Drug – bio-affecting and body treating compositions – Preparations characterized by special physical form – Liposomes
Reexamination Certificate
1999-04-13
2003-09-02
Kishore, Gollamudi S. (Department: 1615)
Drug, bio-affecting and body treating compositions
Preparations characterized by special physical form
Liposomes
C264S004100, C264S004300
Reexamination Certificate
active
06613352
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to a liposomal formulation that is capable of fusing with cells.
BACKGROUND OF THE INVENTION
Encapsulation of bioactive compounds in natural or synthetic matrixes has been extensively studied over the past decades. Advantages of encapsulation are numerous. First, it provides protection from the inactivation or degradation of the bioactive compound. Secondly, it controls the kinetics of compound release, allowing the optimization of the blood concentration profile. This optimization diminishes the deleterious effects of bioactive compounds with short half lives. In addition, it permits a reduction in toxicity, where relevant.
Liposomes are closed microscopic vesicles that form spontaneously from phospholipids above their transition temperature, in the presence of excess water. Vesicles with diameters ranging from 20 nanometers to several micrometers can be prepared. Multilamellar liposomes are made of concentric phospholipid bilayers separated by aqueous layers. Unilamellar liposomes consist of a single phospholipid bilayer surrounding an aqueous core. Liposomes can accommodate hydrophilic molecules in the aqueous spaces and lipophilic molecules in the lipid bilayers.
The potential of liposomes as vehicles for antimicrobial agents, or therapeutic liposomal formulations, has been studied by several investigators. Successful treatments with liposomes against intracellular bacteria have been demonstrated (Lopez-Berestein et al., 1987, U.S. Pat. No. 4,981,692). A number of studies have also shown that liposome-entrapped antibacterial agents increase the therapeutic indices of these agents as a result of decreased toxicity, modification of pharmacokinetics and tissue distribution parameters (Lagacé et al., 1991, J. Microencapsulation 8:53-61 and references therein; Omri et al., 1994, Antimicrob. Agents Chemother. 38:1090-1095)
Microorganism resistance to antibiotics is an important health problem world-wide. According to estimates from the United States Center for Disease Control and Prevention, for the period 1980 to 1992, approximately two million hospital-acquired infections occurred annually in the United States, accounting for more than eight million days extended hospital stay and generating more that $4 billion in additional health care costs each year. While overall per capita mortality rates for all diseases declined in the United States from 1980 to 1992, the per capita mortality rate due to infectious disease increased 58% over this period, making infectious diseases the third leading cause of death in the United States. Microbial infections, especially infections caused by difficult-to-treat, antibiotic-resistant microbes, such as bacteria and fungi, cause or contribute to a substantial majority of these deaths.
Microbes use different mechanisms to resist antibiotics. These mechanisms include prevention of the penetration, and/or extrusion, of the drugs from the microbial cells, enzymatic inactivation of the drugs, or alteration of the molecular target. Frequently, multiple mechanisms are present in a synergistic way, thus increasing the degree of resistance. Increasing evidence suggests that acquired antibiotic resistance is often due to a balance between outer membrane penetration rate and the subsequent enzyme inactivation rate. Thus, the outer membrane barrier and the antibiotic-degrading enzymes are strongly synergistic. New generation antibiotics, which can overcome strain-based enzymatic degradation, still do not solve the significant hurdle of penetration through the impermeable microbial membrane or through an exopolysaccharide layer of the microorganism and to its site of action. Recent research reports have also indicated that membranes with a low level of permeability, combined with a multiple drug efflux, play a dominant role in many antibiotic-resistant microorganisms, including
Pseudomonas aeruginosa
(Poole, K. et al., 1996, Antimicrob. Agents Chemother. 40:2021-2028).
The problem of increased resistance to antibiotics is compounded by the misuse of these agents (Merck manual, 1992, 16th Edition, Merck Res. Lab.). For example, because of the antibiotic resistance of microorganisms, which is more acute with older types of antibiotics, practitioners are often prompted to use a newer generation antibiotic, contributing to the increased resistance of microorganisms to these antibiotics. The large scale use of antibiotics in animals, including but not limited to dairy cows, and the presence of these antibiotics in milk, or in the environment, is yet another contributor to increases in microbial resistance to antibiotics.
Although antibiotics are useful for treating infections, their use can be accompanied by concentration-dependent toxicity and side effects. It is, therefore, important to ensure that their plasma concentrations do not exceed toxic levels. It is equally important to ensure that fear of toxicity does not result in a therapeutically inadequate dosage.
The encapsulation of antibiotics into liposomal formulations has been described (Lagacé et al., 1991, J. Microencapsulation 8:53-61; Boswell et al., J. Clinical Pharmacology 38:583-592 and references therein; Da Cruz et al., 1993, WO 93/23015 and Proffitt et al., 1994, WO 94/12155). Nevertheless, these formulations fail to display a very drastic enhancement of the therapeutic activity of the antibiotic as compared to its activity in the free form. Indeed, the preferred aminoglycoside (netilmicin) liposomal formulation of Da Cruz et al., which comprises phosphatidylcholine (PC), cholesterol and phosphatidylinositol (PI), only shows a modest increase activity in vivo with the aminoglycoside as part of the liposomal formulation as compared to free aminoglycoside (at best by a factor of three). Proffitt et al., disclose a different aminoglycoside (amikacin) liposomal formulation comprising PC, cholesterol and distearoyl phosphatidylglycerol (DSPG). Although the Proffitt et al., formulation appears to be superior at enhancing the in vivo therapeutic activity of the aminoglycoside as compared to that of Da Cruz, this increase is still relatively low and dependent on the tissue (10-fold in lung).
In view of the therapeutic, diagnostic, and research benefits incurring therefrom, it would be useful to have liposomes capable of fusing with pathogenic microbes and other cells.
SUMMARY OF THE INVENTION
We have now discovered that a liposomal formulation, previously known only to fuse with bacteria to deliver antimicrobials, is capable of delivering virtually any agent to a microbial cell, including a non-bacterial cell, or a macrophage. Accordingly, the invention provides compositions and methods for liposomal delivery of compounds to cells.
In the first aspect, the invention features a low-rigidity liposomal formulation, which is characterized as being free of cholesterol, and including neutral and anionic phospholipids at a molar ratio of 5:1 to 20:1, having a phase transition temperature (T
c
) below 42° C. as measured by differential scanning calorimetry (DSC), where said T
c
is below about 42° C., such that the formulation enhances fusion of the neutral and anionic phospholipids with a cell, where the formulation either does not include an antibacterial compound or where the formulation does not enhance penetration inside a bacterial cell.
In preferred embodiments of the first aspect, the formulation further comprises an agent, preferably an antimicrobial agent. In other preferred embodiments the cell is a macrophage or a non-bacterial microbial cell, preferably a fungus (e.g. a yeast), or the agent to be delivered is not an antimicrobial agent, for example, a nucleic acid encoding a commercially useful protein.
In other embodiments of the invention, the neutral and anionic phospholipids are present at ratios of about 8:1 to 18:1 or 10:1 to 15:1. The preferred neutral phospholipid may be dipalmitoylphosphatidylcholine (DPPC) or 1,2-di-o-hexadecyl-sn-glycero-3-phosphocholine (DHPC) and the preferred anionic phospholipid may be dimirystoylphosphatidylglycerol (
Beaulac Christian
Clement-Major Sebastien
Lagace Jacqueline
Bieker-Brady Kristina
Clark & Elbing LLP
Kishore Gollamudi S.
Universite de Montreal
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