The Laryngoscope
`Lippincott Williams & Wilkins, Inc., Philadelphia
`© 2001 The American Laryngological,
`Rhinological and Otological Society, Inc.
`
`Classification of Cilio-Inhibiting Effects of
`Nasal Drugs
`
`Paul Merkus, MD; Stefan G. Romeijn; J. Coos Verhoef, PhD; Frans W. H. M. Merkus, PharmD, PhD;
`Paul F. Schouwenburg, MD, PhD
`
`Objective/Hypothesis: Nasal drug formulations
`are widely used for a local therapeutic effect, but are
`also used for systemic drug delivery. In the develop-
`ment of new nasal drugs, the toxic effects on the mu-
`cociliary clearance and therefore on the ciliated tis-
`sue is of importance. In this study, the effect of nasal
`drugs and their excipients on the ciliary beat fre-
`quency (CBF) is investigated. Study Design: Experi-
`mental, in vitro. Methods: CBF is measured by a
`photograph–electric registration method. Excised cil-
`iated chicken trachea tissue is incubated for 15 min-
`utes in the formulation, followed by a reversibility
`test. To estimate the ciliostatic potential, a classifica-
`tion is given of all tested formulations. According to
`the CBF, after 60 minutes every drug or excipient
`could be classified as follows: cilio-friendly: after 60
`minutes the CBF has regained 75% or more of its ini-
`tial frequency; cilio-inhibiting: after 60 minutes the
`CBF has regained between 25% and 75% of its initial
`frequency; or ciliostatic: after 60 minutes the CBF has
`regained 25% or less of its initial frequency. Results:
`Most formulations used are cilio-friendly or cilio-
`inhibiting. Only some are ciliostatic. Preservatives
`have a major role in the cilio-inhibiting effect of the
`drug. Also, other additives can contribute to the tox-
`icity profile of nasal drug formulations. Conclusion:
`This classification of the cilio-inhibiting potential of
`nasal drug formulations is a valuable tool in the de-
`sign of safe nasal drugs. The number of animal studies
`in vivo can be reduced substantially by using this in
`vitro screening technique. This study demonstrates
`that the effect on ciliary movement of most drug for-
`mulations is due to the preservatives and/or additives
`and mostly not to the drug itself. Key Words: Nasal
`drug, preservatives, ciliary beat frequency, ciliosta-
`tic, cilio-inhibiting, cilio-friendly.
`Laryngoscope, 111:595–602, 2001
`
`From the Department of Otorhinolaryngology & Head and Neck
`Surgery (P.M., P.F.S.), Academic Medical Center, Amsterdam, The Nether-
`lands, and the Department of Pharmaceutical Technology and Biopharma-
`ceutics, Leiden/Amsterdam Center for Drug Research (S.G.R., J.C.V.,
`F.W.H.M.M.), University of Leiden, Leiden, The Netherlands.
`Editor’s Note: This Manuscript was accepted for publication January
`9, 2001.
`Send Correspondence to Paul Merkus, MD, Department of ENT & Head
`and Neck Surgery, Academic Medical Center, Amsterdam, PO Box 22700, 1100
`DE Amsterdam, The Netherlands. E-mail: P.Merkus@amc.uva.nl
`
`Laryngoscope 111: April 2001
`
`INTRODUCTION
`Nasal drug formulations, for instance, those contain-
`ing decongestants and corticosteroids, are widely used for
`a local therapeutic effect. The nasal mucosa is also an
`attractive site for systemic drug absorption. It is an effec-
`tive alternative for other routes of drug administration
`(oral, injection), for instance, in the case of antimigraine
`substances,1,2 steroids,3 and peptide and protein drugs.4,5
`Nasal drug absorption can be efficient because the nasal
`epithelium has a relatively large permeability and the
`subepithelial layers are highly vascularized.6
`Nasal drug delivery has a number of clear advantages,
`including ease of administration, patient acceptability, and
`prevention of first-pass effect.7 The relatively small surface
`area of the nasal cavity and the mucociliary clearance are
`drawbacks in nasal drug delivery. The residence time of a
`drug formulation in the nose is limited to only approximately
`15 minutes, because of the nasal mucociliary clearance.8 –10
`It is obvious that during acute or chronic nasal drug appli-
`cation, the drug itself and the formulation excipients should
`not disturb the nasal mucociliary clearance, because it is an
`extremely important defense mechanism of the respiratory
`tract. The mucociliary clearance remove bacteria viruses,
`allergens, and dust from the respiratory tract. Because cili-
`ary movement is a major factor in mucociliary clearance, the
`influence of drug formulations on the ciliary beat frequency
`(CBF) is an important issue to establish the safety of nasally
`administered drugs and various formulation excipients
`such as preservatives11–13 and absorption enhancing
`compounds.13,14
`The aim of this study was to test the cilio-inhibiting
`effects of a number of drugs using ciliated chicken embryo
`tracheal tissue. Chicken trachea is a valid substitute for
`human material in studying ciliary activity in vitro.15,16
`Moreover, the reversibility of the observed effects was
`established after exposure of the ciliated tissue to the
`nasal drug formulations during 15 minutes, comparable to
`the situation in vivo. The evaluation of the influence on
`ciliary movement may offer a possibility to classify drugs
`and excipients according to their inhibiting effect.
`
`MATERIALS AND METHODS
`The nasal formulations selected for this study are widely
`prescribed drugs for local and systemic effects, some excipients,
`
`Merkus et al.: Cilio-Inhibiting Effects of Nasal Drugs
`595
`
`Opiant Exhibit 2087
`Nalox-1 Pharmaceuticals, LLC v. Opiant Pharmaceuticals, Inc.
`IPR2019-00688
`Page 1
`
`

`

`Fig. 1. Classification of the effect of
`nasal formulations on ciliary beat fre-
`quency (CBF). CBF is expressed as per-
`centage of the initial frequency (100%).
`After 15 minutes incubation of the cili-
`ated tissue in the nasal formulation, the
`reversibility of the CBF in Locke-Ringer
`solution is measured. At 60 minutes after
`the start of the incubation, the degree of
`reversibility is classified into three cate-
`gories: cilio-friendly, cilio-inhibiting, or
`ciliostatic.
`
`and investigational drug formulations indicated for systemic na-
`sal drug absorption. Products have been selected that are avail-
`able on the market in the United States and Europe, although
`brand names may sometimes differ.
`
`Materials
`Benzalkonium chloride (BAC; USP quality) was from Bro-
`(Maarssen, The Netherlands), chlorobutanol was from
`cacef
`Sigma-Chemie (Dreisenhofen, Germany), and sodium edetate
`(EDTA; PA quality) from Merck (Darmstadt, Germany). Ran-
`domly methylated b-cyclodextrin (RAMEB; degree of substitution
`
`1.8) was obtained from Wacker (Burghausen, Germany). All other
`chemical compounds were from Sigma–Chemie (Dreisenhofen,
`Germany), and the drug substances were from Bufa (Uithoorn,
`The Netherlands).
`The species of chickens used was Hubbard-Golden Comeet
`(Vossensteijn, Groenekan, The Netherlands).
`
`(Non-)Prescription Nasal Drug Formulations
`All nasal formulations selected for the present study are
`widely used prescription and non-prescription drugs for local or
`systemic effects, and were studied for their influence on ciliary
`
`TABLE I.
`The Effect of (Non-)Prescription Nasal Drug Formulations on Ciliary Beat Frequency (CBF) in vitro.
`
`Nasal Product
`
`Aerodiol®
`Flixonase®
`
`Imigran®
`Miacalcic®
`Minrin®
`Nasacort®
`
`Nasivin®
`Nasivin® pur
`Nasonex®
`
`Otriven®
`Otrivin®
`Rhinocort®
`Sinex®
`
`Control
`Locke-Ringer (LR)
`
`Main Constituents
`
`Estradiol, RAMEB
`Fluticasone, BAC,
`phenylethylalcohol
`Sumatriptan, phosphate buffer
`Calcitonin, BAC
`Desmopressin, chlorobutanol
`Triamcinolone acetonide, BAC,
`EDTA
`Oxymetazoline, BAC, EDTA
`Oxymetazoline
`Mometasone fuorate, BAC,
`phenylethylalcohol
`Xylometazoline, citrate, glycerol
`Xylometazoline, BAC, EDTA
`Budesonide, Sorbate, EDTA
`Oxymetazoline, BAC,
`chlorhexidine, EDTA,
`camphor, menthol, eucalyptol
`
`CBF t 5 15
`(SD)
`
`CBF t 5 60
`(SD)
`
`42 (7)
`9 (5)
`
`0 (0)
`12 (9)
`0 (0)
`38 (7)
`
`2 (5)
`25 (4)
`0 (0)
`
`18 (5)
`21 (9)
`25 (13)
`0 (0)
`
`97 (8)
`62 (11)
`
`96 (14)
`58 (20)
`0 (0)
`78 (8)
`
`4 (10)
`97 (13)
`33 (19)
`
`103 (6)
`36 (12)
`98 (22)
`0 (0)
`
`Classification
`
`Cilio-friendly
`Cilio-inhibiting
`
`Cilio-friendly
`Cilio-inhibiting
`Ciliostatic
`Cilio-friendly
`
`Ciliostatic
`Cilio-friendly
`Cilio-inhibiting
`
`Cilio-friendly
`Cilio-inhibiting
`Cilio-friendly
`Ciliostatic
`
`100 (3)
`
`100 (4)
`
`Cilio-friendly
`
`CBF (% of initial frequency) after 15 min incubation in the test formulation (t 5 15) and after reversibility testing
`in Locke-Ringer solution until 60 min (t 5 60). Data are expressed as the mean (6 standard deviation) of 6 – 8
`experiments. Classification according to Figure 1.
`BAC 5 benzalkonium chloride; EDTA 5 sodium edetate; RAMEB 5 randomly methylated b-cyclodextrin.
`
`Laryngoscope 111: April 2001
`596
`
`Merkus et al.: Cilio-Inhibiting Effects of Nasal Drugs
`
`Opiant Exhibit 2087
`Nalox-1 Pharmaceuticals, LLC v. Opiant Pharmaceuticals, Inc.
`IPR2019-00688
`Page 2
`
`

`

`TABLE II.
`The Effect of Investigational Nasal Formulations on Ciliary Beat Frequency (CBF) in vitro.
`
`Investigational Products
`
`Main Constituents
`
`Hydroxocobalamin 2.0% Hydroxocobalamin, Locke-Ringer
`Hydroxocobalamin 1.2% Hydroxocobalamin, acetate buffer
`Melatonin 0.05%
`Melatonin, Locke-Ringer
`Melatonin, b-Cyclodextrin
`Melatonin 0.2%
`Midazolam 3.1%
`Midazolam, benzylalcohol,
`propylene glycol
`Propranolol, Locke-Ringer
`
`Propranolol 1.0%
`
`CBF t 5 15
`(SD)
`
`CBF t 5 60
`(SD)
`
`Classification
`
`90 (13)
`0 (0)
`80 (12)
`42 (5)
`0 (0)
`
`Cilio-friendly
`88 (5)
`79 (12) Cilio-friendly
`99 (4)
`Cilio-friendly
`102 (3)
`Cilio-friendly
`0 (0)
`Ciliostatic
`
`0 (0)
`
`0 (0)
`
`Ciliostatic
`
`CBF (% of initial frequency) after 15 min incubation in the test formulation (t 5 15) and after reversibility testing
`in Locke-Ringer solution until 60 min (t 5 60). Data are expressed as the mean (6 standard deviation) of 6 – 8
`experiments. Classification according to Figure 1.
`BAC 5 benzalkonium chloride; EDTA 5 sodium edetate; RAMEB 5 randomly methylated b-cyclodextrin.
`
`beating in undiluted form. The following formulations were in-
`vestigated: estradiol (Aerodiol®; Servier, Paris, France) 0.2% w/v,
`containing randomly methylated b-cyclodextrin (RAMEB) 2.0%
`w/v; fluticasone (Flixonase®; Glaxo Wellcome B.V., Zeist, The
`Netherlands) 0.05% w/v, containing BAC 0.02% w/v and phenyl-
`ethylalcohol 0.25% w/v; sumatriptan (Imigran®; Glaxo Wellcome
`B.V.) 20% w/v in a phosphate buffer pH 5.4; salmon calcitonin
`(Miacalcic®; Novartis Farmaceutica, Barcelona, Spain) 2200 IU/
`mL, containing benzalkonium chloride (BAC) 0.01% w/v; desmo-
`pressin (Minrin®; Ferring, Malmo¨, Sweden) 0.01% w/v, contain-
`ing chlorobutanol 0.5% w/v; triamcinolone acetonide (Nasacort®;
`Rhoˆne Poulenc Rorer B.V., Amstelveen, The Netherlands) 0.05%
`w/v, containing cellulose, sodium carboxymethylcellulose, poly-
`sorbate 80, BAC, and EDTA; oxymetazoline (Nasivin®; Merck,
`Darmstadt, Germany) 0.05% w/v, containing BAC and EDTA;
`oxymetazoline (Nasivin® pur; Merck) 0.05% w/v, preservative-
`free; mometasone fuorate (Nasonex®; Schering-Plough B.V.,
`
`Maarssen, The Netherlands) 0.05% w/v, containing BAC, polysor-
`bate 80 and phenylethylalcohol; xylometazoline (Otriven®; No-
`vartis Consumer Health, Munich, Germany) 0.1% w/v, containing
`citric acid, sodium citrate and glycerol, preservative-free; xylo-
`metazoline (Otrivin®; Novartis Consumer Health, Breda, The
`Netherlands) 0.1% w/v, containing BAC and EDTA; budesonide
`(Rhinocort®; Astra Pharmaceutica, Zoetermeer, The Nether-
`lands) 0.1% w/v, containing potassium sorbate and sodium ede-
`tate (EDTA); and oxymetazoline (Sinex®; Richardson Vicks B.V.,
`Rotterdam, The Netherlands) 0.05% w/v, containing BAC 0.02%
`w/v, chlorhexidine digluconate, EDTA 0.01% w/v, and also men-
`thol, camphor, eucalyptol, and tyloxapol.
`
`Investigational Nasal Formulations
`The investigational hydroxocobalamin formulation con-
`sisted of hydroxocobalamin 1.2% w/v and NaCl 0.7% w/v in 20
`mmol/L sodium acetate buffer of pH 4.5. Melatonin nasal prepa-
`
`Fig. 2. The effect of three nasal products
`on CBF. After 15 minutes incubation of
`the ciliated tissue in the nasal formula-
`tion,
`the reversibility of
`the CBF in
`Locke-Ringer solution was measured.
`The effect, after reversibility testing at 60
`minutes, of Rhinocort® (l) is classified
`as cilio-friendly, that of Miacalcic® ((cid:130)) as
`cilio-inhibiting, and that of Sinex® (e) as
`ciliostatic. Locke Ringer (e), the control
`solution, has no cilio-inhibiting influence.
`CBF is expressed as percentage of the
`initial
`frequency (100%) and data are
`mean 1 standard deviation.
`
`Laryngoscope 111: April 2001
`
`Merkus et al.: Cilio-Inhibiting Effects of Nasal Drugs
`597
`
`Opiant Exhibit 2087
`Nalox-1 Pharmaceuticals, LLC v. Opiant Pharmaceuticals, Inc.
`IPR2019-00688
`Page 3
`
`

`

`(A and B) Effects of Imigran®
`Fig. 3.
`and Minrin® on CBF: contribution of
`formulation constituents. Its constit-
`uents can explain effects of both na-
`sal products. The effect, after revers-
`ibility testing, of Imigran® (containing
`a phosphate buffer) ((cid:145), A) is probably
`the result of the buffer solution ((cid:145), B).
`The ciliostatic effect of Minrin® (V, A)
`is caused by its preservative chloro-
`butanol 0.5% (V, B). CBF is ex-
`pressed as percentage of the initial
`frequency (100%) and data are mean
`6 standard deviation.
`
`rations contained melatonin 0.2% w/v, NaCl 0.9% w/v, and the
`solubilizer b-cyclodextrin 0.75% w/v in water. The midazolam
`formulation consisted of midazolam hydrochloride 3.1% w/v, ben-
`zylalcohol 1% v/v, and propylene glycol 25% v/v in water. Pro-
`pranolol hydrochloride 1.0% w/v was dissolved in Locke-Ringer.
`
`Excipients
`A number of excipients used in the (non-)prescription and
`investigational nasal drug formulations were measured for their
`effect on ciliary beat frequency, after dissolving these substances
`in Locke-Ringer solution: the solubilizer/absorption enhancer
`
`Laryngoscope 111: April 2001
`598
`
`Merkus et al.: Cilio-Inhibiting Effects of Nasal Drugs
`
`Opiant Exhibit 2087
`Nalox-1 Pharmaceuticals, LLC v. Opiant Pharmaceuticals, Inc.
`IPR2019-00688
`Page 4
`
`

`

`TABLE III.
`The Effect of Excipients on Ciliary Beat Frequency (CBF) in vitro.
`
`Excipient
`
`NaCl 0.9%
`BAC 0.01%
`BAC 0.02%
`BAC 0.01%/EDTA 0.1%
`Benzylalcohol 1%/propylene glycol 25%
`Chlorobutanol 0.5%
`Phenylethylalcohol 0.5%
`Phosphate buffer (120 mM; pH 5.4)
`Potassium sorbate 0.2%/EDTA 0.1%
`RAMEB 2.0%
`Sodium acetate buffer (20 mM; pH 4.5)
`
`CBF t 5 15
`(SD)
`
`CBF t 5 60
`(SD)
`
`74 (12)
`54 (22)
`52 (27)
`35 (14)
`0 (0)
`0 (0)
`0 (0)
`0 (0)
`62 (9)
`61 (17)
`0 (0)
`
`95 (8)
`70 (11)
`20 (19)
`43 (23)
`0 (0)
`0 (0)
`97 (12)
`98 (6)
`99 (5)
`93 (6)
`88 (15)
`
`Classification
`
`Cilio-friendly
`Cilio-inhibiting
`Ciliostatic
`Cilio-inhibiting
`Ciliostatic
`Ciliostatic
`Cilio-friendly
`Cilio-friendly
`Cilio-friendly
`Cilio-friendly
`Cilio-friendly
`
`CBF (% of initial frequency) after 15 min incubation in the test formulation (t 5 15) and after reversibility testing
`in Locke-Ringer solution until 60 min (t 5 60). Data are expressed as the mean (6 standard deviation) of 6 – 8
`experiments. Classification according to Figure 1.
`BAC 5 benzalkonium chloride; EDTA 5 sodium edetate; RAMEB 5 randomly methylated b-cyclodextrin.
`
`RAMEB in concentrations of 2.0% w/v, the preservative BAC in
`concentrations of 0.01% and 0.02% w/v, and the preservatives
`phenylethylalcohol and chlorobutanol in concentrations of 0.5%
`w/v. Additionally, combination preparations of the preservative
`BAC 0.01% and potassium sorbate 0.2% with EDTA 0.1% w/v in
`Locke-Ringer were tested. Three vehicle solutions were investi-
`gated: 120 mmol/L phosphate buffer (adjusted to pH 5.4), 20
`mmol/L sodium acetate buffer containing NaCl 0.9% w/v (adjust-
`ed to pH 4.5), and benzylalcohol 1% v/v with propylene glycol 25%
`v/v in water.
`
`Locke-Ringer (Control Solution)
`Locke-Ringer (LR) is an isotonic solution of the following
`composition per liter of water: NaCl, 7.72 g (132 mmol); KCl,
`0.42 g (5.63 mmol); CaCl20.2H2O, 0.16 g (1.24 mmol); NaHCO3,
`0.15 g (1.79 mmol); glucose, 1.00 g (5.55 mmol). Locke-Ringer
`solution was prepared using Millipore-deionized water, and the
`solution was subsequently sterilized for 20 minutes at 120°C. The
`pH of the Locke-Ringer solution was established at 7.4.
`
`Ciliary Beat Frequency Measurements
`Ciliary beat frequency (CBF) measurements were per-
`formed on the ciliated epithelium of isolated chicken embryo
`trachea as described previously.13,17 Briefly, the chicken embryo
`trachea was dissected from the embryo and sliced into small rings
`of approximately 1 mm thickness. The trachea slices were placed
`in stainless steel supporting rings, and were allowed to recover
`for 30 minutes in Locke-Ringer solution. Thereafter, the tissue
`samples were put in a well containing 1.0 mL of the test solution,
`and placed under an Olympus BH-2 light microscope. The micro-
`scope table was connected with a thermostat to maintain a tem-
`perature of 33°C. The CBF was subsequently monitored using a
`photograph–electric registration device. A light beam was trans-
`mitted through the moving cilia, and after magnification by the
`microscope the flickering light was projected to a photocell. The
`electrical signal generated by this photocell was visualized with a
`computer monitor. The frequency of the signal was calculated
`electronically by Fast-Fourier transform algorithm and displayed
`as a frequency distribution.
`After starting the incubation, the CBF was measured at 5,
`10, and 15 minutes. Thereafter, to test the reversibility of CBF,
`the trachea slices were washed by shaking them vigorously in a
`
`Laryngoscope 111: April 2001
`
`tube with 3 mL Locke-Ringer. Then the slices were replaced in
`pure Locke-Ringer and CBF was measured again every 5 to 10
`minutes until 60 minutes after the start of the incubation. Every
`formulation has been tested using tissue samples of at least six
`different chickens.
`CBF data were calculated as the relative frequency of the
`initial frequency measured in Locke-Ringer solution at the start
`of the experiment, the latter being expressed as 100%.
`
`Classification of Effects on CBF
`The influence of the studied nasal drug formulations and
`excipients on CBF was classified into the following three catego-
`ries (Fig. 1):
`1) Cilio-friendly: after 60 minutes the CBF has regained
`75% or more of its initial frequency.
`2) Cilio-inhibiting: after 60 minutes the CBF has regained
`between 25 and 75% of its initial frequency.
`3) Ciliostatic: after 60 minutes the CBF has regained 25%
`or less of its initial frequency.
`
`RESULTS
`A summary of the results is shown in Tables I, II, and
`III. The CBF of the control solution (Locke-Ringer) re-
`mained 100% of the initial frequency for at least 1 hour in
`all experiments (Table I).
`
`Nasal Products
`Imigran®, Rhinocort®, Nasacort®, and Aerodiol® re-
`duce CBF, and this effect is reversible. Imigran® arrested
`the ciliary beating within 5 minutes, but the mean CBF
`recovered to 96% of the initial frequency at completion of
`the reversibility test. Rhinocort® (Fig. 2), Nasacort®, and
`Aerodiol® resulted in mild effects on the CBF after 15
`minutes incubation: the mean CBF decreased to 25%,
`38%, and 42%, respectively. In the subsequent reversibil-
`ity test CBF increased to 98%, 78%, and 97%, respectively,
`of their initial frequency.
`Miacalcic® (Fig. 2) and Flixonase® appeared to have
`almost identical effects on CBF. Their initial frequency
`dropped to 12% and 9%, respectively, after 15 minutes
`
`Merkus et al.: Cilio-Inhibiting Effects of Nasal Drugs
`599
`
`Opiant Exhibit 2087
`Nalox-1 Pharmaceuticals, LLC v. Opiant Pharmaceuticals, Inc.
`IPR2019-00688
`Page 5
`
`

`

`(A and B) The difference be-
`Fig. 4.
`tween the effects of Otrivin® (with
`preservative) and Otriven® (without
`preservative) on CBF. The cilio-
`inhibiting effect of Otrivin® is likely to
`be caused by its preservative. Note
`the similar profile of Otrivin® (V, A)
`and BAC 0.01%/EDTA 0.1% (l, B)
`compared with the cilio-friendly ef-
`fect of Otriven® (r, A), xylometazo-
`line without any preservative. CBF is
`expressed as percentage of the initial
`frequency (100%) and data are mean
`6 standard deviation.
`
`incubation. After washing and putting the ciliated tissue
`back into pure Locke-Ringer, the CBF regained up to 58%
`and 62%, respectively, of their initial frequency. Both
`products contain BAC as a preservative.
`
`Nasivin® pur, containing oxymetazoline without any
`preservative, decreased the CBF after 15 minutes to 25%,
`but this effect was completely reversible. Nasivin® and
`Sinex® (Fig. 2), containing oxymetazoline and BAC as
`
`Laryngoscope 111: April 2001
`600
`
`Merkus et al.: Cilio-Inhibiting Effects of Nasal Drugs
`
`Opiant Exhibit 2087
`Nalox-1 Pharmaceuticals, LLC v. Opiant Pharmaceuticals, Inc.
`IPR2019-00688
`Page 6
`
`

`

`major constituents, caused a ciliary arrest after 15 min-
`utes incubation, and this effect appeared to be irrevers-
`ible. Otrivin® (containing xylometazoline, BAC, and
`EDTA) and Otriven® (preservative-free xylometazoline)
`decreased the mean CBF to 21% and 18%, respectively,
`after 15 minutes exposure. However, only the effect of the
`preservative-free Otriven® was completely reversible (see
`Table I).
`Nasonex® showed no ciliary beating after 15 min-
`utes, but the ciliated tissue regained its activity to 33 6
`19% at 60 minutes. Minrin® appeared to be ciliostatic,
`showing complete and irreversible ciliary arrest within 5
`minutes after exposure in all experiments (Fig. 3A, n 5 8).
`As an illustration of the classification into three cat-
`egories, the profile of Rhinocort®, Miacalcic®, and Sinex®
`are presented in Figure 2.
`
`Investigational Products
`The effects of some investigational nasal products
`(hydroxocobalamin, melatonin, midazolam, and propran-
`olol) are summarized in Table II.
`
`Excipients
`The effects on CBF of a number of excipients (physi-
`ological saline, preservatives, buffers, and so on) are de-
`scribed in Table III. Sometimes the effect is cilio-friendly,
`but also a ciliostatic effect can be measured, as demon-
`strated in Figure 3B, for the phosphate buffer and the
`preservative chlorobutanol.
`
`DISCUSSION
`The measurement of effects on CBF in vitro is an
`accurate and reproducible technique for testing formula-
`tions that can interfere with normal cilia movement. On
`the basis of the results of this study, it is possible to
`classify nasal drug formulations by their effects on cilia
`movement in vitro.
`However, it is important to emphasize that the ef-
`fects of drugs and excipients as measured in this study are
`only indicational for the effects of nasal drugs on cilia
`activity in vivo. To establish the actual local toxicity of
`nasal drugs, measuring CBF in vitro is probably too sen-
`sitive.10,14 In vitro the excised ciliated tissue is totally
`immersed in the test formulation, whereas in vivo the
`viable ciliated epithelium is protected by a mucus barrier.
`Nevertheless, this in vitro method is a valuable tool for the
`development of safe nasal drug formulations and the se-
`lection of safe excipients. It has been shown that the
`effects on the ciliated tissue of chicken trachea in vitro are
`quite similar to those on human ciliated tissue in
`vitro.15,16 Moreover, use of a large number of animals
`(e.g., rats, rabbits) can be avoided, because one chicken
`trachea allows up to 20 in vitro cilia experiments.
`To evaluate the outcome of the CBF and the revers-
`ibility testing, we have made a classification into three
`categories. The classification of drugs and excipients com-
`pares in relative terms the toxicity potential of constitu-
`ents of nasal drug formulations. Cilio-friendly and cilio-
`inhibiting formulations will give a reversible effect on the
`cilia, whereas ciliostatic formulations have a stronger and
`(almost) irreversible effect on CBF (Figs. 1 and 2).
`
`Laryngoscope 111: April 2001
`
`In the present study we investigated widely used
`nasal products, investigational formulations, and a num-
`ber of excipients used in these products. Locke-Ringer
`(LR) was selected as the control solution, because LR does
`not influence ciliary activity in a time span of at least 60
`minutes (Figs. 2–4). Physiological saline is not a good
`control, because it has a mild inhibiting effect on CBF
`(Table III), as recently reported in this journal.18
`Most nasal products also contain preservatives as a
`major constituent, which appeared to contribute substan-
`tially to the ciliostatic potential of the whole product. For
`example, Minrin®, in a number of countries, containing
`chlorobutanol 0.5% as a preservative, has a ciliostatic
`profile similar to that of the single preservative (compare
`Fig. 3A with 3B).
`Also, all products with BAC as a preservative have a
`cilio-inhibiting effect, most likely caused by the presence
`of this preservative. The corticosteroid nasal sprays tested
`in this study are either cilio-friendly (Nasacort®, Rhino-
`cort®) or cilio-inhibiting (Flixonase®, Nasonex®). The dif-
`ference between these products is the result of the pres-
`ence of different preservatives and probably not the
`different drug compounds. Additives (like NaCl, benzylal-
`cohol, propylene glycol, acetate buffer, and phosphate
`buffer) also have their effect on ciliated tissue, as demon-
`strated in Table III and Figure 3. For example, hydroxo-
`cobalamin 1.2% nasal
`formulation containing acetate
`buffer (pH 4.5) resulted in a completely reversible ciliary
`arrest. This effect can be attributed to the acetate buffer
`(Tables II and III). Similarly, the effect of Imigran® is
`mainly caused by the phosphate buffer (Tables I and III;
`Fig. 3A, B).
`Xylometazoline and oxymetazoline have a similar ef-
`fect on CBF.15 Nasivin® pur, oxymetazoline (without any
`preservative), has a cilio-friendly effect. However, Na-
`sivin® and Sinex®, oxymetazoline with BAC and EDTA as
`main constituents, are classified as ciliostatic. The main
`reason for the ciliostatic effect is the high concentration of
`BAC, which was measured to be 0.02% w/v in both prod-
`ucts. For the products with xylometazoline (Otrivin® and
`Otriven®), a similar explanation is feasible, as shown in
`Figure 4. Additionally, Sinex® contains chlorhexidine,
`camphor, menthol, and eucalyptol, which also enhance the
`ciliostatic effect.13
`It is clear that most nasal products have a reversible
`effect on the ciliated tissue classified as cilio-friendly
`(.75%) or cilio-inhibiting (25%–75%). Only sometimes the
`drug itself (e.g., propranolol 1.0%) is irreversibly ciliostatic,
`but often the presence of the additives, especially preserva-
`tives, is the reason for the observed ciliostatic profile of nasal
`formulations. We recommend preservative-free formula-
`tions, especially those for chronic use. When prescribing
`products with a ciliostatic profile, the effects on the ciliated
`tissue should be taken into account and frequent use should
`be avoided.
`
`CONCLUSION
`This classification, evaluating the influence of nasal
`drug formulations on ciliary movement, is a valuable tool
`in the design of safe nasal drugs. The number of whole
`
`Merkus et al.: Cilio-Inhibiting Effects of Nasal Drugs
`601
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`animal studies in vivo can be reduced substantially by
`using this in vitro screening technique.
`The formulations and excipients investigated in this
`study demonstrate that the effect on ciliary movement of
`most drug formulations is due to the preservatives and/or
`additives, and mostly not to the drug itself.
`
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`Merkus et al.: Cilio-Inhibiting Effects of Nasal Drugs
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`Opiant Exhibit 2087
`Nalox-1 Pharmaceuticals, LLC v. Opiant Pharmaceuticals, Inc.
`IPR2019-00688
`Page 8
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