U.S. patent application number 15/692133 was filed with the patent office on 2017-12-21 for calcium flux agonists and methods therefor.
This patent application is currently assigned to Nant Holdings IP, LLC. The applicant listed for this patent is Nant Holdings IP, LLC. Invention is credited to Oleksandr BUZKO, Justin GOLOVATO, Anne-Laure LE NY, Kayvan NIAZI, Shahrooz RABIZADEH, Patrick Soon-Shiong.
Application Number | 20170360925 15/692133 |
Document ID | / |
Family ID | 50731660 |
Filed Date | 2017-12-21 |
United States Patent
Application |
20170360925 |
Kind Code |
A1 |
NIAZI; Kayvan ; et
al. |
December 21, 2017 |
Calcium Flux Agonists And Methods Therefor
Abstract
Calcium flux agonists are used to enhance a TLR- or NOD-mediated
stimulus and to so increase an immune response of a host and reduce
healing time. Preferred calcium flux agonists include Ca.sup.2+
ionophores and SERCA inhibitors and are used in synergistic
quantities where a ligand to a TLR or NOD receptor is present.
Inventors: |
NIAZI; Kayvan; (Los Angeles,
CA) ; RABIZADEH; Shahrooz; (Los Angeles, CA) ;
GOLOVATO; Justin; (Los Angeles, CA) ; Soon-Shiong;
Patrick; (Los Angeles, CA) ; LE NY; Anne-Laure;
(South Pasadena, CA) ; BUZKO; Oleksandr; (Los
Angeles, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Nant Holdings IP, LLC |
Culver City |
CA |
US |
|
|
Assignee: |
Nant Holdings IP, LLC
Culver City
CA
|
Family ID: |
50731660 |
Appl. No.: |
15/692133 |
Filed: |
August 31, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15264947 |
Sep 14, 2016 |
9750805 |
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15692133 |
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14442392 |
May 12, 2015 |
9463184 |
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PCT/US13/69939 |
Nov 13, 2013 |
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15264947 |
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61725881 |
Nov 13, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 39/39 20130101;
A61K 31/407 20130101; A61K 31/16 20130101; A61K 31/404 20130101;
A61K 31/423 20130101; A61K 31/365 20130101; A61K 47/06 20130101;
A61K 31/66 20130101; A61P 31/04 20180101; A61K 31/12 20130101; A61K
33/24 20130101; A61K 2039/57 20130101; A61K 47/10 20130101; A61P
17/00 20180101; A61K 2039/55511 20130101; A61P 17/02 20180101; A61K
31/341 20130101; A61P 37/04 20180101; A61K 9/0014 20130101; A61K
2039/54 20130101; A61K 31/05 20130101; A61P 43/00 20180101; A61K
39/085 20130101 |
International
Class: |
A61K 39/39 20060101
A61K039/39; A61K 39/085 20060101 A61K039/085 |
Claims
1. A pharmaceutical composition comprising: a calcium flux agonist
in a pharmaceutically acceptable carrier, wherein the
pharmaceutical composition is formulated for topical application to
injured or infected skin; and wherein the calcium flux agonist is
present in an amount that enhances, upon application of the
formulation to the injured or infected skin, an immune response of
an immune competent cell in the injured or infected skin to a
ligand of a pattern recognition receptor.
2. The pharmaceutical composition of claim 1, wherein the calcium
flux agonist is a calcium ionophore or a SERCA inhibitor.
3. The pharmaceutical composition of claim 1, wherein the pattern
recognition receptor is a TLR receptor or a NOD receptor.
4. The pharmaceutical composition of claim 1, wherein the
pharmaceutical composition is formulated for topical application to
injured or infected skin via a solid carrier that is applied to the
injured or infected skin.
5. The pharmaceutical composition of claim 1, wherein the amount of
calcium flux agonist synergistically enhances the immune response
in the presence of the ligand as compared to the immune response in
the absence of the ligand.
6. The pharmaceutical composition of claim 1, wherein the calcium
flux agonist is ionomycin, calcimycin,
2,5-di-tert-butylhydro-quinone (DBHQ), or thapsigargin.
7. The pharmaceutical composition of claim 1, wherein the skin is
infected with a bacterial pathogen that comprises a ligand for a
TLR receptor or a NOD receptor.
8. The pharmaceutical composition of claim 1, wherein the calcium
flux agonist is a calcium ionophore.
9. The pharmaceutical composition of claim 8, wherein the calcium
ionophore is ionomycin, calcimycin, calcium ionophore II, calcium
ionophore IV, calcium ionophore V, or calcium ionophore VI.
10. The pharmaceutical composition of claim 1, wherein the calcium
flux agonist is a SERCA inhibitor.
11. The pharmaceutical composition of claim 10, wherein the SERCA
inhibitor is 2,5-Di-tert-butylhydroquinone, thapsigargin, ruthenium
red, gingerol, paxilline, or cyclopiazonic acid.
12. The pharmaceutical composition of claim 1, wherein the
pharmaceutical composition is formulated for topical application to
injured or infected skin, either prophylactically prior to surgery
or other manipulation, or therapeutically.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 15/264,947, filed Sep. 14, 2016, which is a divisional of U.S.
application Ser. No. 14/442,392, filed May 12, 2015 and issued Jun.
10, 2016 as U.S. Pat. No. 9,463,184, which is in turn a U.S.
national phase application of PCT international application serial
number PCT/US 2013/69939, filed Nov. 13, 2013, which claims
priority to U.S. provisional application Ser. No. 61/725,881, filed
Nov. 13, 2012, all of which contents are incorporated by reference
herein.
FIELD OF THE INVENTION
[0002] The field of the invention is compositions and methods of
pharmaceutical compounds, and especially of topically applied
calcium flux agonists.
BACKGROUND OF THE INVENTION
[0003] Various calcium flux agonists are known in the art and have
vastly different origins. For example, certain compounds act as
ionophores and typically raise intracellular calcium levels by
importing calcium ions into the cell, while other compounds raise
intracellular calcium levels by increasing calcium secretion from
intracellular stores like the endoplasmic reticulum and
mitochondria.
[0004] Among various other known ionophores, calcimycin (A23187)
and ionomycin are natural products (from the Gram+ bacteria
Streptomyces chartreusensis and Streptomyces conglobatus,
respectively) which were initially described as antibiotics decades
ago. More specifically, both A23187 and ionomycin demonstrate
direct antibiotic activity against a variety of potential microbial
pathogens as was reported in U.S. Pat. No. 3,960,667 and U.S. Pat.
No. 3,873,693. Unlike classical antibiotics (e.g., penicillin or
tetracycline) that inhibit biochemical pathways specific to
microbial proliferation such as bacterial cell wall synthesis or
bacterial ribosome function, A23187 and ionomycin belong to a
separate class of antibiotic compounds that bind divalent cations
as substrates with relatively high specificity. For example, A23187
binding affinity is characterized by
Mn.sup.2+>Ca.sup.2+=Mg.sup.2+>Sr.sup.2+>Ba.sup.2+ while
ionomycin is characterized by
Ca.sup.2+>Mg.sup.2+>Ba.sup.2+>Sr.sup.2+.
[0005] On the other hand, thapsigargin is a typical ER secretagogue
and can be characterized as a sesquiterpene lactone. Thapsigargin
is isolated from a plant (Thapsia garganica) and acts as a
non-competitive inhibitor of various sarco/endoplasmic reticulum
Ca2+ ATPases (SERCA). Thapsigargin:SERCA binding demonstrates an
affinity constant in the low picomolar range and is toxic to both
dividing and non-dividing cells. In animals, limited skin contact
with thapsigargin can result in inflammation and chronic repetitive
topical exposure can result in non-malignant papilloma formation
when used in conjunction with a strong DNA damaging agent. In
addition to thapsigargin (and structural analogs like
thapsigargicin, etc.), other SERCA inhibitors include cyclopiazonic
acid (CPA) and 2,5-di-tert-butylhydro-quinone (DBHQ).
[0006] Difficult-to-treat skin infections represent an emerging
public health concern for several reasons including the
ever-increasing number of diabetic patients suffering from chronic
skin ulcers, the presence of antibiotic resistant microbial flora
(e.g., methicillin-resistant Staphylococcus aureus or MRSA), and
the increasing frequency of outbreaks of necrotizing fasciitis (or
"flesh-eating bacteria disease"). In addition to bacteria, many
fungi and viruses can also cause significant infections of the
skin. Although antibiotics remain the best treatment option for
many of these disorders, it would be desirable to stimulate a
patient's own cells and their associated functions to further
improve patient recovery from ongoing infections and possibly even
to stimulate long-term immunity against the offending organism to
limit pathogenesis upon subsequent encounter.
[0007] In higher organisms, epithelial tissues including the skin
serve as a critical barrier against pathogen-based, chemical, and
physical insults. The epidermal layer of skin is comprised of
keratinocytes, immune cells such as Langerhans cells and CD8.sup.+
T cells, Merkel cells and melanocytes. In addition to Langerhans
cells, which are a subtype of dendritic cells (DC) responsible for
disease surveillance, keratinocytes (which account for .about.95%
of the total epidermal population) also serve as immune sentinels
through their expression of various pattern recognition receptors
such as members of the toll-like receptor (TLR) proteins, C-type
lectin receptors (CLR), inflammasomes, etc.
[0008] Activation of these receptors in keratinocytes by their
cognate ligands results in the release of both chemokines such as
interleukin-8 (IL-8), CLL2, and CLL20 to recruit other immune cells
as well as immune-regulating cytokines such as TGF-.beta. and
IL-10. Below the epidermis, the dermis is comprised of a larger
variety of cell types including various subsets of CD4.sup.+ (Th1,
Th2, Th17, etc) and non-classical (e.g. .gamma..delta., NK--, etc.)
T lymphocytes, various antigen presenting cells (such as
macrophages and dermal and plasmacytoid DC), mast cells and
fibroblasts many of which express the various pattern recognition
proteins described above. As such, both epidermal and dermal cells
cooperate to prevent microbial invasion or other physical insults
that could lead to significant disease.
[0009] The TLR proteins (TLR 1-10 in man) are type I membrane
proteins which serve as pattern recognition receptors (PRR) for
specific classes of ligands associated with disease and tissue
homeostasis and as such are expressed on a variety of immune and
non-immune cell types alike. As with all receptors, TLR signal
transduction is triggered by ligand binding, and ligands may be
grouped into pathogen-associated molecular patterns (or PAMP) and
disease-associated molecular patterns (DAMP).
[0010] TLR-recognized PAMP include bacterial
lipoproteins/lipopeptides, liposaccharides, flagellin, and
unmethylated CpG DNA, fungal cell wall components (e.g. zymosan),
and viral nucleic acids (dsRNA, ssRNA, and CpG DNA), while DAMPs
are derived directly from the host, can occur in the absence of
infection, and are recognized predominantly by only two members of
the TLR family of proteins (either TLR2 and/or TLR4).
[0011] Once activated by their appropriate ligands, TLR initiate
signaling cascades which serve both to limit the extent of
infection/disease and to trigger tissue repair. With regards to the
latter, TLR ligand recognition results in the up-regulation of
antimicrobial activity and causes the activation/maturation of
various immunological players to complete the destruction of the
invading pathogen. For example, TLR activation can result in the
release of reactive oxygen species (ROS), antimicrobial peptides,
and upregulation of phagocytic function in innate cells such as
macrophages, neutrophils, keratinocytes, etc. In addition to their
role in combating disease, TLRs are also involved in tissue repair
and regeneration as demonstrated in various models of tissue damage
(including those induced by chemical, radiation, surgical, and
infectious injury).
[0012] A further class of pattern recognition receptors is formed
by the NOD-like receptor protein family, and includes NOD1 and NOD2
as the most prominent members. NOD1 and NOD2 are intracellular
pattern recognition receptors, which are similar in structure to
resistance proteins of plants, and mediate innate and acquired
immunity by recognizing bacterial molecules containing
D-glutamyl-meso-diaminopimelic acid and muramyl dipeptide,
respectively. Following stimulation by their respective ligands,
both NOD proteins interact with RIPK2 through respective
recognition domains, which ultimately results in activation of the
transcription factor NF-.kappa.B.
[0013] Previous efforts to characterize the response of intact skin
to topical application of small molecule agonists of signal
transduction (such as the protein kinase C agonist TPA/PMA or
sustained calcium flux agonists like A23187, ionomycin, or
thapsigargin) demonstrated a spectrum of downstream results. For
example, topical application of the phorbol ester TPA caused
vasodilation, microvascular permeability alterations, inflammatory
cell recruitment, and the release of pro-inflammatory factors from
various cell types. In contrast to PKC agonists, topical treatment
with sustained calcium flux agonists (SCFA) resulted in skin
inflammation and hyper-proliferation. On the other hand, exposure
of cells to the TLR ligand (LPS) in the presence of relatively high
quantities of a calcium ionophore (ionomycin) did not lead to any
measurable immunostimulatory effect (Proc Natl Acad Sci USA. 2012
Jul. 10; 109(28): 11282-7). When applied individually at relatively
high dosages, calcium ionophores and TLR ligands are known to
stimulate differentiation or to activate dendritic cells as
discussed in US 2012/0272700A1 and US 2013/0183343A1.
[0014] Therefore, while numerous compositions and uses for calcium
flux agonists and/or ligands for TLR/NOD are known in the art,
there is still a need to provide compositions and methods that
provide improved immunomodulatory activity.
SUMMARY OF THE INVENTION
[0015] The present inventive subject matter is drawn to various
compositions and methods of calcium flux agonists in which these
compounds are used to modulate an immune response to a TLR- or
NOD-mediated event, and especially to synergistically increase
responses to TLR and/or NOD ligand binding. Notably, synergistic
effect with respect to immune stimulation is observed where the
calcium flux agonist is present in suboptimal concentrations.
[0016] Viewed from a different perspective, the inventors
contemplate use of calcium flux agonists to amplify intracellular
Ca.sup.2+ dependent TLR/NOD signaling. Therefore, and among other
suitable uses, especially contemplated uses include
pre-conditioning of tissue to allow for an enhanced response to a
TLR/NOD-dependent stimulus (e.g., infection or injury), and/or
treatment of topical or other infections of the epithelium with
calcium flux agonists to amplify intracellular Ca.sup.2+ dependent
TLR/NOD signaling. Consequently, the inventors also contemplate
topical pharmaceutical and cosmetic compositions for prevention
and/or treatment of skin infections and other conditions (e.g.,
diabetic ulcers) to stimulate wound healing, and/or to decrease
scarring.
[0017] In one aspect of the inventive subject matter, the inventors
contemplate use of a calcium flux agonist to enhance an immune
response of an immune competent cell to a ligand of a pattern
recognition receptor. For example, where the calcium flux agonist
is a calcium ionophore, preferred agonists include ionomycin,
calcimycin, beauvericin, calcium ionophore II, calcium ionophore
IV, calcium ionophore V, and calcium ionophore VI, and where the
agonist is a SERCA inhibitor, preferred SERCA inhibitors include
DBHQ (2,5-di-tert-butylhydroquinone), thapsigargin, ruthenium red,
gingerol, paxilline, and cyclopiazonic acid. Among other phenomena
observable, the enhanced immune response is typically evidenced by
an increased IL-8 secretion and/or an increased activation of
NF-.kappa.B signaling, and it is preferred that the immune response
is synergistically enhanced by the calcium flux agonist in the
presence of the ligand, particularly where the calcium flux agonist
is used at a suboptimal concentration (with respect to a maximum
effect of the calcium flux agonist in the absence of the
ligand).
[0018] With respect to suitable cells it is generally contemplated
that the cells are immune competent cell, which will preferably
reside in the epidermal or dermal layer of skin. Moreover, the
immune competent cells will generally express a TLR receptor and/or
a NOD receptor as the pattern recognition receptor. Thus, ligands
will typically include PAMP and DAMP ligands.
[0019] Consequently, the inventors also contemplate use of a
calcium flux agonist in the manufacture of a topically applied
medicament to enhance an immune response in skin. In such uses, the
immune response is generally associated with binding of a (PAMP or
DAMP) ligand to a pattern recognition receptor in an immune
competent cell, and the pattern recognition receptor is most
typically a TLR receptor or a NOD receptor. As noted above,
preferred calcium flux agonists include calcium ionophores (e.g.,
ionomycin, calcimycin, calcium ionophore II, calcium ionophore IV,
calcium ionophore V, or calcium ionophore VI), and SERCA inhibitors
(e.g., 2,5-di-tert-butylhydroquinone, thapsigargin, ruthenium red,
gingerol, paxilline, or cyclopiazonic acid, etc.), and/or the
calcium flux agonist is present in the medicament at a
concentration such that the agonist is present in the cell in the
presence of the ligand at a suboptimal concentration.
[0020] Consequently, and viewed from a different perspective, the
inventors also contemplate use of a calcium flux agonist in the
manufacture of a topically applied medicament to enhance wound
healing of skin. In such uses, the calcium flux agonist is
typically present in an amount effective to activate NF-.kappa.B
signaling of cells in the wound. Such uses are particularly
advantageous where the wound is infected with a bacterial pathogen
(typically expressing or producing a ligand for a TLR receptor
and/or a NOD receptor).
[0021] In yet another aspect of the inventive subject matter, the
inventors also contemplate a pharmaceutical composition that
comprises a calcium flux agonist (e.g., calcium ionophore or a
SERCA inhibitor) in a pharmaceutically acceptable carrier, wherein
the pharmaceutical composition is formulated for topical
application to injured or infected skin, and wherein the calcium
flux agonist is present in an amount that enhances, upon
application of the formulation to the injured or infected skin, an
immune response of an immune competent cell in the injured or
infected skin to a ligand of a pattern recognition receptor (e.g.,
TLR receptor or NOD receptor). While such pharmaceutical
compositions may be formulated, for example, as a liquid, a spray,
or a gel, it is also contemplated that the pharmaceutical
composition is formulated for topical application to injured or
infected skin via a solid carrier that is applied to the injured or
infected skin (e.g., wound dressing or band aid impregnated with
the pharmaceutical composition). For example, the skin may be
infected with a (e.g., bacterial) pathogen that comprises a ligand
for a TLR receptor or a NOD receptor.
[0022] In particularly preferred pharmaceutical compositions, the
amount of the calcium flux agonist (e.g., ionomycin, calcimycin, or
thapsigargin) synergistically enhances the immune response in the
presence of the ligand as compared to the immune response in the
absence of the ligand.
[0023] Therefore, the inventors also contemplate a method of
enhancing an immune response of a cell expressing a pattern
recognition receptor (e.g., TLR receptor or a NOD receptor) to a
ligand (e.g., PAMP) of the pattern recognition receptor, wherein
the method includes a step of exposing the cell in the presence of
the ligand to a calcium flux agonist (e.g., SERCA inhibitor or
calcium ionophore) in an amount that enhances the immune
response.
[0024] Most typically, the immune response is evidenced as an
increased IL-8 secretion and/or an increased activation of
NF-.kappa.B signaling, and it is especially preferred that the
ligand and the calcium flux agonist are present in synergistic
quantities. While not limiting to the inventive subject matter, it
is further preferred that the cell is located in a dermal layer or
epidermal layer of skin (e.g., injured or infected skin).
[0025] Viewed from a different perspective, the inventors therefore
also contemplate a method of treating injured or infected skin in
which in one step the injured or infected skin is contacted with a
calcium flux agonist (e.g., calcium ionophore or a SERCA inhibitor)
in an amount that enhances an immune response (e.g., increased IL-8
secretion or increased activation of NF-.kappa.B signaling) and
that increases wound healing.
[0026] Thus, the inventors also contemplate a method of modulating
an immune response to a TLR- or NOD-mediated stimulus in a tissue
(e.g., epithelial tissue) or cell, in which in one step the tissue
or cell is contacted (e.g., topically applied) with a calcium flux
agonist at a concentration effective to increase an intracellular
calcium concentration as compared to an intracellular calcium
concentration without the calcium flux agonist. Most typically, the
concentration of the calcium flux agonist (e.g., calcium ionophore
or SERCA inhibitor) is effective to modulate the immune response to
the TLR-mediated stimulus. In some aspects of the inventive subject
matter, the TLR- or NOD-mediated stimulus is a bacterial, viral, or
fungal infection, while in other aspects the TLR- or NOD-mediated
stimulus is a tissue injury.
[0027] Various objects, features, aspects and advantages of the
inventive subject matter will become more apparent from the
following detailed description of preferred embodiments, along with
the accompanying drawing figures in which like numerals represent
like components.
BRIEF DESCRIPTION OF THE DRAWING
[0028] FIGS. 1A-1C are graphs depicting the dose response curves
for various calcium flux agonists (1A: A23187; 1B: Ionomycin; 1C:
Thapsigargin) with respect to strength of NF-.kappa.B signaling and
IL-8 production.
[0029] FIG. 2 is an exemplary graph depicting synergistic effect of
various calcium flux agonists with an exemplary TLR ligand
(Pam2CYS).
[0030] FIGS. 3A-3E show various graphs illustrating the synergistic
effect of exemplary calcium flux agonists (FIG. 3A: A23187; FIG.
3B: Ionomycin; FIG. 3C: Cyclopiazonic acid; FIG. 3D: DBHQ; FIG. 3E:
Thapsigargin) and exemplary TLR and NOD ligands for respective TLR
and NOD receptors with respect to strength of IL-8 production.
[0031] FIG. 4 shows graphs illustrating the effect of different
Ca.sup.2+ modulating compounds on the synergistic action with
respect to NF-.kappa.B activation.
[0032] FIGS. 5A-5E show graphs depicting the effect of live
bacterial growth on .kappa.B-LUC THP-1 reporter cells (FIG. 5A),
augmentation of NF-.kappa.B response by thapsigargin (FIG. 5B), and
the extracellular requirement of Ca.sup.2+ for calcium flux agonist
signal amplification (FIGS. 5C-5E).
[0033] FIG. 6 shows graphs illustrating the requirement of human
primary monocytes and granulocytes for extracellular Ca.sup.2+ for
optimal killing of phagocytosed S. aureus.
[0034] FIG. 7 shows photographs of wound healing using selected
calcium flux agonists.
[0035] FIG. 8 shows graphs indicating wound size and bacterial
burden following wound treatment with control and selected calcium
flux agonists.
[0036] FIGS. 9A-9C show graphs for antibiotic sensitivity of S.
aureus against A23187, ionomycin, and CPA, respectively.
[0037] FIG. 10 is a graph depicting lack of direct antibiotic
effect of thapsigargin against S. aureus.
[0038] FIG. 11 is a graph showing improved killing of intracellular
S. aureus by thapsigargin-treated cells.
DETAILED DESCRIPTION
[0039] The inventors have discovered that calcium flux agonists
that increase intracellular free Ca.sup.2+ concentration
(especially calcium ionophores and SERCA inhibitors) can be
effectively used to modulate and/or enhance the immune response of
a host to a TLR- or NOD-mediated event. In a particularly notable
aspect, calcium flux agonists synergistically increased host
responses to TLR and/or NOD ligand binding where the calcium flux
agonist is present in a substantial suboptimal concentration.
[0040] Indeed, the inventors have discovered as further detailed
below that innate immune cells help to control infection by
recognizing S. aureus bacteria or their shed products (and other
pathogens and pathogen fragments) in a Ca.sup.2+-dependent manner
to become activated, which can then be effectively augmented by
exposure of the affected cells to suboptimal doses of Ca.sup.2+
flux agonists. Since many common pathogen (e.g., S. aureus)
products are predominantly recognized by TLR and NOD receptors, the
inventors investigated and also confirmed that pathogen products
are indeed activating and that such activation can be further
significantly enhanced using numerous Ca.sup.2+ flux agonists
(which can be abrogated or substantially reduced by calcium
chelating agents). Moreover, the inventors also demonstrated that
Ca.sup.2+ is a critical factor in human monocytes and granulocytes
(especially neutrophils) for effective killing of phagocytosed
bacteria. Such findings also directly translated into a mammalian
(murine) model of skin infection and wound healing following live
infection.
[0041] In one aspect of the inventive subject matter, the inventors
therefore contemplate various compositions and methods for
(typically topical) treatment and/or prophylaxis that are effective
in stimulating the host cells' capability of immune response to a
pathogen and that are effective in reducing scarring and
time-to-closure of a topical wound, especially where the wound is
infected with one or more pathogens that express or otherwise
comprise a ligand for TLR and/or NOD. For example, the inventors
contemplate topical application of a SERCA inhibitor (e.g.,
thapsigargin) and/or an ionophore (e.g., ionomycin and/or A23187
(calcimycin) to prevent or treat a superficial skin infection
(e.g., bacterial infection).
[0042] In that context, the inventors discovered that topical
application of SERCA inhibitors and/or ionophores greatly decreased
bacterial burden in superficial skin infections (e.g., S. aureus)
with concomitant improvement in wound healing kinetics in a live
animal infection model. Remarkably, the antibacterial effect was
not (in the case of the SERCA inhibitor thapsigargin) or not
entirely (in the case of ionophores) attributable to a direct
antibiotic effect where the calcium flux agonist acted as a biocide
against the pathogen, but rather to the role of the calcium flux
agonists as an immunological adjuvant. While not wishing to be
bound by any theory or hypothesis, the inventors contemplate that
the antibacterial effect may be due to synergistic activation of
cytokine release and activation of the nuclear factor-.kappa.B
(NF-.kappa.B) signaling pathway when calcium flux agonists were
provided in the presence of TLR or NOD ligands.
[0043] Even more remarkable, very strong synergy between TLR and/or
NOD activation and calcium flux agonists (ionophores/SERCA
inhibitors) was observed for IL-8 production and activation of
NF-.kappa.B signaling. For example, human promonocytic THP-1 cells
were incubated in the presence or absence of a suboptimal dose of
the TLR2 ligand Pam2CSK4 and compared to similarly treated cells
which also received suboptimal doses of ionophore (here: A23187 and
ionomycin). Interestingly and as further shown in more detail
below, the inventors found that both agonists significantly
increased the amount of IL-8 produced beyond the levels that would
be produced if the individual responses were cumulative, thus
indicating true synergy. In another example, .kappa.B-LUC THP-1
cells (a THP-1-derived transfectant line harboring an
NF-.kappa.B-driven luciferase expression cassette) were cultivated
in the presence or absence of a suboptimal dose of the TLR2 ligand
Pam2CSK4 and compared to similarly treated cells that also received
suboptimal doses of various SERCA inhibitors. Notably, and as also
shown in more detail below, the inventors found that all of the
tested agonists (here: thapsigargin, cyclopiazonic acid)
significantly increased the amount of luciferase produced beyond
levels that would be produced if individual responses were
cumulative and so once more indicate synergy.
[0044] In that context it should be noted that NF-.kappa.B impacts
adaptive immunity through its involvement in mediating cellular
activation, inflammatory cytokine secretion, proliferation, and
survival, while IL-8 is a powerful chemo-attractant for immune
cells like neutrophils, an innate immune cell type critical for
antibacterial and antifungal responses. Interestingly, ligand
activation of TLR4 in the presence of the Ca.sup.2+ flux inducing
agent ionomycin results in the synergistic production of
arachidonic acid-derived eicosanoid lipids in a mouse macrophage
line, indicating the possibility of combining Ca.sup.2+ flux
inducing agents with TLR ligands for preventative and curative
effects.
[0045] The inventors therefore contemplate in one aspect of the
inventive subject matter that ionophores (e.g., A23187 and/or
ionomycin) will produce a synergistic effect in vivo with
infection/disease associated TLR and/or NOD ligands to alter in
situ immune cell response and repair processes. Such effects are
readily ascertained as the inventors demonstrated in more detail
below by use of topically-applied formulations containing various
ionophores to alter the course of superficial skin infection, for
example, induced by S. aureus as a model system, using bacterial
burden and wound size as indicators of prophylactic and/or
therapeutic success.
[0046] Similarly, the inventors also contemplate in another aspect
of the inventive subject matter that thapsigargin (and various
other SERCA inhibitors) produce a synergistic effect in vivo with
infection/disease associated TLR and/or NOD ligands to alter in
situ immune cell and repair processes in vivo. As noted above, such
effects are readily ascertained as the inventors demonstrated in
more detail below by use of topically-applied formulations that
contain various SERCA inhibitors (and especially thapsigargin) to
alter the course of superficial skin infection, for example,
induced by S. aureus as a model system, using bacterial burden and
wound size as indicators of prophylactic and/or therapeutic
success.
[0047] Therefore, it should be appreciated that the inventors
especially contemplate use of a calcium flux agonist to enhance the
immune response of one or more immune competent cells to a ligand
of a pattern recognition receptor. Most typically, suitable immune
competent cells are cells that are part of the cellular immune
system (e.g., a B-cell, a T-cell, an antigen-presenting cell or
innate sentinel cell, and especially dendritic cells, macrophages,
mast cells, monocytes, etc.), and it is generally preferred that
such immune competent cells are in the dermal or epidermal layer of
skin. Consequently, the inventors also contemplate the use of a
calcium flux agonist to manufacture a topically applied medicament
to enhance an immune response and/or wound healing (e.g., reduce
time-to-closure) in skin. In such uses, it is generally
contemplated that the immune response is associated with binding of
a ligand to a pattern recognition receptor in an immune competent
cell. Among other pattern recognition receptors, especially
suitable pattern recognition receptors include those of the TLR and
NOD families, and especially TLR1, TLR2, TLR3, TLR4, TLR5, TLR6,
TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and TLR13, as well as NOD1,
NOD2, NOD3, NOD4, NOD5, and CIITA. Therefore, it should be
appreciated that the ligand may vary considerably, and contemplated
ligands include all ligands known to bind to the TLR and/or NOD
receptors, and most preferably pathogen-associated molecular
pattern ligands (PAMP) and damage/disease-associated molecular
pattern ligands (DAMP). Consequently, suitable PAMP ligands will
include various lipopeptides, glycolipids, lipoteichoic acid, heat
shock proteins, beta-glucans, fibrinogen, heparin sulfate
fragments, hyaluronic acid fragments, RNA (and esp. ssRNA), DNA
(and esp. CpG sequences), profiling, etc. Likewise, suitable DAMPs
will include various nuclear and/or cytosolic proteins, and
especially heat shock proteins, HMGB1, DNA, RNA, and fragments of
proteins derived from extracellular matrix.
[0048] In yet another aspect of the inventive subject matter, the
inventors also contemplate a method of enhancing an immune response
of a cell (that expresses one or more of the pattern recognition
receptors noted above) to a ligand of the pattern recognition
receptor. As noted above, the nature of the ligand will
predominantly depend on the type of receptor, and all receptors and
ligands as discussed above are suitable for contemplated methods.
In especially preferred methods, the cell is exposed in the
presence of the ligand to a calcium flux agonist in an amount that
enhances the immune response. Of course, it should be noted that
the ligand may be delivered (e.g., together with the calcium flux
agonist) as part of a treatment regimen. However, and more
typically, the ligand will be provided by a pathogen that is
present in or near the cell, or provided by the host as a DAMP.
While not limiting to the inventive subject matter, the enhanced
immune response will typically be characterized by at least one of
an increased IL-8 production and an increased NF-.kappa.B signaling
(e.g., increased expression of a gene under the control of
NF-.kappa.B in the presence of the calcium flux agonist as compared
to the expression of the same gene in the absence of the calcium
flux agonist). As is readily evident from the experimental details
below, the enhanced immune response is typically a synergistic
increase.
[0049] Therefore, the inventors also contemplate a method of
(prophylactic) treating injured or infected skin by contacting the
injured or infected skin with a calcium flux agonist in an amount
that enhances an immune response and that increases wound healing
as further shown in more detail below. Viewed from a different
perspective, it should thus be appreciated that one or more calcium
flux agonists can be employed to modulate an immune response to a
TLR- or NOD-mediated stimulus in a tissue or cell.
[0050] Contemplated Calcium Flux Agonists
[0051] Compounds contemplated suitable for use herein are generally
deemed to be useful for prophylaxis and/or treatment of various
infectious diseases and trauma, and especially with infectious
disease of the skin or other epithelium, particularly where the
host response to the disease and/or trauma is associated with a TLR
or NOD response pathway. Therefore, it is generally preferred that
the compounds according to the inventive subject matter will be
calcium flux mediators (and especially agonists) that lead to an at
least temporary, and more typically sustained increase of
intracellular calcium.
[0052] Therefore, especially preferred compounds include ionophores
and SERCA inhibitors well known in the art. Most preferably,
suitable ionophores are calcium ionophores, and especially
ionomycin, calcimycin, calcium ionophore II, calcium ionophore IV,
calcium ionophore V, or calcium ionophore VI, while particularly
preferred SERCA inhibitors include 2,5-di-tert-butylhydroquinone
(DBHQ), thapsigargin, ruthenium red, gingerol, paxilline, or
cyclopiazonic acid. It should also be noted that while the above is
a list of preferred compounds, the list is not exhaustive, and
individual compounds may be combined to form a mixture of two or
more calcium flux agonists (e.g., to provide an extracellular and
intracellular agonist), and/or additional compounds may be
added.
[0053] Of course, it should be appreciated that (where appropriate)
contemplated compounds may have one or more asymmetric centers or
groups that may give rise to isomeric, tautomeric, or other steric
isoforms (e.g., R-, and/or S-configuration, E/Z configuration,
tautomeric isoforms, enantiomers, diastereomers, etc.), and each of
such forms and mixtures thereof are expressly contemplated herein.
Additionally, it should be appreciated that contemplated calcium
flux agonists may be chemically modified to achieve a desired
physicochemical parameter (e.g., solubility in aqueous solvents,
membrane permeability, selectivity towards Ca.sup.2+, etc.)
Therefore, suitable calcium flux agonists may be fully synthetic,
semi-synthetic, or isolated from host strains producing such
ionophores.
[0054] Moreover, contemplated compounds may also be converted to
prodrugs to increase delivery and/or target specificity to an
affected tissue or organ. The term "prodrug" as used herein refers
to a modification of contemplated compounds, wherein the modified
compound exhibits less pharmacological activity (as compared to the
unmodified compound) and wherein the modified compound is converted
within a target cell or target organ back into the unmodified form.
For example, conversion of contemplated compounds into prodrugs may
be useful where the active drug is too toxic for safe systemic
administration, or where the contemplated compound is poorly
absorbed by the digestive tract, or where the body breaks down the
contemplated compound before reaching its target. There are
numerous methods for the preparation of prodrugs known in the art,
and all of those are contemplated herein. For example, suitable
prodrug approaches are described in Prodrugs (Drugs and the
Pharmaceutical Sciences: a Series of Textbooks and Monographs) by
Kenneth B. Sloan (ISBN: 0824786297), or in Hydrolysis in Drug and
Prodrug Metabolism: Chemistry, Biochemistry, and Enzymology by
Bernard Testa (ISBN: 390639025X), which are to the appropriate
extent incorporated by reference herein.
[0055] Similarly, it should be noted that contemplated compounds
may also be less active in the form as described herein, and be
more active as metabolite or metabolites that are formed in vivo.
For example, contemplated compounds may be transformed by the
hepatic phase I and/or phase II enzyme system, or by gastric
acidity, intestinal microbial environment, or other biochemical
process. Thus, suitable compounds may be oxidized, hydroxylated,
ligated to a carbohydrate, etc.
[0056] Contemplated Compositions
[0057] It is generally contemplated that contemplated calcium flux
agonists are provided in a composition that is suitable for
delivery to a cell or tissue. Therefore, suitable compositions will
include liquid compositions, gels, solid compositions, all of which
may be associated or coupled to a carrier, or applied directly to
the cell or tissue. Most typically, such compositions will include
an aqueous solvent or otherwise pharmaceutically acceptable carrier
together with one or more calcium flux agonists. In particularly
preferred aspects of the inventive subject matter, the
pharmaceutical composition is formulated for topical application to
an injured or infected tissue, more preferably epithelial tissue,
and most preferably skin. Moreover, it is generally preferred that
the calcium flux agonist is present in an amount that enhances,
upon application of the formulation to the cell or tissue, an
immune response of an immune competent cell in the injured or
infected cell or tissue to one or more ligands of a pattern
recognition receptor (typically TLR and/or NOD receptor). It is
still further preferred that the calcium flux agonist is present in
an amount that synergistically enhances the immune response in the
presence of the ligand as compared to the immune response in the
absence of the ligand.
[0058] Therefore, it should be appreciated that the compositions
according to the inventive subject matter may be administered using
various routes, including topically, nasally, by inhalation,
orally, parenterally, etc. wherein the term "parenteral" as used
herein includes subcutaneous, intravenous, intramuscular,
intraarticular, intrasynovial, intrathecal, intrahepatic,
intralesional, and intracranial administration (typically injection
or infusion). Most preferably, however, the compositions are
administered topically in a liquid, gel, or solid form.
[0059] For example, the pharmaceutical compositions of this
invention may be administered topically to areas or organs readily
accessible by topical application, including the eye, the skin, the
lower intestinal tract, or areas exposed during surgical
intervention. There are numerous topical formulations known in the
art, and all of such formulations are deemed suitable for use
herein.
[0060] For example, contemplated compositions may be formulated in
a suitable ointment containing the active component suspended or
dissolved in one or more carriers. Carriers for topical
administration of the compounds of this invention include mineral
oil, liquid petrolatum, white petrolatum, propylene glycol,
polyoxyethylene, polyoxypropylene compound, emulsifying wax and
water. Alternatively, the pharmaceutical compositions can be
formulated in a suitable lotion or cream containing the active
components suspended or dissolved in one or more pharmaceutically
acceptable carriers. Suitable carriers include mineral oil,
sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl
alcohol, 2-octyldodecanol, benzyl alcohol and water. As at least
some of the active compounds are highly hydrophobic, it is
contemplated that the formulation will take into account relatively
poor solubility and thus may be prepared as an emulsion, as
nanovesicular particles, or administered in a hydrophobic base
under occlusion.
[0061] Alternatively, contemplated formulations may also be
injected into skin or other site of administration. Most
preferably, sterile injectable forms of contemplated compounds will
include emulsions, aqueous solutions, or oleaginous suspensions.
These suspensions may be formulated according to techniques known
in the art using suitable dispersing or wetting agents and
suspending agents. The sterile injectable preparation may also be
prepared as a sterile injectable solution or suspension in a
non-toxic parenterally acceptable diluent or solvent, for example
as a solution in 1,3-butanediol. Among other acceptable vehicles
and solvents, especially contemplated liquids include water,
Ringer's solution, and isotonic sodium chloride solution. In
addition, sterile, fixed oils may be employed as a co-solvent or
suspending medium (e.g., natural or synthetic mono- or
diglycerides). Fatty acids may also be used, and suitable fatty
acids include oleic acid and its glyceride derivatives, olive oil,
castor oil, especially in their polyoxyethylated versions. Such oil
solutions or suspensions may further contain a long-chain alcohol
diluent or dispersant.
[0062] In another example, contemplated compounds may be orally
administered in any orally acceptable dosage form, including
capsules, tablets, aqueous suspensions, or solutions. In the case
of tablets for oral use, all pharmaceutically acceptable carriers
(e.g., lactose, corn starch, etc) are deemed suitable. Similarly,
various lubricating agents may be added (e.g., magnesium stearate).
For oral administration in a capsule form, useful diluents include
lactose and dried corn starch.
[0063] With respect to the amount of contemplated compounds in the
composition, it should be recognized that the particular quantity
will typically depend on the specific formulation, active
ingredient, and desired purpose. Therefore, it should be recognized
that the amount of contemplated compounds will vary significantly.
However, it is generally preferred that the compounds are present
in a minimum amount effective to deliver prophylactic and/or
therapeutic effect in vitro and/or in vivo. Viewed from another
perspective, the calcium flux agonist is typically present in an
amount effective to activate NF-.kappa.B signaling and/or increase
IL-8 production of cells in vitro, or in a wound or otherwise
diseased (e.g., infected) tissue.
[0064] Moreover, it is generally preferred that the calcium flux
agonist is provided to the cell or tissue such that the agonist
will be present in the cell (in the presence of the TLR or NOD
ligand) at a suboptimal concentration with respect to a maximum
effect of the calcium flux agonist in the absence of the ligand. In
this context, it should be noted that the suboptimal concentration
of the calcium flux agonist is a concentration that is well below
the maximum response (with respect to NF-.kappa.B and/or IL-8
production) obtainable with the calcium flux agonist. Thus,
sub-optimal concentration of the calcium flux agonist will be
concentration that will provide equal or less than 80%, equal or
less than 70%, equal or less than 60%, between 20-60%, or between
10-50% of the dosage that provides a maximum effect (with respect
to NF-.kappa.B and/or IL-8 production) for that calcium flux
agonist. For example, as can be seen from the experimental data
below, an exemplary suboptimal concentration for thapsigargin is
between 10-50 nM (e.g., about 20 nM), which is well below a maximum
effect obtainable for thapsigargin as can be seen from FIG. 1C.
Likewise, an exemplary suboptimal concentration for A23187 is
between 100-500 nM (e.g., about 316 nM), which is well below a
maximum effect obtainable for A23187 as can be seen from FIG. 1A,
and an exemplary suboptimal concentration for ionomycin is between
300 nM-5 .mu.M (e.g., about 1 .mu.M), which is well below a maximum
effect obtainable for ionomycin as can be seen from FIG. 1B. Viewed
from another perspective, suboptimal concentrations will therefore
be characterized as concentrations below which an acute toxic
effect can be observed for a cell or tissue exposed to the calcium
flux agonist.
[0065] As is shown in more detail below, maximum response and
suboptimal concentrations can be readily determined using an IL-8
ELISA test and/or a luminescence test. In preferred aspects of the
inventive subject matter, the suboptimal concentration will be a
concentration at which the response is equal or less than 70% of
the maximum response, more typically equal or less than 50% of the
maximum response, and most typically equal or less than 30% of the
maximum response. Thus, suboptimal concentrations will be in the
range of between 1-20% of the maximum response, between 20-40% of
the maximum response, between 40-60% of the maximum response,
between 60-80% of the maximum response, or between 80-95% of the
maximum response. Likewise, it is noted that the ligand may also be
present in a suboptimal concentration, and the suboptimal
concentration will be a concentration at which the response to the
ligand is equal or less than 70% of the maximum response, more
typically equal or less than 50% of the maximum response, and most
typically equal or less than 30% of the maximum response for the
ligand alone. Thus, suboptimal concentrations will be in the range
of between 1-20% to 1-40% of the maximum response, between 20-40%
to 20-80% of the maximum response, between 30-70% of the maximum
response, between 40-80% of the maximum response, or between 50-95%
of the maximum response for the ligand alone.
[0066] Consequently, in at least some embodiments, contemplated
compounds are present in an amount of between about 0.1 ng/ml to
about 100 mg/ml, more typically in an amount of between about 10
ng/ml to about 10 mg/ml, and most typically between about 1
.mu.g/ml to about 100 .mu.g/ml. Where the formulation is a solid or
a gel, contemplated compounds will be present in an amount of
between about 0.1 ng/g to about 100 mg/g, more typically in an
amount of between about 10 ng/g to about 10 mg/g, and most
typically between about 1 .mu.g/g to about 100 .mu.g/g. Viewed from
a different perspective, the calcium flux agonist will typically be
present in the formulation at a concentration of between 0.1 .mu.M
to 10 .mu.M, between 10 .mu.M to 100 .mu.M, between 100 .mu.M to 1
mM, between 1 mM to 10 mM, or between 10 mM to 100 mM.
Additionally, and with respect to the concentration of the calcium
flux agonist at the cell or tissue ("effective exposure
concentration"), it is generally preferred that the effective
exposure concentration will be between 1 pM and 1 mM, and most
preferably at a suboptimal concentration for the respective calcium
flux agonist. Therefore, thapsigargin will typically have an
effective exposure concentration of between 1 nM to 1 .mu.M, more
typically between 1 nM to 500 nM, and most typically between 1 nM
to 50 nM, while DBHQ and CPA will typically have an effective
exposure concentration of between 100 nM to 500 .mu.M, more
typically between 500 nM to 100 .mu.M, and most typically between 1
.mu.M to 50 .mu.M. On the other hand, ionomycin and A23187 will
typically have an effective exposure concentration of between 10 nM
to 100 .mu.M, more typically between 100 nM to 50 .mu.M, and most
typically between 200 nM to 10 .mu.M.
[0067] Therefore, suitable amounts of contemplated compounds will
be in the range of 0.1 .mu.g per dosage unit to about 0.5 gram per
dosage unit, more typically between 10 .mu.g per dosage unit to
about 0.05 gram per dosage unit, and most typically between 50
.mu.g per dosage unit to about 100 mg per dosage unit. Thus,
suitable dosages will be in the range of about 0.01 .mu.g/kg and
100 mg/kg, more typically between 1 .mu.g/kg and 50 mg/kg, and most
typically between 10 .mu.g/kg and 10 mg/kg.
[0068] With respect to dosage units, it is generally contemplated
that the dosage unit will be such that the dosage unit is effective
to achieve the desired therapeutic and/or prophylactic effect.
Viewed from a different perspective, a dosage unit will preferably
contain sufficient quantities of the calcium flux agonist to
(preferably synergistically) increase IL-8 production and/or
NF-.kappa.B signaling.
[0069] It should further be appreciated that while contemplated
compounds and compositions may be applied topically or in a
pharmaceutical composition (e.g., cream, ointment, etc.) numerous
alternative methods of application to the affected tissue are also
deemed suitable. For example, contemplated compositions may be
coupled to or incorporated into a carrier that is directly and
reversibly applied to the site of treatment or that is implanted or
otherwise placed in proximity or contact with the treatment site.
For example, contemplated compounds and compositions may be
incorporated into one or more portions of topically applied and
removable carriers (e.g., bandages, gauze, etc.) or into covering
films that may or may not dissolve or erode (e.g., via
biodegradable drug-eluting polymers). Alternative carriers include
beads or biodegradable drug-eluting polymers that are implanted
wherein contemplated compounds and compositions may be part of the
surface of the implanted device or coated onto such devices.
[0070] Dose Response of Calcium Flux Agonists
[0071] The inventors performed several studies to identify the
effect of calcium flux agonists on various components of an immune
response, and particularly on the effect of calcium flux agonists
on IL-8 production and activation of NF-.kappa.B signaling. As can
be readily taken from FIGS. 1A-1C, both ionophores and SERCA
inhibitor produced significant increases in IL-8 production and
activation of NF-.kappa.B signaling. The dose response is observed
over relatively large windows for ionophores (e.g., A23187 and
ionomycin) and over at least two orders of magnitude for SERCA
inhibitors (e.g., thapsigargin).
[0072] More specifically, the dose response of ionophores on
NF-.kappa.B and IL-8 is shown in FIGS. 1A-1B using a
monocyte-derived cell line to treatment and A23187 and ionomycin in
the absence of externally added TLR or NOD ligands. A human
monocytic cell line (THP-1) transfectant possessing a stably
integrated NF-.kappa.B/luciferase reporter cassette was treated
with increasing concentrations of A23187 and ionomycin and
NF-.kappa.B reporter-based responses were monitored using
luminometry (RLU=relative light units, filled circles) while human
IL-8 (hIL-8) release was quantified using a sandwich ELISA (empty
circles). FIG. 1C depicts the dose response to thapsigargin on
NF-.kappa.B and IL-8. Here, the human monocytic cell line (THP-1)
transfectant possessing a stably integrated NF-.kappa.B/luciferase
reporter cassette was treated with increasing concentrations of
thapsigargin and NF-.kappa.B reporter-based responses were
monitored using luminometry (RLU=relative light units, filled
circles) while human IL-8 (hIL-8) release was quantified using a
sandwich ELISA (empty circles). As can be taken from both flux
agonist groups, dose response is increasing over orders of
magnitude at significantly increasing NF-.kappa.B and IL-8
responses.
[0073] Human IL-8 activity: A titration of thapsigargin (A.G.
Scientific, San Diego, Calif.), ionomycin (Sigma-Aldrich, St.
Louis, Mo.), and the calcium ionophore A23187 (Sigma-Aldrich) was
added to a 96-well plate (Thermo Matrix, Waltham, Mass.) seeded
with 150,000 THP-1 cells (ATCC TIB-202) grown in RPMI 1640
(Cellgro, Herndon, Va.) supplemented with 10% FBS (Thermo HyClone,
Waltham, Mass.) and 1% penicillin-streptomycin-fungizone (Thermo
HyClone). 24 hours after addition, 25 uL of supernatant was removed
and assayed for human IL-8 using the Meso Scale Discovery Human
IL-8 Tissue Culture Kit (MSD, Rockville, Md.) per manufacturer's
protocol. The results were analyzed using the Meso Scale Discovery
SECTOR Imager 2400 and normalized to a titration of human IL-8
supplied by the MSD Kit.
[0074] NF-.kappa.B activation: A titration of thapsigargin (A.G.
Scientific), ionomycin, and the calcium ionophore A23187 was added
to a 96-well plate (Thermo Matrix) seeded with 150,000 THP-1
.kappa.B-LUC THP-1's (THP-1 cells containing the stable integration
of a NF-.kappa.B Luciferase Reporter) grown in RPMI 1640 (Cellgro,
Herndon, Va.) supplemented with 10% FBS (Thermo HyClone) and 1%
penicillin-streptomycin-fungizone (Thermo HyClone). 24 hours after
addition, 25 uL of Bright-Glo Luciferase Assay System (Promega,
Madison, Wis.) was added to each well and analyzed for luciferase
activity using Perkin Elmer TopCount NXT (Perkin Elmer, Waltham,
Mass.).
[0075] Calcium Flux Agonists as Synergistic Agents with Pattern
Recognition Receptors
[0076] In an initial set of experiments, the TLR2 receptor ligand
Pam2CYS was tested in the presence of various concentrations of
calcium flux agonists and the inventors unexpectedly found that
suboptimal and relatively low concentrations of a variety of
calcium flux agonists in the presence of a TLR ligand produced a
dramatic synergistic response.
[0077] For example, FIG. 2 is a graph depicting a typical
synergistic effect selected calcium flux agonists with an exemplary
TLR ligand. Here, the ionophores A23187 and ionomycin, as well as
the SERCA inhibitor thapsigargin synergize with the TLR2 ligand
(Pam2CYS) in the activation of cytokine release from the human
THP-1 promonocytic leukemia cell line. It should be especially
noted that while the TLR2 ligand alone provided for about 5 ng/ml
IL-8 response, addition of a suboptimal dose of the calcium flux
agonists (e.g. 20 nM for thapsigargin, 316 nM for A23187, and 1
.mu.M for ionomycin) produced more than 10-fold quantities of IL-8
production in the cells.
[0078] To investigate if that observation was also true for other
pattern recognition receptors, and especially for TLR and NOD
receptors and other ligands, the inventors tested numerous TLR and
NOD receptors and various ligands. Notably, as is evidenced from
FIGS. 3A-3E, substantial synergy was observed across a large
selection of types and classes of calcium flux agonists, as well as
various TLR and NOD receptors and ligands, thus establishing that
TLR- and NOD-mediated signals can be (typically synergistically)
enhanced with suboptimal dosages of calcium flux agonists.
[0079] More specifically, FIG. 3A shows graphs depicting the
synergistic effect of suboptimal doses of A23187 to augment
cellular response for toll-like receptor (TLR) family members
(TLR2, TLR4, TLR2, TLR9), and NOD family members (NOD1, NOD2). FIG.
3B shows graphs depicting the synergistic effect of suboptimal
doses of ionomycin to augment cellular response for toll-like
receptor (TLR) family members (TLR2, TLR4, TLR2, TLR9), and NOD
family members (NOD1, NOD2). FIG. 3C shows graphs depicting the
synergistic effect of suboptimal doses of cyclopiazonic acid to
augment cellular response for toll-like receptor (TLR) family
members (TLR2, TLR4, TLR2), and NOD family members (NOD1, NOD2).
FIG. 3D shows graphs depicting the synergistic effect of suboptimal
doses of 2,5-di(tert-butyl) hydroquinone (DBHQ) to augment cellular
response for toll-like receptor (TLR) family members (TLR2, TLR4,
TLR2), and NOD family members (NOD1, NOD2). FIG. 3E shows graphs
depicting the synergistic effect of suboptimal doses of
thapsigargin to augment cellular response for toll-like receptor
(TLR) family members (TLR2, TLR4, TLR5, TLR9), and NOD family
members (NOD1, NOD2). The doses used in this experiment were 20 nM
for thapsigargin, 316 nM for A23187, 1 .mu.M for ionomycin, 10
.mu.M for cyclopiazonic acid, and 31.6 .mu.M for DBHQ.
[0080] As can be readily appreciated, the synergistic effect with
respect to IL-8 production was observed for all of the tested TLR
and NOD family members and suitable ligands, while all of the
tested calcium flux agonists were used at suboptimal concentration.
Thus, a clear pattern of adjuvant activity of calcium flux agonists
on the immune response, and especially on IL-8 production and
NF-.kappa.B signaling is evident where the immune response is
associated with a TLR- or NOD-mediated event.
[0081] Nature of Calcium Flux
[0082] The inventors further investigated if the nature of calcium
flux was of significance as increased intracellular calcium may
have different origins. To that effect, the inventors used various
calcium signaling modulators to investigate the nature of the
calcium flux and contribution. As can be readily taken from the
data shown, extracellular calcium flux into the cell was critical
for synergistic amplification of calcium signaling. More
specifically, FIG. 4 illustrates the effects of different Ca.sup.2+
modulating compounds on synergy with respect to NF-.kappa.B
activation. It should be noted that EGTA is an extracellular
chelator, and that KN-62 is an inhibitor of Cam Kinase II
(important for T-cell activation) that does not have chelator
effect. As can be readily appreciated from FIG. 4, the
extracellular Ca.sup.2+ scavenger EGTA reduces synergistic signal
down to levels in line with what is achieved by TLR ligand alone
while treatment with the Cam Kinase II inhibitor KN-62 does not
alter reporter gene expression levels.
[0083] Previously, it was demonstrated that TLR2 and NOD2 play
important roles in the sensing and control of S. aureus infections
by mammalian cells. Given that THP-1-derived .kappa.B-LUC THP-1
cells were activated by the generic TLR2 and NOD2 ligands Pam2Cys
and MDP, respectively, and that this activation could be further
augmented through the addition of calcium flux agonists (FIGS.
3A-3E), the ability of these cells to recognize bona fide S.
aureus-derived products was evaluated as follows. Briefly, a
culture of logarithmically dividing S. aureus was washed three
times with RPMI 1640 (Cellgro). Concurrently, THP-1 .kappa.B-LUC
THP-1's (THP-1 cells containing the stable integration of a
NF-.kappa.B luciferase reporter gene) cultured in RPMI 1640
(Cellgro) supplemented with 10% FBS (Thermo HyClone, Waltham,
Mass.) and 1% penicillin-streptomycin-fungizone (Thermo HyClone)
were washed three times with RPMI 1640 (Cellgro) and transferred to
the bottom compartment of a 24-well 0.4 .mu.m cutoff transwell
plate (Corning) at a concentration of 1.2 million/mL. Dilutions of
the washed S. aureus culture were then added to the top chamber of
the transwell system at the indicated initial multiplicities of
infection (MOI). The cells were incubated for 18 hours at
37.degree. C. at which time aliquots of 60,000 cells were
transferred to wells in a 384-well white bottom plate (Thermo). To
evaluate luciferase gene expression, Bright-Glo Luciferase Assay
Reagent (Promega) was added to each well and the plates were
analyzed for luciferase activity using the Perkin Elmer TopCount
NXT system (Perkin Elmer). Consistent with the previous results,
the .kappa.B-LUC THP-1 reporter cells demonstrate a MOI-dependent
increase in reporter activity when co-cultured with live bacteria
in an culture system which allows for passive diffusion of
bacterial products but prevents direct contact between whole S.
aureus bacteria and the .kappa.B-LUC THP-1 reporter cells (FIG.
5A).
[0084] To determine whether the recognition of S. aureus-produced
products could be further augmented through modulation of calcium
flux, .kappa.B-LUC THP-1 reporter cells were incubated in the
presence of a 1:100 dilution (V/V) of sterile filtered S. aureus
conditioned (i.e. spent) or unused sterile media and treated with a
suboptimal dose (20 nM) of thapsigargin or dimethyl sulfoxide
(DMSO) vehicle control as described above. Briefly, conditioned
media was produced by culturing S. aureus in RPMI with 10% fetal
bovine serum overnight in a shaking incubator at 37.degree. C.
following which the bacteria were pelleted by centrifugation and
the supernatants filter-sterilized using a 0.2 .mu.m filter (VWR
Radnor, Pa.). The .kappa.B-LUC THP-1 cells were incubated in the
presence of the conditioned/spent media for 18 hours prior to
luciferase assay as described above. Interestingly, the response of
the .kappa.B-LUC THP-1 reporter cells to the conditioned/spent
bacterial culture was significantly enhanced in the presence of
thapsigargin (FIG. 5B).
[0085] To determine whether the observed enhancement in activation
of .kappa.B-LUC THP-1 cells by bacterial products achieved by
suboptimal thapsigargin treatment was also true for the calcium
ionophores A23187 and ionomycin, the experiment was repeated using
these ionophores at the suboptimal doses of 316 nM and 1 .mu.M,
respectively. In all three cases, recognition of S. aureus
conditioned/spent media was significantly enhanced in the presence
of any of the calcium flux agonists (FIGS. 5C-5E). Perhaps of equal
interest, the calcium flux-induced synergy could be inhibited by
the addition of the extracellular chelator EGTA (1 mM) as can be
readily seen form the Figures.
[0086] Role of Extracellular Calcium in Human Cells
[0087] To investigate whether the response in human cells is
affected by the nature of the calcium flux, the inventors tested
primary human monocytes and granulocytes infected with S. aureus.
As can be seen from the data in FIG. 6, there is a clear
requirement of human primary monocytes and granulocytes for
extracellular Ca.sup.2+ for optimal killing of phagocytosed S.
aureus. Briefly, primary human monocytes were purified from a
commercially available peripheral blood mononuclear cell
preparation (Human Buffy Coat Leukocytes, Innovation Research,
Novi, Mich.) using the Dynabeads Untouched Human Monocyte kit (Life
Technologies, Grand Island, N.Y.) per manufacturer's suggestions.
Primary human granulocytes were purified by separating normal donor
blood into two fractions by Ficoll-Paque density centrifugation by
manufacturer's directions (GE Healthcare Life Sciences, Piscataway,
N.J.). The peripheral blood mononuclear cell fraction was then
discarded and the erythrocyte/granulocyte fraction was subjected to
Red Blood Cell Lysis Buffer treatment per manufacturer's protocol
(Biolegend, San Diego, Calif.) followed by two washes in Hank's
Buffered Salt Solution (Cellgro) to remove the lysis reagent and
cell debris. Next, a culture of S. aureus was grown overnight in
tryptic soy broth (Cellgro). The resulting culture was used to
inoculate a new culture to ensure log phase growth prior to the
start of the experiment. The bacterial culture was pelleted
(2000.times.g for 10 min) and washed three times with Hank's
Buffered Salt Solution (Cellgro). Primary monocytes/granulocytes
prepared as above were then supplemented with 10% human serum
(Innovative Research, Novi, Mich.) and 1 mM EGTA (Sigma-Adrich, St.
Louis, Mo.) where applicable and chilled on ice for 30 minutes.
After the 30 minutes, S. aureus (MOI of 3.33) was added and the
mixtures were incubated on ice for an additional 20 minutes to
synchronize binding. Tubes were then incubated for 20 minutes at
37.degree. C. After 20 minutes, 10 U/mL of lysostaphin
(Sigma-Aldrich) was added to each tube to eliminate
non-internalized S. aureus. At the desired time points (20, 30, 60
minutes) 20 uL aliquots were removed and diluted in 10 mL of water.
The dilution tubes were vortex and allowed to sit at room temp for
10 minutes to ensure proper lysis of the primary cells. After 10
minutes, the dilution tubes were centrifuged for 10 minutes at
2000.times.g with the remaining S. aureus was concentrated 20 fold.
The S. aureus-containing samples were then plated in triplicate on
tryptic soy agar (Cellgro) and incubated overnight at 37.degree. C.
to allow colony enumeration the following day.
[0088] In vivo Models on Infection and Wound Healing
[0089] As already noted above, calcium flux agonists synergize with
TLR and NOD ligands to activate pathways relevant to immune
activation. Given the prevalence of TLR and NOD ligands in
infection and injury, the inventors therefore contemplate that
introduction of calcium flux agonists in these contexts will
greatly decrease the duration of disease and/or augment repair
function. To substantiate such model, various calcium flux agonists
were tested in a pre-infection treatment experiment in their
ability to alter wound healing kinetics and impact bacterial
clearance.
[0090] To determine if calcium flux agonists such as the ionophores
A23187 and ionomycin or the SERCA pump inhibitor thapsigargin can
be used as a pre-treatment of bacterial infection, the inventors
treated the shaved dorsal skin of 6-8 week old male C57Bl/6 mice
with vehicle alone, or formulations containing 2 mM A23187,
ionomycin, or thapsigargin. One day later, the inventors
superficially infected the dorsal skin of groups of 5 mice with
2.times.10.sup.6 CFU of S. aureus and evaluated bacterial burden
and wound size over the next week. Using this model, the inventors
investigated if the calcium flux agonists would have a beneficial
effect on wound healing (as measured by size) as demonstrated in
FIG. 7. Interestingly, animals treated with either ionophore or
thapsigargin demonstrated significantly smaller wound sizes
(p<0.01 as determined by Student's T-test) on days 3, 5 and 7 of
infection as compared to control treated animals. Furthermore,
animals pre-treated with formulations containing either ionophore
or thapsigargin possess significantly fewer bacteria at the
infection site on day 7 compared to vehicle controls (FIG. 8).
[0091] Topical delivery: A23187 and ionomycin and thapsigargin were
reconstituted in dehydrated ethanol (Spectrum Chemicals, Gardena,
Calif.) to appropriate concentrations for subsequent formulations.
In the pre-infection in vivo study, topical formulations consisted
of 75% dehydrated ethanol (Spectrum Chemicals), 22% cyclohexane
(Sigma-Aldrich), and 3% dimethyl sulfoxide (Sigma-Aldrich). Each
topical formulation was delivered by submersing a circular piece of
Whatman paper (Whatman, a division of GE Healthcare) with a 1.0
centimeter diameter and applying said circle to the skin of each
recipient for 5 minutes.
[0092] Preparation of S. aureus for skin inoculation: Briefly,
mid-logarithmic phase S. aureus bacteria was washed twice and
resuspended in sterile saline (0.9%) at the noted concentrations.
Mice: Male mice, 6-8 weeks old, on a C57BL/6 genetic background
were used in all experiments (Jackson Laboratories, Bar Harbor,
Me.).
[0093] Mouse model of S. aureus skin wound infection: To prepare
animals for wound infection, the skin of the posterior upper back
and neck of mice was shaved using #40 clippers. Next, three
parallel 8 mm long full-thickness scalpel cuts (no. 11 blade) were
made into the dermis. Resulting wounds were inoculated with 10
.mu.l of S. aureus (2.times.10.sup.8 CFUs per mL) with a
micropipettor. Total lesion size (cm2) measurements were quantified
by determining total pixel count from photographed animals using a
millimeter ruler as a reference.
[0094] Quantification of in vivo S. aureus bacterial burden: To
determine in vivo bacterial burden, infected mice were sacrificed
on day 7 following infection and lesions were harvested surgically.
Harvested tissues were homogenized and bacterial count was
determined following plating on appropriate solid growth media.
[0095] Antibacterial Effect
[0096] Consistent with these and previous results regarding their
inherent antibiotic activity against S. aureus, the inventors
confirmed the direct antibiotic activity of both ionophores for
direct antibiotic activity on methicillin-sensitive S. aureus
(MSSA). As would be predicted, both ionophores were active against
MSSA in a liquid culture assay with A23187 being the more active
compound. Interestingly, both compounds demonstrated significant
antibiotic activity against a methicillin-resistant S. aureus
(MRSA) strain.
[0097] For example, FIGS. 9A and 9B are graphs showing the
antibiotic sensitivity of S. aureus against A23187 and ionomycin,
respectively, and FIG. 9C shows antibiotic sensitivity of S. aureus
against CPA. As can be taken from the graphs, both ionophores had
some direct antibiotic effect, however, that effect was
substantially less than the control using kanamycin as direct
antibiotic. In contrast, FIG. 10 is a graph depicting lack of
direct antibiotic effect of the SERCA inhibitor thapsigargin
against S. aureus, even at high concentrations. To determine the
antibiotic sensitivity of S. aureus, cultures were grown overnight
in tryptic soy broth (Cellgro). The resulting cultures were used to
inoculate new cultures to ensure log phase growth prior to the
start of the experiment. Once the bacteria had reached log phase
the bacteria was diluted to the appropriate OD.sub.600.about.0.05
and transferred to a 24-well plate. Compounds of interest were
added and plates were incubated in a shaking incubator at
37.degree. C. Aliquots were removed at various time points and
their respective OD.sub.600 was measured using a BioTek Synergy2
microplate reader (BioTek, Winooski, Vt.). For wells whose
turbidity was affected by the addition of the compounds of
interest, aliquots were removed and plated on tryptic soy agar
(Cellgro) and incubated overnight in a 37.degree. C. incubated and
counted the following day. Thus, it should be appreciated that the
calcium flux agonists are effective in antimicrobial treatments via
an indirect effect, most likely due to the enhanced production of
IL-8 and increased NF-.kappa.B -driven transcription.
[0098] Moreover, the inventors also discovered that the indirect
antibiotic effect can be used in a prophylactic manner For example,
the inventors demonstrate that the promonocytic THP-1 cells treated
thapsigargin displayed greater antimicrobial activity than
similarly matured cells not treated with thapsigargin (FIG. 11).
Briefly, a logarithmically dividing S. aureus was pelleted
(2000.times.g for 10 min) and washed three times with Hank's
Buffered Salt Solution (Cellgro). Concurrently, 2 day
differentiated THP-1 cells (using 1 uM retinoic acid and 1 uM
cholecalciferaol, Sigma-Aldrich) followed by 1 day with 20 nM
thapsigargin or vehicle were collected and washed in Hank's
Buffered Salt Solution. Differentiated cells were supplemented with
10% human serum (Innovative Research) and chilled on ice for 30
minutes. After the 30 minutes, S. aureus (MOI of 10) was added on
ice for an additional 20 minutes to synchronize binding. Tubes were
then incubated for 20 minutes at 37.degree. C. After 20 minutes, 10
U/mL of lysostaphin (Sigma-Aldrich) was added to eliminate
non-internalized bacteria. At the desired time points (20, 30, 60
minutes) 20 uL aliquots were removed and diluted into 10 mL of
water. The dilution tubes were vortex and allowed to sit at room
temp for 10 minutes to ensure proper lysis of the primary cells.
After 10 minutes, the dilution tubes were centrifuged for 10
minutes at 2000.times.g and the remaining S. aureus was
concentrated 20 fold and plated in triplicate on tryptic soy agar
(Cellgro) and incubated overnight at 37.degree. C. prior to colony
enumeration.
[0099] Consequently, it should be appreciated that one or more
ionophores can be employed as topical prophylactic and/or
therapeutic agents for treatment of skin infections and/or to
improve wound healing. Of particular significance is the use of
such compositions and methods in an immunostimulatory manner rather
than in a direct antibiotic manner, which will advantageously
avoids difficulties otherwise associated with resistance build-up
due to antibiotic therapy. Of course, it should be appreciated that
numerous other pathogens are also contemplated herein, and in fact
include all currently known skin pathogens (bacterial, viral,
parasitical, and fungal).
[0100] It should still further be appreciated that a significant
synergistic effect was observed during treatment with contemplated
compounds, where the synergy was between the ionophores and TLR
ligands in activating immune cells. Such synergy could be of
particular interest for treatments that are already directed to
modification of immune response (e.g., drug therapy using Aldara)
or other immune-activating therapies to combat disease. Therefore,
the inventors contemplate that ionophores can be used
therapeutically to combat superficial skin infections before and
after onset. Moreover, and given the dual physical barrier and
immunological functions of the skin and the wealth of in vitro data
demonstrating the activating/modulating activities of the
ionophores in immune cells, the inventors also contemplate that
topical formulations containing ionophores will be of therapeutic
benefit in patients requiring treatment for acute (e.g., from
injury) or chronic (e.g., as observed in diabetic ulcers, etc)
superficial wounds, burns, and other inflammatory/autoimmune
disorders of the skin (e.g., psoriasis, eczema, etc).
[0101] Besides the skin, epithelial tissues include the cells
lining the gastrointestinal tract (including the alimentary
cavity), the respiratory tract and the urogenital tract. Other than
the latter, the remaining tissues are constantly exposed to
microorganisms and other factors such as pollen and man-made
environmental pollutants, which can cause or otherwise exacerbate
local inflammation and/or lesion formation. Due to the prevalence
of microorganisms which activate TLR receptors throughout the
gastrointestinal tract (including the mouth), it is also
contemplated to utilize ionophores in the treatment of mouth
abscesses and possibly inflammatory bowel disorders.
[0102] Consequently, it should be appreciated that
topically-applied thapsigargin can be used as a treatment agent to
synergize with TLR ligands in activating immune cells. Most
notably, the inventors discovered that skin treated with
thapsigargin or other calcium flux agonists prior to infection
clears live bacteria and heals faster than control-treated skin. Of
equal importance, the inventors showed that the differences
observed in vivo are unlikely to be due to inherent antibiotic
property of thapsigargin. As a result, the inventors contemplate
that thapsigargin or other calcium flux agonists can be used
therapeutically to combat superficial skin infections caused by
pathogens that produce or contain TLR/NOD ligands (e.g., S. aureus)
after their onset. Given the dual physical barrier and
immunological functions of the skin and the wealth of in vitro data
demonstrating the activating/modulating activities of thapsigargin
or other calcium flux agonists in immune cells, the inventors
contemplate that topical formulations containing thapsigargin are
of therapeutic benefit in patients requiring treatment for acute
(e.g., from injury) or chronic (e.g., as observed in diabetic
ulcers, etc.) superficial wounds, burns, and other
inflammatory/autoimmune disorders of the skin (e.g., psoriasis,
eczema, etc).
[0103] Thus, specific embodiments and applications of calcium flux
agonists have been disclosed. It should be apparent, however, to
those skilled in the art that many more modifications besides those
already described are possible without departing from the inventive
concepts herein. The inventive subject matter, therefore, is not to
be restricted except in the spirit of the appended claims.
Moreover, in interpreting both the specification and the claims,
all terms should be interpreted in the broadest possible manner
consistent with the context. In particular, the terms "comprises"
and "comprising" should be interpreted as referring to elements,
components, or steps in a non-exclusive manner, indicating that the
referenced elements, components, or steps may be present, or
utilized, or combined with other elements, components, or steps
that are not expressly referenced.
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