U.S. patent application number 10/911800 was filed with the patent office on 2005-03-31 for infection prophylaxis using immune response modifier compounds.
This patent application is currently assigned to 3M Innovative Properties Company. Invention is credited to Guy, Cynthia A., Hammerbeck, David M..
Application Number | 20050070460 10/911800 |
Document ID | / |
Family ID | 34193164 |
Filed Date | 2005-03-31 |
United States Patent
Application |
20050070460 |
Kind Code |
A1 |
Hammerbeck, David M. ; et
al. |
March 31, 2005 |
Infection prophylaxis using immune response modifier compounds
Abstract
The present invention provides methods of providing prophylaxis
to a subject against an infectious agent. In general, the methods
include topically administering to the respiratory tract of a
subject an IRM compound in an amount effective to reduce infection
by the agent.
Inventors: |
Hammerbeck, David M.;
(Houlton, WI) ; Guy, Cynthia A.; (Little Canada,
MN) |
Correspondence
Address: |
3M INNOVATIVE PROPERTIES COMPANY
PO BOX 33427
ST. PAUL
MN
55133-3427
US
|
Assignee: |
3M Innovative Properties
Company
|
Family ID: |
34193164 |
Appl. No.: |
10/911800 |
Filed: |
August 5, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60493109 |
Aug 5, 2003 |
|
|
|
Current U.S.
Class: |
514/261.1 ;
514/2.4; 514/3.7; 514/4.6 |
Current CPC
Class: |
A61P 31/12 20180101;
A61P 37/02 20180101; A61P 19/02 20180101; A61P 31/04 20180101; A61P
11/06 20180101; A61P 27/16 20180101; A61K 31/437 20130101; A61P
33/00 20180101; A61P 31/00 20180101; A61P 37/04 20180101; A61P
37/08 20180101; A61P 31/16 20180101; A61P 3/12 20180101 |
Class at
Publication: |
514/002 |
International
Class: |
A61K 038/00 |
Claims
What is claimed is:
1. A method of providing prophylaxis to a subject against an
infectious agent comprising topically administering to the
respiratory tract of a subject an IRM compound in an amount
effective to reduce infection by the agent.
2. The method of claim 1 wherein the IRM compound is administered
nasally.
3. The method of claim 1 wherein the IRM compound is administered
orally.
4. The method of claim 1 wherein the IRM compound is administered
from about 72 hours prior to exposure to the infectious agent to
about 72 hours after exposure to the infectious agent.
5. The method of claim 4 wherein the IRM compound is administered
prior to and after exposure to the infectious agent.
6. The method of claim 4 wherein the IRM compound is administered
prior to but not after exposure to the infectious agent.
7. The method of claim 4 wherein the IRM compound is administered
after but not prior to exposure to the infectious agent.
8. The method of claim 4 wherein the IRM compound is administered
in from 1 to about 12 doses.
9. The method of claim 8 wherein the IRM compound is administered
in from 1 to about 3 doses.
10. The method of claim 9 wherein the IRM compound is administered
in 1 dose.
11. The method of claim 10 wherein the infectious agent comprises a
virus, a bacterium, a parasite, or a prion.
12. The method of claim 11 wherein the virus is an influenza
virus.
13. The method of claim 11 wherein the virus is a coronavirus.
14. The method of claim 13 wherein the virus is a SARS coronavirus.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. Provisional
Application Ser. No. 60/493,109, filed Aug. 5, 2003.
BACKGROUND
[0002] Immune response modifiers ("IRMs") include compounds that
possess potent immunomodulating activity including but not limited
to antiviral and antitumor activity. Certain IRMs affect immune
activity by modulating the production and secretion of cytokines.
For example, cytokines that are induced by certain small molecule
IRM compounds include but are not limited to Type I interferons,
TNF-.alpha., IL-1, IL-6, IL-8, IL-10, IL-12, MIP-1, and MCP-1.
Alternatively, certain IRM compounds can inhibit production and
secretion of certain T.sub.H2 cytokines such as IL-4 and IL-5.
[0003] By stimulating certain aspects of the immune system,
suppressing other aspects, or both, IRMs may be used to treat many
diseases and conditions. For example, IRM compounds may be useful
for treating viral diseases, neoplasias, fungal diseases,
neoplastic diseases, parasitic diseases, atopic diseases, and
opportunistic infections and tumors that occur after suppression of
cell-mediated immunity. IRM compounds also may be useful for
promoting healing of wounds and post-surgical scars. Specifically,
but not exclusively, diseases that may be treated using IRM
compounds include, but are not limited to, external genital and
perianal warts caused by human papillomavirus, basal cell
carcinoma, eczema, essential thrombocythaemia, hepatitis B,
multiple sclerosis, neoplastic diseases, atopic dermatitis, asthma,
allergies, psoriasis, rheumatoid arthritis, type I herpes simplex,
and type II herpes simplex.
[0004] Certain formulations of a small molecule imidazoquinoline
IRM compound have been shown to be useful for the therapeutic
treatment of certain cancerous or pre-cancerous lesions (See, e.g.,
Geisse et al., J. Am. Acad. Dermatol., 47(3): 390-398 (2002);
Shumack et al., Arch. Dermatol., 138: 1163-1171 (2002); U.S. Pat.
No. 5,238,944; and WO 03/045391).
[0005] IRM compounds also can modulate humoral immunity by
stimulating antibody production by B cells. Further, various IRMs
have been shown to be useful as vaccine adjuvants (see, e.g., U.S.
Pat. Nos. 6,083,505 and 6,406,705).
[0006] Certain IRM compounds are known to be agonists of at least
one Toll-like receptor (TLR). For example, IRM compounds are known
to be an agonist of TLR6, TLR7, TLR8, or some combination thereof.
Also, for example, certain modified oligonucleotide IRM compounds
are known to be agonists of TLR9.
SUMMARY
[0007] It has been found that certain IRMs can be used to provide
prophylaxis against an infectious agent when topically administered
to the respiratory tract of a subject. Moreover, infection
prophylaxis may be provided regardless of whether the IRM is
administered before or after the subject is exposed to the
infectious agent.
[0008] Accordingly, the present invention provides a method of
providing prophylaxis to a subject against an infectious agent. The
method includes topically administering to the respiratory tract of
a subject an IRM compound in an amount effective to limit infection
by the agent. In some embodiments, the IRM compound can be
administered from about 72 hours prior to exposure to the
infectious agent to about 72 hours after exposure to the infectious
agent.
[0009] Various other features and advantages of the present
invention should become readily apparent with reference to the
following detailed description, examples, claims and appended
drawings. In several places throughout the specification, guidance
is provided through lists of examples. In each instance, the
recited list serves only as a representative group and should not
be interpreted as an exclusive list.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 is a bar graph comparing viral titers in rats after
treatment with vehicle, IFN-.alpha., or IRM compound four hours
before viral challenge.
[0011] FIG. 2 is a bar graph comparing viral titers in rats after
treatment with vehicle, IFN-.alpha., or IRM compound twenty-four
hours and again at four hours before viral challenge.
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS OF THE
INVENTION
[0012] The present invention relates to methods of providing
prophylaxis against an infectious agent by topically administering
an IRM compound to the respiratory system of a subject. In some
embodiments of the present invention, the IRM compound is
administered in a relatively limited dosing regimen that is
specifically initiated upon actual, suspected, or anticipated
possible exposure to an infectious agent. Therefore, certain
methods of the present invention may be particularly suited for
providing infection prophylaxis for those who have been, intend to
be, or suspect that they may be exposed to an infectious agent.
[0013] For purposes of this invention, the following terms shall
have the meanings set forth as follows:
[0014] "Agonist" refers to a compound that can combine with a
receptor (e.g., a TLR) to induce a cellular activity. An agonist
may be a ligand that directly binds to the receptor. Alternatively,
an agonist may combine with a receptor indirectly by, for example,
(a) forming a complex with another molecule that directly binds to
the receptor, or (b) otherwise results in the modification of
another compound so that the other compound directly binds to the
receptor. An agonist may be referred to as an agonist of a
particular TLR (e.g., a TLR6 agonist) or a particular combination
of TLRs (e.g., a TLR 7/8 agonist--an agonist of both TLR7 and
TLR8).
[0015] "Cellular activity" refers to a biological activity (e.g.,
cytokine production), the modulation of which results from an
agonist-receptor interaction or an antagonist-receptor
interaction.
[0016] "Exposure" of a subject to a pathogen refers to actual or
anticipated contact between the subject and the pathogen. "Actual
exposure" refers to exposure in fact, whether known or unknown.
"Anticipated exposure" refers to any level of expected possibility
of being exposed to a pathogen.
[0017] "Induce" and variations thereof refer to any measurable
increase in cellular activity. Thus, as used herein, "induce" or
"induction" may be referred to as a percentage of a normal level of
activity. In the case of a cell product such as, for example, a
cytokine, chemokine, or co-stimulatory marker, "induce" also may
refer to any measurable increase in the amount of the cell product
that is produced in response to a stimulus.
[0018] "Prophylaxis" refers to any degree of limiting an infection
by an infectious agent including (a) preventing or limiting an
initial infection, (b) preventing or limiting the spread of an
existing infection, or both. "Prophylaxis" may be used
interchangeably with "reduce infection" and variations thereof.
[0019] "Respiratory Tract" refers, generally, to the major
structures and passages that permit or provide air flow between the
environment and the lungs of a subject. "Upper Respiratory Tract"
refers, generally, to the nasal cavity, paranasal sinuses,
nasopharynx, oral cavity, pharynx, and larynx. "Lower Respiratory
Tract" refers, generally, to the trachea and lungs, including the
bronchi, bronchioles, and alveoli.
[0020] "TLR-mediated" refers to a biological or biochemical
activity that results, directly or indirectly, from TLR function. A
particular biological or biochemical activity may be referred to as
mediated by a particular TLR (e.g., "TLR6-mediated" or
"TLR7-mediated").
[0021] "Topical" refers to administering the IRM compound to a
surface of the respiratory tract. Topical administration to the
respiratory tract can occur via formulations including but not
limited to an aerosol, a non-aerosol spray, a cream, an ointment, a
gel, a lotion, a mouthwash, and the like.
[0022] The methods of the present invention can provide general
prophylaxis against infection by an infectious agent. One feature
of certain methods of the present invention is that topical
administration of the IRM compound can provide infection
prophylaxis against one or a plurality of known or unknown
infectious agents. Knowledge or even suspicion of the identity of
the particular infectious agent to which one has or might be
exposed is not required. Accordingly, the methods of the present
invention may be particularly useful when exposure to an infectious
agent has occurred, is suspected to have occurred, or is
anticipated, but the identity of the particular infectious agent is
not known. Events in which the methods of the present invention
thus may be particularly useful include, but are not limited to,
outbreaks of new or previously unidentified infectious agents,
biological warfare, bio-terrorism, and exposure to environments
that can have a relatively large number of different infectious
agents (e.g., health care clinics, daycare centers, and the
like).
[0023] In certain embodiments, it may be possible to enhance
infection prophylaxis against a particular infectious agent, if
known, by combining administration of the IRM compound with
separate or co-administration of one or more components of the
infectious agent. However, infection prophylaxis using the methods
of the present invention does not require such combinations or--as
stated above--even knowledge of the identity of the infectious
agent. Another feature of certain embodiments of the present
invention is the ease and relatively limited discomfort associated
with administration of the IRM compound. Topical administration to
the respiratory tract can involve inhalation of an aerosol or
non-aerosol formulation, the inhaled formulation being deposited on
the surface of structures of the upper respiratory tract, the lower
respiratory tract, or both.
[0024] Alternatively, topical administration of the IRM compound to
the respiratory tract may involve contacting a surface of the
respiratory tract--typically, the upper respiratory tract--with a
cream, gel, mouthwash, or the like. The formulation may be left in
place following administration (e.g., a cream or gel applied to a
surface of the oral or nasal cavity) or may be discarded (e.g., a
mouthwash). Administration of the IRM compound can be minimally
invasive, particularly when compared to subcutaneous,
intramuscular, or transdermal vaccination.
[0025] Yet another feature of certain methods of the present
invention is that administration of the IRM compound can be
temporally connected to an exposure event, whether the exposure
event has occurred, is suspected of having occurred, or is expected
to occur. Moreover, infection prophylaxis can be provided without
requiring administration of a prophylactic agent over weeks or
months of treatment. For example, in some embodiments, infection
prophylaxis can be provided by one or two doses of IRM compound
administered about 72 hours or less before an anticipated exposure
to an infectious agent. In some embodiments, infection prophylaxis
can be provided by a single dose of IRM compound administered four
hours before exposure to the infectious agent.
[0026] Alternatively, infection prophylaxis can be provided by
administering the IRM compound after exposure to the infectious
agent has occurred or is suspected to have occurred. For example,
in some embodiments, infection prophylaxis can be provided by one
or two doses of IRM compound administered about 72 hours or less
after an actual or suspected exposure event. In certain
embodiments, infection prophylaxis can be provided with a single
dose of IRM compound provided within 24 hours of the exposure
event. In many embodiments, infection prophylaxis can be provided
without requiring administration of a prophylactic or therapeutic
agent over weeks or months of treatment.
[0027] In certain embodiments, administration of IRM compound
before an anticipated exposure event may be combined with
administration of IRM compound after an actual or suspected
exposure event.
[0028] The features of certain embodiments of the present invention
can be particularly desirable for some applications. For example,
each of: (a) the temporal connection to an exposure event, (b) ease
of administration, and (c) relatively short dosing regimens--alone
and in various combinations--can increase the likelihood and extent
of compliance, thereby increasing the efficacy of the
prophylaxis.
[0029] As another example, the general infection prophylaxis
conferred by administering IRM compound renders certain embodiments
useful for providing infection prophylaxis even when the number and
identity of infectious agent(s) is unknown. Thus, methods of the
present invention may be particularly desirable in circumstances
when, at the time when a standard vaccination would have to be
given to provide infection prophylaxis, it is unclear to which, if
any, infectious agents one may be exposed. Thus, infection
prophylaxis using methods of the present invention can be provided
on an "as needed" basis, thereby reducing the number of
vaccinations administered unnecessarily.
[0030] Additionally, a single course of administering IRM compound
may provide at least as great a scope--and in some cases, even
greater scope--of infection prophylaxis as several vaccinations,
with reduced costs (e.g., less developmental cost, one
administration versus several, etc.), less discomfort, and without
the inherent risk associated with standard vaccinations. All of the
above factors may contribute to increased compliance compared to
traditional vaccinations, which, in turn, may result in a higher
percentage of individuals in a treated population having
efficacious infection prophylaxis.
[0031] Infectious agents against which IRM compounds may provide
infection prophylaxis using methods of the present invention
include, but are not limited to: (a) viruses such as, for example,
variola, HIV, CMV, VZV, rhinovirus, adenovirus, coronavirus
(including, e.g., SARS), influenza, para-influenza, ebola,
hepatitis B, hepatitis C, and West Nile virus; (b) bacteria, such
as those that can cause tuberculosis, anthrax, listeriosis, and
leprosy; (c) other infectious agents such as prions and parasites
(e.g., Leishmania spp.);
[0032] For example, certain methods of the present invention may be
particularly useful for daycare or health care workers who have
been or will be exposed to an infectious agent. Thus, certain
methods of the present invention may secondarily reduce sick time
taken by employees in certain industries.
[0033] As another example, certain methods of the present invention
may be particularly useful for providing prophylaxis against
biological warfare or bio-terrorism agents. Such methods may be
employed by military personnel either (a) before an anticipated
exposure to a biological warfare agent, (b) before entering an area
where a biological warfare agent has previously been used, or (c)
after a suspected or known exposure to a biological warfare agent.
Certain methods of the present invention may be particularly useful
in the event of exposure to a bio-terrorism agent. In some cases,
the methods may be employed by emergency personnel including
police, medical personnel, firefighters, and the like. In other
cases, the methods may be employed by the general population or a
subset of the general population. In either case, certain methods
may provide infection prophylaxis when employed during or after
either (a) exposure to a bio-terrorism agent, or (b) a warning that
a bio-terrorism event may occur.
[0034] As yet another example, the invention may be practiced to
provide infection prophylaxis for those who must--or choose
to--travel to an area experiencing an outbreak of an infectious
agent. For example, health care workers may travel to an area
experiencing such an outbreak to care for those affected by the
infectious agent. Others may choose not to cancel or delay business
or pleasure travel plans. In these cases, practicing the invention
may help provide infection prophylaxis to those traveling into the
area of an outbreak, thereby, for example, facilitating health care
for those affected, minimizing the economic and social cost of the
outbreak, or both.
[0035] Type 1 interferons such as, for example, IFN-.alpha. have
been shown to possess antiviral activity, perhaps by induction of a
nonspecific antiviral state. Administration of IFN-.alpha. can be
effective, as a monotherapy, preventing some viral infections.
However, IFN-.alpha. monotherapy has not gained widespread
acceptance, perhaps at least in part due to associated side-effects
such as, for example, nasal stuffiness, increased sneezing,
bleeding, and nasal erosion.
[0036] FIGS. 1 and 2 summarize data demonstrating the efficacy of
the invention in providing infection prophylaxis against viral
infection. Administration of the IRM compound prior to intranasal
infection with influenza virus dramatically reduced the viral titer
present in the lung and nasal cavity 24 hours after the infection,
even compared to pretreatment with IFN-.alpha..
[0037] Table 3 summarizes data that demonstrates the efficacy of
IRM compound preventing replication of various viruses. IRM
compound was used to inhibit viral replication in each of two ways:
directly and indirectly. Indirect inhibition of viral replication
occurred when IRM compound was used to stimulate a culture of
peripheral blood mononuclear cells (PBMCs). Factors secreted by the
PBMCs into the culture supernatant were collected and tested for
the ability to inhibit viral replication.
[0038] Inhibition of viral replication occurred with PBMC
supernatant solutions at approximate EC.sub.50 values of 0.17 .mu.M
against coronavirus, <0.0015 .mu.M against influenza
A/Panama/2007/99, <0.0015 .mu.M against influenza A/New
Calcdonia/20/99, and 0.15 .mu.M against SARS.
[0039] Direct inhibition of viral replication occurred when IRM
compound itself was tested for the ability to inhibit viral
replication. IRM compound alone added to media was able to inhibit
SARS replication with an approximate EC.sub.50 at 3 .mu.M.
[0040] The results of these studies indicate that IRM compounds
stimulate significant antiviral factors to be secreted from PBMCs
into supernatant solutions. When considered together with the data
from FIGS. 1 and 2, the antiviral properties associated with
practicing the invention are greater than the antiviral properties
of IFN-.alpha. monotherapy. These data support the use of IRM
compounds for providing non-specific prophylactic and/or
therapeutic treatment of viral infections.
[0041] Immune response modifiers ("IRMs") include compounds that
possess potent immunomodulating activity including but not limited
to antiviral and antitumor activity. Certain IRMs modulate the
production and secretion of cytokines. For example, certain IRM
compounds induce the production and secretion of cytokines such as,
e.g., Type I interferons, TNF-.alpha., IL-1, IL-6, IL-8, IL-10,
IL-12, MIP-1, and/or MCP-1. As another example, certain IRM
compounds can inhibit production and secretion of certain T.sub.H2
cytokines, such as IL-4 and IL-5. Additionally, some IRM compounds
are said to suppress IL-1 and TNF (U.S. Pat. No. 6,518,265).
[0042] Certain IRMs are small organic molecules (e.g., molecular
weight under about 1000 Daltons, preferably under about 500
Daltons, as opposed to large biological molecules such as proteins,
peptides, and the like) such as those disclosed in, for example,
U.S. Pat. Nos. 4,689,338; 4,929,624; 4,988,815; 5,037,986;
5,175,296; 5,238,944; 5,266,575; 5,268,376; 5,346,905; 5,352,784;
5,367,076; 5,389,640; 5,395,937; 5,446,153; 5,482,936; 5,693,811;
5,741,908; 5,756,747; 5,939,090; 6,039,969; 6,083,505; 6,110,929;
6,194,425; 6,245,776; 6,331,539; 6,376,669; 6,451,810; 6,525,064;
6,541,485; 6,545,016; 6,545,017; 6,558,951; 6,573,273; 6,656,938;
6,660,735; 6,660,747; 6,664,260; 6,664,264; 6,664,265; 6,667,312;
6,670,372; 6,677,347; 6,677,348; 6,677,349; 6,683,088; 6,756,382;
European Patent 0 394 026; U.S. Patent Publication Nos.
2002/0016332; 2002/0055517; 2002/0110840; 2003/0133913;
2003/0199538; and 2004/0014779; and International Patent
Publication Nos. WO 01/74343; WO 02/46749 WO 02/102377; WO
03/020889; WO 03/043572; WO 03/045391; WO 03/103584; and WO
04/058759.
[0043] Additional examples of small molecule IRMs include certain
purine derivatives (such as those described in U.S. Pat. Nos.
6,376,501, and 6,028,076), certain imidazoquinoline amide
derivatives (such as those described in U.S. Pat. No. 6,069,149),
certain imidazopyridine derivatives (such as those described in
U.S. Pat. No. 6,518,265), certain benzimidazole derivatives (such
as those described in U.S. Pat. No. 6,387,938), certain derivatives
of a 4-aminopyrimidine fused to a five membered nitrogen containing
heterocyclic ring (such as adenine derivatives described in U.S.
Pat. Nos. 6,376,501; 6,028,076 and 6,329,381; and in WO 02/08905),
and certain 3-p-D-ribofuranosylthiazolo[4- ,5-d]pyrimidine
derivatives (such as those described in U.S. Publication No.
2003/0199461).
[0044] Other IRMs include large biological molecules such as
oligonucleotide sequences. Some IRM oligonucleotide sequences
contain cytosine-guanine dinucleotides (CpG) and are described, for
example, in U.S. Pat. Nos. 6,194,388; 6,207,646; 6,239,116;
6,339,068; and 6,406,705. Some CpG-containing oligonucleotides can
include synthetic immunomodulatory structural motifs such as those
described, for example, in U.S. Pat. Nos. 6,426,334 and 6,476,000.
Other IRM nucleotide sequences lack CpG sequences and are
described, for example, in International Patent Publication No. WO
00/75304.
[0045] Other IRMs include biological molecules such as aminoalkyl
glucosaminide phosphates (AGPs) and are described, for example, in
U.S. Pat. Nos. 6,113,918; 6,303,347; 6,525,028; and 6,649,172.
[0046] The IRM compound may be any suitable IRM compound. In some
embodiments of the present invention, the IRM compound may include
a 2-aminopyridine fused to a five membered nitrogen-containing
heterocyclic ring, or a 4-aminopyrimidine fused to a five membered
nitrogen-containing heterocyclic ring. In some embodiments,
suitable IRM compounds include but are not limited to the small
molecule IRM compounds (e.g., molecular weight of less than about
1000 Daltons) described above.
[0047] Certain small molecule IRM compounds--those having a
2-aminopyridine fused to a five membered nitrogen-containing
heterocyclic ring--include, for example, imidazoquinoline amines
including but not limited to substituted imidazoquinoline amines;
tetrahydroimidazoquinolin- e amines including but not limited to
amide substituted tetrahydroimidazoquinoline amines, sulfonamide
substituted tetrahydroimidazoquinoline amines, urea substituted
tetrahydroimidazoquinoline amines, aryl ether substituted
tetrahydroimidazoquinoline amines, heterocyclic ether substituted
tetrahydroimidazoquinoline amines, amido ether substituted
tetrahydroimidazoquinoline amines, sulfonamido ether substituted
tetrahydroimidazoquinoline amines, urea substituted
tetrahydroimidazoquinoline ethers, and thioether substituted
tetrahydroimidazoquinoline amines; imidazopyridine amines including
but not limited to amide substituted imidazopyridine amines,
sulfonamide substituted imidazopyridine amines, urea substituted
imidazopyridine amines, aryl ether substituted imidazopyridine
amines, heterocyclic ether substituted imidazopyridine amines,
amido ether substituted imidazopyridine amines, sulfonamido ether
substituted imidazopyridine amines, urea substituted
imidazopyridine ethers, and thioether substituted imidazopyridine
amines; 1,2-bridged imidazoquinoline amines; 6,7-fused
cycloalkylimidazopyridine amines; imidazonaphthyridine amines;
tetrahydroimidazonaphthyridine amines; oxazoloquinoline amines;
thiazoloquinoline amines; oxazolopyridine amines; thiazolopyridine
amines; oxazolonaphthyridine amines; thiazolonaphthyridine amines;
and 1H-imidazo dimers fused to pyridine amines, quinoline amines,
tetrahydroquinoline amines, naphthyridine amines, or
tetrahydronaphthyridine amines.
[0048] In certain embodiments, the IRM compound may be an
imidazonaphthyridine amine, a tetrahydroimidazonaphthyridine amine,
an oxazoloquinoline amine, a thiazoloquinoline amine, an
oxazolopyridine amine, a thiazolopyridine amine, an
oxazolonaphthyridine amine, or a thiazolonaphthyridine amine.
[0049] In certain embodiments, the IRM compound may be a
substituted imidazoquinoline amine, a tetrahydroimidazoquinoline
amine, an imidazopyridine amine, a 1,2-bridged imidazoquinoline
amine, a 6,7-fused cycloalkylimidazopyridine amine, an
imidazonaphthyridine amine, a tetrahydroimidazonaphthyridine amine,
an oxazoloquinoline amine, a thiazoloquinoline amine, an
oxazolopyridine amine, a thiazolopyridine amine, an
oxazolonaphthyridine amine, or a thiazolonaphthyridine amine.
[0050] As used herein, a substituted imidazoquinoline amine refers
to an amide substituted imidazoquinoline amine, a sulfonamide
substituted imidazoquinoline amine, a urea substituted
imidazoquinoline amine, an aryl ether substituted imidazoquinoline
amine, a heterocyclic ether substituted imidazoquinoline amine, an
amido ether substituted imidazoquinoline amine, a sulfonamido ether
substituted imidazoquinoline amine, a urea substituted
imidazoquinoline ether, a thioether substituted imidazoquinoline
amines, or a 6-, 7-, 8-, or 9-aryl or heteroaryl substituted
imidazoquinoline amine. As used herein, substituted
imidazoquinoline amines specifically and expressly exclude
1-(2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-amine and
4-amino-.alpha.,.alpha.-dimethyl-2-ethoxymethyl-1H-imidazo[4,5-c]quinolin-
-1-ethanol.
[0051] In certain embodiments, the IRM compound can be a
sulfonamide substituted imidazoquinoline amine. In certain specific
embodiments, the IRM compound can be
N-{2-[4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quino-
lin-1-yl]-1,1-dimethylethyl}methane sulfonamide.
[0052] Suitable IRM compounds also may include the purine
derivatives, imidazoquinoline amide derivatives, benzimidazole
derivatives, adenine derivatives, and oligonucleotide sequences
described above.
[0053] In some embodiments, the IRM compound may be a compound
identified as an agonist of one or more TLRs. In some embodiments
of the present invention, the IRM compound may be an agonist of at
least one TLR, preferably an agonist of TLR6, TLR7, or TLR8. The
IRM compound may, in some cases, be an agonist of TLR 9. In certain
embodiments, the IRM compound may be an agonist of at least
TLR7--i.e., may be, for example, a TLR7/8 agonist or a
TLR7-selective agonist.
[0054] As used herein, the term "TLR7/8 agonist" refers to a
compound that acts as an agonist of both TLR7 and TLR8. A
"TLR7-selective agonist" refers to a compound that acts as an
agonist of TLR7, but does not act as an agonist of TLR8. Thus, a
"TLR7-selective agonist" may refer to a compound that acts as an
agonist for TLR7 and for no other TLR, but it may alternatively
refer to a compound that acts as an agonist of TLR7 and a TLR other
than TLR8 such as, for example, TLR6.
[0055] The TLR agonism for a particular compound may be assessed in
any suitable manner. For example, assays for detecting TLR agonism
of test compounds are described, for example, in U.S. patent
application Ser. No. 10/732,563, filed Dec. 10, 2003, and
recombinant cell lines suitable for use in such assays are
described, for example, in U.S. patent application Ser. No.
10/732,796, filed Dec. 10, 2003.
[0056] Regardless of the particular assay employed, a compound can
be identified as an agonist of a particular TLR if performing the
assay with a compound results in at least a threshold increase of
some biological activity mediated by the particular TLR.
Conversely, a compound may be identified as not acting as an
agonist of a specified TLR if, when used to perform an assay
designed to detect biological activity mediated by the specified
TLR, the compound fails to elicit a threshold increase in the
biological activity. Unless otherwise indicated, an increase in
biological activity refers to an increase in the same biological
activity over that observed in an appropriate control. An assay may
or may not be performed in conjunction with the appropriate
control. With experience, one skilled in the art may develop
sufficient familiarity with a particular assay (e.g., the range of
values observed in an appropriate control under specific assay
conditions) that performing a control may not always be necessary
to determine the TLR agonism of a compound in a particular
assay.
[0057] The precise threshold increase of TLR-mediated biological
activity for determining whether a particular compound is or is not
an agonist of a particular TLR in a given assay may vary according
to factors known in the art including but not limited to the
biological activity observed as the endpoint of the assay, the
method used to measure or detect the endpoint of the assay, the
signal-to-noise ratio of the assay, the precision of the assay, and
whether the same assay is being used to determine the agonism of a
compound for both TLRs. Accordingly it is not practical to set
forth generally the threshold increase of TLR-mediated biological
activity required to identify a compound as being an agonist or a
non-agonist of a particular TLR for all possible assays. Those of
ordinary skill in the art, however, can readily determine the
appropriate threshold with due consideration of such factors.
[0058] Assays employing HEK293 cells transfected with an
expressible TLR structural gene may use a threshold of, for
example, at least a three-fold increase in a TLR-mediated
biological activity (e.g., NFKB activation) when the compound is
provided at a concentration of, for example, from about 1 .mu.M to
about 10 .mu.M for identifying a compound as an agonist of the TLR
transfected into the cell. However, different thresholds and/or
different concentration ranges may be suitable in certain
circumstances. Also, different thresholds may be appropriate for
different assays.
[0059] The IRM compound may be provided in a formulation suitable
for topical administration to the respiratory tract of a subject.
The IRM compound may be provided in any suitable form including but
not limited to a solution, a suspension, an emulsion, a dry powder,
or any form of mixture. The IRM compound may be administered in
formulation with any pharmaceutically acceptable excipient,
carrier, or vehicle. The formulation may be administered in any
conventional dosage form for topical delivery to a surface of the
respiratory tract. Such dosage forms include but are not limited to
an aerosol formulation, a non-aerosol spray, a cream, an ointment,
a gel, a lotion, a mouthwash, and the like. Suitable aerosol
formulations are described, for example, in U.S. Pat. No.
6,126,919. Alternative formulations are described, for example, in
U.S. Pat. No. 5,238,944; EP 0 394 026; U.S. Pat. No. 6,365,166; and
U.S. Pat. No. 6,245,776. The formulation may further include one or
more additives including but not limited to adjuvants, penetration
enhancers, colorants, fragrances, moisturizers, thickeners, and the
like.
[0060] In some embodiments, the methods of the present invention
include administering IRM compound to a subject in a formulation
of, for example, from about 0.001% to about typically 10% (unless
otherwise indicated, all percentages provided herein are
weight/weight with respect to the total formulation) to the
subject, although in some embodiments the IRM compound may be
administered using a formulation that provides IRM compound in a
concentration outside of this range. In certain embodiments, the
method includes administering to a subject a formulation that
includes from about 0.01% to about 1.0% IRM compound, for example,
a formulation that includes about 0.375% IRM compound.
[0061] An amount of an IRM compound effective for providing
infection prophylaxis is an amount sufficient to either prevent or
limit (a) an initial infection, or (b) the spread of an existing
infection, or both. One method of determining an amount effective
for providing infection prophylaxis is to determine an amount
effective to reduce--to a statistically significant extent--nasal
or lung titers of the infectious agent 24 hours after the latter
of: (a) exposure to the infectious agent, or (b) administration of
the IRM compound.
[0062] The precise amount of IRM compound in a dose may vary
according to factors known in the art including but not limited to
the physical and chemical nature of the IRM compound, the nature of
the carrier, the intended dosing regimen, the state of the
subject's immune system (e.g., suppressed, compromised,
stimulated), the method of administering the IRM compound, the
species to which the formulation is being administered, and the
infectious agent or agents, if known, to which the subject has been
or is expected to be exposed. Accordingly it is not practical to
set forth generally the amount that constitutes an amount of IRM
compound effective for providing infection prophylaxis for all
possible applications. Those of ordinary skill in the art, however,
can readily determine the appropriate amount with due consideration
of such factors.
[0063] In some embodiments, the methods of the present invention
include administering sufficient IRM compound to provide a dose of,
for example, from about 100 ng/kg to about 50 mg/kg to the subject,
although in some embodiments the methods may be performed by
administering IRM compound in concentrations outside this range. In
some of these embodiments, the method includes administering
sufficient IRM compound to provide a dose of from about 10 .mu.g/kg
to about 5 mg/kg to the subject, for example, a dose of from about
100 .mu.g/kg to about 1 mg/kg.
[0064] The dosing regimen may depend at least in part on many
factors known in the art including but not limited to the physical
and chemical nature of the IRM compound, the nature of the carrier,
the amount of IRM compound being administered, the state of the
subject's immune system (e.g., suppressed, compromised,
stimulated), the method of administering the IRM compound, the
species to which the formulation is being administered, and the
infectious agent or agents, if known, to which the subject has been
or is expected to be exposed. Accordingly it is not practical to
set forth generally the dosing regimen effective to provide
infection prophylaxis for all possible applications. Those of
ordinary skill in the art, however, can readily determine the
appropriate amount with due consideration of such factors.
[0065] In some embodiments of the invention, the IRM compound may
be administered, for example, from once to about twelve times over
the entire course of treatment, although in some embodiments the
methods of the present invention may be performed by administering
the IRM compound at a frequency outside this range. In certain
embodiments, the IRM compound may be administered from once to
about four times over the entire course of treatment. In one
particular embodiment, the IRM compound is administered once. In
another particular embodiment, the IRM compound is administered
twice.
[0066] The IRM compound may be administered before an anticipated
exposure to the infectious agent. In some embodiments, the IRM
compound is administered once per day for a period before an
anticipated exposure to the infectious agent. In certain
embodiments, the IRM compound may be administered once per day for
two days. In other embodiments, the IRM compound may be
administered in a single dose. In some embodiments, at least one
dose of the IRM compound is administered 72 hours or less before an
anticipated exposure to the infectious agent. In one particular
embodiment, for example, the IRM compound is administered in a
single dose, about 4 hours prior to exposure to the infectious
agent.
[0067] In alternative embodiments, the IRM compound may be
administered after a suspected or confirmed exposure to the
infectious agent. In these embodiments, the IRM compound may be
administered once per day for a period after a suspected or actual
exposure to the infectious agent. In certain embodiments, the IRM
compound may be administered once per day for two days. In other
embodiments, the IRM compound may be administered in a single dose.
In some embodiments, at least one dose of the IRM compound is
administered 72 hours or less after the actual or suspected
exposure to the infectious agent. In one particular embodiment, for
example, the IRM compound is administered in a single dose, about 4
hours after exposure to the infectious agent.
[0068] In embodiments in which the IRM compound may be administered
after actual or suspected exposure to the infectious agent,
administration of the IRM can begin prior to the detectable onset
of symptoms resulting from infection by the infectious agent. In
such embodiments, the course of treatment may conclude before the
onset of any symptoms. Alternatively, in some embodiments, the
course of treatment may begin prior to the onset of symptoms and
continue even after the subject experiences symptoms resulting from
infection by the infectious agent. In such cases, it is expected
that the extent of infection will be significantly reduced by
practicing the methods of the invention, thereby limiting the
severity, extent, and/or duration of symptoms.
[0069] In other alternative embodiments, the IRM compound may be
administered before an anticipated exposure to an infectious agent,
and again after the exposure to the infectious agent has, or is
suspected to have, occurred. Again, in such embodiments, the course
of treatment may conclude prior to, or may continue after, the
onset of any symptoms resulting from infection by the infectious
agent.
[0070] The methods of the present invention may be performed on any
suitable subject. Suitable subjects include but are not limited to
animals such as but not limited to humans, non-human primates,
rodents, dogs, cats, horses, pigs, sheep, goats, or cows.
EXAMPLES
[0071] The following examples have been selected merely to further
illustrate features, advantages, and other details of the
invention. It is to be expressly understood, however, that while
the examples serve this purpose, the particular materials and
amounts used as well as other conditions and details are not to be
construed in a matter that would unduly limit the scope of this
invention. Unless otherwise provided, all percentages are given as
w/w %.
[0072] The IRM compound used in the examples is
N-{2-[4-amino-2-(ethoxymet-
hyl)-1H-imidazo[4,5-c]quinolin-1-yl]-1,1-dimethylethyl}methanesulfonamide,
the synthesis of which is described in example 268 of U.S. Pat. No.
6,677,349.
Example 1
[0073] IRM compound was prepared as a 0.375% solution formulation
capable of being nasally administered via a spray pump. The
formulation vehicle was prepared as follows:
1TABLE 1 Excipient w/w % Carboxymethyl cellulose sodium, low
viscosity, USP 0.1 (Spectrum Chemicals and Laboratory Products,
Inc., Gardena, CA,) Benzalkonium chloride, Ph. Eur. (Fluka, Buchs
Switzerland) 0.02 Disodium EDTA, USP (Spectrum Chemicals) 0.1
L-Lactic acid, Purac (Lincolnshire, IL) 1.53 PEG 400, NF (Spectrum
Chemicals) 15 1 N NaOH, NF (Spectrum Chemicals) qs Water qs Total
100.00 pH 4.0
[0074] Carboxymethyl cellulose sodium, low viscosity, (CMC) was
hydrated in water (about 50% of total) for 20 minutes with
stirring. The EDTA was added and dissolved. The CMC/EDTA solution
was mixed with the benzalkonium chloride to form a CMC/EDTA/BAC
solution. Separately, the lactic acid and PEG 400 were mixed with
water. For the IRM formulation, IRM compound was dissolved into the
lactic acid/PEG 400 solution. The CMC/EDTA/BAC solution was mixed
with lactic acid/PEG 400 solution to prepare the Vehicle
formulation. The CMC/EDTA/BAC solution was mixed with lactic
acid/PEG 400/IRM solution to prepare the IRM formulation. 1 N NaOH
was added, as necessary, to adjust each formulation to a pH of 4.0.
Finally, water was added to each formulation to adjust to the final
formulation weight.
Example 2
[0075] Fisher 344 rats (Charles River Laboratories, Raleigh, N.C.)
were divided into six treatment groups. Rats in each group were
infected intranasally with humanized, non-lethal influenza virus.
24 hours after infection, viral titers were measured in nasal
lavage fluid and whole lung homogenates. The influenza virus and
methods for measuring viral titers are described in Burleson, Gary
L., "Influenza Virus Host Resistance Model for Assessment of
Immunotoxicity, Immunostimulation, and Antiviral Compounds,"
Methods in Immunology 2: 181-202, Wiley-Liss Inc., 1995.
[0076] Each of the six treatment groups received a different
pre-infection treatment. Rats in each group received the treatment
indicated in Table 2. The results are summarized in FIG. 1 and FIG.
2.
2TABLE 2 Group Treatment 1 Vehicle formulation (Table 1), 50 .mu.L
(25 .mu.L per nare), 1x* 2 Interferon-.alpha. (rat recombinant
IFN-.alpha., Cat. No. PRP13, Serotec Inc., Raleigh, NC), 10,000 IU,
1x 3 IRM formulation (Table 1), 50 .mu.L (25 .mu.L per nare), 1x 4
Vehicle formulation (Table 1), 50 .mu.L (25 .mu.L per nare), 2x** 5
Interferon-.alpha., 10,000 IU, 2x (Day -1: Product No. RR2030U,
Pierce Biotechnology, Inc., Rockford, IL; Day 0: Serotec Inc. Cat.
No. PRP13) 6 IRM formulation (Table 1), 50 .mu.L (25 .mu.L per
nare), 2x *1x: one dose of treatment provided four hours before
viral infection. **2x: one dose of treatment 24 hours (Day -1)
before viral infection, second treatment four hours before viral
infection (Day 0).
Example 3
[0077] Thirty-five mL of blood from volunteer donors was layered
over 15 mL of room temperature Histopaque-1077 Hybri-Max (Sigma
Chemical Co., St. Lois, Mo.) in an Accuspin tube (Sigma Chemical
Co.) and centrifuged at room temperature, 800.times.g for 15
minutes (Beckman centrifuge). PBMC band (2.sup.nd layer) was
removed and washed twice with Hanks Balanced Salt Solution (HBSS).
Final cell pellet was resuspended in 10% Fetal Bovine Serum and
1.times.Penicillin/Streptomycin in RPMI-1640. Suspension density
was determined using trypan blue and a hemacytometer. Density was
brought to 1.times.10.sup.6 cells/milliliter in growth media, 10
mLs was placed in a T25 tissue culture flask, and suspension was
allowed to equilibrate at 37.degree. C. in a humidified incubator
with 5-7% CO.sub.2 for 1 hour.
[0078] IRM compound was added in growth media to desired final
concentration. Supernatants were harvested after 24 hours and
frozen until tested. Five concentrations of IRM compound in test
media or as PBMC supernatant solutions (sup) were prepared for
testing.
[0079] Viruses:
[0080] Human coronavirus (HcoV), strain OC43: Obtained from the
American Type Culture Collection (ATCC), Manassas, Va. Pools of
virus made up in BSC-1 cells.
[0081] Influenza A/Panama/2007/99 (H.sub.3N.sub.2): Obtained from
the Centers for Disease Control and Prevention (CDC), Atlanta, Ga.
Virus stocks were prepared in MDCK cells.
[0082] Influenza A/New Calcdonia/20/99 (H1N1): Obtained from the
CDC, Atlanta, Ga. Virus stocks were prepared in MDCK cells.
[0083] Severe acute respiratory syndrome (SARS) coronavirus, strain
Urbani. Obtained from the CDC, Atlanta, Ga. A pool was prepared in
African green monkey kidney (Vero 76) cells.
[0084] Cells and Media:
[0085] BSC-1 cells (used for human corona viruses). Growth medium
is MEM with 5% fetal bovine serum (FBS) and 0.1% NaHCO.sub.3. Test
medium is MEM with 2% FBS, 0.1% NaHCO.sub.3 and 50 .mu.g/mL
gentamycin.
[0086] MDCK cells (Maden Darby canine kidney, used for influenza
virus), obtained from the ATCC. Growth medium is MEM with 5% FBS
and 0.1% NaHCO.sub.3. Test medium is MEM without serum, 0.18%
NaHCO.sub.3, 20 .mu.g trypsin/mL, 2.0 .mu.g EDTA/mL, and 50 .mu.g
gentamycin/mL.
[0087] Vero 76 cells (used for SARS coronavirus), obtained from the
ATCC. Growth medium is MEM with 5% fetal bovine serum (FBS) and
0.1% NaHCO.sub.3. Test medium is MEM with 2% FBS, 0.1% NaHCO.sub.3
and 50 .mu.g gentamycin/mL.
[0088] Antiviral Testing Procedure:
[0089] For all viruses, cells were seeded to 96-well flat-bottomed
tissue culture plates (Coming Glass Works, Corning, N.Y.), 0.2
mL/well, at the proper cell concentration, and incubated overnight
at 37.degree. C. in order to establish a cell monolayer. When the
monolayer was established, the growth medium was decanted. Each
dilution of the compound was added in a volume of 0.1 mL to a total
of 5 wells/dilution; approximately 5 min later 0.1 mL of virus in
test medium was added to 3 wells per dilution, and sterile virus
diluent added to the remaining wells. Six wells were exposed to
drug diluent and virus to serve as virus controls, and 6 wells were
exposed to drug diluent and sterile virus diluent to serve as
normal cell controls. The plates were sealed with plastic wrap and
incubated in a humidified incubator with 5% CO.sub.2, 95% air
atmosphere at 37.degree. C. for the time required for viral
cytopathic effect (CPE) to fully develop in the virus control
wells.
[0090] Cells were then examined microscopically for CPE, this being
scored from 0 (normal cells) to 4 (maximal, 100%, CPE). The cells
in the toxicity control wells were observed microscopically for
morphologic changes attributed to cytotoxicity. This cytotoxicity
was also graded at T (100% toxicity, complete cell sloughing from
plate), PVH (60% cytotoxicity), P(40% cytotoxicity), PS (20%
cytotoxicity), and 0 (normal cells). The 50% effective dose
(EC.sub.50) and 50% cytotoxic dose (CC.sub.50) were calculated by
regression analysis of the virus CPE data and the toxicity control
data, respectively. The selective index (SI) for each compound
tested is calculated by the formula:
SI.dbd.CC.sub.50.div.EC.sub.50.
[0091] After the plates were read visually for cytopathology and
toxicity, the cells were then stained with neutral red to verify
the visual determinations. To do this, 0.1 mL of sterile neutral
red (0.034% in physiological saline solution) was added to each
well. The plates were wrapped in aluminum foil to eliminate light
exposure and placed at 37.degree. C. for one to two hours. All
medium was removed and the cells gently washed 2.times.(0.2 mL/well
for each wash) with phosphate buffered saline. The plates were
inverted and allowed to drain on a paper towel. Neutral red was
extracted from the cells by adding 0.2 mL of an equal volume
mixture of absolute ethanol and Sorensen's citrate buffer, pH4, to
each well and placing the plates in the dark at room temperature
for 30 minutes. The contents of each well were mixed gently and the
O.D. values of each well were obtained by reading the plates at 540
nm with a Model EL309 microplate reader (Bio-Tek Instruments, Inc.,
Winooski, Vt.). The EC.sub.50 and CC.sub.50 are calculated by
regression analysis. The SI for each compound tested was again
calculated by the formula: SI.dbd.CC.sub.50.div.EC.sub.50.
[0092] A known active substance was run in the same manner as above
for each batch of compounds tested. For influenza viruses,
ribavirin was used. For human coronavirus (HCOV) and SARS, ALFERON
(Hemispherx Biopharma, Inc., Philadelphia, Pa.) was used. In these
studies, selected cell types were pretreated with supernatant
solutions from IRM-stimulated PBMC cultures or with media
containing solubilized drug and then exposed to different types of
respiratory viruses. Viral replication was then determined by viral
cytopathic effect (CPE) analysis and neutral red (NR) analysis for
confirmation. Cytotoxic concentrations were also determined and
were described by the concentration that produced 50% cell death.
Antiviral activity was only considered to exist when the
concentration for 50% cytotoxicity was at least 3 times greater
than the effective concentration for 50% inhibition in the CPE
assay (i.e, SI>3). Results are summarized in Table 3.
3TABLE 3 Virus Treatment CC.sub.50 (.mu.M) EC.sub.50 (.mu.M) SI
(CC.sub.50/EC.sub.50) Influenza PBMC supernatant >1.5 <0.0015
>1000 A (H3N2) IRM >300 >300 0 Ribavirin >450 24 >19
Influenza PBMC supernatant >1.5 <0.0015 >1000 A (H1N1) IRM
>300 >300 0 Ribavirin >450 14 >32 SARS PBMC supernatant
>1.5 0.15 >10 IRM >150 >3 50 ALFERON >32,000 <32
>1000 HCoV PBMC supernatant >1.5 0.17 >9 IRM >150
>150 0 ALFERON >32,000 32 >1000
[0093] PBMC supernatant solutions from cells stimulated with IRM
compound had significant activity against SARS, both of the
influenza viruses tested, and human coronavirus. IRM compound,
alone, possessed significant direct activity against SARS.
[0094] The complete disclosures of the patents, patent documents
and publications cited herein are incorporated by reference in
their entirety as if each were individually incorporated. In case
of conflict, the present specification, including definitions,
shall control.
[0095] Various modifications and alterations to this invention will
become apparent to those skilled in the art without departing from
the scope and spirit of this invention. Illustrative embodiments
and examples are provided as examples only and are not intended to
limit the scope of the present invention. The scope of the
invention is limited only by the claims set forth as follows.
* * * * *