United States Patent [19]
`Ghio et al.
`
`lllllllllllllllllllllllllllllllll||l|llllllllllllllllllllllllllllllllllllll
`US005512270A
`[11] Patent Number:
`5,512,270
`[45] Date of Patent:
`Apr. 30, 1996
`
`[54] METHOD OF INHIBITING OXIDANTS
`USING ALKYLARYL POLYETHER
`ALCOHOL POLYMERS
`
`[75] Inventors: Andrew J. Ghio; Claude A.
`Piantadosi, both of Durham, N .C.;
`Thomas P. Kennedy, Richmond, Va.
`
`[73] Assignee: Duke University, Durham, NC.
`
`[21] Appl. No.: 299,316
`[22] Filed:
`Aug. 31, 1994
`
`Related US. Application Data
`
`[63] Continuation of Ser. No. 39,732, Mar. 30, 1993, abandoned.
`
`............................................ ._ A61K 31/045
`[51] rm. (:1.6
`[52] US. Cl. ................... .. 424/45; 424/78.05; 424/78.06;
`424/78.08; 424/78.37; 514/887
`[58] Field of Search ............................... .. 424/45, DIG. l,
`424/78.03, 78.05, 78.06, 78.37, 78.08; 514/78,
`887, 969, 975
`
`[56]
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`2,454,541 11/1948 Bock et a1. ........................... .. 525/507
`3,663,230
`5/1972 Sato et al. ..... ..
`430/550
`4,039,669
`8/1977 Beyler et a1.
`514/178
`4,826,821
`5/1989 Clements
`.... .. 514/78
`4,944,941
`7/1990 Ammann
`424/85.5
`5,110,806
`5/1992 Clements
`.. 514/78
`5,134,129
`7/1992 Lichtenberger ......................... .. 514/78
`
`OTHER PUBLICATIONS
`
`. epithelioma”, British J-Can
`Pimm et al., “In?uence of . .
`cer, vol. 32(1) pp. 62-67 (1975).
`Hashimoto et al., “Antimetastatic elfect .
`.
`. tumors”
`Tohoku—J—Exp.-Med. 128(3) pp. 259-265 (1979).
`McCarty-M F, “An antithrombotic role for. . . pathologies”
`Med-Hypotheses, 19(4) pp. 345-357 (Apr. 1986).
`Wiseman et al., “The Structural mimicry .
`.
`. anti-cancer
`agent” Biochem-Biophys-ACTA, 1138(3) pp. 197-202
`(1992).
`Wiseman et al., “Droloxifene .
`
`. breast cancer” Cancer
`
`.
`
`-Lett, 66(1) pp. 61-68 (1992).
`. reduction method” J.
`.
`Kim et al., “Reevaluation of the .
`Korean Ag. Chem. Society, 36(5) pp. 364-369 (1993).
`Kondo et al., “Triton . . . Infection” Japan J. Med. Sci. Biol.,
`39(2) pp. 35-47 (1986).
`,Matalon et al., “Mitigation of Pulmonary . . . Surfactant”, J.
`Appl. Physiol., vol. 62, No. 2, pp. 756-761, (Feb. 1987).
`Tooley et al., “Lung Function .
`.
`. Surfactant,” AM. Rev.
`Respir-Dis., vol. 136, No. 3, pp. 651-656, (Sep. 1987).
`Notter, R. H., “Biophysical Behavior . . . Pathophysiology,”
`Semin-Perinatol., vol. 12, No. 3, pp. 180-212 (Jul. 1988).
`Baker et al., “Development .
`.
`. Enzymes,” J.—J.-Appl
`-Physiology, vol. 66, No. 4, pp. 1679-1684, (Apr. 1989).
`Cornforth et al. Nature,“Antituberculous ElTect of Certain
`Surface-Active Polyoxyethylene Ethers in Mice” vol. 168,
`pp. 150-153 (1951).
`Floyd et al. Journal of Biochemical and Biophysical Meth
`ods, “Sensitive Assay of Hydroxyl Free Radical Formation
`.
`. .” vol. 10, pp. 221-235 (1984).
`Floyd et al. Journal of Free Radicals in Biology & Medicine,
`“Use of Salicylate With High Pressure Liquid Chromatog
`raphy .
`.
`. ”, V0. 2, pp. 13-18 (1986).
`
`(List continued on next page.)
`Primary Examiner—Melvyn I. Marquis
`Assistant Examiner—Robert H. Harrison
`Attorney, Agent, or Firm—Richard E. Jenkins
`
`[57]
`
`ABSTRACT
`
`The present invention relates to use of alkylaryl polyether
`alcohol polymers as antioxidants to suppress certain oxidant
`chemical reactions that cause tissue injury and disease in
`mammals. Disclosed is a method of inhibiting oxidants
`using alkylaryl polyether alcohol polymers. More particu
`larly, disclosed is a method for the treatment of mammalian
`disease entities related to overproduction of partially
`reduced oxygen species comprising administering to a mam
`mal a treatment e?ective amount of an alkylaryl polyether
`alcohol polymer. The mammalian disease entities include,
`but are not limited to, myocardial infarction, stroke, adult
`respiratory distress syndrome, oxygen toxicity of the lung,
`lung injury from asbestos, Parkinson’s disease, thermal and
`solar burns of the skin, and injury to the gastrointestinal tract
`from nonsteroidal anti-in?ammatory agents.
`
`8 Claims, 3 Drawing Sheets
`
`[32,5 DIHYDROXYBENZOIC ACID
`I 2,3 DIHYDROXYBENZOIC ACID
`
`.5 O J
`
`S0 o
`
`8.0 '
`
`XIO2 SAL] CYLATE PRODUCT pM/ml
`
`
`
`
`
`NORMAL TYLOXAPOL TYLOXAPOL TYLOXAPOL
`SALINE O.Img/ml lOmg/ml l0.0mg/ml
`
`Page 1
`
`

`
`5,512,270
`Page 2
`
`OTHER PUBLICATIONS
`
`Glassrnan, Science, “Hemolytic Activity of Some Nonionic
`Surface~Active Agents”, vol. 111, pp. 688—689 (Jun. 23,
`1950).
`Halliwell et a1. Method In Enzymology, “Role of Free
`Radicals .
`.
`. ”, vol. 186, pp. 1-83 (1990).
`Turrens et 211., Journal of Clinical Investigation, “Protection
`Against Oxygen Toxicity”, v01. 73, pp. 87-95 (Jan., 1984)
`
`Tainter et al. The New England Journal of Medicine,
`“Alevaire as a Mucolytic Agent”, vol. 253, pp. 764—767
`(1955).
`Ghio et a1. American Journal of Physiology, Lung Cellular
`and Molecular Physiology 7, “Role of Surface Complexed
`Iron in Oxidant Generation .
`.
`. " vol. 263, pp. L511-L518
`(29 Jun. 1992) by Ghio et a1.
`
`Page 2
`
`

`
`US. Patent
`
`Apr. 30, 1996
`
`Sheet 1 0f 3
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`5,512,270
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`Page 3
`
`

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`US. Patent
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`Apr. 30, 1996
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`Page 4
`
`

`
`US. Patent
`
`Apr. 30, 1996
`
`Sheet 3 of 3
`
`5,512,270
`
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`
`Page 5
`
`

`
`5,512,270
`
`1
`METHOD OF INHIBITING OXIDANTS
`USING ALKYLARYL POLYETHER
`ALCOHOL POLYMERS
`
`This is a continuation of application Ser. No. 08/039,732
`?led on Mar. 30, 1993, now abandoned.
`
`5
`
`BACKGROUND OF THE INVENTION
`
`The present invention relates to use of alkylaryl polyether
`alcohol polymers as antioxidants to suppress certain oxidant
`chemical reactions that cause tissue injury and disease in
`mammals and plants.
`Oxygen is life-giving to aerobic plants and animals who
`depend on it for energy metabolism. It can also be lethal to
`those same organisms when it is altered from its stable
`dioxygen (02) state to any one of three partially reduced
`species: a) the one electron reduced form superoxide anion
`(02'); b) the two electron reduced form hydrogen peroxide
`(H202); or the deadly three electron reduced form hydroxyl
`radical ('OH). In biologic systems 02- and H202 are meta
`bolic byproducts of a host of enzymes (oxygenases) that use
`oxygen as a cofactor. H2O2 is also produced from Oz'by the
`enzymatic action of superoxide dismutases. However, ‘OH is
`generally produced only when O2_and H2O2 interact with
`transitional ions of metals such as iron and copper in
`dangerous cyclical redox reactions:
`
`25
`
`30
`
`2
`biologic molecules. Not all oxidizable compounds can per
`form an antioxidant function. To successfully protect chemi
`cal and biologic systems from oxidants, the antioxidant must
`have a higher reactivity for the oxidant than the chemical or
`biologic molecule which it seeks to protect. It is theoretically
`possible to synthesize a multitude of compounds with anti
`oxidant properties. However, the factor limiting use of these
`antioxidants as treatments in biologic systems is the inherent
`toxicity of the antioxidant compounds themselves. Thus, it
`is a major advantage to discover that a class of commonly
`used and nontoxic ingredients in medicinal pharmacologic
`preparations are also potent antioxidants. Not only can such
`compounds react with partially reduced 02 species, but they
`can be used as treatments for oxidant mediated diseases
`without themselves causing toxicity to biologic systems.
`
`SUMMARY OF THE INVENTION
`
`As explained below, this invention describes how alky
`laryl polyether alcohol polymers are useful as antioxidants
`in blocking oxidant reactions and biologic injury from
`partially reduced 02 species. Alkylaryl polyether alcohol
`polymers are known and used commerically as surface
`active detergents and wetting agents (U.S. Pat. No. 2,454,
`541). A structure representative of the class of compounds is
`
`The above reaction is termed the superoxide driven Fenton
`reaction. The Fenton reaction can also be initiated by other
`reducing substances such as ascorbate in the presence of
`ferric ion and H202.
`While 02- and H202 are each toxic for biologic systems,
`‘OH (and its alternate hypothesized form the ferryl interme
`diate FeO2+) is a highly reactive species that can oxidize
`unsaturated membrane lipids, damage cellular proteins and
`cause mutagenic strand breaks in DNA. To prevent injury
`from partially reduced 02 species under normal conditions,
`cells have evolved an elaborate system of antioxidant
`enzymes (superoxide dismutase, catalase, glutathione per
`oxidase) and antioxidant molecules (glutathione, alpha-to
`copherol, beta carotene). However, when production of
`partially reduced 02 species exceeds the capacity of cellular
`antioxidant defenses to contain them, oxidant injury occurs.
`A growing number of mammalian disease entities are now
`thought to be related to overproduction of partially reduced
`02 species, including the reperfusion injury syndromes,
`myocardial infarction and stroke, adult respiratory distress
`syndrome, oxygen toxicity of the lung, lung injury from
`asbestos, Parkinson’s disease, thermal and solar burns of the
`skin, and injury to the gastrointestinal tract from nonsteroi
`dal anti-in?ammatory agents (see Table IV, page 60, Halli
`well B and Gutteridge JMC. Methods in Enzymology (1990)
`186:1-85). Treatment of these conditions is increasingly
`directed either toward strategies that prevent enzymatic
`production of partially reduced 02 species or toward the
`introduction of exogenous antioxidant compounds that
`restore oxidant-antioxidant balance in biologic and chemical
`systems.
`Antioxidants are compounds that can be easily oxidized to
`stable chemical forms. They can protect chemical and bio
`logic systems by sacri?cing themselves to oxidation in
`preference to oxidation of critically important chemical and
`
`35
`
`45
`
`50
`
`55
`
`60
`
`65
`
`where, R=ethylene, R1=tertiary octyl, x is greater than 1, and
`y=8 to 18. The best known of this class is tyloxapol, a
`polymer of 4-(1,l,3,3-tetramethylbutyl)phenol with formal
`dehyde and oxirane. Tyloxapol has been used in human
`pharrnacologic formulations for over 30 years (Tainter ML
`et al. New England Journal of Medicine (1955)
`253:764-767). Tyloxapol is relatively nontoxic and does not
`hemolyze red blood cells in a thousand times the concen
`trations at which other detergents are hemolytic (Glassman
`HN. Science (1950) 111:688-689).
`It is the object of the present invention to provide a
`method to inhibit oxidant chemical reactions caused by
`partially reduced 02 species.
`It is a further object of the present invention to provide a
`method to protect mammalian tissues against injury from
`partially reduced 02 species.
`It is a further object of the present invention to provide a
`method for inhibiting oxidant chemical reactions caused by
`partially reduced 02 species by aerosol treatment with the
`therapeutic agent.
`It is a further object of the present invention to provide a
`method for inhibiting oxidant chemical reactions caused by
`partially reduced 02 species by topical application of the
`therapeutic agent to the skin.
`It is an advantage of the present invention that the
`therapeutic agent is produced from a toxicologically char
`acterized class of compounds with low toxicologic potential
`to biologic systems.
`Consideration of the speci?cation, including the several
`?gures and examples to follow will enable one skilled in the
`art to determine additional objects and advantages of the
`invention.
`
`Page 6
`
`

`
`5,512,270
`
`3
`The present invention provides a medicament for the
`inhibition of injurious eiTects of partially reduced 02 species
`in chemical and biologic systems comprising a treatment
`effective amount of tyloxapol and related alkylaryl polyether
`alcohol polymers. In preferred embodiments of the inven
`tion, the medicament is directly instilled into the respiratory
`system and administered by aerosolization. In this embodi
`ment, the medicament preferably includes a physiologically
`acceptable carrier which may be selected from the group
`consisting of physiologically bulfered saline, isotonic saline,
`and normal saline and an additional treatment effective
`amount of cetyl alcohol. The pH of the alkylaryl polyether
`alcohol polymer and carrier mixture is preferably greater
`than 6.5 but equal to or less than 7.4. In other preferred
`embodiments of the invention, the medicament is applied
`topically to the skin. In this embodiment, the medicament
`preferably includes a physiologic carrier selected from a
`commercially available petrolatum based ointment or U.S.P.
`cold cream.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`Reference to the following detailed description may help
`to better explain the invention in conjunction with the
`drawings which:
`FIG. 1 shows a graph of the inhibitory effect of tyloxapol
`on ‘OH generation by the Fenton reaction, as measured by
`hydroxylation of salicylate.
`FIG. 2 shows a graph of the inhibitory eifect of tyloxapol
`on ‘OH generation by the Fenton reaction, as measured by
`oxidation of the sugar 2-deoxyribose.
`FIG. 3 shows lung wet/dry weight ratios in rats exposed
`to 100% oxygen and treated with normal saline, tyloxapol,
`and tyloxapol plus cetyl alcohol.
`FIG. 4 shows pleural ?uid in rats exposed to 100%
`oxygen and treated with normal saline, tyloxapol, and tylox
`apol plus cetyl alcohol.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`Alkylaryl polyether alcohol polymers can in general be
`synthesized by condensing alkylaryl alcohols with formal
`dehyde, as described by Bock and Rainey in U.S. Pat. No.
`2,454,541 (1948 to Rohm & Haas). Several speci?c alky
`laryl polyether alcohol polymers can be easily synthesized
`by methods previously described (J .W. Cornforth et al.
`Nature (1951) 168:150-153). The prototype compound of
`this class tyloxapol can be conveniently purchased in phar
`macologically acceptable purity from Rohm and Haas Co.,
`Philadelphia, Pa.
`For treatment of mammalian respiratory conditions
`related to overproduction of partially reduced 02 species, the
`alkylaryl polyether alcohol polymer is dissolved in sterile
`0.9% NaCl for injection, and the pH is adjusted to approxi
`mately 7.0 by addition of NaOH or HCl. A nonpolymeric
`alkyl or aryl alcohol such as cetyl alcohol (hexadecanol)
`may be added equivalent to 1—1.5 times the weight of
`tyloxapol to increase the eifectiveness (see FIG. 5 and Table
`H below) of the mixture in protecting against oxidant injury.
`This mixture is then administered to the lung by direct
`instillation into the respiratory system. The mixture may also
`be administered by aerosolization using a clinically avail
`able positive pressure driven nebulizer that produces respi
`rable particles of less than 5 microns mass median diameter.
`As an example, a 0.125% solution of tyloxapol is made in
`sterile 0.9% NaCl and double glass distilled deionized water
`
`10
`
`20
`
`25
`
`30
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`4
`to make it isotonic with respect to respiratory secretions. The
`pH is adjusted to approximately 7.0 to prevent broncho
`spasm from extremes of acidity or alkalinity. This mixture is
`sterilized by vacuum ?ltration through a 0.22 micron Mil
`lipore ?lter and 3.3 ml each is packaged into 5 ml unit dose
`glass vials with rubber stoppers fastened with aluminum
`crimp-on “?ip-tear” seals. To provide additional sterilization
`of product, unit dose vials are terminally autoclaved 12-14
`minutes at 125 degrees Centigrade. A 5% concentration of
`glycerol may be optionally added to the above mixture to
`stabilize droplet size during aerosolization. For administra
`tion of treatment eifective doses, 3 ml of sterile tyloxapol
`solution is inhaled as an aerosol every 4-6 hours using a
`clinically available positive pressure driven’ nebulizer
`(Acorn or deVilbiss). Alternatively, the mixture can be
`nebulized into the respiratory delivery circuit of a mechani
`cal ventilator. A beta sympathetic agonist bronchodilator _
`(such as 1.25 to 2.5 mg of albuterol) can be mixed with the
`tyloxapol solution and nebulized concommitantly to prevent
`any transient bronchospasm that might occur from the
`tyloxapol solution itself.
`For treatment of cutaneous oxidant-mediated disorders
`such as solar burn, a 0.5 to 5% mixture (w/w) is made with
`an alkylaryl polyether alcohol such as tyloxapol in a com
`mercially available petrolatum based ointment such as
`Aquaphor (Beiersdorf, Inc., Norwalk, Conn.), white petro
`latum or U.S.P. cold cream as the base vehicle. This mixture
`is rubbed lightly onto the affected skin area 3 to 4 times
`daily.
`In order to facilitate a further understanding of the inven
`tion, the following examples primarily illustrate certain
`more speci?c details thereof.
`Example I demonstrates the potent activity of alkylaryl
`polyether alcohol polymers as ‘OH inhibitors in chemical
`systems. Example H demonstrates the therapeutic bene?t of
`using alkylaryl polyether alcohol polymers to prevent mam
`malian lung injury from exposure to 100% oxygen.
`
`EXAMPLE I
`
`Inhibition of Oxidants Generated by the Fenton
`Reaction
`
`The ?rst chemical system used to test the antioxidant
`activity of alkylaryl polyether alcohol polymers employed
`salicylate as the target molecule of oxidants. Hydroxyl
`radical reacts with salicylic acid (2-hydroxybenzoic acid) to
`produce two dihydroxybenzoic acid products, 2,3- and 2,5—
`dihydroxybenzoic acid. These hydroxylated products pro
`vide evidence of ‘OH generation (R.A. Floyd et al. Journal
`of Biochemical and Biophysical Methods (1984)
`10:221-235; R.A. Floyd et al. Journal of Free Radicals in
`Biology & Medicine (1986) 2:13-18). The detection of 2,3
`and 2,5-dihydroxybenzoic acid was performed using high
`performance liquid chromatography with electrochemical
`detection. Suspensions of 10 uM FeCl3, 1.0 mM H202, 1.0
`mM ascorbate, and 10.0 uM salicylic acid were employed to
`generate and detect vOH. Either 0.1 ml of normal saline or
`tyloxapol (?nal concentrations of 0.0 to 10 mg/ml) were
`added. The reaction mixtures were incubated at 45 degrees
`Centigrade for 30 min and centrifuged at 1200 g for 10 min.
`Supernatant was centrifuged (Beckman Microfuge E)
`through a 0.22 uM microfuge tube ?lter (PGC Scienti?c No.
`352—118) at 15,000 g. A 100 uL sample of the eluate was
`injected onto a C18 RP HPLC column (250X4.7 mm,
`Beckman No. 235329). Hydroxylated products of salicylate
`were quanti?ed with a Coulochem electrochemical detector
`
`Page 7
`
`

`
`5,512,270
`
`10
`
`25
`
`30
`
`35
`
`5
`(ESA model 5100A) with the detector set at a reducing
`potential of —0.40 VDC. The guard cell (used as a screen)
`was set at an oxidizing potential of +0.40 VDC. Measure
`ments were done in duplicate. FIG. 1 shows that the addition
`of tyloxapol to the reaction mixture inhibited 'OH generation
`in a concentration dependent manner.
`The second chemical system used to test the antioxidant
`activity of alkylaryl polyether alcohol polymers employed
`2-deoxyribose as the target molecule of oxidants. This
`pentose sugar reacts with oxidants to yield a mixture of
`products. On heating with thiobarbituric acid (TBA) at low
`pH, these products form a pink chromophore that can be
`measured by its absorbance at 532 nm (B. Halliwell and
`J.M.C. Gutteridge. Methods in Enzymology (1990)
`186:1-85). The chemical system employed to generate oxi
`dants was a reaction mixture containing 10.0 uM FeCl3, 1.0
`mM ascorbate, 1.0 mM H202, and 1.0 mM deoxyribose in
`Hanks Balanced Salt Solution. This system is useful for
`measuring site-speci?c ‘OH generation on biologic mol
`ecules, as described by Halliwell and Gutteridge in the
`reference immediately above. Either 0.1 ml of normal saline
`or tyloxapol (?nal concentrations of 0.0 to 10.0 mg/ml) were
`added. The reaction mixtures were incubated at 45 degrees
`Centigrade for 30 min'and centrifuged at 1200 g for 10 min.
`One ml of both 1.0% (w/v) TBA and 2.8% (w/v) trichloro
`acetic acid were added to 1.0 ml of supernatant, heated at
`100 degrees Centrigrade for 10 min, cooled in ice, and the
`chromophore determined in triplicate by its absorbance at
`532 nm. FIG. 2 shows that the addition of 10 mg/ml
`tyloxapol to the reaction mixture causes marked inhibition
`of the oxidation of deoxyribose, as measured by absorbance
`of the oxidant reaction product at 532 nm.
`The third system used to test the antioxidant activity of
`alkylaryl polyether alcohol polymers employed asbestos as
`the source of iron for oxidant generation and Z-deoxyribose
`as the target molecule of oxidants. The generation of oxi
`dants by asbestos has been described previously (A.J. Ghio
`et al. American Journal of Physiology (Lung Cellular and
`Molecular Physiology 7) (1992) 263:L511—L518). The reac
`tion mixture, in a total volume of 2.0 ml phosphate~buifered
`saline (PBS), contained the following reagents: 1.0 mM
`deoxyribose, 1.0 mM H202, 1.0 mM ascorbate, and 1.0
`mg/ml crocidolite asbestos. The mixture was incubated at 37
`degrees Centigrade for 1 h with agitation and then centri
`fuged at 1,200 g for 10 min. Oxidant generation was
`assessed by measuring TBA reactive products of deoxyri
`bose as detailed in the paragraph above. Measurements were
`done in triplicate. TABLE I below shows that the addition of
`tyloxapol inhibited in a concentration dependent manner the
`generation of oxidants by asbestos, as measured by absor'
`bance 0f the oxidant reaction product at 532 nm.
`
`45
`
`50
`
`TABLE I
`
`Effect of Tyloxavol on Oxidant Generation by Asbestos
`
`Tyloxapol 0.0 mg/ml
`Tyloxapol 0.1 rug/m1
`Tyloxapol 1.0 mg/ml
`Tyloxapol 10.0 mg/ml
`
`Absorbance at 532 nm
`
`0.93 i 0.02
`0.89 i 0.04
`0.75 i 0.01
`0.53 i 0.04
`
`EXAMPLE H
`Protection from Mammalian Lung Injury by 100%
`Oxygen
`To determine if alkylaryl polyether alcohol polymers
`could protect against oxidant injury to intact biologic sys
`
`55
`
`60
`
`65
`
`6
`tems, this treatment was studied in a well established model
`of oxygen toxicity to the lung (IF. Turrens et al. Journal of
`Clinical Investigation (1984) 73:87-95). Sixty-day old male
`Sprague-Dawley rats (Charles River, Inc., Wilmington,
`Mass.) were tracheally instilled with 0.5 ml of either normal
`saline, tyloxapol (6.0 mg) or tyloxapol (6.0 mg) and cetyl
`alcohol (hexadecanol, 11.0 mg). 'Ihese rats (n=10 in each
`treatment group) were then exposed to either air or 100%
`oxygen in plexi glass chambers at a ?ow rate of 10 liters/min.
`Oxygen percentage was monitored by a polarographic elec
`trode and maintained continuously above 98%. Temperature
`was maintained between 20 and 22 degrees Centigrade.
`Survival times were determined by checking animals every
`4 hours. Separate groups of rats treated similarly (n=10 in
`each treatment group) were exposed to 100% oxygen for 61
`hours, and then were euthanized with 100 mg/kg intraperi
`toneal pentobarbital. Pleural ?uid volume was measured by
`aspirating pleural ?uid from the chest cavity through a small
`incision in the diaphragm. Lung wet/dry weight ratios were
`calculated from the left lung after drying the tissue for 96
`hours at 60 degrees Centigrade. Survival data in shown in
`TABLE II below. Rats receiving intratracheal tyloxapol had
`markedly improved survival compared to placebo control
`animals instilled with saline. The protective effect of tylox
`apol was further enhanced by combining it with cetyl
`alcohol.
`
`TABLE H
`
`Effect of Tyloxapol on Oxygen Toxicity in Rats
`Percent Survival
`
`Hours
`
`Saline
`
`Tyloxapol
`
`Tyloxapol/cetyl Alcohol
`
`0
`58
`62
`66
`70
`72
`76
`80
`84
`88
`92
`96
`
`100
`100
`83
`42
`17
`17
`8
`8
`8
`8
`0
`0
`
`100
`100
`100
`100
`75
`75
`58
`58
`58
`58
`58
`58
`
`100
`100
`100
`100
`100
`100
`100
`100
`100
`100
`100
`100
`
`Lung wet/dry weight ratios were substantially lower in rats
`treated with tyloxapol or tyloxapol and cetyl alcohol (FIG.
`3), demonstrating that tyloxapol or the combination of
`tyloxapol and cetyl alcohol protect against edema formation
`from oxidant injury. Rats treated with tyloxapol or the
`combination of tyloxapol and cetyl alcohol also had less
`pleural ?uid accumulation than saline treated controls (FIG.
`4). These results demonstrate the ability of alkyaryl poly
`ether alcohol polymers such as tyloxapol to protect against
`oxidant tissue injury. The survival studies (TABLE 11)
`further demonstrate that the protective eifect of the medi
`cament is enhanced by combining it with alcohols such as
`cetyl alcohol.
`The appended claims set forth various novel and useful
`features of the invention.
`What is claimed is:
`1. A method for the treatment of mammalian disease
`entities resultant from oxidant species consisting essentially
`of administering to a mammal an amount of alkylaryl
`polyether alcohol polymer of the formula
`
`Page 8
`
`

`
`5,512,270
`
`R1
`
`R1
`
`where, R=ethylene, R1=tertiary octyl, x is greater than 1, and
`y:8 to 18, effective to inhibit oxidant chemical reactions
`caused by the oxidant species in the mammal, thereby
`treating the mammalian disease entities.
`2. The method of claim 1, wherein said carrier is selected
`from physiologically buifered solutions.
`'
`3. The method of claim 2, wherein the physiologically
`buifered solutions are selected from the group consisting of
`isotonic saline, normal saline, and combinations thereof.
`4. The method of claim 1, wherein the mammalian disease
`entities are selected from the group consisting of myocardial
`infarction, stroke, adult respiratory distress syndrome oxy
`
`10
`
`20
`
`8
`gen toxicity of the lung, lung injury from asbestos Parkin
`son’s disease, thermal and solar burns of the skin, injury to
`the gastrointestinal tract from nonsteroidal anti-in?amma
`tory agents, and combinations thereof.
`5. The method of claim 1, wherein said administering of
`said alkylaryl polyether alcohol polymer is directly into the
`mammal’s respiratory tract.
`6. The method of claim 1, wherein said administering of
`said alkylaryl polyether alcohol polymer is by aerosoliza
`tion.
`7. The method of claim 1, wherein said administering of
`said alkylaryl polyether alcohol polymer is by application to
`the mammal’s skin.
`8. The method of claim 1, wherein said administering of
`said alkylaryl polyether alcohol polymer includes a physi
`ologically acceptable carrier.
`
`*
`
`*
`
`*
`
`*
`
`>l<
`
`Page 9
`
`

`
`UNITED STATES PATENT AND TRADEMARK OFFICE
`CERTIFICATE OF CORRECTION
`
`PATENTNQ. 5,512,270
`DATED
`IApril 30, 1996
`INVENTOR(S) : Andrew J_ Ghio, et al.
`
`It is certi?ed that error appears in the above-indenti?ed patent and that said Letters Patent is hereby
`corrected asshown below:
`
`On the Cover page, Column 2, Publication References,
`last line, delete "V0." and insert ——Vol.—— therefor.
`
`Column 3, line 59
`
`, delete "FIG. 5 and".
`
`Column 4, line 19, delete "concommitantly" and
`insert -—concomitantly—— therefor.
`
`Column 5, line 14, after "Gutteridge" delete the
`period ( .) and insert a comma (, ) therefor.
`
`Column 5, line 53, in the sub-heading in Table I,
`delete "Tyloxavol" and insert —-Tyloxapol—- therefor.
`
`Column 6, line 21, delete "in" (first occurrence)
`and insert ——is-— therefore.
`
`(,)
`
`(I > '
`
`Column '7, last line, after "syndrome" insert a comma
`
`Column 8, line 1, after "asbestos" insert a comma
`
`Signed and Sealed this
`
`Seventeenth Day of December, 1996
`
`Arresting O?icer
`
`Commissioner of Parents and Trademarks
`
`BRUCE LEHMAN
`
`Page 10
`
`

`
`UNITED sTATEs PATENT AND TRADEMARK OFFICE
`CERTIFICATE OF CORRECTION
`5,512,270
`April 30, 1996
`
`PATENT ND. :
`DATED
`:
`
`INVENTQMS) :
`
`Ghio etal.
`
`It is certified that error appears in the above-identi?ed patent and that said Letters Patent is hereby
`corrected as shown below:
`
`Column 2, lines 25-35 and column 7, lines 1-10, before the formula, each occurrence, insert --[-- such that the
`
`formulas appear as:
`
`O(RO)yH
`
`O(RO))IH
`
`Q A
`
`R]
`
`RI
`
`In the Claims:
`
`Column 7, claim 2, line 1, "claim 1" should read --claim 8--.
`
`Signed and Sealed this
`Twenty-eighth Day of September, 1999
`
`Arresting Officer
`
`Arring Cmnmisxirmer of Purem‘s and Trademarks
`
`Q. TODD DICKINSON
`
`Page 11