U.S. patent application number 15/639575 was filed with the patent office on 2017-12-21 for trizol-1-ol analogs anti-retroviral latency drugs.
The applicant listed for this patent is University of Utah Research Foundation. Invention is credited to Alberto Bosque-Pardos, Mary Kay Harper-Ireland, Chris M. Ireland, John Alan Maschek, Vincente Planelles, Ryan Van Wagoner.
Application Number | 20170360784 15/639575 |
Document ID | / |
Family ID | 52022952 |
Filed Date | 2017-12-21 |
United States Patent
Application |
20170360784 |
Kind Code |
A1 |
Planelles; Vincente ; et
al. |
December 21, 2017 |
TRIZOL-1-OL ANALOGS ANTI-RETROVIRAL LATENCY DRUGS
Abstract
In one aspect, the invention relates to triazol-1-ol compounds,
analogs thereof, compositions comprising same, and methods of using
same, alone or in combination with other agents, to reactivate
latent retroviruses, and more particularly to reactivate latent
HIV-1. Such compounds, compositions, and methods can be used, for
example, in connection with diagnosing and/or treating a
retrovirus, and more specifically HIV-1. This abstract is intended
as a scanning tool for purposes of searching in the particular art
and is not intended to be limiting of the present invention.
Inventors: |
Planelles; Vincente; (Salt
Lake City, UT) ; Bosque-Pardos; Alberto; (Salt Lake
City, UT) ; Ireland; Chris M.; (Salt Lake City,
UT) ; Van Wagoner; Ryan; (Salt Lake City, UT)
; Harper-Ireland; Mary Kay; (Salt Lake City, UT) ;
Maschek; John Alan; (Salt Lake City, UT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Utah Research Foundation |
Salt Lake City |
UT |
US |
|
|
Family ID: |
52022952 |
Appl. No.: |
15/639575 |
Filed: |
June 30, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14896906 |
Dec 8, 2015 |
9730928 |
|
|
PCT/US2014/042418 |
Jun 13, 2014 |
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15639575 |
|
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|
61835297 |
Jun 14, 2013 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/4192 20130101;
A61K 31/416 20130101; A61K 45/06 20130101; C07D 249/04 20130101;
C07D 253/08 20130101; C07D 403/04 20130101; C07D 231/54 20130101;
A61K 31/53 20130101; C07D 471/04 20130101; A61K 31/437 20130101;
A61K 31/437 20130101; A61K 31/4192 20130101; A61K 31/501 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; C07D 249/18 20130101;
A61K 2300/00 20130101; A61K 31/53 20130101 |
International
Class: |
A61K 31/501 20060101
A61K031/501; C07D 403/04 20060101 C07D403/04; C07D 253/08 20060101
C07D253/08; C07D 249/18 20060101 C07D249/18; C07D 231/54 20060101
C07D231/54; A61K 31/416 20060101 A61K031/416; A61K 45/06 20060101
A61K045/06; A61K 31/53 20060101 A61K031/53; A61K 31/437 20060101
A61K031/437; A61K 31/4192 20060101 A61K031/4192; C07D 471/04
20060101 C07D471/04; C07D 249/04 20060101 C07D249/04 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under Grant
No. R01 AI 087508-01A1 awarded by the National Institutes of
Health. The United States government has certain rights in the
invention.
Claims
1. A method of activating a latent retrovirus in a subject, the
method comprising the step of administering to the subject an
effective amount of a compound represented by a formula:
##STR00063## wherein n is 0 or 1; wherein R.sup.1 is selected from
H, C1-C4 alkyl, C1-C6 aryl, C(O)Ar, C(O)N(CH.sub.3).sub.2,
SO.sub.2N(CH.sub.3).sub.2, fluorenylmethyloxycarbonyl,
N-((dimethylamino)methylene)-N-methylmethanaminium
tetrafluoroborate,
N-((dimethylamino)methylene)-N-methylmethanaminium
hexafluorophosphate (V), tri(pyrrolidin-1-yl)phosphonium
hexafluorophosphate (V), tris(dimethylamino)phosphonium
hexafluorophosphate (V),
1-(pyrrolidin-1-ylmethylene)pyrrolidin-1-ium hexafluorophosphate
(V), and 1-(piperidin-1-ylmethylene)piperidin-1-ium
hexafluorophosphate (V); wherein R.sup.2 is selected from H and
C1-C4 alkyl; and wherein R.sup.3 is selected from H and C1-C4
alkyl; or wherein R.sup.2 and R.sup.3 are covalently bonded and,
together with the intermediate atoms, comprise an optionally
substituted fused six-membered aryl or heteroaryl ring.
2-11. (canceled)
12. The method of claim 1, wherein the compound is represented by a
formula: ##STR00064## wherein R.sup.1 is selected from H, C1-C4
alkyl, C1-C6 aryl, C(O)Ar, C(O)N(CH.sub.3).sub.2,
SO.sub.2N(CH.sub.3).sub.2, fluorenylmethyloxycarbonyl,
N-((dimethylamino)methylene)-N-methylmethanaminium
tetrafluoroborate,
N-((dimethylamino)methylene)-N-methylmethanaminium
hexafluorophosphate (V), tri(pyrrolidin-1-yl)phosphonium
hexafluorophosphate (V), tris(dimethylamino)phosphonium
hexafluorophosphate (V),
1-(pyrrolidin-1-ylmethylene)pyrrolidin-1-ium hexafluorophosphate
(V), and 1-(piperidin-1-ylmethylene)piperidin-1-ium
hexafluorophosphate (V); and wherein each of R.sup.4, R.sup.5, and
R.sup.6 is independently selected from H, Cl, CH.sub.3, and
NO.sub.2.
13. The method of claim 1, wherein the compound is represented by a
formula: ##STR00065## wherein R.sup.1 is selected from H and
N-((dimethylamino)methylene)-N-methylmethanaminium
hexafluorophosphate (V); and wherein each of R.sup.4, R.sup.5, and
R.sup.6 is independently selected from H, Cl, CH.sub.3, and
NO.sub.2.
14. The method of claim 1, wherein the compound is represented by a
formula: ##STR00066## wherein each of R.sup.4, R.sup.5, and R.sup.6
is independently selected from H, Cl, CH.sub.3, and NO.sub.2.
15. The method of claim 1, wherein the compound is represented by a
formula: ##STR00067##
16. The method of claim 1, wherein the compound is represented by a
formula: ##STR00068##
17. The method of claim 1, wherein the compound is represented by a
formula: ##STR00069## wherein each of R.sup.4, R.sup.5, and R.sup.6
is independently selected from H, Cl, CH.sub.3, or NO.sub.2.
18. The method of claim 1, wherein the compound is represented by a
formula: ##STR00070##
19. The method of claim 1, wherein the compound is represented by a
formula: ##STR00071## wherein R.sup.1 is selected from H, C1-C4
alkyl, C1-C6 aryl, C(O)Ar, C(O)N(CH.sub.3).sub.2,
SO.sub.2N(CH.sub.3).sub.2, fluorenylmethyloxycarbonyl,
N-((dimethylamino)methylene)-N-methylmethanaminium
tetrafluoroborate,
N-((dimethylamino)methylene)-N-methylmethanaminium
hexafluorophosphate (V), tri(pyrrolidin-1-yl)phosphonium
hexafluorophosphate (V), tris(dimethylamino)phosphonium
hexafluorophosphate (V),
1-(pyrrolidin-1-ylmethylene)pyrrolidin-1-ium hexafluorophosphate
(V), and 1-(piperidin-1-ylmethylene)piperidin-1-ium
hexafluorophosphate (V).
20-21. (canceled)
22. The method of claim 1, wherein the compound is represented by a
formula: ##STR00072## wherein R.sup.1 is selected from H, C1-C4
alkyl, C1-C6 aryl, C(O)Ar, C(O)N(CH.sub.3).sub.2,
SO.sub.2N(CH.sub.3).sub.2, fluorenylmethyloxycarbonyl,
N-((dimethylamino)methylene)-N-methylmethanaminium
tetrafluoroborate,
N-((dimethylamino)methylene)-N-methylmethanaminium
hexafluorophosphate (V), tri(pyrrolidin-1-yl)phosphonium
hexafluorophosphate (V), tris(dimethylamino)phosphonium
hexafluorophosphate (V),
1-(pyrrolidin-1-ylmethylene)pyrrolidin-1-ium hexafluorophosphate
(V), and 1-(piperidin-1-ylmethylene)piperidin-1-ium
hexafluorophosphate (V); and wherein each of R.sup.7, R.sup.8, and
R.sup.9 is independently selected from H, Cl, CH.sub.3, and
NO.sub.2.
23. The method of claim 1, wherein the compound is represented by a
formula: ##STR00073## wherein R.sup.1 is selected from H and
N-((dimethylamino)methylene)-N-methylmethanaminium
hexafluorophosphate (V); and wherein each of R.sup.7, R.sup.8, and
R.sup.9 is independently selected from H, Cl, CH.sub.3, and
NO.sub.2.
24. (canceled)
25. The method of claim 1, wherein the compound is represented by a
formula: ##STR00074##
26. The method of claim 1, wherein the compound is represented by a
formula: ##STR00075## wherein R.sup.1 is selected from H, C1-C4
alkyl, C1-C6 aryl, C(O)Ar, C(O)N(CH.sub.3).sub.2,
SO.sub.2N(CH.sub.3).sub.2, fluorenylmethyloxycarbonyl,
N-((dimethylamino)methylene)-N-methylmethanaminium
tetrafluoroborate,
N-((dimethylamino)methylene)-N-methylmethanaminium
hexafluorophosphate (V), tri(pyrrolidin-1-yl)phosphonium
hexafluorophosphate (V), tris(dimethylamino)phosphonium
hexafluorophosphate (V),
1-(pyrrolidin-1-ylmethylene)pyrrolidin-1-ium hexafluorophosphate
(V), and 1-(piperidin-1-ylmethylene)piperidin-1-ium
hexafluorophosphate (V).
27-41. (canceled)
42. The method of claim 1, further comprising treating the patient
with at least one agent that can be used to treat HIV-1.
43. The method of claim 42, wherein the at least one agent is
selected from entry inhibitors, nucleoside reverse transcriptase
inhibitors (NRTIs), nucleotide reverse transcriptase inhibitors
(NtRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs),
protease inhibitors, integrase inhibitors, or maturation
inhibitors, or a mixture thereof.
44. The method of claim 42, wherein the at least one agent is
selected from nucleoside reverse transcriptase inhibitors (NRTIs),
nucleotide reverse transcriptase inhibitors (NtRTIs),
non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease
inhibitors, or integrase inhibitors, or a mixture thereof.
45. The method of claim 42, wherein the at least one agent is
selected from Maraviroc, Enfuvirtide, Zidovudine, Didanosine,
Zalcitabine, Stavudine, Lamivudine, Abacavir, Emtricitabine,
Entecavir, Tenofovir, Adefovir, Efavirenz, Nevirapine, Delavirdine,
Rilpivirine, Raltegravir, Saquinavir, Ritonavir, Indinavir,
Nelfinavir, or Amprenavir, or a mixture thereof.
46. The method of claim 42, wherein the at least one agent is
HAART.
68. A pharmaceutical composition comprising a compound represented
by a formula: ##STR00076## wherein n is 0 or 1; wherein R.sup.1 is
selected from H, C1-C4 alkyl, C1-C6 aryl, C(O)Ar,
SO.sub.2N(CH.sub.3).sub.2, fluorenylmethyloxycarbonyl,
N-((dimethylamino)methylene)-N-methylmethanaminium
tetrafluoroborate,
N-((dimethylamino)methylene)-N-methylmethanaminium
hexafluorophosphate (V), tri(pyrrolidin-1-yl)phosphonium
hexafluorophosphate (V), tris(dimethylamino)phosphonium
hexafluorophosphate (V),
1-(pyrrolidin-1-ylmethylene)pyrrolidin-1-ium hexafluorophosphate
(V), and 1-(piperidin-1-ylmethylene)piperidin-1-ium
hexafluorophosphate (V); wherein R.sup.2 is selected from H and
C1-C4 alkyl; wherein R.sup.3 is selected from H and C1-C4 alkyl; or
wherein R.sup.2 and R.sup.3 are covalently bonded and, together
with the intermediate atoms, comprise an optionally substituted
fused six-membered aryl or heteroaryl ring; and a pharmaceutically
acceptable carrier or diluent.
69. (canceled)
70. The composition of claim 68, further comprising a therapeutic
agent that can be used to treat a retrovirus.
71-74. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This Application claims the benefit of U.S. Provisional
Application No. 61/835,297, filed on Jun. 14, 2013, which is
incorporated herein by reference in its entirety.
BACKGROUND
[0003] The advent of highly active anti-retroviral therapy (HAART),
which involves the use of three or more antiretroviral drugs, has
led to a significant improvement in the care and survival of
patients infect with HIV-1. In patients not infected with resistant
strains of the virus, HAART typically results in a dramatic
decrease in viral load often from levels of 10,000-100,000 RNA
copies/mL of plasma to less than 50 copies/mL.
[0004] Given the dramatic effects of HAART, it was proposed that
complete elimination of the virus might be possible within 2 to 3
years. However, even after long-term suppression of viral
replication with HAART, the virus rapidly rebounds after therapy is
discontinued. A key contributor to viral rebound appears to be a
reservoir of latently infected cells, including CD4 memory T cells.
The half-life of the latently infected population is quite long,
and it is estimated that it would take over 60 years of HAART to
eliminate this population. Therefore, life-long HAART would be
required to control infection in patients
[0005] Retroviruses, including HIV-1, are RNA viruses that
replicate through a DNA intermediate and integrate very efficiently
into the genome of an infected cell, forming a provirus. Once the
provirus is formed, it is maintained in the genome of the infected
cell and transferred to daughter cells in the same fashion as any
other genetic element within the cellular genome. Thus, the virus
has the potential to persist if it infects long-lived cells such as
memory T cells. It has been known since 1986 that HIV-1 can
establish a latent infection in culture. It was found that a human
T cell line infected with replication-competent virus could develop
a latent infection in which the provirus was dormant but could be
reactivated upon stimulation. Since then it has been established
that a number of cytokines can reactivate latent proviruses.
[0006] The role that latency is playing in preventing clearance of
the virus infection has become evident in recent years. Patients
that had been successfully treated with HAART in which viral RNA
was maintained at levels below 50 copies/mL in the plasma for
years, experienced rapid virus rebound upon withdrawal of therapy.
Moreover, it was found that after T cell activation, virus could be
isolated from CD4 T cells taken from these patients making it clear
that to eradicate the virus it will be necessary to eliminate the
latently infected cells.
[0007] There have been attempts to flush the latent virus from
infected individuals by nonspecific activation of T cells to "turn
on" latent proviruses. As part of this approach, the patients
remain on HAART to prevent new infections, and the infected cells
from which the latent proviruses are activated should die due to
cytotoxic effects of viral expression and/or because of targeting
by the immune system which can recognize the cells once they begin
to express the viral proteins. Agents that can dissociate latent
virus activation from global T cell activation, however, are
extremely rare. Thus, there remains a need for new drugs capable of
selectively activating latent viruses.
SUMMARY
[0008] In accordance with the purpose(s) of the invention, as
embodied and broadly described herein, the invention, in one
aspect, relates to methods of activating a latent retrovirus in a
subject, the method comprising the step of administering to the
subject an effective amount of a compound represented by a
formula:
##STR00001##
wherein n is 0 or 1; wherein R.sup.1 is selected from H, C1-C4
alkyl, C1-C6 aryl, C(O)Ar, C(O)N(CH.sub.3).sub.2,
SO.sub.2N(CH.sub.3).sub.2, fluorenylmethyloxycarbonyl,
N-((dimethylamino)methylene)-N-methylmethanaminium
tetrafluoroborate,
N-((dimethylamino)methylene)-N-methylmethanaminium
hexafluorophosphate (V), tri(pyrrolidin-1-yl)phosphonium
hexafluorophosphate (V), tris(dimethylamino)phosphonium
hexafluorophosphate (V),
1-(pyrrolidin-1-ylmethylene)pyrrolidin-1-ium hexafluorophosphate
(V), and 1-(piperidin-1-ylmethylene)piperidin-1-ium
hexafluorophosphate (V); wherein R.sup.2 is selected from H and
C1-C4 alkyl; and wherein R.sup.3 is selected from H and C1-C4
alkyl, or R.sup.2 and R.sup.3 are covalently bonded and, together
with the intermediate atoms, comprise an optionally substituted
fused six-membered aryl or heteroaryl ring.
[0009] Also disclosed are methods for the manufacture of a
medicament for treatment of a retrovirus in a subject, the method
comprising the step of combining an effective amount of a compound
represented by a formula:
##STR00002##
wherein n is 0 or 1, wherein R.sup.1 is selected from H, C1-C4
alkyl, C1-C6 aryl, C(O)Ar, SO.sub.2N(CH.sub.3).sub.2,
fluorenylmethyloxycarbonyl,
N-((dimethylamino)methylene)-N-methylmethanaminium
tetrafluoroborate,
N-((dimethylamino)methylene)-N-methylmethanaminium
hexafluorophosphate (V), tri(pyrrolidin-1-yl)phosphonium
hexafluorophosphate (V), tris(dimethylamino)phosphonium
hexafluorophosphate (V),
1-(pyrrolidin-1-ylmethylene)pyrrolidin-1-ium hexafluorophosphate
(V), or 1-(piperidin-1-ylmethylene)piperidin-1-ium
hexafluorophosphate (V); wherein R.sup.2 is selected from H and
C1-C4 alkyl; wherein R.sup.3 is selected from H and C1-C4 alkyl, or
R.sup.2 and R.sup.3 are covalently bonded and, together with the
intermediate atoms, comprise an optionally substituted fused
six-membered aryl or heteroaryl ring; with a pharmaceutically
acceptable carrier or diluent.
[0010] Also disclosed are kits comprising at least one compound
represented by a formula:
##STR00003##
wherein n is 0 or 1; wherein R.sup.1 is selected from H, C1-C4
alkyl, C1-C6 aryl, C(O)Ar, C(O)N(CH.sub.3).sub.2,
SO.sub.2N(CH.sub.3).sub.2, fluorenylmethyloxycarbonyl,
N-((dimethylamino)methylene)-N-methylmethanaminium
tetrafluoroborate,
N-((dimethylamino)methylene)-N-methylmethanaminium
hexafluorophosphate (V), tri(pyrrolidin-1-yl)phosphonium
hexafluorophosphate (V), tris(dimethylamino)phosphonium
hexafluorophosphate (V),
1-(pyrrolidin-1-ylmethylene)pyrrolidin-1-ium hexafluorophosphate
(V), and 1-(piperidin-1-ylmethylene)piperidin-1-ium
hexafluorophosphate (V); wherein R.sup.2 is selected from H and
C1-C4 alkyl; and wherein R.sup.3 is selected from H and C1-C4
alkyl, or R.sup.2 and R.sup.3 are covalently bonded and, together
with the intermediate atoms, comprise an optionally substituted
fused six-membered aryl or heteroaryl ring; and one or more of
[0011] a) at least one agent known to treat a retrovirus; [0012] b)
instructions for detecting a retrovirus; and [0013] c) instructions
for treating a retrovirus.
[0014] Also disclosed are pharmaceutical compositions comprising a
compound represented by a formula:
##STR00004##
wherein n is 0 or 1; wherein R.sup.1 is selected from H, C1-C4
alkyl, C1-C6 aryl, C(O)Ar, SO.sub.2N(CH.sub.3).sub.2,
fluorenylmethyloxycarbonyl,
N-((dimethylamino)methylene)-N-methylmethanaminium
tetrafluoroborate,
N-((dimethylamino)methylene)-N-methylmethanaminium
hexafluorophosphate (V), tri(pyrrolidin-1-yl)phosphonium
hexafluorophosphate (V), tris(dimethylamino)phosphonium
hexafluorophosphate (V),
1-(pyrrolidin-1-ylmethylene)pyrrolidin-1-ium hexafluorophosphate
(V), and 1-(piperidin-1-ylmethylene)piperidin-1-ium
hexafluorophosphate (V); wherein R.sup.2 is selected from H and
C1-C4 alkyl; wherein R.sup.3 is selected from H and C1-C4 alkyl, or
R.sup.2 and R.sup.3 are covalently bonded and, together with the
intermediate atoms, comprise an optionally substituted fused
six-membered aryl or heteroaryl ring; and a pharmaceutically
acceptable carrier or diluent.
[0015] While aspects of the present invention can be described and
claimed in a particular statutory class, such as the system
statutory class, this is for convenience only and one of skill in
the art will understand that each aspect of the present invention
can be described and claimed in any statutory class. Unless
otherwise expressly stated, it is in no way intended that any
method or aspect set forth herein be construed as requiring that
its steps be performed in a specific order. Accordingly, where a
method claim does not specifically state in the claims or
descriptions that the steps are to be limited to a specific order,
it is no way intended that an order be inferred, in any respect.
This holds for any possible non-express basis for interpretation,
including matters of logic with respect to arrangement of steps or
operational flow, plain meaning derived from grammatical
organization or punctuation, or the number or type of aspects
described in the specification.
BRIEF DESCRIPTION OF THE FIGURES
[0016] The accompanying figures, which are incorporated in and
constitute a part of this specification, illustrate several aspects
and together with the description serve to explain the principles
of the invention.
[0017] FIG. 1A shows the results of a screen of the Ireland Natural
Product Collection, a set of natural products isolated from marine
invertebrate animals and microorganisms. Plate II well C07 was
identified as a positive result and was taken on for further
analysis.
[0018] FIG. 1B shows that Plate II well C07 activates latent HIV-1
as indicated by an increase in fluorescence compared to the
control.
[0019] FIG. 1C shows the % of positive cells and the % of
reactivation compared to the positive control for 20 fractions of
C07. The fractions were separated using HPLC with a gradient of
10-100% in acetonitrile with 0.1% formic acid. The activity of
CD3/CD28 is shown as a positive control. The ability of C07 (MNP-II
C07) to activate latent HIV-1 is also displayed.
[0020] FIG. 1D shows that the compound responsible for the activity
of C07 is located in fraction MS. The major component of this
fraction is 1-hydroxybenzotriazole (HOBt).
[0021] FIG. 1E shows the structure of HOBt.
[0022] FIG. 1F shows that HOBt reactivates HIV-1 latency in a dose
dependent manner.
[0023] FIG. 1G shows that HOBt is able to reactive HIV-1 latency
with slower kinetics compared to anti-CD3/CD28.
[0024] FIG. 2A shows the ability of HOBt and HOBt analogs to
activate latent HIV-1. In addition to HOBt, HOAt also activates
HIV-1 latent cells.
[0025] FIG. 2B shows that both HOBt and HOAt activate latent
HIV-1.
[0026] FIG. 2C shows that HOBt and HOAt reactivate HIV-1 latency in
a dose dependent manner.
[0027] FIG. 2D shows the ability of a panel of 7 derivatives of
HOBt to activate latent HIV-1. HOAt demonstrates increased activity
compared to HOBt. Two additional analogs, HATU and HO-DhBt, also
illustrate an increased ability to induce viral reactivation in a
dose dependent manner.
[0028] FIG. 3 shows that HOBt and HOAt synergize with several
.gamma.c-cytokines to reactivate HIV-1 latency. HOBt and HOAt
increase viral reactivation in the presence of IL-2, IL-7 and IL-5.
IL-4 did not induce viral reactivation by itself; however,
combining IL-4 with either HOBt or HOAt did induce significant
viral reactivation. IL-9 and IL-21 show no effect.
[0029] FIG. 4A shows that HOAt induces viral reactivation in a
STAT5 dependent manner. Inhibition of either NFAT or NF-.kappa.B
did not have an effect on viral reactivation by HOAt. Inhibition of
JAK selectively blocked viral reactivation by HOAt but not by
.alpha.CD3/.alpha.CD28. While the STAT3 inhibitor did not have a
significant effect, the STAT5 inhibitor was able to completely
prevent activation of latent HIV-1 by HOAt.
[0030] FIG. 4B shows that HOAt alone is able to induce an increase
in STAT5 phosphorylation over untreated cells. While cells
incubated with IL-2 alone increase the levels of STAT5
phosphorylation at 30 min, at 24 hrs the level has decreased to
basal levels. Incubation of cultured T.sub.CM cells with a
combination of IL-2 and HOAt lead to a drastic increase in STAT5
phosphorylation.
[0031] FIG. 5A shows that HOAt increases the nuclear localization
of STAT5. pSTAT5 and STAT5 levels increased in the nucleus of the
cells treated with HOAt at the time points indicated. Histone H3
and alpha Tubulin were used as controls for purity of the
fractionation.
[0032] FIG. 5B shows that IL-2 alone or in the presence of HOAt
increases phosphorylation of JAK1 and JAK3.
[0033] FIG. 5C shows that HOAt increases the transcription of
SOC3S, a target gene for STAT5.
[0034] FIG. 6A shows that HOBt does not induce general T cell
activation.
[0035] FIG. 6B shows that HOBt does not induce T cell
differentiation measured as lost CCR7 and CD27 expression.
[0036] FIG. 6C shows that HOBt only induces some degree of T cell
proliferation at the higher concentration tested.
[0037] FIG. 61D shows that HOBt only induces an increase of 1.4
times of CD25 levels at the maximum concentration used. This level
is increased by 37 times after TCR engagement with
.alpha.CD3/.alpha.CD28.
[0038] FIG. 6E shows that HOAt slightly increases the levels of T
cell activation when compared with .alpha.CD3/.alpha.CD28.
[0039] FIG. 7 shows that HOAt increases viral reactivation mediated
by PMA, ionomycin and SAHA. This indicates that HOAt and additional
analogs can be used to synergize the activity of other anti-latency
drugs.
[0040] FIG. 8 shows representative data pertaining to the ability
of HOAt to reactivate latent HIV-1 in cells isolated from aviremic
HIV-1 patients. Briefly, cells for two patients suppressed with
antiretroviral were isolated and treated with either DMSO
(Control), HOAt or the mitogen PHA. As it is shown, HOAt can induce
regrowth of latent viruses in both patients in vitro above the
treatment control (DMSO).
[0041] FIG. 9A shows representative data demonstrating that HOAt
increases the levels of pSTAT5 in the inputs. FIG. 9B shows
representative data illustrating the ladder pattern normally
observed in ubiquitinilated proteins. Without wishing to be bound
by theory, this may suggest that HOAt and derivatives may block
ubiquitation and subsequent degradation of pSTAT5. Briefly, cells
were treated with DMSO or HOAt for 30 min, and subsequently treated
with IL-2 for another 30 min. STAT5 was immunoprecipitated with
antibodies specific for the phosphorylated protein and western blot
was performed against pSTAT5.
[0042] FIG. 10 shows representative data pertaining to the ability
of HOBt, HOAt, HODHBt and HATU to maintain pSTAT5 levels in a dose
dependent manner. Cells were treated with IL-2 and several
derivatives of HOAt. Levels of pSTAT5 were measured 48 h later by
flow cytometer.
[0043] Additional advantages of the invention will be set forth in
part in the description which follows, and in part will be obvious
from the description, or can be learned by practice of the
invention. The advantages of the invention will be realized and
attained by means of the elements and combinations particularly
pointed out in the appended claims. It is to be understood that
both the foregoing general description and the following detailed
description are exemplary and explanatory only and are not
restrictive of the invention, as claimed.
DESCRIPTION
[0044] The present invention can be understood more readily by
reference to the following detailed description of the invention
and the Examples and Figures included herein.
[0045] Before the present compounds, compositions, articles,
systems, devices, and/or methods are disclosed and described, it is
to be understood that they are not limited to specific synthetic
methods unless otherwise specified, or to particular reagents
unless otherwise specified, as such may, of course, vary. It is
also to be understood that the terminology used herein is for the
purpose of describing particular aspects only and is not intended
to be limiting. Although any methods and materials similar or
equivalent to those described herein can be used in the practice or
testing of the present invention, example methods and materials are
now described.
[0046] While aspects of the present invention can be described and
claimed in a particular statutory class, such as the system
statutory class, this is for convenience only and one of skill in
the art will understand that each aspect of the present invention
can be described and claimed in any statutory class. Unless
otherwise expressly stated, it is in no way intended that any
method or aspect set forth herein be construed as requiring that
its steps be performed in a specific order. Accordingly, where a
method claim does not specifically state in the claims or
descriptions that the steps are to be limited to a specific order,
it is no way intended that an order be inferred, in any respect.
This holds for any possible non-express basis for interpretation,
including matters of logic with respect to arrangement of steps or
operational flow, plain meaning derived from grammatical
organization or punctuation, or the number or type of aspects
described in the specification.
[0047] Throughout this application, various publications are
referenced. The disclosures of these publications in their
entireties are hereby incorporated by reference into this
application in order to more fully describe the state of the art to
which this pertains. The references disclosed are also individually
and specifically incorporated by reference herein for the material
contained in them that is discussed in the sentence in which the
reference is relied upon. Nothing herein is to be construed as an
admission that the present invention is not entitled to antedate
such publication by virtue of prior invention. Further, the dates
of publication provided herein may be different from the actual
publication dates, which can require independent confirmation.
A. DEFINITIONS
[0048] As used herein, nomenclature for compounds, including
organic compounds, can be given using common names, IUPAC, IUBMB,
or CAS recommendations for nomenclature. When one or more
stereochemical features are present, Cahn-Ingold-Prelog rules for
stereochemistry can be employed to designate stereochemical
priority, E/Z specification, and the like. One of skill in the art
can readily ascertain the structure of a compound if given a name,
either by systemic reduction of the compound structure using naming
conventions, or by commercially available software, such as
CHEMDRAW.TM. (Cambridgesoft Corporation, U.S.A.).
[0049] As used in the specification and the appended claims, the
singular forms "a," "an" and "the" include plural referents unless
the context clearly dictates otherwise. Thus, for example,
reference to "a functional group," "an alkyl," or "a residue"
includes mixtures of two or more such functional groups, alkyls, or
residues, and the like.
[0050] Ranges can be expressed herein as from "about" one
particular value, and/or to "about" another particular value. When
such a range is expressed, a further aspect includes from the one
particular value and/or to the other particular value. Similarly,
when values are expressed as approximations, by use of the
antecedent "about," it will be understood that the particular value
forms a further aspect. It will be further understood that the
endpoints of each of the ranges are significant both in relation to
the other endpoint, and independently of the other endpoint. It is
also understood that there are a number of values disclosed herein,
and that each value is also herein disclosed as "about" that
particular value in addition to the value itself. For example, if
the value "10" is disclosed, then "about 10" is also disclosed. It
is also understood that each unit between two particular units are
also disclosed. For example, if 10 and 15 are disclosed, then 11,
12, 13, and 14 are also disclosed.
[0051] References in the specification and concluding claims to
parts by weight of a particular element or component in a
composition denotes the weight relationship between the element or
component and any other elements or components in the composition
or article for which a part by weight is expressed. Thus, in a
compound containing 2 parts by weight of component X and 5 parts by
weight component Y, X and Y are present at a weight ratio of 2:5,
and are present in such ratio regardless of whether additional
components are contained in the compound.
[0052] A weight percent (wt. %) of a component, unless specifically
stated to the contrary, is based on the total weight of the
formulation or composition in which the component is included.
[0053] As used herein, the terms "optional" or "optionally" means
that the subsequently described event or circumstance can or can
not occur, and that the description includes instances where said
event or circumstance occurs and instances where it does not.
[0054] As used herein, the term "subject" can be a vertebrate, such
as a mammal, a fish, a bird, a reptile, or an amphibian. Thus, the
subject of the herein disclosed methods can be a human, non-human
primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig
or rodent. The term does not denote a particular age or sex. Thus,
adult and newborn subjects, as well as fetuses, whether male or
female, are intended to be covered. In one aspect, the subject is a
mammal. A patient refers to a subject afflicted with a disease or
disorder. The term "patient" includes human and veterinary
subjects.
[0055] As used herein, the term "treatment" refers to the medical
management of a patient with the intent to cure, ameliorate,
stabilize, or prevent a disease, pathological condition, or
disorder. This term includes active treatment, that is, treatment
directed specifically toward the improvement of a disease,
pathological condition, or disorder, and also includes causal
treatment, that is, treatment directed toward removal of the cause
of the associated disease, pathological condition, or disorder. In
addition, this term includes palliative treatment, that is,
treatment designed for the relief of symptoms rather than the
curing of the disease, pathological condition, or disorder,
preventative treatment, that is, treatment directed to minimizing
or partially or completely inhibiting the development of the
associated disease, pathological condition, or disorder, and
supportive treatment, that is, treatment employed to supplement
another specific therapy directed toward the improvement of the
associated disease, pathological condition, or disorder. In various
aspects, the term covers any treatment of a subject, including a
mammal (e.g., a human), and includes: (i) preventing the disease
from occurring in a subject that can be predisposed to the disease
but has not yet been diagnosed as having it; (ii) inhibiting the
disease, i.e., arresting its development; or (iii) relieving the
disease, i.e., causing regression of the disease. In one aspect,
the subject is a mammal such as a primate, and, in a further
aspect, the subject is a human. The term "subject" also includes
domesticated animals (e.g., cats, dogs, etc.), livestock (e.g.,
cattle, horses, pigs, sheep, goats, etc.), and laboratory animals
(e.g., mouse, rabbit, rat, guinea pig, fruit fly, etc.).
[0056] As used herein, the term "prevent" or "preventing" refers to
precluding, averting, obviating, forestalling, stopping, or
hindering something from happening, especially by advance action.
It is understood that where reduce, inhibit or prevent are used
herein, unless specifically indicated otherwise, the use of the
other two words is also expressly disclosed.
[0057] As used herein, the term "diagnosed" means having been
subjected to a clinical interview and/or a physical examination by
a person of skill, for example, a physician, and found to have a
condition that can be diagnosed or treated by the compounds,
compositions, or methods disclosed herein. For example, "diagnosed
with a depression disorder" means having been subjected to a
clinical interview and/or physical examination by a person of
skill, for example, a physician, and found to have a condition that
can be diagnosed or treated by a compound or composition that can
cure, alleviate, prevent, or otherwise treat a depression
disorder.
[0058] As used herein, the terms "administering" and
"administration" refer to any method of providing a pharmaceutical
preparation to a subject. Such methods are well known to those
skilled in the art and include, but are not limited to, oral
administration, transdermal administration, administration by
inhalation, nasal administration, topical administration,
intravaginal administration, ophthalmic administration, intraaural
administration, intracerebral administration, rectal
administration, sublingual administration, buccal administration,
and parenteral administration, including injectable such as
intravenous administration, intra-arterial administration,
intramuscular administration, and subcutaneous administration.
Administration can be continuous or intermittent. In various
aspects, a preparation can be administered therapeutically; that
is, administered to treat an existing disease or condition. In
further various aspects, a preparation can be administered
prophylactically; that is, administered for prevention of a disease
or condition.
[0059] The term "contacting" as used herein refers to bringing a
disclosed compound and a cell, target receptor, or other biological
entity together in such a manner that the compound can affect the
activity of the target, either directly; i.e., by interacting with
the target itself, or indirectly; i.e., by interacting with another
molecule, co-factor, factor, or protein on which the activity of
the target is dependent.
[0060] As used herein, the term "effective amount" refers to an
amount that is sufficient to achieve the desired result or to have
an effect on an undesired condition. For example, a
"therapeutically effective amount" refers to an amount that is
sufficient to achieve the desired therapeutic result or to have an
effect on undesired symptoms, but is generally insufficient to
cause adverse side affects. The specific therapeutically effective
dose level for any particular patient will depend upon a variety of
factors including the disorder being treated and the severity of
the disorder, the specific composition employed; the age, body
weight, general health, sex and diet of the patient; the time of
administration; the route of administration; the rate of excretion
of the specific compound employed; the duration of the treatment;
drugs used in combination or coincidental with the specific
compound employed and like factors well known in the medical arts.
For example, it is well within the skill of the art to start doses
of a compound at levels lower than those required to achieve the
desired therapeutic effect and to gradually increase the dosage
until the desired effect is achieved. If desired, the effective
daily dose can be divided into multiple doses for purposes of
administration. Consequently, single dose compositions can contain
such amounts or submultiples thereof to make up the daily dose. The
dosage can be adjusted by the individual physician in the event of
any contraindications. Dosage can vary, and can be administered in
one or more dose administrations daily, for one or several days.
Guidance can be found in the literature for appropriate dosages for
given classes of pharmaceutical products. In further various
aspects, a preparation can be administered in a "prophylactically
effective amount"; that is, an amount effective for prevention of a
disease or condition.
[0061] The term "pharmaceutically acceptable" describes a material
that is not biologically or otherwise undesirable, i.e, without
causing an unacceptable level of undesirable biological effects or
interacting in a deleterious manner.
[0062] As used herein, the term "derivative" refers to a compound
having a structure derived from the structure of a parent compound
(e.g., a compound disclosed herein) and whose structure is
sufficiently similar to those disclosed herein and based upon that
similarity, would be expected by one skilled in the art to exhibit
the same or similar activities and utilities as the claimed
compounds, or to induce, as a precursor, the same or similar
activities and utilities as the claimed compounds. Exemplary
derivatives include salts, esters, amides, salts of esters or
amides, and N-oxides of a parent compound.
[0063] As used herein, the term "pharmaceutically acceptable
carrier" refers to sterile aqueous or nonaqueous solutions,
dispersions, suspensions or emulsions, as well as sterile powders
for reconstitution into sterile injectable solutions or dispersions
just prior to use. Examples of suitable aqueous and nonaqueous
carriers, diluents, solvents or vehicles include water, ethanol,
polyols (such as glycerol, propylene glycol, polyethylene glycol
and the like), carboxymethylcellulose and suitable mixtures
thereof, vegetable oils (such as olive oil) and injectable organic
esters such as ethyl oleate. Proper fluidity can be maintained, for
example, by the use of coating materials such as lecithin, by the
maintenance of the required particle size in the case of
dispersions and by the use of surfactants. These compositions can
also contain adjuvants such as preservatives, wetting agents,
emulsifying agents and dispersing agents. Prevention of the action
of microorganisms can be ensured by the inclusion of various
antibacterial and antifungal agents such as paraben, chlorobutanol,
phenol, sorbic acid and the like. It can also be desirable to
include isotonic agents such as sugars, sodium chloride and the
like. Prolonged absorption of the injectable pharmaceutical form
can be brought about by the inclusion of agents, such as aluminum
monostearate and gelatin, which delay absorption. Injectable depot
forms are made by forming microencapsule matrices of the drug in
biodegradable polymers such as polylactide-polyglycolide,
poly(orthoesters) and poly(anhydrides). Depending upon the ratio of
drug to polymer and the nature of the particular polymer employed,
the rate of drug release can be controlled. Depot injectable
formulations are also prepared by entrapping the drug in liposomes
or microemulsions which are compatible with body tissues. The
injectable formulations can be sterilized, for example, by
filtration through a bacterial-retaining filter or by incorporating
sterilizing agents in the form of sterile solid compositions which
can be dissolved or dispersed in sterile water or other sterile
injectable media just prior to use. Suitable inert carriers can
include sugars such as lactose. Desirably, at least 95% by weight
of the particles of the active ingredient have an effective
particle size in the range of 0.01 to 10 micrometers.
[0064] The term "stable," as used herein, refers to compounds that
are not substantially altered when subjected to conditions to allow
for their production, detection, and, in certain aspects, their
recovery, purification, and use for one or more of the purposes
disclosed herein.
[0065] By "virlon," "viral particle," or "retroviral particle" is
meant a single virus minimally composed of an RNA or DNA genome,
Pol protein (for reverse transcription of the RNA genome following
infection), Gag protein (structural protein present in the
nucleocapsid), and an envelope protein. As used herein, the RNA
genome of the retroviral particle is usually a recombinant RNA
genome, e.g., contains an RNA sequence exogenous to the native
retroviral genome and/or is defective in an andogenous retroviral
sequence (e.g., is defective in pol, gag, and/or env, and, as used
herein, is normally defective in all three genes).
[0066] Compounds described herein comprise atoms in both their
natural isotopic abundance and in non-natural abundance. The
disclosed compounds can be isotopically-labeled or
isotopically-substituted compounds identical to those described,
but for the fact that one or more atoms are replaced by an atom
having an atomic mass or mass number different from the atomic mass
or mass number typically found in nature. Examples of isotopes that
can be incorporated into compounds of the invention include
isotopes of hydrogen, carbon, nitrogen, oxygen, such as .sup.2H,
.sup.3H, .sup.13C, .sup.14C, .sup.15N, .sup.18O, .sup.17O,
respectively. Compounds further comprise prodrugs thereof and
pharmaceutically acceptable salts of said compounds or of said
prodrugs which contain the aforementioned isotopes and/or other
isotopes of other atoms are within the scope of this invention.
Certain isotopically-labeled compounds of the present invention,
for example those into which radioactive isotopes such as .sup.3H
and .sup.14C are incorporated, are useful in drug and/or substrate
tissue distribution assays. Tritiated, i.e., .sup.3H, and
carbon-14, i.e., .sup.14C, isotopes are particularly preferred for
their ease of preparation and detectability. Further, substitution
with heavier isotopes such as deuterium, i.e., .sup.2H, can afford
certain therapeutic advantages resulting from greater metabolic
stability, for example increased in vivo half-life or reduced
dosage requirements and, hence, may be preferred in some
circumstances. Isotopically labeled compounds of the present
invention and prodrugs thereof can generally be prepared by
carrying out the procedures below, by substituting a readily
available isotopically labeled reagent for a non-isotopically
labeled reagent.
[0067] The compounds described in the invention can be present as a
solvate. In some cases, the solvent used to prepare the solvate is
an aqueous solution, and the solvate is then often referred to as a
hydrate. The compounds can be present as a hydrate, which can be
obtained, for example, by crystallization from a solvent or from
aqueous solution. In this connection, one, two, three or any
arbitrary number of solvate or water molecules can combine with the
compounds according to the invention to form solvates and hydrates.
Unless stated to the contrary, the invention includes all such
possible solvates.
[0068] The term "co-crystal" means a physical association of two or
more molecules which owe their stability through non-covalent
interaction. One or more components of this molecular complex
provide a stable framework in the crystalline lattice. In certain
instances, the guest molecules are incorporated in the crystalline
lattice as anhydrates or solvates, see e.g. "Crystal Engineering of
the Composition of Pharmaceutical Phases. Do Pharmaceutical
Co-crystals Represent a New Path to Improved Medicines?"
Almarasson, O., et. al., The Royal Society of Chemistry, 1889-1896,
2004. Examples of co-crystals include p-toluenesulfonic acid and
benzenesulfonic acid.
[0069] It is known that chemical substances form solids which are
present in different states of order which are termed polymorphic
forms or modifications. The different modifications of a
polymorphic substance can differ greatly in their physical
properties. The compounds according to the invention can be present
in different polymorphic forms, with it being possible for
particular modifications to be metastable. Unless stated to the
contrary, the invention includes all such possible polymorphic
forms.
[0070] Certain materials, compounds, compositions, and components
disclosed herein can be obtained commercially or readily
synthesized using techniques generally known to those of skill in
the art. For example, the starting materials and reagents used in
preparing the disclosed compounds and compositions are either
available from commercial suppliers such as Aldrich Chemical Co.,
(Milwaukee, Wis.), Acros Organics (Morris Plains, N.J.), Fisher
Scientific (Pittsburgh, Pa.), or Sigma (St. Louis, Mo.) or are
prepared by methods known to those skilled in the art following
procedures set forth in references such as Fieser and Fieser's
Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons,
1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and
Supplementals (Elsevier Science Publishers, 1989); Organic
Reactions, Volumes 1-40 (John Wiley and Sons, 1991); March's
Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition); and
Larock's Comprehensive Organic Transformations (VCH Publishers Inc,
1989).
[0071] Unless otherwise expressly stated, it is in no way intended
that any method set forth herein be construed as requiring that its
steps be performed in a specific order. Accordingly, where a method
claim does not actually recite an order to be followed by its steps
or it is not otherwise specifically stated in the claims or
descriptions that the steps are to be limited to a specific order,
it is no way intended that an order be inferred, in any respect.
This holds for any possible non-express basis for interpretation,
including: matters of logic with respect to arrangement of steps or
operational flow; plain meaning derived from grammatical
organization or punctuation; and the number or type of embodiments
described in the specification.
[0072] Disclosed are the components to be used to prepare the
compositions of the invention as well as the compositions
themselves to be used within the methods disclosed herein. These
and other materials are disclosed herein, and it is understood that
when combinations, subsets, interactions, groups, etc. of these
materials are disclosed that while specific reference of each
various individual and collective combinations and permutation of
these compounds can not be explicitly disclosed, each is
specifically contemplated and described herein. For example, if a
particular compound is disclosed and discussed and a number of
modifications that can be made to a number of molecules including
the compounds are discussed, specifically contemplated is each and
every combination and permutation of the compound and the
modifications that are possible unless specifically indicated to
the contrary. Thus, if a class of molecules A, B, and C are
disclosed as well as a class of molecules D, E, and F and an
example of a combination molecule, A-D is disclosed, then even if
each is not individually recited each is individually and
collectively contemplated meaning combinations, A-E, A-F, B-D, B-E,
B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any
subset or combination of these is also disclosed. Thus, for
example, the sub-group of A-E, B-F, and C-E would be considered
disclosed. This concept applies to all aspects of this application
including, but not limited to, steps in methods of making and using
the compositions of the invention. Thus, if there are a variety of
additional steps that can be performed it is understood that each
of these additional steps can be performed with any specific
embodiment or combination of embodiments of the methods of the
invention.
[0073] It is understood that the compositions disclosed herein have
certain functions. Disclosed herein are certain structural
requirements for performing the disclosed functions and it is
understood that there are a variety of structures that can perform
the same function that are related to the disclosed structures, and
that these structures will typically achieve the same result.
B. PHARMACEUTICAL COMPOSITIONS
[0074] In one aspect, the invention relates to pharmaceutical
compositions comprising a compound represented by a formula:
##STR00005##
wherein n is 0 or 1; wherein R.sup.1 is selected from H, C1-C4
alkyl, C1-C6 aryl, C(O)Ar, SO.sub.2N(CH.sub.3).sub.2,
fluorenylmethyloxycarbonyl,
N-((dimethylamino)methylene)-N-methylmethanaminium
tetrafluoroborate,
N-((dimethylamino)methylene)-N-methylmethanaminium
hexafluorophosphate (V), tri(pyrrolidin-1-yl)phosphonium
hexafluorophosphate (V), tris(dimethylamino)phosphonium
hexafluorophosphate (V),
1-(pyrrolidin-1-ylmethylene)pyrrolidin-1-ium hexafluorophosphate
(V), and 1-(piperidin-1-ylmethylene)piperidin-1-ium
hexafluorophosphate (V); wherein R.sup.2 is selected from H and
C1-C4 alkyl; wherein R.sup.3 is selected from H and C1-C4 alkyl, or
R.sup.2 and R.sup.3 are covalently bonded and, together with the
intermediate atoms, comprise an optionally substituted fused
six-membered aryl or heteroaryl ring, and a pharmaceutically
acceptable carrier or diluent.
[0075] In certain aspects, the disclosed pharmaceutical
compositions comprise the disclosed compounds and pharmaceutically
acceptable salt(s) thereof as an active ingredient, a
pharmaceutically acceptable carrier, and, optionally, other
therapeutic ingredients or adjuvants. The instant compositions
include those suitable for oral, rectal, topical, and parenteral
(including subcutaneous, intramuscular, and intravenous)
administration, although the most suitable route in any given case
will depend on the particular host, and nature and severity of the
conditions for which the active ingredient is being administered.
The pharmaceutical compositions can be conveniently presented in
unit dosage form and prepared by any of the methods well known in
the art of pharmacy.
[0076] As used herein, the term "pharmaceutically acceptable salts"
refers to salts prepared from pharmaceutically acceptable non-toxic
bases or acids. When the compound of the present invention is
acidic, its corresponding salt can be conveniently prepared from
pharmaceutically acceptable non-toxic bases, including inorganic
bases and organic bases. Salts derived from such Inorganic bases
include aluminum, ammonium, calcium, copper (-ic and -ous), ferric,
ferrous, lithium, magnesium, manganese (-ic and -ous), potassium,
sodium, zinc and the like salts. Particularly preferred are the
ammonium, calcium, magnesium, potassium and sodium salts. Salts
derived from pharmaceutically acceptable organic non-toxic bases
include salts of primary, secondary, and tertiary amines, as well
as cyclic amines and substituted amines such as naturally occurring
and synthesized substituted amines. Other pharmaceutically
acceptable organic non-toxic bases from which salts can be formed
Include ion exchange resins such as, for example, arginine,
betaine, caffeine, choline, N,N'-dibenzylethylenediamine,
diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol,
ethanolamine, ethylenediamine, N-ethylmorpholine,
N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine,
isopropylamine, lysine, methylglucamine, morpholine, piperazine,
piperidine, polyamine resins, procaine, purines, theobromine,
triethylamine, trimethylamine, tripropylamine, tromethamine and the
like.
[0077] As used herein, the term "pharmaceutically acceptable
non-toxic acids," includes inorganic acids, organic acids, and
salts prepared therefrom, for example, acetic, benzenesulfonic,
benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric,
gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic,
maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic,
pantothenic, phosphoric, succinic, sulfuric, tartaric,
p-toluenesulfonic acid and the like. Preferred are citric,
hydrobromic, hydrochloric, maleic, phosphoric, sulfuric, and
tartaric acids.
[0078] In practice, the compounds of the invention, or
pharmaceutically acceptable salts thereof of this invention can be
combined as the active ingredient in intimate admixture with a
pharmaceutical carrier according to conventional pharmaceutical
compounding techniques. The carrier can take a wide variety of
forms depending on the form of preparation desired for
administration, e.g., oral or parenteral (including intravenous).
Thus, the pharmaceutical compositions of the present invention can
be presented as discrete units suitable for oral administration
such as capsules, cachets or tablets each containing a
predetermined amount of the active ingredient. Further, the
compositions can be presented as a powder, as granules, as a
solution, as a suspension in an aqueous liquid, as a non-aqueous
liquid, as an oil-in-water emulsion or as a water-in-oil liquid
emulsion. In addition to the common dosage forms set out above, the
compounds of the invention, and/or pharmaceutically acceptable
salt(s) thereof can also be administered by controlled release
means and/or delivery devices. The compositions can be prepared by
any of the methods of pharmacy. In general, such methods include a
step of bringing into association the active ingredient with the
carrier that constitutes one or more necessary ingredients. In
general, the compositions are prepared by uniformly and intimately
admixing the active ingredient with liquid carriers or finely
divided solid carriers or both. The product can then be
conveniently shaped into the desired presentation.
[0079] Thus, the pharmaceutical compositions of this invention can
include a pharmaceutically acceptable carrier and a compound or a
pharmaceutically acceptable salt of the compounds of the invention.
The compounds of the invention, or pharmaceutically acceptable
salts thereof; can also be included in pharmaceutical compositions
in combination with one or more other therapeutically active
compounds.
[0080] The pharmaceutical carrier employed can be, for example, a
solid, liquid, or gas. Examples of solid carriers include lactose,
terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium
stearate, and stearic acid. Examples of liquid carriers are sugar
syrup, peanut oil, olive oil, and water. Examples of gaseous
carriers include carbon dioxide and nitrogen.
[0081] In preparing the compositions for oral dosage form, any
convenient pharmaceutical media can be employed. For example,
water, glycols, oils, alcohols, flavoring agents, preservatives,
coloring agents and the like can be used to form oral liquid
preparations such as suspensions, elixirs and solutions; while
carriers such as starches, sugars, microcrystalline cellulose,
diluents, granulating agents, lubricants, binders, disintegrating
agents, and the like can be used to form oral solid preparations
such as powders, capsules and tablets. Because of their ease of
administration, tablets and capsules are the preferred oral dosage
units whereby solid pharmaceutical carriers are employed.
Optionally, tablets can be coated by standard aqueous or nonaqueous
techniques
[0082] A tablet containing the composition of this invention can be
prepared by compression or molding, optionally with one or more
accessory ingredients or adjuvants. Compressed tablets can be
prepared by compressing, in a suitable machine, the active
ingredient in a free-flowing form such as powder or granules,
optionally mixed with a binder, lubricant, inert diluent, surface
active or dispersing agent. Molded tablets can be made by molding
in a suitable machine, a mixture of the powdered compound moistened
with an inert liquid diluent.
[0083] The pharmaceutical compositions of the present invention
comprise a compound of the invention (or pharmaceutically
acceptable salts thereof) as an active ingredient, a
pharmaceutically acceptable carrier, and optionally one or more
additional therapeutic agents or adjuvants. The instant
compositions include compositions suitable for oral, rectal,
topical, and parenteral (including subcutaneous, intramuscular, and
intravenous) administration, although the most suitable route in
any given case will depend on the particular host, and nature and
severity of the conditions for which the active ingredient is being
administered. The pharmaceutical compositions can be conveniently
presented in unit dosage form and prepared by any of the methods
well known in the art of pharmacy.
[0084] Pharmaceutical compositions of the present invention
suitable for parenteral administration can be prepared as solutions
or suspensions of the active compounds in water. A suitable
surfactant can be included such as, for example,
hydroxypropylcellulose. Dispersions can also be prepared in
glycerol, liquid polyethylene glycols, and mixtures thereof in
oils. Further, a preservative can be included to prevent the
detrimental growth of microorganisms.
[0085] Pharmaceutical compositions of the present invention
suitable for injectable use include sterile aqueous solutions or
dispersions. Furthermore, the compositions can be in the form of
sterile powders for the extemporaneous preparation of such sterile
injectable solutions or dispersions. In all cases, the final
injectable form must be sterile and must be effectively fluid for
easy syringability. The pharmaceutical compositions must be stable
under the conditions of manufacture and storage; thus, preferably
should be preserved against the contaminating action of
microorganisms such as bacteria and fungi. The carrier can be a
solvent or dispersion medium containing, for example, water,
ethanol, polyol (e.g., glycerol, propylene glycol and liquid
polyethylene glycol), vegetable oils, and suitable mixtures
thereof.
[0086] Pharmaceutical compositions of the present invention can be
in a form suitable for topical use such as, for example, an
aerosol, cream, ointment, lotion, dusting powder, mouth washes,
gargles, and the like. Further, the compositions can be in a form
suitable for use in transdermal devices. These formulations can be
prepared, utilizing a compound of the invention, or
pharmaceutically acceptable salts thereof via conventional
processing methods. As an example, a cream or ointment is prepared
by mixing hydrophilic material and water, together with about 5 wt
% to about 10 wt % of the compound, to produce a cream or ointment
having a desired consistency.
[0087] Pharmaceutical compositions of this invention can be in a
form suitable for rectal administration wherein the carrier is a
solid. It is preferable that the mixture forms unit dose
suppositories. Suitable carriers include cocoa butter and other
materials commonly used in the art. The suppositories can be
conveniently formed by first admixing the composition with the
softened or melted carrier(s) followed by chilling and shaping in
moulds.
[0088] In addition to the aforementioned carrier ingredients, the
pharmaceutical formulations described above can include, as
appropriate, one or more additional carrier ingredients such as
diluents, buffers, flavoring agents, binders, surface-active
agents, thickeners, lubricants, preservatives (including
anti-oxidants) and the like. Furthermore, other adjuvants can be
included to render the formulation isotonic with the blood of the
intended recipient. Compositions containing a compound of the
invention, and/or pharmaceutically acceptable salts thereof, can
also be prepared in powder or liquid concentrate form.
[0089] It is understood that the disclosed compositions can be
prepared from the disclosed compounds. It is also understood that
the disclosed compositions can be employed in the disclosed methods
of using.
[0090] In a further aspect, the disclosed compositions are for oral
administration.
[0091] In a further aspect, the disclosed compositions further
comprise a therapeutic agent that can be used to treat a
retrovirus. In a still further aspect, the therapeutic agent can be
used to treat a retrovirus selected from the group comprising
HIV-1, HIV-2, SIV, XMRV, HTLV-1, HTLV-2, HTLV-3, or HTLV-4. In yet
a further aspect, the therapeutic agent can be used to treat HIV-1.
In an even further aspect, the therapeutic agent is selected from
entry inhibitors, nucleoside reverse transcriptase inhibitors
(NRTIs), nucleotide reverse transcriptase inhibitors (NtRTIs),
non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease
inhibitors, integrase inhibitors, or maturation inhibitors, or a
mixture thereof. In a still further aspect, the therapeutic agent
is selected from nucleoside reverse transcriptase inhibitors
(NRTIs), nucleotide reverse transcriptase inhibitors (NtRTIs),
non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease
inhibitors, or integrase inhibitors, or a mixture thereof.
C. METHODS OF USING THE COMPOUNDS AND COMPOSITIONS
[0092] The compounds disclosed herein are useful for treating or
detecting a retrovirus, and more particularly for treating or
detecting HIV-1. Thus, provided is a method of treating or
detecting a retrovirus in a subject comprising the step of
administering to the subject at least one compound represented by a
formula:
##STR00006##
wherein n is 0 or 1; wherein R.sup.1 is selected from H, C1-C4
alkyl, C1-C6 aryl, C(O)Ar, C(O)N(CH.sub.3).sub.2,
SO.sub.2N(CH.sub.3).sub.2, fluorenylmethyloxycarbonyl,
N-((dimethylamino)methylene)-N-methylmethanaminium
tetrafluoroborate,
N-((dimethylamino)methylene)-N-methylmethanaminium
hexafluorophosphate (V), tri(pyrrolidin-1-yl)phosphonium
hexafluorophosphate (V), tris(dimethylamino)phosphonium
hexafluorophosphate (V),
1-(pyrrolidin-1-ylmethylene)pyrrolidin-1-ium hexafluorophosphate
(V), and 1-(piperidin-1-ylmethylene)piperidin-1-ium
hexafluorophosphate (V); wherein R.sup.2 is selected from H and
C1-C4 alkyl; and wherein R.sup.3 is selected from H and C1-C4
alkyl, or R.sup.2 and R.sup.3 are covalently bonded and, together
with the intermediate atoms, comprise an optionally substituted
fused six-membered aryl or heteroaryl ring; and/or at least one
disclosed pharmaceutical composition in an effective dosage and
amount.
[0093] The pharmaceutical compositions and methods of the present
invention can further comprise other therapeutically active
compounds as noted herein which are usually applied in the
treatment of retroviruses, for example HIV-1, HIV-2, SIV, XMRV,
HTLV-1, HTLV-2, HTLV-3, or HTLV-4.
[0094] In various aspects, the disclosed treatment methods can be
applied to a subject, for example, a patient. In further aspects,
the subject is a mammal, for example, a human.
[0095] 1. Treatment Methods
[0096] In one aspect, the invention relates to methods for the
treatment of a patient diagnosed with a retrovirus, the method
comprising the step of administering to the patient, together in an
effective amount, a compound represented by a formula:
##STR00007##
wherein 0 is 1; wherein R.sup.1 is selected from H, C1-C4 alkyl,
C1-C6 aryl, C(O)Ar, C(O)N(CH.sub.3).sub.2,
SO.sub.2N(CH.sub.3).sub.2, fluorenylmethyloxycarbonyl,
N-((dimethylamino)methylene)-N-methylmethanaminium
tetrafluoroborate,
N-((dimethylamino)methylene)-N-methylmethanaminium
hexafluorophosphate (V), tri(pyrrolidin-1-yl)phosphonium
hexafluorophosphate (V), tris(dimethylamino)phosphonium
hexafluorophosphate (V),
1-(pyrrolidin-1-ylmethylene)pyrrolidin-1-ium hexafluorophosphate
(V), and 1-(piperidin-1-ylmethylene)piperidin-1-ium
hexafluorophosphate (V); wherein R.sup.2 is selected from H and
C1-C4 alkyl; and wherein R.sup.3 is selected from H and C1-C4
alkyl, or R.sup.2 and R.sup.3 are covalently bonded and, together
with the intermediate atoms, comprise an optionally substituted
fused six-membered aryl or heteroaryl ring, and at least one agent
known to treat a retrovirus.
[0097] a. Activation of a Latent Retrovirus
[0098] In one aspect, the invention relates to methods of
activating a latent retrovirus, and more specifically HIV-1, in a
subject, the method comprising the step of administering to the
subject an effective amount of a compound represented by a
formula:
##STR00008##
wherein n is 0 or 1; wherein R is selected from H, C1-C4 alkyl,
C1-C6 aryl, C(O)Ar, C(O)N(CH.sub.3).sub.2,
SO.sub.2N(CH.sub.3).sub.2, fluorenylmethyloxycarbonyl,
N-((dimethylamino)methylene)-N-methylmethanaminium
tetrafluoroborate,
N-((dimethylamino)methylene)-N-methylmethanaminium
hexafluorophosphate (V), tri(pyrrolidin-1-yl)phosphonium
hexafluorophosphate (V), tris(dimethylamino)phosphonium
hexafluorophosphate (V),
1-(pyrrolidin-1-ylmethylene)pyrrolidin-1-ium hexafluorophosphate
(V), and 1-(piperidin-1-ylmethylene)piperidin-1-ium
hexafluorophosphate (V); wherein R.sup.2 is selected from H and
C1-C4 alkyl; and wherein R.sup.3 is selected from H and C1-C4
alkyl, or R.sup.2 and R.sup.3 are covalently bonded and, together
with the intermediate atoms, comprise an optionally substituted
fused six-membered aryl or heteroaryl ring.
[0099] The use of antiretroviral therapy in human immunodeficiency
virus type 1 (HIV-1) infected patients does not lead to virus
eradication. This is due, to a significant degree, to the fact that
HIV-1 can establish a highly stable reservoir of latently infected
cells. The principle reservoir of HIV-1 latency is thought to
reside in resting, CD4+ memory T cells, which harbor integrated
HIV-1 (Finzi et al. 1997). The low frequency of latently infected
cells (1 in 10.sup.6 resting CD4+ T cells (Chun et al. 1997)), for
which known phenotypic markers are not available, poses a great
challenge to the study of latency in vive.
[0100] Previous studies on HIV-1 latency were based on the
generation of chronically infected cell lines, such as the ACH2
(Folks at al. 1989), J.DELTA.K (Antoni at al. 1994), and J-Lat
(Jordan et al. 2003) T-cell lines, and the U1 promonocytic cell
line (Folks at al. 1987). In these systems, latency was defined as
a state in which integrated proviruses failed to drive efficient
gene expression. However, these systems do not necessarily reflect
the latency state in vivo because the lack of viral gene expression
is due to mutations in that (ACH2 and U1 (Folks at al. 1989, Folks
at al. 1987)) or mutations in the LTR (J.DELTA.K T-cell line
(Antoni at al. 1994)). While these latency models recapitulate a
plethora of mechanisms that can underlie viral latency, the focus
of this study was in developing a more general model that did not
rely on clonal probiral integration sites, and which utilized
non-transformed, primary human T-cells.
[0101] A model using human fetal liver tissue in SCID-hu mice has
also generated a great deal of interest in the field of HIV-1
latency (Brooks at al. 2001). This model relies upon infection of
thymocytes and the vast majority of latently infected cells in this
system are mature, quiescent CD4+ single positive naive T cells.
This is in contrast with findings in HIV-1 patients, where the
majority of latently infected cells are CD4+ memory T cells (Finzi
at al. 1997). Although naive and memory cells share the
characteristic of being quiescent, a likely requirement for HIV-1
latency in T cells (Finzi at al. 1997), there are important
differences between these cell types that impact latency and
reactivation.
[0102] More recently, a model using human, primary cells was
disclosed in which relevant signaling pathways involved in viral
reactivation are dissected from latently infected memory CD4+ cells
(Planelles et al. 2010). Replacement of the nef gene with one
encoding the green fluorescent protein (GFP) and using GFP as a
readout allows for this primary cell model for the study of HIV-1
latency to be suitable for high throughput screening (HTS) of
compounds that reactivate HIV-1 from its latency state. Disclosed
herein are methods of activating a latent retrovirus, and more
particularly latent HIV-1, using compounds identified in this
screen.
[0103] In a further aspect, the invention relates to a method of
activating a latent retrovirus in a subject, the method comprising
the step of administering to the subject an effective amount of a
compound represented by a formula*
##STR00009##
wherein n is 0 or 1; wherein R.sup.1 is selected from H,
N-((dimethylamino)methylene)-N-methylmethanaminium
tetrafluoroborate, and
N-((dimethylamino)methylene)-N-methylmethanaminium
hexafluorophosphate (V); wherein R.sup.2 is selected from H and
C1-C4 alkyl; and wherein R.sup.3 is selected from H and C1-C4
alkyl, or R.sup.2 and R.sup.3 are covalently bonded and, together
with the intermediate atoms, comprise an optionally substituted
fused six-membered aryl or heteroaryl ring.
[0104] In a further aspect, the invention relates to a method of
activating a latent retrovirus in a subject, the method comprising
the step of administering to the subject an effective amount of a
compound represented by a structure depicted in Table 1.
TABLE-US-00001 TABLE 1 Compound Name Structure
1-hydroxybenzotriazole ##STR00010##
6-chloro-1H-benzo[d][1,2,3]triazol-1-ol ##STR00011##
1-hydroxy-7-azabenzotriazole ##STR00012##
4,5-dichloro-1-(cyclopentyloxy)-1H-1,2,3- benzotriazole
##STR00013## 1-(benzoyloxy)-1H-1,2,3-benzotriazole ##STR00014##
9-fluorenylmethyl 1-benzotriazolyl carbonate ##STR00015##
1-{[(dimethylamino)carbonyl]oxy}-6-nitro- 1H-1,2,3-benzotriazole
##STR00016## 1-{[(dimethylamino)sulfonyl]oxy}-6-methyl-
1H-1,2,3-benzotriazole ##STR00017##
1-{[(dimethylamino)sulfonyl]oxy}-5-methyl- 1H-1,2,3-benzotriazole
##STR00018## O-(benzotriazole-1-yl)-N,N,N',N'- tetramethyluronium
tetrafluoroborate ##STR00019##
O-(6-chlorobenzotriazol-1-yl)-N,N,N',N'- tetramethyluronium
tetrafluoroborate ##STR00020## (benzotriazole-1-
yloxy)dipiperidineocarbenium hexafluorophosphate ##STR00021##
O-(benzotriazole-1-yl)-N,N,N',N'- tetramethyluronium
hexafluorophosphate ##STR00022##
O-(6-chlorobenzotriazol-1-yl)-N,N,N',N'- hexafluorophosphate
##STR00023## (benzotriazol-1- yloxy)tripyrrolidinophosphonium
hexafluorophosphate ##STR00024## O-(benzotriazole-1-yl-N,N,N',N'-
bis(pentamethylene)uranium hexafluorophosphate ##STR00025##
(benzotriazole-1- yloxy)tris(dimethylamino)phosphonium
hexafluorophosphate ##STR00026## (benzotriazole-1-
yloxy)tripyrrolidinophosphonium hexafluorophosphate ##STR00027##
(6-chlorobenzotriazol-1- yloxy)tripyrrolidiniophosphonium
hexafluorophosphate ##STR00028## (7-azabenzotriazol-1-
yloxy)tripyrrolidinophosphonium hexafluorophosphate ##STR00029##
3-hydroxybenzo[d][1,2,3]triazin-4-(3H)-one ##STR00030##
[0105] In a further aspect, the invention relates to a method of
activating a latent retrovirus in a subject, the method comprising
the step of administering to the subject an effective amount of a
compound represented by a formula:
##STR00031##
wherein n is 0 or 1; wherein R.sup.1 is selected from H, C1-C4
alkyl, C1-C6 aryl, C(O)Ar, C(O)N(CH.sub.3).sub.2,
SO.sub.2N(CH.sub.3).sub.2, fluorenylmethyloxycarbonyl,
N-((dimethylamino)methylene)-N-methylmethanaminium
tetrafluoroborate,
N-((dimethylamino)methylene)-N-methylmethanaminium
hexafluorophosphate (V), tri(pyrrolidin-1-yl)phosphonium
hexafluorophosphate (V), tris(dimethylamino)phosphonium
hexafluorophosphate (V),
1-(pyrrolidin-1-ylmethylene)pyrrolidin-1-ium hexafluorophosphate
(V), and 1-(piperidin-1-ylmethylene)piperidin-1-ium
hexafluorophosphate (V); wherein R.sup.2 and R.sup.3 are covalently
bonded and, together with the intermediate atoms, comprise an
optionally substituted fused six-membered aryl or heteroaryl ring.
In a still further aspect, R.sup.2 and R.sup.3 are covalently
bonded and, together with the intermediate atoms, comprise an
optionally substituted fused six-membered aryl ring. In yet a
further aspect, R.sup.2 and R.sup.3 are covalently bonded and,
together with the intermediate atoms, comprise an optionally
substituted fused phenyl ring. In an even further aspect, R.sup.2
and R.sup.3 are covalently bonded and, together with the
intermediate atoms, comprise an optionally substituted fused
six-membered heteroaryl ring. In a still further aspect, R.sup.2
and R.sup.3 are covalently bonded and, together with the
intermediate atoms, comprise an optionally substituted fused
pyridine ring.
[0106] In a further aspect, the invention relates to a method of
activating a latent retrovirus in a subject, the method comprising
the step of administering to the subject an effective amount of a
compound represented by a formula:
##STR00032##
wherein n is 0, R.sup.1 is selected from H, C1-C4 alkyl, C1-C6
aryl, C(O)Ar, C(O)N(CH.sub.3).sub.2, SO.sub.2N(CH.sub.3).sub.2,
fluorenylmethyloxycarbonyl,
N-((dimethylamino)methylene)-N-methylmethanaminium
tetrafluoroborate,
N-((dimethylamino)methylene)-N-methylmethanaminium
hexafluorophosphate (V), tri(pyrrolidin-1-yl)phosphonium
hexafluorophosphate (V), tris(dimethylamino)phosphonium
hexafluorophosphate (V),
1-(pyrrolidin-1-ylmethylene)pyrrolidin-1-ium hexafluorophosphate
(V), and 1-piperidin-1-ylmethylene)piperidin-1-ium
hexafluorophosphate (V); wherein R.sup.2 is selected from H and
C1-C4 alkyl; and wherein R.sup.3 is selected from H and C1-C4
alkyl, or R.sup.2 and R.sup.3 are covalently bonded and, together
with the intermediate atoms, comprise an optionally substituted
fused six-membered aryl or heteroaryl ring. In a still further
aspect, n is 1.
[0107] In a further aspect, the invention relates to a method of
activating a latent retrovirus in a subject, the method comprising
the step of administering to the subject an effective amount of a
compound represented by a formula:
##STR00033##
wherein R is selected from H, C1-C4 alkyl, C1-C6 aryl, C(O)Ar,
C(O)N(CH.sub.3).sub.2, SO.sub.2N(CH.sub.3).sub.2,
fluorenylmethyloxycarbonyl,
N-((dimethylamino)methylene)-N-methylmethanaminium
tetrafluoroborate,
N-((dimethylamino)methylene)-N-methylmethanaminium
hexafluorphosphate (V), tri(pyrrolidin-1-yl)phosphonium
hexafluorophosphate (V), tris(dimethylamino)phosphonium
hexafluorophosphate (V),
1-(pyrrolidin-1-ylmethylene)pyrrolidin-1-ium hexafluorophosphate
(V), and 1-(piperidin-1-ylmethylene)piperidin-1-ium
hexafluorophosphate (V); and wherein each of R.sup.4, R.sup.5, and
R.sup.6 is independently selected from H, Cl, CH.sub.3, and
NO.sub.2.
[0108] In a still further aspect, the invention relates to a method
of activating a latent retrovirus in a subject, the method
comprising the step of administering to the subject an effective
amount of a compound represented by a formula:
##STR00034##
wherein R.sup.1 is selected from H and
N-((dimethylamino)methylene)-N-methylmethanaminium
hexafluorophosphate (V); and wherein each of R.sup.4, R.sup.5, and
R.sup.6 is independently selected from H, Cl, CH.sub.3, and
NO.sub.2.
[0109] In yet a further aspect, the invention relates to a method
of activating a latent retrovirus in a subject, the method
comprising the step of administering to the subject an effective
amount of a compound represented by a formula:
##STR00035##
wherein each of R.sup.4, R.sup.5, and R.sup.6 is independently
selected from H, Cl, CH.sub.3, and NO.sub.2.
[0110] In an even further aspect, the invention relates to a method
of activating a latent retrovirus in a subject, the method
comprising the step of administering to the subject an effective
amount of a compound represented by a formula:
##STR00036##
[0111] In a still further aspect, the invention relates to a method
of activating a latent retrovirus in a subject, the method
comprising the step of administering to the subject an effective
amount of a compound represented by a formula:
##STR00037##
[0112] In a further aspect, the invention relates to a method of
activating a latent retrovirus in a subject, the method comprising
the step of administering to the subject an effective amount of a
compound represented by a formula:
##STR00038##
wherein each of R.sup.4, R.sup.5, and R.sup.6 is independently
selected from H, Cl, CH.sub.3, or NO.sub.2.
[0113] In a still further aspect, the invention relates to a method
of activating a latent retrovirus in a subject, the method
comprising the step of administering to the subject an effective
amount of a compound represented by a formula:
##STR00039##
[0114] In a further aspect, the invention relates to a method of
activating a latent retrovirus in a subject, the method comprising
the step of administering to the subject an effective amount of a
compound represented by a formula:
##STR00040##
wherein R.sup.1 is selected from H, C1-C4 alkyl, C1-C6 aryl,
C(O)Ar, C(O)N(CH.sub.3).sub.2, SO.sub.2N(CH.sub.3).sub.2,
fluorenylmethyloxycarbonyl,
N-((dimethylamino)methylene)-N-methylmethanaminium
tetrafluoroborate,
N-((dimethylamino)methylene)-N-methylmethanaminium
hexafluorophosphate (V), tri(pyrrolidin-1-yl)phosphonium
hexafluorophosphate (V), tris(dimethylamino)phosphonium
hexafluorophosphate (V),
1-(pyrrolidin-1-ylmethylene)pyrrolidin-1-ium hexafluorophosphate
(V), and 1-(piperidin-1-ylmethylene)piperidin-1-ium
hexafluorophosphate (V).
[0115] In a still further aspect, the invention relates to a method
of activating a latent retrovirus in a subject, the method
comprising the step of administering to the subject an effective
amount of a compound represented by a formula:
##STR00041##
wherein R.sup.1 is selected from H and
N-((dimethylamino)methylene)-N-methylmethanaminium
hexafluorophosphate (V).
[0116] In yet a further aspect, the invention relates to a method
of activating a latent retrovirus in a subject, the method
comprising the step of administering to the subject an effective
amount of a compound represented by a formula:
##STR00042##
[0117] In a further aspect, the invention relates to a method of
activating a latent retrovirus in a subject, the method comprising
the step of administering to the subject an effective amount of a
compound represented by a formula:
##STR00043##
wherein R is selected from H, C1-C4 alkyl, C1-C6 aryl, C(O)Ar,
C(O)N(CH.sub.3).sub.2, SO.sub.2N(CH.sub.3).sub.2,
fluorenylmethyloxycarbonyl,
N-((dimethylamino)methylene)-N-methylmethanaminium
tetrafluoroborate,
N-((dimethylamino)methylene)-N-methylmethanaminium
hexafluorophosphate (V), tri(pyrrolidin-1-yl)phosphonium
hexafluorophosphate (V), tris(dimethylamino)phosphonium
hexafluorophosphate (V),
1-(pyrrolidin-1-ylmethylene)pyrrolidin-1-ium hexafluorophosphate
(V), and 1-(piperidin-1-ylmethylene)piperidin-1-ium
hexafluorophosphate (V); and wherein each of R.sup.7, R.sup.8, and
R.sup.9 is independently selected from H, Cl, CH.sub.3, and
NO.sub.2.
[0118] In a still further aspect, the invention relates to a method
of activating a latent retrovirus in a subject, the method
comprising the step of administering to the subject an effective
amount of a compound represented by a formula:
##STR00044##
wherein R.sup.1 is selected from H and
N-((dimethylamino)methylene)-N-methylmethanaminium
hexafluorophosphate (V); and wherein each of R.sup.7, R.sup.8, and
R.sup.9 is independently selected from H, Cl, CH.sub.3, and
NO.sub.2.
[0119] In yet a further aspect, the Invention relates to a method
of activating a latent retrovirus in a subject, the method
comprising the step of administering to the subject an effective
amount of a compound represented by a formula:
##STR00045##
wherein each of R.sup.7, R.sup.8, and R.sup.9 is independently
selected from H, Cl, CH.sub.3, and NO.sub.2.
[0120] In an even further aspect, the invention relates to a method
of activating a latent retrovirus in a subject, the method
comprising the step of administering to the subject an effective
amount of a compound represented by a formula:
##STR00046##
[0121] In a further aspect, the invention relates to a method of
activating a latent retrovirus in a subject, the method comprising
the step of administering to the subject an effective amount of a
compound represented by a formula:
##STR00047##
wherein R.sup.1 is selected from H, C1-C4 alkyl, C1-C6 aryl,
C(O)Ar, C(O)N(CH.sub.3).sub.2, SO.sub.2N(CH.sub.3).sub.2,
fluorenylmethyloxycarbonyl,
N-((dimethylamino)methylene)-N-methylmethanaminium
tetrafluoroborate,
N-((dimethylamino)methylene)-N-methylmethanaminium
hexafluorophosphate (V), tri(pyrrolidin-1-yl)phosphonium
hexafluorophosphate (V), tris(dimethylamino)phosphonium
hexafluorophosphate (V),
1-(pyrrolidin-1-ylmethylene)pyrrolidin-1-ium hexafluorophosphate
(V), and 1-(piperidin-1-ylmethylene)piperidin-1-ium
hexafluorophosphate (V).
[0122] In a still further aspect, the invention relates to a method
of activating a latent retrovirus in a subject, the method
comprising the step of administering to the subject an effective
amount of a compound represented by a formula:
##STR00048##
wherein R.sup.1 is selected from H and
N-((dimethylamino)methylene)-N-methylmethanaminium
hexafluorophosphate (V).
[0123] In yet a further aspect, the invention relates to a method
of activating a latent retrovirus in a subject, the method
comprising the step of administering to the subject an effective
amount of a compound represented by a formula:
##STR00049##
[0124] In a further aspect, the subject is a population of cells.
In a still further aspect, the subject is a population of cells
latently infected with a retrovirus. In yet a further aspect, the
subject is a population of cells latently infected with HIV-1.
[0125] In a further aspect, the subject is a mammal. In a still
further aspect, the subject is a human. In yet a further aspect,
the subject is a patient. In an even further aspect, the subject is
a patient who has been diagnosed with a need for treatment of a
retrovirus prior to the administering step. In a still further
aspect, the subject is a patient who has been diagnosed with a need
for treatment of HIV prior to the administering step.
[0126] b. Agent Known to Treat a Retrovirus
[0127] In one aspect, the invention relates to methods of
activating a latent retrovirus in a subject, the method further
comprising treating the patient with at least one agent that can be
used to treat a retrovirus.
[0128] In various aspects, the invention relates to methods of
activating latent HIV-1 in a subject, the method further comprising
treating the patient with at least one agent that can be used to
treat HIV-1. In a further aspect, the at least one agent is
selected from entry inhibitors, nucleoside reverse transcriptase
inhibitors (NRTIs), nucleotide reverse transcriptase inhibitors
(NtRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs),
protease inhibitors, integrase inhibitors, or maturation
inhibitors, or a mixture thereof. In a further aspect, the at least
one agent is selected from nucleoside reverse transcriptase
inhibitors (NRTIs), nucleotide reverse transcriptase inhibitors
(NtRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs),
protease inhibitors, or integrase inhibitors, a mixture thereof. In
yet a further aspect, the at least one agent is selected from
Maraviroc, Enfuvirtide, Zidovudine, Didanosine, Zalcitabine,
Stavudine, Lamivudine, Abacavir, Emtricitabine, Entecavir,
Tenofovir, Adefovir, Efavirenz, Nevirapine, Delavirdine,
Rilpivirine, Raltegravir, Saquinavir, Ritonavir, Indinavir,
Nelfinavir, or Amprenavir, or a mixture thereof. In an even further
aspect, the at least one agent is HAART.
[0129] Typically, an agent is administered in an effective amount,
per its normal dosing instructions. In one aspect, the effective
amount is a therapeutically effective amount.
[0130] 2. Manufacture of a Medicament
[0131] In one aspect, the invention relates to methods for the
manufacture of a medicament for the treatment of a retrovirus in a
subject, the method comprising the step of combining an effective
amount of a compound represented by a formula:
##STR00050##
wherein n is 0 or 1, wherein R.sup.1 is selected from H, C1-C4
alkyl, C1-C6 aryl, C(O)Ar, SO.sub.2N(CH.sub.3).sub.2,
fluorenylmethyloxycarbonyl,
N-((dimethylamino)methylene)-N-methylmethanaminium
tetrafluoroborate,
N-((dimethylamino)methylene)-N-methylmethanaminium
hexafluorophosphate (V), tri(pyrrolidin-1-yl)phosphonium
hexafluorophosphate (V), tris(dimethylamino)phosphonium
hexafluorophosphate (V),
1-(pyrrolidin-1-ylmethylen)pyrrolidin-1-ium hexafluorophosphate
(V), or L-(piperidin-1-ylmethylene)piperidin-1-ium
hexafluorophosphate (V); wherein R.sup.2 is selected from H and
C1-C4 alkyl; wherein R.sup.3 is selected from H and C1-C4 alkyl, or
R.sup.2 and R.sup.3 are covalently bonded and, together with the
intermediate atoms, comprise an optionally substituted fused
six-membered aryl or heteroaryl ring, alone or in combination with
another agent, with a pharmaceutically acceptable carrier or
diluent.
[0132] In a further aspect, the subject is a population of cells.
In a still further aspect, the subject is a population of cells
latently infected with a retrovirus. In yet a further aspect, the
subject is a population of cells latently infected with HIV-1.
[0133] In a further aspect, the subject is a mammal. In a still
further aspect, the subject is a human. In yet a further aspect,
the subject is a patient. In an even further aspect, the subject is
a patient who has been diagnosed with a need for treatment of a
retrovirus prior to the administering step. In a still further
aspect, the subject is a patient who has been diagnosed with a need
for treatment of HIV-1 prior to the administering step.
[0134] In a further aspect, the invention further comprises
treating the subject with at least one agent that can be used to
treat a retrovirus. In a still further aspect, the invention
further comprises treating the subject with at least one agent that
can be used to treat HIV-1. In yet a further aspect, the at least
one agent is selected from entry inhibitors, nucleoside reverse
transcriptase inhibitors (NRTIs), nucleotide reverse transcriptase
inhibitors (NtRTIs), non-nucleoside reverse transcriptase
inhibitors (NNRTIs), protease inhibitors, integrase inhibitors, or
maturation inhibitors, or a mixture thereof. In an even further
aspect, the at least one agent is selected from nucleoside reverse
transcriptase inhibitors (NRTIs), nucleotide reverse transcriptase
inhibitors (NtRTIs), non-nucleoside reverse transcriptase
inhibitors (NNRTIs), protcase inhibitors, or integrase inhibitors,
or a mixture thereof. In a still further aspect, the at least one
agent is selected from Maraviroc, Enfuvirtide, Zidovudine,
Didanosine, Zalcitabine, Stavudine, Lamivudine, Abacavir,
Emtricitabine, Entecavir, Tenofovir, Adefovir, Efavirenz,
Nevirapine, Delavirdine, Rilpivirine, Raltegravir, Saquinavir,
Ritonavir, Indinavir, Nelfinavir, or Amprenavir, or a mixture
thereof. In yet a further aspect, the at least one agent is
HAART.
[0135] 3. Use of Compounds and Compositions
[0136] Also provided are the uses of compounds represented by a
formula:
##STR00051##
wherein n is 0 or 1, wherein R.sup.1 is selected from H, C1-C4
alkyl, C1-C6 aryl, C(O)Ar, SO.sub.2N(CH.sub.3).sub.2,
fluorenylmethyloxycarbonyl,
N-((dimethylamino)methylene)-N-methylmethanaminium
tetrafluoroborate,
N-((dimethylamino)methylene)-N-methylmethanaminium
hexafluorophosphate (V), tri(pyrrolidin-1-yl)phosphonium
hexafluorophosphate (V), tris(dimethylamino)phosphonium
hexafluorophosphate (V),
1-(pyrrolidin-1-ylmethylene)pyrrolidin-1-ium hexafluorophosphate
(V), or 1-(piperidin-1-ylmethylene)piperidin-1-ium
hexafluorophosphate (V); wherein R.sup.2 is selected from H and
C1-C4 alkyl; wherein R.sup.3 is selected from H and C1-C4 alkyl, or
R.sup.2 and R.sup.3 are covalently bonded and, together with the
intermediate atoms, comprise an optionally substituted fused
six-membered aryl or heteroaryl ring, and compositions comprising
the same.
[0137] In various aspects, the use relates to the activation of a
latent retrovirus in a subject. In a further aspect, the use
relates to the activation of latent HIV-1 in a subject. In a still
further aspect, the subject is a population of cells. In yet a
further aspect, the subject is a population of cells latently
infected with a retrovirus. In an even further aspect, the subject
is a population of cells latently infected with HIV-1. In a still
further aspect, the subject is a mammal. In a still further aspect,
the subject is a human. In yet a further aspect, the subject is a
patient.
[0138] 4. Kits
[0139] In one aspect, the invention relates to a kit comprising a
compound represented by a formula:
##STR00052##
wherein n is 0 or 1; wherein R.sup.1 is selected from H, C1-C4
alkyl, C-C6 aryl, C(O)Ar, C(O)N(CH.sub.3).sub.2,
SO.sub.2N(CH.sub.3).sub.2, fluorenylmethyloxycarbonyl,
N-((dimethylamino)methylene)-N-methylmethanaminium
tetrafluoroborate,
N-((dimethylamino)methylene)-N-methylmethanaminium
hexafluorophosphate (V), tri(pyrrolidin-1-yl)phosphonium
hexafluorophosphate (V), tris(dimethylamino)phosphonium
hexafluorophosphate (V),
1-(pyrrolidin-1-ylmethylene)pyrrolidin-1-ium hexafluorophosphate
(V), and 1-(piperidin-1-ylmethylene)piperidin-1-ium
hexafluorophosphate (V); wherein R.sup.2 is selected from H and
C1-C4 alkyl; and wherein R.sup.3 is selected from H and C1-C4
alkyl, or R.sup.2 and R.sup.3 are covalently bonded and, together
with the intermediate atoms, comprise an optionally substituted
fused six-membered aryl or heteroaryl ring and one or more of:
[0140] (a) at least one agent that can be used to treat a
retrovirus;
[0141] (b) instructions for detecting a retrovirus; and
[0142] (c) instructions for treating a retrovirus.
[0143] In a further aspect, the at least one compound and the at
least one agent are co-formulated. In a further aspect, the at
least one compound and the at least one agent are co-packaged.
[0144] In a further aspect, the retrovirus is selected from a group
comprising HIV-1, HIV-2, SIV, XMRV, HTLV-1, HTLV-2, HTLV-3, or
HTLV-4. In a still further aspect, the retrovirus is HIV-1.
[0145] The kits can also comprise compounds and/or products
co-packaged, co-formulated, and/or co-delivered with other
components. For example, a drug manufacturer, a drug reseller, a
physician, a compounding shop, or a pharmacist can provide a kit
comprising a disclosed compound and/or product and another
component for delivery to a patient.
[0146] It is contemplated that the disclosed kits can be used in
connection with the disclosed methods of making, the disclosed
methods of using, and/or the disclosed compositions.
[0147] 5. Non-Medical Uses
[0148] Also provided are the uses of compounds represented by a
formula:
##STR00053##
wherein n is 0 or 1; wherein R.sup.1 is selected from H, C1-C4
alkyl, C1-C6 aryl, C(O)Ar, C(O)N(CH.sub.3).sub.2,
SO.sub.2N(CH.sub.3).sub.2, fluorenylmethyloxycarbonyl,
N-((dimethylamino)methylene)-N-methylmethanaminium
tetrafluoroborate,
N-((dimethylamino)methylene)-N-methylmethanaminium
hexafluorophosphate (V), tri(pyrrolidin-1-yl)phosphonium
hexafluorophosphate (V), tris(dimethylamino)phosphonium
hexafluorophosphate (V),
1-(pyrrolidin-1-ylmethylene)pyrrolidin-1-ium hexafluorophosphate
(V), and L-(piperidin-1-ylmethylene)piperidin-1-ium
hexafluorophosphate (V); wherein R.sup.2 is selected from H and
C1-C4 alkyl; and wherein R.sup.3 is selected from H and C1-C4
alkyl, or R.sup.2 and R.sup.3 are covalently bonded and, together
with the intermediate atoms, comprise an optionally substituted
fused six-membered aryl or heteroaryl ring as pharmacological tools
in the development and standardization of in vitro and in vivo test
systems for the evaluation of the effects of anti-retroviral
latency drugs, and more particularly anti HIV-1 latency drugs, in
laboratory animals such as cats, dogs, rabbits, monkeys, rats and
mice, as part of the search for new therapeutic agents that
activate latent retroviruses, and more specifically that activate
latent HIV-1.
D. REFERENCES
[0149] Antoni B, Rabson A B, Kinter, A, Bodkin M, Poli G (1994)
NF-kappa B-dependent and -independent pathways of HIV activation in
a chronically infected T cell line. Virology 202:684-694. [0150]
Bosque A, Planelles V (2009) Induction of HIV-1 latency and
reactivation in primary memory CD4+ T cells. Blood 113:58-65.
[0151] Bosque A, Planelles V (2010) "Studies of HIV-1 latency in an
ex vivo model that uses primary central memory T cells". Methods.
[0152] Bosque A, Famiglietti M, Weyrich A S, Goulston C, Planelles
V (2011) Homeostatic Proliferation Fails to Efficiently Reactivate
HIV-1 Latently Infected Central Memory CD4+ T Cells. PLOS Pathog.
7(10). [0153] Brooks D G, Kitchen S G, Kitchen C M, Scripture-Adams
D D, Zack J A (2001) Generation of HIV latency during thymopoiesis.
Nat Med 7:459-464. [0154] Chun T W, Carruth L, Finzi D, et al.
(1997) Quantification of latent tissue reservoirs and total body
viral load in HIV-1 infection. Nature 387:183-188. [0155] Finzi D,
Hermankova M, Pierson T, et al. (1997) Identification of a
reservoir for HIV-1 in patients on highly active antiretroviral
therapy. Science 278:1295-1300. [0156] Folks T M, Clouse K A,
Justement J, et al. (1989) Tumor necrosis factor alpha Induces
expression of human immunodeficiency virus in a chronically
infected T-cell clone. Proc Natl Acad Sci USA 86:2365-2368. [0157]
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 238:800-802. [0158] Jordan A,
Bisgrove D, Verdin E (2003) HIV reproducibly establishes a latent
infection after acute infection of T cells in vitro. Embo J
22:1868-1877. [0159] Planelles V, Bosque A, Methods and
compositions relating to viral latency, U.S. Patent Application
2010/0291067 A1, November 2010.
E. EXPERIMENTAL
[0160] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how the compounds, compositions, articles, devices
and/or methods claimed herein are made and evaluated, and are
intended to be purely exemplary of the invention and are not
intended to limit the scope of what the inventors regard as their
invention. Efforts have been made to ensure accuracy with respect
to numbers (e.g., amounts, temperature, etc.), but some errors and
deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, temperature is in .degree. C. or is at
ambient temperature, and pressure is at or near atmospheric.
[0161] 1. General Methods
[0162] A novel primary cell model for the study of HIV-1 latency
was adapted so as to be suitable for high-throughput screening
(HTS) of new compounds that may reactivate HIV-1 from its latency
state (Bosque and Planelles, 2009; Bosque and Planelles, 2010; and
patent application: Methods and compositions relating to viral
latency, Planelles V, Bosque A, U.S. Patent Application
2010/0291067 A1, November 2010).
[0163] To facilitate HTS, the nef gene was replaced with that
encoding the green fluorescent protein (GFP). It was confirmed that
deletion of nef has no effect in latency/reactivation (Bosque and
Planelles, 2009) and that using GFP provides a faster, more
economical, and equally sensitive, as compared to intracellular p24
detection, readout.
[0164] The reactivation experiments were modified to be performed
in 96-well plates, utilizing 100,000 cells per well. After
reactivation, flow cytometric analysis was performed with Becton
Dickinson FACSCanto II with a High Throughput Sampler (HTS) for
microtiter plates that is available through the Flow Cytometry Core
Facility at the University of Utah. It takes about 30 minutes to
analyze a plate, when acquiring 10,000 cells per well. Analysis for
each well includes (a) green fluorescence as a direct indicator of
viral reactivation; and (b) forward and side scatter as general
measures of cell morphology, which is a gross indication of
viability.
[0165] 2. Activation of Latent HIV-1 in Latently Infected Cultured
T.sub.CM
[0166] Latently infected cells were generated using healthy,
uninfected DONOR 78 cells that were infected with DHIV virus.
Reactivation was monitored by analysis of GFP by Flow Cytometry 72
hours after stimulation. Beads coated with anti-CD3/anti CD28
(CD3/CD28) were used as a positive reactivation stimulus.
Reactivation was done in a 96-well plate round bottom. Screening of
the Ireland Natural Product Collection, a set of natural products
isolated from marine invertebrate animals and microorganisms,
resulted in a positive hit in Plate 2 well C7 (FIGS. 1A and 1B).
This compound is an intermediate en route to synthesis of a cyclic
peptide isolated from a tunicate.
[0167] HPLC was used to generate 20 fractions based on elution time
using a gradient from 10-100% acetonitrile in 0.1% formic acid
using a Phenomenex Kinetic C18 50.times.2.10 mm column (2.6 .mu.M,
100 A). The ability of these fractions to activate HIV-1 was tested
and compared in parallel with the original hit. FIG. 1C shows the %
of p24 positive cells in the Y-axis and the % of reactivation
compared to the positive control (beads coated with
anti-CD3/anti-CD28) at the top of each bar. This data reveals that
the active compound is enriched in fractions 2 and 3. Further
fractionation of C7 was performed and each fraction tested at the
concentration indicated in the graph (FIG. 1D). The active
component is found in fraction MS. This fraction was found to be
enriched in 1-hydroxybenzotriazol (HOBt) (FIG. 1E). The ability of
HOBt to activate latent HIV-1 in a dose-dependent manner compared
to C7 was also established (FIG. 1F). Finally, HOBt was tested at
different concentrations and analyzed on either Day 3, Day 5, or
Day 7. When compared with anti-CD3/CD28, a non-druggable treatment
due to its side effects, HOBt was found to reactive HIV-1 latency
with slower kinetics (FIG. 1G).
[0168] 3. Activation of Latent HIV-1 by Additional Analogs
[0169] The ability of additional triazole analogs including
benzotriazole (HBt), 1-hydroxy-7-amino benzotriazole (HOAt),
triazole (Ht) and 1-aminobenzotriazole (ABt) to activate latent
HIV-1 was also tested (Table 2). Latently infected cells were
generated using healthy, uninfected DONOR 78 cells that were
infected with DHIV virus. Reactivation was monitored by analysis of
p24Gag by Flow Cytometry 72 hours after stimulation. It was
demonstrated that both HOBt and HOAt were able to reactivate HIV-1
latency (FIGS. 2A and 2C). The activity of both analogs is dose
dependent (FIG. 2C).
TABLE-US-00002 TABLE 2 Compound Structure HOBt ##STR00054## HOAt
##STR00055## 6C1-HOBt ##STR00056## HBt ##STR00057## ABt
##STR00058## Triazole ##STR00059## HO-DhBt ##STR00060## HATU
##STR00061## Pyridozine ##STR00062##
[0170] A panel of 7 derivatives of the original hit, HOBt, were
tested for their ability to activate latent HIV-1 (FIG. 2D). HOAt
is a more potent analog than the original hit. Two additional
compounds, HATU and HO-DhBt, were also able to induce viral
reactivation in a dose dependent manner and with increased
efficiency.
[0171] 4. Synergistic Effect of Several r-Cytokines
[0172] Latently infected cells were generated using healthy,
uninfected DONOR 78 cells that were infected with DHIV virus.
Reactivation was monitored by analysis of p24Gag by Flow Cytometry
72 hours after stimulation. HOBt was tested in the presence or
absence of IL-2, a .gamma.c-cytokine required for survival of the
cells in vivo. The ability of HOBt and HOAt to activate latent
HIV-1 in the absence of IL-2 or in the presence of different
.gamma.c-cytokines, e.g. IL-4, IL-7, IL-15, IL-9, and IL-21, was
tested (FIG. 3). In the absence of .gamma.c-cytokines both analogs
have the ability to significantly reactivate HIV-1 latency. When
compared with incubation of the cells in the absence of cytokines,
IL-2 and IL-7 trend towards increasing viral reactivation. HOBt and
HOAt dramatically increase viral reactivation in the presence of
all, IL-2, IL-4, and IL-15. IL-4 was not able to induce significant
viral reactivation by itself; however, combination of IL-4 with
either HOBt or HOAt induced significant viral reactivation.
Finally, IL-9 and IL-21 did not have an effect in viral
reactivation wither alone or in combination with HOBt and HOAt.
[0173] 5. Activation of Latent HIV-1 in a STAT5 Dependent
Manner
[0174] The ability of chemical inhibitors of known signaling
pathways to block viral reactivation mediated by HOAt was examined
(FIG. 4A). Blocking either NFAT or NF-.kappa.B with either
Cyclosporine A (CsA) or BMS345541 did not have a significant effect
on viral reactivation induced by HOAt. CsA is able to block viral
reactivation induced by .alpha.CD3/.alpha.CD28. The JAK inhibitor
DBI strongly blocks viral reactivation mediated by HOAt but not by
.alpha.CD3/.alpha.CD28. Activation of JAK kinases lead to the
activation of the transcription factors STATs. Specific inhibitors
of STAT3 and STAT5 were thus also studied. The STAT3 inhibitor
peptide used did not have a significant effect in viral
reactivation compared with an inactive control peptide. In the
other hand, the STAT5 inhibitor completely abrogates viral
reactivation induced by HOAt.
[0175] The levels of STAT5 phosphorylation were analyzed by flow
cytometry after incubating cultured T.sub.CM with HOAt, IL-2, or a
combination of both, during a 24 h period (FIG. 4B). STAT5
phosphorylation could be only slightly detected in the absence of
either IL-2 or HOAt at 30 min. HOAt alone is able to induce an
increase in STAT5 phosphorylation over untreated cells. Cells
incubated with IL-2 alone dramatically increase the levels of STAT5
phosphorylation at 30 min, but these levels decrease to basal
levels after 24 hrs of incubation. Incubation of cultured T.sub.CM
with a combination of IL-2 and HOAt induce a drastic increase in
STAT5 phosphorylation. Moreover, the levels of STAT5
phosphorylation do not significantly decrease after 24 hrs of
incubation with IL-2 and HOAt.
[0176] 6. Effect on the Nuclear Localization of STAT5
[0177] The levels of nuclear localization of STAT5 after incubation
of the cells with IL-2 alone or in the presence of HOAt were
analyzed. Nuclear translocation of STAT5 is required for its
transcriptional activity. Both pSTAT5 and STAT5 levels increased in
the nucleus of cells treated with HOAt at the time points indicated
(FIG. 5A). Histone H3 and alpha Tubulin were used as controls for
purity of the fractionation.
[0178] Engagement of .gamma.c-cytokines to its receptors leads to a
signaling cascade initiated by the phosphorylation and activation
of the janus kinases JAK1 and JAK3. These kinases then
phosphorylate and activate the transcription factors STATs. IL-2
alone or in the presence of HOAt increases phosphorylation of JAK1
and JAK3 at similar levels (FIG. 5B). This indicates that HOAt
alters STAT5 phosphorylation without altering the phosphorylation
pattern of the JAK kinases.
[0179] STAT5 is a transcriptional activator of multiple genes. To
address whether HOAt will increase the transcription of any STAT5
target genes, the expression levels of SOCS3, a target gene for
STAT5, were analyzed. Incubation of cells with IL-2 and HOAt was
found to increase the levels of SOCS3 at 24 h post-treatment (FIG.
5C).
[0180] 7. Analysis of T Cell Activation
[0181] Memory cells were generated from 3 different donors and
treated with either HOBt or anti-CD3/CD28 to analyze the effect of
HOBt on T cell activation (FIG. 6A). Analysis of T cell activation
was done by analyzing the upregulation of the activation markers
CD25 and CD69 three days post activation. HOBt does not induce
general T cell activation. Analysis of T cell differentiation was
done by analyzing the levels of CCR7 and CD27 three days post
activation (FIG. 6B). This revealed that HOBt does not induce T
cell differentiation measured as loss of CCR7 and CD27
expression.
[0182] In order to determine whether HOAt is inducing T cell
proliferation, culter T.sub.CM were incubated with HOAt in a dose
dependent manner. HOAt only induces some degree of cell
proliferation at the higher concentration tested (FIG. 6C). It has
been shown previously that both cultured T.sub.CM and ex vivo
T.sub.0c express low levels of CD25 (Bosque et al. 2011). This
level is increased by 37 times after TCR engagement with
.alpha.CD3/.alpha.CD28, measured as mean intensity of fluorescence
(values between parenthesis, FIG. 6D). HOAt only induced an
increase of 1.4 times at the maximum concentration used.
[0183] CD69 upregulation is concomitant with T cell activation.
HOAt slightly increases the levels of T cell activation when
compared with .alpha.CD3/.alpha.CD28 (FIG. 6E). These results
indicate that HOAt can reactivate latent HIV-1 in the absence of
cellular proliferation of cellular activation.
[0184] 8. Vial Reactivation Mediated by Other Stimuli
[0185] To test whether HOAt can enhance viral reactivation mediated
by other stimuli, cells were reactivated with three different
agonists: PMA, a PKC agonist that has been shown to reactivate
latent HIV in a NF-.kappa.B dependent manner, ionomycine, a
ionophore that increases the intracellular levels of Ca.sup.2+ and
induces the activation of the transcription factor NFAT; and SAHA,
a histone deacetylase inhibitor that has been shown to reactive
latent HIV-1 In vitro and ax vivo (FIG. 7). HOAt is able to
increase viral reactivation mediated by these three stimuli. These
results indicate that HOAt and its derivatives can be used to
synergize the activity of other anti-latency drugs.
[0186] It will be apparent to those skilled in the art that various
modifications and variations can be made in the present invention
without departing from the scope or spirit of the invention. Other
embodiments of the invention will be apparent to those skilled in
the art from consideration of the specification and practice of the
invention disclosed herein. It is intended that the specification
and examples be considered as exemplary only, with a true scope and
spirit of the invention being indicated by the following
claims.
* * * * *