Drug – bio-affecting and body treating compositions – Preparations characterized by special physical form – Liposomes
Reexamination Certificate
1999-02-17
2001-04-24
Kishore, Gollamudi S. (Department: 1615)
Drug, bio-affecting and body treating compositions
Preparations characterized by special physical form
Liposomes
C424S234100, C424S236100, C424S241100, C424S252100, C424S257100, C424S258100, C424S260100, C424S812000, C436S829000, C514S054000, C514S885000
Reexamination Certificate
active
06221386
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a liposomal delivery of a therapeutic agent and is particularly concerned with the use of virulence factors of pathogens to improve the liposomal delivery of the therapeutic agent.
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. swis, 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 has 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 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 eat. 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 Jan. 24, 1995) wherein liposome encapsulated ciprofloxacin was found to be more effective in the prevention and treatment of
Francisella tularensis
infection than the nonencapsulated 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 will liposomes have proven effective against some strains of Brucella (e.g.
B. canis
and
B. abortus
) but less so against another strain (e.g.
B. melitensis
) (Hernandez-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 Dec. 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.
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
It is an object of the present invention to provide a new microsphere formulation, which includes the virulence factors such as O-polysaccharide or lipopolysaccharide of bacteria. Preferably, the microsphere is a liposome consisting of phosphatidylcholine, cholesterol, and phosphatidylserine in a molar ratio of 7:2:1. Suitable bacteria are selected from the group consisting of
Vibrio cholorae, Yersinia entierocolitica
O:9, toxigenic
Escherichia coli
O:157H:7,
Salmonella landau, Pseudomonas maltophilia
555 and Brucella such as
B. abortus
or
B. melitensis.
The microsphere formulation according to the present invention has enhanced effectiveness as delivery systems for antibiotics in the treatment of disease.
Thus, the invention provides for the use of virulence factor in the formulation (in combination with or in the replacement of lipids) of microspheres and specifically in liposomes.
Further, the invention provide for a pharmaceutical formulation for preventing or treating infections wherein a therapeutic agents are encapsulated by liposomes comprised in part by virulence factors such as bacterial components.
The invention also provides a method of increasing the uptake of a composition in an mammal comprising administering to the mammal the composition, wherein the composition comprises a therapeutic agent entrapped within a microsphere, for example, a liposome comprising a bacterial component selected from the group consisting of O-polysaccharide and lipopolysaccharide. Preferably, the bacterial component is incorporated into or onto the membrane of the microsphere.
In one embodiment of the invention, the composition is taken-up by non-phagacytic cells.
In accordance with another aspect of the invention, there is provided a method of treating a mammal against brucellosis comprising administering to said mammal a liposomal formulation comprising a bacterial component selected from the group consisting of O-polysaccharide and lipopolysaccharide incorporated into or onto the membrane of said liposome to enhance the uptake of said liposomal formulation by non-phagocytic cells of said mammal.
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 pathogenic bacteria have the same rare sugar
Cherwonogrodzky John
Dininno Vincent L.
Wong Jonathan P.
Her Majesty the Queen in right of Canada as represented by the
Kishore Gollamudi S.
Nixon & Vanderhye P.C.
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