as
`Er‘;
`*2»
`
`%,‘\\fl?’f4‘é& !AMERICAN JOURNAL OF
`‘ATS a Respiratory and
`Critical Care Medicine
`
`1%)
`
`Volume 154 ° Number 3 ° September 1996
`(part I of 2 parts‘)
`
`§.E%§%é%RV
`%%E@aL‘FE=€ S$§.E§€$E$
`University of Wéeconsin
`
`SE? 2 57
`
`i3§6
`
`1305 Linden Drive
`Mamson, W: 53706
`
`AN OFFICIAL JOURNAL OF THE AMERICAN THORACIC SOCIETY
`MEDICAL SECTION OF THE AMERICAN LUNG ASSOCIATION
`
`|PR2015—01099
`
`|PR2015-01097
`
`|PR2015—01100
`
`|PR2015—01105
`
`Lupin EX1173
`
`Page 1
`
`Page 1
`
`

`
`FORMERLY THE AMERICAN REVIEW OF RESPIRATORY DISEASE
`
`AN OFFICIAL JOURNAL OF THE AMERICAN THORACIC SOCIETY
`Medical Section of the American Lung Association
`1
`
`
`EDITORIAL BOARD
`
`JEAN RINALDO, M.D.
`Nashville, TN
`
`ROBERT RODRIGUEZ-ROISIN, M.D.
`Barcelona, Spain
`ANDREA ROSSI, M.D.
`Verona, Italy
`CATHERINE SASSOON, M.D.
`Long Beach, CA
`GREGORY SCHMIDT, M.D.
`Chicago,
`IL
`
`HEALTHSQIEMBESLIBRARYURIVGFSIWCIWIESGIIEIII
`
`SE?25
`
`RICHARD ALBERT, M.D.
`Seattle, WA
`RALPH ALTIERE, PH.D.
`Denver, CO
`PETER BARNES, D.M., D.SC.
`London, England
`ROBERT BAUGHMAN, M.D.
`_Cincinnati, OH
`JUDITH BLACK, M.B. B.S., PH.D.
`Sydney, NS W, Australia
`JEAN BOUSQUET, M.D.
`Montpellier, France
`LAURENT BROCHARD, M.D.
`Creteil, France
`WILLIAM J. CALHOUN, M.D.
`Pittsburgh, PA
`RICHARD CASABURI, PH.D., M.D.
`Torrance, CA
`NEIL CHERNIACK, M.D.
`Cleveland, OH
`JOHN W. CHRISTMAN, M.D.
`Nashville, TN
`KIAN FAN CHUNG, M.B.B.S.
`London, England
`MICHAEL CYNAMON, M.D.
`' Syracuse, NY
`JAMES DAUBER, M.D.
`Pittsburgh, PA
`ANDRE DE TROYER, M.D.
`Brussels, Belgium
`CLAIRE DOERSCHUK, M.D.
`Boston, MA
`
`JEFFREY DRAZEN, M.D.
`Boston, MA
`
`DIDIER DREYFUSS, M.D.
`Columbes, France
`JOHN EVANS, PH.D.
`Burlington, VT
`LEONARDO FABBRI, M.D.
`Ferrara, Italy
`
`NICHOLAS J. GROSS, M.D., PH.D.
`Hines,
`IL
`NICHOLAS HILL, M.D.
`Providence, RI
`J. M. B. HUGHES, D.M.
`London, England
`ALAN JOBE, M.D., PH.D.
`Torrance, CA
`GARY LARSEN, M.D.
`Denver, CO
`MICHAEL LEBOWITZ, PH.D.
`Tucson, AZ
`FRANCOIS LEMAIRE, M.D.
`Creteil, France
`JAMES MARTIN, M.D.
`Montreal, PQ, Canada
`RICHARD MARTIN, M.D.
`Denver, CO
`THOMAS MARTIN, M.D.
`Seattle, WA
`WILLIAM MARTIN ll, M.D.
`Indianapolis, IN
`MICHAEL MATTHAY, M.D.
`San Francisco, CA
`RENE MICHEL, M.D.
`Montreal, PQ, Canada
`EDwARD NARDELL, M.D.
`Cambridge, MA
`JOHN NEWMAN, M.D.
`Nashville, TN
`MICHAEL NIEDERMAN, M.D.
`Mineola, NY
`'
`ALLAN PACK, M.D., PH.D.
`Philadelphia, PA
`JAMES PENNINGTON, M.D.
`Berkeley CA
`0
`THOMAS PLATTS-MILLS, M.D., PH.D.
`Charlottesville, VA
`MICHAEL B. REID, PH.D.
`Houston, TX
`STEPHEN RENNARD, M.D.
`Omaha, NE
`
`‘
`
`DANIEL SCHUSTER, M.D.
`St. Louis, MO
`STEPHANIE SHORE, PH.D.
`Boston, MA
`
`ARTHUR SLUTSKY, M.D.
`Toronto, ON, Canada
`LEWIS SMITH, M.D.
`Chicago,
`IL
`
`ROGER G. SPRAGG, M.D.
`San Diego, CA
`PETER STERK, M.D., PH.D.
`Leiden, The Netherlands
`JACOB I. SZNAJDEFI, M.D.
`Chicago,
`IL
`IRA TAGER, M.D.
`Berkeley, CA
`ANNE TATTERSFIELD, M.D.
`Nottingham, England
`AUBREY E. TAYLOR, PH.D.
`Mobile, AL
`
`WILLIAM VOLLMER, PH.D.
`Portland, OF?
`
`KEITH R. WALLEY, M.D.
`Vancouver, BC, Canada
`SCOTT WEISS, M.D.
`Boston, MA
`
`DAVID WHITE, M.D.
`Denver, CO
`
`STEVEN WHITE, M.D.
`Chicago,
`IL
`
`
`
`
`
`
`
`Pétiifiiiaiiifiigi‘
`
`
`
`THE AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE (ISSN 1073-449X) is published by the American'Lung Association and issued monthly about
`the first of the month. The JOURNAL is the official journal of the American Thoracic Society. A volume includes six numbers and begins with the January and July issues.
`Subscription price is $150 per year for individuals in the USA, Canada, and Mexico, and $220 for libraries and institutions. Subscription rate for individuals outside North
`America is $190 per year, and for libraries and institutions, $260 per year The price of single copies is,'$2O in USA, Canada, and Mexico; $22 Outside North America.
`The contents of this journal are included in Index Medicus, Current Contentsll_ile Sciences‘? Current ContentslC|inical MediCine®, Current ContentslLife Sciences on Dis-
`kette“-", Medlars@, Medline@, Radline, and CABS.
`Published monthly at 1740 Broadway, New York, NY 10019 by the American Lung Association, Periodicals postage paid at New York, NY and at additional mailing office
`at Montpelier, Vermont. Postmaster: Send address Changes to American Journal of Respiratory and Critical Care Medicine, 1740 Broadway, New York, NY 10019.
`Copyright © 1996, by the American Lung Association.
`Permission to photocopy for internal or personal use or the internal or personal use of specific clients is granted by the AMERICAN JOURNAL OF RESPIRATORY AND
`CRITICAL CARE MEDICINE for libraries and other users registered with the Copyright Clearance Center (CCC), provided that the base fee of $2.00 per copy of the article
`is paid directly to CCC, 21 Congress St., Salem, MA 01970. Special requests should be addressed to the AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE
`MEDICINE,.Attn: Managing Editor, 1740 Broadway, New York, NY 10019. 0003-0805/83/1271—0001$2.00
`
`Page 2
`
`Page 2
`
`

`
`(cid:1) (cid:1)(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:9)(cid:10)(cid:3)(cid:7)(cid:11)(cid:5)(cid:6)(cid:7)(cid:12)(cid:5)(cid:13)(cid:9)(cid:5)(cid:14)(cid:10)(cid:15)(cid:8)(cid:9)(cid:16)(cid:8)(cid:9)(cid:17)(cid:5)(cid:13)(cid:12)(cid:5)(cid:18)(cid:15)(cid:14)(cid:12)(cid:10)(cid:3)(cid:19)(cid:2)(cid:8)(cid:5)(cid:11)(cid:7)(cid:20)(cid:5)(cid:21)(cid:1)(cid:3)(cid:8)(cid:11)(cid:9)(cid:5)(cid:22)(cid:23)(cid:5)(cid:24)(cid:25)(cid:26)(cid:25)(cid:5)(cid:18)(cid:15)(cid:17)(cid:9)(cid:27)(cid:1)
`
`Page 3
`
`

`
`784
`
`AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 154
`
`I996
`
`centration of 50 pg/ml). Tyloxapol was tested in triplicate at four con-
`centrations in either the presence or the ‘absence of 100 ng/ml Salmonella
`ryphosa lipopolysaccharide (LPS; 250 pl of 4 X desired final concentra-
`tion added) and incubated at 37° C in humidified 5% CO2 for 16 h.
`At this time, culture supernatants are aspirated off and the unattached
`cells and cell debris were removed by filtration. The release of TNF-ot;
`IL-11}, IL-6, and IL-8; and the growth factor GM-CSF was determined
`in the cell-free supernatants using enzyme linked immunosorbent
`(ELISA) capture assays.
`'
`
`Monocyte Release of LTB4, Platelet Activating Factor,
`and Thromboxane A2
`
`Monocytes were washed and resuspended in RPMI containing 5 mg/ml
`bovine serum albumin (BSA) at 2 X 10“ cells/ml, and were added to
`wells of a 48-well plate. The cells were allowed to adhere for 2 h and
`then washed in Hanks’ balanced salt solution (HBSS)-BSA-I-IEPES
`buffer. Tyloxapol was added at four concentrations for 60 min and the
`monocytes were then stimulated by addition of 300 mg/ml zymosan A
`(175 pl of 2x desired final concentration). Supernatant medium was
`collected from the wells after 90 min incubation and stored at — 20° C
`until assayed. Supernatants were assayed for leukotriene B4 (LTB.,), plate-
`let activating factor (PAF), or thromboxane A, (TXAZ) using specific
`scintillation proximity assays. Experiments were performed in triplicate
`for each concentration of tyloxapol.
`
`Electrophoretic Mobility Shift Assays
`A549 human pulmonary epithelial cells were cultured in Ham’s F-12
`medium supplemented with 10% heat-inactivated fetal calf serum (FCS),
`L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 ug/ml),
`and amphotericin B (250 ng/ml). Confluent cells were stimulated with
`10 U/ml IL-1B or 100 p.M H202. In some cultures l00'ug/ml tyloxapol
`was added at the same time as the stimulators. After 2 h of incubation,
`nuclear extracts were isolated as described by Dignam and colleagues
`(16), with minor modifications (17). In brief, after removal of the super-
`natant, cells were scraped gently in 20 to 30 ml ofPBS containing 1 mM
`phenylmethylsuflonyl fluoride (PMSF) and 1 mM dithiothreitol (DTT).
`The cell suspensions were centrifuged and the pellets were resuspended
`and incubated for 15 min in 1 ml buffer A containing 10 mM HEPES,
`1.5 1nM MgCl,, 10 mM KCl,
`1 mM PMSF,
`1 mM DTT, 10 mM B-glyc-
`erolphosphate, 2.5 mM benzamidine, 1 mM NaF, 1 mM NaVO.., 1 mg/ml
`leupeptin, and 1 mg/ml pepstatin A, and were then sheared by five pas-
`sages of the suspensions through a 25-gauge needle. After centrifuga-
`tion, the pellets were suspended and stirred for 30 min in buffer C con-
`taining 25% vol/vol glycerol, 0.25 M NaCl, 1.5 mM MgCl2, 0.2 mM
`ethylenediamine tetraacetic acid (EDTA),
`1 mM PMSF,
`1 mM DTT,
`10 mM B-glycerophosphate, 2.5 mM benzamidine,
`1 mM NaF, 1 mM
`NaVO.., 1 mg/ml leupeptin, and 1 mg/ml pepstatin A. After centrifuga-
`tion, nuclear extracts were obtained by dialysis of the supernatants in
`buffer D containing 20 mM HEPES, 20% vol/vol glycerol, 100 mM KCl,
`0.2 mM EDTA, 1 mM PMSF, and 1 mM DTT. Utilizing the wild-type
`consensus sequences for AP-1 (18) and NF-KB (19) loci, the following
`Oligonucleotides were synthesized (binding sites underlined):
`AP-1:
`5’—TTCCGGCTGACTCATCAAGCG—3’
`3’~AAGGCCGACTGAGTAGTTCGC-5'
`5’-AGTTGAGGGGACTTTCCCAGGC-3’
`3’-TCAACTCCCCTGAAAGGGTCCG-5’
`
`NK—KB:
`
`The oligonucleotides were purified by denaturing polyacrylamide gel
`electrophoresis (PAGE) followed by passage over Sep-Pak C18 columns.
`Each complementary strand was end‘-labeled by phosphorylation with
`[«/“Pjadenosine triphosphate ([y“P]-ATP) and T4 polynucleotide ki-
`nase. Double-stranded DNA probes were generated by annealing the com-
`plementary end-labeled oligonucleotide strands, boiling for 3 min, and
`slow-cooling to room temperature in a water bath. Unincorporated ra-
`dionucleotides were removed by Sephadex G-25 column chromatogra-
`phy. Binding reactions were performed for 20 min on ice with 5 to 10
`ug total protein in a 20—ul volume containing 300 ng BSA,
`1 to 2 pg
`poly deoxyinosine-deoxycytosine (dl-dC), 50 mM DTT, 0.5 mM PMSF,
`and 1 to 2 X 10“ cpm of“P-labeled probes. In addition, a concentration
`of 6 mM MgCl, was used for AP-1 binding reactions. In selected sam-
`ples, a 100-fold molar excess of unlabeled DNA probe was included in
`the binding reaction in order to confirm the specificity of DNA—protein
`
`interactions. DNA-protein complexes were separated from unbound DNA
`probe on 4.5% polyacrylamide gels under high-ionic-strength conditions
`in 50 mM tris-(hydroxymethyl) aminomethane (Tris), 0.4 M glycine, 2
`mM EDTA, and 2.5% vol/vol glycerol, pH 8.5. Electrophoresis was done
`at 4° C at a constant current of 20 mA. Gels were dried under vacuum
`and exposed to film at — 70° C for 6 to 24 h with an intensifier screen.
`
`Investigation of.Tyloxapo| as an HOCI Scavenger In Vitro
`
`The activity of tyloxapol as an HOCI scavenger was tested by its ability
`to prevent HOCI-mediated conversion of diethanolamine to its corre-
`sponding chloramine, diethanolchloramide (20). The reaction mixture
`(total volume: 1.2 ml) comprised 10.0 mM diethanolamine in 0.1 N so-
`dium acetate buffer. To this were added varying concentrations (wt/vol%)
`of tyloxapol (0, 0.05, 0.1%) in 0.1 M NaCl, and the baseline absor-
`bance was read at 280 nm. NaOCl (1.0 to 7.5 mM) was then added, the
`reaction mixture was incubated for 15 min at 37° C, and the absorbance
`was again measured. The difference in Am before and after addition
`of NaOCl‘ was used as a measure of ‘concentration of the stable chlora-
`mine, Experiments were performed in triplicate at each concentration
`of NaOCl and tyloxapol.
`
`Measurement of Acute Lung Inflammation
`
`The ability of tyloxapol to protect against lung injury from HOCI was
`studied in 60-d-old male Sprague-Dawley rats'(n = 6 per treatment group)
`weighing 250 to 300 g (Charles River Breeding Labs, Wilmington, MA).
`After anesthesia with halothane (2 to 5%), rats were injected in-
`tratracheally with either 0.3 ml of 2.0 mM NaOCl in normal saline (buf-
`fered to pH 6.0), or with normal saline alone. The rats were allowed to
`recover, and 1 h later were closed intratracheally with either 6.0 mg tylox-
`apol in normal saline or with normal saline. Twenty-four hours after
`NaOCl instillation, all rats were euthanized with sodium pentobarbital.
`The tracheas were cannulated and the lungs were lavaged wtih normal
`saline (35 ml/kg body weight). After staining of the lavage fluid with
`a modified Wright’s stain (Diff—Quick stain; ASP, McGaw Park, IL),
`the cell differentials were determined on 500 cells/sample. Values were
`expressed as the percentage of total cells recovered. Lavage protein was
`measured using the Bio-Rad method for total protein determination as
`modified for use on the centrifugal analyzer.
`
`Collection of Sputum
`
`CF sputum was collected from hospitalized young adult CF patients of
`the Duke Adult Cystic Fibrosis Center and frozen at — 20° C until used.
`The diagnosis of CF had been previously made in all subjects by a posi-
`tive sweat chloride test and compatible clinical course of disease. No
`patient had received recombinant deoxyribonuclease (rhDNase) for at
`least 2 wk prior to the time of sputum collection.
`
`Measurement of Sputum Viscosity
`Sputum viscosity was studied with a model LVT Brookfield cone/plate
`viscometer (Brookfield Engineering Laboratories, Inc., Stoughton, MA)
`with a calibrated torque of 673.7 dynes-cm at full scale. Sputum (750
`pl) was mixed 3:1 with 0.9% saline or 0.125% tyloxapol (wt/vol) in sa-
`line (250 ul), vortexed for 30 s, and then incubated for 15 min at 37° C.
`Viscosity of the 1-ml preparation was measured at 0.3 rpm (shear rate
`of 2.25/s) and 37° C, using a CP-40 cone spindle (cone angle of 0.8 degrees
`and radius of 2.4 cm), and was expressed as milli-Poiseville (mPa). Control
`measurements of saline or saline and tyloxapol alone showed that they
`behaved similarly to non-Newtonian pseudoplastic fluids, with respece
`tive viscosities of 20.6 and 10.3 centipoises, respectively, at a shear rate
`of 2.25/sec, and 0.78 and 0.83 centipoises, respectively, at a shear rate
`of 225/sec. Because the viscosity of saline-treated sputum from Patients
`1 and 9 was too high for measurement, sputum ofthese individuals wafé
`diluted 1:3 and 121, respectively, with saline or tyloxapol in saline.
`
`Statistics
`
`Data are expressed as mean values 1 SEM. The difference between tylox-
`apol and saline—treated sputum was analyzed with the paired {test (21)-
`Differences among groups in animal experiments and human monocyte
`incubations were analyzed with analysis of Variance (ANOVA) and
`SchefI"e’s post hoc test (21). Significance was assumed at p < 0.05-
`
`Page 4
`
`Page 4
`
`

`
`785
`
`no
`m
`to
`
`:2
`2:
`,8 so
`'.‘
`'°
`:1 I0
`so20
`..
`"
`
`o.aoi
`
`o.m
`
`moo
`
`moo
`
`I10
`‘°,§
`on
`,0
`2;‘.
`,,
`2
`11)
`'
`
`tr
`=1
`
`
`
`u.no1
`
`o.m
`
`moo
`
`i no
`
`Tyloxapol (mglml)
`
`mm
`
`mm;
`
`mm
`
`moo
`
`um
`
`nmo
`
`man
`
`man
`
`‘-51
`
`0''
`
`ll‘
`
`‘.1
`
`Tyloxapol (mglml)
`
`"°
`‘W
`'1
`
`X
`u.
`I:
`5 ’’
`no
`so
`T2
`
`0
`go,
`'2
`I)
`
`‘"1
`:
`‘:3
`as
`so
`B
`N
`.i
`°“
`«v
`as
`g =- 2
`-5
`:
`‘B
`10
`Q
`"
`
`0 E
`
`9-«
`
`1
`-:
`1:
`§ 12
`5'
`I0
`<9
`1:
`I
`
`Figure 2. Tyloxapol significantly (p < 0.01) reduces secretion of the
`cytokines TN F—a,
`|L—1B, IL-6, IL-8, and of the growth factor GM-CSF
`by LPS-stimulated human monocytes in a dose-dependent manner.
`Methods are detailed in text.
`
`In Vitro Scavenging of HOCI
`NaOCl oxidized diethanolamine to its corresponding chloramine
`in a dose-dependent fashion (Table 2). Tyloxapol dramatically
`reduced chloramine formation (Table 2), suggesting that tylox-
`apol is an avid scavenger of HOCI.
`
`Acute Lung Inflammation from HOCI
`
`Intratracheal instillation of NaOCl caused acute lung injury, as
`demonstrated by a marked increase in protein concentration and
`the percentage of PIVINS in lung lavage fluid (Figure 5). Postex-
`posure treatment with tyloxapol significantly reduced lavage pro-
`tein concentration (p < 0.001) and the percentage of PMNs (p <
`0.01), demonstrating that tyloxapol also protects against HOCI-
`mediated cytotoxicity in viva Although not significantly different
`from saline control, tyloxapol alone increased the percentage of
`PMNS in lavage fluid, a finding that will require additional work
`to confirm.
`
`TABLE I
`
`EFFECTIVE CONCENTRATIONS OF TYLOXAPOL FOR 50% INHIBITION
`(EC5o) OF MONOCYTE CYTOKINE RELEASE
`
`Cytokine
`TN F-(1
`IL-I I3
`IL-6
`IL-8
`
`.
`
`ECso
`(pg/m/)
`30
`60
`30
`70
`
`Cytokine secretion was studied in cultured human monocytes stimulated with Sal-
`monella typhosa Iipopolysaccharide (LPS). Levels were measured with ELISA capture as-
`says. Details of methods are found in text.
`
`Page 5
`
`Ghio, Marshall, Diaz, et aI.: Tyloxapol and Cystic Fibrosis
`
`RESULTS
`
`Inhibitory Activity on Cytokine and Mediator Secretion
`
`The concentration of endotoxin in all buffers and tyloxapol was
`below the level of detection (25 pg/ml). Incubations of mono-
`cytes in concentrations of tyloxapol of 100 ug/ml or below were
`not associated with significant elevations in the concentration
`of lactate dehydrogenase (LDH) in the supernatant, supporting
`a lack of cytotoxicity and suggesting that the inhibition of
`cytokine secretion by tyloxapol was not due to an injurious de-
`tergent effect on monocytes.
`Tyloxapol had no effect on thevbaseline release of any media-
`tor except for IL-8, but significantly decreased secretion of this
`Cytokine in unstimulated cells (Figure 1). However, release of sev-
`eral mediators by LPS-stimulated monocytes was significantly
`diminished at low concentrations of tyloxapol. Secretion of
`TNF—or, IL-1 B, IL-6, and IL-8 (Figure 2) was significantly (p <
`0.01) decreased by tyloxapol in a dose-dependent manner, with
`effective concentrations for 50070 inhibition (EC50) ranging from
`30 to 70 ug/ml (Table 1). As in the case of the cytokines, release
`of GM—CSF (Figure 2) by stimulated monocytes decreased with
`increasing concentrations of tyloxapol in the cell suspension. Fi-
`nally, secretion of the eicosanoids LTB4'and TXA, (Figure 3) was
`also significantly (p < 0.01) reduced by tyloxapol. However, tylox-
`apol did not change PAF release from LPS-stimulated mono-
`cytes (data not shown).
`An effect of a second detergent, Triton X, on mediator re-
`lease by human monocytes was also examined. Triton X was cyto-
`toxic at lower concentrations than tyloxapol, with significant ele-
`vations in supernatant [LDH] after 1.0 ug/ml. Therefore, the
`concentrations of Triton X requried to inhibit 50% of the medi-
`ator release by stimulated human monocytes probably reflected
`the cytotoxicity of this detergent.
`
`Inhibitory Activity on Activation of Nuclear
`Transcription Factors
`
`IL—1B or H202 treatment of A549 human pulmonary epithelial
`cells increased DNA binding of both NF-KB and AP—l, as mea-
`sured by gel shift assays (arrows, Lanes 2 and 4, Figure 4). Con-
`comitant treatment of cells with 100 ug/ml tyloxapol substan-
`tially blocked the increase in NF-KB-binding activity induced by
`IL-1 [3 and H202 (Lanes 3 and 5, Figure 4), but had no significant
`effect on AP—1 binding activity.
`
`150
`
`releaseofIL-8 0.010
`
`Percentmaximal
`
`0.100
`Tyloxapol (mglml)
`
`Figure 1. Tyloxapol reduces baseline secretion of IL-8 by unstimu-
`lated human monocytes. IL-8 release was significantly (p < 0.01) re-
`duced by tyloxapol concentrations greaterthan 0.01 mg/ml. Methods
`are detailed in text.
`
`Page 5
`
`

`
`786
`
`AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 154
`
`1996
`
`120
`110
`100
`90
`80
`70
`60
`50
`40
`30
`20
`10
`
`120
`1 10
`100
`90
`80
`70
`60
`50
`40
`30
`20
`10
`0
`
`LaukotrieneB4
`
`ThromboxaneA2
`
`
`
`Percentofmaximalrelease
`
`0.001
`
`0.010
`
`0.100
`
`1.000
`
`0.001
`
`0.100
`0.010
`Tyloxapol (mg/ml)
`
`1.000
`
`Figure 3. Tyloxapol significantly (p < 0.01) reduces secretion of the
`eicosanoids LTB4 and TXA2 by LPS-stimulated human monocytes in
`a dose-dependent manner. Methods are detailed in text.
`
`5
`
`Effect on Sputum Viscosity
`Tyloxapol significantly (p < 0.01) reduced the viscosity of CF
`sputum in vitro (Table 3).
`
`DISCUSSION
`
`Tyloxapol, or Triton WR-1339, is a nonionic detergent that has
`been in common use in the pharmaceutical industry for over 40
`yr. It is relatively nontoxic for human cells (22), has an LD50 in
`mice of > 10 g/kg orally and 3.8 to 5.0 g/kg intravenously (23),
`and has no demonstrable respiratory toxicity when administered
`chronically to rhesus monkeys in a high-dose aerosol for a year
`(23). Until 1981, tyloxapol was marketed in the United States as
`an aerosolized mucolytic treatment
`for chronic bronchitis
`(Alevaire, a 0.125% aqueous solution of tyloxapol in 2% Na-
`HCO3 and 5°70 glycerol) (14, 15). However, with passage of the
`Harris-Kefauver amendment to the Food and Drug Act, older
`drugs previously tested only for safety were required to demon-
`strate efficacy to retain their drug approval status. Instead of per-
`forming the requisite clinical trials, the sponsoring pharmaceu-
`tical company withdrew tyloxapol from the market (24). Available
`literature does not suggest that Alevaire or tyloxapol in any form
`was ever tested as an antioxidant or treatment for CF.
`We have shown that tyloxapol is a potent inhibitor of mono-
`cyte secretion of cytokines (Figures 1 and 2), GM-CSF (Figure
`2), the cyclooxygenase product TXA;, and the 5-lipoxygenase
`product LTB4 (Figure 3). Although complete mechanistic under-
`standing of the effect of tyloxapol will require additional studies
`in monocytes, inhibition of cytokine secretion is perhaps ex-
`plained by the ability of tyloxapol to block the activation of NF-
`KB (Figure 4), a transcription factor influencing the expression
`of TNF—0t, IL—1B, IL-6, IL-8 and GM-CSF (15). The inhibition
`of cytokine and mediator secretion by tyloxapol was probably
`not from an injurious detergent effect on monocytes. Tyloxapol
`concentrations used in these studies caused no LDH release from
`monocytes, in contrast to the prompt increase in [LD H] seen when
`cells were incubated with much lower concentrations of another
`detergent, Triton X. Also, tyloxapol failed to decrease LPS-
`‘induced monocyte secretion of PAF, or to prevent IL—lB- or H202-
`enhanced DNA binding of the transcription factor AP-1 (Figure
`
`""5
`o.
`:6
`
`'5
`o.
`cu‘
`
`xc
`><
`-3.9
`-:
`PE
`1-
`+0:
`‘l’
`¢3__q:;__NN€J.
`
`1 NNO
`T31
`0 :-'. E I'IO
`
`-4--NF""KB
`
`-I--AP"‘1
`
`Figure 4. Tyloxapol prevents IL-1|3— or H202-induced DNA binding
`of NF-KB, but notAP-1 . Confluent A549 human pulmonary epithelial
`cells were incubated without (Lane 1 ) or with 10 U/ml of IL-1 [3 (Lanes
`2 and 3) or 100 uM H202 (Lanes 4 and 5). Tyloxapol (100 ug/ml,
`Lanes 3 and 5) was added at the same time as stimulators. After 3 h
`of incubation, nuclear extracts were prepared. Aliquots of the extracts
`were incubated with 32P—|abe|ed NF-i<B— and AP-1-specific o|igonu—
`cleotides and analyzed in electrophoretic mobility shift assays. Posi-
`tions of the specific DNA-protein complexes are indicated by the ar-
`rowheads. A 100-fold molar excess of the appropriate unlabeled DNA
`probe was included in the binding reactions for the samples shown
`in the competition lanes.
`
`4), suggesting selective pharmacologic rather than toxic effects
`as the explanation for our observations.
`Tyloxapol also scavenges HOCI in vitro (Table 2) and pro-
`tects against HOCI-induced lung injury (Figure 5). We have pre-
`viously demonstrated that tyloxapol is an antioxidant that pro-
`tects against Fenton generation of hydroxyl radicals (-OH) in vitro
`and the toxic effects of 100% O2 in rats (12). Tyloxapol is an
`alkylaryl polyether alcohol polymer. Alcohols can function as
`antioxidants and are oxidized to aldehydes (25). The detergent
`
`TYLOXAPOL IS AN ANTIOXIDANT THAT SCAVENGES HOCI
`
`TABLE 2
`
`NaOC| (mM)
`
`0
`
`1.0
`2.5
`5.0
`7.5
`
`0.087 1 0.005
`0.219 t 0.001
`0.482‘: 0.001
`0.786 :1: 0.004
`
`Tyloxapol
`(wt/voI%)
`
`0.05
`
`O
`0.048 t 0.03
`031610.008
`0.561 1 0.024
`
`0.10
`
`O
`0
`0.191 10.101
`0.464 t 0.007
`
`Values represent difference in A230 of reaction mixture before and after addition Oi
`NaOCl. The activity of tyloxapol as an HOCI scavenger was tested by its ability to prevem
`HOC|—mediated oxidation of diethanolamine to its corresponding chloramine (20). Ty-
`loxapol
`in 0.1 M NaCl was added in varying concentrations (wt/vo|%) to 10.0 mM
`dletnanolamine in 0.1 N sodium acetate buffer, and baseline absorbance was read at
`280 nm. NaOCl (1.0 to 7.5 mM) was added, the reaction mixture was incubated lor 15
`min at 37° C, and the absorbance was again measured. The difference in A250 before
`and after addition of NaOC| was used as a measure of chloramine concentration.
`
`Page 6
`
`Page 6
`
`

`
`Ghio, Marshall, Diaz, et a/.: Tyloxapol and Cystic Fibrosis
`
`787
`
`040:
`0354
`
`9 E
`o Non I
`
`
`
`
`
`Lavageprotein(mglml)5E
`
`‘I
`
`EFFECT OF TYLOXAPOL ON VISCOSITY OF CF SPUTUM
`
`TABLE 3
`
`Viscosity in Centipoise (rnPa)
`
`Patient
`1
`2
`3
`4
`5
`6
`7
`8
`9
`10
`11
`MeaniSEM
`
`Saline
`198
`507
`531
`918
`1,586
`364
`270
`118
`80
`159
`358
`4631-133
`
`Tyloxapol
`16
`63
`317
`325
`515
`16
`71
`24
`16
`32
`16
`128:52*
`
`Methods are detailed in text.
`* p < 0.01 compared with CF sputum + saline.
`
`
`
`Salinelsaline
`
`HOClISaline
`salineffyloxapoi
`Instillations
`
`Hoclffyloxapol
`
`005
`
`0.11)
`
`10
`
`
`
`
`
`LavagePMNs('/0)
`
` Saline/Saline
`
`HOCI/Tyloxapol
`
`HOClISaline
`Salineflvloxnpol
`lnslillations
`
`Figure 5. Tyloxapol prevents HOCl-induced lung injury in rats. Rats
`were anesthetized, dosed intratracheally with 0.3 ml of 2.0 mM NaOCl
`in normal saline or normal saline, allowed to recover, and 1 h later
`dosed intratracheally with either 6.0 mg tyloxapol in normal saline
`or normal saline. Twenty-four hours after NaOC| instillation, all rats
`were euthanized, lungs were lavaged with normal saline, and injury
`was assessed by measuring the protein concentration and percent-
`age of neutrophils in the cellular differential of lung lavage fluid. As
`compared with NaOC|-exposed rats treated with saline alone, postex—
`posure treatment with tyloxapol significantly reduced protein con-
`centration (p < 0.001) and the percentage of neutrophils (p < 0.01)
`in lung lavage fluid.
`
`retains functional groups of the original alcohol monomer, and
`these may confer a capacity to perform as an antioxidant against
`a variety of oxidant species. Antioxidant activity may best ex-
`plain the ability of tyloxapol to inhibit eicosanoid secretion by
`human monocytes. Antioxidants are well known to inhibit mam-
`malian 5-lipoxygenase and cyclooxygenase, perhaps by intercept-
`ing peroxyl radicals generated at the active sites of these enzymes,
`or by reducing or chelating the active Fe“ forms of the enzymes
`(26). Antioxidant activity could also account for the ability of
`tyloxapol to inhibit NF-KB. NF—i<B activation is controlled in
`some (27) but not all cell systems (28) by intracellular redox regu-
`lation of tyrosine kinase-mediated phosphorylation events within
`the common step of the NF-KB signal transduction pathway (27).
`A number of antioxidants prevent activation of NF-KB by LPS
`(13-15) and other stimuli (15), prevent increases in correspond-
`ing messenger ribonucleic acids (RNAs) for inflammatory cyto-
`kines (15), and decrease levels of TNF—u and IL-16 in the circula-
`tion of mice following LPS injection (15).
`
`We have also shown that tyloxapol reduces the viscosity of
`CF sputum in vitro. Although the precise molecular mechanism
`for this result is uncertain, we speculate that tyloxapol acts as
`a detergent or dispersing agent. This is a preliminary observa-
`tion, the significance of which can be assessed only after more
`sophisticated measurements of mucus rheology and transport
`(9, 29). It has been recently reported that the synthetic surfac-
`tant Exosurf® (Burroughs Wellcome, Research Triangle Park,
`NC) improves mucus clearability in neonatal respiratory distress
`syndrome (29) and CF (30), as well as lung function in patients
`.with chronic bronchitis (31). Exosurf® also decreases the release
`of inflammatory mediators (32, 33) and their corresponding mes-
`senger RNAs (33) after exposure to diverse agents such as endo-
`toxin and interleukins. These effects of Exosurf® have been
`attributed to the surface-active component dipalmitoylphos—
`phatidylcholine (29, 30, 33), but could conceivably be due to the
`tyloxapol added as a dispersing agent for the phospholipid.
`Recently, rhDNasc has been approved as a mucolytic therapy
`for CF, largely because patients treated with this mucolytic agent
`had an average 30% reduction in exacerbations of disease dur-
`ing treatment (34). However, at a factory cost of $27 per dose,
`rhDNasc is extraordinarily expensive, with a $9,855 wholesale
`cost for once daily and $19,710 cost for twice daily treatment
`per year per patient (34). Tyloxapol might decrease lung inflam-
`mation in CF by suppressing airway production of cytokines (2-4)
`and by scavenging HOCI (5, 6). By inhibiting TNF secretion,
`tyloxapol could ameliorate the cachexia suffered by patients with
`CF lung disease (35). If additional studies confirm antiinflam-
`matory activity and a beneficial effect on mucus rheology, tylox-
`apol might represent a less expensive treatment for CF lung
`disease.
`
`Acknowledgment: The authors greatly appreciate the technical assistance of
`Lise Sorenson in performing the electrophoretic mobility shift assays and the
`technical assistance of Lynn Tatro in performing the scavenging studies of tylox-
`apol for HOCI and thank Drs. John Hoidal, Marvin McCall, and Richard Cor-
`bin for their helpful comments on the manuscript.
`
`References
`
`1. Konstan, M. W., and M. Berger. 1993. Infection and inflammation of
`the lung in cystic fibrosis. In P. B. Davis, editor. Cystic Fibrosis. Marcel
`Dekker, New York. 219-276.
`2. Bonfield, T. L., J. R. Panuska, M. W. Konstan, K. A. Hilliard, J. B.
`Hilliard, H. Ghnaim, and M. Berger. 1995. Inflammatory cytokines
`in cystic fibrosis lungs. Am. J. Respir. Crit. Care Med. 152:2! 1 1-21 18.
`3. Nakamura, H., K. Yoshimura, N. G. McElvaney, and R. G. Crystal.
`
`Page 7
`
`Page 7
`
`

`
`788
`
`AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 154
`
`1996
`
`1992. Neutrophil elastase in respiratory epithelial lining fluid of indi-
`viduals with cystic fibrosis induces interleukin-8 gene expression in
`a human bronchial epithelial cell line. J. Clin. Invest. 89:l478—1484.
`. McElvaney, N. G., H. Nakamura, P. Birrer, C. A. Hebert, W. L. Wong,
`M. Alphonso, J. B. Baker, M. A. Catalano, and R. G. Crystal. 1992.
`Modulation of airway inflammation in cystic fibrosis: in vivo sup-
`pression of interleukin-8 levels on the respiratory epithelial surface
`by aerosolization of recombinant secretory leukoprotease inhibitor.
`J. Clin. Invest. 90:1296-1301.
`. Mohammed, J. R., B. S. Mohammed, L. J. Pawluk, D. M. Bucci, N. R.
`Baker, and W. B. Davis. 1988. Purification and cytotoxic potential
`of myeloperoxidase in cystic fibrosis sputum. J. Lab. Clin. Med. 112:
`711-720.
`. Cantin, A., and D. E. Woods. 1993. Protection by antibiotics against
`myeloperoxidase-dependent cytotoxicity to lung epithelial cells in vitro.
`J. Clin. Invest. 91:38-45.
`. Lethem, M. 1., S. L. James, and C. Marriott. 1990. The role of mucous
`glycoproteins in the rheologic properties of cystic fibrosis sputum.
`Am. Rev. Respir. Dis. 142:1053—l058.
`. Vasconcellos, C. A., P. G. Allen, M. E. Wohl, J. M. Drazen, P. A.
`Janmey, and T. P. Stossel. 1994. Reduction in viscosity of cystic fibro-
`sis sputum in vitro by gelsolin. Science 263:969—97l.
`Zahm, J.-M., S. Girod de Bentzmann, E. _Deneuville, C. Pcrrot-Minnot,
`A. Dabadie, F. Pennaforte, M. Roussey, S. Shak, and E. Puchelle.
`1995. Dose—dependent in vitro effect of recombinant human DNase
`on rheological and transport properties of cystic fibrosis respiratory
`mucus. Eur. Respir. J. 8:381-386.
`Tainter, M. L., F. C. Nachod, and J. G. Bird. 1955. Alevaire as a muco-
`lytic agent. N. Eng]. J. Med. 253:764-767.
`Paez, P. N., and W. F. Miller. 1971. Surface active agents in sputum
`evacuation: a blind comparison with normal saline solution and dis-
`tilled water. Chest 60:312-317.
`‘
`’
`Ghio, A. H., P. J. Fracica, S. L. Young, and C. A. Piantadosi. 1994.
`Synthetic surfactant scavenges oxidants and protects against hyperoxic
`lung injury. J. Appl. Physio]. 7721217-1223.
`Schreck, R., P. Rieber, and P. A. Baeuerle. 1991. Reactive oxygen in-
`termediates as apparently widely used messengers in the activation
`of the NF-KB transcription factor and HIV-1. EMBO J. 10:2247—2258.
`Schreck, R., B. Meier, D. N. Mannel, W. Droge, and P. A. Baeuerlc.
`1992. Dithiocarbamates as potent inhibitors of nuclear factor KB ac-
`tivation in intact cells. J. Exp. Med. 17521181-1194.
`Eugui, E. M., B. DeLustsro, S. Rouhafza, M. Ilnicka, S. W. Lee, R.
`Wilhelm, and A. C. Allison. 1993. Some antioxidants inhibit, in a
`co—ordinate fashion, the production of tumor necrosis factor-alpha,
`IL-beta, and IL-6 by human peripheral blood mononuclear cells. Int.
`J. Immunol. 6:409-422.
`Dignam, J. D., R. M. Lebovitz, and R. G. Roeder. 1983. Accurate tran-
`scription initiation by RNA polymerase 11 in a soluble extract from
`isolated mammalian nuclei. Nucleic Acids Res. 11:l475-1489.
`Gunther, C. V., and B. J. Graves. 1994. Identification of ETS domain
`proteins in murine T lymphocytes that interact with the Moloney mu-
`rine leukemia virus enhancer. Mol. Cell. Biol. 14:7569-7580.
`
`10.
`
`ll.
`
`12.
`
`13.
`
`14.
`
`15.
`
`16.
`
`17.
`
`18.
`
`Lee, W., P. Mitchell, and R. Tijan. 1987. Purified transcription factor
`AP-1 interacts with TPA-inducible enhancer elements. Cell 49:741-752.
`
`19
`
`20.
`
`21.
`
`22.
`
`23.
`24.
`
`25.
`
`26.
`
`27.