U.S. patent application number 15/159444 was filed with the patent office on 2016-11-24 for treatment agents for inhibiting hiv and cancer in hiv infected patients.
This patent application is currently assigned to University of Maryland, Baltimore. The applicant listed for this patent is Charles E. Davis, Ronald B. Gartenhaus, Alonso Heredia, Robert R. Redfield, Edward A. Sausville. Invention is credited to Charles E. Davis, Ronald B. Gartenhaus, Alonso Heredia, Robert R. Redfield, Edward A. Sausville.
Application Number | 20160339030 15/159444 |
Document ID | / |
Family ID | 57324090 |
Filed Date | 2016-11-24 |
United States Patent
Application |
20160339030 |
Kind Code |
A1 |
Redfield; Robert R. ; et
al. |
November 24, 2016 |
TREATMENT AGENTS FOR INHIBITING HIV AND CANCER IN HIV INFECTED
PATIENTS
Abstract
Methods are provided for treating HIV and cancer in a subject in
need thereof by administering to the subject therapeutically
effective amounts of an mTOR inhibitor. Other methods are provided
for treating subjects infected with HIV by administering to the
subject therapeutically effective amounts of the mTOR inhibitor
INK128, GSK2126458, AZD2014 or Torin-2.
Inventors: |
Redfield; Robert R.;
(Baltimore, MD) ; Heredia; Alonso; (Washington,
DC) ; Davis; Charles E.; (Laurel, MD) ;
Gartenhaus; Ronald B.; (Pikesville, MD) ; Sausville;
Edward A.; (Edgewater, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Redfield; Robert R.
Heredia; Alonso
Davis; Charles E.
Gartenhaus; Ronald B.
Sausville; Edward A. |
Baltimore
Washington
Laurel
Pikesville
Edgewater |
MD
DC
MD
MD
MD |
US
US
US
US
US |
|
|
Assignee: |
University of Maryland,
Baltimore
Baltimore
MD
|
Family ID: |
57324090 |
Appl. No.: |
15/159444 |
Filed: |
May 19, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62163408 |
May 19, 2015 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/5377 20130101;
A61K 31/4745 20130101; A61K 31/519 20130101; A61K 31/501 20130101;
A61K 45/06 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 31/5377
20130101; A61K 31/4745 20130101; A61K 31/501 20130101; A61K 31/519
20130101 |
International
Class: |
A61K 31/519 20060101
A61K031/519; A61K 45/06 20060101 A61K045/06; A61K 31/5377 20060101
A61K031/5377; A61K 31/4745 20060101 A61K031/4745; A61K 31/501
20060101 A61K031/501 |
Claims
1. A method of treating Human Immunodeficiency Virus and cancer in
a subject in need thereof comprising administering to the subject a
therapeutically effective amount of an mTOR inhibitor.
2. The method of claim 1, wherein the mTOR inhibitor is selected
from the group consisting of INK128, GSK2126458, AZD2014, and
Torin-2.
3. The method of claim 2, wherein the mTOR inhibitor is INK128 or
Torin-2.
4. The method of claim 3, wherein INK128 is administered in a
therapeutically effective amount from about 0.5 mg to about 4 mg to
achieve a plasma concentration of about 200 nM in the subject.
5. The method of claim 3, wherein Torin-2 is administered in a
therapeutically effective amount from 0.05 mg to about 10 mg.
6. The method of claim 2 wherein GSK2126458 is administered in a
therapeutically effective amount from 0.05 mg to about 0.25 mg.
7. The method of claim 2, wherein AZD2014 is administered in a
therapeutically effective amount from 5 mg to about 50 mg.
8. The method of claim 2, wherein the mTOR inhibitor is
administered orally, intravenously, intramuscularly, intrathecally,
or subcutaneously, sublingually. buccally, rectally, vaginally, by
ocular route or by otic route, nasally, by inhalation, by
nebulization, cutaneously, topically or systemically, and
transdermally.
9. The method of claim 2, wherein the mTOR inhibitor is
administered alone, or in combination with a second mTOR
inhibitor.
10. The method of claim 9, wherein the mTOR inhibitor is
INK128.
11. The method of claim 10, wherein INK128 is administered in
combination with Torin-2.
12. The method of claim 2, wherein the mTOR inhibitor is
administered alone, or in combination with a second antiretroviral
agent.
13. The method of claim 12, wherein the mTOR inhibitor and the
second antiretroviral agent are administered at the same time, at
different times, or sequentially.
14. The method of claim 12, wherein the second antiretroviral agent
is selected from the group consisting of CCR5 antagonists, reverse
transcriptase inhibitors, integrase inhibitors, protease
inhibitors, and any combination thereof.
15. The method of claim 12, wherein the wherein the mTOR inhibitor
and the second antiretroviral agent are administered in combination
with a cancer therapeutic agent selected from the group consisting
of DNA damaging agents, microtubule agents, and signal transduction
agents.
16. The method of claim 15, wherein the cancer therapeutic agents
are selected from the group consisting of carboplatin, BCNU,
cytosine arabinoside, paclitaxel, vinblastine, sorafenib,
pazopanib, erlotinib, and imatinib.
17. The method of claim 1, wherein the cancer is selected from the
group consisting of non-small cell lung, small cell lung, prostate,
breast, liver, Hodgkin lymphoma, non-Hodgkin lymphoma, Kaposi
sarcoma, B cell acute lymphoblastic leukemia, bone sarcoma, and
soft tissue sarcoma.
18. A method of treating Human Immunodeficiency Virus in a subject
in need thereof comprising administering to the subject a
therapeutically effective amount of a an INK128 having a chemical
structure: ##STR00005## or an mTOR inhibitor Torin-2 having a
chemical structure: ##STR00006## or a salt or hydrate thereof.
19. The method of claim 18, wherein INK128 is administered in a
therapeutically effective amount from about 0.5 mg to about 4 mg to
achieve a plasma concentration of about 200 nM in the subject.
20. The method of claim 18, wherein Torin-2 is administered in a
therapeutically effective amount from about 0.05 mg to about 10
mg.
21. The method of claim 18, wherein the mTOR inhibitor is
administered alone, or in combination with a second antiretroviral
agent.
22. The method of claim 21, wherein the antiretroviral agent is
selected from the group consisting of an entry inhibitor, a reverse
transcriptase inhibitor, a protease inhibitor and an integrase
inhibitor.
23. The method of claim 22, wherein the antiretroviral agent is
selected from the group consisting of CCR5 antagonists, reverse
transcriptase inhibitors, integrase inhibitors, protease inhibitors
and any combination thereof.
24. The method of claim 23, wherein the antiretroviral agents are
selected from the group consisting of maraviroc, efavirenz,
raltegravir, indinavir, and any combination thereof.
25. The method of claim 21, wherein the mTOR inhibitor and the
second antiretroviral agent are administered in combination with a
cancer therapeutic agent selected from the group consisting of DNA
damaging agents, microtubule agents, and signal transduction
agents.
26. The method of claim 25, wherein the cancer therapeutic agents
are selected from the group consisting of: carboplatin, BCNU,
cytosine arabinoside, paclitaxel, vinblastine, sorafenib,
pazopanib, erlotinib, and imatinib.
27. A method of claim 1, wherein the therapeutically effective
amount of mTOR inhibitor inhibits both R5 and X4 HIV
replication.
28. A method of claim 1, wherein the therapeutically effective
amount of mTOR inhibitor inhibits entry of R5 Human
Immunodeficiency Virus in vitro.
29. A method of claim 1, wherein the therapeutically effective
amount of mTOR inhibitor inhibits LTR gene expression.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of provisional
application 62/163,408, entitled "A Single Treatment Agent for
Inhibiting HIV and Cancer in HIV Infected Patients," filed May 19,
2015, the entire contents of which are incorporated herein.
BACKGROUND
[0002] Human Immunodeficiency Virus (HIV), the virus that causes
AIDS, is one of the world's most serious health and development
challenges. According to the World Health Organization (WHO) there
were approximately 36.9 million people worldwide living with
HIV/AIDS in 2014. Of these, 2.6 million were children (<15 years
old).
[0003] In the United States, the CDC estimates that 1,218,400
persons aged 13 years and older are living with infection,
including 156,300 (12.8%) who are unaware of their infection. Over
the past decade, the number of people living with HIV has
increased, while the annual number of new HIV infections has
remained relatively stable. Still, the pace of new infections
continues at far too high a level--particularly among certain
groups. In 2013, an estimated 47,352 people were diagnosed with HIV
infection in the United States. In that same year, an estimated
26,688 people were diagnosed with AIDS. Overall, an estimated
1,194,039 people in the United States have been diagnosed with
AIDS.
[0004] Despite significant advances in the treatment of HIV
infection with the use of antiretroviral therapies, a strong need
for the development of alternative antiviral agents exists. The HIV
virus has been difficult to treat in view of the emergence of
drug-resistant viral strains, the need for sustained adherence to
complex treatment regimens, and the toxicity of currently used
antivirals agents.
SUMMARY
[0005] It has been discovered that certain mTOR inhibitors (e.g.,
INK128, Torin-2, GSK2126458, and AZD2014)128 and Torin-2) are
effective in treatment of the HIV virus as well as in HIV-infected
subjects diagnosed with cancer. In certain embodiments, methods are
provided for treating the HIV virus and cancer in a subject in need
by administering to the subject a therapeutically effective amount
of an mTOR inhibitor. The mTOR inhibitor is selected from the group
consisting of INK128, GSK2126458, AZD2014, and Torin-2. Preferably,
the mTOR inhibitor is INK128 or Torin-2.
[0006] INK128 is administered in certain embodiments in a
therapeutically effective amount from about 0.5 mg to about 4 mg to
achieve a plasma concentration of about 200 nM in the subject.
Torin-2 may be administered in a therapeutically effective amount
from 0.05 mg to about 10 mg. GSK2126458 is administered in a
therapeutically effective amount from 0.05 mg to about 0.25 mg in
certain embodiments. In other embodiments, AZD2014 is administered
in a therapeutically effective amount from 5 mg to about 50 mg.
[0007] The mTOR inhibitor (e.g., INK128, Torin-2, GSK2126458, and
AZD2014)128 or Torin-2) may be administered orally, intravenously,
intramuscularly, intrathecally, or subcutaneously, sublingually.
buccally, rectally, vaginally, by ocular route or by otic route,
nasally, by inhalation, by nebulization, cutaneously, topically or
systemically, and transdermally in certain embodiments.
[0008] In other embodiments, the mTOR inhibitor (e.g., INK128,
Torin-2, GSK2126458, and AZD2014)128 or Torin-2) is administered
alone, or in combination with a second mTOR inhibitor or a second
antiretroviral agent. The mTOR inhibitor and the second
antiretroviral agent are administered at the same time, at
different times, or sequentially in certain embodiments. The second
antiretroviral agent may be selected from the group consisting of
CCR5 antagonists, reverse transcriptase inhibitors, integrase
inhibitors, protease inhibitors, and any combination thereof. The
mTOR inhibitor and the second antiretroviral agent can also be
administered in combination with a cancer therapeutic agent
selected from the group consisting of DNA damaging agents,
microtubule agents, and signal transduction agents. For example,
the cancer therapeutic agents are selected from the group
consisting of carboplatin, BCNU, cytosine arabinoside, paclitaxel,
vinblastine, sorafenib, pazopanib, erlotinib, and imatinib.
[0009] In certain embodiments, the type of cancer is selected from
the group consisting of non-small cell lung, small cell lung,
prostate, breast, liver, Hodgkin lymphoma, non-Hodgkin lymphoma,
Kaposi sarcoma, B cell acute lymphoblastic leukemia, bone sarcoma,
and soft tissue sarcoma.
[0010] Methods of treating HIV in a subject in need thereof are
also provided comprising administering to the subject a
therapeutically effective amount of INK128 having a chemical
structure described herein or Torin-2 having a chemical structure
described herein or salts or hydrates thereof. INK128 may be
administered in a therapeutically effective amount from about 0.5
mg to about 4 mg to achieve a plasma concentration of about 200 nM
in the subject. Torin-2 may be administered in a therapeutically
effective amount from about 0.05 mg to about 10 mg.
[0011] In other embodiments, the mTOR inhibitor (e.g., INK128,
Torin-2, GSK2126458, and AZD2014)128 or Torin-2) is administered
alone, or in combination with a second antiretroviral agent. The
antiretroviral agent may be selected from the group consisting of
an entry inhibitor, a reverse transcriptase inhibitor, a protease
inhibitor and an integrase inhibitor. Preferably, the
antiretroviral agent is selected from the group consisting of CCR5
antagonists, reverse transcriptase inhibitors, integrase
inhibitors, protease inhibitors and any combination thereof. The
antiretroviral agents are selected from the group consisting of
maraviroc, efavirenz, raltegravir, indinavir, and any combination
thereof.
[0012] The mTOR inhibitor and the second antiretroviral agent may
be administered in combination with a cancer therapeutic agent
selected from the group consisting of DNA damaging agents,
microtubule agents, and signal transduction agents in certain
embodiments. The cancer therapeutic agents are selected from the
group consisting of: carboplatin, BCNU, cytosine arabinoside,
paclitaxel, vinblastine, sorafenib, pazopanib, erlotinib, and
imatinib.
[0013] Methods are provided in certain embodiments with a
therapeutically effective amount of mTOR inhibitor that inhibits
both R5 and X4 HIV replication. Other methods are provided with a
therapeutically effective amount of mTOR inhibitor that inhibits
entry of R5 HIV in vitro. In yet other methods, the therapeutically
effective amount of mTOR inhibitor inhibits LTR gene
expression.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] The following drawings form part of the present
specification and are included to further demonstrate certain
embodiments of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0015] FIG. 1A-1C are representations that illustrate that INK128
inhibits replication of R5 HIV BaL and X4 HIV HXB2 in primary
cells. FIG. 1A is a diagram that illustrates the chemical structure
of INK128. FIG. 1B is a graph illustrating the effect of INK128 on
cell viability. PBLs from healthy donors were activated with
antibodies against CD3 and CD8 for 3 days. Activated cells were
cultured in medium containing IL-2 and the indicated concentrations
of INK128 for 5 days. On day 5, cell viability was measured by MTT.
FIG. 1C is a graph illustrating the effect of INK128 on HIV
replication. Activated PBLs were infected with R5 HIV BaL or X4 HIV
HXB2 for 2 hours using a MOI of 0.001. Infected cells were cultured
in IL-2 medium and various dilutions of INK128. On day 7, virus
production was measured by p24 ELISA in the culture supernatants
according to an embodiment.
[0016] FIG. 2A-2B are bar graphs that illustrate how INK128
inhibits replication of multidrug resistant HIV molecular clone
NL4329129-2. Activated PBLs were infected for 2 hours using a MOI
of 0.001. Infected cells were cultured in IL-2 medium and the
indicated concentrations of INK128. On day 7, virus production was
measured by p24 ELISA in the culture supernatants (FIG. 2A), and
cell viability was measured by MTT (FIG. 2B) according to an
embodiment.
[0017] FIG. 3A-3F are graphs that illustrate that INK128 inhibits
entry of R5, but not X4, HIV in primary lymphocytes. FIG. 3A-3B are
bar graphs that shows cell-cell fusion between effector cells
expressing R5 HIV JRFL Env or X4 HIV HXB2 Env and primary CD4+ T
target cells. FIG. 3C-3D are graphs that depict early products of
reverse transcription (R/U5 region) in PBLs infected with R5 (JRFL)
or X4 (HXB2) HIV and treated with INK128 for 16 hours FIG. 3E-3F
are graphs that illustrates integrated HIV DNA in PBLs infected
with R5 (JRFL) or X4 (HXB2) HIV and treated with INK128 for 72
hours according to an embodiment.
[0018] FIG. 4A-4C are bar graphs that reflect INK128's reduction of
CCR5, but not CXCR4 or CD4, levels on PBLs. PBLs were cultured in
IL-2 medium and various concentrations of INK128 for 7 days before
Flow Cytometry Analysis. FIG. 4A and FIG. 4B represent measurement
of CCR5 (FIG. 4A) and CXCR4 (FIG. 4B), where lymphocytes were first
gated on CD3 and CD4. FIG. 4C is a bar graph that illustrates
measurement of CD4 where lymphocytes were gated using CD3 in
combination with CD8 Immunofluorescence intensity was measured as
an estimate of the average number of molecules on the cell surface
using Quantibrite-phycoerythrin (PE) beads according to an
embodiment.
[0019] FIG. 5A-5C are bar graphs that illustrate INK128's
inhibition of HIV activation in chronically infected cells.
Latently HIV infected U1 cells were cultured in the presence of
various concentrations of INK128 for 1 hour. Cultures were then
untreated (FIG. 5A), or treated with HIV inducers PMA (FIG. 5B) and
Tat protein (FIG. 5C). HIV production was measured by p24 ELISA in
the culture supernatants on day 3 according to an embodiment.
[0020] FIG. 6 is a bar graph that depicts how INK128 inhibits HIV
transcription in U1 cells. U1 cells were cultured in the presence
of 10 nM PMA and the indicated concentrations of INK128. After 2
days, cells were collected, mRNA isolated, quantified, reverse
transcribed and amplified by real time PCR using unspliced HIV cDNA
primer pair US.1a/US.2a and housekeeping [beta]-actin primers. For
quantification, standard curves for unspliced HIV cDNA and
[beta]-actin sequences were generated by performing 10-fold serial
dilutions of mRNA isolated from PBLs acutely infected with HIV BaL.
PCR amplification was performed using Quantitect.RTM. SYBR Green
PCR Kit in a LightCycler.RTM.. Negative controls consisted of
mixture reactions without the reverse transcription step according
to an embodiment.
[0021] FIG. 7A-7B are graphs showing how INK128 treatment does not
result in weight loss in humanized mice. FIG. 7A is a graph
illustrating mice weight at the beginning of experiment (day 0) and
after 14 days of daily treatment (i.p.) with INK128. FIG. 7B is a
graph that shows the comparison of changes in body weight (day 0 to
day 14) between INK128-treated mice according to an embodiment.
[0022] FIG. 8A-8B are graphs that illustrate INK128's reduction of
plasma HIV RNA in humanized mice. Five- to seven week-old NSG mice
were intraperitoneally (i.p.) injected with PBLs (10.sup.7 per
mouse) from healthy donors. Three weeks later, successfully
engrafted mice were i.p. injected with 15,000 TCID50s of HIV BaL
Immediately after virus challenge, i.p. treatment with INK128 or
PBS was initiated and continued daily for 14 days. Plasma HIV RNA
(copies per mL) was measured by quantitative RT PCR on days 7 and
14 (FIG. 8A). CD4/CD8 cell ratios on days 7 and 14 retro orbital
blood samples were determined by Flow Cytometry Analysis (FIG. 8B)
according to an embodiment.
[0023] FIG. 9A-9B are graphs showing how Torin-2 inhibits HIV. PHA
activated PBMCs were infected with HIV-1 for 2 hours. Infected
cells were washed to remove non-adsorbed virus, and plated in
culture medium containing IL-2 and various dilutions of Torin-2.
Cultures were evaluated for HIV production by measuring HIV p24
levels in the culture supernatants by ELISA (FIG. 9A). Also on day
7, cell viability was measured by MTT assays (FIG. 9B) according to
an embodiment.
[0024] FIG. 10A-10C are graphs illustrating how INK-128 inhibits
tumor growth in NSG mice. A xenograft tumor was induced by
subcutaneous injection of 5.times.10.sup.6 non small cell lung
cancer (NSCLC) A549 cells in mice according to an embodiment.
[0025] In the following description, for the purposes of
explanation, numerous specific details are set forth in order to
provide a thorough understanding of the present invention. It will
be apparent, however, to one skilled in the art that the present
invention may be practiced without these specific details. In order
that the invention may be readily understood and put into practical
effect, particular preferred embodiments will now be described by
way of the following non-limiting examples.
DETAILED DESCRIPTION
[0026] It has been discovered that targeting an mTOR catalytic site
with the mTORC1/mTORC2 inhibitor INK128 results in inhibition of
multiple steps of the HIV-1 lifecycle and suppression of HIV-1
viremia in vivo. mTOR is a conserved serine/threonine kinase that
forms two complexes, mTORC1 and mTORC2. The natural product
isolated from Streptomyces hygroscopicus known as Rapamycin (RAPA,
Rapamune, sirolimus) targets mTOR in mammals and is an allosteric
mTOR inhibitor but only selectively inhibits mTORC1. In doing so,
Rapamycin interferes with viral entry of CCR5 (R5)-tropic HIV and
with basal transcription of the HIV LTR, potently inhibiting
replication of R5 HIV but not CXCR4 (X4)-tropic HIV in primary
cells.
[0027] In an effort to overcome this drawback, ATP-competitive mTOR
kinase inhibitors (TOR-KIs) have been developed to inhibit both
mTORC1 and mTORC2. Using INK128 as a prototype TOR-KI, it has been
discovered that potent inhibition of both R5 HIV and X4 HIV in
primary lymphocytes (EC50<50 nM) can occur in the absence of
toxicity. Experimental results described herein demonstrate that
INK128 inhibited R5 HIV entry by reducing CCR5 levels. INK128 also
inhibited both basal and induced transcription of HIV genes,
consistent with inhibition of mTORC2, whose activity is critical
for phosphorylation of PKC isoforms and, in turn, induction of
NF-.kappa.B. INK128 enhanced the antiviral potency of the CCR5
antagonist Maraviroc in a synergistic manner, and interacted
favorably with antivirals such as HIV inhibitors of reverse
transcriptase, integrase and protease. In vivo, INK128 decreased
plasma HIV RNA by >2 log.sup.10 units and partially restored
CD4/CD8 cell ratios. As a result, targeting of cellular mTOR with
INK128 (and perhaps others TOR KIs, e.g., Torin-2) provides a
strategy to inhibit HIV, especially in patients with drug resistant
HIV strains.
1. DEFINITIONS
[0028] For the purpose of interpreting this specification, the
following definitions will apply and whenever appropriate, terms
used in the singular will also include the plural and vice versa.
In the event that any definition set forth below conflicts with the
usage of that word in any other document, including any document
incorporated herein by reference, the definition set forth below
shall always control for purposes of interpreting this
specification and its associated claims unless a contrary meaning
is clearly intended (for example in the document where the term is
originally used). The use of "or" means "and/or" unless stated
otherwise. The use of "a" herein means "one or more" unless stated
otherwise or where the use of "one or more" is clearly
inappropriate. The use of "comprise," "comprises," "comprising,"
"include," "includes," and "including" are interchangeable and
intended to be non-limiting. Furthermore, where the description of
one or more embodiments uses the term "comprising," those skilled
in the art would understand that, in some specific instances, the
embodiment or embodiments can be alternatively described using the
language "consisting essentially of" and/or "consisting of."
[0029] Unless otherwise defined, all technical and scientific terms
used herein are intended to have the same meaning as commonly
understood in the art to which this invention pertains and at the
time of its filing. Although various methods and materials similar
or equivalent to those described herein can be used in the practice
or testing of the present invention, suitable methods and materials
are described below. However, the skilled should understand that
the methods and materials used and described are examples and may
not be the only ones suitable for use in the invention. Moreover,
it should also be understood that as measurements are subject to
inherent variability, any temperature, weight, volume, time
interval, pH, salinity, molarity or molality, range, concentration
and any other measurements, quantities or numerical expressions
given herein are intended to be approximate and not exact or
critical figures unless expressly stated to the contrary. Hence,
where appropriate to the invention and as understood by those of
skill in the art, it is proper to describe the various aspects of
the invention using approximate or relative terms and terms of
degree commonly employed in patent applications, such as: so
dimensioned, about, approximately, substantially, essentially,
consisting essentially of, comprising, and effective amount.
[0030] Generally, nomenclature used in connection with, and
techniques of, cell and tissue culture, molecular biology,
immunology, microbiology, genetics, protein, and nucleic acid
chemistry and hybridization described herein are those well-known
and commonly used in the art. The methods and techniques of the
present invention are performed generally according to conventional
methods well known in the art and as described in various general
and more specific references that are cited and discussed
throughout the present specification unless otherwise indicated.
See, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual,
2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y. (1989); Ausubel et al., Current Protocols in Molecular
Biology, Greene Publishing Associates (1992, and Supplements to
2002); Harlow and Lan, Antibodies: A Laboratory Manual, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990);
Principles of Neural Science, 4th ed., Eric R. Kandel, James H.
Schwartz, Thomas M. Jessell editors. McGraw-Hill/Appleton &
Lange: New York, N.Y. (2000). Unless defined otherwise, all
technical and scientific terms used herein have the same meaning as
commonly understood by one of ordinary skill in the art.
[0031] The term "administering" as used herein, means delivery, for
example of an mTOR inhibitor alone, or in combination with a
antiretroviral agent, or in further combination with a cancer
therapeutic agent.
[0032] The term "cancer therapeutic agent" as used herein, means an
anticancer agent, an antineoplastic agent, and antitumor agent,
used in the treatment of HIV-infected subjects diagnosed with
cancer.
[0033] The term "HIV," as used herein means a genetically related
member of the Lentivirus genus of the Retroviridae family that
shows a particular tropism for CD4+ T cells.
[0034] The term "HART", or "highly active antiretroviral treatment
as used herein refers to a treatment consisting of a combination of
different therapeutic agents that inhibit HIV replication.
[0035] The term "mTOR" or "Mechanistic Target of Rapamycin" as used
herein means a serine/threonine protein kinase that regulates cell
growth, cell proliferation, cell motility, cell survival, protein
synthesis, autophagy, transcription. mTOR forms two conserved,
structurally distinct kinase complexes termed mTOR complex 1
(mTORC1) and mTORC2. Each complex phosphorylates a different set of
substrates to regulate cell growth.
[0036] The term "mTOR inhibitor" as used herein refers to a
compound or ligand, or a pharmaceutically acceptable salt thereof,
which inhibits the mTOR kinase in a cell. In an embodiment an mTOR
inhibitor is an allosteric inhibitor. In an embodiment an mTOR
inhibitor is a catalytic inhibitor. Preferable mTOR inhibitors
include (i) INK128 (i.e., Sapanisertib, MLN0128) shown in FIG. 1A,
having the molecular formula C.sub.15H.sub.15N.sub.7O identified as
CAS No: 1224844-38-5, having the structure,
##STR00001##
(*Adapted from PubChem) (ii) Torin-2 a potent and selective
inhibitor of cellular mTOR activity, having the molecular formula
C.sub.24H.sub.15F.sub.3N.sub.4O identified as CAS No: 1223001-51-1,
and identified as
9-(6-Amino-3-pyridinyl)-1-[3-(trifluoromethyl)phenyl]-benzo[h]-1,6-naphth-
yridin-2(1H)-one, having the structure,
##STR00002##
(*Adapted from PubChem) (iii) GSK2126458, having the chemical name,
2,4-Difluoro-N-{2-(methyloxy)-5-[4-(4-pyridazinyl)-6-quinolinyl]-3-pyridi-
nyl}benzenesulfonamide (GSK2126458, 1) and identified as a highly
potent, orally bioavailable inhibitor of PI3K.alpha. and mTOR with
in vivo activity in both pharmacodynamic and tumor growth efficacy
models, having the structure,
##STR00003##
(*Adapted from PubChem), and (iv) AZD2014, a potent (IC50 2.81 nM),
selective (inactive against 220 other kinases) inhibitor of mTOR
kinase, having the structure,
##STR00004##
(*Adapted from PubChem).
[0037] The term "primary cells" as used herein refers to cells
established for growth in vitro. Cells that are cultured directly
from a subject are known as primary cells. With the exception of
some derived from tumors, most primary cell cultures have limited
lifespan.
[0038] The term "subject," as used herein refers to animals, such
as mammals. For example, mammals contemplated include humans,
primates, rats, mice, dogs, and cats. The terms "subject" and
"patient" are used interchangeably.
[0039] The term "therapeutically effective amount" as used herein
refers to an amount administered to achieve a therapeutic effect
obtained by reduction, suppression, remission, or eradication of a
disease state.
2. OVERVIEW
[0040] Antiretroviral agents have transformed HIV infection into a
chronic condition that can require life-long therapy. Patients on
therapy can fail treatment, among other factors, because of the
emergence of drug resistance. In view of the drug resistance
problem, a strong need exists for identification of therapeutic
agents that can target and inhibit HIV through different cellular
mechanisms. This need for the development of alternative
antiretroviral agents is primarily due to drug resistance but also
for sustained adherence to complex treatment regimens and toxicity
of currently used antiviral drugs. Current antiretroviral agents
against HIV target several different steps in the viral lifecycle
(1, 2). However, there is a continuous need for novel classes of
antiretroviral agents targeting additional stages of viral
replication, primarily due to emergence of drug resistance (1, 3).
There are currently several antiretroviral classes against reverse
transcription, integration and maturation (4). Within each class,
the availability of several drugs with distinct resistance profiles
make it possible to switch to an alternative drug from the same
class in the event of resistance.
[0041] In contrast, the HIV lifecycle steps of entry and
transcription are underrepresented in current therapy. First, there
are only two licensed entry inhibitors: the CCR5 antagonist
Maraviroc (5) and the fusion inhibitor Enfuvirtide (6). However,
the virus tropism specificity of Maraviroc (7) and the need for
twice-daily s.c. injection of Enfuvirtide (8) limit their clinical
potential. Second, there are no licensed inhibitors of HIV
transcription. Therefore, new approaches for targeting entry and
transcription may provide alternative treatment options for
HIV-infected patients and HIV-infecting patients diagnosed with
cancer, especially those with drug-resistant HIV strains.
mTOR
[0042] Targeting cellular proteins that HIV requires in its
lifecycle is an attractive approach to overcome HIV drug resistance
because cellular proteins have lower mutations rates than do HIV
proteins under drug pressure, and because there are so many host
proteins needed by HIV for its replication. A downside, of course,
is the possibility of side effects from inhibition of a cellular
protein.
[0043] One mode of potential attack is the targeting of cellular
proteins that HIV requires in its lifecycle to help overcome HIV
drug resistance because cellular proteins have lower mutation rates
than do HIV proteins. mTOR is a serine/threonine protein kinase
that regulates cell growth, cell proliferation, cell motility, cell
survival, protein synthesis, autophagy, and transcription. mTOR
belongs to the phosphatidylinositol 3-kinase-related kinase protein
family, and fibrosis. As previously discussed, mTOR comprises two
complexes-mTORC1 and mTORC2--which are involved in regulating
protein translation and transduction signaling. mTORC1 promotes
translation initiation and synthesis of cellular proteins whereas
mTORC2 regulates full activation of the AKT pathway and also
regulates PKC signaling.
HIV-1 Co-Receptors CCR5 and CXCR4
[0044] HIV entry into target cells occurs by a multi-step process
that culminates with the fusion of viral and cellular membranes.
HIV-1 utilizes CD4 as its primary receptor. HIV tropism (the type
of CD4 cell that the virus will be able to infect) is determined by
the type of coreceptor recognized by gp120. Binding to CCR5 is
known as CCR5 (or R5) tropism, while binding to CXCR4 is known as
CXCR4 (or X4) tropism. Binding to CD4 is followed by conformational
changes in the viral envelope that lead to the engagement of one of
the viral co-receptors (CCR5 or CXCR4). Based on their
functionality in vitro, other chemokine receptors may also work as
HIV-1 co-receptors. CCR5 and CXCR4 constitute the major
co-receptors in vivo. The observation that high level of CCR5
expression on CD4-positive primary T cells is associated with high
viral loads and accelerated disease progression further highlights
the contribution of CCR5 to disease progression.
[0045] Rapamycin, which targets mTORC1 but not mTORC2 (10),
interferes with the HIV steps of CCR5-mediated entry and with basal
(but not induced) transcription of the HIV LTR (11-14). These
activities of rapamycin effectively inhibit replication of CCR5
(R5)-tropic HIV, but not CXCR4 (X4)-tropic HIV, in primary
lymphocytes (11, 13, 15). The recently developed ATP-competitive
mTOR kinase inhibitors (TOR-KIs) inhibit both mTORC1 and mTORC2
complexes (16-19). mTORC1 controls CCR5 expression and basal HIV
transcription (11-13), and because mTORC2 controls phosphorylation
of PKC (9, 20-22), required for NF-.kappa.B induction of HIV
transcription (23, 24).
3. EMBODIMENTS
[0046] Most HIV antiretroviral agents target viral proteins.
Unfortunately, HIV mutates under drug pressure, which can lead to
drug resistance. Targeting cellular proteins that HIV requires in
its lifecycle may help overcome HIV drug resistance because
cellular proteins have lower mutations rates than do HIV proteins.
Methods are provided for treatment of HIV infected subjects
diagnosed with cancer with an mTOR inhibitor. Other methods are
provided for treatment of HIV using the mTOR inhibitor INK128,
Torin-2, GSJ 2126458, or AZD2014. The present inventors demonstrate
that dual targeting of mTORC-1/2 with the catalytic inhibitor
INK128 blocks HIV by interfering with entry and with transcription
(basal and induced) Importantly, INK128 suppressed HIV in a
preclinical animal model, suggesting that mTORC-1/2 catalytic
inhibitors may help control HIV in patients, particularly in those
patients diagnosed with cancer and with drug-resistant HIV.
A. Methods of Treatment
[0047] Subjects with HIV and Cancer
[0048] Patients undergoing long-term drug therapies (e.g., highly
active antiretroviral therapy treatment) are at a higher risk of
various HIV-related complications. Hyperactivation of mTOR has been
found to contribute to dysregulated apoptosis and autophagy which
determine CD4+-T-cell loss, impaired function of innate immunity
and development of neurocognitive disorders. Dysregulated mTOR
activation has also been shown to play a key part in the
development of nephropathy and in the pathogenesis of
HIV-associated malignancies. These studies strongly support a
multifunctional key role for mTOR in the pathogenesis of
HIV-related disorders and suggest that specific mTOR inhibitors
could represent a novel approach for the prevention and treatment
of these pathologies. In one embodiment, methods are provided for
treating a subject infected with HIV and diagnosed as having cancer
with a therapeutically effective amount of an mTOR inhibitor (e.g.,
INK128, Torin-2, GSK2126458, and AZD2014) as described in, but not
limited to those in Table 1 and Table 2.
mTOR Inhibitors
[0049] Currently two classes of mTOR inhibitors exist: allosteric
inhibitors (Table 1) and catalytic inhibitors (Table 2).
[0050] Allosteric mTOR inhibitors include the neutral tricyclic
compound rapamycin (sirolimus), rapamycin-related compounds, that
is compounds having structural and functional similarity to
rapamycin including, e.g., rapamycin derivatives, rapamycin analogs
(also referred to as rapalogs) and other macrolide compounds that
inhibit mTOR activity.
TABLE-US-00001 TABLE 1 Allosteric mTOR Inhibitors Compound Chemical
Name (IUPAC) Rapamycin
40-[3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate] (sirolimus)
AY-22989 Rapamycin (1R,2R,4S)-4-{(2R)-2- (temsirolimus)
[(3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R,34aS)- CCT779
9,27-dihydroxy-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-
1,5,11,28,29-pentaoxo-
1,4,5,6,9,10,11,12,13,14,21,22,23,24,25,26,27,28,29,31,32,33,34,34a-
tetracosahydro-3H-23,27-epoxypyrido[2,1-
c][1,4]oxazacyclohentriacontin-3-yl]propyl}-2-methoxycyclohexyl 3-
hydroxy-2-(hydroxymethyl)-2-methylpropanoate Ridaforolimus
1R,2R,4S)-4-[(2R)-2- (AP-23573/
[(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S,35R)-
MK-8669)
1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-
2,3,10,14,20-pentaoxo-11,36-dioxa-4-
azatricyclo[30.3.1.0.sup.4,9]hexatriaconta-16,24,26,28-tetraen-12-
yl]propyl]-2-methoxycyclohexyl dimethylphosphinate Rapamycin
dihydroxy-12-[(2R)-1-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3- derivatives
methoxycyclohexyl]propan-2-yl]-19,30-dimethoxy- (e.g., everolimus)
15,17,21,23,29,35-hexamethyl-11,36-dioxa-4-azatricyclo[30.3.1.0
hexatriaconta-16,24,26,28-tetraene-2,3,10,14,20-pentone Rapamycin
analogs Various (rapalogs) Zotarolimus
(3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R,34aS)-9,27-
(ABT578)
dihydroxy-10,21-dimethoxy-3-{(1R)-2-[(1S,3R,4S)-3-methoxy-4-(1H-
tetrazol-1-yl)cyclohexyl]-1-methylethyl}-6,8,12,14,20,26-hexamethyl-
4,9,10,12,13,14,21,22,23,24,25,26,27,32,33,34,34a-heptadecahydro-3H-
23,27-epoxypyrido[2,1-c][1,4]oxazacyclohentriacontine-
1,5,11,28,29(6H,31H)-pentone Umirolimus Various
*Adapted from PubChem
[0051] Alternatively or additionally, catalytic, ATP-competitive
mTOR inhibitors have been found to target the mTOR kinase domain
directly and target both mTORC1 and mTORC2. These are also more
complete inhibitors of mTORC1 than such allosteric mTOR inhibitors
as rapamycin, because they modulate rapamycin-resistant mTORC1
outputs such as 4EBP1-T37/46 phosphorylation and cap-dependent
translation.
TABLE-US-00002 TABLE 2 Catalytic mTOR Inhibitors Compound Chemical
Name INK-128 Sapanisertib, 5-(4-amino-1-propan-2-ylpyrazolo[3,4-
MLN0128 d]pyrimidin-3-yl)-1,3-benzoxazol-2- amine BEZ235
2-methyl-2-[4-(3-methyl-2-oxo-8-
quinolin-3-yl-2,3-dihydro-imidazo[4,5-
c]quinolin-1-yl)-phenyl]-propionitrile AZD2014
3-[2,4-bis[(3S)-3-methylmorpholin-4-
yl]pyrido[2,3-d]pyrimidin-7-yl]-N- methylbenzamide PKI-587
1-[4-[4-[(dimethylamino)piperidine-1- carbonyl]phenyl]-3-[4-(4,6-
dimorpholino-1,3,5-triazin-2- yl)phenyl]urea GSK-2126458
2,4-difluoro-N-{2-methoxy-5-[4-(4- pyridazinyl)-6-quinolinyl]-3-
pyridinyl}benzenesulfonamide TORIN 2 9-(6-Amino-3-pyridinyl)-1-[3-
(trifluoromethyl)phenyl]-benzo[h]1,6- naphthyridin-2(1H)-one
PALOMID 529 8-(1-Hydroxyethyl)-2-methoxy-3-((4-
methoxybenzypoxy)-6H- benzo[c]chromen-6-one
*Adapted from PubChem
[0052] Further examples of catalytic mTOR inhibitors include
8-(6-methoxy-pyridin-3-yl)-3-methyl-1-(4-piperazin-1-yl-3-trifluoromethyl-
-phenyl)-1,3-dihydro-imidazo[4,5-c]quinolin-2-one (WO2006/122806)
and Ku-0063794 (Garcia-Martinez J M, et al., Biochem J., 2009,
421(1), 29-42. Ku-0063794 is a specific inhibitor of the mammalian
target of rapamycin (mTOR).) WYE-354 is another example of a
catalytic mTOR inhibitor (Yu K, et al. (2009). Biochemical,
Cellular, and In vivo Activity of Novel ATP-Competitive and
Selective Inhibitors of the Mammalian Target of Rapamycin. Cancer
Res. 69(15): 6232-6240).
[0053] mTOR inhibitors useful according to the present invention
also include prodrugs, derivatives, pharmaceutically acceptable
salts, or analogs thereof.
[0054] mTOR inhibitors such as INK128 are useful in cancer therapy
where mTOR is upregulated. Unfortunately, a growing population of
HIV infected patients with cancer exists. HIV-infected patients
with cancer potentially face a higher cancer-specific mortality
compared to those without HIV. Recent literature suggests that
cancer-specific mortality was significantly higher in HIV-infected
patients for a variety of cancers, including colorectal,
pancreatic, laryngeal, lung, melanoma, breast and prostate. There
is a pressing need to identify mTOR agents that can target and
inhibit HIV entry and transcription as well as aid in treating
those HIV patients with cancer. Identification of such mTOR
inhibitors would provide an approach for concomitant treatment of
HIV and cancer without having to disrupt anti-HIV therapy in HIV
infected patients with certain kinds of cancers. Such an approach
would simplify treatment by reducing the number of therapeutic
agents administered and avoiding the potentially detrimental
interactions between cancer drugs and HIV drugs.
HIV Infected Subjects
[0055] In certain embodiments, methods are provided for treating a
subject infected with HIV by administering a therapeutically
effective amount of the mTOR inhibitor INK128, Torin-2, GSJ
2126458, or Torin-2). In a preferred aspect mTOR targets HIV and
(i) inhibits its entry into the cell, (ii) inhibits LTR gene
expression, (iii) inhibits replication, and thereby treats
HIV-infected subjects. Identification of mTOR agents that provide
an approach for treating subjects infected with HIV is desirable.
Therefore, methods of inhibiting entry of R5 HIV in vitro, methods
of inhibiting HIV LTR gene expression, and methods of inhibiting R4
and X4 HIV replication are provided by administering the mTOR
inhibitor INK128.
[0056] It has been discovered that INK128 inhibits entry of R5, but
not X4, HIV in primary lymphocytes. Unlike rapamycin, the newly
developed TOR-KIs including INK128 inhibit both mTORC1 and mTORC2.
Where INK128 is used, inhibition of CCR5 expression and inhibition
of R5 HIV entry were demonstrated. Methods are provided in certain
embodiments for inhibiting R5 and X4 HIV replication by
administering a therapeutically effective amount of the mTOR
inhibitor INK128.
[0057] In other embodiments, INK128 potently inhibited basal
transcription as well as transcription induced by PMA and by Tat.
This broad anti-transcriptional activity of INK128 is consistent
with inhibition of mTORC2, whose activity is important for
phosphorylation of PKC isoforms (including isoforms .alpha. and
.beta.) (9, 20-22), and, in turn, induction of NF-.kappa.B. By
interfering with NF-.kappa.B induction, INK128 may prevent
recruitment of the host transcription factor P-TEFb to the HIV LTR,
thereby decreasing virus transcription. In infectivity assays using
primary PBLs, INK128 inhibited both R5 and X4 HIV,
laboratory-adapted and primary isolates, with EC50 values <50
nM. Moreover, INK128 enhanced the antiviral potency of the CCR5
antagonist Maraviroc, probably by decreasing CCR5 levels, and had
favorable antiviral interactions with inhibitors of reverse
transcription, integration and protease.
[0058] Administration of INK128 in the present invention reduces
plasma viremia by more than 2 log.sup.10 units in humanized mice.
This magnitude of virus suppression is similar to that achieved by
EFdA, a potent NRTI in clinical development, in humanized mice
(38). Thus, INK128, and perhaps other TOR-KIs, may have anti-HIV
activity in vivo. A counterintuitive, yet important, property of
TOR-KIs is that their inhibition of both mTORC1 and mTORC2 is
better tolerated by normal PBLs than targeting of mTORC1 alone with
allosteric inhibitors.
[0059] Without being bound by theory, it is possible that mTOR may
have a noncatalytic scaffolding function that is suppressed by
allosteric inhibition, but not with the catalytic inhibitor. It is
also possible that catalytic inhibitors may have a more transient
effect on blocking the kinase activity of mTOR, sufficient for
anti-HIV activity but not for cellular toxicity. The data reflect
that INK128 did not decrease proliferation of primary PBLs at
concentrations of up to 1 .mu.M in the assays. Moreover, daily
administration of INK128 inhibited HIV viremia in humanized mice
without obvious toxicity, as determined by changes in body weight,
over a 2-wk period. Mechanistically, mTOR controls host protein
synthesis mainly at the translation level (9). In the present
invention, TOR-KI inhibition of HIV gene expression occurs at the
transcription level, suggesting an indirect effect of the drug.
B. Combination Treatments
[0060] Antiretroviral agents are referred to as ARV. Combination
ARV therapy (cART) is referred to as highly active ART(HAART). It
has been discovered that the mTOR inhibitor INK128 has favorable
drug interactions with current antiretroviral classes that target
the HIV lifecycle steps of entry, reverse transcription,
integration, and maturation. Therefore, in certain aspects,
antiretroviral agents (Table 3) or cancer therapeutic agents (Table
4) in combination with INK128 (or other mTOR inhibitors) are
preferred because they increase the antiviral potency of current
antiretroviral regimens. Each type, or "class," of ARV drugs
attacks HIV in a different way.
[0061] In some embodiments, it may be advantageous to administer an
mTOR inhibitor, e.g., an mTOR inhibitor described herein, with one
or more therapeutic agents, preferably a second antiretroviral
agent, and/or a second cancer therapeutic agent. For example,
synergistic effects can occur with other antiretroviral agents,
other cancer therapeutic agents. Suitable therapeutic agents are
known to one of ordinary skill in the art and can be found listed
in the Physicians' Desk Reference.
Antiretroviral Agents
[0062] The first class of anti-HIV drugs was the nucleoside reverse
transcriptase inhibitors (also called NRTIs or "nukes".) These
drugs block the step, where the HIV genetic material is used to
create DNA from RNA. The following antiretroviral agents preferable
in certain embodiments in this class include but are not limited to
those set forth in Table 3.
TABLE-US-00003 TABLE 3 Antiretroviral Agents. Nucleoside reverse
transcriptase inhibitors (NRTIs, nukes) Zidovudine (Retrovir, AZT)
Didanosine (Videx, Videx EC, ddI) Stavudine (Zerit, d4T) Lamivudine
(Epivir, 3TC) Abacavir (Ziagen, ABC) Tenofovir, a nucleotide analog
(Viread, TDF) Combivir (combination of zidovudine and lamivudine)
Trizivir (combination of zidovudine, lamivudine and abacavir)
Emtricitabine (Emtriva, FTC) Truvada (combination of emtricitabine
and tenofovir) Epzicom (combination of abacavir and lamivudine)
Non-nucleoside reverse transcriptase inhibitors (non-nukes, NNRTIs)
Nevirapine (Viramune, NVP) Delavirdine (Rescriptor, DLV) Efavirenz
(Sustiva or Stocrin, EFV, also part of Atripla) Etravirine
(Intelence, ETR) Rilpivirine (Edurant, RPV, also part of Complera
or Epivlera). Protease inhibitors (PIs) Saquinavir (Invirase, SQV)
Indinavir (Crixivan, IDV) Ritonavir (Norvir, RTV) Nelfinavir
(Viracept, NFV) Amprenavir (Agenerase, APV) Lopinavir/ritonavir
(Kaletra or Aluvia, LPV/RTV) Atazanavir (Reyataz, ATZ)
Fosamprenavir (Lexiva, Telzir, FPV) Tipranavir (Aptivus, TPV)
Darunavir (Prezista, DRV) Entry inhibitors (Enfuvirtide, Fuzeon,
ENF, T-20) Maraviroc (Selzentry or Celsentri, MVC) HIV integrase
inhibitors Raltegravir (Isentress, RAL) Elvitegravir (EVG, part of
the combination Stribild) Dolutegravir (Tivicay, DTG)
Cancer Therapeutic Agents
[0063] The decision to use cancer therapeutic agents (e.g.,
anticancer antineoplastic, and antitumor), such as DNA damaging
agents, microtubule agents, and signal transduction agents used in
the treatment of HIV-infected subjects diagnosed with cancer (Table
4) depends on the type of tumor to be treated, the stage of
malignancy, the condition of the subject, and financial
considerations. In certain embodiments, cancer may be non-small
cell lung cancer, small cell lung cancer, prostate cancer, breast
cancer, liver cancer, Hodgkin lymphoma, non-Hodgkin lymphoma,
Kaposi sarcoma, B cell acute lymphoblastic leukemia, bone sarcoma,
and soft tissue sarcomas. Chemotherapy can be used as an adjuvant
to surgery and irradiation and can be administered immediately
after or before the primary treatment. Neoadjuvant therapy is
administered before surgery or irradiation and is intended to
improve the effectiveness of the primary therapy by possibly
decreasing tumor size, stage of malignancy, or the presence of
micro metastatic lesions. Responses to cancer chemotherapy can
range from palliation (remission of secondary signs, generally
without increase in survival time) to complete remission (in which
clinically detectable tumor cells and all signs of malignancy are
absent). The percentage and duration of complete remissions are
criteria for the success of a particular chemotherapeutic
protocol.
TABLE-US-00004 TABLE 4 Cancer Therapeutic Agents Abiraterone ABVD
Hydroxyurea Omacetaxine Acetate Mepesuccinate Abitrexate ABVE
Hyper-CVAD Oncaspar (Methotrexate) (Pegaspargase) Abraxane ABVE-PC
Ibrance (Palbociclib) Ondansetron (Paclitaxel Hydrochloride
Albumin-stabilized Nanoparticle Formulation) AC Afatinib Dimaleate
Ibritumomab Tiuxetan Onivyde (Irinotecan Hydrochloride Liposome)
AC-T Afinitor (Everolimus) Ibrutinib Ontak (Denileukin Diftitox)
Adcetris Akynzeo ICE Opdivo (Brentuximab (Netupitant and
(Nivolumab) Vedotin) Palonosetron Hydrochloride) ADE Alecensa
Iclusig (Ponatinib OPPA (Alectinib) Hydrochloride) Ado-Trastuzumab
Alectinib Idamycin Osimertinib Emtansine (Idarubicin Hydrochloride)
Adriamycin Alemtuzumab Idarubicin Oxaliplatin (Doxorubicin
Hydrochloride Hydrochloride) Aldara Alkeran for Idelalisib
Paclitaxel (Imiquimod) Injection (Melphalan Hydrochloride)
Aldesleukin Alkeran Tablets Ifex (Ifosfamide) Paclitaxel
(Melphalan) Albumin- stabilized Nanoparticle Formulation Aloxi
Alimta Ifosfamide PAD (Palonosetron (Pemetrexed Hydrochloride)
Disodium) Ambochlorin Anastrozole Ifosfamidum Palbociclib
(Chlorambucil) (Ifosfamide) Amboclorin Aprepitant IL-2
(Aldesleukin) Palifermin (Chlorambucil) Aminolevulinic Aredia
Imatinib Mesylate Palonosetron Acid (Pamidronate Hydrochloride
Disodium) Arsenic Trioxide Arimidex Imbruvica Palonosetron
(Anastrozole) (lbrutinib) Hydrochloride and Netupitant Arzerra
Aromasin Imiquimod Pamidronate (Ofatumumab) (Exemestane) Disodium
Asparaginase Arranon Imlygic Panitumumab Erwinia (Nelarabine)
(Talimogene chrysanthemi Laherparepvec) Avastin Axitinib Inlyta
(Axitinib) Panobinostat (Bevacizumab) BEP Azacitidine Interferon
Alfa-2b, Paraplat Recombinant (Carboplatin) Bevacizumab BEACOPP
Interleukin-2 Paraplatin (Aldesleukin) (Carboplatin) Bexarotene
Becenum Intron A Pazopanib (Carmustine) (Recombinant Hydrochloride
Interferon Alfa-2b) Bexxar Beleodaq Iodine 1131 PCV (Tositumomab
and (Belinostat) Tositumomab and Iodine I 131 Tositumomab
Tositumomab) Bicalutamide Belinostat Ipilimumab Pegaspargase BiCNU
Bendamustine Iressa (Gefitinib) Peginterferon (Carmustine)
Hydrochloride Alfa-2b Bleomycin Bortezomib Irinotecan PEG-Intron
Hydrochloride (Peginterferon Alfa-2b) Blinatumomab Bosulif
(Bosutinib) Irinotecan Pembrolizumab Hydrochloride Liposome
Blincyto Bosutinib Istodax Pemetrexed (Blinatumomab) (Romidepsin)
Disodium Busulfex Brentuximab Ixabepilone Perjeta (Busulfan)
Vedotin (Pertuzumab) Cabazitaxel Busulfan Ixazomib Citrate
Pertuzumab Cabozantinib-S- Capecitabine Ixempra Platinol Malate
(Ixabepilone) (Cisplatin) CAF CAPDX Jakafi (Ruxolitinib Platinol-AQ
Phosphate) (Cisplatin) Campath Carac Jevtana Plerixafor
(Alemtuzumab) (Fluorouracil-- (Cabazitaxel) Topical) Camptosar
Carboplatin Kadcyla (Ado- Pomalidomide (Irinotecan Trastuzumab
Hydrochloride) Emtansine) CARBOPLATIN- CEM Keoxifene Pomalyst TAXOL
(Raloxifene (Pomalidomide) Hydrochloride) Carfilzomib Ceritinib
Kepivance Ponatinib (Palifermin) Hydrochloride Carmubris Cerubidine
Keytruda Portrazza (Carmustine) (Daunorubicin (Pembrolizumab)
(Necitumumab) Hydrochloride) Carmustine Cervarix Kyprolis
Pralatrexate (Recombinant HPV (Carfilzomib) Bivalent Vaccine)
Carmustine Implant Cetuximab Lanreotide Acetate Prednisone Casodex
Chlorambucil Lapatinib Procarbazine (Bicalutamide) Ditosylate
Hydrochloride CeeNU CHLORAMBUCIL Lenalidomide Proleukin (Lomustine)
-PREDNISONE (Aldesleukin) Cisplatin CHOP Lenvatinib Prolia Mesylate
(Denosumab) Clafen COPDAC Lenvima Promacta (Cyclophosphamide)
(Lenvatinib (Eltrombopag Mesylate) Olamine) Clofarabine COPP
Letrozole Provenge (Sipuleucel-T) Clofarex COPP-ABV Leucovorin
Purinethol (Clofarabine) Calcium (Mercaptopurine) Clolar Cosmegen
Leukeran Purixan (Clofarabine) (Dactinomycin) (Chlorambucil)
(Mercaptopurine) CMF Cotellic Leuprolide Acetate Radium 223
(Cobimetinib) Dichloride Cobimetinib Crizotinib Levulan Raloxifene
(Aminolevulinic Hydrochloride Acid) Cometriq Cytosar-U Linfolizin
Ramucirumab (Cabozantinib-S- (Cytarabine) (Chlorambucil) Malate)
CVP Cytoxan LipoDox Rasburicase (Cyclophosphamide) (Doxorubicin
Hydrochloride Liposome) Cyclophosphamide Dabrafenib Lomustine
R-CHOP Cyfos (Ifosfamide) Dacarbazine Lonsurf R-CVP (Trifluridine
and Tipiracil Hydrochloride) Cyramza Dacogen Lupron (Leuprolide
Recombinant (Ramucirumab) (Decitabine) Acetate) Human
Papillomavirus (HPV) Bivalent Vaccine Cytarabine Dactinomycin
Lupron Depot Recombinant (Leuprolide Human Acetate) Papillomavirus
(HPV) Nonavalent Vaccine Cytarabine Daratumumab Lupron Depot-Ped
Recombinant Liposome (Leuprolide Human Acetate) Papillomavirus
(HPV) Quadrivalent Vaccine Dasatinib Darzalex Lupron Depot-3
Recombinant (Daratumumab) Month (Leuprolide Interferon Alfa-
Acetate) 2b Daunorubicin Doxorubicin Lupron Depot-4 Regorafenib
Hydrochloride Hydrochloride Month (Leuprolide Liposome Acetate)
Decitabine Dox-SL Lynparza R-EPOCH (Doxorubicin (Olaparib)
Hydrochloride Liposome) Defibrotide Sodium DTIC-Dome Margibo
Revtimid (Dacarbazine) (Vincristine Sulfate (Lenalidomide)
Liposome) Defitelio Efudex Matulane Rheumatrex (Defibrotide
(Fluorouracil-- (Procarbazine (Methotrexate) Sodium) Topical)
Hydrochloride) Degarelix Elitek Mechlorethamine Rituxan
(Rasburicase) Hydrochloride (Rituximab) Denileukin Diftitox Ellence
(Epirubicin Megestrol Acetate Rituximab Hydrochloride) Denosumab
Elotuzumab Mekinist Rolapitant (Trametinib) Hydrochloride DepoCyt
Eloxatin Melphalan Romidepsin (Cytarabine (Oxaliplatin) Liposome)
Dexamethasone Eltrombopag Melphalan Romiplostim Olamine
Hydrochloride Dexrazoxane Emend Mercaptopurine Rubidomycin
Hydrochloride (Aprepitant) (Daunorubicin Hydrochloride) Dinutuximab
Empliciti Mesna Ruxolitinib (Elotuzumab) Phosphate Docetaxel
Enzalutamide Mesnex (Mesna) Sclerosol Intrapleural Aerosol (Talc)
Doxil (Doxorubicin Epirubicin Methazolastone Siltuximab
Hydrochloride Hydrochloride (Temozolomide) Liposome) Doxorubicin
EPOCH Methotrexate Sipuleucel-T Hydrochloride Erbitux Everolimus
Methotrexate LPF Somatuline (Cetuximab) (Methotrexate) Depot
(Lanreotide Acetate) Eribulin Mesylate Evista (Raloxifene Mexate
Sonidegib Hydrochloride) (Methotrexate) Erivedge Evomela Mexate-AQ
Sorafenib (Vismodegib) (Melphalan (Methotrexate) Tosylate
Hydrochloride) Erlotinib Exemestane Mitomycin C Sprycel
Hydrochloride (Dasatinib) Erwinaze 5-FU (Fluorouracil Mitoxantrone
STANFORD V (Asparaginase Injection) Hydrochloride Erwinia
chrysanthemi) Etopophos 5-FU (Fluorouracil-- Mitozytrex Sterile
Talc (Etoposide Topical) (Mitomycin C) Powder (Talc) Phosphate)
Etoposide Fareston MOPP Sternalc (Talc) (Toremifene) Etoposide
Farydak Mozobil Stivarga Phosphate (Panobinostat) (Plerixafor)
(Regorafenib) Evacet Faslodex Mustargen Sunitinib Malate
(Doxorubicin (Fulvestrant) (Mechlorethamine Hydrochloride
Hydrochloride) Liposome) FEC Fluorouracil-- Mutamycin Sutent
Topical (Mitomycin C) (Sunitinib Malate) Femara (Letrozole)
Flutamide Myleran (Busulfan) Sylatron (Peginterferon Alfa-2b)
Filgrastim Folex Mylosar Sylvant (Methotrexate) (Azacitidine)
(Siltuximab) Fludara Folex PFS Mylotarg Synovir (Fludarabine
(Methotrexate) (Gemtuzumab (Thalidomide) Phosphate) Ozogamicin)
Fludarabine FOLFIRI Nanoparticle Synribo Phosphate Paclitaxel
(Omacetaxine (Paclitaxel Mepesuccinate) Albumin-stabilized
Nanoparticle Formulation) Fluoroplex FOLFIRI- Navelbine Tabloid
(Fluorouracil-- BEVACIZUMAB (Vinorelbine (Thioguanine) Topical)
Tartrate) Fluorouracil FOLFIRI- Necitumumab TAC Injection CETUXIMAB
FOLFIRINOX Gazyva Nelarabine Tafinlar (Obinutuzumab) (Dabrafenib)
FOLFOX Gefitinib Neosar Tagrisso (Cyclophosphamide) (Osimertinib)
Folotyn Gemcitabine Netupitant and Talc
(Pralatrexate) Hydrochloride Palonosetron Hydrochloride FU-LV
GEMCITABINE- Neupogen Talimogene CISPLATIN (Filgrastim)
Laherparepvec Fulvestrant GEMCITABINE- Nexavar (Sorafenib Tamoxifen
OXALIPLATIN Tosylate) Citrate Gardasil Gemtuzumab Nilotinib
Tarabine PFS (Recombinant HPV Ozogamicin (Cytarabine) Quadrivalent
Vaccine) Gardasil 9 Gemzar Ninlaro (Ixazomib Tarceva (Recombinant
HPV (Gemcitabine Citrate) (Erlotinib Nonavalent Hydrochloride)
Hydrochloride) Vaccine) Gilotrif (Afatinib Halaven (Eribulin
Nivolumab Targretin Dimaleate) Mesylate) (Bexarotene) Gleevec
(Imatinib Herceptin Nolvadex Tasigna Mesylate) (Trastuzumab)
(Tamoxifen (Nilotinib) Citrate) Gliadel HPV Bivalent Nplate Taxol
(Carmustine Vaccine, (Romiplostim) (Paclitaxel) Implant)
Recombinant Gliadel wafer HPV Nonavalent Obinutuzumab Taxotere
(Carmustine Vaccine, (Docetaxel) Implant) Recombinant Glucarpidase
HPV Quadrivalent Odomzo Temodar Vaccine, (Sonidegib) (Temozolomide)
Recombinant Goserelin Acetate Hycamtin OEPA Temozolomide (Topotecan
Hydrochloride) Thioguanine Hydrea Ofatumumab Temsirolimus
(Hydroxyurea) Thiotepa Topotecan OFF Thalidomide Hydrochloride
Tolak Toremifene Olaparib Thalomid (Fluorouracil-- (Thalidomide)
Topical) Trabectedin Trifluridine and Torisel Velban Tipiracil
(Temsirolimus) (Vinblastine Hydrochloride Sulfate) Trametinib
Trisenox (Arsenic Tositumomab and Velcade Trioxide) Iodine I 131
(Bortezomib) Tositumomab Trastuzumab Tykerb (Lapatinib Totect
Velsar Ditosylate) (Dexrazoxane (Vinblastine Hydrochloride)
Sulfate) Treanda Unituxin TPF Vandetanib (Bendamustine
(Dinutuximab) Hydrochloride) Vemurafenib Uridine Triacetate
Vectibix VAMP (Panitumumab) Venclexta VAC VeIP Vidaza (Venetoclax)
(Azacitidine) Venetoclax Vorinostat Vinorelbine Vinblastine
Tartrate Sulfate Viadur (Leuprolide Votrient VIP Vincasar PFS
Acetate) (Pazopanib (Vincristine Hydrochloride) Sulfate) Varubi
(Rolapitant Wellcovorin Vismodegib Vincristine Hydrochloride)
(Leucovorin Sulfate Calcium) XELIRI Xalkori Vistogard (Uridine
Vincristine (Crizotinib) Triacetate) Sulfate Liposome XELOX Xeloda
Voraxaze Xofigo (Radium (Capecitabine) (Glucarpidase) 223
Dichloride) Xgeva Xtandi Yervoy Yondelis (Denosumab) (Enzalutamide)
(Ipilimumab) (Trabectedin) Zaltrap (Ziv- Zinecard Zoledronic Acid
Zykadia Aflibercept) (Dexrazoxane (Ceritinib) Hydrochloride) Zarxio
(Filgrastim) Ziv-Aflibercept Zolinza Zytiga (Vorinostat)
(Abiraterone Acetate) Zelboraf Zofran Zometa (Vemurafenib)
(Ondansetron (Zoledronic Acid) Hydrochloride) Zevalin Zoladex
(Goserelin Zydelig (Idelalisib) (Ibritumomab Acetate) Tiuxetan)
[0064] Combination cancer therapeutic agents with an mTOR inhibitor
offer many advantages. Drugs with different target sites or
mechanisms of action are used together to enhance destruction of
tumor cells. If the adverse effects of the component agents are
different, the combination may be no more toxic than the individual
agents given separately. Combinations that include a
cycle-nonspecific drug administered first, followed by a
phase-specific drug, may offer the advantage that cells surviving
treatment with the first drug are provoked into mitosis and,
therefore, are more susceptible to the second drug. Another
advantage of combination therapy is the decreased possibility of
development of drug resistance.
[0065] Special considerations associated with administration of
cancer therapeutic agents include evaluation of the subject's
quality of life, medical and nutritional support, control of pain,
and psychologic comfort.
C. Administration
[0066] Where the compounds of the invention are administered in
conjunction with other therapies, dosages of the co-administered
compounds will of course vary depending on the type of co-drug
employed, on the specific drug employed, on the condition being
treated and so forth.
[0067] Accordingly, an mTOR inhibitor, e.g., an mTOR inhibitor
described herein, may be used at low, immune enhancing, dose in
combination with other known agents and therapies. Administered "in
combination", as used herein, means that two (or more) different
treatments are delivered to the subject during the course of the
subject's affliction with the disorder, e.g., the two or more
treatments are delivered after the subject has been diagnosed with
the disorder and before the disorder has been cured or eliminated
or treatment has ceased for other reasons. In some embodiments, the
delivery of one treatment is still occurring when the delivery of
the second begins, so that there is overlap in terms of
administration. This is sometimes referred to herein as
"simultaneous" or "concurrent delivery". In other embodiments, the
delivery of one treatment ends before the delivery of the other
treatment begins. In some embodiments of either case, the treatment
is more effective because of combined administration. For example,
the second treatment is more effective, e.g., an equivalent effect
is seen with less of the second treatment, or the second treatment
reduces symptoms to a greater extent, than would be seen if the
second treatment were administered in the absence of the first
treatment, or the analogous situation is seen with the first
treatment. In some embodiments, delivery is such that the reduction
in a symptom, or other parameter related to the disorder is greater
than what would be observed with one treatment delivered in the
absence of the other. The effect of the two treatments can be
partially additive, wholly additive, or greater than additive. The
delivery can be such that an effect of the first treatment
delivered is still detectable when the second is delivered.
[0068] An mTOR inhibitor, e.g., an mTOR inhibitor described herein,
at a preferred dose, and the at least one additional therapeutic
agent (e.g., a second mTOR inhibitor, a second antiretroviral
agent, or a cancer therapeutic agent) can be administered
simultaneously, in the same or in separate compositions, or
sequentially. For sequential administration, the mTOR inhibitor can
be administered first, and the additional agent can be administered
second, or the order of administration can be reversed. In some
embodiments, the mTOR inhibitor is administered as a pretreatment,
e.g., 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks,
8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks or more, before
treatment with the at least one additional therapeutic agent.
[0069] INK128 may be administered alone, or in combination in a
therapeutically effective amount from about 0.5 mg to about 4 mg to
achieve a plasma concentration of about 200 nM in the subject. The
mTOR inhibitor, Torin-2 may be administered in a therapeutically
effective amount from about 0.05 mg to about 10 mg. GSK2126458 may
be administered in a therapeutically effective amount from about
0.04 mg to about 0.25 mg. In other embodiments, AZD2014 may be
administered in a therapeutically effective amount from about 5 mg
to about 50 mg.
[0070] In some embodiments, an mTOR inhibitor, e.g., an mTOR
inhibitor described herein, is administered to a HIV-infected
subject who also has cancer, e.g., a cancer described herein. The
subject may receive treatment with an additional therapeutic
agent.
[0071] A dose of an mTOR inhibitor, can allow for more aggressive
administration of the additional treatment. Thus, in an embodiment,
the unit dosage, total dosage, frequency of administration, or
number of administrations, is increased. In an embodiment, the
increase is relative to a reference administration, e.g., the
standard of care that is provided in the absence of a low, immune
enhancing, dose of mTOR inhibitor. In an embodiment, the increase
is relative to an administration that would give the maximum
tolerable or acceptable levels of immune suppression, in the
absence of a low, immune enhancing, dose of mTOR inhibitor. In
another embodiment, the immune enhancing dose of an mTOR inhibitor,
can allow for less aggressive administration of the additional
treatment. Thus, in an embodiment, the unit dosage, total dosage,
frequency of administration, or number of administrations, is
decreased. In an embodiment, the decrease is relative to a
reference administration, e.g., the standard of care that is
provided in the absence of a low, immune enhancing, dose of mTOR
inhibitor. In an embodiment, the decrease is relative to an
administration that would give the maximum tolerable or acceptable
levels of immune suppression, in the absence of a low, immune
enhancing, dose of mTOR inhibitor.
[0072] In some embodiments, an antiretroviral agent as described
herein, is administered to a HIV-infected subject who also has
cancer, e.g., a cancer described herein. The subject may receive
treatment with an additional therapeutic agent. Thus,
antiretroviral agents could include the following at daily
doses:
TABLE-US-00005 Integrase inhibitors (daily doses) Raltegravir
(400-800 mg) Elvitegravir (85-150 mg) Dolutegravir (50-100 mg)
Reverse transcriptase inhibitors (daily doses) Lamivudine (150-300
mg) Abacavir (600 mg) Emtricitabine (200 mg) Tenofovir (300 mg)
Efavirenz (600 mg) Nevirapine (200-400 mg) Etravirine (400 mg)
Rilpivirine (25 mg) Protease inhibitors (daily doses) Indinavir
(1200-2400 mg) Atazanavir (150-300 mg) Darunavir (800-1200 mg) CCR5
antagonists (daily doses) Maraviroc (150-600 mg)
[0073] Dosing strategies for anticancer drugs are known in the art
and can be found in Physicians Desk Reference and other references
such as "Dosing strategies for anticancer drugs: the good, the bad
and body-surface area," A Felici, J Verweij, A Sparreboom--European
Journal of Cancer, 2002. For example, AZD2014 has been administered
orally to solid tumor cancer patients in single doses up to 100 mg
and multiple doses up to 100 mg twice daily (BID). In other
examples, patients receiving oral GSK458 once or twice daily in a
dose escalation design to define the maximally tolerated dose
(MTD). Expansion cohorts evaluated pharmacodynamics (PD),
pharmacokinetics (PK), and clinical activity in histologically- and
molecularly-defined cohorts. 170 patients received doses ranging
from 0.1 to 3 mg once or twice daily. See, Munster P., et al.,
"First-in-Human Phase I Study of GSK2126458, an Oral Pan-Class I
Phosphatidylinositol-3-Kinase Inhibitor, in Patients with Advanced
Solid Tumor Malignancies," Clin Cancer Res. 2016 Apr. 15;
22(8):1932-9. doi: 10.1158/1078-0432.CCR-15-1665. Epub 2015 Nov.
24.
[0074] Dosing is dependent on severity and responsiveness of the
condition to be treated, with course of treatment lasting from
several days to several months or until a reduction in HIV viral
titre (routinely measured by Western blot, ELISA, RT-PCR, or RNA
(Northern) blot) is effected or a diminution of disease state is
achieved. Optimal dosing schedules are easily calculated from
measurements of drug accumulation in the body. Persons of ordinary
skill can easily determine optimum dosages, dosing methodologies,
and repetition rates. Therapeutically or prophylactically effective
amounts (dosages) may vary depending on the relative potency of
individual compositions, and can generally be routinely calculated
based on molecular weight and EC50s in in vitro and/or animal
studies. For example, given the molecular weight of drug compound
(derived from sequence and chemical structure) and an
experimentally derived effective dose such as an IC.sub.50, for
example, a dose in mg/kg is routinely calculated. In general,
dosage is from 0.001 .mu.g to 100 g and may be administered once or
several times daily, weekly, monthly, yearly, or even every
decade.
D. Routes of Administration
[0075] In some embodiments, an mTOR inhibitor, alone or in
combination with a therapeutic agent is administered orally,
intravenously, intramuscularly, intrathecally, subcutaneously,
sublingually, buccally, rectally, vaginally, by ocular route or by
otic route, nasally, by inhalation, by nebulization, cutaneously,
topically, or systemically, and transdermally. Preferable
administration may be parenterally. Parenteral vehicles include
sodium chloride solution, Ringer's dextrose, dextrose and sodium
chloride, lactated Ringer's or fixed oils and other vehicle known
to one of skill in the art. Intravenous vehicles may include fluid
and nutrient replenishers, electrolyte replenishers (such as those
based on Ringer's dextrose). In some embodiments, an mTOR
inhibitor, for parenteral administration may be in the form of a
sterile injectable preparation, such as a sterile injectable
aqueous or non-aqueous solutions, suspensions, and emulsions.
Examples of non-aqueous solvents are propylene glycol, polyethylene
glycol, vegetable oils such as olive oil, and injectable organic
esters such as ethyl oleate. Carriers or occlusive dressings can be
used to increase skin permeability and enhance antigen absorption.
Suspensions may be formulated according to methods well known in
the art using suitable dispersing or wetting agents and suspending
agents. The sterile injectable preparation may also be a sterile
injectable solution or suspension in a parenterally acceptable
diluent or solvent, such as a solution in 1, 3-butanediol. Suitable
diluents include, for example, water, Ringer's solution and
isotonic sodium chloride solution. In addition, sterile fixed oils
may be employed conventionally as a solvent or suspending medium.
For this purpose, any bland fixed oil may be employed including
synthetic mono- or diglycerides. In addition, fatty acids such as
oleic acid may likewise be used in the preparation of injectable
preparations.
[0076] Liquid dosage forms for oral administration may generally
comprise a liposome solution containing the liquid dosage form.
Suitable forms for suspending liposomes include emulsions,
suspensions, solutions, syrups, and elixirs containing inert
diluents commonly used in the art, such as purified water. Besides
the inert diluents, such compositions can also include adjuvants,
wetting agents, emulsifying and suspending agents, or sweetening,
flavoring, or perfuming agents.
[0077] In some embodiments, the mTOR inhibitors in combination with
a therapeutic agent is provided as a liquid suspension or as a
freeze-dried product. Suitable liquid preparations include, e.g.,
isotonic aqueous solutions, suspensions, emulsions, or viscous
compositions that are buffered to a selected pH. Transdermal
preparations include lotions, gels, sprays, ointments or other
suitable techniques. If nasal or respiratory (mucosal)
administration is desired (e.g., aerosol inhalation or
insufflation), compositions can be in a form and dispensed by a
squeeze spray dispenser, pump dispenser or aerosol dispenser.
Aerosols are usually under pressure by means of a hydrocarbon. Pump
dispensers can preferably dispense a metered dose or a dose having
a particular particle size, as discussed below.
[0078] In certain embodiments, the mTOR inhibitors in combination
with a therapeutic agent may be provided in the form of a solution,
suspension and gel. In other embodiments, formulation of the
conjugate vaccine may contain a major amount of water that may be
purified in addition to the active ingredient. Minor amounts of
other ingredients such as pH adjusters, emulsifiers, dispersing
agents, buffering agents, preservatives, wetting agents, jelling
agents, colors, and the like can also be present.
[0079] Compositions comprising mTOR inhibitors, may be administered
in a number of ways either alone or in combination with other
treatments, at the same time, at different times, either
simultaneously or sequentially depending on the condition to be
treated and whether local or systemic treatment is desired.
Administration may be by direct injection, or by intrathecal
injection, or intravenously, or by stereotaxic injection. The route
of administration can be selected based on the disease or
condition, the effect desired, and the nature of the cells being
used. Actual methods of preparing dosage forms are known, or will
be apparent, to those skilled in the art. (See Remington: The
Science and Practice of Pharmacy, 22nd edition, 2012,
Pharmaceutical Press.) Where a composition as described herein is
to be administered to an individual, administration is preferably
in a "prophylactically effective amount" or a "therapeutically
effective amount," this being sufficient to show benefit to the
individual.
[0080] The number of administrations can vary. Alternatively,
administration may be, for example, daily, weekly, or monthly. The
actual amount administered, and rate and time-course of
administration, will depend on the age, sex, weight, of the
subject, the stage of the disease, and severity of what is being
treated (including prophylactic treatment). Prescription of
treatment, e.g., decisions on dosage is within the responsibility
of general practitioners and other medical doctors.
[0081] Effective clinical use of use cancer therapeutic agents
depends on the ability to balance the killing of tumor cells
against the inherent toxicity of many of these drugs to host cells.
Because of the narrow therapeutic indices of cancer therapeutic
agents, dosages are frequently calculated based on body surface
area (BSA) rather than body mass. Correlation is better between
body weight and these toxicities. Cancer therapeutic agents can be
administered by intraperitoneal, transdermal, intratumor injection,
IV, SC, IM, topical, intracavitary, intralesional, intravesicular,
intrathecal, or intra-arterial routes. The route chosen depends on
the individual agent and is determined by drug toxicity; location,
size, and type of tumor; and physical constraints.
[0082] Cancer therapeutic agents are commonly administered in
various combinations of dosages and timing; the specific regimen is
referred to as a protocol. A protocol may use one or as many as
five or six different antineoplastic agents. Selection of an
appropriate protocol should be based on type of tumor, grade or
degree of malignancy, stage of the disease, condition of the
animal, and financial considerations. Preferences of individual
clinicians for treatment of specific neoplastic conditions may also
vary. Regardless of the protocol chosen, a thorough knowledge of
the mechanism of action and toxicities of each therapeutic agent is
essential.
4. SUMMARY OF EXPERIMENTAL RESULTS
[0083] The following is a summary of results of experiments
described in the Examples of this application: [0084] INK128
inhibits R5 and X4 HIV replication in primary cells; [0085] INK128
inhibits entry of R5, but not X4, HIV in primary cells; [0086]
INK128 inhibits HIV gene expression; [0087] INK128 has favorable
drug interactions with current antiretroviral classes; [0088]
INK128 inhibits HIV replication in vivo; [0089] Torin-2 inhibits
HIV in vitro in the absence of cell toxicity; and [0090] INK128
inhibits tumor growth in NSG mice.
5. EXAMPLES
[0091] The invention is illustrated herein by the experiments
described by the following examples, which should not be construed
as limiting. The contents of all references, pending patent
applications and published patents, cited throughout this
application are hereby expressly incorporated by reference. Those
skilled in the art will understand that this invention may be
embodied in many different forms and should not be construed as
limited to the embodiments set forth herein. Rather, these
embodiments are provided so that this disclosure will fully convey
the invention to those skilled in the art. Many modifications and
other embodiments of the invention will come to mind in one skilled
in the art to which this invention pertains having the benefit of
the teachings presented in the foregoing description. Although
specific terms are employed, they are used as in the art unless
otherwise indicated.
Example 1
Materials and Methods
Cell Proliferation and Infectivity Assays
[0092] PBLs were isolated from buffy coats of HIV seronegative
donors (New York Blood Center). The laboratory-adapted strains HIV
BaL and HIV HXB2; primary isolates HIV 92BR020, 92UG031, 93BR029,
and 93UG082; and multidrug resistant molecular clone NL4329129-2
were obtained from the NIH AIDS Repository. Primary isolate 2044
was from Paul Clapham (Windeyer Institute), and 1633 and 1638 from
the Institute of Human Virology (University of Maryland School of
Medicine). Maraviroc, efavirenz, raltegravir, and indinavir were
from the NIH AIDS Repository. INK128 was purchased from ApexBio.
Proliferation of PBLs was measured by the MTT kit (Roche),
following the manufacturer's directions. PBL infectivity assays
were performed as described herein.
Cell Fusion Assay
[0093] CD4 cells were isolated from PBL cultures maintained for 7
days in the presence of different concentrations of INK128.
Isolation of CD4 cells was done by positive selection using
immunomagnetic beads following the manufacturer's directions
(Invitrogen.TM.). Cell fusion was analyzed by measuring cytoplasmic
dye exchange between fluorescent dye-labeled CD4 lymphocytes
(targets) and transfected 293T cells expressing the R5 HIV Env or
X4 HIV Env (effectors) using flow cytometry analysis. This
methodology is described in detail herein.
Quantitation of CD4, CCR5, and CXCR4
[0094] Quantitation of CCR5, CXCR4, and CD4 was done as described
(59) using the following antibody clones: clone 45531 (CCR5), clone
12G5 (CXCR4), and clone RPA-T4 (CD4). For CCR5 and CXCR4,
lymphocytes were first gated on CD3 (clone UCHT1) and CD4 (clone
RPA-T4). For CD4, lymphocytes were gated using CD3 (clone UCHT1) in
combination with CD8 (clone SK1). All antibodies were from BD
Biosciences except for the CCR5 antibody, which was from R&D
Systems. The methodology is fully described herein.
Real-Time PCR for Detection of Early Products of Reverse
Transcription and of Integrated HIV DNA
[0095] Activated PBLs were infected with R5 and X4 HIV strains
using a MOI of 0.01. Infected cells were cultured in the presence
of IL-2 and different concentrations of INK128. Cell aliquots were
collected at 16 and 72 hours for analyses of early products of
reverse transcription and of integration, respectively, by real
time PCR. Detection of early products of reverse transcription was
done with primers specific for the R/U5 region (60). For detection
of integrated HIV DNA, a real-time nested Alu-HIV PCR assay was
used as previously described (61, 62), with the modifications
described herein.
[0096] DNA was isolated using Miniblood kit (Qiagen.TM.). PCR
amplification was performed using Quantitect SYBR Green.TM. PCR Kit
(Qiagen.TM.) in a LightCycler.TM. (Biorad.TM.). Detection of early
products of reverse transcription was done in reactions containing
100 ng of DNA and the primer pair 5'-GCTCTCTGGCTAACTAGGGAAC-3' (SEQ
ID NO. 1) and 5'-TGACTAAAAGGGTCTGAGGGAT-3' (SEQ ID NO. 2) (R/U5
region) (60). Samples were also amplified with primers for the
housekeeping gene .alpha.-tubulin. Both sets of PCR reactions were
done at an annealing temperature of 56.degree. C. Amplified
products were analyzed by denaturation/renaturation to verify the
specific Tm. The PCR cycle at which the signal entered the
exponential range was used for quantification, and HIV copy numbers
were corrected for those of .alpha.-tubulin. Standard curves for
HIV and .alpha.-tubulin copy numbers were generated by analyzing
serial dilutions of plasmids carrying the corresponding sequences.
For detection of integrated HIV DNA, we used a real-time nested
Alu-HIV PCR assay previously described (61, 62), with the following
modifications. The first PCR used 100 ng of DNA template and
primers Alu (5'-GCCTCCCAAAGTGCTGGGATTACAG-3') SEQ ID NO. 3 and Gag
(5'-GCTCTCGCACCCATCTCTCTCC-3') (SEQ ID NO. 4) for 25 cycles. From
this reaction, 1/20 of amplified product was used as template for
the nested PCR with primers LTR-R (5'-GCCTCAATAAAGCTTGCCTTGA-3')
(SEQ ID NO. 5) and LTR-U5 (5'-TCCACACTGACTAAAAGGGTCTGA-3') (SEQ ID
NO. 6), as described (61, 62) except for the annealing temperature,
which was 61.degree. C. in our reactions. As a standard curve for
relative quantification of integrated DNA, the Alu-gag was first
run using serial dilutions of DNA isolated from HIV infected PBLs
(diluted in HIV-negative DNA).
[0097] CD4 cells were isolated from PBL cultures maintained for 7
days in the presence of different concentrations of INK128.
Isolation of CD4 cells was done by positive selection using
immunomagnetic beads (Invitrogen.TM.) following the manufacturer's
directions. HIV JRFL-Env (R5) and HXB2-Env (X4) expressing 293T
cells were prepared by calcium phosphate transfection, using 5
.mu.g of Env-expressing plasmid and 2.5 .mu.g of cRev plasmid per
60-mm dish. CD4 lymphocytes (targets) were labeled with calcein
acetoxymethyl ester (Calcein AM), and HIV Env-expressing 293T cells
(effector cells) were labeled with orange dye, 5- and
6-([(4-chloromethyl)benzoyl]-amino) tetramethylrhodamine (CMTMR) on
day 2 after transfection. Calcein AM and CMTMR dyes (both from
Invitrogen) were used at final concentrations of 75 nM and 1 .mu.M,
respectively. Target cells (1.times.105) and effector cells
(3.times.105) were cocultured in triplicate wells of a 96-well
plate in Hepes-buffered DMEM (pH 7.2) supplemented with 1 mg/mL BSA
(Sigma) for 2.5 h at 37.degree. C. to allow fusion. INK128 was
added at different concentrations at the beginning of the 2.5-h
incubation. Cells were washed with PBS, and incubated with
Trypsin/EDTA for 5 min at 37.degree. C. to stop the fusion reaction
and to disrupt cell clusters. Trypsin was neutralized by adding
DMEM containing 10% (vol/vol) FBS and cells were washed with PBS.
Cells were then analyzed on a FACSCalibur.TM. (BD Biosciences.TM.)
after collecting 40,000 events from each well using Cellquest.TM.
software (BD Biosciences.TM.). Fusion was scored as a number of
cells positive for both dyes in the histogram. The results (fusion
events) were normalized for the total number of target cells in the
histogram. Background was determined by analyzing fusion in the
presence of the fusion inhibitor, C34 peptide at 1 .mu.M, a
concentration that abrogates fusion. The obtained background signal
(false positive fusion events) was subtracted from the signal
obtained in the absence of C34. Quantification of CD4, CCR5, and
CXCR4. Before staining, PBLs were washed twice with PBS and
incubated in blocking buffer (PBS containing 2% human serum, 5%
horse serum, and 0.1% sodium azide) for 30 min at room temperature.
Cells were then stained with the antibodies for 30 min at room
temperature, washed twice with PBS, and acquired on a FACS
Calibur.TM. (BD Biosciences.TM.) using Cellquest.TM. software (BD
Biosciences) Immunofluorescence intensity was measured as an
estimate of the average number of molecules on the cell surface.
Fluorescence was measured using the Quantiquest.TM. system (BD
Biosciences.TM.), which produces a regression line from a series of
Quantibrite-phycoerythrin (PE).TM. bead standards (BD
Biosciences.TM.). The mean number of surface molecules for a cell
labeled with a PE antibody was then determined from the FL-2 value
of the cell using this linear regression and taking into account
the PE/antibody ratio for each antibody (1:1 in our reagents).
Semiquantitative RT-PCR for Detection of Cellular HIV mRNA
[0098] Total cellular RNA was isolated using the Qiagen.TM. RNA
Isolation Kit (Qiagen.TM.) RNA was then treated with DNase I,
Amplification Grade (Invitrogen.TM.), and reverse transcribed with
SuperScript III First-Strand Synthesis Supermix.TM.
(Invitrogen.TM.) using hexamer primers. An aliquot of the cDNA was
used as a template for PCR amplification of full length, unspliced
HIV cDNA using primers US.1 a and US.2a, and another aliquot for
amplification with primer pairs specific for housekeeping
[beta]-actin sequences (63).
Generation of Humanized Mice, Quantitation of Plasma HIV RNA and
Lymphocyte Subsets
[0099] Animal protocols were approved by the Institutional Animal
Care and Use Committee, University of Maryland School of Medicine.
NSG mice (5-7 weeks) were intraperitoneally (i.p) injected with 107
PBLs isolated from buffy coats of healthy donors. Three weeks later
mice were screened for human lymphocytes in peripheral blood
samples. Successfully reconstituted animals were i.p injected with
15,000 units of 50% tissue culture infective dose (TCID50) of HIV
BaL, followed by daily i.p. treatment with INK128 [1-5 mg/kg/day,
prepared in a 1-methyl-2-pyrrolidinone (NMP)/polyvinylpyrrolidone
k30 (PVP) solution as described (26)] or PBS (in NMP/PVP solution)
for 14 days. Animals were monitored daily for external signs of
clinical deterioration. Blood samples, drawn from the retroorbital
vein on days 7 and 14 after infection, were analyzed for plasma HIV
RNA copy number by quantitative RT PCR using HIV gag primers (64)
and for human CD4/CD8 ratios (flow cytometry analysis).
MTT Assays
[0100] Cell proliferation was measured by a colorimetric MTT test
(Roche.TM.). This test is based on the reduction of the yellow
colored MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide] to blue formazan by mitochondrial dehydrogenases. The
quantity of formazan produced (absorbance at 490 nm) is directly
proportional to the number of living cells. Briefly, cell aliquots
were seeded in 96-well plates (100 .mu.L) and incubated with 10
.mu.L of MTT solution for 4 hours at 37.degree. C. A solubilization
solution (50 .mu.L) was added and plates incubated overnight at
37.degree. C. MTT conversion to formazan by mitochondrial
dehydrogenase was assayed by optical density at 490 nm measured in
an ELISA plate reader.
Infectivity Assays
[0101] PBLs were separated from buffy coats by density
centrifugation over Ficoll-Hypaque (Sigma.TM.), and activated by
culture in the presence of 1 .mu.g/mL anti-human CD3 antibody
(clone x35, Fisher Scientific.TM.) and 2 .mu.g/mL anti-human CD28
antibody (Clone CD28.6, eBioscience.TM.) for 3 days. Activated
cells were infected by incubation with virus at a multiplicity of
infection (MOI) of 0.001 for 2 hours. Infected cells were washed
three times with PBS and cultured in 5% CO2 at 37.degree. C., in
RPMI/10% FBS supplemented with 100 units/mL IL-2 (Roche.TM.) and
antiviral drugs, in 96-well flat-bottom plates at a density of
2.times.105 PBLs per 200 .mu.L. Following 3 d of culture, half of
the medium was replaced with fresh medium containing IL-2 and
antiviral drugs. On day 7, viral replication was measured by p24
ELISA (Coulter) in culture supernatants and cell viability was
measured by MTT assays.
Quantification of Plasma HIV RNA
[0102] For quantification of HIV RNA, viral RNA was extracted from
40 .mu.L of plasma samples using Qiagen viral RNA Minikit.TM.
(Qiagen.TM.). RNA was converted to cDNA using SuperScript III
Supermix.TM. (Invitrogen.TM.). cDNA was amplified with HIV gag
consensus primers (64), using Quantitec.TM. SYBR Green PCR kit
(Qiagen.TM.) in a LightCycler.TM. (BioRad.TM.). Reactions were
heated at 50.degree. C. for 2 minutes, 95.degree. C. for 15
seconds, followed by 35 amplification cycles (94.degree. C. for 15
seconds, 58.degree. C. for 30 seconds, 72.degree. C. for 30
seconds). A standard curve was prepared by serial dilutions of RNA
extracted from plasma of an HIV patient with known HIV RNA copy
number (HIV VQA RNA Quantification Standard; NIH AIDS Repository,
catalog no. 3443). Peripheral blood CD4/CD8 ratios were determined
by staining of whole blood with FITC-conjugated mouse anti-CD4 and
APC-conjugated mouse anti-CD8 monoclonal antibodies (BD
Pharmingen.TM.), followed by flow cytometry analysis.
Statistics Analyses
[0103] EC50 values were determined by variable slope nonlinear
regression analysis. Unpaired two-tailed t tests were used to check
for statistical significant differences between INK128 EC50 values
of R5 HIV versus X4 HIV in infectivity assays. Nonparametric
Mann-Whitney tests were used to compare each treatment group and
control group in animal studies. Statistical analyses were
performed using GraphPad Prism.TM. (version 4.0). P<0.05 was
considered significant.
Example 2
INK128 Inhibits R5 and X4 HIV Replication in Primary Cells
[0104] The chemical structure of INK128 is shown in FIG. 1A. The
effect of INK128 was evaluated on proliferation of peripheral blood
lymphocytes (PBLs) from four different donors. For each donor, PBLs
were activated by treatment with anti-CD3/CD28 antibodies for 3
days, cultured in the presence of IL-2 and various dilutions of
INK128 for 5 days, followed by measurement of cell proliferation by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
assays (FIG. 1B). INK128 did not inhibit cell proliferation at
concentrations of up to 100 nM. Therefore, 100 nM was selected as
the highest INK128 concentration in subsequent experiments
evaluating antiviral activity.
[0105] The antiviral activity of INK128 was investigated in PBLs
infected with CCR5 (R5)-tropic and CXCR4 (X4)-tropic HIV reference
strains BaL and HXB2, respectively. In experiments with PBLs from
three donors, INK128 inhibited replication of both viruses, but it
inhibited BaL more potently than HXB2 (EC50s of 10.5 vs. 38 nM;
P=0.007 by two-tailed, unpaired t test) (FIG. 1C). Similarly, in
primary isolates evaluated in three donors, INK128 was more potent
against R5 (EC50s ranging 2.9-10.1 nM) than against X4 (EC50s
ranging 17.5-36.7 nM) (Table 6).
TABLE-US-00006 TABLE 6 Activity of INK128 against primary isolates
of HIV-1 in PBMCs Primary Isolate Coreceptor tropism Geometric mean
INK128 EC50, nM (95% CI)* HIV-1 93BR029 R5 4.73 (2.78-8.05) HIV-1
92UG031 R5 10.15 (3.73-27.60) HIV-1 93UG082 R5 2.89 (0.82-10.13)
HIV-1 92BR020 R5 1.03 (0.48-2.19) HIV-1 2044 X4 36.72 (13.38-100.8)
HIV-1 1633 X4 17.47 (6.89-44.24) HIV-1 1638 X4 21.20 (3.25-138.4)
*EC50 values were determined by variable slope nonlinear regression
analysis using Graph Pad Prism software. Data are from three
experiments, each with a different donor.
[0106] EC50 values were determined by variable slope nonlinear
regression analysis. The difference in INK128 potency against R5
vs. X4 primary isolates was again significant (P=0.01 by
two-tailed, unpaired t test). In addition, INK128 inhibited a
multidrug-resistant HIV molecular clone NL4329129-2, which carries
the RT gene amplified from plasma of a patient with multidrug
resistant HIV (28), with an EC50 of 10.9 nM (FIG. 2A-2B). Together,
these data show that INK128 inhibits replication of R5 and X4
strains of HIV, both laboratory adapted and primary isolates, in
PBLs.
Example 3
INK128 Inhibits Entry of R5, but not X4, HIV in Primary
Lymphocytes
[0107] The mechanism of INK128 inhibition of HIV was evaluated.
Because INK128 activity was more potent against R5 than against X4
HIV, it was hypothesized that INK128 affects entry of these viruses
differently. To test this, cell-cell fusion assays were performed
between 293T cells expressing R5 or X4 HIV envelopes (effectors)
and INK128-treated primary CD4+ T cells (targets). In this assay,
targets are labeled with the fluorescent dye calcein (green) and
effectors with CMTMR (red) before co-culture. Fused cells score
positive for both dyes. INK128 inhibited fusion of CD4+ T target
cells with R5 HIV JRFL Env, but not with X4 HIV HXB2 Env (FIG. 3A).
These data, obtained with CD4 targets from two different donors,
suggested inhibition at an early step of the R5, but not X4, HIV
lifecycle. Downstream steps in HIV infection were evaluated by
measuring early products of reverse transcription and integrated
provirus using real-time PCR in PBLs from two donors. As expected
from the cell-cell fusion data, PBLs infected with R5 HIV (JRFL)
and treated with INK128 had decreased levels of early products of
reverse transcription (R/U5 transcripts) (FIG. 3B) and integrated
provirus (FIG. 3C).
[0108] In contrast, INK128 did not decrease R/U5 transcripts or
integrated provirus on infection with X4 HIV (HXB2). Together these
data demonstrate that INK128 inhibits entry of R5 HIV, but it does
not inhibit X4 HIV infection before, or at the level of,
integration. To gain insight into the mechanism of INK128
inhibition of R5 HIV entry, we evaluated the effects of INK128 on
the receptor CD4 and the coreceptors CCR5 and CXCR4 by flow
cytometry analysis in PBLs from three donors. In these experiments
PBLs were stimulated with IL-2 alone (without previous activation
with anti-CD3/CD28), in the presence and absence of INK128, for 7
days. INK128 reduced percentages of CCR5 expressing cells in both
the CD4+ and CD8+ subsets of T cells (FIG. 4A). INK128 also
decreased CCR5 receptor density (molecules/cell). In contrast,
INK128 did not change CXCR4 levels, either in terms of percentage
or density (FIG. 4B). In addition, INK128 did not impact CD4
receptor levels (FIG. 4C). Together, these data suggest that INK128
inhibits R5 HIV entry by decreasing CCR5 levels, consistent with
the observation that CCR5 levels are limiting for R5 HIV infection
(29-32).
Example 4
INK128 Inhibits HIV Gene Expression
[0109] The effects of INK128 on activation of HIV in the
chronically HIV-infected U1 cell line were examined. The U1 cell
line carries two copies of the HIV provirus per cell (33). Under
basal conditions, U1 cells express low levels of HIV, but HIV
expression is enhanced by stimulation with the phorbol ester PMA or
by exogenous addition of Tat (34, 35). U1 cells were cultured in
the presence of INK128 in the absence and presence of 10 nM PMA or
1 .mu.g/mL Tat. INK128 was used at concentrations .ltoreq.10 nM
because optimization experiments showed inhibition of cell
proliferation at higher concentrations, consistent with the
increased drug sensitivity of U937 cells (parent cell line of U1)
to TOR-KIs compared with primary cells (36). As expected,
unstimulated U1 cells produced low levels of HIV p24, but
production was increased by addition of PMA or Tat. INK128
inhibited p24 production in untreated cells as well as in cells
treated with PMA or Tat (FIG. 5A-5C). RT-PCR analyses showed that
INK128 inhibits synthesis of full-length unspliced HIV mRNA (FIG.
6). Together, these data suggest that INK128 inhibits
transcription, both basal and induced, of the HIV LTR.
Example 5
INK128 has Favorable Drug Interactions with Current Antiretroviral
Classes
[0110] Available ARTs target the HIV lifecycle steps of entry,
reverse transcription, integration, and maturation. The observation
that INK128 reduces CCR5 density and inhibits R5 HIV entry
suggested that INK128 could enhance the antiviral activity of the
CCR5 antagonist Maraviroc. In addition, by targeting virus
transcription, INK128 could have favorable interactions with
inhibitors of reverse transcription (RTIs), integration (Hs), and
protease (PIs). We therefore evaluated the antiviral potency of
each ART class in the presence and absence of INK128. We conducted
these assays in activated PBLs infected with R5 HIV BaL and treated
with various dilutions of Maraviroc (CCR5 antagonist), Efavirenz
(RTI), Raltegravir (II), and Indinavir (PI). INK128 was used at low
concentrations (<EC50) to better detect changes in ART potency
(Table 7).
TABLE-US-00007 TABLE 7 Fifty percent effective concentrations
(EC50) of Maraviroc (MVC), Efavirenz (EFV), Raltegravir (RAL), and
Indinavir (IND) against HIV-1 BaL, in the absence and presence of
INK128 in PBMCs MVC EC.sub.50,.sup.a EFV EC50,.sup.a RAL
EC.sub.50,.sup.a IND EC.sub.50,.sup.a Treatment (95% CI) (95% CI)
(95% CI) (95% CI) No INK128 1.78 nM 0.52 nM 0.65 nM 3.84 nM
(0.67-4.76) (0.31-0.87) (0.34-1.13) (3.21-4.57) +3 nM INK128 0.54
nM 0.51 nM 0.81 nM 3.14 nM (0.20-1.50) (0.34-0.75) (0.49-1.14)
(2.31-4.27) +10 nM INK128 0.30 nM 0.51 nM 0.57 nM 3 nM (0.20-0.45)
(0.33-0.78) (0.24-1.34) (1.10-8.18) .sup.aEC.sub.50 values are
Geometric Means, determined by variable slope non-linear regression
analysis using GraphPad Prism software. Data are from 2
experiments, each with a different donor.
[0111] In experiments with two donors, INK128 enhanced the
antiviral potency of Maraviroc by five- to six fold, and had no
negative effect on the potency of the other tested ARTs. Together,
these data suggest that INK128 enhances the antiviral activity of
Maraviroc, and it has favorable, non-antagonist drug interactions
with the other existing ART classes.
Example 6
INK128 Inhibits HIV Replication in Humanized Mice
[0112] Anti-HIV activity of INK128 was evaluated in vivo, using
NOD/SCID/IL-2R.gamma.null (NSG) mice reconstituted with human PBLs
and infected with HIV BaL. In pilot experiments, in which
uninfected huPBLNSG mice were treated with daily i.p. injections of
INK128 at 0.5, 1, 3, 5, and 7 mg/kg for 2 wk, the 7 mg/kg dose was
associated with wasting and death. Thus, the antiviral activity of
INK128 was evaluated at 0 (PBS) (n=6 mice), 1 mg/kg (n=5), 3 mg/kg
(n=5), and 5 mg/kg (n=5). Treatment was initiated immediately after
virus injection and continued once daily for 14 days. Treatment had
no adverse effects on the weight of the animals compared with
controls (FIG. 6A-6B). Two mice, one in the control group and one
in the 5 mg INK128/kg group, died in the course of the experiment.
We could not determine the cause of death in the two animals, but
incidental death, often the result of graft-versus-host disease
from the transplanted human cells, is frequent in this animal model
(37). On day 7 after infection, control mice (n=6) had mean plasma
HIV RNA (copies per mL) of 3.3.times.10.sup.6 (range,
2.1.times.10.sup.6 to 5.2.times.10.sup.6)(FIG. 7A). In
INK128-treated mice, mean HIV RNA (copies/mL) were 1.2.times.107
(range, 1.7.times.106 to 4.5.times.107; n=5; P=0.3), 8.5.times.105
(range, 3.5.times.105 to 1.7.times.106; n=5; P=0.008) and
3.8.times.104 (range, 1.times.104 to 1.times.105; n=4; P=0.009), at
1, 3, and 5 mg/kg/day doses, respectively. On day 14 after
infection, mean plasma HIV RNA values were 1.2.times.106 (range,
2.4.times.105 to 2.4.times.106) in controls; and 1.1.times.106
(range, 5.2.times.105 to 2.1.times.106; P=0.9), 2.5.times.105
(range, 1.4.times.105 to 3.8.times.105; P=0.03) and 5.times.103
(range, 1.3.times.103 to 8.times.103; P=0.01), at 1, 3, and 5
mg/kg/day doses, respectively. Consistent with reductions in
viremia, infected mice treated with INK128 had higher CD4/CD8
ratios than did controls (FIG. 7B). Although CD4/CD8 ratios on day
7 were somewhat variable, day 14 ratios were significantly higher
that controls. Day 14 mean CD4/CD8 cell ratios were 0.04 (range,
0.03-0.06) in control mice and 0.11 (range, 0.06-0.18; P=0.01),
0.18 (range, 0.14-0.24; P=0.01), and 0.76 (range, 0.5-1.14;
P=0.01), at 1, 3, and 5 mg/kg/day doses, respectively. Together,
these data demonstrate that INK128 suppresses viremia of the HIV
reference strain BaL in a preclinical animal model. INK128 reduced
plasma viremia by more than 2 log 10 units, a decrease in viral
load comparable to that achieved with EFdA, a potent NRTI in
clinical trials, in a similar experimental setting (38).
Example 7
Torin-2 Inhibits HIV
[0113] PHA activated PBMCs were infected with HIV-1 for 2 hours.
Infected cells were washed to remove non-adsorbed virus, and plated
in culture medium containing IL-2 and various dilutions of Torin-2.
On day 3, fresh medium with fresh drug was added to the cultures.
On day 7, cultures were evaluated for HIV production by measuring
HIV p24 levels in the culture supernatants by ELISA (FIG. 9A). Also
on day 7, cell viability was measured by MTT assays (FIG.
9B)/Example
Example 8
INK-128 Inhibits Tumor Growth in NSG Mice
[0114] A xenograft tumor was induced by subcutaneous injection of
5.times.10.sup.6 non small cell lung cancer (NSCLC) A549 cells in
mice. After 3 weeks, tumors became visible (.about.200 mm.sup.3, as
measured with calipers). Mice were treated with INK128, or vehicle
control (FIG. 10A), daily (via ip) for 4 weeks. INK-128 was used at
5 mg/kg (FIG. 10B). Tumor volume was measured at the indicated time
points (FIG. 10C).
REFERENCES
[0115] 1. Giinthard H F, et al.; International Antiviral
Society-USA Panel (2014) Antiretroviral treatment of adult HIV
infection: 2014 recommendations of the International Anti-viral
Society-USA Panel. JAMA 312(4):410-425. [0116] 2. Wong A (2014) The
HIV pipeline. Nat Rev Drug Discov 13(9):649-650. [0117] 3. Pennings
P S (2013) HIV Drug Resistance: Problems and Perspectives. Infect
Dis Rep 5 (Suppl 1):e5. [0118] 4. De Clercq E (2013) The nucleoside
reverse transcriptase inhibitors, nonnucleoside reverse
transcriptase inhibitors, and protease inhibitors in the treatment
of HIV infections (AIDS). Adv Pharmacol 67:317-358. [0119] 5. Dorr
P, et al. (2005) Maraviroc (UK-427,857), a potent, orally
bioavailable, and selective small-molecule inhibitor of chemokine
receptor CCR5 with broad-spectrum anti-human immunodeficiency virus
type 1 activity. Antimicrob Agents Chemother 49(11):4721-4732.
[0120] 6. Dilby J M, et al. (1998) Potent suppression of HIV-1
replication in humans by T-20, a peptide inhibitor of gp41-mediated
virus entry. Nat Med 4(11):1302-1307. [0121] 7. Fatkenheuer G, et
al. (2005) Efficacy of short-term monotherapy with maraviroc, a new
CCR5 antagonist, in patients infected with HIV-1. Nat Med
11(11):1170-1172. [0122] 8. Kilby J M, et al. (2002) The safety,
plasma pharmacokinetics, and antiviral activity of subcutaneous
enfuvirtide (T-20), a peptide inhibitor of gp41-mediated virus
fusion, in HIV-infected adults. AIDS Res Hum Retroviruses
18(10):685-693. [0123] 9. Chi H (2012) Regulation and function of
mTOR signalling in T cell fate decisions. Nat Rev Immunol
12(5):325-338. [0124] 10. Zoncu R, Efeyan A, Sabatini D M (2011)
mTOR: From growth signal integration to cancer, diabetes and
ageing. Nat Rev Mol Cell Biol 12(1):21-35. [0125] 11. Heredia A, et
al. (2008) Reduction of CCR5 with low-dose rapamycin enhances the
antiviral activity of vicriviroc against both sensitive and
drug-resistant HIV-1. Proc Natl Acad Sci USA 105(51):20476-20481.
[0126] 12. Roy J, Paquette J S, Fortin J F, Tremblay M J (2002) The
immunosuppressant rapamycin represses human immunodeficiency virus
type 1 replication. Antimicrob Agents Chemother 46(11):3447-3455.
[0127] 13. Heredia A, et al. (2003) Rapamycin causes
down-regulation of CCR5 and accumulation of anti-HIV
beta-chemokines: An approach to suppress R5 strains of HIV-1. Proc
Natl Acad Sci USA 100(18):10411-10416. [0128] 14. Rai P, et al.
(2013) Rapamycin-induced modulation of HIV gene transcription
attenuates progression of HIVAN. Exp Mol Pathol 94(1):255-261.
[0129] 15. Heredia A, et al. (2007) Rapamycin reduces CCR5 density
levels on CD4 T cells and this effect results in potentiation of
Enfuvirtide (T-20) against R5 HIV-1 in vitro. Anti-microb Agents
Chemother 51(7):2482-2496. [0130] 16. Garcia-Martinez J M, et al.
(2009) Ku-0063794 is a specific inhibitor of the mammalian target
of rapamycin (mTOR). Biochem J 421(1):29-42. [0131] 17. Thoreen C
C, et al. (2009) An ATP-competitive mammalian target of rapamycin
inhibitor reveals rapamycin-resistant functions of mTORC1. J Biol
Chem 284(12): 8023-8032. [0132] 18. Yu K, et al. (2009)
Biochemical, cellular, and in vivo activity of novel
ATP-competitive and selective inhibitors of the mammalian target of
rapamycin. Cancer Res 69(15): 6232-6240. [0133] 19. Feldman M E, et
al. (2009) Active-site inhibitors of mTOR target
rapamycin-resistant outputs of mTORC1 and mTORC2. PLoS Biol
7(2):e38. [0134] 20. Moschetta M, Reale A, Marasco C, Vacca A,
Carrat M R (2014) Therapeutic targeting of the mTOR-signalling
pathway in cancer: Benefits and limitations. Br J Pharmacol
171(16):3801-3813. [0135] 21. Facchinetti V, et al. (2008) The
mammalian target of rapamycin complex 2 controls folding and
stability of Akt and protein kinase C. EMBO J 27(14):1932-1943.
[0136] 22. Ikenoue T, Inoki K, Yang Q, Zhou X, Guan K L (2008)
Essential function of TORC2 in PKC and Akt turn motif
phosphorylation, maturation and signalling. EMBO J 27(14):
1919-1931. [0137] 23. Chan J K, Greene W C (2012) Dynamic roles for
NF-.kappa.B in HTLV-I and HIV-1 retroviral pathogenesis. Immunol
Rev 246(1):286-310. [0138] 24. Nabel G, Baltimore D (1987) An
inducible transcription factor activates expression of human
immunodeficiency virus in T cells. Nature 326(6114):711-713. [0139]
25. Hsieh A C, et al. (2012) The translational landscape of mTOR
signalling steers cancer initiation and metastasis. Nature
485(7396):55-61. [0140] 26. INK128 in models of B-cell acute
lymphoblastic leukemia. Leukemia 27(3):586-594. [0141] 27. Slotkin
E K, et al. (2015) MLN0128, an ATP-competitive mTOR kinase
inhibitor with potent in vitro and in vivo antitumor activity, as
potential therapy for bone and soft-tissue sarcoma. Mol Cancer Ther
14(2):395-406. [0142] 28. Johnston E, et al. (2005) Panel of
prototypical infectious molecular HIV-1 clones containing multiple
nucleoside reverse transcriptase inhibitor resistance mutations.
AIDS 19(7):731-733. [0143] 29. Platt E J, Wehrly K, Kuhmann S E,
Chesebro B, Kabat D (1998) Effects of CCR5 and CD4 cell surface
concentrations on infections by macrophagetropic isolates of human
immunodeficiency virus type 1. J Virol 72(4):2855-2864. [0144] 30.
Reeves J D, et al. (2002) Sensitivity of HIV-1 to entry inhibitors
correlates with envelope/coreceptor affinity, receptor density, and
fusion kinetics. Proc Natl Acad Sci USA 99(25): 16249-16254. [0145]
31. Reynes J, et al. (2001) CD4 T cell surface CCR5 density as a
host factor in HIV-1 disease progression. AIDS 15(13):1627-1634.
[0146] 32. Reynes J, et al. (2000) CD4+ T cell surface CCR5 density
as a determining factor of virus load in persons infected with
human immunodeficiency virus type 1. J Infect Dis 181(3):927-932.
[0147] 33. Folks T M, Justement J, Kinter A, Dinarello C A, Fauci A
S (1987) Cytokine-induced expression of HIV-1 in a chronically
infected promonocyte cell line. Science 238(4828): 800-802. [0148]
34. Michael N L, et al. (1991) Induction of human immunodeficiency
virus type 1 expression in chronically infected cells is associated
primarily with a shift in RNA splicing patterns. J Virol 65
(12):7084. [0149] 35. Pomerantz R J, Trono D, Feinberg M B,
Baltimore D (1990) Cells nonproductively infected with HIV-1
exhibit an aberrant pattern of viral RNA expression: A molecular
model for latency. Cell 61(7):1271-1276. [0150] 36. Altman J K, et
al. (2011) Dual mTORC2/mTORC1 targeting results in potent
suppressive effects on acute myeloid leukemia (AML) progenitors.
Clin Cancer Res 17(13): 4378-4388. [0151] 37. Akkina R (2013) New
generation humanized mice for virus research: Comparative aspects
and future prospects. Virology 435(1):14-28. [0152] 38. Hattori S,
et al. (2009) Potent activity of a nucleoside reverse transcriptase
inhibitor, 4'-ethynyl-2-fluoro-2'-deoxyadenosine, against human
immunodeficiency virus type 1 infection in a model using human
peripheral blood mononuclear cell-transplanted NOD/SCID Janus
kinase 3 knockout mice. Antimicrob Agents Chemother 53(9):
3887-3893. [0153] 39. Deeks S G, Lewin S R, Havlir D V (2013) The
end of AIDS: HIV infection as a chronic disease. Lancet
382(9903):1525-1533. [0154] 40. Gilliam B L, et al. (2007)
Rapamycin reduces CCR5 mRNA levels in macaques: Potential
applications in HIV-1 prevention and treatment. AIDS
21(15):2108-2110. [0155] 41. Nicoletti F, et al. (2009) Inhibition
of human immunodeficiency virus (HIV-1) infection in human
peripheral blood leucocytes-SCID reconstituted mice by rapamycin.
Clin Exp Immunol 155(1):28-34. [0156] 42. Stock P G, et al.; for
Solid Organ Transplantation in HIV Study Investigators (2014)
Reduction of HIV persistence following transplantation in
HIV-infected kidney transplant recipients. Am J Transplant 14(5):
1136-1141. [0157] 43. Fiume G, et al. (2012) Human immunodeficiency
virus-1 Tat activates NF-.kappa.B physical interaction with
I.kappa.B-.alpha. and p65. Nucleic Acids Res 40(8):3548-3562.
[0158] 44. Barboric M, Nissen R M, Kanazawa S, Jabrane-Ferrat N,
Peterlin B M (2001) NF-kappaB binds P-TEFb to stimulate
transcriptional elongation by RNA polymerase I I. Mol Cell
8(2):327-337. [0159] 45. Janes M R, et al. (2010) Effective and
selective targeting of leukemia cells using a TORC1/2 kinase
inhibitor. Nat Med 16(2):205-213. [0160] 46. Wei P, Garber M E,
Fang S M, Fischer W H, Jones K A (1998) A novel CDK9-associated
C-type cyclin interacts directly with HIV-1 Tat and mediates its
high-affinity, loop-specific binding to TAR RNA. Cell
92(4):451-462. [0161] 47. He N, et al. (2010) HIV-1 Tat and host
AFF4 recruit two transcription elongation factors into a
bifunctional complex for coordinated activation of HIV-1
transcription. Mol Cell 38(3):428-438. [0162] 48. Sobhian B, et al.
(2010) HIV-1 Tat assembles a multifunctional transcription
elongation complex and stably associates with the 7S K snRNP. Mol
Cell 38(3):439-451. [0163] 49. Simioni C, et al. (2014) Activity of
the novel mTOR inhibitor Torin-2 in B-precursor acute lymphoblastic
leukemia and its therapeutic potential to prevent Akt
re-activation. Oncotarget 5(20):10034-10047. [0164] 50. Takeuchi C
S, et al. (2013) Discovery of a novel class of highly potent,
selective, ATP-competitive, and orally bioavailable inhibitors of
the mammalian target of rapamycin (mTOR). J Med Chem
56(6):2218-2234. [0165] 51. Venkatesha V A, et al. (2014) P7170, a
novel inhibitor of mTORC1/mTORC2 and Activin receptor-like Kinase 1
(ALK1) inhibits the growth of non small cell lung cancer. Mol
Cancer 13:259. [0166] 52. Gravina G L, et al. (2014) Torc1/Torc2
inhibitor, Palomid 529, enhances radiation response modulating
CRM1-mediated survivin function and delaying DNA repair in prostate
cancer models. Prostate 74(8):852-868. [0167] 53. Lin F, Buil L,
Sherris D, Beijnen J H, van Tellingen 0 (2013) Dual mTORC1 and
mTORC2 inhibitor Palomid 529 penetrates the blood-brain barrier
without restriction by ABCB1 and ABCG2. Int J Cancer
133(5):1222-1233. [0168] 54. Liao H, et al. (2015) Dramatic
antitumor effects of the dual mTORC1 and mTORC2 inhibitor AZD2014
in hepatocellular carcinoma. Am J Cancer Res 5(1):125-139. [0169]
55. Araki K, et al. (2009) mTOR regulates memory CD8 T-cell
differentiation. Nature 460(7251):108-112. [0170] 56. Pearce E L,
et al. (2009) Enhancing CD8 T-cell memory by modulating fatty acid
metabolism. Nature 460(7251):103-107. [0171] 57. Haidinger M, et
al. (2010) A versatile role of mammalian target of rapamycin in
human dendritic cell function and differentiation. J Immunol
185(7):3919-3931. [0172] 58. Deeks S G, Phillips A N (2009) HIV
infection, antiretroviral treatment, ageing, and non AIDS related
morbidity. BMJ 338:a3172. [0173] 59. Salkowitz J R, et al. (2003)
CCR5 promoter polymorphism determines macrophage CCR5 density and
magnitude of HIV-1 propagation in vitro. Clin Immunol 108(3):
234-240. [0174] 60. Lin Y L, et al. (2002) Cell surface CCR5
density determines the post entry efficiency of R5 HIV-1 infection.
Proc Natl Acad Sci USA 99(24):15590-15595. [0175] 61. Butler S L,
Hansen M S, Bushman F D (2001) A quantitative assay for HIV DNA
integration in vivo. Nat Med 7(5):631-634. [0176] 62. Konig R, et
al. (2008) Global analysis of host-pathogen interactions that
regulate early-stage HIV-1 replication. Cell 135(1):49-60.
Hermankova M, et al. (2003) Analysis of human immunodeficiency
virus type 1 gene expression in latently infected resting CD4+ T
lymphocytes in vivo. J Virol 77(13): 7383-7392. [0177] 63. Ou C Y,
et al. (1988) DNA amplification for direct detection of HIV-1 in
DNA of peripheral blood mononuclear cells. Science
239(4837):295-297.
Sequence CWU 1
1
6122DNAArtificial SequenceSynthetic primer 1gctctctggc taactaggga
ac 22222DNAArtificial SequenceSynthetic primer 2tgactaaaag
ggtctgaggg at 22325DNAArtificial SequenceSynthetic primer Alu
3gcctcccaaa gtgctgggat tacag 25422DNAArtificial SequenceSynthetic
primer Gag 4gctctcgcac ccatctctct cc 22522DNAArtificial
SequenceSynthetic primer LTR-R 5gcctcaataa agcttgcctt ga
22624DNAArtificial SequenceSynthetic primer LTR-U5 6tccacactga
ctaaaagggt ctga 24
* * * * *