U.S. patent application number 17/685661 was filed with the patent office on 2022-06-16 for use of cyp26-resistant rar alpha selective agonists in the treatment of cancer.
The applicant listed for this patent is Io Therapeutics, Inc., The Johns Hopkins University. Invention is credited to Salvador Alonso, Roshantha A. Chandraratna, Gabriel Ghiaur, Richard J. Jones.
Application Number | 20220184013 17/685661 |
Document ID | / |
Family ID | |
Filed Date | 2022-06-16 |
United States Patent
Application |
20220184013 |
Kind Code |
A1 |
Ghiaur; Gabriel ; et
al. |
June 16, 2022 |
USE OF CYP26-RESISTANT RAR ALPHA SELECTIVE AGONISTS IN THE
TREATMENT OF CANCER
Abstract
Disclosed herein are methods for treating a cancer comprising
administering to a subject in need thereof an effective dose of a
CYP26-resistant retinoic acid receptor (RAR) alpha (RAR.alpha.)
selective agonist, whereby as a result of the treatment the tumor
burden is reduced in the subject and cancer stem cells resident in
the bone marrow are substantially reduced.
Inventors: |
Ghiaur; Gabriel; (Baltimore,
MD) ; Jones; Richard J.; (Baltimore, MD) ;
Alonso; Salvador; (Baltimore, MD) ; Chandraratna;
Roshantha A.; (Spring, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Io Therapeutics, Inc.
The Johns Hopkins University |
Spring
Baltimore |
TX
MD |
US
US |
|
|
Appl. No.: |
17/685661 |
Filed: |
March 3, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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17136428 |
Dec 29, 2020 |
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17685661 |
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15777868 |
May 21, 2018 |
10940127 |
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PCT/US16/63659 |
Nov 23, 2016 |
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17136428 |
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62260098 |
Nov 25, 2015 |
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International
Class: |
A61K 31/196 20060101
A61K031/196; A61K 45/06 20060101 A61K045/06; A61K 31/192 20060101
A61K031/192; A61K 31/4166 20060101 A61K031/4166; A61K 31/454
20060101 A61K031/454; A61K 31/4196 20060101 A61K031/4196; A61K
31/138 20060101 A61K031/138; A61K 31/58 20060101 A61K031/58; A61K
31/4545 20060101 A61K031/4545; A61K 31/5685 20060101 A61K031/5685;
A61K 31/277 20060101 A61K031/277; A61P 35/00 20060101 A61P035/00;
A61K 31/69 20060101 A61K031/69 |
Goverment Interests
STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH
[0002] This invention was made with government support under
HL127269 and CA015396 awarded by the National Institutes of Health.
The government has certain rights in the invention.
Claims
1. A method for treating multiple myeloma consisting of
administering to a subject in need thereof an effective dose of a
CYP26-resistant retinoic acid receptor alpha (RAR.alpha.) selective
agonist and a proteasome inhibitor, wherein the CYP26-resistant
RAR.alpha. selective agonist is a selected from: ##STR00006## and
optionally, at least one additional anti-cancer agent selected from
etoposide, an anthracycline, idarubicin, daunorubicin,
mitoxantrone, cytarabine, a combination of an anthracycline,
cytarabine and etoposide, a demethylating agent, 5-azacytidine,
decitabine, a tyrosine kinase inhibitor, a BCR-ABL inhibitor, a
Flt3 inhibitor, a cKit inhibitor, an IDH1/2 inhibitor, a JAK2
inhibitor, a BTK inhibitor, an immunotherapeutic agent, an
anti-CD33 monoclonal antibody (mAb), an anti-CD20 mAb, an anti-CD19
mAb, an anti-CD30 mAb, an anti-PD1 mAb, an anti-CTL4 mAb,
lenalidomide, pomalidomide, cyclophosphamide, bevacizumab,
vincristine, a corticosteroid, bleomycin, adriamycin, bendamustin,
fludarabine, G-CSF, GM-CSF, EPO, and combinations thereof, whereby
as a result of the treatment the tumor burden, including multiple
myeloma (MM) B cell burden, is reduced in the subject.
2. The method according to claim 1, wherein the CYP26-resistant
RAR.alpha. selective agonist is tamibarotene (AM80).
3. The method according to claim 1, wherein the CYP26-resistant
RAR.alpha. selective agonist is AM580.
4. The method according to claim 1, wherein the CYP26-resistant
RAR.alpha. selective agonist is Re-80.
5. The method according to claim 1, wherein administration of an
effective dose of the CYP26-resistant RAR.alpha. selective agonist
and the proteasome inhibitor results in the elimination of minimal
residual disease or cancer stem cells from the bone marrow niche of
the subject, thereby improving the disease-free survival of the
subject.
6. The method according to claim 1, wherein administration of the
effective dose of the CYP26-resistant RAR.alpha. selective agonist
results in sensitization of minimal residual disease to the at
least one additional anticancer agent, whereby combination of the
CYP26-resistant RAR.alpha. selective agonist with the at least one
additional anticancer agent results in improvement of the
disease-free survival of the subject.
7. The method according to claim 1, whereby, as a result of the
treatment, reduction of tumor burden is greater than the additive
effect of the CYP26-resistant RAR.alpha. selective agonist and the
proteasome inhibitor acting alone.
8. The method according to claim 1, wherein the proteasome
inhibitor is bortezomib.
9. The method according to claim 1, wherein the subject has minimal
residual disease.
10. A method of sensitizing multiple myeloma cells in bone marrow
to treatment with a proteasome inhibitor, consisting of
administering to a subject in need thereof an effective dose of a
CYP26-resistant RAR.alpha. selective agonist, wherein the
CYP26-resistant RAR.alpha. selective agonist is a selected from:
##STR00007## and wherein the subject in need thereof is receiving
treatment for multiple myeloma with a proteasome inhibitor, and
optionally, at least one additional anti-cancer agent selected from
etoposide, an anthracycline, idarubicin, daunorubicin,
mitoxantrone, cytarabine, a combination of an anthracycline,
cytarabine and etoposide, a demethylating agent, 5-azacytidine,
decitabine, a tyrosine kinase inhibitor, a BCR-ABL inhibitor, a
Flt3 inhibitor, a cKit inhibitor, an IDH1/2 inhibitor, a JAK2
inhibitor, a BTK inhibitor, an immunotherapeutic agent, an
anti-CD33 monoclonal antibody (mAb), an anti-CD20 mAb, an anti-CD19
mAb, an anti-CD30 mAb, an anti-PD1 mAb, an anti-CTL4 mAb,
lenalidomide, pomalidomide, cyclophosphamide, bevacizumab,
vincristine, a corticosteroid, bleomycin, adriamycin, bendamustin,
fludarabine, G-CSF, GM-CSF, EPO, and combinations thereof.
11. The method according to claim 10, wherein the CYP26-resistant
RAR.alpha. selective agonist is tamibarotene (AM80).
12. The method according to claim 10, wherein the CYP26-resistant
RAR.alpha. selective agonist is AM580.
13. The method according to claim 10, wherein the CYP26-resistant
RAR.alpha. selective agonist is Re-80.
14. The method according to claim 10, wherein administration of an
effective dose of the CYP26-resistant RAR.alpha. selective agonist
while receiving the proteasome inhibitor results in the elimination
of minimal residual disease or cancer stem cells from the bone
marrow niche of the subject, thereby improving the disease-free
survival of the subject.
15. The method according to claim 10, wherein administration of an
effective dose of the CYP26-resistant RAR.alpha. selective agonist
while receiving the proteasome inhibitor and the at least one
additional anti-cancer agent, results in sensitization of minimal
residual disease to the at least one additional anticancer agent,
whereby combination of the CYP26-resistant RAR.alpha. selective
agonist with the at least one additional anticancer agent results
in improvement of the disease-free survival of the subject.
16. The method according to claim 10, wherein the proteasome
inhibitor is bortezomib.
17. The method according to claim 10, wherein the subject has
minimal residual disease.
18. The method according to claim 10, whereby, as a result of
treatment, reduction of tumor burden is greater than the additive
effect of the CYP26-resistant RAR.alpha. selective agonist and the
proteasome inhibitor acting alone.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 17/136,428, filed Dec. 29, 2020, which is a divisional of
U.S. patent application Ser. No. 15/777,868, filed May 21, 2018,
now U.S. Pat. No. 10,940,127 issued Mar. 9, 2021, which is a 35
U.S.C. 371 national phase entry of PCT/US2016/063659, filed Nov.
23, 2016, which claims the benefit of U.S. provisional patent
application 62/260,098 filed Nov. 25, 2015; the entire contents of
all of which are each incorporated by reference herein.
FIELD
[0003] Disclosed herein are methods of treating cancer with
CYP26-resistant retinoic acid receptor alpha (RAR.alpha.) selective
agonists.
BACKGROUND
[0004] Normal hematopoietic stem cells (HSCs) are primed to be
highly sensitive to retinoids but are maintained in a retinoid
signaling-naive state by isolating them from physiologic levels of
retinoids. The bone marrow microenvironment, by expression of the
enzyme CYP26 metabolically inactivates retinoic acid, regulates the
exposure of the bone marrow to retinoids. This mechanism
(CPY26-mediated retinoid metabolism) is dynamic and used by the
bone marrow stroma to match HSC behavior to physiological needs.
For example, steady state low levels of retinoids in the bone
marrow niche maintains HSCs in a quiescent state, while during
situations of stress (i.e., exposure to radiation or chemotherapy)
higher retinoid levels are maintained to recruit HSCs into cell
division and rescue hematopoiesis.
[0005] In subjects with hematologic malignancies, cancer HSCs are
protected from retinoids by stromal CYP26, in a similar fashion to
the normal situation. However, because of other alterations in the
bone marrow niche in hematologic malignancies, such as differences
in aldehyde dehydrogenase (ALDH) activity, there exists a
therapeutic window for retinoids to be useful in the treatment of
hematologic malignancies. Expression of CYP26 by the bone marrow
microenvironment contributes to the protection of immature acute
myeloid leukemia (AML) cells from all-trans retinoic acid (ATRA)
and may explain why ATRA is not effective in treating AML. Exposure
to pharmacological concentrations of ATRA acting through retinoic
acid receptor gamma (RAR.gamma.), induces CYP26 expression in the
bone marrow microenvironment, thus protecting the cancer stem cells
therein from retinoid activity. However, the use of retinoid
analogs which are not inactivated by CYP26 enables such retinoids
to terminally differentiate and thus eliminate the cancer HSCs from
the protective bone marrow niche. Since such differentiation is
mediated by RAR.alpha., and the use of RAR.alpha. specific analogs,
which are CYP26 resistant, enables the therapeutic
differentiation-inducing activity without inactivation by the CYP26
enzyme.
SUMMARY
[0006] Disclosed herein are methods of treating cancer with
CYP26-resistant, retinoic acid receptor (RAR) alpha (RAR.alpha.)
selective agonists and their use in the treatment of malignancies
by acting upon cancer stem cells resident in the bone marrow.
[0007] Thus, provided herein are methods for treating a hematologic
malignancy comprising administering to a subject in need thereof an
effective dose of a CYP26-resistant RAR.alpha. selective agonist,
whereby as a result of the treatment the tumor burden is reduced in
the subject.
[0008] In some embodiments, administration of an effective dose of
the CYP26-resistant RAR.alpha. selective agonist results in the
elimination of minimal residual disease or cancer stem cells from
the bone marrow niche of the subject, thereby rendering the subject
substantially free of cancer. In certain embodiments,
administration of an effective dose of the CYP26-resistant
RAR.alpha. selective agonist results in sensitization of minimal
residual disease to other anticancer agents, whereby combination of
the CYP26-resistant RAR.alpha. selective agonist with other
anticancer agents results in elimination of minimal residual
disease or cancer stem cells from the bone marrow niche of the
subject, thereby rendering the subject substantially free of
cancer.
[0009] In certain embodiments, the cancer stem cell is a
hematologic cancer stem cell (HSC). In some embodiments, the
hematologic malignancy is acute myeloid leukemia (AML), chronic
myelogenous leukemia (CML), accelerated CML, CML blast phase
(CML-BP), acute lymphoblastic leukemia (ALL), chronic lymphocytic
leukemia (CLL), Hodgkin's disease (HD), non-Hodgkin's lymphoma
(NHL), follicular lymphoma, mantle cell lymphoma, B-cell lymphoma,
T-cell lymphoma, multiple myeloma (MM), Waldenstrom's
macroglobulinemia, a myelodysplastic syndrome (MDS), refractory
anemia (RA), refractory anemia with ringed siderblasts (RARS),
refractory anemia with excess blasts (RAEB), RAEB in transformation
(RAEB-T), or a myeloproliferative syndrome.
[0010] In certain embodiments, the CYP26-resistant RAR.alpha.
selective agonist is
##STR00001##
[0011] In other embodiments, the CYP26-resistant RAR.alpha.
selective agonist is tamibarotene (AM80), AM580, or Re 80.
##STR00002##
[0012] In some embodiments, as a result of the administration, the
subject remains in remission longer than 5 years.
[0013] Also disclosed herein are methods for treating a solid tumor
malignancy comprising administering to a subject in need thereof an
effective dose of a CYP26-resistant RAR.alpha. selective agonist,
and at least one additional anti-cancer agent, whereby as a result
of the treatment, the tumor burden is reduced in the subject.
[0014] In certain embodiments, administration of an effective dose
of a RAR.alpha. agonist which is not metabolized by CYP26 results
in the elimination of minimal residual disease or cancer stem cells
in the bone marrow niche of the subject, thereby rendering the
subject substantially free of cancer.
[0015] In some embodiments, the solid tumor malignancy is a type of
cancer which typically metastasizes to the bone marrow. In some
embodiments, the additional anti-cancer agent is an agent listed in
Table 1. In certain embodiments, the solid tumor malignancy is
pancreatic cancer, bladder cancer, colorectal cancer, breast
cancer, prostate cancer, renal cancer, hepatocellular cancer, lung
cancer, ovarian cancer, cervical cancer, gastric cancer, esophageal
cancer, head and neck cancer, melanoma, neuroendocrine cancer,
brain cancer, bone cancer, or soft tissue sarcoma.
[0016] In some embodiments, the additional anti-cancer agent is
selected from the combinations in Table 1. In certain embodiments,
the additional anti-cancer agent is trastuzumab, tamoxifen,
anastrazole, exemestrane, letrozole, crizotinib, aberatrone,
enzalutamide, bicalutemide, bortezomib, or thalidomide.
[0017] In some embodiments, as a result of the administration, the
subject remains in remission longer than 1 year, longer than 2
years, longer than 3 years, longer than 4 years, or longer than 5
years.
[0018] In some embodiments, as a result of the administration, the
subject remains in remission for at least 1 year (e.g., 1-2 years,
1-3 years, 1-4 years, 1-5 years, 2-3 years, 2-4 years, 2-5 years,
3-4 years, 3-5 years, or 4-5 years).
[0019] In some embodiments, as a result of the administration, the
subject remains in remission for 1-2 years, 1-3 years, 1-4 years,
or 1-5 years.
[0020] Also disclosed herein are methods for treating cancer,
comprising administering to a subject in need thereof a
CYP26-resistant retinoic acid receptor alpha (RAR.alpha.) selective
agonist and bortezomib.
[0021] Also disclosed herein are methods for treating cancer,
comprising administering to a subject in need thereof an effective
dose of a CYP26-resistant retinoic acid receptor alpha (RAR.alpha.)
selective agonist and bortezomib.
[0022] Also disclosed herein are methods for treating cancer,
comprising administering to a subject in need thereof an effective
dose of a CYP26-resistant retinoic acid receptor alpha (RAR.alpha.)
selective agonist and bortezomib, whereby as a result of the
treatment the tumor burden is reduced in the subject.
[0023] Also disclosed herein are methods for treating multiple
myeloma comprising administering to a subject in need thereof an
effective dose of a CYP26-resistant retinoic acid receptor alpha
(RAR.alpha.) selective agonist and bortezomib, whereby as a result
of the treatment the tumor burden is reduced in the subject.
[0024] Also disclosed herein are methods for treating multiple
myeloma comprising administering to a subject in need thereof an
effective dose of a IRX5183 and bortezomib, whereby as a result of
the treatment the tumor burden is reduced in the subject.
[0025] Also disclosed herein are methods for treating cancer,
comprising administering to a subject in need thereof IRX5183 and
bortezomib.
[0026] Also disclosed herein are methods for treating cancer,
comprising administering to a subject in need thereof an effective
dose of a IRX5183 and bortezomib.
[0027] Also disclosed herein are methods for treating cancer,
comprising administering to a subject in need thereof an effective
dose of a IRX5183 and bortezomib, whereby as a result of the
treatment the tumor burden is reduced in the subject.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1A-H depicts the relative concentration of plasma
markers BCL6 (FIGS. 1A and E), BLIMP-1 (FIGS. 1B and F), XBPS-1
(FIGS. 1C and G), and CHOP (FIGS. 1D and H) and in multiple myeloma
(MM) cell lines H929 (FIG. 1A-D) or CD138+ MM cells from three
different patient samples (FIG. 1E-H) incubated for 5 days either
in the absence of stroma (Liquid), with or without AGN (RA receptor
antagonist AGN194310, 1 .mu.M), or cocultured with BM mesenchymal
cells (Stroma), with or without R115 (CYP26 inhibitor R115866, 1
.mu.M) or IRX (CYP26-resistant retinoid IRX5183, 1 .mu.M).
Expression in untreated liquid conditions was set at 1. Data are
representative of 3 independent experiments with similar results
and represent the mean.+-.SEM. *P.ltoreq.0.05 and **P.ltoreq.0.01,
by repeated-measures 1-way ANOVA for determination of statistical
significance between groups; P values were corrected for multiple
comparisons using Dunnett's test. Ctrl, control; max, maximum.
[0029] FIG. 2A-B depicts the clonogenic recovery (CFU) of H929
cells (FIG. 2A) or cellular recovery of primary CD138+ MM cells
from 3 different patient samples (FIG. 2B). MM cells were treated
with BTZ (2.5 nM) for 48 hours after being incubated for 5 days
either in the absence of stroma (Liquid), with or without the
pan-RAR inhibitor AGN (1 .mu.M), or in the presence of BM
mesenchymal cells (Stroma), with or without the CYP26 inhibitor
R115 (1 .mu.M) or the CYP26-resistant retinoid IRX (1 .mu.M).
Clonogenic or cellular recovery was normalized to each condition in
the absence of BTZ.
[0030] FIG. 3 depicts clonogenic recovery of H929 cells treated
with BTZ (2.5 nM). MM cells were incubated for 5 days in the
absence (Liquid) or presence of BM mesenchymal cells (Stroma), with
or without R115 (1 .mu.M). Following this preincubation, H929 cells
were separated from BM stroma, cultured in fresh media for 0 to 48
hours, and then treated with BTZ (2.5 nM) for 48 hours. Clonogenic
recovery was normalized to each condition in the absence of
BTZ.
[0031] FIG. 4 depicts bioluminescent images of systemic MM
xenografts. Following engraftment of H929 Luc+ cells, mice were
treated with IRX (n=4), BTZ (n=5), or a combination of both (n=5)
for 4 weeks. Data represent the mean.+-.SEM of the fold change in
bioluminescence (photons/second) from day 0.
[0032] FIG. 5A-C depicts the effects of MM cells on the expression
of CYP26A1 in BM stroma. Relative quantification of CYP26A1 mRNA in
human BM mesenchymal cells incubated for 24 hours either in the
absence (Ctrl) or presence (Coculture or Transwell) of MM cells
(H929 [FIG. 5A], MM1s [FIG. 5B], U266 [FIG. 5C]). Expression in
untreated BM stroma (Ctrl) was arbitrarily set at 1.
[0033] FIG. 6A-C depicts the relative quantification of CYP26A1
mRNA in mouse wild type (WT) or Smo-KO BM stroma incubated for 24
hr in the absence (Ctrl) or presence (Coculture or Transwell) of MM
cells (H292, MM1s, U266). Expression in untreated WT or Smo-KO
stroma was arbitrarily set at 1 for the respective treated
conditions. Data represent the mean.+-.SEM of 3 independent
experiments. *P.ltoreq.0.05 and **P.ltoreq.0.01, by unpaired,
2-tailed Student's t test.
[0034] FIG. 7 depicts bioluminescent images of mice showing tumor
burden during 4 weeks of treatment with IRX (10 mg/kg), BTZ (0.5
mg/kg), or the combination. Anterior tumors consisted of a
combination of MM1S luciferase.sup.+ cells and Smo.sup.FI/FI BM
stroma cells transduced with a control vector (WT BM stroma).
Posterior tumors consisted of a combination of MM1S luciferase+
cells and Smo.sup.FI/FI BM stroma cells transduced with
Cre-recombinase (Smo KO BM stroma).
[0035] FIG. 8 depicts the fold change in bioluminescence
(photons/second) of tumors during 4 weeks of treatment. The change
in bioluminescence for each tumor at day 1 was normalized to the
change in bioluminescence at day 14 and at the end of treatment
(day 28).
[0036] FIG. 9A-H depicts the relative quantification of BCL6 (B
cell marker), BLIMP, XBP1s, and CHOP (PC markers) in H929 cells
(FIG. 9A) and primary CD138+ MM cells (FIG. 9B) from 3 different
patient samples incubated for 5 days either in the absence of
stroma (Ctrl) or cocultured with WT or Smo-KO stromal cells.
Expression in untreated liquid conditions was set at 1. Data
represent the mean.+-.SEM. *P.ltoreq.0.05 and **P.ltoreq.0.01, by
repeated-measures 1-way ANOVA to determine statistical significance
between treatment groups; P values were corrected for multiple
comparisons using Dunnett's test.
[0037] FIG. 10A-C depicts stroma blockage of ATRA-mediated, but not
AM80- or IRX5183-induced, differentiation and elimination of AML.
(FIG. 10A) CFU experiments with NB4 cells treated with 10.sup.-7 M
ATRA, IRX5183, or 10.sup.-8 M AM80; (FIG. 10B) OCI-AML3 cells and
(FIG. 10C) Kasumi-1 cells treated with 10.sup.-6 M ATRA, IRX5183,
or 10.sup.-7 M AM80 showed a decrease in clonogenic growth compared
to control with AM80 and IRX5183 both off and on stroma. Data
across three independent experiments.
DETAILED DESCRIPTION
[0038] Disclosed herein are methods of treating cancer with
CYP26-resistant, retinoic acid receptor (RAR) alpha (RAR.alpha.)
selective agonists and their use in the treatment of malignancies
by acting upon cancer stem cells resident in the bone marrow.
[0039] Many, if not most, malignancies arise from a rare population
of cells that exclusively maintain the ability to self-renew and
sustain the tumor. These cancer stem cells are often biologically
distinct from the bulk of differentiated cancer cells that
characterize the disease. For example, chronic myeloid leukemia
(CML) occurs at the level of hematopoietic stem cells and, like
their normal counterparts, CML stem cells undergo orderly
differentiation. Thus, the bulk of the leukemic mass in CML
consists of differentiated blood cells, whereas the rare cells
responsible for disease maintenance resemble normal hematopoietic
stem cells. Similarly, in multiple myeloma, which is characterized
by neoplastic plasma cells, these cells appear to be terminally
differentiated like their normal counterparts. The myeloma plasma
cells that form the bulk of the tumor arise from a population of
less differentiated cancer stem cells that resemble post-germinal
center B cells. Other cancers, including but not limited to,
hematological malignancies, myelodysplastic syndrome, breast
cancer, prostate cancer, pancreatic cancer, colon cancer, ovarian
cancer, melanoma, non-melanoma skin cancers, and brain cancers have
been demonstrated to arise from corresponding cancer stem
cells.
[0040] Thus, disclosed herein are methods of treating cancer with
agents which can target cancer stem cells in the protected bone
marrow niche by inducing differentiation of the cancer stem cells
into mature cancer cells that are susceptible to standard
therapies. Administration of CYP26-resistant, RAR.alpha. selective
agonists which can act on the cancer stem cells in the bone marrow
niche (because they are not inactivated by CYP26) is one such
approach. In certain embodiments, effectiveness of therapy with a
RAR.alpha. selective agonist disclosed herein leads to a
substantial decrease in the number of cancer stem cells in the bone
marrow.
[0041] The cancer stem cells can be enumerated by various
mechanisms and reduction in their numbers as a result of
administration of a CYP26-resistant RAR.alpha. selective agonist
measured thereby. In embodiments disclosed herein, as a result of
administration of a RAR.alpha. selective agonist, the cancer stem
cells in the bone marrow are reduced by more than about 0.5 log,
more than about 1 log, more than about 1.5 log, more than about 2.0
log, more than about 2.5 log, more than about 3.0 log, more than
about 3.5 log, more than about 4.0 log, more than about 4.5 log, or
more than about 5.0 log.
[0042] Compounds with retinoid activity (vitamin A and its
derivatives) have activity in cell proliferation and
differentiation processes. Many biological effects of retinoids are
mediated by modulating the nuclear retinoic acid receptors (RARs).
The RARs activate transcription by binding to DNA sequence
elements, known as RAR response elements (RARE), in the form of a
heterodimer with one of the retinoid X receptors (known as RXRs).
Three subtypes of human RARs have been identified and described:
RAR.alpha., RAR.beta., and RAR.gamma..
[0043] As used herein, the term "RAR.alpha. agonist", is synonymous
with "RAR.alpha. selective agonist" and refers to a compound that
selectively binds RAR.alpha.. As used herein, the term "selectively
binds," when made in reference to a RAR.alpha. selective agonist,
refers to the discriminatory binding of a RAR.alpha. selective
agonist to the indicated target RAR.alpha. such that the RAR.alpha.
selective agonist does not substantially bind with non-target
receptors like a RAR.beta. or a RAR.gamma..
[0044] Selective binding of a RAR.alpha. selective agonist to a
RAR.alpha. includes binding properties such as, e.g., binding
affinity and binding specificity. Binding affinity refers to the
length of time a RAR.alpha. selective agonist resides at its a
RAR.alpha. binding site, and can be viewed as the strength with
which a RAR.alpha. selective agonist binds its a RAR.alpha..
Binding specificity is the ability of a RAR.alpha. selective
agonist to discriminate between a RAR.alpha. and a receptor that
does not contain its binding site, such as, e.g., a RAR.beta. or a
RAR.gamma.. One way to measure binding specificity is to compare
the association rate of a RAR.alpha. selective agonist for its
RAR.alpha. relative to the association rate of a RAR.alpha. agonist
for a receptor that does not contain its binding site; for example,
comparing the association rate constant of a RAR.alpha. selective
agonist for its RAR.alpha. relative to a RAR.beta. and/or a
RAR.gamma..
[0045] In some embodiments, a RAR.alpha. selective agonist will
have a ratio of activity at a RAR.alpha. relative to a RAR.beta.
and/or a RAR.gamma. of, e.g., at least 5 times greater, at least 10
times greater, at least 15 times greater, at least 20 times greater
or at least 10,000 times greater. A RAR pan agonist will have
activity at a RAR.alpha., a RAR.beta., and a RAR.gamma., i.e.,
similar potency at a RAR.alpha., a RAR.beta., and a RAR.gamma..
[0046] The binding specificity of a RAR.alpha. selective agonist
that selectively binds to a RAR.alpha. can also be characterized as
an activity ratio that such a RAR.alpha. selective agonist can
exert through binding to its RAR.alpha. relative to a receptor not
comprising its binding site, such as, e.g., a RAR.beta. or a
RAR.gamma.. In some embodiments, a RAR.alpha. selective agonist
that selectively binds to a RAR.alpha. has an activity ratio
through its RAR.alpha. relative to a receptor not comprising its
binding site of, e.g., at least 2:1, at least 3:1, at least 4:1, at
least 5:1, at least 64:1, at least 7:1, at least 8:1, at least 9:1,
at least 10:1, at least 15:1, at least 20:1, at least 25:1, at
least 30:1, at least 35:1, or at least 40:1. In some embodiments, a
RAR.alpha. selective agonist that selectively binds to a RAR.alpha.
has an activity ratio through its RAR.alpha. relative to a
RAR.beta. and/or a RAR.gamma. of, e.g., at least 2:1, at least 3:1,
at least 4:1, at least 5:1, at least 64:1, at least 7:1, at least
8:1, at least 9:1, at least 10:1, at least 15:1, at least 20:1, at
least 25:1, at least 30:1, at least 35:1, or at least 40:1. In some
embodiments, a RAR.alpha. selective agonist that selectively binds
to a RAR.alpha. has an activity ratio through its RAR.alpha.
relative to a receptor not comprising its binding site of, e.g., at
least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 6:1,
at least 7:1, at least 8:1, at least 9:1, at least 10:1, at least
15:1, at least 20:1, at least 25:1, at least 30:1, at least 35:1,
or at least 40:1. In some embodiments, a RAR.alpha. selective
agonist that selectively binds to a RAR.alpha. has an activity
ratio through its RAR.alpha. relative to a RAR.beta. and/or a
RAR.gamma. of, e.g., at least 2:1, at least 3:1, at least 4:1, at
least 5:1, at least 6:1, at least 7:1, at least 8:1, at least 9:1,
at least 10:1, at least 15:1, at least 20:1, at least 25:1, at
least 30:1, at least 35:1, or at least 40:1.
[0047] RAR.alpha. selective agonists useful in the methods
disclosed herein are RAR.alpha. selective agonists which are not
metabolized by CYP26. CYP26 is a cytochrome P450 monooxygenase that
metabolizes retinoic acid into inactive or less active substances
which can also be readily eliminated from cells and regulates
cellular levels of retinoic acid. RAR.alpha. selective agonists
that are readily metabolized by CYP26 are not within the scope of
the present methods.
[0048] In an aspect of this embodiment, a CYP26-resistant
RAR.alpha. selective agonist is a compound having the structure of
formula I,
##STR00003##
[0049] wherein R.sup.1 is H or C.sub.1-6 alkyl;
[0050] R.sup.2 and R.sup.3 are independently H or F; and
[0051] R.sup.4 is a halogen.
[0052] In some embodiments of formula I, halogen is F, CI, Br or I.
In some embodiments, of formula I, halogen is F. In some
embodiments, of formula I, halogen is Cl. In some embodiments, of
formula I, halogen is Br. In some embodiments, of formula I,
halogen is I.
[0053] In an aspect of this embodiment, a CYP26-resistant
RAR.alpha. selective agonist is a compound having a structure of
formula II
##STR00004##
[0054] wherein R.sup.1 is H or C.sub.1-16 alkyl.
[0055] In another aspect of this embodiment, a CYP26-resistant
RAR.alpha. selective agonist is the compound having the structure
of formula III
##STR00005##
[0056] In another embodiment, a CYP26-resistant RAR.alpha.
selective agonist is tamibarotene (AM80;
4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carbamoyl]benzo-
ic acid). In another embodiment, a CYP26-resistant RAR.alpha.
selective agonist is AM580
(4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido]be-
nzoic acid). In another embodiment, a CYP26-resistant RAR.alpha.
selective agonist is Re 80
(4-[1-hydroxy-3-oxo-3-(5,6,7,8-tetrahydro-3-hydroxy-5,5,8,8-tetramethyl-2-
-naphthalenyl)-1-propenyl]benzoic acid).
[0057] As used herein, the term "CYP26-resistant" refers to
RAR.alpha. selective agonists which are not metabolized, degraded,
or otherwise inactivated by the CYP26 enzyme and have activity
within the bone marrow.
[0058] Assays by which a compound can be tested and established
whether or not it is an RAR.alpha. selective agonist are described
in numerous prior art publications and patents. For example, a
chimeric receptor transactivation assay which tests for
agonist-like activity in the RAR.alpha., RAR.beta., RAR.gamma.,
RXR.alpha. receptor subtypes, is described in detail in U.S. Pat.
No. 5,455,265, which is hereby incorporated by reference for all it
discloses regarding receptor transactivation assays.
[0059] The compounds and pharmaceutical compositions disclosed
herein are particularly useful for the treatment of cancer. As used
herein, the term "cancer" refers to a cellular disorder
characterized by uncontrolled or disregulated cell proliferation,
decreased cellular differentiation, inappropriate ability to invade
surrounding tissue, and/or ability to establish new growth at
ectopic sites. The term "cancer" includes, but is not limited to,
solid tumors and hematologic tumors. The term "cancer" encompasses
diseases of skin, tissues, organs, bone, cartilage, blood, and
vessels. The term "cancer" further encompasses primary and
metastatic cancers. Included within the term "cancer" are cancer
stem cells.
[0060] In certain embodiments, the cancer is a hematologic
malignancy. Non-limiting examples of hematologic malignancy include
acute myeloid leukemia (AML), chronic myelogenous leukemia (CML),
including accelerated CML and CML blast phase (CML-BP), acute
lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL),
Hodgkin's disease (HD), non-Hodgkin's lymphoma (NHL), including
follicular lymphoma and mantle cell lymphoma, B-cell lymphoma,
T-cell lymphoma, multiple myeloma (MM), Waldenstrom's
macroglobulinemia, myelodysplastic syndromes (MDS), including
refractory anemia (RA), refractory anemia with ringed siderblasts
(RARS), (refractory anemia with excess blasts (RAEB), and RAEB in
transformation (RAEB-T), and myeloproliferative syndromes. In
certain embodiments, when the cancer is a hematologic malignancy,
the RAR.alpha. selective agonist could be administered either as a
stand-alone therapy in the absence of other anti-cancer treatments
or in combination with other therapies, including but not limited
to those listed below in Table 1.
[0061] In some embodiments, the cancer is a solid tumor. In other
embodiments, the cancer is a solid tumor which can metastasize to
the bone. Non-limiting examples of solid tumors that can be treated
by the disclosed methods include 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. In certain embodiments, when the
cancer is a solid tumor, the RAR.alpha. selective agonist is used
in combination with a cytotoxic agent or other anti-cancer
agent.
[0062] A compound disclosed herein, or a composition comprising
such a compound, is generally administered to an individual as a
pharmaceutical composition. Pharmaceutical compositions may be
prepared by combining a therapeutically effective amount of at
least one compound as disclosed herein, or a pharmaceutically
acceptable acid addition salt thereof, as an active ingredient,
with conventional acceptable pharmaceutical excipients, and by
preparation of unit dosage forms suitable for therapeutic use. As
used herein, the term "pharmaceutical composition" and refers to a
therapeutically effective concentration of an active compound, such
as, e.g., any of the compounds disclosed herein. Preferably, the
pharmaceutical composition does not produce an adverse, allergic,
or other untoward or unwanted reaction when administered to an
individual. A pharmaceutical composition disclosed herein is useful
for medical and veterinary applications. A pharmaceutical
composition may be administered to an individual alone, or in
combination with other supplementary active compounds, agents,
drugs or hormones. The pharmaceutical compositions may be
manufactured using any of a variety of processes, including,
without limitation, conventional mixing, dissolving, granulating,
drage-making, levigating, emulsifying, encapsulating, entrapping,
and lyophilizing. The pharmaceutical composition can take any of a
variety of forms including, without limitation, a sterile solution,
suspension, emulsion, lyophilizate, tablet, pill, pellet, capsule,
powder, syrup, elixir, or any other dosage form suitable for
administration.
[0063] Liquid dosage forms suitable for parenteral injection may
comprise physiologically acceptable sterile aqueous or nonaqueous
solutions, dispersions, suspensions or emulsions and sterile
powders for reconstitution into sterile injectable solutions or
dispersions. Examples of suitable aqueous and nonaqueous carriers,
diluents, solvents or vehicles include water, ethanol, polyols
(propylene glycol, polyethyleneglycol (PEG), glycerol, and the
like), suitable mixtures thereof, vegetable oils (such as olive
oil) and injectable organic esters such as ethyl oleate. Proper
fluidity can be maintained, for example, by the use of a coating
such as lecithin, by the maintenance of the required particle size
in the case of dispersions and by the use of surfactants. In liquid
formulations, a therapeutically effective amount of a compound
disclosed herein typically is between about 0.0001% (w/v) to about
50% (w/v), about 0.001% (w/v) to about 10.0% (w/v), or about 0.01%
(w/v) to about 1.0% (w/v).
[0064] Solid dosage forms suitable for oral administration include
capsules, tablets, pills, powders and granules. In such solid
dosage forms, the active compound may be admixed with at least one
inert customary excipient (or carrier) such as sodium citrate or
dicalcium phosphate or (a) fillers or extenders, as for example,
starches, lactose, sucrose, glucose, mannitol and silicic acid, (b)
binders, as for example, carboxymethylcellulose, alginates,
gelatin, polyvinylpyrrolidone, sucrose and acacia, (c) humectants,
as for example, glycerol, (d) disintegrating agents, as for
example, agar-agar, calcium carbonate, potato or tapioca starch,
alginic acid, certain complex silicates and sodium carbonate, (e)
solution retarders, as for example, paraffin, (f) absorption
accelerators, as for example, quaternary ammonium compounds, (g)
wetting agents, as for example, cetyl alcohol and glycerol
monostearate, (h) adsorbents, as for example, kaolin and bentonite,
and (i) lubricants, as for example, talc, calcium stearate,
magnesium stearate, solid polyethylene glycols, sodium lauryl
sulfate or mixtures thereof. In the case of capsules, tablets and
pills, the dosage forms may also comprise buffering agents.
[0065] A pharmaceutical composition disclosed herein can optionally
include a pharmaceutically acceptable carrier that facilitates
processing of an active compound into pharmaceutically acceptable
compositions. As used herein, the term "pharmaceutically
acceptable" refers to those compounds, materials, compositions,
and/or dosage forms which are, within the scope of sound medical
judgment, suitable for contact with the tissues of human beings and
animals without excessive toxicity, irritation, allergic response,
or other problem complications commensurate with a reasonable
benefit/risk ratio. As used herein, the term "pharmacologically
acceptable carrier" is synonymous with "pharmacological carrier"
and refers to any carrier that has substantially no long term or
permanent detrimental effect when administered and encompasses
terms such as "pharmacologically acceptable vehicle, stabilizer,
diluent, additive, auxiliary, or excipient." Such a carrier
generally is mixed with an active compound or permitted to dilute
or enclose the active compound and can be a solid, semi-solid, or
liquid agent. It is understood that the active compounds can be
soluble or can be delivered as a suspension in the desired carrier
or diluent. Any of a variety of pharmaceutically acceptable
carriers can be used including, without limitation, aqueous media
such as, e.g., water, saline, glycine, hyaluronic acid and the
like; solid carriers such as, e.g., starch, magnesium stearate,
mannitol, sodium saccharin, talcum, cellulose, glucose, sucrose,
lactose, trehalose, magnesium carbonate, and the like; solvents;
dispersion media; coatings; antibacterial and antifungal agents;
isotonic and absorption delaying agents; or any other inactive
ingredient. Selection of a pharmacologically acceptable carrier can
depend on the mode of administration. Except insofar as any
pharmacologically acceptable carrier is incompatible with the
active compound, its use in pharmaceutically acceptable
compositions is contemplated. Non-limiting examples of specific
uses of such pharmaceutical carriers can be found in Pharmaceutical
Dosage Forms and Drug Delivery Systems (Howard C. Ansel et al.,
eds., Lippincott Williams & Wilkins Publishers, 7.sup.th ed.
1999); Remington: The Science and Practice of Pharmacy (Alfonso R.
Gennaro ed., Lippincott, Williams & Wilkins, 20.sup.th ed.
2000); Goodman & Gilman's The Pharmacological Basis of
Therapeutics (Joel G. Hardman et al., eds., McGraw-Hill
Professional, 10.sup.th ed. 2001); and Handbook of Pharmaceutical
Excipients (Raymond C. Rowe et al., APhA Publications, 4.sup.th
edition 2003). These protocols are routine and any modifications
are well within the scope of one skilled in the art and from the
teaching herein.
[0066] A pharmaceutical composition disclosed herein can optionally
include, without limitation, other pharmaceutically acceptable
components (or pharmaceutical components), including, without
limitation, buffers, preservatives, tonicity adjusters, salts,
antioxidants, osmolality adjusting agents, physiological
substances, pharmacological substances, bulking agents, emulsifying
agents, wetting agents, sweetening or flavoring agents, and the
like. Various buffers and means for adjusting pH can be used to
prepare a pharmaceutical composition disclosed herein, provided
that the resulting preparation is pharmaceutically acceptable. Such
buffers include, without limitation, acetate buffers, borate
buffers, citrate buffers, phosphate buffers, neutral buffered
saline, and phosphate buffered saline. It is understood that acids
or bases can be used to adjust the pH of a composition as needed.
Pharmaceutically acceptable antioxidants include, without
limitation, sodium metabisulfite, sodium thiosulfate,
acetylcysteine, butylated hydroxyanisole, and butylated
hydroxytoluene. Useful preservatives include, without limitation,
benzalkonium chloride, chlorobutanol, thimerosal, phenylmercuric
acetate, phenylmercuric nitrate, a stabilized oxy chloro
composition, such as, e.g., sodium chlorite and chelants, such as,
e.g., DTPA or DTPA-bisamide, calcium DTPA, and CaNaDTPA-bisamide.
Tonicity adjustors useful in a pharmaceutical composition include,
without limitation, salts such as, e.g., sodium chloride, potassium
chloride, mannitol or glycerin and other pharmaceutically
acceptable tonicity adjustor. The pharmaceutical composition may be
provided as a salt and can be formed with many acids, including but
not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric,
malic, succinic, etc. Salts tend to be more soluble in aqueous or
other protonic solvents than are the corresponding free base forms.
It is understood that these and other substances known in the art
of pharmacology can be included in a pharmaceutical composition
useful in the claimed methods.
[0067] "Administering", as used herein, refers to providing a
pharmaceutical agent or composition to a subject, and includes, but
is not limited to, administering by a medical professional and
self-administering. Administration includes, but is not limited to,
oral administration, nasal administration, pulmonary
administration, subcutaneous administration, intravenous
administration, intramuscular administration, intratumoral
administration, intracavity administration, intravitreal
administration, dermal administration, and transdermal
administration, etc.
[0068] Depending on the type of cancer, and the patient to be
treated, as well as the route of administration, the disclosed
RAR.alpha. selective agonists may be administered at varying
therapeutically effective doses to a patient in need thereof.
[0069] However, the dose administered to a mammal, particularly a
human, in the context of the present methods, should be sufficient
to effect a therapeutic response in the mammal over a reasonable
timeframe. One skilled in the art will recognize that the selection
of the exact dose and composition and the most appropriate delivery
regimen will also be influenced by inter alia the pharmacological
properties of the formulation, the nature and severity of the
condition being treated, and the physical condition and mental
acuity of the recipient, as well as the potency of the specific
compound, the age, condition, body weight, sex and response of the
patient to be treated, and the stage/severity of the disease.
[0070] As a non-limiting example, when administering a RAR.alpha.
selective agonist disclosed herein to a mammal, a therapeutically
effective amount generally may be in the range of about 1
mg/m.sup.2/day to about 100 mg/m.sup.2/day. In some embodiments, an
effective amount of a RAR.alpha. selective agonist disclosed herein
may be about 5 mg/m.sup.2/day to about 90 mg/m.sup.2/day, about 10
mg/m.sup.2/day to about 80 mg/m.sup.2/day, about 15 mg/m.sup.2/day
to about 70 mg/m.sup.2/day, about 20 mg/m.sup.2/day to about 65
mg/m.sup.2/day, about 25 mg/m.sup.2/day to about 60 mg/m.sup.2/day,
or about 30 mg/m.sup.2/day to about 55 mg/m.sup.2/day. In some
embodiments, a therapeutically effective amount of a compound or a
composition disclosed herein may be at least 10 mg/m.sup.2/day, at
least 15 mg/m.sup.2/day, at least 20 mg/m.sup.2/day, at least 25
mg/m.sup.2/day, at least 30 mg/m.sup.2/day, at least 35
mg/m.sup.2/day, at least 40 mg/m.sup.2/day, at least 45
mg/m.sup.2/day, at least 50 mg/m.sup.2/day, at least 55
mg/m.sup.2/day, at least 60 mg/m.sup.2/day, at least 65
mg/m.sup.2/day ,or at least 75 mg/m.sup.2/day. In some embodiments,
a therapeutically effective amount of a RAR.alpha. selective
agonist disclosed herein may be at most 15 mg/m.sup.2/day, at most
20 mg/m.sup.2/day, at most 25 mg/m.sup.2/day, at most 30
mg/m.sup.2/day, at most 35 mg/m.sup.2/day, at most 40
mg/m.sup.2/day, at most 45 mg/m.sup.2/day, at most 50
mg/m.sup.2/day, at most 55 mg/m.sup.2/day, at most 60
mg/m.sup.2/day, at most 65 mg/m.sup.2/day, at most 70
mg/m.sup.2/day, at most 80 mg/m.sup.2/day, at most 90
mg/m.sup.2/day, or at most 100 mg/m.sup.2/day.
[0071] Administration may be continuous or intermittent. The dosage
may also be determined by the timing and frequency of
administration. Thus, the RAR.alpha. selective agonists disclosed
herein can be given on a daily, weekly, or monthly basis for a
period of time, followed by an optional drug holiday (drug free
period) and that this drug administration/drug holiday cycle can be
repeated as necessary.
[0072] In certain embodiments, the RAR.alpha. selective agonist is
administered in combination with one or more additional anti-cancer
agents. Anti-cancer agents include cytotoxic drugs, including, but
not limited to, paclitaxel, docetaxel, and the like and mixtures
thereof. Additional anti-cancer agents include adriamycin,
dactinomycin, bleomycin, vinblastine, cisplatin, acivicin,
aclarubicin, acodazole hydrochloride, acronine, adozelesin,
aldesleukin, altretamine, ambomycin, ametantrone acetate,
aminoglutethimide, amsacrine, anastrozole, anthramycin,
asparaginase, asperlin, azacitidine, azetepa, azotomycin,
batimastat, benzodepa, bicalutamide, bisantrene hydrochloride,
bisnafide dimesylate, bizelesin, bleomycin sulfate, brequinar
sodium, bropirimine, busulfan, cactinomycin, calusterone,
caracemide, carbetimer, carboplatin, carmustine, carubicin
hydrochloride, carzelesin, cedefmgol, chlorambucil, cirolemycin,
cladribine, crisnatol mesylate, cyclophosphamide, cytarabine,
dacarbazine, daunorubicin hydrochloride, decitabine, dexormaplatin,
dezaguanine, dezaguanine mesylate, diaziquone, doxorubicin,
doxorubicin hydrochloride, droloxifene, droloxifene citrate,
dromostanolone propionate, duazomycin, edatrexate, eflornithine
hydrochloride, elsamitrucin, enloplatin, enpromate, epipropidine,
epirubicin hydrochloride, erbulozole, esorubicin hydrochloride,
estramustine, estramustine phosphate sodium, etanidazole,
etoposide, etoposide phosphate, etoprine, fadrozole hydrochloride,
fazarabine, fenretinide, floxuridine, fludarabine phosphate,
fluorouracil, flurocitabine, fosquidone, fostriecin sodium,
gemcitabine, gemcitabine hydrochloride, hydroxyurea, idarubicin
hydrochloride, ifosfamide, ilmofosine, interleukin 2, interferon
alfa-2a, interferon alfa-2b, interferon alfa-nl, interferon
alfa-n3, interferon beta-Ia, interferon gamma-Ib, iproplatin,
irinotecan hydrochloride, lanreotide acetate, letrozole, leuprolide
acetate, liarozole hydrochloride, lometrexol sodium, lomustine,
losoxantrone hydrochloride, masoprocol, maytansine, mechlorethamine
hydrochloride, megestrol acetate, melengestrol acetate, melphalan,
menogaril, mercaptopurine, methotrexate, methotrexate sodium,
metoprine, meturedepa, mitindomide, mitocarcin, mitocromin,
mitogillin, mitomalcin, mitomycin, mitosper, mitotane, mitoxantrone
hydrochloride, mycophenolic acid, nocodazole, nogalamycin,
ormaplatin, oxisuran, pegaspargase, peliomycin, pentamustine,
peplomycin sulfate, perfosfamide, pipobroman, piposulfan,
piroxantrone hydrochloride, plicamycin, plomestane, porfimer
sodium, porfiromycin, prednimustine, procarbazine hydrochloride,
puromycin, puromycin hydrochloride, pyrazofurin, riboprine,
rogletimide, safingol, safingol hydrochloride, semustine,
simtrazene, sparfosate sodium, sparsomycin, spirogermanium
hydrochloride, spiromustine, spiroplatin, streptonigrin,
streptozocin, sulofenur, talisomycin, tecogalan sodium, tegafur,
teloxantrone hydrochloride, temoporfin, teniposide, teroxirone,
testolactone, thiamiprine, thioguanine, thiotepa, tiazofurin,
tirapazamine, toremifene citrate, trestolone acetate, triciribine
phosphate, trimetrexate, trimetrexate glucuronate, triptorelin,
tubulozole hydrochloride, uracil mustard, uredepa, vapreotide,
verteporfin, vinblastine sulfate, vincristine sulfate, vindesine,
vindesine sulfate, vinepidine sulfate, vinglycinate sulfate,
vinleurosine sulfate, vinorelbine tartrate, vinrosidine sulfate,
vinzolidine sulfate, vorozole, zeniplatin, zinostatin, zorubicin
hydrochloride, and bortezomib.
[0073] In some embodiments, the anti-cancer agent includes, but is
not limited to: 5-fluorouracil, 20-epi-I,25 dihydroxyvitamin D3,
5-ethynyluracil, abiraterone, aclarubicin, acylfulvene, adecypenol,
adozelesin, aldesleukin, ALL-TK antagonists, altretamine,
ambamustine, amidox, amifostine, aminolevulinic acid, amrubicin,
amsacrine, anagrelide, anastrozole, andrographolide, angiogenesis
inhibitors, antagonist D, antagonist G, antarelix, anti-dorsalizing
morphogenetic protein-1, antiandrogen, prostatic carcinoma,
antiestrogen, antineoplaston, antisense oligonucleotides,
aphidicolin glycinate, apoptosis gene modulators, apoptosis
regulators, apurinic acid, ara-CDP-DL-PTBA, arginine deaminase,
asulacrine, atamestane, atrimustine, axinastatin 1, axinastatin 2,
axinastatin 3, azasetron, azatoxin, azatyrosine, baccatin III
derivatives, balanol, batimastat, BCR/ABL antagonists,
benzochlorins, benzoylstaurosporine, beta lactam derivatives,
beta-alethine, betaclamycin B, betulinic acid, bFGF inhibitor,
bicalutamide, bisantrene, bisaziridinylspermine, bisnafide,
bistratene A, bizelesin, breflate, bropirimine, budotitane,
buthionine sulfoximine, calcipotriol, calphostin C, camptothecin
derivatives, capecitabine, carboxamide-amino-triazole,
carboxyamidotriazole, CaRest M3, CARN 700, cartilage derived
inhibitor, carzelesin, casein kinase inhibitors (ICOS),
castanospermine, cecropin B, cetrorelix, chlorins,
chloroquinoxaline sulfonamide, cicaprost, cis-porphyrin,
cladribine, clomifene analogues, clotrimazole, collismycin A,
collismycin B, combretastatin A4, combretastatin analogue,
conagenin, crambescidin 816, crisnatol, cryptophycin 8,
cryptophycin A derivatives, curacin A, cyclopentanthraquinones,
cycloplatam, cypemycin, cytarabine ocfosfate, cytolytic factor,
cytostatin, dacliximab, decitabine, dehydrodidemnin B, deslorelin,
dexamethasone, dexifosfamide, dexrazoxane, dexverapamil,
diaziquone, didemnin B, didox, diethylnorspermine,
dlhydro-5-azacytidine, 9-dioxamycin, diphenyl spiromustine,
docosanol, dolasetron, doxifturidine, droloxifene, dronabinol,
duocarmycin SA, ebselen, ecomustine, edelfosine, edrecolomab,
eflornithine, elemene, emitefur, epirubicin, epristeride,
estramustine analogue, estrogen agonists, estrogen antagonists,
etanidazole, etoposide phosphate, exemestane, fadrozole,
fazarabine, fenretinide, filgrastim, finasteride, flavopiridol,
flezelastine, fluasterone, fludarabine, fluorodaunorunicin
hydrochloride, forfenimex, formestane, fostriecin, fotemustine,
gadolinium texaphyrin, gallium nitrate, galocitabine, ganirelix,
gelatinase inhibitors, gemcitabine, glutathione inhibitors,
hepsulfam, heregulin, hexamethylene bisacetamide, hypericin,
ibandronic acid, idarubicin, idoxifene, idramantone, ilmofosine,
ilomastat, imidazoacridones, imiquimod, immunostimulant peptides,
insulin-like growth factor-1 receptor inhibitor, interferon
agonists, interferons, interleukins, iobenguane, iododoxorubicin,
ipomeanol, iroplact, irsogladine, isobengazole, isohomohalicondrin
B, itasetron, jasplakinolide, kahalalide F, lamellarin-N
triacetate, lanreotide, leinamycin, lenograstim, lentinan sulfate,
leptolstatin, letrozole, leukemia inhibiting factor, leukocyte
alpha interferon, leucovorin, leuprolide+estrogen+progesterone,
leuprorelin, levamisole, liarozole, linear polyamine analogue,
lipophilic disaccharide peptide, lipophilic platinum compounds,
lissoclinamide 7, lobaplatin, lombricine, lometrexol, lonidamine,
losoxantrone, lovastatin, loxoribine, lurtotecan, lutetium
texaphyrin, lysofylline, lytic peptides, maitansine, mannostatin A,
marimastat, masoprocol, maspin, matrilysin inhibitors, matrix
metalloproteinase inhibitors, menogaril, merbarone, meterelin,
methioninase, metoclopramide, MIF inhibitor, mifepristone,
miltefosine, mirimostim, mismatched double stranded RNA,
mitoguazone, mitolactol, mitomycin analogues, mitonafide, mitotoxin
fibroblast growth factor-saporin, mitoxantrone, mofarotene,
molgramostim, human chorionic gonadotrophin, monophosphoryl lipid
A+myobacterium cell wall sk, mopidamol, multiple drug resistance
gene inhibitor, multiple tumor suppressor 1-based therapy, mustard
anticancer agent, mycaperoxide B, mycobacterial cell wall extract,
myriaporone, N-acetyldinaline, N-substituted benzamides, nafarelin,
nagrestip, naloxone+pentazocine, napavin, naphterpin, nartograstim,
nedaplatin, nemorubicin, neridronic acid, neutral endopeptidase,
nilutamide, nisamycin, nitric oxide modulators, nitroxide
antioxidant, nitrullyn, O6-benzylguanine, octreotide, okicenone,
oligonucleotides, onapristone, ondansetron, oracin, oral cytokine
inducer, ormaplatin, osaterone, oxaliplatin, oxaunomycin,
palauamine, palmitoylrhizoxin, pamidronic acid, panaxytriol,
panomifene, parabactin, pazelliptine, pegaspargase, peldesine,
pentosan polysulfate sodium, pentostatin, pentrozole, perflubron,
perfosfamide, perillyl alcohol, phenazinomycin, phenylacetate,
phosphatase inhibitors, picibanil, pilocarpine hydrochloride,
pirarubicin, piritrexim, placetin A, placetin B, plasminogen
activator inhibitor, platinum complex, platinum compounds,
platinum-triamine complex, porfimer sodium, porfiromycin,
prednisone, propyl bis-acridone, prostaglandin J2, proteasome
inhibitors, protein A-based immune modulator, protein kinase C
inhibitor, microalgal, protein tyrosine phosphatase inhibitors,
purine nucleoside phosphorylase inhibitors, purpurins,
pyrazoloacridine, pyridoxylated hemoglobin polyoxyethylene
conjugate, raf antagonists, raltitrexed, ramosetron, ras farnesyl
protein transferase inhibitors, ras inhibitors, ras-GAP inhibitor,
retelliptine demethylated, rhenium Re 186 etidronate, rhizoxin,
ribozymes, RII retinamide, rogletimide, rohitukine, romurtide,
roquinimex, rubiginone BI, ruboxyl, safmgol, saintopin, SarCNU,
sarcophytol A, sargramostim, Sdi 1 mimetics, semustine, senescence
derived inhibitor 1, sense oligonucleotides, signal transduction
inhibitors, signal transduction modulators, single chain
antigen-binding protein, sizofiran, sobuzoxane, sodium borocaptate,
sodium phenylacetate, solverol, somatomedin binding protein,
sonermin, sparfosic acid, spicamycin D, spiromustine, splenopentin,
spongistatin 1, squalamine, stem cell inhibitor, stem-cell division
inhibitors, stipiamide, stromelysin inhibitors, sulfinosine,
superactive vasoactive intestinal peptide antagonist, suradista,
suramin, swainsonine, synthetic glycosaminoglycans, tallimustine,
tamoxifen methiodide, tauromustine, tazarotene, tecogalan sodium,
tegafur, tellurapyrylium, telomerase inhibitors, temoporfin,
temozolomide, teniposide, tetrachlorodecaoxide, tetrazomine,
thaliblastine, thiocoraline, thrombopoietin, thrombopoietin
mimetic, thymalfasin, thymopoietin receptor agonist, thymotrinan,
thyroid stimulating hormone, tin ethyl etiopurpurin, tirapazamine,
titanocene bichloride, topsentin, toremifene, totipotent stem cell
factor, translation inhibitors, tretinoin, triacetyluridine,
triciribine, trimetrexate, triptorelin, tropisetron, turosteride,
tyrosine kinase inhibitors, tyrphostins, UBC inhibitors, ubenimex,
urogenital sinus-derived growth inhibitory factor, urokinase
receptor antagonists, vapreotide, variolin B, vector system,
erythrocyte gene therapy, velaresol, veramine, verdins,
verteporfin, vinorelbine, vinxaltine, vitaxin, vorozole,
zanoterone, zeniplatin, zilascorb, and zinostatin stimalamer.
[0074] In some embodiments, the anti-cancer agent is one or more of
an antracycline (e.g., doxorubicin or epirubicin),
cyclophosphamide, a platinum agent (e.g., cisplatin, carboplatin,
oxaliplatin), a taxane (e.g., paclitaxel or docetaxel),
methotrexate, 5-fluorouracil, trastuxumab, pertuzumab, vinorelbine,
capecitabine, gemcitabine, mitoxantrone, ixabepilone, reibulin, an
anti-hormone therapy (e.g., tamoxifen, anastrazole, exemestrane,
letrozole), etoposide, irinotecan, vinblastine, pemetrexed,
bevacizumab, cetuximab, an EGFR inhibitor (e.g., erlotinib), an
EML4-ALK kinase inhibitor (e.g., crixotinil), estramustine,
cytarabine, a demethylating agent (e.g., 5-azacytidine,
decitabine), an immunomodulator (e.g., lenolidamide, pomalidomide),
a corticosteroid, bleomycin, adriamycin, benamustin, fludarabine, a
growth factor (GOSH, GM-CSF, EPO), and bortezomib.
[0075] Specific combinations of RAR.alpha. selective agonists and
additional anti-cancer agents useful in the treatment of cancer are
listed below in Table 1. The listed drug combinations can be
administered at the same time, at different times, in the same
composition, in different compositions, in alternating times (1
week of RAR.alpha. selective agonist followed by 1 week of another
anti-cancer agent, etc.), or at any administration schedule
established by a healthcare professional.
TABLE-US-00001 TABLE 1 Cancer Exemplary Drug combination Breast
RAR.alpha. selective agonist + an anthracycline (e.g., doxorubicin
or epirubicin) + cyclophosphamide with or without a platinum agent
(e.g., cisplatin or carboplatin) RAR.alpha. selective agonist + an
anthracycline + taxane (e.g., paclitaxel or docetaxel) with or
without a platinum agent RAR.alpha. selective agonist +
cyclophosphamide + methotrexate + 5-fluorouracil with or without a
platinum agent RAR.alpha. selective agonist + an anthracycline +
cyclophosphamide + 5-fluorouracil with or without a platinum agent
RAR.alpha. selective agonist + trastuzumab + a taxane RAR.alpha.
selective agonist + pertuzumab + trastuzumab + a platinum agent
RAR.alpha. selective agonist + vinorelbine with or without a
platinum agent RAR.alpha. selective agonist + capecitabine with or
without a platinum agent RAR.alpha. selective agonist + gemcitabine
with or without a platinum agent RAR.alpha. selective agonist +
mitoxantrone with or without a platinum agent RAR.alpha. selective
agonist + ixabepilone with or without a platinum agent RAR.alpha.
selective agonist + eribulin with or without a platinum agent
RAR.alpha. selective agonist + an anthracycline with or without a
platinum agent RAR.alpha. selective agonist + a platinum agent
RAR.alpha. selective agonist + a taxane with or without a platinum
agent RAR.alpha. selective agonist + trastuzumab or pertuzumab with
or without a platinum agent RAR.alpha. selective agonist +
cyclophosphamide with or without a platinum agent RAR.alpha.
selective agonist + methotrexate with or without a platinum agent
RAR.alpha. selective agonist + 5-fluorouracil with or without a
platinum agent RAR.alpha. selective agonist + a combination of two
or more of a platinum agent, a taxane, gemcitabine, vinorelbine,
capecitabine, cyclophosphamide, metotrexate, 5-fluorourocil, an
anthracycline, trastuzumab, pertuzumab, mitoxantrone, ixabepilone,
or eribulin. RAR.alpha. selective agonist + trastuzumab RAR.alpha.
selective agonist + anti-hormone therapy (e.g., tamoxifen,
anastrazole, exemestrane, letrozole) Lung RAR.alpha. selective
agonist + a platinum agent RAR.alpha. selective agonist + etoposide
RAR.alpha. selective agonist + irinotecan RAR.alpha. selective
agonist + a platinum agent + etoposide RAR.alpha. selective agonist
+ a platinum agent + irinotecan RAR.alpha. selective agonist + a
taxane RAR.alpha. selective agonist + gemcitabine RAR.alpha.
selective agonist + vinorelbine RAR.alpha. selective agonist +
capecitabine RAR.alpha. selective agonist + vinblastine RAR.alpha.
selective agonist + pemetrexed RAR.alpha. selective agonist +
bevacizumab RAR.alpha. selective agonist + cetuximab RAR.alpha.
selective agonist + a combination of two or more of a platinum
agent, etoposide, irinotecan, a taxane, gemcitabine, vinorelbine,
capecitabine, vinblastine, pemetrexed, bevacizumab, or cetuximab
RAR.alpha. selective agonist + EGFR inhibitor (e.g., erlotinib)
RAR.alpha. selective agonist + EML4-ALK kinase inhibitor (e.g.,
crizotinil) Pancreas RAR.alpha. selective agonist + gemcitabine
RAR.alpha. selective agonist + erlotinib RAR.alpha. selective
agonist + 5-fluourouracil RAR.alpha. selective agonist + irinotecan
RAR.alpha. selective agonist + a platinum compound RAR.alpha.
selective agonist + oxaliplatin RAR.alpha. selective agonist +
capecitabine RAR.alpha. selective agonist + a taxane RAR.alpha.
selective agonist + a combination of two or more of a platinum
agent, irinotecan, a taxane, gemcitabine, capecitabine, erlotinib,
5-fluorouracil, or oxaliplatin. Prostate RAR.alpha. selective
agonist + etoposide RAR.alpha. selective agonist + a platinum agent
RAR.alpha. selective agonist + a taxane RAR.alpha. selective
agonist + vinorelbine RAR.alpha. selective agonist + vinblastine
RAR.alpha. selective agonist + mitoxantrone RAR.alpha. selective
agonist + cabazitaxel RAR.alpha. selective agonist + estramustine
RAR.alpha. selective agonist + an anthracycline RAR.alpha.
selective agonist + a combination of two or more of a platinum
agent, etoposide, a taxane, vinorelbine, vinblastine, mitoxantrone,
cabazitaxel, estramustine, or an anthracycline Hematological
RAR.alpha. selective agonist + etoposide Malignancies RAR.alpha.
selective agonist + an anthracycline (e.g., idarubicin,
daunorubicin, mitoxantrone) RAR.alpha. selective agonist +
cytarabine RAR.alpha. selective agonist + a combination of an
anthracycline, cytarabine and etoposide RAR.alpha. selective
agonist + demethylating agent (5-azacytidine or decitabine)
RAR.alpha. selective agonist + small molecule inhibitors (e.g.,
thyrosine kinase inhibitors including BCR-ABL inhibitors, Flt3
inhibitors or cKit inhibitor, IDH1/2 inhibitors, JAK2 inhibitors,
BTK inhibitors) RAR.alpha. selective agonist + immunotherapeutic
agents (monoclonal antibodies such as anti-CD33, anti-CD20,
anti-CD19, anti-CD30or with PD1 inhibitors or CTL4 inhibitors)
RAR.alpha. selective agonist + immunomodulatory drugs such as
lenolidamide, pomalidomide, and their derivatives RAR.alpha.
selective agonist + cyclophosphamide RAR.alpha. selective agonist +
bevacizumab RAR.alpha. selective agonist + vincristine RAR.alpha.
selective agonist + a corticosteroid RAR.alpha. selective agonist +
bleomycin RAR.alpha. selective agonist + adriamycin RAR.alpha.
selective agonist + bendamustin RAR.alpha. selective agonist +
fludarabine RAR.alpha. selective agonist + growth factors including
GCSF, GM-CSF and EPO RAR.alpha. selective agonist + a combination
of two or more of combinations listed above. RAR.alpha. selective
agonist + bortezomib
[0076] The effectiveness of cancer therapy is typically measured in
terms of "response." The techniques to monitor responses can be
similar to the tests used to diagnose cancer such as, but not
limited to: [0077] A lump or tumor involving some lymph nodes can
be felt and measured externally by physical examination. [0078]
Some internal cancer tumors will show up on an x-ray or CT scan and
can be measured with a ruler. [0079] Blood tests, including those
that measure organ function can be performed. [0080] A tumor marker
test can be done for certain cancers.
[0081] Regardless of the test used--whether blood test, cell count,
or tumor marker test, it is repeated at specific intervals so that
the results can be compared to earlier tests of the same type.
[0082] Response to cancer treatment is defined several ways: [0083]
Complete response--all of the cancer or tumor disappears; there is
no evidence of disease. A tumor marker (if applicable) may fall
within the normal range. [0084] Partial response--the cancer has
shrunk by a percentage but disease remains. A tumor marker (if
applicable) may have fallen but evidence of disease remains. [0085]
Stable disease--the cancer has neither grown nor shrunk; the amount
of disease has not changed. A tumor marker (if applicable) has not
changed significantly. [0086] Disease progression--the cancer has
grown; there is more disease now than before treatment. A tumor
marker test (if applicable) shows that a tumor marker has
risen.
[0087] There are two standard methods for the evaluation of solid
cancer treatment response with regard to tumor size (tumor burden),
the WHO and RECIST standards. These methods measure a solid tumor
to compare a current tumor with past measurements or to compare
changes with future measurements and to make changes in a treatment
regimen. In the WHO method, the solid tumor's long and short axes
are measured with the product of these two measurements is then
calculated; if there are multiple solid tumors, the sum of all the
products calculated. In the RECIST method, only the long axis is
measured. If there are multiple solid tumors, the sum of all the
long axes measurements is calculated. However, with lymph nodes,
the short axis is measured instead of the long axis.
[0088] In some embodiments of the current method, the tumor burden
of a treated patient is reduced by about 5%, about 10%, about 15%,
about 20%, about 25%, about 30%, about 35%, about 40%, about 45%,
about 50%, about 55% about 60%, about 65%, about 70%, about 75%,
about 80%, about 90%, about 95%, about 100%, or any other range
bound by these values.
[0089] In other embodiments, the 1-year survival rate of treated
individual is increased by about 5%, about 10%, about 15%, about
20%, about 25%, about 30%, about 35%, about 40%, about 45%, about
50%, about 55% about 60%, about 65%, about 70%, about 75%, about
80%, about 90%, about 95%, about 100%, or any other range bound by
these values.
[0090] In other embodiments, the 5-year survival rate of treated
individual is increased by about 5%, about 10%, about 15%, about
20%, about 25%, about 30%, about 35%, about 40%, about 45%, about
50%, about 55% about 60%, about 65%, about 70%, about 75%, about
80%, about 90%, about 95%, about 100%, or any other range bound by
these values.
[0091] In other embodiments, the 10-year survival rate of treated
individual is increased by about 5%, about 10%, about 15%, about
20%, about 25%, about 30%, about 35%, about 40%, about 45%, about
50%, about 55% about 60%, about 65%, about 70%, about 75%, about
80%, about 90%, about 95%, about 100%, or any other range bound by
these values.
[0092] In yet other embodiments, the subject has a sustained
remission of at least 6 months, at least 7 months, at least 8
months, at least 9 months, at least 10 months, at least 11 months,
at least 12 months, at least 14 months, at least 16 months, at
least 18 months, at least 20 months, at least 22 months, at least
24 months, at least 27 months, at least 30 months, at least 33
months, at least 36 months, at least 42 months, at least 48 months,
at least 54 months, or at least 60 months or more.
[0093] As used herein, the term "substantially free of cancer"
refers to a subject which does not have clinical evidence of cancer
or cancer cells, or cancer stem cells.
[0094] In other embodiments, the method may help to treat or
alleviate conditions, symptoms, or disorders related to cancer. In
some embodiments, these conditions or symptoms may include, but are
not limited to, anemia, asthenia, cachexia, Cushing's Syndrome,
fatigue, gout, gum disease, hematuria, hypercalcemia,
hypothyroidism, internal bleeding, hair loss, mesothelioma, nausea,
night sweats, neutropenia, paraneoplastic syndromes, pleuritis,
polymyalgia rheumatica, rhabdomyolysis, stress, swollen lymph
nodes, thrombocytopenia, Vitamin D deficiency, or weight loss. In
other embodiments, the administration of the RAR.alpha. selective
agonist prolongs the survival of the individual being treated.
EXAMPLES
Example 1
The Bone Marrow Niche Induces a Bortezomib Resistance in Multiple
Myeloma
[0095] Multiple myeloma (MM) is characterized by the proliferation
of malignant plasma cells (PCs) within the BM and their production
of monoclonal immunoglobulin (Ig). Novel therapies, including
proteasome inhibitors, have significantly extended the survival of
patients with MM but have failed to achieve a cure. Increasing
evidence demonstrates that interactions with the BM
microenvironment play a critical role in the survival of MM cells
during chemotherapy. However, the mechanisms mediating this BM
niche-dependent chemoprotection are incompletely understood and
remain a critical area of research.
[0096] There exist MM cells that resemble mature B cells and are
resistant to bortezomib (BTZ). Like their normal B cell
counterparts, these CD138-MM cells are capable of clonogenic growth
and differentiation into CD138+ PCs. Moreover, these cells are
enriched during minimal residual disease (MRD), suggesting a
critical role in disease relapse. Differential BTZ sensitivity of
CD138+ and CD138-MM cells may be explained by their secretory
activity. As a result of their abundant Ig production, CD138+ PCs
are highly dependent on an intact proteasome pathway to degrade
improperly folded proteins. Conditions that disrupt protein
degradation by the proteasome activate a cellular stress pathway
known as the unfolded protein response (UPR), which counteracts ER
stress by decreasing protein synthesis and promoting protein
degradation. If homeostasis cannot be reestablished, UPR activation
eventually leads to apoptosis. On the other hand, CD138-MM cells
exhibit limited Ig production and low ER stress and are less
dependent on proteasome-mediated degradation of misfolded
proteins.
[0097] Methods
[0098] Cell cultures. All cell lines were purchased from the
American Type Culture Collection. H929, MM1s, and U266 cells were
cultured in RPMI 1640 with 10% FCS (Sigma-Aldrich), 2 mM
L-glutamine, and 100 .mu.g/ml penicillin-streptomycin (P/S). OP-9
cells were cultured in .alpha.-MEM, 20% FCS, L-glutamine, and P/S.
Cell lines were authenticated by short-tandem repeat profiling.
[0099] Primary MM cells were obtained from patients with newly
diagnosed or relapsed MM under an IRB-approved protocol at The
Johns Hopkins Universtiy. Briefly, mononuclear cells were isolated
from fresh BM aspirates by density gradient centrifugation
(Ficoll-Paque); CD138+ cells were then selected via magnetic beads
and columns and incubated in RPMI 1640, 10% FCS, L-glutamine, and
P/S at 37.degree. C.
[0100] Primary human BM stromal cells were derived from aspirates
collected from healthy donors under an IRB-approved protocol at
Johns Hopkins. Briefly, total mononuclear cells isolated from BM
aspirates were cultured in Iscove's modified Dulbecco's medium
(IMDM) supplemented with 10% horse serum, 10% FCS, 10-5 M
hydrocortisone 21-hemisuccinate, P/S, and 0.1 mM
.beta.-mercaptoethanol (.beta.-ME) (FBMD1 media). The following
day, cells in suspension were removed by washing twice with PBS,
and the media were replaced. Attached stromal cells were incubated
at 33.degree. C. until a confluent monolayer was obtained. Mouse
primary BM stromal cells were isolated following the same protocol,
after isolation of total BM mononuclear cells from mouse
femurs.
[0101] Vectors and viral supernatants. To generate Smo-KO and WT
stroma, BM stromal cells were derived from Smo.sup.fl/fl mice and
transduced with the retroviral vector PIG-Cre encoding
Cre-recombinase (Addgene; catalog 50935) or a control vector
(Addgene; catalog 18751), respectively. Successfully infected cells
were selected using 4 .mu.g/ml puromycin for 5 days and confirmed
by expression of GFP via flow cytometry. The pLenti-CMV-LUC-Puro
lentiviral vector (plasmid 17477) was used to generate H929 Luc+
cells.
[0102] To generate CYP26A1-overexpressing stromal cells, WT and
Smo-KO stromal cells were transduced with the lentiviral vector
pBABE-neo (Addgene; catalog 1767) that had been engineered to
encode CYP26A1. Briefly, Cyp26a1 cDNA (Origene) was amplified via
PCR using primers incorporating the restriction sites BamHl and
EcoRl and cloned into the pCR2.1 vector. Cyp26a1 cDNA was confirmed
via Sanger sequencing, and the fragment was isolated after
digestion with the restriction enzymes BamHl and EcoRl and
subcloned into the corresponding sites of the pBABE vector.
Lentiviral particles were produced as previously described.
Successfully infected stromal cells were selected using 3 .mu.g/ml
G-418 for 10 days, and expression of Cyp26a1 was confirmed by
qRT-PCR.
[0103] Coculture experiments. 24-well plates were coated with 0.1%
gelatin in PBS for 30 min at 37.degree. C. The gelatin solution was
removed, and the stromal cells were cultured overnight at a density
of 5.times.10.sup.4 cells/well to obtain a confluent monolayer. At
that time, MM cell lines or primary MM cells (1.times.10.sup.5 in 2
ml) were added to the stroma cultures. The stroma cocultures were
incubated at 37.degree. C. in RPMI containing 10% FCS, L-glutamine,
and P/S, with or without AGN19194310 (1 pM for 5 days), R115866 (1
.mu.M for 5 days), IRX5183 (1 .mu.M for 5 days), or BTZ (2.5 nM for
48 hr).
[0104] Transwell experiments. For Transwell experiments, 6-well
plates were coated with 0.1% gelatin in PBS for 30 min at
37.degree. C. The gelatin solution was removed, and the stromal
cells were cultured overnight in FBMD1 media at a density of
10.times.10.sup.4 cells/well in 2 ml of media to obtain a confluent
monolayer. At that time, Transwell inserts (Corning) were placed
over the stroma cultures, and MM cell lines (1.times.10.sup.6 in 1
ml) were seeded in the Transwell for 24 hr at 37.degree. C.
Following this incubation, Transwell and MM cells were removed, and
stromal cells were detached from the wells and analyzed by qRT-PCR
for CYP26 expression.
[0105] Mobilization experiments. MM cells were separated from BM
stromal cells by gently pipetting several times around the well.
Detached cells were centrifuged, resuspended in fresh media, and
incubated in a 24-well plate for 1 hr at 37.degree. C. During this
short incubation period, contaminating stromal cells attached to
the well, while MM cells remained in suspension. MM cells were then
recovered by gently pipetting. This protocol was used for qRT-PCR
and CFU coculture experiments. The purity achieved using this
protocol was confirmed by flow cytometry to be 98%-99% MM cells and
less than 2% contaminating stroma.
[0106] Clonogenic assays. After treatment, MM cells were collected,
washed with PBS, and plated at a density of 5,000 cells/ml in 1 ml
of 1.32% methylcellulose supplemented with 30% FBS, 10% BSA,
L-glutamine, P/S, and 0.1 mM .beta.-ME. Cells were plated in
triplicate in 35-mm culture dishes, incubated at 37.degree. C., and
scored for the presence of colonies 14 days later.
[0107] qRT-PCR. Total RNA was extracted using the RNeasy Mini Kit
(QIAGEN) according to the manufacturer's instructions. cDNA was
synthesized by reverse transcription using the iScript cDNA
Synthesis Kit (Bio-Rad). qRT-PCR was performed with iTaq SYBR Green
Supermix (Bio-Rad) using sequence specific primers. Gene expression
was normalized to GAPDH, and relative quantification was calculated
using .DELTA..DELTA.Ct. All experiments were performed in duplicate
and run on the Bio-Rad CFX96 machine.
[0108] Flow cytometry. Following treatment, MM cells were
collected, washed with PBS, and stained for 15 min at room
temperature with phycoerythrin-conjugated (PE-conjugated)
anti-CD138. Cells were washed to remove unbound antibody and
evaluated in a FACSCalibur system (BD Biosciences). Stromal cells
were identified by GFP expression, and viable cells were identified
using 7-aminoactinomycin D (7-AAD). To calculate cell numbers, live
GFP-cells were normalized to calibration beads.
[0109] Mouse xenografts. 1.times.10.sup.6 H929 Luc+ cells and
1.times.10.sup.6 mouse BM stromal cells were resuspended in 100
.mu.l Matrigel, diluted with RPMI (1:1), and injected
subcutaneously into 16-week-old male NSG mice. After 4 days,
treatment with BTZ (0.5 mg/kg i.p. twice weekly) and IRX (10 mg/kg
i.p. daily) was initiated. Tumor burden was assessed by
bioluminescence using the In Vivo Imaging System (PerkinElmer). For
imaging, mice were exposed to 120 mg/kg D-luciferin via
intraperitoneal injection 10-5 min before imaging and were
anesthetized using isoflurane. Images were analyzed with Living
Image Software, version 2.5 (PerkinElmer), and data were quantified
as photons/second.
[0110] For the systemic MM model, 2.times.10.sup.6 Luc+/GFP+H929
cells were injected via the tail vein into 16-week-old male NSG
mice. After engraftment, as determined by an exponential increase
in bioluminescence, mice were treated with BTZ (0.5 mg/kg i.p.)
twice weekly and with IRX (10 mg/kg) once daily. Tumor burden was
assessed by bioluminescence, as above.
[0111] Statistics. First evaluated was whether the treatment groups
were different from the controls using 1-way ANOVA. If the ANOVA
test yielded a statistically significant result, then the
difference between the control group and each treatment group was
evaluated, with the P values adjusted for multiple comparisons
using Dunnett's test. For experiments in which only 2 sets of data
were analyzed, statistical significance was evaluated using an
unpaired, 2-tailed Student's t test. Pearson's R value for
correlation and P values were calculated using GraphPad Prism 7
(GraphPad Software).
[0112] Results
[0113] The BM niche limits PC differentiation by modulating
retinoid signaling. A population of MM progenitors, phenotypically
similar to B cells, is intrinsically resistant to BTZ and
contributes to MRD and relapse. To investigate whether the BM niche
plays a role in determining the phenotype of MM cells, the mRNA
expression of B cell and PC markers in MM H929 cell lines (FIG.
1A-D) and MM CD138+ primary cells (FIG. 1E-H) was analyzed
following coculture with mouse BM stroma using human-specific
primers. B cell lymphoma 6 (BCL6), a transcriptional repressor that
promotes self-renewal of germinal center B cells and prevents PC
differentiation, was upregulated in the presence of BM stromal
cells (FIG. 1A, 1E). In contrast, coculture of MM cells with BM
stroma decreased the mRNA expression of B lymphocyte-induced
maturation protein 1 (BLIMP1) and spliced X box-binding protein 1
(XBP1s) (FIG. 1B, C, F, G), which are critical mediators of PC
differentiation. Similarly, C/EBP homologous protein (CHOP), a key
component of the UPR pathway, was downregulated in the presence of
BM stromal cells (FIG. 1D, H).
[0114] The BM niche regulates hematopoietic stem cell (HSC)
differentiation by expressing the retinoid-inactivating enzyme
CYP26. CYP26 enzymes were highly expressed in BM mesenchymal cells,
while their expression was barely detectable in MM cells. Since
retinoid signaling promotes PC differentiation and potentiates Ig
secretion, it was determined whether stromal CYP26 is responsible
for inducing a B cell phenotype in MM cells. To this end, coculture
conditions were treated with the CYP26 inhibitor R115866 (R115) or
the CYP26-resistant RA receptor .alpha.-selective
(RAR.alpha.-selective) retinoid IRX5183 (IRX). Incubation of stroma
cocultures with either R115 or IRX restored all markers to levels
comparable to those of liquid control conditions (FIG. 1A-H).
Moreover, treatment of MM cells with the pan-RAR antagonist
AGN194310 (AGN) mimicked the changes induced by BM stromal cells
(FIG. 1A-H), limiting PC differentiation.
[0115] Expression of CD138 is a hallmark of normal PC
differentiation as well MM PCs. Consistent with mRNA levels of PC
markers, surface CD138 expression was markedly decreased by
coculture with BM stromal cells or incubation with AGN. Incubation
of BM stromal cell cocultures with R115 or IRX restored CD138
expression in MM cells. R115 did not significantly affect the
expression of differentiation markers in liquid conditions by
quantitative reverse transcription-PCR (qRT-PCR) or flow cytometry,
while IRX induced comparable changes, irrespective of the presence
or absence of BM stroma. Taken together, these data suggest that
retinoid signaling promotes PC differentiation of MM cells and that
this process is blocked by stromal CYP26-mediated metabolism of
RA.
[0116] A RA-low microenvironment induces BTZ resistance. To
determine whether decreased retinoid signaling contributes to BTZ
resistance within the BM niche, MM cell lines and MM CD138+ primary
cells were incubated with BM stroma for 5 days, followed by BTZ
treatment. In the absence of BM stroma (liquid), MM cells were
highly sensitive to BTZ (FIG. 2A-B). However, incubation with BM
stroma induced BTZ resistance, which was overcome by CYP26
inhibition via R115 or by the CYP26-resistant retinoid IRX.
Moreover, treatment of MM cells with the pan-RAR antagonist AGN
mimicked the changes induced by BM stromal cells (FIG. 3),
decreasing BTZ sensitivity.
[0117] Strategies to overcome microenvironment-dependent
chemoprotection have focused on mobilization of cancer cells from
the BM niche into the peripheral circulation. It was analyzed
whether the change in phenotype and subsequent BTZ resistance of MM
cells were lost upon separation from the BM stroma, a process that
mimics mobilization. To this end, H929 cells were separated from BM
mesenchymal cells following a 5-day stroma coculture, incubated in
fresh media (RPMI with 10% FBS) for 0 to 48 hr, and then treated
with BTZ. Interestingly, MM cells remained partially resistant to
BTZ for up to 48 hr following detachment from stroma (FIG. 3).
Moreover, treatment of the coculture conditions with R115 prevented
the development of a BTZ-resistant phenotype (FIG. 3). Thus,
microenvironment-dependent BTZ resistance induced by the change in
MM cell phenotype may not immediately be reversed by tumor
mobilization.
[0118] To test whether retinoids can enhance BTZ activity in MM, a
systemic MM xenograft was developed by injecting 2.times.10.sup.6
H929 luciferase+ (Luc+) cells via the tail vein of nonobese,
diabetic, severe combined immunodeficiency IL-2 receptor .gamma.-KO
(NSG) mice. The animals were randomized to receive IRX, BTZ, or a
combination of both, and disease burden was followed weekly by
bioluminescence imaging (FIG. 4). Mice treated with BTZ showed
decreased tumor growth compared with untreated controls; however,
some MM cells remained resistant to BTZ, as demonstrated by the
continued increase in bioluminescence. Similarly, mice treated with
IRX monotherapy showed a decrease in tumor burden compared with
untreated mice. Most important, IRX sensitized MM cells to BTZ,
leading to a significant (P<0.01) decrease in disease burden.
Collectively, these data suggest that an RA-low microenvironment
created by stromal CYP26 induces a BTZ-resistant phenotype, which
is maintained even after displacement from the BM niche.
[0119] MM cells induce stromal CYP26. Recent studies have
demonstrated the existence of a bi-directional crosstalk, in which
not only stromal cells provide a protective microenvironment, but
also cancer cells actively adapt and build a reinforced niche.
Thus, it was determined whether MM cells reinforce a protective
microenvironment by strengthening the ability of BM stroma to
inactivate retinoids. Stromal CYP26 expression was analyzed by
qRT-PCR in BM mesenchymal cells following a 24-hr coculture with MM
cells. The isoenzyme CYP26A1 was highly upregulated by all 3 MM
cell lines tested (FIG. 5A-C). In contrast, the isoenzyme CYP26B1
showed little to no changes in mRNA levels. Conditioned media
derived from MM cells also upregulated CYP26A1 in BM stromal cells,
although to a lesser extent. This could be explained by the
presence of physical interactions in coculture experiments, or the
lack of continuous production of soluble ligands by MM cells in
conditioned media experiments. Consistent with the latter, stromal
CYP26A1 was highly upregulated when MM and BM stromal cells were
separated by a Transwell that prevented physical contact but
allowed the diffusion of soluble factors (FIG. 5A-C).
[0120] MM cells produce a variety of soluble factors including
cytokines (IL-1, IL-3, IL-6, TNF-.alpha.) as well as Hedgehog
ligands such as sonic hedgehog (SHH), which could impact the BM
stromal compartment. Therefore, it was determined whether any of
these factors was responsible for the observed upregulation of
CYP26A1 on BM stromal cells. Of the soluble factors tested, only
SHH produced a sustained overexpression of CYP26A1, while IL-1,
IL-3, IL-6, and TNF-.alpha. had no significant effects. Whereas SHH
is expressed by BM stromal cells and thus may be able to activate
the Hedgehog pathway in an autocrine manner, its expression was
considerably higher in MM cells compared with that detected in BM
stroma, suggesting that paracrine activation may play a dominant
role. Consistent with this, there was a statistically significant
correlation between the mRNA levels of SHH in MM cells and
activation of stromal Hedgehog signaling as determined by protein
patched homolog 1 (PTCH1) expression. Moreover, activation of
stromal Hedgehog significantly correlated with CYP26A1
upregulation. Specifically, MM1S cells with the highest expression
of SHH also induced the highest expression of both PTCH1 and CY26A1
in stromal cells. SHH has a half-life of less than 1 hr, which may
explain the reduced effect of MM-conditioned media on stromal
CYP26A1 expression compared with that observed in coculture and
Transwell experiments.
[0121] To confirm the role of paracrine Hedgehog on this
interaction, smoothened (Smo), a membrane receptor that transduces
SHH signaling, was knocked out at the genomic level in the
mesenchymal compartment. For this, BM mesenchymal cells derived
from Smo.sup.fl/fl mice were transduced with a retroviral vector
encoding Cre recombinase (Smo-KO stroma). Mouse Smo.sup.fl/fl
0stromal cells transduced with an empty retroviral vector were used
as a control (WT stroma). The transduced BM stromal cells were
cocultured with MM cells for 24 hr. As expected, Smo-KO stroma had
a decreased ability to upregulate Cyp26a1 in response to MM cells
compared with WT stroma (FIG. 6A-C). Similarly, the SMO inhibitor
cyclopamine partially overcame stromal Cyp26a1 upregulation by MM
cells. These data suggest that MM cells modulate stromal CYP26
expression at least in part via paracrine SHH.
[0122] Paracrine Hedgehog produced by MM cells reinforces a
protective microenvironment. Given the observations that stromal
CYP26 activity may be responsible for BTZ resistance in MM cells,
it was assessed whether paracrine Hedgehog secreted by MM cells
reinforces a chemoprotective niche by regulating retinoid
metabolism. It was first investigated whether modulation of
Hedgehog signaling paralleled the retinoid-dependent phenotypes
observed previously. Disruption of paracrine Hedgehog signaling in
Smo-KO stroma cocultures partially restored PC differentiation
(downregulation of BCL6 and upregulation of BLIMP1, XBP1, and CHOP)
in H929 (FIG. 9A-D) and primary CD138+ MM cells (FIG. 9E-H).
Surface expression of CD138 was also restored in the presence of
Smo-KO stroma. As expected, these findings were associated with an
increased sensitivity to BTZ of MM cells treated in the presence of
Smo-KO stroma compared with WT stroma.
[0123] To demonstrate that paracrine Hedgehog indeed induces a
BTZ-resistant phenotype by increasing the ability of BM stroma to
inactivate retinoids, Cyp26a1 expression in Smo-KO stroma was
rescued via lentivirus-mediated gene transfer (pBABE-Cyp26a1) in
order to achieve comparable CYP26A1 levels in WT (WT-Cyp26a1) and
Smo-KO (Smo-KO-Cyp26a1) stromal cells. If the role of paracrine
Hedgehog was independent of retinoid signaling, the relative
inability of Smo-KO stroma to induce a B cell phenotype and BTZ
resistance should have persisted even after Cyp26a1 upregulation.
However, Cyp26a1 overexpression rescued the ability of Smo-KO
stroma to induce a B cell phenotype and restored the expression of
differentiation markers and BTZ resistance to levels comparable to
those detected in WT and WT-Cyp26a1 stroma coculture conditions.
This finding is consistent with the hypothesis that paracrine
Hedgehog reinforces a protective niche via Cyp26a1
upregulation.
[0124] To study to what extent an RA-low environment created by the
BM stroma and enhanced by MM cells via paracrine Hedgehog signaling
contributes to BTZ resistance, a xenograft model of MM-niche
interactions was developed. Each mouse carried 2 subcutaneous
tumors consisting of H929 Luc+ cells and either WT (anterior
tumors) or Smo-KO stroma (posterior tumors). Mice were treated with
IRX (10 mg/kg i.p. daily), BTZ (0.5 mg/kg i.p. twice weekly), or a
combination of both. The growth of tumors bearing WT or Smo-KO
stroma was not different in untreated or IRX-treated groups (FIGS.
7 and 8). Consistent with in vitro data, tumors with WT stroma were
refractory to BTZ treatment, as determined by an exponential
increase in bioluminescence, while tumors carrying Smo-KO stroma
showed a significant response (FIG. 8). Moreover, the combination
of IRX and BTZ resulted in a significant and equivalent response,
regardless of the phenotype of the stromal compartment (FIG. 8).
While some tumors in the treatment group receiving combined IRX and
BTZ appeared to have regressed completely, even after anatomical
study, this was not the case for all the mice in this group. Flow
cytometric analyses of the tumors after treatment revealed no
differences in the in vivo growth of WT or Smo-KO stroma. Taken
together, these data suggest that paracrine Hedgehog secreted by MM
cells modulates retinoid signaling and BTZ sensitivity in the BM
niche via CYP26A1 upregulation.
[0125] Given their high secretion of Ig, PCs are particularly
sensitive to proteasome inhibition, and this accounts for the high
remission rates achieved in MM patients treated with this family of
drugs. Nonetheless, BTZ has failed to achieve a cure. A population
of MM cells, phenotypically similar to B cells, survive BTZ
treatment and are able to differentiate into PCs and recapitulate
the original disease. Despite efficient elimination of MM PCs,
these MM B cells survive BTZ treatment and become the predominant
cell population during MRD. Consequently, new therapeutic
strategies targeting MM B cells are required. A retinoid-low
microenvironment created by stromal CYP26 maintained an immature,
BTZ-resistant phenotype in MM. Thus, these data reveal a
therapeutic opportunity to overcome BTZ resistance in the MM
microenvironment using CYP26-resistant retinoids.
[0126] Despite being extensively studied in many hematological
malignancies, the use of retinoids as differentiation therapy has
proved beneficial only in patients with acute promyelocytic
leukemia (APL). CYP26 expression by BM stromal cells may explain
the lack of a clinical benefit of natural retinoids, despite their
in vitro activity. Recent studies have highlighted the efficacy of
CYP-resistant synthetic retinoids in differentiating cancer cells
and sensitizing them to targeted therapy. For instance, AM80
differentiates FMS-like tyrosine kinase 3/internal tandem
duplication (FLT3/ITD) acute myeloid leukemia (AML) cells and
increase their sensitivity to FLT3 inhibitors. Similarly, synthetic
retinoids reverse a stem cell phenotype in BCR-ABL1+ leukemic
lymphoblasts and substantially increase their responsiveness to
tyrosine kinase inhibitor (TKI) therapy in vivo. Such strategies to
bypass stromal CYP26 could expand the clinical effectiveness of
retinoid therapy.
[0127] MM cells utilize physical contacts to maintain drug
resistance and survive within the BM niche. Thus, therapeutic
strategies to overcome stromal chemoprotection have focused on
mobilization of malignant cells from the BM niche by targeting
adhesion molecules or chemokines such as CXCR4. MM cells exposed to
a retinoid-low microenvironment acquire a BTZ-resistant phenotype
that is maintained even after these cells are displaced from their
niche. Initial clinical studies have shown improved response rates
in relapse/refractory patients receiving the CXCR4 inhibitor
plerixafor in combination with BTZ; however, this data suggest that
such mobilization approaches may be insufficient to eliminate MM B
cells.
[0128] Recent studies have demonstrated the existence of a
bidirectional communication, in which not only stromal cells
provide a chemoprotective niche, but also cancer cells actively
shape and reinforce their microenvironment. The role of paracrine
Hedgehog has been studied extensively in solid malignancies. In
this system, ligands secreted by cancer cells activate the Hedgehog
pathway in neighboring stromal cells, enhancing their
chemoprotective properties via incompletely understood mechanisms.
This data suggest that paracrine Hedgehog may work at least in part
by increasing the ability of stroma to inactivate retinoids through
upregulation of CYP26 and thus to maintain a BTZ-resistant
phenotype in MM. Interestingly, CYP26 upregulation is associated
with an "activated stromal subtype" and a significantly worse
prognosis in patients with pancreatic cancer, a disease in which
paracrine Hedgehog signaling is well established. The extent to
which Hedgehog ligands produced by cancer cells contribute to this
"activated" stromal phenotype and high CYP26 levels is unknown.
Moreover, BM mesenchymal cells migrate and become a relevant cell
population in the stromal compartment of these tumors.
[0129] The endosteal region is the primary niche of MM, AML, and
micrometastatic disease from solid tumors. Within the osteoblastic
region, these cancer cells maintain a quiescent, stem cell
phenotype and are protected from chemotherapy-induced apoptosis. It
is likely that these cancer cells rely on the same cues from the BM
microenvironment as normal hematopoietic stem cells do to survive
chemotherapy and perpetuate the disease. The BM microenvironment
protected MM and AML cells by directly inactivating various
chemotherapy agents via expression of CYP3A4 and other detoxifying
enzymes. Another potential mechanism of microenvironment-mediated
drug resistance is now demonstrated: creation of a retinoid-low
niche that maintains a drug-resistant B cell phenotype. A
CYP26-resistant retinoid potentiated the activity of BTZ against MM
in the BM niche provides a therapeutic opportunity to bypass this
mechanism of resistance.
Example 2
CYP26-Resistant RAR.alpha. Agonists Overcome Bone Marrow (BM)
Protection of AML by CYP26
[0130] All-trans retinoic acid (ATRA), acting through RAR.alpha.,
causes terminal differentiation and apoptosis of non-APL AML cells
in vitro but is clinically ineffective against AML. The BM stroma,
which expresses inducibly high levels of CYP26, metabolically
inactivates ATRA and provides a protected environment for AML
cancer stem cells. Synthetic, CYP26-resistant RAR.alpha. agonists,
such as IRX5183 and AM80, should be able to bypass stroma-mediated
protection of AML cells and differentiate the cancer stem cells
resident in the protected environment of bone marrow niche. To test
these hypotheses, clonogenic growth experiments were conducted in
the non-APL AML cell lines OCI-AML 3 and Kasumi-1 and APL NB4
cells, which were treated with ATRA, IRX5183, and AM80 in the
presence or absence of stroma. ATRA inhibited clonogenic growth
only in the absence of stroma. In contrast, IRX5183 inhibited
clonogenic growth to a similar extent in the presence or absence of
stroma (FIG. 10A-C). AM80 also inhibited clonogenic growth in the
presence of stroma but not as effectively as IRX5183. These data
indicate that CYP26-resistant, RAR.alpha. agonists such as IRX5183
may be effective treatments for AML by overcoming stromal
mechanisms of drug resistance.
Example 3
Phase I/II Clinical Study of IRX5183 in Relapsed and Refractory
Myeloid Malignancies
[0131] Acute myeloid leukemia (AML) is successfully treated in only
30-40% of younger patients and very few older patients with
standard chemotherapy regimens. Given the clinical activity of
all-trans retinoic acid (ATRA; retinoic acid, RA) in acute
promyelocytic leukemia (APL), ATRA was considered an attractive
therapeutic strategy for other AML subtypes. APL, and most non-APL
AMLs undergo terminal differentiation and are therefore
successfully treated by ATRA in vitro. However, ATRA has not proven
effective in non-APL AMLs in clinical trials.
[0132] Retinoic acid (RA) plays a significant role in the
differentiation of hematopoietic stem cells (HSCs). The cytochrome
P450 enzyme CYP26, expressed in bone marrow (BM) stromal cells,
inactivates RA, thereby limiting differentiation of HSCs. Several
AML cell lines, both APL and non-APL, are sensitive to RA-induced
differentiation, but this effect was abrogated in the presence of
BM stroma. Thus, it may be useful to treat AML with a retinoid that
is resistant to metabolism by the CYP26 pathway. IRX5183 is a
RAR.alpha. selective agonist which is resistant to CYP26
metabolism. Use of IRX5183 in AML provides a novel targeted
approach to this disease, which has the potential to change the
prognosis of this and other hematologic malignancies. Thus, a phase
I/II clinical trial will be conducted of IRX5183 in
relapsed/refractory AML and high risk myelodysplastic syndrome
(HR-MDS).
[0133] Study Objectives
[0134] Dose escalation phase primary objectives:
[0135] 1) Evaluate safety and toxicity associated with
administration of IRX5183 in patients with relapsed and refractory
AML by determining the dose limiting toxicities (DLT) and
maximally-tolerated dose (MTD).
[0136] 2) Determine pharmacokinetic (PK) parameters of IRX5183 in
the peripheral blood.
[0137] Dose escalation phase secondary objectives:
[0138] 1) Determine the PK parameters of IRX5183 in the bone
marrow.
[0139] 2) Define differentiation profiles associated with IRX5183,
BM cellular retinoid concentrations, blast counts, and cytogenetics
at different dose levels.
[0140] Dose expansion phase primary objectives:
[0141] 1) Define differentiation markers, BM retinoid
concentrations, blast counts, and cytogenetics in AML and HR-MDS
patients at the optimal dose level.
[0142] 2) Obtain preliminary efficacy data of IRX5183 in terms of
complete response (CR), partial response (PR), and hematological
improvement (HI) in both cohorts of patients.
[0143] Dose expansion phase secondary objectives:
[0144] 1) Define toxicity profiles of IRX5183 at the optimal dose
in both patient cohorts.
[0145] 2) Obtain data on correlations between IRX5183-induced
differentiation and toxicity and clinical responses.
[0146] Eligibility criteria--dose escalation/determination. This
phase will only recruit patients with AML:
[0147] 1. Patients must be able to understand and voluntarily sign
an informed consent form.
[0148] 2. Age 18-70 years at the time of signing the informed
consent.
[0149] 3. Able to adhere to the study visit schedule and other
protocol requirements.
[0150] 4. Life expectancy of greater than 6 months.
[0151] 5. Must have pathologically confirmed AML with one or two
prior courses of induction chemotherapy or hypomethylating agent
therapy or relapsed after complete remission, before or after
allogeneic bone marrow transplant, AND no plans for further
intensive chemotherapy.
[0152] 6. Patients must not have received any other treatment for
their disease (aside from hydroxyurea for control of blast count in
AML patients), including hematopoietic growth factors, within three
weeks of beginning the trial, and should have recovered from all
toxicities of prior therapy (to grade 0 or 1).
[0153] 7. ECOG performance status of 2 at study entry, or Karnofsky
.gtoreq.60%.
[0154] 8. Laboratory test results within these ranges: [0155] a.
Calculated creatinine clearance by MDRD (CrCL)>50 ml/min/1.73
squared meter [0156] b. Total bilirubin.ltoreq.2.0 mg/dL unless due
to Gilbert's syndrome, hemolysis, or ineffective hematopoiesis AST
(SGOT) and ALT (SGPT) 3 x ULN [0157] 9. Females of childbearing
potential must have negative pregnancy test. [0158] 10. Patients
must have no clinical evidence of CNS or pulmonary leukostasis,
disseminated intravascular coagulation, or CNS leukemia.
[0159] 11. Patients must have no serious or uncontrolled medical
conditions.
[0160] Eligibility criteria--dose expansion. This phase will
recruit patients with relapsed/refractory AML (cohort 1) and
patients with HR-MDS not responding to hypomethylating agents
(cohort 2) and will follow the noted eligibility criteria above
(aside from #5 above in MDS patients), including pathologically
confirmed CMML or MDS with high risk features at the time of
referral as defined by:
[0161] 1. INT-2 or high IPSS score
[0162] 2. Secondary MDS
[0163] 3. INT-1 MDS with excess blasts 5% blasts in BM) or RBC or
platelet transfusion-dependency 4. CMML with 5% marrow blasts, or
RBC or platelet transfusion-dependency, abnormal karyotype, or
proliferative features
[0164] All HR-MDS patients are required to have failed or relapsed
after an initial response to hypomethylating agents or have refused
to receive hypomethylating therapy. Failure to respond is defined
as failing to achieve a CR, PR or HI after at least 4 cycles of
hypomethylating therapy.
[0165] Treatment Plan
[0166] For the dose escalating phase, IRX5183 is administered
orally in daily doses continually in 28 day cycles until toxicity
or disease progression. Bone marrow testing during each of the
first 4 cycles determines marrow status and response. Only patients
with relapsed or refractory AML are enrolled in the dose escalation
phase. The starting dose (DL1) of single agent IRX5183 is 30
mg/m.sup.2/day, and the individual dosing levels are noted
below:
TABLE-US-00002 Dose level (DL) Daily dose (mg/m.sup.2) DL(-1) 15
DL1 30 DL2 45 DL3 60 DL4 75
[0167] The phase-expansion part of the study uses the optimal dose
identified in the phase-escalation part of the study and includes
two separate arms; one for AML patients and another for HR-MDS, and
each of these two arms will recruit 26 patients.
[0168] Dose levels are explored according to a traditional 3+3
design, with an aim to enroll 3 subjects at a time to determine the
toxicity profile of IRX5183 in AML patients. If none of the three
patients receiving DL1 experiences a DLT, another three patients
will be treated at the next higher dose level. However, if one of
the first three patients experiences a DLT, three more patients
will be treated at the same dose level. The dose escalation will
continue until at least two patients among a cohort of 3-6 patients
experience DLTs. If two or more patients experience DLT on DL1, the
next patient will be recruited to DL(-1). The MTD of single agent
IRX5183 will be the highest dose at which 0 or 1 DLT are seen in a
cohort of six subjects.
[0169] For the phase 2 dose expansion cohort, patients with AML are
continued to be enrolled at the MTD, with goal of enrolling 26
patients (inclusive of patients treated at the MTD in first phase
of the trial). Patients continue on single agent IRX5183 until they
experience toxicity or disease progression. If patients achieve a
complete remission, they have the option to consolidate with
transplant, chemotherapy, and/or continue on maintenance IRX5183.
If patients achieve a partial response or hematologic improvement
they have the option to obtain salvage therapy in combination with
IRX5183. For MDS patients, the phase 2 dose expansion cohort will
recruit 26 patients who receive single agent IRX5183 at the MTD
from the first phase of the trial. They also continue until undue
toxicity or disease progression. Patients who achieve hematologic
improvement or better have the option to consolidate with
transplant, combine with demethylating agent therapy, or continue
on maintenance IRX5183.
[0170] Pharmacokinetics Analyses
[0171] Plasma concentrations of IRX5183 are evaluated for the
escalation and expansion phases, targeting safe and effective
retinoid levels by pharmacokinetics using LCM-MS (liquid
chromatography-mass spectroscopy tandem). Targeting peak levels of
1 .mu.M should avoid systemic toxicity, while presumably preserving
local BM niche retinoid levels. The plasma concentration of IRX5183
are obtained using a single 2 mL blood sample, pre-dose on day 14.
Samples are shipped to and analyzed by the designated analytical
laboratory.
[0172] Pharmacodynamics Analyses
[0173] In addition to assessing standard clinical response
criteria, BM cellular (normal HSCs and LSCs) concentrations,
peripheral blood and bone marrow blast counts, markers of
differentiation, apoptosis, and clonogenic growth are determined. A
bone marrow aspirate and biopsy are obtained at baseline, on day
14, and at the end each of the first 4 cycles of therapy.
Differentiation is assessed using flow cytometry, comparing
expression of differentiation markers on CD45 positive cells and
ALDHint LSCs on day 14 marrow versus baseline. FISH analysis is
also conducted after each cycle for patients with baseline
abnormalities to determine if leukemic clone still present on day
14.
[0174] Expected outcomes: Patients receiving RAR.alpha. selective
agonist are monitored for response criteria based on hematological
parameters including complete blood counts and percentage of
leukemia blasts in the peripheral blood and in the bone marrow.
Patients with improved neutrophil count, decreased transfusion
requirements of red blood cells and platelets together with
decreased percentage of blasts in the bone marrow and induction of
differentiation and apoptosis of these malignant blasts are deemed
responsive to therapy. Quality of life parameters such as pain,
performance status and participation in activity of daily living
and instrumental activities of daily living are assessed to
evaluate the impact of this therapy on study patients. Use of this
RAR.alpha. selective agonist is expected to improve hematological
and quality of life parameters in patients with MDS/AML and solid
malignancies. In addition, the use of the RAR.alpha. agonist which
is CYP26 resistant may result in differentiation and thus
elimination of minimal residual disease in the bone marrow of these
patients.
[0175] The ultimate goal is to develop better treatments for
patients with relapsed/refractory AML and this study will provide
valuable insights into the use of novel retinoids in this
setting.
[0176] Unless otherwise indicated, all numbers expressing
quantities of ingredients, properties such as molecular weight,
reaction conditions, and so forth used in the specification and
claims are to be understood as being modified in all instances by
the term "about." As used herein the terms "about" and
"approximately" means within 10 to 15%, preferably within 5 to 10%.
Accordingly, unless indicated to the contrary, the numerical
parameters set forth in the specification and attached claims are
approximations that may vary depending upon the desired properties
sought to be obtained by the present invention. At the very least,
and not as an attempt to limit the application of the doctrine of
equivalents to the scope of the claims, each numerical parameter
should at least be construed in light of the number of reported
significant digits and by applying ordinary rounding techniques.
Notwithstanding that the numerical ranges and parameters setting
forth the broad scope of the invention are approximations, the
numerical values set forth in the specific examples are reported as
precisely as possible. Any numerical value, however, inherently
contains certain errors necessarily resulting from the standard
deviation found in their respective testing measurements.
[0177] The terms "a," "an," "the" and similar referents used in the
context of describing the invention (especially in the context of
the following claims) are to be construed to cover both the
singular and the plural, unless otherwise indicated herein or
clearly contradicted by context. Recitation of ranges of values
herein is merely intended to serve as a shorthand method of
referring individually to each separate value falling within the
range. Unless otherwise indicated herein, each individual value is
incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein is intended
merely to better illuminate the invention and does not pose a
limitation on the scope of the invention otherwise claimed. No
language in the specification should be construed as indicating any
non-claimed element essential to the practice of the invention.
[0178] Groupings of alternative elements or embodiments of the
invention disclosed herein are not to be construed as limitations.
Each group member may be referred to and claimed individually or in
any combination with other members of the group or other elements
found herein. It is anticipated that one or more members of a group
may be included in, or deleted from, a group for reasons of
convenience and/or patentability. When any such inclusion or
deletion occurs, the specification is deemed to contain the group
as modified thus fulfilling the written description of all Markush
groups used in the appended claims.
[0179] Certain embodiments of this invention are described herein,
including the best mode known to the inventors for carrying out the
invention. Of course, variations on these described embodiments
will become apparent to those of ordinary skill in the art upon
reading the foregoing description. The inventor expects skilled
artisans to employ such variations as appropriate, and the
inventors intend for the invention to be practiced otherwise than
specifically described herein. Accordingly, this invention includes
all modifications and equivalents of the subject matter recited in
the claims appended hereto as permitted by applicable law.
Moreover, any combination of the above-described elements in all
possible variations thereof is encompassed by the invention unless
otherwise indicated herein or otherwise clearly contradicted by
context.
[0180] Specific embodiments disclosed herein may be further limited
in the claims using consisting of or consisting essentially of
language. When used in the claims, whether as filed or added per
amendment, the transition term "consisting of" excludes any
element, step, or ingredient not specified in the claims. The
transition term "consisting essentially of" limits the scope of a
claim to the specified materials or steps and those that do not
materially affect the basic and novel characteristic(s).
Embodiments of the invention so claimed are inherently or expressly
described and enabled herein.
[0181] Furthermore, numerous references have been made to patents
and printed publications throughout this specification. Each of the
above-cited references and printed publications are individually
incorporated herein by reference in their entirety.
[0182] In closing, it is to be understood that the embodiments of
the invention disclosed herein are illustrative of the principles
of the present invention. Other modifications that may be employed
are within the scope of the invention. Thus, by way of example, but
not of limitation, alternative configurations of the present
invention may be utilized in accordance with the teachings herein.
Accordingly, the present invention is not limited to that precisely
as shown and described.
* * * * *