ORIGINAL ARTICLE
`Influence of Preservatives and Topical Steroids
`on Ciliary Beat Frequency In Vitro
`Thiemo Hofmann, MD; Markus Gugatschga, Cand Med; Bemd Koidl, MD; Gerald Wolf, MD
`
`Objective: To measure the influence of topical ste­
`roids and the preservative potassium sorbate on the cili­
`ary beat frequency (CBF) of human nasal mucosa in vitro.
`
`Design: In vitro study of cultured ciliated cells of hu­
`man nasal mucosa.
`
`Methods: Human nasal mucosa was removed endo-
`scopically and cultured for 10 days. Cell cultures with
`ciliated cells grown on an object slide were exposed
`to benzalkonium chloride and topical steroids in an
`exposure chamber. The CBF was measured with a pho­
`tometer.
`
`Results: The preservative potassium sorbate did not in­
`fluence CBF in different concentrations. The glucocor­
`ticoid budesonide spray containing potassium sorbate did
`
`not affect CBF at 10% dilution and showed moderate re­
`versible decrease of CBF at 50% dilution. The glucocor­
`ticoid sprays fluticasone propionate and mometasone fu-
`orate containing the preservative benzalkonium chloride
`caused a reversible decrease of CBF at 10% dilution and
`a complete irreversible standstill at 50% dilution.
`
`Conclusions: In vitro, tbe steroid sprays containing flu­
`ticasone or mometasone, both with benzalkonium chlo­
`ride, caused slowing or standstill of CBF depending on
`the concentration. The isolated preservative potassium
`sorbate and the budesonide nasal spray containing this
`preservative did not have negative influence on CBF in
`vitro. Potassium sorbate can therefore be considered harm­
`less to the motility of ciliated cells.
`
`Arch Otolaryngol Head Neck Surg. 2004; 130:440-445
`
`Mucociliary clear-
`ance of the nose and
`paranasal sinuses is
`based on normal ana­
`tomic structures, physi­
`ologic composition of mucus, and healthy
`ciliated cells with coordinated and con­
`stant ciliary beat frequency (CBF). Messer-
`klinger''^ identified genetically deter­
`mined pathways transporting the mucus
`from the paranasal sinuses toward the choa-
`nae and the epipharynx. Ciliary function
`can be influenced by temperature,^ pH,"* os­
`motic pressure,^ infections, genetic fac­
`tors, and iatrogenical factors after surgery.
`In recent years, studies have shown alter­
`ations of ciliated cells associated with topi­
`cal medications and preservatives such as
`benzalkonium chloride.*'* We were inter­
`ested in the possible influence of topical glu­
`cocorticoids and the preservative potas­
`sium sorbate on CBF, which has not
`heretofore been investigated in vitro.
`
`METHODS
`During routine endonasal endoscopic sinus sur­
`gery for chronic sinusitis or nasal polyposis,
`
`mucosa was harvested from the uncinate pro­
`cess or middle turbinate if there was no sign
`of polyps, mucosal edema, or pathologic mu­
`cus. No patient had previous endonasal sur­
`gery, The samples were immediately placed in
`isotonic sodium chloride solution (normal sii-
`line) and transported to the laboratory. The tis­
`sue sample of each patient was sufficient for 8
`to 10 cell cultures, which were kept in nutri­
`tive medium (M199; Sigma, St Louis, Mo) with
`penicillin and streptomycin and incubated in
`an atmosphere of 5% carbon dioxide, 95% air,
`and 100% humidity.
`After 10 days, cells were spread on an ob­
`ject slide to a field size up to 4 X 8 mm. On av­
`erage, 3 of 10 cell cultures contained ciliated
`cells. For visualization, we used a Zeiss Axi-
`omat reversed microscope (Carl Zeiss, Oberko-
`chen, Germany). The object slide with the cul­
`tivated cells fixed on the surface was placed
`in a 30-mm-diameter waterproof chamber on
`the microscope (Figure 1). With a special
`minipump it was possible to rinse the cell cul­
`ture with fluid and maintain a constant fluid
`level of 1.5 to 2 mm in the exposure chamber.
`With this system, the fluid surrounding the cell
`culture could be changed without drying, and
`therefore damage to the cilia was prevented.
`Thus it was possible to rinse the cell culture
`with either neutral Tyrode solution (137mM
`sodium chloride, 2.7mM potassium chloride.
`
`From the Departments of
`Otorhinolaryngology
`(Drs Hofmann, Gugatschga,
`and Wolf) and Physics
`(Dr Koidl), University Medical
`School of Graz, Graz, Austria.
`The authors have no relevant
`financial interest in this article.
`
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`

`Table 2. Preservative Concentrations*
`
`Nasal Spray
`Test Series 1
`Preservative
`Potassium sorbate
`1.2 (0.12)
`0.6 (0.06)
`Benzalkonium chloride
`0.2 (0.02)
`0.1 (0.01)
`♦Data are milligrams per milliliter (percentage).
`
`Test Series 2
`0.12 (0.012)
`0.05 (0.005)
`
`Table 3. Nasal Spray Dilutions*
`
`Nasal Spray
`Test Series 1
`Test Series 2
`1:9 (10)
`1:1 (50)
`Mometasone
`1:9 (10)
`1:1 (50)
`Fluticasone
`1:9 (10)
`1:1 (50)
`Budesonide
`‘Data are ratio of nasal spray to Tyrode solution (percentage dilution)
`
`According to Deitmer,“ who summarized the reports of
`various authors, the average mucociliary transport time from
`the anterior part of the inferior turbinate to the pharynx is be­
`tween 4 and 20 minutes measured with the saccharine test. To
`simulate as closely as possible in vivo conditions in this in vitro
`experiment, we exposed the cell cultures to test substances for
`a time period of 7 minutes because during this time in vivo, a
`large amount of nasal spray would have been transported to
`the epipharynx. Each cell culture was used for 1 measurement
`only. Each substance was tested at least 5 times.
`
`The preservative potassium sorbate did not influence CBF.
`No alteration of CBF was found at either 0.6-mg/mL or
`0.12-mg/mL concentration (Figure 2). The preserva­
`tive benzalkonium chloride showed a decrease in CBF
`at a concentration of 0,05 mg/mL and irreversible stand­
`still at 0.1 mg/mL (Figure 3).
`Budesonide nasal spray containing potassium sor­
`bate showed no alteration of CBF at 10% dilution and
`minor reversible slowing of CBF at 50% percent dilu­
`tion (Figure 4). The topical glucocorticoid mometa-
`sone fuorate spray containing benzalkonium chloride as
`preservative showed a slight decrease in CBF at 10% di­
`lution and irreversible standstill during rinsing with 50%
`dilution (Figure 5)
`These results are comparable with previously pub­
`lished experiments® on the topical steroid fluticasone pro­
`pionate spray, also containing benzalkonium chloride,
`which caused reversible decrease of CBF after exposure
`at 10% percent dilution and complete standstill of cilia
`after rinsing with a 50% dilution (Figure 6).
`
`COMMENT
`
`In the present in vitro study, the preservative potassium
`sorbate did not show an influence on CBF (Figure 2).
`Benzalkonium chloride caused concentration-
`dependent ciliary inhibitory effect (Figure 3). The topi­
`cal glucocorticoid nasal spray with aqueous budesonide
`containing potassium sorbate as preservative did not show
`a decrease of CBF at 10% dilution and showed minimal
`reversible decrease at 50% dilution (Figure 4). The topi-
`
`Figure 1. A Zeiss Axiomat reversed microscope (Carl Zeiss, Oberkochen,
`Germany) with a connected photometer was used to measure the ciliary beat
`frequency of cultured human cells from the nasal mucosa. The cell cultures
`were fixed on an object slide in the exposure chamber. By using a minipump,
`it was possible to rinse the cell culture with either neutral Tyrode solution or
`test substance under constant temperature.
`
`Table 1. Compounds of Nasal Sprays
`
`Nasal Spray
`Budesonide
`
`Mometasone
`
`Fluticasone
`
`Corllcoid Concentration
`1.26 mg/mL Budesonide
`0.5 mg/mL Mometasone
`fuorate
`0.5 mg/mL Ruticasone
`propionate
`
`Preservative
`1.2 mg/mL (0.12%)
`Potassium sorbate
`0.2 mg/mL (0.02%)
`Benzalkonium chloride
`0.2 mg/mL (0.02%)
`Benzalkonium chloride
`
`1.8 mM calcium chloride, 2.2mM sodium bicarbonate, 0.4mM
`sodium hydrogen phosphate, 1, ImM magnesium chloride, and
`5.6mM glucose at pH 7.2), which does not influence the CBF,
`or with a solution containing nasal spray or preservative alone.
`The nasal sprays (Table 1) and the isolated preserva­
`tives were tested in 2 series of different dilutions with Tyrode
`solution (Table 2 and Table 3). The dilutions were de­
`signed to imitate the in vivo situation, where topical medica­
`tions are dispersed on the surface of the nasal mucosa and are
`therefore diluted.The nasal sprays were tested in 10% di­
`lution (1 part nasal spray to 9 parts Tyrode solution) and 50%
`dilution (1 part nasal spray to 1 part Tyrode solution). These
`dilutions were chosen according to Deitmer and Scheffler’ and
`other authors who used the same dilutions in similar in vitro
`experiments on
`The concentrations of isolated po­
`tassium sorbate and beirzalkonium chloride in test series 1 and
`2 were lower than the concentrations in the original nasal sprays
`(Table 2).
`Neither Tyrode solution nor normal saline influence CBF.
`The fluids were kept at a constant temperature of 35°C in our
`system and could be changed at any time without interrupting
`the cell culture rinsing process. With this system, it was pos­
`sible to create constant conditions such as temperature and os-
`molarity during measurements. A photometer was used to mea­
`sure CBF.
`Every measurement started with a run-in period of 20 min­
`utes during rinsing with neutral Tyrode solution to measure
`the baseline CBF. After 20 minutes, the Tyrode solution was
`changed to test solution for 7 minutes, which was followed by
`a washout period with Tyrode solution for 30 minutes.
`
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`

`■ Test Series 1
`• Test Series 1
`A Test Series 2
`□ Test Series 2
`A Test Series 2
`
`Figure 2. For 20 minutes, ciliary beat frequency (CBF) was measured during rinsing with Tyrode solution For 7 minutes, the cell cultures were exposed to
`potassium sorbate at a concentration of 0.12 mg/mL (test series 1) or 0.6 mg/mL (test series 2). A washout period with Tyrode solution followed. No influi
`influence on
`CBF was found at either concentration.
`
`■ Test Series 1
`A Test Series 1
`□ Test Series 1
`A Test Series 2
`• Test Series 2
`
`Figure 3. For 20 minutes, ciliary beat frequency (CBF) was measured during rinsing with Tyrode solution. For 7 minutes, the cell cultures were exposed to
`benzalkonium chloride (BKCHL) at a concentration of 0.05 mg/mL (test series 1) or 0.1 mg/mL (test series 2). A washout period with Tyrode solution followed.
`Test series 2 caused a complete standstill of the cilia, which was irreversible during washout with Tyrode solution. During test series 1, slowing of the cilia was
`observed.
`
`cal glucocorticoid sprays with fluticasone propionate and
`mometasone fuorate, both containing benzalkonium chlo­
`ride as preservative, showed reversible decrease of CBF
`at 10% dilutions and complete irreversible standstill at
`50% dilutions (Figures 5 and 6).
`A highly significant correlation between CBF and
`mucus transport time has been observed by Duchateau
`et al.'"* Previous in vitro experiments found inhibitory ef­
`fects on cilia function through nasal deconges­
`tants,antifungal solutions,*^ topical steroids,** and
`
`the preservative benzalkonuim chloride.*’ *’ In vivo inves­
`tigations have found controversial results: Berg et al**’ con­
`firmed the presence of squamous cell metaplasia in an
`in vivo animal experiment after 21 days of exposure to a
`topical steroid containing benzalkonium chloride, while
`this effect was not observed after exposure to 0.9% so­
`dium chloride or steroid without benzalkonium chlo­
`ride. Boek et al*’’ used a gamma scintigraphy technique
`on 15 healthy volunteers whose noses were treated with
`technetium Tc 99-marked xylometazoline spray with ben-
`
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`

`♦ Test Series 1
`■ Test Series 1
`A Test Series 1
`□ Test Series 1
`A Test Series 1
`• Test Series 2
`O Test Series 2
`V Test Series 2
`▼ Test Series 2
`
`Fijura 4. For 20 minutes, ciliary beat frequency (CBF) was measured during rinsing with Tyrone solution. For 7 minutes, the cell cultures were exposed to
`budesonide spray at 10% dilution (lest series 1) or 50% dilution (test series 2). Only short reversible slowing of CBF was measured during test series 2.
`
`A Test Series 1
`o Test Series 1
`A Test Series 1
`♦ Test Series 2
`*• Test Series 2
`
`Figure 5. For 20 minutes, ciliary beat frequency (CBF) was measured during rinsing with Tyrode solution. For 7 minutes, the cell cultures were exposed to
`mometasone fuorafe spray at 10% dilution (test series 1) or 50% dilution (test series 2). Test series 1 caused short reversible slowing of CBF After test series 2
`complete and irreversible standstill of the cilia was observed.
`
`zalkonium chloride. A nonsignificant mean reduction of
`mucociliary transport time was found. Storaas et al'** in­
`vestigated the effect of benzalkonitim chloride on glan­
`dular secretion and nasal symptoms of healthy volun­
`teers. During 10 days of repeated exposure to commonly
`used dosages of benzalkonitim ehloride, short-term glan­
`dular secretion or nasal pain occurred, but no exudative
`hyperresponsiveness or airway inflammation was found.
`Long-term in vivo studies of topical corticoste­
`roids with and without benzalkonium chloride have not
`confirmed damage to the ciliated cells of the nasal mu­
`cosa; placebo-controlled randomized studies have proven
`
`the efficacy and safety of topical steroid therapy for al­
`lergic rhinilis^'*-^ and nasal polyposis.^-^ Other than mild
`local adverse effects such as nasal dryness, irritation, or
`mild nasal bleeding, topical corticosteroids are well tol­
`erated over years. According to M inshall et al,^^ the per­
`centage of ciliated epithelium tentled to increase, aitd the
`cell metaplasia decreased, after 12 months of treatment
`with 200 pg of mometasone fuorate once daily. After 1
`and 5 yeare' adminisiration of budesonide nasal aerosol,
`healthy nasal epithelium has been documented by his­
`topathologic evaluation ” Biopsy specimens of nasal mu­
`cosa did not show nasal mucosa atrophy after once-
`
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`

`Figure 6. For 20 mlnules, dliSry baal frequency (CBF) was measured during rinsing with Tyrode soiution. For 7 minutes, the ceil cultures were exposed to
`fluticasone propionate spray at 10% dilution (test series 1) or 50% dilution (test series 2). In test series 1, CBF slowed during testing but recovered during
`washout with Tyrode solution. After lest series 2. complete irreversible standstill of the cilia was observed.
`
`daily treatment with 200 jtg of fluticasone over 1 year
`but, instead, showed improved epithelial thickness un­
`der evaluation with light and electron microscopy.-®
`The diverse findings of in vitro and in vivo studies
`can be explained by the lack of the mucus layer in vitro,
`which protects the ciliated epithelium of the human na­
`sal mucosa. In vivo measurements with saccharin or in­
`digo blue tests are subjective; the technetium Tc 99m test
`is objective; but many factors, such as circadian and na­
`sal rhythms, anatomic variations, potential infections, in­
`dividual variations of mucus transport time, and the re­
`quirement of absolute constant breathing throughout the
`measurements, influence the results. Because of indi­
`vidual variations in probands, it is necessary to conduct
`a high number of in vivo experiments to get reliable re­
`sults. In vitro experiments simulate in vivo conditions
`as closely as possible, but there will always be a differ­
`ence from human physiologic characterisLies. Nasal sprays
`are placed mainly in the region of the head of the infe­
`rior and middle turbinate when used in vivo. A possible
`influence on CBF is more likely to occur in vivo only in
`these parts of the nasal mucosa, where the concentra­
`tion of the topical drug is high. Because of the lack of
`the mucus layer, in vitro cells are less resistant to toxic
`substances, and the ability for tissue repair is limited.®
`Nevertheless, in our opinion, in vitro experiments
`on CBF are important to evaluate the safety of topically
`administered drugs because only in vitro experiments
`guarantee constant conditions and exclude other fac­
`tors, such as stress, hormone secretion, or inflamatory
`mediators, which may influence the sensitive ciliated cells.
`Osmolarity, pH, and temperature, which influence CBF,‘*
`can be controlled under in vitro conditions. If there is a
`reduction in CBF found by different investigators in vitro
`under standardized conditions and exclusion of cofac­
`tors, it can be assumed that also in vivo an influence on
`
`CBF is present. The degree of this effect in vivo is diffi­
`cult to quantify, but in the present study a clear differ­
`ence between the tested substances was found. As Bach-
`erC® discussed recently, one must be critical about the
`results of in vitro studies and should not overreact, as
`the German Federal Institute for Drugs and Medical De­
`vices is now doing as it considers reducing the period of
`administration ofbeiizalkoniiim chloride-containing na­
`sal sprays to 5 to 7 days.
`Because of the missing protective mucus layer, in
`vitro experiments cannot precisely duplicate in vivo con­
`ditions, but in our opinion they do help to objectify the
`influence of different substances on CBF. Substances that
`do not harm ciliated cells in vitro can be considered safe
`in vivo and in our opinion should be preferred by the
`pharmaceutical industry.
`In conclusion, the preservative potassium sorbate
`and the topical nasal spray containing this preservative
`did not show a significant influence on CBF in vitro. This
`preservative can therefore be considered harmless to the
`motility of ciliated cells.
`
`Submitted for publication March 25,2003; final revision re­
`ceived July 10, 2003; accepted September 9, 2003.
`This study was funded by the University Medical School
`of Graz, Graz, Austria.
`Corresponding author and reprints: Thiemo Hofmann,
`MD, Deparimcnl oj Otorhinolaryngology, f/niVErsity Hospi­
`tal Graz, Aut:nbntggerplaiz26. 8036 Graz, Austria (e-mail.-
`thiemo.hofmann@kfunigraz.ac.at).
`
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