U.S. patent application number 16/371193 was filed with the patent office on 2019-07-25 for guanosine as an immune potentiator mediated through toll receptors.
This patent application is currently assigned to Nitor Therapeutics. The applicant listed for this patent is NITOR THERAPEUTICS. Invention is credited to Shanta Bantia.
Application Number | 20190224224 16/371193 |
Document ID | / |
Family ID | 54359125 |
Filed Date | 2019-07-25 |
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United States Patent
Application |
20190224224 |
Kind Code |
A1 |
Bantia; Shanta |
July 25, 2019 |
Guanosine as an Immune Potentiator Mediated through Toll
Receptors
Abstract
Methods and Compositions involving the administration of
guanosine, identified as Toll like receptor (TLR) 2 and 4 agonists,
that will be useful for enhancing the potency of vaccine and cancer
immunotherapies are disclosed. Method of preventing and treating
cancer and infection by administration of guanosine or pro-drugs of
guanosine, or a precursor of guanosine are also disclosed.
Compositions of guanosine or a pro-drug of guanosine or precursor
of guanosine may be formulated as pharmaceutical dosage forms and
components can be assembled as kits. Methods for activating TLRs
with guanosine to enhance an immune response and to
potentiate/augment antiviral, antibacterial or anticancer effects
of other antiviral, anti-bacterial and anticancer therapeutic
agents are also disclosed.
Inventors: |
Bantia; Shanta; (Birmingham,
AL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NITOR THERAPEUTICS |
Birmingham |
AL |
US |
|
|
Assignee: |
Nitor Therapeutics
Birmingham
AL
|
Family ID: |
54359125 |
Appl. No.: |
16/371193 |
Filed: |
April 1, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15306876 |
Oct 26, 2016 |
10278984 |
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PCT/US2014/071499 |
Dec 19, 2014 |
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16371193 |
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61987567 |
May 2, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/4745 20130101;
A61K 31/513 20130101; A61K 31/4025 20130101; A61K 31/4025 20130101;
A61K 2039/55511 20130101; A61K 39/08 20130101; A61K 31/522
20130101; A61K 31/4745 20130101; A61K 31/4725 20130101; A61K
2300/00 20130101; A61K 31/513 20130101; A61K 2300/00 20130101; A61K
2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 31/439 20130101;
A61P 35/00 20180101; A61K 45/06 20130101; A61K 31/4725 20130101;
A61K 31/522 20130101; A61K 31/519 20130101; A61K 31/708 20130101;
A61K 31/7072 20130101; A61P 31/04 20180101; A61K 31/439 20130101;
A61P 31/00 20180101; A61K 2300/00 20130101; A61K 31/536 20130101;
A61K 2300/00 20130101; A61K 39/39558 20130101; A61K 31/519
20130101; A61K 31/536 20130101; A61K 31/7072 20130101; A61K 31/675
20130101; A61K 31/708 20130101; A61K 2039/55561 20130101; A61K
31/675 20130101; A61K 39/39 20130101; A61P 31/12 20180101; A61K
2300/00 20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 31/708 20060101
A61K031/708; A61K 31/675 20060101 A61K031/675; A61K 45/06 20060101
A61K045/06; A61K 39/395 20060101 A61K039/395; A61K 39/08 20060101
A61K039/08; A61K 31/7072 20060101 A61K031/7072; A61K 39/39 20060101
A61K039/39; A61K 31/4745 20060101 A61K031/4745 |
Claims
1-8. (canceled)
9. A method for treating infectious disease in a subject, the
method comprising administering a pharmaceutically effective amount
of one or more of: guanosine, a pro-drug thereof, and a precursor
thereof.
10. The method according to claim 9 wherein the guanosine precursor
is guanosine mono phosphate (GMP) or a salt or pro-drug
thereof.
11. A method for treating cancer in a subject, the method
comprising administering a pharmaceutically effective amount of
guanosine or a pro-drug thereof or a precursor of guanosine.
12. The method according to claim 11 wherein the guanosine
precursor is guanosine mono phosphate (GMP) or a salt thereof, or a
prodrug thereof.
13. A method for enhancing a potentiating treatment with an
anti-cancer, anti-viral or anti-bacterial agent, the method
comprising administering guanosine or a pro drug thereof or a
precursor of guanosine to the subject in conjunction with the
anti-cancer, anti-viral, or anti-bacterial agent.
14. The method according to claim 13 wherein an anti-viral agent is
selected from the group consisting of simeprevir, sofosbuvir,
ledipasavir, ABT-267, ABT-333, tenofovir, entecavir, and
atripla.
15. The method according to claim 13 wherein the guanosine
precursor is guanosine mono phosphate (GMP) or a salt thereof, or a
prodrug thereof.
16-17. (canceled)
18. A method for activating toll like receptor (TLR) 2 and TLR 4 in
a subject, the method comprises administering a pharmaceutically
effective amount of guanosine or a pro-drug of guanosine, or a
precursor of guanosine to the subject in conjunction with the
vaccine or immunotherapy.
19. The method according to claim 18 wherein the guanosine
precursor is guanosine mono phosphate (GMP) or a salt thereof, or a
prodrug thereof.
20. The method according to claim 13 wherein the anti-viral agent
is selected from the group consisting of compounds targeting viral
polymerases, compounds targeting viral proteases, and compounds
targeting other structural and non-structural viral proteins.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority to U.S. Provisional
application Ser. No. 61/987,567 filed 2 May 2014, the entirety of
which is incorporated by reference herein in its entirety.
TECHNICAL FIELD
[0002] The invention relates to preventing and treating infectious
diseases and cancer, enhancing an immune response and augmenting
the potency of vaccines and immunotherapies by activation of toll
like receptor 2 (TLR2) and toll like receptor 4 (TLR4). Embodiments
include activation by administration of guanosine alone or in
combination with an antigen, vaccine or immunotherapy to provide an
adjuvant effect.
BACKGROUND
[0003] An adjuvant is an agent administered to potentiate the
immune response to an antigen and/or modulate it toward a desired
immune response. An endogenous adjuvant is a compound or molecule
naturally occurring within the cell or tissue that likewise
enhances an immune response by stimulating innate immunity, thus
possessing the capacity to potentiate an effect of some triggering
event or agent. Endogenous adjuvants play a central role in
alerting the immune system to potential danger and promote response
to infection, transplantation, tumor, and autoimmunity.
[0004] Vaccines attempt to safely elicit an immunity to pathogens
that is ideally robust, protective and long-lived. However, current
formulations of many subunit vaccines provide weaker and
shorter-lived immunity than natural infection. While it is clear
that adjuvants can be used to boost immunity, the adjuvants that
are currently approved for use in licensed vaccines are limited.
Alum, a mixture of aluminum salts, was the first vaccine adjuvant
that was widely utilized in vaccine preparations. Alum is a weak
adjuvant and one that biases responses to effector responses (Th2)
that are not protective against many pathogens. It was the only
vaccine adjuvant in use in the United States until 2009, when the
U.S. Food and Drug Administration approved Cervarix, a human
papillomavirus vaccine that contains an adjuvant designated as
AS04. This adjuvant is a mixture of alum and a bacterial lipid
(fat) molecule that has been modified so that it does not cause
disease.
[0005] Endogenous adjuvants generally have not been evaluated for
their potential use in vaccines. In theory, they may allow
vaccinations to safely mimic the pathway that naturally triggers
immunity to many pathogens. These agents also promote CD8+ T cell
immune responses which are important to developing immunity to many
pathogens such as viruses and tumors, but which are not elicited by
most subunit vaccines (Rock et al. in Springer Seminars in
Immunopathology (2005) 26:231-246).
[0006] Clearly, identification of endogenous adjuvants, triggered
in response to certain pathogens, may provide novel exogenous
adjuvants, which may thereafter be administered exogenously to
enhance an immune response and augment the potency of vaccines and
cancer immunotherapies.
SUMMARY OF DISCLOSURE
[0007] Cancer and infection can be treated with some success by
boosting the immune system to eliminate the malignant cells and/or
pathogens by means of natural physiological process. The present
disclosure is based on the surprising discovery that guanosine, a
purine nucleoside, can act as exogenous adjuvant and can activate
the immune system in the presence of an infection or tumor cells to
minimize the deleterious effects and alleviate the disease.
[0008] Accordingly, it is an object of the instant invention to
provide compositions of one or more agents identified as endogenous
adjuvants, for example one or more purine nucleosides and more
specifically guanosine. Methods comprising administering the
compositions to enhance an immune response and augment the potency
of vaccine and immune therapies are also provided.
[0009] According to specific embodiments, the present disclosure
describes compositions and methods for administering guanosine to a
subject. Guanosine can act as immune-potentiator in the presence of
an antigen or vaccine and enhance the potency of vaccines and
immunotherapies. Because guanosine has immune-potentiating activity
it can be used as anti-infective and anti-cancer agent. Such
compositions and methods were not previously appreciated in the
art.
[0010] The present disclosure further describes administration of
compositions comprising guanosine to activate toll like receptors
(TLR), which are known to activate the immune system. TLRs, in
turn, act as immune-enhancers in the presence of an antigen or
vaccine and enhance the potency of the vaccine and immunotherapies.
Such compositions and methods were not previously appreciated in
the art.
[0011] These and other embodiments and aspects of the present
invention will be expanded and clarified by reference to the
Drawings and Detailed Description set forth below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1. Depicts a schematic illustration of the relationship
between PNP inhibition and levels of guanosine.
[0013] FIG. 2. Illustrates the activity of NTR001, inosine and
guanosine as single agents, and in combination, on seven different
human TLRs (TLR2, 3, 4, 5, 7, 8 and 9) as a potential agonist.
[0014] FIG. 3. Data showing serum tetanus toxoid antibody titers on
day 38 in vehicle- and PNP inhibitors NTR001- and NTR002-treated
mice groups in the tetanus toxoid mouse model.
[0015] FIG. 4. Depicts serum interferon-g levels on day 30 in
vehicle and PNP inhibitors NTR001 and NTR002 treated mice groups in
the tetanus toxoid mouse model.
[0016] FIG. 5. Demonstrates effect of PNP inhibitor NTR001 and
chemotherapeutic agent cyclophosphamide on tumor volume in the
mouse melanoma model.
[0017] FIG. 6. Demonstrations effect of PNP inhibitor NTR001 and
chemotherapeutic agent cyclophosphamide on survival in the mouse
melanoma model.
[0018] FIG. 7. Demonstrates effect of PNP inhibitor NTR001 on
weight loss in the mouse model of L. Monocytogenes infection.
[0019] FIG. 8. Demonstrates effect of PNP inhibitor NTR001 on
survival in the mouse model of L. Monocytogenes infection.
[0020] FIG. 9. Demonstrates effect of PNP inhibitor NTR002 on
weight loss in the mouse model of L. Monocytogenes infection.
[0021] FIG. 10. Demonstrates effect of PNP inhibitor NTR002 on
survival in the mouse model of L. Monocytogenes infection.
[0022] FIG. 11. Demonstrates effect of PNP inhibitor NTR001 and
CTLA4-Ab on tumor volume in the mouse melanoma model.
[0023] FIG. 12. Demonstrates effect of PNP inhibitor NTR001 and
CTLA4-Ab on survival in the mouse melanoma model.
DETAILED DESCRIPTION
[0024] New generation vaccines will increasingly comprise highly
purified recombinant proteins. Unfortunately, these antigens are
often poorly immunogenic. Therefore, adjuvants may be required to
enable these proteins to become effective as vaccines. Stimulation
of the innate immune response is now known to have an important
role in the evolution of the adaptive immune response. Inclusion of
immune potentiators (also termed adjuvants), which trigger an early
innate immune response central to the generation of robust and long
lasting adaptive immune responses, is crucial to vaccine
effectiveness.
[0025] In the past, antiviral, antibacterial and anti-cancer
research has focused mainly on viral, bacterial and tumor cells as
targets. As the search for effective and differentiated
antiviral/antibacterial/anticancer therapies continues, modulation
of cellular targets that can activate the immune system to fight
the infection and/or cancer and alleviate the disease is gaining
attention. Toll-like receptors (TLRs) are pattern recognition
receptors (PRR) and can provide a unique mechanism by which one
could harness the host immune system to combat infection and
cancer. Agonists of TLRs trigger activation of the innate immune
system and could potentially be used as anti-infective and
anticancer agent. In addition, it can act as an adjuvant to enhance
the potency of vaccines and immunotherapies.
[0026] TLRs play a critical role in the early innate immune
response to invading pathogens by sensing both microorganisms and
endogenous danger signals. TLRs recognize highly conserved
structural motifs including pathogen-associated microbial patterns
(PAMPs), which are exclusively expressed by microbial pathogens,
and danger-associated molecular patterns (DAMPs), which are
endogenous molecules released from necrotic or dying cells.
Stimulation of TLRs by the corresponding PAMPs or DAMPs initiates
signaling cascades leading to the activation of transcription
factors, such as AP-1, NF-.kappa.B and interferon regulatory
factors (IRFs). Signaling by TLRs results in a variety of cellular
responses that direct the adaptive immune response, including but
not limited to the producing interferon (IFN), pro-inflammatory
cytokine and effector cytokine.
[0027] The present disclosure is based on the surprising discovery
that activation of the innate immune response by guanosine can
regulate an adaptive immune response through activation of TLRs.
Embodiments of the present invention therefore provide compositions
and methods effective for enhancing the potency of vaccines and
immunotherapies, and/or preventing or treating cancer, infection,
and other diseases or conditions amenable to treatment by
immunotherapy, wherein the compositions comprise guanosine or a
pro-drug thereof, or a guanosine precursor (a substance from which
guanosine is derived in the body, for example, guanosine mono
phosphate (GMP)), and optionally, one or more vaccine and/or
immunotherapy agents. According to other specific embodiments, the
methods comprise administering compositions comprising guanosine or
a pro-drug thereof or a guanosine precursor in conjunction with one
or more vaccine and/or immunotherapy agents. "In conjunction with,"
in accordance with the instant disclosure, means "as part of the
same treatment cycle" and encompasses administration via the same
composition or via independent compositions.
[0028] As used herein, "guanosine" is interpreted to include
pharmacological functional equivalents such as guanosine
monophosphate (GMP), a precursor of guanosine. Pro-drugs of
guanosine are also contemplated as within the scope, may be readily
developed. Suitable pro-drugs of guanosine and synthesis thereof
are set forth in Ray, Adrian S. et al. "Novel Use of a Guanosine
Prodrug Approach To Convert 2',3'-Didehydro-2',3'-Dideoxyguanosine
into a Viable Antiviral Agent" Antimicrob Agents Chemother. 2002
March; 46(3): 887-891, Zhang, Youxi et al. "Current prodrug
strategies for improving oral absorption of nucleoside analogues"
Asian Journal of Pharmaceutical Sciences. 2014 April; 9(2): 65-74,
and Bourdin, C. et al. "Synthesis and evaluation against hepatitis
C virus of 7-deaza analogues of 2'-C-methyl-6-O-methyl guanosine
nucleoside and L-Alanine ester phosphoramidates" Bioorg Med Chem.
Lett 2013 Apr. 20; 23(7):2260-4, the entire disclosures of which
are incorporated herein by this reference. According to specific
embodiments, the pro-drug of guanosine is selected from 6-O-methyl
guanosine, 6-cyclopropyl amino guanosine, and combinations
thereof.
[0029] According to specific embodiments, immunotherapeutic agents
comprise TLR agonists. Non-limiting examples of TLR agonists
include TLR7 agonist imiqimod, GS-9620, TLR7/8 agonist resiquimod,
and TLR9 agonists, and CpG oligodeoxynucleotides (CPG ODN's) that
are either approved or in clinical trials.
[0030] CpG ODNs are short synthetic single-stranded DNA molecules
containing unmethylated CpG dinucleotides in particular sequence
contexts (CpG motifs). CpG ODNs possess a partially or completely
phosphorothioated (PS) backbone, as opposed to the natural
phosphodiester (PO) backbone found in genomic bacterial DNA. Three
major classes of stimulatory CpG ODNs have been identified based on
structural characteristics and activity on human peripheral blood
mononuclear cells (PBMCs), in particular B cells and plasmacytoid
dendritic cells (pDCs). These three classes are Class A (Type D),
Class B (Type K) and Class C. CpG-A ODNs are characterized by a PO
central CpG-containing palindromic motif and a PS-modified 3'
poly-G string. They induce high IFN-.alpha. production from pDCs
but are weak stimulators of TLR9-dependent NF-.kappa.B signaling
and pro-inflammatory cytokine (e.g. IL-6) production. CpG-B ODNs
contain a full PS backbone with one or more CpG dinucleotides. They
strongly activate B cells and TLR9-dependent NF-.kappa.B signaling
but weakly stimulate IFN-.alpha. secretion. CpG-C ODNs combine
features of both classes A and B. They contain a complete PS
backbone and a CpG-containing palindromic motif. C-Class CpG ODNs
induce strong IFN-.alpha. production from pDC as well as B cell
stimulation. Exemplary TLR9 Agonists include Class A (ODN 1585, ODN
2216, ODN 2216, and ODN 2336), Class B (ODN BW006. ODN D-SL01, ODN
1668, ODN 1826, ODN 2006, ODN 2007, and Class C (ODN D-SL03, ODN
2395, ODN M362, and Bacterial DNA (E. coli DNA).
[0031] In other specific embodiments the immunotherapy comprises
cancer immunotherapy, and non-limiting examples of
immunotherapeutic agents effective in the treatment of cancer
include checkpoint protein modulators. Non-limiting examples of
checkpoint protein modulators include CTLA-4 antagonists, GITR
agonists, OX40 agonists, LAG-3 antagonists, TIM-3 antagonists and
PD-1 antagonists, PDL-1 antagonists and CD-27 agonist. In
accordance with specific embodiments a checkpoint protein modulator
is an antibody to a checkpoint protein, which antibody may be
monoclonal or polyclonal. In another embodiment, immunotherapeutic
agents comprise of indoleameine 2,3 dioxygenase inhibitors. The
methods comprise administration of compositions comprising
guanosine or its pro-drug, or a guanosine precursor, and one or
more of these agents, or administration of a composition of
guanosine or its pro-drug, or a guanosine precursor in conjunction
with these agents. According to yet another embodiment, the present
disclosure provides pharmaceutical compositions effective for
treatment or prevention of cancer and/or infection comprising
guanosine or its pro-drug, or a guanosine precursor in conjunction
with other anti-infective and/or anti-cancer agents. Non-limiting
examples of anti-infective agents include direct antiviral agents
(DAA) targeting viral polymerases, proteases and other structural
and non-structural viral proteins as well as antibacterial agents.
DAAs in market or clinical trials include simeprevir, sofosbuvir,
ledipasavir, ABT-267, ABT-333 for hepatitis C, tenofovir, entecavir
for hepatitis B and atripla for HIV.
[0032] Guanosine may be administered as an oral, parenteral, or
topical formulation, or via any other known method of
administration in the literature. A person of ordinary skill in the
art may prepare formulations according to the requirements and the
procedures reported in the literature without undue
experimentation. Methods of formulating guanosine are not within
the scope of the instant invention.
[0033] In one embodiment, useful dosages of the guanosine can be
determined by comparing in vitro activity and in vivo activity of
guanosine in accepted animal models.
[0034] Methods for the extrapolation of effective dosages in mice
and other animals to humans are known to the art; for example, as
disclosed in U.S. Pat. No. 4,938,949, the entire content of which
is incorporated herein by reference.
EXAMPLES
[0035] The following Examples are set forth to illustrate certain
aspects and features of the instant in results in immune
potentiating effects as it leads to expression of transcription
factors (like NF-.kappa.B and IRF-3) resulting in expression of
inflammatory cytokines and other cellular activation events.
Inhibition or deficiency of purine nucleoside phosphorylase (PNP)
enzyme results in a large elevation of guanosine in the plasma
(FIG. 1. Markert in Immunodeficiency Review (1991) 3:45-81). Hence,
PNP inhibitors (PNPi) NTR001 and NTR002 (see U.S. provisional
patent application Ser. Nos. 61/887,625 and 61/934,094, the entire
disclosures of which are incorporated herein by this reference) was
used in mouse models of vaccine, infectious disease and cancer to
determine the immunepotentiating activity of guanosine. Examples 2,
3, 4 and 5 demonstrate the immune potentiating activity of PNP
inhibitor related to elevation of TLR2 and TLR4 agonist, guanosine,
in various in-vivo mouse models.
Example 1
[0036] Toll-Like Receptor (TLR) Ligand Screening: Activity of the
PNPi, guanosine and inosine on seven different human TLRs (TLR2, 3,
4, 5, 7, 8 and 9) as a potential agonist.
[0037] Background: TLRs play a critical role in the early innate
immune response to invading pathogens by sensing microorganism and
are involved in sensing endogenous danger signals. TLRs recognize
highly conserved structural motifs known as pathogen-associated
microbial patterns (PAMPs), which are exclusively expressed by
microbial pathogens, or as danger-associated molecular patterns
(DAMPs) that are endogenous molecules released from necrotic or
dying cells. Stimulation of TLRs by the corresponding PAMPs or
DAMPs initiates signaling cascades leading to the activation of
transcription factors, such as AP-1, NF-.kappa.B and interferon
regulatory factors (IRFs). Signaling by TLRs results in a variety
of cellular responses including the production of interferons
(IFNs), pro-inflammatory cytokines and effector cytokines that
direct the adaptive immune response.
[0038] Objective: The objective of this study is to determine the
activity of the NTR001, Inosine and guanosine as single agent and
in combination on seven different human TLRs (TLR2, 3, 4, 5, 7, 8
and 9) as a potential agonist.
[0039] Method: TLR stimulation is tested by assessing NF-.kappa.B
activation in HEK293 cells expressing a given TLR. The Secreted
Embryonic Alkaline Phosphatase (SEAP) reporter is under the control
of a promoter inducible by the transcription factor NF-.kappa.B.
This reporter gene allows the monitoring of signaling through the
TLR based on the activation of NF-.kappa.B. The activity of the
compounds is tested on seven different human TLRs (TLR2, 3, 4, 5,
7, 8 and 9) as a potential agonist. The compounds are evaluated at
one concentration and compared to control ligands. This step is
performed in triplicate.
[0040] Results: Guansoine (100 uM) exhibits a significant
stimulatory effect on human TLR2 and TLR4, alone or in combination
with article NTR001 (10 uM) and/or Inosine (100 uM). NTR001,
Inosine, and NTR001+Inosine do not directly exhibit a stimulatory
effect on human TLR2, 3, 4, 5, 7, 8 or 9. (FIG. 2).
[0041] Conclusion: Guanosine is an agonist of TLR2 and TLR4
receptors. Activation of TLR2 and TLR4 results in immune activation
and hence guanosine would be beneficial for the prevention and
treatment of cancer and infections.
Example 2
[0042] Evaluation of PNPi as an adjuvant in Tetanus Toxoid Vaccine
Efficacy Study.
[0043] Background: Aluminium based mineral salts (Alum) have been
used as adjuvants in licensed vaccines for many years. Although
alum has been shown to be safe and effective in traditional
vaccines where eliciting antibody response is necessary, it is a
weak adjuvant for protein subunits, which is one of the major
drawbacks. Another limitation of alum is that it fails to induce
the Th1 response associated with the induction of interferon-gamma
(interferon-g) and cytotoxic T lymphocytes (CTL). Natural control
of infectious diseases such as HIV, malaria and tuberculosis that
cause the most global mortality are either entirely or partially
dependent on the generation of Th1-type immunity. Hence, there is
sufficient interest to develop new vaccine adjuvants. PNP
inhibitors can elevate purine nucleosides, more specifically
guanosine, which is TLR2 and TLR4 agonist (Example 1) and
activation of TLR2 and TLR4 can have adjuvant like effect and
enhance the potency of the vaccines.
[0044] Objective: One objective of this study is to investigate
whether the PNP inhibitors NTR001 and NTR002 can enhance the
potency of the tetanus toxoid vaccine by increasing the antibody
titers. Another objective is to investigate whether the PNP
inhibitors can induce Th1 responses associated with the induction
of interferon-g.
[0045] Method: Tetanus toxoid (TT) was used to vaccinate mice
thrice, two weeks apart. Mice were treated by oral administration
of compounds NTR001 and NTR002 and serum was collected at various
time points for antibody titer and interferon-g analysis.
Treatments are done as shown in Table 1. Mice in Groups 2-6 (Table
2) are vaccinated subcutaneously with 0.1 ml tetanus toxoid vaccine
on DAYS 0, 14 and 28. Mice in Group 1 (Table 1) received no
vaccine. Antibody titers for DAYS 38 are determined by ELISA using
tetanus toxoid coated microtiter plates and anti-mouse conjugate.
Sera from DAY 30 are assayed by ELISA for interferon-g.
TABLE-US-00001 TABLE 1 Group Compound Treatments No. Test Dose
Group Mice Material ROA (mg/kg) Dose Frequency 1 6 Vehicle p.o*.
N/A Days 0, 14, 28 No Vaccine 2 6 Vehicle p.o. N/A Days 0, 14, 28
Vaccinated 3 6 NTR001 p.o. 30 Days 0, 1, 14, 15, 28, 29 4 6 NTR001
p.o. 60 Days 0, 14, 28 5 6 NTR002 p.o. 30 Days 0, 1, 14, 15, 28, 29
6 6 NTR002 p.o. 60 Days 0, 14, 28 *p.o. = oral dose
[0046] Results: Both NTR001 and NTR002 PNP inhibitors significantly
elevated the tetanus toxoid antibody titers compared to the vehicle
treated group. The two dosing regimens, 30 mg/kg (given on the day
of vaccination and the following day with a total of 6 days of
treatment) and 60 mg/kg (given on the day of the vaccination with a
total of 3 days of treatment), were effective in increasing the
antibody titers (FIG. 3). The interferon-g was elevated in the high
dose group (60 mg/kg) for both PNP inhibitors compared to the
vehicle treated group (FIG. 4).
[0047] Conclusion: PNP inhibitors NTR001 and NTR002 elevates
guanosine levels and enhances the potency of the tetanus toxoid
vaccine by increasing the antibody titers, and importantly, the PNP
inhibitors induced Th1 responses associated with the induction of
interferon-g. Thus, guanosine and/or PNP inhibitors represent a
novel approach to enhancing both cellular and humoral immunity and
will be useful as a vaccine adjuvant.
Example 3
[0048] Evaluation of PNPi as anticancer agent in Mouse Melanoma
model.
[0049] Background: Chemotherapy is used to treat diverse cancers,
but chemotherapy alone is insufficient to cure many advanced
cancers owing to side effects and the limited efficacy against
chemo-resistant or relapsing tumors. The need for establishing more
efficacious anticancer strategies led to the development of
immunotherapies. PNP inhibitors can elevate purine nucleosides,
more specifically guanosine, which is TLR2 and TLR4 agonist
(Example 1) and activation of TLR2 and TLR4 can have
immune-potentiating agents that may translate into benefit in
cancer treatment.
[0050] Objective: The objective of this study is to investigate
whether PNP inhibitor, a small molecule immune enhancer,
demonstrates efficacy in reducing tumor volume and/or increasing
survival in a syngeneic mouse model of B16 tumors in C57BL/6
mice.
[0051] Method: Cancer cells were injected subcutaneously in right
flank of each mouse, 1.times.10.sup.4 cells in 0.1 ml PBS with 20%
Matrigel. Treatment with the NTR001 was initiated on day 6 after
injection of tumor cells. Tumor volume and survival were recorded
every 3-4 days. Treatment arms were as shown in Table 2.
TABLE-US-00002 TABLE 2 Group Treatments Group #Mice Material Dose
(mg/kg) ROA Frequency 1 10 Vehicle 0 PO 4 wks (week on/off)* 2 10
NTR001 30 PO 4 wks (week on/off)* 3 10 Cyclophosphamide 100 IP
Single dose 4 10 Cyclophosphamide 100 IP Single dose and NTR001 30
PO 4 wks qd/week on/off* 5. 10 NTR001 5 drinking water 28 days 6 10
Cyclophosphamide 100 IP Single dose NTR001 5 drinking water 28 days
*one week on treatment and one week off treatment
[0052] Results: Treatment with NTR001 resulted in a significant
decrease in tumor volume (FIG. 5). Treatment with NTR001
demonstrated 0-20% survival as single agent (FIG. 6).
Cyclcophosphamide and combination of cyclophosphamide with NTR001
at 5 mg/kg dose demonstrated 30% survival whereas there were no
survivors in the vehicle treated group.
[0053] Conclusion: PNP inhibitor NTR001, which elevates guanosine,
demonstrated significant efficacy in the syngeneic mouse melanoma
model. Combinations of guanosine and/or NTR001 with other
anticancer agents and cancer immunotherapies such as checkpoint
agonist, Yervoy, anti-PD1, etc. will be beneficial. Treatment with
alternate doses and dose schedule is also warranted.
Example 4
[0054] Evaluation of antibacterial activity of PNPi in Mouse Model
of L. Monocytogenes Infection.
[0055] Background: In the past, antiviral and antibacterial
research has focused mainly on viral and bacterial targets. Due to
continued growth of drug resistant organisms the search for
effective and differentiated antiviral and antibacterial therapies
continues. Development of immune-potentiating agent is one of the
strategies being pursued to identify new anti-infective agents. PNP
inhibitors can elevate purine nucleoside, more specifically
guanosine, which is TLR2 and TLR4 agonist (Example 1) and
activation of TLR2 and TLR4 can have immune-potentiating effect and
hence will benefit in viral and bacterial infections.
[0056] Objective: The objective of this study is to investigate
whether PNP inhibitors NTR001 and NTR002 administered by oral and
intraperitoneal routes demonstrate antibacterial effect in the
mouse model of Listeria monocytogenes infection.
[0057] Method: Balb/c mice are infected with 1.times.10.sup.6 CFU
of L. monocytogenes (ATCC Strain35152, hemolytic substrain) by
intravenous route. The treatment of various groups is initiated -4
hr prior to infection except for Groups 3 and 7 for which treatment
was initiated 2 days prior to infection and group 6 and 10 for
which treatment was initiated 5 days prior to infection. Weight and
survival are the end points of the study. Treatment arms were as
shown in Table 3.
TABLE-US-00003 TABLE 3 Treatment Groups Dose Group # mice Treatment
(mg/kg) Route Frequency 1 5 Vehicle 10 ml/kg PO DAYS 0, 1, 2 2 10
Vehicle '' PO DAYS 0, 1, 2 3 '' NTR001 30 PO DAYS -2, -1, 0, 1, 2 4
'' '' '' PO DAYS 0, 1, 2 5 '' '' '' IP DAYS 0, 1, 2 6 '' '' 2 DW
DAY -5 thru end 7 '' NTR002 30 PO DAYS -2, -1, 0, 1, 2 8 '' '' ''
PO DAYS 0, 1, 2 9 '' '' '' IP DAYS 0, 1, 2 10 '' '' 2 DW DAY -5
thru end PO = oral gavage; IP = intraperitoneal injection; DW =
drinking water
[0058] Results: Treatment with NTR001 and NTR002 resulted in a
significant decrease in weight loss (FIGS. 7 and 9) and protection
of 10-20% of the animals (FIGS. 8 and 10).
[0059] Conclusion: PNP inhibitors NTR001 and NTR002, which elevates
guanosine in vivo, demonstrated significant benefit in mouse model
of L. monocytogenes infection. Combinations of guanosine and/or
NTR001 and NTR002 with other antibacterial agents will be
beneficial. Treatment with alternate doses and dose schedule is
also warranted.
Example 5
[0060] Evaluation of PNPi NTR001 in combination with CTLA4-Ab in
Mouse Melanoma model.
[0061] Chemotherapy is used to treat diverse cancers, but
chemotherapy alone is insufficient to cure many advanced cancers,
owing to side effects and the limited efficacy against
chemo-resistant or relapsing tumors. The need for establishing more
efficacious anticancer strategies led to the development of
immunotherapies that can harness host immune system to combat
cancer. Immunotherapeutic agent, CTLA4-ab, has demonstrated
anti-tumor effects both in preclinical and clinical studies. In
this example, PNPi in combination with CTLA4-Ab demonstrates
efficacy in reducing tumor volume and increasing survival in a
syngeneic mouse model of B16 tumors in C57BL/6 mice.
[0062] Method: Cancer cells were injected subcutaneously in right
flank of each mouse, 1.times.10.sup.4 cells in 0.1 ml PBS with 20%
Matrigel. Tumor volume and survival were recorded every 2-3 days.
Treatment arms were as follows.
TABLE-US-00004 TABLE 1 GROUP TREATMENT Group #Mice Test Material
Dose (mg/kg) ROA Frequency 1 10 Vehicle 0 PO* DAYS 0, 3 and 6
Hamster IgG 100, 50, 50 ug** IP*** DAYS 0, 3 and 6 2 10 NTR001 60
PO DAYS 0, 3 and 6 Hamster IgG 100, 50, 50 ug IP DAYS 0, 3 and 6 3
10 CTLA Ab 100, 50, 50 ug IP DAYS 0, 3 and 6 4 10 NTR001 60 PO DAYS
0, 3 and 6 CTLA Ab 100, 50, 50 ug IP DAYS 0, 3 and 6
[0063] Results: Treatment with NTR001 and CTLA4-Ab (clone 9H10) by
itself showed decrease in tumor volume but was not statistically
significant. Combination of NTR001 and CTLA4-Ab demonstrated
significant decrease in tumor volume (FIG. 11). Combination of
NTR001 and CTLA4-Ab and CTLA4-Ab by itself also demonstrated
significant improvement in survival (FIG. 12).
[0064] Conclusion: Combination of PNP inhibitor, NTR001, and
CTLA4-Ab demonstrates significant decrease in tumor volume and
significant improvement in survival. Additional dose and dose
schedule to be pursued.
[0065] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. The scope of the present invention is not intended to be
limited to the above Description, but rather is as set forth in the
appended claims. It will be appreciated that the invention is in no
way dependent upon particular results achieved in any specific
example or with any specific embodiment. Articles such as "a", "an"
and "the" may mean one or more than one unless indicated to the
contrary or otherwise evident from the context. Claims or
descriptions that include "or" between one or more members of a
group are considered satisfied if one, more than one, or all of the
group members are present in, employed in, or otherwise relevant to
a given product or process unless indicated to the contrary or
otherwise evident from the context. The invention includes
embodiments in which exactly one member of the group is present in,
employed in, or otherwise relevant to a given product or process.
The invention also includes embodiments in which more than one, or
all of the group members are present in, employed in, or otherwise
relevant to a given product or process. Furthermore, it is to be
understood that the invention encompasses all variations,
combinations, and permutations in which one or more limitations,
elements, clauses, descriptive terms, etc., from one or more of the
listed claims or from the description above is introduced into
another claim. For example, any claim that is dependent on another
claim can be modified to include one or more elements, limitations,
clauses, or descriptive terms, found in any other claim that is
dependent on the same base claim. Furthermore, where the claims
recite a composition, it is to be understood that methods of using
the composition for any of the purposes disclosed herein are
included within the scope of the invention, unless otherwise
indicated or unless it would be evident to one of ordinary skill in
the art that a contradiction or inconsistency would arise. Methods
can include a step of providing a subject suffering from a targeted
disease or condition, or being at risk of developing a disease or
condition, a step of diagnosing a subject as having a targeted
disease or condition or as being at risk of a disease or condition,
and/or a step of selecting a subject for which an inventive
composition or method would be suitable.
[0066] Where elements are presented as lists, it is to be
understood that each subgroup of the elements is also disclosed,
and any element(s) can be removed from the group. For purposes of
conciseness only some of these embodiments have been specifically
recited herein, but the invention includes all such embodiments. It
should also be understood that, in general, where the invention, or
aspects of the invention, is/are referred to as comprising
particular elements, features, etc., certain embodiments of the
invention or aspects of the invention consist, or consist
essentially of, such elements, features, etc.
[0067] Where ranges are given, endpoints are included. Furthermore,
it is to be understood that unless otherwise indicated or otherwise
evident from the context and understanding of one of ordinary skill
in the art, values that are expressed as ranges can assume any
specific value or subrange within the stated ranges in different
embodiments of the invention, to the tenth of the unit of the lower
limit of the range, unless the context clearly dictates otherwise.
Any particular embodiment, aspect, element, feature, etc., of the
present invention, or any combination thereof, may be explicitly
excluded from any one or more claims whether or not such exclusion
is expressly recited herein. Applicants reserve the right to
proviso out of the claims any specific agent or combination
thereof, whether or not such agent or combination thereof, is
recited herein.
* * * * *