I Reduces Lymphadenopathy in CLL Patient
[0354] This example provides evidence of the reduction in size of a
bulky lymphadenopathy in a CLL patient with a del[17p]. A patient
with del(17p) had an axillary lymphadenopathy, which was imaged by
computed tomography (CT) to provide a baseline measurement of 5.9
cm.times.4.1 cm, FIG. 40A. After one cycle of treatment with
compound I, the lymphadenopathy was reduced to a dimension of
3.8.times.1.8 cm, FIG. 40B. A cycle treatment was 28 days of
continuous oral dosing at either 200 mg BID or 350 mg BID of
compound I.
Example 26
Limited Effect of Compound I on Glucose and Insulin Levels of a
Subject
[0355] This example demonstrates that treatment with compound I has
little or no effect on glucose and insulin levels. Compound I was
administered at 50-200 mg amounts BID to a subject over a period of
up to 10 days. Blood glucose and insulin concentrations were
measured over time and compared to placebo results as shown in
FIGS. 25A-B.
[0356] Blood glucose concentration remained steady after 10 days of
treatment with even the highest dosage amount of compound I.
Insulin levels remained within the normal range after 7 days of
treatment with compound I. This provides evidence that compound I
has little or no effect on glucose and insulin levels.
Example 27
Materials and Methods
[0357] This example provides information on materials and methods
of carrying out the experiments described in Examples 28-35 which
relate to the use of compound I in the treatment of multiple
myeloma.
[0358] Materials
[0359] p110.delta. inhibitor compound I and compound II were
provided by Calistoga Pharmaceuticals, (Seattle, Wash.). The sample
of compound I and II used was over 95% the S enantiomer. Compound I
was dissolved in Dimethyl sulphoxide at 10 mM and stored at
-20.degree. C. for in vitro study. Compound II was dissolved in 1%
carboxyl methylcellulose (CMC)/0.5% Tween 80 and stored at
4.degree. C. for in vivo study. Recombinant human P110.alpha.,
.beta., .gamma., and .delta. were reconstituted with sterile
phosphate-buffered saline (PBS) containing 0.1% BSA. bortezomib was
provided by Millennium Pharmaceuticals (Cambridge, Mass.).
3-Methyladenine was purchased from Sigma-Aldrich (St. Louis,
Mo.).
[0360] Cell Culture
[0361] Dex-sensitive (MM.1S) and resistant (MM.1R) human MM cell
lines were kindly provided by Dr. Steven Rosen (Northwestern
University, Chicago, Ill.). H929, RPMI8226, and U266 human MM cell
lines were obtained from American Type Culture Collection
(Manassas, Va.). Melphalan-resistant RPMI-LR5 and Doxorubicin
(Dox)-resistant RPMI-Dox40 cell lines were kindly provided by Dr.
William Dalton (Lee Moffitt Cancer Center, Tampa, Fla.). OPM1
plasma cell leukemia cells were provided by Dr. Edward Thompson
(University of Texas Medical Branch, Galveston). IL-6-dependent
human MM cell line INA-6 was provided by Dr. Renate Burger
(University of Kiel, Kiel, Germany). LB human MM cell line was
established in the laboratory. Phenotypic analysis revealed no
cytogenetic abnormalities. Phenotypic analysis is shown in table 6.
CD expression profile of LB cell line, defined by flow-cytometric
analysis.
TABLE-US-00010 TABLE 6 LB expression CD marker % expression CD3
5.5% CD19 61.7% CD20 97.2% CD38 54.1% CD40 96.8% CD49e 5.9% CD70
98.0% CD138 96.3%
[0362] All MM cell lines were cultured in RPMI1640 medium. Bone
marrow stromal cells (BMSCs) were cultured in Dulbecco's
modification of Eagle's medium (DMEM) (Sigma) containing 15% fetal
bovine serum, 2 mM L-glutamine (Life Technologies), 100 U/mL
penicillin, and 100 .mu.g/mL streptomycin (Life Technologies).
Blood samples collected from healthy volunteers were processed by
Ficoll-Paque.TM. gradient to obtain peripheral blood mononuclear
cells (PBMNCs). Patient MM and BM cells were obtained from BM
samples after informed consent was obtained per the Declaration of
Helsinki and approval by the Institutional Review Board of the
Dana-Farber Cancer Institute (Boston, Mass.). BM mononuclear cells
were separated using Ficoll-Paque.TM. density sedimentation, and
plasma cells were purified (>95% CD138+) by positive selection
with anti-CD138 magnetic activated cell separation micro beads
(Miltenyi Biotec, Auburn, Calif.). Tumor cells were also purified
from the BM of MM patients using the RosetteSep negative selection
system (StemCell Technologies, Vancouver, BC, Canada).
[0363] Growth Inhibition Assay
[0364] The growth inhibitory effect of compound I on growth of MM
cell lines, PBMCs, and BMSCs was assessed by measuring
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetra-sodium bromide
(MTT; Chemicon International, Temecula, Calif.) dye absorbance.
[0365] Effect of Compound I on Paracrine MM Cell Growth in the
BM
[0366] MM cells (2.times.104 cells/well) were cultured for 48 h in
BMSC coated 96-well plates (Costar, Cambridge, Mass.), in the
presence or absence of drug. DNA synthesis was measured by
[3H]-thymidine (Perkin-Elmer, Boston, Mass.) uptake, with
[3H]-thymidine (0.5 .mu.Ci/well) added during the last 8 h of 48 h
cultures. All experiments were performed in quadruplicate.
[0367] Transient Knockdown of P110.delta. Expression
[0368] INA-6 cells and LB cells were transiently transfected with
siRNA ON-TARGET plus SMART pool P110.delta. or nonspecific control
duplex (Dharmacon Lafayette, Co) using Cell Line Nucleofector Kit V
(Amaxa BIosystems Gaitherburg, Md.).
[0369] Immunofluorescence
[0370] Viable MM cells (2.5.times.104) were pelleted on glass
slides by centrifugation at 500 rpm for 5 minutes using a cytospin
system (Thermo Shandon, Pittsburgh, Pa.). Cells were fixed in cold
absolute acetone and methanol for 10 min. Following fixation, cells
were washed in phosphate-buffered saline (PBS) and then blocked for
60 min with 5% FBS in PBS. Slides were then incubated with
anti-CD138 antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.)
at 4.degree. C. for 24 h, washed in PBS, incubated with goat
anti-mouse IgG for 1 h at 4.degree. C., and analyzed using Nikon
E800 fluorescence microscopy.
[0371] Detection and Quantification of Acidic Vesicular Organelles
(AVO) with Acridine Orange Staining.
[0372] Autophagy was characterized by sequestration of cytoplasmic
proteins and development of AVOs. To detect and quantify AVOs in
compound I or 3MA-treated cells, vital staining was performed for
15 min with acridine orange at a final concentration of 1 .mu.g/ml.
Samples were examined under a fluorescence microscope.
[0373] Angiogenesis Assay
[0374] The anti-angiogenic activity of compound I was determined
using an in vitro Angiogenesis Assay Kit (Chemicon, Temecula,
Calif.). HUVEC and endothelial growth media were obtained from
Lonza (Walkersville, Md., USA). HUVEC were cultured with compound I
on polymerized matrix gel at 37.degree. C. After 8 h, tube
formation was evaluated using Leika DM IL microscopy (Leica
Microsystems, Wetzlar, Germany) and analyzed with IM50 software
(Leica Microsystems Imaging Solutions, Cambridge, UK). HUVEC cell
migration and rearrangement was visualized, and the number of
branching points counted.
[0375] Western Blotting
[0376] MM cells were cultured with or without compound I;
harvested; washed; and lysed using radioimmuno precipitation assay
(RIPA) buffer, 2 mM Na.sub.3VO.sub.4, 5 m M NaF, 1 mM
phenylmethylsulfonyl fluoride (5 mg/ml). Whole-cell lysates were
subjected to sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) separation, transferred to Pure
Nitrocellulose membranes (Bio-Rad Laboratories, Hercules, Calif.),
and immunoblotted with anti-AKT, phospho(p)-AKT (Ser473, Thr 308),
ERK1/2, P-ERK1/2, P-PDK1, STAT, P-STAT, P-FKRHL, P-70S6K, LC3, and
PI3K/p110.alpha. Abs (Cell Signaling Danvers, Mass.);
anti-p110.beta., PI3K/p110.delta., Glyceraldehyde 3-phosphate
dehydrogenase (GAPDH), .alpha.-tubulin, and actin Abs (Santa Cruz
Biotechnology, CA); and anti-p110.gamma. Ab (Alexis, San Diego,
Calif.): and anti-LC3 Ab (Abgent, San Diego, Calif.).
[0377] ELISA
[0378] Cytokine secretion by human BMSCs cocultured with MM cells
was assessed by ELISA. BMSCs were cultured in 96-well plates with
varying concentrations of compound I, with or without INA-6 cells.
After 48 h, supernatants were harvested and stored at -80.degree.
C. Cytokines were measured using Duo set ELISA Development Kits
(R&D Systems, Minneapolis, Minn.). All measurements were
carried out in triplicate.
[0379] Human Cytokine Array
[0380] The cytokine levels in culture supernatants were assessed
using Proteome Profiler Antibody Arrays Panel A (R&D Systems,
Minneapolis, Minn.), Supernatants from co-cultures with BMSCs were
incubated for 4 hours with membranes arrayed with Abs against 37
cytokines, according to manufacturer's instructions.
[0381] Murine Xenograft Models of Human MM
[0382] CB17 SCID mice (48-54 days old) were purchased from Charles
River Laboratories (Wilmington, Mass.). All animal studies were
conducted according to protocols approved by the Animal Ethics
Committee of the Dana-Farber Cancer Institute. Mice were inoculated
subcutaneously in the right flank with 3.times.106 LB cells in 100
.mu.L RPMI-1640. When tumors were palpable, mice were assigned into
the treatment groups receiving 10 mg/kg or 30 mg/kg gavages twice
daily; and 7 mice in the control group receiving vehicle alone.
Caliper measurements of the longest perpendicular tumor diameters
were performed every alternate day to estimate the tumor volume
using the following formula representing the 3D volume of an
ellipse: 4/3.times.(width/2)2.times.(length/2). Animals were
sacrificed when tumors reached 2 cm or the mice appeared moribund.
Survival was evaluated from the first day of treatment until death.
Tumor growth was evaluated using caliper measurements from the
first day of treatment until day of first sacrifice, which was day
12 for the control group and days 17 and 19 for the treatment
groups. The images were captured with a canon IXY digital 700
camera. Ex vivo analysis of tumor images was captured with a LEICA
DM IL microscope and LEICA DFC300 FX camera at 40 u/0.60 (Leica,
Heidelberg, Germany).
[0383] Human fetal bone grafts were implanted into CB17 SCID-mice
(SCID-hu). Four weeks following bone implantation, 2.5.times.106
INA-6 cells were injected directly into the human BM cavity in the
graft in a final volume of 100 .mu.l of RPMI-1640 medium. An
increase in the levels of soluble human IL-6 receptor (shuIL-6R)
from INA-6 cells was used as an indicator of MM cell growth and
burden of disease in SCID-hu mice. Mice developed measurable serum
shuIL-6R approximately 4 weeks following INA-6 cell injection, and
then received either 10 or 30 mg/kg drug or vehicle alone daily for
7 weeks. Blood samples were collected and assessed for shuIL-6R
levels using an enzyme-linked immunosorbent assay (ELISA, R&D
Systems. Minneapolis Minn.).
[0384] Statistical Analysis
[0385] Statistical significance was determined by Dunn's multiple
comparison tests. The minimal level of significance was p<0.05.
Survival was assessed using Kaplan-Meier curves and log-rank
analysis. The combined effect of compound I and bortezomib was
analyzed by isobologram analysis using the CalcuSyn software
program (Biosoft, Ferguson, Mo.); a combination index (CI)<0.7
indicates a synergistic effect.
Example 28
Expression of p110 Delta in MM Cells
[0386] This example demonstrates that p110.delta. is highly
expressed in patient MM cells. To assess PI3K/p110 expression, Abs
was used against recombinant human PI3K/p110.alpha., .beta.,
.gamma., and .delta. proteins with specific immunoreactivity
against these isoforms. The expression of p110.delta. in 11 MM cell
lines (MM.1S, OPM1, OPM2, RPMI8226, DOX40, LR5, MM.1R, U266, INA-6,
H929, and LB), as well as 24 patient MM samples were evaluated and
immunoblots shown in FIGS. 30A and 30B. FIG. 30A shows expression
of p110-.alpha., -.beta., -.gamma., and -.delta. in MM cell lines
detected by immunoblotting using specific antibodies.
Anti-.alpha.-Tubulin MAb served as a loading control. p110.delta.
in patient MM cells was detected by immunoblotting using
anti-P110.delta. Ab (FIG. 30B).
[0387] Anti-GAPDH MAb served as a loading control. INA-6 and LB
cells strongly expressed p110.delta., whereas MM.1S, OPM1, MM.1R,
Dox40, U266 or H929 lacked p110.delta. expression (FIG. 30A).
[0388] p110.delta. expression in MM.1S and LB cells was confirmed
by immunofluorescence analysis (FIG. 30C). Human recombinant
P110-.alpha., -.beta., -.gamma., -.delta. proteins in SDS sample
buffer were heated for 3 min prior to loading on gel. (10-20 .mu.g
per lane.) Recombinant human P110-.alpha., -.beta., -.gamma.,
-.delta. proteins were detected by Immunoblot analysis. Levels of
P110.delta. were measured in MM1S and LB cells using P110.delta.
specific FITC conjugated secondary antibodies. P110.delta. stained
green, and nucleic acids (DAPI) stained blue.
[0389] Western blotting revealed no correlation of between
p110.delta. expression and expression of the other isoforms
(.alpha., .beta. and .gamma.). Importantly, all patient MM cells
also expressed p110.delta., (FIG. 30B).
Example 29
Cytotoxicity of Compound I on MM Cells
[0390] This example demonstrates that compound I has selective
cytotoxicity against cells with p110.delta.. Specifically, compound
I potently induced cytotoxicity in p110.delta. positive MM cells as
well as in primary patient MM cells without cytotoxicity in
peripheral blood mononuclear cells from healthy donors, suggesting
a favorable therapeutic index.
[0391] The growth inhibitory effect of p110.delta. knockdown in MM
cells was evaluated. LB and INA-6 cells were transfected with
P110.delta. siRNA (Si) or control siRNA (Mock). After 24 h,
expression of P110.delta. was determined by western blot analysis,
see FIG. 31A. INA-6 cells were transfected with p110.delta. siRNA
or control siRNA, and then cultured for 72 hours. Cell growth was
assessed by MTT assay, see FIG. 31B. Data indicates mean.+-.SD of
triplicate cultures, expressed as fold of control. Transfection
with p110.delta. siRNA, but not mock siRNA, down-regulated
p110.delta. and inhibited MM cell growth at 72 h (FIGS. 31A and
31B). The growth inhibitory effect of p110.delta. specific small
molecule inhibitor compound I in MM cell lines, PBMCs, and patient
MM cells was evaluated.
[0392] Compound I induced cytotoxicity against LB and INA-6 MM
cells (p110.delta.-positive) in a dose- and time-dependent fashion;
in contrast, minimal cytotoxicity was noted in p110.delta.-negative
cell lines (FIG. 31C). The legend for FIG. 31C: LB (.quadrature.),
INA-6 (.DELTA.), RPMI 8226 (.largecircle.), OPM2 (.diamond.), H929
( ), U266 (.diamond-solid.), RPMI-LR5 (.tangle-solidup.) and OPM1
(.box-solid.) MM cells were cultured with or without compound I for
48 h.
[0393] Importantly, compound I also induced cytotoxicity against
patient MM cells (FIG. 31D), without cytotoxicity in PBMCs from 4
healthy volunteers at concentrations up to 20 .mu.M (FIG. 31E).
Patients MM cells isolated from BM by negative selection were
cultured with compound I for 48 h. Peripheral blood mononuclear
cells isolated from healthy donors were cultured with compound I
for 72 h. Data represent mean.+-.SD viability, assessed by MTT
assay of triplicate cultures, expressed as percentage of untreated
controls. These results strongly suggest that sensitivity to
compound I is associated with P110.delta. expression, and suggest a
favorable therapeutic window.
[0394] To determine whether the cytotoxicity induced by compound I
is via apoptosis, the cleavage of caspases and PARP by western blot
analysis was examined. INA-6 cells were cultured with compound I
(0-5 .mu.M) for 120 h. Total cell lysates were subjected to
immunoblotting using anti-caspase-3, -8, -9, PARP, and
.alpha.-tubulin Abs. FL indicates full-length protein, and CL
indicates cleaved protein. Significantly increased cleavage of
caspase-8, caspase-9, caspase-3, and PARP was observed in INA-6 MM
cells treated with compound I for 120 h (FIG. 31F). These results
indicate that cytotoxicity triggered by compound I is mediated, at
least in part, via caspase-dependent (both intrinsic and extrinsic)
apoptosis.
Example 30
Inhibition of AKT and ERK Phosphorylation by Compound I
[0395] This example demonstrates the Inhibition of AKT and ERK
phosphorylation by compound I.
[0396] An important downstream effector of PI3K is the
serine/threonine protein kinase AKT, which is activated by
phosphorylation of Thr308 in the activation loop of the kinase
domain and Ser473 in the C-terminal tail. Phosphorylation of both
sites requires an interaction between the N-terminal pleckstrin
homology domain of AKT and membrane phosphoinositide generated by
PI3K. It was shown that compound I inhibits both domains,
suggesting that P110.delta. is the predominant isoform responsible
for PI3K signaling in MM cell lines.
[0397] Inhibition of AKT and ERK pathways in INA-6 cells by
compound I was examined. INA-6 cells were cultured with Compound I
or LY294002 for 12 h, FIG. 32A. Actin Ab was used as a loading
control. INA-6 and MM.1S cells were cultured with Compound I (0,
0.25, 1.0, 5.0 .mu.M) for 6 hours, FIG. 32B. LB and INA-6 cells
were cultured with compound I for 0-6 hours, FIG. 32C. Whole cell
lysates were subjected to immunoblotting using AKT, P-AKT (Ser473
and Thr308), ERK1/2, P-ERK1/2, P-PDK1, and P-FKRHL antibodies.
.alpha.-tubulin is used as a loading control.
[0398] Compound I significantly blocked phosphorylation of AKT and
ERK1/2 in p110.delta. positive INA-6 cells (FIG. 32A), but did not
affect phosphorylation of AKT or ERK in MM.1S cells with low
expression of P110.delta. (FIG. 32B). Compound I also significantly
inhibited phosphorylation of upstream PDK-1 and downstream FKHRL in
INA-6 and LB MM cells in a time- and dose-dependent fashion (FIG.
32C), further confirming inhibition of a both PI3K/AKT and ERK
pathways in these cells.
Example 31
Compound I Induces AVO Development and Autophagy
[0399] This example demonstrates the ability of compound Ito
trigger both apoptosis and autophagy.
[0400] AKT regulates autophagy, thus investigation of compound I in
inducing autophagy in LB and INA-6 MM cells was carried out.
[0401] INA-6 and LB MM cells were treated with 5 .mu.M Compound I
for 6 h. Compound I treatment induced LC3 accumulation in LB and
INA-6 cells, evidenced by fluorescence microscopy or transmission
electron microscopy. Autophagosome formation was defined by the
accumulation of LC3; arrows indicate autophagosomes, FIG. 33A.
[0402] INA-6 cells were treated with 5 .mu.M Compound I or serum
starvation for 6 h, stained with 1 .mu.g/mL acridine orange for 15
min, and analyzed by fluorescence microscopy, FIG. 33B.
[0403] LC3 and beclin-1 protein levels were determined by western
blotting using LC3 and beclin-1 antibodies of lysates from INA-6
cells treated with Compound I, with or without 3-MA, FIG. 33C.
GAPDH served as a loading control.
[0404] Immunofluorescence analysis showed markedly increased LC 3
staining in INA-6 and LB cells triggered by compound I (5 .mu.M, 6
h) treatment (FIG. 33A). Electron microscopic analysis also showed
increased autophagic vacuoles (arrows) in MM cells treated with
compound I. Since autophagy is characterized as acidic vesicular
organelle (AVO) development, acridine orange staining was carried
out. As shown in FIG. 33B, vital staining with acridine orange
revealed development of AVOs in compound I-treated LB and INA-6
cells. Moreover, markedly increased LC3-II and Beclin 1 protein
were detected in INA-6 MM cells after 6 h treatment with compound
I, which was blocked by 3-MA autophagic inhibitor (FIG. 33C).
[0405] No cytotoxicity in INA-6 and LB cells was induced by 3-MA at
concentrations up to 100 .mu.M, FIG. 33D. P110.delta. positive LB
cells (.diamond-solid.) were treated with 3-MA (0-100 .mu.M) for 24
h. Data represent means (.+-.SD) of triplicate cultures.
[0406] These results indicate that compound I induces development
of AVOs and autophagy at earlier time points than induction of
caspase/PARP cleavage.
[0407] Autophagy degrades cellular components, recycles cellular
constituents, and responds to various cellular stress. In this
example, LC3-II, a hallmark of autophagy, is induced by compound I
treatment in p110.delta. positive MM cell lines. Importantly,
compound I treatment resulted in a marked increase in autophagy,
evidenced by the presence of autophagic vacuoles in the cytoplasm,
formation of AVOs, membrane association of microtubule-associated
protein I of LC3 with autophagosomes, and a marked induction of
LC3-II protein. Electron microscopic analysis confirmed that
compound I induced autophagosomes. LC3-II was expressed through
LC3-I conversion. Conversely, autophagy induced by compound I was
suppressed by 3-MA, a specific inhibitor of autophagy. These
studies suggest that early cytotoxic effects of compound I are
associated with autophagy.
Example 32
Compound I Inhibits Cell Growth in the Presence of BMSC
[0408] This example demonstrates the ability of compound Ito
inhibit paracrine MM cell growth with BMSCs.
[0409] Since IL-6 and IGF-1 induces growth and anti-apoptosis in MM
cells, compound I was examined in overcoming the effects of these
cytokines in INA-6 and LB MM cells. LB and INA-6 cells were
cultured for 48 h with control media (.box-solid.); or with
compound I at 5.0 .mu.M (.box-solid.) or 10 .mu.M (.quadrature.),
in the presence or absence of IL-6 (1 and 10 ng/ml), FIG. 34A, or
IGF-1 (10 and 100 ng/mL), FIG. 34B. DNA synthesis was determined by
measuring [3H]-thymidine incorporation during the last 8 h of 72 h
cultures. Data represent means (.+-.SD) of triplicate cultures.
Neither IL-6 nor IGF-1 protected against the growth inhibition
induced by compound I (FIGS. 34A and 34B).
[0410] The BM microenvironment confers proliferation and
drug-resistance in MM, thus MM cell growth inhibitory effect of
compound I in the presence of BMSCs was examined.
[0411] LB and INA-6 MM cells were cultured for 48 h with control
media (.quadrature.), and with 2.5 .mu.M (), 5 .mu.M (.box-solid.),
and 10 .mu.M (.box-solid.) of Compound I, in the presence or
absence of BMSCs, FIG. 34C. DNA synthesis was determined by
[3H]-thymidine incorporation. Data represent means (.+-.SD) of
triplicate cultures.
[0412] IL-6 in culture supernatants from BMSCs treated with
compound I (0-2.5 .mu.M) was measured by ELISA, FIG. 34D. Error
bars indicate SD (.+-.).
[0413] BMSCs were cultured with 1.0 .mu.M compound I or control
media for 48 h; cytokines in culture supernatants were detected
using cytokine arrays, FIG. 34E.
[0414] INA-6 cells cultured with or without BMSCs were treated with
compound for 48 h. Total cell lysates were subjected to
immunoblotting using indicated antibodies, FIG. 34F. Actin was used
as a loading control.
[0415] BMSCs from 2 different patients (.quadrature., .diamond.)
were cultured with compound I (0-20 .mu.M) for 48 h. Cell viability
was assessed by MTT assay, FIG. 34G. Values represent mean.+-.SD of
triplicate cultures.
[0416] Importantly, compound I inhibited growth and cytokine
secretion (FIG. 34C-E), as well as phosphorylation of AKT and ERK
(FIG. 34F), induced by BMSCs. In contrast, no significant growth
inhibition in BMSCs was noted (FIG. 34G). These results indicate
that compound I blocks paracrine MM cell growth in the context of
the BM microenvironment.
Example 33
Compound I Inhibits Angiogenic HuVEC Tubule Formation
[0417] This example demonstrates the ability of Compound Ito
inhibit HuVEC tubule formation. The role of PI3K, specifically p110
isoform, in angiogenesis was investigated. Endothelial cells are an
essential regulator of angiogenesis for tumor growth. Both Akt and
ERK pathways are associated with endothelial cell growth and
regulation of angiogenesis; and importantly, endothelial cells
express p110.delta.. This example also demonstrates that compound I
blocks in vitro capillary-like tube formation, associated with down
regulation of Akt phosphorylation.
[0418] The effect of P110.delta. inhibition on angiogenesis was
investigated. HuVECs were treated with 0, 1.0, or 10 .mu.M of
compound I for 8 h, and tube formation by endothelial cells was
evaluated (FIG. 35A). HuVEC cells were plated on Matrigel-coated
surfaces and allowed to form tubules for 8 h, in the presence or
absence of Compound I. Endothelial cell tube formation was measured
by microscopic analysis, FIG. 35B. *P<0.005.
[0419] HuVECs were cultured with Compound I (0-20 .mu.M) 48 h, and
viability was assessed by MIT assay, FIG. 35C. Data shown are
mean.+-.SE of triplicate wells from a representative experiment.
Thus, compound I inhibited capillary-like tube formation in a
dose-dependent fashion (p<0.05) (FIG. 35B), without associated
cytotoxicity (FIG. 35C).
[0420] Phosphorylation and expression of AKT and ERK1/2 was
markedly down regulated in HuVEC cells by compound I treatment.
HuVECs were cultured with compound I (0-200 .mu.M) for 8 h, and
cell lysates were analyzed by immunoblotting using the indicated
antibodies, FIG. 35D. Actin was used as a loading control.
[0421] These findings suggest that compound I can inhibit
angiogenesis, associated with down regulation of AKT and ERK
activity.
Example 34
Compound II Inhibits MM Cell Growth In Vivo
[0422] This example demonstrates the ability of compound II to
inhibit human MM cell growth in vivo.
[0423] The in vivo efficacy of P110.delta. inhibitor was evaluated
in a xenograft model in which SCID mice are injected subcutaneously
with human MM cells.
[0424] Mice injected with 5.times.10.sup.6 LB cells were treated
orally twice a day with control vehicle ( ), and compound II 10
mg/kg (.quadrature.) or 30 mg/kg (.largecircle.). Mean tumor volume
was calculated as in Materials and Methods, FIG. 36A. Error bars
represent SD (.+-.).
[0425] Representative whole-body images from a mouse treated for 12
d with control vehicle (top panel) or Compound II (30 mg/kg)
(bottom panel), FIG. 36B.
[0426] Tumors harvested from Compound II (30 mg/kg) treated mouse
(right panel) and control mouse (left panel) were subjected to
immuno-histochemistric analysis using CD31 and P-AKT Abs. CD31 and
P-AKT positive cells are dark brown, FIG. 36D.
[0427] Mice were treated with Compound II 10 mg/kg (- -), 30 mg/kg
( . . . ) or Control vehicle (-). Survival was evaluated from the
first day of treatment until sacrifice using Kaplan-Meier curves,
FIG. 36C.
[0428] Tumor tissues were harvested from mice treated with control
vehicle or Compound II (30 mg/kg). Protein levels of phosphorylated
of PDK-1 and AKT (Ser473) were determined by western blotting of
cell lysates, FIG. 36E. Actin was used as a loading control.
[0429] Growth of INA-6 cells engrafted in human bone chips in SCID
mice was monitored by serial serum measurements of shuIL-6R. Mice
were treated with Compound II 10 mg/kg (.quadrature.), 30 mg/kg
(.DELTA.) or control vehicle ( ), and shuIL-6R levels were
determined weekly by ELISA, FIG. 36F. Error bars indicate SD
(.+-.).
[0430] Compound II (p110.delta. inhibitor) significantly reduced MM
tumor growth in the treatment group (n=7) compared with control
mice (n=7). Comparison of tumor volumes showed statistically
significant differences between control versus treatment groups (vs
10 mg/kg, P<0.05; vs 30 mg/kg, P<0.01) (FIG. 36A). Marked
decrease in tumor growth in treated versus in control mice was
observed at day 12. (FIG. 36B) Kaplan-Meier curves and log-rank
analysis showed a mean Overall Survival (OS) of 15 days (95%
confidence interval, 12-17 days) in control mice versus 23 days
(95% CI, 15-34 days) and 32 days (95% CI, 27-49 days) in the 10
mg/kg and 30 mg/kg compound II treated groups, respectively.
Statistically significant prolongation in mean OS compared with
control mice was also observed in treatment groups (vs 10 mg/kg,
P=0.086; vs 30 mg/kg, P=0.056) (FIG. 36C). Importantly, treatment
with either the vehicle alone or compound II did not affect body
weight. In addition, immunohistochemical (FIG. 36D) and immunoblot
(FIG. 36E) analysis confirmed that compound II treatment (30 mg/kg)
significantly inhibited p-Akt and p-PDK-1, as well as significantly
decreased CD31 positive cells and microvessel density (p<0.01)
(FIG. 36D). This suggests that compound II can inhibit angiogenesis
in vivo via suppression of the Akt pathway.
[0431] In order to examine the activity of compound II on MM cell
growth in the context of the human BM microenvironment in vivo, a
SCID-hu model was used in which IL-6 dependent INA-6 cells are
directly injected into a human bone chip implanted subcutaneously
in SCID-mice. This model recapitulates the human BM
microenvironment with human IL-6/BMSC-dependent growth of INA-6
human MM cells. These SCID-hu mice were treated with compound II or
vehicle alone daily for 4 weeks, and serum shuIL-6R monitored as a
marker tumor burden. As shown in FIG. 36F, compound II treatment
significantly inhibited tumor growth compared with vehicle control.
Significant tumor growth inhibition in this model was observed,
evidenced by decreased serum shuIL-6R levels released by INA-6
cells, confirming that p110.delta. inhibition blocks the MM growth
promoting activity of the BM microenvironment in vivo. Taken
together, these data demonstrate that inhibition of p110.delta. by
compound II significantly inhibits MM growth in vivo and prolongs
survival.
Example 35
Compound I in Combination with Bortezomib Exhibits Synergistic
Cytotoxicity
[0432] This example demonstrates the effect of Compound I in
combination with bortezomib to mediate synergistic MM
cytotoxicity.
[0433] The effects of combining compound I with bortezomib in
inducing synergistic MM cytotoxicity was investigated. LB and INA-6
MM cells were cultured with medium (.box-solid.) and with compound
I, 1.25 .mu.M (.box-solid.), 2.5 .mu.M () or 5.0 .mu.M
(.quadrature.), in the presence or absence of bortezomib (0-5 nM).
Cytotoxicity was assessed by MTT assay; data represent the
mean.+-.SD of quadruplicate-cultures, FIG. 37A.
[0434] INA-6 cells were treated with Compound I (5 .mu.M) and/or
bortezomib (5 nM) for 6 h. Phosphorylation of AKT was determined by
western blotting of cell lysates using phospho-AKT (ser473)
antibody, FIG. 37B. Actin served as a loading control.
[0435] Compound I enhances cytotoxicity of bortezomib. Increasing
concentrations of compound I (1.5-5.0 .mu.M) added to bortezomib
(2.5, 5.0 nM) triggered synergistic cytotoxicity in LB and INA-6 MM
cells (FIG. 37A and Table 7). Importantly, induction of phospho-Akt
by bortezomib treatment was inhibited in the presence of compound I
(FIG. 37B).
TABLE-US-00011 TABLE 7 Combination index (CI) Bortezomib Compound I
(nM) (.mu.M) Fa CI LB 2.5 1.25 0.39 0.57 2.5 2.5 0.52 0.58 2.5 5
0.57 0.67 5 1.25 0.42 0.88 5 2.5 0.60 0.25 5 5 0.67 0.22 INA-6 2.5
1.25 0.49 0.31 2.5 2.5 0.58 0.48 2.5 5 0.69 0.54 5 1.25 0.56 0.73 5
2.5 0.66 0.42 5 5 0.75 0.31
Example 36
Compound I Effective in Follicular Lymphoma Cell Lines
[0436] This example provides evidence that compound I blocks PI3K
signaling and induces apoptosis in follicular lymphoma cells.
P110.delta. is expressed in FL cell lines as shown in FIG. 38A.
Certain cell lines show reduction in the production of pAkt, Akt,
pS6 and S6 when the cell is exposed to compound I, FIG. 38B.
Cleavage of PARP and Caspase-3 is observed after exposure to
compound I in a dose dependent fashion after 24 hours at 0.1 .mu.M
and 0.5 .mu.M, FIG. 38C.
Example 37
Compound I Effective in Primary MCL Cells
[0437] This example demonstrates that compound I is effective
against MCL. Compound I was found to block constitutive PI3K
signaling in primary MCL cells of two patients in a dose dependent
manner when exposed to 0.1 .mu.M or 1 .mu.M of compound I, FIG.
39A. Compound I is also observed to inhibit survival factor and
chemokine signaling in MCL cell lines. FIG. 39B shows a significant
reduction of pAkt in MCL lines exposed to different survival
factors in the presence of compound I.
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