Rostrum
`
`Is the use of benzalkonium chloride as a
`preservative for nasal formulations a
`safety concern? A cautionary note based
`on compromised mucociliary transport
`
`I. Leonard Bernstein, MD Cincinnati, Ohio
`
`Background: Topical nasal solution and suspension delivery
`systems are available for short- and long-acting vasoconstric-
`tors, ipratropium, cromolyn, azelastine, and glucocortico-
`steroids. The use of intranasal glucocorticosteroids has
`increased substantially because the efficacy of these agents has
`been well established for the treatment of perennial and sea-
`sonal allergic rhinitis. Adverse local effects of burning, irrita-
`tion, and dryness are occasionally associated with glucocorti-
`costeroid nasal preparations. Benzalkonium chloride (BKC) is
`a quaternary ammonium antimicrobial agent included in some
`nasal solutions (including glucocorticosteroids) to prevent the
`growth of bacteria. Some reports suggest that BKC in nasal
`sprays may cause adverse effects, including reduced mucocil-
`iary transport, rhinitis medicamentosa, and neutrophil dys-
`function.
`Objective: This article summarizes recent literature about pos-
`sible adverse biologic effects associated with BKC as a nasal
`spray preservative by examining its effects on the following
`properties of mucociliary transport: ciliary motion, ciliary
`form, ciliary beat frequency, electron microscopy, and particle
`movement/saccharin clearance tests.
`Conclusion: Both animal and human in vitro data suggest that
`BKC promotes ciliostasis and reduction in mucociliary trans-
`port that may be partially masked by absorption and dilution
`effects occurring in respiratory mucus. These possible con-
`founding factors may account for several disparate human in
`vivo results. The use of BKC-free glucocorticosteroid formula-
`tions should be considered, particularly in patients who com-
`plain of nasal burning, dryness, or irritation. (J Allergy Clin
`Immunol 2000;105:39-44.)
`
`Key words: Allergic rhinitis, topical nasal drugs, preservatives,
`benzalkonium chloride, mucociliary transport
`
`Topical nasal aqueous and suspension delivery sys-
`tems are available for vasoconstrictors, ipratropium, cro-
`molyn, azelastine, and glucocorticosteroids (Table I).
`Intranasal corticosteroid therapy is recognized as the
`
`From the Division of Immunology, University of Cincinnati College of Med-
`icine, Cincinnati, Ohio.
`Received for publication Aug 2, 1999; revised Sept 15, 1999; accepted for
`publication Sept 16, 1999.
`Reprint requests: I. Leonard Bernstein, MD, University of Cincinnati College
`of Medicine, 231 Bethesda Ave, ML 563, Cincinnati, OH 45267.
`Copyright © 2000 by Mosby, Inc.
`0091-6749/2000 $12.00 + 0 1/1/103181
`
`Abbreviation used
`BKC: Benzalkonium chloride
`
`most significant advance in the treatment of allergic
`rhinitis since the advent of antihistamines.1 Treatment
`with corticosteroid-containing nasal sprays is recom-
`mended when nasal symptoms are not controlled with
`antihistamines.1 Topical corticosteroids have proved
`highly effective by decreasing cellular inflammation in
`the nasal mucosa.1,2
`Formulation of nonpropellant mucosal delivery sys-
`tems often requires preservatives for inhibition of micro-
`bial growth. Both toxic and allergic effects of these agents
`have been reported.3,4 Among available antimicrobial
`preservatives, benzalkonium chloride (BKC) is common-
`ly used to prevent bacterial contamination in various nasal
`formulations (Table I). Adverse local effects of these
`products (eg, rhinitis medicamentosa, burning, irritation,
`dryness, and epistaxis) have generally been attributed to
`the active therapeutic agents. Nevertheless, the possibility
`that BKC could contribute to adverse responses should be
`reconsidered further in view of recent reports that this
`agent may affect mucociliary function.
`
`BKC AS A PRESERVATIVE:
`STRUCTURE/FUNCTION AND MECHANISM
`OF ACTION
`
`BKC has been used to prevent bacterial contamination
`and to preserve pharmacologic activity in multidose top-
`ical aqueous drops and sprays.5,6 BKC is a mixture of
`quaternary benzyldimethylalkylammonium chlorides. Its
`germicidal activity is attributed to surface-active proper-
`ties of its hydrophobic and cationic groups, which are
`bactericidal against a wide variety of gram-positive and
`gram-negative bacteria at low concentrations.5 This
`activity is accomplished by altering the permeability of
`cell walls of microorganisms.7 EDTA enhances penetra-
`tion of BKC into the bacterial cell wall and is often rec-
`ommended as a BKC coadditive.7 It is postulated that the
`cationic surfactant properties of BKC promote strong
`binding to nasal tissue, thereby increasing the viscoelas-
`tic nature of the mucus gel with attendant toxicity.8,9
`
`39
`
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`40 Bernstein
`
`J ALLERGY CLIN IMMUNOL
`JANUARY 2000
`
`TABLE I. Excipient profiles of nasal preparations used in the United States
`Class of drugs
`Generic name
`Trade name
`BKC CFC EDTA Surf Alc
`
`Hg
`
`PG
`
`G PS-80 BA MC
`
`Vasoconstrictor-
`short
`
`Phenylephrine HCl
`
`Neosynephrine
`
`+
`
`+
`
`+
`
`+
`
`+
`+
`
`+
`–
`
`–
`
`–
`
`–
`
`–
`
`–
`–
`
`–
`+
`
`–
`
`–
`
`+
`
`–
`
`+
`+
`
`+
`–
`
`–
`
`–
`
`–
`
`–
`
`–
`–
`
`–
`–
`
`–
`
`–
`
`+
`
`–
`
`–
`–
`
`–
`+
`
`–
`–
`
`+
`
`–
`
`–
`
`–
`
`–
`–
`
`–
`–
`
`–
`–
`
`–
`
`–
`
`+
`
`–
`
`–
`–
`
`–
`–
`
`–
`–
`
`–
`
`–
`
`–
`
`–
`
`–
`–
`
`–
`–
`
`–
`–
`
`–
`
`–
`
`+
`
`–
`
`–
`–
`
`–
`–
`
`+
`–
`
`+
`
`+
`
`+
`
`+
`
`+
`+
`
`–
`–
`
`+
`–
`
`–
`
`–
`
`–
`
`–
`
`+
`–
`
`–
`–
`
`+
`–
`
`Vasoconstrictor-
`long
`
`Antihistamine
`Anticholinergic
`
`Phenylephrine HCl,
`naphzoline HCl,
`pyrilamine maleate
`Oxymetazoline
`
`4-Way Fast Acting
`nasal spray
`
`Afrin
`
`Oxymetazoline
`
`4-Way Long Lasting
`nasal spray
`Astelin
`Azelastine
`Ipratropium bromide Atrovent nasal spray
`0.03% and 0.06%
`Nasalcrom solution
`Nasacort inhaler
`
`Mast cell stabilizer Cromolyn sodium
`Glucocortico-
`Triamcinolone
`steroid
`acetonide
`
`Flunisolide
`
`Nasacort AQ
`Nasalide nasal
`solution
`Nasarel nasal
`solution
`Decadron Turbinaire
`Beconase AQ
`
`Dexamethasone
`Beclomethasone
`dipropionate
`
`Beconase aerosol
`inhaler
`Vancenase AQ
`Vancenase nasal
`inhaler
`Flonase
`
`Fluticasone
`proprionate
`Rhinocort
`Budesonide
`Mometasone furoate Nasonex nasal
`suspension
`
`+
`+
`
`+
`
`–
`+
`
`–
`
`+
`–
`
`+
`
`–
`
`+
`
`–
`–
`
`–
`
`+
`–
`
`+
`
`–
`+
`
`–
`
`+
`
`–
`
`+
`+
`
`+
`
`–
`–
`
`–
`
`–
`–
`
`–
`
`–
`
`–
`
`–
`–
`
`–
`
`–
`+
`
`–
`
`–
`–
`
`–
`
`+
`
`–
`
`–
`
`+
`–
`
`–
`
`+
`–
`
`+
`
`–
`
`+
`
`–
`
`–
`–
`
`–
`
`–
`–
`
`–
`
`–
`
`–
`
`–
`
`–
`–
`
`–
`
`–
`–
`
`–
`
`–
`
`–
`
`–
`
`–
`–
`
`–
`
`–
`–
`
`–
`
`–
`
`+
`
`–
`
`–
`–
`
`–
`
`–
`–
`
`+
`
`–
`
`+
`
`–
`
`–
`–
`
`–
`
`–
`–
`
`–
`
`–
`
`+
`
`–
`
`–
`–
`
`–
`
`–
`–
`
`+
`
`–
`
`+
`
`BKC, Benzalkonium chloride; CFC, chlorofluorocarbons; Surf, surfactant; Alc, alcohol; Hg, mercurial preservatives; PG, propylene glycol; G, glycerin; PS-80,
`polysorbate-80; BA, buffering agents; MC, methylcellulose compounds; +, present; –, not present.
`
`Although ophthalmic mucositis10 and contact der-
`matitis11 resulting from BKC have been documented,
`allergic effects of BKC in nasal sprays have not been
`reported. In the current article the possibility that BKC
`may exert adverse effects on mucociliary transport is
`discussed.
`
`ADVERSE MUCOCILIARY EFFECTS OF BKC
`
`Three techniques are available to assess mucociliary
`function. These include (1) ciliary form and motion, (2)
`mucus production and properties, and (3) measurement
`of the efficiency of the combined ciliary activity and
`mucus production.12 All these parameters have been
`examined to investigate biologic effects of BKC. How-
`ever, the effect of BKC on rheologic properties of mucus
`has not been investigated because a reliable clinical test
`is not available.12
`
`Ciliary motion
`The effects of BKC on nasal mucosal motility have
`been studied in several animal and human models.13-16 A
`
`major strength of this method is the ability to harvest
`cilia readily from the inferior turbinate for in vitro exam-
`ination in a mucus-free environment12 that excludes con-
`founding factors such as stress, hormone secretion, or
`inflammatory mediators.16 In contrast, in vitro cultures
`of cilia may not be representative of how they behave in
`the body. For example, ciliary dysfunction in patients
`with cystic fibrosis may not be demonstrated in culture.17
`To measure ciliary motion, the ciliary beat frequency of
`randomly selected areas is measured photometrically in
`the presence and absence of BKC for periods ranging
`from minutes to hours. The photosensitive cell converts
`the reflected light from the beating cilia to an electric
`current that is then amplified to an oscilloscope dis-
`play.12,18 In vitro, ciliary beating occurs in a coordinated
`manner at 10 to 20 Hz with an effective stroke perpen-
`dicular to the mucosal surface.18
`Animal studies. BKC was chosen as a prototypical
`polar compound to examine the in vitro ciliary beat fre-
`quency of chicken embryo tracheas compared with
`lipophilic (eg, chlorocresol) and mercuric compounds
`(eg, thimerosal).13 BKC (0.1% wt/vol) in the presence of
`
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`J ALLERGY CLIN IMMUNOL
`VOLUME 105, NUMBER 1, PART 1
`
`Bernstein 41
`
`EDTA (0.05% wt/vol) caused a reduction of beat fre-
`quency of about 30% after a 20-minute exposure. Longer
`BKC exposure led to more pronounced reductions in beat
`frequency and ciliostatic effects. The t50% (frequency
`time 50% of control) and t0% (complete loss of motility)
`was 1.13 and >2 hours, respectively. Chlorhexidine glu-
`conate, a nonquaternary ammonium polar compound,
`was more toxic. Nevertheless, van de Donk et al13 rec-
`ommended BKC (0.01% wt/vol) and EDTA (0.05%
`wt/vol) as nasal drop additives because these agents had
`fewer ciliotoxic effects at these concentrations than did
`chlorhexidine gluconate or thimerosal.
`The effect of BKC on the ciliary beat frequency in rat
`and guinea pig tracheal epithelial tissues was studied by
`Joki et al.15 In rat tracheal explants, decrease in ciliary
`beat frequency was dependent on both duration and con-
`centration of BKC exposure. At concentrations of 0.01%
`(wt/vol) ciliary activity ceased by 5 minutes, whereas
`about 30 minutes were required to abrogate beat fre-
`quency at lower concentrations (0.00125% wt/vol and
`0.0025% wt/vol).
`In guinea pig tracheal preparations a dilute concentration
`of BKC (ie, 0.0025% wt/vol) led to a 27% decrease in cil-
`iary beat frequency by 60 minutes, whereas a slightly high-
`er concentration of 0.005% (wt/vol) irreversibly stopped
`ciliary activity after 40 minutes. In fact, these in vitro stud-
`ies revealed that BKC had higher toxicity than three other
`common preservatives used in nasal formulations.
`Human studies. Stanley et al19 demonstrated a dose-
`dependent ciliostatic response of BKC in ciliated epithe-
`lial preparations obtained by brushing the inferior nasal
`turbinates of healthy human volunteers. A maximal cilio-
`static response was observed by 5 minutes in the pres-
`ence of 0.01% to 0.02% (wt/vol) and by 60 minutes in
`the presence of 0.002% (wt/vol). At a concentration of
`0.0008% (wt/vol) the ciliostatic response was 57% of
`control by 60 minutes. These data convincingly indicat-
`ed the in vitro ciliotoxic nature of BKC.
`Significant retardation of ciliary beat frequency of
`human nasal mucosa by BKC was confirmed in patients
`with nasal polyposis, chronic sinusitis, or sinus pseudo-
`cysts.15 Reduction of ciliary beat frequency by BKC
`(0.0012%-0.005% wt/vol) was detected in mucosal
`explants for periods of 10 to 60 minutes. At 0.005%
`(wt/vol), BKC irreversibly reduced the ciliary beat fre-
`quency by 60%. However, the magnitude of inhibition of
`ciliary beat frequency by BKC was not as pronounced as
`that found in mucosal preparations from rat and guinea pig.
`In a recent study the effect of several BKC-containing
`nasal sprays on ciliary beat frequency of human nasal
`mucosa in vitro was compared with one without BKC.16
`Ciliary beat frequency was slowed down dramatically or
`irreversibly arrested by 3 of the 4 agents (ie, fluticasone
`propionate, azelastine, and levocabastine) that contained
`BKC. In contrast, only mild and transient reduction in
`beat frequency was observed in the one preparation with-
`out BKC, (ie, budesonide). These investigators also
`demonstrated that BKC alone caused an irreversible
`arrest of ciliary beat frequency.
`
`Other preservatives used in nasal aqueous solutions of
`corticosteroids similarly have been shown to be harmful.
`Propylene glycol (20% wt/vol), a main preservative of
`flunisolide, caused reversible inhibition of human nasal
`ciliary beat frequency in vitro.20 Thimerosal (0.005%
`wt/vol), a preservative found in some formulations of
`glucocorticosteroidal nose drops containing either beta-
`methasone alone or betamethasone with neomycin, was cil-
`iostatic within 5 minutes.19
`
`Nasal and ciliary histologic features
`In addition to routine nasal mucosal morphologic
`examination, ciliary histologic features can be assessed
`quantitatively by electron microscopy. A normal colum-
`nar cell displays 200 whip-like cilia 6 to 8 µm in length
`on its surface.12 These assessments include the number
`of compound cilia, central and peripheral microtubule
`defects, inner and outer dynein arms per cilium, and cil-
`iary orientation.12 A ciliary angle may be determined
`against a perpendicular ciliary axis line drawn through
`the centers of the 2 central microtubules.12
`Animal in vivo and ex vivo studies. Intranasal admin-
`istration of BKC in rats has been associated with the
`development of nasal lesions.21 BKC (0.01, 0.05, and
`0.10% wt/vol) was administered to a nasal cavity of rats
`8 times for 1 day. Epithelial desquamation, degeneration,
`edema, or neutrophilic cellular infiltration were demon-
`strated in the nasal mucosa of rats administered BKC at
`higher concentrations (0.05 and 0.10% wt/vol) than are
`used in many nasal sprays (0.01% wt/vol).
`Berg et al22 found that BKC is potentially toxic to
`nasal mucosa in vivo. In this study rats were exposed to
`nasal steroids with and without BKC twice daily for 21
`days. The structure of the mucosal lining of all parts of
`the nose was then investigated in serial frontal sections.
`Squamous cell metaplasia was observed in rats exposed
`to either beclomethasone or flunisolide containing BKC
`(0.031% and 0.022%, respectively). These alterations
`were found in anterior regions of the nose and included
`reduced epithelial cell height, pleomorphism of individ-
`ual epithelial cells, fewer cilia, reduced number of goblet
`cells, and loss of mucus covering the epithelial cell layer.
`In contrast, pathologic changes were not found in nasal
`tissues exposed to either nasal steroid without preserva-
`tive (ie, budesonide) or saline solution alone.
`Ainge et al23 studied the in vivo effect of corticoste-
`roid sprays on monkey and rat nasal ciliated epithelia for
`28 days. Cynomolgus monkeys were treated intranasally
`8 × 0.1 mL per day with fluticasone propionate (0.05%
`wt/vol) containing BKC (0.02% wt/vol) or control (5%
`glucose). Rats received either beclomethasone dipropi-
`onate aqueous spray containing BKC (0.01% wt/vol) in a
`inhalation chamber for 1 hour per day or air. A BKC con-
`trol group was not studied. At the end treatment periods
`ciliated cells were counted, and both scanning and trans-
`mission electron microscopy were performed on monkey
`inferior turbinate and rat intermediate turbinate sites,
`respectively. There was no difference in the number of cil-
`iated cells in the corticosteroid-treated group versus the
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`42 Bernstein
`
`J ALLERGY CLIN IMMUNOL
`JANUARY 2000
`
`control group. Scanning electron microscopy revealed
`intact turbinate surfaces with normal ciliated respiratory
`epithelia. Likewise, transmission electron microscopy
`analysis showed no ultrastructural abnormalities, suggest-
`ing that the effects of BKC-containing corticosteroid
`sprays were not deleterious.
`Human studies. Steinsvag et al6 demonstrated destruc-
`tion of human in vitro nasal mucosa preparations by
`topical nasal steroids containing BKC. In explant cul-
`tures of adenoid tissue, corticosteroid nasal sprays (ie,
`beclomethasone dipropionate, fluticasone propionate,
`and flunisolide) containing BKC or BKC alone de-
`creased the number of beating cilia in chronic studies of
`once-daily administration. Assessment by inverted-phase
`microscopy and scanning electron microscopy showed a
`gradual transition from a continuous epithelial lining to
`a rough, irregular surface. After 10 days of exposure,
`cilia were no longer present on the exposed tissue frag-
`ments. The kinetics of morphologic changes seemed to
`correlate with the levels of BKC present in various nasal
`sprays. For example, tissue explants exposed to flu-
`nisolide containing 0.031% (wt/vol) BKC exhibited his-
`togic changes earlier than preparations of beclometha-
`sone and fluticasone propionate containing 0.022% and
`0.02% (wt/vol) BKC, respectively.6 In contrast, tissue
`fragments exposed to steroid sprays in the absence of
`BKC retained beating cilia at all times during the 10-day
`treatment period. These data support the hypothesis that
`BKC is responsible for in vitro toxic effects on human
`respiratory mucosa.
`Berg et al24 studied the in vitro effects of exposure
`time and concentration on human respiratory mucosa
`treated with BKC-containing nasal spray. Ten-day cul-
`tures of adenoid tissue fragments were exposed to a 14-
`day treatment of oxymetazoline with BKC (0.015%
`wt/vol) for 1, 3, 10, or 30 minutes and neat, 30%, 10%,
`and 3% concentrations of oxymetazoline with BKC
`(0.015% wt/vol) for 10 minutes. Morphologic and func-
`tional assessments were done by electron microscopy
`and ciliary beat frequency, respectively, for each trial
`regimen. In the group exposed to daily oxymetazoline
`for 30 minutes, adenoid fragments exhibited a black,
`nonpellucid appearance within 3 days. When daily expo-
`sure was continued for 14 days, scanning electron
`microscopy revealed absence of epithelium, basal lami-
`na, defects in cell and nuclear membranes, stromal com-
`ponents of collagen fibrils, autophagosomes, dying
`cells, and cell remnants. Similar but later-onset findings
`were observed after shorter exposure times or with more
`dilute preparations. In addition, the number of beating
`cilia corresponded to the loss of the continuous epithe-
`lial lining in a concentration- and exposure time–depen-
`dent manner.
`In contrast, Braat et al25 failed to demonstrate an effect
`of BKC on the function and morphologic features of cilia
`in human nasal epithelium. This double-blinded study
`included 22 patients with allergic rhinitis who received
`one of the following for 6 weeks: fluticasone propionate
`aqueous nasal spray (200 µg) with BKC (0.02% wt/vol),
`
`BKC (0.02% wt/vol) alone, or placebo. Functional
`assessment was performed by a saccharin transport test
`performed before, during, and after treatment. Anatomic
`evaluation was also performed by electron microscopy of
`biopsy specimens taken 2 cm behind the anterior tip of
`the inferior turbinate before and after treatment. The cil-
`iary transport time, as measured by the saccharin test,
`was unchanged at all time points examined. No differ-
`ence was observed in the number of ciliated cells
`between treatment groups, although there were large
`variations within and between treatment arms. Consider-
`able variation was also observed in the ultrastructure of
`the ciliated epithelial cells, including the appearance of
`swollen mitochondria and cytoplasmic vacuoles. Howev-
`er, significant differences were not noted between treat-
`ment groups, possibly because of the small number of
`cohorts in each treatment arm. Despite these variations
`that may reflect the allergic rhinitis population under
`study, this negative BKC in vivo and ex vivo study con-
`trasts with previous in vitro observations.
`As suggested by Berg et al,22 the study by Braat et al25
`was not directly comparable because (1) the biopsy sam-
`ples were limited to the inferior turbinates, (2) significant
`variation was present in the control populations, and (3)
`the patients under study had perennial rhinitis with
`excessive mucus, which may have masked detrimental
`effects of BKC on the nasal mucosa.
`
`Measurement of the combined effects of the
`mucus and ciliary systems
`As recently summarized by Lale et al,12 when study-
`ing the combined effects of the mucus and ciliary sys-
`tems it is necessary to distinguish between mucociliary
`transport and mucociliary clearance. Mucociliary trans-
`port represents the movement of particles in an anatom-
`ically defined location in an animal model. This may be
`measured by movement of graphite particles over a
`defined length of frog palate.9 Mucociliary clearance
`measures elimination of inhaled or insufflated aerosols
`and is typically performed by a saccharin test.12 The test
`involves placing one fourth of a saccharin tablet on the
`anterior end of the inferior turbinate.17 The patient is
`asked to sit quietly (without sniffing, sneezing, eating,
`drinking, or moving the head forward) until the first per-
`ception of sweet taste is experienced. Normal saccharin
`clearance times range from 7 to 15 minutes, with greater
`than 20 minutes indicative of pathologic mucociliary
`transport.12
`Animal in vitro studies. In a model designed to exam-
`ine the mucociliary transport rate of the frog palate, Batts
`et al9 showed BKC to be tolerated poorly. In this model
`a small volume of the solution was applied to the palate
`for 10 minutes, followed by measurement of the trans-
`port rate of graphite particles over a given distance of the
`palate for up to 200 minutes. BKC (0.01% wt/vol) halted
`transport irreversibly after 1 or 2 applications. Unlike
`other in vitro models, the frog palate system has a ciliat-
`ed epithelium protected by mucus that may better reflect
`the in vivo situation in that damage attributable to BKC
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`J ALLERGY CLIN IMMUNOL
`VOLUME 105, NUMBER 1, PART 1
`
`Bernstein 43
`
`may include alterations in the viscoelastic nature of the
`protective mucus layer.
`Human in vivo studies. The immediate and short-term
`effect of BKC on human nasal mucosa in vivo was exam-
`ined by McMahon et al.26 In 34 healthy volunteers subject-
`ed to a saccharin clearance test, a 10-minute exposure to
`BKC (0.02% wt/vol) led to a significant increase in the
`mean clearance time (762.7 ± 459 seconds) versus that
`observed with saline solution alone (620 ± 437 seconds).
`Despite these abnormal single-dose findings in healthy vol-
`unteers, these investigators did not detect a difference of
`saccharin clearance in a double-blind multidose study com-
`paring saline solution, fluticasone propionate plus BKC,
`and BKC alone administered for 2 weeks at 2 puffs per day.
`Van de Donk et al27 examined the effect of BKC on
`human nasal mucociliary clearance but failed to see an
`increase in transport time, indicating normal ciliary beat
`frequency. These data may not be comparable because the
`BKC concentrations used were at least 2 log orders more
`dilute than concentrations used in clinical preparations.
`
`OTHER ADVERSE BIOLOGIC EFFECTS OF
`BKC
`Bronchoconstriction
`
`In addition to its ability to retard ciliary beat frequency
`and mucociliary clearance, BKC as a preservative has
`been associated with other adverse biologic effects. This
`was first reported when paradoxic bronchoconstriction
`occurred after inhalation of nebulizer solutions of iprat-
`ropium bromide and several β2agonists.28 In preservative-
`free formulations of ipratropium bromide, the expected
`increase in FEV1 was observed after inhalation, whereas
`in the presence of BKC patients actually had bron-
`choconstriction with a fall in FEV1 after inhalation. The
`mechanism of action by which BKC promotes bron-
`choconstriction may involve histamine release because an
`H1 antihistamine blocked BKC-induced bronchoconstric-
`tion.28 Inclusion of BKC (0.01% wt/vol) in albuterol sul-
`fate and metaproterenol sulfate solutions may also coun-
`teract the bronchodilating properties of these drugs in
`some asthmatic patients.28 In contrast, the presence of
`BKC did not cause bronchoconstriction in single-dose
`nebulizer solutions of salbutamol, albeit at lower concen-
`trations than that found in ipratropium bromide (0.01% vs
`0.025% wt/vol, respectively).28
`
`Rhinitis medicamentosa
`BKC has been shown to potentiate rhinitis medica-
`mentosa in healthy volunteers treated with a deconges-
`tant nasal spray. Graf et al29 found an increase of mucos-
`al swelling after use of BKC-containing oxymetazoline 3
`times daily for 30 days. In a subsequent study the same
`investigators determined that the nasal swelling caused
`by BKC may last for 3 months.30
`
`Neutrophil dysfunction
`Human neutrophil function was compromised by expo-
`sure to BKC-preserved glucocorticosteroid nasal sprays
`
`such as flunisolide and beclomethasone.6 Several index
`values of neutrophil function, including actin polymeriza-
`tion, degranulation of azurophilic granules, and oxidative
`burst, were depressed in a time- and concentration-depen-
`dent manner. Flow cytometric analysis demonstrated neu-
`trophil disintegration at concentrations of BKC greater
`than 0.001% (wt/vol). In contrast, exposure of neutrophils
`to flunisolide or beclomethasone dipropionate in the
`absence of BKC did not affect neutrophil function.
`BKC has been demonstrated to affect leukocyte
`response to local inflammation. Hakansson et al8 found
`that human granulocyte migration was inhibited by BKC
`at a concentration as low as 0.00008% (wt/vol). In that
`study, BKC was found to be more deleterious than
`thimerosal at respective preservative concentrations in
`nasal drops (ie, BKC [0.02% wt/vol] and thimerosal
`[0.0024% wt/vol]).8
`
`LIMITATIONS OF STUDIES
`
`Discordant results between in vitro and in vivo studies
`may reflect the protective mechanisms of the respiratory
`mucosa against topically applied toxic substances,
`including BKC, found in vivo.6,26 The preservative may
`be diluted by nasal secretions covering the nasal mucosa
`or removed by absorption to the mucociliary layer.19
`Ainge et al23 postulate that proteins in the mucous blan-
`ket may inactivate BKC on the basis of the ability of pro-
`tein and organic material to rapidly inactivate quaternary
`ammonium compounds. These naturally occurring pal-
`liative events may vary from individual to individual
`depending on the volume and the physiochemical prop-
`erties of secretions.
`The pH of a BKC solution may be an important factor
`in accounting for disparate results of different in vitro
`studies. In this regard, van de Donk et al13 suggested that
`a modest decrease in the pH of BKC may markedly
`reduce ciliary movement of guinea pig trachea. More-
`over, a reduction of BKC solution pH from 7.4 to 6.0
`decreased the duration of ciliary movement from 1.33 to
`1 hour. Whether pH is indeed an important variable in
`BKC toxicity will require further study.
`The extent of BKC toxicity may vary from one site to
`another within the nasal bed. Areas directly challenged
`by the impact of the nasal spray, particularly the anterior
`part of the nasal septum, the tips of the inferior and mid-
`dle turbinate, and the anterior aspect of a polyp, would be
`exposed to high concentrations of preservative.20 This
`could lead to reduction in ciliary beat frequency and
`mucosal clearance in specific nasal loci.
`The failure to demonstrate deleterious effects of BKC
`in morphologic studies may also be due to biopsy site.22
`For example, Ainge et al23 limited their study to the
`examination of the inferior turbinate and were unable to
`demonstrate BKC toxicity. In contrast, Berg et al22 found
`BKC to be harmful in vivo when the total nasal mucosal
`lining was investigated histologically by serial frontal
`sections.
`The toxic effects of BKC may be more prevalent in
`
`Opiant Exhibit 2070
`Nalox-1 Pharmaceuticals, LLC v. Opiant Pharmaceuticals, Inc.
`IPR2019-00688
`Page 5
`
`

`

`44 Bernstein
`
`J ALLERGY CLIN IMMUNOL
`JANUARY 2000
`
`patients with rhinitis and sinusitis because mucociliary
`clearance is already impaired.20 This is of clinical signif-
`icance because recent studies suggest that ciliary beat
`frequency is the main factor affecting nasal mucociliary
`clearance.12 In fact, patients with atopic rhinitis, vaso-
`motor rhinitis, or an early state of chronic maxillary
`sinusitis display a doubling in the nasal mucociliary
`clearance time. Moreover, Graf and Hallen30 observed a
`potentiation of rhinitis medicamentosa in the presence of
`BKC. Further study of the acute and chronic adverse bio-
`logic effects induced by BKC in these patient popula-
`tions is warranted.
`
`FUTURE DIRECTIONS
`
`Although in vitro and in vivo data are somewhat con-
`flicting, the use of BKC as a preservative in nasal sprays
`may lead to adverse biologic effects directed at local
`nasal mucociliary transport. Currently, the only available
`nasal delivery systems in the United States that do not
`contain BKC are propellant-driven devices that are used
`chiefly for glucocorticosteroid nasal products.24 Because
`pharmacologically beneficial action has not been
`ascribed to BKC apart from its antibacterial effect, drug
`manufacturers should be encouraged to investigate other
`antibacterial agents that have minimal effects on
`mucociliary transport. Meanwhile, physicians have the
`option of recommending BKC-free formulations, partic-
`ularly in patients who have side effects of burning, irrita-
`tion, or dryness when using glucocorticosteroid nasal
`devices that contain BKC. A worthy technologic goal for
`the new millennium would be a universally adaptable
`nasal delivery system that would eschew the need for
`antibacterial additives, thereby eliminating potential
`adverse biologic effects on the nasal mucosa.
`
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`
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`20. Stafanger G. In v