United States Patent [19]
`-Ghio et al.
`
`[54] METHOD OF INHIBITING OXIDANTS
`USING ALKYLARYL POLYETHER
`ALCOHOL POLYMERS
`_
`[75] Inventors: Andrew J: Ghlo; Claude A‘
`Plantadosli both of Durham’ NC‘;
`Th°mas P- Kennedy’ Rlchmond, Va-
`[73] Assigneez Duke University’ Durham, NC‘
`
`[21] Appl No_ 219 770
`
`.
`
`..
`
`,
`
`|||l||lllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllll
`US005474760A
`[11] Patent Number:
`5,474,760
`[45] Date of Patent:
`Dec. 12, 1995
`
`Cantin et al.; The Journal of Clinical Investigation, Inc.
`Protection
`by
`Antibiotics
`agains
`Myeloperoxidase~dependent Cytotoxicity to Lung Ephithe
`lial Cells in Vitro; vol. 91, pp. 38-45 (Jan, 1993).
`Ramsey, et al. The New England Journal of Medicine;
`Efficacy of Aerosolized Tobrarnycin in Patients with Cystic
`Fibrosis; vol. 328 No. 24 pp. 1740-1746 (Jun. 17, 1993).
`Vasconcellos, et al. Science; Reduction in Viscosity of
`Cystic Fibrosis Sputum in Vitro by Gelsolin; vol. 263, pp.
`969-971 (Feb. 18, 1994).
`
`Primary Examiner-Jyothsna Venkat
`Attorney, Agent, or Firm—Richard E. Jenkins
`
`ABSTRACT
`[57]
`_
`_
`.
`A method and medicament for the mhrbitron of oxidants
`comprising administering a treatment effective amount of
`alkylaryl polyether alcohol polymers to a chemical or bio_
`logic system in need thereof The medicament is preferably
`administered by aerosolization into the mammalian respira
`[cry System. The medicament may also be
`[0 the
`mammalian skin. Preferably, the medicament includes a
`physiologically acceptable carrier which may be selected
`from the group consisting of physiologically buifered saline,
`isotonic saline, normal saline, petrolatum based ointrnents
`and U.S.P. cold cream.
`
`5 Claims, 5 Drawing Sheets
`
`[22] Filed;
`
`Mar, 29, 1994
`
`Related US. Application Data
`
`63 Cti ti-'-artfS.N.39,732,M.30,1993,
`[
`]
`a?iarildlgiidin m p
`0
`er
`0
`at
`
`[51] Int. Cl.6 ......................... .. A61K 9/12; A61K 31/765
`[52] US. Cl. ....................... .. 424/45; 424/7837; 514/828;
`514/851
`
`Of Search ................................. ..
`
`514/351’ 828
`
`[56]
`
`_
`References Cited
`PUBLICATIONS
`
`1
`Robert A. Greenwald, CRC Handbook of Methods for Oxy
`gen Radical Research; Deterrnination of HOCl Production
`by Micloperoxidase; p. 300 (1987).
`
`0(R0lyH
`
`"
`
`MOW
`
`0112
`
`l
`
`

`
`‘ US. Patent
`
`Dec. 12,1995
`
`Sheet 1 of 5
`
`5,474,760
`
`> cuz
`
`%
`
`%
`
`/
`
`I
`
`\
`R‘
`
`R
`
`Page 2
`
`

`
`U.S. Patent
`
`Dec. 12, 1995
`
`Sheet 2 of 5
`
`5,474,760
`
` 3:: E12,?»
`
`
`I 2,5
`
`89
`
`DIHYDROXYBENZOIC new
`DIHYDROXYBENZOIC ACID
`
`7.0 -
`2;;
` §§ 6.0
`
`G-><
`
`E; 5.0
`:33 4.0
`5 3.0
`
`2.0
`
`1.0
`
`o
`
`TYLOXAPOL TYLOXAPOL TYLOXAPOL
`NORMAL
`SALINE O.lmg/ml |.Omg/ml
`|0.0mg/ml
`
`F|G. 2
`
`Page 3
`
`Page 3
`
`

`
`US. Patent
`
`Dec. 12, 1995
`
`Sheet 3 of 5
`
`5,474,760
`
`E 0.00 /; 7
`¢ / f"
`
`2 / / /
`2 0.20% Z Z Z
`
`QOO
`
`0.0
`
`
`
`./ 0|
`
`:0
`
`10.0
`
`FIG. 3
`
`Page 4
`
`

`
`US. Patent
`
`Dec. 12, 1995
`
`Sheet 4 of 5
`
`5,474,760
`
`
`
`2:: ..O.. 9. AMQWEFMOCMM pm;
`
`
`
`
`
`0.0.0.0.
`321.0
`
`“ //////////
`
`“ ////////////////
`
`.250:
`
`2.2m
`
`,//////////
`
`492x35
`
`FIG. 4
`
`Page 5
`
`

`
`US. Patent
`
`Dec. 12, 1995
`
`Sheet 5 of 5
`
`5,474,760
`
`O. m
`mu. 9
`
`2:: 2x3:
`
`
`
`7654.32
`Z CEV
`//
`/%4
`// % 0.0.
`10
`425%;
`.252
`wzz?
`s2 5&2:
`5:2: :5
`
`FIG. 5
`
`Page 6
`
`

`
`5 ,474,7 60
`
`1
`METHOD OF INHIBITING OXIDANTS
`USING ALKYLARYL POLYETHER
`ALCOHOL POLYMERS
`
`RELATED APPLICATION(S)
`This application is a continuation-in-part of applicants’
`application U.S. Ser. No. 039,732, ?led Mar. 30, 1993, now
`abandoned.
`
`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 02- by the
`enzymatic action of superoxide dismutases. However, -OH
`is generally produced only when 02- and H202 interact with
`transitional ions of metals such as iron and copper in
`dangerous cyclical redox reactions:
`
`15
`
`20
`
`30
`
`35
`
`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 iron and H202.
`While 02- and H202 are each toxic for biological sys
`tems, -OH (and its alternate hypothesized form the ferryl
`intermediate FeOZ‘“) is a highly reactive species that can
`oxidize unsaturated membrane lipids, damage cellular pro-.
`teins and cause mutagenic strand breaks in DNA. To prevent
`45
`injury from partially reduced 02 species under normal
`conditions, cells have evolved an elaborate system of anti
`oxidant enzymes (superoxide dismutase, catalase, glu
`tathione peroxidase) and antioxidant molecules (glutathione,
`alpha-tocopherol, beta carotene). However, when produc
`tion 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 syn
`dromes 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
`nonsteroidal anti-in?ammatory agents (see Table IV, page
`60, Halliwell B and Gutteridge JMC. Methods in Enzymol
`ogy (1990) l86:l—85). Also, studies suggest that airway cells
`in cystic ?brosis patients are at risk of oxidant-mediated
`injury. The reason is that the leukocyte-derived enzyme,
`myeloperoxidase, present in large amounts in the bronchial
`secretions of cystic ?brosis patients, converts with H2O2
`produced by polymorphonuclear leukocytes to HOCl/OCl,
`
`55
`
`65
`
`2
`the major leukocyte~de1ived oxidant. See, for instance, Can
`tin et al. “Protection by Antibiotics Against Myeloperoxi
`dase-Dependent Cytotoxicity to Lung Epithelial Cells in
`Vitro,” Journal of Clinical Investigation (January, 1993)
`91:38-45; Ramsey et al., “E?ieacy of Aerosolized Tobra
`mycin in Patients with Cystic Fibrosis,” The New England
`Journal of Medicine (June, 1993) 328:1740-1746; Vascon
`cellos et al., “Reduction In Viscosity of Cystic Fibrosis
`Sputum in Vitro by Gelsolin," Science (February, 1994)
`263:969-971. Treatment of these conditions is increasingly
`directed either toward strategies that prevent enzymatic
`production of partially reduced 02 species and to the intro
`duction of exogenous antioxidant compounds that restore
`oxidant-antioxidant balance in biologic and chemical sys
`tems.
`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
`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 pharrnacologic
`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 can be explained below, this invention describes how
`alkylaryl polyether alcohol polymers are useful as antioxi
`dants in blocking oxidant reactions and biologic injury from
`partially reduced 02 species. Alkylaryl polyether alcohol
`polymers are known and used commercially as surface
`active detergents and wetting agents (U.S. Pat. No. 2,454,
`541), the disclosure of which is incorporated herein by
`reference. All alkylaryl polyether alcohol polymers dis
`closed in this patent should work in the present invention.
`A structure representative of the class of compounds is
`shown in FIG. 1. The best known of this class is tyloxapol,
`a polymer of 4-(1,1,3,3-tetramethylbutyl)phenol with form
`aldehyde 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 and a medicament for the treatment of cystic ?brosis
`in patients having the disease to protect the patients from
`airway injury by HOCl/OCl, which for convenience, is
`
`Page 7
`
`

`
`5,474,760
`
`3
`referred to herein also as HOCl.
`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.
`The present invention provides a medicament for the
`inhibition of injurious eifects 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 buffered 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 the proposed structure of the class of
`compounds known as alkylaryl polyether alcohol polymers
`wherein R=ethylene; R1=tertiary octyl; x is greater than 1;
`and y=8 to 18.
`FIG. 2 shows a graph of the inhibitory eifect of tyloxapol
`on .OH generation by the Fenton reaction, as measured by
`hydroxylation of salicylate.
`FIG. 3 shows a graph of the inhibitory e?ect of tyloxapol
`on ‘OH generation by the Fenton reaction, as measured by
`oxidation of the sugar 2-deoxyribose.
`FIG. 4 shows lung wet/dry weight ratios in rats exposed
`to 100% oxygen and treated with normal saline, tyloxapol,
`and tyloxapol plus cetyl alcohol.
`FIG. 5 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 US. Pat. No.
`2,454,541 (1948 to Rohm & Haas). Several speci?c alky
`
`l0
`
`20
`
`25
`
`4
`laryl polyether alcohol polymers can be easily synthesized
`by methods previously described (J. W. Conforth 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 l—l.5 times the weight of
`tyloxapol to increase the effectiveness of the mixture in
`protection 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 available positive pres
`sure driven nebulizer that produces respirable 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 to make it isotonic
`with respect to respiratory secretions. The pH is adjusted to
`approximately 7.0 to prevent bronchospasm from extremes
`of acidity or alkalinity. This mixture is sterilized by vacuum
`?ltration through a 0.22 micron Millipore ?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 option
`ally added to the above mixture to stabilize droplet size
`during aerosolization. For administration of treatment elfec
`tive 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). Alterna
`tively, the mixture can be nebulized into the respiratory
`delivery circuit of a mechanical ventilator. A beta sympa
`thetic agonist bronchodilator (such as 1.25 to 2.5 mg of
`albuterol) can be mixed with the tyloxapol solution and
`nebulized concomitantly to prevent any transient broncho
`spasm 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 II demonstrates the therapeutic bene?t of
`using alkylaryl polyether alcohol polymers to prevent mam
`malian lung injury from exposure to 100% oxygen. Example
`HI demonstrates the potent activity of alkylaryl polyether
`alcohol polymers as scavengers of HOCl in chemical sys~
`terns.
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`EXAMPLE I
`
`65
`
`Inhibitions of Oxidants Generated by the Fenton Reaction
`The ?rst chemical system used to test the antioxidant
`activity of alkylaryl polyether alcohol polymers employed
`
`Page 8
`
`

`
`5,474,760
`
`5
`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)
`l0:221—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 salycylic acid were employed
`to generate and detect ~OH. 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
`(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. 2 shows that the addition
`of tyloxapol to the reaction mixture inhibited -OH genera
`tion 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 Centigrade for 10 min, cooled in ice, and the
`chromophore determined in triplicate by its absorbance at
`532 nm. FIG. 3 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 produced 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 2-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 :L5 ll-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 l h with agitation and then centri
`fuged at 1,200 g for 10 min. Oxidant generation was
`
`45
`
`50
`
`55
`
`60
`
`65
`
`20
`
`25
`
`35
`
`40
`
`6
`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 of the oxidant reaction product at 532.
`
`TABLE I
`
`Effect of Tyloxapol on Oxidant Generation by Asbestos
`
`Tyloxapol 0.0 mg/ml
`Tyloxapol 0.1 mg/ml
`Tyloxapol 1.0 mg/ml
`Tyloxapol 10.0 rug/m1
`
`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 11
`Protection from Mammalian Lung Injury by 100% Oxygen
`To determine if alkylaryl polyether alcohol polymers
`could protect against oxidant injury to intact biologic sys
`tems, this treatment was studied in a well established model
`of oxygen toxicity to the lung (J. F. 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). These rats (n=l0 in each
`treatment group) were then exposed to either air or 100%
`oxygen in plexiglass 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=l0 in
`each treatment group) were exposed to 100% oxygen for 61 '
`hours, and then were euthanized with lOOmg/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
`TABLE II below. Rats receiving intratracheal tyloxapol had
`markedly improved survival compared to placebo control
`animals instilled with saline. The protective eifect of tylox
`apol was further enhanced by combining it with cetyl
`alcohol.
`
`TABLE H
`
`Etfect Of Tyloxapol On Oxygen Toxicity In Rats
`
`Percent Siurvival
`
`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
`S8
`
`100
`100
`100
`100
`100
`100
`100
`100
`100
`100
`100
`100
`
`Lungs wet/dry weight ratios were substantially lower in rats
`
`Page 9
`
`

`
`5,474,760
`
`7
`treated with tyloxapol or tyloxapol and cetyl alcohol (FIG.
`4), 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.
`5). These results demonstrate the ability of alkyaryl poly
`ether alcohol polymers such as tyloxapol to protect against
`oxidant tissue injury. The survival studies (TABLE H)
`further demonstrate that the protective effect of the medi
`cament is enhanced by combining it with alcohols such as
`cetyl alcohol.
`
`EXAMPLE III
`
`scavenging of HOCl
`The activity of tyloxapol to scavenge 0C1-1 was tested
`studying its ability to prevent OCl“1-medicated oxidant
`conversion of diethanolamine to its corresponding chloram
`ine (“Determination of HOCl Production by Micloperoxi
`dase” Robert A Greenwald, editor, Handbook of Methods for
`Oxygen Radical Research, CRC Press, Boca Raton, Fla.
`(1987), page 300). The reaction mixture comprised 0.9 ml of
`10.0rnM diethanolamine in 0.1M sodium acetate buffer, pH
`of 4.5. To this resultant was added either 100 microliters of
`0.1M NaCl or tyloxapol in 0.1M NaCl, and the baseline
`absorbance was read at 280 nm. NaOCl was added to a ?nal
`concentration of 10 mM. The reaction mixture was incu
`bated 15 minutes, and the absorbance was measured at 280
`nm. The difference in 80 before and after addition of N aOCl
`was used as a measure of concentration of the stable
`chloramine. Experiments were performed in triplicate.
`Results are summarized in Table H1 below.
`
`TABLE III
`
`Microliters of
`Tyloxapol (10 mg/ml)
`
`0
`25
`50
`75
`
`Absorbance
`(Mean i SD)
`
`0505 i 0.002
`0.468 t 0.008
`0.444 1- 0.023
`0377 i 0.010
`
`8
`
`TABLE III-continued
`
`Microliters of
`Tyloxapol (10 mg/ml)
`
`100
`
`Absorbance
`(Mean i SD)
`
`0.319 i 0.025
`
`Thus, tyloxapol is a potent inhibitor of the oxidant activity
`of HOCl, and should be useful in preventing HOCl~medi
`cated oxidant injury of the airway in diseases such as cystic
`?brosis. Administration of tyloxapol by tracheal installation
`to cystic ?brosis patients should inhibit HOCl produced in
`these patients and therefore protect them from oxidant
`injury. The result should be even better if some cetyl alcohol
`is admixed with the tyloxapol; preferably, the cetyl alcohol
`is added in 1 to 1.5 times the weight of the tyloxapol.
`Preparation of samples for administration to the patient
`should be the same as described above in the second
`paragraph of the “DETAILED DESCRIPTION OF THE
`INVENTIO ” section herein, most preferably inhalation of
`3 ml of a 0.125% solution of tyloxapol by jet aerosol every
`4 to 6 hours.
`The appended claims set forth various novel and useful
`features of the invention.
`What is claimed is:
`1. A method for the treatment of cystic ?brosis diesease
`resultant from overproduction of HOCl, said method com
`prising administering to a mammal having cystic ?brosis
`disease an effective amount of tyloxapol to inhibit oxidant
`chemical reactions caused by the HOCl in the mammal.
`2. The method of claim 1, wherein said administering
`comprises administering the tyloxapol directly into the
`mammal‘s respiratory tract.
`3. The method of claim 1, wherein said administering
`comprises administering the tyloxapol by aerosolization.
`4. The method of claim 1, wherein said administering of
`the tyloxapol comprises a physiologically acceptable carrier.
`5. The method of claim 4, wherein said physiologically
`acceptable carrier is selected from the group consisting of
`isotonic saline, normal saline, and combinations thereof.
`
`20
`
`25
`
`30
`
`35
`
`40
`
`*****
`
`Page 10
`
`

`
`UNITED STATES PATENT AND TRADEMARKZOFFICE
`CERTIFICATE OF CORRECTION
`PATENT N0. ; 5,474,760
`Page 1 of 2
`
`;
`
`
`
`DATED mvemrums) ; Ghio et al. ; Decenber 12, 1995
`
`
`
`It is certified that error appears in the above-identified patent and that said Letters Patent is hereby
`corrected asshown below:
`
`At the last line of column 1 on the Title page, in item
`[56] on the front page, change "Micloperoxidase" instead to
`read as -— Myeloperoxidase --.
`‘
`
`At line 1 of column 2 on the front page, in item [56]
`on the front page, delete ", Inc." and replace with -— ; --.
`
`At line 2 of column 2 on the front page, in item [56]
`on the front page, delete "agains" and replace with —
`against --.
`
`At line 12 of column 5, delete "salycylic" and replace
`with -— salicylic —-.
`
`At line 17 of column 7, delete "OCI‘" and replace with
`—- HOC]. ——.
`
`At line 18 of column 7, delete "Ocl'l-medicated" and
`replace with -- HOCl-mediated —-.
`
`Page 11
`
`

`
`UNITED STATES PATENT AND TRADEMARK OFFICE
`CERTIFICATE OF CORRECTION
`PATENT N0. : 5,474,760
`Page 2 of 2
`DATED
`; December 12, 1995
`
`INVENTOFHS) ; Ghio et al.
`
`It is certified that error appears in the above-identified patent and that said Letters Patent is hereby
`corrected asshown below:
`
`At line 20 of column 7, delete "Micloperoxi—" and
`replace with —— Myeloperoxi— ——.
`
`At line 21 of column 7, delete "Robert A " and replace
`with --, Robert A. ——.
`
`At line 10 of column 8, delete "cated" and replace with
`-— ated ——.
`
`a
`
`Signed and Sealed this
`
`Ninth Day ofApril, 1996
`
`Arresting O?icer
`
`Cummiumner 0/ Palenn and Trademarks
`
`BRUCE LEI-{MAN
`
`Page 12