CA2179986A1 - Treatment using arachidonic acid metabolite and particulate formulations - Google Patents

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

Wo 9S/18620 ~17 9 9 8 ~ r~ /u 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 clld-,llklul~ic acid' ' ' 'pdlli..ulate fommulations.
Arachidonic acid, and other twenty carbon "essential" fatty acids having at 10 least three double bonds, can be used to make jnuolclyldll~lil la (for a review, see, eg., Goodman and Gilman's The Pl~dlllla~.UIo~ .dl Basis of TheraPeutics (A.
Goodman Gilman et al., eds.), Pergamon Press, New York (1990), pp. 600-611);
L. Stryer, Biu~ ioll~ (2nd edition), W. H. Freeman and Co., New York (1981), pp. 8'i3-854)). The various ulualdyldlldills are grouped into several categories15 (A-l), which are distinguished by varying substituents on the five-carbon ring introduced into the twenty-carbon fatty acid precursor during ,u, ualdyldl l~il l synthesis. These groups can be further subdivided based upon the number, and position, of double bonds in the IJluDkluldlldillo' carbon chains.
The ~lualdyldlldillo can have a broad spectnum of biological activities. E
series ,uluo~dyldll~;lls, for example, can affect smooth vascular muscle, e.g., arterioles, ,u,~ s, sphincters and F ~ , " y venules, and can be potent vaso.iil..`u,~. PGD2, PGFalpha and PG12 can also have ~_ "' ' /c effects.
r~ualdyldlldil la, and related denvabves, can affect the functioning of blood cells, 25 particularly neutrophils and platelets. PGI2, for example, can inhibit platelet dyyl~, ' I at cullcelltl - hS as low as 1 nM (see Goodman Gilman's The Fhdll~ nl~ l Basis of TheraPeutics, supfa). Uterine ~.ull~ld~.liuiis can be aflected by PGE, PGF and PGI action. rlualdyldlldills can also affect renal, central nervous system and afferent nerve function. Vanous endocrine tissues 30 typically respond to plua~dyldlldills. Furthermore, pluolduldlldilla can modulate "nd""".A""y responses and can ameliorate toxemic disorders. r~Ualdyldll.lills , are believed to act on their target cells by way of cellular surface receptors; these receptors are believed to be coupled to second Illeaa~:lly~l systems by which ~uluald~ldlldill action is mediated.
Mizishuma et al. (J. Rheumatol. 14:97 U987)) and Hoshi et al. (Drugs.
Exptl. Clin. Res. 12(8):681 (1986)) describe lipid Illi~,lua,ul~ s containing ~uoldyldll~ill E1 (PGE1). However, as disclosed in Mizishuma et al. (U.S.

Wo 95/18620 2 1 7 9 ~ 8 6 . ~ u Patent No. 4,493,847) and Imagawa et al. (U.S. Patent No. 4,684,633), these iUIua,uliele:l" are actually uluaLayldlIdil~-containing fat emulsions. The emulsions are not particles, and do not offer the same therapeutic advantages asthe uu,,,, " s comprising a particle and an dldullidullil, acid metabolite 5 provided herein. These references do not disclose any phammaceutical or therapeutic activity for the emulsions themselves. Shell and See (U.S. Patent Nos. 4,82û,732 and 4,955,878) disclose treatments for reducing dysfunction during dlIyiOplaal~ procedures which involve ad~ eli,l~ ,uluaLa~l containing c~", - ' ns to patients. These CulllpOai~iùl~5 also contain a carrier.
10 However, the liquid carriers disclosed, e.g., ~ellyvla~ed alcohols and salinesolutions, in these patents do not comprise particles. The fat-laden Illiulu~,ul~éle 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. AdlllilliaIIa~ivll of Illiulua,ullél~s of such large size to animals can cause difficulties because the 1~ Illi-,lu~,ullel~s 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 dd~ leled to animals.

SUMMARY OF THE INVENTION
This invention provides a method of ad~,,i,,i~Ie,i,,y to an animal a free cl,d-,l ,i-lu"i~. acid metabolite, the method comprising ad",i"i~ ,i"~ to the animal a 2~ cu",, ' , comprising a pharrr~ ' 'Iy acceptable camer, the free metabolite and an elldoc~/~uadLIle particle. Preferably, the cu"., I is ad~llilli:.leled to a human. Preferably, the metabolite is a pluaId~u,lalldill, more preferably a ~lualaulalldill of the E series or of the I senes, and still more preferably, ,uluaIaykllldill E1. The par~icle, which can be any e,~dùcytv~dLle particle, for30 example, a liposome, latex rllil.lualullelel puly;,ly,ei,e, xymozin, starch or gelatin particle, is preferably a liposome.
?
The method can be used therapeutically to administer free dldl,llid~l1ic acid ". ~.L ' to animals afflicted with disorders lldld-.leli~dd by cell activation 35 and adhesion, i,lnd"", ', or toxemia including, without limitation, vaso-occlusive, toxemic, athritic and auto-immune disorders such as- reperfusion injury, post-traumatic shock, restenosis, myocardial infarction, vasculitis, acute respiratory distress syndrome, systemic i"nd"",...'vly response syndrome, .. . : . . .. . .. .

wo 95118620 ~ 1 ~ 9 9 8 6 ~I/U~ lu rheumatoid arthrits, gout, systemic lupus t:ly~;lellldLusus~ juvenile dianetes, multiple sclerosis and Ha~llilllulu's thyroiditis. Particularly preferred therapeutic indications are acute respiratory distress syndrome and systemic il,rldlllllld~uly response syndrome. The disorder treated can be a disorder that is e~d~ l LJdL~d 5 by exposure of the animal to a free ala~.llidulliu acid metabolite in the absence of an endocytosable particle.
Accordingly, an amount of the cr~ ,uu:9Liull comprisin3 an anti-disorder effective amount of the metabolite and particle is a.l~,,i,,iDL.;,ùd to the animal.
o Typically, the anti-disorder effective amount of the c~lllr ' , comprises fromabout 10-12 9 of the metabolite per kg of body wei3ht of the animal to about 10~3 g per ks; desirably the anti-disorder effective amûunt comprises from about 10-8g to about 10-4 9, and more desirably, about 1 o-6 g Of the metabolite per kg.
Preferably, but not necessarily, the ~ ocy'v~aL,le 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 eflective amount of the uu,,,, ", comprises from about 101 of such el~ducy~uadL,le particles per kg of body weight of the animal to about 1014 of 2û such particles per kg, or about 10-6 g of such particles per kg. Preferably, the anti-disorder effective amount of the ~ 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 101 to about 1014 Of the particles per kg.
25 ~ Also provided herein is a pharmaceubcal cu,,,r " , comprising per dose a pham ArPI ~' 'Iy acceptable carrier and an anti-disorder effective amount of afree dl a-,l li~ul li-, acid metabolite and an el ,docy~u ,dL,le particle.
Further provided is a method of treating an animal afflicted with a disorder 30 ullald~ d by cell activation and adhesion, i~nd~",~iv~1 or toxemia; the method comprises adlllilli~ lillg to the animal a, , ', comprising a pham Acel~ acceptable carrier and an anti-disorder eflective amount of an ~,,docytu ,dL,le particle, for example, a liposome, latex ,,,k.,u~ul,e,t,, polystyrene, - xymozin, starch or gelatin particle, is preferably a liposome. Preferably, the anti-disorder effective amount comprises from about 101 to about 1014 ttlldocy~v~.dL,le particles, or about 10-6 9 of ~,)doc~'uaabl~ particles, per kg of body weight of the animal. Preferred therapeutic indications are acute respiratory distress syndrome and systemic illnallllllaLuly response syndrome.

WO 95/18620 ~ ~ ~ 8 ~ r~l~u~ u BRIEF DESCRIPTION QF THE DRAWINGS
Figure 1. Rat Air Pouch Studies. X-axis: saline control, free PGE1, free 15-M-PGE1 (15-methyl-PGE1), LUV-PGE1 formulation ("C-53"; unilamellar liposomal PGE1, prepared in accu,d~",,e with the procedures described in Example 1, below), LUV placebo liposomes ("C-53 placebo"; liposomes prepared o in accu,da"c~ with the procedures used to prepare LUV-PGE1, but not having PGE1), LUV placebo liposomes plus free PGE1, MLV-PGE1 (multilamellar liposomes, prepared in accG,~al,ce with the procedures described in Example 1, below), MLV placebo liposomes (liposomes prepared in ac.,u,.la,,ce with the procedures used to prepare MLV-PGE1, but not having PGE1), MLV placebo liposomes plus free PGE1; y-axis: cells/ml times 10,000 in exudate.
Figure 2. Inhibition of Leukocyte Subset EX~ 1. The graph is scaled for ,cluala~lall-lill~. 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 20 (unshaded): pol~.,,u,,ul~,,ucleocytes (PMNS); second column (lightly shaded):platelets; third column (darkly shaded): I~."~,lloc~tcc.; fourth column (unshaded):
monocytes. Y-axis: cellslml x 10,000 in exudate.
Figure 3. Dose Response of Inhibition of Leukocyte Extravasation. The 25 graph is scaled for ,clu~la~lalldills. The value for neutrophils in the saline control was of ~ 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 liposomes, MLV placebo liposomes plus free PGE1; y-axis: cellslml x 10,000 in exudate. Darkly shaded columns: 25 glkg 30 PGE1 or equivalent amount of placebo liposomes; unshaded: 50 g/kg; lightly shaded: 10 g/kg.
FIGURE 4. Inhibition of Ex~ by Altemative Liposomal Formulations.
X- axis: saline control, free PGE1, LUV-PGE1, LUV placebo liposomes, MLV-35 PGE1, MLV placebo liposomes, SPLV-PGE1 (stable plunlamellar 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)lPOPE-GA (1-palmitoyl-2-oleoyl-pll~a,ul ~ h ' ,e--glutanc acid)-PGE1, wo 95118620 2 ~ 7 ~ 9 8 6 ~ u EPC/Chol/POPE-GA (no PGE1) EPC/Chol/POPE-GA/DOPE-PGE1 (dioleoyl a~)ll iyk ;:,c",olc""i"e covalently linked to PGE1) EPC/Chol/DOPE-PGE
EPC/Chol/POPE-GA placebo plus free PGE1; y-axis: cells/ml x 10 000.
5 Figure 5. Adjuvant A,ll"i'kn'Fr~e PGE1. X-axis: time (days); y-axis: %
change in jaw size. Filled squares: adjuvant control (no PGE1); open squares:
adjuvant and PGE1 cldlllilliatt~ d at day 0; filled diamonds: adjuvant adlllilliaLel~d at day 0 PGE1 at day 10; open diamonds: saline control (no adjuvant).

Figure 6. Adjuvant Al~ /r" ~ PGE1. X-axis: time (days) post-adjuvant ad",i~,iaL, ~ y-axis: percent weight change. Filled squares: adjuvant control;
open squares: adjuvant plus PGE1 at day 0; filled diamonds: adjuvant plus PGE
at day 10; open diamonds: saline control.
Figure 7. Rat General I l~ ,/r ' ~y. 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.
20 FIGURE 8. Rat [n~LUA~lllid Model. X-axis: time (days) post-LPS
~d~ l;aLI ~ 1, y-axis: percent survival in treatment group. Filled squares: ratsa~lllilliak~ d saline control (0 g/kg LPS); open squares: rats eldlll;lliaLellt:d 10 g/kg LPS; hlled diamonds: 15 g/kg LPS; open diamonds: 25 g/kg LPS; filled triangles: 50 g/kg LPS; open triangles: 75 g/kg LPS; filled cirdes: 100 g/kg LPS.
Figure 9. Placebo Liposomes Inhibit Secretion of Human Monocyte TNF and11-1 in Response to Liuu~,~ly~ (LPS). X-axis: free PGE1 LUV-PGE1 ~
placebo LUVs placebo LUVs and saline control (no liposomes or PGE1); y-axis:
percent inhibition of TNF and IL-1 secretion; unshaded: TNF; shaded: IL-1.
Figure 10. Placebo Liposomes Attenuate LPS-lnduced Mortality and Synergize with Free PGE1 to Increase Survival. X-axis: time (days) post-LPS
adlll;~liaLI. ' ~, y-axis: percent survival in treatment group. Filled squares: saline - control (no LPS au,.,i"iaL~ d); filled diamonds: LUV-PGE1; open diamonds:
35 placebo LUVs plus free PGE1; filled triangle: placebo LUVs; open triangles: LPS
control (no liposomes or PGE1); open squares: free PGE1.

WO 95/18620 ~ 8 ~ r~
Figure 11. LUVs and Latex ~r ua,ui~ S 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 PGE1), LUV-PGE1, placebo LUVs plus free PGE1, LATEX ,,,iu,ua,ul,e,~s plus 5 free PGE1, placebo LUVs, LATEX Illk,lua,ull~ltls; y-axis: percent survival in treatment group.
Figure 12. Increasin3 the Particle Number Effects an Increased Survival in the Preâence of a Conâtant Dosage of Free PGE1. Saline and LPS controls are 10 not shown. The survival for these groups was 1ûû% and 5û%"~ ,au~ cly. X-axis: 0, 20, 30, 40, 50 g/kg; y-axis: percent survival in treatment group; z-axis: no particles ad~";";al~ d, LUV-PGE1 a.ll";";a~ d at a particle equivalency of 40 g/kg plus free PGE1 at the indicated doâe.
15 Figure 13. Increasing the Dosa3e of Free PGE1 Alone Increases Mortality Vvhile Increasing Free PGE1 in the Presence of a Constant Particle Number Increaseâ Survival. Saline and LPS controls are not shown. The âurvival for these groups was 100% and 50%, 1~ .ly. X-axis: 0, 20, 30, 40, 50 g/kg; y-axis: percent survival in treatment group; z-axis: no particles d~ liaL~ d, LUV-20 PGE1 ~Idlllilliatl~ at a particle equivalency of 40 g/kg plus free PGE1 at theindicated dose.
FIGURE 14. Leukocyte Pl~dyu~tuaia of Particles is Obligate for the Protective Effect of Particles and PGE1 During Rat [ndutuA_.,,i~. X-axis: LPS control; free25 PGE1; pansorbin; pansorbin plus free PGE1; pansorbin plus free PGE1 plus anti-CR3 ,,,ù,~oclu,~al antibody. Y-axis: percent âurvival.
DETAILED DESC~" I ION
This invention provides a method of dd~llilliaL~Iillg a free clld-~llidu~, acid metabolite to an animal, which comprises ad~"i"ia~:,i"y to the animal a Culll,uOai~iul~ comprising a pharmA~ P~ acceptable carrier, the free metabolite and an e,~docy'uadL,le particle. Preferably, the animal is a human.
"Phann~ t "~ acceptable carriers" are any of the âtandard camers, diluents, excipients and the like generally intended for use in connection with the ddlllilliatlclLiul~ of biologically active agents to animals. Such carners are well .. ~ .:: .. .. .

~ WO95/1~620 ~179~6 r~llU..,~ /u 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 ddlllillia~
well understood by the ordinarily skilled artisan, or within his purview to determine without undue exp~li",t:" " ~. Suitable caniers include, but are not limited to 5 salt solutions such as physiological saline, aqueous buffered solutions, and the like. The pharmaceutical uulllr " , 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 phammaceutical c~ can be provided as a unit dosage form "Arachidonic acid ,Il '~L ' " are ~luala~lalldilla, or compounds which can be wnverted to ,uluala~lall~illa, e.g., artificially or in the body of an animal.
PluaLdulalldills are a group of twenty-carbon fatty acids containing a five-carbon ring, plus seven- and eight-carbon chains, that are made from ala~ idolli~ acid 15 and other twenty-carbon fatty acids having at least three double bonds (e.g., the "essential" fatty acids 8,11,14-eico~ "oic acid, 5,8,11,14-~iw~ dt:llUiL acid or 5,8,11,14,17-~il,ùsa,ut:llLalluic acid; see, e.g., Goodman and Gilman's The Fllallllacolo~icdl Basis of ThPri~r~ c~ supra). Arachidonic acid is the most abundant of these twenty-carbon ,ulualdglalldill precursors in humans.
The twenty-carbon essential fatty acid uluaLdyldlldill precursors, i"L~"" " fommed dunn3 pluaLd~ldlldill synthesis, e.g., prostanoic acid, and structural analogs which can be converted to these compounds, are "clla~.l ,i.lu, ,i-, acid ", ' " " ''P~uala~la,,.li,,-related wmpounds,'' e.g., leukotnenes, 25 ~l ll u" ILJU~dl ,es, lipoxins and ,u, ualacy~lil 1S, include those compounds which are functionally related to ,ClUaLd~U,Idll-lillD and which can also be derived from the twenty carbon essential fatty acid ~luaLaulalldill precursors; ,ulua~a~lalldills and ,C I ua~a~làl Idil ,-related compounds are also "dl dul lidUI liU acid " ' " ' - "
Preferably, the dlaullidulli-, acid metabolite a-JIllillia~ d to animals in accu,da"u~ with the practice of this invention is a ulua~a~lalldill, more preferably, - an E or I series UlU~Ld!JIall-lill, and most preferably, ,uluaLayldlldill E1. The Ul ua~a~lal idil I can also be 1 5-methyl-PGE1 or PGD2.
The temm "free", as used herein with respect to an ala~.llidclllil, acid metabolite, describes the l~laliull~lli,u between the metabolite and a particle coexisting in the same aqueous medium. The metabolite and particle are generally not found at a greater local ~.UI~Ce~ ld;;ui~ in the medium in connection s Wo 95/18620 ` r~1,u~ ~ . /u ~7~98~ _ with each other than the cu, ,c~:"l, " s at which they will be found elsewhere in the medium. That is, the metabolite and particle are not pl~ lll';.A"y associated with each other in the aqueous medium.
An ~lldoc~ ~uadbl~ particle is a particle that can be e, Iducytvaed by endocytic cells typically found in animals. [ndoc~vais (see, e.g., J.E. Darnell et al., Molecular Cell Bioloqv, Scientific American Books, Inc. (New York), 1987, pp.
638-644, the contents of which are ill~.OI,uuld~ed herein by reference) is the process by which a region of a cell's plasma membrane binds to, and envelops, a o particle in the eAtemal medium. As used herein, "~:, Iducytuaia" includes,ullagùcy~uDia, the process by which cells take in relatively large particles in the sunrounding medium.
Md~.,uul,a~ s and monocytes are typical phagocytic cells (pl~auuc~
Particles ,u: ,agoc~tuaed by such cells are generally first coated with serum antibodies on their eAtemal surfaces. Surface receptors on the IJI ,ayùc~ bind to a region common to the various antibodies present in an animal's senum.
Such binding between proteins on particle surfaces and cellular receptors sUmulates plld~u,u~uaia. The process generaily proceeds with the sequential binding of phagocyte surface receptors to antibodies bound to the particle's eAtemal surface, until the particle is completely engulfed by the plasma membrane.
Accordingly, the ~lld~cy~uadL,le particles of this invention are particles having surfaces that can be coated with the senum proteins generally believed tobe lua,uùllaible for recognition of particles by animal endocytic cells.
[nduc~iuad~le pariicles of this invention generally have sizes, and cullt:a,uulldillg surface areas, eflective for coating with a suflficient number of antibody molecules to allow for ,ùI)agoc~tvaia of the particle, but are generally not so lar~qe that they tend to accumulate in small blood vessels. Typically, the most desirable e.~duc~tuadlJlc: particle is about spherical and has a size (diameter) of at most about 5000 nm. More desirably, the c I~doc~tusdùl~ particle has a size of from about 50 nm to about 1000 nm, and still more desirably, about 100 nm. Methods of dt,l~""i"i"g 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 ~A,UFIilll~,ll 1.

wo 95118620 ~ ~ 7 9 9 8 6 P~"~ u The e,1ducytùadl,le particle can be any ~"ducy~u~dl-le, pharmaceutically acceptable particle which can be safely adl"i"ial~":d to animals, and which can be combined with an dldullidol1ic acid metabolite such that the metabolite is free and is capable of being therapeutically effectiYe when ad"li"iaI~,~d to animals. It 5 can be solid, or it can have one or more internal non-solid, e.g., aqueous, ..u",~.alll,le,Ib~ Suitable el~duc~:uadLlt: particles include, without limitation:
liposomes, latex Illi~,lu~ s, pansorbinTM, xymozin, starch, gelatin or ~ol~lylc,i,e particles. Additional ~:llducy~uaa~le particles can readily be identified by ordinanly skilled artisans given the teachings of this invention, e.g., by use in o Yitro in connection with a free dld-,lli~ulliu acid metabolite, or by adl~,i"iaL~dliul- to suitable animal models, such as those descnbed herein.
Liposomes are preferred herein as ~lldoc~w:,dLlt: particles. These are self-ass~",' ' ,9 structures comprising one or more lipid bilayers, each of which 15 t~ c~ an aqueous C~llludl~ . 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 5û nm. Liposomes having more than one lipid bilayer are nl~"' "_"~.. liposomes (MLVs). MLVs used in the practice of this 20 invention preferably comprise a solute entrapped in their aqueou s ,UIll~Jdl~lll~llI~, wherein the cu"~,~"~, , of the solute in each of the aqueous c~ Jall",c"la is substantially equal; i.e., the MLVs have substantially equal illl~lldlll~ . solute distribution. Liposomes ddlllilliaI~ d in a suspension in connection with a free dl~ullidulliu acid metabolite, but not entrapping, or having 25 associated with them, the free metabolite at a greater ..u"ce"t, " ~ than that at which the metabolite may be found elsewhere in the suspension are e,~doc~:uaaLle particles and can be referred to as "placebo" liposomes.
Liposomes can be produced by a variety of processes known in the art.
30 (for a review, see, e.g., Cullis et al., in: Liposomes, From BioDhysics 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"
m~"' I,~lllell liposomes (MLVs). "Ordinary" MLVs can have unequal solute distribution amongst their aqueous Culll,udlllllt:llIa and thereby, osmotic stress between.. u",~.c"I",t~ a. Lenketal.(U.S.PatentNos.4,522,8û3,5,030,453and 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 n~ lk,l liposomes having substantially equal ill~lldlll~llcll solute distribution. Having substantially WO95/18620 2~ 79986 equal ill~elldlllelldl solute distribution means that there will be less osmoticstress amongst the aqueous cclll,udlLlllel,Ia of these MLVs, which will therefore generally be more stable than ordinary MLVs. Unilamellar liposomes can be produced from MLVs by sonication (see Pa,ull~rljol~o~llnc 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 illu~ uldLed herein by reference.
Liposomes and other el Idoc~tuadL,lè particles, can contain, or have lo associated with them, one or more biologically active agents, i.e., compounds which can be ad~ lia~el ed to animals and which may have biological or diagnostic activity therein, or on an animal's cells jn vitro. Biolo~ic~:'y active, or bioactive, agents include, but are not limited to: antiviral, dll~iL~duLelidl, anUfungal, dllLi~ual ~ dll" li '~L ', antiglaucomic, anti-i"nd"""~:u,y or dll" leO~IdaliU compounds, sterols, cdlL~ulljdl ' , amino acids, peptides, proteins, immunoglobulins, immur~nmn~ tnrs, dyes, toxins, enzymes, hommones, ne~"ut,d"~",i~ , g,y~u~,u~ei"a, radiolabels, radiopaque compounds, fluorescent compounds, cell receptor proteins, cell receptor ligands,mydriatic compounds, L~lull..llO.i;:~.'Ula, local dllealll~ ,a, growth promoting~o agents, leyellel~.~;lc agents and the like. Gi~,logic..l'y active agents used in the practice of this invention include, but are not limited to, dll" li~.luLJiàl, anti-illlldlllllld~uly and vas~di'..';I~ agents.
The method of this invention can be used to treat animals afFlicted with a 25 disorder ulldldl,Ieli~ed by cell activation and adhesion, i"na"", " ~ or toxemia which comprises adlllilliaIelillg to the animal an amount of the cu",r ' , whichcomprises an anU-disorder effective amount of the free dl aul ,i,lo"iu acid metabolite and the ellduc~l'usdL~lé particle.
Cell a~ . " ,/adl,esiu" disorders are ul lalauIeli~ed by abnommal activaUon of cells in the blood; the activated cells can adhere to each other, or to activated cells in surrounding vascular endothelium. Such adhesions can lêad to the blockage of small blood vessels, e.g., lung capillaries, consequent stoppage of blood flow, and subsequent damage to surrounding tissue. Cell d~.';:." I/a~llèsiull disorders are a significant problem in a wide variety of medical, " lulu~ies. Endothelial cells, for example vascular, plural, pericardial or abdominal endothelial cells, can be activated by cytokinês, e.g., interleukin-1 (IL-1), tumor necrosis factor-alpha (iTNF-alpha) or bacterial endotoxins. In like _ _ _ _ _ _ _ _ _ _ _ _: . _ . _ _ . _ _ _ _ _ _ _ _ _ _ _ _ _ _ . _ _ . . ...

WO 95118620 2 1 7 ~ ~ 8 6 ~ c ~u /~
manner, blood cells, particularly neutrophils and platelets, can be activated bya3ents such as GM-CSF, bacterial endotoxins, bacterial ~ llud~llduLdll~a, TNF-alpha and the C5a c~" ,uu"e"~ of cu"I,ul~,"~"L.
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 therebyreduce blood flOw to surrounding tissue. The activated cells can also adhere to activated vascular endothelial cells; such adhesion can lead to subsequent o degranulation of vascular endothelium, or to the release of mediators of cell damage such as superoxide anion (2-) and proteolytic enzymes. Accordingly, cell activation/adhesion can lead to the circulatory problems seen in animals affflicted 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 l~lllp~ldl;ly stopped (see, e.g., Se .. ' ' Brcker 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: Leukocyte Adhesion Molecules,Springer-Verlag, New York (1990), pp. 85-156). When there is a blockage in a blood vessel, surrounding endothelial cells, as well as ,' . ,al":d", 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 tum induce activation in neutrophils and platelets after restoration of blood flow to the affected areas.
The same cells which become activated and subsequently undergû
intracellular adhesion can have surface receptors for aldulliduilic acid ~,, 'c,L '' ~ ~Ithout intending to be limited by theory, it is believed that treatment with ald~ idul~ic acid ",: .L "' s, by binding to these receptors, canreduce cell a~,.i; ~/ddl1135iO~l disorder-associated damage by ded~,Li:. ' ,9 the cell surface receptors, ~a,uul ,aiL,le for the elevated levels of intercellular adhesion.
Il,rldlllll ., is a process of cytological and lliaLulOui-,dl reactions occurring in affected blood vessels, and surrounding tissues, in response to an injury or abnommal physical,`chemical or biological stimulation (see, e.g., W0 951~8620 2 1 79 98 ~ C I ~u .
~ .
~tedman's Medical DictionarY (Illustrated) (24th edition, J. V. Basmajian et al., eds.), Williams and Wilkins, Baltimore, MD (1982), pp. 707-708). Illnd,,lllld~uly responses include local reactions and resulting ~o~ul,ological chan3es, destruction or removal of injurious materials and activation of repair ",e~.l ,d"i .,~,s.
5 Agents, includin3 dlaullidul~ic acid ", ' ' '' s, thât can inhibit illndlllllld~uly responses to injuries or abnormal ,ul,;_;alogical stimuli can be used to treat i"nd"""dlu,y disorders, e.g., gout, and various arthntic conditions, including rheumatoid arthritis.
Humoral il~ndlllllldluly responses can be ~lldlduleli~ed 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 ,lldld-,leli~ed by a relatively low number of large cells, or by a relatively large number of neutrophils, eu~i,,u~,l,il~,, monocytes, Iymphocytes and plasma cells.
15 Arachidonic acid r" '.cL "', e.g., Inualdyldll~ills, can inhibit e~ 'lal~, without intending to be limited by theory, it is believed that this is a means by which such agents can inhibit i, Indl 1111 IdlUI y responses.
The rat air pouch exudate model, a well-accepted model for studying i"nd"", ' ~ (see Tate et al, Laboratory In~ , , 59: 192 (1988), the contents of which are il~col,uul~ ,d herein by reference, and the Examples below), can be used to study the ability of dldulli~uilic acid ll, '-.L "' - to inhibit eAt~d. " ,. Rats acclimated to their ell:i.u"",c"t~ are d~eaUleli~ed, and pouches are fommed in their backs by subcutaneous injection of air therein.
25 II~ndlllllldliUI~ is then induced in the pouch by injection therein of an i"" "" ' ~-inducing amount of an i"na"", ' y agent, for example, fMLP. Cells are then expected to enter the air pouch by e,~t~ , from the surrounding vasculature. The ability of an anti-i,,nd,,,,,,dtuly agent to inhibit such extravasation can be examined, for example, by injecting such agents into the 30 animals and then comparing the number of cells entering the air pouch in agent-treated vs. control/untreated animals.

For example, as described below, rats were acclimated, then dl 16~ ed and injected subcutaneously with 2û cc of ambient air. Six days thereafter, 35 i"nd""" 'i~:1 was induced by direct injection of 2.15 Illil.lUUldlll~ of fMLP into the air pouch. Free ~,, Uald!JIdl l~il l E1, free PGE-1 plus placebo liposomes, free PGE-plus latex I l li..l u ,,ul~el es, placebo liposomes alone and latex I l li-,l u:"ul lel es alone were then injected into the animals via the tail vein. Six hours post-.

~ WO 9S118~20 2 ~ 7 ~ 3 ~ ~ r~
i~,llc""" ', induction, the total exudate fluid was recoverered from the air pouch. fMLP ~dlllilliall , induced a substantial number of leukocytes to invade the air pouches; the data~show that ad",il,ial, " ~ of free PGE-1 (2.5, 5and 10 Illiulu~ldllla) plus el~01ucy~uadLle particles ai~ tly (greater than 50%,5 and as much as about 80C/o), reduced extravasation (as measured by cell uullcell~ in the air pouch fluid) in cOIllUdl;aUll to the level of reduction achieved by a.ll,,il,i:.~,dliull of PGE-1 alone. It should be noted that the same il~ndll~ tUIy stimulator and anti~ nc.,llllldluly agent can be used in similar eApeli~llell~a, but in amounts varying within ranges, and according to reasons, l0 well within the purview of ordinarily skilled artisans to detemmine and contrûl, siven the teachings of this invention. Other stimulators and agents can be substituted for those specifically described herein, by ordinarily skilled artisans given the teachings of this invention, and can be used within ranges of amounts well within the purview of such artisans to determine.

Illndllllllc~liull and anti-illndl"",dtu,y action can further be studied by a number of models directed to arthritis induction, and inhibition by anti-arthritic agents, well accepted in the art for such purposes, for example, the rat adjuvant arthritis model described below. Rats are a~",i"iale,ed an arthritis inducer, for ~o example, Freund's adjuvant, by well accepted means, such as intra-dural injection at the base of the tail vein. Ordinarily skilled artisans are well able to determine the d,U~JlU,UI amount of arthritis inducer, for example 1û ,,,i-,,u~,d,,,a of Freund's adjuvant per kg of rat body weight, to use. As joint i"L"""~ , is typically 1l Idl dLlel i~ed by joint ~el~clel, leaa, pain and swelling, inhibition of 25 swelling can be another measure of the effectiveness of an anti-arthritic agent, for example free PGE-1 plus èl~duc~uadLle particles, as can the increased general health and motility of the treated animals.
Toxemia is the clinical Illdll:' ,s observed during the course of 30 infections by infectious agents, e.g., microbes, which contain toxins and other substances poisonous to host animals and which release such toxins inside tha - animals (see, e.g., Stedman's Medical Dictionary (Illustrated). pages 1274-1275 and 1464). Such infections frequently result from physical or chemical trauma.
Fever, I Ijputel laiul 1, changes in leukocyte counts and diarrhea are frequent 35 symptoms ac~.u"l~all~:.,g toxemia induced by gram negative bacterial infections.
Such infections can lead to di~aelllilldled intravascular ro~ ' ', and irreversible shock.

WO95/18620 ~ ~ 7~986 ~ u ~
~s~
Toxemia, arld 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 ~lldU~UA-_.llid model described below. An LDso, that is, a dose of the endotoxin, e.g., E coli li~,u~.olysa..l,l~d,ide (LPS), lethal to about ~0% ûf the test group, is first 5 e:.ldLli~.l,e.l, subsequently, animals are a.li"i"i~ d 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 IlliUlU,U,ldllls per kg of the animal's body weight) or PGE-1 plus endocytosableparticles (at a cumulative dose equivalent to the 40 microgram per kg dose of lo free PGE-1 alone). The ability of the anti-toxemia agent to inhibit endûtoxin-induced mortality is then d~'~"";"ed. Furthermore, inhibition of interleukin-1, interleukin-6 and tumor necrûsis factor alpha secretion, a marker ûf toxemic conditions, can also be assessed; ill~ d~illyly effective anti-toxemia agents are expected to decrease the ~IUt~ dy~, vs. ûf untreated control, of the inhibition ûf 15 secretion of these proteins that is induced by the endotoxin.
Data denved from the l~,ul~a~l,ld~ examples described herein, and presented below, indicate that free PGE-1 plus e,ldûcy.u:.aL~le particles was ~iyl ,iri.,d";'y mûre effective than free PGE-1 alone in increasing the survial rate in 20 endotoxin-treated animals, and was siy" ' I l~ more effective in decreasing the pc~ lllayc: of endotoxin-induced secretion inhibition. For example, the data presented below show that ddlllil liall I of free PGE-1 plus placebo liposomes, in cr "~Ud~i~UI ~ to adl"i"i~l, ' ) 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 4û%
25 decrease in secretion inhibition.
An "anti-disorder effective" amount of a free dld~,lli~Ulli~, acid metabolite and a particle, is any amount of the metabolite plus the particle effective, prophylactically or therapeutically, to ameliorate, alleviate, inhibit, eliminate or 30 prevent a disorder ~,lldldult~ d by cell activation and adhesion, il~rld"", " , or toxemia. The amount of the free dld~ idol~ic acid metabolite is generally equivalent to known therapeutic amounts of free dldullid~lliu acid " ' L ' -alone, in conditions treatable with dldullidùllic acid ~ .L ' However, conditions ordinarily eAdCt:l~dl~d by exposure to dldulli~ol~k. acid ", lL ' s,35 such as illndlllllldlUIy conditions induced or aggravated by ylualddldllJills~ can be treated by coa.l",i";al, - ~ of the same ", 'L " with dllducytu~dLII~
particles. Accordingly, for such conditions, there is a therapeutically effective amount of a free dld~,llidulli~, acid ", ~L " It~, used in connection with an : ~ : . . , .. . ~ .

Wo 95118620 ~ 1 ~ 9 9 8 ~ P~/u~
~,,.luc~tu .dLle particle, where there is 3enerally nû such amount for the metabolite used alûne.
Amounts of free dldulli-:lo~, acid Ill ~L " - included in anti-disorder5 effective amounts of C.Clllr~ "' )S comprisin3 the ", ' ' " - and endocytosable particles are typically dependent upûn 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 andel,~ucytuDdLle particle used; the particular disorder with which the subject is o afflicted; and the age, size, weight and 3eneral condition of the subject treated, including other disorders with which the subject may be aflflicted. Typically the anti-disorder effective amount of the c~ comprises at least about 10-12 g of the free alclullidullic acid metabolite per kg of body weight of the animaltreated with the ,,ulll,u~ ;ùl~.
As described above, a "free" metabolite is a metabolite generally not found in 3reater ..UllUt~ l ,s with el~docyt~saLld particles than elsewhere in the same aqueous medium. Accordin31y, the amount of free metabolite described can include some metabolite which is randomly located in the medium 20 in proximity with the particles, or which is randomly associated with the particle's`
surfaces. Desirably, the anti-disorder effective amount comprises from about 10-12 5 of the metabolite per kg of body weight to about 10-3 9 per kg; more desirably, the anti-disorder effective amount of the ..u,,,, ~ " ., comprises from about 10-8 9 of the metabolite per kg of body weight to about 10-4 9 per kg.
25 Most desirably, the anti-disorder effective amount of the ~,ulllr " , comprises about 1 o-6 9 of the metabolite per kg of body weight.
Anti-disorder effective amounts of free dl d~.l IidUI liC acid 1ll ' ' 'ulldul,ytu.aLle particle-containing c~lllr~ ,s typically comprise a 30 number of e,~doc~u~aL~1,3 particles at least sufficient to enhance the therapeutic response of animals to d~d~ idc)l liu acid ", 'L ~ ' , for conditions responsive to such Ill 'aL " ., or, a number of particles sufficient to elicit a therapeutic response when ad",i"iab,~d with dld~.llidullic acid ", '..L ' , in conditions exract:,Ld~.:d by exposure to such l" 'L " in the absence of ~"doc~.u:.dLle 35 particles.
As described above, "endocytosable particles" typically have surfaces which become coated with senum proteins; it is generally believed that such Wo 95/18620 ~ 1 7 9 9 8 6 F~ JL,...'t- ~u 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 ala~l li.lulliu acid metabolite interaction with target cells.
5 Accordingly, the amount of particles included in anti-disorder effective amounts of uulllr " .15 used herein is typically an amount of particles having a surFace srea suffcient for coating with an amount of serum proteins effedive to enhance e,~ducy'ua;s. As described above, the elldocytuadL/le particles prefenred for use herein are typically about spherical, although variations in shape are ~f~ le, l0 the sperical particles typically have diameters of from about 50 nm to about 1 000nm.
Ordinarily skilled artisans given the teachings of this invention can detemmine the number and weight of el~doc~tu:-aL,le particles to employ in order15 to formulate and anti-disorder effective amount of the uull.r " ~. As discussed herein, an amount of el IdùL,ytu ,r~Lle particles at least sufflcient to elicit, or enhance, a therapeutic response, is used to fommulate the L,u",r " ,. The weight of this amount of particles is generally within the range of about 10-8 to about 10-4 9 per kg of the animal's body weight, and is usually about 10-6 9 or 20 less, but can be outside this range if necesary. Ordinarily skilled artisans given the teachings of this invention can, without undue e,q~e,i",e"' " .~, detemmine if a higher or lower weight of a particular e, ,.lucytu~dLle particle should be used. For examples, such artisans can use the rat air pouch, adjuvant arthntis and el~dULUA~..llia models described herein to assess the anti-disorder effectiveness of a particular weight of the el ,doc~Jtu~al,le particle, either alone or in connection with an amount of a free al dul ,i.lul ,i-, acid metabolite. The number of ~::"~uc~tu:,aL,le particles in the amount selected varies according to a variety of factors, such as the eflective weight ûf particles selected, as well as the size, shape and densiy of the particles. For the preferred el,.lucj.u:.aLle partiles herein, this number is typically from about 101 to about 1014, and can be about Preferably, the anti-disorder effective amount of the c~.",,- ", comprises about 10-6 9 of the metabolite plus the particle per kg, that is 10-6 9 j5 35 about their combined weight, or about 10-6 9 of the metabolite per kg and from about 101 ~:lldûC~tuadLld particles to about 1014 particles. For example, the data presented herein show that both 40 Illiulu~lallli~, or 40 x 10-6 g, of an el IdOcyLu~aL ld particle alone, a liposome or a latex " ,;~,, u:,,ul ,e, e, or 40 2~79g8~
WO 95/18620 P~,ll~l.,,~,.
~ /7 Illil..lUyldlll::l combined oF a free dld-,llidùl~iu acid metabolite and an ~, ,docytuad~le particle can be more therapeutically effective in the models described herein.
Anti-disorder effective amounts of c~"",o:,itiù"s described herein can be used to treat animals afflicted with disorders ulldldu~ d by cell activation andadhesion, i,,lla,,,,,,d~iù,~ or toxemia. Accordingly, such amounts include anti-cell activation and adhesion, i"nd"" ' , or toxemia amounts of a free dldullidulliu acid metabolite and an e~doc~ytuadble particle. An "anti-cell activation/adhesion 0 effective amount" of a free a,d-,l,idu"iu acid metabolite and an ~,n~uc~usdl,le particle is any amount effective to ameliorate, inhibit or prevent the activation of adhesion sites on cells in the blood, or in sun-ounding vascular tissue, and/or the adhesion of such activated cells to other cells in the blood or sun ounding 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-il,nd",ll,d~uly disorder effective amount" of a free dld-,llidulli-, acid metabolite and an e,~;lu~,~tu ,dl,le particle is any amount effective to ameliorate, inhibit or prevent abnommal illndlllllld~UIy responses or readions in animals afflicted with conditions ,lldldulcni~ttd by abnommal i~nd~ , i.e., il~nd~ which is in response to abnormal plly_;Jl~yiudl 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 abnommal ill~la,l, " ~. An "anti-toxemic disorder effective amount" of the Free dld~llidOl)iC acid metabolite andthe particle is any amount effective to ameliorate, inhibit or prevent abnommal i"nd"""d~u,y 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, post-- traumatic shock, acute respiratory distress syndrome, systemic illlldlllllld~uly response syndrome, rheumatoid arthritis, gout, systemic lupus e~yll,e."~'Jaus, juvenile diabetes, multiple sclerosis or I là~llilllu~u'~ thyroiditis. Particularly preFenred therapeutic indications are systemic illndlllllld~UIy response syndrome (SIRS) acute respiratory distress syndrome (ARDS).

Wo 95/18620 ~ ~ 7 9 9 8 6 p~l/~ r . ,~, ~
Treatable disorders according to the method of this invention include those exact:,Ldlùd by exposure of the animal to a free dldullklulli., acid metabolite. As used herein, a disorder "e~dL.t:lllJdk:d" by a free dldullidol~ic acid metabolite i5 a disorder the severity of whose signs, symptoms or effects is 5 increased by adl"i"i:,~, " , of a free dlc...lli.lUlliC acid metabolite, but which can be dlll~l;ul~ ,d, alleviated, eliminated, inhibited, or prevented, therapeutically or ulupllJ~...,~k.~.'ly, by adi"i"i,l,_" ~ of a free dl~lullidollic acid metabolite in connection with an elldocytuadL,l~ particle.
lo Also provided herein is a ~u,,,~u_;';u,) comprising per dose a phammaceutically acceptable carrier and an anti-disorder effective amount of a free ~ li.lol~ic acid metabolite and an ~"docytu~dLle particle.
Further provided is a method of treating an animal afflicted with a disorder 15 ~,lldld~ ed by cell activation and adhesion, il~ndlllllldi;UI~ or toxemia; the method comprises a.li"i"i~ ,i"g to the animal a cu".r- ", comprising a phammaceutically acceptable carrier and an anti-disorder effective amount of an e,~ducyt~,adl,le particle, for example, a liposome, latex rllk,lua,ullt:l~, pol~Ly~ e~
xymozin, starch or gelatin particle, is preferably a liposome. The anti-disorder20 effective amount of the ~"~ocytu~L,l~ particle alone can be about the same asthe anti-disorder effective amount of the free dl d~.l IidUI li-, 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, detemmine the ~5 ~ ,U~UU~i~.~ amount of a particular l:llducy'u:,dL,I~ 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 101 to about 1014, or about 10-6 9, of e,,docy'u~dL,le particles, per kg of body weight of the animal.Preferred therapeutic indications are acute respiratory distress syndrome and 30 systemic illndllllll~.~JIy 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.

~8~
W1~95/18620 EXAM PLES
Example 1 5 Preparation Placebo liPosomes ~ ~" ' "~::a, Liposomes (MLVs) An egg pl~aul~aLi.ly'~,l, " ,~ (EPC) stock solution (20 mg/ml in ethanol) was prepared as follows: 1 9 of dried EPC was dissolved in 50 ml of absolute 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 EPC5 stock solution was added to a round-bottom flask, from which the ethanol was removed by rotocv~."u, " , at 30 deg. C. for at least two hours. The dried EPC
was resuspended in a citrate buffer (50 mM citrate, 15û mM NaCI, brought to pH
4.5 with 10 N NaOH) so as to form a suspension of MLVs. For the ~I~,Udl - I of DPPC-Gontaining MLVs, a DPPC stock solution was prepared as described 20 above using 1.û35 9 of dipalmitoyl phospl, 'y~ (DPPC).
Unilamellar Liposomes (LUVs) Eg3 pllOa,ul '~!~,I, " ,e (20û mg) in ht:,~all~ llallOI (95:5) was rotary 25 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 2001 of ethanol, and the ethanolic solution was drawn into a 1.û ml tuberculin synnge and injected through a 21-gauge needle, at a rate of about 2 drops per second, into 4.0 ml ofa stirring solution at pH 7.0, comprising 1û% weight by volume of aqueous 30 dextrose and 0.01% weight by volume aqueous EDTA. The solution became cloudy upon the addition of the ~l lal IGI,'I;,Ji~ mixture; the solution was extruded 5 times through a 0.1 micron pOl~ -,al L~, ' straight-path filter, followed by a second series of five extnusion through the same type of filter.
35 Pl uaLayla~ Idil l E1 Solutions A PGE1 stock solution (1 mg/ml in ethanol) was prepared as follows: 2û
mg of dried PGE1 was transferred to a 20-ml vial, to which 2û ml of absolute WO 95/18620 2 1 7 9 ~ ~ 6 P~ c ,., ~C~
ethanol was added. The PGE1 was dissolved 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 5 of latex ll,iulua,ul~ s, were mixed with the requisite amount of the PGE1 solution.
Plu~dyld~ldi~l E1 Liposomal Formulations lo For multilamellar liposomal PGE1 formulations, an aliquot of the EPC or DPPC âtOck solutions (9.5 ml) and an aliquot of the PGE1 stock solution (û.5 ml)were combined in the round-bottom flask prior to removal of the ethanol. For unilamellar liposomal PGE1 fommulations, 1.0 mg of PGE1 was added to the ethanol used to resuspend the dried lipid prior to ethanol injection.

ExamPle 2 20 Rat Air Pouch Studies The rat subcutaneous air pouch, a model for acute i"na""" ", and leukocyte eAI,.,.~ ", from the peripherdl vasculature to sites of i"nd""" 'io,~
(Tate, et al., Laboratory l"~ , ", 59:192 (1988), the contents of which are 25 ill1U~U~ herein by reference), was used to study the effect of systemic PGE1 liposomes, no-liposomal PGE1, and particles, in mediating flMLP induced i~nd~ i;UIl.
Male Sprague-Dawley rats, weighing 126-150 9 each, were obtained from 30 Charles River LalJu,d~u,ies. Upon receipt, the rats were acclimated in the animal facility for 2 days. Throughout the ex~."i",~"~;,, the rats were watered and fed ad libitum. For air pouch fommation, the rats were dll~all~t:Li~t:d via inhalent, their backs shaved, and swabbed with ethyl alcohol. Twenty cc of ambient air was injected subcutaneously into the animal's back to form an air pouch, and the 35 animal was retumed to it's cage. The air pouches were monitored to determine integrity, and additional air was injected, if wanranted. For each treatment andcontrol group, the number of rats (n) in each was six. At six days following airpouch fommation, intra-air pouch i"nd""" " , was induced by direct injection into ~ Wo 95/18620 ~ ~ ~ 9 9 ~ 6 r~l~u~
~, the air pouch of 2.15 9 fMLP. Free PGE1, PGE1-placebo liposome, PGE1-latex ",i.,,u,~ s, placebo liposome and latex Illiulu:,ul,~,~ formulations, were simultaneously injected i.v. via the tail vein, and the animals returned to their ca3es. Six hours after flMLP stimulation, the rats were sacrificed by CO2 5 inhalation, and the total exudate fluid was recovered from the air pouch via syringe. The results of these expa,i",t:"~:, are presented in Figures 1-4.
Visual eAd",i" , of the post-stimulation air pouch lining indicated that flMLP effected a thickening of the lining and a large number of invasive lo 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 vascularreactivity and a concunrent 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 e~l,d.- -, from the vasculature to the 15 lumenlexudate fluid of the air pouch. Since the leukocytes were transiently crossing the air pouch lining, subsequent analysis was conflned to those cells present in the aspirated exudate fluid. The free stable ulus~auldll-lill analog 15-methyl-PGE1 was included in these expe,i",~"~a due to its longer bioavailabilityof > 8 hours, as compared to the < 15 min. bioa. ' ' ' `y of free PGE1.

Examole 3 25 Adjuvant Arthritis Male Lewis rats, weighing 126-15û 9 each, were obtained from Charles River Ldbuld~uli~s. Upon receipt, the rats were acclimated in the animal facility for 2 days. Throughout the e~p~,i",t"l~ the rats were watered and fed ad 30 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 glkg, be3inning at day 10, and repeated every second day. Also 35 c~dlllilli~ d was an adjuvant control (no PGE1) and a saline control (no adjuvant). For each treatment group, n=6.

2~79~8G
Wo 95/18620 ` . : r~ /u ~ .
The onset of arthritis was abrupt, occurring between days 10 and 14 in the Freund's-induced anima~s. The symptoms exhibited by untreated control animals were lelld~ll ,ess upon palpation in most active joints, symmetric edemainvolving the joints of the paws, ankles and knees, flexation contractures of the 5 forepaws, malaise, and weight loss attributable to both primary disease as well as inability or diail)vlillaLiùll to access food supplies, due to pain and decreased mobility.
EA,Uel il l lel ILa were conducted to assess the efficacy of free PGE1 1 as well10 as ~,uala~lal~di,, E1 in connection with placebo liposomes and latex Illi~,lv~ eles, placebo liposomes alone and latex Illi~,luapilele5 alone, in mediating the rluyleaSiûl~ of adjuvant arthritis. The pdldllle~la assessed in these eA~,i",a"ts were changes in joint size measured at the rear knee, dSSeaalllell~ of which were made on a weekly basis, changes in body weight, 15 and a subjective scoring of general health, vigor and motility. The results from these eAp~lilll_.~ta are shown in Figures 5-7.
20 ExamPle 4 Rat Cn ;IU~VA611lid Fever, IlJlJUtellaiUI~, changes in leukocyte counts and dianhea are 25 symptoms of gram-negative baderial infections. These infections may lead to diss~",i"~t~,l intravascular rn~ and irreversible shock. A large volume of literature indicates the involvement of leukocyte derived !L-1, IL-6 and TNF in mediatin3 the ,ulu~leaSiùll of endotoxic shock. Because our in vitro data indicated an inhibition of these cytokines from cultured monocytes, we developed30 an in vivo model of rat elldutuA_.Ilia, using mortality as an end point, to assess the e~re~ _. ,ess of PGE1 and particulate fommulations in attenuating LPS-induced death.
[x~ llel~ta were designed to establish an LDso for E. coli LPS
35 (li,vupûlysd~,~;llalide, serotype û55:B5) in Sprague-Dawley rats. The data from these eA~elil~ are shown in Figure 8, and indicate that the LDso is at 50 g/kg. This LPS dosage was used in subsequent expelill.~,.,Lal unless otherwise indicated .

~ WO 9!i/18620 2 ~ ~ g g 8 6 r~ u a3 Male Sprague-Dawley rats, weighing 126-150 9 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 li,uouolysd-,ul,d,i~c as a single 5 bolus, or with a saline (no LPS) control. Mortality was assessed at various times (days) post-LPS ad",;"ial,~l~iol~.
10 ~
Inhibition of Tumor Necrosis Factor AlPha rTNF) and Interleukin-1 Beta (IL-1) SYnthesis in Response to Lil)u,Ju~vsdu-,l ,a~kie (LPS) and PGE1 Adherent human monocytes were stimulated with LPS (1g/mll1û6 cells) at time û. Free PGE1 (not entrapped in liposomes), LUV-PGE1 (large unilamellar liposomes (LUVs) plus free PGE1), LUV-PGE1 "placebo" liposomes (LUVs not containing PGE1) plus free PGE1, placebo liposomes or a saline control (no PGE1) were injected simultaneously (10 M PGE1). Secreted TNF and IL-1 were assayed at three hours. Results from these cA~Je~illlCII~ are presented in Figure 9.
EAam~le 6 Rat CnduluAc.,,ic~ Model Male Sprague-Dawley rats were injected i.v. with 50 9 LPS/kg of body weight at time 0. Free PGE1, LUV-PGE1 (40 g/kg PGE1), placebo LUVs (LUVs not containing PGE1; 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 eq~ cy) 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 35 post-LPS c.dlllil li~ll ,. Results are presented in Figure 10.

~ ~9986 WO 95/18620 2 ~ ~ ~ r~ J. /u : aY
Examole 7 Rat Cn-lu~u~e",ia/Particle Ad~ liaLIdLiol~
Male Sprague-Dawley rats were injected i.v. with 5û g/kg LPS at time û
Free PGE1 (4ûglkg) LUV-PGE1 (4û g/kg PGE1) placebo LUVs (40 g/kg lipid equivalency i e, the number of placebo LUVs was equal to the number of LUVs presentinconnectionwithadoseof40I~ .luyldll~7ofulua~ldl~dillE1 perkgof body weight) latex ~ uaul,~lea (the number equivalent to the number of 10 placebo LUVs) or latex I~ luaul~elea plus free PGE1 (4ûgAcg) 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 Cndutu~elllia ModellParticle Adlll;l~ial, Male Sprague-Dawley rats were d-Jlllillia~eled cu,,, ,s containing PGE1 and a particle in this case either 100 nm LUVs (LUVs having 1ûO nm diameters) or 1ûû nm LATEX Illi,.lua~ les in which either the PGE1 cu, ,ce" 1 or particle number was vaned while the co~c~, IL c.Liu,l or number ofthe other ~ u",uulle"~ remained constant. FIu~Ld~ldlldill E1 dose ranged from 25-40 " ,iu, uu, dl I la per kg; the number of particles d~ll 11;1 lia~e~ ed was 1.2-2 x 1 û1 2 per kg. The rats were injected i.v. at time û with 5û g/kg of LPS. The c~"" - ls were adlll;llia~eled simultaneously by i.v. injection. Survival in each treatment group (n=16) was assessed at 24 hours. Results of these expe,i",e"b are presented in Figures 12-13.
:~

~ WO 95/18620 2 1 7 9 ~ 8 6 F~, I/U~, _. . /u Example 9 Leukocvte Plla,,ùc~tu .i~
Male Sprague-Dawley rats were injected i.v. with 75 mg/kg LPS at time 0.
PGE1 (40 glkg), 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.
o 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 prokin A.
They are not ~ lld~ocy~uDed by leukocytes in their prepared state, but are ~,I,agùcytuDed when antibody directed to the leukocyte phagocytic receptor CR3 15 (CD11a/CD18) are coupled to the particles via a protein A-Fc linkage (see Fallman, M., R. Andersson and T. Andersson. 1993. J. Immunology 151:330-338, the contents of which are i, ,uu, ~,u, ~ ,d herein by reference).

Claims (25)

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.