Note: Descriptions are shown in the official language in which they were submitted.
2171369
The Use Of Virulence Factors Of Pathogens To Improve
Liposomal Delivery Of Antibiotics And/Or Vaccines
Background of the Invention
Brucellosis is a zoonotic disease that afflicts, depending on the region,
about 5% of
the livestock around the world. Although cattle, swine, sheep, goats and dogs
are the usual
hosts (with B. abortus, B. suis, B. ovis, B. melitensis and B. canis being the
usual agents,
respectively), the impact of brucellosis may be far greater as it can also
infect other animals
such as poultry and marine mammals. The manifestation of these bacteria in
animals are
usually reproductive complications (aborted fetuses, inflammed uterus or
orchitis, sterility).
Brucella is a bacterium that can become a facultative parasite, invading cells
of the blood,
bone marrow, organs and skeletal tissue. It is difficult to eliminate and
relapses of infections
may occur once antibiotic treatment ceases. Vaccination in animals have proven
partially
effective in offering protection, though these vaccines are pathogenic for
other animals and
humans.
Because it is a highly infective organism that causes debilitating symptoms,
Brucella
can persist in the environment for months under the right conditions, and as
there are no
effective vaccines or therapeutic recourses, it is potentially a bacterial
warfare agent. There
is, therefore, an urgent need to develop a means for protecting or treating
people at risk.
Although antibiotics are effective in inhibiting or killing pathogens, they
are less
effective against pathogens that infect and then become intracellular
parasites within animal
or human hosts. Rather than being destroyed by white blood cells, the Brucella
species, for
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example, thrive within these cells. Antibiotics are available that will
inactivate Brucella
species, but these are effective only in the test tube. In vivo, the bacterium
will invade cells
of the reticulo-endothelial system and become a facultative parasite,
rendering it protected and
difficult to treat. Antibiotics are limited in their effectiveness due to the
following reasons:
- only a small portion of the antibiotic may reach the infected cell due to
its
dilution throughout the body;
- some antibiotics may not be able to cross the mammalian cell membrane
barrier;
- the antibiotic may be excreted through the urine; and,
- some antibiotics may become inactivated by serum or cellular enzymes.
Current research in liposome encapsulation of antibiotics has brought in a new
era in
the therapy of disease. Liposomes are microscopic pockets of lipids that can
be used to
entrap antibiotics and to deliver these into phagocytic cells. The advantages
of such a process
are:
- liposomes contain the antibiotic and prevent its dilution within the body or
secretion in the urine;
- these lipid vesicles are also phagocytized and will be delivered to the site
where the pathogen has sequestered; and,
- the liposomes are made of bio-degradable lipids and are non-toxic. Indeed,
these may shield the body from the harmful side-effects of toxic antibiotics.
The use of liposomes as an antibiotic delivery system is described in the
inventors' co-
pending Canadian application no. 2,101,241 (published January 24, 1995)
wherein liposome
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encapsulated ciprofloxacin was found to be more effective in the prevention
and treatment of
Francisella tularensis infection than the non-encapsulated antibiotic.
Further, the use of multiple doses of negatively charged liposomes as carriers
of
gentamicin into cells have been reported but these were only partially
effective in vivo (Dees,
C. et al., 1985, Vet. Immunol. Immunopathol., 8, 171-182), possibly because
liposomes require
phagocytosis for delivery and Brucella can invade even non-phagocytic cells
(Detilleux, P.G.
et al., 1990, Infect. Immun., 58, 2320-2328). Non-phagocytic cells are
unlikely to engulf
liposomal antibiotics and so will protect their intra-cellular parasites from
these therapeutic
agents. Other antibiotics within liposomes have proven effective against some
strains of
Brucella (eg. B. canis and B. abortus) but less so against another strain (eg.
B. melitensis)
(Hemandez-Caselles, T. et al., 1989, Am. J. Vet. Res., 50, 1486-1488). The
treatment of the
latter strain with antibiotics requires liposomes of a positive rather than
negative charge,
requires multiple treatments to be effective and although the organism may
appear eliminated
in mice 5 days after treatment, relapses are a possibility.
Gregoriadis, in Canadian application no. 2,109,952 (published December 23,
1992),
describes the use of polysaccharide coated liposomes as drug delivery agents.
It is described
that such polysaccharide coating is used to increase the residence time of
liposomes in vivo
thereby prolonging the availability of the drug. However, this reference does
not address the
issue of such liposomes entering non-phagocytic cells. The use of
lipopolysaccharide (LPS)
with liposomes has been described by Djikstra et al. (1988, J. Immunol. Meth.,
114, 197-205)
but the LPS was typically water-soluble and housed within the liposome rather
than part of
the liposome's composition.
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Thus, antibiotic therapy of some diseases is very limited due to the
protection offered
when the facultative parasites are intracellular. Liposome encapsulation of
these antibiotics
enhances their effectiveness, but the indication is that there is a need for
"designer" liposomes,
or specific formulations of liposomes for different diseases.
Summary of the Invention
Accordingly, it is an object of the present invention to provide a new
liposome or
microsphere formulation which includes the virulence factors of infectious
agents.
Liposomes according to the present invention have enhanced effectiveness as
delivery systems
for antibiotics in the treatment of disease.
Thus, the invention provides for the use of virulence factors in the
formulation (in
combination with or in the replacement of lipids) of microspheres and
specifically in
liposomes.
Further, the invention provides for a pharmaceutical formulation for
preventing or
treating infections wherein antibiotics are encapsulated by liposomes
comprised in part by
virulence factors such as bacterial components.
The invention also provides a method of manufacturing liposomes formulated
with
virulence factors.
Detailed Description of the Preferred Embodiment
Since some bacteria have mechanisms for invading host cells (Kuhn, M. and W.
Goebel, 1989, Infect. Immun., 57, 55-61) it was speculated that these
virulence factors could
be used to improve liposomes for the delivery of antibiotics into mammalian
cells. As several
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pathogenic bacteria have the same rare sugar on their cell surfaces as
Brucella
(Cherwonogrodzky, J.W. et al., 1989, in Animal Brucellosis, K. Nielson and
J.R. Duncan
(ed.), 19-81), it is likely that their smooth-lipopolysaccharides (S-LPS) have
something to do
with their invasiveness into cells.
Virulence factors include enzymes (e.g proteases of the flesh-eating
bacterium), toxins
(e.g. diphtheria toxin), binding components (e.g. Protein A of Staphlococcus
aureus) and
invasive factors (e.g. the protein "invasin" from Listeria monocytogenous, the
lipopolysaccharide of Brucella, and the glycoproteins of some viruses).
Further, the invention will be described with specific reference to liposomes.
However, the invention is applicable to any microsphere. By microsphere it is
meant any
microscopic vesicle capable to carrying antibiotics, drugs etc. Some examples
are nylon
beads, polymers of amino acids or carbohydrates, globules of detergents and
"bubbles" of
lipids or liposomes.
Thus, it will be shown that virulence factors (such as bacterial components)
can be
included in the composition of liposomes to improve their effectiveness as
delivery systems
of therapeutic agents against specific diseases. Proof of this concept is
given in the following
study that uses the bacterial component lipopolysaccharide with exceptional
properties
(hydrophobic rather than hydrophilic, associated with invasive properties, low
toxicity).
Material and Methods
i) Bacterium: Brucella melitensis 16M was acquired from Agriculture Canada,
Animal Diseases Research Institute, Nepean, Ontario. It was maintained on
Brucella agar
with 1.4 ppm crystal violet, 370C, 5% COz. The strain used was passed once
through a
mouse and isolated from its spleen. Prior to use, a single colony of bacteria
was sub-cultured
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CA 02171369 2007-08-07
.,.~. .
on tiypticase soy agar witliout crystal violet, incubated for three days, and
then this fresh
growth, suspended in sterile ]% saline, was used to infect mice. previous
studies showed that
a suspension having an O.D.. of 0,1 bad 1 x 10 colony forming units (CFU) /
mi. Dilutions
of 2.5 x 10' CFU/ml were made and 0.2 ml of this was used to infect each
mouse.
ii) S-LFS; Bnrcella melitensis 16 M S-LFS was purified by the rnethod of
Cherwonogrodzky et al. (Antigens of Brucella. In Anirnal brucellosis, CRC
Press, Boca
Raton, pp. 19-81, [1990]). Briefly, a culture was grown in Bruoalls broth,
killed with 2%
phenol, then following centrifugation, the supetnatant was saved and crudc S-
LPS was
prccipitated with 5 volumes of inethanol with 1% sodium acetate (w/v). The
precipitate was
collected by centrifugation, dialysed against TRIS buffer (pH 7), then
digested with
lysozyme, RNAse, DNAse and proteinase K. A phenol-water extraction was done,
the
phenol layer was removed and the S-LPS was precipitated and washed with
=thanol-acetate.
The preparation was dialysed, ultraoentrifuged overnight at 120,000 x g and
the pellet was re-
suspended in distilled water, then lyophilizcd,
iii) Preparation of liposomes with S-LPS: The basis liposome was prepared by
dissolving in 2:1 chloroform:ntethanol the lipids
phosphatidylcholine; cholesterol:phosphatidylserino(AvAntiLipidsInc.,
Birmingham,Alabama.)
in a molar ratio of 7:2:1 (a total of 20 moles were used, respective
molecular weights are
810:387:745, amounts used were 11.34 mg:1.55 mg-1,49 mg a1a in 1 ml). The
lipid solution
was dried to a thin film on the bvttam of a large screw-capped tube by heating
at 45oC in
a heating block. Throughout this procedure, the content of the tube was purged
with a gentle
stream of dry nitrogen. The lipids were then further dried for 30 min. in a
vacuum chamber
to remove any remaining organic solvent.
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The S-LPS (10 mg in 1 ml phosphate buffered saline, pH 7) was sonicated in a
bath-
type sonicator for approximately 3 min. or until the S-LPS solution became
homogenous.
Part of the dispersed S-LPS solution (40 l) was added to the tube containing
the dried lipid
mixture. Ciprofloxacin (Miles Canada Inc., Etobicoke, Ontario) or tetracycline
(Sigma
Chemical Co., St. Louis, MO) was made at 20 mg/ml distilled water and of this
2 ml was
added to the tube. The contents of the tube were mixed vigorously by vortexing
and heating
at 45 oC. The vortexing-heating cycle was repeated about 15-25 times, under
dry nitrogen,
until the dried lipids were completely dislodged from the sides of the tube
(about 20 min. is
required for this procedure). The lipid-antibiotic-LPS mixture was gently
sonicated as before
for approximately 2 min. and was then rapidly frozen in a dry ice-methanol
mixture. The
sample was then freeze-dried overnight in a lyophilizer (Virtis Company Inc.,
Gardiner, NY).
The freeze dried mixture was reconstituted in 0.5 ml of distilled water. The
reconstituted liposomes were then vortexed for 2 to 3 minutes under nitrogen
and were left
undisturbed at room temperature for 1 hour. The liposomes were washed with 8
ml of
distilled water then ultra-centrifuged at 125,000 x g for 30 min at 4 OC. The
pellet was
re-suspended in distilled water, ultra-centrifuged as before, and then
reconstituted in distilled
water. Encapsulation was about 50% efficient and so 4 ml of water gave a
suspension of 5
mg/ml (1 mg/0.2 ml was used to treat each mouse). The preparation was used
immediately.
iv) Animal Studies: 5 week old BALB/c mice (15 - 16g) were obtained from
Charles River Canada Inc. (St. Constant, Quebec) and were left in quarantine
at least a week
before use. Intravenous injections were done at the tail vein, intramuscular
injections were
at a thigh muscle. Throughout the study, the mice were housed in HorsfalTM
units and cared
for under the Canadian Council on Animal Care guidelines. At the end of the
study, the
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CA 02171369 2007-08-07
animals were sacrificed by cervicat dislocation, their spleens were
aseptir,ally removed,
crushed in 2 mt sterile saline with a manual tissue grinder (Wheaton,
Mi1lville, N.3.), then
diluted in saline and plated onto Brueella agar with crystal violet. The
plates were lacubuteci
as before and inspected 3 and 7 days later.
gesults gnd Dilcusslnn
Upon testing negatively-abarged liposomes in the delivery of ciprofloxacin
(LIP-Cipro,
or liposome encapsulated ciprofloxaain) for the prophylaxis and treatment of
mice given 10
LDja of Francisella tulamnsis LVS, the animals were rescued from certain
death, even when
only a single dose of LIP-Cipro was given (as described in Canadian patent
number
2,101,a41).
Upon testing the sama formulation against B. selitensia 16M, it appeared to
be neither
protective nor therapeutic against this disease as illustrated in Tables 1 and
2 respectively.
This supports the study by Dees et al. (1985) who showed that even multiple
doses of .._ ,. .
antibiotic within liposomes cannot eliminate brucellosis in vivo. Thie
limftation is
understandable in that Brncella has been found to have the ability to invade
even non- .
pba$oc.ytic celfs (Hernandez-Caselles et al., 1989). It theretbre can invade,
sequestor and
grow within tissues that liposomes of the usval formulation caanot reach.
The mechanism by which Brucella speaies can penetrate the noted cells is
unknown.
However, it has been obaerved that several invasive pathogens (e.g. Yibrio
eholerae, Yersinia
enterocolitica 0:9, togigenic Escherichia coli 0:15yH.=7, Salmonella landau,
Pseudomonas
maltophilia 555) have derivatives of a rare sugar (4-amino-4,6-alpha-D-
mannopyrannose
(Cberwonogrodzky et al., Antigens of Brucella. In Animal brucellosis, CRC
Press, Boca
Raton, pp. 19-81, [1990]), also fotmd on Brucella, on their 0-polysaccharide
which forms
part of the smooth-lipopolysaccharide (S-LPS) that coats these bactcria. On
the chance
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that liposomes, with this antigen as part of their composition, would gain an
advantage in
being delivered to similar sites as viable Brucella, we formulated a novel
liposome that had
B. melitensis S-LPS as part of its composition. It should be noted that this S-
LPS differs
from the S-LPS of several other bacteria in that it is hydrophobic and is
readliy incorporated
with the other lipids in liposome formulation. Table 3, for the protection
against diseases, and
Table 4, for the treatment of infected mice, show that multiple doses of
liposomes having S-
LPS in their composition and used to encapsulated antibiotics, such as
ciprofloxacin or
tetracycline, were effective in greatly reducing the number of bacteria. Table
3 shows that
this result is temporary, possibly due to other sites in the animal providing
a source of
infection. There is also some protection given by S-LPS given with
tetracycline, in the
absence of liposomal formulation. This latter observation may be due to the
ability of S-LPS
to spontaneously form structures that may entrap or associate with
tetracycline. The evidence
suggests that virulence factors, in this case Brucella S-LPS, may replace part
or all of the
lipids used in liposome formulation.
Although the embodiment described herein relates to the use of S-LPS in the
formulation of liposomes, the same results may also be obtained by using other
virulence
factors (i.e. bacterial components such as rough-lipopolysaccharide, outer-
polysaccharide,
lipids, or proteins) or bacterial components linked to carriers (i.e. O-
polysaccharide linked to
proteins such as bovine serum albumin) or modified bacterial components (i.e.
alkaline treated
S-LPS, detoxified LPS, cloned protein fragments). Further, the virulence
factors can be used
with, or replace part or all of the lipids used in the formulation of
liposomes.
It is believed that the present invention may have several applications:
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T 2171369
1) For diseases (bacteria, rickettsiae, viruses, fungi, parasites) which are
difficult to
treat, "designer" liposomes may be formulated by extracting components from
these pathogens
and incorporating these in the formulation of delivery systems. The components
could be
used intact, fragmented, or coupled to carriers before being used. The
components do not
have to be characterized, and if cross-reactive, may be used in the treatment
or protection
against more than one pathogen. Thus, by incorporating whole, modified, or
fragments of
cellular components of the pathogen into the liposome formulation, one may
improve the
effectiveness of this delivery system.
2) Potentially, a liposome or microsphere formulated with this technology
could be
multi-functional (ie. the invasive factor in the liposome composition may
assist delivery
within cells as well as serve as a vaccine, this novel liposomal formulation
may be used to
entrap antibiotics, immuno-modulators or drugs). In the embodiment described
herein, the S-
LPS component is used to enhance either the stability or delivery of the
liposome to sites of
infection. The S-LPS is also a strong antigen. One could therefore have a
liposome or
microsphere that serves as a more effective delivery system, provides antigen
as a vaccine,
encapsulates antibiotics for treatment and may have some immuno-modulation
effect. For
example, this type of multi-functional liposome or microsphere has an invasive
factor and/or
a vaccinating agent incorporated within its structure and is used to deliver
antibiotics, drugs,
antibodies and/or immuno-modulators. The use of this new formulation may
greatly enhance
prophylaxis against disease or its treatment.
3) Further, such multi-functional liposomes may have significant impact on
difficult
diseases. In the example of AIDS research, inactivated HIV coupled to B.
abortus gives 6-
fold better immunization than inactivated HIV alone (Golding, G. et al., 1991,
AIDS Res.
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Hum. Retrovir., 7, 435-446). Potentially, a liposome, with B. abortus or B.
melitensis LPS
as part of its formulation, that encapsulates inactivated HIV, anti-viral
agents, antibodies from
HIV-positive/AIDS-negative patients, immuno-modulators or a combination
thereof may be
even more effective. Also, one may have the HIV antigen as part of the
liposome formulation
that encapsulates anti-viral antibiotics such as AZT. It should be noted that
the Brucella LPS
is about 1000-fold less toxic than other bacterial S-LPS (Goldstein et al.,
1992, Infect.
Immun., 60, 1385-1389) and would be ideal for this formulation.
Although the invention has been described with reference to certain specific
embodiments, various modifications thereof will be apparent to those skilled
in the art without
departing from the spirit and scope of the invention as outlined in the
appended claims.
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Table 1
Protection studies of BALB/c mice given single doses of antibiotics at
different times before
infection with Brucella melitensis 16M'
Antibiotic Time Before Infection B. melitensis 16M counts
(days) in spleens 7 days after
infection
Control 1.0 0.3 x 10'
Ciprofloxacin 7 1.9 0.4 x 104
3 1.4 f 0.2 x 104
2 2.8f1.0x104
1 1.5 f 0.3 x 104
Liposome Encapsulated 7 3.4 1.0 x 10'
Ciprofloxacin (LIP-Cipro) 3 2.3 0.5 x 104
2 3.9f1.4x104
1 2.9f1.1x104
A total of 1 mg ciprofloxacin in 0.2 ml saline/mouse (3 mice/set) was given
intramuscularly
at the times noted. The mice were then infected with 5 x 104 colony forming
units (CFU) of
B. melitensis 16M.
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Table 2
Treatment studies of BALB/c mice given single doses of antibiotics at
different times after
infection with Brucella melitensis 16M'
Antibiotic Time After Infection B. melitensis 16M counts
(days) in spleens 7 days after
treatment
Control 1.0 0.3 x 104
Ciprofloxacin 1 1.6 0.3 x 104
2 3.0 f 0.6 x 104
3 3.5f0.9x104
7 3.9f1.6x104
Liposome Encapsulated 1 2.8 f 0.9 x 104
Ciprofloxacin (LIP-Cipro) 2 2.0 0.7 x 104
3 2.4f0.8x104
7 2.6 f 0.7 x 104
Same procedure as in Table 1.
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Table 3
Protection studies of BALB/c mice given multiple doses of antibiotics at
different times
before infection with Brucella melitensis 16M'
11 B. melitensis 16M counts in spleens
Antibiotic2 Time Before 3 days after 11 days after
Infection (Days) infection infection
Control: 3.6 0.9 x 104 1.0 0.4 x 105
no treatment
Control: 7,3,2,1 1.2 f 0.4 x 103 2.1 0.7 x 104
LIP-LPS 3,2,1 2.3 0.7 x 104 4.3 0.3 x 104
2,1 8.0f2.0x103 1.1f0.8x105
1 5.4f0.8x104 1.6f0.6x105
Control: 7,3,2,1 4.3 0.3 x 102 3.6 f 1.8 x 103
TETRA-LPS 3,2,1 7.0 3.8 x 103 1.2 f 1.0 x 104
2,1 1.8f0.4x104 1.7f0.5x104
1 7.9f2.5x104 3.5f0.3x104
LIP-CIPRO-LPS 7,3,2,1 0 1.2 f 1.2 x 103
3,2,1 0 1.0 f 0.8 x 103
2,1 1.0 1.0 x 10' 3.3 f 0.9 x 103
1 1.8t0.8x103 1.0f0.4x103
LIP-TETRA-LPS 7,3,2,1 7.5 0.9 x 102 1.8 f 1.8 x 103
3,2,1 1.9f0.2x103 9.0t1.2x103
2,1 3.1f1.1x103 1.9f0.1x104
1 3.0t0.6x103 2.8f1.6x104
Same as for Table 1, except 2 mice were used per set. Protection is defined as
causing a
log,o less CFU as controls.
2 Antibiotic abbreviations are:
a) LIP-LPS for liposomes made with B. melitensis smooth-lipopolysaccharide (S-
LPS)
and no antibiotic;
b) TETRA-LPS is non-encapsulated tetracycline and S-LPS;
c) LIP-CIPRO-LPS is ciprofloxacin encapsulated in liposomes made with S-LPS;
and,
d) LIP-TETRA-LPS is tetracycline encapsulated in liposomes with S-LPS.
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Table 4
Treatment studies of BALB/c mice given multiple doses of antibiotics at
different times after
infection with Brucella melitensis 16M'
Spleen counts after antibiotic was given by the
following routes
Antibiotic Intramuscular Intravenous
Control: no treatment 2.4 f 0.3 x 104 2.4 0.3 x 104
Control: LIP-LPS 2.1 ~ 0.5 x 104 2.1 0.5 x 104
TetracyclineZ 4.8 f 0.8 x 103 4.1 0.6 x 103
TETRA-LIP 3.2 ~ 1.4 x 103 4.4 0.3 x 103
TETRA-LIP-LPS - 4.7 0.9 x 102
Ciprofloxacin 1.3 0.4 x 104 2.0 0.6 x 104
CIPRO-LIP 1.1 0.1 x 104 1.4 0.2 x 104
CIPRO-LIP-LPS - 8.1 4.9 x 103
For each set, three mice were infected with 5 x 104 colony forming units (CFU)
of Brucella
melitensis intravenously on day 0. Treatments consisted of a dose of 1 mg
antibiotic/0.2 ml
and were given on days 7, 8 and 9. The mice were sacrificed on day 12, the
spleens were
harvested, crushed in saline and then plated.
2 All antibiotics were given at 1 mg/mouse. TETRA = tetracycline, CIPRO =
ciprofloxacin,
LIP = liposome encapsulated, LPS = B. melitensis 16M smooth-lipopolysaccharide
in
liposome formulation.
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