The general physiological phenomenon of inflammation at the site of a wound or infection has been recognized for centuries. It is also well understood that while this phenomenon may be of positive value in response to such stimuli, the extent of this response must often be controlled in order properly to secure the comfort of the human or animal subject. In addition, inflammation may occur as a chronic inflammatory disorder, such as, for example, rheumatoid arthritis or systemic lupus erythomatosis. Such conditions are debilitating in themselves-and can result in often life threatening, acute episodes. The inflammation associated with another such disorder, asthma, may also result in death due to constriction of the bronchia.
Within the last few decades a more detailed picture of the biochemical events associated with inflammation has been accumulated. The picture is a complex one. Part of the initiation process is mediated by peptide kinins, such as bradykinin, which are liberated by kallikrein proteases upon tissue destruction. The kinins or other peptide messengers, act on specific cell receptors at the inflammation site to activate the phospholipase enzymes A2 and/or C, to initiate the arachidonate cascade.
The "arachidonate cascade" is of singular importance in maintaining the inflammatory response. Within this complex of reactions, illustrated in FIG. 1, arachidonic acid is liberated from membrane phospholipids of a subject cell, and converted to a variety of products known collectively as eicosanoids. The eicosanoids include the leukotrienes and prostaglandins, which are released into the extracellular environment to exert their effects directly on the inflammatory site. They have relatively short half lives. However, their physiological effects are varied and dramatic, and include vasodilation (e.g., prostacylin and leukotrienes LTC.sub.4 and LTD.sub.4), vasoconstriction (e.g., thromboxane and LTB.sub.4), and histamine release.
Most of the members of the current repertoire of anti-inflammatory pharmaceuticals are directed against some aspect of the arachidonate cascade. From this standpoint, the significant features of the cascade, as shown in FIG. 1, are that production of all products begins with the liberation of arachidonic acid from cellular phospholipids, catalyzed by the phospholipases, and the reaction pathway then branches into several reaction series: the cyclooxygenase pathways which generate the prostaglandins and the lipoxygenase pathways which generate leukotrienes.
Most of the commonly used non-steroid anti-inflammatory drugs, such as aspirin or indomethacin, inhibit cyclooxygenase, and hence only some of the pathways by which arachidonic acid is converted to end products. Other pathways of the arachidonate cascade are not affected. Steroid, or glucocorticoid, hormones, on the other hand, generally exert their effect on the production of arachidonic acid from the phospholipid membrane sources, and thus directly affect the entire cascade. Hong, S. et al, Proc Natl Acad Sci (USA) (1976) 73:1730-1734. However, the disadvantages of steroid therapy are well known. Side effects such as water retention, hyperglycemia, hyperlipidemia, osteoporosis, glaucoma, and increased risk of coronary and large-vessel atherosclerosis are among the undesirable responses which may accompany such treatment.
It has recently been shown that the anti-inflammatory effect of the steroids is due, at least in part, to their ability to induce the secretion of proteins which bind to and inhibit the phospholipase enzymes which are responsible for the release of arachidonic acid. Hirata, F., J Biol Chem (1981) 256:7730-7733. This inhibitor has been designated macrocortin (Blackwell, R. J., et al, Nature (1980) 287:147-149); renocortin (Russo-Marie, F., et al, Biochim Biophys Acta (1982) 712:177-185); or lipomodulin (Hirata, F., et al, Proc Natl Acad Sci (USA) (1980) 77:2533-2536). The proteins have been partially purified from rat and rabbit cells, and appear to be immunologically cross-reactive (Hirata, F., et al, Biochem Biophys Res Comm (1982) 109:223-230; Rothhut, B., et al, ibid (1983) 117:878-884).
Recently, a human form of the inhibitor termed lipocortin has been identified in human fibroblasts (Errasfa, M., et al, Biochim Biophys Acta (1985) 847:247-254. Also Wallner, B. P., et al, Nature (1986) 320:77-81 and Pepinsky, R. B., et al, J Biol Chem (1986) 261:4239-4261, have reported the isolation and sequencing of rat lipocortin, and the cloning of its human analog, which is a PA2 inhibitor in vitro.
Direct administration of the proteins which inhibit arachidonic acid formation rather than of the glucocorticoids, which stimulate the production of these proteins, would result in the advantages of steroid control of inflammation without exposing the subject to the risk of the attendant side effects. However, the human form of this protein is not available in purified form. It would, of course, be highly desirable to have pure human phospholipase inhibiting protein (PIP) in sufficient purity and amount to permit such direct treatment of unwanted inflammatory response.