EP1133278A2 - Verabreichung liposomaler antioxidantien zur therapeutischen verwendung - Google Patents

Verabreichung liposomaler antioxidantien zur therapeutischen verwendung

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Publication number
EP1133278A2
EP1133278A2 EP00947706A EP00947706A EP1133278A2 EP 1133278 A2 EP1133278 A2 EP 1133278A2 EP 00947706 A EP00947706 A EP 00947706A EP 00947706 A EP00947706 A EP 00947706A EP 1133278 A2 EP1133278 A2 EP 1133278A2
Authority
EP
European Patent Office
Prior art keywords
composition
liposomal
antioxidant
compnses
nac
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00947706A
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English (en)
French (fr)
Inventor
Pang N. Shek
Zacharias E. Suntres
Jie Fan
Ori D. Rotstein
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Minister of National Defence of Canada
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Minister of National Defence of Canada
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Filing date
Publication date
Application filed by Minister of National Defence of Canada filed Critical Minister of National Defence of Canada
Publication of EP1133278A2 publication Critical patent/EP1133278A2/de
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A61K31/355—Tocopherols, e.g. vitamin E
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/08—Plasma substitutes; Perfusion solutions; Dialytics or haemodialytics; Drugs for electrolytic or acid-base disorders, e.g. hypovolemic shock

Definitions

  • 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 va ⁇ ous types and etiologies
  • MODS multiple organ dysfunction syndrome
  • 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.
  • pro-inflammatory mediators and cells are known to be involved.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • the invention provides a liposomal composition containing a hydrophilic sulfhydryl agent and a lipophilic antioxidant
  • 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
  • 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
  • free-radical is defined as a reactive chemical intermediate form of an oxygen molecule.
  • a free-radical is [O 2 ] which, due to its high reactivity, can irreversibly damage organic compounds within cells.
  • antioxidant as utilized herein, is defined as a chemical compound which possesses the ability to mitigate oxidation.
  • 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 , ⁇ -tocopherol (vitamin E); vitamin C, and ⁇ -carotene (vitamin A)) which function to bind and sequester free radicals
  • SOD superoxide dimutase
  • glutathione peroxidase glutathione peroxidase
  • exogenously- gested nutnents or agents e g , ⁇ -tocopherol (vitamin E); vitamin C, and ⁇ -carotene (vitamin A)
  • the antioxidants to be administered are substantially pure, i.
  • 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.
  • 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
  • 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
  • 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.
  • 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 inte ⁇ or 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 ⁇ -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.
  • the composition preferably does not contain a metal such as Zn, Se, Cr, Cu, or Mn
  • 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
  • the phosphohpid is dipalmitoylphosphatyidylchohne and the approximate molar ratio of dipalmitoylphosphatyidylchohne cholesterol is 7 3
  • the approximate molar ratio of phosphohpid cholesterol hydrophilic sulfhydryl agent a hydrophilic sulfhydryl agent, a phosphohpid, and cholesterol
  • 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 .
  • compositions contain at least 10% cholesterol
  • the composition is formulated to contain at least 20%, 25%, 30%, 35% or 40% cholesterol
  • the percentage of cholesterol in the composition does not exceed 45%
  • compositions are therapeutically acti e and have been demonstrated to produce clinical benefits in subjects suffenng from oxidative tissue damage
  • 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
  • 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, mtraartal, mtrapentoneal, or mtratissue route
  • FIGS 1 A-B are line graphs showing the recovery of ⁇ -tocopherol and N-acetyl cysteine (NAC) from lung homogenates following the intratracheal instillation of liposomal ⁇ -tocopherol (FIG 1 A) or free NAC, liposomal NAC (L-NAC) or liposomal ⁇ -tocopherol and NAC (L- ⁇ T-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 ⁇ -tocopherol (L- ⁇ T), or liposomal ⁇ -tocopherol and NAC (L- ⁇ T-NAC), administered intratracheally to the lungs of shocked animals
  • FIG 2A shows changes on the LPS-mduced changes in hpid peroxidation.
  • 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
  • animals were intratracheally instilled with salme or different antioxidant preparations
  • animals were challenged intratreacheally with LPS (300 ug/kg body eight) and killed 4 hours later
  • LPS 300 ug/kg body eight
  • 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 ⁇ -tocopherol (L- ⁇ T), or liposomal ⁇ -tocopherol and NAC (L- ⁇ T-NAC), administered intratracheally to the lungs of shocked animals
  • NAC free N-acetyl cysteine
  • L-NAC liposomal NAC
  • L- ⁇ T liposomal ⁇ -tocopherol
  • NAC liposomal ⁇ -tocopherol and NAC
  • FIG 3A shows changes on the LPS-induced changes in pulmonary myeloperoxidase concentration
  • FIG 3B shows the number of polymorphonuclear leukocytes (PMN) in the bronchoalv eolar lavage (BAL) fluid
  • PMN polymorphonuclear leukocytes
  • BAL bronchoalv eolar lavage
  • FIG 4 is a bar graph showing the effects of free N-acetyl cysteine (NAC), liposomal NAC (L-NAC), liposomal ⁇ -tocopherol (L- ⁇ T), or liposomal ⁇ -tocopherol and NAC (L- ⁇ T-NAC), administered via the circulation to shocked animals, on the LPS-
  • NAC free N-acetyl cysteine
  • L-NAC liposomal NAC
  • L- ⁇ T liposomal ⁇ -tocopherol
  • L- ⁇ T-NAC liposomal ⁇ -tocopherol and NAC
  • SUBSTITL TE SHEET (RULE 26) induced changes in the number of polymorphonuclear leukocytes (PMN) in the bronchoalveolar lavage (BAL) fluid.
  • PMN polymorphonuclear leukocytes
  • BAL bronchoalveolar lavage
  • 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
  • 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 ⁇ -tocopherol (L- ⁇ T), or liposomal ⁇ -tocopherol and NAC (L- ⁇ T-NAC), administered to shocked animals, on the LPS-induced changes in cytokine-induced neutrophil chemoattractant (CINC) expression in lung tissue.
  • NAC free N-acetyl cysteine
  • L-NAC liposomal NAC
  • L- ⁇ T liposomal ⁇ -tocopherol
  • NAC liposomal ⁇ -tocopherol and
  • FIG. 5C 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).
  • FIGS 6A-B are bar graphs showing the effects of free N-acetyl cysteine (NAC), liposomal XA.C (L-NAC), liposomal ⁇ -tocopherol (L- ⁇ T), oi liposomal ⁇ -tocopherol and NAC (L- ⁇ T-NAC), administered to shocked animals, on the LPS-induced changes in angiotensm con erting enzyme (ACE) activity, an injury marker of pulmonary endothelial cells
  • ACE angiotensm con erting enzyme
  • FIGS 6A-B are bar graphs showing the effects of free N-acetyl cysteine (NAC), liposomal XA.C (L-NAC), liposomal ⁇ -tocopherol (L- ⁇ T), oi liposomal ⁇ -tocopherol and NAC (L- ⁇ T-NAC)
  • ACE angiotensm con erting enzyme
  • FIGS 6A-B are bar graphs
  • FIGS 7A-B are bar graphs showing the effects of free N-acetyl cysteine (NAC).
  • L-NAC liposomal ⁇ A.C
  • L- ⁇ T liposomal ⁇ -tocopherol
  • NAC liposomal ⁇ -tocopherol and NAC
  • FIG 8 is a bar graph showing the effects of free N-acetyl cysteine (NAC), liposomal NAC (L-NAC), liposomal ⁇ -tocopherol (L- ⁇ T), or liposomal ⁇ -tocopherol and NAC (L- ⁇ T-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 Animal
  • NAC free N-acetyl cysteine
  • L-NAC liposomal NAC
  • L- ⁇ T liposomal ⁇ -tocopherol
  • NAC liposomal ⁇ -tocopherol and NAC
  • 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"
  • 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
  • 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
  • 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
  • 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 h
  • the liposomal antioxidant formulation may be administered to a vertebrate host by acceptable conventional methods, including, but not limited to, intratracheal, intravenous, intraartal, 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
  • Liposome preparations consisted of either DPPCxholesterol 7:3 with NAC entrapped; DPPC: ⁇ -tocopherol:cholesterol 7:2: 1 with NAC entrapped: or DPPC: ⁇ - 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.
  • PBS phosphate- buffered saline
  • Table 1 illustrates particle sizing and the entrapment efficiencies of the liposomal antioxidants of the present invention.
  • Liposome Composition Vesicle Size Entrapment Efficiency (molar ratio) (mean + MEM) N-acetyl cysteine ⁇ -Tocopherol
  • ⁇ -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, ⁇ -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 ⁇ -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.
  • NAC N-acetyl cysteine
  • N-acetyl cysteine was encapsulated in the vesicles at an entrapment efficiency of about 21-28%.
  • the methods described herein co-entrap a hydrophilic and lipophilic agent such as tocopherol and NAC.
  • Preferred Liposome Size (prepared by the extrusion method) a) For intratracheal administration
  • Liposomes containing ⁇ -tocopherol and N-acetylcysteme 477 - 7 0 nm b) For intravenous administration Liposomes containing ⁇ -tocopherol and N-acetylcysteine 149 - 0 3 nm
  • ketamine 80 mg/kg
  • 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.
  • MAP mean arterial pressure
  • 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.
  • RL Ringer's Lactate
  • Withdrawn blood was collected into a solution of 0.1 ml citrate/ml of blood, to prevent clotting.
  • 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 ⁇ -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 ⁇ l of a liposomal preparation which contained 9.4 mg/kg body weight of NAC and/or ⁇ - 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 ⁇ g/kg in 200 ml saline) was administered intratrachealy. Animals were sacrificed in 4-6 hours by a pentobarbital overdose.
  • 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.
  • Lipid peroxidation products in lung homogenates i.e., malonaldehyde (MDA) and 4-hydroxyalkenals (4-HNE)
  • MDA malonaldehyde
  • 4-HNE 4-hydroxyalkenals
  • 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 Na 2 HP0 4 solution followed by addition of 0.5 ml of 0.04% 5,5-dithiobis-[2- nitrobenzoic acid] (NbS 2 ) dissolved in 10% sodium citrate. The absorbance at 412 nm was measured immediately after mixing. VII. Calculation of Pulmonary NAC Content
  • 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).
  • pre-treatment of rats with NAC- or ⁇ -T-containing liposomes or liposomes containing both ⁇ -T and NAC were found to partially protect against the LPS-induced lipid peroxidation at levels of 55%, 38%, and 62%, respectively.
  • NP-SH non-protein sulphydryls
  • glutathione and NAC are known to play an important role in protecting cells against oxidant-induced tissue injury
  • concentration of non-protein sulphydryls in lung tissues were also measured
  • 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
  • BAL Bronchoalveolar Lavage Preparation
  • 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
  • PBS cold phosphate-buffered saline
  • 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
  • RNA from lungs was obtained using the guanidium-isothiocyanate method
  • 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.
  • MPO myeloperoxidase
  • FIGS. 5A-D it was determined that antecedent shock primed the increase in CINC mRNA in response to a subsequent LPS challenge.
  • the administration of liposome-associated NAC with or w ithout ⁇ -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.
  • 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).
  • 1 ml of blood was withdrawn by cardiac puncture for scintillation counting by the following procedure.
  • 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:
  • angiotensin converting enzyme has been used as an injury marker of pulmonary endothelial cells
  • NAC, L-NAC, L ⁇ -T, or L- ⁇ T-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)
  • pre-treatment of animals with L- ⁇ T or L- ⁇ T-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
  • transpulmonary albumin flux was meausred 24 hours after hemorrhage-resuscitation and 6 hours after intratracheal administration of LPS
  • 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. ⁇ -tocopherol, and NAC/ ⁇ -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
  • Plasma Alanine Aminotransferase (ALT) Enzvme The measurement of hepatic enzymes such as plasma alanine aminotransferase
  • ALT 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
  • bifunctional liposome formulation containing both ⁇ -tocopherol and N-acetyl cy steme is effective in providing a therapeutic benefit for treating lung and liver mjunes
  • the concept of bifunctional liposomes can be further exploited to coentrap other pairs of related antioxidants in therapeutic applications

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GB0111279D0 (en) * 2001-05-10 2001-06-27 Nycomed Imaging As Radiolabelled liposomes
EP3015101B1 (de) 2005-10-03 2019-08-21 PINSKY, Mark A. Non-phospholipid liposomen enthaltend hyaluronsäure
WO2008089408A1 (en) * 2007-01-18 2008-07-24 Pinsky Mark A Materials and methods for delivering antioxidants into the skin
ES2710498T3 (es) 2007-12-26 2019-04-25 Mark A Pinsky Formulaciones de colágeno para la mejora del cuidado de la piel
GB0815435D0 (en) * 2008-08-22 2008-10-01 Camurus Ab Formulations
JP2012521389A (ja) * 2009-03-25 2012-09-13 ノバルティス アーゲー 薬物及びｓｉＲＮＡを含有する医薬組成物
CN101829075B (zh) * 2010-04-27 2011-07-20 中国人民解放军第三军医大学第一附属医院 苦柯胺a和苦柯胺b的用途
WO2021203143A1 (en) * 2020-04-03 2021-10-07 Vivacelle Bio, Inc. Compositions and methods for treating or preventing multiple organ dysfunction syndrome
PH12022552818A1 (en) * 2020-04-24 2024-03-25 Ashvattha Therapeutics Inc Dendrimer compositions and methods for treatment of severe acute respiratory distress syndrome
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US5637315A (en) * 1993-01-04 1997-06-10 Thomas Jefferson University Treatment of disease states induced by oxidative stress

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JURIMA-ROMET M. ET AL: "Lung uptake of liposome-entrapped glutathione after intratracheal administration", J. PHARM. PHARMACOL., vol. 43, 1991, pages 6 - 10 *
See also references of WO0103669A3 *
SUNTRES Z.E. ET AL: "Incorporation of alpha-tocopherol in liposomes promotes the retention of liposome-encapsulated glutathione in rat lung", J. PHARM. PHARMACOL., vol. 46, 1994, pages 23 - 28 *

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