WO2001003669A2

WO2001003669A2 - Delivery of liposomal-encapsulated antioxidants and applications thereof

Info

Publication number: WO2001003669A2
Application number: PCT/CA2000/000806
Authority: WO
Inventors: Pang N. Shek; Zacharias E. Suntres; Jie Fan; Ori D. Rotstein
Original assignee: Her Majesty The Queen As Represented By The Minister Of National Defence Of Her Majesty's Canadian Government
Priority date: 1999-07-07
Filing date: 2000-07-07
Publication date: 2001-01-18
Also published as: CA2340757A1; AU6142700A; EP1133278A2; JP2003504322A; WO2001003669A3

Abstract

A liposomal composition comprises a hydrophilic sulfhydryl agent and a lipophilic antioxidant, e.g., N-acetyl cysteine and/or α-tocopherol, and a method of delivery by contacting a pulmonary tissue of a mammal with the liposomal composition to achieve a prolonged antioxidant effect.

Description

DELIVERY OF LIPOSOMAL-ENCAPSULATED ANTIOXIDANTS AND PPLICATIONS THEREOF

FIELD OF THE INVENTION

The present invention is related to the field of posome-encapsulation of hydrophilic and hydrophobic agents More specifically, the present invention relates to the use of hposome-encapsulated antioxidants m the amelioration of pulmonary and hepatic damage in multiple organ dysfunction syndrome (MODS), as well as respiratory distress syndromes of vaπous types and etiologies

BACKGROUND OF THE INVENTION

Massive hemorrhage is one of the leading causes of mortality in cases of penetrating trauma For example, the hypoxia within tissues which is associated with hemorrhage and hemorrhagic shock can cause seπous damage to endothelial cells Reperfusion of such lschemic tissue can actually exacerbate the condition, by further promoting the generation of reactive oxygen species, initially by the enzymatic action of xanthine oxidase on xanthine, and later by the recruitment of neutrophils See e g , Granger, 1988 Am J Physwl 255 H1269-H1275, Weiss, 1989 New Engl J Med 320 365-376 Although the body is equipped with an anti-oxidant system which possesses the ability to counteract a limited oxidative msult, if the oxidative burden is overwhelming (e g , in the case of extensive reperfusion injury) the endogenous antioxidant defense simply cannot cope with the extraordinary damaging oxidative load This condition commonly referred to as oxidant stress

In addition, in cases of penetrating trauma (e g , such as those encountered in accident victims or combat casualties), microbial- and toxin-based contamination is frequently encountered This contamination causes a "double-insult" type of clinical scenario, namely concomitant massive hemorrhage and microbial toxic contamination Despite interv ention with blood replacement and aggressive antibiotic administration, the patient may still develop an uncontrolled, systemic inflammatorv response, the se\ eπty of w hich appears to correlate with the dev elopment of multiple organ dysfunction syndrome (MODS) See Marshall and Sweeney, 1990 Arch Surg 125 17- 23 The lung is among the earliest and most frequently affected organ in critically ill patients developing MODS The seventy of dysfunction ranges from mild hypoxemia to a profound respiratory failure designated - Adult Respiratory Distress Syndrome (ARDS).

ARDS is clinically-characterized by hypoxemia, reduced lung compliance, and diffuse alveolar infiltrates. The syndrome usually manifests itself within 2-3 days of the initial underlying disease process, where pro-inflammatory mediators and cells are known to be involved. For example, infiltrating alveolar neutrophils may release reactive oxygen species (i.e., free radicals) and various proteolytic enzymes, causing damage to the endothelium and the epithelium. The invading neutrophils may also either directly release, or initiate the release of, a large number of pro-inflammatory molecules, thus promoting further cellular sequestration and injury. See, Murray, et al, 1988. Am. Rev. Respir. Dis. 138: 720-723.

ARDS is a frequent complication of sepsis and trauma. The mortality rate associated with this acute lung injury is in the range of 50-70%, and the overall annual incidence of ARDS within the United States has been reported to be no less than 150,000. See. Kirkpatrick, et al, 1996. Shock 6: S17-S22, 1996. The terminal mediators of the pathophysiological changes associated with ARDS are believed to be reactive oxygen species, which are either generated by de novo synthesis at injured tissue sites or released in large concentrations by infiltrating neutrophils. In humans, the extent of neutrophil influx and the presence of neutrophil products in the alveolar lavage fluid have been correlated with the severity of the lung injury. See, Hanson, et al, 1984. Fed. Proc. 43: 2799.

These aforementioned observations have led to the development of several therapeutic strategies which are designed to both reduce the influx of neutrophils and to counteract the damaging effects of reactive oxygen species. N-acetyl cysteine (NAC), a known free-radical scavenger and anti-oxidant (see, e.g., Gressier, et al, 1994. Methods Find. Exp. Clin. Pharmacol. 16: 9-13) has been shown to confer protective effects in endotoxemia {see, e.g., Zhang, et al, 1994. Am. J. Physiol. 266: H1746-H1754); to reduce neutrophil influx and lung leaks (see, e.g., Leff. et al, 1993. Am. J. Physiol 265: L501-L506; and attenuate LPS-induced acute lung injury (see, e.g., Davreux, et al, 1997. Shock 6: 432-438) in animals. In each of the above-referenced scenarios, however, a very high dose of N-acetyl cysteine (i.e., 150 mg/kg intravenously, or 1 g/kg intraperitoneally) was required in order to achieve a demonstrable biological effect. Moreover, the potential therapeutic application of N-acetylceyteine in humans has been examined in at least two clinical tnals, with markedly different expenmental results The results of one human tnal sho ed no beneficial treatment effect (see, Jepsen, et al 1992 Crit Care Med 20 918-923), whereas the other tnal demonstrated that the administration of NAC significantly reduced the extent of pulmonary dysfunction (see, Suter, et al , 1994 Chest 105 190-194)

Another therapeutic strategy has been to use a very potent lipophilic antioxidant (e g α-tocopherol) to quench reactive oxygen species, which mediate the injury at the site of inflammation It has been demonstrated that α-tocopherol protects against oxidant-mduced tissue injury by inhibiting membrane hpid peroxidation and hpid peroxide formation, via scavenging singlet oxygen and other reactive oxygen species and by exerting a stabilizing effect on membranes See, e g , Burton and Ingold, 1989 Ann N Y Acad Sci 570 7-22 However, α-tocopherol, in its free-form, is too viscous for parenteral administration and emulsifiers which are utilized to solubhlize this antioxidant are generally found to be toxic to tissues Thus, there remains an, as yet, unfulfilled need, for the development of a therapeutic for the treatment of Adult Respiratory Distress Syndrome (ARDS) by mitigating the influx of neutrophils and the damaging effects of pro-inflammatory mediators (e g , reactive oxygen species and vanous proteolytic enzymes)

SUMMARY OF THE INVENTION

The invention features a therapeutic liposomal formulation, which is useful to ameliorate oxidativ e tissue damage Such pathological conditions may be induced by hemorrhagic shock and endotoxin msult The liposomal formulation of the invention is suitable for circulatory (e g , intrav enous) or mtratracheal administration, and has been quantitatively demonstrated to ameliorate lung and liver mjunes caused by neutrophil infiltration and reactive oxygen species The compositions and methods are also useful for reducing the extent of complications associated with the clinical condition of ARDS and liver mjunes

Accordingly, the invention provides a liposomal composition containing a hydrophilic sulfhydryl agent and a lipophilic antioxidant Preferably the composition contains at least 1%, more preferably at least 10%, more preferably at least 20%, more preferably at least 25% by w eight of the hydrophilic agent For example, the composition contains 28%o by eight of a hydrophilic sulfhydryl agent The amount of hydrophilic sulfhydryl agent in the composition does not exceed 50% by w ght

The composition is charactenzed as having free radical scavenging activity and antioxidant activity The term "free-radical", as utilized herein, is defined as a reactive chemical intermediate form of an oxygen molecule. For example, a free-radical is [O₂] which, due to its high reactivity, can irreversibly damage organic compounds within cells. The term "antioxidant", as utilized herein, is defined as a chemical compound which possesses the ability to mitigate oxidation. For example, an antioxidant reduces the level of oxidation of biological tissues by highly reacti e free radicals by neutralizing free radicals Antioxidants fall into at least two classes, (i) endogenously-produced enzyme anti-oxidants (e g , superoxide dimutase (SOD), glutathione peroxidase) which can catalytically alter or destroy free radicals, and (u) exogenously- gested nutnents or agents (e g , α-tocopherol (vitamin E); vitamin C, and β-carotene (vitamin A)) which function to bind and sequester free radicals Each of the antioxidants listed above may be incorporated into the therapeutic compositions descnbed herein. The antioxidants to be administered are substantially pure, i.e., purified from substances with which they naturally occur.

The compositions contain liposomes. e.g., in the form of unilamellar and ohgolamellar hposome vesicles. The range of size of liposomes in the composition is within 25% of the mean size of the liposomes. The liposomes of the composition are relatively uniform in size. For example, the range of size of liposomes in the composition is preferably within 20%, more preferably within 15%, more preferably within 10%, and most preferably within 5% of the mean size of the liposomes For example, at least 85% (more preferably 90%, more preferably 95%, and most preferably 99-100%) of the liposomes in the composition are with a defined size range, e.g , between 100-400 nm in size To produce uniformly-sized liposomes, the vesicles are produced by extrusion rather than sonication Liposomes are extruded to be approximately 150 nm in size. In another example, the liposomes are extruded to be approximately 450 nm in size. Unlike other methods of hposome manufacture (e g , sonication which method yields a heterogeneous population of liposomes which vary widely m size), extrusion yields a population of liposomes that are relatively uniform in size. Uniformity of size allows more reproducible pharmacokinetics than other methods in the art. The hydrophilic sulfhydryl agent is encapsulated in an aqueous inteπor of a liposomal vesicle and the lipophilic antioxidant is incorporated an outer membrane of the liposomal vesicle

The hydrophilic sulfhydryl agent preferably is an antioxidant such as N-acetyl cysteine, and the lipophilic antioxidant is preferably α-tocopherol The composition contains at least 1%, more preferably at least 5%, more preferably at least 7%, and most preferably at least 9% by weight of the lipophilic antioxidant The composition may also contain a phosphohpid such as a phosphatidylchohne, a dipalmitoylphosphatidylchohne, a lysophosphatidylchohne, a phosphatidylsenne, a phosphatidyl-ethanolamme, a phosphatidylglycerol. or a phosphatidylmositol Cholesterol may also be present in the composition However, the composition preferably does not contain a metal such as Zn, Se, Cr, Cu, or Mn For example, the composition is substantially free of such metals which may contaminate a liposomal preparation as a consequence of the sonication process Since the liposomal compositions descnbed herein are prepared by extrusion rather than sonication, contamination by trace amounts of metals is avoided The liposomal composition contains a hydrophilic sulfhydryl agent, a phosphohpid, and cholesterol, and the approximate molar ratio of dipalmitoylphosphatyidylchohne cholesterol is 7 3, 6 4, or 9 1 For example, the phosphohpid is dipalmitoylphosphatyidylchohne and the approximate molar ratio of dipalmitoylphosphatyidylchohne cholesterol is 7 3 In another example, the the approximate molar ratio of phosphohpid cholesterol hydrophilic sulfliydryl agent is 7 3 15 The liposomal composition is formulated to contain hydrophilic sulfhydryl agent, a phosphohpid, cholesterol, and a lipophilic antioxidant, with an approximate molar ratio of phosphohpid lipophilic antioxidant. cholesterol hydrophilic sulfhydryl agent of 7 2 1 15 The approximate molar ratio of phosphohpid to cholesterol is altered to achieve a desired pharmacokmetic effect The rate of antioxidant release from the composition is indirectly proportionate to the concentration of cholesterol in the composition, I e . a higher percentage of cholesterol yields a composition with a slower pharmacokmetic release profile compared to a composition with a lo er percentage of cholesterol Increasing the amount of cholesterol in the composition results in production of liposomes w ith a more ngid membrane A more ngid membrane indicates a relatively more stable hposome A composition formulated w ith an approximate molar ratio of dipalmitoylphosphatyidylchohne cholesterol of 7 3 is systemically released over a longer penod of time compared to formulations with a lower relative amount of cholesterol The compositions contain at least 10% cholesterol To tailor the kinetics of drug release, the composition is formulated to contain at least 20%, 25%, 30%, 35% or 40% cholesterol Preferably, the percentage of cholesterol in the composition does not exceed 45%

The compositions are therapeutically acti e and have been demonstrated to produce clinical benefits in subjects suffenng from oxidative tissue damage Accordingly, the invention provides a method of dehvenng an antioxidant to a vertebrate (e g , a mammal) by contacting a pulmonary tissue of the mammal with a liposomal composition containing a hydrophilic sulfhydryl agent and a lipophilic antioxidant as descnbed abov e Preferably, the mammal is a human The subjects to be treated include those which have been identified as suffenng from or at nsk of developing a pulmonary injury, a hepatic injury, hemorrhagic shock, endotoxic insult, reperfusion injury, or adult respiratory distress syndrome Methods of diagnosing such ailments are known in the art The compositions are administered orally or parenterally, e g , by an intratracheal, intravenous, mtraartenal, mtrapentoneal, or mtratissue route The invention also includes a method of treatment for insults of oxidative stress and neturophil infiltration induced by hemorrhagic shock and bactenal hpopolysacchande challenge The methods result in a demonstrable reduction of lung and liver mjunes Other features and advantages of the invention will be apparent from the following detailed descnption. and from the claims

DESCRIPTION OF THE DRAWINGS

FIGS 1 A-B are line graphs showing the recovery of α-tocopherol and N-acetyl cysteine (NAC) from lung homogenates following the intratracheal instillation of liposomal α-tocopherol (FIG 1 A) or free NAC, liposomal NAC (L-NAC) or liposomal α-tocopherol and NAC (L-αT-NAC) (FIG IB) The liposomal preparations were formulated as descnbed in "Preparation of hposome-associated antioxidants", and lungs of treated animals were removed at vanous time penods after intratracheal instillation as indicated in the figure Each point represents the mean percentage of recovered dose + SEM of 4 animals

FIGS 2 A-B are bar graphs showing the effects of free N-acetyl cysteine (NAC), liposomal NAC (L-NAC), liposomal α-tocopherol (L-αT), or liposomal α-tocopherol and NAC (L-αT-NAC), administered intratracheally to the lungs of shocked animals FIG 2A shows changes on the LPS-mduced changes in hpid peroxidation. an indicator of oxidative stress, and FIG 2B shows changes m non-protein thiol concentration, a group of protectiv e agents against oxidant-induced injury Animals were maintained in the hemorrhagic shock state for 60 minuets, followed by reperfusion with shed blood over a 2 hour penod Thirty minutes after reperfusion, animals were intratracheally instilled with salme or different antioxidant preparations Following a penod of 18 hours after the initiation of shock, animals were challenged intratreacheally with LPS (300 ug/kg body eight) and killed 4 hours later Each data point represents the mean + SEM of 6 animals The symbol "*" represents significantly different (p<0 05) from the corresponding v alue obtained from shocked animals treated with saline and challenged with LPS

FIGS 3 A-B are bar graphs showing the effects of free N-acetyl cysteine (NAC), liposomal NAC (L-NAC), liposomal α-tocopherol (L-αT), or liposomal α-tocopherol and NAC (L-αT-NAC), administered intratracheally to the lungs of shocked animals FIG. 3A shows changes on the LPS-induced changes in pulmonary myeloperoxidase concentration, and FIG 3B shows the number of polymorphonuclear leukocytes (PMN) in the bronchoalv eolar lavage (BAL) fluid Animals were maintained in the hemorrhagic shock state for 60 minuets, followed by reperfusion with shed blood over a 2 hour penod Thirty minutes after reperfusion, animals were intratracheally instilled with saline or different antioxidant preparations Following a penod of 18 hours after the initiation of shock, animals were challenged intratracheally with LPS (300 g/kg body eight) and killed 4 hours later Each data point represents the mean + SEM of 6 animals The symbol "*" represents significantly different (p<0 05) from the corresponding v alue obtained from shocked animals treated with salme and challenged with LPS

FIG 4 is a bar graph showing the effects of free N-acetyl cysteine (NAC), liposomal NAC (L-NAC), liposomal α-tocopherol (L-αT), or liposomal α-tocopherol and NAC (L-αT-NAC), administered via the circulation to shocked animals, on the LPS-

SUBSTITL TE SHEET (RULE 26) induced changes in the number of polymorphonuclear leukocytes (PMN) in the bronchoalveolar lavage (BAL) fluid. Animals were maintained in the hemorrhagic shock state for 60 min, followed by reperfusion with shed blood and an equal volume of Ringer's lactate with or without liposomal antioxidants, over a 2-h period. Following a period of 18 h after the initiation of shock, animals were challenged intratracheally with LPS (300 micrograms/kg body weight) and killed 4 h later. Each data point represents the mean + SEM of 3 animals. The symbol "*" represents significantly different (p<0.05) from the corresponding value obtained from shocked animals treated with saline and challenged with LPS.

FIGS. 5 A-B are photographs showing the results of a Northern blot assay. FIG. 5A shows expression of cytokine-induced neutrophil chemoattractant (CLNC) mRNA, and FIG. 5 B shows expression of G3PDH mRNA (as a control). FIGS. 5C-D are bar graphs showing the effects of free N-acetyl cysteine (NAC), liposomal NAC (L-NAC), liposomal α-tocopherol (L-αT), or liposomal α-tocopherol and NAC (L-αT-NAC), administered to shocked animals, on the LPS-induced changes in cytokine-induced neutrophil chemoattractant (CINC) expression in lung tissue. Thirty minutes after reperfusion, animals were intratracheally instilled with saline or different antioxidant preparations (FIG. 5C). Alternatively, the saline or antioxidant preparations were administered via the circulation during the 2 hour reperfusion period (FIG. 5D). Animals were maintained in the hemorrhagic shock state for 60 min, followed by reperfusion with shed blood over a 2-h period. Thirty minutes after reperfusion, animals were intratracheally instilled with saline or different antioxidant preparations (Panel A). Alternatively, the saline or antioxidant preparations were administered via the circulation during the 2-h reperfusion period (Panel B). Following a period of 18 h after the initiation of shock, animals were challenged intratreacheally with LPS (300 micrograms/kg body weight) and 4 h later, animals were killed and their lung tissues harvested for the Northern blot procedure. Corresponding G3PDH mRNA bands (in lanes 1 - 6, each representing the corresponding treatment group shown in FIGS. 5C-D) are shown as evidence of comparable loading. Scanning densitometry of Northern blots for CINC mRNA was normalized by densitometry of corresponding G3PDH mRNA bands and expressed as mean +SEM of 4 animals per group. The symbol "*" represents significantly different (p<0.01) from the corresponding value obtained from shocked animals treated with salme and challenged with LPS

FIGS 6A-B are bar graphs showing the effects of free N-acetyl cysteine (NAC), liposomal XA.C (L-NAC), liposomal α-tocopherol (L-αT), oi liposomal α-tocopherol and NAC (L-αT-NAC), administered to shocked animals, on the LPS-induced changes in angiotensm con erting enzyme (ACE) activity, an injury marker of pulmonary endothelial cells Thirtv minutes after reperfusion animals were intratracheally instilled with salme or different antioxidant preparations (FIG 6A) Alternatively, the salme or antioxidant preparations were administered via the circulation dunng the 2 hour reperfusion penod (FIG 6B) Animals were maintained in the hemorrhagic shock state for 60 min, followed by reperfusion with shed blood over a 2-h penod Thirty minutes after reperfusion, animals were intratracheally instilled with saline or different antioxidant preparations (FIG 6A) Alternatively, the saline or antioxidant preparations were administered via the circulation dunng the 2-h reperfusion penod (FIG 6B) Following a penod of 18 h after the initiation of shock, animals were challenged intratracheally with LPS (300 micrograms/kg body weight) and killed 4 h later Each data point represents the mean + SEM of 6 animals The symbol "*" represents

different (p<0 05) from the corresponding value obtained from shocked animals treated with saline and challenged with LPS

FIGS 7A-B are bar graphs showing the effects of free N-acetyl cysteine (NAC). liposomal \ A.C (L-NAC), liposomal α-tocopherol (L-αT), or liposomal α-tocopherol and NAC (L-αT-NAC), administered intratracheally to the lungs of shocked animals, on the LPS-induced changes in transpulmonary albumin flux Animals were maintained in the hemorrhagic shock state for 60 min, followed by reperfusion with shed blood over a 2-h penod Thirty minutes after reperfusion. animals were intratracheally instilled with saline or different antioxidant preparations Follo ing a penod of 18 h after the initiation of shock, animals were challenged intratracheally with LPS (300 micrograms kg body weight) and 4 h later, their transpulmonary albumin flux was assessed as descnbed in the text Each data point represents the mean ^SEM of 3 animals in each group The symbol "*" represents significantly different (p<0 01 ) from the corresponding value obtained from shocked animals treated with saline and challenged w ith LPS FIG 8 is a bar graph showing the effects of free N-acetyl cysteine (NAC), liposomal NAC (L-NAC), liposomal α-tocopherol (L-αT), or liposomal α-tocopherol and NAC (L-αT-NAC), administered via the circulation to shocked animals, on the LPS- mduced changes in the plasma levels of alanme ammotransf erase (ALT), an indicator of liver damage Animals were maintained in the hemorrhagic shock state for 60 min, follo ed by reperfusion with shed blood and an equal volume of Ringer's lactate with or without liposomal antioxidants, over a 2-h penod Following a penod of 18 h after the initiation of shock, animals were challenged intratracheally with LPS (300 micrograms/kg body weight) and killed 4 h later Each data point represents the mean + SEM of 6 animals The symbol "*" represents significantly different (p<0 05) from the corresponding value obtained from shocked animals treated with saline and challenged with LPS

DETAILED DESCRIPTION OF THE INVENTION Methods and compositions are disclosed herein for the production of a therapeutic agent for the treatment of inflammatory complications associated with pulmonary and hepatic mjunes, induced by hemorrhagic shock and endotoxemia The compositions compnse bifunctional liposomal vesicles containing a hydrophilic sulfhydryl agent encapsulated in the aqueous intenor of the vesicle and a lipophilic antioxidant incorporated in the vesicle membrane One of the novel charactenstic of the present invention resides in its sustained release property, which enables the initial delivery and subsequent retention of the active therapeutic agent( s) at the injured tissue "target site" For example, antioxidants are release shortly after administration (e g, within 30 minutes) and continue to be released for a prolonged period of time, e g , for 6 hours, 12 hours, 24 hours, and up to several days post-admmistration

The compositions are prepared by producing liposomes with a specific combination of bilayer- forming hpids, which are compatible with, and non-toxic to, pulmonary tissues. A wide vanety of hpids including, but not limited to, phosphatidyl esters and ethers (e g , phosphatidylchohne, phosphatidylethanolamme, etc ), glycendes, cerebrosides; sphingomyelm; ganghosides; steroids (e g , cholesterol), and the like, may be utilized in the production of the liposomes disclosed herein One or more hpid entities may be present in the hposome, with a bilayer-forming hpid constituting the major liposomal component and the other hpid (e g cholesterol) constituting the minor component

The biologically-active, therapeutic ingredients are incorporated in the liposomal microcapsules and do not interfere with the integnty, nor the stability of the hpid earner Moreover, the therapeutic agents compnsmg the liposomal formulations of the present invention may also serve to increase the overall structural and/or chemical stability of said formulations Typically, the hydrophilic component of the liposomal preparation is compnsed of a compound possessing a sulfhydryl group with free-radical scavenging and antioxidant properties The hydrophobic therapeutic agent, also possessing strong antioxidant properties, is incorporated into the liposomal bi layer The v esicles were prepared b combining the selected hpids, in appropnate ratios, in the presence of the lipophilic antioxidant, followed by the subsequent entrapment of the hydrophilic antioxidant The procedure is conducted in such a manner so as not to denature, inactivate, or compromise the therapeutic efficacy of said antioxidants Numerous methodologies may be utilized for hposome production, including, but not limited to, Shek, et al 1985 ImmunologΛ 57 153-157, 1985, Junma-Romet and Shek, 1991 J

Pharm Pharmacol 43 6-10, Suntres and Shek, 1994 J Pharm Pharmacol 46 23-28, whose disclosures are incorporated herein by reference in their entirety

The liposomal antioxidant formulation may be administered to a vertebrate host by acceptable conventional methods, including, but not limited to, intratracheal, intravenous, intraartenal, and intrapentoneal procedures The administered dose will v ary depending upon the specific antioxidant composition and the recipient A therapeutic regimen can be established by determining the antioxidant retention time at the target body-site and the extent of local inflammation and injury The liposomal antioxidant preparation may be used for the treatment of inflammatory complications associated with sepsis, trauma, and adult respiratory distress syndrome

In contrast to the aforementioned results for NAC, the administration of hposome- entrapped α-tocopherol, alone, has been reported to significantly attenuate endotoxin- mduced tissue injury in the liver and the lung See, Suntres and Shek, 1996 Shock 6 S57-64, 1996, Suntres and Shek, 1996 J Endotoxin Res 3 505-512 Lrposome- entrapped α-tocopherol also has been shown to reduce the toxic effects of reactive oxygen species released from phorbol mynstate acetate-stimulated pulmonary target cells and infiltrating neutrophils See, Suntres and Shek, 1995 J Drug Targeting 3 201-208 The doses of NAC administered according to the mvetion are at least 1-2 logs less than the doses administered using previously described therapeutic methods. The methods described herein are therefore safer than earlier methods.

DETAILED DESCRIPTION OF THE INVENTION I. Preparation of Liposome-Associated Anti-Oxidants

Liposome preparations consisted of either DPPCxholesterol 7:3 with NAC entrapped; DPPC:α-tocopherol:cholesterol 7:2: 1 with NAC entrapped: or DPPC:α- tocopherohcholesterol 7:2:1 without NAC. The lipids were dissolved in chloroform:methanol (2:1, v/v) and the lipid solution was dried in a water-bath at 40°C under a stream of helium gas to a thin film, coating the interior surface of a round- bottomed glass vessel. Any residual solvent was removed by placing the vessel under vacuum for at least 18 hours. The dried lipid was then hvdrated with either 1 ml of 200 mg/ml NAC for every 100 mg of lipid or 1 ml of phosphate-buffered saline for every 100 mg of lipid at 51 ^°C. The glass vessel was vortexed periodically and kept at this temperature for one hour to form multilamellar vesicles. The multilamellar vesicles were subjected to a total of 5 freeze/thaw cycles using liquid nitrogen and a 40°C water- bath. The multilamellar vesicles were then extruded a total of 10-times with an extruder (Lipex Biomolecules; Vancouver, BC) fitted with two, stacked polycarbonate filters of various pore sizes (e.g., 100 nm, 400 nm) under a helium pressure of 100 to 200 p.s.i. Non-entrapped NAC was removed by washing the liposomes twice in phosphate- buffered saline (PBS) and pelleting by centrifugation at 105,000 x g for 1 hour at 5^°C in a Beckman L8-70 ultracentrifuge. Supernatant and resuspended pellet fractions were then assayed to determine overall NAC entrapment, and liposomal vesicle size was determined with the use of a Coulter N4SD particle size analyser. The final NAC liposome preparations were diluted to a concentration of 25.5 mg/ml before use.

Table 1 illustrates particle sizing and the entrapment efficiencies of the liposomal antioxidants of the present invention.

Table 1

Liposome Composition Vesicle Size Entrapment Efficiency (molar ratio) (mean + MEM) N-acetyl cysteine α-Tocopherol

DPP Chol (7:3) 337.3 + 15.0 27.9 + 4.5% 100%

DPPC: α-T:Chol (7:2: 1) 477.0 + 7.00 21.2 + 0.5% 100% 7o

Data represent Mean + SEM of three experimental determinations. Abbreviations: Choi = cholesterol; α-T = α-tocopherol

As shown in Table 1, α-tocopherol incorporated with a high degree of efficiency into the liposomal bilayers, with 100%o entrapment at the lipid molar ratio used. The anti-oxidant, α-tocopherol is an extremely viscous and highly insoluble liquid, which renders it very difficult, if not impossible, to administer parenterally. The liposomal formulation disclosed herein provides a vehicle to facilitate the incorporation of α-tocopherol and its parenteral delivery. Additionally, the same liposome vehicle also enables the encapsulation of N-acetyl cysteine (NAC) for concomitant delivery of both anti-oxidants. N-acetyl cysteine was encapsulated in the vesicles at an entrapment efficiency of about 21-28%. Unlike previously described methods which describe liposomes which entrap a hydrophilic agent or those which entrap a lipophilic agent, the methods described herein co-entrap a hydrophilic and lipophilic agent such as tocopherol and NAC.

Preferred Liposomal Antioxidant Composition For intratracheal administration:

Formula 1

Dipalmitoylphosphatidylcholine 7.56 mg α-Tocopherol 1.27 mg

Cholesterol 0.57 mg

Isotonic saline 94 μl Formula 2

Dipalmitoylphosphatidylchohne 7.67 mg

Cholesterol 1.73 mg

N-acetylcysteme 3.76 mg

Isotonic salme 94 μl

Formula 3

Dipalmitoylphosphatidylchohne 7 56 mg α-Tocopherol 1 27 mg

Cholesterol 0 57 mg

N-acetylcysteme 3 76 mg

Isotonic saline 94 μl

For intravenous administration:

Formula 4

Dipalmitoylphosphatidylchohne 37 82 mg α-Tocopherol 6.34 mg

Cholesterol 2.85 mg

Isotonic salme 470 μl

Formula 5

Dipalmitoylphosphatidylchohne 38.34 mg

Cholesterol 8.66 mg

N-acetylcysteine 18 8 mg

Isotonic salme 470 μl

Formula 6

Dipalmitoylphosphatidylchohne 37 82 mg α-Tocopherol 6 34 mg

Cholesterol 2 85 mg

N-acetylcysteine 18 8 mg

Isotonic saline 470 μl

Preferred Lipid and Antioxidant Molar Ratio

Formula 7

Dipalmitoylphosphatidylchohne 7.00

Cholesterol 3.03

N-acetylcysteine 15.45 Formula 8

Dipalmitoylphosphatidylchohne 7 00 α-Tocopherol 1 97

Cholesterol 1 02 N-acetylcysteme 15.59

Preferred Liposome Size (prepared by the extrusion method) a) For intratracheal administration

Liposomes containing α-tocopherol and N-acetylcysteme 477 - 7 0 nm b) For intravenous administration Liposomes containing α-tocopherol and N-acetylcysteine 149 - 0 3 nm

Preferred Liposomal Antioxidant Dosage for Effective Therapy a) For intratracheal administration

N-acetylcysteine m α-tocopherol liposomes 9 4 mg/kg body eight b) For intravenous administration N-acetylcysteme in α-tocopherol liposomes 47 0 mg/kg body weight

II. Prolonged Anti-Oxidant Retention in the Lung

The therapeutic efficacy of an antioxidant in treating oxidant stress-induced lung injury, to a large extent, depends upon the availability of the antioxidant in sufficient quantities in the pulmonary milieu Most, if not all of the published studies have disclosed very limited (e g , 0 5-5% of initial dose) pulmonary uptake of α-tocopherol following intragastnc and parenteral administration In contrast, the results disclosed herein demonstrate that the intratracheal administration of liposomal α-tocopherol resulted in a total pulmonary level of about 1 5 mg or a retention of about 79% of the administered α-tocopherol dose at 24 hours post-administration (see, FIG 1A)

The retention of NAC within the lungs of normal rats treated intratracheally with free NAC, liposomal NAC, or α-tocopherol liposomal NAC is shown in FIG IB Recovery of NAC in the lung was approximately 1% of the initial dose 1 hour after the administration of free NAC, and subsequently declined to 0 2% of initial dose approximately 3 hours, later In direct contrast, the recovery of NAC after the administration of L-NAC was found to be approximately 8% and 3% of initial dose at 1 hour and 24 hours post-admmistration. respectively The pulmonary retention of NAC following the administration of α-tocopherol liposomal NAC followed a similar retention characteristics to that of liposomal NAC, but it was lower.

III. Animal Model of Hemorrhagic Shock and Lung Injury

Male Sprague-Dawley rats (300-350 grams in weight) were anesthetized with intraperitoneally administration of ketamine (80 mg/kg) and xylazine (8 mg/kg). The right carotid artery was cannulated with a 22-gauge angiocath (Becton Dickinson; Franklin Lakes. NJ) for monitoring of mean arterial pressure (MAP), blood sampling and resuscitation. Hemorrhagic shock was then initiated by blood withdrawal and reduction of the MAP to 40 mm Hg within 15 minutes. This blood pressure was subsequently maintained by further blood withdrawal if the MAP rose to a level of greater-than 45 mm Hg, and by infusion of 0.5 ml of Ringer's Lactate (RL) if the MAP dropped to a level greater-than 35 mm Hg. Withdrawn blood was collected into a solution of 0.1 ml citrate/ml of blood, to prevent clotting. After a hypotensive period of 60 minutes, animals were resuscitated by transfusion of the withdrawn blood and RL in a volume equal to that of withdrawn blood, over a period of two hours. The catheter was then removed, the carotid artery ligated, and the cervical incision sutured. Control (sham) animals underwent the same surgical procedures, but hemorrhage was not induced. NAC delivery occurred in the control animals at an equivalent time to that received by the experimental animals in which shock was induced. Thirty minutes after resuscitation, the endotracheal intubation of liposome- associated NAC and/or α-tocopherol was performed. The animal was placed on a slanted board (20° from the vertical) and was supported by an elastic band under its' upper incisors. A microscope lamp, with its beam directed at the neck area, provided transillumination during the procedure. By opening the mouth of the animal and depressing the tongue, the larynx could be easily visualized. The liposomal suspension was delivered to the lung, via the intratracheal administration, using PE-50 polythylene tubing (6.5 cm) connected to a 25-gauge epidural catheter. The endotracheal tube was introduced into the trachea using gentle pressure. All animals received 150 μl of a liposomal preparation which contained 9.4 mg/kg body weight of NAC and/or α- tocopherol, followed by 20 mechanically-ventilated breaths using a rodent ventilator. Eighteen hours after hemorrhage-resuscitation, lipopolysaccharide (LPS; Escherichia coli strain 01 1B4; at a concentration of 300 μg/kg in 200 ml saline) was administered intratrachealy. Animals were sacrificed in 4-6 hours by a pentobarbital overdose.

IV. Lung Tissue Preparation

The lungs were removed from animals immediately after decapitation and rinsed with ice-cold saline to remove residual blood. All subsequent steps were carried out at 0-4°C. Approximately 1 g of lung sample was homogenized with a Brinkmann Polytron in a sufficient volume of ice-cold 50 mM potassium phosphate buffer, pH 7.4, to produce a 20% homogenate.

V. Lipid Peroxidation Determination

Lipid peroxidation products in lung homogenates (i.e., malonaldehyde (MDA) and 4-hydroxyalkenals (4-HNE)), were measured by the use of an assay kit (R&D Systems; Minneapolis. MN). This assay is based upon the reaction of a chromogenic reagent (N- methyl-2-phenylindole), with MDA and 4-HNE at 45°C. One molecule of either MDA or 4-HNE reacts with 2 molecules of the chromogenic reagent to yield a stable chromophore with maximal absorbance at 586 nm. The concentration of MDA and 4- HNE is then quantitated by the absorbance at this wavelength.

VI. Determination of Pulmonary Non-Protein Sulphydryl Concentration

The non-protein sulphydryl concentration, which includes glutathione and NAC, in pulmonary homogenates was determined as described by Suntres and Shek (1994. J. Pharm. Pharmacol. 46: 23-28). Briefly, the tissue was homogenized in 20% (w v) trichloroacetic acid and centrifuged at 600 x g for 20 minutes in a refrigerated Beckman GS-6R centrifuge. An aliquot of the deproteinized supernatant fraction was added to 2 ml of 0.3 M Na₂HP0₄ solution followed by addition of 0.5 ml of 0.04% 5,5-dithiobis-[2- nitrobenzoic acid] (NbS₂) dissolved in 10% sodium citrate. The absorbance at 412 nm was measured immediately after mixing. VII. Calculation of Pulmonary NAC Content

Since the assay performed above measures the total non-protein sulphydryl content (GSH + NAC) in the lungs of normal animals, the NAC values were obtained by subtracting the values for GSH (460 μg/lung) from the total non-protein sulfhydryl values. VIII. Enzyme Measurements The activity of angiotensin converting enzyme (ACE) m lung horhogenates as determined by using a kit (Sigma Chemical Company, St. Louis, MO) according to the manufacturer's protocol. The activity of myeloperoxidase (MPO) in sonicated whole lung homogenates was performed using an assay kit (R&D Systems; Minneapolis, MN) according to the manufacturer's directions. Plasma alanme aminotransf erase (ALT) activity, expressed as Sigma Frankel (SF) umts/ml, was determined with a diagnostic kit (No. 505, Sigma Chemical Company; St. Louis, MO).

IX. Reduction of Pulmonary Oxidant-Stress After Treatment with Liposomal

Anti-Oxidants Pulmonary Lipid Peroxidation .

The level of hpid peroxidation has been used as an indicator of oxidative stress Challenge of shocked with LPS produced a significant increase in hpid peroxidation in pulmonary homogenates (/ e , an 18-fold increase), as measured by the formation of MDA and 4-HNE. Pre-treatment of rats with NAC did not significantly alter the LPS- induced increases in hpid peroxidation (see, FIG. 2A). Conversely, pre-treatment of rats with NAC- or α-T-containing liposomes or liposomes containing both σ-T and NAC, were found to partially protect against the LPS-induced lipid peroxidation at levels of 55%, 38%, and 62%, respectively.

Pulmonary Non-Protein Thiols As the non-protein sulphydryls (NP-SH), glutathione and NAC. are known to play an important role in protecting cells against oxidant-induced tissue injury , the concentration of non-protein sulphydryls in lung tissues were also measured The administration of LPS in shocked animals was found to result in a significant reduction (43%) in NP-SH concentration (see, FIG. 2B). Intratracheal administration of NAC did not significantly increase the NP-SH content of the lung

In contrast, intratracheal administration instillation of liposomes containing NAC or NAC and α-tocopherol was shown to result in a significant increase in the pulmonary NP-SH content, which may be attnbuted to the retention of NAC within the lung.

X. Bronchoalveolar Lavage Preparation For Bronchoalveolar Lavage Preparation (BAL), the lungs were lavaged with cold phosphate-buffered saline (PBS. 8 mM sodium phosphate. 2 mM potassium phosphate, 0 14 M sodium chlonde, 0 01 M potassium chlonde, pH 7 4 with 0 1 mM EDTA) using an intratracheal angiocath The PBS was instilled in 10 ml aliquots, and gently w ithdrawn with a 10 ml synnge, so as to provide a total administered volume of 40 ml The collected BAL fluid was then centnfuged at 300 x g for 10 minutes to pellet cells The supernatant was discarded, and the pelleted cells were resuspended in a small volume of serum- free DMEM culture medium (Gibco, Burlington, Ontano) Total cell counts were determined on a grid hemocytometer Differential cell counts were enumerated on cytospin-prepared slides that were stained with Wnght-Giemsa stam A total of 500 cells were counted in cross-section per sample and the number of polymorphonuclear leukocytes (PMN) and alveolar macrophages were calculated as the total cell count multiplied by the percentage of the respectiv e cell type in the BAL fluid (BALF) sample

XI. Quantitation of CINC mRNA Expression by Northern Blot Analysis Total RNA from lungs was obtained using the guanidium-isothiocyanate method

See Chomczynski and Sacchi, 1987 Anal Biochem 162 156-160 In bnef, the lungs were harvested from treated animals and immediately frozen in liquid nitrogen The lungs were then thawed and homogenized m 10 ml of 4 M guamdine-isothiocyanate containing 25 mM sodium citrate, 0 5%> sarcosyl, and 100 mM β-mercaptoethanol RNA was denatured, electrophoresed through a 1 2% formaldehyde-agarose gel and transferred to nylon membrane Hybndization was earned out using a [³²P]ATP-end- labeled 30-base ohgonucleotide probe for the cytokine-induced neutrophil chemoattractant (CINC) possessing the with the nucleotide sequence

₅ -GCGGCATCACCTTCAAACTCTGGATGTTCT-3 , [SEQ ID NO 1] which is complementary to nucleotides 134 to 164 of CINC cDNA (see Balckwell, et al 1994 Am J Respir Cell Mol Biol U 464-472), kindly provided by Dr Timothy S Blackwell, \ anderbilt University School of Medicine, Nashville, TN Blots were then washed under conditions of high stnngency and specific mRNA bands were detected by autoradiography in the presence of intensifying screens as previously reported Blots were stnpped and reprobed for glyceraldehyde 3-phosphate dehydrogenase (G3PDH), which is a ubiquitously expressed housekeeping gene to control for loading (see Tso, et al Nucl Acids Res 3 2485-2490) Expression of mRNA was quantitated using a phosphoimager and accompanying ImageQuant software (Molecular Dynamics; Sunnyvale, CA) and was normalized to the G3PDH signal

XII. Reduction of Neutrophil Infiltration in the Lung After Treatment with

Liposomal Anti-Oxidants Lung M\ eloper oxidase ActivitΛ

Lung injury in shocked animals subsequently challenged with LPS, is generally associated with the infiltration and activation of neutrophils This neutrophihc infiltration in the lungs of shocked animals challenged with LPS, was assessed by measunng the activity of myeloperoxidase (MPO), an enzyme localized pnmanly in neutrophils As shown m FIG. 3 A, the MPO activity in shocked animals was increased by 16-fold, following LPS administration This increase is suggestive of neutrophil infiltration w ithin the lungs A very similar increase in MPO activity was also observed in LPS-challenged animals pretreated with NAC Although L-αT liposomal treatment prevented some neutrophil infiltration (i e , 16% reduction), L-NAC and L-αT-NAC had a more pronounced suppressive effect against neutrophil infiltration in the lung, with a 30% and 35% reduction, respectively

Neutrophil Infiltration

Hemorrhage-resuscitation followed by LPS administration caused a 14-fold increase in polymoφhonuclear leukocyte (PMN) infiltration, in companson to that in the control (sham) animals ( ee, FIG 3B) The increase in PMN m the shock/LPS animal group was attenuated to 71 2%, 80 0%, and 58 9% by hposome-associated NAC, hposome-associated α-tocopherol, and hposome-associated NAC/α-tocopherol, respectively Empty liposome alone, did not alter PMN infiltration Similarly, intratracheal administration of NAC alone, did not decrease PMN influx after shock/LPS administration A very similar pattern of liposomal antioxidant-mediated reduction in PMN infiltration was also evident upon anti-oxidant administration via the circulation (see FIG 4)

CINC mRNA Expression

In order to determine whether the alteration in polymoφhonuclear leukocyte (PMN) infiltration is associated with changes m cytokine-induced neutrophil chemoattractant (CINC) expression, total RNA was extracted from whole lung tissue 4 hours after LPS administration. Northern blot analysis for CINC mRNA was then performed.

As shown in FIGS. 5A-D. it was determined that antecedent shock primed the increase in CINC mRNA in response to a subsequent LPS challenge. However, the administration of liposome-associated NAC with or w ithout α-tocopherol via the trachea (see, FIG. 5C) or the circulation (see, FIG. 5D), significantly decreased the CINC mRNA expression, in comparison to those mRNA expression levels found in shocked animals treated with saline and challenged with LPS. Since the inventors of the present invention have previously shown that CINC is the major chemokine, which contributes to PMN influx into alveoli in a two-hit model (see, Fan, et al, 1998. J. Immunol 161 : 440-447), the sustained effect of NAC on preventing PMN infiltration may be mediated by a decrease in CINC expression.

XIII. Assessment of Transpulmonary Albumin Flux

125 Transpulmonary albumin flux was assessed by injecting 1 mCi of I-albumin, in a total volume of 0.2 ml saline, into the tail vein of the rat immediately following intratracheal administration of LPS or saline (see, Nathens, et al, 1996. Surgery 120: 360-366). Six hours after LPS administration, 1 ml of blood was withdrawn by cardiac puncture for scintillation counting by the following procedure. Following exsanguination, the lungs were perfused via a cannula /^'// situ with 10 ml of PBS. The perfused PBS was withdrawn gently and a volume of 1 ml/tube was aliquoted for counting. The transpulmonary albumin flux was normalized to blood cpm using the following formula:

Transpulmonary Albumin Flux = BALF cpm ml Blood cpm ml

XIV. Evidence of Reduced Lung Damage by Treatment with Liposomal Anti-Oxidants

Lung Angiotensin Converting Enzyme:

Due to the fact that angiotensin converting enzyme (ACE) has been used as an injury marker of pulmonary endothelial cells, the effect of LPS on the activity of this enzyme in lung homogenates of shocked animals pre-treated with saline. NAC, L-NAC, Lα-T, or L-αT-NAC, was measured As shown in FIGS 6A-B, the challenge of shocked animals with LPS produced a significant reduction in ACE (35%) in lung homogenates of sahne-pretreated animals Treatment of animals with NAC failed to attenuate the LPS-induced decreases in ACE activity, whereas treatment of animals with NAC-contaimng liposomes conferred a protective effect (19% of salme-pre-treated animals) Additionally, pre-treatment of animals with L-αT or L-αT-NAC also ameliorated the LPS-induced changes in ACE activity, to approximately the same level as that which was observed following L-NAC treatment The administration of the liposomal preparations via the tracheal (see FIG 6A) or the circulation (see FIG 6B) were effective in maintaining ACE activities in the lung, and therefore in reducing the extent of associated pulmonary endothelial cell damage

Transpulmonan Albumin Flux

In order to evaluate w hether hposome-associated NAC and/or α-tocopherol could prevent lung injury, transpulmonary albumin flux was meausred 24 hours after hemorrhage-resuscitation and 6 hours after intratracheal administration of LPS As shown in FIGS 7A-B, the antecedent shock and the subsequent challenge with LPS, markedly increased lung permeability index (PI) The intratracheal administration of hposome-associated NAC. α-tocopherol, and NAC/α-tocopherol significantly attenuated the increase in PI to 27 7° o, 50 9%, and 20 4%, respectively, as compared to that of shocked animals treated w ith salme and challenged with LPS

XV. Reduced Hepatic Damage in Animals Treated with Liposomal

Anti-Oxidants

Plasma Alanine Aminotransferase (ALT) Enzvme The measurement of hepatic enzymes such as plasma alanine aminotransferase

(ALT) released into the blood has been shown to be a reliable indicator of hepatic injury Plasma ALT activities w ere found to be elevated by greater than 8-fold in shocked animals subsequently challenged w ith LPS, thus indicating a rather substantial hepatic injury ( ee FIG 8) The administration of free antioxidant reduced the ALT down to about 6-fold, but the most effectiv e treatment was mediated by the administration of liposomal antioxidants, hich essentially prevented hepatic injury as indicated by the presence of normal plasma ALT activities

It is evident from the above observations that the said bifunctional liposome formulation containing both α-tocopherol and N-acetyl cy steme is effective in providing a therapeutic benefit for treating lung and liver mjunes Furthermore, the concept of bifunctional liposomes can be further exploited to coentrap other pairs of related antioxidants in therapeutic applications

Equivalents

From the foregoing detailed descnption of the specific embodiments of the present invention, it should be readily apparent that a unique compositions and methods of treatment involving the use of uni- and multi-lamellar liposomes as a vehicle to provide systemic delivery of an antioxidant, via administration to the pulmonary system, have been disclosed Although particular embodiments have been disclosed herein in detail, this has been done by way of example for puφoses of illustration only, and is not intended to be limiting with respect to the scope of the appended claims which follow In particular, it is contemplated by the inventor that vanous substitutions, alterations, and modifications may be made to the invention without departing from the spint and scope of the invention as defined by the claims

Other embodiments are within the following claims

Claims

WHAT IS CLAIMED IS

1 A liposomal composition compnsmg hydrophilic sulfhydryl agent and a lipophilic antioxidant. wherein said composition compnses at least 1% by weight of said hydrophilic agent 2 The composition of claim 1. wherein said composition compnses free radical scavenging activity and antioxidant activity

3 The composition of claim 1 , herein said composition compnses at least 10% by weight of said hydrophilic sulfhydryl agent

4 The composition of claim 1, wherein said composition compnses at least 20% by weight of said hydrophilic sulfhydryl agent

5 The composition of claim 1, wherein said composition compnses at least 25% by weight of said hydrophilic sulfhydryl agent

6 The composition of claim 1, wherein said composition compnses at least 28% by weight of said hydrophilic sulfhydryl agent 7 The composition of claim 1, wherein said hydrophilic sulfhydryl agent is encapsulated in an aqueous intenor of a liposomal vesicle and said lipophilic antioxidant is mcoφorated an outer membrane of said liposomal vesicle

8 The composition of claim 1 , wherein said hydrophilic sulfhydryl agent is an antioxidant 9 The composition of claim 8, wherein said antioxidant is N-acetyl cysteine

10 The composition of claim 1, wherein said composition compnses at least 1% by weight of said lipophilic antioxidant

1 1 The composition of claim 1, wherein said composition compnses at least 5% by weight of said lipophilic antioxidant 12 The composition of claim 1, wherein said composition compnses at least 7% by weight of said lipophilic antioxidant

13 The composition of claim 1, wherein said composition compnses at least 9% by weight of said lipophilic antioxidant

14 The composition of claim 1, wherein said lipophilic antioxidant is α-tocopherol 15 The composition of claim 1 wherein the range of size of liposomes in said composition is within 25% of the mean size of said liposomes

16 The composition of claim 1. wherein the size of liposomes in said composition is between 100-400 nm 17 The composition of claim 1, wherein the size of liposomes in said composition is uniform

18 The composition of claim 1, wherein said composition compnses a phosphohpid selected from the group consisting of a phosphatidylchohne, a dipalmitoylphosphatidylchohne, a lysophosphatidylchohne, a phosphatidylsenne. a phosphatidyl-ethanolamine, a phosphatidylglycerol, and a phosphatidvlmositol

19 The composition of claim 18, wherein said composition further compnses cholesterol

20 The composition of claim 1, wherein said composition does not compnse a metal

21 The composition of claim 1, wherein said composition substantially lacks Zn, Se, Cr, Cu, or Mn

22 A liposomal composition compnsmg hydrophilic sulfhydryl agent, a phosphohpid, and cholesterol, wherein the approximate molar ratio of phosphohpid cholesterol is selected from the group consisting of 7 3, 6 4, and 9 1

23 The liposomal composition of claim 22, wherein said phosphohpid is dipalmitoylphosphatyidylchohne and wherein the approximate molar ratio of dipalmitoylphosphatyidylchohne cholesterol is 7 3

24 The liposomal composition of claim 21, wherein the approximate molar ratio of phosphohpid cholesterol hydrophilic sulfhydryl agent is 7 3 15

25 \ liposomal composition compnsmg hydrophilic sulfhydryl agent, a phospholipd. cholesterol, and a lipophilic antioxidant, wherein the approximate molar ratio of phosphohpid lipophilic antioxidant cholesterol hydrophilic sulfhydryl agent is 7 2 1 15

26 A method of delivering an antioxidant to a vertebrate compnsmg contacting a pulmonary tissue of said vertebrate with a liposomal composition compnsmg hydrophilic sulfhydryl agent and a lipophilic antioxidant, wherein said composition compnses at least 1% by weight of said hydrophilic agent 27 The method of claim 26, wherein said vertebrate is a mammal

28 The method of claim 26, wherein said vertebrate is a human

29 The method of claim 26, wherein said vertebrate is identified as suffenng from or at nsk of developing a pulmonary injury, a hepatic injury, hemorrhagic shock, endotoxic insult, reperfusion injury, or adult respiratory distress syndrome

30 The method of claim 26, wherein said composition is administered to said vertebrate by a intratracheal. intravenous, intraartenal, intrapentoneal, or mtratissue route.