Original Paper
`
` ORL 2012;74:149–153
` DOI: 10.1159/000337830
`
` Received: December 20, 2011
` Accepted after revision: February 22, 2012
` Published online: April 13, 2012
`
` Effects of Benzalkonium Chloride and
`Potassium Sorbate on Airway Ciliary
`Activity
`
` Chengshuo Wang a Qiu Deng b Demin Han a, b Luo Zhang a, b
`
` a Department of Otolaryngology, Head and Neck Surgery, Beijing TongRen Hospital, Capital Medical University,
`and b Key Laboratory of Otolaryngology, Head and Neck Surgery (Ministry of Education), Beijing Institute of
`Otolaryngology, Beijing , China
`
` Key Words
` Preservatives ⴢ Ciliary beat frequency ⴢ Benzalkonium
`chloride ⴢ Potassium sorbate
`
`CBF after 10 min of exposure, respectively. Conclusions: PS
`could be considered as a safer and more promising preserva-
`tive than BKC for use in topical formulations.
` Copyright © 2012 S. Karger AG, Basel
`
` Abstract
` Objectives: Preservatives are indispensable components of
`aqueous multidose topical formulations. The purpose of our
`study was to investigate the effects of two representative
`preservatives, benzalkonium chloride (BKC) and potassium
`sorbate (PS), on rabbit tracheal ciliary beat frequency (CBF).
` Methods: Rabbit tracheal ciliated cell culture was estab-
`lished and CBF was determined using high-speed digital im-
`aging methods. The effects of preservatives at different con-
`centrations on CBF were observed over a 10-min exposure
`period. Results: BKC induced inhibition of CBF in a concen-
`tration-dependent manner. Ciliary beating was stopped by
`0.01% BKC after 5 min of exposure. A low concentration of PS
`(0.12%) only resulted in a mild decrease in CBF during a 10-
`min exposure. The CBF decreased by 13.0% from baseline
`after 10 min. However, there was no statistically significant
`difference compared with the corresponding control condi-
`tion. Application of 0.24, 0.48 and 0.96% of PS to rabbit tra-
`cheal cells resulted in an increase in CBF, with an increase of
`105 8 9.8, 107.6 8 4.0, and 117.1 8 9.5% relative to baseline
`
` Introduction
`
` The administration of drugs via the upper airway (na-
`sal) or lower airway (tracheal and bronchial) has become
`a common method of medication in recent years. In par-
`ticular, nasal sprays or drops have been widely used for
`respiratory tract disorders of the nasal cavity and para-
`nasal sinuses in the field of otorhinolaryngology. A pre-
`requisite of nasally applied preparations is that drugs and
`additives in the dosage form do not interfere with normal
`nasal functioning, such as the nasal mucociliary clear-
`ance system [1] . Mucociliary clearance is one of the most
`important local defense mechanisms of the respiratory
`tract. The coordinated beating of cilia plays an essential
`role in efficient mucociliary clearance, and ciliary beat
`frequency (CBF) is one of the basic functional ciliary pa-
`
` D.H. and L.Z. contributed equally to the study.
`
`Fax +41 61 306 12 34
`E-Mail karger@karger.ch
`www.karger.com
`
` © 2012 S. Karger AG, Basel
`0301–1569/12/0743–0149$38.00/0
`
` Accessible online at:
`www.karger.com/orl
`
` Luo Zhang, MD
` Beijing Institute of Otolaryngology
` No. 17, Hou Gou Hu Tong, Dong Cheng District
` Beijing, 100005 (China)
` Tel. +86 10 6514 1136, E-Mail dr.luozhang   @   gmail.com
`
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`

`rameters [2] . Hence, it is important to investigate the in-
`fluence of active drugs and additives in the dosage form
`on CBF to evaluate the safety of nasally administered
`drugs and various additives such as preservatives [1] .
` Preservatives are indispensable components of tradi-
`tional multidose nasal formulations due to the necessity
`for repeated administration and their aqueous nature,
`which is susceptible to microorganism infestation. Ben-
`zalkonium chloride (BKC) is a quaternary ammonium
`compound, which is by far the most commonly used pre-
`servative for the prevention of bacterial contamination in
`nasal sprays or drops. Although it has been recognized
`that excipients in nasal formulations should be harmless
`to nasal tissues, several studies have demonstrated the im-
`pairment of mucociliary clearance by BKC [3–5] . Potassi-
`um sorbate (PS) is a white crystalline powder which is used
`in foods, cosmetics and drug preparations to inhibit mold,
`yeast and bacterial growth. Recent research data suggest
`that PS may be a safer preservative for nasal ciliated epi-
`thelium [6, 7] . The purpose of the present study was to
`determine the effects of BKC and PS on CBF in rabbit tra-
`cheal mucosa cultures and to compare their ciliotoxicities.
`
` Materials and Methods
`
` Chemicals and Materials
` Dulbecco’s modified Eagle medium (DMEM) and sterile
`Hanks’ balanced salt solution (sHBSS) were purchased from Sig-
`ma Co. Ltd. 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid
`and flavin mononucleotide were purchased from Merck. Strepto-
`mycin sulfate was purchased from Amresco, penicillin G sodium
`salt and amphotericin B solubilized from Sigma. BKC and PS were
`purchased from Sigma.
`
` Tissue Isolation and Culture
` The establishment of rabbit tracheal epithelium primary cul-
`tures has been described in detail previously [8] . Briefly, tracheas
`were removed aseptically from 2-month-old New Zealand rab-
`bits. The tracheal epithelium was separated from the underlying
`cartilage and cut into 1-mm 2 explants. As needed, explants were
`placed onto slides precoated with a thin film of rat tail collagen,
`which were placed into four-well dishes in advance (one or two
`explants per slide). Petri dishes were then added to a minimal
`amount of DMEM/NaHCO 3 culture medium supplemented with
`10% fetal calf serum and antibiotics (penicillin, 100 U/ml; strep-
`tomycin, 100 U/ml, and amphotericin B, 0.25 ␮ g/ml). After a few
`minutes, explant adherence had occurred and 0.5 ml culture me-
`dium was added to each culture dish. Cultures were incubated in
`humidified 95% air/5% CO 2 at 37   °   C, and the culture medium was
`changed every 2 days.
`
` Measurement of CBF
` Six- to eight-day tissue cultures were used for measurements
`of CBF. Fields of view with well-grown rabbit tracheal cilia which
`
`swung regularly were selected using an inverted microscope
`(Olympus IX 71, Tokyo, Japan) at a magnification of ! 400. The
`images of cilia were captured by a high-speed digital camera cap-
`ture system (PULNiX High-Speed Digital) at a frame rate of 240
`frames per second with a sampling interval of 3 s and results were
`transmitted to a computer workstation. A region of interest was
`selected and analyzed with IPLab4.0 software. CBF was calcu-
`lated by determination of fluctuations in light intensity [8, 9] . All
`experiments were performed at a constant temperature of 25 8
`1   °   C.
` The effect of preservatives on CBF was determined over a 10-
`min period of exposure. The four-well culture plates were re-
`moved from the incubator at least 1 h before the start of the ex-
`periment in order to allow the medium to adapt to ambient tem-
`perature. The cell culture medium was replaced by 1 ml of sHBSS
`and the culture left to stabilize for at least 10 min, after which CBF
`was recorded as baseline values of CBF (prior to exposure). The
`sHBSS was then replaced by 1 ml of a solution of the preservative
`to be tested and CBF was determined at 1, 2, 3, 4, 5, 6, 7, 8, 9 and
`10 min after exposure. The test concentrations were 0.005, 0.0075
`and 0.01% for BKC and 0.12, 0.24, 0.48 and 0.96% for PS. All con-
`centrations of preservatives were prepared in sHBSS (pH 7.4).
`
` Data Presentation and Statistical Analysis
` All data were expressed as percentages relative to the baseline
`values, which were considered as 100% and presented as mean
`percentage 8 SD. The differences of data between and within
`groups were statistically analyzed by analysis of variance followed
`by Student’s t test using Microsoft Excel 2002. A p value ! 0.05 was
`considered as significant.
`
` Results
`
` Effect of BKC on CBF
` Figure 1 shows the effects of BKC (0.0, 0.005, 0.0075
`and 0.01%) on the CBF of rabbit tracheal cilia. The abso-
`lute value of CBF in test conditions ranged from ⬃ 8 to
` ⬃ 14 Hz and the value of controls remained constant over
`the 10 min of the experiments. The CBF was significantly
`increased by up to 42.2% at 3 min after exposure to 0.005%
`BKC, which was statistically significant compared with
`the corresponding control condition (p ! 0.01). However,
`CBF gradually decreased and the difference between
`0.005% BKC and the control group was reduced to 13.4%
`at 10 min after exposure (p ! 0.05). At a concentration of
`0.0075% BKC, CBF dramatically decreased over time.
`Mean CBF decreased by up to 90.3% of the baseline level
`after 10 min of exposure, in which 76.9% of cilia were ob-
`served to be in stasis. At a concentration of 0.01% BKC,
`there were 2 out of 13 ciliated cells (15.4%) in ciliostasis
`after a 3-min exposure period, and all ciliary beat activity
`had completely ceased at 6 min after exposure. Overall,
`BKC demonstrated significant concentration-dependent
`inhibitory effects on CBF ( table 1 ).
`
`150
`
`ORL 2012;74:149–153
`
` Wang   /Deng   /Han   /Zhang
`
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`

`0
`
`1
`
`2
`
`3
`
`0.00% (n = 14)
`0.12% (n = 10)
`0.24% (n = 11)
`
`4
`
`5
`Time (min)
`
`6
`
`0.48% (n = 10)
`0.96% (n = 14)
`
`7
`
`8
`
`9
`
`10
`
`140
`
`120
`
`100
`
`80
`
`60
`
`40
`
`20
`
`CBF (% baseline)
`
`sHBSS
`0.005% BKC
`
`0.0075% BKC
`0.01% BKC
`
`200.0
`
`150.0
`
`100.0
`
`50.0
`
`CBF (% baseline)
`
`0
`
`0
`
`1
`
`2
`
`3
`
`4
`
`6
`5
`Time (min)
`
`7
`
`8
`
`9
`
`10
`
` Fig. 1. Effect of BKC at different concentrations on the CBF of rab-
`bit tracheal cilia. CBF values were calculated as percentages of
`CBF values at baseline (immediately before exposure). The expo-
`sure period was 10 min. BKC showed strongly concentration-de-
`pendent inhibitory effects on the CBF. In the 0.01% group, muco-
`ciliary activities were completely inhibited by 0.01% BKC by 5 min
`after exposure.
`
` Fig. 2. Effect of PS at different concentrations on the CBF of rabbit
`tracheal cilia. CBF values were calculated as percentages of CBF
`values at baseline (immediately before exposure). The exposure
`period was 10 min. A trend of increase in CBF as a function of
`concentration was observed. There were significant differences
`compared with the corresponding control condition at 10 min
`after exposure at concentrations of 0.24, 0.48 and 0.96% (p ! 0.01).
`
`Table 1. Changes in CBF measurements for BKC at different concentrations tested over 10 min in rabbit trachea in vitro
`
`Concentration
`
`C BF, %
`
`0 min
`
`1 min
`
`2 min
`
`3 min
`
`4 min
`
`5 min
`
`6 min
`
`7 min
`
`8 min
`
`9 min
`
`10 min
`
`100810.2 110.9815.8 98.1813.9 96.9811.4 95.3812.1 95.6812.6 95.086.8
`
`94.686.6
`
`sHBSS (n = 14)
`BKC
`0.005% (n = 13) 100810.5 131.5816.0 136.6818.3 137.5818.0*137.2818.9*133.5820.9 129.5823.9 123.7828.3 116.5832.2 111.8835.0 105.8836.1
`0.0075% (n = 13) 10089.3
`139.8816.9 112.9835.9 79.2844.0 54.3839.3 42.6839.3 34.3834.6 24.7830.1 18.9826.3 11.4821.9
`9.3817.9
`10088.7
`90.6832.1 51.5830.4 23.5830.5
`7.4818.3
`0.080.0
`0.01% (n = 12)
`–
`–
`–
`–
`–
`
`95.187.3
`
`92.987.4
`
`91.686.8
`
`Al l data are expressed as percentages relative to baseline values (immediately before exposure, i.e. 0 min) which were considered as 100% and are presented
`as mean percentage 8 SD. * p < 0.01, compared with the sHBSS group (control).
`
`Table 2. Changes in CBF measurements for PS at different concentrations tested over 10 min in rabbit trachea in vitro
`
`Concentration
`
`C BF, %
`
`sHBSS (n = 14)
`PS
`0.12% (n = 10)
`0.24% (n = 11)
`0.48% (n = 10)
`0.96% (n = 14)
`
`0 min
`
`1 min
`
`2 min
`
`3 min
`
`4 min
`
`5 min
`
`6 min
`
`7 min
`
`8 min
`
`9 min
`
`10 min
`
`100810.2 110.9815.8
`
`98.1813.9
`
`96.9811.4
`
`95.3812.1
`
`95.6812.6
`
`95.086.8
`
`94.686.6
`
`95.187.3
`
`92.987.4
`
`91.686.8
`
`10089.5
`10087.6
`10087.5
`10088.3
`
`85.5811.9
`87.5810.9
`89.888.1
`90.2819.9
`96.3817.2
`88.1815.1
`100.3813.7 100.3815.5 103.9811.3 104.9811.3 104.7811.1 103.3814.5
`113.486.4
`108.783.4
`110.285.9
`106.988.0
`108.686.6
`107.286.6
`122.188.2
`117.187.6
`117.387.6
`117.587.9
`117.288.6
`117.088.9
`
`83.6815.7
`10589.1
`107.085.7
`116.589.0
`
`86.5810.8
`106.389.8
`106.486.2
`116.589.6
`
`87.0811.1
`83.6812.4
`104.7811.2 105.589.8*
`106.985.1
`107.684.0*
`116.689.6
`117.189.5*
`
`All data are expressed as percentages relative to baseline values (immediately before exposure, i.e. 0 min) which were considered as 100% and are presented
`as mean percentage 8 SD. * p < 0.01, compared with the sHBSS group (control).
`
` Effects of Preservatives on Rabbit
`Tracheal Ciliary Movement
`
`ORL 2012;74:149–153
`
`151
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`well as the difference between in vitro and in vivo condi-
`tions [13, 14] . Particularly, there was a lack of a mucus
`layer in vitro, which protects the ciliated epithelium of the
`nasal cavity. It is worth noting that most results showing
`no deleterious effects on CBF were observed in in vivo
`studies. Given the healthy human nose has about 0.4 ml
`mucus [15] and generally spray dose volumes are of the
`order of 0.1 ml in clinical practice, a fivefold dilution of
`nasal preparations may, in fact, be more realistic when
`considering ciliotoxic potential. Thus, in establishing the
`actual toxicity of preservatives, measuring CBF in vitro
`is probably too sensitive an approach. In our study, CBF
`demonstrated extreme sensitivity to changes in the con-
`centration of BKC, as shown by changes from a strong
`inhibition of mucociliary activity by 0.01% BKC to a slight
`and positive stimulation by 0.005%, which was only half
`the original level. Given that the most commonly used
`concentration of BKC as a preservative in clinical practice
`is 0.01%, the actual concentration of BKC in the nasal cav-
`ity after administration of nasal drops or sprays is ex-
`pected to be below 0.005% due to dilution. This could
`therefore explain the fact that the deleterious effects on
`nasal ciliated epithelium in vitro are not reflected in the
`findings in vivo or in the clinic.
` On the other hand, PS concentrations in the range of
`0.12–0.96% showed no significant effects on CBF in this
`study. As discussed above, although in vitro experiments
`could not precisely duplicate in vivo conditions due to the
`lack of a protective mucus layer, in vitro experiments on
`CBF are a very useful tool for studying the ciliotoxicity of
`various compounds due to their controllable properties
`without other interfering factors such as stress, hormone
`secretion or inflammatory mediators. If there is an effect
`on CBF observed in vitro under standardized conditions
`and the exclusion of cofactors, it can be assumed that
`such an effect does exist in vivo. As our study shows, PS
`was a safer preservative than BKC on CBF at the com-
`monly used concentrations (0.01% for BKC and 0.12% for
`PS, fig. 3 ). There was only a mild increase in CBF even
`when the concentration of PS tested was up to 0.96%
`(about 5 times the commonly used concentration).
` In conclusion, the potential ciliotoxicity of BKC at the
`concentration commonly used in nasal formulations
`(0.01%) was proven. However, a slight, positive effect on
`CBF was observed for BKC when the concentration was
`diluted to half of the original value, i.e. 0.005%, implying
`that BKC may be a cilia-friendly preservative in the hu-
`man nasal cavity. By contrast, PS showed no effect on
`mucociliary activity and is thus considered as a safer and
`more promising preservative.
`
`sHBSS (n = 14)
`0.12% PS (n = 10)
`0.01% BKC (n = 12)
`
`140
`
`120
`
`100
`
`80
`
`60
`
`40
`
`20
`
`CBF (% baseline)
`
`0
`
`0
`
`2
`
`4
`Time (min)
`
`6
`
`8
`
`10
`
` Fig. 3. Comparison of effects of BKC and PS at the concentration
`used in nasal formulations (0.01 and 0.12%, respectively) on CBF.
`As shown in the graph, mucociliary activities were completely
`inhibited by 0.01% BKC at 5 min, while there was no notable effect
`of PS at 0.12% during 10 min of exposure compared with the cor-
`responding control condition.
`
` Effect of PS on CBF
` Generally, there was no notable effect of PS on CBF. As
`shown in figure 2 , a trend of CBF increase with PS con-
`centration increase from 0.12 to 0.96% was observed. A
`low concentration of PS (0.12%) only resulted in a mild
`decrease in CBF during the 10 min of exposure. The CBF
`decreased by 13.0% from baseline after 10 min, although
`this was not statistically significant compared with the
`corresponding control condition. Application of 0.24,
`0.48 and 0.96% PS to rabbit tracheal cilia cells resulted in
`an increase in CBF, with increases of 105 8 9.8, 107.6 8
`4.0, and 117.1 8 9.5% relative to baseline CBF after 10
`min of exposure, respectively (all p ! 0.01 compared with
`the corresponding control condition, table 2 ).
`
` Discussion
`
` BKC is a preservative widely used in topical multidose
`formulations to prevent contamination. Its effect on nasal
`mucociliary clearance has not been clearly established.
`Some studies, including both in vitro and in vivo studies
` [3–5] , have suggested that BKC does produce adverse ef-
`fects on mucociliary activity. Other data, however, sug-
`gest BKC lacks deleterious effects [10–13] . The discrep-
`ancy in these results, particularly between in vitro and in
`vivo findings, could be explained by the possibility of
`problems in the experimental design and methodology as
`
`152
`
`ORL 2012;74:149–153
`
` Wang   /Deng   /Han   /Zhang
`
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`

` Acknowledgments
`
` Disclosure Statement
`
` This work was supported by grants from the National Science
`Fund for Distinguished Young Scholars (81025007), National
`Natural Science Foundation of China (30973282), Beijing Natural
`Science Foundation (7102030) and the Special Fund of Sanitation
`Elite Reconstruction of Beijing (2009-2-007) to L.Z.
`
` The authors declare that they have no conflict of interest.
`
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` Effects of Preservatives on Rabbit
`Tracheal Ciliary Movement
`
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