WO1995018620A1

WO1995018620A1 - Treatment using arachidonic acid metabolite and particulate formulations

Info

Publication number: WO1995018620A1
Application number: PCT/US1995/000370
Authority: WO
Inventors: Andrew S. Janoff; David F. Eierman
Original assignee: The Liposome Company, Inc.
Priority date: 1994-01-11
Filing date: 1995-01-11
Publication date: 1995-07-13
Also published as: CA2179986A1; NO962911L; JPH10507737A; AU682753B2; EP0738151A1; AU1601895A; FI962806A0; FI962806A; NO962911D0

Abstract

Provided herein is a method of administering a free arachidonic acid metabolite to an animal, the method involving administration of the free metabolite and an endocytosable particle. This method can be used to treat animals afflicted with disorders characterized by cell activation and adhesion, inflammation or toxemia. Also provided is a method of treating an animal for such disorders by administration to the animals of a composition containing an anti-disorder effective amount of an endocytosable particle.

Description

TREATMENT USING ARACHIDONIC ACID METABOLITE
AND PARTICULATE FORMULATIONS
This application is a continuation-in-part of U. S. Serial No. 08/179,739
January 11,1994 and is directed to the therapeutic uses of arachidonic acid metabolite/particulate formulations.

Arachidonic acid, and other twenty carbon"essential"fatty acids having at least three double bonds, can be used to make prostaglandins (for a review, see, e. g., Goodman and Gilman's The Pharmacological Basis of Therapeutics (A.

Goodman Gilman et al., eds.), Pergamon Press, New York (1990), pp. 600-611);
L. Stryer, Biochemistrv (2nd edition), W. H. Freeman and Co., New York (1981), pp. 853-854)). The various prostaglandins are grouped into several categories (A-I), which are distinguished by varying substituents on the five-carbon ring introduced into the twenty-carbon fatty acid precursor during prostaglandin synthesis. These groups can be further subdivided based upon the number, and position, of double bonds in the prostaglandins'carbon chains.

The prostaglandins can have a broad spectrum of biological activities. E series prostaglandins, for example, can affect smooth vascular muscle, e. g., arterioles, precapillaries, sphincters and postcapillary venules, and can be potent vasodilators. PGD2, PGFalpha and PG12 can also have vasodilative effects.

Prostaglandins, and related derivatives, can affect the functioning of blood cells, particularly neutrophils and platelets. PGI2, for example, can inhibit platelet aggregation at concentrations as low as 1 nM (see Goodman Gilman's The
Pharmacological Basis of Therapeutics, supra). Uterine contractions can be affected by PGE, PGF and PGI action. Prostaglandins can also affect renal, central nervous system and afferent nerve function. Various endocrine tissues typically respond to prostaglandins. Furthermore, prostaglandins can modulate inflammatory responses and can ameliorate toxemic disorders. Prostaglandins are believed to act on their target cells by way of cellular surface receptors; these receptors are believed to be coupled to second messenger systems by which prostaglandin action is mediated.

Mizishuma et al. (J. Rheumatol. 14: 97 (1987)) and Hoshi et al. (Drugs.

Exptl. Clin. Res. 12 (8): 681 (1986)) describe lipid microspheres containing prostaglandin E1 (PGE1). However, as disclosed in Mizishuma et al. (U. S.

Patent No. 4,493,847) and Imagawa et al. (U. S. Patent No. 4,684,633), these "microspheres"are actually prostaglandin-containing fat mulsions. The mulsions are not particles, and do not offer the same therapeutic advantages as the compositions comprising a particle and an arachidonic acid metabolite provided herein. These references do not disclose any pharmaceutical or therapeutic activity for the emulsions themselves. Shell and See (U. S. Patent
Nos. 4,820,732 and 4,955,878) disclose treatments for reducing dysfunction during angioplasty procedures which involve administering prostaglandincontaining compositions to patients. These compositions also contain a carrier.

However, the liquid carriers disclosed, e. g., dehydrated alcools and saline solutions, in these patents do not comprise particles. The fat-laden microsphere carriers disclosed are taught to be at least as large as a red blood cell, i. e, at least 7 microns in diameter, and can be much larger. Administration of microspheres of such large size to animals can cause difficulties because the microspheres can become stuck in, and clog, small blood vessels, e. g., lung capillaries. The particles disclosed in the Shell and See patents are larger than the particles used herein; the particles employed in the present invention, by contrast, can be safely administered to animals.

SUMMARY OF THE INVENTION
This invention provides a method of administering to an animal a free arachidonic acid metabolite, the method comprising administering to the animal a composition comprising a pharmaceutically acceptable carrier, the free metabolite and an endocytosable particle. Preferably, the composition is administered to a human. Preferably, the metabolite is a prostaglandin, more preferably a prostaglandin of the E series or of the I series, and still more preferably, prostaglandin E1. The particle, which can be any endocytosable particle, for example, a liposome, latex microsphere, polystyrene, xymozin, starch or gelatin particle, is preferably a liposome.

The method can be used therapeutically to administer free arachidonic acid metabolites to animals afflicted with disorders characterized by cell activation and adhesion, inflammation or toxemia including, without limitation, vasoocclusive, toxemic, athritic and auto-immune disorders such as: reperfusion injury, post-traumatic shock, restenosis, myocardial infarction, vasculitis, acute respiratory distress syndrome, systemic inflammatory response syndrome, rheumatoid arthrits, gout, systemic lupus erythematosus, juvenile dianetes, multiple sclerosis and Hashimoto's thyroiditis. Particularly preferred therapeutic indications are acute respiratory distress syndrome and systemic inflammatory response syndrome. The disorder treated can be a disorder that is exacerbated by exposure of the animal to a free arachidonic acid metabolite in the absence of an endocytosable particle.

Accordingly, an amount of the composition comprising an anti-disorder effective amount of the metabolite and particle is administered to the animal.

Typically, the anti-disorder effective amount of the composition comprises from about 10-12 g of the metabolite per kg of body weight of the animal to about 10-3 g per kg; desirably the anti-disorder effective amount comprises from about 10-8 g to about 10-4 g, and more desirably, about 10-6 g of the metabolite per kg.

Preferably, but not necessarily, the endocytosable particle is about spherical in shape. More preferably, the particle is a spherical particle having a diameter of from about 50 nm to about 1000 nm. Typically, the anti-disorder effective amount of the composition comprises from about 101 of such endocytosable particles per kg of body weight of the animal to about 1014 of such particles per kg, or about 10-6 g of such particles per kg. Preferably, the anti-disorder effective amount of the composition comprises about 10-6 g of the metabolite plus the particle per kg of body weight of the animal, or about 10-6 g of the metabolite and from about 1010 to about 1014 of the particles per kg.

Also provided herein is a pharmaceutical composition comprising per dose a pharmaceutically acceptable carrier and an anti-disorder effective amount of a free arachidonic acid metabolite and an endocytosable particle.

Further provided is a method of treating an animal afflicted with a disorder characterized by cell activation and adhesion, inflammation or toxemia; the method comprises administering to the animal a composition comprising a pharmaceutically acceptable carrier and an anti-disorder effective amount of an endocytosable particle, for example, a liposome, latex microsphere, polystyrene, xymozin, starch or gelatin particle, is preferably a liposome. Preferably, the antidisorder effective amount comprises from about 101 to about 1014 endocytosable particles, or about 10-6 g of endocytosable particles, per kg of body weight of the animal. Preferred therapeutic indications are acute respiratory distress syndrome and systemic inflammatory response syndrome.

BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Rat Air Pouch Studies. X-axis: saline control, free PGE1, free 15
M-PIGEZ (1 5-methyl-PGE1), LUV-PGE1 formulation ("C-53" ; unilamellar liposomal PGE1, prepared in accordance with the procedures described in
Example 1, below), LUV placebo liposomes ("C-53 placebo" ; liposomes prepared in accordance with the procedures used to prepare LUV-PGE1, but not having
PGE1), LUV placebo liposomes plus free PGE1, MLV-PGE1 (multilamellar liposomes, prepared in accordance with the procedures described in Example 1, below), MLV placebo liposomes (liposomes prepared in accordance with the procedures used to prepare MLV-PGE1, but not having PGE1), MLV placebo liposomes plus free PAGEL; y-axis: cells/ml times 10,000 in exudate.

Figure 2. Inhibition of Leukocyte Subset Extravasation. The graph is scaled for prostaglandins. The value for neutrophils in the saline control was off the scale used, and was 1.62 x 106 neutrophils/ml. First column in each set (unshaded): polymorphonucleocytes (PMNS); second column (lightly shaded): platelets; third column (darkly shaded): lymphocytes; fourth column (unshaded): monocytes. Y-axis: cells/ml x 10,000 in exudate.

Figure 3. Dose Response of Inhibition of Leukocyte Extravasation. The graph is scaled for prostaglandins. The value for neutrophils in the saline control was off the scale used, and was 1.62 x 106 neutrophils/ml. X-axis: saline control, free PGE1, LUV-PGE1, LUV placebo liposomes, LUV placebo liposomes plus free PGE1, MLV-PGE1, MLV placebo iiposomes, MLV placebo liposomes plus free PAGEL; y-axis: cells/ml x 10,000 in exudate. Darkly shaded columns: 25 g/kg
PGE1 or equivalent amount of placebo liposomes; unshaded: 50 g/kg; lightly shaded: 10 g/kg.

FIGURE 4. Inhibition of Extravasation by Alternative Liposomal Formulations.

X-axis: saline control, free PGE1, LUV-PGE1, LUV placebo liposomes, MLV
PGE1, MLV placebo liposomes, SPLV-PGE1 (stable plurilamellar vesicle, see
Lenk et al., U. S. Patent Nos. 4,522,803,5,030,453 and 5,169,637), SPLV placebo liposomes, SPLV placebo plus free PGE1, EPC/Cholesterol
(Chol)/POPE-GA (1-palmitoyl-2-oleoyl-phosphatidylcholine--glutaric acid)-PGE1,
EPC/Chol/POPE-GA (no PIGE1), EPC/Chol/POPE-GA/DOPE-PGE1 (dioleoyl phosphatidylethanolamine covalently linked to PIGE1), EPC/Chol/DOPE-PGE1,
EPC/Chol/POPE-GA placebo plus free PAGEL; y-axis: cells/ml x 10,000.

Figure5. Adjuvant Arthritis/Free PGE1. X-axis: time (days); y-axis: % change in jaw size. Filled squares: adjuvant control (no PAGEL); open squares: adjuvant and PGE1 administered at day 0; filled diamonds: adjuvant administered at day 0, PGE1 at day 10; open diamonds: saline control (no adjuvant).

Figure 6. Adjuvant Arthritis/Free PGE1. X-axis: time (days) post-adjuvant administration; y-axis: percent weight change. Filled squares: adjuvant control; open squares: adjuvant plus PGE1 at day 0; filled diamonds: adjuvant plus PIGE at day 10; open diamonds: saline control.

Figure 7. Rat General Health/Motility. X-axis: adjuvant control, adjuvant plus
PGE1 at day 0, adjuvant plus PGE1 at day 10, adjuvant plus PGE1 at day 14, saline control (no adjuvant); y-axis: subjective score.

FIGURE 8. Rat Endotoxemia Model. X-axis: time (days) post-LPS administration; y-axis: percent survival in treatment group. Filled squares: rats administered saline control (0 g/kg LPS); open squares: rats administered 10 g/kg LPS; filled diamonds: 15 g/kg LPS; open diamonds: 25 g/kg LPS; filled triangles: 50 g/kg LPS; open triangles: 75 g/kg LPS; filled circles: 100 g/kg LPS.

Figure 9. Placebo Liposomes inhibit Secretion of Human Monocyte TNF and 11-1 in Response to Lipopolysaccharide (LPS). X-axis: free PGE1, LUV-PGE1, placebo LUVs, placebo LUVs and saline control (no liposomes or PAGEL); y-axis: percent inhibition of TNF and IL-1 secretion; unshaded: TNF; shaded: IL-1.

Figure 10. Placebo Liposomes Attenuate LPS-induced Mortality and
Synergize with Free PGE1 to Increase Survival. X-axis: time (days) post-LPS administration; y-axis: percent survival in treatment group. Filled squares: saline control (no LPS administered); filled diamonds: LUV-PGE1; open diamonds: placebo LUVs plus free PAGEL; filled triangle: placebo LUVs; open triangles: LPS control (no liposomes or PAGEL); open squares: free PGE1.

Figure 11. LUVs and Latex Microspheres Synergize with Free PGE1 to
Eliminate Free PGE1-lnduced Mortality and to Increase Survival. X-axis: Saline control (no LPS, PGE1 or liposomes), LPS control (LPS, but no liposomes or
PIGE1), LUV-PGE1, placebo LUVs plus free PGE1, LATEX microspheres plus free PGE-), placebo LUVs, LATEX microspheres; y-axis: percent survival in treatment group.

Figure 12. Increasing the Particle Number Effects an Increased Survival in the Presence of a Constant Dosage of Free PGE1. Saline and LPS controls are not shown. The survival for these groups was 100% and 50%, respectively. Xaxis: 0,20,30,40,50 g/kg; y-axis: percent survival in treatment group; z-axis: no particles administered, LUV-PGE1 administered at a particle equivalency of 40 g/kg plus free PGE1 at the indicated dose.

Figure 13. Increasing the Dosage of Free PGE1 Alone Increases Mortality
While Increasing Free PIGEZ in the Presence of a Constant Particle Number
Increases Survival. Saline and LPS controls are not shown. The survival for these groups was 100% and 50%, respectively. X-axis: 0,20,30,40,50 g/kg; yaxis: percent survival in treatment group; z-axis: no particles administered, LUV
PGE1 administered at a particle equivalency of 40 g/kg plus free PGE1 at the indicated dose.

FIGURE 14. Leukocyte Phagocytosis of Particles is Obligate for the Protective
Effect of Particles and PGE1 During Rat Endotoxemia. X-axis: LPS control; free
PIGEZ; pansorbin; pansorbin plus free PAGEL; pansorbin plus free PGE1 plus anti
CR3 monoclonal antibody. Y-axis: percent survival.

DETAILED DESCRIPTION
This invention provides a method of administering a free arachidonic acid metabolite to an animal, which comprises administering to the animal a composition comprising a pharmaceutically acceptable carrier, the free metabolite and an endocytosable particle. Preferably, the animal is a human.

"Pharmaceutically acceptable carriers"are any of the standard carriers, diluents, excipients and the like generally intended for use in connection with the administration of biologically active agents to animals. Such carriers are well known in the art and are generally chosen with regards to a number of factors, such as the particular drug being used and the intended route of administration, well understood by the ordinarily skilled artisan, or within his purview to determine without undue experimentation. Suitable carriers include, but are not limited to salt solutions such as physiological saline, aqueous buffered solutions, and the like. The pharmaceutical composition can further comprise auxiliary agents such as preservatives, anti-oxidants and the like in amounts, and for reasons, well known to the ordinarily skilled artisan.

The pharmaceutical composition can be provided as a unit dosage form
"Arachidonic acid metabolites"are prostaglandins, or compounds which can be converted to prostaglandins, e. g., artificially or in the body of an animal.

Prostaglandins are a group of twenty-carbon fatty acids containing a five-carbon ring, plus seven-and eight-carbon chains, that are made from arachidonic acid and other twenty-carbon fatty acids having at least three double bonds (e. g., the "essential"fatty acids 8,11,14-eicosatrienoic acid, 5,8,11,14-eicosatetraenoic acid or 5,8,11,14,17-eicosapentanoic acid; see, e. g., Goodman and Gilman's The
Pharmacological Basis of Therapeutics, supra). Arachidonic acid is the most abundant of these twenty-carbon prostaglandin precursors in humans.

The twenty-carbon essential fatty acid prostaglandin precursors, intermediates formed during prostaglandin synthesis, e. g., prostanoic acid, and structural analogs which can be converted to these compounds, are"arachidonic acid metabolites. ""Prostaglandin-related compounds,"e. g., leukotrienes, thromboxanes, lipoxins and prostacyclins, include those compounds which are functionally related to prostaglandins and which can also be derived from the twenty carbon essential fatty acid prostaglandin precursors; prostaglandins and prostaglandin-related compounds are also"arachidonic acid metabolites."
Preferably, the arachidonic acid metabolite administered to animals in accordance with the practice of this invention is a prostaglandin, more preferably, an E or I series prostaglandin, and most preferably, prostaglandin E1. The prostaglandin can also be 15-methyl-PGE1 or PGD2.

The term"free", as used herein with respect to an arachidonic acid metabolite, describes the relationship between the metabolite and a particle coexisting in the same aqueous medium. The metabolite and particle are generally not found at a greater local concentration in the medium in connection with each other than the concentrations at which they will be found elsewhere in the medium. That is, the metabolite and particle are not preferntially associated with each other in the aqueous medium.

An endocytosable particle is a particle that can be endocytosed by endocytic cells typically found in animals. Endocytosis (see, e. g., J. E. Darnell et al., Molecular Cell Bioloay, Scientific American Books, Inc. (New York), 1987, pp.

638-644, the. contents of which are incorporated herein by reference) is the process by which a region of a cell's plasma membrane binds to, and envelops, a particle in the external medium. As used herein,"endocytosis"includes phagocytosis, the process by which cells take in relatively large particles in the surrounding medium.

Macrophages and monocytes are typical phagocytic cells (phagocytes).

Particles phagocytosed by such cells are generally first coated with serum antibodies on their external surfaces. Surface receptors on the phagocytes bind to a region common to the various antibodies present in an animal's serum.

Such binding between proteins on particle surfaces and cellular receptors stimulates phagocytosis. The process generally proceeds with the sequential binding of phagocyte surface receptors to antibodies bound to the particle's external surface, until the particle is completely engulfed by the plasma membrane.

Accordingly, the endocytosable particles of this invention are particles having surfaces that can be coated with the serum proteins generally believed to be responsible for recognition of particles by animal endocytic cells.

Endocytosable particles of this invention generally have sizes, and corresponding surface areas, effective for coating with a sufficient number of antibody molecules to allow for phagocytosis of the particle, but are generally not so large that they tend to accumulate in small blood vessels. Typically, the most desirable endocytosable particle is about spherical and has a size (diameter) of at most about 5000 nm. More desirably, the endocytosable particle has a size of from about 50 nm to about 1000 nm, and still more desirably, about 100 nm. Methods of determining particle size, e. g., by dynamic light scattering, are well known to those of ordinary skill in the art and can readily be practiced by them without undue experimentation.

The endocytosable particle can be any endocytosable, pharmaceutically acceptable particle which can be safely administered to animals, and which can be combined with an arachidonic acid metabolite such that the metabolite is free and is capable of being therapeutically effective when administered to animals. It can be solid, or it can have one or more internal non-solid, e. g., aqueous, compartments. Suitable endocytosable particles include, without limitation: liposomes, latex microspheres, pansorbinTM, xymozin, starch, gelatin or polystyrene particles. Additional endocytosable particles can readily be identified by ordinarily skilled artisans given the teachings of this invention, e. g., by use in vitro in connection with a free arachidonic acid metabolite, or by administration to suitable animal models, such as those described herein.

Liposomes are preferred herein as endocytosable particles. These are self-assembling structures comprising one or more lipid bilayers, each of which encompasses an aqueous compartment. Liposomes with a single lipid bilayer are unilamellar. Preferably in the practice of this invention, the unilamellar liposome is a large unilamellar liposome (LUV), i. e., a unilamellar liposome with a diameter of greater than about 50 nm. Liposomes having more than one lipid bilayer are multilamellar liposomes (MLVs). MLVs used in the practice of this invention preferably comprise a solute entrapped in their aqueou s compartments, wherein the concentration of the solute in each of the aqueous compartments is substantially equal; i. e., the MLVs have substantially equal interlamellar solute distribution.

Liposomes administered in a suspension in connection with a free arachidonic acid metabolite, but not entrapping, or having associated with them, the free metabolite at a greater concentration than that at which the metabolite may be found elsewhere in the suspension are endocytosable particles and can be referred to as"placebo"liposomes.

Liposomes can be produced by a variety of processes known in the art.

(for a review, see, e. g., Cullis et al., in: Liposomes, From Biophysics to
Therapeutics (M. Ostro, ed.), Marcel Dekker (New York), 1987, pp. 39-72).

Bangham's procedure (J. Mol. Biol. 13: 238-252 (1965)) produces"ordinary" multilamellar liposomes (MLVs)."Ordinary"MLVs can have unequal solute distribution amongst their aqueous compartments and thereby, osmotic stress between compartments. Lenk et al. (U. S. Patent Nos. 4,522,803,5,030,453 and 5,169,637), Fountain et al. (U. S. Patent No. 4,588,578) and Cullis et al. (U. S.

Patent No. 4,975,282) disclose methods for producing multilamellar liposomes having substantially equal interlamellar solute distribution. Having substantially equal interlamellar solute distribution means that there will be less osmotic stress amongst the aqueous compartments of these MLVs, which will therefore generally be more stable than ordinary MLVs. Unilamellar liposomes can be produced from MLVs by sonication (see Paphadjopoulos et al. (1968)) or extrusion (Cullis et al. (U. S. Patent No. 5,008,050) and Loughrey et al. (U. S.

Patent No. 5,059,421)). The contents of these disclosures are incorporated herein by reference.

Liposomes and other endocytosable particles, can contain, or have associated with them, one or more biologically active agents, i. e., compounds which can be administered to animals and which may have biological or diagnostic activity therein, or on an animal's cells in vitro. Biologically active, or bioactive, agents include, but are not limited to: antiviral, antibacterial, antifungal, antiparasitic, antimetabolic, antiglaucomic, anti-inflammatory or antineoplastic compounds, sterols, carbohydrates, amino acids, peptides, proteins, immunoglobulins, immunomodulators, dyes, toxins, enzymes, hormones, neurotransmitters, glycoproteins, radiolabels, radiopaque compounds, fluorescent compounds, cell receptor proteins, cell receptor ligands, mydriatic compounds, bronchodilators, local anesthetics, growth promoting agents, regenerative agents and the like.

Biologically active agents used in the practice of this invention include, but are not limited to, antimicrobial, antiinflammatory and vasodilative agents.

The method of this invention can be used to treat animals afflicted with a disorder characterized by cell activation and adhesion, inflammation or toxemia which comprises administering to the animal an amount of the composition which comprises an anti-disorder effective amount of the free arachidonic acid metabolite and the endocytosable particle.

Cell activation/adhesion disorders are characterized by anormal activation of cells in the blood; the activated cells can adhere to each other, or to activated cells in surrounding vascular endothelium. Such adhesions can lead to the blockage of small blood vessels, e. g., lung capillaries, consequent stoppage of blood flow, and subsequent damage to surrounding tissue. Cell activation/adhesion disorders are a significant problem in a wide variety of medical pathologies. Endothelial cells, for example vascular, plural, pericardial or abdominal endothelial cells, can be activated by cytokines, e. g., interleukin-1 (IL1), tumor necrosis factor-alpha (TNF-alpha) or bacterial endotoxins.

In like manner, blood cells, particularly neutrophils and platelets, can be activated by agents such as GM-CSF, bacterial endotoxins, bacterial chemoattractants, TNFalpha and the C5a component of complement.

Activated cells have adhesion sites on their surfaces by which they can adhere to each other. Activated and adhered cells can form clumps, which can clog small blood vessels such as those found in the lungs and heart, and thereby reduce blood flow to surrounding tissue. The activated cells can also adhere to activated vascular endothelial cells; such adhesion can lead to subsequent degranulation of vascular endothelium, or to the release of mediators of cell damage such as superoxide anion (02') and proteolytic enzymes. Accordingly, cell activation/adhesion can lead to the circulatory problems seen in animals afflicted with vaso-occlusive disorders.

Amongst the cell activation/adhesion disorders to which the present invention is directed are reperfusion disorders, such as those related to the reperfusion of occluded blood vessels, or incidental to surgery in which blood flow is temporarily stopped (see, e. g., Seewaldt-Becker et al.,"Effect of Anti-Adhesive
Antibodies on Reperfusion Injury," (Springer et al., eds.) in: Leukocyte Adhesion
Molecules, Springer-Verlag, New York (1990) pp. 138-148; and"Adhesion in
Disease and Therapy," (Springer et al., eds.), in: Leukocvte Adhesion Molecules,
Springer-Veriag, New York (1990), pp. 85-156). When there is a blockage in a blood vessel, surrounding endothelial cells, as well as downstream ischemic tissue, can be damaged. There can even be further damage to nearby endothelial cells when the occlusion is cleared.

Such damaged cells can in turn induce activation in neutrophils and platelets after restoration of blood flow to the affected areas.

The same cells which become activated and subsequently undergo intracellular adhesion can have surface receptors for arachidonic acid metabolites. Without intending to be limited by theory, it is believed that treatment with arachidonic acid metabolites, by binding to these receptors, can reduce cell activation/adhesion disorder-associated damage by deactivating the cell surface receptors responsible for the elevated levels of intercellular adhesion.

Inflammation is a process of cytological and histological reactions occurring in affected blood vessels, and surrounding tissues, in response to an injury or anormal physical,'chemical or biological stimulation (see, e. g.,
Stedman's Medical Dictionarv (Illustrated) (24th edition, J. V. Basmajian et al., eds.), Williams and Wilkins, Baltimore, MD (1982), pp. 707-708). Inflammatory responses include local reactions and resulting morphological changes, destruction or removal of injurious materials and activation of repair mechanisms.

Agents, including arachidonic acid metabolites, that can inhibit inflammatory responses to injuries or anormal physiological stimuli can be used to treat inflammatory disorders, e. g., gout, and various arthritic conditions, including rheumatoid arthritis.

Humoral inflammatory responses can be characterized by the extravasation of cells to the location of the agent inducing the response, that is, exit of cells from the circulatory system to the site of attack. The exudate can be characterized by a relatively low number of large cells, or by a relatively large number of neutrophils, eosinophils, monocytes, lymphocytes and plasma cells.

Arachidonic acid metabolites, e. g., prostaglandins, can inhibit extravasation; without intending to be limited by theory, it is believed that this is a means by which such agents can inhibit inflammatory responses.

The rat air pouch exudate model, a well-accepted model for studying inflammation (see Tate et al, Laboratory Investigation 59: 192 (1988), the contents of which are incorporated herein by reference, and the Examples below), can be used to study the ability of arachidonic acid metabolites to inhibit extravasation. Rats acclimated to their environments are anesthetized, and pouches are formed in their backs by subcutaneous injection of air therein.

Inflammation is then induced in the pouch by injection therein of an inflammationinducing amount of an inflammatory agent, for example, fMLP. Cells are then expected to enter the air pouch by extravasation from the surrounding vasculature. The ability of an anti-inflammatory agent to inhibit such extravasation can be examined, for example, by injecting such agents into the animals and then comparing the number of cells entering the air pouch in agenttreated vs. control/untreated animals.

For example, as described below, rats were acclimated, then anesthetized and injected subcutaneously with 20 cc of ambient air. Six days thereafter, inflammation was induced by direct injection of 2.15 micrograms of fMLP into the air pouch. Free prostaglandin E1, free PGE-1 plus placebo liposomes, free PGE1 plus latex microspheres, placebo liposomes alone and latex microspheres alone were then injected into the animals via the tail vein.

Six hours post inflammation induction, the total exudate fluid was recoverered from the air pouch. fMLP administration induced a substantial number of leukocytes to invade the air pouches; the data show that administration of free PGE-1 (2.5,5 and 10 micrograms) plus endocytosable particles significantly (greater than 50%, and as much as about 80%), reduced extravasation (as measured by cell concentration in the air pouch fluid) in comparison to the level of reduction achieved by administration of PGE-1 alone. It should be noted that t

Toxemia, and the ability of agents to inhibit toxemia, can be studied by a number of models accepted by the art for such studies, for example, the rat endotoxemia model described below. An LD50, that is, a dose of the endotoxin, e. g., E coli lipopolysachharide (LPS), lethal to about 50% of the test group, is first established; subsequently, animals are administered about this dose of the endotoxin. Animals are then either not treated with an anti-toxemia agent (control animals), or with such an agent, for example PGE-1 (e. g., at a dose of 40 micrograms per kg of the animal's body weight) or PGE-1 plus endocytosable particles (at a cumulative dose equivalent to the 40 microgram per kg dose of free PGE-1 alone). The ability of the anti-toxemia agent to inhibit endotoxininduced mortality is then determined.

Furthermore, inhibition of interleukin-1, interleukin-6 and tumor necrosis factor alpha secretion, a marker of toxemic conditions, can also be assessed; increasingly effective anti-toxemia agents are expected to decrease the percentage, vs. of untreated control, of the inhibition of secretion of these proteins that is induced by the endotoxin.

Data derived from the representative examples described herein, and presented below, indicate that free PGE-1 plus endocytosable particles was significantly more effective than free PGE-1 alone in increasing the survial rate in endotoxin-treated animals, and was significantly more effective in decreasing the percentage of endotoxin-induced secretion inhibition. For example, the data presented below show that administration of free PGE-1 plus placebo liposomes, in comparison to administration of free PGE-1 alone, can lead to about a five-fold increase in the rate of survival in LPS-treated animals, and to about a 40% decrease in secretion inhibition.

An"anti-disorder effective"amount of a free arachidonic acid metabolite and a particle, is any amount of the metabolite plus the particle effective, prophylactically or therapeutically, to ameliorate, alleviate, inhibit, eliminate or prevent a disorder characterized by cell activation and adhesion, inflammation or toxemia. The amount of the free arachidonic acid metabolite is generally equivalent to known therapeutic amounts of free arachidonic acid metabolites alone, in conditions treatable with arachidonic acid metabolites. However, conditions ordinarily exacerbated by exposure to arachidonic acid metabolites, such as inflammatory conditions induced or aggravated by prostaglandins, can be treated by coadministration of the same metabolites with endocytosable particles.

Accordingly, for such conditions, there is a therapeutically effective amount of a free arachidonic acid metaboliute, used in connection with an endocytosable particle, where there is generally no such amount for the metabolite used alone.

Amounts of free arachidonic acid metabolites included in anti-disorder effective amounts of compositions comprising the metabolites and endocytosable particles are typically dependent upon a number of factors well within the purview of the ordinarily skilled artisan to understand and control, given the teachings of this invention. These include, without limitation: the particular metabolite and endocytosable particle used; the particular disorder with which the subject is afflicted; and the age, size, weight and general condition of the subject treated, including other disorders with which the subject may be aflflicted. Typically the anti-disorder effective amount of the composition comprises at least about 10-12 g of the free arachidonic acid metabolite per kg of body weight of the animal treated with the composition.

As described above, a"free"metabolite is a metabolite generally not found in greater concentrations with endocytosable particles than elsewhere in the same aqueous medium. Accordingly, the amount of free metabolite described can include some metabolite which is randomly located in the medium in proximity with the particles, or which is randomly associated with the particle's' surfaces. Desirably, the anti-disorder effective amount comprises from about 1012 9 of the metabolite per kg of body weight to about 10-3 g per kg; more desirably, the anti-disorder effective amount of the composition comprises from about 10-8 g of the metabolite per kg of body weight to about 10-4 g per kg.

Most desirably, the anti-disorder effective amount of the composition comprises about 10-6 g of the metabolite per kg of body weight.

Anti-disorder effective amounts of free arachidonic acid metabolite/endocytosable particle-containing compositions typically comprise a number of endocytosable particles at least sufficient to enhance the therapeutic response of animals to arachidonic acid metabolites, for conditions responsive to such metabolites; or, a number of particles sufficient to elicit a therapeutic response when administered with arachidonic acid metabolites, in conditions exacerbated by exposure to such metabolites in the absence of endocytosable particles.

As described above,"endocytosable particles"typically have surfaces which become coated with serum proteins; it is generally believed that such coating enhances the recognition of the particles, and their uptake, by endocytic cells. Without intending to be limited by theory, it is also believed that such particle endocytosis can have a therapeutic effect alone, as well as in connection with stimulating beneficial arachidonic acid metabolite interaction with target cells.

Accordingly, the amount of particles included in anti-disorder effective amounts of compositions used herein is typically an amount of particles having a surface area sufficient for coating with an amount of serum proteins effective to enhance endocytosis. As described above, the endocytosable particles preferred for use herein are typically about spherical, although variations in shape are acceptable; the sperical particles typically have diameters of from about 50 nm to about 1000nm.

Ordinarily skilled artisans given the teachings of this invention can determine the number and weight of endocytosable particles to employ in order to formulate and anti-disorder effective amount of the composition. As discussed herein, an amount of endocytosable particles at least sufficient to elicit, or enhance, a therapeutic response, is used to formulate the composition. The weight of this amount of particles is generally within the range of about 10-8 to about 10-4 g per kg of the animal's body weight, and is usually about 10-6 g or less, but can be outside this range if necesary. Ordinarily skilled artisans given the teachings of this invention can, without undue experimentation, determine if a higher or lower weight of a particular endocytosable particle should be used.

For examples, such artisans can use the rat air pouch, adjuvant arthritis and endotoxemia models described herein to assess the anti-disorder effectiveness of a particular weight of the endocytosable particle, either alone or in connection with an amount of a free arachidonic acid metabolite. The number of endocytosable particles in the amount selected varies according to a variety of factors, such as the effective weight of particles selected, as well as the size, shape and densiy of the particles. For the preferred endocytosable parties herein, this number is typically from about 101 to about 1014, and can be about 1012.

Preferably, the anti-disorder effective amount of the composition comprises about 10-6 g of the metabolite plus the particle per kg, that is 10-6 g is about their combined weight, or about 10-6 g of the metabolite per kg and from about 1010 endocytosable particles to about 1014 particles. For example, the data presented herein show that both 40 micrograms, or 40 x 10-6 g, of an endocytosable particle alone, a liposome or a latex microsphere, or 40 micrograms combined of a free arachidonic acid metabolite and an endocytosable particle can be more therapeutically effective in the models described herein.

Anti-disorder effective amounts of compositions described herein can be used to treat animals afflicted with disorders characterized by cell activation and adhesion, inflammation or toxemia. Accordingly, such amounts include anti-cell activation and adhesion, inflammation or toxemia amounts of a free arachidonic acid metabolite and an endocytosable particle. An"anti-cell activation/adhesion effective amount"of a free arachidonic acid metabolite and an endocytosable particle is any amount effective to ameliorate, inhibit or prevent the activation of adhesion sites on cells in the blood, or in surrounding vascular tissue, and/or the adhesion of such activated cells to other cells in the blood or surrounding vascular tissue.

The anti-cell activation/adhesion amount is generally effective to inhibit or lessen vascular occlusion resulting from such activation and intracellular adhesion. An"anti-inflammatory disorder effective amount"of a free arachidonic acid metabolite and an endocytosable particle is any amount effective to ameliorate, inhibit or prevent anormal inflammatory responses or reactions in animals afflicted with conditions characterized by anormal inflammation, i. e., inflammation which is in response to anormal physiological stimuli or injury.

Such amounts are effective, for example, to prevent extravasation of cells from the circulation of the animal to the site of the anormal inflammation. An"antitoxemic disorder effective amount"of the free arachidonic acid metabolite and the particle is any amount effective to ameliorate, inhibit or prevent anormal inflammatory responses or reactions in animals afflicted with toxemic conditions, e. g. toxemic conditions resulting from microbial infection or physical or chemical trauma.

Disorders treatable according to the method of this invention include, without limitation, vaso-occlusive, arthritic, traumatic and auto-immune disorders, such as: reperfusion injury, restenosis, myocardial infarction, vasculitis, posttraumatic shock, acute respiratory distress syndrome, systemic inflammatory response syndrome, rheumatoid arthritis, gout, systemic lupus erythematosus, juvenile diabetes, multiple sclerosis or Hashimoto's thyroiditis. Particularly
preferred therapeutic indications are systemic inflammatory response syndrome
(SIRS) acute respiratory distress syndrome (ARDS).

Treatable disorders according to the method of this invention include those exacerbated by exposure of the animal to a free arachidonic acid metabolite. As used herein, a disorder"exacerbated"by a free arachidonic acid metabolite is a disorder the severity of whose signs, symptoms or effects is increased by administration of a free arachidonic acid metabolite, but which can be ameliorated, alleviated, eliminated, inhibited, or prevented, therapeutically or prophylactically, by administration of a free arachidonic acid metabolite in connection with an endocytosable particle.

Also provided herein is a composition comprising per dose a pharmaceutically acceptable carrier and an anti-disorder effective amount of a free arachidonic acid metabolite and an endocytosable particle.

Further provided is a method of treating an animal afflicted with a disorder characterized by cell activation and adhesion, inflammation or toxemia; the method comprises administering to the animal a composition comprising a pharmaceutically acceptable carrier and an anti-disorder effective amount of an endocytosable particle, for example, a liposome, latex microsphere, polystyrene, xymozin, starch or gelatin particle, is preferably a liposome. The anti-disorder effective amount of the endocytosable particle alone can be about the same as the anti-disorder effective amount of the free arachidonic acid metabolite plus the particle. The effective amount of the particle can also be greater or less than this amount, as necessary.

Ordinarily skilled artisans given the techings of this invention can, for example using the models described herein, determine the appropriate amount of a particular endocytosable particle to use, and whether this amount is outside of the range described as preferred herein. Preferably, the anti-disorder effective amount comprises from about 1010 to about 1014, or about 10-6 g, of endocytosable particles, per kg of body weight of the animal.

Preferred therapeutic indications are acute respiratory distress syndrome and systemic inflammatory response syndrome.

This invention is further described in the following Examples. However, those of ordinary skill in the art will readily understand that these examples are merely illustrative of the invention as defined in the claims which follow thereafter.

EXAMPLES
Example 1
Preparation
Placebo liposomes
Multilamellar Liposomes (MLVs)
w
An egg phosphatidylcholine (EPC) stock solution (20 mg/ml in ethanol) was prepared as follows: 1 g of dried EPC was dissolved in 50 ml of absoute ethanol, with gentle swirling, in a 50-ml brown bottle with a Teflon-lined lid. The resulting solution was stored at minus 20 degrees Celsius. An aliquot of the EPC stock solution was added to a round-bottom flask, from which the ethanol was removed by rotoevaporation at 30 deg. C. for at least two hours. The dried EPC was resuspended in a citrate buffer (50 mM citrate, 150 mM NaCI, brought to pH 4.5 with 10 N NaOH) so as to form a suspension of MLVs.

For the preparation of
DPPC-containing MLVs, a DPPC stock solution was prepared as described above using 1.035 g of dipalmitoyl
Unilamellar Liposomes (LUVs)
Egg phosphatidylcholine (200 mg) in hexane: ethanol (95: 5) was rotary evaporated under reduced pressure in a water bath set at 37 degrees Celsius to a thin film on the sides of the flask. The film was resuspended in 200 I of ethanol, and the ethanolic solution was drawn into a 1.0 ml tuberculin syringe and injected through a 21-gauge needte, at a rate of about 2 drops per second, into 4.0 ml of a stirring solution at pH 7.0, comprising 10% weight by volume of aqueous dextrose and 0.01% weight by volume aqueous EDTA.

The solution became cloudy upon the addition of the ethanol/lipid mixture; the solution was extruded 5 times through a 0.1 micron polycarbonate straight-path filter, followed by a second series of five extrusion through the same type of filter.

Prostaglandin E1 Solutions
A PGE1 stock solution (1 mg/ml in ethanol) was prepared as follows: 20 mg of dried PGE1 was transferred to a 20-ml vial, to which 20 ml of absoute ethanol was added. The PGE1 was dissolve in the ethanol with gentle swirling; the resulting solution was stored at minus 20 degrees Celsius.

Placebo liposomes suspensions, or suspensions of an equivalent number of latex microspheres, were mixed with the requisite amount of the PIGE solution.

Prostagiandin E1 Liposomal Formulations
For multilamellar liposomal PGE formulations, an aliquot of the EPC or
DPPC stock solutions (9.5 ml) and an aliquot of the PGE1 stock solution (0.5 ml) were combined in the round-bottom flask prior to removal of the ethanol. For unilamellar liposomal PGE1 formulations, 1.0 mg of PGE1 was added to the ethanol used to resuspend the dried lipid prior to ethanol injection.

Example 2
Rat Air Pouch Studies
The rat subcutaneous air pouch, a model for acute inflammation and leukocyte extravasation from the peripheral vasculature to sites of inflammation (Tate, et al., Laboratory Investigation 59: 192 (1988), the contents of which are incorporated herein by reference), was used to study the effect of systemic PGE1 liposomes, no-liposomal PGE1, and particles, in mediating fMLP induced inflammation.

Male Sprague-Dawley rats, weighing 126-150 g each, were obtained from
Charles River Laboratories. Upon receipt, the rats were acclimated in the animal facility for 2 days. Throughout the experiments, the rats were watered and fed ad libitum. For air pouch formation, the rats were anesthetized via inhalent, their backs shaved, and swabbed with ethyl alcool. Twenty cc of ambient air was injected subcutaneously into the animal's back to form an air pouch, and the animal was returned to it's cage. The air pouches were monitored to determine integrity, and additional air was injected, if warranted. For each treatment and control group, the number of rats (n) in each was six. At six days following air pouch formation, intra-air pouch inflammation was induced by direct injection into the air pouch of 2.15 g fMLP.

Free PGE1, PGE1-placebo liposome, PGE1-latex microspheres, placebo liposome and latex microsphere formulations, were simultaneously injected i. v. via the tail vein, and the animals returned to their cages. Six hours after fMLP stimulation, the rats were sacrificed by CO2 inhalation, and the total exudate fluid was recovered from the air pouch via syringe. The results of these experiments are presented in Figures 1-4.

Visual examination of the post-stimulation air pouch lining indicated that fMLP effected a thickening of the lining and a large number of invasive leukocytes, as compared to control animals, in which saline alone was injected into the air pouch. Treatment with free PGE1 resulted in a reduction in vascular reactivity and a concurrent reduction in the number of leukocytes invading the pouch lining. The neutrophil population evident in the lining was transient, i. e., the leukocytes were in the process of extravasation from the vasculature to the lumen/exudate fluid of the air pouch. Since the leukocytes were transiently crossing the air pouch lining, subsequent analysis was confined to those cells present in the aspirated exudate fluid.

The free stable prostaglandin analog 15methyl-PGE1 was included in these experiments due to its longer bioavailability of > 8 hours, as compared to the < 15 min. bioavailability of free PGE1.

Example 3
Adjuvant Arthritis
Male Lewis rats, weighing 126-150 g each, were obtained from Charles
River Laboratories. Upon receipt, the rats were acclimated in the animal facility for 2 days. Throughout the experiments the rats were watered and fed ad libitum. Chronic bilateral arthritis was induced by the i. d. (intra-dural) injection of complete Freund's adjuvant at the base of the tail. Free PGE1 was injected into one group of rats, at a dose of 10 g/kg, beginning at day 0, with the injections repeated every second day. Free PGE1 was also injected into another group, at a dose of 10 g/kg, beginning at day 10, and repeated every second day. Also administered was an adjuvant control (no PAGEL) and a saline control (no adjuvant). For each treatment group, n=6.

The onset of arthritis was abrupt, occurring between days 10 and 14 in the Freund's-induced animals. The symptoms exhibited by untreated control animals were tenderness upon palpation in most active joints, symmetric edema involving the joints of the paws, ankles and knees, flexation contractures of the forepaws, malaise, and weight loss attributable to both primary disease as well as inability or disinclination to access food supplies, due to pain and decreased mobility.

Experiments were conducted to assess the efficacy of free PGE1, as well as prostaglandin E1 in connection with placebo liposomes and latex microspheres, placebo liposomes alone and latex microspheres alone, in mediating the progression of adjuvant arthritis. The parameters assessed in these experiments were changes in joint size measured at the rear knee, assessments of which were made on a weekly basis, changes in body weight, and a subjective scoring of general health, vigor and motility. The results from these experiments are shown in Figures 5-7.

Example 4
Rat Endotoxemia
Fever, hypotension, changes in leukocyte counts and diarrhea are symptoms of gram-negative bacterial infections. These infections may lead to disseminated intravascular coagulation and irreversible shock. A large volume of literature indicates the involvement of leukocyte derived IL-1, IL-6 and TNF in mediating the progression of endotoxic shock. Because our in vitro data indicated an inhibition of these cytokines from cultured monocytes, we developed an in vivo model of rat endotoxemia, using mortality as an end point, to assess the effectiveness of PGE1 and particulate formulations in attenuating LPSinduced death.

Experiments were designed to establish an LDso for E. coli LPS (lipopolysaccharide; serotype 055: B5) in Sprague-Dawley rats. The data from these experiments are shown in Figure 8, and indicate that the LDso is at 50 g/kg. This LPS dosage was used in subsequent experiments, unless otherwise indicated.

Male Sprague-Dawley rats, weighing 126-150 g each, were acclimated for two days in an animal facility with food and water ad libitum. At time 0, groups of rats (n=16) were injected i. v. with either E. coli lipopolysaccharide as a single bolus, or with a saline (no LPS) control. Mortality was assessed at various times (days) post-LPS administration.

Example 5
Inhibition of Tumor Necrosis Factor Alpha (TNF) and Interleukin-1 Beta (IL-1)
Svnthesis in Response to Lipopolvsaccharide (LPS) and PGE1
Adherent human monocytes were stimulated with LPS (1g/ml/106 cells) at time 0. Free PGE1 (not entrapped in liposomes), LUV-PGE1 (large unilamellar liposomes (LUVs) plus free PGE1), LUV-PGE1"placebo"liposomes (LUVs not containing PAGEL) plus free PGE1, placebo liposomes or a saline control (no
PAGEL) were injected simultaneously (10 M PGE1). Secreted TNF and IL-1 were assayed at three hours. Results from these experiments are presented in Figure 9.

Example 6
Rat Endotoxemia Model
Male Sprague-Dawley rats were injected i. v. with 50 g LPS/kg of body weight at time 0. Free PGE1, LUV-PGE1 (40 g/kg PIGE1), placebo LUVs (LUVs not containing PIGEZ; equivalent particle number to the number of liposomes given with the 40 g/kg PGE1--LUV-PGE1 dose) or placebo LUVs (40 g/kg lipid equivalency) plus free PGE1 (40 g/kg). There were 12 rats were in each treatment group. Survival was assessed in each group at 6,12,18 and 24 days post-LPS administration. Results are presented in Figure 10.

Example 7
Rat Endotoxemia/Particle Administration
Male Sprague-Dawley rats were injected i. v. with 50 g/kg LPS at time 0.

Free PIGEZ (40g/kg), LUV-PGE1 (40 g/kg PGE1), placebo LUVs (40 g/kg lipid equivalency, i. e., the number of placebo LUVs was equal to the number of LUVs present in connection with a dose of 40 micrograms of prostaglandin E1 per kg of body weight), latex microspheres (the number equivalent to the number of placebo LUVs) or latex microspheres plus free PGE1 (40g/kg) were simultaneously injected i. v. Survival in each treatment group (16 rats) was assessed at 24 hours. Results are presented in Figure 11.

Example 8
Rat Endotoxemia Model/Particle Administration
Male Sprague-Dawley rats were administered compositions containing
PGE1 and a particle, in this case either 100 nm LUVs (LUVs having 100 nm diameters) or 100 nm LATEX microspheres, in which either the PGE1 concentration or particle number was varied while the concentration or number of the other component remained constant. Prostaglandin E1 dose ranged from 2540 micrograms per kg; the number of particles administered was 1.2-2 x 1012 per kg. The rats were injected i. v. at time 0 with 50 g/kg of LPS. The compositions were administered simultaneously by i. v. injection. Survival in each treatment group (n=16) was assessed at 24 hours. Results of these experiments are presented in Figures 12-13.

Example 9
Leukocyte Phagocvtosis
Male Sprague-Dawley rats were injected i. v. with 75 mg/kg LPS at time 0.

PGE1 (40 g/kg), non-phagocytosable Pansorbin particles (1.2x1012/kg),
Pansorbin particles (1.2x1012/kg) + PGE1 (40 g/kg), or Pansorbin particles (1.2x1012/kg) to which anti-CR3 (MoAb OX42) was linked, thus rendering the particle phagocytosable, + PGE1 (40 g/kg) were simultaneously injected i. v.

Survival was assessed at 48 hours. Survival in saline controls was 100%. For each treatment group, the number of animals ("n") was 16. Pansorbin particles (Calbiochem) are hardened S. aureus which have been coated with protein A.

They are not phagocytosed by leukocytes in their prepared state, but are phagocytosed when antibody directed to the leukocyte phagocytic receptor CR3 (CD11 a/CD18) are coupled to the particles via a protein A-Fc linkage (see
F llman, M., R. Andersson and T. Andersson. 1993. J. Immunology 151: 330338, the contents of which are incorporated herein by reference).

Claims

What is claimed is: ¯ 1. A method of administering a free arachidonic acid metabolite to an anmal which comprises administering to the animal a composition comprising a pharmaceutically acceptable carrier, the free metabolite and an endocytosable particle.

2. The method of claim 1, wherein the metabolite is a prostaglandin.

3. The method of claim 2, wherein the prostaglandin is a prostaglandin of the E series or a prostaglandin of the I series.

4. The method of claim 3, wherein the prostaglandin is prostaglandin E1.

5. The method of claim 1, wherein the animal is a human.

6. The method of claim 1, wherein the particle is a liposome, latex microsphere, polystyrene, xymozin, starch or gelatin particle.

7. The method of claim 6, wherein the particle is a liposome.

8. The method of claim 1, wherein the animal is afflicted with a disorder characterized by cell activation and adhesion, inflammation or toxemia and wherein an amount of the composition comprising an anti-disorder effective amount of the free arachidonic acid metabolite and the particle is administered.

9. The method of claim 8, wherein the disorder comprises a vaso-occlusive, arthritic, traumatic or auto-immune disorder.

10. The method of claim 8, wherein the disorder comprises reperfusion injury, restenosis, myocardial infarction, vasculitis, post-traumatic shock, acute respiratory distress syndrome, systemic inflammatory response syndrome, rheumatoid arthritis, gout, systemic lupus erythematosus, juvenile diabetes, multiple sclerosis or Hashimoto's thyroiditis.

11. The. method of claim 8, wherein the disorder comprises systemic inflammatory response syndrome.

12. The method of claim 8, wherein the disorder comprises acute respiratory distress syndrome.

13. The method of claim 8, wherein the disorder is exacerbated by exposure of the animal to a free arachidonic acid metabolite in the absence of an endocytosable particle.

14. The method of claim 8, wherein the anti-disorder effective amount of the composition comprises from about 10-12 g of the metabolite per kg of body weight of the animal to about 10-3 g per kg.

15. The method of claim 14, wherein the anti-disorder effective mount comprises about 10-6 g of the metabolite per kg.

16. The method of claim 8, wherein the endocytosable particle is about spherical and wherein the particle has a diameter of from about 50 nm to about 1000nm.

17. The method of claim 16, wherein the anti-disorder effective amount of the composition comprises from about 1010 of the endocytosable particles per kg of body weight of the animal to about 1014 of the endocytosable particles per kg.

18. The method of claim 16, wherein the anti-disorder effective amount of the composition comprises about 10-6 g of the particle per kg.

19. The method of claim 8, wherein the anti-disorder effective amount of the composition comprises about 10-6 g of the metabolite and from about 1010 of the endocytosable particles per kg of body weight of the animal to about 1014 endocytosable particles per kg and wherein the particle is about spherical and has a diameter of from about 50 nm to about 1000 nm.

20. The method of claim 8, wherein the anti-disorder effective amount of the composition comprises about 10-6 g of the metabolite plus the particle per kg of body weight 21. A pharmaceutical composition comprising per dose a pharmaceutically acceptable carrier and an anti-disorder effective amount of a free arachidonic acid metabolite and an endocytosable particle.

22. A method of treating an animal afflicted with a disorder characterized by cell activation and adhesion, inflammation or toxemia which comprises administering to the animal a pharmaceutical composition comprising per dose a pharmaceutically acceptable carrier and an anti-disorder effective amount of an endocytosable particle.

23. The method of claim 22, wherein the disorder comprises systemic inflammatory response syndrome or acute respiratory distress syndrome.

24. The method of claim 22, wherein anti-disorder effective amount of the composition comprises from about 1010 endocytosable particles per kg of body weight of the animal to about 1014 particles per kg.

25. The method of claim 22, wherein the endocytosable particle is a liposome, latex microsphere, polystyrene, xymozin, starch or gelatin particle.