Benzalkonium chloride and nasal mucociliary clearance:
`A randomized, placebo-controlled, crossover,
`double-blind trial
`
`Jose´ Aˆ ngelo Rizzo, M.D., De´cio Medeiros, M.D., Almerinda Rego Silva, M.D., and
`Emanuel Sarinho, Ph.D. (Brazil)
`
`ABSTRACT
`Background: Benzalkonium chloride (BKC) has been considered an innocuous preservative for prescription drugs.
`Methods: We performed a double-blind, placebo-controlled, randomized, crossover, single-center trial with a 3-week washout period in 43
`healthy volunteers comparing the effect of 3-week use of saline nasal spray containing BKC 0.01% to preservative-free saline t.i.d. on nasal
`mucociliary clearance rate. Evaluations were done at the beginning and the end of each period by ␥-scintigraphy with technetium99m-labeled
`strontium.
`Results: Nasal mucociliary clearance rate was significantly impaired by BKC with a difference of 1.23 mm䡠min⫺1 (p ⬍ 0.01) between
`periods.
`Conclusion: BKC in the concentration used in nasal preparations impaired mucociliary clearance in healthy individuals after 3 weeks of
`use. Presently, when preservative-free alternatives are available, BKC could be a risk without benefit.
`(Am J Rhinol 20, 243–247, 2006; doi: 10.2500/ajr.2006.20.2867)
`
`Among available antimicrobial preservatives, benzalko-
`
`nium chloride (BKC) was first introduced in 1935 as an
`antiseptic agent for clinical use and later was approved by the
`Federal Drug Administration, in 1982, as an innocuous ingre-
`dient for prescription drugs at concentrations up to 0.1%. It is
`the most commonly used agent to prevent bacterial contam-
`ination and to preserve pharmacologic activity in a wide
`range of prescription and over-the-counter products for a
`large array of indications—including several topical formula-
`tions for nasal use—with millions of units sold worldwide
`annually.1–3
`Although considered inert, there still is a large uncovered
`debate in the literature about potential harmful effects of BKC.
`Recently, BKC effects on nasal mucociliary clearance (NMC),
`mucosal histology, ciliotoxicity, and neutrophil function were
`reviewed with conflicting findings.2,3 Most studies evaluating
`the effect of BKC on NMC—one of the main nasal defense
`mechanisms—were done with methodologies that depend on
`subjective perception as the saccharine test, usually in solu-
`tions also containing topical steroids or oxymetazoline and in
`patients with atopic rhinitis, all of which can introduce serious
`bias on evaluation. The objective of this study was to inves-
`tigate the effect of BKC 0.01% saline solution on NMC rate in
`healthy subjects in a controlled clinical trial.
`
`From the Research Center in Allergy and Immunology, Clinical Hospital, Federal
`University of Pernambuco, Recife, Brazil
`JAR has been consultant for GSK in the last year, paid for lectures by GSK, MSD,
`Altana, Novartis, and Libbs. ES has been paid for lectures by Libbs. ARS has been paid
`for lectures by ScheringPlough do Brasil. JAR, DM, ARS, and ES have received
`research funds for clinical trial performance from Astra/Zeneca, Altana, Libbs, No-
`vartis, and ScheringPlough Intl. None of the authors have stock ownership or commer-
`cial royalties in any of these companies. This research was unrestrictedly funded by
`Libbs Farmaceutica do Brasil
`Address correspondence and reprint requests to Emanuel Sarinho, Ph.D., Av Par-
`namirim 327, apto 202 Parnamirim, Recife, 52.060-000 PE, Brazil
`E-mail address: emanuelsarinho@uol.com.br
`Copyright © 2006, OceanSide Publications, Inc., U.S.A.
`
`METHODS
`
`Study Design and Population
`This was a double-blind, placebo-controlled, randomized,
`crossover, single-center trial devised to investigate the effects
`of 3-week t.i.d. use of 0.9% saline spray with or without BKC
`0.01% (BKC free) on NMC rate. A 3-week washout interval
`was adopted between both periods. We planned our study in
`accordance with CONSORT statements.4 The Institutional Re-
`view Board of the Federal University of Pernambuco ap-
`proved the study and written informed consent was obtained
`from all participants.
`Healthy volunteers, 13–50 years of age, were recruited
`among relatives of children attending the Pediatrics Allergy
`Clinic at the University Clinical Hospital. Participants re-
`ceived no payment except transport and meal allowances.
`Inclusion and exclusion criteria are listed in Table 1.
`
`Interventions
`Subjects were screened according to selection criteria (Table
`1) and submitted to a basal NMC rate determination. Then,
`they were submitted to a sequence of two periods of blinded
`medication use of 3 weeks each (periods 1 and 3), with a 3
`weeks washout period of no medication use between them
`(period 2). At the beginning of periods 1 and 3 subjects
`received the solution containing atomizers and were in-
`structed to use 1 spray in each nostril t.i.d. NMC rate deter-
`minations were done at the end of periods 1, 2, and 3. Any
`complaints were questioned at each visit, especially those
`related to symptoms of upper airway infection (upper respi-
`ratory infection [URI]), such as fever, sore throat, cough,
`stuffy/runny nose, and malaise.
`BKC saline (Sorine Infantil; batch 0302304; Ache´ Laborato´-
`rios Farmaceˆuticos SA, Sa˜o Paulo, Brazil) and preservative-
`free (Salsep; batch 31023; Libbs Farmaceutica do Brasil, Sa˜o
`Paulo, Brazil) solutions were purchased from the market as
`commercial formulations, both approved by the Brazilian reg-
`
`American Journal of Rhinology
`
`243
`
`Opiant Exhibit 2049
`Nalox-1 Pharmaceuticals, LLC v. Opiant Pharmaceuticals, Inc.
`IPR2019-00694
`Page 1
`
`

`

`Table 1 Inclusion and exclusion
`Inclusion Criteria
`No respiratory symptoms
`Never had asthma (wheezing and shortness of breath
`associated with sputum) or rhinitis (recurrent
`sneezing and itching and watery discharge)
`No influenza symptoms in the past 60 days
`No topical nasal medicine use
`NMC ⬎ 6 mm䡠min⫺1
`
`No septum deviation occluding the nostril at anterior
`rhinoscopy
`Signed informed consent
`␤-HCG ⫽ Beta human chorionic gonadotrophin.
`
`ulatory agency (ANVISA) and conditioned in identical sealed
`atomizers labeled “A” or “B” according to randomization. No
`difference in the appearance, taste, viscosity, or smell could be
`detected between the preparations.
`
`Compliance
`To check for compliance, bottles were weighed before and
`after use. Subjects were not informed of this procedure. Ex-
`pected weight consumption was 7 g in each period based on
`the mean weight difference in bottles after 126 actuations of 5
`atomizers.
`
`Primary Outcome and Assessment
`The primary outcome was the difference between NMC
`rates after 3-week use t.i.d. of nasal spray with BKC or pre-
`servative-free saline solutions. Image acquisitions and analy-
`sis for NMC rate determinations were done through a
`StarCam Gamma Camera (General Electric, Milwaukee, WI)
`3.200 AC/T equipped with a general purpose, parallel holes,
`low energy collimator using a 128/128 pixels matrix. Radio-
`active solutions were prepared immediately before the exam
`by diluting 2.5 mCi of colloidal strontium labeled with
`tecnetium99m in BKC-free 0.9% saline, resulting in 0.12–0.15
`mCi of radioactivity per drop. The strontium (with a mean
`particle diameter of 0.03 ␮m) and technetium were purchased
`from Instituto de Pesquisas Energe´ticas e Nucleares, Sa˜o
`Paulo, Brazil.
`After explaining the procedure to the volunteer, a droplet
`of the radioactive solution was placed on the floor of the most
`unobstructed nostril through a pipette calibrated to deliver a
`0.05-mL drop, 1.5 cm from the mucocutaneous junction. If no
`nostril was more patent than the other, the right was chosen.
`One drop of solution also was placed at the tip of the nose to
`create a fixed reference mark for NMC rate calculations. Im-
`mediately after, the subject was seated with the chosen side of
`the face in contact with the vertically positioned collimator,
`with the head slightly bent forward, with a chin support to
`minimize movements, and was instructed not to move the
`head, not to talk, or not to sniff.
`Images were acquired in dynamic mode at 15-second inter-
`vals for 15 minutes, (total, 60 frames). At the end of acquisi-
`
`Exclusion Criteria
`Rhinosinusal complaints
`Important septum deviation at anterior rhinoscopy
`
`Divers
`Smokers or exsmokers ⬍5 yr
`Pregnant woman (fertile woman had ␤-HCG in
`urine)
`Refusal to sign informed consent
`
`tion, the NMC rate was calculated in millimeters per minute,
`measuring the length and time that elapsed between the
`starting point of the drop displacement and the point imme-
`diately before its fall into the pharynx. To avoid errors caused
`by minor head movements, the mark at the tip of the nose was
`used as the reference for drop displacement assessment. The
`calculations were performed by the same investigator and, to
`ascertain for accuracy, a comparison was made with a second
`NMC rate measurement done in 20 exams stored in the com-
`puter, randomly, and blindly chosen by a coinvestigator, be-
`fore breaking the randomization codes. An example of images
`chosen for analysis is depicted in Fig. 1.
`
`Sample Size
`The sample size was estimated based on a pilot study
`undertaken in nine volunteers, which showed a mean (⫾SD)
`difference of 1.1 (⫾1.58) mm䡠min⫺1 between measurements of
`NMC rate taken after two periods of 2 weeks each of nasal use
`of 0.01% BKC saline or BKC-free control saline, with an inter-
`vening 2-week washout period (Rizzo JA, unpublished data).
`Accepting an ␣- and ␤-errors of 0.05 and 0.1 (two-tailed),
`respectively, the sample size calculated to detect a difference
`on NMC of 1.0 mm䡠min⫺1 between periods was 33 individu-
`als for a paired clinical trial.5 A total of 43 participants were
`selected to compensate for dropouts or losses because of URI,
`a well-known NMC rate interference cause.6 URIs were de-
`fined based on common symptoms of cold.
`
`Figure 1. Example of NMC rate calculation. Displacement dis-
`tance ⫽ AC ⫺ AB; Time ⫽ final chosen image ⫺ initial chosen
`image; Rate ⫽ Distance/Time.
`
`244
`
`May–June 2006, Vol. 20, No. 3
`
`Opiant Exhibit 2049
`Nalox-1 Pharmaceuticals, LLC v. Opiant Pharmaceuticals, Inc.
`IPR2019-00694
`Page 2
`
`

`

`Randomization
`Randomization was accomplished using the restricted
`shuffle approach5 by a third party and the sequence was kept
`blind to the investigators. A pharmacist conditioned the so-
`lutions according to the randomization sequence in identical
`sealed atomizer bottles labeled “A” and “B,” which were
`enclosed in numbered boxes from 1 to 46. The randomization
`list and the atomizers were prepared at another institution
`and each box was intended for use by only one volunteer,
`bottle A first, followed by bottle B in entry sequence. Con-
`cealment was attained because it was not possible for subjects
`or investigators to distinguish any difference among the
`bottles.
`
`Statistical Analysis
`Paired t-test was performed on log-transformed data for
`comparison between NMC rates after solutions use. Unpaired
`t-test was used for comparison between groups. Log transfor-
`mation was needed to normalize NMC rate distribution. Pro-
`portions were compared by chi-squared or Fisher’s exact tests.
`Subjects who had URI symptoms during the study period
`were excluded from final analysis. Period and carryover ef-
`fects were evaluated according to Pocock.7
`
`RESULTS
`
`Volunteers Information
`Forty-three volunteers were randomized into the study.
`Demographics and distribution characteristics are depicted in
`Table 2. Twenty-one subjects were allocated to group A (to
`use preservative-free saline first) and 22 to group B (receiving
`BKC saline first).
`During the trial nine subjects had URI: four subjects had
`URI in the first period (one on preservative-free and three on
`BKC saline solutions), three subjects had URI during the
`washout period (all after BKC-free saline), and two subjects
`had URI in the second period (both on preservative-free sa-
`line). Of these, all but one subject—who refused final NMC
`evaluation—completed the planned observations. At the end,
`17 individuals in each group (our accrual goal) completed the
`study period without URIs. There was no association between
`solutions and respiratory infection (p ⬎ 0.05, Fisher’s exact
`test).
`
`Baseline and Postwashout NMC Rate Measurements
`The distributions of baseline and postwashout NMC rate
`measurements of the 34 subjects included in the analysis are
`
`Table 2 Baseline characteristics of participants
`No. of patients
`43
`30
`13–54
`18/25
`21
`22
`8.8 mm䡠min⫺1
`7.9, 9.5
`
`Age (yr)
`Median and limits
`Gender (M/F)
`BKC-free solution First (group A)
`Saline with BKC first (group B)
`Basal NMC
`Mean (CI 95%)
`
`depicted in the steam-and-leaf plot in Fig. 2. There was no
`difference (p ⬎ 0.05) between these periods, because there
`were no carryover effects. When subjects who had URI were
`included in this analysis, a statistical significant difference
`was observed between these two periods (8.7 mm䡠min⫺1 ver-
`sus 7.6 mm䡠min⫺1; p ⬍ 0.05), which demanded their exclusion
`from the final analysis.
`
`Effect of BKC on NMC Rate
`There was no difference between basal NMC rate com-
`pared with that after preservative-free solution period
`(mean ⫾ SD, respectively: 8.5 ⫾ 1.3 mm䡠min⫺1 and 8.5 ⫾ 1.4
`mm䡠min⫺1; p ⬎ 0.05). After BKC-containing saline, NMC
`mean rate was 6.9 ⫾ 1.3 mm䡠min⫺1, p ⬍ 0.01 compared to
`basal and BKC-free saline period (Fig. 3).
`
`Compliance Evaluation
`In the first period of nasal spray use, 2/34 volunteers used
`⬍50% of the expected dose (both on BKC saline) and 5/34
`used ⬎150% of prescribed dose (two on preservative-free
`solutions and three on BKC saline). In the subsequent cross-
`over period 2/34 subjects used ⬍50% (both on preservative-
`free saline) and 7/34 used ⬎150% of expected dose (four on
`preservative-free solution and three on BKC saline; Fig. 4). It
`is interesting to note that all individuals that used ⬎150% of
`medication in the first did so in the second period. In the last
`period two more subjects exceeded the recommended dose,
`one in each solution. There was no significant difference
`between groups in medication use (p ⬎ 0.05, Fisher’s exact
`test). Difference in NMC rate remained significant even ex-
`cluding these overusers from analysis (p ⬍ 0.05).
`
`NMC Rate Measurement Consistency
`The test–retest consistency of NMC rate determination was
`very high in the 20 repeated measurements by the investiga-
`
`Figure 2. Steam-and-leaf plot of log of NMC rates at basal (left) and
`after washout period (right). Numbers in parenthesis are antilogged
`values.
`
`American Journal of Rhinology
`
`245
`
`Opiant Exhibit 2049
`Nalox-1 Pharmaceuticals, LLC v. Opiant Pharmaceuticals, Inc.
`IPR2019-00694
`Page 3
`
`

`

`A variety of methods, end points, and different techniques
`were used in the few studies on adverse effects of BKC on
`nasal function, generating conflicting data and opinions.2,3
`The radioactive method we adopted is considered to provide
`the most physiological and reliable information about NMC
`in humans.8 In fact, Naclerio et al., using a similar method to
`compare NMC in allergic rhinitis patients on topical steroid
`solutions for 2 weeks, with or without BKC, also found a
`significant reduction on nasal clearance in the group using
`preservative-containing solutions.9
`A recent authoritative review of the literature10 discussing
`the adverse effects of BKC as a preservative in topical nasal
`preparations concluded that this conservant causes no signif-
`icant damage to the nasal mucosal, even with prolonged use,
`although based in an in vivo small amount of data and recog-
`nizing that the in vitro data suggest deleterious effects. Unfor-
`tunately, the great majority of those in vivo studies deserve a
`critical look.
`Since 1995, Graf et al. have shown, in a randomized double-
`blind parallel study in 20 healthy volunteers, that BKC added
`to oxymetazoline in nasal spray for 30 days accentuated the
`severity of rhinitis medicamentosa with a mean increase in
`rebound swelling and worse evening symptoms score.11 He
`also showed that 28-day use of BKC nasal spray without
`oxymetazoline also was capable of inducing mucosal swell-
`ing.12
`Lebe13 performed an experimental in vivo study to investi-
`gate symptom manifestations (sneezing and nasal rubbing)
`and histological changes induced by administration of BKC
`0.01% to the nasal mucosa of rats for 1 and 4 weeks. Symp-
`toms were more intense after the 6th day and both light and
`electron microscopy showed mucosal lesions that were more
`pronounced with prolonged administration.
`Recently, Riechelmann et al.14 assessed the ciliotoxicity of
`BKC in isolated human nasal epithelium from 15 donors.
`They also measured the effects of nasal 0.05% BKC saline (4 ⫻
`200 ␮L/day for 8 days) on saccharin transport time, inflam-
`matory cells populations, cytokine levels in nasal secretions,
`and nasal symptom scores in 16 healthy volunteers, in a
`randomized, double-blind crossover trial. BKC exposure
`showed ciliotoxicity (p ⬍ 0.0001) in vitro but, in vivo, BKC
`containing solution did not alter saccharin transport time (p ⬎
`0.8) and no proinflammatory effects were observed. The
`short-term BKC exposure could not be sufficient to reflect the
`observed histological ciliotoxic changes. In contrast, a well-
`designed human nasal mucosa in vitro study has established
`that steroid nasal sprays containing fluticasone or mometa-
`sone, both with BKC, caused slowing or paralysis of ciliary
`movements, depending on the concentration.15
`It is very important to emphasize that the statistically sig-
`nificant differences found in our research may not be clini-
`cally relevant. However, some studies indicate that BKC in
`nasal decongestant sprays affects the nasal mucosa even after
`short-term use (10 days) and sustained use of BKC alone can
`induce nasal mucosal swelling.16,17
`Nasal saline spray with BKC also was toxic to human
`neutrophils at concentrations far lower than those found in
`commercially available formulations.18 Bernstein,2 in a less
`recent review article, concluded that both animal and human
`in vitro data suggest that BKC promotes ciliostasis and reduc-
`tion in NMC that may be partially masked by absorption and
`
`Figure 3. NMC rate (mean ⫾ SD) at basal evaluation (8.5 ⫾ 1.3
`mm䡠min⫺1) and after BKC-free solution (8.5 ⫾ 1.4 mm䡠min⫺1) or
`BKC containing solution periods (6.9 ⫾ 1.3 mm䡠min⫺1).
`
`Figure 4. Use of Solutions by volunteers measured as difference in
`weight of bottles before and after use period. (A) First period and (B)
`second period. Expected differences, 7 mg (full line). Fifty percent
`use (3.5 mg) and 150% use (10.5 mg) are represented as dotted
`lines. There was a value of p ⬎ 0.05 between solution weights at
`periods A and B.
`
`tor, with a mean difference of 0.04 mm䡠min⫺1 (95% CI, ⫺0.44,
`0.52).
`
`DISCUSSION
`This randomized, placebo-controlled, crossover, double-
`blind trial in healthy volunteers showed that 1 puff of 0.01%
`BKC saline in each nostril t.i.d. for 3 weeks impaired the NMC
`rate. The huge difference in NMC rates observed before and
`after URIs in the nine subjects who had the infection during
`the trial (6.1 mm䡠min⫺1) interfered in NMC after washout,
`leading to a period effect and demanded their exclusion from
`final analysis, although their inclusion in an intention-to-treat
`basis did not change the results.
`
`246
`
`May–June 2006, Vol. 20, No. 3
`
`Opiant Exhibit 2049
`Nalox-1 Pharmaceuticals, LLC v. Opiant Pharmaceuticals, Inc.
`IPR2019-00694
`Page 4
`
`

`

`dilution effects because of respiratory mucus. His recommen-
`dation is that the use of BKC-free glucocorticosteroid formu-
`lations should be considered, particularly in patients who
`complain of nasal burning, dryness, or irritation.
`Possible confounding factors that may account for the dis-
`cordances are inconsistent methods of study, poor compli-
`ance, insufficient length of exposure, and variation in solution
`concentrations. We tried to minimize confounding factors by
`adopting a reliable NMC rate determination method, check-
`ing consistency of measurements and compliance. In addition,
`concentration, doses, and length of use of the study solutions
`were planned to replicate real-world prescriptions.
`Nasal medications containing BKC are used worldwide
`and topical nasal steroids containing this preservative are
`prescribed for months, sometimes for years. BKC-containing
`saline solutions are commonly prescribed as adjuvants in the
`treatment of rhinitis and sinusitis for nasal irrigation, often
`many times a day and sometimes for long periods. In Brazil,
`an epidemiological study showed that by the 3rd month of life
`20% of all infants had used some medicine for 1 month or
`longer, among which the most frequently prescribed was
`0.9% nasal saline containing BKC.19
`In conclusion, our work shows that 3 weeks use of saline
`nasal spray containing BKC as preservative slows down NMC
`rate. The clinical significance of these findings remains to be
`established but the potential risk of short- and long-term use
`of BKC-containing solutions needs considerations, especially
`when we have enough device technologies that make it pos-
`sible to deliver nasal medicines without preservatives and
`represent a more reasonable alternative.3 We agree with Verse
`et al.’s20 opinion that, nowadays, when preservative-free al-
`ternatives are available, preserved nasal sprays are obsolete.
`
`REFERENCES
`1. Grosselin RE, Smith RP, Hodge H (Eds.) Benzalkonium chlo-
`ride. In Clinical Toxicology of Commercial Products, 5th ed.
`Baltimore: Williams & Wilkins, 63–66, 1984.
`2. Bernstein IL. Is the use of benzalkonium chloride as a pre-
`servative for nasal formulations a safety concern? A caution-
`ary note based on compromised mucociliary transport. J
`Allergy Clin Immunol 106:595–596, 2000.
`3. Graf P. Benzalconium chloride as a preservative in nasal
`solutions: Re-examining the data. Respir Med 95:728–733,
`2001.
`4. Moher D, Schulz KF, and Altman DG. The CONSORT state-
`ment: Revised recommendations for improving the quality
`of reports of parallel-group randomises trials. Lancet 357:
`1191–1194, 2001.
`
`5. Friedman LM, Furberg CD, and DeMets DL (Eds.). Sample
`size. In Clinical Trials, 3rd ed. St. Louis, MO: Mosby-Year
`Book, Inc., 94–129, 1996.
`6. Doyle WJ, Skoner DP, Hayden F, et al. Nasal and otologic
`effects of experimental influenza A virus infection. Ann Otol
`Rhinol Laryngol 103:59–69, 1994.
`7. Pocock SJ (Ed). Crossover trials. In Clinical Trials. A Practi-
`cal Approach. Chichester, England: Wiley & Sons, Ltd., 110–
`122, 1983.
`8. Lale AM, Mason JDT, and Jones NS. Mucociliary transport
`and its assessment: A review. Clin Otolaryngol 23:388–396,
`1998.
`9. Naclerio RM, Baroody FM, Bidani N, et al. A comparison of
`nasal clearance after treatment of perennial allergic rhinitis
`with budesonide and mometasone. Otolaryngol Head Neck
`Surg 128:220–227, 2003.
`10. Marple B, Roland P, and Benninger M. Safety Review of
`benzalkonium chloride used as preservative in intranasal
`solutions: An overview of conflicting data and opinions.
`Otolaryngol Head Neck Surg 130:131–141, 2004.
`11. Graf P, Hallen H, and Juto JE. Benzalkonium chloride in a
`decongestant nasal spray aggravates rhinitis medicamentosa
`in healthy volunteers. Clin Exp Allergy 25:395–400, 1995.
`12. Graf P, and Hallen H. Effect on the nasal mucosa of long-
`term treatment with oxymetazoline, benzalkonium chloride,
`and placebo nasal sprays. Laryngoscope 106:605–609, 1996.
`13. Lebe E, Baka M, Yavasoglu A, et al. Effects of preservatives
`in nasal formulations on the mucosal integrity: An electron
`microscopic study. Pharmacology 72:113–120, 2004.
`14. Riechelmann H, Deutschle T, Stuhlmiller A, et al. Nasal
`toxicity of benzalkonium chloride. Am J Rhinol 18:291–299,
`2004.
`15. Hofmann T, Gugatschga M, Koidl B, et al. Influence of
`preservatives and topical steroids on ciliary beat frequency
`in vitro. Arch Otolaryngol Head Neck Surg 130:440–445,
`2004.
`16. Graf P, Enerdal J, and Hallen H. Ten days’ use of oxymeta-
`zoline nasal spray with or without benzalkonium chloride in
`patients with vasomotor rhinitis. Arch Otolaryngol Head
`Neck Surg 125:1128–1132, 1999.
`17. Graf P. Adverse effects of benzalkonium chloride on the
`nasal mucosa: Allergic rhinitis and rhinitis medicamentosa.
`Clin Ther 21:1749–1755, 1999.
`18. Boston M, Dobratz EJ, Buescher ES, et al. Effects of nasal
`saline spray on human neutrophils. Arch Otolaryngol Head
`Neck Surg 129:660–664, 2003.
`19. Weiderpass E, Beria JU, Barros FC, et al. Epidemiology of
`drug use during the first three months of life in a urban area
`of southern Brazil. Rev Saude Publica 32:335–344, 1998.
`20. Verse T, Sikora C, Rudolph P, et al. The tolerability of nasal
`drugs with special regard to preservatives and physico-
`chemical parameter. Laryngorhinootologie
`82:782–789,
`e
`2003.
`
`American Journal of Rhinology
`
`247
`
`Opiant Exhibit 2049
`Nalox-1 Pharmaceuticals, LLC v. Opiant Pharmaceuticals, Inc.
`IPR2019-00694
`Page 5
`
`