Phospholipase A2 inhibitors as mediators of gene expression

Details Phospholipase A2 inhibitors as mediators of gene expression Phospholipase A2 inhibitors as mediators of gene expression

1994-05-24

2002-11-26

Wityshyn, Michael G. (Department: 1651)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

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

C435S242000, C435S173100, C514S747000, C514S263370, C514S291000, C514S297000, C514S169000

Reexamination Certificate

active

06486170

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the fields of radiation biology and cell biology. More particularly, it concerns the attenuation of the effect of ionizing radiation induced activation of tumor necrosis factor by inhibitors of extranuclear signal transduction.
2. Description of the Related Art
Signaling pathways activated by DNA damage contribute to survival of prokaryotes and eukaryotic cells following exposure to x-rays or UV light. In irradiated
E. coli,
damaged DNA forms a complex with the Rec A protease resulting in the transcriptional induction of a variety of genes including those encoding DNA repair enzymes (Walker, 1985). In yeast, UV light and x-rays result in the induction of genes which participate in the repair of damaged DNA (Jones et al., 1991, Cole et al., 1987). Genes whose products are proposed to recognize damaged or un-replicated DNA and to participate in intracellular signaling that regulates cell cycle progression and DNA repair have been identified in
S. cerevisiae
and
S. pombe
(House et al., 1992, Enoch et al., 1992). The complexity of this signaling pathway is demonstrated by the number of genes involved in sensing DNA damage and transmitting the signal (Enoch et al., 1992). DNA damage is presumed to be the initiating event in mammalian cell induction of stress response genes following x-ray or UV exposure (Herrlich et al., 1992, Kastan et al., 1992). However, the mechanisms of DNA damage recognition have not been identified in mammalian cells.
Signal transduction pathways activated by ionizing radiation include increased phosphotransferase activity of cytoplasmic protein kinases (Hallahan et al., 1991a, Hallahan et al., 1991b, Uckun et al., 1992). Moreover, inhibition of protein kinases blocks radiation-mediated gene induction and effects diverse biological endpoints such as apoptosis (Uckun et al., 1992), radiation survival (Hallahan et al., 1992)) and induction of the cytokine tumor necrosis factor (TNF) (Hallahan et al., 1991b). The calcium/phospholipid-dependent protein kinase (PKC) is activated within 15 seconds of ionizing radiation exposure and is extinguished by 90 seconds in human leukemia HL-60 cells (Hallahan- et al., 1991b).
Phospholipase A2 inhibitors used in clinical radiotherapy to ameliorate acute and subacute sequelae include glucocorticoids and pentoxifylline,(Bianco et al., 1991, Phillips et al., 1975). Glucocorticoids are used to treat radiation induced proctitis, pneumonitis, conjunctivitis, external otitis, CNS syndromes and occasionally mucositis. Pentoxifylline is effective in preventing pneumonitis and mucositis following total body irradiation prior to bone marrow transplantation (Bianco et al., 1991). Taken together, these findings implicate phospholipase A2 in radiation induced TNF induction and the acute sequelae of radiotherapy.
Since phospholipase A2 hydrolyses phosphatidylcholine to arachidonic acid, the effects of the phospholipase A2 inhibitors mepacrine (Rao et al., 1993), and bromphenylbromide (BPB) (Peppelenbosch et al., 1993) were investigated. In addition, the effects of dexamethasone and pentoxifylline on radiation-induced fatty acid hydrolysis were studied, as these agents have been shown to inhibit phospholipase A2, reduce the production of cellular mediators of inflammation and tissue injury, and inhibit lipopolysaccharide-induced TNF production in monocytes (Strieter et al., 1988, Han et al., 1990). Moreover, glucocorticoids and pentoxifylline are employed clinically to prevent some acute toxicities of radiotherapy (Bianco et al., 1991, Phillips et al., 1975). The inventors determined that each agent attenuated arachidonic acid release into the medium of cells treated with X-rays or H
2
O
2
. Thus, extranuclear second messengers are in part responsible for radiation-mediated signal transduction and inhibition of this pathway may provide a means of attenuating the inflammatory-like response observed in irradiated tissues through the inhibition of TNF gene induction.
SUMMARY OF THE INVENTION
The present invention, in a general and overall sense, concerns methods of inhibiting the production of cytokines, for example tumor necrosis factor (TNF), following exposure of cells to ionizing radiation. In accordance with these methods, cells or tissues are contacted with phospholipase A2 inhibitors prior to exposure to ionizing radiation, reducing the production of cellular mediators of inflammation and tissue injury, and inhibiting the radiation-induced TNF production.
As used herein, “cytokine”refers to a class of molecules that are secreted by cells that affect the functions of other cells. More specifically, the cytokines of the present invention are secreted in response to ionizing radiation, and are produced as a result of or are otherwise involved in the arachidonic acid metabolic pathway.
Preferred inhibitors of phospholipase A2 include, but are not limited to mepacrine, bromphenylbromide (BPB), dexamethasone, or pentoxifylline. It will be recognized by-those skilled in the art that compounds with similar activity or derivatives of these inhibitors does not depart from the scope or spirit of the invention.
The present invention thus encompasses the use of any phospholipase inhibitor derivative that has a significant (i.e., consistently above background) inhibitory effect on phospholipase activity.
In certain embodiments of the invention, methods are provided for the treatment of acute radiation sequelae that mimic local inflammatory reactions, such as pneumonitis, proctitis, mucositis, dermatitis, and esophagitis. These consequences of radiotherapy may be associated with cytokine production, such as tumor necrosis factor, or with arachidonic acid metabolites. The methods described herein are designed to ameliorate these acute side effects associated with radiation therapy.
On other embodiments, the methods of the present invention are useful in inhibiting cytokine production in vitro following ionizing radiation exposure. The methods allow the production of certain polypeptides operatively linked to radiation inducible promoters unaffected by phospholipase A2 inhibitors, while reducing the effect of cytokines and arachadonate metabolic products produced following radiation exposure.
In this aspect, the present invention contemplates a pharmaceutical composition comprising an inhibitor of phospholipase A2 in a therapeutically effective amount and a physiologically acceptable carrier. The terms “contacted” and “exposed,” when applied to a cell, are used herein to describe the process by which a phospholipase inhibitor is delivered to a target cell. Any method may be used to contact a cell with a phospholipase inhibitor, as long as the method results in decreased phospholipase A2 activity within the cell
The phrases “pharmaceutically or pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a human. The appropriate doses for treating persons with radiation induced sequelae may be determined from a consideration of the condition to be treated and the properties of the composition being administered. This will be readily understood by those of skill in the art when in possession of the present disclosure.
A therapeutically effective amount of an inhibitor of phospholipase A2 that is combined with a carrier to produce a single dosage form varies depending upon the host treated and the particular mode of administration. A “therapeutically effective amount” is an amount of a phospholipase inhibitor or similar agent that, when administered to an animal, is effective to reduce or eliminate phospholipase A2 activity within the animal.
As is well known in the art, a specific dose level for any particular patient depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, and the se

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