WO1988006445A1

WO1988006445A1 - Prostaglandin b1, macromolecules as anti-inflammatory agents

Info

Publication number: WO1988006445A1
Application number: PCT/US1987/000408
Authority: WO
Inventors: William Regelson; Richard Franson; Marc W. Fariss
Original assignee: Individual
Current assignee: Individual
Priority date: 1987-02-24
Filing date: 1987-02-24
Publication date: 1988-09-07
Anticipated expiration: 1989-08-24

Abstract

This invention describes novel uses for macromolecular derivatives of a fatty acid such as prostaglandin B1, which include polymers, and oligomeric compounds thereof such as dimers, trimers and tetramers of the same. When used for therapeutic purposes in vivo and in vitro, such macromolecules have been discovered to be anti-inflammatory and anti-oxidant in effect. The class of macromolecules, as described above, are generically referred to as PGBx. They are discovered to be excellent membrane stabilizers and cytoprotective agents. They act to protect cells by increasing the resistance of each cell against injury from phospholipases and free radical induced oxidations, particularly the mediation of phospholipase A2 enzymes. PGBx is differentiated over the prior art in at least that characteristic which makes it an effective dual agent acting as both an anti-phospholipase enzyme inhibitor and an anti-oxidant.

Description

PROSTAGLANDIN B^ MACROMOLECULES AS ANTI-INFLAMMATORY AGENTS

Background of the Invention

The methods for the preparation of a new class of polymeric derivati of prostaglandin B₁ , referred to herein as PGBx, are described in US Pate 4,153,808 and 4,245,111 issued 5/1979 and 1/1981 respectively to D. Polis al. It was taught in these patents that PGBx had the unique property restoring _jm vitro phosphorylating ability of degraded liver mitochondria also reversed the degenerative effects of myocardial ischemia in monkeys brain ischemia in rabbits. The present invention describes the developme by the inventors, of something not taught in the Polis patent above, i.e., molecular mechanism of action of PGBx. We have invented new uses based on discovery that PGBx stabilizes membrane phospholipid composition and mainta cell function in situations of ischemia and injury because it possesses bo anti-phospholipase and anti-oxidant activities. By virtue of the discovery these dual properties, we have invented unique, widespread and fundament uses for PGBx as an anti-inflammatory, anti-oxidant, and cytoprotective agen In general terms, PGBx enables cell membranes to resist injury and destructi by preserving membrane integrity and permits tissue repair mechanisms assist in stabilization and/or return to normal function of previously damag or inflamed tissues.

Prior Art: Anti-Inflammatory Agents

In relation to the teachings of the above, in the prior art, since Poli original observations and despite numerous subsequent reports of organ a mitochondrial protection from injury afforded by PGBx, no fundamental teachi of PGBx's mode of action has been made, prior to our invention. For purposes of describing our invention, the term PGBx shall refer to the above polymeric derivatives of prostaglandin B. including macromolecules thereof, such as dimers, trimers, tetramers, and polymers, etc., known to the inventors. In the present case, the term macromolecule shall also mean dimers, trimers, tetramers and polymers of prostaglandin B... The term PGBx shall further include that mixture made by Polis and taught in the above patents. We have discovered that this PGBx contains macromolecules in the form of polymers and oligomers of prostaglandin B.. The term oligomers describes dimers, trimers, tetramers, etc., of prostaglandin B, . The major presently used clinically effective prior art anti-inflammatory drugs are corticosteroids and non-steroidal anti-inflammatory agents (NSAIAs) . These drugs act to control inflammation and to minimize cell injury by regulating the formation of prostaglandins and leukotrienes (see figure 1) which are produced in increased quantities in inflammation and promote cell dysfunction and injury. The mechanism of action of these drugs is to either suppress the release of free fatty acid (such as arachidonate) from the 2-position of membrane phospholipid by inhibiting cellular phospholipases (the mechanism of corticosteroid action), or for NSAIAs, to inhibit the further conversion of arachidonate, released by the action of cellular phospholipases, to prostaglandins. In addition, recent studies have demonstrated that cellular and extra cellular phospholipases may be activated by the generation of oxygen free radicals. This can establish a "vicious cycle" as phospholipase activation can release free radicals which, in turn, activate more phospholipases. In this regard, oxygen free radicals are produced from free fatty acids, released by the action of phospholipases, which are then converted to prostaglandins and leukotrienes. (Fig. 1) These oxygen free radicals generated from free fatty acids and tissue injury are inhibited by PGBx. Fatty acids and free radicals are known to be prime mediators in the cascade of reactions that result in membrane injury and cell death. One of the hallmarks of inflammation and cell injury is the breakdown o cellular membrane phospholipid. Phospholipids are the major structural building blocks of the cell membrane; they give rise to the barrier structural and functional properties of membranes and their integrity is crucial to normal cell responsiveness and function. Phospholipid changes in cell membrane integrity alter the fluidity of cell membranes, their receptor availability and the leakiness or availability of cellular contents to the external environment.

During inflammation, phospholipases, from whatever cause, that are normally under the control of natural suppressor systems are activated to degrade membrane phospholipid which, in turn, generates oxygen free radicals. A key enzyme which is activated in inflammation is phospholipase A„ which acts on phospholipids as enzyme targets to release free fatty acids. These fatty acids (i.e., arachidonate) released by phospholipase A_ degradation are converted to potent biologically active metabolites, prostaglandins and leukotrienes, with the concomitant generation of oxygen free radicals. These metabolites, fatty acids and free radicals are powerful mediators of pathophysiology which propagate injury and cell death.

The position of phospholipases, particularly phospholipase A„, as mem- brane targeted enzymes, makes them veritable "death triggers" as the ex¬ pression of their degradative activity results in further production of inflammatory mediators leading to further membrane injury which propagates damage within the cell itself or into adjacent tissues. Thus, the spread of injury from the initial site to contiguous or distant sites can be promoted by the activation and/or release of phospholipase A_.

In addition to the intrinsic membrane-related tissue breakdown via the activation of phospholipase A„, phospholipases, and particularly phospholipase A„ are part of the normal host defensive system of the body. Phospholipase A„ is found in particularly high levels in human white blood cells (WBCs or PMNs) or phagocytic cells. WBCs play a role in resisting infection, but when these cells are mobilized to ward off injury and infection, phospholipases A_ are released from adherent and circulating WBCs and produce local tissue necrosis which increases the extent of initial injury. In addition, WBCs adhere to blood vessel walls where they release enzymes such as phospholipase A„. WBC's also generate free radicals and thus promote damage to the vascular endothelium, lung alveoli or to tissue sites contiguous with WBC infiltration or concentration. This WBC adherence to vascular endothelium with release of phospholipase A_ activation results in damage to vascular integrity during shock and ischemia. Thus, in addition to being a prime defensive system of the body against infection, WBC's can also damage the body by propagating injury and inflammation beyond their normal defensive role. PGBx, by inhibiting WBC adherence and phospholipase activity, and by scavenging or neutralizing WBC free radical generating activity protectively modulate the toxic inflammatory activity of WBC's to permit the body to develop a reparative equilibrium which permits adequate host defense while minimizing or putting an end to WBC mediated tissue damage and inflammation.

In addition, PGBx prevents platelet induced vascular occlusion and tissue injury. Platelets are circulating non-nucleated particulates that induce blood clotting, promote phagocytosis and plug holes in blood vessels and thus are necessary to the integrity of vessel function and host defense. Like WBC's, platelets stimulated by infection, ischemia or trauma can propagate injury by perverting their normal protective role. PGBx, by inhibiting platelet aggregation and platelet release of reactive products, can prevent propagation of platelet mediated occlusive injury or injury related to the release of platelet contained vasoactive toxins permitting the body to restore functional equilibrium to inflamed or damaged tissues. Among the platelet factors released are those which stimulate smooth muscle proliferation, thus PGBx can be of value in the control of atherosclerosis as smooth muscle hyperplasia is part of the pathology that narrows blood vessels arteriosclerosis ("hardening of the arteries") .

This invention is based on our finding that PGBx has the dual action o inhibiting phospholipases, particularly phospholipase A_ as well as possessin antioxidant activity. In addition, PGBx protects lysosomal membranes intracellular digestive vacuoles (called "suicide bags") whose rupture ca result in cell injury and death from autodigestion. PGBx protects previousl oxidized phospholipid membranes from further phospholipase degradation

Because of the above, PGBx is of value as an anti-inflammatory agent and wil be useful wherever anti-inflammatory agents have been shown to be of value.

The classical description of inflammation is "redness and swelling wit heat and pain (Celsus, 100 AD)." Inflammation has been defined as th reaction of irritated and damaged tissues which still retain vitalit

(Grawitz, 1980). Inflammation is a process which, at one level, can go on t cell death, tissue necrosis and scarring and at another level, inflammatio can resolve with return to normalcy and no apparent injury or with minima changes, i.e., pigmentation, fibrosis or tissue thickening with collage formation related to healing and scarring. The process is dynamic with cel death as one consequence and recovery, healing and scarring as another. Fo inflammation to occur as a process, cells must retain their vitality, dead o severely compromised cells do not respond with inflammatory reactions. Injur in inflammation can also relate to the late results of fibrosis and scarrin with the loss of blood vessels, tissue elasticity and cosmetic quality.

Inflammation, while a normal process of the body's resistance to injur and infection can become aberrant leading to propagated injury with extensiv scarring, tissue death and/or the death of the organism. "Within certai limits, the inflammatory reaction is stereotyped and it cannot distinguis between those instances in which the process protects the host and those in which the host is harmed." (Ebert, 1965) Microscopically inflammation is characterized by vasodilation, vascular leakage, enhanced lymphatic flow, platelet vascular adherence and clumping and white blood cell infiltration and vascular adherence and phagocytosis with slowing of blood flow, red cell aggregation on the formation of blood clots. Clinically, these local phenomenon can be associated with pain, fever and swelling which can lead to local tissue destruction (granulation, caseation and necrosis) healing or scarring or to systemic symptoms of fever, shock (prostration) hypotension, leading to death or recovery.

Microscopically, Cohnheim (1967) first described inflammation as related o:

(1) Atony of the muscle coat of the blood vessel wall.

(2) Increased resistance to blood flow related to friction and adhesiveness of blood elements (i.e., red blood cells, proteins, white blood cells, platelets). (3) Enhanced permeability, i.e., loss of red blood cells, white blood cells and blood fluid through the vascular wall.

In physiologic terms, these are described as hyperemia, edema, blood stasis, thrombosis, and hemorrhage. Inflammation can be mediated by humoral substances produced by tissue elements or infectious agents or by changes in H (acidity) or oxygen concentration. Clinically, pain, fever, malaise, muscle, arterial and visceral spasm as well as headache can accompany inflammation from whatever primary cause.

It is our thesis, which has led to our invention, that the above events are mediated by phospholipase activation, followed by fatty acid release and the formation of free radicals. These events can be endogenous to the matrix of the body, the cells and tissue that are functionally or systemically integrated or exogenously induced by white blood cell or platelet activity which respond as part of the body's defenses and which release phospholipases or free radicals as part of their role in resistance to infection, or their place in maintenance of coagulative integrity, i.e., the prevention hemorrhage.

Free radicals, produced by white blood cells or tissue injury, are high reactive chemical species which, in the case of tissue injury, are most oft derived from respiratory oxygen. Oxygen, while necessary for the energeti of life, is also a toxin which, as the highly chemically related superoxid or as peroxides, can damage tissue instead of supporting it. Free radica derived from oxygen are critical to damage produced by radiation, inflammati ischemia (loss of blood supply) or through excess oxygen inhalation and, stated previously, free radicals are used by white blood cells to destr infecting organisms, but can, under circumstances of shock, infection a ischemia, damage or destroy the tissue they were meant to protect.

Free radicals, induced by radiation, oxygen inhalation, chemical agen

(i.e., dioxin, paraquat) or white blood cell reaction can or may be tiss damaging or important to mutational changes associated with aging or t development of cancer and hyperimmune proliferative diseases such rheumatoid arthritis.

Inflammation is associated with trauma, infection and host defen reactions, i.e., fever, malaise, related to direct bacterial or virus killi o associated immune responses. Immune responses can be both beneficia protective or tissue damaging as can be seen in their being responsible f resistance or cure of infection on the one hand, or capable of produci autoimmune phenomenon that result in allergy, i.e., asthma, urticaria, ho versus graft disease, glomerular nephritis, rheumatic fever, lupus a rheumatoid arthritis.

In regard to the current treatment of inflammation, corticosteroids ar effective anti-inflammatories, but must be used with caution clinicall because they are powerful immunosuppressants and inhibitors of fibroblas activity necessary for wound and bone repair. In addition, corticosteroid are diabetogenic drugs and their toxic side effects involve interference with wound repair, bone matrix formation, sodium retention, potassium loss, resis¬ tance to infection, as well as effects on sex steroid formation, blood pres¬ sure and body habitus. Alternatively, the clinically active, non-steroidal anti-inflammatory agents (NSAIAs), such as aspirin and indomethacin, (ibuprofen, etc.) work by inhibiting the conversion of free fatty acids to prostaglandins. The side effects of NSAIA's include gastric ulceration and metabolites of prostaglandin can be either damaging or protective to cells depending on the structure of the prostaglandin produced or utilized pharmacologically and the cell or tissue affected.

In conjunction with fatty acid release, as part of phospholipid cell membrane mediated injury produced by phospholipase activation, leukotrienes are generated (See Fig. 1). These leukotrienes produced from membrane phospholipid breakdown, damage tissue through direct toxic action, and associated free radical formation; or by indirect effects on vascular smooth muscle or vascular endothelial lining via platelet, WBC, endothelial (blood vessel lining) or smooth muscle constricting interactions.

Leukotrienes are responsible for smooth muscle constriction leading to bronchospasm and the asthmatic attacks seen in allergy or infectious asthma. There is an active search for leukotriene inhibitors for clinical application In the treatment of allergy, asthma and tissue injury and inflammation.

PGBx is both an inhibitor of leukotriene production and a leukotriene antagonist through its dual action as a phospholipase A„ Inhibitor and anti-oxidant. PGBx inhibits both leukotriene generation and acts directly on its free radical tissue damaging activity. For this reason, PGBx has activity as an agent to relieve bronchospasm and smooth muscle constriction involving leukotriene action on blood vessel, bowel or ureteral smooth muscle.

As seen in Figure 1, because the phospholipase activated biochemical pathway for the formation of prostaglandins and leukotrienes from free fatt acid is branched, inhibition of one branch of this pathway, as with NSAIAs can create an imbalance in these reactive metabolites. This imbalance ma actually aggravate inflammation and promote cell injury as evidenced by th gastric ulceration side effects of NSAIA's. Due to the adverse effects of both steroid and NSAIA's, there i currently much clinical medical interest to identify phospholipase A inhibiting agents that do not have steroidal side effects, but lik corticosteroids modulate the first step leading to the production of injuriou metabolites and free radicals, i.e., (See Fig. 1). Again, lik corticosteroids, PGBx blocks the initial reaction leading to the release o free fatty acid from membrane phospholipid activation by phospholipase A„, which is the primary trigger to the induction of membrane injury, inflammatio and cell death.

In regard to the above, we have discovered that PGBx inhibits bot phospholipases A„ and C in_ vitro which are the major cellular phospholipases that release arachidonate from membrane phospholipid. We have also discovered that PGBx inhibits the release of arachidonic acid from membrane phospholipid in intact human endothelial cells. This effect is selective in that PGBx inhibits preferentially when arachidonic acid production is stimulated, but levels are minimally effected in non-stimulated endothelial cells. In addition, we have discovered that PGBx inhibits the auto-oxidation of membrane phospholipid and, therefore, is a powerful anti-oxidant. Finally, we have discovered that PGBx has potent anti-inflammatory activity in _in vivo animal studies. Through stabilization of cell membrane integrity, depending on cell type and dosage, PGBx can inhibit the release of humoral and inflammatory agents (i.e., histamine, slow reactive substance) as well as free radical release involved in the process of injury and the body's reaction to injury or infection. PGBx is unique in having dual action as a phospholipase inhibitor and an anti-oxidant thus simultaneously acting protectively at two sites whic simultaneously prevent the production of substances injurious and inflammatory to tissue. PGBx also is unique in protecting previously damaged tissue; i.e., containing already oxidized lipld in the cell membrane from further injury by phospholipase action (see Fig. 6), as well as in distinguishing betwee stimulated and normal cells in effecting the release of arachidonic acid fro blood vessel endothelial cells.

As stated previously, PGBx will have value wherever steroidal and non-steroidal anti-inflammatory agents have been shown to be of benefit. PGBx will be equal or superior to known clinically useful anti-inflammatories because of its dual membrane protective action. In this regard, PGBx has been shown to block mast cell proliferation to IL-3 mast cells which are specialized cells in vessel walls and connective tissue that release histamine and phospholipases, increasing vascular permeability wherever injury or immune responses occur (see fig. 9).

The membrane protective action of PGBx enables it to be clinically useful in the broad pathophysiology of injury and disease associated with cell membrane destruction and dissolution. In that regard, PGBx is a pivotally active modulator of the key pathways that lead to cell injury and death. In effect, the pathophysiology of injury and death is a phospholipid mediated event.

In summary, the pathophysiology of tissue injury and the body's respons to injury is primarily mediated by phospholipase activation and free radica formation. Cell membranes, which provide for functional and structural integrity necessary for life are made up of phospholipids and the destructio of phospholipid integrity leads to not only organizational and functiona change, but also the formation of free radical chemical destructive agents which propagate further phospholipase activity and membrane destruction. Fre radicals are responsible for protein denaturation and nucleic acid stra breaks which propagate injury beyond the initial insult. In this regard, t pattern of injury functions in a manner analogous to a breeder reactor whi results in destructive energy formation beyond the initial input of energy the system. PGBx can be likened to the graphite rods that absorb neutrons stop an atomic reactor, i.e., PGBx inhibits both phospholipases and the fr radicals generated from phospholipase or host reactivity (white blood cell that enhance or propagate the initial infectious, ischemic hypoxic, chemic or traumatic insult to the host. Cell injury is membrane mediated and if y protect the cell membranes, you prevent or limit injury and permit recover PGBx stabilizes the lysosomal enzyme containing phospholipid envelope to blo the release of proteolytic hydrolases that destroy intracellular extracellular protein or connective tissue matrices necessary for cell a tissue integrity. In addition, PGBx can distinguish between stimulated a unsti ulated cells to block the release of prostaglandin and leukotrie precursors that play a role in inflammation.

By virtue of PGBx possessing anti-phospholipase, anti-oxidant, an anti-inflammatory activities, the following list represents clinical appli cations for PGBx in those diseases currently treated by steroidal and NSAI agents. These include:

(1) Topical application to inflamed skin and mucous membrane, i.e. poison ivy, allergy, thermal, actinic, chemical and radiation burns conjunctivitis and mucositis.

(2) Systemic administration in the treatment or prevention of ischemi injury and shock from sepsis, blood loss or trauma or syndromes secondary t ischemic or septic injury.

(3) Treatment of cerebral edema, cord and brain injury; multipl sclerosis, amyotrophic lateral sclerosis, encephalitis. (4) Treatment and prevention of localized ischemic injury to brain and heart (i.e., stroke, myocardial Infarction) and to peripheral limbs.

(5) Treatment of acute respiratory distress syndrome, endotoxin shock and acute renal failure.

(6) Treatment of contusions and reactions secondary to injury of muscle, bone and skin.

(7) Treatment of peptic ulcer, acute and chronic pancreatitis, ileitis, ulcerative colitis and cystitis.

(8) Treatment of toxicity and inflammation from infection or chemical injury.

(9) Treatment of symptoms related to inflammation or infection, i.e., pain, swelling, fever, malaise, muscle aches, headache.

(10) Treatment of asthma, laryngeal spasm, bowel, bladder or ureteral spasm and edema secondary to leukotriene formation and allergy or chemical injury.

(11) Treatment of rheumatoid arthritis, lupus erythematosis, rheumatic fever, glomerular nephritis and related hyperimmune or transplantation re¬ jection syndromes.

(12) Treatment of osteoarthritis

(13) Treatment of inflammatory reactions secondary to viral, ricketsial, bacterial, fungal and parasitic infection where inflammation or granulomas are major components.

(14) Treatment and prevention of radiation injury.

(15) Treatment and modulation of toxicity of drugs or radiation, i.e., agricultural, industrial and anti-tumor agents with free radical generating toxicity.

(16) Anti-snake and insect venom activity

(17) Anti-tumor activity In regard to free radical formation and clinical injury:

"Radiation damage to phospholipid membranes involves free radical chai reactions which propagate on their own. These reactions oxidize th constituent fatty acids to alkyl radicals which, upon oxygenation form lipi hydroperoxides" (Petkau, 1980). Peroxidation products have direct mutageni effects on DNA and, in addition, lipid hydroperoxides which result fro membrane injury or radiation can activate carcinogens such as N-hydroxy-N Acetyl 2-aminofluorene which can lead to tumor formation or effects on hos resistance, i.e., host response to infection and tumor growth via immunity o macrophage response. In addition, autoimmune disease, i.e., lupus, rheumatoi arthritis is associated with free radical related clastogenic product produced by lymphocyte reaction in serum which have chromosomal breaking o mutational effects. PGBx can be expected to block this because of its fre radical inhibiting action. In similar fashion to radiation induced free radical formation, there i evidence that acute cerebral or spinal cord injury from trauma or ischemia i also associated with lipid peroxidation. For this reason, PGBx has a place i stroke, spinal cord injury and infectious encephalitides. Although the dat is less clear for the role of free radicals or phospholipases in th demyelinating diseases of multiple sclerosis and amyotrophic lateral sclerosi as they represent the end stage of demyelinating syndromes and the myeli nerve sheath is a prime phospholipid membrane complex, it is to be expecte that PGBx will be of value in arresting or reversing the pathology of this disease. In this regard, corticosteroids are of short term, acute value i the treatment of CNS injury and multiple sclerosis and similar results for PGBx are based on the similarity of their action in inhibiting phospholipases. Polymorphonuclear leukocytes and macrophages which are mobilized during tissue injury and infection release the superoxide anion radical into the tissue space. These give rise to activated oxygen species such as hydrogen peroxide, hydroxyl radicals and singlet oxygen all of which have tissu damaging properties and increase microvascular permeability destroying th integrity of the blood supply governing oxygen and carbon dioxide diffusio tissue nutrition and the fluid mechanics responsible for tissue turgor an homeostasis.

Shock is simply defined "as the loss of effective circulating bloo volume" and the causes of shock can be multiple: trauma, blood and fluid loss sepsis, endotoxemia, ischemia and hypoxia, but the final common pathway i associated with damage to the microcirculation which loses its integrity t allow blood cells and fluid to leave the vascular system with decline i oxygenation and a fall in pH (increased acidity) . The latter activate phospholipase A„ to destroy phospholipid membranes and vascular and cellula integrity. Again, as mentioned previously, phospholipases release fatty acid to propagate free radical formation resulting in further injury an phospholipase activation.

Shock states are reversible depending on the extent and type of injur or circulatory loss, but in time become irreversible, despite efforts t improve circulatory tone or replace fluid and blood loss. In microcirculator terms, the irreversible stage of shock has been called "stagnant anoxia" state associated with sludging of blood and intravascular coagulation.

For many years, platelet aggregation was felt to be a primary culprit the irreversible circulatory and tissue damage of shock. PGBx inhibi platelet phospholipase C involved in the platelets production of prostagland and leukotrienes which mediate platelet tissue injury (Fig. 3) . More recentl the adherence of polymorphonuclear (PMN's) leukocytes to the endothelial wal of the blood vessels of the microcirculation are currently felt to be t primary contributing agents to irreversible tissue injury in shock. The PMN' adherence to blood vessel walls on reperfusion during attempts to resto circulatory function with administration of oxygen, colloids, blood and flu replacement provide the post-shock environment for PMN release of fr radicals and phospholipases. These punch holes in the blood vessel walls whi prevent adequate tissue perfusion denying oxygen, nutrients and preventing t elimination of wastes which block tissue recovery producing irreversib shock, despite efforts to restore fluid loss and blood pressure.

PGBx, as a phospholipase A„ and C inhibitor and a free radica scavenger, permits recovery from shock by blocking the primary steps whic lead to the loss of microcirculatory integrity and tissue injury and, i addition, block the damaging action of platelets or WBC's which propagat injury during fluid or blood replacement on perfusion efforts to promot recovery.

In summary, inflammation, shock and ischemia have related final commo pathways of pathophysiology based on platelet, cellular or WBC phospholipas activation, vascular injury and free radical formation which propagate injury.

In the case of thrombosis or ischemia as seen in stroke, coronar insufficiency with myocardial infarction or peripheral vascular disease, th loss of blood perfusion decreases oxygen availability with increased fall i pH (acidity) and/or PMN adherence which results in tissue damage or death unless the process can be arrested. PGBx arrests the physiologic instrument of injury and, in addition, even after membrane phospholipid oxidation afte injury, PGBx protects from further phospholipid destruction and permits tissu recovery. In this regard, PGBx stabilizes lysosomal membranes which protect cells from the action of endogenous proteolytic and lipolytic enzyme mediating autolysis and death.

In the case of skeletal or cardiac muscle ischemic injury (coronar occlusion or peripheral vascular occlusion) PGBx stabilizes the sarcoplasmi reticulum, maintaining the integrity of the muscle's contractile protei through its inhibition of phospholipases A„ and C and free radical action. I addltion, this action protects mitochondria, as described by Polis (1981) necessary for survival of the muscle and its tissue energy system.

As discussed previously, there is an added advantage to PGBx as, becaus of its phospholipase inhibition, it also interferes with platelet release o toxic factors and in addition, PGBx blocks the primary stages of intravascula coagulation to maintain the vascular patency which is lost in shock, ischemi and tissue injury.

PGBx is also of value in hyperimmune states such as is seen in allergy anaphylaxis, tissue transplant rejection and autoimmune disease. Immun reactions are associated with the same tissue events found in inflammation an at extreme levels, i.e., anaphylaxis, Arthus's phenomenon, tissue or orga rejection and immune reactions, can result in shock or tissue death.

As in the phenomenon of inflammation, immune reactions necessary for hos defense can exceed their protective role, mobilize WBC's and releas Inflammatory substances from tissue as well as host defensive mast cells an

WBC's .I.e., histamine, and leukotrienes whose release or toxic actions ar inhibited by PGBx.

Of interest to shock, allergy and inflammation, the corticosteroids whic produce inhibitors of phospholipase A_ have clinical value and have been use with varied but significant success for the last 35 years. More recently, th NSAIA compounds have been shown effect experimental endotoxic or septic shock but in contrast to corticosteroids, have had little success in the clinica treatment of allergy or acute trauma.

PGBx inhibits mast cell proliferation and inhibitors of mast cel degranulation, I.e., chromolyn, have been of value in allergic asthma. I addition, PGBx has been shown (Fig.7) to inhibit lymphocyte respons indicating that, in similar fashion to corticosteroids, it has a primary rol for its effects on inhibiting the effector lymphocytes from producing thei immune responses which provides a place for PGBx in blocking tissue rejectio as well as the symptoms of allergy, asthma and other hyperimmune states. mentioned previously, the anti-inflammatory and immune modulating action o PGBx provide a place for it in rheumatoid arthritis, rheumatic fever glomerular nephritis, lupus erythematosis, periarteritis and encephalitide and/or neuropathies (i.e., multiple sclerosis, amyotrophic lateral sclerosis which may have an immune basis, as well as to prevent rejection in tissu transplantation. The effects of PGBx on these immune mediated syndromes ar provided through its anti-inflammatory action as well as its action i directly modulating lymphocyte response. There is evidence that prostacyclin activity correlates with macrophag response and phagocytosis, antiviral (vaccine) and anti-tumor activit (Schultz and Chirigos, 1980; Stringfellow, DA, et.al., 1978). In contrast cyclo-oxygenase inhibitors have inhibited macrophage mediated suppression of cell blastogenesis. The role of PGBx as an antiinflammatory agent may b inhibitory to macrophage function as inhibition of this kind is seen for PG and cyclo-oxygenase inhibitors. However, PGBx may have direct modulatin action of its own on host resistance as the anti-viral modulator interfero induces prostaglandin biosynthesis (Fitzpatrick and Stringfellow, 1980) i association with viral infection. There is evidence that phospholipase A„ activation occurs on endotoxi administration. Endotoxins are toxic bacterial products responsible fo platelet aggregation, neutrophil vascular adherence, fever and shock. PGBx, as a phospholipase A„ inhibitor, will alter shock states secondary t infection. Inflammation characterizes viral and rickettsial infection (i.e., hepatitis, encephalitis, enterovirus, colitis and respiratory infection), bacterial and fungal infection (i.e., pneumonia, abscess formation, granulomas) as well as parasitic disease of protozoal or helminthic origin. PGBx, as is true for corticosteroids, can modulate and moderate the degree of toxic inflammatory reaction to infectious agents. Snake venoms are lethal because as hemolytic or neurotoxic poisons the function as phospholipase activators to destroy cell membranes and this can b inhibited by PGBx action. Insect venoms release tissue damaging substance which can behave like snake venoms for local toxic action or can produc inflammation or allergic responses, i.e., bee stings, tick and mosquit reactions.

Muscle fatigue from excess exercise is associated ^' with a pH fall anoxemia, heat generation and muscle tissue destruction in similar fashion t what is seen in ischemia. In contrast to ischemia, muscle fatigue can b reversible, but under some clinical circumstances in marathon runners and rac horses fatigue and heat buildup, can result in diffuse Irreversible damage This is similar to what can occur in coronary ischemia when cardiac output i increased. PGBx can relieve symptoms and protect from damage related t muscle fatigue, spasm, ischemia, trauma and sprain because of it anti-inflammatory action and protective action in ischemic injury.

Prostaglandins have been shown to be cytoprotective in blocking pepti ulceration, as well as preventing GI tract and bladder injury to chemotherapy PGBx, because of its membrane stabilizing action, has value in the preventio of ulcerogenic and autolytic activity. Burns: Thermal and radiation: While anti-inflammatory actions of PGB may be a factor in burn injury, the activation of leukotrienes may also be factor in effecting the extent of tissue injury. PGBx may be useful i treatment of both first and secondary burns, i.e., sunburn, which have a inflammatory component. There is evidence that phospholipase A„ activity is associated wit metastases and nidation of tumor cells (Liotta, 1986) . The action of PGBx a a phospholipase A„ inhibitor will be useful in preventing metastatic tumo spread or local tumor growth by direct extension. Tumor growth is modulate by effecting platelet, vascular or inflammatory events. In further support of this is the work of Stringfellow and Fitzpatri (1979) : Prostaglandin D2 is inversely associated with lung metastases of B melanoma in a rodent model and indomethacin increases metastatic spread. possible mechanism for this is related to the action of prostaglandin D2 blocking platelet adherence and release of coagulation factors which similar to the action of PGBx.

There is also evidence that phospholipase A_ activity is associated wi oncogene expression. For this reason, the action of PGBx may also serve inhibit the expression of tumor virus related cancer development. Another area of PGBx action involved in the prevention or modulation inflammation is its effects on blood coagulation. Platelets respond physiologic stimuli, i.e., thrombin, collagen and ADP, by aggregating wi other platelets and blood cell elements and, in this process, degranulating releasing the contents of intracellular granules. The degranulation o release phase of the platelet reaction recruits more platelets and blood cell into the region to promote clot formation or thrombosis. The release o arachidonic acid from phospholipid and its conversion to the prostaglandin thromboxane, is absolutely necessary for platelet aggregation, degranulatio and clot formation. Thus, nonsteroidal agents such as aspirin inhibit th synthesis of thromboxane from free fatty acid and suppress the coagulativ response. Similarly then, agents which inhibit the release of free fatt acids from membrane phospholipid would be expected to inhibit the productio of thromboxane by blocking the release of the precursor, arachidonic acid Thus, phospholipase inhibitors such as PGBx will affect the aggregatio release reaction of human platelets.

As indicated previously, we have direct evidence that PGBx inhibits th platelet aggregation release reaction and evidence that PGBx also affect clotting time (See Fig. 9). Pain, hyperalgesic states elicited by inflammation or injury t peripheral nerves is mediated by prostaglandins, particularly, prostaglandi E_. Prostaglandin effects on pain production occur via bradykinin o noradrenalin action, mediators which effect pain nerve afferents and ar released in inflammation or injury. Corticosteroids can suppress bradykini or noradrenalin related pain and, in similar fashion to PGBx, suppres prostaglandin synthesis and work through effects on inhibition o phospholipase A„. Thus, in similar fashion to corticosteroids, PGBx wil inhibit not only the primary inflammatory response, but suppress the action o post ganglionic neurons, small diameter afferents responsible for pai perception and generation.

Additional roles for PGBx include:

(18) Tissue stabilization: Use as food additives to prolong th shelf-life of foods or pharmaceutical preparations. Subject to oxidativ injury, i.e., rancidity, discoloration, loss of taste.

(19) Prolongation of half life of cells in tissue culture.

(20) Embalming

(21) Anti-autolysis: (a) prolongation of half life of muscle at higher temperatures o in pigs with the autolytic muscle destroying syndrome.

(b) preservation of blood or tissue for transfusion, transplan tation or grafting.

(c) preservation of pathologic or embryonic tissue specimens. PGBx can prolong the shelf life of foods, or pharmaceutical preparation subject to oxidative injury, i.e., rancidity, discoloration, odor and loss o taste.

PGBx can provide a substitute to sodium bisulfite (NaBiSulfite) , vitami E, vitamin C, BHT and other antioxidants in use by the Food and Pharmaceutica Industry (common food preservative used to maintain the color and taste vegetables, fruit, meat and dairy products in restaurants and shops as well preserving potency of antibiotic, vaccines or chemical drug preparation).

In regard to the above, PGBx can substitute for BHT, vit. E, vit. phenols and related anti-oxidant preservatives. PGBx, a naturally deriv product can extend the shelf life of milk, cream, butter, bacon and oth preserved meats by reducing the endogenous breakdown of phospholipid membran and the action of free radicals.

PGBX can be used in embalming for both immediate and long te preservation of tissue. Because of its phospholipase inhibiting and fr radical scavenging activity, PGBx can be useful in tissue culture and isolat organ maintenance (kidneys, heart, lung, liver) to prolong the half life cultures so important to monoclonal antibody, or leukokine production, i.e

IL2, interferons, or organ transplantation. The anti-autolytic, cytoprotective action of PGBx will be of value i pig strains whose meat is unusable following slaughter because their muscl autolyze (dissolve) too rapidly. PGBx stabilizes meat to provide for long half life at higher temperatures. PGBx can be administered prior slaughter, injected post mortem via arteries under pressure, or appli directly to meat preparations.

In similar fashion, as mentioned previously, PGBx is useful in preservi organs for transplantation, i.e., kidney, heart, lung, liver, skin, as well a preserving pathology specimens that autolyze rapidly, i.e., stomach and G tract, epithelium, brain following surgical biopsy or autopsy. Brief Description of the Drawings

Fig 1 is a chart showing fatty acid release from membrane phospholipid and the action of anti-inflammatory agents.

Fig 2 is a chart showing inhibition of phospholipase A_ by PGBx and PGB.. on phospholipase A„ activity. Fig 3 is a chart showing the inhibitory effect of PGBx on lysosomal phospho¬ lipase A_ and phosphatidyl inositol-specific phospholipase C. Fig 4 is a chart showing the specific interaction of PGBx with highly purified snake venom phospholipase A„. Fig 5 is a chart showing inhibitory effect of PGBx on auto-oxidation of phosphatidylethanolamine. Fig 6 is a chart showing inhibitory effect of PGBx on the preferential hydrol¬ ysis of peroxidized phosphatidylethanolamine by dog sarcoplasmic reticulum phospholipase C. Fig 7 is a chart showing the inhibitory effect of PGBx on the generation of cytotoxic T lymphocytes. Fig 8 is a chart showing the inhibitory effect of PGBx on IL-3 dependent proliferation of mast cells. Fig 9 is a chart showing the inhibitory effect of PGBx on human platelet aggregation.

Description of the Preferred Embodiment

Previous studies had indicated that PGBx could protect ischemic brain and heart from tissue damage and could restore the ^ vitro phosphorylating ability of degraded rat liver mitochondria (patent 4,245,111 to D. Polis 1/1981), but no mechanism of action (affecting cell membranes) had been taught prior to this invention. We found that PGBx affected fundamental events at the level of the cell membrane. Two such fundamental discoveries of ours were that PGBx inhibits fatty acid release from membrane phospholipid by activation of cellular phospholipases (see figure 1) and oxidation of membrane phospholipids by oxygen free radicals which are universally associated with cell injury and membrane dysfunction. Indeed, these phenomena represent the predominate mode of membrane injury leading to cell death. In addition, it has been observed that oxygen free radicals peroxidize unsaturated fatty acids in membrane phospholipid and these modified phospholipids are consequently les functional and more susceptible to attack by cellular phospholipases which w have shown is inhibited or prevented by PGBx.

EXAMPLE 1:

Phospholipase Inhibition:

We demonstrated the effect of PGBx, the polymer, and PGB.. , the monomer, on the hydrolysis of radiolabelled phospholipid by a highly purified huma white blood cell phospholipase A„ that was Ca2 -dependent and most active at physiologic pH (figure 2). We showed that the PGBx ingredients, but not the monomer PGB.. was a potent inhibitor of phospholipase A„ activity in_ vitro; and as shown in table I, the inhibitory activity of PGBx toward a human plasma phospholipase A„ is stable to autoclaving. PGBx as used herein, is to be understood to include any and all macromolecules including a polymer, a dimer, a trimer or tetramer, etc., of the monomer PGB₁.

TABLE I

Effect of Autoclaving on the Phospholipase A„ Inhibitory Activity of PGBx

Percent Inhibition of Human Plasma PLA„ Activity

PGBx (uM) Untreated Autoclaved 5 13.2% 11.3%

10 67.3% 60.0%

20 71.8% 80.9% The dose required for 50% inhibition of the human white blood cell (H>_cn was 4 uM and this inhibition was independent of the calcium concentration i the reaction mixture.

In order to demonstrate how PGBx inhibited phospholipase A_ activity reaction mixtures containing a fixed concentration of drug and varyin concentrations of either enzyme (snake venom phospholipase A„) or phospholipi were studied. The results indicated that the inhibition by PGBx wa independent of the phospholipid substrate and therefore the drug probabl associates with the enzyme itself. Direct binding of PGBx to a highl purified snake venom phospholipase A„ is shown in figure 4. PGBx, at 10 u concentrations completely inhibited tryptophan fluoresence that is eviden with the enzyme alone. A highly purified snake venom enzyme was necessary fo this experiment because adequate quantities of human phospholipase A„ neede to produce sufficient fluoresence signal are not available. The above data demonstrate that the polymer and/or other macromolecule of PGBx has unique inhibitory activity toward phospholipases A_ Ln vitro Phospholipase A_ (PLA„) specifically releases the cis-unsaturated fatty acid predominately esterified in the 2-position of mammalian phospholipids. Th 2-acyl position, therefore, contains most of the arachidonic acid which mus be released by the action of phospholipase A„ to generate the "free" precurso for prostaglandin and leukotriene synthesis. From the above study, w recognized that the mechanism by which PGBx interacted with membran phospholipid to inhibit the phospholipases A„ could produce generalize stabilization of membrane phospholipid composition and, therefore, function by inhibiting other important cellular phospholipases as well.

Cells contain lysosomal phospholipases that are most active at acid p (pH 4 to 5) . These enzymes contained within an organelle, the lysosome degrade ingested intracellular membrane phospholipids such as those fro phagocytized bacteria. These enzymes are also released externally o interstitially by phagocytic cells in response to inflammatory stimuli a contribute to adjacent, or contiguous membrane breakdown in inflammato fluids. An additional lipolytic enzyme, phosphatidylinositol (PI) specif phospholipase C, plays an important role in membrane signal transduction specifically hydrolyzing PI, a membrane phospholipid present in sma quantities but highly enriched in arachidonic acid. Thus, the Pi-specif phospholipase C also contributes to arachidonic acid release from membra phospholipid during inflammation. Based on the above, and its importance inflammation, we tested the effect of PGBx on both lysosomal phospholipase and human platelet Pi-specific phospholipase C (figure 3) and we discover that PGBx not only produced dose-dependent inhibition of the human white blo cell and plasma phospholipases A„, but it also inhibited the lysosom phospholipase A„ and the Pi-specific phospholipase C.

Despite effects on other phospholipases, the neutral-active a Ca2 -dependent phospholipase A„ inhibited by PGBx is the major lipolyt enzyme in the pathway leading to the generation of ^' inflammatory metabolite prostaglandins and leukotrienes. Because of this, it is important to no that the dose required for 50% inhibition of the white cell phospholipase

(4 uM) was 10-fold less than that required to inhibit the lysosom phospholipase A„ and the Pi-specific phospholipase C (PLC) (approx. 40 uM)

These _jLn vitro data clearly support our concept that PGBx functions stabilize membranes by suppressing phospholipid turnover mediated by cellul phospholipases, particularly phospholipase A„. The differences phospholipase inhibitory sensitivities, i.e., PLA„ versus PLC indicate tha PGBx can be used clinically to inhibit different phospholipases in a dos response or regionalized manner to obtain clinical results with a controlle therapeutic index.

The inhibition of _in_ vitro phospholipase activity indicated to us tha

PGBx could inhibit the cellular turnover of arachidonic acid and this, i turn, would suppress the production of further pro-inflammatory metabolites prostaglandins, leukotrienes and free radicals. Therefore, human umbilica

14 cord endothelial cells were cultured in the presence of C-arachidonic aci to allow for incorporation of the radiolabelled fatty acid into cell membran phospholipid. Washed cells were challenged with histamine and A23187 neurotransmitter or ionophore stimulants known to cause the mobilization o arachidonic acid from cell membrane phospholipid.

EXAMPLE 2

^"4 Effect of PGBx on Release of C-Arachidonic Acid from Prelabelled

Human Endothelial Cells

TABLE II

14 C-Arachidonate released (cpm)

Cultured Endothelial Cells Control Histamine A23187

Cells alone 922 2594 3080

Cells alone + PX (10 uM) 699 1141 1232

Percent Inhibition 24% 56% 60% _:

As indicated by the in_ vitro experiments, Table II shows that 10 uM PGB

14 inhibited the histamine and A23187-induced release of C-arachidonate, 56 and 60%, respectively. That the same dose of PGBx had less effect on th control rate of turnover (24%) indicates that PGBx has a preferential effec on the stimulus-induced turnover of membrane phospholipid and this observatio suggested to us that PGBx will have beneficial effects on the fundament inflammatory reactions in which arachidonate mobilization and ^"prostagland and leukotriene production are markedly increased. Thus, we have discovered basic molecular mechanism for PGBx: inhibition of phospholipase A„ media fatty acid mobilization _in vitro and _in_ situ; this property alone w stabilize membrane phospholipid composition and, therefore, protect cellu function.

EXAMPLE 3:

Inhibition of Lipid Peroxidation:

The carbon-carbon double bonds of unsaturated fatty acids fo predominately in the 2 position of mammalian phospholipids are particula susceptible to attack by oxygen free radicals. Radical-induced peroxidat of membrane phospholipid is a well-known mediator of cell injury from expos to toxic drugs and chemical agents as well as oxidative injury to foo pharmaceutical agents and biological specimens. Therefore, anti-oxidants h widespread industrial and pharmacologic uses as preservatives. Peroxidati of membrane phospholipid initiates a self-propagating free-radical reacti that results in membrane dysfunction with concomitant alterations phospholipid composition mediated by activated phospholipases. The fact th PGBx interacted with membrane phospholipid to inhibit _in vitro and _in. si phospholipase activities and contains peroxidizable double bonds itsel indicated to us that PGBx might also influence the susceptibility phospholipid to auto-oxidation.

In experiments testing the effect of PGBx on peroxidation of the membra phospholipid, phosphatidylethanolamine, we discovered that PGBx h anti-oxidant activity. Figure 5 shows that PGBx protected phospholipid fr auto-oxidation in the concentration range in which it inhibits phospholipa activity _in_ vitro and _in_ situ.

The demonstration that PGBx is a powerful anti-oxidant is shown in Tab III which compares the anti-oxidant activity of PGBx with sodi metabisulfite, a widely used commercial anti-oxidant. In these experiment the phospholipid was incubated for 24 hrs at 37 C at pH 5.0 with and without

PGBx or bisulfite; 51.5% of the phospholipid was peroxidized by this treatment. Both PGBx and bisulfite inhibited the peroxidation of phospholipid, but PGBx was 100-times more effective as an anti-oxidant than bisulfite; 10 uM PGBx provided 23.9% protection while 1000 uM bisulfite afforded 19.8% protection. Thus, we have discovered that PGBx has potent anti-oxidant activity and, therefore, can be used as a commercial preservative to enhance the shelf-life of drugs, vaccines, and other chemicals susceptible to oxidative damage. In addition, because PGBx also inhibits phospholipas activities which contribute to autolysis of foods and biological tissu samples, PGBx can be used as a preservative for foods and meats as well as for blood and blood products, organs for transplantation and pathologic an embryonic tissue samples. PGBx survives autoclaving (Table I) and filtratio and, thus, can be a useful preservative or stabilizer in situations wher sterility is required.

TABLE III Comparison of the Anti-Oxidant Activity of PGBx and Sodium Meta Bisulfite

Percent Peroxidized Percent Phosphatidylethanolamine Protection

Control 51.5%

Control + PGBx 10 uM 39.2% 23.9%

Control + PGBx 100 uM 14.7% 71.5%

Control + NaBisulfite

1000 uM 41.3% 19.8%

Control +- NaBisulfite

5000 uM 36.1% 29.9% In recent studies in our laboratory, we have discovered a highly unusu phospholipase C in preparations of dog sarcoplasmic reticulum that degrad membrane phospholipid as free radical production is increased with concomita loss of membrane organelle function. This enzyme is unique because hydrolyzes peroxidized, but not native or intact phospholipid when both a present in equimolar mixtures. The oxidized membrane phospholipid preferentially degraded. This is the first phospholipase to our knowledg that selectively hydrolyzes peroxidized phospholipid. The pivotal role oxygen free radicals in membrane injury suggested to us that an agent li PGBx capable of inhibiting peroxidation and phospholipid turnover would extremely beneficial in the management of free radical induced inflammati and injury. Therefore, we tested the effect of PGBx on the hydrolysis previously peroxidized phosphatidylethanolamine using the sarcoplasm reticulum phospholipase C. Figure 5 shows that PGBx produces a dose-depende inhibition of the hydrolysis of peroxidized phospholipid. Thus, we ha discovered that PGBx can inhibit the hydrolysis of membrane phospholipid ev after it has been injured by peroxidation. Thus, PGBx can not only inhib peroxidation itself, but can also intervene in the injury process aft initiation of the oxidative insult. Naturally occurring anti-oxidants such vitamins A, E, and C, B-carotene, superoxide dismutase (SOD) and catala function as cellular radical scavengers and SOD have known protective effec against cell injury or inflammation.

That PGBx has both intrinsic anti-phospholipase and anti-oxida activities provides a synergistic advantage via simultaneous inhibition of t fundamental reactions stimulated in inflammatory processes. Suppression o both phospholipase and oxidant activity by PGBx indicated to us that PGB would function as an ideal broad spectrum anti-inflammatory agent. EXAMPLE 4 :

The anti-inflammatory activity of PGBx was assessed using th carrageenan-induced rat paw edema model. This model of inflammation ha provided the best correlation to date of _in vivo anti-inflammatory activity i an animal model with clinical effectiveness in man. Table IV shows that PGB given intraperitoneally (1.5 mg/kg) 1 hr prior to the injection of carrageena into the foot pad inhibited edema 50% as measured 4 hours after the injectio of the irritant.

TABLE IV

Anti-Inflammatory Activity of PGBx Using Carrageenan-induced Rat Paw Edema

Foot Width (mM) % of Control

Control 4.2 (n=10) 100%

Control + carrageenan 8.7 (n=8) 207% Control + carrageenan + PGBx 6.4 (n=5) 152%

* SD is equal to or less than 0.1 mM in each group. ** PGBx (1.5 mg/kg) injected IP 1 hr prior to irritant.

Table V compares the effectiveness of PGBx with a wide variety o anti-rheumatic nonsteroidal and steroidal anti-inflammatory agents. PGBx is potent anti-inflammatory agent and is clearly more effective than all th NSAIA's currently in use and better than hydrocortisone and cortisone, th over-the-counter steroids, although not equal to paramethasone. TABLE V

Comparison of Anti-Inflammatory Effectiveness of PGBx and 14 Anti-Rheumatic

Drugs Using the Carrageenan-induced Rat Paw Edema Model

Substance ^ED50 (^ms/^k§ injected IP)

Indomethacin 2.2

Mefenamin Acid 9

Flufenamic Acid 10

Phenylbutazone 25 Amidopyrine 31

Phenacetin 57

Aspirin 72

Phenazone 87

Acetaminophen 88

Cinchophen 92

Sodium Salicylate 98

PGBx 1.5

Paramethasone 0.06

Hydocortisone 30

Cortisone 40

EXAMPLE 5.

We have examined the effects of PGBx on the generation of cytolytic lymphocytes (CTL) in mixed leukocyte cultures (MLC) , historically one of t most useful systems for the _in_ vitro analysis of the cellular arm of t immune response. Splenic leukocytes from one genetically inbred strain mice (C57BL/6; H-2 ) were cultured with irradiated allogeneic leukocytes fr a genetically disparate strain (DBA/2; H-2 ). PGBx was tested at concentra¬ tions ranging from 1 to 50 uM by adding it to the culture medium (RPMI 1640 supplemented with 5% fetal calf serum, 2 mM L-glutamine, 50 uM 2-mercapto- ethanol, and antibiotics). At the initiation of MLC, after 3 days of incubation, the level of CTL activity against "target" cells homologous with

2 51 the alloantigen (P815 mastocytoma cells; H-2 ) was measured in a standard Cr release assay. Results are shown in Figure 7. PGBx significantly inhibited

CTL induction in a dose dependent manner at dosages from 10 to 50 uM.

The effect of PGBx has been tested on activated CTL by its addition to the cytotoxicity assay. In contrast to the effect of PGBx on the initiation of CTL, the drug did not affect the cytolytic capacity of functional CTL.

Development of CTL in MLC requires complex interactions between CTL precursors, helper T cells, and macrophages. The macrophages and helper T cells produce the immune system hormones (leukokines) interleukin 1, interleukin 2, and gamma-interferon. The suppressive effect of PGBx on CTL development could be exerted at one or more levels by inhibiting the production and/or activity of interleukin 1, interleukin 2 or gamma-inter- feron. These immunologic hormones also contribute to the pathogenesis of chronic inflammatory disease (i.e., rheumatoid arthritis). From the foregoing, the following advantages and novel features of this invention should be apparent: First, the essence of this invention is the recognition and experimental demonstration that PGBx affects fundamental membrane phospholipid reactions of phospholipase-induced degredation and free radical peroxidation. The discovery of these dual properties, anti-phospho- lipase and anti-oxidant activities of PGBx, establishes a sound scientific basis for the molecular action of PGBx in protecting the cell and its membrane from injury. It predicts in theory, and confirms with experimental results that PGBx is a potent anti-inflammatory agent. Of particular advantage is that PGBx works at the site of inhibitory action for the arachidonate cascade, and the preferential effect on stimulus-induced mobilization of arachidonate

Inhibition of phospholipase A„ will depress the production of both prosta glandins and leukotrienes in stimulated or inflamed cells. Importantly, PGB had a much more pronounced effect on stimulus-induced, than control release o arachidonate indicating a selective effect on the former. Moreover, whe phospholipid was peroxidized, PGBx inhibited the degradation of this lipid b lysosomal phospholipase C indicating that PGBx can protect already damage (oxidized) membranes. Whatever PGBx multiple actions are responsible fo anti-inflammatory action, on the basis of the rat carrageenan model, it i evident that PGBx can effectively rival or replace both currently availabl steroids and NSAIAs in the treatment of inflammation and injury making it candidate for clinical application and usefulness in localized and systemic injury and disease. In addition, the action of PGBx in inhibiting mast cell and lymphocyte response indicates that PGBx is useful in effecting the primary effector systems that are associated with inflammation and tissue injury in hypersensitivity states.

The effect of PGBx was tested on the proliferation of mast cells. Propagation of mast cells _in_ vitro requires a mast cell growth hormone, interleukin 3. Ten micromolar PGBx was added to cultures of mast cells in medium containing serial dilutions of interleukin 3 (Fig. 8). Proliferation

3 was assessed by measuring incorporation of H-thymidine into DNA. The proliferative response of mast cells was inhibited over the entire dose range of IL-3. Thus, PGBx may be useful in preventing or modulating immunologic reactions involving mast cells (e.g., allergic reactions) by inhibition of mast cell development.

The fact that production of the prostaglandin, thromboxane, from free arachidonic acid is required for platelet function indicated to us that PGBx should affect the platelet aggregation release reaction. Figure 9 shows that

10 uM PGBx inhibits human platelet aggregation release reaction by approximately 50% compared to control. These data are consistent with previously described studies using prelabelled human neutrophils endothelial cells. PGBx inhibits stimulus response release of arachid acid from platelets to minimize the production of powerful mediators inflammation and injury.

REFERENCES

Chemistry:

1. Polis BD, Kwong S, Polis E, Nelson G and Shmukler HW: Studies of PGBx. polymeric derivative of prostaglandin B. : I Synthesis and purification PGBx. Physiological Chemistry and Physics 11, 109-123, 1979.

2. Polis DB, Kwong S, Polic E and Nelson GL: PGBx, an oligomeric derivati of prostaglandin B₁ : Physical and chemical and spectral propertie Physiological Chemistry and Physics 12, 167-177, 1980.

3. Shmukler HW, Kwong S, Polis E, Zawryt MG and Soffer. Fractionation PGBx, An oligomeric prostaglandin, by Column Chromatography Hydroxylapatite. Physiological Chemistry and Physics 12, 551-557, 1980

4. Shmukler HW: Cation complex formation with PGBx, A prostagland oligomer, as measured by fluroescence quenching. Physiological Chemist and Physics 12, 558-563, 1980.

13

5. Polis BD, Polis E and Kwong S: C Nuclear magnetic resonance studies prostaglandin B monomers as an approach to elucidation of the structu of PGBx, a prostaglandin B polymer. Physiological Chemistry and Physi 13, 111-119, 1981.

6. Polis DB, Polis E and Kwong S: Structutal features of PGBx prostaglandin polymer) deduced by analogies with dimers derived fr 15-keto-prostaglandin B... Physiological Chemistry and Physics 1 531-548, 1981.

7. Nelson GL and Verdine GL: The base promoted oligomerization 15-dehydro-prostaglandin B- : Dimer formation and structural implicatio for a complex mixture termed PGBx. Tetrahedron Letters 24, 991-99 1983.

Biochemical Studies:

1. Polis BD, Grandizio AM and Polis E: Some in vitro effects of a n prostaglandin derivative. Advances in Experimental Medicine and Biolo 33, 213-220, 1973.

2. Polis BD, Polis E and Kwong S: Protection and reactivation of oxidati phosphorylation in mitochondria by a stable free-radical prostagland polymer (PGBx). Proc. Natl. Acad. Sci. U.S.A., 76, 1598-1602, 1979.

3. Ohnishi ST and Devlin TM: Calcium ionophore activity of a prostagland B₁ derivative (PGBx). Biochemical and Biophysical Research Communic tions 89, 240-245, 1979.

4. Aronson CE and Guerrero NH: Effects of PGBx on the isolated perfused r heart. Gen. Pharmac. 10, 303-308, 1979.

5. Devlin MT, Devlin TM and Ohnishi ST: Anti-sickling effect of prost glandin B- derivatives. Biophysical Journal 33, 91 (abstract) 1981. 6. Kreutter DK and Devlin TM: Inhibition of mitochondrial F.F -ATPase PGBx, A derivative of prostaglandin B.. (abstract). Fed. Proc. 39, 215 1980.

7. Pieper GM, Salhany JM, Wu ST, and Eliot RS: Prostaglandin B (PGB protects the adenine nucleotide pool during myocardial ischem (abstract), Clinical Research 14, 193, 1966.

8. Aronson CE: Relationships between PGBx and isoproterenol in the isolat perfused rat heart. Gen. Pharmac. 10, 103-107, 1980.

9. Aronson CE: Interactions between PGBx and disophenol in the isolat perfused rat heart. Gen. Pharmac. 12, 249-252, 1980.

2+

10. Kreutter DK, and Devlin TM: Modulation of mitochondrial Ca retention

PGBx, an oligomeric derivative of prostaglandin B.. (abstract) Fed. Pro 40, 1562, 1981.

11. Kometani T, Devlin MT and Ohnishi ST: Mechanism of divalent cati ionophoretic activity of PGBx (abstract) (not available) . Internation Biophysics Congress, Mexico, 1981.

12. Shmukler HW, Zawryt MG, Soffer E, Feely W and Polis E: Interaction bovine serum albumin with PGBx (Polymeric 15-keto-prostaglandin B.. Physiological Chemistry and Physics 13, 241-250, 1981.

13. Shmukler HW, Soffer E, Zawryt MG, Polis E, Feely WM, Kwong SF and Cope Mechanism of polymeric prostaglandin PGBx for _jLn vitro stabilization rat liver mitochondrial oxidative phosphorylation. Physiol. Chem. Phy 14, 445-469, 1982.

14. Shmukler HW, Zawryt MG, Soffer EF, Polis E, Feely Wm, Kwong SW and Co FW: Studies on the mechanism of mitochondrial protection by plymer prostaglandin PGBx. Physiological Chemistry and Physics 14, 471-48 1982.

15. Kruger M and Booyens J: The effect of the prostaglandin derivative PG on calcium uptake and release by skeletal muscle sarcoplasmic reticulu Sa Mediese Tydskrif Deel 62, 855-858, 1982.

16. Kreutter DK, "and Devlin TM: Inhibition of oxidative phosphorylation an oligomer of prostaglandin B.., PGBx. Archives of Biochemistry a Biophysics 221, 216-226, 1983.

17. Aronson CE: Effects of PGBx on glucose utilization and glycogen conte of the isolated rat diaphragm. Gen. Pharmac. 14, 519-523, 1983.

2+

18. Uribe S, Israelite C and Devlin TM: Ca ionophoretic properties prostaglandin B.. cligomers. Fed. Proc. 43, 1576, 1984. Animal Studies:

1. Polis BD and Polis E: Normalization of the diabetic syndrome hereditary diabetic mice by PGBx, a polymeric derivative of prostaglan B. Physiological Chemistry and Physics 8, 429-436, 1976.

2. Yamazaki H, Bodenheimer M, Banka U, Lewandowski J and Helfant R: effect of a new prostaglandin, PGBx, on length-tension relations following partial coronary occlusion and reperfusion. Abstracts Americ Heart Association 51st Scientific Session, Dallas, TX. , 1978.

3. Moss G, Magliocchetti T and Quarmby R: Immediate restoration of C autonomic cardio-pulmonary control: Survival of "lethal" cerebral hypox by treatment with PGBx. Surgical Forum 29, 1-3, 1978.

4. Polis BD and Polis E: Dose dependence of PGBx. A polymeric derivative prostaglandin B, for normalization of hereditary diabetics of the mous Physiological Chemistry and Physics 11, 3-8, 1979.

5. Angelakos ET, Riley RL and Polis BD: Recovery of monkeys after myocardi infarction with ventricular fibrillation. Effects of PGBx. Physiologic Chemistry and Physics, 12, 81-96, 1980.

6. Kolata RJ and Polis BD: Facilitation of recovery from ischemic bra damage in rabbits by polymeric prostaglandin PGBx, a mitochondri protective agent. Physiological Chemistry and Physics 12, 545-550, 1980

7. Polis E and Cope FW: Dose-dependent reduction of hereditary obesity the non-diabetic mouse by polymeric prostaglandin PGBx. Physiologic Chemistry and Physics 12, 564-568, 1980.

8. Walls AP, Himori N and Burkman AM: Comparison of the effects prostaglandin Bx and verapamil on changes in myocardial function th occur during ischemia (abstract). Federation Proceedings 40, 692, 1981.

9. Polis E, and Cope FW: Chronic effects of PGBx, A polymeric prostaglandi on blood leukocyte counts. Physiological Chemistry and Physics 1 431-437, 1982.

10. Polis E and Cope FW: Polymeric prostaglandin PGBx and other prostagland polymers prolong survival of the heart of the hypoxic mouse. Avia Space Environ. Med. 54, 420-424, 1983.

Claims

We claim:

1. A membrane protective agent comprising a macromolecular derivative of fatty acid.

2. The membrane protective agent of claim 1 further comprising a capabilit for stabilizing membranes against degradation.

3. The membrane stabilizing and protective agent of claim 2 comprising polymers, and oligomers of prostaglandin Bl.

4. The multifunctional membrane stabilizing and protective agent of claim comprising a macromolecular derivative of a fatty acid.

5. The membrane stabilizing agent of claim 2 comprising a macromolecular derivative of a prostaglandin.

6. The membrane protective agent of claim 1 comprising a macromolecular derivative of prostaglandin Bl.

7. The multifunctional membrane stabilizing and protective agent of claim comprising a macromolecular derivative of a prostaglandin.

8. The membrane stabilizing agent of claim 2 comprising a macromolecular derivative of prostaglandin Bl.

9. The membrane protective agent of claim 1 comprising a macromolecular derivative of. prostaglandin Bl.

10. The membrane stabilizing and protective agent of claim 2 comprising PGB containing a macromolecular derivative of prostaglandin Bl.

11. The multifunctional membrane stabilizing and protective agent of claim comprising multiple macromolecular derivatives of prostaglandin Bl.

12. The multifunctional agent of claim 2 for use as a membrane stabilizing and cytoprotective agent.

13. An anti-pathophysiological agent comprising PGBx.

14. The anti-pathophysiological agent of claim 13 further comprising macromolecules of prostaglandin Bl in said PGBx.

15. The anti-pathophysiological agent of claim 13 for use in vivo.

16. The anti-pathophysiological agent of claim 13 for use as an inhibitor o phospholipase action jLn vivo.

17. The anti-pathophysiological agent of claim 13 for use in protection of cell membranes in vivo against degradation by free radicals.

18. The anti-pathophysiological agent of claim 13 for protection of cell membranes, in vitro, against degradation by free radicals.

19. The anti-pathophysiological agent of claim 13 for protection of cell membranes against degradation by phospholipase _in_ vivo.

20. The anti-pathophysiological agent of claim 13 for protection of cell membranes against degradation by phospholipase in vitro.

21. The anti-pathophysiological agent of claim 13 for inhibiting cell dama caused by phospholipase.

22. The anti-pathophysiological agent of claim 13 for inhibiting cell dama caused by free radicals.

23. The anti-pathophysiological agent of claim 13 for inhibiting cell damag produced by photoactivation or chemically exogenously mediated free radical forming agents.

24. The anti-pathophysiological agent of claim 13 for inhibiting cell damag caused by oxidation.

25. The anti-pathophysiological agent of claim 13 for inhibiting cell damag induced by endogenous free radical or oxidizing agents such as porphyrins.

26. The anti-pathophysiological agent of claim 13 for inhibiting cell damag caused by phospholipase and free radical synergistic damaging effects.

27. The anti-pathophysiological agent of claim 13 for inhibiting cell damag caused by cell membrane free fatty acid release.

28. The anti-pathophysiological agent of claim 13 for inhibiting cell damag caused by leukotriene action.

29. The anti-pathophysiological agent of claim 13 for inhibiting cell damag caused by prostaglandin imbalance.

30. The anti-pathophysiological agent of claim 13 for inhibiting platelet aggregation, release and vascular endothelial adhesion.

31. The anti-pathophysiological agent of claim 13 for inhibiting WBC aggregation, release and vascular endothelial adhesion.

32. The anti-pathophysiological agent of claim 13 for inhibiting undesirabl endothelial-vascular reactions.

33. The anti-pathophysiological agent of claim 13 for inhibiting auto oxidation of phospholipid and other lipids.

34. The anti-pathophysiological agent of claim 13 for inhibiting inflammation.

35. The anti-pathophysiological agent of claim 13 for inhibiting lymphocyte and mast cell responses involved in immune or inflammatory reactions.

36. The anti-pathophysiological agent of claim 13 for inhibiting the symptom of inflammation from whatever primary or secondary cause, i.e., relief or prevention of swelling, redness, pain, fever, malaise, headache, muscle, vascular and visceral spasm.

37. The anti-pathophysiological agent of claim 13 for the treatment of pain primarily through its effect on blocking hyperalgesia pain generation and perception mediated by sympathetic post ganglionic fibers and small diameter neuronal afferents.

38. The anti-pathophysiological agent of claim 13 for the treatment of allergies, i.e., asthma, hay fever.

39. The anti-pathophysiological agent of claim 13 for inhibiting cell damag associated with contact dermatitis producing inflammed skin and mucous membranes, and chemical burns, keratitis, conjunctivitis and rhinitis.

40. The anti-pathophysiological agent of claim 13 for inhibiting cell damag from insect bites or helminithic infections.

41. The anti-pathophysiological agent of claim 13 for inhibiting cell damag and debility from viral, rickettsial, bacterial and fungal and protozoal infections.

42. The anti-pathophysiological agent of claim 13 for the treatment of asthma, naso-pharyngeal or laryngeal edema secondary to hypersensitivity, allergic states or leukotriene formation.

43. The anti-pathophysiological agent of claim 13 for the treatment of bowel bladder or uretral spasm secondary to leukotriene formation, hypersensitivit or inflammation.

44. The anti-pathophysiological agent of claim 13 for the treatment of rheumatoid arthritis.

45. The anti-pathophysiological agent of claim 13 for the treatment of rheumatic fever.

46. The anti-pathophysiological agent of claim 13 for the treatment of glomular nephritis, lupus erythematosis, periarteritis nodosa dermatomyositis and related hyperimmune syndromes.

47. The anti-pathophysiological agent of claim 13 for the treatment of chronic or acute ileitis.

48. The anti-pathophysiological agent of claim 13 for the treatment of ulcerative colitis.

49. The anti-pathophysiological agent of claim 13 for the treatment of cystitis.

50. The anti-pathophysiological agent of claim 13 for the treatment or prevention of acute and chronic organ or skin transplant rejection.

51. The anti-pathophysiological agent of claim 13 for use as a topical or systemic treatment for thermal, actinic burns or cold injury ("frostbite").

52. The anti-pathophysiological agent of claim 13 of oxygen poisoning (exces oxygen exposure) .

53. The anti-pathophysiological agent of claim 13 for use as a treatment fo systemic administration in the treatment or prevention of ischemic injury an shock from sepsis, blood and circulatory fluid loss or trauma or syndromes secondary to ischemic, thrombotic or septic injury.

54. The anti-pathophysiological agent of claim 13 for the treatment of myocardial infarction or coronary insufficiency.

55. The anti-pathophysiological agent of claim 13 for peripheral vascular ischemic or thrombotic injury.

56. The anti-pathophysiological agent of claim 13 for superior mesenteric o renal arterial bowel and kidney ischemic injury.

57. The anti-pathophysiological agent of claim 13 for the treatment of acut respiratory distress syndrome or pulmonary edema secondary to inhalation injury.

58. The anti-pathophysiological agent of claim 13 for the treatment of endotoxin or septic shock.

59. The anti-pathophysiological agent of claim 13 for the treatment of acute renal failure.

60. The anti-pathophysiological agent of claim 13 for the treatment of contusions and inflammation of muscles, bone and skin, i.e., osteoarthritis, myositis.

61. The anti-pathophysiological agent of claim 13 for the treatment of pepti ulcer or ulcers related to acute and chronic inflammation.

62. The anti-pathophysiological agent of claim 13 for the treatment of acute and chronic pancreatitis.

63. The anti-pathophysiological agent of claim 13 for the treatment of cerebral edema and glial responses secondary to stroke, hemorrhage or trauma.

64. The anti-pathophysiological agent of claim 13 for the treatment of spina cord and brain injury due to trauma or infection.

65. The anti-pathophysiological agent of claim 13 for the treatment of localized brain injury, abscess or reaction to tumor growth or metastases.

66. The anti-pathophysiological agent of claim 13 for the treatment of multiple sclerosis or other demyelinating diseases.

67. The anti-pathophysiological agent of claim 13 for the treatment of amyotrophic lateral sclerosis.

68. The anti-pathophysiological agent of claim 13 for the treatment and prevention of radiation injury from radionucleides or external radiation.

69. The anti-pathophysiological agent of claim 13 for the treatment and modulation of toxicity of drugs, anti-cancer agents, agricultural and industrial chemical agents, cancer chemotherapy and chemical warfare agents with free radical generating toxicity.

70. The anti-pathophysiological agent of claim 13 for use as a food additive to prolong the shelf life of both chemical, animal and plant derived foods, medicines and vaccines from degradation due to oxidative injury such as rancidity, discoloration and loss of taste and potency.

71. The anti-pathophysiological agent of claim 13 for preservation of decorative flowers, fruits or agricultural products.

7-2. The anti-pathophysiological agent of claim 13 to protect _in vitro froze tissue from ice crystal injury or thawing.

73. The anti-pathophysiological agent of claim 13 for preservation of vaccines, biologicals and drugs.

74. The anti-pathophysiological agent of claim 13 for use to prolong the hal life of meat at above refrigeration temperatures.

75. The anti-pathophysiological agent of claim 13 for use as an additive fo administration to live stock prior to slaughter or immediately after for the purpose of extending the integrity of muscle, against the autolytic muscle destroying syndrome.

76. The anti-pathophysiological agent of claim 13 as an additive to blood an blood derivatives for use as a storage extender or preservative.

77. The anti-pathophysiological agent of claim 13 as an additive to tissue for use as a preservative or extender for storage prior to transplantation.

78. The anti-pathophysiological agent of claim 13 as an additive for the preservation of pathology tissue specimens or for embalming.

79. The anti-pathophysiological agent of claim 13 as an additive for embryonic tissue specimens for preservation.

80. The anti-pathophysiological agent of claim 13 for extension of tissue culture life and productivity.

81. The anti-pathophysiological agent of claim 13 for use as an anti-tumor treatment for cancer to prevent metastases, oncogene expression, nidation and growth.

82. The anti-pathophysiological agent of claim 13 for use as a medication to enhance or synergize with anti-coagulation therapy.

83. The anti-pathophysiological agent of claim 13 for use as an anti-snake o insect, venom medication or for the treatment of plant, jelly fish, or fish toxins..

84. The anti-pathophysiological agent of claim 13 for maintenance of muscle integrity and function under exercise stress to enhance performance, tissue recovery and conditioning under conditions of acute or chronic fatigue, dehydration and internal and external heat elevation from sustained muscle action or environmental exposure.