Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing – Hydrolases
Reexamination Certificate
2000-12-18
2002-11-05
Saidha, Tekchand (Department: 1652)
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
Hydrolases
C435S195000, C435S253400
Reexamination Certificate
active
06475484
ABSTRACT:
This application claims priority under 35 U.S.C. §119 to provisional application No. 60/172,467 filed Dec. 17, 1999.
FIELD OF THE INVENTION
This invention pertains to a novel recombinant mutant protein of human Group IIA Phospholipase A2 (PLA2) which has significantly enhanced antibacterial activity compared to the wild-type human Group IIA PLA2, pharmaceutical formulations comprising the protein and methods of use thereof.
BACKGROUND OF THE INVENTION
The growing prevalence of antibiotic resistance in bacterial pathogens has stimulated renewed interest in the discovery of novel antibiotics. U.S. Pat. No. 5,874,079 discloses that a “Group IIA” 14 kDa Phospholipase A2 (PLA2), mobilized during inflammation expresses potent bactericidal activity toward a broad range of clinically important Gram-positive bacteria and enhances the activity of the host defense mechanisms toward many Gram-negative bacteria.
The phospholipase A2 (PLA2) family of enzymes hydrolyze the sn-2 ester of glycerophospholipids to produce a fatty acid and a lysophospholipid (Dennis, J.Biol. Chem. 269:13057-13060, 1994; Gelb et al, Ann. Rev. Biochem. 64, 653-688, 1995; Waite, The phospholipases, Plenum Press, New York, 1987). Based on amino acid sequences, 10 groups of PLA2s have been identified, including eight from mammals (Dennis, Trends Biochem. Sci. 22: 1-2, 1997; Cupillard et al., J. Biol. Chem. 272: 15745-15752, 1997). Group IIA PLA2 in mammals are produced by many different cell types including phagocytic cells, platelets, Paneth cells and lacrimal cells. It has been shown that both rabbit and human Group IIA PLA2 can, in concert with other host defense mechanisms, increase the destruction of gram-negative bacteria (Wright et al., J. Clin. Invest. 85: 1925-1935, 1990; Weiss et al., J. Biol. Chem. 269: 26331-26337, 1994 Elsbach et al., Trends Microbiol. 2: 324-328, 1994 and Madsen et al., Infect. Immun. 64: 2425-2430, 1996) and by itself, kill many gram-positive bacteria (Weinrauch et al., J. Clin. Invest. 97: 250-257, 1996). The antibacterial activity of Group IIA PLA2 appears to be a specific attribute of the mammalian 14 kDa isoform. This is further exemplified in experimentally induced local inflammatory (ascitic) fluid in rabbits, whereby the mobilization of Group IIA PLA2 is fully responsible for the potent bactericidal activity expressed in the fluid toward
S. aureus
and several other gram-positive bacteria (Weiss et al., en supra). Normal plasma, by contrast contains low levels of PLA2 and antistaphylococcal activity. It has recently been shown that the mobilization of this enzyme in baboons during inflammation may play an important role in host defense mechanisms against invading bacteria (Weinrauch et al., J. Clin. Invest. 102 (3): 633-638, 1998).
In biological fluids, as little as 100 ng/ml of the human Group II A PLA2 is sufficient to kill greater than 99% of 10
6
Staphylcoccus aureus
cells/ml, including all multi-drug resistant clinical isolates tested. The bactericidal activity of the PLA2 was dependent on catalytic activity and was enhanced synergistically by the co-treatment with sub-inhibitory doses of &bgr;-lactam antibiotics. The potent antibacterial activity of the mammalian Group IIA PLA2 is not expressed by other closely related PLA2s reflecting the presence and localization of a high density of basic residues in the Group IIA PLA2 that is absent in all other subsets of related PLA2s.
U.S. Pat. No. 5,874,079 discloses that the rabbit Group IIA PLA2 possesses 10 fold greater antibacterial activity than the human enzyme. Since it is preferable to treat humans with human-derived therapeutic proteins, what is needed is a human PLA2 with activity similar to the rabbit counterpart.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides methods for treating Gram-positive bacterial infections in humans, by administering bactericidal-effective amounts of mutant human Group IIA PLA2.
In another aspect, the invention provides pharmaceutical formulations having bactericidal activity against Gram-positive bacteria. These formulations comprise bactericidal-effective concentrations of mutant human Group IIA PLA2 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 Group IIA PLA2 to promote bacterial killing.
These and other aspects of the present invention will be apparent to those of ordinary skill in the art in light of the present description, claims and drawings.
REFERENCES:
patent: 5874079 (1999-02-01), Weinrauch et al.
Kramer Ruth M. et al. Structure and Properties of a Human Non-pancreatic Phospholipase A2, J. Biol. Chem. 1989, 264, 5768-5775.*
Dennis, J. Biol. Chem. 269: 13057-13060, 1994.
Gelb et al., Ann. Rev. Biochem, 64, 653-688, 1995.
Dennis, Trends Biochem. Sci. 22: 1-2, 1997.
Cupillard, et al., J. Biol. Chem. 272: 15745-15752, 1997.
Wright et al., J. Clin. Invest. 85: 1925-1935, 1990.
Weiss et al., J. Biol. Chem. 269: 26331-26337, 1994.
Elsbach et al., Trends Microbiol. 2: 324-328, 1994.
Madsen et al., Infect. Immune. 64: 2425-2430, 1996.
Weinrauch et al., J. Clin. Invest. 102 (3): 633-638, 1998.
Scott D.L., White, S.P., Browning, J.L., Rosa, J.J., Gelb, M.H., Sigler, P.B. 1997. Science 254: 1007-1010.
Bomalaski et al., J. Immunol. 146: 3904, 1997.
Cirino et al., J. Rheumatol. 21: 824, 1994.
Thannhauser et al., Biochemistry 24: 7681-7688., 1985.
Liang, N.S. et al., FEBS Letters 334: 55-59, 1993.
Fourcade et al., Cell 80: 919-923, 1995.
Hochkeppel et al.,J. Clin. Microbiol, 25:526-530.
McKenney et al.,Science284: 1523-1527.
Xu et al.,Infect. Immun.60: 1358-1362; Table I.
Elsbach Peter
Liang Ning-Sheng
Weiss Jerrold
Darby & Darby
New York University
Saidha Tekchand
Walicka Malgorzata A.
LandOfFree
Recombinant antibacterial group IIA phospholipase A2 and... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Recombinant antibacterial group IIA phospholipase A2 and..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Recombinant antibacterial group IIA phospholipase A2 and... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2981630