ELSEVIER International Journal of Pharmaccutics 152 (1997) 67-73 intemational journal of pharmaceutics Reversibility and clinical relevance of morphological changes after nasal application of ephedrine nasal drops 1% Erik Bechgaard a,,, Erling Bindseil b Morten Bagger a, Hanne Wulff Nielsen ~,~ d Royal Danish School of Pharmacy, Department of Pharmaceutics, Universitetsparken 2, DK-2100 Copenhagen O, Denmark b The Royal Veterinary and Agricultural University, Department of Pharmacology and Pathobiology, Biilowsvej 13, DK-1870 Frederiksberg C, Copenhagen, Denmark Peptech (Europe) A IS, Herredsvejen 2, DK-3400 Hillerod, Denmark Received 30 August 1996; received in revised form 5 February 1997; accepted 12 February 1997 Abstract To predict the toxicity of nasal formulations, various in vitro and in vivo techniques have been used. Many of these techniques are very sensitive and it is a general problem to extrapolate the results to the clinical situation. The aim of the present study was to establish a clinically well known nasal formulation, Ephedrine Nasal Drops 1%, DAK 63 (EBE), as a relevant reference for other nasal formulations with respect to histological changes to and reversibility of the nasal rnucosa after repeated short-term nasal application to rabbits. This ephedrine formulation also contains the well known local irritants, benzalkonium chloride and EDTA, which is why it is abbreviated to EBE. Seventy five ill was applied in one nasal cavity of rabbits (n = 3) four times per day for 1 week, while the other cavity served as a control. Twelve rabbits were divided into four groups and were sacrificed at 4 h and 1, 7 and 21 days after last nasal application, respectively. The macro- and microscopical changes of the nasal mucosa were recorded. Except for minor greyish exudates seen at 4 h, and a slight congestion of the mucosa seen at 4 and 24 h after application, there were no gross changes of the nasal mucosa. The microscopical examination, however, showed an extended infiltration of the mucosa by eosinophils, a general inflammatory reaction and a pronounced atrophy and disorganisation of the epithelium, which was furthermore void of goblet cells and cilia. These microscopical changes were seen after 4 h and, to a minor extent, 24 h after application. After 7 days, no changes could be found, indicating that they were reversible in less than 1 week. It is concluded that EBE may be a good reference in the predictive testing of local toxicity, with respect to a cost/benefit evaluation of nasal formulations, meant for acute or short-term treatment. © 1997 Elsevier Science B.V. Keywords: Ephedrine; Nasal drops; Morphological changes; Nasal application * Corresponding author. 0378-5173/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. Pll S0378-51 73(97)0491 7-X
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`68 E. Bechgaard et al./ International Journal of Pharmaceutics 152 (1997) 67-73 1. Introduction In recent years, much attention has been paid to the systemic delivery of drugs by the nasal route (Chien, 1994). The ease of administration and fast absorption without first pass metabolism makes nasal application an attrac- tive alternative to the parenteral route. How- ever, with nasal application it is very important to evaluate and reduce the local toxicity at the site of application. Therefore, it is of great im- portance to know the clinical relevance of vari- ous predictive tests for local toxicity. To predict the toxicity of nasal formulations, various in vitro and in vivo techniques have been used (Gizurarson, 1992; Pereswetoff- Morath et al., 1996) including measurement of the mucociliary clearance (CBF) (Hermens and Merkus, 1987; Gizurarson, 1990), of electro- physiological properties, e.g. using chamber technique (Bechgaard et al., 1993; Vermehren et al., 1996), of erythrocyte haemolysis (Hirai et al., 1981), influence on the viability of cell cul- tures (Jorgensen et al., 1993), and histological studies (Bj6rk et al., 1991; Chandler et al., 1994; Bindseil et al., 1995). The techniques are also used in combination with scanning electron microscopy (SEM) and evaluation of drug bioavailability or with measurement of release of biochemical markers from the nasal mucosa (Marttin et al., 1995). In establishing the actual local toxicity of nasal drug formulation, most of these predictive techniques, i.e. measurement of CBF in vitro (Romeijn et al., 1996), is probably a too sensitive approach. Primarily, these meth- ods give valuable information on the toxic po- tency of a nasal drug or excipient. There is a need for further correlations to studies where the clinical situation is resembled. To establish such correlations, nasal drugs or formulations which are well known from clinical practice may be tested and compared with other substances with respect to local toxicity. The use of Ephedrine Nasal Drops 1% (EBE), may be a relevant reference, as this formulation has been used for many years as a nasal deconges- tant and since it is known to be irritating to the nasal mucosa. The irritative effect of the formu- lation, EBE, is probably not caused by ephedrine alone, but also by the preservative benzalkonium chloride 0.01%, which has been shown to exert histological changes in rabbit nasal mucosa (Marttin et al., 1996) and by EDTA, well known for disruption of epithelial tight junctions (Lee, 1991). Pure ephedrine solu- tions, 0.5 and 1.0%, have also been shown to cause a decrease in the ciliary beat function (CBF) in vitro (Su and Po, 1993). Another in vitro study on CBF has shown the effect of ephedrine 0.5% to be reversible within approxi- mately 1 h (van de Donk et al., 1982). Ephedrine solutions for use in the nasal cav- ity is still accepted, e.g. the British Pharmaco- poeia (Vol. 2, 1993, p. 896) and USP (23, 1995, p. 594). In Denmark, however, EBE was with- drawn in 1994 primarily due to the introduction of new drugs with a prolonged effect and with less systemic and local side effects. The local irritation from EBE is well known, which is the reason why the use of the formula- tion has been restricted to five daily applications for 1 week. A registration of the toxic effect on the nasal mucosa after such a dosage regime with EBE is therefore clinically relevant and may serve as a toxicity reference for the correla- tion of other drugs, absorption enhancers or ve- hicles for use in the nasal cavity. Therefore, the effects of single applications in vivo of EBE were studied 4 h after application by a combined macro- and microscopical inves- tigation of the rabbit nasal septae and conchae (Bindseil et al., 1995). Surprisingly, a mild toxic- ity was observed after only one application indi- cated by histological changes. The aim of the present study was to establish EBE as a clinically relevant reference for nasal formulations with respect to the histological changes of the nasal mucosa and their reversib- lity after repeated short-term nasal applications to rabbits. The study was intended to be rele- vant to the discussion of nasal formulations, with or without the use of absorption en- hancers.
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`E. Bechgaard et al.//International Journal of Pharmaeeuties 152 (1997) 67 73 69 2. Materials and methods 2.5. Post mortem procedures 2. I. Materials Ephedrine hydrochloride, Na-EDTA and NaC1 of Ph. Eur. (2nd Ed.) quality and benzalkonium chloride of Ph. Nord. 63 quality were obtained from Nomeco (Copenhagen, Denmark). Destilled water of Ph. Eur. (2nd Ed.) quality was used throughout the experiments. 2.2. Animals Twelve male New Zealand White rabbits were obtained from Hvidesten (Denmark). They were housed individually with free access to water and given approximately 100 g Altromin No. 2123 from Chr. Pedersen (Ringsted, Denmark) daily. The rabbits were approximately 1 year old and weighed 3.6 4.7 kg at the beginning of the exper- iments. 2.3. Nasal jormulation The nasal formulation, EBE, consisted of 1.0 g ephedrine, HC1, 10.0 mg benzalkonium chloride, 50.0 mg Na-EDTA and 0.6 g NaCI dissolved in 98.5 g destilled water as described for Ephedrine Nasal Drops 1% in DAK 63. The formulation was stored at 4°C. 2.4. Experimental design Seventy five ~tl of EBE was administered with an Eppendorph Multipipette ® to the right nasal cavity of the conscious rabbits four times daily for 1 week. The animals were observed closely during the dosing period, as unnormal behaviour of the rabbits was not accepted. The rabbits were killed 4 h and 1, 7 and 21 days after the last nasal application by anaesthesia with a sodium pentobarbital injection (50 mg/ml) and exsanguination by an incision in the carotid arter- ies. Immediately after exsanguination the nasal cavities were opened by removing the nasal and maxillary bones. 2.5.1. Gross examination After removal of the nasal and maxillary bones, the nasal mucosa was inspected and recordings were made of the gross appearance. 2.5.2. Light microscopy After opening of the nasal cavity, the septum and the ventral nasal conchae were removed sepa- rately and fixed in 10% neutral, buffered formalin. After fixation the conchae were decalcified in a 14% solution of ethylenedinitrotetraacetic acid for at least 2 3 days, and each septum was transected at three different levels to obtain tissue blocks from the anterior, middle, and posterior regions. The decalcified conchae and the septal tissue blocks were processed routinely by imbedding in paraffin and sectioning at 5 pm. The conchae were sectioned longitudinally. The sections were stained with haematoxylin- eosin, with Luna's stain for eosinophilic granulo- cytes, with periodic acid-Schiff reagent (PAS) for ground substance, and with Van Gieson's stain for collagen. 3. Results A summary of the results appears in Table 1, where score values for various histological fea- tures are displayed. A detailed description is as follows: 3.1. Clinical signs No clinical signs of respiratory disease, e.g. dyspnoea and exudative discharge from the nares, were observed in any of the rabbits. The activity and food consumption was also normal, and no signs of discomfort were observed. 3.2. Gross examination At 4 h, minor greyish exudates were found in the right, applicated nasal cavity between the ventral conchae and the septum in 2 of the 3 rabbits. No changes were observed in the left,
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`70 E. Bechgaard et al./International Journal of Pharmaceutics 152 (1997) 67 73 Table 1 Score of main histopathological features in the nasal septae of rabbits (n = 3 in each group) at various times after repeated (1 week 4 times/day) nasal application of Ephedrine Nasal Drops 1% (EBE) in the right nasal cavity (The left cavity was used as a control) Histopathological feature Control 4 h 24 h 7 days and 3 weeks Exudate on mucosa Cellular infiltration in epithelium Inflammation in lamina propria Epithelial atrophy Epithelial hypertrophy Epithelial metaplasia Loss of goblet cells and cilia 0 + 0 0 0 ++ 0 0 0 + 0 0 0 +++ 0 0 0 0 +++ 0 0 +++ +++ 0 0 +++ +++ 0 o, not present or negligible; +, slight; + +, moderate; + + +, marked. untreated nasal cavity. The septum appeared nor- mal in all three animals, whereas there was a slight congestion of the right conchae in all three animals. At 24 h a slight, bilateral congestion of the treated conchal mucosa was observed in all three animals. At 7 and 21 days, no changes of the treated nasal mucosa were observed. In the left nasal cavities, serving as untreated controls, no macroscopical changes of the nasal mucosa were observed. pronounced as to leave an unilayered, flat epithe- lium almost like an endo- or mesothelium (Fig. 2). The metaplastic epithelium was of the same thick- ness as the epithelium on the untreated, normal side, but consisted of a layer of disorganised epithelial cells without a distinct, regular basal layer (Fig. 3). Regardless of whether the epithe- lium was atrophic or metaplastic, it was almost void of goblet cells and cilia. Either type of changed epithelium was infiltrated of eosinophils which were also seen in 3.3. Light microscopy At 4 h, the right applicated side of the nasal cavity of all three animals, scattered strands and clumps of exudate with fibrin, neutrophils, eosinophils and desquamated epithelial cells were seen on the conchal mucosa (Fig. 1). Particularly in the rostral parts of the conchae, the epithelium was slightly to heavily infiltrated of neutrophils and eosinophils, and patchy epithelial disorgani- sation and necrosis were seen. Inflammatory reac- tions dominated by eosinophils, oedema, and hyperaemia were seen in the lamina propria. Many small blood vessels contained eosinophils. There appeared to be a slight increase in the number of activated osteoblasts along the bony spicules. The septal epithelium of the right, treated nasal cavity showed, in all three regions, a marked patchy atrophy or metaplasia (substitution of one type of cells for another). The atrophy could be so Fig. 1. Marked cellular exudate (E) on the conchal mucosa in the right nasal cavity of a rabbit 4 h after repeated nasal application of Ephedrine Nasal Drops 1% DAK 63 (EBE), for 1 week 4 times/day. Conchal epithelium (C) and bony spicule in conchal lamella (B). Bar = 75 ~m.
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`E. Bechgaard et al./International Journal of Pharmaceutics 152 (1997) 67 73 b Fig. 2. (I) Normal features of septal respiratory epithelium (between arrows) of the left, untreated side of the nasal cavity of a rabbit. Vascular plexus (V) in lamina propria, goblet cells (G) and septal cartilage (S). The vague zone observed on the luminal surface of the epithelial cells is a brim of cilia. Bar = 75 pm. (II) Marked atrophy of septal, respiratory epithelium (arrow) in the right, treated side of the nasal cavity of a rabbit 4 h after repeated (1 week) nasal application of EBE. Note lack of goblet cells and cilia. Vascular plexus (V) and septal cartilage (S). Bar = 75 pro. the lamina propria and in the lumen of small blood vessels. Minute strands of exudate contain- ing fibrin, eosinophils, and desquamated epithelial cells were occasionally seen on the surface of the epithelium, Fig. 3. Metaplastic, septal, respiratory epithelium (E) in the right, treated side of the nasal cavity of a rabbit 4 h after repeated nasal application of EBE. The epithelium is of the same height as normal respiratory epithelium (Fig. 2I), but it is completely void of goblet cells and cilia. Septal cartilage (S) and lamina propria (L). Bar = 75 /~m. At 24 h, the conchal epithelium in the right nasal cavity showed less of the histopathological features described in rabbits sacrificed at 4 h, but they were principally identical. The vestibular squamous epithelium of the sep- turn in the right side was hyperplastic (increase in the number of cells) compared with the epithelium of the left side (Fig. 4). The epithelium of the respiratory and olfactory regions was metaplastic in places as described in the animals sacrificed at 4 h, and was almost void of goblet cells and cilia. A few scattered mitoses were observed in the epithelium, but epithelial atrophy was not a fea- ture in this group of animals. The inflammatory reaction in the lamina pro- pria was negligible, and only a few eosinophils were detected in the epithelium and the underly- ing tissues. At 7 and 21 days, no essential pathological changes were observed in the right, applicated nasal cavity in any of the rabbits. The nasal mucosa of the untreated left nasal cavities, displayed no pathological changes in any of the animals. Scattered eosinophils and minor foci of lymphoid tissue, which are normal features in the nasal mucosa of animals, were occasionally observed.
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`72 E. Bechgaard et al./ International Journal of Pharmaceutics 152 (1997) 67-73 Fig. 4. Tranverse section of the nasal septum of a rabbit 24 h after repeated (1 week) application of EBE. Normal vestibular epithelium (EL) in the left, untreated nasal cavity, and hyperplastic epithelium (ER) in the opposite, right, treated side. Septal cartilage (S). Bar = 75/lm. 4. Discussion The experimental results were considered to be successful, as pronounced but reversible changes were observed in the mucosa of the treated nasal cavity caused by the clinically well known nasal formulation, EBE. The effect on the nasal mucosa was predominantly histological in terms of infiltration of eosinophils, epithelial atrophy, meta- and hyperplasia and absence of cilia and goblet cells. However, macroscopical findings such as minute exudates on and slight congestion of the mucosa were noted. This observed toxicity was surprisingly high even though some changes of the nasal mucosa were seen after a single application (Bindseil et al., 1995). It was therefore remarkable that the rabbits behaved normally and showed no sign of discomfort during the 1 week period of application. The observed local toxicity was found reversible in less than 24 h and 1 week for macroscopical and histological findings, respectively. The num- ber of experimental animals (n = 3) used in each group is relatively low, but the results were very reproducible. Furthermore, it is interesting that one nasal cavity could be used for application and one as a control. As mentioned in the results and in Table 1, no pathological changes were seen in the controls. It would be interesting, in further investiga- tions, to reveal a more precise time of histiological reversibility by adding observations after 2 or 3 days to the experiment. The seperate effects of 1% ephedrine, 0.01% benzalkonium chloride or 0.05% Na-EDTA after repeated application could have been studied as well, but it was chosen to use the DAK 63 formulation, since it is well known from clinical use. The significance of the findings in the present study is primarily that the observed local toxicity has been accepted for many years. Therefore, EBE may be used as a clinically relevant reference in the evaluation of other formulations or sub- stances, e.g. new drugs or absorption enhancers for nasal application. The degree of acceptable local toxicity is depen- dant upon the cost-benefit ratio as well as the extent of the period and frequency of application. In this sense, the acceptance of local toxicity to the nasal mucosa would be very low for chronical treatments, whereas a formulation meant for acute treatment in a life-threatening situation could be accepted, despite a high local toxicity. EBE may be a good reference for nasal formula- tions meant for acute or short-term treatment, while other clinically well known substances with lower irritating effects, may be used as standards for long-term or chronic treatment. Acknowledgements We are grateful to Mrs. Lisbet Kiorboe for her excellent technical assistance with the histopatho- logical work.
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`E. Bechgaard et al./ International Journal of Pharmaceutics 152 (!997) 67 73 73 References Bechgaard, E., Jorgensen, L., Larsen, R., Gizurarson, S., Carstensen, J. and Hvass, A., Insulin and didecanoyl-L-~- phosphatidylcholine: in vitro study of the transport through rabbit nasal mucosal tissue. Int. J. Pharm., 89 (1993) 147 153. Bindseil, E., Bechgaard, E., Jorgensen, L. and Larsen, R., Morphological examination of rabbit nasal mucosa after exposure to acetylsalicylic acid, glycofurol 75 and ephedrine. Int. J. Pharm., 119 (1995) 37-46. Bj6rk, E., Bjurstr6m, S. and Edman, P., Morphologic exami- nation of rabbit nasal mucosa after nasal administration of degradable starch microspheres, h,t. J. Pharm., 75 (1991) 73 80. Chandler, S.G., Thomas, N.W. and lllum, L., Nasal absorp- tion in the rat. lII. Effect of lysophospholipids on insulin absorption and nasal histology. Pharm. Res., 11(11) (1994) 1623 1630. Chien, Y.W., Nasal drug delivery and delivery systems. In Y.W. Chien (Eds.), Novel Drug Deliver)' Systems, 2nd Ed. Marcel Dekker, New York, 1994, pp. 229 268. van de Donk, H.J.M., Jadoenath, S., Zuidema, J. and Merkus, F.W.H.M., The effects of drugs on ciliary motility. I. Decongestants. Int. J. Pharm., 12 (1982) 57 65. Gizurarson, S., The influence of insulin and some excipients used in nasal insulin preparations on mucociliary clear- ance. Int. J. Pharm., 65 (1990) 243-247. Gizurarson, S., In situ method to estimate local effect of nasal preparations. Stand. J. Lab. Anim. Sci., 19(3) (1992) 129 35. Hermens, W.A.J.J. and Merkus, F.W.H.M., The influence of drugs on nasal ciliary movement. Pharm. Res., 4(6) (1987) 445 9. Hirai, S., Yashiki, T. and Mima, H., Mechanisms for the enhancement of the nasal absorption of insulin by surfac- tants. Int. J. Pharm., 9 (1981) 173 84. Jorgensen, L., Artursson, P. and Bechgaard, E., Toxicological and absorption enhancing effects of glycofurol 75 and sodium glycocholate in monolayers of human intestinal epithelial (Caco-2) cells. Int. J. Pharm., 95 (1993) 209-17. Lee, V.H.L. (Ed), Peptide and Protein Drug Delivery, Marcel Dekker, New York, 1991, pp. 1 -56. Marttin, E., VerhoL J.C., Romeijn, S.G. and Merkus, F.W.H.M., Effects of absorption enhancers on rat nasal epithelium in vivo: release of marker compounds in the nasal cavity. Pharm. Res., 12(8)(1995) 1150- 1157. Marttin, E., Verhof, J.C., Romeijn, S.G., Zwart, P. and Merkus, F.W.H.M.. Acute histopathological affects of benzalkonium chloride and absorption enhancers on rat epithelium in vivo. Int. J. Pharm., 141 (1996) 151 160. Pereswetoff-Morath, L., Bjurstrom, S., Khan. R., Dahlin, M. and Edman, P., Toxicological aspects of the use of dextran microspheres and thermogelling ethyl(hydroxyethyl) cellu- lose (EHEC) as nasal drug delivery systems. Int. J. Pharm., 128 (1996) 9 21. Romeijn, S.G.. Verhoef, J.C., Marttin, E. and Merkus, F.W.H.M., The effect of nasal drug formulations on ciliary beating in vitro. Int. J. Pharm., 135 (1996) 137 145. Su, X.Y. and Po, A.L., The effects of some commercially available antihistamine and decongestant intra-nasal for- mulations on ciliary beat frequency. J. CTin. Pharm. Ther., 8 (1993) 219-222. Vermehren, C., Hansen, H.S. and Thomsen, M.K., Time-de- pendent effects of two absorption enhancers on the nasal absorption of growth hormone in rabbits. Int. J. Pharm., 128 (1996) 239 250.
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