U.S. patent application number 16/914703 was filed with the patent office on 2020-12-17 for formulations of milciclib and therapeutic combinations of the same for use in the treatment of cancer.
The applicant listed for this patent is Tiziana Life Sciences PLC. Invention is credited to Kunwar SHAILUBHAI.
Application Number | 20200390769 16/914703 |
Document ID | / |
Family ID | 1000005059414 |
Filed Date | 2020-12-17 |
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United States Patent
Application |
20200390769 |
Kind Code |
A1 |
SHAILUBHAI; Kunwar |
December 17, 2020 |
FORMULATIONS OF MILCICLIB AND THERAPEUTIC COMBINATIONS OF THE SAME
FOR USE IN THE TREATMENT OF CANCER
Abstract
This application relates to methods of treating and/or
preventing cancer (e.g., non-small cell lung cancer, renal cell
carcinoma, hepatocellular carcinoma, thyroid carcinoma, colorectal
cancer, gastrointestinal stromal tumors, breast cancer, prostate
cancer, pancreatic cancer, or thymoma) in patients in need thereof
comprising administering to the patient a therapeutically effective
amount of a CDK inhibitor (e.g., milciclib) in combination with a
therapeutically effective amount of another anticancer drug (e.g.,
sorafenib, lenvatinib, regorafenib, sunitinib, nivolumab,
gemcitabine, and palbociclib).
Inventors: |
SHAILUBHAI; Kunwar; (Line
Lexiginton, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Tiziana Life Sciences PLC |
London |
|
GB |
|
|
Family ID: |
1000005059414 |
Appl. No.: |
16/914703 |
Filed: |
June 29, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16182036 |
Nov 6, 2018 |
10758541 |
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16914703 |
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62582288 |
Nov 6, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/404 20130101;
A61K 31/47 20130101; A61K 9/48 20130101; A61K 9/0053 20130101; A61K
9/20 20130101; A61K 31/4412 20130101; A61K 2300/00 20130101; A61P
35/00 20180101; A61K 31/519 20130101 |
International
Class: |
A61K 31/519 20060101
A61K031/519; A61P 35/00 20060101 A61P035/00; A61K 9/00 20060101
A61K009/00; A61K 31/404 20060101 A61K031/404; A61K 31/4412 20060101
A61K031/4412; A61K 31/47 20060101 A61K031/47 |
Claims
1. A method of treating or preventing cancer in a patient in need
thereof, comprising administering to the patient a therapeutically
effective amount of a CDK inhibitor, or a pharmaceutically
acceptable salt, isomer, or tautomer thereof, in combination with a
therapeutically effective amount of another anticancer drug.
2. The method of claim 1, wherein the cancer is non-small cell lung
cancer, renal cell carcinoma, hepatocellular carcinoma, thyroid
carcinoma, melanoma, multiple myeloma, mantle cell lymphoma,
non-Hodgkin's lymphoma, colorectal cancer, acute lymphocytic
leukemia, chronic lymphocytic leukemia, chronic myelogenous
leukemia, skin cancer, ovarian cancer, gastrointestinal stromal
tumors, breast cancer, prostate cancer, pancreatic cancer, or
thymoma.
3. The method of claim 1, wherein the CDK inhibitor is milciclib or
a pharmaceutically acceptable salt thereof, and the other
anticancer drug is sorafenib, lenvatinib, regorafenib, sunitinib,
nivolumab, gemcitabine, palbociclib, afatinib, alectinib, axitinib,
bortezomib, bosutinib, cabozantinib, carfilzomib, ceritinib,
cobimetinib, crizotinib, dabrafenib, erlotinib, gefitinib,
ibrutinib, idelalisib, imatinib, ixazomib, lapatinib, nilotinib,
nintedanib, niraparib, osimertinib, pazopanib, pegaptanib,
ponatinib, rucaparib, ruxolitinib, sonidegib, tofacitinib,
trametinib, vandetanib, vemurafenib, vismodegibor, or a
pharmaceutically acceptable salt thereof.
4. The method of claim 3, wherein the other anticancer drug is
sorafenib or a pharmaceutically acceptable salt thereof.
5. The method of claim 4, wherein the therapeutically effective
amount of sorafenib is 400 mg twice daily, 200 mg twice daily, or
200 mg once daily.
6. The method of claim 4, wherein the cancer is renal cell
carcinoma or thyroid carcinoma.
7. The method of claim 3, wherein the other anticancer drug is
lenvatinib or a pharmaceutically acceptable salt thereof.
8. The method of claim 7, wherein the therapeutically effective
amount of lenvatinib is 8, 10, 12, 14, 18, 20, 22, 24, 26, 28, 30,
32, or 34 mg once daily.
9. The method of claim 7, wherein the cancer is renal cell
carcinoma or thyroid carcinoma.
10. The method of claim 3, wherein the other anticancer drug is
regorafenib or a pharmaceutically acceptable salt thereof.
11. The method of claim 10, wherein the therapeutically effective
amount of regorafenib is 80, 100, or 120 mg once daily for three
weeks, followed by one week of no administration, wherein the cycle
is optionally repeated.
12. The method of claim 10, wherein the cancer is colorectal cancer
or gastrointestinal stromal tumors.
13. The method of claim 3, wherein the other anticancer drug is
sunitinib or a pharmaceutically acceptable salt thereof.
14. The method of claim 13, wherein the therapeutically effective
amount of sunitinib is 12.5, 25, 37.5, 50, 62.5, 75, 87.5, or 100
mg once daily continuously or for 4 weeks followed by two weeks of
no administration, wherein the cycle is optionally repeated.
15. The method of claim 13, wherein the cancer is renal cell
carcinoma or gastrointestinal stromal tumors.
16. The method of claim 3, wherein the other anticancer drug is
nivolumab.
17. (canceled)
18. The method of claim 3, wherein the other anticancer drug is
gemcitabine or a pharmaceutically acceptable salt thereof.
19. The method of claim 18, wherein the therapeutically effective
amount of gemcitabine is 1000 mg/m.sup.2 over 30 minutes once
weekly for seven weeks, followed by one week of no administration,
wherein the cycle is optionally repeated.
20. (canceled)
21. The method of claim 3, wherein the other anticancer drug is
palbociclib or a pharmaceutically acceptable salt thereof.
22. The method of claim 21, wherein the therapeutically effective
amount of palbociclib is 75, 100, or 125 mg once daily for 3 weeks
followed by one week of no administration, wherein the cycle is
optionally repeated.
23. (canceled)
24. The method of claim 3, wherein the therapeutically effective
amount of milciclib is 50, 75, 100, 125, or 150 mg once daily for
four consecutive days, followed by non-administration for 3
consecutive days, wherein the cycle is optionally repeated.
25.-52. (canceled)
53. The method of claim 1, wherein the CDK inhibitor and the other
anticancer drug are administered in temporal proximity.
54.-62. (canceled)
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 16/182,036, filed Nov. 6, 2018, now allowed, which claims the
benefit of, and priority to, U.S. Provisional Application No.
62/582,288, filed Nov. 6, 2017, each of which are incorporated by
reference herein in their entireties for all purposes.
TECHNICAL FIELD
[0002] This application relates generally to the treatment of
cancers, and more particularly relates to the treatment of cancers
with a combination of a cyclin-dependent kinase (CDK) inhibitor and
at least one additional anticancer drug. The invention finds
utility in the fields of medicine and pharmacotherapy.
BACKGROUND
[0003] Milciclib, which may be referred herein to as Compound 1, or
N,1,4,4-tetramethyl-8-((4-(4-methylpiperazin-1-yl)phenyl)amino)-4,5-dihyd-
ro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide, has the following
structure:
##STR00001##
[0004] Milciclib is a small molecule inhibitor of multiple CDKs,
including CDK1, CDK2, CDK4, CDK5, CDK7, and CDK9, and TRKs (TPKA
and TRKC), has shown efficacy in several preclinical tumor models
(Albanese C et al. (2010) Mol Cancer Ther 9:2243-2254.). In a phase
I study, oral treatment with milciclib was found to be
well-tolerated and the drug showed promising clinical responses in
patients with advanced solid malignancies such as in thymic
carcinoma, pancreatic carcinoma and colon cancer (Weiss G J et al.
(2013) Invest New Drugs 31:136-144.) The major toxicity profile
consisted of tremors and gastrointestinal toxicity which was
reversible upon treatment suspension. Results from this study
recommended a RP2D of 150 mg/day.
[0005] Particularly, hepatocellular carcinoma (HCC) is an extremely
complex multi-factorial condition associated with many confounding
factors affecting disease course and patient prognosis. A broad
range of mechanisms, including telomere dysfunction, activation of
oncogenic pathways, abrogation of DNA damage checkpoints,
activation of pro-inflammatory and metastatic pathways, and
induction of the oxidative stress response. Thus, an effective
therapy for HCC needs to control proliferation of hepatocytes and
also suppress their metastatic potential. Milciclib, exhibiting
broad-spectrum inhibitory activities against CDKs, effectively
retards proliferation of cancer cells. Therefore, it is reasonable
to propose that anticancer activity of milciclib may be potentiated
by an inhibitor of tyrosine kinase to produce synergistic
anti-tumorigenic activity.
[0006] There is a need for novel therapies by using milciclib in
combination with a second anticancer drug or agent for the
treatment of cancer. The present application addresses such a
need.
SUMMARY OF THE INVENTION
[0007] In one aspect, this application pertains to a method of
treating or preventing cancer in a patient in need thereof,
comprising administering to the patient a therapeutically effective
amount of a CDK inhibitor, or a pharmaceutically acceptable salt,
isomer, or tautomer thereof, in combination with a therapeutically
effective amount of another anticancer drug.
[0008] In one aspect, this application pertains to a method of
treating or preventing cancer in a patient in need thereof, wherein
the cancer is non-small cell lung cancer, renal cell carcinoma,
hepatocellular carcinoma, thyroid carcinoma, melanoma, multiple
myeloma, mantle cell lymphoma, non-Hodgkin's lymphoma, colorectal
cancer, acute lymphocytic leukemia, chronic lymphocytic leukemia,
chronic myelogenous leukemia, skin cancer, ovarian cancer,
gastrointestinal stromal tumors, breast cancer, prostate cancer,
pancreatic cancer, or thymoma.
[0009] In one aspect, this application pertains to a method of
treating or preventing cancer in a patient in need thereof, wherein
the CDK inhibitor is milciclib or a pharmaceutically acceptable
salt thereof, and the other anticancer drug is sorafenib,
lenvatinib, regorafenib, sunitinib, nivolumab, gemcitabine,
palbociclib, afatinib, alectinib, axitinib, bortezomib, bosutinib,
cabozantinib, carfilzomib, ceritinib, cobimetinib, crizotinib,
dabrafenib, erlotinib, gefitinib, ibrutinib, idelalisib, imatinib,
ixazomib, lapatinib, nilotinib, nintedanib, niraparib, osimertinib,
pazopanib, pegaptanib, ponatinib, rucaparib, ruxolitinib,
sonidegib, tofacitinib, trametinib, vandetanib, vemurafenib,
vismodegibor, or a pharmaceutically acceptable salt thereof.
[0010] In one aspect, this application pertains to a method of
treating or preventing cancer in a patient in need thereof, wherein
the other anticancer drug is sorafenib or a pharmaceutically
acceptable salt thereof.
[0011] In one aspect, this application pertains to a method wherein
the therapeutically effective amount of sorafenib is 400 mg twice
daily, 200 mg twice daily, or 200 mg once daily.
[0012] In one aspect, this application pertains to a method of
treating or preventing cancer in a patient in need thereof, wherein
the cancer is renal cell carcinoma, hepatocellular carcinoma, or
thyroid carcinoma.
[0013] In one aspect, this application pertains to a method of
treating or preventing cancer in a patient in need thereof, wherein
the other anticancer drug is lenvatinib or a pharmaceutically
acceptable salt thereof.
[0014] In one aspect, this application pertains to a method of
treating or preventing cancer in a patient in need thereof, wherein
the therapeutically effective amount of lenvatinib is 8, 10, 12,
14, 18, 20, 22, 24, 26, 28, 30, 32, or 34 mg once daily.
[0015] In one aspect, this application pertains to a method of
treating or preventing cancer in a patient in need thereof, wherein
the cancer is renal cell carcinoma or thyroid carcinoma.
[0016] In one aspect, this application pertains to a method of
treating or preventing cancer in a patient in need thereof, wherein
the other anticancer drug is regorafenib or a pharmaceutically
acceptable salt thereof.
[0017] In one aspect, this application pertains to a method of
treating or preventing cancer in a patient in need thereof, wherein
the therapeutically effective amount of regorafenib is 80, 100, or
120 mg once daily for three weeks, followed by one week of no
administration, wherein the cycle is optionally repeated.
[0018] In one aspect, this application pertains to a method of
treating or preventing cancer in a patient in need thereof, wherein
the cancer is colorectal cancer or gastrointestinal stromal
tumors.
[0019] In one aspect, this application pertains to a method of
treating or preventing cancer in a patient in need thereof, wherein
the other anticancer drug is sunitinib or a pharmaceutically
acceptable salt thereof.
[0020] In one aspect, this application pertains to a method of
treating or preventing cancer in a patient in need thereof, wherein
the therapeutically effective amount of sunitinib is 12.5, 25,
37.5, 50, 62.5, 75, 87.5, or 100 mg once daily continuously or for
4 weeks followed by two weeks of no administration, wherein the
cycle is optionally repeated.
[0021] In one aspect, this application pertains to a method of
treating or preventing cancer in a patient in need thereof, wherein
the cancer is renal cell carcinoma or gastrointestinal stromal
tumors.
[0022] In one aspect, this application pertains to a method of
treating or preventing cancer in a patient in need thereof, wherein
the other anticancer drug is nivolumab.
[0023] In one aspect, this application pertains to a method of
treating or preventing cancer in a patient in need thereof, wherein
the cancer is non-small cell lung cancer or renal cell
carcinoma.
[0024] In one aspect, this application pertains to a method of
treating or preventing cancer in a patient in need thereof, wherein
the other anticancer drug is gemcitabine or a pharmaceutically
acceptable salt thereof.
[0025] In one aspect, this application pertains to a method of
treating or preventing cancer in a patient in need thereof, wherein
the therapeutically effective amount of gemcitabine is 1000
mg/m.sup.2 over 30 minutes once weekly for seven weeks, followed by
one week of no administration, wherein the cycle is optionally
repeated.
[0026] In one aspect, this application pertains to a method of
treating or preventing cancer in a patient in need thereof, wherein
the cancer is breast cancer.
[0027] In one aspect, this application pertains to a method of
treating or preventing cancer in a patient in need thereof, wherein
the other anticancer drug is palbociclib or a pharmaceutically
acceptable salt thereof.
[0028] In one aspect, this application pertains to a method of
treating or preventing cancer in a patient in need thereof, wherein
the therapeutically effective amount of palbociclib is 75, 100, or
125 mg once daily for 3 weeks followed by one week of no
administration, wherein the cycle is optionally repeated.
[0029] In one aspect, this application pertains to a method of
treating or preventing cancer in a patient in need thereof, wherein
the cancer is breast cancer.
[0030] In one aspect, this application pertains to a method of
treating or preventing cancer in a patient in need thereof, wherein
the therapeutically effective amount of milciclib is 50, 75, 100,
125, or 150 mg once daily for four consecutive days, followed by
non-administration for 3 consecutive days, wherein the cycle is
optionally repeated.
[0031] In one aspect, this application pertains to any of the
methods described herein, wherein milciclib and the other
anticancer drug are administered to the patient simultaneously.
[0032] In one aspect, this application pertains to any of the
methods described herein, wherein milciclib and the other
anticancer drug are administered in a single pharmaceutical
formulation that further includes a pharmaceutically acceptable
excipient.
[0033] In one aspect, this application pertains to any of the
methods described herein, wherein the pharmaceutical formulation is
in a controlled release form.
[0034] In one aspect, this application pertains to any of the
methods described herein, wherein milciclib and the other
anticancer drug are each administered in separate pharmaceutical
formulations, wherein each formulation further includes a
pharmaceutically acceptable excipient.
[0035] In one aspect, this application pertains to any of the
methods described herein, wherein one or both of the pharmaceutical
formulations is in a controlled release form.
[0036] In one aspect, this application pertains to any of the
methods described herein, wherein milciclib and the other
anticancer drug are administered to the patient sequentially.
[0037] In one aspect, this application pertains to any of the
methods described herein, wherein administration of milciclib
begins before administration of the other anticancer to the
patient.
[0038] In one aspect, this application pertains to any of the
methods described herein, wherein administration of milciclib
begins after administration of the other anticancer to the
patient.
[0039] In one aspect, this application pertains to any of the
methods described herein, wherein milciclib is administered in a
single pharmaceutical formulation that further includes a
pharmaceutically acceptable excipient.
[0040] In one aspect, this application pertains to any of the
methods described herein, wherein the pharmaceutical formulation is
formulated for oral administration.
[0041] In one aspect, this application pertains to any of the
methods described herein, wherein the pharmaceutical formulation is
in the form of a tablet, pill, or capsule.
[0042] In one aspect, this application pertains to a method of
treating or preventing renal cell carcinoma in a patient in need
thereof comprising administering to the patient a therapeutically
effective amount of milciclib, or a pharmaceutically acceptable
salt, isomer, or tautomer thereof, in combination with a
therapeutically effective amount of sorafenib, or a
pharmaceutically acceptable salt, isomer, or tautomer thereof.
[0043] In one aspect, this application pertains to a method of
treating or preventing hepatocellular carcinoma in a patient in
need thereof comprising administering to the patient a
therapeutically effective amount of milciclib, or a
pharmaceutically acceptable salt, isomer, or tautomer thereof, in
combination with a therapeutically effective amount of sorafenib,
or a pharmaceutically acceptable salt, isomer, or tautomer
thereof.
[0044] In one aspect, this application pertains to a method of
treating or preventing thyroid carcinoma in a patient in need
thereof comprising administering to the patient a therapeutically
effective amount of milciclib, or a pharmaceutically acceptable
salt, isomer, or tautomer thereof, in combination with a
therapeutically effective amount of sorafenib, or a
pharmaceutically acceptable salt, isomer, or tautomer thereof.
[0045] In one aspect, this application pertains to any of the
methods described herein, wherein the therapeutically effective
amount of sorafenib is 400 mg twice daily, 200 mg twice daily, or
200 mg once daily.
[0046] In one aspect, this application pertains to any of the
methods described herein, wherein the therapeutically effective
amount of milciclib is 50, 75, 100, 125, or 150 mg once daily for
four consecutive days, followed by non-administration for 3
consecutive days, wherein the cycle is optionally repeated.
[0047] In one aspect, this application pertains to any of the
methods described herein, wherein milciclib and sorafenib are
administered to the patient simultaneously.
[0048] In one aspect, this application pertains to any of the
methods described herein, wherein milciclib and sorafenib are
administered in a single pharmaceutical formulation that further
includes a pharmaceutically acceptable excipient.
[0049] In one aspect, this application pertains to any of the
methods described herein, wherein the pharmaceutical formulation is
in a controlled release form.
[0050] In one aspect, this application pertains to any of the
methods described herein, wherein milciclib and sorafenib are
administered in separate pharmaceutical formulations, wherein each
formulation further includes a pharmaceutically acceptable
excipient.
[0051] In one aspect, this application pertains to any of the
methods described herein, wherein one or both of the pharmaceutical
formulations is in a controlled release form.
[0052] In one aspect, this application pertains to any of the
methods described herein, wherein milciclib and sorafenib are
administered to the patient sequentially.
[0053] In one aspect, this application pertains to any of the
methods described herein, wherein administration of milciclib
begins before administration of sorafenib to the patient.
[0054] In one aspect, this application pertains to any of the
methods described herein, wherein administration of milciclib
begins after administration of sorafenib to the patient.
[0055] In one aspect, this application pertains to any of the
methods described herein, wherein milciclib and sorafenib are each
administered in separate pharmaceutical formulations that each
further include a pharmaceutically acceptable excipient.
[0056] In one aspect, this application pertains to any of the
methods described herein, wherein one or both pharmaceutical
formulations are formulated for oral administration.
[0057] In one aspect, this application pertains to any of the
methods described herein, wherein each pharmaceutical formulation
is independently in the form of a tablet, pill, or capsule.
[0058] In one aspect, this application pertains to any of the
methods described herein, wherein milciclib and sorafenib are
administered in temporal proximity.
[0059] In one aspect, this application pertains to any of the
methods described herein, wherein the CDK inhibitor and the other
anticancer drug are administered in temporal proximity.
[0060] In one aspect, this application pertains to a pharmaceutical
composition comprising milciclib or a pharmaceutically acceptable
salt, isomer, or tautomer thereof, and another anticancer drug.
[0061] In one aspect, this application pertains to a pharmaceutical
composition comprising milciclib or a pharmaceutically acceptable
salt, isomer, or tautomer thereof, and another anticancer drug for
use in the treatment or prevention of non-small cell lung cancer,
renal cell carcinoma, hepatocellular carcinoma, thyroid carcinoma,
melanoma, multiple myeloma, mantle cell lymphoma, non-Hodgkin's
lymphoma, colorectal cancer, acute lymphocytic leukemia, chronic
lymphocytic leukemia, chronic myelogenous leukemia, skin cancer,
ovarian cancer, gastrointestinal stromal tumors, breast cancer,
prostate cancer, pancreatic cancer, or thymoma.
[0062] In one aspect, this application pertains to the use of a
pharmaceutical composition comprising milciclib or a
pharmaceutically acceptable salt, isomer, or tautomer thereof, and
another anticancer drug in the manufacture of a medicament for the
treatment or prevention of non-small cell lung cancer, renal cell
carcinoma, hepatocellular carcinoma, thyroid carcinoma, melanoma,
multiple myeloma, mantle cell lymphoma, non-Hodgkin's lymphoma,
colorectal cancer, acute lymphocytic leukemia, chronic lymphocytic
leukemia, chronic myelogenous leukemia, skin cancer, ovarian
cancer, gastrointestinal stromal tumors, breast cancer, prostate
cancer, pancreatic cancer, or thymoma.
[0063] In one aspect, this application pertains to milciclib for
use in the treatment or prevention of non-small cell lung cancer,
renal cell carcinoma, hepatocellular carcinoma, thyroid carcinoma,
melanoma, multiple myeloma, mantle cell lymphoma, non-Hodgkin's
lymphoma, colorectal cancer, acute lymphocytic leukemia, chronic
lymphocytic leukemia, chronic myelogenous leukemia, skin cancer,
ovarian cancer, gastrointestinal stromal tumors, breast cancer,
prostate cancer, pancreatic cancer, or thymoma, by
co-administration with another anticancer drug.
[0064] In one aspect, this application pertains to sorafenib,
lenvatinib, regorafenib, sunitinib, nivolumab, gemcitabine,
palbociclib, afatinib, alectinib, axitinib, bortezomib, bosutinib,
cabozantinib, carfilzomib, ceritinib, cobimetinib, crizotinib,
dabrafenib, erlotinib, gefitinib, ibrutinib, idelalisib, imatinib,
ixazomib, lapatinib, nilotinib, nintedanib, niraparib, osimertinib,
pazopanib, pegaptanib, ponatinib, rucaparib, ruxolitinib,
sonidegib, tofacitinib, trametinib, vandetanib, vemurafenib,
vismodegibor, or a pharmaceutically acceptable salt thereof, for
use in the treatment or prevention of non-small cell lung cancer,
renal cell carcinoma, hepatocellular carcinoma, thyroid carcinoma,
melanoma, multiple myeloma, mantle cell lymphoma, non-Hodgkin's
lymphoma, colorectal cancer, acute lymphocytic leukemia, chronic
lymphocytic leukemia, chronic myelogenous leukemia, skin cancer,
ovarian cancer, gastrointestinal stromal tumors, breast cancer,
prostate cancer, pancreatic cancer, or thymoma, by
co-administration with milciclib.
[0065] In one aspect, this application pertains to the use of
milciclib in the manufacture of a medicament for the treatment or
prevention of non-small cell lung cancer, renal cell carcinoma,
hepatocellular carcinoma, thyroid carcinoma, melanoma, multiple
myeloma, mantle cell lymphoma, non-Hodgkin's lymphoma, colorectal
cancer, acute lymphocytic leukemia, chronic lymphocytic leukemia,
chronic myelogenous leukemia, skin cancer, ovarian cancer,
gastrointestinal stromal tumors, breast cancer, prostate cancer,
pancreatic cancer, or thymoma, by co-administration with another
anticancer drug.
[0066] In one aspect, this application pertains to use of
sorafenib, lenvatinib, regorafenib, sunitinib, nivolumab,
gemcitabine, palbociclib, afatinib, alectinib, axitinib,
bortezomib, bosutinib, cabozantinib, carfilzomib, ceritinib,
cobimetinib, crizotinib, dabrafenib, erlotinib, gefitinib,
ibrutinib, idelalisib, imatinib, ixazomib, lapatinib, nilotinib,
nintedanib, niraparib, osimertinib, pazopanib, pegaptanib,
ponatinib, rucaparib, ruxolitinib, sonidegib, tofacitinib,
trametinib, vandetanib, vemurafenib, vismodegibor, or a
pharmaceutically acceptable salt thereof, in the manufacture of a
medicament for the treatment or prevention of non-small cell lung
cancer, renal cell carcinoma, hepatocellular carcinoma, thyroid
carcinoma, melanoma, multiple myeloma, mantle cell lymphoma,
non-Hodgkin's lymphoma, colorectal cancer, acute lymphocytic
leukemia, chronic lymphocytic leukemia, chronic myelogenous
leukemia, skin cancer, ovarian cancer, gastrointestinal stromal
tumors, breast cancer, prostate cancer, pancreatic cancer, or
thymoma, by co-administration with milciclib.
[0067] In one aspect, this application pertains to a product
comprising milciclib, or a pharmaceutically acceptable salt
thereof, and sorafenib, lenvatinib, regorafenib, sunitinib,
nivolumab, gemcitabine, palbociclib, afatinib, alectinib, axitinib,
bortezomib, bosutinib, cabozantinib, carfilzomib, ceritinib,
cobimetinib, crizotinib, dabrafenib, erlotinib, gefitinib,
ibrutinib, idelalisib, imatinib, ixazomib, lapatinib, nilotinib,
nintedanib, niraparib, osimertinib, pazopanib, pegaptanib,
ponatinib, rucaparib, ruxolitinib, sonidegib, tofacitinib,
trametinib, vandetanib, vemurafenib, vismodegibor, or a
pharmaceutically acceptable salt thereof, as a combined preparation
for simultaneous, separate, or sequential use in the treatment or
prevention of non-small cell lung cancer, renal cell carcinoma,
hepatocellular carcinoma, thyroid carcinoma, melanoma, multiple
myeloma, mantle cell lymphoma, non-Hodgkin's lymphoma, colorectal
cancer, acute lymphocytic leukemia, chronic lymphocytic leukemia,
chronic myelogenous leukemia, skin cancer, ovarian cancer,
gastrointestinal stromal tumors, breast cancer, prostate cancer,
pancreatic cancer, or thymoma.
[0068] In one aspect, this application pertains to kit
comprising:
[0069] (a) a pharmaceutical composition comprising milciclib, or a
pharmaceutically acceptable salt thereof;
[0070] (b) a pharmaceutical composition comprising sorafenib,
lenvatinib, regorafenib, sunitinib, nivolumab, gemcitabine,
palbociclib, afatinib, alectinib, axitinib, bortezomib, bosutinib,
cabozantinib, carfilzomib, ceritinib, cobimetinib, crizotinib,
dabrafenib, erlotinib, gefitinib, ibrutinib, idelalisib, imatinib,
ixazomib, lapatinib, nilotinib, nintedanib, niraparib, osimertinib,
pazopanib, pegaptanib, ponatinib, rucaparib, ruxolitinib,
sonidegib, tofacitinib, trametinib, vandetanib, vemurafenib,
vismodegibor, or a pharmaceutically acceptable salt thereof;
and
[0071] (c) instructions for the use thereof in the treatment and/or
prevention of cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0072] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0073] FIG. 1 is a graph showing the IC50 value of milciclib in
MHCC97H cells.
[0074] FIG. 2 is a graph showing the IC50 value of milciclib in
MHCC97L cells.
[0075] FIG. 3 is a graph showing the IC50 value of milciclib in
HepG2.2.15 cells.
[0076] FIG. 4 is a series of graphs depicting the IC50 of
sorafenib, regorafenib, sunitinib, lenvatinib, and palbociclib in a
MHCC97H cell proliferation assay.
[0077] FIG. 5 is a set of two graphs showing the IC50 value of
sorafenib and the combination of sorafenib and milciclib in MHCC97H
cells.
[0078] FIG. 6 is a set of two graphs showing the IC50 value of
sunitinib and the combination of sunitinib and milciclib in MHCC97H
cells.
[0079] FIG. 7 is a set of two graphs showing the IC50 value of
regorafenib and the combination of regorafenib and milciclib in
MHCC97H cells.
[0080] FIG. 8 is a set of two graphs showing the IC50 value of
palbociclib and the combination of palbociclib and milciclib in
MHCC97H cells.
[0081] FIG. 9 is a set of two graphs showing the IC50 value of
lenvatinib and the combination of lenvatinib and milciclib in
MHCC97H cells.
[0082] FIG. 10 is a graph showing the IC50 value of sunitinib in
MHCC97L cells.
[0083] FIG. 11 is a graph showing the IC50 value of sorafenib in
MHCC97L cells.
[0084] FIG. 12 is a graph showing the IC50 value of regorafenib in
MHCC97L cells.
[0085] FIG. 13 is a graph showing the IC50 value of lenvatinib in
MHCC97L cells.
[0086] FIG. 14 is a graph showing the IC50 value of palbociclib in
MHCC97L cells.
[0087] FIG. 15 is a set of two graphs showing the IC50 value of
sorafenib and the combination of sorafenib and milciclib in MHCC97L
cells.
[0088] FIG. 16 is a set of two graphs showing the IC50 value of
sunitinib and the combination of sunitinib and milciclib in MHCC97L
cells.
[0089] FIG. 17 is a set of two graphs showing the IC50 value of
regorafenib and the combination of regorafenib and milciclib in
MHCC97L cells.
[0090] FIG. 18 is a set of two graphs showing the IC50 value of
lenvatinib and the combination of lenvatinib and milciclib in
MHCC97L cells.
[0091] FIG. 19 is a set of two graphs showing the IC50 value of
palbociclib and the combination of palbociclib and milciclib in
MHCC97L cells.
[0092] FIG. 20 is a graph showing the IC50 value of sunitinib in
HepG2.2.15 cells.
[0093] FIG. 21 is a graph showing the IC50 value of sorafenib in
HepG2.2.15 cells.
[0094] FIG. 22 is a graph showing the IC50 value of regorafenib in
HepG2.2.15 cells.
[0095] FIG. 23 is a graph showing the IC50 value of lenvatinib in
HepG2.2.15 cells.
[0096] FIG. 24 is a graph showing the IC50 value of palbociclib in
HepG2.2.15 cells.
[0097] FIG. 25 is a set of two graphs showing the IC50 value of
sorafenib and the combination of sorafenib and milciclib in
HepG2.2.15 cells.
[0098] FIG. 26 is a set of two graphs showing the IC50 value of
lenvatinib and the combination of lenvatinib and milciclib in
HepG2.2.15 cells.
[0099] FIG. 27 is a set of two graphs showing the IC50 value of
regorafenib and the combination of regorafenib and milciclib in
HepG2.2.15 cells.
[0100] FIG. 28 is a set of two graphs showing the IC50 value of
sunitinib and the combination of sunitinib and milciclib in
HepG2.2.15 cells.
[0101] FIG. 29A is a heat map depicting synergism between milciclib
and sorafenib in MHCC97H cells. Milciclib concentration is varied
on the y-axis and sorafenib concentration is depicted along the
x-axis. Red depicts 100% inhibition while green depicts 0%
inhibition.
[0102] FIG. 29B is a heat map depicting synergism between milciclib
and lenvatinib in MHCC97H cells. Milciclib concentration is varied
on the y-axis and lenvatinib concentration is depicted along the
x-axis. Red depicts 100% inhibition while green depicts 0%
inhibition.
[0103] FIG. 29C is a heat map depicting synergism between milciclib
and regorafenib in MHCC97H cells. Milciclib concentration is varied
on the y-axis and regorafenib concentration is depicted along the
x-axis. Red depicts 100% inhibition while green depicts 0%
inhibition.
[0104] FIG. 30 is a graph showing changes in expression of
alphafetoprotein (AFP) in MHCC97H cells treated with vehicle of
milciclib.
[0105] FIG. 31 is a series of graphs from the data collected in the
Promega Triplex Assay of milciclib in MHCC97H cells.
[0106] FIG. 32 is a series of graphs from the data collected in the
Promega Triplex Assay of sorafenib in MHCC97H cells.
[0107] FIG. 33 is a series of graphs from the data collected in the
Promega Triplex Assay of regorafenib in MHCC97H cells.
[0108] FIG. 34 is a series of graphs from the data collected in the
Promega Triplex Assay of sunitinib in MHCC97H cells.
[0109] FIG. 35 is a series of graphs from the data collected in the
Promega Triplex Assay of lenvatinib in MHCC97H cells.
[0110] FIG. 36 is a series of graphs from the data collected in the
Promega Triplex Assay of the combination of milciclib and sorafenib
in MHCC97H cells.
[0111] FIG. 37 is a series of graphs from the data collected in the
Promega Triplex Assay of the combination of milciclib and
regorafenib in MHCC97H cells.
[0112] FIG. 38 is a series of graphs from the data collected in the
Promega Triplex Assay of the combination of milciclib and
lenvatinib in MHCC97H cells.
[0113] FIG. 39 is a series of graphs from the data collected in the
Promega Triplex Assay of the combination of milciclib and sunitinib
in MHCC97H cells.
[0114] FIG. 40 is a series of graphs from the data collected in the
Promega Triplex Assay of milciclib in MHCC97L cells.
[0115] FIG. 41 is a series of graphs from the data collected in the
Promega Triplex Assay of regorafenib in MHCC97L cells.
[0116] FIG. 42 is a series of graphs from the data collected in the
Promega Triplex Assay of sunitinib in MHCC97L cells.
[0117] FIG. 43 is a series of graphs from the data collected in the
Promega Triplex Assay of sorafenib in MHCC97L cells.
[0118] FIG. 44 is a series of graphs from the data collected in the
Promega Triplex Assay of lenvatinib in MHCC97L cells.
[0119] FIG. 45 is a series of graphs from the data collected in the
Promega Triplex Assay of palbociclib in MHCC97L cells.
[0120] FIG. 46 is a series of graphs from the data collected in the
Promega Triplex Assay of the combination of milciclib and sorafenib
in MHCC97L cells.
[0121] FIG. 47 is a series of graphs from the data collected in the
Promega Triplex Assay of the combination of milciclib and
regorafenib in MHCC97L cells.
[0122] FIG. 48 is a series of graphs from the data collected in the
Promega Triplex Assay of the combination of milciclib and sunitinib
in MHCC97L cells.
[0123] FIG. 49 is a series of graphs from the data collected in the
Promega Triplex Assay of the combination of milciclib and
lenvatinib in MHCC97L cells.
[0124] FIG. 50 is a series of graphs from the data collected in the
Promega Triplex Assay of the combination of milciclib and
palbociclib in MHCC97L cells.
[0125] FIG. 51 is a series of photographs depicting the results of
a wound-healing assay with milciclib in MHCC97H cells.
[0126] FIG. 52 is a series of photographs depicting the results of
a wound-healing assay with sorafenib and the combination of
sorafenib and milciclib in MHCC97H cells.
[0127] FIG. 53 is a series of photographs depicting the results of
a wound-healing assay with sunitinib and the combination of
sunitinib and milciclib in MHCC97H cells.
[0128] FIG. 54 is a series of photographs depicting the results of
a wound-healing assay with lenvatinib and the combination of
lenvatinib and milciclib in MHCC97H cells.
[0129] FIG. 55 shows is a series of photographs depicting the
results of a wound-healing assay with regorafenib and the
combination of regorafenib and milciclib in MHCC97H cells.
[0130] FIG. 56 is a series of photographs depicting the results of
a wound-healing assay with milciclib in MHCC97L cells.
[0131] FIG. 57 is a series of photographs depicting the results of
a wound-healing assay with sorafenib and the combination of
sorafenib and milciclib in MHCC97L cells.
[0132] FIG. 58 is a series of photographs depicting the results of
a wound-healing assay with sorafenib and the combination of
regorafenib and milciclib in MHCC97L cells.
[0133] FIG. 59 is a series of photographs depicting the results of
a wound-healing assay with sorafenib and the combination of
sunitinib and milciclib in MHCC97L cells.
[0134] FIG. 60 is a series of photographs depicting the results of
a wound-healing assay with sorafenib and the combination of
lenvatinib and milciclib in MHCC97L cells.
[0135] FIG. 61 is a series of photographs depicting the results of
a wound-healing assay with milciclib in HepG2.2.15 cells.
[0136] FIG. 62 is a series of photographs depicting the results of
a wound-healing assay with sorafenib and the combination of
sorafenib and milciclib in HepG2.2.15 cells.
[0137] FIG. 63 is a series of photographs depicting the results of
a wound-healing assay with regorafenib and the combination of
regorafenib and milciclib in HepG2.2.15 cells.
[0138] FIG. 64 is series of bar graphs displaying the results of an
EMT assay with milciclib (A), regorafenib (B), sorafenib (C),
sunitinib (D), and lenvatinib (E) in MHCC97L cells.
[0139] FIG. 65 is series of bar graphs displaying the results of an
EMT assay with milciclib (A), regorafenib (B), sorafenib (C),
sunitinib (D), and lenvatinib (E) in MHCC97H cells.
[0140] FIG. 66 is a schematic depicting the experimental design of
in vivo studies wherein athymic mice with livers injected with
MHCC97H cells were treated with vehicle, sorafenib, milciclib, or
milciclib+sorafenib.
[0141] FIG. 67 is a graph showing weight of mice livers following
treatment via oral administration with sorafenib, milciclib,
sorafenib+milciclib.
[0142] FIG. 68 is a graph showing weight of mice liver tumors
following treatment via oral administration with sorafenib,
milciclib, sorafenib+milciclib.
[0143] FIG. 69 is a series of photographs depicting changes in
MHCC97H orthotopic HCC mouse liver tumor burden following treatment
with vehicle, milciclib, sorafenib, or milciclib+sorafenib.
[0144] FIG. 70 is a graph depicting changes in AFP serum levels in
athymic mice with livers injected with MHCC97H cells were treated
with vehicle, sorafenib, milciclib, or milciclib+sorafenib.
[0145] FIGS. 71A and 71B are a series of graphs depicting relative
expression of miR-221 (71A) and miR-222 (71B) miRNAs in athymic
mice with livers injected with MHCC97H cells treated with vehicle,
sorafenib, milciclib, or milciclib+sorafenib.
[0146] FIG. 72 is a series of graphs depicting relative expression
of p27.sup.kip1(A), p21 (B), p57 (C), and p53 (D) in athymic mice
with livers injected with MHCC97H cells following treatment with
vehicle, sorafenib, milciclib, or milciclib+sorafenib.
[0147] FIGS. 73A and 73B are a series of graphs depicting relative
expression of Cyclin D1 (73A) and Cyclin E2 (73B) in athymic mice
with livers injected with MHCC97H cells following treatment with
vehicle, sorafenib, milciclib, or milciclib+sorafenib.
[0148] FIGS. 74A, 74B, and 74C are a series of graphs depicting
relative expression of MKI67 (74A), c-Myc (74B) and Cdc6 (74C) in
athymic mice with livers injected with MHCC97H cells following
treatment with vehicle, sorafenib, milciclib, or
milciclib+sorafenib.
[0149] FIG. 75 is a series of western blots depicting changes in
expression of pAKT.sup.Ser473, AKT, Cyclin D1, c-Myc, and PTEN in
cells cultured from orthotopic HCC model mice following treatment
with vehicle, sorafenib, milciclib, or milciclib+sorafenib. Actin
is used as a loading control.
[0150] FIG. 76 is a schematic depicting milciclib mechanism of
action in hepatocellular carcinoma.
DETAILED DESCRIPTION OF THE INVENTION
[0151] It must be noted that, as used in this specification and the
appended claims, the singular forms "a," "an" and "the" include
plural referents unless the context clearly dictates otherwise.
Thus, for example, "a CDK inhibitor" refers not only to a single
inhibitor but also to a combination of two or more different
inhibitors, "a dosage form" refers to a combination of dosage forms
as well as to a single dosage form, and the like.
[0152] Unless defined otherwise, all technical and scientific terms
used herein have the meaning commonly understood by one of ordinary
skill in the art to which the invention pertains. Specific
terminology of particular importance to the description of the
present invention is defined below.
[0153] As used herein, the term "patient" or "individual" or
"subject" refers to any person or mammalian subject for whom or
which therapy is desired, and generally refers to the recipient of
the therapy to be practiced according to the invention.
[0154] As used herein, the term "CDK inhibitor" refers to a
compound that inhibits the enzyme in humans referred to as
cyclin-dependent kinase. Examples include, without limitation,
milciclib, palbociclib, dinaciclib, P276-00, roniciclib,
ribociclib, P1446A-05, AT7519M, SNS-032, SCH 727965, AG-024322,
hygrolidin, fascaplysin, abemaciclib, arcyriaflavin A, CINK4,
AM-5992, CDK4 Inhibitor (CAS #546102-60-7), CDK4 Inhibitor III (CAS
#265312-55-8), Cdk4/6 Inhibitor IV (CAS #359886-84-3), MM-D37K, NSC
625987, ON-123300, or any pharmaceutically acceptable salt thereof.
(See Law, M. E. et al. "Cyclin-Dependent Kinase Inhibitors as
Anticancer Therapeutics" Mol. Pharmacol. 88:846-852 (2015), which
is incorporated by reference herein in its entirety.). In one
embodiment, the CDK inhibitor is milciclib.
[0155] As used here, the term "anticancer drug" or "anticancer
agent" includes kinase inhibitor drugs which refers to any member
of the group of anticancer drugs that specifically targets protein
kinases that are altered in cancer cells and account for some of
their abnormal growth. In one embodiment, the anticancer drug is
selected from the group consisting of sorafenib, lenvatinib,
regorafenib, sunitinib, palbociclib, afatinib, alectinib, axitinib,
bortezomib, bosutinib, cabozantinib, carfilzomib, ceritinib,
cobimetinib, crizotinib, dabrafenib, erlotinib, gefitinib,
ibrutinib, idelalisib, imatinib, ixazomib, lapatinib, nilotinib,
nintedanib, niraparib, osimertinib, pazopanib, pegaptanib,
ponatinib, rucaparib, ruxolitinib, sonidegib, tofacitinib,
trametinib, vandetanib, vemurafenib, vismodegibor, or any
pharmaceutically acceptable salt thereof. In one embodiment, the
anticancer drug is sorafenib. In one embodiment, the anticancer
drug is lenvatinib. In one embodiment, the anticancer drug is
regorafenib. In one embodiment, the anticancer drug is sunitinib.
In one embodiment, the anticancer drug is palbociclib.
[0156] Other anticancer drugs, include, without limitation, an
alkylating agent, an antibiotic, an anti-metabolite, a detoxifying
agent, an interferon, a polyclonal or monoclonal antibody, an EGFR
inhibitor, a HER2 inhibitor, a histone deacetylase inhibitor, a
hormone; a mitotic inhibitor, an MTOR inhibitor, a multi-kinase
inhibitor, a serine/threonine kinase inhibitor, a tyrosine kinase
inhibitors, a VEGF/VEGFR inhibitor, a taxane or taxane derivative,
an aromatase inhibitor, an anthracycline, a microtubule targeting
drug, a topoisomerase poison drug, an inhibitor of a molecular
target or enzyme (e.g., a kinase inhibitor), a cytidine analogue
drug, or any chemotherapeutic, anti-neoplastic or
anti-proliferative agent listed in
www.cancer.org/docroot/cdg/cdg_0.asp. In one embodiment, the
anticancer drug is nivolumab. In one embodiment, the anticancer
drug is gemcitabine.
[0157] When referring to an active agent, applicant intends the
term "active agent" to encompass not only the specified molecular
entity but also its pharmaceutically acceptable, pharmacologically
active analogs, including, but not limited to, salts, esters,
amides, prodrugs, conjugates, active metabolites, crystalline forms
(including polymorphs), enantiomers, and other such derivatives,
analogs, and related compounds.
[0158] The terms "treating" and "treatment" include the following
actions: (i) preventing a particular disease or disorder from
occurring in a subject who may be predisposed to the disease or
disorder but has not yet been diagnosed as having it; (ii)
inhibiting the disease, i.e., arresting its development; or (iii)
relieving the disease by reducing or eliminating symptoms and/or by
causing regression of the disease.
[0159] The term "unit dosage forms" as used herein refers to
physically discrete units suited as unitary dosages for the
individuals to be treated. That is, the compositions are formulated
into discrete dosage units each containing a predetermined, "unit
dosage" quantity of an active agent calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specifications of unit dosage forms of
the invention are dependent on the unique characteristics of the
active agent to be delivered. Dosages can further be determined by
reference to the usual dose and manner of administration of the
ingredients. It should be noted that, in some cases, two or more
individual dosage units in combination provide a therapeutically
effective amount of the active agent, e.g., two tablets or capsules
taken together may provide a therapeutically effective dosage of
milciclib, such that the unit dosage in each tablet or capsule is
approximately 50% of the therapeutically effective amount.
[0160] By the terms "effective amount" and "therapeutically
effective amount" of a compound is meant a nontoxic but sufficient
amount of an active agent to provide the desired effect, i.e.,
treatment of cancer.
[0161] As used herein, a "subject in need thereof" is a subject
having cancer, or a subject having an increased risk of developing
cancer relative to the population at large.
[0162] The term "cancer" includes solid tumors, as well as,
hematologic tumors and/or malignancies. A "cancer cell" or
"cancerous cell" is a cell manifesting a cell proliferative
disorder that is a cancer. Any reproducible means of measurement
may be used to identify cancer cells. Cancer cells can be
identified by histological typing or grading of a tissue sample
(e.g., a biopsy sample). Cancer cells can be identified through the
use of appropriate molecular markers.
[0163] Exemplary cancers include, but are not limited to,
adrenocortical carcinoma, AIDS-related cancers, AIDS-related
lymphoma, anal cancer, anorectal cancer, cancer of the anal canal,
appendix cancer, childhood cerebellar astrocytoma, childhood
cerebral astrocytoma, basal cell carcinoma, skin cancer
(non-melanoma), biliary cancer, extrahepatic bile duct cancer,
intrahepatic bile duct cancer, bladder cancer, urinary bladder
cancer, bone and joint cancer, osteosarcoma and malignant fibrous
histiocytoma, brain cancer, brain tumor, brain stem glioma,
cerebellar astrocytoma, cerebral astrocytoma/malignant glioma,
ependymoma, medulloblastoma, supratentorial primitive
neuroectodeimal tumors, visual pathway and hypothalamic glioma,
breast cancer, triple negative breast cancer, bronchial
adenomas/carcinoids, carcinoid tumor, gastrointestinal, nervous
system cancer, nervous system lymphoma, central nervous system
cancer, central nervous system lymphoma, cervical cancer, childhood
cancers, chronic lymphocytic leukemia, chronic myelogenous
leukemia, chronic myeloproliferative disorders, colon cancer,
colorectal cancer, cutaneous T-cell lymphoma, lymphoid neoplasm,
mycosis fungoides, Seziary Syndrome, endometrial cancer, esophageal
cancer, extracranial germ cell tumor, extragonadal germ cell tumor,
extrahepatic bile duct cancer, eye cancer, intraocular melanoma,
retinoblastoma, gallbladder cancer, gastric (stomach) cancer,
gastrointestinal carcinoid tumor, gastrointestinal stromal tumor
(GIST), germ cell tumor, ovarian germ cell tumor, gestational
trophoblastic tumor glioma, head and neck cancer, hepatocellular
carcinoma, hepatocellular (liver) cancer, Hodgkin lymphoma,
hypopharyngeal cancer, intraocular melanoma, ocular cancer, islet
cell tumors (endocrine pancreas), Kaposi's sarcoma, kidney cancer
(renal cell carcinoma), renal cancer, laryngeal cancer, acute
lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic
leukemia, chronic myelogenous leukemia, hairy cell leukemia, lip
and oral cavity cancer, liver cancer, lung cancer, non-small cell
lung cancer, small cell lung cancer, AIDS-related lymphoma,
non-Hodgkin lymphoma, primary central nervous system lymphoma,
Waldenstram macroglobulinemia, medulloblastoma, melanoma,
intraocular (eye) melanoma, merkel cell carcinoma, mesothelioma
malignant, mesothelioma, metastatic squamous neck cancer, mouth
cancer, cancer of the tongue, multiple endocrine neoplasia
syndrome, mycosis fungoides, myelodysplastic syndromes,
myelodysplastic/myeloproliferative diseases, chronic myelogenous
leukemia, acute myeloid leukemia, multiple myeloma, chronic
myeloproliferative disorders, nasopharyngeal cancer, neuroblastoma,
oral cancer, oral cavity cancer, oropharyngeal cancer, ovarian
cancer, ovarian epithelial cancer, ovarian low malignant potential
tumor, pancreatic cancer, islet cell pancreatic cancer, paranasal
sinus and nasal cavity cancer, parathyroid cancer, penile cancer,
pharyngeal cancer, pheochromocytoma, pineoblastoma and
supratentorial primitive neuroectodermal tumors, pituitary tumor,
plasma cell neoplasm/multiple myeloma, pleuropulmonary blastoma,
prostate cancer, rectal cancer, renal pelvis and ureter,
transitional cell cancer, retinoblastoma, rhabdomyosarcoma,
salivary gland cancer, Ewing family of sarcoma tumors, Kaposi
Sarcoma, uterine cancer, uterine sarcoma, skin cancer
(non-melanoma), skin cancer (melanoma), merkel cell skin carcinoma,
small intestine cancer, soft tissue sarcoma, squamous cell
carcinoma, stomach (gastric) cancer, supratentorial primitive
neuroectodermal tumors, testicular cancer, throat cancer, thymoma,
thymoma and thymic carcinoma, thyroid cancer, thyroid carcinoma,
transitional cell cancer of the renal pelvis and ureter and other
urinary organs, gestational trophoblastic tumor, urethral cancer,
endometrial uterine cancer, uterine sarcoma, uterine corpus cancer,
vaginal cancer, vulvar cancer, and Wilm's Tumor.
Methods of Treatment
[0164] The present application provides methods of treating cancer,
comprising administering to a subject in need thereof a
therapeutically effective amount of milciclib, or a
pharmaceutically acceptable salt thereof, with one or more
pharmaceutically acceptable carriers or excipients, in combination
with a therapeutically effective amount of a second agent, i.e., an
anticancer drug, with one or more pharmaceutically acceptable
carriers or excipients, wherein the cancer is treated. In one
embodiment, the anticancer drug is any compound disclosed herein
other than milciclib.
[0165] The cancer can be a hematologic tumor or malignancy, or a
solid tumor (or tumors), or a refractory solid tumor.
[0166] In one embodiment, the cancer is selected from the group
consisting of non-small cell lung cancer, renal cell carcinoma,
hepatocellular carcinoma, thyroid carcinoma, colorectal cancer,
gastrointestinal stromal tumors, breast cancer (e.g., triple
negative breast cancer), prostate cancer, pancreatic cancer, or
thymoma (i.e., thymic carcinoma).
[0167] This method of treating cancer includes a reduction in tumor
size. Alternatively, or in addition, the cancer is metastatic
cancer and this method of treatment includes inhibition of
metastatic cancer cell invasion.
[0168] The other anticancer drug or agent can be an alkylating
agent; an antibiotic; an anti-metabolite; a detoxifying agent; an
interferon; a polyclonal or monoclonal antibody; an EGFR inhibitor;
a HER2 inhibitor; a histone deacetylase inhibitor; a hormone; a
mitotic inhibitor; an MTOR inhibitor; a multi-kinase inhibitor; a
serine/threonine kinase inhibitor; a tyrosine kinase inhibitors; a
VEGF/VEGFR inhibitor; a taxane or taxane derivative, an aromatase
inhibitor, an anthracycline, a microtubule targeting drug, a
topoisomerase poison drug, an inhibitor of a molecular target or
enzyme (e.g., a kinase inhibitor), a cytidine analogue drug or any
chemotherapeutic, anti-neoplastic or anti-proliferative agent
listed in www.cancer.org/docroot/cdg/cdg_0.asp.
[0169] In one embodiment, the other anticancer agent is an
anti-metabolite or a nucleoside analog. Exemplary anti-metabolites
or nucleoside analogs include, but are not limited to, fluorouracil
(Adrucil); capecitabine (Xeloda); hydroxyurea (Hydrea);
mercaptopurine (Purinethol); pemetrexed (Alimta); fludarabine
(Fludara); nelarabine (Arranon); cladribine (Cladribine Novaplus);
clofarabine (Clolar); cytarabine (Cytosar-U); decitabine (Dacogen);
cytarabine liposomal (DepoCyt); hydroxyurea (Droxia); pralatrexate
(Folotyn); floxuridine (FUDR); gemcitabine (Gemzar); cladribine
(Leustatin); fludarabine (Oforta); methotrexate (MTX; Rheumatrex);
methotrexate (Trexall); thioguanine (Tabloid); TS-1 or cytarabine
(Tarabine PFS).
[0170] In one embodiment, the other anticancer drug or agent is
selected from the group consisting of sorafenib, lenvatinib,
regorafenib, sunitinib, nivolumab, gemcitabine, and
palbociclib.
[0171] Milciclib or a pharmaceutically acceptable salt thereof,
and/or the other anticancer drug, can be incorporated into
pharmaceutical compositions suitable for administration. Such
compositions typically comprise the compound (i.e. including the
active compound), and a pharmaceutically acceptable excipient or
carrier. As used herein, "pharmaceutically acceptable excipient" or
"pharmaceutically acceptable carrier" is intended to include any
and all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like, compatible with pharmaceutical administration. Suitable
carriers are described in the most recent edition of Remington's
Pharmaceutical Sciences, a standard reference text in the field.
Preferred examples of such carriers or diluents include, but are
not limited to, water, saline, ringer's solutions, dextrose
solution, and 5% human serum albumin.
[0172] Pharmaceutically acceptable carriers include solid carriers
such as lactose, terra alba, sucrose, talc, gelatin, agar, pectin,
acacia, magnesium stearate, stearic acid and the like. Exemplary
liquid carriers include syrup, peanut oil, olive oil, water and the
like. Similarly, the carrier or diluent may include time-delay
material known in the art, such as glyceryl monostearate or
glyceryl distearate, alone or with a wax, ethylcellulose,
hydroxypropylmethylcellulose, methylmethacrylate or the like. Other
fillers, excipients, flavorants, and other additives such as are
known in the art may also be included in a pharmaceutical
composition according to this application. Liposomes and
non-aqueous vehicles such as fixed oils may also be used. The use
of such media and agents for pharmaceutically active substances is
well known in the art. Except insofar as any conventional media or
agent is incompatible with the active compound, use thereof in the
compositions is contemplated. Supplementary active compounds can
also be incorporated into the compositions.
[0173] In one aspect, milciclib, or a pharmaceutically acceptable
salt thereof, and/or the other anticancer drug, is administered in
a suitable dosage form prepared by combining a therapeutically
effective amount (e.g., an efficacious level sufficient to achieve
the desired therapeutic effect through inhibition of tumor growth,
killing of tumor cells, etc.) of milciclib, or a pharmaceutically
acceptable salt thereof (as an active ingredient) and/or the other
anticancer drug or agent, with standard pharmaceutical carriers or
diluents according to conventional procedures (i.e., by producing a
pharmaceutical composition of the application). These procedures
may involve mixing, granulating, and compressing or dissolving the
ingredients as appropriate to attain the desired preparation.
[0174] As used herein, "treating" describes the management and care
of a subject for the purpose of combating a disease, condition, or
disorder and includes decreasing or alleviating the symptoms or
complications, or eliminating the disease, condition or
disorder.
[0175] As used herein, "preventing" describes stopping the onset of
the symptoms or complications of the disease, condition or
disorder.
[0176] In one aspect, treating cancer results in a reduction in
size of a tumor. A reduction in size of a tumor may also be
referred to as "tumor regression." Preferably, after treatment,
tumor size is reduced by 5% or greater relative to its size prior
to treatment; more preferably, tumor size is reduced by 10% or
greater; more preferably, reduced by 20% or greater; more
preferably, reduced by 30% or greater; more preferably, reduced by
40% or greater; even more preferably, reduced by 50% or greater;
and most preferably, reduced by greater than 75% or greater. Size
of a tumor may be measured by any reproducible means of
measurement. In a preferred aspect, size of a tumor may be measured
as a diameter of the tumor.
[0177] In another aspect, treating cancer results in a reduction in
tumor volume. Preferably, after treatment, tumor volume is reduced
by 5% or greater relative to its size prior to treatment; more
preferably, tumor volume is reduced by 10% or greater; more
preferably, reduced by 20% or greater; more preferably, reduced by
30% or greater; more preferably, reduced by 40% or greater; even
more preferably, reduced by 50% or greater; and most preferably,
reduced by greater than 75% or greater. Tumor volume may be
measured by any reproducible means of measurement.
[0178] In another aspect, treating cancer results in a decrease in
number of tumors. Preferably, after treatment, tumor number is
reduced by 5% or greater relative to number prior to treatment;
more preferably, tumor number is reduced by 10% or greater; more
preferably, reduced by 20% or greater; more preferably, reduced by
30% or greater; more preferably, reduced by 40% or greater; even
more preferably, reduced by 50% or greater; and most preferably,
reduced by greater than 75%. Number of tumors may be measured by
any reproducible means of measurement. In a preferred aspect,
number of tumors may be measured by counting tumors visible to the
naked eye or at a specified magnification. In a preferred aspect,
the specified magnification is 2.times., 3.times., 4.times.,
5.times., 10.times., or 50.times..
[0179] In another aspect, treating cancer results in a decrease in
number of metastatic lesions in other tissues or organs distant
from the primary tumor site. Preferably, after treatment, the
number of metastatic lesions is reduced by 5% or greater relative
to number prior to treatment; more preferably, the number of
metastatic lesions is reduced by 10% or greater; more preferably,
reduced by 20% or greater; more preferably, reduced by 30% or
greater; more preferably, reduced by 40% or greater; even more
preferably, reduced by 50% or greater; and most preferably, reduced
by greater than 75%. The number of metastatic lesions may be
measured by any reproducible means of measurement. In a preferred
aspect, the number of metastatic lesions may be measured by
counting metastatic lesions visible to the naked eye or at a
specified magnification. In a preferred aspect, the specified
magnification is 2.times., 3.times., 4.times., 5.times., 10.times.,
or 50.times..
[0180] In another aspect, treating cancer results in an increase in
average survival time of a population of treated subjects in
comparison to a population receiving carrier alone. Preferably, the
average survival time is increased by more than 30 days; more
preferably, by more than 60 days; more preferably, by more than 90
days; and most preferably, by more than 120 days. An increase in
average survival time of a population may be measured by any
reproducible means. In a preferred aspect, an increase in average
survival time of a population may be measured, for example, by
calculating for a population the average length of survival
following initiation of treatment with an active compound. In
another preferred aspect, an increase in average survival time of a
population may also be measured, for example, by calculating for a
population the average length of survival following completion of a
first round of treatment with an active compound.
[0181] In another aspect, treating cancer results in an increase in
average survival time of a population of treated subjects in
comparison to a population of untreated subjects. Preferably, the
average survival time is increased by more than 30 days; more
preferably, by more than 60 days; more preferably, by more than 90
days; and most preferably, by more than 120 days. An increase in
average survival time of a population may be measured by any
reproducible means. In a preferred aspect, an increase in average
survival time of a population may be measured, for example, by
calculating for a population the average length of survival
following initiation of treatment with an active compound. In
another preferred aspect, an increase in average survival time of a
population may also be measured, for example, by calculating for a
population the average length of survival following completion of a
first round of treatment with an active compound.
[0182] In another aspect, treating cancer results in increase in
average survival time of a population of treated subjects in
comparison to a population receiving monotherapy with a drug that
is not milciclib, or a pharmaceutically acceptable salt, prodrug,
metabolite, analog or derivative thereof. Preferably, the average
survival time is increased by more than 30 days; more preferably,
by more than 60 days; more preferably, by more than 90 days; and
most preferably, by more than 120 days. An increase in average
survival time of a population may be measured by any reproducible
means. In a preferred aspect, an increase in average survival time
of a population may be measured, for example, by calculating for a
population the average length of survival following initiation of
treatment with an active compound. In another preferred aspect, an
increase in average survival time of a population may also be
measured, for example, by calculating for a population the average
length of survival following completion of a first round of
treatment with an active compound.
[0183] In another aspect, treating cancer results in a decrease in
the mortality rate of a population of treated subjects in
comparison to a population receiving carrier alone. In another
aspect, treating cancer results in a decrease in the mortality rate
of a population of treated subjects in comparison to an untreated
population. In a further aspect, treating cancer results a decrease
in the mortality rate of a population of treated subjects in
comparison to a population receiving monotherapy with a drug that
is not milciclib, or a pharmaceutically acceptable salt, prodrug,
metabolite, analog or derivative thereof. Preferably, the mortality
rate is decreased by more than 2%; more preferably, by more than
5%; more preferably, by more than 10%; and most preferably, by more
than 25%. In a preferred aspect, a decrease in the mortality rate
of a population of treated subjects may be measured by any
reproducible means. In another preferred aspect, a decrease in the
mortality rate of a population may be measured, for example, by
calculating for a population the average number of disease-related
deaths per unit time following initiation of treatment with an
active compound. In another preferred aspect, a decrease in the
mortality rate of a population may also be measured, for example,
by calculating for a population the average number of
disease-related deaths per unit time following completion of a
first round of treatment with an active compound.
[0184] In another aspect, treating cancer results in a decrease in
tumor growth rate. Preferably, after treatment, tumor growth rate
is reduced by at least 5% relative to number prior to treatment;
more preferably, tumor growth rate is reduced by at least 10%; more
preferably, reduced by at least 20%; more preferably, reduced by at
least 30%; more preferably, reduced by at least 40%; more
preferably, reduced by at least 50%; even more preferably, reduced
by at least 50%; and most preferably, reduced by at least 75%.
Tumor growth rate may be measured by any reproducible means of
measurement. In a preferred aspect, tumor growth rate is measured
according to a change in tumor diameter per unit time.
[0185] In another aspect, treating cancer results in a decrease in
tumor regrowth. Preferably, after treatment, tumor regrowth is less
than 5%; more preferably, tumor regrowth is less than 10%; more
preferably, less than 20%; more preferably, less than 30%; more
preferably, less than 40%; more preferably, less than 50%; even
more preferably, less than 50%; and most preferably, less than 75%.
Tumor regrowth may be measured by any reproducible means of
measurement. In a preferred aspect, tumor regrowth is measured, for
example, by measuring an increase in the diameter of a tumor after
a prior tumor shrinkage that followed treatment. In another
preferred aspect, a decrease in tumor regrowth is indicated by
failure of tumors to reoccur after treatment has stopped.
[0186] One skilled in the art may refer to general reference texts
for detailed descriptions of known techniques discussed herein or
equivalent techniques. These texts include Ausubel et al., Current
Protocols in Molecular Biology, John Wiley and Sons, Inc. (2005);
Sambrook et al., Molecular Cloning, A Laboratory Manual (3d ed.),
Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2000); Coligan
et al., Current Protocols in Immunology, John Wiley & Sons,
N.Y.; Enna et al., Current Protocols in Pharmacology, John Wiley
& Sons, N.Y.; Fingl et al., The Pharmacological Basis of
Therapeutics (1975), Remington's Pharmaceutical Sciences, Mack
Publishing Co., Easton, Pa., 18th edition (1990). These texts can,
of course, also be referred to in making or using an aspect of the
application.
[0187] The term "controlled release" or "controlled release form"
refers to a drug-containing formulation or fraction thereof in
which release of the drug is not immediate, i.e., with a
"controlled release" formulation, administration does not result in
immediate release of the drug into an absorption pool. The term is
used interchangeably with "non-immediate release" as defined in
Remington: The Science and Practice of Pharmacy, Nineteenth Ed.
(Easton, Pa.: Mack Publishing Company, 1995). In general, the term
"controlled release" as used herein includes sustained release and
delayed release formulations.
[0188] The term "sustained release" (synonymous with "extended
release") is used in its conventional sense to refer to a drug
formulation that provides for gradual release of a drug over an
extended period of time, and that preferably, although not
necessarily, results in substantially constant blood levels of a
drug over an extended time period. The term "delayed release" is
also used in its conventional sense, to refer to a drug formulation
which, following administration to a patient, provides a measurable
time delay before drug is released from the formulation into the
patient's body.
[0189] By "pharmaceutically acceptable" is meant a material that is
not biologically or otherwise undesirable, i.e., the material may
be incorporated into a pharmaceutical composition administered to a
patient without causing any undesirable biological effects or
interacting in a deleterious manner with any of the other
components of the composition in which it is contained. When the
term "pharmaceutically acceptable" is used to refer to a
pharmaceutical carrier or excipient, it is implied that the carrier
or excipient has met the required standards of toxicological and
manufacturing testing or that it is included on the Inactive
Ingredient Guide prepared by the U.S. Food and Drug administration.
"Pharmacologically active" (or simply "active") as in a
"pharmacologically active" derivative or analog, refers to a
derivative or analog having the same type of pharmacological
activity as the parent compound and approximately equivalent in
degree.
[0190] Administration of the active agents may be carried out using
any appropriate mode of administration. Thus, administration can
be, for example oral or parenteral, although oral administration is
preferred.
[0191] Depending on the intended mode of administration, the
pharmaceutical formulation may be a solid, semi-solid or liquid,
such as, for example, a tablet, a capsule, a caplet, a liquid, a
suspension, an emulsion, a suppository, granules, pellets, beads, a
powder, or the like, preferably in unit dosage form suitable for
single administration of a precise dosage. Suitable pharmaceutical
formulations and dosage forms may be prepared using conventional
methods known to those in the field of pharmaceutical formulation
and described in the pertinent texts and literature, e.g., in
Remington: The Science and Practice of Pharmacy (Easton, Pa.: Mack
Publishing Co., 1995). Oral administration and therefore oral
dosage forms are generally preferred, and include tablets,
capsules, caplets, solutions, suspensions and syrups, and may also
comprise a plurality of granules, beads, powders, or pellets that
may or may not be encapsulated. Preferred oral dosage forms are
capsules and tablets.
[0192] As noted above, it is especially advantageous to formulate
compositions of the invention in unit dosage form for ease of
administration and uniformity of dosage. The term "unit dosage
forms" as used herein refers to physically discrete units suited as
unitary dosages for the individuals to be treated. That is, the
compositions are formulated into discrete dosage units each
containing a predetermined, "unit dosage" quantity of an active
agent calculated to produce the desired therapeutic effect in
association with the required pharmaceutical carrier. The
specifications of unit dosage forms of the invention are dependent
on the unique characteristics of the active agent to be delivered.
Dosages can further be determined by reference to the usual dose
and manner of administration of the ingredients. It should be noted
that, in some cases, two or more individual dosage units in
combination provide a therapeutically effective amount of the
active agent, e.g., two tablets or capsules taken together may
provide a therapeutically effective dosage of each active agent,
such that the unit dosage in each tablet or capsule is
approximately 50% of the therapeutically effective amount.
[0193] Tablets may be manufactured using standard tablet processing
procedures and equipment. Direct compression and granulation
techniques are preferred. In addition to the active agent, tablets
will generally contain inactive, pharmaceutically acceptable
carrier materials such as binders, lubricants, disintegrants,
fillers, stabilizers, surfactants, coloring agents, and the
like.
[0194] Capsules are also preferred oral dosage forms, in which case
the active agent-containing composition may be encapsulated in the
form of a liquid or solid (the latter including particulates such
as granules, beads, powders or pellets). Suitable capsules may be
either hard or soft, and are generally made of gelatin, starch, or
a cellulosic material, with gelatin capsules preferred. Two-piece
hard gelatin capsules are preferably sealed, such as with gelatin
bands or the like. See, for example, Remington: The Science and
Practice of Pharmacy, cited earlier herein, which describes
materials and methods for preparing encapsulated
pharmaceuticals.
[0195] Generally, as will be appreciated by those of ordinary skill
in the art, sustained release dosage forms are formulated by
dispersing the active agents within a matrix of a gradually
hydrolyzable material such as a hydrophilic polymer, or by coating
a solid, drug-containing dosage form with such a material.
Hydrophilic polymers useful for providing a sustained release
coating or matrix include, by way of example: cellulosic polymers
such as hydroxypropyl cellulose, hydroxyethyl cellulose,
hydroxypropyl methyl cellulose, methyl cellulose, ethyl cellulose,
cellulose acetate, and carboxymethylcellulose sodium; acrylic acid
polymers and copolymers, preferably formed from acrylic acid,
methacrylic acid, acrylic acid alkyl esters, methacrylic acid alkyl
esters, and the like, e.g. copolymers of acrylic acid, methacrylic
acid, methyl acrylate, ethyl acrylate, methyl methacrylate and/or
ethyl methacrylate; and vinyl polymers and copolymers such as
polyvinyl pyrrolidone, polyvinyl acetate, and ethylene-vinyl
acetate copolymer.
[0196] Sustained release dosage forms herein may be composed of the
acrylate and methacrylate copolymers available under the tradename
"Eudragit" from Rohm Pharma (Germany). The Eudragit series E, L, S,
RL, RS, and NE copolymers are available as solubilized in organic
solvent, in an aqueous dispersion, or as a dry powder. Preferred
acrylate polymers are copolymers of methacrylic acid and methyl
methacrylate, such as the Eudragit L and Eudragit S series
polymers. In one embodiment, any of the pharmaceutical formulations
may be formulated for sustained release, i.e., in a sustained
release dosage form.
[0197] Preparations according to this invention for parenteral
administration include sterile aqueous and non-aqueous solutions,
suspensions, and emulsions. Injectable aqueous solutions contain
the active agent in water-soluble form. Examples of non-aqueous
solvents or vehicles include fatty oils, such as olive oil and corn
oil, synthetic fatty acid esters, such as ethyl oleate or
triglycerides, low molecular weight alcohols such as propylene
glycol, synthetic hydrophilic polymers such as polyethylene glycol,
liposomes, and the like. Parenteral formulations may also contain
adjuvants such as solubilizers, preservatives, wetting agents,
emulsifiers, dispersants, and stabilizers, and aqueous suspensions
may contain substances that increase the viscosity of the
suspension, such as sodium carboxymethyl cellulose, sorbitol, and
dextran. Injectable formulations are rendered sterile by
incorporation of a sterilizing agent, filtration through a
bacteria-retaining filter, irradiation, or heat. They can also be
manufactured using a sterile injectable medium. The active agent
may also be in dried, e.g., lyophilized, form that may be
rehydrated with a suitable vehicle immediately prior to
administration via injection.
[0198] Each of the active agents may in addition be administered
through the skin using conventional transdermal drug delivery
systems, wherein the active agent or agents are contained within a
laminated structure that serves as a drug delivery device to be
affixed to the skin. In such a structure, the drug composition is
contained in a layer, or "reservoir," underlying an upper backing
layer. The laminated structure may contain a single reservoir, or
it may contain multiple reservoirs. In one embodiment, the
reservoir comprises a polymeric matrix of a pharmaceutically
acceptable contact adhesive material that serves to affix the
system to the skin during drug delivery. Alternatively, the
drug-containing reservoir and skin contact adhesive are present as
separate and distinct layers, with the adhesive underlying the
reservoir which, in this case, may be either a polymeric matrix as
described above, or it may be a liquid or hydrogel reservoir, or
may take some other form. Transdermal drug delivery systems may in
addition contain a skin permeation enhancer.
[0199] In addition to the formulations described previously, the
active agents may be formulated in a depot preparation for
controlled release of the active agents, preferably sustained
release over an extended time period. These sustained release
dosage forms are generally administered by implantation (e.g.,
subcutaneously or intramuscularly or by intramuscular
injection).
[0200] A "daily dose" of a particular material refers the amount of
the material administered in a day. A daily dose can be
administered as a single dose or as multiple doses. When a daily
dose is administered as multiple doses, the daily dose is the sum
of the amount of material administered in all of the multiple doses
that are administered over the course of one day. For example, a
daily dose of 12 mg can be administered in a single 12 mg dose once
per day, in 6 mg doses administered twice per day, in 4 mg doses
administered three times per day, in 2 mg doses administered six
times per day, etc. The multiple doses can be the same or different
doses of the material, unless otherwise specified. When a daily
dose is administered as multiple doses, the multiple doses can be
administered by the same or different route of administration,
unless otherwise specified. Thus, a daily dose of 12 mg can
include, for example, a 10 mg intramuscular dose and a 2 mg oral
dose administered over the course of one day.
[0201] Administration of one compound "with" a second compound, as
used herein, includes but is not limited to cases where the two
compounds are administered simultaneously or substantially
simultaneously. For example, administration of a first compound
with a second compound can include administering the first compound
in the morning and administering the second compound in the
evening, as well as administering the first and second compounds in
the same dosage form or in two different dosage forms that at the
same or nearly the same time.
[0202] In combining the active agents disclosed herein, i.e.,
milciclib with another anticancer drug or agent disclosed herein,
milciclib will generally reduce the quantity of the second drug or
agent needed to achieve a therapeutic effect when administered as a
monotherapy, and, conversely, the other anticancer drug or agent
will generally reduce the quantity of milciclib required.
[0203] As the method of the application involves combination
therapy, the active agents may be administered separately, at the
same or at different times of day, or they be administered in a
single pharmaceutical formulation.
[0204] In some embodiments, "temporal proximity" means that
administration of the other anticancer drug occurs within a time
period before or after the administration of the CDK inhibitor
(e.g., milciclib), such that the therapeutic effect of the other
kinase inhibitor drug overlaps with the therapeutic effect of the
CDK inhibitor (e.g., milciclib). In some embodiments, the
therapeutic effect of the other kinase inhibitor drug completely
overlaps with the therapeutic effect of the CDK inhibitor (e.g.,
milciclib). In some embodiments, "temporal proximity" means that
administration of the other kinase inhibitor drug occurs within a
time period before or after the administration of the CDK inhibitor
(e.g., milciclib), such that there is a synergistic effect between
the other kinase inhibitor drug and the CDK inhibitor.
[0205] "Temporal proximity" may vary according to various factors,
including but not limited to, the age, gender, weight, genetic
background, medical condition, disease history, and treatment
history of the subject to which the therapeutic agents are to be
administered; the disease or condition to be treated or
ameliorated; the therapeutic outcome to be achieved; the dosage,
dosing frequency, and dosing duration of the therapeutic agents;
the pharmacokinetics and pharmacodynamics of the therapeutic
agents; and the route(s) through which the therapeutic agents are
administered. In some embodiments, "temporal proximity" means
within 15 minutes, within 30 minutes, within an hour, within two
hours, within four hours, within six hours, within eight hours,
within 12 hours, within 18 hours, within 24 hours, within 36 hours,
within 2 days, within 3 days, within 4 days, within 5 days, within
6 days, within a week, within 2 weeks, within 3 weeks, within 4
weeks, with 6 weeks, or within 8 weeks. In some embodiments,
multiple administration of one therapeutic agent can occur in
temporal proximity to a single administration of another
therapeutic agent. In some embodiments, temporal proximity may
change during a treatment cycle or within a dosing regimen.
Summary of Data/Examples
[0206] IC50 values by cell proliferation assay were determined for
MHCC97H and MHCC97L (highly metastatic hepatocellular carcinoma
cell line, derived from humans) and HepG2.2.15 cells (derived from
the human hepatoblastoma cell line HepG2). The cells were treated
with milciclib, sorafenib, regorafenib, sunitinib, and lenvatinib,
individually or in combination. Each inhibitor exhibited a dose
dependent decrease in cell proliferation with comparable half
maximal inhibitory concentration (IC50) across the three cell
lines: (FIGS. 1 and 4--MHCC97H cells; FIGS. 2 and 10-14--MHCC97L
cells; and FIGS. 3 and 20-24, HepG2.2.15 cells.)
[0207] A synergistic effect on inhibition of cell proliferation was
observed upon treating MHCC97H, MHCC97L, and HepG2.2.15 cells with
increasing concentration of TKIs (tyrosine kinase inhibitors) in
the presence of fixed concentration corresponding to milciclib
IC.sub.50 value. In all cases, the IC.sub.50 value of each TKI was
reduced by .about.50% (MHCC97H: FIGS. 5-9; MHCC97L: FIGS. 15-19;
HepG2.2.15: FIGS. 25-28).
[0208] Increasing concentration of inhibitors with a fixed
concentration of milciclib was tested on MHCC97L and MHCC97H cells
to determine the synergistic effect on inhibition of cell
proliferation. For sorafenib, the individual IC.sub.50 was 12 .mu.M
but with the combination with milciclib the IC.sub.50 was 6.7 .mu.M
in MHCC97H (FIG. 29A). For lenvatinib, the individual IC.sub.50 was
0.28 .mu.M but with the combination with milciclib the IC.sub.50
was 0.12 .mu.M in MHCC97H (FIG. 29B). For regorafenib, the
individual IC.sub.50 was 4.7 .mu.M but with the combination with
milciclib the IC.sub.50 was 1.9 .mu.M in MHCC97H (FIG. 29C).
[0209] MHCC97H cells were shown to produce human Alphafetoprotein
(AFP)
AFP ELISA Assay.
[0210] Appreciably lower levels of AFP were detected in milciclib
treated cells as compared to vehicle control (FIG. 30).
[0211] Promega ApoTox-Glo.TM. Triplex assays were also performed.
Milciclib in combination with other TKIs at various concentrations
decreased the cell viability and increased caspase 3/7 activity in
MHCC97H cells (FIGS. 31-39) and MHCC97L cells (FIGS. 40-50) in a
dose-dependent manner compared to those in vehicle-treated
cells.
[0212] Wound healing experiments were also performed. Treatment of
scratched monolayer of MHCC97H cells with TKIs (tyrosine kinase
inhibitors) in combination with milciclib (1.3 .mu.M) for 96 h,
reduced cell migration as compared to corresponding vehicle control
(FIGS. 51-55). Treatment of scratched monolayer of MHCC97L cells
with milciclib alone or in combination with other TKIs for 96
hours, reduced cell migration as compared to corresponding vehicle
control (FIGS. 56-60). Treatment of scratched monolayer of
HepG2.2.15 cells with milciclib alone or in combination with other
TKIs for 96 hours, reduced cell migration as compared to
corresponding vehicle control (FIGS. 61-63).
[0213] EMT (Epithelial to Mesenchymal Transition) assays were also
performed. Regorafenib, sorafenib, sunitinib and lenvatinib in
combination with milciclib reduced the invasion potential to a
greater extent as compared to individual treatment (P<0.005)
(FIG. 64). The inclusion of milciclib or TKIs alone resulted in
statistically significant inhibition (P<0.05) in cell migration
in MHCC97H cells. Regorafenib, sorafenib, sunitinib and lenvatinib
in combination with milciclib reduced the invasion potential to a
greater extent as compared to individual treatment (P<0.005),
demonstrative of the anti-invasive potential of milciclib (FIG.
65).
[0214] Mouse experiments (tumor induction) were performed. Oral
administration of milciclib (30 mg/kg/day) either alone or in
combination with sorafenib (20 mg/kg/day) produced synergistic
effect in reducing tumor growth [milciclib -20% (p<0.002) or
sorafenib -21% (p<0.001) vs combination -38% (p<0.0002) as
compared to vehicle (FIG. 67). Vehicle group had more liver weight
but with combination the liver weight goes down (FIG. 68). Pictures
were taken showing the difference in tumor burden with the
treatment of milciclib, sorafenib, and the combination. Vehicle
group has an enlarged tumor but with the combination, the tumor
burden goes down (FIG. 69). A steady increase in serum AFP was
observed in vehicle administered animals until the end of the
study. Significantly lower serum AFP levels were recorded for
animals treated with milciclib (30 mg/kg), sorafenib (20 mg/kg)
alone or in combination (FIG. 70).
[0215] It was determined that milciclib acts via specifically
downregulating miR221/222. Gene expression studies suggest that
milciclib possibly exerts its action through downregulation of
miR-221 and miR-222. Data suggest that oral treatment with
milciclib exerts its activity via downregulation of miR-221 and
miR-222 (FIGS. 71A and 71B). These data suggest that milciclib
specifically acts via reducing expression of miR-221 and miR-222,
which are known to be major culprits of hepatocarcinogenesis.
[0216] Mechanism of action studies with milciclib were also
performed. The mechanism of action of milciclib appears to be
distinct from the mechanism of action of sorafenib as it
upregulated the expression of tumor suppressors such as p27, p21,
p53 and p57 (FIGS. 72A, B, C, D). Oral administration of milciclib
alone or in combination with sorafenib downregulated expression of
cyclins such as cyclin E2 and cyclin D1 (FIGS. 73A and 73B). Oral
administration of milciclib alone or in combination with sorafenib
downregulated expression of cell proliferation genes such as MKI67,
cdc6, c-Myc (FIGS. 74A, 74B, 74C). The mechanism of action of
milciclib appears to be distinct from the mechanism of action of
sorafenib.
[0217] Mechanistic studies revealed a reduction in pAKT, c-Myc and
cyclin D1 expression and upregulation of PTEN in liver samples
derived from milciclib and milciclib and sorafenib administered
animals as compared to vehicle treated group (FIG. 75). Data from
cell culture studies and from orthotopic HCC model in nude mice
suggest that oral treatment with milciclib exerts its activity via
a new mechanism.
Hepatocellular Carcinoma (HCC)
[0218] Hepatocellular carcinoma (HCC) is an extremely complex
multi-factorial condition associated with many confounding factors
affecting disease course and patient prognosis. A broad range of
mechanisms, including telomere dysfunction, activation of oncogenic
pathways, abrogation of DNA damage checkpoints, activation of
pro-inflammatory and metastatic pathways, and induction of the
oxidative stress response. Consequently, HCC is typically
associated with overexpression of receptor tyrosine kinases (RTK)
and excessive oxidative stress (ROS). Collectively, overexpression
of RTK and ROS lead to increased expression of c-myc, resulting in
high metastatic potentials of hepatocytes. Thus, metastatic
potential of hepatocytes can be reduced with specific inhibitors of
RTK. On the other hand, HCC is also associated with overexpression
of miR-221, miR-222 and CDKs, resulting in dysregulation of cell
cycle, which leads to excessive proliferation of hepatocytes.
Treatment with milciclib is known to inhibit miR-221/miR-222 and a
number of CDKs and it can effectively reduce proliferation of
hepatocytes. Therefore, collectively combination of milciclib with
an inhibitor of RTK may produce synergistic effect in reducing
expression of c-myc and in total tumor growth and progression.
Thus, an effective therapy for HCC needs to control proliferation
of hepatocytes and also suppress their metastatic potential. (FIG.
76)
Pharmaceutical Compositions and Formulations
[0219] A pharmaceutical composition of the application is
formulated to be compatible with its intended route of
administration. Examples of routes of administration include
parenteral, e.g., intravenous, intradermal, subcutaneous, oral
(e.g., inhalation), transdermal (topical), and transmucosal
administration. Solutions or suspensions used for parenteral,
intradermal, or subcutaneous application can include the following
components: a sterile diluent such as water for injection, saline
solution, fixed oils, polyethylene glycols, glycerin, propylene
glycol or other synthetic solvents; antibacterial agents such as
benzyl alcohol or methyl parabens; antioxidants such as ascorbic
acid or sodium bisulfite; chelating agents such as
ethylenediaminetetraacetic acid; buffers such as acetates, citrates
or phosphates, and agents for the adjustment of tonicity such as
sodium chloride or dextrose. The pH can be adjusted with acids or
bases, such as hydrochloric acid or sodium hydroxide. The
parenteral preparation can be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic.
[0220] A compound or pharmaceutical composition of the application
can be administered to a subject in many of the well-known methods
currently used for chemotherapeutic treatment. For example, for
treatment of cancers, a compound of the application may be injected
directly into tumors, injected into the blood stream or body
cavities or taken orally or applied through the skin with patches.
The dose chosen should be sufficient to constitute effective
treatment but not so high as to cause unacceptable side effects.
The state of the disease condition and the health of the patient
should preferably be closely monitored during and for a reasonable
period after treatment.
[0221] The term "therapeutically effective amount," as used herein,
refers to an amount of a pharmaceutical agent to treat, ameliorate,
or prevent an identified disease or condition, or to exhibit a
detectable therapeutic or inhibitory effect. The effect can be
detected by any assay method known in the art. The precise
effective amount for a subject will depend upon the subject's body
weight, size, and health; the nature and extent of the condition;
and the therapeutic or combination of therapeutics selected for
administration. Therapeutically effective amounts for a given
situation can be determined by routine experimentation that is
within the skill and judgment of the clinician. In a preferred
aspect, the disease or condition to be treated is cancer. In
another aspect, the disease or condition to be treated is a cell
proliferative disorder.
[0222] The therapeutically effective amount of milciclib is 1-500
mg administered one or more times over a day for up to 30 or more
days, followed by 1 or more days of non-administration of
milciclib. This type of treatment schedule, i.e., administration of
milciclib on consecutive days followed by non-administration of
milciclib on consecutive days may be referred to as a treatment
cycle. A treatment cycle may be repeated as many times as necessary
to achieve the intended affect.
[0223] In one embodiment, the therapeutically effective amount of
milciclib is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40,
45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115,
120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180,
185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245,
250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310,
315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375,
380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440,
445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, or 500 mg
once or twice daily for one, two, three, four, five, six, seven,
eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen
consecutive days, followed by non-administration for one, two,
three, four, five, six, or seven consecutive days, wherein the
cycle is optionally repeated 1, 2, or 3 times.
[0224] In one embodiment, the therapeutically effective amount of
milciclib is 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,
90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155,
160, 165, 170, 175, 180, 185, 190, 195, or 200 mg once or twice
daily for one, two, three, four, five, six, seven, eight, nine, or
ten consecutive days, followed by non-administration for one, two,
three, four, five, six, or seven consecutive days, wherein the
cycle is optionally repeated 1, 2, or 3 times.
[0225] In one embodiment, the therapeutically effective amount of
milciclib is 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,
105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 mg once or
twice daily for one, two, three, four, five, six, or seven
consecutive days, followed by non-administration for one, two,
three, four, five, six, or seven consecutive days, wherein the
cycle is optionally repeated 1, 2, or 3 times.
[0226] In one embodiment, the therapeutically effective amount of
milciclib is 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, or 125 mg
once daily for four consecutive days, followed by
non-administration for three consecutive days, wherein the cycle is
optionally repeated 1, 2, or 3 times.
[0227] For any compound, the therapeutically effective amount can
be estimated initially either in cell culture assays, e.g., of
neoplastic cells, or in animal models, usually rats, mice, rabbits,
dogs, or pigs. The animal model may also be used to determine the
appropriate concentration range and route of administration. Such
information can then be used to determine useful doses and routes
for administration in humans. Therapeutic/prophylactic efficacy and
toxicity may be determined by standard pharmaceutical procedures in
cell cultures or experimental animals, e.g., ED50 (the dose
therapeutically effective in 50% of the population) and LD50 (the
dose lethal to 50% of the population). The dose ratio between toxic
and therapeutic effects is the therapeutic index, and it can be
expressed as the ratio, LD50/ED50. Pharmaceutical compositions that
exhibit large therapeutic indices are preferred. The dosage may
vary within this range depending upon the dosage form employed,
sensitivity of the patient, and the route of administration.
[0228] Dosage and administration are adjusted to provide sufficient
levels of the active agent(s) or to maintain the desired effect.
Factors which may be taken into account include the severity of the
disease state, general health of the subject, age, weight, and
gender of the subject, diet, time and frequency of administration,
drug combination(s), reaction sensitivities, and tolerance/response
to therapy. Long-acting pharmaceutical compositions may be
administered every 3 to 4 days, every week, or once every two weeks
depending on half-life and clearance rate of the particular
formulation.
[0229] The pharmaceutical compositions containing active compounds
of the present application may be manufactured in a manner that is
generally known, e.g., by means of conventional mixing, dissolving,
granulating, dragee-making, levigating, emulsifying, encapsulating,
entrapping, or lyophilizing processes. Pharmaceutical compositions
may be formulated in a conventional manner using one or more
pharmaceutically acceptable carriers comprising excipients and/or
auxiliaries that facilitate processing of the active compounds into
preparations that can be used pharmaceutically. Of course, the
appropriate formulation is dependent upon the route of
administration chosen.
[0230] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringeability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyethylene glycol, and the like), and suitable
mixtures thereof. The 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 dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0231] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle that contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, methods of preparation are vacuum
drying and freeze-drying that yields a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0232] Oral compositions generally include an inert diluent or an
edible pharmaceutically acceptable carrier. They can be enclosed in
gelatin capsules or compressed into tablets. For the purpose of
oral therapeutic administration, the active compound can be
incorporated with excipients and used in the form of tablets,
troches, or capsules. Oral compositions can also be prepared using
a fluid carrier for use as a mouthwash, wherein the compound in the
fluid carrier is applied orally and swished and expectorated or
swallowed. Pharmaceutically compatible binding agents, and/or
adjuvant materials can be included as part of the composition. The
tablets, pills, capsules, troches and the like can contain any of
the following ingredients, or compounds of a similar nature: a
binder such as microcrystalline cellulose, gum tragacanth or
gelatin; an excipient such as starch or lactose, a disintegrating
agent such as alginic acid, Primogel, or corn starch; a lubricant
such as magnesium stearate or Sterotes; a glidant such as colloidal
silicon dioxide; a sweetening agent such as sucrose or saccharin;
or a flavoring agent such as peppermint, methyl salicylate, or
orange flavoring.
[0233] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from pressured container
or dispenser, which contains a suitable propellant, e.g., a gas
such as carbon dioxide, or a nebulizer.
[0234] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0235] In one aspect, the active compounds are prepared with
pharmaceutically acceptable carriers that will protect the compound
against rapid elimination from the body, such as a controlled
release formulation, including implants and microencapsulated
delivery systems. Biodegradable, biocompatible polymers can be
used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic
acid, collagen, polyorthoesters, and polylactic acid. Methods for
preparation of such formulations will be apparent to those skilled
in the art. The materials can also be obtained commercially from
Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal
suspensions (including liposomes targeted to infected cells with
monoclonal antibodies to viral antigens) can also be used as
pharmaceutically acceptable carriers. These can be prepared
according to methods known to those skilled in the art, for
example, as described in U.S. Pat. No. 4,522,811.
[0236] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the application are dictated by and directly dependent on the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved.
[0237] In therapeutic applications, the dosages of the
pharmaceutical compositions used in accordance with the application
vary depending on the agent, the age, weight, and clinical
condition of the recipient patient, and the experience and judgment
of the clinician or practitioner administering the therapy, among
other factors affecting the selected dosage. Generally, the dose
should be sufficient to result in slowing, and preferably
regressing, the growth of the tumors and also preferably causing
complete regression of the cancer. Dosages can range from about
0.01 mg/kg per day to about 3000 mg/kg per day. In preferred
aspects, dosages can range from about 1 mg/kg per day to about 1000
mg/kg per day. In an aspect, the dose will be in the range of about
0.1 mg/day to about 50 g/day; about 0.1 mg/day to about 25 g/day;
about 0.1 mg/day to about 10 g/day; about 0.1 mg to about 3 g/day;
or about 0.1 mg to about 1 g/day, in single, divided, or continuous
doses (which dose may be adjusted for the patient's weight in kg,
body surface area in m2, and age in years). An effective amount of
a pharmaceutical agent is that which provides an objectively
identifiable improvement as noted by the clinician or other
qualified observer. For example, regression of a tumor in a patient
may be measured with reference to the diameter of a tumor. Decrease
in the diameter of a tumor indicates regression. Regression is also
indicated by failure of tumors to reoccur after treatment has
stopped. As used herein, the term "dosage effective manner" refers
to amount of an active compound to produce the desired biological
effect in a subject or cell.
[0238] The pharmaceutical compositions can include co-formulations
of milciclib and any of the compounds described herein.
[0239] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[0240] The present application also relates to the following:
[0241] A. A method of treating or preventing cancer in a patient in
need thereof, comprising administering to the patient a
therapeutically effective amount of milciclib, or a
pharmaceutically acceptable salt, isomer, or tautomer thereof, in
combination with a therapeutically effective amount of another
anticancer drug selected from the group consisting of sorafenib,
lenvatinib, regorafenib, sunitinib, nivolumab, gemcitabine, and
palbociclib, or a pharmaceutically acceptable salt thereof.
[0242] B. A method of treating or preventing non-small cell lung
cancer, renal cell carcinoma, hepatocellular carcinoma, thyroid
carcinoma, colorectal cancer, gastrointestinal stromal tumors,
breast cancer, prostate cancer, pancreatic cancer, or thymoma in a
patient in need thereof, comprising administering to the patient a
therapeutically effective amount of milciclib, or a
pharmaceutically acceptable salt, isomer, or tautomer thereof, in
combination with a therapeutically effective amount of another
anticancer drug.
[0243] C. A method of treating or preventing non-small cell lung
cancer, renal cell carcinoma, hepatocellular carcinoma, thyroid
carcinoma, colorectal cancer, gastrointestinal stromal tumors,
breast cancer, prostate cancer, pancreatic cancer, or thymoma in a
patient in need thereof, comprising administering to the patient a
therapeutically effective amount of milciclib, or a
pharmaceutically acceptable salt, isomer, or tautomer thereof, in
combination with a therapeutically effective amount of another
anticancer drug selected from the group consisting of sorafenib,
lenvatinib, regorafenib, sunitinib, nivolumab, gemcitabine, and
palbociclib.
[0244] D. Any of methods disclosed herein, wherein the other
anticancer drug is sorafenib or a pharmaceutically acceptable salt
thereof. In one embodiment, the therapeutically effective amount of
sorafenib is 400 mg twice daily, 200 mg twice daily, or 200 mg once
daily. In one embodiment, the cancer is renal cell carcinoma,
hepatocellular carcinoma, or thyroid carcinoma.
[0245] E. Any of methods disclosed herein, wherein the other
anticancer drug is lenvatinib or a pharmaceutically acceptable salt
thereof. In one embodiment, the therapeutically effective amount of
lenvatinib is 8, 10, 12, 14, 18, 20, 22, 24, 26, 28, 30, 32, or 34
mg once daily. In one embodiment, the cancer is renal cell
carcinoma or thyroid carcinoma.
[0246] F. Any of methods disclosed herein, wherein the other
anticancer drug is regorafenib or a pharmaceutically acceptable
salt thereof. In one embodiment, the therapeutically effective
amount of regorafenib is 80, 100, or 120 mg once daily for three
weeks, followed by one week of no administration, wherein the cycle
is optionally repeated. In one embodiment, the cancer is colorectal
cancer or gastrointestinal stromal tumors.
[0247] G. Any of methods disclosed herein, wherein the other
anticancer drug is sunitinib or a pharmaceutically acceptable salt
thereof. In one embodiment, the therapeutically effective amount of
sunitinib is 12.5, 25, 37.5, 50, 62.5, 75, 87.5, or 100 mg once
daily continuously or for 4 weeks followed by two weeks of no
administration, wherein the cycle is optionally repeated. In one
embodiment, the cancer is renal cell carcinoma or gastrointestinal
stromal tumors.
[0248] H. Any of methods disclosed herein, wherein the other
anticancer drug is nivolumab. In one embodiment, the cancer is
non-small cell lung cancer or renal cell carcinoma.
[0249] I. Any of methods disclosed herein, wherein the other
anticancer drug is palbociclib or a pharmaceutically acceptable
salt thereof. In one embodiment, the therapeutically effective
amount of palbociclib is 75, 100, or 125 mg once daily for 3 weeks
followed by one week of no administration, wherein the cycle is
optionally repeated. In one embodiment, the cancer is breast
cancer.
[0250] J. Any of methods disclosed herein, wherein the other
anticancer drug is gemcitabine. In one embodiment, the
therapeutically effective amount of gemcitabine is 1000 mg/m.sup.2
over 30 minutes once weekly for seven weeks, followed by one week
of no administration, wherein the cycle is optionally repeated. In
one embodiment, the cancer is breast cancer.
[0251] K. Any of methods disclosed herein, wherein the
therapeutically effective amount of milciclib is 50, 75, 100, 125,
or 150 mg once daily for four consecutive days, followed by
non-administration for 3 consecutive days, wherein the cycle is
optionally repeated. For example, the therapeutically effective
amount of milciclib is 50, 75, 100, 125, or 150 mg once daily for
four consecutive days, followed by non-administration for 3
consecutive days, wherein the cycle is repeated as multiple times,
e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more times. For example, the
therapeutically effective amount of milciclib is about 100 mg once
daily for four consecutive days, followed by non-administration for
3 consecutive days, wherein the cycle is optionally repeated. For
example, the therapeutically effective amount of milciclib is 100
mg once daily for four consecutive days, followed by
non-administration for 3 consecutive days, wherein the cycle is
optionally repeated.
[0252] L. Any of methods disclosed herein, wherein milciclib and
the other anticancer drug are administered to the patient
simultaneously.
[0253] M. Milciclib and the other anticancer drug are administered
in a single pharmaceutical formulation that further includes a
pharmaceutically acceptable excipient. In one embodiment, wherein
the pharmaceutical formulation is in a controlled release form.
[0254] N. Milciclib and the other anticancer drug are each
administered in separate pharmaceutical formulations, wherein each
formulation further includes a pharmaceutically acceptable
excipient. In one embodiment, one or both of the pharmaceutical
formulations is in a controlled release form.
[0255] O. Any of methods disclosed herein, wherein milciclib and
the other anticancer drug are administered to the patient
sequentially. In one embodiment, the administration of milciclib
begins before administration of the other anticancer drug to the
patient. In one embodiment, the administration of milciclib begins
after administration of the other anticancer drug to the
patient.
[0256] P. Milciclib is administered in a single pharmaceutical
formulation that further includes a pharmaceutically acceptable
excipient. In one embodiment, the other anticancer drug is
administered in a single pharmaceutical formulation that further
includes a pharmaceutically acceptable excipient. In one
embodiment, any of the pharmaceutical formulations are formulated
for oral administration. For example, in one embodiment, the
pharmaceutical formulation is in the form of a tablet, pill, or
capsule.
[0257] Q. Any of methods disclosed herein, wherein the method of
treating or preventing renal cell carcinoma in a patient in need
thereof comprising administering to the patient a therapeutically
effective amount of milciclib, or a pharmaceutically acceptable
salt, isomer, or tautomer thereof, in combination with a
therapeutically effective amount of sorafenib.
[0258] R. Any of methods disclosed herein, wherein the
therapeutically effective amount of sorafenib is 400 mg twice
daily, 200 mg twice daily, or 200 mg once daily.
[0259] S. Any of methods disclosed herein, wherein the
therapeutically effective amount of milciclib is 50, 75, 100, 125,
or 150 mg once daily for four consecutive days, followed by
non-administration for 3 consecutive days, wherein the cycle is
optionally repeated.
[0260] T. Any of methods disclosed herein, wherein the method of
treating or preventing hepatocellular carcinoma in a patient in
need thereof comprising administering to the patient a
therapeutically effective amount of milciclib, or a
pharmaceutically acceptable salt, isomer, or tautomer thereof, in
combination with a therapeutically effective amount of
sorafenib.
[0261] U. Any of methods disclosed herein, wherein the
therapeutically effective amount of sorafenib is 400 mg twice
daily, 200 mg twice daily, or 200 mg once daily.
[0262] V. Any of methods disclosed herein, wherein the
therapeutically effective amount of milciclib is 50, 75, 100, 125,
or 150 mg once daily for four consecutive days, followed by
non-administration for 3 consecutive days, wherein the cycle is
optionally repeated.
[0263] W. A method of treating or preventing thyroid carcinoma in a
patient in need thereof comprising administering to the patient a
therapeutically effective amount of milciclib, or a
pharmaceutically acceptable salt, isomer, or tautomer thereof, in
combination with a therapeutically effective amount of
sorafenib.
[0264] X. Any of methods disclosed herein, wherein the
therapeutically effective amount of sorafenib is 400 mg twice
daily, 200 mg twice daily, or 200 mg once daily.
[0265] Y. Any of methods disclosed herein, wherein the
therapeutically effective amount of milciclib is 50, 75, 100, 125,
or 150 mg once daily for four consecutive days, followed by
non-administration for 3 consecutive days, wherein the cycle is
optionally repeated.
[0266] Z. Any of methods disclosed herein, wherein milciclib and
sorafenib are administered to the patient simultaneously.
[0267] AA. Any of methods disclosed herein, wherein milciclib and
sorafenib are administered in a single pharmaceutical formulation
that further includes a pharmaceutically acceptable excipient.
[0268] BB. Any of methods disclosed herein, wherein the
pharmaceutical formulation is in a controlled release form.
[0269] CC. Any of methods disclosed herein, wherein milciclib and
sorafenib are administered in separate pharmaceutical formulations,
wherein each formulation further includes a pharmaceutically
acceptable excipient. In one embodiment, one or both of the
pharmaceutical formulations is in a controlled release form.
[0270] DD. Any of methods disclosed herein, wherein milciclib and
sorafenib are administered to the patient sequentially.
[0271] EE. Any of methods disclosed herein, wherein, the
administration of milciclib begins before administration of
sorafenib to the patient.
[0272] FF. Any of methods disclosed herein, wherein the
administration of milciclib begins after administration of
sorafenib to the patient.
[0273] GG. Any of methods disclosed herein, wherein milciclib and
sorafenib are each administered in separate pharmaceutical
formulations that each further include a pharmaceutically
acceptable excipient. In one embodiment, one or both pharmaceutical
formulations are formulated for oral administration. For example,
in one embodiment, each pharmaceutical formulation is independently
in the form of a tablet, pill, or capsule.
[0274] HH. Milciclib, or a pharmaceutically acceptable salt,
isomer, or tautomer thereof, for use in the treatment or prevention
of cancer in a patient in need thereof, further comprising the use
of another anticancer drug that is selected from the group
consisting of sorafenib, lenvatinib, regorafenib, sunitinib,
nivolumab, gemcitabine, and palbociclib.
[0275] II. Milciclib, or a pharmaceutically acceptable salt,
isomer, or tautomer thereof, for use in the treatment or prevention
of non-small cell lung cancer, renal cell carcinoma, hepatocellular
carcinoma, thyroid carcinoma, colorectal cancer, gastrointestinal
stromal tumors, breast cancer, prostate cancer, pancreatic cancer,
or thymoma in a patient in need thereof, further comprising the use
of another anticancer drug.
[0276] JJ. Milciclib, or a pharmaceutically acceptable salt,
isomer, or tautomer thereof, for use in the treatment or prevention
of non-small cell lung cancer, renal cell carcinoma, hepatocellular
carcinoma, thyroid carcinoma, colorectal cancer, gastrointestinal
stromal tumors, breast cancer, prostate cancer, pancreatic cancer,
or thymoma in a patient in need thereof, further comprising the use
of another anticancer drug that is selected from the group
consisting of sorafenib, lenvatinib, regorafenib, sunitinib,
nivolumab, gemcitabine, and palbociclib.
[0277] KK. Milciclib, or a pharmaceutically acceptable salt,
isomer, or tautomer thereof, for use in the manufacture of a
medicament for the treatment or prevention of cancer in a patient
in need thereof, further comprising the use of another anticancer
drug that is selected from the group consisting of sorafenib,
lenvatinib, regorafenib, sunitinib, nivolumab, gemcitabine, and
palbociclib.
[0278] LL. Milciclib, or a pharmaceutically acceptable salt,
isomer, or tautomer thereof, for use in the manufacture of a
medicament for the treatment or prevention of non-small cell lung
cancer, renal cell carcinoma, hepatocellular carcinoma, thyroid
carcinoma, colorectal cancer, gastrointestinal stromal tumors,
breast cancer, prostate cancer, pancreatic cancer, or thymoma in a
patient in need thereof, further comprising the use of another
anticancer drug.
[0279] MM. Milciclib, or a pharmaceutically acceptable salt,
isomer, or tautomer thereof, for use in the manufacture of a
medicament for the treatment or prevention of non-small cell lung
cancer, renal cell carcinoma, hepatocellular carcinoma, thyroid
carcinoma, colorectal cancer, gastrointestinal stromal tumors,
breast cancer, prostate cancer, pancreatic cancer, or thymoma in a
patient in need thereof, further comprising the use of another
anticancer drug that is selected from the group consisting of
sorafenib, lenvatinib, regorafenib, sunitinib, nivolumab,
gemcitabine, and palbociclib.
[0280] All patents, patent applications, and publications mentioned
herein are hereby incorporated by reference in their entireties.
However, where a patent, patent application, or publication
containing express definitions is incorporated by reference, those
express definitions should be understood to apply to the
incorporated patent, patent application, or publication in which
they are found, and not to the remainder of the text of this
application, in particular the claims of this application.
[0281] It is to be understood that while the invention has been
described in conjunction with the preferred specific embodiments
thereof, that the foregoing description is intended to illustrate
and not limit the scope of the invention. It will be understood by
those skilled in the art that various changes may be made and
equivalents may be substituted without departing from the scope of
the invention, and further that other aspects, advantages and
modifications will be apparent to those skilled in the art to which
the invention pertains.
EXAMPLES
Example 1. IC50 Values by Cell Proliferation Assay
[0282] See Shailubhai, K et al. "Atiprimod is an inhibitor of
cancer cell proliferation and angiogenesis." J Exp Ther Oncol.
2004; 4: 267-279; and Choudhari et al. "Deactivation of Akt and
STAT3 signaling promotes apoptosis, inhibits proliferation, and
enhances the sensitivity of hepatocellular carcinoma cells to an
anticancer agent, atiprimod" Molecular Cancer Therapeutics. 2007;
6: 112-121. References are incorporated herein in their
entireties.
[0283] MHCC97H and MHCC97L (highly metastatic hepatocellular
carcinoma cell line, derived from humans)
[0284] HepG2.2.15 cells are derived from the human hepatoblastoma
cell line HepG2.
[0285] Cells (MHCC97H, MHCC97L, and HepG2.2.15) were cultured for
24 hours in 2% FBS and then were trypsinized, resuspended in 2% FBS
and seeded in a rat collagen coated 96-well plate at a density of
10,000 cells/100 .mu.l/well, a day before the experiment and
cultured at 37.degree. C. in 5% CO.sub.2. The cells were treated
the next day with milciclib, sorafenib, regorafenib, sunitinib, and
lenvatinib, individually or in combination in DMEM/F12+2% FBS+for
72 hours prior to the addition of WST-1 reagent. About 100 .mu.L
media was added to control well. After cells had been cultured for
72 hours with different drug concentrations, cells were washed 3
times with sterile 1.times. Phosphate buffered saline (PBS). To
determine cell proliferation by the colorimetric test, 10 .mu.L of
WST-1 reagent (Sigma Aldrich, St Louis, Mo.) was added in 100 .mu.L
cell culture media and incubated for 2 hours in 5% CO.sub.2 at
37.degree. C. At the end of incubation period, the plate was placed
on a plate shaker for 1 minute and was monitored using a
spectrophotometer at an optical density of 450 nm with a reference
wavelength of 600 nm using Tecan F200 and iControl software.
IC.sub.50 values were determined using GraphPad Prism (GraphPad
Software, La Jolla, Calif.). Each experiment was performed in
duplicate and repeated 2 times.
[0286] Each inhibitor exhibited a dose dependent decrease in cell
proliferation with comparable half maximal inhibitory concentration
(IC50) across the three cell lines: FIGS. 1 and 4--MHCC97H cells;
FIGS. 2 and 10-14--MHCC97L cells; and FIGS. 3 and 20-24--HepG2.2.15
cells.
[0287] Lenvatinib exhibited the lowest IC.sub.50 value followed by
milciclib, regorafenib, sorafenib and sunitinib.
TABLE-US-00001 CELL LINE PROTEIN KINASE MHCC97H MHCC97L HepG2.2.15
INHIBITORS IC.sub.50, .mu.M IC.sub.50, .mu.M IC.sub.50, .mu.M
MILCICLIB 1.3 1 1.16 SORAFENIB 12.01 8.8 6.48 REGORAFENIB 4.7 3.5
3.94 LENVATINIB 0.28 0.14 0.24 SUNITINIB 30.48 8.2 25.43
PABLOCICLIB 14.5 8.2 11.11
Example 2. IC.sub.50 Values of Inhibitors in Combination with
Milciclib by Cell Proliferation Assay in MHCC97H Cells
Synergy Studies
TABLE-US-00002 [0288] IC.sub.50 (.mu.M) IC.sub.50 (.mu.M) OF KINASE
PROTEIN KINASE KINASE INHIBITORS + INHIBITORS INHIBITORS 1.3 .mu.M
MILCICLIB SORAFENIB 12.01 6.7 REGORAFENIB 4.7 1.9 LENVATINIB 0.28
0.12 SUNITINIB 30.4 17.2 PALBOCICLIB 14.5 6.7
[0289] A synergistic effect on inhibition of cell proliferation was
observed upon treating MHCC97H, MHCC97L, and HepG2.2.15 cells with
increasing concentration of TKIs in the presence of fixed
concentration corresponding to milciclib IC.sub.50 value. In all
cases, the IC.sub.50 value of each TKI was reduced by .about.50%
(MHCC97H: FIGS. 5-9; MHCC97L: FIGS. 15-19; HepG2.2.15: FIGS.
25-28).
[0290] Increasing concentration of inhibitors with a fixed
concentration of milciclib was tested on MHCC97L and MHCC97H cells
to determine the synergistic effect on inhibition of cell
proliferation.
[0291] In the synergy studies the arrow represents the mid-point of
combination of TKIs with milciclib. For sorafenib, the individual
IC.sub.50 was 12 .mu.M but with the combination with milciclib the
IC.sub.50 was 6.7 .mu.M in MHCC97H (FIG. 29A).
[0292] For lenvatinib, the individual IC.sub.50 was 0.28 .mu.M but
with the combination with milciclib the IC.sub.50 was 0.12 .mu.M in
MHCC97H (FIG. 29B).
[0293] For regorafenib, the individual IC.sub.50 was 4.7 .mu.M but
with the combination with milciclib the IC.sub.50 was 1.9 .mu.M in
MHCC97H (FIG. 29C).
Example 3. MHCC97H Cells Produce Human Alphafetoprotein (AFP)
AFP ELISA Assay
[0294] MHCC97H cells were seeded at a density of 500,000 cells/2
mL/well on a 6 well culture plate and incubated overnight at
37.degree. C. in a humidified CO.sub.2 incubator. Cells were then
treated with 1.3 .mu.M milciclib or vehicle in DMEM/F12+2% FBS for
72 hours. Subsequently, cells were lysed in RIPA buffer, the
supernatant was collected and used to perform ELISA assay as per
the manufacturer's instructions. In orthotopic HCC mouse model,
levels of serum marker alpha-fetoprotein (AFP) were determined on
day 0, 6, 12, 18, 24, 30, 36, 42 and 48 using High Range AFP kit as
per manufacturer's instructions.
[0295] Freshly cultured MHCC97H cells were treated with 1.3 .mu.M
milciclib for 72 hours to determine the AFP levels. Appreciably
lower levels of AFP were detected in milciclib treated cells as
compared to vehicle control (FIG. 30).
Example 4. Promega Apotox-Glo.TM. Triplex Assay
[0296] The HCC cells were seeded at a density of 10,000 cells/100
.mu.L in each well of a rat collagen coated 96 well plate and
allowed to grow overnight in 5% CO.sub.2 at 37.degree. C. The cells
were then treated with different concentrations of each agent alone
or in combination with 1.3 milciclib in MHCC97H and 1.16 .mu.M
milciclib in MHCC97L cells for 48 hours. Promega ApoTox-Glo Triplex
assay (Madison, Wis.) was used according to manufacturer's
instructions to determine the number of viable cells, cell death
because of apoptosis and cytotoxic effect on cells. After 48 hours
the viability/cytotoxicity reagent, containing both the GF-AFC
substrate and the bis-AAF-R110 substrate, was added to all wells
and incubated for 30 minutes and was measured at an optical density
of 400EX/505EM for viability and 485EX/520EM for cytotoxicity. For
apoptosis, caspase-glo 3/7 was added to all wells, mixed briefly at
500 rpm for 30 seconds, then incubated at room temperature for 30
minutes and luminescence was measured which is proportional to the
amount of caspase activity present. See Ito H, Uchida T, Makita K.
Ketamine causes mitochondrial dysfunction in human induced
pluripotent stem cell-derived neurons. PLoS One. 2015; 10:
e0128445) for experimental details. Reference is incorporated
herein in its entirety.
[0297] Milciclib in combination with other TKIs at various
concentrations decreased the cell viability and increased caspase
3/7 activity in MHCC97H cells (FIGS. 31-39) and MHCC97L cells
(FIGS. 40-50) in a dose-dependent manner compared to those in
vehicle-treated cells.
Example 5. Wound Healing (Using 0.5.times.10.sup.6 Cells)
[0298] Cells were seeded at a density of 500,000 cells/2 mL/well on
to a collagen-coated 6-well culture plate and incubated overnight
in a humidified CO.sub.2 incubator at 37.degree. C. to form a
uniform monolayer. The monolayer was then scratched in a straight
line with a new 104, pipette tip across the center of the well.
After scratching, cells were washed with sterile PBS once to remove
detached cells. Subsequently, wells were replenished with fresh
DMEM/F12 containing 2% FBS and the test article either alone or in
combination with milciclib. Photos were taken of the scratched
monolayer immediately T.sub.0 and after various times 24, 48, and
72 hours using an Olympus IX81 microscope. Images were analyzed
using SlideBook.TM. 5 software. The values were expressed as a
percentage of migration. See Saxena N K, Sharma D, Ding X, et al.
Concomitant activation of the JAK/STAT, PI3K/AKT, and ERK signaling
is involved in leptin-mediated promotion of invasion and migration
of hepatocellular carcinoma cells. Cancer Res. 2007; 67:
2497-2507.
[0299] Treatment of scratched monolayer of MHCC97H cells with TKIs
(tyrosine kinase inhibitors) in combination with milciclib (1.3
.mu.M) for 96 h, reduced cell migration as compared to
corresponding vehicle control (FIGS. 51-55).
[0300] Treatment of scratched monolayer of MHCC97L cells with
milciclib alone or in combination with other TKIs for 96 hours,
reduced cell migration as compared to corresponding vehicle control
(FIGS. 56-60).
[0301] Treatment of scratched monolayer of HepG2.2.15 cells with
milciclib alone or in combination with other TKIs for 96 hours,
reduced cell migration as compared to corresponding vehicle control
(FIGS. 61-63).
Example 6 EMT Assays
[0302] EMT (Epithelial to Mesenchymal Transition) induction
employing kit from R&D systems (Cat #CCM017)
[0303] For transwell invasion assay HCC cells were seeded in the
top chamber in 6 well transwell plates (Sigma-Aldrich, St. Louis,
Mo.) at a density of 100,000 cells/500 .mu.L/well in standard
culture media containing 100.times. StemXVivo.RTM. EMT Inducing
Media Supplement (R&D systems, Minneapolis, Minn.) and was
incubated overnight at 37.degree. C. in humidified CO.sub.2
incubator. Next day, EMT inducing media with or without the test
articles was added and incubation was continued for 10 days with
media changes every 3 days. On the 10th day, the number of cells
migrated to the lower chamber was determined using Bio-Rad
(Hercules, Calif.) automatic cell counter. Values obtained were
expressed as a percentage of invasion and the cell counts of
control cells were considered 100%.
[0304] The invasive potential of cancer cells is dependent on
losing epithelial characteristics and acquiring a migratory
mesenchymal property referred to as Epithelial to Mesenchymal
Transition (EMT).
[0305] The inclusion of milciclib or TKIs alone resulted in
statistically significant inhibition (P<0.05) in cell migration
in MHCC97L cells. Regorafenib, sorafenib, sunitinib and lenvatinib
in combination with milciclib reduced the invasion potential to a
greater extent as compared to individual treatment (P<0.005)
(FIG. 64).
[0306] The inclusion of milciclib or TKIs alone resulted in
statistically significant inhibition (P<0.05) in cell migration
in MHCC97H cells. Regorafenib, sorafenib, sunitinib and lenvatinib
in combination with milciclib reduced the invasion potential to a
greater extent as compared to individual treatment (P<0.005),
demonstrative of the anti-invasive potential of milciclib (FIG.
65).
Example 7: Orthotopic Tumor Induction in Athymic Nude Mice
[0307] Mouse experiments were performed in accordance with the
guidelines approved by the Institutional Animal Care and use
committee of Washington Biotechnology Inc (Baltimore, Md.) where
the studies were conducted. (FIG. 66) Test agents were dissolved in
chremophor/ethanol (1:1) to make a 5.times. stock solution and
diluted in water when used. MHCC97H human liver cells (5.times.106)
in PBS were mixed with 20% Matrigel and then inoculated
orthotopically into the right flank of female Balb/c nude mice.
Seven days after cell inoculation, the mice were randomly allocated
to either the treatment group (n=12) or the control group (n=12)
based on the levels of AFP. The daily animal inspection was
conducted for general appearance and tumor growth. Milciclib (30
mg/Kg), sorafenib (20 mg/Kg), milciclib+sorafenib or the
corresponding vehicle was given orally to individual mice once
daily from day 12 until day 47. After completion of the treatment
at day 48, animals were euthanized and blood, liver tissues and
tumor were collected for gene expression and mechanistic assays.
Liver tumors developed 100% of animals challenged with orthotopic
MHCC97H injection.
[0308] Oral administration of milciclib (30 mg/kg/day) either alone
or in combination with sorafenib (20 mg/kg/day) produced
synergistic effect in reducing tumor growth [milciclib -20%
(p<0.002) or sorafenib -21% (p<0.001) vs combination -38%
(p<0.0002) as compared to vehicle (FIG. 67). Vehicle group had
more liver weight but with combination the liver weight goes down
(FIG. 68).
[0309] Pictures showing the difference in tumor burden with the
treatment of milciclib, sorafenib, and the combination are
provided. Vehicle group has an enlarged tumor but with the
combination, the tumor burden goes down (FIG. 69).
[0310] A steady increase in serum AFP was observed in vehicle
administered animals until the end of the study. Significantly
lower serum AFP levels were recorded for animals treated with
milciclib (30 mg/kg), sorafenib (20 mg/kg) alone or in combination
(FIG. 70).
Example 8: Milciclib Acts Via Specifically Downregulating
MIR221/222
[0311] miRNA Isolation and Expression Analysis
[0312] miRNA from athymic nude mice following treatment with
vehicle, milciclib, sorafenib, or milciclib+sorafenib was isolated
from Total RNA using the TaqMan Advanced miRNA cDNA Synthesis Kit
(Thermo Fisher Scientific, Rockford, Ill.) according to the
manufacturer's instructions. miRNA-221 and miR-222 quantifications
were performed in duplicates using TaqMan Advanced miRNA Assay
(Thermo Fisher Scientific, Rockford, Ill.) with a sample dilution
of 1:10. The PCR mixture was incubated at 95.degree. C. for 20
seconds, followed by 40 cycles of 95.degree. C. for 3 seconds and
60.degree. C. for 30 seconds. Results were normalized to
hsa-miR-192-5p as reference miRNA and the relative gene expression
calculated as 2-.DELTA.CT was expressed as fold increase over
control samples.
[0313] Gene expression studies suggest that milciclib possibly
exerts its action through downregulation of miR-221 and
miR-222.
[0314] Data suggest that oral treatment with milciclib exerts its
activity via downregulation of miR-221 and miR-222 (FIGS. 71A and
71B).
[0315] Tumor tissues from mice treated with vehicle, sorafenib,
milciclib, milciclib+sorafenib and normal liver tissues from naive
untreated mice were collected and levels of miR-221 and miR-222
were determined. The levels of both of miR-221 and miR-222 were
elevated in tumors from vehicle treated mice. Treatment with
sorafenib alone modestly reduced the expression of these miRs but
treatment with milciclib alone significantly reduced expression of
both miR-221 and miR-222. These data suggest that milciclib
specifically acts via reducing expression of miR-221 and miR-222,
which are known to be major culprits of hepatocarcinogenesis (See
Park J K, et al. "miR-221 silencing blocks hepatocellular carcinoma
and promotes survival." Cancer Res. 2011; 71:7608-76.) These data
also imply that oral treatment with milciclib reduced tumor growth
via a mechanism distinct from orally administered sorafenib.
Example 9: Milciclib Mechanism of Action Studies
Western Blot Analysis
[0316] The tumor tissues from a thymic nude mice were weighed and
homogenized with RIPA lysis buffer (Sigma-Aldrich, St. Louis, Mo.)
and protease inhibitor cocktail (Sigma-Aldrich, St. Louis, Mo.).
Tumor lysates were cleared by centrifugation and the protein
concentration was determined using Bradford reagent (Sigma-Aldrich,
St. Louis, Mo.). Equal amounts of protein (30 .mu.g) were resolved
on precast polyacrylamide gels (Thermo Fisher Scientific, Rockford,
Ill.) and transferred to nitrocellulose membrane, 0.2 .mu.m pore
size (Thermo Fisher Scientific, Rockford, Ill.). The blots were
blocked with 5% (w/v) nonfat dry milk for 2 h at room temperature
and then probed with primary antibody overnight at 4.degree. C. The
primary antibodies were directed against the following proteins:
human PTEN, human AKT and phospho-AKT (Ser473), human c-Myc, human
CyclinD1 and human (3-actin (Cell Signaling Technology, Beverly,
Mass.). After three washes, incubation was followed by the reaction
with horseradish peroxidase-conjugated secondary antibody for 1 h
at room temperature. The immunoreactive bands were visualized using
Image Studio 4.0-Western Analysis Ribbon (Li-Cor, Lincoln,
Nebr.).
Reverse Transcription and Quantitative Real-Time PCR
[0317] Total RNA was extracted using the RNeasy Mini kit according
to the manufacturer's instructions (Qiagen, Germantown, Md.). RNA
was quantified using Nanodrop Lite (Thermo Scientific, Wilmington,
Del.). Complementary DNA (cDNA) synthesis was performed by reverse
transcription of total RNA using the High Capacity cDNA Reverse
Transcription Kit (Thermo Fisher Scientific, Rockford, Ill.).
Real-time quantitative PCR was employed using the LightCycler 480
Instrument II (Roche Diagnostics Corporation, Indianapolis, Ind.)
using TaqMan Fast Advanced Master Mix and TaqMan Gene Expression
probes for human p27, cyclin E2, cyclin A2, CdC6, MKI67, cyclin D1,
p21, p57, c-Myc and p53 (Thermo Fisher Scientific). The expression
of target genes was normalized to the housekeeping gene GAPDH in
each sample. All samples were run in duplicate and the relative
gene expression calculated as 2-.DELTA.CT was expressed as fold
increase over control samples.
[0318] The mechanism of action of milciclib appears to be distinct
from the mechanism of action of sorafenib as it upregulated the
expression of tumor suppressors such as p27, p21, p53 and p57
(FIGS. 72A, B, C, D).
[0319] Oral administration of milciclib alone or in combination
with sorafenib downregulated expression of cyclins such as cyclin
E2 and cyclin D1 (FIGS. 73A and 73B).
[0320] Oral administration of milciclib alone or in combination
with sorafenib downregulated expression of cell proliferation genes
such as MKI67, cdc6, c-Myc (FIGS. 74A, 74B, 74C).
[0321] The mechanism of action of milciclib appears to be distinct
from the mechanism of action of sorafenib.
[0322] Mechanistic studies revealed a reduction in pAKT, c-Myc and
cyclin D1 expression and upregulation of PTEN in liver samples
derived from milciclib and milciclib and sorafenib administered
animals as compared to vehicle treated group (FIG. 75).
[0323] Data from cell culture studies and from orthotopic HCC model
in nude mice suggest that oral treatment with milciclib exerts its
activity via a new mechanism.
Example 10: AFP ELISA Assay
[0324] MHCC97H cells were seeded at a density of 500,000 cells/2
mL/well on a 6 well culture plate and incubated overnight at
37.degree. C. in a humidified CO.sub.2 incubator. Cells were then
treated with 1.3 .mu.M milciclib or vehicle in DMEM/F12+2% FBS for
72 hours. Subsequently, cells were lysed in RIPA buffer, the
supernatant was collected and used to perform ELISA assay as per
the manufacturer's instructions. In orthotopic HCC mouse model,
levels of serum marker alpha-fetoprotein (AFP) were determined on
day 0, 6, 12, 18, 24, 30, 36, 42 and 48 using High Range AFP kit as
per manufacturer's instructions.
Example 11: Mechanism of Action
[0325] Hepatocellular carcinoma (HCC) is an extremely complex
multi-factorial condition associated with many confounding factors
affecting disease course and patient prognosis. A broad range of
mechanisms, including telomere dysfunction, activation of oncogenic
pathways, abrogation of DNA damage checkpoints, activation of
pro-inflammatory and metastatic pathways, and induction of the
oxidative stress response. Consequently, HCC is typically
associated with overexpression of receptor tyrosine kinases (RTK)
and excessive oxidative stress (ROS). Collectively, overexpression
of RTK and ROS lead to increased expression of c-myc, resulting in
high metastatic potentials of hepatocytes. Thus, metastatic
potential of hepatocytes can be reduced with specific inhibitors of
RTK. On the other hand, HCC is also associated with overexpression
of miR-221, miR-222 and CDKs, resulting in dysregulation of cell
cycle, which leads to excessive proliferation of hepatocytes.
Treatment with milciclib is known to inhibit miR-221/miR-222 and a
number of CDKs and it can effectively reduce proliferation of
hepatocytes. Therefore, collectively combination of milciclib with
an inhibitor of RTK may produce synergistic effect in reducing
expression of c-myc and in total tumor growth and progression.
Thus, an effective therapy for HCC needs to control proliferation
of hepatocytes and also suppress their metastatic potential. (FIG.
76)
* * * * *
References