CA2171369C - The use of virulence factors of pathogens to improve liposomal delivery of antibiotics and/or vaccines - Google Patents

Abstract

Liposome encapsulated antibiotic therapy has limited application against infectious organisms which can sequester in non-phagocytic cells. Virulence factors of these infectious organisms, for example bacterial components, when used in the formulation of liposomes can enhance the effectiveness of liposomes as delivery systems in the treatment of disease. In this manner, multi-functional liposomes can be developed to treat target diseases. In addition to serving as antibiotic delivery systems, such liposomes also have an immunization effect. Thus, the liposomes can be used for both the prevention and treatment of diseases.

Description

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 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 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.

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 _ 2171369 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 .,.~. .

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.

=6-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 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 .g.

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:

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.

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.

_ 2171369 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.

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.

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.

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.

Claims (13)

1. A liposome for the delivery of a therapeutic agent to an animal, the liposome having a smooth lipopolysaccharide from Brucella melitensis incorporated into its structure, and wherein the therapeutic agent is entrapped within the liposome.

2. The liposome according to claim 1 wherein the therapeutic agent is an antibiotic.

3. The liposome according to claim 2 wherein the antibiotic is ciprofloxacin.

4. A method of preparing a liposome comprising the steps of:
- dissolving lipids in a suitable solvent and drying;
- thoroughly dissolving the dried lipids in a solution comprising a smooth lipopolysaccharide from Brucella melitensis and a therapeutic agent;
- sonicating and freeze drying the resulting solution; and - reconstituting the liposome with water.

5. The method of claim 4 wherein the therapeutic agent is an antibiotic.

6. The method of claim 5 wherein the antibiotic is ciprofloxacin.

7. The method of any one of claims 4 to 6 wherein the lipids are phophatidylcholine, cholesterol and phophatidylserine in the molar ratio of 7:2:1.

8. A composition for the delivery of a therapeutic agent to an animal, the composition comprising a liposome; and a smooth lipopolysaccharide from Brucella melitensis incorporated into the structure of the liposome; and a therapeutic agent, wherein the therapeutic agent is entrapped within the liposome.

9. The composition of claim 8 wherein the therapeutic agent is an antibiotic.

10. The composition of claim 9 wherein the antibiotic is ciprofloxacin.

11. Use of a liposome for the delivery of a therapeutic agent to a non-phagocytic cell in an animal, the liposome having a smooth lipopolysaccharide from Brucella melitensis incorporated into its structure, and wherein the therapeutic agent is entrapped within the liposome.

12. The liposome according to claim 11 wherein the therapeutic agent is an antibiotic.

13. The liposome according to claim 12 wherein the antibiotic is ciprofloxacin.