U.S. patent application number 16/924539 was filed with the patent office on 2020-11-05 for tailored liposomes for the treatment of bacterial infections.
This patent application is currently assigned to UNIVERSITAET BERN. The applicant listed for this patent is UNIVERSITAET BERN. Invention is credited to Eduard BABIYCHUK, Annette DRAEGER.
Application Number | 20200345639 16/924539 |
Document ID | / |
Family ID | 1000004957511 |
Filed Date | 2020-11-05 |
United States Patent
Application |
20200345639 |
Kind Code |
A1 |
BABIYCHUK; Eduard ; et
al. |
November 5, 2020 |
TAILORED LIPOSOMES FOR THE TREATMENT OF BACTERIAL INFECTIONS
Abstract
The invention relates to the use of empty liposomes of defined
lipid composition or mixtures of empty liposomes of defined lipid
composition and to the use of other lipid bilayers or monolayers of
defined lipid composition for the treatment and prevention of
bacterial infections. It has been found that such liposomes, in
particular a two-and a four-component mixture of liposomes
comprising cholesterol and sphingomyelin, liposomes consisting of
sphingomyelin, liposomes comprising sphingomyelin and
phosphatidylcholine, and liposomes comprising cholesterol and
phosphatidylcholine efficiently sequestrate a variety of toxins
secreted by bacteria, thus preventing binding of bacterial toxins
to target cells and toxin-induced lysis of the target cells.
Injected intravenously, liposome mixtures prevented death of
laboratory mice infected with lethal doses of Staphylococcus aureus
or Streptococcus pneumoniae.
Inventors: |
BABIYCHUK; Eduard; (Bern,
CH) ; DRAEGER; Annette; (Bern, CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UNIVERSITAET BERN |
Bern |
|
CH |
|
|
Assignee: |
UNIVERSITAET BERN
Bern
CH
|
Family ID: |
1000004957511 |
Appl. No.: |
16/924539 |
Filed: |
July 9, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14404985 |
Dec 2, 2014 |
10744089 |
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PCT/EP2013/062207 |
Jun 13, 2013 |
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16924539 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/688 20130101;
A61K 9/1271 20130101; A61K 9/127 20130101; A61K 9/10 20130101; A61K
31/575 20130101 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 9/10 20060101 A61K009/10; A61K 31/575 20060101
A61K031/575; A61K 31/688 20060101 A61K031/688 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 14, 2012 |
EP |
12171924.9 |
Jan 29, 2013 |
EP |
13153039.6 |
Claims
1. Empty liposomes comprising cholesterol and sphingomyelin, and
mixtures of empty liposomes comprising cholesterol and
sphingomyelin, or phosphatidylcholine and sphingomyelin, with other
empty liposomes of defined lipid composition for use in the
treatment and prevention of bacterial infections.
2. Empty liposomes comprising cholesterol and sphingomyelin, and
mixtures of empty liposomes comprising cholesterol and
sphingomyelin, or phosphatidylcholine and sphingomyelin, with other
empty liposomes comprising lipids or phospholipids selected from
the group consisting of cholesterol, sphingomyelins, ceramides,
phosphatidyl-cholines, phosphatidylethanolamines,
phosphatidylserines, diacylglycerols, and phosphatidic acids
containing one or two saturated or unsaturated fatty acids longer
than 4 carbon atoms and up to 28 carbon atoms, for use in the
treatment and prevention of bacterial infections according to claim
1.
3. Empty liposomes comprising sphingomyelin and 30% (w/w) or more
cholesterol, and mixtures of empty liposomes comprising
sphingomyelin and 30% (w/w) or more cholesterol with other empty
liposomes of defined lipid composition, for use in the treatment
and prevention of bacterial infections according to claim 1 or
2.
4. Liposome mixture of empty liposomes comprising or consisting of
cholesterol and sphingomyelin, with other empty liposomes
comprising or consisting of sphingomyelin, for use in the treatment
and prevention of bacterial infections according to any one of
claims 1 to 3.
5. Liposome mixture of empty liposomes comprising or consisting of
phosphatidylcholine and sphingomyelin, with other empty liposomes
comprising or consisting of sphingomyelin, for use in the treatment
and prevention of bacterial infections according claim 1 or 2.
6. Liposome mixture of empty liposomes comprising or consisting of
cholesterol and sphingomyelin with other empty liposomes comprising
or consisting of phosphatidyl-choline and sphingomyelin, and with
empty liposomes consisting of sphingomyelin, for use in the
treatment and prevention of bacterial infections according to any
one of claims 1 to 5.
7. Liposome mixture of empty liposomes comprising or consisting of
cholesterol and sphingomyelin with other empty liposomes comprising
or consisting of phosphatidyl-choline and sphingomyelin, with empty
liposomes consisting of sphingomyelin, and with empty liposomes
comprising or consisting of cholesterol and phosphatidylcholine,
for use in the treatment and prevention of bacterial infections
according to any one of claims 1 to 6.
8. Empty liposomes comprising cholesterol and sphingomyelin, and
mixtures of empty liposomes comprising cholesterol and
sphingomyelin, or phosphatidylcholine and sphingomyelin, with other
empty liposomes of defined lipid composition, wherein some or all
liposomes are modified with polyethylene glycol, for use in the
treatment and prevention of bacterial infections according to any
one of claims 1 to 7.
9. Empty liposomes comprising cholesterol and sphingomyelin, and
mixtures of empty liposomes comprising cholesterol and
sphingomyelin, or phosphatidylcholine and sphingomyelin, with other
empty liposomes of defined lipid composition, for use in the
treatment and prevention of bacteremia, bacterially infected skin
lesions, meningitis and respiratory tract infections, according to
any one of claims 1 to 8.
10. Lipid bilayers or lipid monolayers comprising cholesterol and
sphingomyelin, and optionally phosphatidylcholine, covering
non-lipid surfaces, for use in the treatment and prevention of
bacterial infections.
11. A mixture of empty liposomes comprising cholesterol and
sphingomyelin, or phosphatidylcholine and sphingomyelin, with other
empty liposomes of defined lipid composition.
12. A mixture according to claim 11 of empty liposomes comprising
cholesterol and sphingomyelin, or phosphatidylcholine and
sphingomyelin, with empty liposomes consisting of
sphingomyelin.
13. A mixture according to claim 11 or 12 of empty liposomes
comprising cholesterol and sphingomyelin with other empty liposomes
comprising phosphatidylcholine and sphingomyelin, and with empty
liposomes consisting of sphingomyelin.
14. A mixture according to any one of claims 11 to 13 of empty
liposomes comprising cholesterol and sphingomyelin with other empty
liposomes comprising phosphatidyl-choline and sphingomyelin, with
empty liposomes consisting of sphingomyelin, and with empty
liposomes comprising cholesterol and phosphatidylcholine.
15. A mixture according to any one of claims 11 to 14 of empty
liposomes wherein some or all liposomes are modified with
polyethylene glycol.
16. A method of treating bacterial infections comprising
administering to a patient in need thereof a therapeutically
effective amount of empty liposomes comprising cholesterol and
sphingomyelin, or of a mixture of empty liposomes comprising
cholesterol and sphingomyelin, or phosphatidylcholine and
sphingomyelin, with other empty liposomes of defined lipid
composition.
17. A method of preventing a bacterial infection comprising
administering to a subject exposed to the risk of infection a
preventive amount of empty liposomes comprising cholesterol and
sphingomyelin, or of a mixture of empty liposomes comprising
cholesterol and sphingomyelin, or phosphatidylcholine and
sphingomyelin, with other empty liposomes of defined lipid
composition effective for protection.
18. A method of treating bacterial infections comprising
administering to a patient in need thereof a therapeutically
effective amount of empty liposomes comprising cholesterol and
sphingomyelin, or of a mixture of empty liposomes comprising
cholesterol and sphingomyelin, or phosphatidylcholine and
sphingomyelin, with other empty liposomes of defined lipid
composition before, after, together or in parallel with a standard
antibiotic medication against the bacterial infection.
Description
FIELD OF THE INVENTION
[0001] The invention relates to the use of empty liposomes or
liposome mixtures and to the use of other lipid bilayers or
monolayers of defined lipid composition for the treatment and
prevention of bacterial infections. Likewise the invention relates
to a treatment of such bacterial infections comprising
administering empty liposomes or liposome mixtures, alone or in
combination with standard antibiotic treatment. Furthermore the
invention relates to new liposome mixtures as such.
BACKGROUND OF THE INVENTION
[0002] Bacterial infections remain one of the major threats to
human lives. As bacterial resistance to even the most potent
antibiotics increases, so too must the efforts to identify novel
anti-bacterial strategies. Among other virulence factors, many
pathogenic bacteria secrete toxins that kill eukaryotic cells by
disturbing their plasma membrane. Bacterial pore-forming toxins are
active on the cell surface, causing pore formation and disruption
of the plasma membrane followed by either lysis or apoptosis of
host target cells, whereas bacterial phospholipases induce the
death of host cells by enzymatic degradation of plasmalemmal
phospholipids.
[0003] Bacterial membrane-destabilizing toxins, such as
cholesterol-dependent cytolysins (CDCs: pneumolysin O, streptolysin
O, tetanolysin), .alpha.-hemolysin or bacterial phospholipases
(phospholipase C, sphingomyelinase) play a critical role in the
establishment and progression of infectious diseases. Such diseases
are pneumonia, a major cause of death among all age groups and the
leading cause of death in children in low income countries;
bacteremia, a severe complication of infections or surgery, which
is characterized by high mortality due to sepsis and septic shock;
and meningitis, a life-threatening disease, which also leads to
serious long-term consequences such as deafness, epilepsy,
hydrocephalus and cognitive deficits.
[0004] To target host cells bacterial membrane-destabilizing toxins
either bind to individual membrane lipids (lipid head groups) or
exploit the non-homogenous nature of the lipid bilayer of
eukaryotic cells' plasma membrane, interacting with microdomains
enriched in certain lipid species (Gonzales M. R. et al., Cell.
Mol. Life Sci. 2008, 65:493-507). The non-homogenous distribution
of lipids within the bilayer is not favored by in vivo conditions
since transmembrane proteins and the presence of a multitude of
individual lipid species with variable lengths and saturation
status of their acyl chain oppose lipid de-mixing and thus the
formation of stable lipid microdomains (Simons K. and Gerl M. J.,
Nat. Rev. Mol. Cell Biol. 2010, 11:688-99). However, lipid
de-mixing can be taken to its extremes in artificial protein-free
liposomes, manufactured from a limited number of carefully selected
lipid species, where extended, stable lipid microdomains can be
created (Klose C. et al., J. Biol. Chem. 2010, 285:30224-32).
Moreover, artificial liposomes allow for much higher relative
concentrations of a particular lipid than those ever likely to
occur in vivo. Therefore, liposomes displaying stable lipid
microdomains of defined biochemical properties and possessing high
relative concentrations of particular lipids can be produced.
Liposomes are currently used in the cosmetic and pharmaceutical
industries as carriers for topical and systemic drug delivery and
are considered to be non-toxic.
SUMMARY OF THE INVENTION
[0005] The invention relates to the use of empty liposomes of
defined lipid composition or mixtures of empty liposomes of defined
lipid composition for the treatment and prevention of bacterial
infections, in particular skin lesions, bacteremia, meningitis,
respiratory tract infections, such as pneumonia, and abdominal
infections, such as peritonitis.
[0006] The invention furthermore relates to lipid bilayers or lipid
monolayers of defined lipid composition covering non-lipid
surfaces, for use in the treatment and prevention of bacterial
infections.
[0007] Likewise the invention relates to a treatment of such
bacterial infections comprising administering to a patient in need
thereof a therapeutically effective amount of empty liposomes of
defined lipid composition or mixtures of empty liposomes of defined
lipid composition, and to a method of prevention of such bacterial
inventions in a subject at risk. Furthermore the invention relates
to a treatment of bacterial infections comprising administering to
a patient in need thereof a therapeutically effective amount of
empty liposomes of defined lipid composition or mixtures of empty
liposomes of defined lipid composition, before, after, together or
in parallel with a standard antibiotic treatment of the bacterial
infection.
[0008] Furthermore the invention relates to new mixtures of empty
liposomes of defined lipid composition.
BRIEF DESCRIPTION OF THE FIGURES
[0009] FIG. 1. Liposomes composed of cholesterol and sphingomyelin
protect monocytes from cholesterol-dependent cytolysins,
.alpha.-hemolysin and phospholipase C.
[0010] A-D) Liposomes (1:1 w/w mixtures) containing cholesterol in
combination with PC (3), Sm (4) or PS (5) but not with PE (6)
protected THP-1 cells from cholesterol-dependent cytolysins:
pneumolysin 0 (A), streptolysin 0 (B), tetanolysin (C) as well as
from phospholipase C (D).
[0011] E) Liposomes (1:1 w/w) containing cholesterol in combination
with Sm (4) but not with PC (3), PS (5) or PE (6) protected THP-1
cells from .alpha.-hemolysin.
[0012] F) Liposomes without cholesterol (7-9) were ineffective.
[0013] c (r.u.)=number of cells, maintained in the presence of a
toxin (1, 3-9) related to the number of cells maintained in the
absence of a toxin (2), is given in relative units (r.u.).
PLY=pneumolysin O; SLO=streptolysin O; TL=tetanolysin;
PLC=phospholipase C; HML=.alpha.-hemolysin. 1=Control (no
liposomes); 2=Control (no toxin), 3=Ch:PC (1:1 w/w) liposomes;
4=Ch:Sm (1:1 w/w) liposomes; 5=Ch:PS (1:1 w/w) liposomes; 6=Ch:PE
(1:1 w/w) liposomes; 7=PC:Sm (1:1 w/w) liposomes; 8=Sm liposomes;
9=PC liposomes. Ch=cholesterol; PC=phosphatidylcholine;
PS=phosphatidylserine; Sm=sphingomyelin;
PE=phosphatidylethanolamine.
[0014] FIG. 2. Liposomes composed of cholesterol and sphingomyelin
(1:1 w/w) protect monocytes from cholesterol-dependent cytolysins
at microgram amounts, whereas 25-100 micrograms of the liposomes is
required for protection against Staphylococcus aureus
.alpha.-hemolysin and Clostridium perfringens phospholipase C.
[0015] A) Protection against 0.2 microgram of PLY. B) Protection
against 0.4 microgram of SLO. C) Protection against 0.2 microgram
of TL. D) Protection against 1.2 microgram of HML. E) Protection
against 4.5 microgram of PLC.
[0016] c (r.u.)=number of cells, maintained in the presence of a
toxin related to the number of cells maintained in the absence of a
toxin, given in relative units. X-axis: LP (mkg)=amount of
liposomes in micrograms. PLY=pneumolysin O; SLO=streptolysin O;
TL=tetanolysin; HML=.alpha.-hemolysin; PLC=phospholipase C.
[0017] FIG. 3. Cholesterol in concentrations above 30% (w/w) is
required for liposomes composed of cholesterol and sphingomyelin to
protect monocytes from pneumolysin, tetanolysin or
.alpha.-hemolysin.
[0018] A) Protection against 0.2 microgram of PLY. B) Protection
against 0.2 microgram of TL. C) Protection against 1.2 microgram of
HML. c (r.u.)=number of cells, maintained in the presence of a
toxin related to the number of cells maintained in the absence of a
toxin, given in relative units. Ch (%)=percentage of cholesterol
(w/w) in liposomes composed of cholesterol and sphingomyelin.
PLY=pneumolysin; TL=tetanolysin; HML=.alpha.-hemolysin.
[0019] FIG. 4. Liposomes composed of cholesterol and sphingomyelin
protect monocytes from a combination of cholesterol-dependent
cytolysins and S. aureus .alpha.-hemolysin.
[0020] A) Ch:Sm (1:1 w/w) liposomes (6) exerted fully protective
effects against the combined action of .alpha.-hemolysin (HML; 1.2
microgram), streptolysin O (SLO; 0.4 microgram) and tetanolysin
(TL; 0.2 microgram), whereas Ch:PC (1:1 w/w) liposomes were
ineffective (7). c (r.u.)=number of cells, maintained in the
presence of toxins (2-7) related to the number of control cells
maintained in the absence of toxins (1), in relative units.
1=Control (no toxins); 2=SLO, no liposomes; 3=TL, no liposomes;
4=HML, no liposomes; 5=SLO+TL+HML, no liposomes; 6=SLO+TL+HML,
Ch:Sm liposomes; 7=SLO+TL+HML, Ch:PC liposomes. Ch=cholesterol;
PC=phosphatidylcholine; Sm=sphingomyelin. B) The full protective
effect against the combined action of HML (1.2 microgram), SLO (0.4
microgram) and TL (0.2 microgram) was observed at 25 microgram of
liposomes composed of Ch:Sm (1:1 w/w). LP (mkg)=amount of liposomes
in micrograms. C) Centrifugation experiments confirmed that all
three toxins bind directly to Ch:Sm (1:1 w/w) liposomes. The toxins
were pre-incubated with (6) or without (2-5) Ch:Sm liposomes. After
centrifugation, the supernatants were added to the cells. 1=Control
(no toxins); 2=SLO, no liposomes; 3=TL, no liposomes; 4=HML, no
liposomes; 5=SLO+TL+HML, no liposomes; 6=SLO+TL+HML, Ch:Sm
liposomes.
[0021] FIG. 5. Liposomes composed of cholesterol and sphingomyelin
(1:1 w/w) protect monocytes from Streptococcus pyogenes toxins.
[0022] A,B) THP-1 cells proliferate in the presence of BHI broth
(squares; dashed line in (A)). Culture supernatants of 5
Streptococcus pyogenes strains (GAS 1-5=clinical isolates; all
grown in BHI broth) effectively killed THP-1 cells (triangles),
however the cells were protected from the effect of streptococcal
toxins by liposomes composed of cholesterol and sphingomyelin
(circles). 10.sup.5c=number of cells.times.10.sup.5. t(d)=time
after treatment (days).
[0023] C) Liposomes composed of cholesterol and sphingomyelin
protect monocytes from culture supernatants of Streptococcus
pyogenes in microgram amounts. c(%)=percentage of cells, maintained
in the presence of bacterial supernatants related to cells
maintained in the absence of the bacterial supernatants (100%). LP
(mkg)=amount of liposomes in micrograms.
[0024] FIG. 6. Liposomes composed of cholesterol and sphingomyelin
(1:1 w/w) in combination with sphingomyelin-only liposomes
completely protect monocytes from Streptococcus pneumoniae
toxins.
[0025] A,B) THP-1 cells proliferate in the presence of BHI broth
(squares). Culture supernatants of 2 Streptococcus pneumoniae
strains (Pneumo 1=clinical isolate and Pneumo 2=D39 strain; both
grown in BHI broth) effectively killed THP-1 cells (triangles),
however the cells were partially protected from the effect of
Streptococcus pneumoniae toxins by liposomes composed of
cholesterol and sphingomyelin (1:1 w/w) (circles). 10.sup.5c=number
of cells.times.10.sup.5. t(d)=time after treatment (days).
[0026] C) The mixture of cholesterol-containing and
cholesterol-free, sphingomyelin-only liposomes was fully protective
against Streptococcus pneumoniae toxins. The graph shows the
protective effect of the liposomal mixtures composed of constant
(400 .mu.g) amount of cholesterol:sphingomyelin (1:1 w/w) liposomes
and varying amounts (0-400 .mu.g) of sphingomyelin-only
liposomes.
[0027] c (r.u.)=number of cells, maintained in the presence of a
bacterial supernatant related to the number of cells maintained in
the absence of the supernatant, is given in relative units (r.u.).
Sm LP (mkg)=amounts of sphingomyelin-only liposomes in
micrograms.
[0028] FIG. 7. Cholesterol:phosphatidylcholine liposomes (1:1 w/w)
and a mixture of cholesterol: phosphatidylcholine (1:1 w/w) and
sphingomyelin liposomes protect monocytes from toxins secreted by
Staphylococcus aureus strain MRSA 2040.
[0029] THP-1 cells proliferate in the presence of BHI broth
(squares). Culture supernatants of Staphylococcus aureus (grown in
BHI broth) effectively kill THP-1 cells in the absence of liposomes
(triangles). (A) 900 microgram (diamonds) of
cholesterol:phosphatidylcholine (1:1 w/w) liposomes provide
significant protection against bacterial toxins, whereas only
limited protection was observed at 600 microgram (circles). (B) The
full protection was observed for a mixture of 600 microgram of
cholesterol:phosphatidylcholine (1:1 w/w) liposomes with 75
microgram of Sm liposomes (diamonds). 900 microgram of Sm liposomes
used alone was ineffective (circles). 10.sup.5c=number of
cells.times.10.sup.5. t (d)=time after treatment (days).
[0030] FIG. 8. Cholesterol-free, sphingomyelin-containing liposomes
and a mixture of cholesterol:phosphatidylcholine (1:1 w/w) and
sphingomyelin-only liposomes protect monocytes from toxins secreted
by Staphylococcus aureus Doppelhof strain.
[0031] A,B) THP-1 cells proliferate in the presence of BHI broth
(squares). Culture supernatants of Staphylococcus aureus (grown in
BHI broth) effectively kill THP-1 cells in the absence of liposomes
(triangles). (A) 1200 microgram (diamonds) of sphingomyelin
liposomes provided significant protection against bacterial toxins.
(B) When used at 600 microgram, the most potent protection was
observed for a mixture of sphingomyelin and
sphingomyelin:phosphatidylcholine (1:1 w/w) (diamonds), whereas
sphingomyelin liposomes alone (circles) and
sphingomyelin:phosphatidylcholine (1:1 w/w) liposomes alone
(asterisks) were less effective. C) A mixture of
cholesterol-containing and cholesterol-free, sphingomyelin-only
liposomes was fully protective against toxins secreted by the
Staphylococcus aureus Doppelhof strain. The graph shows the
protective effect of the liposomal mixtures composed of constant
(600 .mu.g) amount of cholesterol:phosphatidylcholine (1:1 w/w)
liposomes and varying amounts (0-1200 .mu.g) of sphingomyelin-only
liposomes.
[0032] 10.sup.5c=number of cells.times.10.sup.5. t (d)=time after
treatment (days). c (r.u.)=number of cells, maintained in the
presence of a bacterial supernatant related to the number of cells
maintained in the absence of the supernatant, is given in relative
units (r.u.). Sm LP (mkg)=amounts of sphingomyelin-only liposomes
in micrograms.
[0033] FIG. 9. A 4-component mixture of cholesterol:sphingomyelin
(1:1 w/w), sphingomyelin-only, sphingomyelin:phosphatidylcholine
(1:1 w/w) and cholesterol:phosphatidylcholine (1:1 wt/wt) liposomes
protect monocytes from toxins secreted by both Doppelhof and MRSA
2040 strains of Staphylococcus aureus.
[0034] THP-1 cells proliferate in the presence of BHI broth
(squares). Culture supernatants of MRSA 2040 (A) or Doppelhof (B)
Staphylococcus aureus strains (grown in BHI broth) effectively kill
THP-1 cells in the absence of liposomes (triangles), however the
cells are completely protected from either MRSA 2040 (A) or
Doppelhof (B) toxins by 1200 microgram of the 4-component liposomal
mixture (1:1:1:1).
[0035] 10.sup.5c=number of cells.times.10.sup.5. t (d)=time after
treatment (days).
[0036] FIG. 10. Liposomes protect mice from Staphylococcus aureus
bacteremia, from Streptococcus pneumoniae pneumonia and from
Streptococcus pneumoniae bacteremia.
[0037] A) Laboratory mice were injected intravenously with a lethal
dose of the Doppelhof Staphylococcus aureus strain. At 1, 5 and 24
hours after injection of bacteria, the mice were injected with
either 25-50 microliter of normal saline (diamonds) or 25
microliter (1 mg) of cholesterol:sphingomyelin (1:1 w/w) liposomes
(squares) or 50 microliter (2 mg) of a 1:2:2 mixture of
cholesterol:sphingomyelin (1:1 w/w) liposomes+sphingomyelin-only
liposomes+sphingomyelin:phosphatidylcholine (3:1 w/w) liposomes
(triangles).
[0038] B) Mice were infected intranasally with the Streptococcus
pneumoniae strain D39. 30 minutes following injection of bacteria,
the mice received either an injection of 50 microliter of normal
saline (diamonds) or a single intranasal injection of 50 microliter
(2 mg) of a 1:1:1:1 mixture of cholesterol:sphingomyelin (1:1
w/w)+cholesterol:phosphatidylcholine (1:1
w/w)+sphingomyelin-only+sphingomyelin:phosphatidylcholine (3:1 w/w)
liposomes (triangles).
[0039] C) Mice were injected intravenously with a lethal dose of
the S. pneumoniae strain D39. At 8 and 12 hours following injection
of bacteria, the mice received intravenously 75
microliter/injection (3 mg) of the following liposomes: 1) a 1:1
mixture of cholesterol:sphingomyelin (1:1 w/w)+sphingomyelin-only
liposomes (triangles); 2) cholesterol:sphingomyelin (1:1 w/w)
liposomes (squares); 3) sphingomyelin-only liposomes (circles) or
4) normal saline (diamonds).
[0040] S (%)=percent surviving mice. t (d)=time after infection
(days)
DETAILED DESCRIPTION OF THE INVENTION
[0041] Engineered to possess higher than in vivo affinities for
membrane-targeting toxins, inhaled or intravenously injected or
infused empty liposomes and liposome mixtures serve as traps for
bacterial toxins residing in blood or airways of infected patients,
paving a way for a novel anti-bacterial toxin-sequestrating
therapy.
[0042] The invention relates to the use of empty liposomes of
defined lipid composition or mixtures of empty liposomes of defined
lipid composition for the treatment and prevention of bacterial
infections, in particular bacteremia, meningitis, bacterial skin
infections, respiratory tract infections, such as pneumonia, and
abdominal infections, such as peritonitis.
[0043] Liposomes of the invention are empty liposomes, i.e.
liposomes not encapsulating any antibiotic or other drug. They may,
if desired, be used in combination with known or novel liposomes
carrying drugs.
[0044] The present study shows that artificial liposomes of
precisely defined lipid composition or mixtures of liposomes of
precisely defined lipid composition efficiently sequestrate
purified pore-forming toxins and phospholipase C, thereby
preventing their binding to the target cells. Consequently, the
application of liposomes or their mixtures prevent the lysis of
cultured epithelial cells and monocytes induced by the application
of purified toxins or culture supernatants of Streptococcus
pneumonia, Streptococcus pyogenes and Staphylococcus aureus and
protect laboratory mice from death due to an experimentally induced
bacteremia or pneumonia.
[0045] The invention relates to the use of empty liposomes of
defined lipid composition or mixtures of empty liposomes of defined
lipid composition for the treatment and prevention of bacterial
infections.
[0046] The invention furthermore relates to lipid bilayers or lipid
monolayers of defined lipid composition covering non-lipid
surfaces, for use in the treatment and prevention of bacterial
infections. Non-lipid surfaces considered are, for example, medical
appliances, biodegradable beads, and nanoparticles.
[0047] Liposomes considered are artificial liposomes of 20 nm to 10
.mu.m, preferably 20 to 500 nm, comprising lipids or phospholipids
selected from the group of sterols, sphingolipids and
glycerolipids, in particular selected from the group consisting of
cholesterol, sphingomyelins, ceramides, phosphatidylcholines,
phosphatidylethanolamines, phosphatidylserines, diacylglycerols,
and phosphatidic acids containing one (lyso-) or two (diacyl-),
saturated or unsaturated fatty acids longer than 4 carbon atoms and
up to 28 carbon atoms.
[0048] The composition of lipid bilayers or lipid monolayers
considered is the same as indicated for liposomes.
[0049] Fatty acids comprising between 4 and 28 carbon atoms are,
for example, saturated linear alkanecarboxylic acids, preferably
with an even number of carbon atoms, such as between 12 and 26
carbon atoms, for example lauric, myristic, palmitic, stearic,
arachidic, or behenic acid, or unsaturated linear alkenecarboxylic
acids, preferably with an even number of between 12 and 26 carbon
atoms and one, two or more, preferably up to six double bonds in
trans or, preferably cis configuration, for example oleic acid,
linoleic acid, alpha-linoleic acid, arachidonic acid, or erucic
acid.
[0050] Empty liposomes means that the liposomes considered in the
present invention do not incorporate antibiotic or other drugs.
"Incorporated" as used herein means encapsulated into the cavity of
the liposome, within the potential double layer of the liposome, or
as part of the membrane layer of the liposome. Liposomes as used
herein also exclude liposomes modified with binding agents such as
antibodies and mono- or oligosaccharides, e.g. as in glycolipids.
However, liposomes modified with polyethylene glycol (PEG) are
considered as part of this invention. PEG is known to modify the
circulation time of liposome.
[0051] In particular, the invention relates to the use of empty
liposomes comprising cholesterol and sphingomyelin, and of mixtures
of empty liposomes comprising cholesterol and sphingomyelin, or
phosphatidylcholine and sphingomyelin, with other empty liposomes
of defined lipid composition, such as liposomes comprising lipids
or phospholipids selected from the group consisting of sterols,
sphingolipids and glycerolipids, in particular selected from the
group consisting of cholesterol, sphingomyelins, ceramides,
phosphatidyl-cholines, phosphatidylethanolamines,
phosphatidylserines, diacylglycerols, and phosphatidic acids
containing one or two saturated or unsaturated fatty acids longer
than 4 carbon atoms and up to 28 carbon atoms, for the treatment
and prevention of bacterial infections.
[0052] In one embodiment, the invention relates to the use of empty
liposomes comprising sphingomyelin and 30% (w/w) or more
cholesterol, and of mixtures of empty liposomes comprising
sphingomyelin and 30% (w/w) or more cholesterol with other empty
liposomes as defined herein, for the treatment and prevention of
bacterial infections. In particular, the invention relates to the
use of empty liposomes consisting of sphingomyelin and of 30% (w/w)
or more cholesterol, and of mixtures of empty liposomes consisting
of sphingomyelin and of 30% (w/w) or more cholesterol with other
empty liposomes as defined herein, for the treatment and prevention
of bacterial infections. More particularly, the invention relates
to the use of empty liposomes consisting of sphingomyelin and of
between 35% and 65% (w/w) cholesterol, preferably between 40% and
55% (w/w) cholesterol, in particular between 45% and 55% (w/w)
cholesterol, such as around 50% (w/w) cholesterol, and of mixtures
of empty liposomes consisting of sphingomyelin and of between 35%
and 65%, or 40% and 55%, or 45% and 55%, e.g. around 50% (w/w)
cholesterol with other empty liposomes as defined herein, for the
treatment and prevention of bacterial infections.
[0053] In a particular embodiment, the invention relates to the use
of a liposome mixture of empty liposomes comprising or consisting
of cholesterol and sphingomyelin, with other empty liposomes
comprising or consisting of sphingomyelin, for the treatment and
prevention of bacterial infections.
[0054] In another embodiment, the invention relates to the use of a
liposome mixture of empty liposomes comprising or consisting of
phosphatidylcholine and sphingomyelin with other empty liposomes as
defined herein, for the treatment and prevention of bacterial
infections. In particular, the invention relates to the use of a
liposome mixture of empty liposomes comprising or consisting of
phosphatidylcholine and sphingomyelin with other empty liposomes
comprising or consisting of sphingomyelin, for the treatment and
prevention of bacterial infections.
[0055] In a particular embodiment, the invention relates to the use
of a three-component liposome mixture of empty liposomes comprising
or consisting of cholesterol and sphingomyelin with other empty
liposomes comprising or consisting of phosphatidylcholine and
sphingomyelin, and with empty liposomes consisting of
sphingomyelin, for the treatment and prevention of bacterial
infections.
[0056] In yet another particular embodiment, the invention relates
to the use of a four-component liposome mixture of empty liposomes
comprising or consisting of cholesterol and sphingomyelin with
other empty liposomes comprising or consisting of
phosphatidylcholine and sphingomyelin, with empty liposomes
consisting of sphingomyelin, and with empty liposomes comprising or
consisting of cholesterol and phosphatidylcholine, for the
treatment and prevention of bacterial infections.
[0057] The particular composition of lipid bilayers or lipid
monolayers considered is the same as indicated for liposomes,
preferably comprising cholesterol and sphingomyelin, and optionally
phosphatidylcholine.
[0058] It is understood that the empty liposomes as defined above,
the mixtures of empty liposomes as defined above, and the lipid
bilayers or lipid monolayers may be used together with further
compounds. For example, it is possible to add components to prepare
standard pharmaceutical compositions. It is also considered to add
drugs or drug-like compounds, or to add further liposomes
incorporating drugs or drug-like compounds in the liposome
interior.
[0059] Drugs considered are, in particular, antibiotics. Such
antibiotics are, for example, carbapenems, such as
imipenem/cilastatin, meropenem, ertapenem, and doripenem; 1.sup.st
generation cephalosporins, such as cefadroxil and cefalexin;
2.sup.nd generation cephalosporins, such as cefuroxime, cefaclor,
and cefprozil; 3.sup.rd generation cephalosporins, such as
ceftazidime, ceftriaxone, cefixime, cefdinir, cefditoren,
cefotaxime, cefpodoxime, and ceftibuten, 4.sup.th generation
cephalosporins, such as cefepime; 5.sup.th generation
cephalosporins, such as ceftaroline fosamil and ceftobiprole;
glycopeptides, such as vancomycin, teicoplanin, and telavancin;
macrolides, such as clarithromycin, azithromycin, dirithromycin,
erythromycin, roxithromycin, troleandomycin, telithromycin,
spectinomycin, and spiramycin; penicillins, such as amoxicillin,
flucloxacillin, oxacillin, carbenicllin, and piperacillin;
penicillin combinations, such as amoxicillin/clavulanate,
piperacillin/tazobactam, ampicillin/sulbactam, and
ticarcillin/clavulanate; quinolones, such as ciprofloxacin (e.g.
Aradigm's liposomal ciprofloxacin) and moxifloxacin; drugs against
mycobacteria, such as rifampicin (rifampin in US), clofazimine,
dapsone, capreomycin, cycloserine, ethambutol, ethionamide,
isoniazid, pyrazinamide, rifabutin, rifapentine, and streptomycin;
other antibiotics, such as metronidazole, arsphenamine,
chloramphenicol, fosfomycin, fusidic acid, linezolid, mupirocin,
platensimycin, quinupristin/dalfopristin, rifaximin, thiamphenicol,
tigecycline, and tinidazole; aminoglycosides, such as amikacin,
gentamicin, kanamycin, neomycin, netilmicin, tobramycin (e.g.
Axentis' fluidosomes.TM. tobramycin) and paromomycin; sulfonamides,
such as mafenide, sulfonamidochrysoidine, sulfacetamide,
sulfadiazine, silver sulfadiazine, sulfamethizole,
sulfamethoxazole, sulfanilamide, sulfasalazine, sulfisoxazole,
trimethoprim, and trimethoprim-sulfamethoxazole (co-trimoxazole,
TMP-SMX); tetracyclines, such as demeclocycline, doxycycline,
minocycline, oxytetracycline, and tetracycline; lincosamides, such
as clindamycin, and lincomycin; and lipopeptides, such as
daptomycin.
[0060] Further drugs considered are anti-cancer agents, for example
vincristine sulfate, vincristine, cytarabine, daunorubicin, and
doxorubicin.
[0061] Still other drugs considered are anti-inflammatory drugs,
for example corticosteroids (glucocorticoids), such as
hydrocortisone (cortisol), cortisone, prednisone, prednisolone,
methylprednisolone, dexamethasone, betamethasone, triamcinolone,
beclometasone, fludrocortisone acetate, deoxycorticosterone acetate
(DOCA), aldosterone, budesonide, desonide, and fluocinonide;
non-steroidal anti-inflammatory drugs, e.g. salicilates, such as
aspirin (acetylsalicylic acid), diflunisal, and salsalate;
propoinic acid derivatives, such as ibuprofen, dexibuprofen,
naproxen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen,
oxaprozin, and loxoprofen; acetic acid derivatives, such as
indomethacin, tolmetin, sulindac, etodolac, ketorolac, diclofenac,
and nabumetone; enolic acid (oxicam) derivatives, such as
piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, and
isoxicam; fenamic acid derivatives (fenamates), such as mefenamic
acid, meclofenamic acid, flufenamic acid, and tolfenamic acid;
selective COX-2 inhibitors (coxibs), such as Celecoxib; and others,
such as licofelone.
[0062] Further drugs considered are vasopressors and
vasoconstrictors, for example vasopressin, oxymetazoline,
phenylephrine, propylhexedrine, pseudoephedrine, epinephrine,
norepinephrine, dopamine, and antihistamines.
[0063] Also considered are other type of drugs, for example
paracetamol (pain killer), amphotericin B (against fungal
infections), bupivacaine (post-surgical pain control), vaccines
against hepatitis A, influenza, tetanus, evasive MRSA, pertussis,
diphtheria, meningococcus, cholera, typhoid, anthrax, pneumococcus
(e.g. Prevnar 13.RTM.), and other antibacterial vaccines, morphine
(pain killer), verteporfin (ophthalmological diseases), estradiol
(menopausal disturbances), aganocide.RTM. compounds, e.g.
auriclosene (NVC-422, N,N-dichloro-2,2-dimethyltaurine
(anti-bacterials), M Bio Technology's liposome particles comprising
specific lipid bacterial antigens, e.g. bacterium mimic particles
as vaccines (mycoplasma infections including pneumonia), and
bacteriophages.
[0064] Further drugs considered are antitoxins, for example tetanus
antitoxin, such as tetanus immunoglobulin, nanosponges, polymeric
nanoparticles, biomimetic polymeric nanoparticle core surrounded by
host cell membranes (such as red blood cell membranes),
toxin-targeting monoclonal antibodies and antibody fragments,
natural compounds inhibiting specific toxin productions, inhibitors
of the bacterial toxin secretion system, such as T3SS inhibitors,
toxin-binding mucin-type fusion proteins, soluble T cell receptors
acting as decoy to neutralize toxins, and peptides that inhibit the
processing of toxins.
[0065] Furthermore the invention relates to new mixtures of empty
liposomes of defined lipid composition. In particular, the
invention relates to mixtures of empty liposomes comprising
cholesterol and sphingomyelin, or phosphatidylcholine and
sphingomyelin, with other empty liposomes of defined lipid
composition, such as liposomes comprising lipids or phospholipids
selected from the group of sterols, sphingolipids and
glycerolipids, in particular selected from the group consisting of
cholesterol, sphingomyelins, ceramides, phosphatidylcholines,
phosphatidylethanolamines, phosphatidylserines, diacylglycerols,
and phosphatidic acids containing one or two saturated or
unsaturated fatty acids longer than 4 carbon atoms and up to 28
carbon atoms, for the treatment and prevention of bacterial
infections.
[0066] Particular mixtures considered are mixtures of empty
liposomes comprising or consisting of sphingomyelin and cholesterol
with other empty liposomes as defined herein, such as mixtures of
empty liposomes comprising or consisting of cholesterol and
sphingomyelin, with other empty liposomes comprising or consisting
of sphingomyelin.
[0067] Other particular mixtures considered are mixtures of empty
liposomes comprising or consisting of phosphatidylcholine and
sphingomyelin with other empty liposomes as defined herein, such as
mixtures of empty liposomes comprising or consisting of
phosphatidylcholine and sphingomyelin with other empty liposomes
comprising or consisting of sphingomyelin.
[0068] Other particular mixtures considered are three-component
liposome mixtures of empty liposomes comprising or consisting of
cholesterol and sphingomyelin with other empty liposomes comprising
or consisting of phosphatidylcholine and sphingomyelin, and with
empty liposomes consisting of sphingomyelin.
[0069] Further particular mixtures considered are four-component
liposome mixtures of empty liposomes comprising or consisting of
cholesterol and sphingomyelin with other empty liposomes comprising
or consisting of phosphatidylcholine and sphingomyelin, with empty
liposomes consisting of sphingomyelin, and with empty liposomes
comprising or consisting of cholesterol and
phosphatidylcholine.
[0070] Within the liposomes, the components may be present in
different amounts, depending on the tendency to form liposomes, the
stability of the liposomes of different composition, and the
intended use. Examples are liposomes consisting of two components
in approximately 1:1, 2:1, 3:1, 4:1, or 5:1 (weight per weight)
composition. Further components may be admixed in approximately 10,
20 or 25% (w/w) amounts.
[0071] In the preferred liposomes of the invention cholesterol is
present in an amount of 30-70%, preferably 40-60%, e.g. 45-55%, in
particular around 50% (w/w), phosphatidylcholine is present in an
amount of 10-60%, preferably 20-60%, preferably 40-60%, e.g.
45-55%, more preferably around 50% (w/w); and sphingomyelin is
present in an amount of 10-100%, preferably 20-60% or 100%,
preferably 40-60%, e.g. 45-55%, more preferably around 50% (w/w) or
100%, the cholesterol:sphingomyelin ratio is between 5:1 and 1:2,
preferably 2:1 and 1:2, in particular around 1:1 (w/w), and the
cholesterol: phosphatidylcholine or
phosphatidylcholine:sphingomyelin ratio is between 5:1 and 1:5,
preferably between 2:1 and 1:2, in particular around 1:1 (w/w).
[0072] In the liposomal mixtures individual liposome components
with different composition are mixed at proportions defined by
treatment needs. Examples are approximately 1:1, 2:1, or 3:1 (w/w)
for 2-component mixtures; approximately 1:1:1, 2:1:1, or 2:2:1
(w/w) for 3-component mixtures; and approximately 1:1:1:1, 2:1:1:1,
2:2:1:1, or 2:2:2:1 (w/w) for 4-component mixtures.
[0073] The liposomes considered consist of one or more phospholipid
bilayers. Preferred are large unilamellar vesicles (LUVs) and
multilamellar vesicles (MLVs). Most preferred are small unilamellar
vesicles (SUVs).
[0074] The liposomes are manufactured according to extrusion or
sonication or microfluidization (e.g. high pressure homogenization)
methods known in the art. For example, the lipids are mixed in an
organic solvent such as chloroform. Chloroform is evaporated and
the dry lipid film is hydrated in an aqueous solution such as
normal saline (0.9% NaCl), Krebs solution, or Tyrode's solution and
further sonicated to produce liposomes. If necessary, the size of
the liposomes can be controlled by their extrusion through membrane
filters of fixed pore diameter. Individually produced liposomes of
different lipid compositions are mixed in the required proportions
just before application.
[0075] Epithelial cells constitute the physical barrier to
pathogens. Artificial liposomes are able to protect human embryonic
kidney (HEK 293) epithelial cells from streptolysin O (SLO) induced
lysis. The SLO pores formed within the plasma membrane are large
enough to cause an efflux of cytoplasmic proteins with M.sub.r up
to 100 kDa. The direct binding of SLO to cholesterol-containing
liposomes was confirmed by pre-incubation of the liposomes with the
toxin followed by centrifugation. After centrifugation, liposomes,
recovered in the pellet, were discarded and the liposome-free
supernatants were added to the cells. The supernatants did not
inflict any damage on the exposed cells, suggesting that the toxin
was efficiently removed from the solution due to its binding to the
liposomes.
[0076] Liposomes protected not only epithelial cells but also cells
of the innate immune system against a variety of pore-forming
toxins (PFTs). The effects of PFTs on the proliferation of THP-1
human monocytic cell line were assessed in the presence or absence
of liposomes of various lipid compositions. Proliferation of THP-1
cells was completely inhibited in the presence of 200 ng of
pneumolysin (PLY), 400 ng of streptolysin O (SLO), 200 ng of
tetanolysin (TL), 1.2 .mu.g S. aureus .alpha.-hemolysin (HML), or
4.5 .mu.g Clostridium perfringens phospholipase C. As shown in FIG.
1A-D, liposomes containing cholesterol in combination (1:1 w/w)
with either phosphatidylcholine (PC), sphingomyelin (Sm) or
phosphatidylserine (PS) but not with phosphatidylethanolamine (PE)
protected THP-1 cells from cholesterol-dependent cytolysins (PLY,
SLO, TL) or phospholipase C (PLC), whereas liposomes, which
contained no cholesterol, were ineffective (FIG. 1F).
[0077] In contrast, Ch:PC liposomes (1:1 w/w) and Ch:PS liposomes
(1:1 w/w) displayed no protective effect against S. aureus
.alpha.-hemolysin, which belongs to the group of small pore forming
toxins (FIG. 1E). Liposomes, which contained no cholesterol, were
also ineffective (FIG. 1F). However, liposomes containing Ch:Sm
(1:1 w/w) were able to exert a fully protective effect against
.alpha.-hemolysin (FIG. 1E). Thus only liposomes composed of
cholesterol and sphingomyelin were able to protect THP-1 cells from
any of the tested toxins.
[0078] FIG. 2 shows that the full protective effect of Ch:Sm (1:1
w/w) liposomes against 200 ng PLY was observed at 3 .mu.g; against
400 ng of SLO at 1.5 .mu.g; against 200 ng TL at 3 .mu.g; against
1.2 .mu.g HML at 25-50 .mu.g, and against 4.5 .mu.g PLC at 100
.mu.g.
[0079] FIG. 3 demonstrates that cholesterol in concentrations equal
or above 30% (w/w) was required for Ch:Sm liposomes to protect
monocytes from PLY, TL or HML. The maximal protection was observed
at 50% (w/w) of cholesterol which corresponds to 66 mol % of
cholesterol.
[0080] Since liposomes composed of cholesterol and sphingomyelin
were able to protect cells from either cholesterol-dependent
cytolysins or from .alpha.-hemolysin, it was investigated whether
these liposomes were effective against a combination of both toxin
classes. Indeed, 25 .mu.g of Ch:Sm (1:1 w/w) liposomes exerted
fully protective effects against the combined action of
.alpha.-hemolysin (1.2 .mu.g), SLO (400 ng) and TL (200 ng),
whereas liposomes composed of Ch:PC (1:1 w/w) had no effect (FIG.
4A,B). Centrifugation experiments confirm that all three toxins
bind directly to Ch:Sm liposomes (FIG. 4C).
[0081] Ch:Sm liposomes are able to protect cultured cells from the
entire palette of toxins secreted by clinically relevant strains of
bacterial pathogens. The proliferation of THP-1 cells was assessed
in the presence of the lytic concentrations of the bacterial
culture supernatants and in the presence or absence of
liposomes.
[0082] As shown in FIG. 5A,B, Ch:Sm (1:1 w/w) liposomes protected
the cells from the effect of toxins secreted by Streptococcus
pyogenes. The full protective effect against Streptococcus pyogenes
toxin(s) was observed at microgram amounts of the liposomes (FIG.
5C). These amounts are similar to those required for neutralization
of lytic concentrations of purified cholesterol-dependent
cytolysins, but are much lower than those required for the
neutralization of either purified .alpha.-hemolysin or purified
phospholipase C (FIG. 2) suggesting that cholesterol-dependent
cytolysins are solely responsible for the cytolytic action of
Streptococcus pyogenes.
[0083] Ch:Sm liposomes also protected the cells from the effect of
toxins secreted by Streptococcus pneumonia (FIG. 6A-C). Whereas
only limited protection was achieved with cholesterol-containing
(1:1 w/w) liposomes (FIG. 6A-C), the mixture of
cholesterol-containing (400 .mu.g) and cholesterol-free, Sm-only
liposomes (400 .mu.g) was fully protective against this pathogen
(FIG. 6C).
[0084] Staphylococcus aureus is notorious for its resistance to the
most potent antibiotics. The liposomal toxin-sequestration provides
protection even against this pathogen. Ch:Sm (1:1 w/w) liposomes
showed only limited protection against toxins secreted by the
methicillin-resistant strain of Staphylococcus aureus (MRSA 2040).
Similar results were obtained for Ch:PC (1:1 w/w) liposomes: as
high as 900 .mu.g of Ch:PC liposomes was required to achieve
significant protection, whereas 600 .mu.g of these liposomes showed
only slight effect (FIG. 7A). However, the detailed analysis of
different liposomal mixtures demonstrated that addition of as
little as 75 .mu.g of sphingomyelin-only liposomes to 600 .mu.g of
Ch:PC (1:1 w/w) liposomes achieved full protection against this
pathogen (FIG. 7B). Sm liposomes alone or PC liposomes alone had no
protective effect at amounts as high as 900 .mu.g (FIG. 7B).
[0085] The liposomal treatment was also efficient against a
clinically relevant "Doppelhof" strain of Staphylococcus aureus,
isolated from a septic patient. Neither Ch:Sm (1:1 w/w) nor Ch:PC
(1:1 w/w) liposomes nor their combination with Sm-only liposomes
were effective when used at concentrations which were protective
against MRSA 2040 strain. However, a detailed analysis of the
protective action of various liposomal compositions and their
combinations demonstrated that--in contrast to MRSA 2040
strain--the cytolytic toxins secreted by the Doppelhof strain were
efficiently sequestrated by Sm-only liposomes (FIG. 8A). Whereas
1200 .mu.g of Sm liposomes alone showed significant protection
against the Doppelhof strain, at lower concentrations the mixture
containing Sm liposomes and Sm:PC liposomes was more potent than
the same amounts of either Sm liposomes or Sm:PC liposomes (FIG.
8B). The mixture of cholesterol-containing (1:1 w/w; 600 .mu.g) and
cholesterol-free, sphingomyelin-only liposomes (1,200 .mu.g) was
fully protective against toxins secreted by the Staphylococcus
aureus Doppelhof strain (FIG. 8C).
[0086] Thus, not only Staphylococcus aureus or Streptococcus
pneumonia secrete multiple cytolytic toxins but also the relative
amounts of secreted toxins varies significantly between different
strains necessitating the use of complex liposomal mixtures to
achieve high-affinity toxin binding for their full neutralization.
However, due to non-ideal selectivity of the toxins-liposomes
interactions, significant partial protection can be already
achieved with single liposomes, provided their concentration is
high enough to promote their low-affinity toxin-binding.
[0087] Detailed analysis of different liposomal mixtures
demonstrated that 1,200 .mu.g (total lipid) of a 1:1:1:1 mixture of
Ch:Sm (1:1 w/w) liposomes+Ch:PC (1:1 w/w) liposomes+Sm-only+Sm:PC
(1:1 w/w) liposomes was required for protection against both MRSA
2040 and Doppelhof strains of `Staphylococcus aureus (FIG. 9). Of
importance, owing to the presence of cholesterol-containing
liposomes, the 4-component mixture also protects against
Streptococcal toxins (see FIGS. 1-5). Thus, the four-component
liposomal mixture (1200 .mu.g of total lipid) is able to protect
cultured cells from a combined action of Streptococcal and
Staphylococcal toxins. The two-component mixture consisting of
cholesterol-containing (50% w/w cholesterol) and sphingomyelin-only
liposomes was also protective against all bacterial supernatants
tested (FIGS. 6-8), however slightly higher amount (1'800 pg of
total lipid) of this mixture was required for the full protection
against toxins secreted by the Staphylococcus aureus Doppelhof
strain (FIG. 8 C).
[0088] The bacterial species tested (Streptococcus pneumoniae,
Staphylococcus aureus and Streptococcus pyogenes) are known to
induce or contribute to the development of life-threatening
conditions such as bacteremia. The preferred three- or
four-component mixture of liposomes is able to protect laboratory
mice from experimentally induced bacteremia or pneumonia.
[0089] Mice were injected intravenously with a lethal dose of
Staphylococcus aureus Doppelhof strain, a clinical isolate from a
septic patient. At 1, 5 and 24 hours following injection of
bacteria, mice were injected intravenously either with normal
saline (control), with 1 mg/injection of Ch:Sm (1:1 w/w) liposomes,
or with 2 mg/injection of a 1:2:2 mixture of Ch:Sm (1:1 w/w)
liposomes; Sm-only and Sm:PC (1:1 w/w) liposomes. No control mice
survived beyond day 7, with 90% of deaths occurring within 36 hours
(FIG. 10A). Mice treated with cholesterol-sphingomyelin liposomes
survived 2-3 days longer than control ones but did not recover
after bacteremia. However, treatment with the 3-component liposomal
mixture resulted in complete recovery of 6 out of 8 mice.
[0090] In the pneumococcal pneumonia model, mice were infected
intranasally with the S. pneumoniae strain D39. 30 minutes
following injection of bacteria, the mice received a single
intranasal injection of 2 mg of a 1:1:1:1 mixture of Ch:Sm (1:1
w/w) liposomes+Ch:PC (1:1w/w) liposomes+Sm-only liposomes+Sm:PC
(3:1 w/w) liposomes. FIG. 10B shows that the liposomal mixture
provided protection against pneumonia.
[0091] In the pneumococcal bacteremia model, mice were injected
intravenously with a lethal dose of the S. pneumoniae strain D39.
At 8 and 12 hours following injection of bacteria, the mice
received intravenously 3 mg/injection of the following liposomes:
1) a 1:1 mixture of Ch:Sm (1:1 w/w) liposomes+Sm-only liposomes; 2)
Ch:Sm (1:1 w/w) liposomes; 3) Sm-only liposomes or 4) normal
saline. FIG. 10C shows that no control or Sm-only mice survived
beyond 32 hours. However, 6 out of 8 mice that received
Ch:Sm+Sm-only liposomal mixture and 3 out of 8 mice that received
Ch:Sm liposomes were still alive after 56 hours of bacteremia.
[0092] The doses of liposomes (50-150 mg/kg) required for the
protection of mice against Staphylococcal bacteremia are known to
be non-toxic when used as carriers for intravenous delivery of
antibiotics in rats (400 mg/kg; Bakker-Woudenberg I. A. J. M. et
al., Antimicrobial Agents and Chemotherapy, 2001, 45:1487-1492).
Moreover, the recommended doses of lipid emulsions (e.g
"Intralipid", "Lipovenos"), which are infused intravenously in
patients suffering from dysfunctions in fatty acid metabolism,
contain, in addition to 2.7 g/kg of fatty acids, approximately 300
mg/kg of egg phospholipids; i.e. the phospholipids used in the
liposomal preparations of the present invention. Thus, it is safe
to administer liposomes for the treatment of bacterial infections
in human patients, and liposomes will not elicit adverse
events.
[0093] The efficiency of liposomal toxin-sequestration can be
further improved. Since the liposomes used in this study were
mostly multilamellar liposomes and therefore at least half of their
lipid content was unavailable for toxin binding, the
toxin-sequestrating capacity of unilamellar liposomes is predicted
to be at least twice as high. Liposomes composed of selected
synthetic lipids, containing uniform acyl chains, and additional
lipid species (e.g. ceramide), known to dramatically enhance
bilayer lipid de-mixing, provide a better target for bacterial
toxins than the liposomes manufactured from natural lipids, which
were used in this study. Using PEG-derivatives of
phosphatidylethanolamine, the circulation time of liposomes and
thus their efficacy can be likewise significantly increased.
[0094] The lipid surface (bilayer) of liposomes forms spontaneously
in water-based solvents and therefore traps water and other
water-soluble inorganic and organic molecules, which might be
present during liposome production, inside the liposome. The empty
liposomes, used in the present study, are liposomes produced in
buffers containing water and simple organic or inorganic molecules
(for example NaCl, KCl, MgCl.sub.2, glucose, HEPES, and/or
CaCl.sub.2). However, complex organic molecules (antibiotics,
vitamins, adjuvants, and others) can likewise be included during
liposome production (loaded liposomes). These complex organic
molecules are not expected to interfere with the
toxin-sequestrating properties of the liposomes; however they will
provide additional therapeutic effects.
[0095] The invention further relates to a treatment of bacterial
infections comprising administering to a patient in need thereof a
therapeutically effective amount of empty liposomes of defined
lipid composition or mixtures of empty liposomes of defined lipid
composition, as described hereinbefore.
[0096] Likewise the invention relates to the prevention of
bacterial infections comprising administering to a subject exposed
to the risk of infection a preventive amount of empty liposomes of
defined lipid composition or mixtures of empty liposomes of defined
lipid composition effective for protection.
[0097] Bacterial infections considered are infections of the
respiratory tract, gastrointestinal tract, urogenital tract,
cardiovascular tract, or of the skin, as well as systemic
infections caused by bacteria that produce pore-forming toxins and
phospholipases, for example caused by Aeromonas hydrophila,
Arcanobacterium pyogene, Bacillus thurgiensis, Bacillus anthracis,
Bacillus cereus, Clostridium botulinum, Clostridium perfringens,
Clostridium septicum, Clostridium sordellii, Clostridium tetani,
Corynebacterium diphtheriae, Escherichia coli, Listeria
monocytogenes, Pseudomonas aeruginosa, Staphylococcus aureus
(including Methicillin-resistant Staphylococcus aureus (MRSA)),
Streptococcus pneumonia, Streptococcus pyogenes (also known as
Group A Streptococcus (GAS)), Streptococcus equisimilis,
Streptococcus agalactiae, Streptococcus suis, Streptococcus
intermedius or Vibrio cholera.
[0098] Further bacterial infections considered are infections of
the nasopharynx system, CNS system, meningeal membranes, vagina,
bones (for example osteomyelitis) and joints, kidney, skeletal
muscles, outer ear (for example otitis externa), and eye, for
example infectious conjunctivitis, bacterial keratitis, and
inner-eye infections.
[0099] Particular bacterial infections considered as target for a
treatment with the liposomes as described above are bacteremia,
bacterially infected skin lesions, meningitis, respiratory tract
infections, for example pneumonia, and abdominal infections, such
as peritonitis.
[0100] Dosages considered for the treatment or prevention of
infections are 1 mg to 300 g of liposomes (total lipid) per
inhalation/injection/infusion once or several times per day,
preferably 100 mg to 10 g once to three times per day. A HED (human
equivalent dose) is between 100 and 1000 mg/m.sup.2, preferably
around 300 mg/m.sup.2, or around 8 mg/kg in humans.
[0101] Liposomes can be administered as aerosol for the treatment
of respiratory tract infections. The preparation of aerosols from
liposomes such as the empty liposomes of the invention is known in
the art. For example the liquid suspension of liposomes can be
delivered with a metered-dose inhaler (MDI), i.e. a device that
delivers a specific amount of medication to the airways or lungs,
in the form of a short burst of aerosolized medicine that is
inhaled by the patient.
[0102] For the treatment of bacterial infections of the skin,
application of the liposomes of the invention is considered in the
form of topical pharmaceutical compositions, such as liquid
suspensions and the like. The preparation of a suspension from
liposomes such as the empty liposomes of the invention is known in
the art. For example, the liposome suspension prepared in normal
saline or any other aqueous solution can be applied directly to the
skin.
[0103] For the treatment of systemic bacterial infections, empty
liposomes of the invention are applied in the form of intravenous,
intramuscular or subcutaneous injections. Injection solutions are
prepared by standard methods known in the art, for example as
suspensions of the liposomes in sterile normal saline. Such
suspensions can be directly injected. It is also considered to
apply the liposomes of the invention in a formulation useful for
sublingual or buccal application. For the treatment of peritonitis,
intraperitoneal application is considered. Eye drops may be used
for bacterial infection of the eyes.
[0104] The empty liposomes of the invention will sequestrate
bacterial toxins and thus prevent bacteria from penetrating the
host's epithelia or their systemic propagation. The development of
systemic disease can thus be averted or slowed; and the pathogens
can be efficiently cleared by cells of the host's innate immune
system, which are likewise protected from toxins by the
liposomes.
[0105] The liposomes themselves are not cytotoxic, nor are they
bactericidal. Therefore, it is unlikely that they will exert
selective antibacterial pressure, which would further the emergence
of drug-resistant bacteria. The empty liposomes of the invention
mimic structures that already exist in the host's cells, in order
to bait bacterial toxins. Therefore it is inconceivable that
bacteria will adapt to the liposomal challenge: every attempt to
escape the bait by decreasing the affinity of their toxins to the
liposomes inevitably leads to the emergence of toxins which are
likewise ineffective against the host's cells.
[0106] Liposome-based chemotherapy is an appealing alternative to
both antibiotic therapy and to a treatment with toxin-sequestrating
antibodies.
[0107] The invention also relates to a treatment of bacterial
infections comprising administering to a patient in need thereof a
therapeutically effective amount of empty liposomes before, after,
together or in parallel with a standard antibiotic treatment of the
bacterial infection. In combination with antibiotic treatment,
apart from neutralization of actively secreted bacterial toxins
during the active phase of an infection, the liposomal treatment
will provide additional benefits for a patient by sequestrating the
toxins which are released during antibiotic treatment by lysed
bacteria, a condition which is known to be detrimental, for
example, during meningitis, in Streptococcus pneumonia infection
(acute pneumolysin release) and Streptococcus pyogenes infection
(acute release of streptolysin O).
[0108] In this combination treatment the empty liposomes and
liposome mixtures may be considered as adjuvants, and the
corresponding method of treatment as adjunct treatment.
[0109] A limitation for liposomal therapy is its restricted
efficiency in immunocompromised individuals since the clearance of
bacteria lies not with the chemotherapeutic agent but with the
host's own immune system. However, even in immunodeficient
patients, liposomal treatment in combination with bactericidal
chemotherapy will be beneficial: slowing down the development of
systemic disease, and thus will provide the organism with the much
needed time to allow antibiotics to unfold their full bactericidal
potential.
[0110] Antibiotic treatment considered together with the treatment
using empty liposomes according to the invention is, for example,
treatment with cephalosporins and other .beta.-lactam antibiotics,
glycopeptides, lincosamides, lipopeptides, macrolides, penicillins
and penicillin combinations, quinolones, sulfonamides,
chloramphenicol and chloramphenicol analogues, tetracyclines,
clindamycin, and folate inhibitors, as listed above. Particular
antibiotics considered in a treatment together with empty liposomes
of the invention are carbapenems such as imipenem, cilastin and
meropenem, 2.sup.nd generation cephalosporins such as cefuroxime,
3.sup.rd generation cephalosporins such as ceftazidime and
ceftriaxone, 4.sup.th generation cephalosporins such as cefepime,
glycopeptides such as vancomycin, macrolides such as
clarithromycin, penicillins such as amoxicillin and flucloxacyllin,
penicillin combinations such as amoxicillin/clavulanate and
piperacillin/tazobactam combinations, and quinolones such as
ciprofloxacin and moxifloxacin, and fluoro-quinolones such as
levofloxacin and gemifloxacin.
[0111] All components of the empty liposomes of the invention are
substances, which occur naturally in humans. These liposomes are
therefore well tolerated and cleared from the body via
physiological pathways. Liposome aerosols should be used for the
prevention of pneumonia and other diseases of the respiratory tract
by the general population during seasonal influenza epidemics. Most
importantly, prophylactic measures based on liposome aerosols or
other liposome applications will be helpful in the prophylaxis of
MRSA pneumonia or bacteremia in hospitals, Pseudomonas aeruginosa,
S. aureus or S. pneumonia infections, and in other settings, which
favor the spread of infectious diseases.
EXAMPLES
Toxins
[0112] Streptolysin O (SLO) from Streptococcus pyogenes,
.alpha.-hemolysin from Staphylococcus aureus, tetanolysin (TL) from
Clostridium tetani and phospholipase C from Clostridium perfringens
were purchased from Sigma. Pneumolysin was obtained from Prof.
Kadioglu (Cruse G. et al., J. Immunol. 2012; 184:7108-7115). Other
toxins include Panton-Valentine leukocidin (PVL) from S. aureus,
listeriolysin O (LLO) from Listeria monocytogenes, perfringolysin O
(PFO) from Clostridium perfringens, suilysin (SLY) from S. suis,
intermedilysin (ILY) from S. intermedius, cereolysin O (CLO) from
B. cereus, thuringiolysin O (TLO) from B. thuringiensis,
botulinolysin (BLY) from C. botulinum, sordellilysin (SDL) from C.
sordelli, pyolysin (PLO) from Arcanobacterium pyogenes. Culture
supernatants from Streptococcus pneumoniae, Streptococcus pyogenes
and Staphylococcus aureus were obtained from Profs. K. Muhlemann
(Bern) and E. Gulbins (Essen).
Cell Culture
[0113] The human embryonic kidney cell line (HEK 293) was
maintained as described by Monastyrskaya K et al., Cell Calcium.
2007, 41:207-219. The human acute monocytic leukemia cell line
(THP-1) was maintained in RPMI 1640 medium containing 10% FBS, 2 mM
L-glutamine and 100 U/ml penicillin, 100 .mu.g/ml streptomycin.
Transfections
[0114] CFP (cyan-fluorescent protein) was transiently expressed in
HEK 293 cells (Monastyrskaya et al., loc. cit.). CFP-expressing HEK
293 cells were used for laser scanning module (LSM) imaging
experiments 2 days after transfection.
Liposomes
[0115] Cholesterol (Ch) (C-8667), Sphingomyelin (Sm) from chicken
egg yolk (S0756), phosphatidylcholine (PC) from soybean (P7443),
phosphatidylethanolamine (PE) from bovine brain (P9137) and
phospatidylserine (PS) sodium salt from bovine brain (P5660) were
purchased from Sigma. The lipids were individually dissolved in
chloroform at 1 mg/ml concentrations and stored at -20.degree. C.
For the preparation of liposomes the chloroform solutions of
individual lipids were mixed in the composition and the
proportions, which are given in the text, to produce routinely
50-500 .mu.l of the final solution. Chloroform was completely
evaporated for 20-50 min at 60.degree. C. 50 .mu.l or 100 .mu.l of
Tyrode's buffer (140 mM NaCl, 5 mM KCl, 1 mM MgCl.sub.2, 10 mM
glucose, 10 mM HEPES; pH=7.4) containing 2.5 mM CaCl.sub.2 was
added to the tubes containing films of dried lipids and vigorously
vortexed. The lipid suspensions were incubated for 20-30 min at
45.degree. C. in an Eppendorf thermomixer with vigorous shaking. To
produce liposomes, the final lipid suspensions were sonicated
3.times.5 sec at 6.degree. C. in a Bandelin Sonopuls sonicator at
70% power. The liposomal preparations were left for at least 1 hour
at 6.degree. C. before they were used in experiments. The
concentration of individual lipids in the liposomes is always given
as the weight per weight (w/w) ratio. In liposomes containing
cholesterol and sphingomyelin, the 1:1 (w/w) ratio corresponds to
50% (w/w) or to 66 mol % cholesterol. The amounts of liposomes are
given as the amount of total lipids used for their preparation.
[0116] In an alternative method, about 25 ml of each formulation
was made by the ethanol hydration and extrusion method. The final
formulations were sterile filtered and filled in autoclave serum
glass vials (final concentration: 40 mg/ml). The liposome particle
sizes are in the range of 80-150 nm with good PDI (polydispersity
index). The results for the osmolality measurement are also
included. They are all in the range of around 400 mmol/kg which is
pretty close to the desired physiological level.
TABLE-US-00001 TABLE 1 Specifications Mean Half- Poly- Zeta
diameter width dispersity potential SD Osmolality Liposomes (nm)
(nm) index (mV) (mV) (mmol/kg) pH Ch:Sm 130 46 0.13 -1.40 0.6 371
7.02 Sm 81 31 0.15 -3.35 0.5 346 7.02 Ch:Sm:PEG2% 116 37 0.11 -9.83
0.7 401 7.03 Ch:Sm:PEG5% 122 43 0.12 -15.70 0.5 392 7.04 Sm:PEG2%
96 42 0.19 -9.5 0.5 398 7.02 Sm:PEG5% 111 45 0.17 -15.10 0.5 387
7.02
Toxin-Induced Cell Lysis and Protective Effects of Liposomes
[0117] In human embryonic kidney epithelial cells (HEK 293),
toxin-induced lysis was monitored as a decline of cytoplasmic
fluorescence due to a pore-induced efflux of intracellular CFP.
Confluent HEK 293 cells seeded on 15 mm glass coverslips
(2.5.times.10.sup.5 cells per coverslip) were mounted in a
perfusion chamber at 25.degree. C. in Tyrode's buffer containing
2.5 mM CaCl.sub.2 and their fluorescence was recorded in an
Axiovert 200 M microscope with a laser scanning module LSM 510 META
(Zeiss, Germany) using a .times.63 oil immersion lens
(Monastyrskaya et al., loc. cit.). At time-point=0, the buffer was
replaced by 100 .mu.l or 200 .mu.l of the same buffer containing
additionally a cytolytic quantity of a given toxin (e.g. 120 ng of
SLO from Streptococcus pyogenes) and 20 mM/L dithiotreitol (DTT).
To investigate the protective effect of liposomes on toxin-induced
cell lysis, at time-point=0, cells were routinely challenged with
100 .mu.l of a mixture containing toxin/DTT and liposomes of
various concentrations and of various lipid composition. The
toxin-liposome mixture was prepared immediately before addition to
the cells (with 20 to 30 sec of handling delay). In some cases, 100
.mu.l of solution containing liposomes alone was added first to the
cells followed (with 20 to 30 sec of handling delay) by 100 .mu.l
of toxin-containing solution. The protective effects of the
liposomes were similar under either experimental condition. The
images were analyzed using the "Physiology evaluation" software
package (Zeiss, Germany).
[0118] The effects of purified PFTs or bacterial culture
supernatants on the proliferation of a human monocyte cell line
(THP-1) were assessed in the presence or absence of liposomes of
various lipid compositions. Routinely, 100-600 .mu.l of
toxin-containing solution (Ca.sup.2+-Tyrode's buffer or BHI broth)
was added to 100 .mu.l (5.times.10.sup.4 cells) of cells maintained
in culture medium and pre-mixed with 50-150 .mu.l of liposomes of
various lipid-composition. After incubation for 3 hours, 1-2 ml of
fresh culture medium was added to the tubes. The cells were counted
each day or every second day for 8-12 days. The toxins and the
liposomes were present for the whole duration of an experiment. The
data presented in the diagrams correspond to day 5 or day 6, when
the cell growth was still in the linear phase.
Comparison of "Empty" and "Filled" Liposomes
[0119] The protection against culture supernatants of S. aureus or
S. pneumoniae by the mixture of "empty" Ch:Sm+Sm-only liposomes is
compared with that of mixture of Ch:Sm+Sm-only liposomes filled
with a fluorescent dye such as Fluorescein, Oregon Green 488,
Rhodamine, or Texas Red.
[0120] The protection against culture supernatants of S. aureus or
S. pneumoniae by the mixture of "empty" Ch:PC liposomes is compared
with that of mixture of Ch:PC liposomes filled with fluorescent dye
such as Fluorescein, Oregon Green 488, Rhodamine, or Texas Red.
Protection by Lipid-Coated Surfaces
[0121] Beads coated by cholesterol and sphingomyelin are tested for
their toxin-sequestrating activity against culture supernatants of
S. aureus or S. pneumoniae.
In Vivo Effect in Combination with Antibiotic Treatment on
Bacteremia Induced by S. aureus or S. pneumoniae.
[0122] The 2-component mixture of Ch:Sm and Sm-only liposomes;
3-component mixture of Ch:Sm; Sm-only and Sm:PC (1:2:2) liposomes
and of a 4-component mixture of Ch:Sm, Sm-only, Sm:PC and Ch:PC
(1:1:1:1) are tested in a mouse models of bacteremia induced by
either a penicillin-susceptible strain Streptococcus pneumonia or
by Methicillin-resistant Staphylococcus aureus (MSSA). Two types of
MSSA strains are considered, characterized by their ability to
secrete or not the toxin Panton-Valentine leukocidin (PVL). In
addition, 2-component mixture of Ch:Sm and Sm-only pegylated
liposomes (2% PEG or 5% PEG) are also tested.
[0123] Laboratory mice are inoculated by intraperitoneal (i.p.),
intravenous (i.v.) or intranasal (i.n.) injection of approximately
10.sup.7 or 10.sup.8 cfu/ml of bacteria.
[0124] For each bacteria strain, each infection route, and for each
liposome mixture (LP mixture), intravenous injections of two
different doses (2 mg/kg or 6 mg/kg) of the LP mixture is started
either six hours (t=6), twelve hours (t=12), eighteen hours (t=18),
or twenty four hours (t=24) after the bacterial challenge (in each
case, the injection of LP mixture is followed by either one
additional injection 12 hours, or two additional injections 4 hours
and 24 hours after the initial injection), with or without
penicillin treatment (30 mg/kg). Antibiotic treatment is initiated
at the same time of liposome treatment. Two types of controls were
performed: infection without treatment and infection treated with
antibiotic alone (at t=6, t=12, t=18, or t=24).
[0125] For each bacteria strain and for each liposome mixture, and
for each dose of liposome and each route of infection, there were
10 groups of animals.
TABLE-US-00002 Group 1 No treatment (control) 2 Penicillin alone 3
LP mixture at t = 6 4 LP mixture at t = 6 + penicillin 5 LP mixture
at t = 12 6 LP mixture at t = 12 + penicillin 7 LP mixture at t =
18 8 LP mixture at t = 18 + penicillin 9 LP mixture at t = 24 10 LP
mixture at t = 24 + penicillin
[0126] In group 1, the survival of 50% of the group were followed
for at least 8 days, 25% of the group were euthanized one hour
after the bacterial challenge and the remaining 25% 6 h after the
bacterial challenge.
[0127] Bacterial counts were determined in blood and several organs
such as lung, spleen, and kidney.
[0128] Read out: survival, signs of infections, metabolism (serial
measurements of weight loss and recovery, O.sub.2 consumption and
CO.sub.2 production rates measured by indirect calorimetry, resting
energy expenditure (REE) calculated with the modified Weir
formula); inflammation cytokines profile (ELISA was performed on
serum for tumor necrosis factor (TNF)-alpha, macrophage
inflammatory protein (MIP)-2, and IL-1b).
Minimal Bactericidal Concentration (MBC)
[0129] No activity of sphingomyelin/cholesterol liposomes against
usual strains:
TABLE-US-00003 Strain MBC (mg/mL) ATCC 27853 P. aeruginosa >16
S. aureus >16 762 >16
[0130] The testing of the minimum bactericidal concentration (MBC)
was carried out following the guidelines proposed by the Clinical
and Laboratory Standards Institute (CLSI).
[0131] For the broth microliter dilution tests, 96-well plates
supplemented with 50 .mu.L of 0.5-16 mg/mL liposomes were
inoculated with 50 .mu.L of Mueller Hinton broth containing a
bacterial cell suspension of 1-5.times.10.sup.5 colony-forming
units (CFU) per mL of S. aureus. The plates were incubated for 24 h
at 36.degree. C. MBC was determined by transferring 10 .mu.L
aliquots from the wells broth microtitre dilution plates onto
Columbia blood agar (Oxoid, Wesel, Germany). The inoculated plates
were further incubated for 24 h at 36.degree. C. and then colonies
were counted.
* * * * *