U.S. patent application number 16/091768 was filed with the patent office on 2019-03-28 for francisella lipids as broad anti-inflammatory therapeutics and associated methods of use.
This patent application is currently assigned to The U.S.A., as represented by the Secretary, Department of Health and Human Services. The applicant listed for this patent is The U.S.A., as represented by the Secretary, Department of Health and Human Services, The U.S.A., as represented by the Secretary, Department of Health and Human Services. Invention is credited to Catharine Mans Bosio, Robin M. Ireland, Glenn A. Nardone.
Application Number | 20190091151 16/091768 |
Document ID | / |
Family ID | 58671896 |
Filed Date | 2019-03-28 |
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United States Patent
Application |
20190091151 |
Kind Code |
A1 |
Bosio; Catharine Mans ; et
al. |
March 28, 2019 |
FRANCISELLA LIPIDS AS BROAD ANTI-INFLAMMATORY THERAPEUTICS AND
ASSOCIATED METHODS OF USE
Abstract
Embodiments of the present disclosure include anti-inflammatory
compositions and methods of use thereof. The compositions include
purified lipids from Francisella, for example, virulent strains of
Francisella. Other features and advantages of the invention will be
apparent from the detailed description, and from the claims.
Inventors: |
Bosio; Catharine Mans;
(Hamilton, MT) ; Ireland; Robin M.; (Hamilton,
MT) ; Nardone; Glenn A.; (Rockville, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The U.S.A., as represented by the Secretary, Department of Health
and Human Services |
Bethesda |
MD |
US |
|
|
Assignee: |
The U.S.A., as represented by the
Secretary, Department of Health and Human Services
Bethesda
MD
|
Family ID: |
58671896 |
Appl. No.: |
16/091768 |
Filed: |
April 6, 2017 |
PCT Filed: |
April 6, 2017 |
PCT NO: |
PCT/US2017/026467 |
371 Date: |
October 5, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62319692 |
Apr 7, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/1271 20130101;
A61K 39/0208 20130101; A61K 31/685 20130101; A61P 29/00 20180101;
A61K 31/6615 20130101; A61K 31/6615 20130101; A61K 2039/58
20130101; A61K 2300/00 20130101; A61K 2039/55555 20130101; A61K
2300/00 20130101; C12N 1/20 20130101; A61K 31/685 20130101; A61P
11/00 20180101 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 31/685 20060101 A61K031/685; A61K 39/02 20060101
A61K039/02; A61P 11/00 20060101 A61P011/00; A61P 29/00 20060101
A61P029/00 |
Goverment Interests
GOVERNMENT FUNDING
[0002] Research supporting this application was carried out by the
United States of America as represented by the Secretary,
Department of Health and Human Services.
Claims
1-15. (canceled)
16. A liposome comprising: (i) a purified lipid from Francisella
tularensis or a modified form thereof, wherein the purified lipid
comprises a phosphatidylethanolamine (PE); or (ii) a synthetic
PE.
17. The liposome of claim 16, wherein the Francisella tularensis is
a virulent strain.
18-20. (canceled)
21. The liposome of claim 16, further comprising
phosphatidylcholine (PC).
22. The liposome of claim 21, wherein at least one of the PE, the
PC, or a combination thereof, comprises an acyl chain with a length
in a range of from 5 to 13 or from 20 to 28 carbons.
23. The liposome of claim 21, wherein at least one of the PE, the
PC, or a combination thereof, comprises two acyl chains, wherein at
least one acyl chain has a length in the range of from 5 to 13
carbons or from 20 to 28 carbons.
24. (canceled)
25. The liposome of claim 21, wherein at least one of the PE, the
PC, or a combination thereof, comprises two acyl chains, wherein
one acyl chain has a length in a range of from 5 to 13 carbons, and
the other acyl chain has a length in a range of from 20 to 28
carbons.
26. The liposome of claim 21, wherein the ratio of PE:PC is in a
range of from about 50:50 to about 95:5.
27. The liposome of claim 16, further comprising another purified
lipid from Francisella tularensis.
28. The liposome of claim 16, wherein the liposome is an emulsified
liposome.
29. The liposome of claim 28, wherein the liposome has a diameter
in a range of from about 20 nm to about 1,500 nm.
30-45. (canceled)
46. A pharmaceutical composition comprising a pharmaceutically
effective amount of the liposome of claim 16.
47. The pharmaceutical composition of claim 46, further comprising
a pharmaceutically acceptable carrier.
48. The pharmaceutical composition of claim 47, wherein the
pharmaceutically acceptable carrier is a gel or cream.
49. A method of treating or inhibiting a microbial infection or
inflammation resulting from a microbial infection in a patient in
need thereof, the method comprising administering an effective
amount of the liposome of claim 16.
50. The method of claim 49, wherein the microbial infection is a
bacterial or a viral infection.
51-55. (canceled)
56. A method of treating or inhibiting inflammation in a patient in
need thereof, the method comprising administering an effective
amount of the liposome of claim 16 to the patient.
57. The method of claim 56, wherein the inflammation is related to
at least one of a bacterial infection, a viral infection, an
autoimmune disease or disorder, an allergy, or a combination
thereof.
58-65. (canceled)
66. A synthetic liposome comprising phosphatidylethanolamine (PE)
and phosphatidylcholine (PC), wherein the ratio of PE:PC is from
about 50:50 to about 95:5.
67. The synthetic liposome of claim 66, wherein the PE comprises at
least acyl chain with a length of 5 to 13 carbons or 20 to 28
carbons, and wherein the PC comprises at least one acyl chain with
a length of 5 to 13 carbons or 20 to 28 carbons.
68. The synthetic liposome of claim 66, wherein the PE comprises at
least one acyl chain with a length of 5 to 13 carbons and at least
one acyl chain with a length of 20 to 28 carbons, and wherein the
PC comprises at least one acyl chain with a length of 5 to 13
carbons and at least one acyl chain with a length of 20 to 28
carbons.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 62/319,692, filed 7 Apr. 2016, entitled FRANCISELLA
LIPIDS AS BROAD ANTI-INFLAMMATORY THERAPEUTICS AND ASSOCIATED
METHODS OF USE, the contents of which is incorporated herein by
reference in its entity for all purposes.
INCORPORATION BY REFERENCE
[0003] All documents cited or referenced herein, together with any
manufacturer's instructions, descriptions, product specifications,
and product sheets for any products mentioned herein or in any
document incorporated herein by reference, and may be employed in
the practice of the invention.
BACKGROUND
1. Field of the Invention
[0004] The present disclosure relates generally to the field of
anti-inflammatories and to methods and compositions for treating
and/or preventing inflammation related diseases and disorders,
including viral and bacterial infections. In particular, the
present disclosure relates to a therapeutic or pharmaceutical
composition/agent effective to ameliorate pro-inflammatory
responses, while not disrupting the immune system's ability to
produce adaptive (i.e., acquired) immune responses.
2. Background
[0005] Anti-inflammatories, especially those used during viral
infection, often have a deleterious side effect of greatly
inhibiting the overall immune response, thereby disrupting the
induction of adaptive immune responses (e.g., B cell and T cell
activation). As a result, patients taking anti-inflammatories are
plagued with the induction of poor immunity to pathogens, which can
results in a failure to clear and/or control infections.
[0006] Interestingly, infections with certain subspecies (ssp.) of
Francisella tularensis have been shown to induce an
anti-inflammatory response/environment. Francisella tularensis is a
highly infectious. Gram-negative, rod-shaped, coccobacillus aerobe
bacterium. F. tularensis is a non-spore forming, non-motile,
facultative intracellular bacterium with four major subspecies that
are capable of infecting and proliferating in a variety of host
cell types (including hepatocytes, endothelial cells, fibroblasts
and mononuclear phagocytes). F. tualarensis is a widespread
zoonosis that affects humans, causing the fatal disease tularemia
(also known as "rabbit fever"). F. tularensis ssp. novicida and
spp. mediasiatica are generally considered attenuated for humans.
F. tularensis ssp. holarctica (Type B; F. holarctica) causes
serious disease in humans, but is not typically fatal. F.
tularensis ssp. tularensis (Type A; F. tularensis) is highly
infectious and can cause a lethal infection (mortality rate of
approximately 30% when untreated) following inhalation of as few as
10 organisms in both humans and rodent models. Type A strains are
geographically distributed in North America, while Type B strains
are found throughout the northern hemisphere.
[0007] Human cases of tularemia usually result from a bite from a
vector such as biting flies, ticks, and mosquitoes that have
recently fed on an infected animal. There have been reported cases,
however, of infections cause by contact with the dead, animals,
infectious aerosols, and ingestion of contaminated food and water.
Hunters, veterinarians, walkers and farmers are at the greatest
risk of contracting tularemia because they are likely to come into
contact with infected animals. The incidence of tularemia in humans
is usually low, but an increase in the number of cases is observed
when there is an epidemic in the local animal reservoir.
[0008] In vitro and in vivo studies have demonstrated that
infection with virulent F. tularensis ssp. tularensis does not
induce the pro-inflammatory response that attenuated strains of F.
tularensis ssp. tularensis and subspecies of Francisella do. It has
been shown that lipids isolated from virulent F. tularensis strain
SchuS4 inhibit innate immune responses, e.g. the inflammatory
response (Robin Ireland, et al. Francisella tularensis SchuS4 and
SchuS4 Lipids Inhibit IL-12p40 in Primary Human Dendritic Cells by
Inhibition of IRF1 and IRF8. J Immunol. 2013; 191: 1276-1286).
Furthermore, this inhibition of the innate immune response is not
observed with the attenuated live vaccine strain (LVS) isolated
lipids. Id.
[0009] Moreover, when inflammatory responses are present during a
Francisella infections, it has been shown to not be effective at
controlling or clearing the bacterial infection, and in some cases,
has contributes to morbidity and may contribute to the death of the
infected individual. F. tularensis ssp. tularensis has been used as
a biological weapon because of its highly infectious nature and
ability to cause severe disease. The LVS is no longer licensed for
use against tularemia, and antibiotic treatment of F. tularensis
infected individuals does not always result in complete clearance
of the infection.
[0010] Accordingly, there exists in the art an ongoing need for
improved methods of treating or preventing infectious diseases and
inflammatory diseases and disorders.
SUMMARY
[0011] As described below, the present disclosure features
compositions and uses thereof for the treatment or prevention of a
pathogen infection, inflammation, or a disease or disorder
associated with inflammation. In particular, it was surprisingly
and unexpectedly discovered that effective amounts of purified
lipid from Francisella, for example Francisella tularensis, is
efficacious as an anti-inflammatory.
[0012] Thus, in an aspect, the present disclosure provides an
anti-inflammatory composition comprising an effective amount of
purified lipid from Francisella, for example Francisella
tularensis. In some embodiments, the Francisella tularensis is a
virulent Francisella tularensis strain, e.g. Francisella tularensis
ssp. tularensis. In another embodiment, the purified lipid
comprises a phosphatidylethanolamine (PE), e.g. a PE having an acyl
chain with a length in a range of from 5 to 13 or from 20 to 28
carbons. In an embodiment, the purified lipid is enriched for the
PE. In a particular embodiment, the purified lipid is PE, for
example, a PE having two acyl chains wherein at least one acyl
chain has a length of from 20 to 28 carbons. In a particular
embodiment, the purified lipid is PE, for example, a PE having two
acyl chains wherein at least one acyl chain has a length of from 5
to 13 carbons. In certain embodiments, the purified lipid is PE
having two acyl chains, wherein one chain is from 5 to 13 carbons,
and the other chain is in the range of from 20 to 28 carbons.
[0013] In certain embodiments, the composition further comprises
phosphatidylcholine (PC), e.g., in effective amounts. In a
particular embodiment, the ratio of PE:PC is in a range of from
about 50:50 to about 95:5. In a further embodiment, the composition
further comprises cholesterol. In some embodiments, the composition
further comprises about 5% to about 20% cholesterol. In other
embodiments, the PEPC composition comprises about 5% to about 20%
cholesterol.
[0014] In another embodiment, the composition further comprises
another purified lipid from Francisella. In a further embodiment,
the another purified lipid is from F. tularensis, which can be a
virulent strain of F. tularensis, for example ssp. tularensis. In a
particular embodiment, the another purified lipid has
anti-inflammatory properties, i.e. inhibits inflammation.
[0015] In other embodiments, the composition is a liposome, e.g. an
emulsified liposome, comprising an effective amount of PE from
Francisella, e.g., Francisella tularensis., as described herein. In
certain embodiments, the liposome can have a diameter in a range of
from about 20 nm to about 1,500 nm. In additional embodiments, the
liposome comprises about 5% to about 20% cholesterol. Cholesterol
further stabilizes the liposomes.
[0016] In any of the aspects or embodiments described herein, a
liposome composition as described herein may further comprises
phosphatidylcholine (PC), e.g., in an effective amount. In certain
embodiments, the liposome composition can have a PE:PC ratio in a
range of from about 50:50 to about 95:5. The composition can, in
some embodiments, further include another purified lipid from
Francisella, for example F. tularensis.
[0017] In another aspect, the present disclosure provides a
liposome comprising purified lipid from Francisella, for example a
virulent strain of Francisella tularensis such as Francisella
tularensis ssp. tularensis. In another embodiment, the disclosure
provides a liposome comprising at least one of a purified lipid
from Francisella, for example a virulent strain of Francisella
tularensis such as Francisella tularensis ssp. tularensis, a
synthetic phosphatidylethanolamine (PE) or a combination of both.
In certain embodiments, the PE and/or the PC comprises an acyl
chain with a length in a range of from 5 to 13 carbons. In
additional embodiments, the PE and/or the PC comprises an acyl
chain of from 20 to 28 carbons. In a particular embodiment, the PE
and/or PC has two acyl chains wherein at least one acyl chain has a
length of from 20 to 28 carbons. In a particular embodiment, the PE
and/or the PC has two acyl chains wherein at least one acyl chain
has a length of from 5 to 13 carbons. In certain embodiments, the
PE and/or PC has two acyl chains, wherein one chain is from 5 to 13
carbons, and the other chain is in the range of from 20 to 28
carbons.
[0018] In a particular embodiment, the liposome is an emulsified
liposome. The liposome or the emulsified liposome, according to an
embodiment, has a diameter in a range of from about 20 nm to about
1,500 nm.
[0019] In an additional aspect, the present disclosure provides an
anti-inflammatory liposome composition produced by the following
process: adding Francisella to a mixture of chloroform/methanol
(e.g., 2:1) and mixing; adding water to the organic mixture;
separating the organic phase and aqueous phase; drying the organic
phase; and reconstituting the dried organic phase, wherein the
reconstituted organic phase is the anti-inflammatory composition.
In some embodiments, the Francisella is a virulent Francisella
tularensis, such as Francisella tularensis ssp. tularensis. In a
particular embodiment, the chloroform/methanol is at a ratio of
about 2:1. In a particular embodiment, the dried organic phase is
reconstituted in ethanol.
[0020] In other embodiments, the process further comprises adding
PC to the isolated lipid of the reconstituted organic phase or the
isolated lipid. In certain embodiments, the ratio of the
reconstituted organic phase or isolated lipid to PC is in a range
of from about 50:50 to about 95:5 (e.g., about 60:40 to about
95:5).
[0021] In further embodiments, the process further comprises
separating complex Francisella lipid via thin layer chromatography
(TLC). In an embodiment, a band close to the solvent front was
isolated to produce enriched lipid. In some embodiment, the
isolated lipid is enriched for PE. In an embodiment, at least one
of the PE, the PC, or a combination thereof, comprises at least one
acyl chain with a length in a range of from 5 to 13. In certain
embodiments, at least one of the PE, the PC, or a combination
thereof, comprises at least one acyl chain with a length in a range
of from 20 to 28 carbons. In a particular embodiment, the purified
lipid is PE, for example, a PE having two acyl chains, wherein at
least one acyl chain has a length of from 20 to 28 carbons and/or
from 5 to 13 carbons. In a particular embodiment, the purified
lipid is PE, for example, a PE having two acyl chains, wherein at
least one acyl chain has a length of from 5 to 13 carbons. In
certain embodiments, the purified lipid is PE having two acyl
chains, wherein one chain is from 5 to 13 carbons, and the other
chain is in the range of from 20 to 28 carbons.
[0022] In yet other embodiments, the process further comprises
adding another purified lipid from Francisella, for example
Francisella tularensis.
[0023] In another embodiment, the process further comprises
producing a liposome from the reconstituted organic phase, isolated
lipid, or isolated lipid-PC mixture, which can be, for example, an
emulsified liposome. In an embodiment, the liposome has a diameter
in a range of from about 20 nm to about 1,500 nm.
[0024] In another aspect, the present disclosure provides a
pharmaceutical composition comprising a pharmaceutically effective
amount of a liposome (or the anti-inflammatory composition) as
described herein. In an embodiment, the pharmaceutical composition
further comprises a pharmaceutically acceptable carrier or
excipient. In certain embodiments, the composition is in the form
of at least one of a liquid, a gel or a cream.
[0025] In another aspect, the disclosure provides a liposome
composition comprising a modified form of PE, e.g., modified PE
isolated from Francisella tularensis. There are a wide range of
lipid modifications known to those skilled in the art, all of which
are expressly contemplated herein, including, e.g., mannosylation.
In certain embodiments, the PE is conjugated with another agent,
e.g., to another biologically active agent, a homing agent, or to a
molecular entity for increasing at least one of half-life,
stability, bioavailability or a combination thereof. In certain
embodiments, the agent is an antibody, or a PEG molecule.
[0026] In another aspect, the disclosure provides a therapeutic
composition comprising a liposome (or the anti-inflammatory
composition) as described herein in combination with another active
agent. In certain embodiments, the agent can be conjugated or
associated with the lipids themselves. In still additional
embodiments, the agents can be encompassed within the continuous
phase of the interior of the liposomes.
[0027] In yet a further aspect, the present disclosure provides a
method of treating or preventing a microbial infection or
inflammation resulting from a microbial infection in a subject,
e.g., a patient, in the need thereof. The method comprising
administering a composition comprising an effective amount of the
liposome (or the anti-inflammatory composition) of present
disclosure to a subject in need thereof, wherein the composition or
liposome is effective in treating or preventing the microbial
infection or the inflammation resulting from a microbial infection.
In certain embodiments, the composition further comprises a
pharmaceutically acceptable excipient or carrier.
[0028] In an embodiment, the microbial infection is a bacterial or
a viral infection, for example a bacterial infection or a viral
infection that causes dermatological inflammation and/or
respiratory inflammation. In a particular embodiment, the bacterial
infection or viral infection is selected from the group consisting
of Staphylococcus aureus, Streptococcus pyogenes, Clostridiumn
perfringens, Bacillus anthracis, Francisella tularensis, measles,
rubella, varicella zoster, parvovirus, herpes simplex virus 6,
herpes simplex virus 7, herpes simplex virus 8, Epstein Barr virus,
enterovirus, coxsackie virus, togavirus, bunyavirus, arenavirus,
smallpox, cowpox, monkey pox, zika virus, dengue virus, nairovirus,
arenavirus, filovirus, west nile virus, molluscum contagiosm, and
human papillomavirus.
[0029] In some embodiments, the composition or the liposome is
administered prior to exposure to the microbial infection, while in
other embodiments, the composition or the liposome is administered
post exposure to the microbial infection. In still additional
embodiments, the composition is administered before and after
microbial infection. In certain embodiments, the method further
comprises co-administering the composition or the liposome with one
or more additional therapeutic agents.
[0030] In a further aspect, the present disclosure provides for a
method of treating or preventing inflammation in a subject, e.g., a
patient, in the need thereof. The method comprises administering a
composition having an effective amount of the liposome (or the
anti-inflammatory composition) of the present disclosure to the
subject, wherein the composition is effective in alleviating,
ameliorating, treating and/or preventing at least one symptom of
inflammation in the subject. In an embodiment, the inflammation is
related to at least one of a bacterial infection, a viral
infection, an autoimmune disease or disorder, and an allergy.
[0031] In an additional aspect, the present disclosure provides a
method of modulating an immune response in a subject. The method
comprises administering a composition comprising an effective
amount of the liposome (or the anti-inflammatory composition) of
the present disclosure to a subject in need thereof, e.g., a
subject having inflammation, wherein the composition is effective
in modulating the immune response in the subject. In some
embodiments, the modulation comprises enhancing the
immunocompetence in the subject, e.g. by suppressing an
inflammatory response. In an embodiment, the suppressing an
inflammatory response does not affect the patients ability to
produce an adaptive immune response.
[0032] In a further embodiment, the modulation of an immune
response comprises inhibiting bacterial replication in the subject.
In other embodiments, the modulation of an immune response
comprises inhibiting viral replication in the subject. In some
embodiments, the bacterial infection or viral infection is a
respiratory infection and/or a dermatological infection.
[0033] In certain embodiments, the inflammation is caused by at
least one of an autoimmune disease, an autoimmune disorder, an
allergy, or a combination thereof. In other embodiments, the
subject has dermatological or respiratory inflammation.
[0034] The present disclosure provides compositions or liposomes
featuring purified lipid from Francisella and methods of using such
compositions/liposomes for the treatment or prevention of an
infectious disease and/or inflammation. Other features and
advantages of the invention will be apparent from the detailed
description, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] The following detailed description, given by way of example,
but not intended to limit the invention solely to the specific
embodiments described, may best be understood in conjunction with
the accompanying drawings.
[0036] FIG. 1 provides a schematic diagram of a typical
Gram-negative outer membrane.
[0037] FIGS. 2A, 2B, and 2C demonstrate that SchuS4 inhibits
pro-inflammatory responses among resting primary human cells.
Differential induction of pro-inflammatory cytokines by attenuated
and virulent strains of F. tularensis. Primary human dendritic
cells (hDC) were infected at a multiplicity of infection of 50 with
the indicated strains of F. tularensis. (A) Intracellular bacteria
were enumerated at the indicated times postinfection. *p<0.01,
compared with SchuS4-infected hDC. (B) Supernatants were harvested
from uninfected or F. tularensis-infected cultures at 24 hours
postinfection and analyzed for IL-12p40 by ELISA. hDC stimulated 24
h prior to harvest with ultrapure E. coli LPS (10 ng/ml) served as
positive controls. *p<0.01, compared with uninfected and
SchuS4-infected hDC; **p<0.001, compared with all samples. (C)
SchuS4- or mock-infected hDC cultures were stimulated 24 hours
postinfection with ultrapure E. coli LPS. Concentrations of
IL-12p40 in culture supernatants were determined an additional 24 h
after LPS treatment. *p<0.01, compared with uninfected,
LPS-treated samples. Error bars represent SEM. Each data point
represents the mean of triplicate samples. Data in (A) are the mean
of eight experiments; data in (B) and (C) are representative of
three experiments of similar design.
[0038] FIG. 3 demonstrates that SchuS4 lipids inhibit inflammatory
responses in vitro. hDC were treated with the indicated
concentration of lipids isolated from SchuS4 or LVS for 18 hours
followed by addition of LPS for an additional 20 hours. EtOH served
as vehicle control. Culture supernatants were assessed for IL-12p40
by ELISA. ns=not significantly different. *=significantly less than
EtOH+LPS treated controls (p<0.05). In each experiment, each
condition was tested in triplicate. Error bars represent SEM. Data
is representative of three experiments of similar design using
different donors.
[0039] FIGS. 4A and 4B demonstrate that SchuS4 lipids inhibit
pulmonary inflammation. Mice (n=5/group) were intranasally
inoculated with 25 .mu.g/25 .mu.l SchuS4 lipids or 25 .mu.l diluted
EtOH. Eighteen hours later, mice were treated with 200 ng/25 .mu.l
E. coli LPS. Five hours after administration of LPS, mice were
euthanized and fluid and cells from the airways were collected by
bronchoalveolar lavage. Completely unmanipulated (-) mice served as
negative controls. (A) Infiltration of neutrophils was evaluated by
flow cytometry. (B) BAL fluid was assessed for TNF-.alpha., IL-6,
and KC by ELISAs. Error bars, SEMs. *, P<0.05, compared to
EtOH-treated controls. Data are representative of two experiments
of similar design.
[0040] FIGS. 5A, 5B, and 5C identifies an active portion of the
SchuS4 lipid preparation. Crude SchuS4 lipids were separated by TLC
(A) and the indicated bands were scraped from the silica plate and
assessed for their ability to inhibit inflammatory responses in hDC
(B and C). Areas of the TLC plate at the same part of the solvent
front, but not containing lipid were scraped as used as negative
controls. hDC were treated and supernatants were assessed for the
indicated cytokines as described in FIG. 3. Following incubation
with lipid and LPS supernatants were assessed for IL-12p40 (B) or
TNF-.alpha. (C) by ELISA. Error bars represent SD. Band 4, which is
the band close to the solvent front, from the TLC plate was
analyzed by LC-MS for lipid content and speciation.
[0041] FIGS. 6A and 6B. Phospholipids from crude SchuS4 lipid
preparations were isolated and fractionated into PE (1) or PC (2)
containing fractions. These samples, or crude lipid, were added to
hDC at the indicated concentrations. Cells were treated with LPS as
described in FIG. 3 (black bars) or treated with media alone (white
bars). Supernatants were assessed for IL-12p40 (A) or TNF-.alpha.
(B) by ELISA. Error bars represent SD. *=p<0.05.
[0042] FIGS. 7A, 7B, and 7C contain images of the liposomes created
by the synthetic 24:10 PE and phosphatidylcholine (PC). Synthetic
PE 24:10 was mixed with commercially available PC 16:18 at a ratio
of 80:20 (PEPC) and imaged by cryo-electron microscopy to reveal
lipid structure (A). PEPC liposomes form varied sized multilaminar
structures. (B) PEPC liposomes are taken up and persist in the
intracellular compartment. BMDM or hDC were treated with PEPC
liposomes. At the indicated time points, the cells were fixed and
stained for PE using duramycin. The nuclei were counterstained with
DAPI. Intracellular PEPC liposomes are detected within one hour
after exposure and accumulated over the 24 hour incubation. PE is
detected on the surface of viable F. tularensis SchuS4. (C) BMDM or
hDC were infected with MOI=50 SchuS4 and fixed at 6 and 8 hours,
respectively. Cells were stained with Alexa 488 conjugated anti-F.
tularensis LPS antibody to detect bacteria (arrow), duramycin to
detect PE (arrow head), and DAPI to detect nuclei. Colocalization
of intense PE staining and SchuS4 was observed in both BMDM and
hDC.
[0043] FIG. 8 demonstrates that the synthetic PC:PC liposomes
inhibit LPS mediated inflammation in hDC. PEPC liposomes or crude
SchuS4 lipid were incubated with hDC, followed by treatment with
LPS as described in FIG. 3. Cells treated with 5% dextrose water
(vehicle) served as a negative control. Supernatants were evaluated
for IL-12p40 by ELISA. Error bars represent SD and *=p<0.05.
[0044] FIGS. 9A and 9B demonstrates that the synthetic PE:PC
liposomes inhibit viral mediated inflammation. A549 human
epithelial cells were treated with crude Schus4 lipid or synthetic
PEPC liposomes overnight. Cells treated with 5% dextrose water
served as negative controls for lipid treatment. Cells were then
infected with West Nile Virus at an MOI=0.01. Forty eight hours
later supernatants were collected and assessed for IL-6 (A) and
IFN-.beta. (B) by ELISA. Error bars represent SD. *=p<0.05.
[0045] FIG. 10 demonstrates that the synthetic PE:PC liposomes
inhibit viral replication. A549 human epithelial cells were treated
with crude Schus4 lipid or synthetic PEPC liposomes overnight.
Cells treated with 5% dextrose water served as negative controls
for lipid treatment. Cells were then infected with West Nile Virus
at an MOI=0.001. Forty eight hours later cells were assessed for
viral load as plaque forming units (PFU) by immunostaining. Error
bars represent SD. *=p<0.05.
[0046] FIGS. 11A, 11B, 11C, 11D, 11E, and 11F demonstrate that the
inhibition of inflammatory responses by PE and PC is dependent on
acyl chain length. BMDM were treated with crude SchuS4 lipids, the
indicated PE lipids, the indicated PC lipids, or PEPC liposomes
overnight. Cells were then stimulated with R848 (TLR8 agonist) and
supernatants were assessed for IL-12p40, as an indicator of
induction of an inflammatory response. Crude SchuS4 lipid (FIG.
11A), PE2410 (FIG. 11B), PC2424 (FIG. 11C) and liposomes comprised
of PE2410 and PC2424 (FIG. 11D) all inhibited inflammatory
responses in a dose dependent manner. PC and PE lipids comprised of
1816 acyl chains (FIGS. 11E and 11F, respectively) did not
significantly impair IL-12p40 secretion. Error bars represent SD.
*=p<0.05 compared to vehicle (-) control treated samples. ns=not
significant. Data are representative of three experiments of
similar design.
DETAILED DESCRIPTION
[0047] The disclosure features compositions and methods that are
useful for the treatment or prevention of a pathogen infection
and/or inflammatory diseases and disorders. The disclosure is
based, at least in part, on the discovery that treatment or
administration of a purified lipid from Francisella, for example
Francisella tularensis, modulates the immune system, e.g., induces
an anti-inflammatory response/environment, while not disrupting the
immune system's ability to induce a pathogen specific immune
response, and is effective to treat or prevent microbial infection,
e.g. a bacterial infection or a viral infection. The composition of
the disclosure is a novel non-toxic anti-inflammatory that does not
rely upon cell destruction. The compositions of the disclosure do
not have long term deleterious effects, thereby making the
composition an affective long-term and short-term
anti-inflammatory. This is especially true because the composition
of the disclosure does not adversely affect the immune system's
ability to produce an effective adaptive immune response, e.g. the
function of B and T cells or the ability to induce a new B cell
response and/or T cell response. As such, the compositions of the
disclosure may be utilized to ameliorate pathogenic inflammation
without impairing the immune system's ability to develop an immune
response directed to the invading pathogen, e.g., bacteria, virus,
or fungus. The composition of the disclosure can also be utilized
to treat inflammation associated diseases and disorders, e.g.
allergies and/or autoimmunity.
[0048] The articles "a", "an", and "the" as used herein and in the
appended claims are used herein to refer to one or to more than one
(i.e., to at least one) of the grammatical object of the article
unless the context clearly indicates otherwise. By way of example,
"an element" means one element or more than one element.
[0049] The phrase "and/or," as used herein in the specification and
in the claims, should be understood to mean "either or both" of the
elements so conjoined, i.e., elements that are conjunctively
present in some cases and disjunctively present in other cases.
Multiple elements listed with "and/or" should be construed in the
same fashion, i.e., "one or more" of the elements so conjoined.
Other elements may optionally be present other than the elements
specifically identified by the "and/or" clause, whether related or
unrelated to those elements specifically identified. Thus, as a
non-limiting example, a reference to "A and/or B", when used in
conjunction with open-ended language such as "comprising" can
refer, in one embodiment, to A only (optionally including elements
other than B); in another embodiment, to B only (optionally
including elements other than A); in yet another embodiment, to
both A and B (optionally including other elements); etc.
[0050] As used herein in the specification and in the claims. "or"
should be understood to have the same meaning as "and/or" as
defined above. For example, when separating items in a list, "or"
or "and/or" shall be interpreted as being inclusive, i.e., the
inclusion of at least one, but also including more than one, of a
number or list of elements, and, optionally, additional unlisted
items. Only terms clearly indicated to the contrary, such as "only
one of" or "exactly one of," or, when used in the claims.
"consisting of," will refer to the inclusion of exactly one element
of a number or list of elements. In general, the term "or" as used
herein shall only be interpreted as indicating exclusive
alternatives (i.e., "one or the other but not both") when preceded
by terms of exclusivity, such as "either," "one of," "only one of,"
or "exactly one of."
[0051] In the claims, as well as in the specification above, all
transitional phrases such as "comprising," "including," "carrying,"
"having," "containing," "involving," "holding," "composed of," and
the like are to be understood to be open-ended, i.e., to mean
including but not limited to. Only the transitional phrases
"consisting of" and "consisting essentially of" shall be closed or
semi-closed transitional phrases, respectively, as set forth in the
United States Patent Office Manual of Patent Examining Procedures,
Section 2111.03.
[0052] As used herein in the specification and in the claims, the
phrase. "at least one," in reference to a list of one or more
elements, should be understood to mean at least one element
selected from anyone or more of the elements in the list of
elements, but not necessarily including at least one of each and
every element specifically listed within the list of elements and
not excluding any combinations of elements in the list of elements.
This definition also allows that elements may optionally be present
other than the elements specifically identified within the list of
elements to which the phrase "at least one" refers, whether related
or unrelated to those elements specifically identified. Thus, as a
non-limiting example, "at least one of A and B" (or, equivalently,
"at least one of A or B," or, equivalently "at least one of A
and/or B") can refer, in one embodiment, to at least one,
optionally including more than one, A, with no B present (and
optionally including elements other than B); in another embodiment,
to at least one, optionally including more than one, B, with no A
present (and optionally including elements other than A); in yet
another embodiment, to at least one, optionally including more than
one, A, and at least one, optionally including more than one, B
(and optionally including other elements); etc.
[0053] It should also be understood that, in certain methods
described herein that include more than one step or act, the order
of the steps or acts of the method is not necessarily limited to
the order in which the steps or acts of the method are recited
unless the context indicates otherwise.
[0054] The term "compound" is to be understood to include any
composition or liposome of the present disclosure.
[0055] The term "patient" or "subject" is used throughout the
specification to describe an animal, including human, nonhuman
primates (e.g., ape or monkey), or a wild/domesticated animals, to
whom treatment, including prophylactic treatment, with the
compositions according to the present disclosure is provided. For
treatment of those infections, conditions or disease states which
are specific for a specific animal, such as a human patient, the
term patient refers to that specific animal, including a wild or
domesticated animal, such as a dog, a cat, a mouse, a hamster, or a
farm animal such as a horse, cow, sheep, donkey, pig, chicken,
etc.
[0056] As used herein, the term "treatment" or "treating" includes
any process, action, application, therapy, or the like, wherein a
subject (or patient), including a human being, is provided with or
administered an agent or composition, e.g., a composition including
at least one Francisella lipid, with the aim of improving the
subject's condition, directly or indirectly, or slowing the
progression of a condition or disorder in the subject (e.g., fever
or shortness of breath due to infection or an inflammation related
disease or disorder), or ameliorating at least one symptom of the
disease or disorder under treatment (e.g., cough or diarrhea caused
by an infection or disease or disorder related to an inflammatory
response--i.e., inflammation). As used in the context of disease
caused by a bacteria or viral pathogen, the terms "treat,"
"treatment," and the like, refer to relief from or alleviation of a
pathological process mediated by an inflammatory response or
directly affecting the pathogen, for example inhibiting
replication.
[0057] The terms "co-administration" and "co-administering" or
"combination therapy" refer to both concurrent administration
(administration of two or more therapeutic agents at the same time)
and time varied administration (administration of one or more
therapeutic agents at a time different from that of the
administration of an additional therapeutic agent or agents), as
long as the therapeutic agents are present in the patient to some
extent, preferably at effective amounts, at the same time. In
certain preferred aspects, the composition described herein is
coadministered in combination with at least one additional
bioactive agent, especially including at least one of an antiviral,
antibacterial, anti-mycotic, anti-inflammatory, or another agent
that ameliorates signs and/or symptoms of the infection or
inflammation related disease or disorder. In particularly preferred
aspects, the co-administration of compounds results in synergistic
activity and/or therapy. One therapy can be based on the
composition of the disclosure. A second (or third, fourth, fifth,
sixth, etc.) therapy can be based on a known therapy for the
disease/disorder or infection being treated. For example,
alternative antiviral or antibacterial drugs may be co-administered
with the composition of the disclosure or therapeutic agents to
ameliorate symptoms or conditions caused by the infection or
disease/disorder being treated or prevented. The order of
administration of two or more sequentially co-administered
therapeutic agents is not limited. The administration of the two or
more therapeutic agents may also be administered by different
routes, e.g., by a local route (e.g., mucosal delivery of a dual
vaccine of the disclosure) and a systemic route (e.g., parenteral
delivery of an anti-rabies or anti-coronavirus small molecule
inhibitor).
[0058] The term "effective" is used to describe an amount of a
compound, composition or component which, when used within the
context of its intended use, effects an intended result. The term
effective subsumes all other effective amount or effective
concentration terms, which are otherwise described or used in the
present application.
[0059] As used herein, the phrases "therapeutically effective
amount" and "prophylactically effective amount" refer to an amount
that provides a therapeutic benefit in the treatment, prevention,
or management of pathological processes mediated by an infection
with a pathogen, or an overt symptom of a pathological processes
mediated by an inflammatory response. The specific amount that is
therapeutically effective can be readily determined by ordinary
medical practitioner, and may vary depending on factors known in
the art, such as, e.g. the type of pathological processes mediated
by a viral/bacterial infection or the disease/disorder, the
patient's history and age, the stage of pathological processes
mediated by the infection, disease and/or disorder, and the
administration of other anti-pathological agents.
[0060] As used herein, a "pharmaceutical composition" comprises a
pharmacologically effective amount of a therapeutic agent of the
disclosure (such as the anti-inflammatory composition or liposome
of the present disclosure) and a pharmaceutically acceptable
carrier. As used herein, "pharmacologically effective amount,"
"therapeutically effective amount" or simply "effective amount"
refers to that amount of a composition effective to produce the
intended pharmacological, therapeutic or preventive result. For
example, if a given clinical treatment is considered effective when
there is at least a 25% reduction in a measurable parameter
associated with a disease or disorder, a therapeutically effective
amount of a drug for the treatment of that disease or disorder is
the amount necessary to effect at least a 25% reduction in that
parameter. Further, the pharmaceutical composition can be designed
to enhance targeting cells involved in the underlying infection
such as dendritic cells, macrophages, hepatocytes, and other
parenchymal cells, and/or inhibit an inflammatory response
associated with an infection, disease and/or disorder. As used
herein, the term "pharmaceutically acceptable" means that the
subject item is appropriate for use in a pharmaceutical
product.
[0061] As used herein, the term "isolated" or "purified" lipid or
biologically-active portion of lipids thereof is substantially free
of other cellular material from the cells that the lipid is
obtained.
[0062] As used herein, "pharmaceutically acceptable carrier"
includes any material which, when combined with an active
ingredient of a composition, allows the ingredient to retain
biological activity and without causing disruptive reactions with
the subject's immune system. Examples include, but are not limited
to, any of the standard pharmaceutical carriers such as a phosphate
buffered saline solution, water, emulsions such as oil/water
emulsion, and various types of wetting agents. Exemplary diluents
for aerosol or parenteral administration are phosphate buffered
saline or normal (0.9%) saline. Compositions comprising such
carriers are formulated by well-known conventional methods (see,
for example. Remington's Pharmaceutical Sciences, Chapter 43, 14th
Ed., Mack Publishing Col, Easton Pa. 18042, USA). Pharmaceutically
acceptable excipients have been amply described in a variety of
publications, including, for example, A. Gennaro (2000) "Remington:
The Science and Practice of Pharmacy," 20th edition, Lippincott,
Williams, & Wilkins; Remington's Pharmaceutical Sciences, 14th
Ed. or latest edition, Mack Publishing Col, Easton Pa. 18042, USA;
Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H. C.
Ansel et al., eds., 7th ed., Lippincott, Williams, & Wilkins;
and Handbook of Pharmaceutical Excipients (2000) A. H. Kibbe et
al., eds., 3rd ed. Amer. Pharmaceutical Assoc. Further discussion
is provided herein.
[0063] As used herein, "ameliorate" is meant decrease, suppress,
attenuate, diminish, arrest, or stabilize the development or
progression of a disease.
[0064] As used herein, "antigen" is meant an agent that induces a
humoral and/or cellular immune response.
[0065] As used herein, "disease" is meant any condition or disorder
that damages or interferes with the normal function of a cell,
tissue, or organ. Examples of diseases include bacterial invasion
or colonization of a host cell.
[0066] As used herein, "liposome" is meant a microscopic vesicle
comprising an aqueous core enclosed in one or more phospholipid
layers.
[0067] As used herein, "pathogen" is meant any bacteria, viruses,
fungi, or protozoans capable of interfering with the normal
function of a cell. Exemplary bacterial pathogens include, but are
not limited to, Aerobacter, Aeromonas, Acinetobacter Agrobacterium,
Bacillus, Bacteroides, Bartonella, Bordtella, Brucella,
Burkholderia, Calymmatobacterium, Campylobacter, Citrobacter,
Clostridium, Cornyebacterium, Enterobacter Escherichia,
Francisella, Haemophilus, Hafnia, Helicobacter, Klebsiella,
Legionella, Listeria, Morganella, Moraxella, Proteus, Providencia,
Pseudomonas, Salmonella, Serratia, Shigella, Staphylococcus,
Streptococcus, Treponema, Xanthomonas, Vibrio, and Yersinia.
[0068] By "protective immune response" is meant an immune response
sufficient to ameliorate a pathogen infection in a mammal.
[0069] By "reference" is meant a standard or control condition.
[0070] In an aspect, the present disclosure provides an
anti-inflammatory composition comprising an effective amount of
purified lipid from Francisella. The lipid can be purified from a
virulent strain from Francisella tularensis, which has been shown
to induce an anti-inflammatory response. The virulent strain can be
Francisella tularensis ssp. tularensis. In a particular embodiment,
the purified lipid has anti-inflammatory properties. In certain
embodiments, the purified lipid inhibits inflammation. In an
additional embodiment, the purified lipid modulates an immune
response. i.e. inhibits inflammation, but does not disrupt the
immune system's ability to produce an adaptive immune response. In
a particular embodiment, the purified lipid enhances the immune
system's ability to produce an adaptive immune response, as
compared to a patient that does not receive the purified lipid. In
a further embodiment, the purified lipid is a synthetic PE.
[0071] In another embodiment, the purified lipid comprises a
phosphatidylethanolamine (PE). It was surprising and unexpected to
discover that the PE of the purified lipid of Francisella has an
acyl chain with a length in a range of from 5 to 13 carbons (e.g.,
C5:0-C13:0). It was also surprising and unexpected to discover that
the PE of the purified lipid of Francisella has an acyl chain with
a length in a range of from 20 to 28 carbons, as PE typically has
acyl chains with a length in a range of from 16 to 18 carbons
(i.e., C16:0-C18:0). In an embodiment, the purified lipid is
enriched for PE. In a particular embodiment, the purified lipid is
PE, for example, a PE having two acyl chains, wherein at least one
acyl chain has a length of from 20 to 28 carbons. In another
embodiment, the purified lipid is PE, for example a PE having two
acyl chains wherein at least one acyl chain has a length of from 5
to 13 carbons. In an additional embodiments, the purified lipid is
PE, for example a PE having two acyl chains, wherein at least one
acyl chain has a length of from 5 to 15 carbons and at least one
acyl chain has a length of from 20 to 28 carbons. In certain
embodiments, the purified lipid is PE having two acyl chains,
wherein one chain is from 5 to 13 carbons (e.g., C5:0-C13:0), and
the other chain is in the range of from 20 to 28 carbons (e.g.,
C20:0-C28:0). In other embodiments, the length of the acyl chain is
5 (e.g., C5:0), 6 (e.g., C6:0), 7 (e.g., C7:0), 8 (e.g. C8:0), 9
(e.g., C9:0), 10 (e.g., C10:0), 11 (e.g., C11:0), 12 (e.g., C12:0),
13 (e.g., C13:0), 20 (e.g., C20:0), 21 (e.g., C21:0), 22 (e.g.,
C22:0), 23 (e.g., C23:0), 24 (e.g., C24:0), 25 (e.g., C25:0), 26
(e.g., C26:0), 27 (e.g., C27:0), or 28 (e.g., C28:0) carbons. In
yet other embodiments, the purified lipid is PE having two acyl
chains, wherein one acyl chain is 5 (e.g., C5:0), 6 (e.g., C6:0), 7
(e.g., C7:0), 8 (e.g., C8:0), 9 (e.g., C9:0), 10 (e.g., C10:0), 11
(e.g., C11:0), 12 (e.g., C12:0), or 13 (e.g., C13:0) carbons, and
the other acyl chain is 20 (e.g., C20:0), 21 (e.g., C21:0), 22
(e.g., C22:0), 23 (e.g., C23:0), 24 (e.g., C24:0), 25 (e.g.,
C25:0), 26 (e.g., C26:0), 27 (e.g., C27:0), or 28 (e.g., C28:0)
carbons. In a particular embodiment, the PE has the following
structure:
##STR00001##
[0072] In certain embodiments, the composition further comprises
phosphatidylcholine (PC). In a particular embodiment, the ratio of
PE:PC is in a range of from about 50:50 to about 95:5. In an
additional embodiment, the ratio of PE:PC is in a range of from:
about 55:45 to about 95.5, about 60:40 to about 95:5, about 65:35
to about 95:5, about 70:30 to about 95:5, about 75:25 to about
95:5, about 80:20 to about 95.5, about 85:15 to about 95.5, about
90:10 to about 95:5, about 50:50 to about 90:10, about 55:45 to
about 90:10, about 60:40 to about 90:10, about 65:35 to about
90:10, about 70:30 to about 90:10, about 75:25 to about 90:10,
about 80:20 to about 90:10, about 85:15 to about 90:10, about 50:50
to about 85:15, about 55:45 to about 85:15, about 60:40 to about
85:15, about 65:35 to about 85:15, about 70:30 to about 85:15,
about 75:25 to about 85:15, about 80:20 to about 85:15, about 50:50
to about 80:20, about 55:45 to about 80:20, about 60:40 to about
80:20, about 65:35 to about 80:20, about 70:30 to about 80:20,
about 75:25 to about 80:20, about 50:50 to about 75:25, about 55:45
to about 75:25, about 60:40 to about 75:25, about 65:35 to about
75:25, about 70:30 to about 75:25, about 50:50 to about 70:30,
about 55:45 to about 70:30, about 60:40 to about 70:30, about 65:35
to about 70:30, about 50:50 to about 65:35, about 55:45 to about
65:35, about 60:40 to about 65:35, about 50:50 to about 60:40, or
about 55:45 to about 60:40. In certain embodiments, the ratio of
PE:PC is about 50:50, about 55:45, about 60:40, about 61:39, about
62:38, about 63:37, about 64:36, about 65:35, about 66:34, about
67:33, about 68:32, about 69:32, about 70:30; about 71:29, about
72:28, about 73:27, about 74:26, about 75:25, about 76:24, about
77:23, about 78:22, about 79:21, about 80:20, about 81:19, about
82:18, about 83:17, about 84:16, about 85:15, about 86:14, about
87:13, about 88:12, about 89:11, about 90:10, about 91:9, about
92:8, about 93:7, about 94:6, or about 95:5.
[0073] It was surprising and unexpected to discover that the PC
with an acyl chain with a length in a range of from 20 to 28
carbons had inhibitory activity. In a particular embodiment, the PC
has two acyl chains, wherein at least one acyl chain has a length
of from 20 to 28 carbons. In an additional embodiment, the PC has
two acyl chains, wherein each of the acyl chains has a length of
from 20 to 28 carbons. In other embodiments, the length of the acyl
chain is 20 (e.g., C20:0), 21 (e.g., C21:0), 22 (e.g., C22:0), 23
(e.g., C23:0), 24 (e.g., C24:0), 25 (e.g., C25:0), 26 (e.g.,
C26:0), 27 (e.g., C27:0), or 28 (e.g., C28:0) carbons. In yet other
embodiments, the PC has two acyl chains, wherein one acyl chain is
20 (e.g., C20:0), 21 (e.g., C21:0), 22 (e.g., C22:0), 23 (e.g.,
C23:0), 24 (e.g., C24:0), 25 (e.g., C25:0), 26 (e.g., C26:0), 27
(e.g., C27:0), or 28 (e.g., C28:0) carbons, and the other acyl
chain is 20 (e.g., C20:0), 21 (e.g., C21:0), 22 (e.g., C22:0), 23
(e.g., C23:0), 24 (e.g., C24:0), 25 (e.g., C25:0), 26 (e.g.,
C26:0), 27 (e.g., C27:0), or 28 (e.g., C28:0) carbons.
[0074] In a particular embodiment, the PC has two acyl chains,
wherein at least one acyl chain has a length of from 5 to 13
carbons. In an additional embodiment, the PC having two acyl
chains, wherein at least one acyl chain has a length of from 5 to
15 carbons and at least one acyl chain has a length of from 20 to
28 carbons. In certain embodiments, the PC having two acyl chains,
wherein one chain is from 5 to 13 carbons (e.g., C5:0. C6:0, C7:0,
C8:0, C9:0, C10:0, C11:0, C12:0, or C13:0), and the other chain is
in the range of from 20 to 28 carbons (e.g., C20:0-C28:0), as
discussed above.
[0075] In some embodiments, the composition further comprises about
5% to about 20% cholesterol. In other embodiments, the PEPC
composition comprises about 5% to about 20% cholesterol.
Cholesterol further stabilizes the liposomes. In another
embodiment, the composition/PEPC composition/liposome of the
present disclosure comprises about 5% to about 20%, about 5% to
about 15%, about 5% to about 10%, about 10% to about 20%, about 10%
to about 15%, or about 15% to about 20% cholesterol.
[0076] In another embodiment, the composition further comprises at
least one additional purified lipid (e.g., a second, a third, a
fourth, a fifth, a sixth, a seventh, an eighth, and/or a ninth
lipid) from Francisella. The at least one additional purified lipid
can be from F. tularensis, for example a virulent strain of F.
tularensis. The virulent strain of F. tularensis can be ssp.
tularensis. In a particular embodiment, the at least one additional
purified lipid has anti-inflammatory properties. In certain
embodiments, the at least one additional purified lipid inhibits
inflammation. In an additional embodiment, the at least one
additional purified lipid modulates an immune response, i.e.
inhibits inflammation, but does not disrupt the immune system's
ability to produce an adaptive immune response. In a particular
embodiment, the at least one additional purified lipid enhances the
immune system's ability to produce an adaptive immune response, as
compared to a patient that does not receive the at least one
additional purified lipid.
[0077] In other embodiments, the composition is a liposome, e.g. an
emulsified liposome. The liposome can have a diameter in a range of
from about 20 nm to about 1,500 nm. In certain embodiments, the
diameter of the liposome is in a range of: from about 20 nm to
about 1,500 nm; from about 100 nm to about 1,500 nm; from about 200
nm to about 1,500 nm; from about 300 nm to about 1,500 nm; from
about 400 nm to about 1,500 nm; from about 500 nm to about 1,500
nm; from about 600 nm to about 1,500 nm; from about 700 nm to about
1,500 nm; from about 800 nm to about 1,500 nm; from about 900 nm to
about 1,500 nm; from about 1,000 nm to about 1,500 nm; from about
1,100 nm to about 1,500 nm; from about 1,200 nm to about 1,500 nm;
from about 1,300 nm to about 1,500 nm; from about 1,400 nm to about
1,500 nm; from about 20 nm to about 1,400 nm; from about 100 nm to
about 1,400 nm; from about 200 nm to about 1,400 nm; from about 300
nm to about 1,400 nm; from about 400 nm to about 1,400 nm; from
about 500 nm to about 1,400 nm; from about 600 nm to about 1,400
nm; from about 700 nm to about 1,400 nm; from about 800 nm to about
1,400 nm; from about 900 nm to about 1,400 nm; from about 1,000 nm
to about 1,400 nm; from about 1,100 nm to about 1,400 nm; from
about 1,200 nm to about 1,400 nm; from about 1,300 nm to about
1,400 nm; from about 20 nm to about 1,300 nm; from about 100 nm to
about 1,300 nm; from about 200 nm to about 1,300 nm; from about 300
nm to about 1,300 nm; from about 400 nm to about 1,300 nm; from
about 500 nm to about 1,300 nm; from about 600 nm to about 1,300
nm; from about 700 nm to about 1,300 nm; from about 800 nm to about
1,300 nm; from about 900 nm to about 1,300 nm; from about 1,000 nm
to about 1,300 nm; from about 1,100 nm to about 1,300 nm; from
about 1,200 nm to about 1,300 nm; from about 20 nm to about 1,200
nm; from about 100 nm to about 1,200 nm; from about 200 nm to about
1,200 nm; from about 300 nm to about 1,200 nm; from about 400 nm to
about 1,200 nm; from about 500 nm to about 1,200 nm; from about 600
nm to about 1,200 nm; from about 700 nm to about 1,200 nm; from
about 800 nm to about 1,200 nm; from about 900 nm to about 1,200
nm; from about 1,000 nm to about 1,200 nm; from about 1,100 nm to
about 1,200 nm; from about 20 nm to about 1,100 nm; from about 100
nm to about 1,100 nm; from about 200 nm to about 1,100 nm; from
about 300 nm to about 1,100 nm; from about 400 nm to about 1,100
nm; from about 500 nm to about 1,100 nm; from about 600 nm to about
1,100 nm; from about 700 nm to about 1,100 nm; from about 800 nm to
about 1,100 nm; from about 900 nm to about 1,100 nm; from about
1,000 nm to about 1,100 nm; from about 20 nm to about 1,000 nm;
from about 100 nm to about 1,000 nm; from about 200 nm to about
1,000 nm; from about 300 nm to about 1,000 nm; from about 400 nm to
about 1,000 nm; from about 500 nm to about 1,000 nm; from about 600
nm to about 1,000 nm; from about 700 nm to about 1,000 nm; from
about 800 nm to about 1,000 nm; from about 900 nm to about 1,000
nm; from about 20 nm to about 900 nm; from about 100 nm to about
900 nm; from about 200 nm to about 900 nm; from about 300 nm to
about 900 nm; from about 400 nm to about 900 nm; from about 500 nm
to about 900 nm; from about 600 nm to about 900 nm; from about 700
nm to about 900 nm; from about 800 nm to about 900 nm; from about
20 nm to about 800 nm; from about 100 nm to about 800 nm; from
about 200 nm to about 800 nm; from about 300 nm to about 800 nm;
from about 400 nm to about 800 nm; from about 500 nm to about 800
nm; from about 600 nm to about 800 nm; from about 700 nm to about
800 nm; from about 20 nm to about 700 nm; from about 100 nm to
about 700 nm; from about 200 nm to about 700 nm; from about 300 nm
to about 700 nm; from about 400 nm to about 700 nm; from about 500
nm to about 700 nm; from about 600 nm to about 700 nm; from about
20 nm to about 600 nm; from about 100 nm to about 600 nm; from
about 200 nm to about 600 nm; from about 300 nm to about 600 nm;
from about 400 nm to about 600 nm; from about 500 nm to about 600
nm; from about 20 nm to about 500 nm; from about 100 nm to about
500 nm; from about 200 nm to about 500 nm; from about 300 nm to
about 500 nm; from about 400 nm to about 500 nm; from about 20 nm
to about 400 nm; from about 100 nm to about 400 nm; from about 200
nm to about 400 nm; from about 300 nm to about 400 nm; from about
20 nm to about 300 nm; from about 100 nm to about 300 nm; from
about 200 nm to about 300 nm; from about 20 nm to about 200 nm;
from about 100 nm to about 200 nm; or from about 100 nm to about
200 nm.
[0078] In another aspect, the present disclosure provides a
liposome comprising purified lipid from Francisella, for example a
virulent strain of Francisella tularensis such as Francisella
tularensis ssp. tularensis. In another embodiment, the purified
lipid comprises a phosphatidylethanolamine (PE) as described
above.
[0079] In further embodiments, the composition further comprises
phosphatidylcholine (PC) as described above. The composition can,
in some embodiments, further include another purified lipid from
Francisella as described above.
[0080] In a particular embodiment, the liposome is an emulsified
liposome. The liposome or the emulsified liposome, according to an
embodiment, has a diameter in a range of from about 20 nm to about
1,500 nm.
[0081] In an additional aspect, the present disclosure provides an
anti-inflammatory composition produced by the following process:
adding Francisella to a mixture of chloroform/methanol (e.g., 2:1)
and mixing, adding water to the organic mixture; separating the
organic phase and aqueous phase; drying the organic phase; and
reconstituting the dried organic phase, wherein the reconstituted
organic phase is the anti-inflammatory composition. In some
embodiments, the Francisella is a virulent Francisella tularensis,
such as Francisella tularensis ssp. tularensis.
[0082] In another aspect, the present disclosure provides a method
of making an anti-inflammatory composition. The method comprises:
adding Francisella to a mixture of chloroform/methanol (e.g., 2:1)
and mixing; adding water to the organic mixture; separating the
organic phase and aqueous phase; drying the organic phase; and
reconstituting the dried organic phase, wherein the reconstituted
organic phase is the anti-inflammatory composition. In some
embodiments, the Francisella is a virulent Francisella tularensis,
such as Francisella tularensis ssp. tularensis.
[0083] In a particular embodiment, the chloroform/methanol is at a
ratio in a range of from about 3:1 to about 1:1, such as about 2:1.
For example the chloroform/methanol ratio can be about 3:1 to about
1:1, about 2.75:1 to about 1:1, about 2:5 to about 1:1, about
2.25:1 to about 1:1, about 2:1 to about 1:1, about 1.75:1 to about
1:1, about 1.5:1 to about 1:1, about 1.25:1 to about 1:1, about 3:1
to about 1.25:1, about 2.75:1 to about 1.25:1, about 2:5 to about
1.25:1, about 2.25:1 to about 1.25:1, about 2:1 to about 1.25:1,
about 1.75:1 to about 1.25:1, about 1.5:1 to about 1.25:1, about
3:1 to about 1.5:1, about 2.75:1 to about 1.5:1, about 2:5 to about
1.5:1, about 2.25:1 to about 1.5:1, about 2:1 to about 1.5:1, about
1.75:1 to about 1.5:1, about 3:1 to about 1.75:1, about 2.75:1 to
about 1.75:1, about 2:5 to about 1.75:1, about 2.25:1 to about
1.75:1, about 2:1 to about 1.75:1, about 3:1 to about 2:1, about
2.75:1 to about 2:1, about 2:5 to about 2:1, about 2.25:1 to about
2:1, about 3:1 to about 2.25:1, about 2.75:1 to about 2.25:1, about
2:5 to about 2.25:1, about 3:1 to about 2.5:1, about 2.75:1 to
about 2.5:1, or about 3:1 to about 2:0.75. In certain embodiments,
the ratio is about 2.75:1, about 2.5:1, about 2.25:1, about 2:1,
about 1.75:1, about 1.5:1, about 1.25:1, or about 1:1.
[0084] In some embodiment, mixing of Francisella and
chloroform/methanol is performed, e.g. vigorously, for about 5
minutes to about 25 minutes. In certain embodiments, the mixing of
Francisella and chloroform/methanol is performed for about 5 to
about 25 minutes, about 5 to about 22.5 minutes, about 5 to about
20 minutes, about 5 to about 17.5 minutes, about 5 to about 15
minutes, about 5 to about 12.5 minutes, about 5 to about 10
minutes, about 5 to about 7.5 minutes, about 7.5 to about 25
minutes, about 7.5 to about 22.5 minutes, about 7.5 to about 20
minutes, about 7.5 to about 17.5 minutes, about 7.5 to about 15
minutes, about 7.5 to about 12.5 minutes, about 7.5 to about 10
minutes, about 10 to about 25 minutes, about 10 to about 22.5
minutes, about 10 to about 20 minutes, about 10 to about 17.5
minutes, about 10 to about 15 minutes, about 10 to about 12.5
minutes, about 12.5 to about 25 minutes, about 12.5 to about 22.5
minutes, about 12.5 to about 20 minutes, about 12.5 to about 17.5
minutes, about 12.5 to about 15 minutes, about 15 to about 25
minutes, about 15 to about 22.5 minutes, about 15 to about 20
minutes, about 15 to about 17.5 minutes, about 17.5 to about 25
minutes, about 17.5 to about 22.5 minutes, about 17.5 to about 20
minutes, about 20 to about 25 minutes, about 20 to about 22.5
minutes, or about 22.5 to about 25 minutes. In other embodiments,
mixing of Francisella and chloroform/methanol is performed for
about 5, about 6, about 7, about 8, about 9, about 10, about 11,
about 12, about 13, about 14, about 15, about 16, about 17, about
18, about 19, about 20, about 21, about 22, about 23, about 24, or
about 25 minutes. In an embodiment, mixing is performed for about
15 minutes.
[0085] In an embodiment, the adding of water to the organic mixture
includes mixing the mixture. In further embodiments, the mixing of
the aqueous and organic mixture is performed, e.g. mixed
vigorously, for about 5 to about 15 minutes. In other embodiments,
the mixing of the aqueous and organic mixture is performed (i.e.,
mixed) for about 5 to about 15 minutes, about 5 to about 12.5
minutes, about 5 to about 10 minutes, about 5 to about 7.5 minutes,
about 7.5 to about 15 minutes, about 7.5 to about 12.5 minutes,
about 7.5 to about 10 minutes, about 10 to about 15 minutes, about
10 to about 12.5 minutes, or about 12.5 to about 15 minutes. In
particular embodiments, the mixing of the aqueous and organic
mixture is performed for about 5, about 5.5, about 6, about 6.5,
about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about
10, about 10.5, about 11, about 11.5, about 12, about 12.5, about
13, about 13.5, about 14, about 14.5, or about 15 minutes.
[0086] In other embodiments, separating the organic phase and the
aqueous phase includes centrifuging the mixture at about
3,500.times.g to about 4,500.times.g for about 5 to about 15
minutes. For example, the mixture may be centrifuged at about
3500.times.g to about 4500.times.g, about 3500.times.g to about
4250.times.g, about 3500.times.g to about 4000.times.g, about
3500.times.g to about 3750.times.g, about 3750.times.g to about
4500.times.g, about 3750.times.g to about 4250.times.g, about
3750.times.g to about 4000.times.g, about 4000.times.g to about
4500.times.g, about 4000.times.g to about 4250.times.g, or about
4250.times.g to about 4500.times.g. In certain embodiments, the
mixture is centrifuged at about 3500.times.g, about 3600.times.g,
about 3700.times.g, about 3800.times.g, about 3900.times.g, about
4000.times.g, about 4100.times.g, about 4200.times.g, about
4300.times.g, about 4400.times.g, or about 4500.times.g. In some
embodiments, the mixture is centrifuged for about 5 to about 15
minutes, about 5 to about 12.5 minutes, about 5 to about 10
minutes, about 5 to about 7.5 minutes, about 7.5 to about 15
minutes, about 7.5 to about 12.5 minutes, about 7.5 to about 10
minutes, about 10 to about 15 minutes, about 10 to about 12.5
minutes, or about 12.5 to about 15 minutes. In certain embodiments,
the mixture is centrifuged for about 5, about 6, about 7, about 8,
about 9, about 10, about 11, about 12, about 13, about 14, or about
15 minutes.
[0087] In yet other embodiments, separating the aqueous phase and
organic phase comprises removing the organic phase and placing the
organic phase into another container. In further embodiments,
drying the organic phase is performed under nitrogen. In an
embodiment, the dried organic phase is reconstituted in an alcohol
(e.g. methanol, ethanol, butanol, and/or pentanol) or chloroform.
In some embodiments, the alcohol is at least 90% alcohol, at least
about 91% alcohol, at least about 92% alcohol, at least about 93%
alcohol, at least about 94% alcohol, at least about 95% alcohol, at
least about 96% alcohol, at least about 97% alcohol, at least about
98%, or at least about 99% alcohol. In other embodiments, the
alcohol is about 90% alcohol, about 90.5% alcohol, about 91%
alcohol, about 91.5% alcohol, about 92% alcohol, about 92.5%
alcohol, about 93% alcohol, about 93.5% alcohol, about 94% alcohol,
about 94.5% alcohol, about 95% alcohol, about 95.5% alcohol, about
96% alcohol, about 96.5% alcohol, about 97% alcohol, about 97.5%
alcohol, about 98% alcohol, about 98.5% alcohol, about 99% alcohol
or about 100% alcohol.
[0088] In further embodiments, the process further comprises
isolating a band of the reconstituted organic phase that runs near
the solvent front of thin layer chromatography to produce an
isolated lipid. In some embodiment, crude lipid is fractioned to
enrich phospholipids and then further fractionated using column
chromatography to enrich for PE. As such, the isolated lipid is
enriched for PE. In an embodiment, the PE is as described in detail
above.
[0089] In other embodiments, the process further comprises adding
PC to the isolated lipid of the reconstituted organic phase, the
isolated lipid, or synthetic PE of the present disclosure. The
ratio of the reconstituted organic phase or isolated lipid to PC is
as described above in detail.
[0090] In yet other embodiments, the process further comprises
adding at least one additional purified lipid from Francisella, for
example Francisella tularensis.
[0091] In another embodiment, the process further comprises
producing a liposome from the reconstituted organic phase, isolated
lipid, or isolated lipid-PC mixture, which can be, for example, an
emulsified liposome. As described above, PE and PC can be combined
in varying ratios from about 50:50 to about 95:5 to obtain
liposomes. In some embodiments, the method further comprises adding
cholesterol in an amount such that cholesterol comprises about 5 to
about 20% of the composition. In other embodiments, the method
further comprises adding cholesterol in an amount such that
cholesterol comprises about 5 to about 20% of the PEPC liposome.
Cholesterol further stabilizes the liposomes. In an embodiment, the
liposome has a diameter as described herein, such as a diameter in
a range of from about 20 nm to about 1,500 nm.
[0092] In another aspect, the present disclosure provides a
pharmaceutical composition comprising a pharmaceutically effective
amount of the composition (e.g. the anti-inflammatory composition)
or the liposome of the present disclosure. In an embodiment, the
pharmaceutical composition further comprises a pharmaceutically
acceptable carrier, for example a gel or a cream.
[0093] Certain embodiments of the disclosure provide pharmaceutical
compositions containing one or more other additional therapeutic
agents, for example, anti-viral small molecule drug inhibits some
aspect of a bacterial or viral infection and/or inflammation
related disease/disorder, or which helps to mitigate one or more
symptoms of a bacterial or viral infection and/or an inflammation
related disease/disorder.
[0094] In another aspect, the disclosure provides a therapeutic
composition comprising the anti-inflammatory composition or a
liposome as described herein in combination with another
therapeutic or biologically active agent. In certain embodiments,
the agent can be conjugated or associated with the lipids
themselves. In still additional embodiments, the agents can be
encompassed within the continuous phase of the interior of the
liposomes.
[0095] The one or more additional therapeutic or biologically
active agent can be any standard therapy known in the art. For
example, the compositions of the disclosure may, if desired, be
administered in combination with an agent that reduces the survival
of a pathogen, including but not limited to Aztreonam;
Chlorhexidine Gluconate; Imidurea; Lycetamine; Nibroxane;
Pirazmonam Sodium; Propionic Acid; Pyrithione Sodium; Sanguinarium
Chloride; Tigemonam Dicholine; Acedapsone; Acetosulfone Sodium;
Alamecin; Alexidine; Amdinocillin; Amdinocillin Pivoxil;
Amicycline; Amlfloxacin; Amifloxacin Mesylate; Amikacin; Amikacin
Sulfate; Aminosalicylic acid; Amninosalicylate sodium; Amoxicillin;
Amphomycin Ampicillin; Ampicillin Sodium; Apalcillin Sodium;
Apramycin; Aspartocin; Astromicin Sulfate; Avilamycin; Avoparcm;
Azithromycin; Azlocillin; Azlocillin Sodium; Bacampicillin
Hydrochloride; Bacitracin; Bacitracin Methylene Disalicylate;
Bacitracin Zinc; Bambermycins; Benzoylpas Calcium; Berythromycin;
Betamicin Sulfate; Biapenem; Biniramycin; Biphenamine
Hydrochloride; Bispyrithione Magsulfex; Butikacin; Butirosin
Sulfate; Capreomycin Sulfate; Carbadox; Carbenicillin Disodium;
Carbenicillin Indanyl Sodium; Carbenicillin Phenyl Sodium;
Carbenicillin Potassium; Carumonam Sodium; Cefaclor; Cefadroxil;
Cefamandole; Cefamandole Nafate; Cefamandole Sodium; Cefaparole;
Cefatrizine; Cefazaflur Sodium; Cefazolin; Cefazolin Sodium;
Celbuperazone; Cefdinir; Cefepime; Cefepime Hydrochloride;
Cefetecol; Cefixime; Cefinenoxime Hydrochloride; Cefmetazole;
Cefmetazole Sodium; Cefonicid Monosodium; Cefonicid Sodium;
Cefoperazone Sodium; Ceforanide; Cefotaxime Sodium; Cefotetan;
Cefotetan Disodium; Cefotiam Hydrochloride; Cefoxitin; Cefoxitin
Sodium; Cefpimizole; Cefpimizole Sodium; Cefpiramide; Cefpiramide
Sodium; Cefpirome Sulfate; Cefpodoxime Proxetil; Cefprozil;
Cefroxadine; Cefsulodin Sodium; Ceflazidime; Ceftibuten;
Ceftiioxime Sodium; Cefiriaxone Sodium; Cefuroxime; Cefuroxime
Axetil; Cefuroxime Pivoxetil; Cefuroxime Sodium; Cephacetrile
Sodium; Cephalexin; Cephalexin Hydrochloride; Cephaloglycin;
Cephaloridine; Cephalothin Sodium; Cephapirin Sodium; Cephradine;
Cetocycline Hydrochloride; Cetophenicol; Chloramphenicol;
Chloramphenicol Palmitate; Chloramphenicol Pantothenate Complex;
Chloramphenicol Sodium Succinate; Chlorhexidine Phosphanilale;
Chloroxylenol; Chlortetracycline Bisulfate; Chlortetracycline
Hydrochloride; Cinoxacin; Ciprofloxacin; Ciprofloxacin
Hydrochloride; Cirolemycin; Clarithromycin; Clinafloxacin
Hydrochloride; Clindamycin; Clindamycin Hydrochloride; Clindamycin
Palmitate Hydrochloride; Clindamycin Phosphate; Clofazimine;
Cloxacillin Benzathine; Cloxacillin Sodium; Cloxyquin;
Colistimethate Sodium; Colistin Sulfate; Coumermycin; Coumermycin
Sodium; Cyclacillin; Cycloserine; Dalfopristin; Dapsone;
Daptomycin; Demeclocycline; Demeclocycline Hydrochloride;
Demecycline; Denofungin; Diaveridine; Dicloxacillin; Dicloxacillin
Sodium; Dihydrostreptomycin Sulfate; Dipyrithione; Dirithromycin;
Doxycycline; Doxycycline Calcium; Doxycycline Fosfatex; Doxycycline
Hyclate; Droxacm Sodium; Enoxacin; Epicillin; Epitetracycline
Hydrochloride; Erythromycin; Erythromycin Acistrate; Erythromycin
Estolate; Erythromycin Ethylsuccinate; Erythromycin Gluceptate;
Erythromycin Lactobionate; Erythromycin Propionate; Erythromycin
Stearate; Ethambutol Hydrochloride; Ethionamide; Fleroxacin;
Floxacillin; Fludalanine; Flumequine; Fosfomycin; Fosfomycin
Tromethamine; Fumoxicillin; Furazolium Chloride; Furazolium
Tartrate; Fusidate Sodium; Fusidic Acid; Gentamicin Sulfate;
Gloximonam; Gramicidin; Haloprogin; Hetacillin; Hetacillin
Potassium; Hexedine; Ibafloxacin; Imipenem; Isoconazole;
Isepamicin; Isoniazid; Josamycin; Kanamycin Sulfate; Kitasamycin;
Levofuraltadone; Levopropylcillin Potassium; Lexithromycin;
Lincomycin; Lincomycin Hydrochloride; Lomefloxacin; Lomefloxacin
Hydrochloride; Lomefloxacin Mesylate; Loracarbef; Mafenide;
Meclocycline; Meclocycline Sulfosalicylate; Megalomicin Potassium
Phosphate; Mequidox; Meropenem; Methacycline; Methacycline
Hydrochloride; Methenamine; Methenamine Hippurate; Methenamine
Mandelate; Methicillin Sodium; Metioprim; Metronidazole
Hydrochloride; Metronidazole Phosphate; Mezlocillin; Mezlocillin
Sodium; Minocycline; Minocycline Hydrochloride; Mirincamycin
Hydrochloride; Monensin; Monensin Sodium; Nafcillin Sodium;
Nalidixate Sodium; Nalidixic Acid; Natamycin; Nebramycin; Neomycin
Palmitate; Neomycin Sulfate; Neomycin Undecylenate; Netilmicin
Sulfate; Neutramycin; Nifuradene; Nifuraldezone; Nifuratel;
Nifuratrone; Nifurdazil; Nifurimide; Nifurpinnol; Nifurquinazol;
Nifurthiazole; Nitrocycline; Nitrofurantoin; Nitromide;
Norfloxacin; Novobiocin Sodium; Ofloxacin; Ormetoprim; Oxacillin
Sodium; Oximonam; Oximonam Sodium; Oxolinic Acid; Oxytetracycline;
Oxytetracycline Calcium; Oxytetracycline Hydrochloride;
Paldinmycin; Parachlorophenol; Paulomycin; Pefloxacin; Pefloxacin
Mesylate; Penamecillin; Penicillin G Benzathine; Penicillin G
Potassium; Penicillin G Procaine; Penicillin G Sodium; Penicillin
V; Penicillin V Benzathine; Penicillin V Hydrabamine; Penicillin V
Potassium; Pentizidone Sodium; Phenyl Aminosalicylate; Piperacillin
Sodium; Pirbenicillin Sodium; Pindicillm Sodium; Pirlimycin
Hydrochloride; Pivampicillin Hydrochloride; Pivampicillin Pamoate
Pivampicillin Probenate; Polymyxin B Sulfate; Porfiromycin;
Propikacin; Pyrazinamide; Pyrithione Zinc; Quindecamine Acetate;
Quinupristin; Racephenicol; Ramoplanin; Ranimycin; Relomycin;
Repromicin; Rifabutin; Rifametane; Rifamexil; Rifamide; Rifampin;
Rifapentine; Rifaximin; Rolitetracycline; Rolitetracycline Nitrate;
Rosaramicin; Rosaramicin Butyrate; Rosaramicin Propionate;
Rosaramicin Sodium Phosphate; Rosaramicin Stearate; Rosoxacil;
Roxarsone; Roxithromycin; Sancycline; Sanfetrinem Sodium;
Sarmoxicillin; Sarpicillin; Scopafungin; Sisomicin; Sisomicin
Sulfate; Sparfloxacin; Spectinomycin Hydrochloride; Spiramycin;
Stallimycin Hydrochloride; Steffimycin; Streptomycin Sulfate;
Streptonicozid; Sulfabenz; Sulfabenzamide; Sulfacetamide;
Sulfacetamide Sodium; Sulfacytine; SulTfdiazine; Sulfadiazine
Sodium; Sulfadoxine; Sulfalene Sulfamerazine; Sulfameter;
Sulfamethazine; Sulfamethizole; Sulfamethoxazole;
Sulfamonomethoxine; Sulfamoxole; Sulfanilate Zinc; Sulfanitran;
Sulfasalazine; Sulfasomizole; Sulfathiazole; Sulfazamet;
Sulfisoxazole; Sulfisoxazole Acetyl; Sulfisoxazole Diolamine;
Sulfomyxin; Sulopenem; Sultamicillin; Suncillin Sodium;
Talampicillin Hydrochloride; Teicoplanin; Temafloxacin
Hydrochloride; Temocillin; Tetracycline; Tetracycline
Hydrochloridc; Tetracycline Phosphate Complex; Tetroxoprim;
Thiamphenicol; Thiphencillin Potassium; Ticarcillin Cresyl Sodium;
Ticarcillin Disodium; Ticarcillin Monosodium; Ticlatone; Tiodonium
Chloride; Tobramycin; Tobramycin Sulfate; Tosufloxacin;
Trimethoprim; Trimethoprim Sulfate; Trisulfapyrimidines;
Troleandomycin; Trospectomycin Sulfate; Tyrothricin; Vancomycin;
Vancomycin Hydrochloride; Virginiamycin; Zorbamycin; Difloxacin
Hydrochloride; Lauryl Isoquinolinium Bromide; Moxalactam Disodium;
Omidazole; Pentisomicin; and Sarafloxacin Hydrochloride. See,
generally, The Merck Manual of Diagnosis and Therapy, 15th Ed.
1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. When used
with the composition of the disclosure, such therapeutic agents may
be used individually, sequentially, or in combination with one or
more other such therapeutic agents. Anti-inflammatory drugs,
including but not limited to nonsteroidal anti-inflammatory drugs
(e.g., naproxen dodium, celecoxib, sulindac, oxaprozin, salsalate,
diflunisal, piroxicam, indomethacin, etodolac, meloxicam, naproxen,
nabumetone, ketorolac tromethamine, naproxen/esomeprazole,
diclofenac, ibuprofine, and/or aspirin) and corticosteroids (e.g.,
bethamethasone, prednisone, prednisolone, triamcinolone,
methylprednisolone, dexamethasone, hydrocortisone, cortisone,
ethamethasoneb, and/or fludrocortisone), and antiviral drugs
(including but not limited to amantadine, rimantadine, oseltamivir,
zanamivi, acyclovir, brivudine, docosanol, famiciclovir,
idoxuridine, penciclovir, valacyclovir, ribavirin, gangciclovir,
trifluoridine, zidovudine, didanosine, zalcitabine, lamivudine,
abacavir, atazanavir, atripla, idovudine, combivir, darunavir,
didanosine, delavirdine, dolutegravir, efavirenz, elvitegravir,
enfuvirtide, etravirine, eviplera, fosamprenavir, emtricitabine,
indinavir, kivexa, lopinavir, ritonavir, maraviroc, nelfinavir,
nevirapine, raltegravir, rilpivirine, ritonavir, saquinavir,
stribild, tenofovir, tenofovir, tipranavir, triomine, trizivir,
truvada, and .alpha.-interfereon) may also be combined in
compositions of the disclosure. See, generally, The Merck Manual of
Diagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway,
N.J., pages 2499-2506 and 46-49, respectively). Other therapeutic
agents are also within the scope of this disclosure. Two or more
combined compounds may be used together or sequentially. Such
compounds may be administered using a separate administration
schedule relative to the administration schedule of the active
agents of the disclosure. The administration schedules may also be
the same or have overlap.
[0096] In yet a further aspect, the present disclosure provides a
method of treating or preventing a microbial infection or
inflammation resulting from a microbial infection in a patient in
the need thereof. The method comprises administering a composition
comprising an effective amount of the liposome (or the
anti-inflammatory composition) of present disclosure, wherein the
composition or liposome is effective in treating or preventing the
microbial infection or the inflammation resulting from a microbial
infection.
[0097] In an embodiment, the microbial infection is a bacterial or
a viral infection, for example a bacterial infection or a viral
infection that causes inflammation, e.g. inflammation of the
epidermis and/or respiratory system.
[0098] In particular embodiments, the invention provides for the
treatment of bacterial infections, including infections with gram
negative and gram positive bacteria. Such gram positive bacteria
include, but are not limited to, Pasteurella species, Staphylococci
species, and Streptococcus species. Gram negative bacteria include,
but are not limited to, Escherichia coli, Pseudomonas species, and
Salmonella species. Specific examples of infectious bacteria
include but are not limited to, Helicobacter pyloris, Burkholderia
sps, Borellia burgdorferi, Legionella pneumophilia, Mycobacteria
sps (e.g. M. tuberculosis, M. avium, M. intracellulare, M. kansaii,
M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae,
Neisseria meningitidis, Listeria monocytogenes, Streptococcus
pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B
Streptococcus), Streptococcus (viridans group), Streptococcus
faecalis, Streptococcus bovis, Streptococcus (anaerobic sps.),
Streptococcus pneumoniae, pathogenic Campylobacter sp.,
Enterococcus sp., Haemophilus influenzae, Bacillus antracis,
corynebacterium diphtheriae, corynebacterium sp., Erysipelothrix
rhusiopathiae, Clostridium perfringers, Clostridium tetani,
Enterobacter aerogenes, Klebsiella pneumoniae, Pasteurella
multocida, Bacteroides sp., Fusobacterium nucleatum, Sluptobacillus
maniliformis, Treponema pallidium, Treponema pertenue, Leptospira,
Rickettsia ssp, Yersinia pestis and Actinomyces israelli.
[0099] In still other embodiments, the methods of the invention can
be used to treat or prevent a viral infection. Exemplary viral
pathogens include but are not limited to: Retroviridae (e.g. human
immunodeficiency viruses, such as HIV-1 (also referred to as
HDTV-III, LAVE or HTLV-II/LAV, or HIV-III; and other isolates, such
as HIV-LP; Picornaviridae (e.g. polio viruses, hepatitis A virus;
enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses);
Calciviridae (e.g. strains that cause gastroenteritis); Togaviridae
(e.g. equine encephalitis viruses, rubella viruses); Flaviridae
(e.g. dengue viruses, encephalitis viruses, yellow fever viruses);
Coronoviridae (e.g. coronaviruses); Rhabdoviridae (e.g. vesicular
stomatitis viruses, rabies viruses); Filoviridae (e.g. ebola
viruses); Paramyxoviridae (e.g. parainfluenza viruses, mumps virus,
measles virus, respiratory syncytial virus); Orthormyxoviridae
(e.g. influenza viruses); Bungaviridae (e.g. Hantaan viruses, bunga
viruses, phleboviruses and Nairo viruses); Arena viridae
(hemorrhagic fever viruses); Reoviridae (e.g. reoviruses,
orbiviurses and rotaviruses); Birnaviridae; Hepadnaviridae
(Hepatitis B virus); Parvovirida (parvoviruses); Papovaviridae
(papilloma viruses, polyoma viruses); Adenoviridae (most
adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2,
varicella zoster virus, cytomegalovirus (CMV), herpes virus;
Poxviridae (variola viruses, vaccinia viruses, pox viruses); and
Iridoviridae (e.g. African swine fever virus); and unclassified
viruses (e.g. the agent of delta hepatitis (thought to be a
defective satellite of hepatitis B virus), the agents of non-A,
non-B hepatitis (class 1=internally transmitted; class
2=parenterally transmitted (i.e. Hepatitis C); Norwalk and related
viruses, and astroviruses).
[0100] In a particular embodiment, the bacterial infection or viral
infection is the bacterial infection or viral infection is selected
from the group consisting of Staphylococcus aureus, Streptrcoccus
pyogenes, Clostridium perfringens, Bacillus anthracis, Francisella
tularensis, Corynebacterium diphtheria, Streptococcus pneumoniae,
Haemophilus influenza, Bordetella pertussis, Mycobacterium
tuberculosis or bovis, Mycoplasma pneumoniae, Legionella
pneumophila, Chlamydia psittaci, Chlamydia pneumoniae, Coxiella
burnetii, measles, rubella, varicella zoster, parvovirus, herpes
simplex virus 6, herpes simplex virus 7, herpes simplex virus 8,
Epstein Barr virus, enterovirus, coxsackie virus, togavirus,
coronavirus, rhinovirus, bunyavirus, arenavirus, smallpox, cowpox,
monkey pox, zika virus, dengue virus, nairovirus, arenavirus,
filovirus, west nile virus, molluscum contagiosm, human
papillomavirus, coronavirus, rhinovirus, respiratory syncytial
virus, and Influenzavirus.
[0101] In some embodiments, the composition or the liposome is
administered prior to exposure to the microbial infection, while in
other embodiments, the composition or the liposome is administered
post exposure to the microbial infection. In certain embodiments,
the composition or the liposome is administered both before and
after infection. In certain embodiments, the method further
comprises co-administering the composition or the liposome with one
or more additional therapeutic agents.
[0102] In a further aspect, the present disclosure provides for a
method of treating or preventing inflammation in a subject, e.g., a
patient, in the need thereof. The method comprises administering a
composition comprising an effective amount of the liposome (or the
anti-inflammatory composition) of the present disclosure to a
subject in need thereof, wherein the composition is effective in
treating or preventing inflammation in the subject. In an
embodiment, the inflammation is related to at least one of a
bacterial infection, a viral infection, an autoimmune disease or
disorder, and an allergy.
[0103] In an additional aspect, the present disclosure provides a
method of modulating an immune response in a subject. The method
comprises administering a composition comprising an effective
amount of the liposome (or the anti-inflammatory composition) of
the present disclosure to a subject in need thereof, e.g., a
subject suffering from inflammation, wherein the composition is
effective in modulating the immune response of the subject. In some
embodiments, the modulation comprises enhancing the
immunocompetence in the subject, e.g. by suppressing an
inflammatory response. In an embodiment, the suppressing an
inflammatory response does not affect the patients ability to
produce an adaptive immune response.
[0104] In a further embodiment, the modulation of an immune
response comprises inhibiting microbial replication, e.g. bacterial
replication or viral replication, in the patient. In some
embodiments, the bacterial infection or viral infection is a
respiratory infection and/or a epidermal infection.
[0105] In certain embodiments, the inflammation is caused by at
least one of an autoimmune (or autoimmune related) disease or
disorder, an inflammatory disease or disorder, an allergy, or a
combination thereof. In other embodiments, the subject has
dermatological or respiratory inflammation. In certain embodiments,
the autoimmune (or autoimmune related) disease or disorder is
selected from the group consisting of Acute Disseminated
Encephalomyelitis (ADEM), acute necrotizing hemorrhagic
leukoencephalitis, Addison's disease, Agammaglobulinemia, Alopecia
areata, Amyloidosis, Ankylosing spondylitis. Anti-GBM/Anti-TBM
nephritis. Antiphospholipid syndrome (APS), autoimmune angioedema,
autoimmune aplastic anemia, autoimmune dysautonomia, autoimmune
hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency,
autoimmune inner ear disease (AIED), autoimmune myocarditis,
autoimmune oophoritis, autoimmune pancreatitis, autoimmune
retinopathy, autoimmune thrombocytopenic purpura (ATP), autoimmune
thyroid disease, autoimmune urticarial, axonal & neuronal
neuropathies. Balo disease, Behcet's disease, Bullous pemphigoid,
cardiomyopathy, Castleman disease, Celiac disease, Chagas disease,
chronic inflammatory demyelinating polyneuropathy (CIDP), chronic
recurrent multifocal ostomyelitis (CRMO), Churg-Strauss syndrome,
Cicatricial pemphigoid/benign mucosal pemphigoid, Crohn's disease,
Cogans syndrome, cold agglutinin disease, congenital heart block,
coxsackie myocarditis, CREST disease, essential mixed
cryoglobulinemia, demyelinating neuropathies, dermatitis
herpetiformis, dermatomyositis, devic's disease (neuromyelitis
optica), discoid lupus, Dressler's syndrome, endometriosis,
eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum,
experimental allergic encephalomyelitis, Evans syndrome, fibrosing
alveolitis, giant cell arteritis (temporal arteritis), giant cell
myocarditis, glomerulonephritis, Goodpasture's syndrome,
Granulomatosis with Polyangiitis (GPA) (formerly called Wegener's
Granulomatosis), Graves' disease, Guillain-Barre syndrome.
Hashimoto's encephalitis, Hashimoto's thyroiditis, Hemolytic
anemia, Henoch-Schonlein purpura, Herpes gestationis,
hypogammaglobulinemia, idiopathic thrombocytopenic purpura (ITP),
IgA nephropathy, IgG4-related sclerosing disease, immunoregulatory
lipoproteins, inclusion body myositis, interstitial cystitis,
juvenile arthritis, juvenile diabetes (Type 1 diabetes), juvenile
myositis, Kawasaki syndrome, Lambert-Eaton syndrome,
leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus,
Ligneous conjunctivitis, Linear IgA disease (LAD), Lupus (SLE),
Lyme disease (chronic), Meniere's disease, microscopic
polyangiitis, mixed connective tissue disease (MCTD), Mooren's
ulcer, Mucha-Habermann disease, multiple sclerosis. Myasthenia
gravis, myositis, narcolepsy, neuromyelitis optica (Devic's),
neutropenia, ocular cicatricial pemphigoid, optic neuritis,
palindromic rheumatism, PANDAS (Pediatric Autoimmune
Neuropsychiatric Disorders Associated with Streptococcus),
paraneoplastic cerebellar degeneration, paroxysmal nocturnal
hemoglobinuria (PNH), Parry Romberg syndrome, Parsonnage-Turner
syndrome, pars planitis (peripheral uveitis), pemphigus, peripheral
neuropathy, perivenous encephalomyelitis, Pernicious anemia, POEMS
syndrome, polyarteritis nodosa, type I, II, & III autoimmune
polyglandular syndromes, polymyalgia rheumatic, polymyositis,
postmyocardial infarction syndrome, postpericardiotomy syndrome,
progesterone dermatitis, primary biliary cirrhosis, primary
sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic
pulmonary fibrosis, pyoderma gangrenosum, pure red cell aplasia,
Raynauds phenomenon, reactive Arthritis, reflex sympathetic
dystrophy, Reiter's syndrome, relapsing polychondritis, restless
legs syndrome, retroperitoneal fibrosis, rheumatic fever,
rheumatoid arthritis, sarcoidosis, schmidt syndrome, scleritis,
scleroderma, Sjogren's syndrome, sperm and/or testicular
autoimmunity, stiff person syndrome, subacute bacterial
endocarditis (SBE), Susac's syndrome, sympathetic ophthalmia,
Takayasu's arteritis, temporal arteritis/giant cell arteritis,
thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, transverse
myelitis, type 1 diabetes, ulcerative colitis, undifflerentiated
connective tissue disease (UCTD), uveitis, vasculitis,
vesiculobullous dermatosis, vitiligo, or Wegener's granulomatosis
(now termed Granulomatosis with Polyangiitis (GPA)).
[0106] In another embodiment, the inflammatory disease is selected
from the group consisting of Alzheimer's, ankylosing spondylitis,
arthritis (including osteoarthritis, rheumatoid arthritis (RA),
and/or psonatic arthritis), asthma, atherosclerosis, Barrett's
esophagus, chronic Lyme disease. Crohn's disease, colitis,
dermatitis, diabetes, diverticulitis, fibromyalgia,
gastroesophageal reflux disease, hepatitis, interstitial cystitis,
irritable bowel syndrome (IBS), Lofgren's syndrome, systemic lupus
erythematous (SLE), multiple sclerosis, nephritis, Parkinson's
disease, polymyalgia rheumatica, prostatitis, psoriasis, psoriatic
arthritis, Sjogren's syndrome, ulcerative colitis, and uveitis.
[0107] In further embodiments, the disease or disorder associated
with inflammation is selected from the group consisting of
allergies, Alzheimer's, anemia, ankylosing spondylitis, asthma,
autism, arthritis, carpal tunnel syndrome, celiac, crohn's disease,
eczema, fibromyalgia, fibrosis, gall bladder disease, GERD,
Guillain-Barre, hashimoto's thyroiditis, lupus, multiple sclerosis,
pancreatitis, psoriasis, polymyalgia rheumatica, rheumatoid
arthritis, scleroderma, and stroke.
[0108] The present disclosure provides compositions or liposomes
featuring purified lipid from Francisella and methods of using such
compositions/liposomes for the treatment or prevention of an
infectious disease and/or inflammation. Other features and
advantages of the invention will be apparent from the detailed
description, and from the claims.
[0109] As used herein, the terms "treat," "treating." "treatment,"
and the like refer to reducing or ameliorating a disorder and/or
symptoms associated therewith. It will be appreciated that,
although not precluded, treating a disorder or condition does not
require that the disorder, condition or symptoms associated
therewith be completely eliminated.
[0110] As used herein, the terms "prevent," "preventing,"
"prevention," "prophylactic treatment" and the like refer to
reducing the probability of developing a disorder or condition in a
subject, who does not have, but is at risk of or susceptible to
developing a disorder or condition.
[0111] The therapeutic methods of the invention (which include
prophylactic treatment) in general comprises administration of a
therapeutically effective amount of the compositions or liposome of
the present disclosure to a subject (e.g., animal, human) in need
thereof, including a mammal, particularly a human. Such treatment
will be suitably administered to subjects, particularly humans,
suffering from, having, susceptible to, or at risk for a disease,
disorder, or symptom thereof. Determination of those subjects "at
risk" can be made by any objective or subjective determination by a
diagnostic test or opinion of a subject or health care provider
(e.g., genetic test, enzyme or protein marker, Marker (as defined
herein), family history, and the like). The compositions and/or
liposomes of the present disclosure may be also used in the
treatment of any other disorders in which a pathogen infection may
be implicated.
[0112] In one embodiment, the invention provides a method of
monitoring treatment progress. The method includes the step of
determining a level of diagnostic marker (Marker) (e.g., any target
delineated herein modulated by the composition described herein, a
protein or indicator thereof, etc.) or diagnostic measurement
(e.g., screen, assay) in a subject suffering from or susceptible to
a disease/disorder or symptoms thereof associated with a pathogen
infection or inflammatory disease/disorder, in which the subject
has been administered a therapeutic amount of a composition herein
sufficient to treat the disease or symptoms thereof. The level of
Marker determined in the method can be compared to known levels of
Marker in either healthy normal controls or in other afflicted
patients to establish the subject's disease status. In preferred
embodiments, a second level of Marker in the subject is determined
at a time point later than the determination of the first level,
and the two levels are compared to monitor the course of disease or
the efficacy of the therapy. In certain preferred embodiments, a
pre-treatment level of Marker in the subject is determined prior to
beginning treatment according to this invention; this pre-treatment
level of Marker can then be compared to the level of Marker in the
subject after the treatment commences, to determine the efficacy of
the treatment.
[0113] Therapeutic Compositions
[0114] In another aspect, the present disclosure provides a
pharmaceutical composition comprising a pharmaceutically effective
amount of a liposome or the anti-inflammatory composition of the
present disclosure. In an embodiment, the pharmaceutical
composition further comprises a pharmaceutically acceptable carrier
or excipient. In certain embodiments, the composition is in the
form of at least one of a liquid, a gel or a cream.
[0115] In another aspect, the disclosure provides a liposome
composition comprising a modified form of PE, e.g., modified PE
isolated from Francisella tularensis. There are a wide range of
lipid modifications known to those skilled in the art, all of which
are expressly contemplated herein, including. e.g., mannosylation.
In certain embodiments, the PE is admixed with or conjugated to
another agent, e.g., to another biologically active agent, a homing
agent, or to a molecular entity, such as a molecular entity for
increasing at least one of half-life, stability, bioavailability or
a combination thereof. In certain embodiments, the agent is an
antibody or a PEG molecule.
[0116] Pharmaceutical compositions comprising combinations of an
effective amount of the composition (e.g., the anti-inflammatory
composition) or liposome of the present disclosure, in combination
with a pharmaceutically effective amount of a carrier, additive
and/or excipient, represents a further aspect of the present
disclosure.
[0117] The compounds as described herein may, in accordance with
the disclosure, be administered in single or divided doses by the
oral, parenteral or topical routes. Administration of the active
composition may range from continuous (intravenous drip) to several
oral administrations per day (for example, Q.I.D.) and may include
oral, topical, parenteral, intramuscular, intravenous,
sub-cutaneous, transdermal (which may include a penetration
enhancement agent), buccal, sublingual and suppository
administration, among other routes of administration. Enteric
coated oral tablets may also be used to enhance bioavailability of
the compounds from an oral route of administration. The most
effective dosage form will depend upon the pharmacokinetics of the
particular agent chosen as well as the severity of disease in the
patient. Administration of composition according to the present
disclosure as sprays, mists, or aerosols for intra-nasal,
intra-tracheal or pulmonary administration may also be used. The
present disclosure therefore also is directed to pharmaceutical
compositions comprising an effective amount of the compositions as
described herein, optionally in combination with a pharmaceutically
acceptable carrier, additive or excipient. Compositions according
to the present disclosure may be administered in immediate release,
intermediate release or sustained or controlled release forms.
Sustained or controlled release forms are preferably administered
orally, but also in suppository and transdermal or other topical
forms. Intramuscular injections in liposomal form may also be used
to control or sustain the release of compound at an injection
site.
[0118] The compositions as described herein may be formulated in a
conventional manner using one or more pharmaceutically acceptable
carriers and may also be administered in controlled-release
formulations. Pharmaceutically acceptable carriers that may be used
in these pharmaceutical compositions include, but are not limited
to, ion exchangers, alumina, aluminum stearate, lecithin, serum
proteins, such as human serum albumin, buffer substances such as
phosphates, glycine, sorbic acid, potassium sorbate, partial
glyceride mixtures of saturated vegetable fatty acids, water, salts
or electrolytes, such as prolamine sulfate, disodium hydrogen
phosphate, potassium hydrogen phosphate, sodium chloride, zinc
salts, colloidal silica, magnesium trisilicate, polyvinyl
pyrrolidone, cellulose-based substances, polyethylene glycol,
sodium carboxymethylcellulose, polyacrylates, waxes,
polyethylene-polyoxypropylene-block polymers, polyethylene glycol
and wool fat.
[0119] The compositions, including pharmaceutical compositions and
liposomes, of the present disclosure may be administered orally,
parenterally, by inhalation spray, topically, rectally, nasally,
buccally, vaginally or via an implanted reservoir. The term
"parenteral" as used herein includes subcutaneous, intravenous,
intramuscular, intra-articular, intra-synovial, intrasternal,
intrathecal, intrahepatic, intralesional and intracranial injection
or infusion techniques. Preferably, the compositions are
administered orally, intraperitoneally or intravenously.
[0120] Sterile injectable forms of the compositions as described
herein may be aqueous or oleaginous suspension. 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 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 the acceptable vehicles and solvents that may be employed are
water, Ringer's solution and isotonic sodium chloride solution. In
addition, sterile, fixed oils are conventionally employed as a
solvent or suspending medium. For this purpose, any bland fixed oil
may be employed including synthetic mono- or di-glycerides. Fatty
acids, such as oleic acid and its glyceride derivatives are useful
in the preparation of injectables, as are natural
pharmaceutically-acceptable oils, such as olive oil or castor oil,
especially in their polyoxyethylated versions. These oil solutions
or suspensions may also contain a long-chain alcohol diluent or
dispersant, such as Ph. Helv or similar alcohol.
[0121] The pharmaceutical compositions as described herein may be
orally administered in any orally acceptable dosage form including,
but not limited to, capsules, tablets, aqueous suspensions or
solutions. In the case of tablets for oral use, carriers which are
commonly used include lactose and corn starch. Lubricating agents,
such as magnesium stearate, are also typically added. For oral
administration in a capsule form, useful diluents include lactose
and dried corn starch. When aqueous suspensions are required for
oral use, the active ingredient is combined with emulsifying and
suspending agents. If desired, certain sweetening, flavoring or
coloring agents may also be added.
[0122] Alternatively, the pharmaceutical compositions, as described
herein, may be administered in the form of suppositories for rectal
administration. These can be prepared by mixing the agent with a
suitable non-irritating excipient, which is solid at room
temperature but liquid at rectal temperature and therefore will
melt in the rectum to release the drug. Such materials include
cocoa butter, beeswax and polyethylene glycols.
[0123] The pharmaceutical compositions as described herein may also
be administered topically. Suitable topical formulations are
readily prepared for each of these areas or organs. Topical
application for the lower intestinal tract can be effected in a
rectal suppository formulation (see above) or in a suitable enema
formulation. Topically-acceptable transdermal patches may also be
used.
[0124] For topical applications, the pharmaceutical 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 disclosure
include, but are not limited to, mineral oil, liquid petrolatum,
white petrolatum, propylene glycol, polyoxyethylene,
polyoxypropylene compound, emulsifying wax and water. In certain
preferred aspects of the disclosure, the compounds may be coated
onto a stent which is to be surgically implanted into a patient in
order to inhibit or reduce the likelihood of occlusion occurring in
the stent in the patient.
[0125] 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, but are not limited
to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl
esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and
water.
[0126] For ophthalmic use, the pharmaceutical compositions may be
formulated as micronized suspensions in isotonic, pH adjusted
sterile saline, or, preferably, as solutions in isotonic, pH
adjusted sterile saline, either with our without a preservative
such as benzylalkonium chloride. Alternatively, for ophthalmic
uses, the pharmaceutical compositions may be formulated in an
ointment such as petrolatum.
[0127] The pharmaceutical compositions as described herein may also
be administered by nasal aerosol or inhalation. Such compositions
are prepared according to techniques well-known in the art of
pharmaceutical formulation and may be prepared as solutions in
saline, employing benzyl alcohol or other suitable preservatives,
absorption promoters to enhance bioavailability, fluorocarbons,
and/or other conventional solubilizing or dispersing agents.
[0128] The amount of the composition or liposome of the present
disclosure in a pharmaceutical composition as described herein that
may be combined with the carrier materials to produce a single
dosage form will vary depending upon the host and disease treated,
the particular mode of administration. Preferably, the compositions
should be formulated to contain between about 0.01 milligram to
about 750 milligrams or more, more preferably about 1 milligram to
about 600 milligrams, and even more preferably about 10 milligrams
to about 500 milligrams of active composition according to the
present disclosure.
[0129] It should also be understood that a specific dosage and
treatment regimen for any particular patient will depend upon a
variety of factors, including the activity of the specific compound
employed, the age, body weight, general health, sex, diet, time of
administration, rate of excretion, drug combination, and the
judgment of the treating physician and the severity of the
particular disease or condition being treated.
[0130] A patient or subject in need of therapy using compositions,
including liposome(s), according to the methods of the present
disclosure can be treated by administering to the patient (subject)
an effective amount of the composition (including liposome)
according to the present disclosure, optionally in a
pharmaceutically acceptable carrier or diluent, either alone, or in
combination with additional agents as otherwise described
herein.
[0131] These compositions can be administered by any appropriate
route, for example, orally, parenterally, intravenously,
intradermally, subcutaneously, or topically, including
transdermally, in liquid, cream, gel, or solid form, or by aerosol
form.
[0132] The composition is included in the pharmaceutically
acceptable carrier or diluent in an amount sufficient to deliver to
a patient a therapeutically effective amount for the desired
indication, without causing serious toxic effects in the patient
treated. A preferred dose of the composition for all of the
herein-mentioned conditions is in the range from about 10 ng/kg to
300 mg/kg, preferably 0.1 to 100 mg/kg per day, more generally 0.5
to about 25 mg per kilogram body weight of the recipient/patient
per day. A typical topical dosage will range from about 0.01 to
about 5% wt/wt in a suitable carrier.
[0133] The composition is conveniently administered in any suitable
unit dosage form, including but not limited to one containing less
than 1 mg, 1 mg to 3000 mg, preferably 5 to 500 mg of active
ingredient per unit dosage form. An oral dosage of about 25-250 mg
is often convenient.
[0134] The composition is preferably administered to achieve peak
plasma concentrations of the active components of the composition
is about 0.00001-30 mM, preferably about 0.1-30 .mu.M. This may be
achieved, for example, by the intravenous injection of a solution
or formulation of the composition, optionally in saline, or an
aqueous medium or administered as a bolus of the active components
of the composition. Oral administration is also appropriate to
generate effective plasma concentrations of active agent.
[0135] The concentration of active components in the drug
composition will depend on absorption, distribution, inactivation,
and excretion rates of the drug as well as other factors known to
those of skill in the an. It is to be noted that dosage values will
also vary with the severity of the condition to be alleviated. It
is to be further understood that for any particular subject,
specific dosage regimens should be adjusted over time according to
the individual need and the professional judgment of the person
administering or supervising the administration of the
compositions, and that the concentration ranges set forth herein
are exemplary only and are not intended to limit the scope or
practice of the claimed composition. The active ingredient may be
administered at once, or may be divided into a number of smaller
doses to be administered at varying intervals of time.
[0136] Oral compositions will generally include an inert diluent or
an edible carrier. They may be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active component(s) can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Pharmaceutically compatible binding agents can be included as part
of the composition.
[0137] The tablets, pills, capsules, troches and the like can
contain any of the following ingredients, or compounds of a similar
nature: a binder such as microcrystalline cellulose, gum tragacanth
or gelatin; an excipient such as starch or lactose, a dispersing
agent such as alginic acid, Primogel, or corn starch; a lubricant
such as magnesium stearate or Sterotes; a glidant such as colloidal
silicon dioxide; a sweetening agent such as sucrose or saccharin or
a flavoring agent such as peppermint, methyl salicylate, or orange
flavoring. When the dosage unit form is a capsule, it can contain,
in addition to material of the above type, a liquid carrier such as
a fatty oil. In addition, dosage unit forms can contain various
other materials which modify the physical form of the dosage unit,
for example, coatings of sugar, shellac, or enteric agents.
[0138] The active components or composition of the disclosure can
be administered as a component of an elixir, suspension, syrup,
wafer, chewing gum or the like. A syrup may contain, in addition to
the active compounds, sucrose as a sweetening agent and certain
preservatives, dyes and colorings and flavors.
[0139] The active component(s) or composition of the disclosure can
also be mixed with other active materials that do not impair the
desired action, or with materials that supplement the desired
action, such as anti-viral agents, antibacterial agents,
anti-inflammatories, among other agents. In certain preferred
aspects of the disclosure, the composition according to the present
disclosure are coadministered with another bioactive agent, such as
an antibiotic, an antiviral, or a known anti-inflammatory, or an
agent to ameliorate a symptom or condition associated with the
diseases or disorders described herein.
[0140] Solutions or suspensions used for parenteral, intradermal,
subcutaneous, or topical application can include the following
components: a sterile diluent such as water for injection, saline
solution, fixed oils, polyethylene glycols, glycerin, propylene
glycol or other synthetic solvents; and/or other agents as
described herein. The parental preparation can be enclosed in
ampoules, disposable syringes or multiple dose vials made of glass
or plastic.
[0141] If administered intravenously, preferred carriers are
physiological saline or phosphate buffered saline (PBS).
[0142] In one embodiment, the compositions of the present
disclosure are prepared with carriers that will protect the active
components against rapid elimination from the body, such as a
controlled release formulation, including implants and
microencapsulated delivery systems. Biodegradable, biocompatible
polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art.
[0143] Liposomal may be prepared according to methods known to
those skilled in the art, for example, as described in U.S. Pat.
No. 4,522,811 (which is incorporated herein by reference in its
entirety). For example, liposome formulations may be prepared by
dissolving the purified lipid(s), isolated lipids, and lipid
compositions as described herein in an inorganic solvent that is
then evaporated, leaving behind a thin film of dried lipid on the
surface of the container. An aqueous solution of the active
compound is then introduced into the container. The container is
then swirled by hand to free lipid material from the sides of the
container and to disperse lipid aggregates, thereby forming the
liposomal suspension.
[0144] A pharmacological composition or formulation refers to a
composition or formulation in a form suitable for administration,
e.g., systemic administration, into a cell or subject, preferably a
human. By "systemic administration" is meant in vivo systemic
absorption or accumulation of drugs in the blood stream followed by
distribution throughout the entire body. Suitable forms, in part,
depend upon the use or the route of entry, for example oral,
transdermal, or by injection. Such forms should not prevent the
composition or formulation from reaching a target cell (i.e., a
cell to which the negatively charged polymer is desired to be
delivered to). For example, pharmacological compositions injected
into the blood stream should be soluble. Other factors are known in
the art, and include considerations such as toxicity and forms
which prevent the composition or formulation from exerting its
effect.
[0145] Administration routes which lead to systemic absorption
include, without limitations: intravenous, subcutaneous,
intraperitoneal, inhalation, oral, intrapulmonary and
intramuscular. The rate of entry of a drug into the circulation has
been shown to be a function of molecular weight or size. The use of
drug carrier comprising the compounds of the present disclosure can
potentially localize the drug, for example, in certain tissue
types, such as the skin or respiratory system or the epidermis. A
liposome formulation can facilitate the association of drug with
the surface of cells, such as, lymphocytes and macrophages.
[0146] The present disclosure also features the use of the
composition comprising surface-modified liposomes containing poly
(ethylene glycol) lipids (PEG-modified, or long-circulating
liposomes or stealth liposomes). Compositions of the present
disclosure can also comprise covalently attached PEG molecules of
various molecular weights. These formulations offer a method for
increasing the accumulation of drugs in target tissues. This class
of drug carriers resists opsonization and elimination by the
mononuclear phagocytic system (MPS or RES), thereby enabling longer
blood circulation times and enhanced tissue exposure for the
encapsulated drug (Lasic et al. Chem. Rev. 1995, 95, 2601-2627;
Ishiwata et al., Chem. Pharm. Bull. 1995, 43, 1005-1011).
[0147] The present disclosure also includes compositions prepared
for storage or administration that include a pharmaceutically
effective amount of the desired components in a pharmaceutically
acceptable carrier or diluent. Acceptable carriers or diluents for
therapeutic use are well known in the pharmaceutical art, and are
described, for example, in Remington's Pharmaceutical Sciences,
Mack Publishing Co. (A. R. Gennaro edit 1985) hereby incorporated
by reference herein. For example, preservatives, stabilizers, dyes
and flavoring agents can be provided. These include sodium
benzoate, sorbic acid and esters of p-hydroxybenzoic acid. In
addition, antioxidants and suspending agents can be used.
[0148] An effective amount, pharmaceutically effective dose,
therapeutically effective amount, or pharmaceutically effective
amount is that dose required to prevent, inhibit the occurrence, or
treat (alleviate a symptom to some extent, preferably all of the
symptoms) of a disease state or pathological condition. The
effective amount depends on the type of disease, the composition
used, the mute of administration, the type of mammal being treated,
the physical characteristics of the specific mammal under
consideration, concurrent medication, and other factors which those
skilled in the medical arts will recognize. Generally, an amount
between 0.1 mg/kg and 1000 mg/kg body weight/day of active
ingredients is administered. In addition, effective amounts of the
compositions of the disclosure encompass those amounts utilized in
the examples to facilitate the intended or desired biological
effect.
[0149] Toxicity and therapeutic efficacy of such compositions or
active ingredients can be determined by standard pharmaceutical
procedures in cell cultures or experimental animals, e.g., for
determining the LD50 (the dose lethal to 50% of the population) and
the ED50 (the dose therapeutically effective in 50% of the
population). The dose ratio between toxic and therapeutic effects
is the therapeutic index and it can be expressed as the ratio
LD50/ED50. Compositions/active ingredients that exhibit large
therapeutic indices are preferred. While compositions/active
ingredients that exhibit toxic side effects may be used, care
should be taken to design a delivery system that targets such
compositions or ingredients to the site of affected tissue in order
to minimize potential damage to uninfected cells and, thereby,
reduce side effects. The data obtained from the cell culture assays
and animal studies can be used in formulating a range of dosage for
use in humans. The dosage of such compositions/active ingredients
lies preferably within a range of circulating concentrations that
include the ED50 with little or no toxicity. The dosage may vary
within this range depending upon the dosage form employed and the
route of administration utilized. For any composition used in the
method of the present disclosure, the therapeutically effective
dose can be estimated initially from cell culture assays. A dose
may be formulated in animal models to achieve a circulating plasma
concentration range that includes the IC50 (i.e., the concentration
of the test compound which achieves a half-maximal inhibition of
symptoms) as determined in cell culture. Such information can be
used to more accurately determine useful doses in humans. Levels in
plasma may be measured, for example, by high performance liquid
chromatography.
[0150] The formulations can be administered orally, topically,
parenterally, by inhalation or spray or rectally in dosage unit
formulations containing conventional non-toxic pharmaceutically
acceptable carriers, adjuvants and vehicles. The term parenteral as
used herein includes percutaneous, subcutaneous, intravascular
(e.g., intravenous), intramuscular, or intrathecal injection or
infusion techniques and the like. In addition, there is provided a
pharmaceutical formulation comprising a peptide of the disclosure
and a pharmaceutically acceptable carrier. One or more peptides of
the disclosure can be present in association with one or more
non-toxic pharmaceutically acceptable carriers and/or diluents
and/or adjuvants, and if desired other active ingredients. The
pharmaceutical compositions of the present disclosure can be in a
form suitable for oral use, for example, as tablets, troches,
lozenges, aqueous or oily suspensions, dispersible powders or
granules, emulsion, hard or soft capsules, or syrups or
elixirs.
[0151] Compositions intended for oral use can be prepared according
to any method known to the art for the manufacture of
pharmaceutical compositions and such compositions can contain one
or more such sweetening agents, flavoring agents, coloring agents
or preservative agents in order to provide pharmaceutically elegant
and palatable preparations. Tablets contain the active ingredient
in admixture with non-toxic pharmaceutically acceptable excipients
that are suitable for the manufacture of tablets. These excipients
can be for example, inert diluents, such as calcium carbonate,
sodium carbonate, lactose, calcium phosphate or sodium phosphate,
granulating and disintegrating agents, for example, corn starch, or
alginic acid; binding agents, for example starch, gelatin or
acacia, and lubricating agents, for example magnesium stearate,
stearic acid or talc. The tablets can be uncoated or they can be
coated by known techniques. In some cases such coatings can be
prepared by known techniques to delay disintegration and absorption
in the gastrointestinal tract and thereby provide a sustained
action over a longer period. For example, a time delay material
such as glyceryl monosterate or glyceryl distearate can be
employed. Formulations for oral use can also be presented as hard
gelatin capsules wherein the active ingredient is mixed with an
inert solid diluent, for example, calcium carbonate, calcium
phosphate or kaolin, or as soft gelatin capsules wherein the active
ingredient is mixed with water or an oil medium, for example peanut
oil, liquid paraffin or olive oil.
[0152] Aqueous suspensions contain the active materials in
admixture with excipients suitable for the manufacture of aqueous
suspensions. Such excipients are suspending agents, for example
sodium carboxymethylcellulose, methylcellulose,
hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone,
gum tragacanth and gum acacia; dispersing or wetting agents can be
a naturally-occurring phosphatide, for example, lecithin, or
condensation products of an alkylene oxide with fatty acids, for
example polyoxyethylene stearate, or condensation products of
ethylene oxide with long chain aliphatic alcohols, for example
heptadecaethyleneoxycetanol, or condensation products of ethylene
oxide with partial esters derived from fatty acids and a hexitol
such as polyoxyethylene sorbitol monooleate, or condensation
products of ethylene oxide with partial esters derived from fatty
acids and hexitol anhydrides, for example polyethylene sorbitan
monooleate. The aqueous suspensions can also contain one or more
preservatives, for example ethyl, or n-propyl p-hydroxybenzoate,
one or more coloring agents, one or more flavoring agents, and one
or more sweetening agents, such as sucrose or saccharin.
[0153] Oily suspensions can be formulated by suspending the active
ingredients in a vegetable oil, for example arachis oil, olive oil,
sesame oil or coconut oil, or in a mineral oil such as liquid
paraffin. The oily suspensions can contain a thickening agent, for
example beeswax, hard paraffin or cetyl alcohol. Sweetening agents
and flavoring agents can be added to provide palatable oral
preparations. These compositions can be preserved by the addition
of an anti-oxidant such as ascorbic acid.
[0154] Dispersible powders and granules suitable for preparation of
an aqueous suspension by the addition of water provide the active
ingredient in admixture with a dispersing or wetting agent,
suspending agent and one or more preservatives. Suitable dispersing
or wetting agents or suspending agents are exemplified by those
already mentioned above. Additional excipients, for example
sweetening, flavoring and coloring agents, can also be present.
Pharmaceutical compositions of the present disclosure can also be
in the form of oil-in-water emulsions. The oily phase can be a
vegetable oil or a mineral oil or mixtures of these. Suitable
emulsifying agents can be naturally-occurring gums, for example gum
acacia or gum tragacanth, naturally-occurring phosphatides, for
example soy bean, lecithin, and esters or partial esters derived
from fatty acids and hexitol, anhydrides, for example sorbitan
monooleate, and condensation products of the said partial esters
with ethylene oxide, for example polyoxyethylene sorbitan
monooleate. The emulsions can also contain sweetening and flavoring
agents.
[0155] Syrups and elixirs can be formulated with sweetening agents,
for example glycerol, propylene glycol, sorbitol, glucose or
sucrose. Such formulations can also contain a demulcent, a
preservative and flavoring and coloring agents. The pharmaceutical
compositions can be in the form of a sterile injectable aqueous or
oleaginous suspension. This suspension can be formulated according
to the known art using those suitable dispersing or wetting agents
and suspending agents that have been mentioned above. The sterile
injectable preparation can also be a sterile injectable solution or
suspension in a non-toxic parentally acceptable diluent or solvent,
for example as a solution in 1,3-butanediol. Among the acceptable
vehicles and solvents that can be employed are water, Ringer's
solution and isotonic sodium chloride solution. In addition,
sterile, fixed oils are conventionally employed as a solvent or
suspending medium. For this purpose any bland fixed oil can be
employed including synthetic mono- or diglycerides. In addition,
fatty acids such as oleic acid find use in the preparation of
injectables.
[0156] For administration to non-human animals, the composition can
also be added to the animal feed or drinking water. It can be
convenient to formulate the animal feed and drinking water
compositions so that the animal takes in a therapeutically
appropriate quantity of the composition along with its diet. It can
also be convenient to present the composition as a premix for
addition to the feed or drinking water. The composition can also be
administered to a subject in combination with other therapeutic
compounds to increase the overall therapeutic effect. The use of
multiple compounds to treat an indication can increase the
beneficial effects while reducing the presence of side effects.
[0157] A further object of the present disclosure is to provide a
kit comprising a suitable container, the therapeutic of the present
disclosure in a pharmaceutically acceptable form disposed therein,
and instructions for its use.
[0158] Preparations for administration of the therapeutic of the
present disclosure include sterile aqueous or non-aqueous
solutions, suspensions, and emulsions. Examples of non-aqueous
solvents are propylene glycol, polyethylene glycol, vegetable oils
such as olive oil, and injectable organic esters such as ethyl
oleate. Aqueous carriers include water, alcoholic/aqueous
solutions, emulsions or suspensions, including saline and buffered
media. Vehicles include sodium chloride solution, Ringer's
dextrose, dextrose and sodium chloride, lactated Ringer's
intravenous vehicles including fluid and nutrient replenishers,
electrolyte replenishers, and the like. Preservatives and other
additives may be added such as, for example, antimicrobial agents,
anti-oxidants, chelating agents and inert gases and the like.
[0159] The composition (which includes "active compounds" or
"active ingredients") of the disclosure can be incorporated into
pharmaceutical compositions suitable for administration. Such
compositions typically comprise the composition of the present
disclosure and a pharmaceutically acceptable carrier. As used
herein, "pharmaceutically acceptable carrier" is intended to
include any and all solvents, dispersion media, coatings,
antibacterial agents, isotonic and absorption delaying agents, and
the like, compatible with pharmaceutical administration. Suitable
carriers are described in the most recent edition of Remington's
Pharmaceutical Sciences, a standard reference text in the field,
which is incorporated herein by reference. Preferred examples of
such carriers or diluents include, but are not limited to, water,
saline, finger's solutions, dextrose solution, and 5% human serum
albumin. Liposomes and non-aqueous vehicles such as fixed oils may
also be used. The use of such media and agents for pharmaceutically
active substances is well known in the art. Except insofar as any
conventional media or agent is incompatible with the active
compound, use thereof in the compositions is contemplated.
Supplementary active compounds can also be incorporated into the
compositions.
[0160] A pharmaceutical composition of the present disclosure is
formulated to be compatible with its intended route of
administration. Examples of routes of administration include
parenteral, e.g., intravenous, intradermal, subcutaneous, oral
(e.g., inhalation), transdermal (i.e., topical), transmucosal,
intraperitoneal, and rectal administration. Solutions or
suspensions used for parenteral, intradermal, or subcutaneous
application can include the following components: a sterile diluent
such as water for injection, saline solution, fixed oils,
polyethylene glycols, glycerine, propylene glycol or other
synthetic solvents, antibacterial agents such as benzyl alcohol or
methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfite; chelating agents such as ethylenediaminetetraacetic acid
(EDTA); buffers such as acetates, citrates or phosphates, and
agents for the adjustment of tonicity such as sodium chloride or
dextrose. The pH can be adjusted with acids or bases, such as
hydrochloric acid or sodium hydroxide. The parenteral preparation
can be enclosed in ampoules, disposable syringes or multiple dose
vials made of glass or plastic.
[0161] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor.TM.. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringeability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyethylene glycol, and the like), and suitable
mixtures thereof. The proper fluidity can be maintained, for
example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial agents, for
example, parabens, chlorobutanol, phenol, ascorbic acid,
thimerosal, and the like. In many cases, it will be preferable to
include isotonic agents, for example, sugars, polyalcohols such as
manitol, sorbitol, sodium chloride in the composition. Prolonged
absorption of the injectable compositions can be brought about by
including in the composition an agent which delays absorption, for
example, aluminum monostearate and gelatin.
[0162] For oral administration, the pharmaceutical compositions may
take the form of, for example, tablets or capsules prepared by
conventional means with pharmaceutically acceptable excipients such
as binding agents (e.g., pregelatinised maize starch,
polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers
(e.g., lactose, microcrystalline cellulose or calcium hydrogen
phosphate); lubricants (e.g., magnesium stearate, talc or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g., sodium lauryl sulphate). The tablets may be
coated by methods well known in the art. Liquid preparations for
oral administration may take the form of, for example, solutions,
syrups, or suspensions, or they may be presented as a dry product
for constitution with water or other suitable vehicle before use.
Such liquid preparations may be prepared by conventional means with
pharmaceutically acceptable additives such as suspending agents
(e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible
fats); emulsifying agents (e.g., lecithin or acacia), non-aqueous
vehicles (e.g., almond oil, oily esters, ethyl alcohol or
fractionated vegetable oils); and preservatives (e.g., methyl or
propyl-p-hydroxybenzoates or sorbic acid). The preparations may
also contain buffer salts, flavoring, coloring, and sweetening
agents as appropriate. Preparations for oral administration may be
suitably formulated to give controlled release of the active
compound. For buccal administration the compositions may take the
form of tablets or lozenges formulated in conventional manner. For
administration by inhalation, the compounds for use according to
the present disclosure are conveniently delivered in the form of an
aerosol spray presentation from pressurized packs or a nebuliser,
with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethan-e, carbon dioxide or other suitable gas.
In the case of a pressurized aerosol the dosage unit may be
determined by providing a valve to deliver a metered amount.
Capsules and cartridges of e.g. gelatin for use in an inhaler or
insufflator may be formulated containing a powder mix of the
compound and a suitable powder base such as lactose or starch. The
compounds may be formulated for parenteral administration by
injection, e.g., by bolus injection or continuous infusion.
Formulations for injection may be presented in unit dosage form,
e.g., in ampoules or in multi-dose containers, with an added
preservative. The compositions may take such forms as suspensions,
solutions, or emulsions in oily or aqueous vehicles, and may
contain formulatory agents such as suspending, stabilizing, and/or
dispersing agents. Alternatively, the active ingredient may be in
powder form for constitution with a suitable vehicle, e.g., sterile
pyrogen-free water, before use. The compounds may also be
formulated in rectal compositions such as suppositories or
retention enemas, e.g., containing conventional suppository bases
such as cocoa butter or other glycerides. In addition to the
formulations described previously, the compounds may also be
formulated as a depot preparation. Such long acting formulations
may be administered by implantation (for example subcutaneously or
intramuscularly) or by intramuscular injection. Thus, for example,
the compounds may be formulated with suitable polymeric or
hydrophobic materials (for example as an emulsion in an acceptable
oil) or ion exchange resins, or as sparingly soluble derivatives,
for example, as a sparingly soluble salt.
[0163] In one embodiment, the compositions are prepared with
carriers that will protect the active ingredients against rapid
elimination from the body, such as a controlled release
formulation, including implants and microencapsulated delivery
systems. Biodegradable, biocompatible polymers can be used, such as
ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, and polylactic acid. Methods for
preparation of such formulations will be apparent to those skilled
in the art. The materials can also be obtained commercially from
Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal
suspensions can be prepared according to methods known to those
skilled in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0164] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the disclosure are dictated by and directly dependent on the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and the limitations inherent in
the art of compounding such an active compound for the treatment of
individuals.
[0165] Toxicity and therapeutic efficacy of such
compounds/compositions can be determined by standard pharmaceutical
procedures in cell cultures or experimental animals, e.g., for
determining the LD50 (the dose lethal to 50% of the population) and
the ED50 (the dose therapeutically effective in 50% of the
population). The dose ratio between toxic and therapeutic effects
is the therapeutic index and it can be expressed as the ratio
LD50/ED50. Compounds which exhibit high therapeutic indices are
preferred.
[0166] The data obtained from cell culture assays and animal
studies can be used in formulation a range of dosage for use in
patients, e.g. humans. The dosage of compositions of the disclosure
lies generally within a range of circulating concentrations that
include the ED50 with little or no toxicity. The dosage may vary
within this range depending upon the dosage form employed and the
route of administration utilized. For any compound/composition used
in the method of the disclosure, the therapeutically effective dose
can be estimated initially from cell culture assays. A dose may be
formulated in animal models to achieve a circulating plasma
concentration range of the compound/composition or, when
appropriate, of the polypeptide product of a target sequence (e.g.,
achieving a decreased concentration of the polypeptide) that
includes the IC50 (i.e., the concentration of the test compound
which achieves a half-maximal inhibition of symptoms), as
determined in cell culture. Such information can be used to more
accurately determine useful doses for a patient, as defined above
(includes, e.g., humans, dogs, cats, etc.). Levels in plasma may be
measured, for example, by high performance liquid
chromatography.
[0167] In addition to their administration individually or as a
plurality, as discussed above, the compositions of the disclosure
can be administered in combination with other known agents
effective in treatment of pathological processes mediated by an
inflammatory response caused by a bacterial infection, viral
infection, or an inflammatory disease or disorder. In any event,
the administering physician can adjust the amount and timing of
composition administration on the basis of results observed using
standard measures of efficacy known in the art or described
herein.
[0168] Kits
[0169] The invention provides kits for the treatment or prevention
of a pathogen infections and/or inflammatory related diseases and
disorders. In one embodiment, the kit includes a therapeutic or
prophylactic composition containing an effective amount of a
liposome formulation or an anti-inflammatory composition
formulation in unit dosage form. In some embodiments, the kit
comprises a sterile container, which contains a therapeutic or
prophylactic cellular composition; such containers can be boxes,
ampules, bottles, vials, tubes, bags, pouches, blister-packs, or
other suitable container forms known in the art. Such containers
can be made of plastic, glass, laminated paper, metal foil, or
other materials suitable for holding medicaments.
[0170] If desired a cell of the invention is provided together with
instructions for administering the agent to a subject having or at
risk of developing a pathogen infection or infectious disease, such
as a bacterial or viral infection, and/or a disease or disorder
related to an inflammatory response, e.g., autoimmunity, asthma,
arthritis, and/or psoriasis. The instructions will generally
include information about the use of the composition for the
treatment or prevention of a pathogen infection or
inflammation-related disease or disorder. In other embodiments, the
instructions include at least one of the following: description of
the therapeutic agent; dosage schedule and administration for
treatment or prevention of a pathogen infection and/or a
disease/disorder related to inflammation, or symptoms thereof;
precautions; warnings; indications; counter-indications; adverse
reactions; animal pharmacology; clinical studies; and/or
references. The instructions may be printed directly on the
container (when present), or as a label applied to the container,
or as a separate sheet, pamphlet, card, or folder supplied in or
with the container.
[0171] The practice of the present disclosure employs, unless
otherwise indicated, conventional techniques of molecular biology
(including recombinant techniques), microbiology, cell biology,
biochemistry and immunology, which are well within the purview of
the skilled artisan. Such techniques are explained fully in the
literature, such as, Sambrook et al., Molecular Cloning: A
Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press,
New York (1989); Kaufman et al., Eds., Handbook of Molecular and
Cellular Methods in Biology in Medicine. CRC Press, Boca Raton
(1995); McPherson, Ed., Directed Mutagenesis: A Practical Approach,
IRL Press, Oxford (1991). Standard reference works setting forth
the general principles of pharmacology include Goodman and Gilman's
The Pharmacological Basis of Therapeutics, 11th Ed., McGraw Hill
Companies Inc., New York (2006). These techniques are applicable to
the production of the polynucleotides and polypeptides of the
invention, and, as such, may be considered in making and practicing
the invention. Particularly useful techniques for particular
embodiments will be discussed in the sections that follow.
[0172] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the assay, screening, and
therapeutic methods of the disclosure, and are not intended to
limit the scope of what the inventors regard as their
invention.
EXAMPLES
Example 1. SchuS4 Inhibits Pro-Inflammatory Responses Among Resting
Primary Human Cells
Example 1A. Secretion of Pro-Inflammatory Cytokines by hDC
Following Infection with F. tularensis
[0173] It was suggested that one difference between virulent and
attenuated strains of F. tularensis is the ability of virulent
strains to evade induction of pro-inflammatory responses. Thus, the
inventors first compared bacterial replication and secretion of
IL-12p40 into culture supernatants following infection of hDC with
either virulent F. tularensis strain SchuS4 or attenuated F.
tularensis strain LVS. Similar numbers of SchuS4 and LVS were
phagocytosed by hDC (FIG. 2A). However, SchuS4 replicated more
quickly over the first 12 hours of infection compared with LVS.
Similar numbers of LVS and SchuS4 were recovered from hDC at 24,
48, and 72 hours postinfection (FIG. 2A). In agreement with our and
others' previous observations, SchuS4 failed to stimulate secretion
of IL-12p40 in concentrations that were significantly different
from uninfected hDC, whereas LVS induced significantly more
IL-12p40 compared with uninfected and SchuS4-infected cells at each
time point tested (FIG. 2B). These data demonstrate that one
difference between SchuS4 and LVS is the ability of SchuS4 to
undergo intracellular replication without provoking inflammatory
cytokines.
Example 1B. SchuS4 Actively Interferes with Secretion of
Pro-Inflammatory Cytokines
[0174] It was confirmed that SchuS4 was actively inhibiting
secretion of pro-inflammatory cytokines in hDC following infection.
Twenty-four hours postinfection. E. coli LPS was added to hDC
cultures, and secretion of IL-12p40 was measured 24 hours later.
Consistent with our previous observations using intracellular
cytokine staining, hDC infected with SchuS4 produced significantly
less IL-12p40 compared with uninfected cells in response to E. coli
LPS (FIG. 2C). Thus, SchuS4 fails to induce inflammatory responses
in hDC and actively inhibits their ability to secrete
proinflammatory cytokines in response to secondary microbial
stimuli.
Example 2. SchuS4 Lipids Inhibit Inflammatory Responses In
Vitro
[0175] hDC were treated with the indicated concentration of lipids
isolated from SchuS4 or LVS for 18 hours followed by addition of
LPS for an additional 20 hours. EtOH served as vehicle control.
Culture supernatants were assessed for IL-12p40 by ELISA (FIG. 3).
ns=not significantly different. *=significantly less than EtOH+LPS
treated controls (p<0.05). In each experiment each condition was
tested in triplicate. Error bars represent SEM. Data is
representative of three experiments of similar design using
different donors.
Example 3. SchuS4 Lipids Inhibit Pulmonary Inflammation In Vivo
[0176] Mice exposed to SchuS4 lipids had significantly fewer
neutrophils in their airways in response to LPS than did
EtOH-treated controls (FIG. 4A). However, neutrophils that were
recruited to the airways of SchuS4 lipid-treated mice expressed
levels of CD11b similar to those observed in EtOH-treated controls.
The inventors did not observe changes in MHC-II expression on
macrophages from any mice treated with LPS at the time point
assessed. Exposure to SchuS4 lipids also significantly reduced the
amounts of TNF-.alpha., IL-6, and KC detected in the airways of
mice following exposure to LPS, compared to EtOH-treated controls
(FIG. 4B). RANTES levels were not significantly increased above
levels in untreated controls for any group (data not shown).
Together, these data suggest that, while SchuS4 lipids fail to
induce inflammatory responses in the lung, these bacterial
components significantly attenuate secondary pulmonary
inflammation.
Example 4. Identification of Active SchuS4 Lipid
[0177] Crude SchuS4 lipids were separated by TLC (FIG. 5A) and the
indicated bands were scraped from the silica plate and assessed for
their ability to inhibit inflammatory responses in hDC (FIGS. 5B
and 5C). Areas of the TLC plate at the same part of the solvent
front, but not containing lipid were scraped as used as negative
controls. hDC were treated and supernatants were assessed for the
indicated cytokines as described in FIG. 3. Following incubation
with lipid and LPS supernatants were assessed for IL-12p40 (B) or
TNF-.alpha. (C) by ELISA. Error bars represent SD. Band 4 from the
TLC plate was analyzed by LC-MS for lipid content and
speciation.
[0178] Phospholipids from crude SchuS4 lipid preparations were
isolated and fractionated into PE (1) or PC (2) containing
fractions. These samples, or crude lipid, were added to hDC at the
indicated concentrations. Cells were treated with LPS as described
in FIG. 3 (black bars) or treated with media alone (white bars).
Supernatants were assessed for IL-12p40 (FIG. 6A) or TNF-.alpha.
(FIG. 6B) by ELISA. Error bars represent SD. *=p<0.05.
Example 5. Generation and Testing of Synthetic 24:10 PE
[0179] Synthetic PE 24:10, as shown below, was mixed with
commercially available PC 16:18 at a ratio of 80:20 (PE:PC) and
imaged by cyro-electron microscopy (FIG. 7A) to reveal lipid
structure. PEPC liposomes form varied sized multilaminar
structures. Bone marrow-derived macrophages (BMDM) and human
dendritic cells (hDC) were treated with PEPC liposomes (FIG. 7B).
At the indicated time points, the cells were fixed and stained for
PE using duramycin and nuclei were counterstained with DAPI.
Intracellular PEPC liposomes were detected within one hour after
exposure and accumulated over a 24 hour incubation. PE is detected
on the surface of viable F. tularensis SchuS4 (FIG. 7B). BMDM or
hDC were infected with MOI=50 SchuS4 and fixed at 6 and 8 hours,
respectively (FIG. 7C). Cells were stained with Alexa 488
conjugated anti-F. tularensis LPS antibody to detect bacteria
(arrow), duramycin to detect PE (arrow head), and DAPI to detect
nuclei. Colocalization of intense PE staining and SchuS4 is
apparent in both BMDM and hDC from the merged in the top left image
for the BMDM and hDC. Data are representative of three
##STR00002##
experiments of similar design.
Example 6. Synthetic PE:PC Liposomes Inhibit LPS Mediated
Inflammation in hDC
[0180] PEPC liposomes or crude SchuS4 lipid were incubated with
hDC, followed by treatment with LPS, as described with regard to
FIG. 3. Cells treated with 5% dextrose water (vehicle) served as a
negative control Supernatants were evaluated for IL-12p40 by ELISA
and shown in FIG. 8. Error bars represent SD and *=p<0.05.
Example 7. Synthetic PE:PC Liposomes Inhibit Viral Mediated
Inflammation
[0181] A549 human epithelial cells were treated with crude Schus4
lipid or synthetic PEPC liposomes overnight. Cells treated with 5%
dextrose water served as negative controls for lipid treatment.
Cells were then infected with West Nile Virus at an MOI=0.01. Forty
eight hours later supernatants were collected and assessed for IL-6
(FIG. 9A) and IFN-.beta. (FIG. 9B) by ELISA. Error bars represent
SD. *=p<0.05.
Example 8. Synthetic PE:PC Liposomes Inhibit Viral Replication
[0182] A549 human epithelial cells were treated with crude Schus4
lipid or synthetic PEPC liposomes overnight. Cells treated with 5%
dextrose water served as negative controls for lipid treatment.
Cells were then infected with West Nile Virus at an MOI=0.001.
Forty eight hours later cells were assessed for viral load as
plaque forming units (PFU) by immunostaining (FIG. 10). Error bars
represent SD. *=p<0.05.
Example 9. Inhibition of Inflammatory Responses by PE and PC is
Dependent on Acyl Chain Length
[0183] BMDM were treated with crude SchuS4 lipids or the indicated
PE or PC lipids or PEPC liposomes overnight. Cells were then
stimulated with R848 (TLR8 agonist) and supernatants were assessed
for IL-12p40 as an indicator of induction of an inflammatory
response. Crude SchuS4 lipid (FIG. 11A), PE2410 (FIG. 11B), PC2424
(FIG. 11C) and liposomes comprised of PE2410 and PC2424 (FIG. 11D)
all inhibited inflammatory responses in a dose dependent manner. PC
and PE lipids comprised of 1816 acyl chains did not significantly
impair IL-12p40 secretion (FIGS. 11E and 11F). Error bars represent
SD. *=p<0.05 compared to vehicle (-) control treated samples.
ns=not significant. Data are representative of three experiments of
similar design. Therefore, these data demonstrate that suppressive
activity of PE and PC originally derived from F. tularensis is
dependent on acyl chain length.
Material and Methods of the Examples
[0184] Generation of Bone Marrow Macrophages (BMMs).
[0185] BMMs were generated as previously described (Crane D et al.
2013, Clin Vaccine Immunol, 20:1531). Briefly, bone marrow was
flushed from B6 femurs and cultured in Dulbecco's modified Eagle's
medium (DMEM) (Life Technologies, Carlsbad, Calif.) supplemented
with 10% heat-inactivated fetal calf serum (Atlas Biologicals, Ft.
Collins, Colo.), 0.2 mM 1-glutamine (Life Technologies, Carlsbad,
Calif.), 1 mM HEPES buffer (Life Technologies), and 0.1 mM
nonessential amino acids (Life Technologies) (cDMEM) plus 10 ng/ml
macrophage colony-stimulating factor (M-CSF) (Peprotech, Rocky
Hill, N.J.), in 75-cm.sup.2 flasks. On day 2 of culture,
non-adherent cells were collected and placed in fresh 75-cm.sup.2
flasks, and the cultures were replenished with fresh cDMEM
containing 10 ng/ml M-CSF. Adherent cells were collected on day 5,
and seeded at 6.times.10.sup.4 per well in a 96-well plate. BMMs
were used on day 6 of culture.
[0186] Generation of Human Monocyte-Derived Dendritic Cells.
[0187] Human monocyte-derived dendritic cells (hDC) were generated
from apheresed monocytes, as previously described (Chase, J. C., J.
Celli. C. M. Bosio. 2009. Direct and indirect impairment of human
dendritic cell function by virulent Francisella tularensis Schu S4.
Infect. Immun. 77: 180-195). Briefly, monocytes were enriched by
apheresis and negative selection using Dynabeads MyPure Monocytes
Kit for untouched human cells, per the manufacturer's instructions
(Invitrogen, Carlsbad, Calif.). Cells were differentiated upon
culture in RPMI 1640 (Invitrogen) supplemented with 10%
heat-inactivated FCS, 0.2 mM L-glutamine, 1 mM HEPES buffer, and
0.1 mM nonessential amino acids (all from Invitrogen) [complete
RPMI (cRPMI)]; 100 ng/ml recombinant human (rh) GM-CSF; and 20
ng/ml rhIL-4 (both from PeproTech, Rocky Hill, N.J.). On day 3 of
culture, cells were replenished with 100 ng/ml rhGM-CSF and 20
ng/ml rhIL-4. All cells were used on day 4 of culture. As
indicated, some hDC were pretreated with 1000 U/ml rhIFN-.beta.
(PBL IFN Source, Piscataway, N.J.) 16 h prior to infection or 1-100
U/ml prior to stimulation with Escherichia coli LPS.
[0188] Bacteria.
[0189] F. tularensis strain LVS were provided by Dr. Jean Celli
(Rocky Mountain Laboratories. Hamilton, Mont.) and F. tularensis
strain SchuS4 was provided by Dr. Jeannine Peterson (Centers for
Disease Control and Prevention, Fort Collins, Colo.). As previously
described, bacterial stocks were generated by growing strains
overnight in modified Mueller-Hinton broth, aliquoted into 1-ml
samples, and frozen at -80.degree. C. (Bosio, C. M., H.
Bielefeldt-Olhmann, J. T. Belisle. 2007. Active suppression of the
pulmonary immune response by Francisella tularensis Schu4. J.
Immunol. 178: 4538-4547; Chase, J. C., J. Celli, C. M. Bosio. 2009.
Direct and indirect impairment of human dendritic cell function by
virulent Francisella tularensis Schu S4. Infect. Immun. 77:
180-195; and Nigrovic, L. E., S. L. Wingerter. 2008. Tularemia.
Infect. Dis. Clin. North Am. 22: 489-504, ix). Immediately prior to
infection, bacterial stocks were thawed, pelleted by
centrifugation, and resuspended in cRPMI. Frozen stocks were
titered by enumerating viable bacteria from serial dilutions plated
on modified Mueller-Hinton agar, as previously described (Bosio, C.
M., H. Bielefeldt-Ohmann. J. T. Belisle. 2007. Active suppression
of the pulmonary immune response by Francisella tularensis Schu4.
J. Immunol. 178: 4538-4547; Chase, J. C., J. Celli, C. M. Bosio.
2009. Direct and indirect impairment of human dendritic cell
function by virulent Francisella tularensis Schu S4. Infect. Immun.
77: 180-195; and Nigrovic, L. E., S. L. Wingerter. 2008. Tularemia.
Infect. Dis. Clin. North Am. 22: 489-504, ix). The number of viable
bacteria in frozen stock vials varied by <1% over a 10-mo
period. Where indicated, SchuS4 was killed by incubation in 2%
paraformaldehyde (PFA) for 30 min at 37.degree. C. washed
extensively in PBS, and resuspended in cRPMI before addition to hDC
cultures.
[0190] Infection of BMDM.
[0191] BMDM were seeded at 2.times.10.sup.5 cells/well in a 24 well
plate containing glass coverslips. Media was removed and reserved.
Bacteria were diluted in cDMEM and added to BMDM at a multiplicity
of infection (MOI) of 50. After 1.5 hours, bacteria-containing
media was removed and cDMEM containing 50 .mu.g/mL gentamicin (Life
Technologies) was added for 45 minutes. Cells were then washed
three times with PBS and reserved media was added back to each
well. Cells were fixed at the indicated times points and coverslips
removed for staining of bacteria with Alexa 488 conjugated
anti-Francisella LSP antibody, duramycin, and DAPI as described
below.
[0192] Infection of hDC.
[0193] hDC were infected at a multiplicity of infection of 50 with
F. tularensis, as previously described (Chase, J. C., J. Celli, C.
M. Bosio. 2009. Direct and indirect impairment of human dendritic
cell function by virulent Francisella tularensis Schu S4. Infect.
Immun. 77: 180-195). Briefly, hDC were removed from their original
cultures, centrifuged, and adjusted to 1-2.times.10.sup.7/ml in
reserved DC medium. Cells treated with medium alone served as
negative controls. Bacteria were added, and cells were incubated at
37.degree. C. in 7% CO.sub.2 for 2 hours, washed once, and then
incubated with 50 .mu.g/ml gentamicin (Invitrogen) for 45 min to
kill extracellular bacteria. Then cells were washed extensively,
adjusted to 5.times.10.sup.5 cells/ml in reserved DC medium, and
plated at 1 or 0.5 ml/well in 24- or 48-well tissue culture plates,
respectively. Intracellular bacteria were enumerated following
lysing of hDC with H.sub.2O and plating serial dilutions of cell
lysate onto modified Mueller-Hinton broth agar plates. Agar plates
were incubated at 37.degree. C./7% CO.sub.2 for 48 h, and
individual colonies were enumerated.
[0194] Where indicated, cells were treated with 10 ng/ml ultrapure
E. coli LPS at the same time that other cells were infected or 24
hours post infection.
[0195] Cytokine Quantification.
[0196] The presence of IL-12p40 in culture supernatants was
quantified using commercially available ELISA, according to the
manufacturer's instructions (R&D Systems, Minneapolis, Minn.).
In experiments measuring secretion of cytokines by SchuS4-infected
hDC in the presence of either mouse IgG (isotype, R&D Systems),
the percentage of cytokine secretion of control (uninfected,
LPS-treated cells) was calculated using the following equation
(concentration of cytokine present in SchuS4-infected
culture/average cytokine concentration in LPS-treated, uninfected
culture).times.100.
[0197] Mice.
[0198] Specific-pathogen-free, 6- to 8-week-old C57BL/6J mice (n=4
or 5/group) were purchased from Jackson Laboratories (Bar Harbor,
Me.). Mice were housed in sterile microisolator cages in the
biosafety level 2 facility at the Rocky Mountain Laboratories. All
mice were provided sterile water and food ad libitum. All research
involving animals was conducted in accordance with animal care and
use guidelines, and animal protocols were approved by the animal
care and use committee at Rocky Mountain Laboratories.
[0199] Isolation of Bacterial Lipids.
[0200] Lipids were isolated from SchuS4 using the standard modified
Folch method for isolation of bacterial lipids (Folch J, Lees M,
Sloane Stanley G H. 1957. A simple method for the isolation and
purification of total lipids from animal tissues. J. Biol. Chem.
226:497-509; Beatty W L, Rhoades E R, Ullrich H J. Chattejee D,
Heuser J E, Russell D G. 2000. Trafficking and release of
mycohacterial lipids from infected macrophages. Traffic 1:235-247;
Dunnick J K, O'Leary W M. 1970. Correlation of bacteria lipid
composition with antibiotic resistance. J. Bacteriol. 101:892-900;
and Liu X, Curtiss R III. 2012. Thermorecovery of cyanobacterial
fatty acids at elevated temperatures. J. Biotechnol. 161:445-449).
Briefly, bacteria were thawed and plated onto 150-mm petri dishes
containing modified Mueller-Hinton agar. Bacteria were incubated
for 48 h at 37.degree. C. in 7% CO.sub.2, scraped from the agar
plates, and added to high performance liquid chromatography
(HPLC)-grade chloroform/methanol (2:1) (both from Sigma). The
resulting mixture was stirred vigorously for 15 min at room
temperature. Then, 20 ml of endotoxin-free water was added and the
mixture was stirred for an additional 10 min. The mixture was
centrifuged at 4,000.times.g for 10 min at room temperature, to
separate the organic and aqueous phases. The organic phase was
pipetted into a separate container and dried under nitrogen. Dried
organic samples were reconstituted in absolute ethanol (EtOH)
(Warner-Graham) to 20 mg/ml. The average yield of lipids from
Francisella was 80 mg/4 g (wet weight) of bacteria, representing
approximately 2% of wet weight. Thus, 10 .mu.g/ml of lipid is
0.00025% (wet weight) of bacteria. Lipid preparations were stored
at 4.degree. C. for up to 2 months. An absence of proteins and
carbohydrates present in the organic phase was confirmed by
analysis of preparations on silver-stained SDS-PAGE gels, protein
quantification using the Bradford assay (Sigma) according to the
manufacturer's instructions, periodate staining of SDS-PAGE gels,
and Western blotting for Francisella O antigen and capsule. No
evidence for proteins or carbohydrates was observed in
organic-phase preparations.
[0201] In Vivo Assessment of Lipid Activity.
[0202] Mice were anesthetized by intraperitoneal injection of 100
.mu.l of 12.5 mg/ml ketamine plus 3.8 mg/m xylazine. Mice were
given 25 .mu.g SchuS4 lipids in 25 .mu.l phosphate-buffered saline
(PBS) intranasally. Mice receiving EtOH alone served as negative
controls for lipid-treated mice. Sixteen hours later, mice were
anesthetized as described above and were given either ultrapure E.
coli K-12 LPS (InvivoGen) or LPS derived from E. coli 0127:B8
(Sigma) (200 ng/25 .mu.l). Similar results were observed regardless
of which LPS type was administered to the animals. At the indicated
time points, mice were euthanized. Tracheas were exposed and
cannulated with disposable 18-gauge catheters. Airways were
repeatedly flushed with 0.5 ml PBS. The first 0.5-ml wash from the
bronchoalveolar lavage (BAL) was collected for assessment of
cytokines, followed by 4 additional 0.5-ml washes to collect cells
for analysis by flow cytometry.
[0203] Detection of Secreted Cytokines and Chemokines.
[0204] TNF-.alpha., IL-6, IL-12p40, and IL-10 (all from BD
Biosciences), and keratinocyte chemoattractant (KC) (R&D
Systems) present in bronchoalveolar lavage fluid were quantitated
using commercially available ELISA kits, following the
manufacturers' instructions.
[0205] Flow Cytometry.
[0206] Populations of cells in bronchoalveolar lavage fluid were
assessed by flow cytometry as described previously (Crane D D,
Scott D P, Bosio C M. 2012. Generation of a convalescent model of
virulent Francisella tularensis infection for assessment of host
requirements for survival of tularemia. PLoS One 7:e33349.
10.1371/journal.pone.0033349). Briefly, the following antibodies in
various combinations were used for flow cytometric analysis:
PerCp/Cy5.5-CD11c, PECy7-CD11b, phycoerythrin (PE)-Ly6C,
fluorescein isothiocyanate (FITC)-Ly6G, and antigen-presenting cell
major histocompatibility complex II (MHC-II) (all from eBioscience.
San Diego, Calif.). Neutrophils were characterized as Ly6G.sup.+
MHC-II.sup.-. Alveolar macrophages were characterized as
CD11c.sup.+ Ly6G.sup.- Ly6C.sup.- MHC-II.sup.-. Staining was
performed in fluorescence-activated cell sorting (FACS) buffer at
room temperature. Following staining, cells were washed and fixed
for 30 min in 1% paraformaldehyde at 4.degree. C. Cells were washed
a final time, resuspended in FACS buffer, and stored at 4.degree.
C. until analysis. Samples were collected using a LSRII flow
cytometer (BD Biosciences). Analysis gates were set on viable
unstained cells and were designed to include all viable cell
populations. Approximately 10,000 gated events were analyzed for
each sample. Isotype control antibodies were included when analyses
were first being performed, to ensure the specificity of staining,
but were not routinely included in each experiment. Data were
analyzed using FlowJo software (TreeStar, Ashland, Oreg.).
[0207] Lipid Analysis.
[0208] Analytical TLC plates were from Sigma-Aldrich-Fluka and
preparative TLC plates (1000.mu., 20.times.20 cm) were from
Analtech. Aminopropyl silica was from Phenomenex and silica gel
solid phase extraction columns from Waters. Primuline, ninhydrin,
copper sulfate, isopropanol, acetone, hexane, ethyl acetate and
triglyceride standards were from Sigma-Aldrich, chloroform from
Macron, methanol from Burdick Jackson. Phospholipid standards were
purchased from Avanti.
[0209] Analytical TLC.
[0210] Silica gel plates were developed in
chloroform:methanol:water, 7:3:0.25 for analysis of fractions I and
III (see solid phase extraction below) and phosphoplipid standards.
Plates were developed in hexane:diethyl ether:acetic acid, 7:3:0.1
for fraction II and fatty acid standards. Visualization of lipids
was done with long wavelength U V after spraying with primuline.
Phospholipids were also visualized by molybdenum blue reaction and
PE by spraying with ninhydrin and heating at 115.degree. C. for 10
to 15 minutes. C.sub.18 silica plates were run in
methanol:dichloromethane:water, 8:2:0.2 and visualization was done
by charring at 120.degree. C. after spraying with copper sulfate in
phosphoric acid. PE was also visualized by ninhydrin spray on these
plates.
[0211] Preparative TLC.
[0212] Crude lipid extract was dissolved in chloroform to a
concentration of approximately 100 mg/ml. Typically, 10 mg of
sample was applied in a narrow zone 8 cm. in length on the origin.
An equal volume of pure solvent was applied in the same way
adjacent to the sample. The plate was placed in
ethylacetate:methanol, 3:2 and the solvent was allowed to move 0.5
cm past the origin. The plate was dried and developed with
chloroform:methanol:water, 7:3:0.25. After drying the plate was
sprayed with primuline and the lipid bands were visualized by
fluorescence with long wavelength UV. Bands were excised with a
scalpel and the silica was soaked, with mixing, in 4 ml
chloroform:methanol, 1:1, for 2 hours in sealed glass vials.
Approximately equal areas of silica adjacent to the sample bands
were excised and processed the same way. The slurry was filtered on
a fritted glass funnel and the silica was washed with 2 ml of ethyl
acetate followed by 2 ml of methanol. The filtrates were combined
and filtered through 0.2 micron regenerated cellulose filters and
the solvent evaporated under an argon stream. The residue was
stored under argon at .sup.-20.degree. C.
[0213] Fractionation by Solid Phase Extraction.
[0214] Fractionation of crude lipid into three classes was based on
the method of Kaluzny (1). One milliliter of 25 mg/ml of crude
lipid in chloroform was gravity loaded on a 2 gram aminopropyl
silica column previously washed with 15 ml of hexane. After
complete entry into the bed the hexane was eluted with brief vacuum
suction. The column was sequentially developed with 14 ml of
chloroform:isopmpanol, 2:1 (1), 2% acetic acid in diethylether (II)
and methanol (III). The three fractions were dried under argon and
stored at .sup.-20.degree. C. Further fractionation of the
phospholipid class was based on the method of Fauland (2) with
modifications. Approximately 10 mg of fraction III dissolved in 10
ml of chloroform:methanol, 2:1, was loaded on to a 1 gram silica
extraction column washed in the same solvent. Another 10 ml of
solvent was applied to the column. Fractions were collected
throughout the loading and washing. Ten milliliters of
chloroform:methanol, 1.5:1, was applied and fractions were
collected. The column was then washed with 10 ml of methanol which
was collected in two fractions. The fractions were dried under
argon, dissolved chloroform:methanol, 1:1, and analyzed by TLC.
Primuline fluorescence revealed the majority of PE was eluted and a
single spot in the first 25 ml. All these fractions were also
ninhydrin positive. PC appeared as a single spot in the methanol
fractions. Both lipid fractions were pooled separately and
dried.
[0215] Separation of Phospholipid Molecular Species by HPLC.
[0216] Chromatography was done at 0.3 mL/min on a 2.0 mm.times.100
mm C.sub.18 Luna column (Phenomenex) in 94% methanol:acetonitrile,
4:1, 6% water. A 1260 Agilent analytical HPLC was used and
detection was done at 208 nm. Dried, pooled PE and PC silica
fractions were dissolved in 0.25 mL chloroform:methanol, 1:1.
Aliquots of 25 .mu.l to 50 .mu.l were diluted with 4 parts
methanol, injected and fractions collected. Typical runs were 60
minutes for PE and 80 minutes for PC in order to elute a highly
retained component. Under these conditions PE(C.sub.10:0).sub.2 and
PE(C.sub.14:0).sub.2 eluted at 7 and 10 minutes respectively.
PE(C.sub.16:0).sub.2 eluted at 22 minutes and PE(C.sub.18:1).sub.2
at 25 minutes. PE(1-C.sub.24:0, 2-C.sub.10:0-) eluted at
approximately 50 minutes. Peaks were broad and asymmetric. The S4
PE was resolved into four major peaks and the PC into six.
[0217] Preparation of Virus Stocks and Assessment of Viral
Load.
[0218] Stocks of West Nile Virus were generated following infection
of VERO cells cultured in DMEM supplemented with 10% heat
inactivated fetal bovine serum. Cell culture supernatants were
collected and centrifuged at 100,000.times.g at 4.degree. C. and
were stored at .sup.-80.degree. C. Virus titers in A549 cells were
enumerated as previously described (Beaty B. J., Calisher C. H.,
Shope R. S. 1989) Arboviruses. in Diagnostic procedures for viral,
rickettsial and chalmydial infections. eds Schmidt N. J., Emmons R.
W. [American Public Health Association, Washington, D.C.] pp
797-856).
[0219] Statistical Analysis.
[0220] Statistical differences between two groups were determined
using an unpaired t test, with significance set at P<0.05. For
comparisons of three or more groups, analysis was done by one-way
analysis of variance (ANOVA) followed by Tukey's
multiple-comparisons test, with significance determined at
P<0.05.
Specific Embodiments
[0221] According to an aspect, the present disclosure provides an
anti-inflammatory composition comprising: an effective amount of
purified lipid from Francisella tularensis or modified form
thereof.
[0222] In any aspect or embodiment described herein, the
Francisella tularensis is a virulent strain.
[0223] In any aspect or embodiment described herein, the purified
lipid comprises a phosphatidylethanolamine (PE).
[0224] In any aspect or embodiment described herein, the purified
lipid is enriched for the PE.
[0225] In any aspect or embodiment described herein, the purified
lipid is PE.
[0226] In any aspect or embodiment described herein, the
composition further comprises phosphatidylcholine (PC).
[0227] In any aspect or embodiment described herein, the PE, and/or
the PC comprises an acyl chain with a length in a range of from 5
to 13 or from 20 to 28 carbons.
[0228] In any aspect or embodiment described herein, the PE and/or
the PC comprises two acyl chains, wherein at least one chain has a
length in the range of from 5 to 13 carbons.
[0229] In any aspect or embodiment described herein, the PE and/or
the PC comprises two acyl chains, wherein at least one chain has a
length in the range of from 20 to 28 carbons.
[0230] In any aspect or embodiment described herein, the PE and/or
the PC comprises two acyl chains, wherein one chain has a length in
the range of from 5 to 13 carbons and the other chain has a length
in the range of from 20 to 28 carbons.
[0231] In any aspect or embodiment described herein, the ratio of
PE:PC is in a range of from about 50:50 to about 95:5.
[0232] In any aspect or embodiment described herein, the
composition further comprises another purified lipid from
Francisella tularensis.
[0233] In any aspect or embodiment described herein, the
composition is a liposome.
[0234] In any aspect or embodiment described herein, the
composition is an emulsified liposome.
[0235] In any aspect or embodiment described herein, the liposome
has a diameter in a range of from about 20 nm to about 1,500
nm.
[0236] According to another aspect, the present disclosure provides
a liposome comprising: purified lipid from Francisella tularensis
or modified form thereof.
[0237] In any aspect or embodiment described herein, the
Francisella tularensis is a virulent strain.
[0238] In any aspect or embodiment described herein, the purified
lipid comprises a phosphatidylethanolamine (PE).
[0239] In any aspect or embodiment described herein, the purified
lipid is enriched for the PE.
[0240] In any aspect or embodiment described herein, the purified
lipid is PE.
[0241] In any aspect or embodiment described herein, the liposome
further comprises phosphatidylcholine (PC).
[0242] In any aspect or embodiment described herein, the PE and/or
the PC comprises an acyl chain with a length in a range of from 5
to 13 or from 20 to 28 carbons.
[0243] In any aspect or embodiment described herein, the PE and/or
the PC two acyl chains, wherein at least one chain has a length in
the range of from 5 to 13 carbons.
[0244] In any aspect or embodiment described herein, the PE and/or
the PC comprises two acyl chains, wherein at least one chain has a
length in a range of from 20 to 28 carbons.
[0245] In any aspect or embodiment described herein, the PE and/or
the PC comprises two acyl chains, wherein one chain has a length in
a range of from 5 to 13 carbons, and the other chain has a length
in a range of from 20 to 28 carbons.
[0246] In any aspect or embodiment described herein, the ratio of
PE:PC is in a range of from about 50:50 to about 95:5.
[0247] In any aspect or embodiment described herein, the liposome
further comprises another purified lipid from Francisella
tularensis.
[0248] In any aspect or embodiment described herein, the liposome
is an emulsified liposome.
[0249] In any aspect or embodiment described herein, the
composition is a liposome or an emulsified liposome.
[0250] In any aspect or embodiment described herein, the liposome
has a diameter in a range of from about 20 nm to about 1,500
nm.
[0251] According to a further aspect, the present disclosure
provides an anti-inflammatory composition comprising an effective
amount of a synthetic PE or modified form thereof, wherein the PE
comprises two acyl chains, wherein at least one chain has a length
in the range of from 5 to 13 carbons and at least one chain has a
length in the range of from 20 to 28 carbons.
[0252] According to yet another aspect, the present disclosure
provides an anti-inflammatory composition produced by the following
process: adding virulent Francisella tularensis to a mixture of
chloroform/methanol and mixing; adding water to the
bacterial-chloroform/methanol mixture; separating the organic phase
and aqueous phase; drying the organic phase, and reconstituting the
dried organic phase.
[0253] In any aspect or embodiment described herein, the
chloroform/methanol is at a ratio of about 3:1 to about 1:1 (such
as, about 2:1).
[0254] In any aspect or embodiment described herein, the dried
organic phase is reconstituted in ethanol.
[0255] In any aspect or embodiment described herein, the process
further comprises isolating a band of the reconstituted organic
phase that runs near the solvent front on thin layer
chromatography.
[0256] In any aspect or embodiment described herein, the
reconstituted dried organic phase or isolated lipid is enriched for
PE.
[0257] In any aspect or embodiment described herein, the process
further comprising adding PC to the isolated lipid of the
reconstituted organic phase or the isolated lipid.
[0258] In any aspect or embodiment described herein, the PE and/or
the PC comprises an acyl chain with a length in a range of from 5
to 13 or from 20 to 28 carbons.
[0259] In any aspect or embodiment described herein, the PE and/or
the PC comprises two acyl chains, wherein at least one has a length
in a range of from 5 to 13 carbons.
[0260] In any aspect or embodiment described herein, the PE and/or
the PC comprises two acyl chains, wherein at least one has a length
in a range of from 20 to 28 carbons.
[0261] In any aspect or embodiment described herein, the PE and/or
the PC comprises two acyl chains, wherein one chain has a length in
the range of from 5 to 13 carbons and the other chain has a length
in the range of 20 to 28 carbons.
[0262] In any aspect or embodiment described herein, a ratio of the
reconstituted organic phase or isolated lipid to PC is in a range
of from about 50:50 to about 95:5.
[0263] In any aspect or embodiment described herein, the process
further comprises adding another purified lipid from Francisella
tularensis.
[0264] In any aspect or embodiment described herein, the process
further comprises producing a liposome from the reconstituted
organic phase, isolated lipid, or isolated lipid-PC mixture.
[0265] In any aspect or embodiment described herein, the liposome
is an emulsified liposome.
[0266] In any aspect or embodiment described herein, the liposome
has a diameter in a range of from about 20 nm to about 1,500
nm.
[0267] According to yet a further aspect, the present disclosure
provides a pharmaceutical composition comprising a pharmaceutically
effective amount of the composition of the present disclosure, or
the liposome of the present disclosure.
[0268] In any aspect or embodiment described herein, pharmaceutical
composition further comprises a pharmaceutically acceptable
carrier.
[0269] In any aspect or embodiment described herein, the
pharmaceutically acceptable carrier is a gel or cream.
[0270] According to an additional aspect a method of treating or
preventing a microbial infection or inflammation resulting from a
microbial infection in a patient in the need thereof, the method
comprising administering an effective amount of the composition of
the present disclosure, or the liposome of the present disclosure,
wherein the composition or liposome is effective in treating or
preventing the microbial infection or the microbial infection
related inflammation.
[0271] In any aspect or embodiment described herein, the microbial
infection is a bacterial or a viral infection.
[0272] In any aspect or embodiment described herein, the bacterial
infection or viral infection causes dermatological inflammation
and/or respiratory inflammation.
[0273] In any aspect or embodiment described herein, the bacterial
infection or viral infection is selected from the group consisting
of Staphylococcus aureus, Streptococcus pyogenes, Clostridium
perfringens, Bacillus anthracis, Francisella tularensis,
Corynebacterium diphtheria. Streptococcus pneumoniae, Haemophilus
influenza, Bordetella pertussis, Mycobacterium tuberculosis or
bovis, Mycoplasma pneumoniae, Legionella pneumophila, Chlamydia
psittaci, Chlamydia pneumoniae, Coxiella burnetii, measles,
rubella, varicella zoster, parvovirus, herpes simplex virus 6,
herpes simplex virus 7, herpes simplex virus 8, Epstein Barr virus,
enterovirus, coxsackie virus, togavirus, coronavirus, rhinovirus,
bunyavirus, arenavirus, smallpox, cowpox, monkey pox, zika virus,
dengue virus, nairovirus, arenavirus, filovirus, west nile virus,
molluscum contagiosm, human papillomavirus, coronavirus,
rhinovirus, respiratory syncytial virus, and Influenzavirus.
[0274] In any aspect or embodiment described herein, the
composition or the liposome is administered prior to exposure to
the microbial infection.
[0275] In any aspect or embodiment described herein, the
composition of the liposome is administered post exposure to the
microbial infection.
[0276] In any aspect or embodiment described herein, the method
further comprises co-administering one or more additional
therapeutic agent.
[0277] According to an aspect, the present disclosure provides a
method of treating or preventing inflammation in a patient in the
need thereof, the method comprising administering an effective
amount of the composition of the present disclosure, or the
liposome of the present disclosure to the patient, wherein the
composition or liposome is effective at treating or preventing
inflammation.
[0278] In any aspect or embodiment described herein, the
inflammation is related to a bacterial infection, a viral
infection, an autoimmune disease or disorder, and/or an
allergy.
[0279] According to yet a further aspect, the present disclosure
provides a method of modulating an immune response in a patient
comprising administering an effective amount of the composition of
the present disclosure, or the liposome of the present disclosure
to the patient, wherein the composition or liposome is effective at
modulating the immune response.
[0280] In any aspect or embodiment described herein, the modulation
comprises enhancing the immunocompetence in the subject.
[0281] In any aspect or embodiment described herein, the modulation
comprises inhibiting bacterial replication in the subject.
[0282] In any aspect or embodiment described herein, the modulation
comprises inhibiting viral replication in the subject.
[0283] In any aspect or embodiment described herein, the bacterial
infection or viral infection is a respiratory infection.
[0284] In any aspect or embodiment described herein, the bacterial
infection or viral infection is a dermatological infection.
[0285] In any aspect or embodiment described herein, the
inflammation is caused by an autoimmune disease or disorder, and/or
an allergy.
[0286] In any aspect or embodiment described herein, the subject
has dermatological or respiratory inflammation.
Other Embodiments
[0287] From the foregoing description, it will be apparent that
variations and modifications may be made to the invention described
herein to adopt it to various usages and conditions. Such
embodiments are also within the scope of the following claims.
[0288] The recitation of a listing of elements in any definition of
a variable herein includes definitions of that variable as any
single element or combination (or subcombination) of listed
elements. The recitation of an embodiment herein includes that
embodiment as any single embodiment or in combination with any
other embodiments or portions thereof.
[0289] All patents and publications mentioned in this specification
are herein incorporated by reference to the same extent as if each
independent patent and publication was specifically and
individually indicated to be incorporated by reference.
* * * * *