Inhibitors of phospholipase enzymes

Details Inhibitors of phospholipase enzymes Inhibitors of phospholipase enzymes

2000-10-11

2002-12-31

Chang, Ceila (Department: 1626)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Having -c-, wherein x is chalcogen, bonded directly to...

C548S509000, C548S250000, C514S339000, C514S381000, C546S277400

Reexamination Certificate

active

06500853

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention relates to chemical inhibitors of the activity of various phospholipase enzymes, particularly phospholipase A
2
enzymes.
Leukotrienes and prostaglandins are important mediators of inflammation, each of which classes contributes to the development of an inflammatory response in a different way. Leukotrienes recruit inflammatory cells such as neutrophils to an inflamed site, promote the extravasation of these cells and stimulate release of superoxide and proteases which damage the tissue. Leukotrienes also play a pathophysiological role in the hypersensitivity experienced by asthmatics [See, e.g. B. Samuelson et al.,
Science,
237:1171-76 (1987)]. Prostaglandins enhance inflammation by increasing blood flow and therefore infiltration of leukocytes to inflamed sites. Prostaglandins also potentiate the pain response induced by stimuli.
Prostaglandins and leukotrienes are unstable and are not stored in cells, but are instead synthesized [W. L. Smith,
Biochem. J.,
259:315-324 (1989)] from arachidonic acid in response to stimuli. Prostaglandins are produced from arachidonic acid by the action of COX-1 and COX-2 enzymes. Arachidonic acid is also the substrate for the distinct enzyme pathway leading to the production of leukotrienes.
Arachidonic acid which is fed into these two distinct inflammatory pathways is released from the sn-2 position of membrane phospholipids by phospholipase A
2
enzymes (hereinafter PLA
2
). The reaction catalyzed by PLA
2
is believed to represent the rate-limiting step in the process of lipid mediated biosynthesis and the production of inflammatory prostaglandins and leukotrienes. When the phospholipid substrate of PLA
2
is of the phosphotidyl choline class with an ether linkage in the sn-1 position, the lysophospholipid produced is the immediate precursor of platelet activating factor (hereafter called PAF), another potent mediator of inflammation [S. I. Wasserman, Hospital Practice, 15:49-58 (1988)].
Most anti-inflammatory therapies have focussed on preventing production of either prostglandins or leukotrienes from these distinct pathways, but not on all of them. For example, ibuprofen, aspirin, and indomethacin are all NSAIDs which inhibit the production of prostaglandins by COX-1/COX-2, but have no effect on the inflammatory production of leukotrienes from arachidonic acid in the other pathways. Conversely, zileuton inhibits only the pathway of conversion of arachidonic acid to leukotriense, without affecting the production of prostaglandins. None of these widely-used anti-inflammatory agents affects the production of PAF.
Consequently the direct inhibition of the activity of PLA
2
has been suggested as a useful mechanism for a therapeutic agent, i.e., to interfere with the inflammatory response. [See, e.g., J. Chang et al,
Biochem. Pharmacol.,
36:2429-2436 (1987)].
A family of PLA
2
enzymes characterized by the presence of a secretion signal sequenced and ultimately secreted from the cell have been sequenced and structurally defined. These secreted PLA
2
s have an approximately 14 kD molecular weight and contain seven disulfide bonds which are necessary for activity. These PLA
2
s are found in large quantities in mammalian pancreas, bee venom, and various snake venom. [See, e.g., references 13-15 in Chang et al, cited above; and E. A. Dennis,
Drug Devel. Res.,
10:205-220 (1987).] However, the pancreatic enzyme is believed to serve a digestive function and, as such, should not be important in the production of the inflammatory mediators whose production must be tightly regulated.
The primary structure of the first human non-pancreatic PLA
2
has been determined. This non-pancreatic PLA
2
is found in platelets, synovial fluid, and spleen and is also a secreted enzyme. This enzyme is a member of the aforementioned family. [See, J. J. Seilhamer et al,
J. Biol. Chem.,
264:5335-5338 (1989); R. M. Kramer et al,
J. Biol. Chem.,
264:5768-5775 (1989); and A. Kando et al,
Biochem. Biophys. Res. Comm.,
163:42-48 (1989)]. However, it is doubtful that this enzyme is important in the synthesis of prostaglandins, leukotrienes and PAF, since the non-pancreatic PLA
2
is an extracellular protein which would be difficult to regulate, and the next enzymes in the biosynthetic pathways for these compounds are intracellular proteins. Moreover, there is evidence that PLA
2
is regulated by protein kinase C and G proteins [R. Burch and J. Axelrod,
Proc. Natl. Acad. Sci. U.S.A.,
84:6374-6378 (1989)] which are cytosolic proteins which must act on intracellular proteins. It would be impossible for the non-pancreatic PLA
2
to function in the cytosol, since the high reduction potential would reduce the disulfide bonds and inactivate the enzyme.
A murine PLA
2
has been identified in the murine macrophage cell line, designated RAW 264.7. A specific activity of 2 mols/min/mg, resistant to reducing conditions, was reported to be associated with the approximately 60 kD molecule. However, this protein was not purified to homogeneity. [See, C. C. Leslie et al,
Biochem. Biophys. Acta.,
963:476-492 (1988)]. The references cited above are incorporated by reference herein for information pertaining to the function of the phospholipase enzymes, particularly PLA
2
.
A cytosolic phospholipase A
2
(hereinafter “cPLA
2
”) has also been identified and cloned. See, U.S. Pat. Nos. 5,322,776 and 5,354,677, which are incorporated herein by reference as if fully set forth. The enzyme of these patents is an intracellular PLA
2
enzyme, purified from its natural source or otherwise produced in purified form, which functions intracellularly to produce arachidonic acid in response to inflammatory stimuli.
It is now desirable to identify pharmaceutically useful compounds which inhibit the actions of these phospholipase enzymes for use in treating or preventing inflammatory conditions, particularly where inhibition of production of prostaglandins, leukotrienes and PAF are all desired results. There remains a need in the art for an identification of such anti-inflammatory agents for therapeutic use in a variety of disease states.
Numerous pieces of evidence have supported the central role of cPLA
2
in lipid mediator biosynthesis: cPLA
2
is the only enzyme which is highly selective for phospholipids containing arachidonic acid in the sn-2 position (Clark et al., 1991, 1995; Hanel & Gelb, 1993); activation of cPLA
2
or its increased expression have been linked with increased leukotriene and prostaglandin synthesis (Lin et al., 1992a, 1992b, 1993); and following activation, cPLA
2
translocates to the nuclear membrane, where it is co-localized with the cyclooxygenase and lipoxygenase that metabolize arachidonate to prostaglandins and leukotrienes (Schievella et al., 1995; Glover et al., 1995). Although these data are compelling, the most definitive evidence for the central role of cPLA
2
in eicosanoid and PAF production came from mice made deficient in cPLA
2
through homologous recombination (Uozumi et al., 1997; Bonventre et al., 1997). Peritoneal macrophages derived from these animals failed to make leukotrienes, prostaglandins, or PAF. The cPLA
2
deficient mice have also been informative of the role of cPLA
2
in disease, since these mice are resistant to bronchial hyperreactivity in an anaphylaxis model used to mimic asthma (Uozumi et al., 1997). Thus, despite the size of the phospholipase A
2
superfamily, cPLA
2
is essential for prostaglandin, leukotriene, and PAF production.
Clark, J. D., Lin, L.-L., Kriz, R. W., Ramesha, C. S., Sultzman, L. A., Lin, A. Y., Milona, N., and Knopf, J. L. (1991). A novel arachidonic acid-selective cytosolic PLA
2
contains a Ca
2+
-dependent translocation domain with homology to PKC and GAP. Cell 65,1043-1051. Hanel, A. M., and Gelb, M. H. (1993). Processive interfacial catalysis by mammalian 85-kilodalton phospholipase A
2
enzymes on product-containing vesicles: application to the determination of substrate

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