Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing – Hydrolases
Reexamination Certificate
1999-01-19
2001-08-28
Lankford, Jr., Leon B. (Department: 1651)
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
Hydrolases
C435S198000
Reexamination Certificate
active
06280726
ABSTRACT:
FIELD OF INVENTION
This invention pertains to methods and compositions for killing Gram-positive bacteria, such as, for example, bacteria of the generi Staphyl Streptococcus, and Bacillus, using as a bactericidal agent Type II phospholipase A2 (PLA2(II)).
BACKGROUND OF THE INVENTION
Gram-positive bacterial infections are widespread and represent a significant proportion of iatrogenic and other infections prevalent in hospitals and medical facilities. The inevitable development of antibiotic-resistant strains necessitates the continuous development of new means of controlling these infections. Thus, there is a need in the art for methods and compositions useful in treating Gram-positive bacterial infections in animals and humans (Waldvogel, in
Principles and Practice of Infectious Diseases
, Mandell et al.,eds., John Wiley, New York, 1995, pp 1754-1775).
In mammals, inflammation is a complex response that involves, at a minimum, the recruitment of immune cells, changes in vascular permeability, and the accumulation of extracellular fluid containing a wide variety of bioactive compounds, including proteins and peptides. Among the bioactivities expressed in such inflammatory fluids are antibacterial activities that are directed against both Gram-positive and Gram-negative bacteria.
In rabbits, sterile injection of glycogen into the peritoneal cavity induces the formation of a cell-rich exudate consisting of polymorphonuclear leukocytes (PMNs) and a cell-free (ascitic) fluid (designated AF). In this model system, potent antibacterial activity accumulates in AF against both Gram-negative and Gram-positive bacteria. The activity against Gram-negative bacteria is attributable to two PMN-derived antibacterial proteins acting synergistically, the Bactericidal/Permeability-Increasing Protein (BPI) and the p15s (Weinrauch et al.,
J. Clin.Invest.
95:1916, 1995). The Gram-negative bactericidal activity of AF is abolished by anti-BPI serum, indicating an absolute dependence on BPI. Antibacterial activity in AF against Staphylococci, by contrast, is unaffected by anti-BPI serum, indicating that BPI is not responsible for Gram-positive killing.
The present inventors have unexpectedly discovered that the Gram-positive bactericidal activity of rabbit AF is due to Type II phospholipase A2 (PLA
2
(II) that is present in the exudate. The bactericidal activity of PLA
2
(II) is independent of other constituents and is specific to Gram-positive bacterial targets. Furthermore, Gram-positive bactericidal activity has been found to be a property of all tested members of the PLA
2
(II) enzyme family, such as those derived from other tissues and mammalian species. Prior to the present invention, no independent bactericidal activity expressed in biological fluids (including unmodified plasma and inflammatory fluids) was ascribed to this protein. The present invention thus provides methods and compositions for killing Gram-positive bacteria that are applicable to many different therapeutic applications and that provide an important therapeutic modality to treat antibiotic-sensitive and antibiotic-resistant Gram-positive bacterial infections.
SUMMARY OF THE INVENTION
The present invention encompasses methods for killing Gram-positive bacteria, which are carried out by exposing the bacteria to a bactericidal-effective amount of Type II phospholipase A2 (PLA
2
(II)). The PLA2(II) may be derived from mammalian species such as human, cow, rabbit, rat, mouse, dog, cat and the like, and may be isolated from a native or recombinant source. Typically, a bactericidal-effective amount of PLA
2
(II) will range from about 0.05 to about 100 ng/ml, depending upon the bacterial species and source of PLA
2
(II).
In another aspect, the present invention provides methods for treating Gram-positive bacterial infections in animals, including humans, by administering bactericidal-effective amounts of PLA
2
(II).
In yet another aspect, the invention provides pharmaceutical formulations having bactericidal activity against Gram-positive bacteria. These formulations comprise bactericidal-effective concentrations of PLA
2
(II) and a pharmaceutically acceptable carrier or diluent. Additionally, the formulations may comprise other bioactive compounds, such as, e.g., conventional antibiotics, that act additively or synergistically with PLA
2
(II) to promote bacterial killing.
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Elsbach Peter
Weinrauch Yvette
Weiss Jerrold
Darby & Darby
Lankford , Jr. Leon B.
New York University
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