Minor changes in the administration technique used for intranasal instillation of clonazepam, have been  found to influence the results significantly. A simple study was performed, where rabbits received 0.5 mg  clonazepam intranasally. One group received the drug while fixed in a sitting position, where the other  group was fixed in a supine position. The results show that both techniques where able to provide a rapid  absorption with a tmax around 3-4 min. The Cmax and AUC, however, were very different. The Cmax was found  to be 40 ng/ml and 86 ng/ml, respectively, and the AUC was found to be 891 and 2249 (ng/ml/min), respectively,  for the sitting and the supine position. The relative bioavailability for sitting/supine was found to be  38%. These results show that the administration technique is very important and should not be underestimated.

Bechgaard, Erik

Gizurarson, Sveinbjörn

Hjortkjær, Rolf K

English

Journals from University of Tartu

IntroductionIntranasal administration of diazepam to rabbits hasbeen shown to provide a rapid absorption with tmaxaround 5 min. The results are reproducible and theonset time for the effect was found to be varyingwithin 1.5-4.5 min (Bechgaard et al., 1997). Thesuccess of each experiment depends highly onwhich techniques and animal species are used andhow one masters the techniques (Gizurarson,1993). Animal models influence the results so muchthat good results may be lost due to a wrongly cho-sen technique or animal species. The influence oftechniques and models should therefore never beunderestimated, even when simple studies are per-formed such as estimating the absolute bioavail-ability. In all experiments it is important to knowthe anatomy and the physiology of the animalmodel chosen and to understand its limitations andhow it may influence the results.The rabbit is a common animal model for intranasaldrug delivery studies due to the size of the nasalcavity; also blood samples may be collected fre-quently and easily (Gizurarson, 1990).  The rabbitnasal cavity has a similar surface area to the humannasal cavity or 60 cm2 and 160 cm2 for rabbit andhuman, respectively. The aim of the study was to investigate how twoadministration techniques, used to instill drug intothe nasal cavity of rabbits, may influence the phar-macokinetics of clonazepam. Two techniques arecompared, the administration to rabbits in either sit-ting or supine position during the application.Material and MethodsClonazepam was kindly provided by Roche A/S(Hvidovre, Denmark), glycofurolum 75 (GF) wasfrom Hoffman-la Roche (Basle, Switzerland),desmethyldiazepam from Ferrosan A/S (Søborg,Denmark) and highly purified tetraethyleneglycol(4EG) was purchased from Fluka Chemie AG(Buchs, Switzerland). All other chemicals were ofScand. J. Lab. Anim. Sci. 2006 Vol. 33 No. 1Published in the Scandinavian Journal of Laboratory Animal Science - an international journal of laboratory animal science35Two Intranasal Administration Techniques Give Two Different Pharmacokinetic Resultsby Sveinbjörn Gizurarson1,*, Erik Bechgaard2 and Rolf K. Hjortkjær31University of Iceland, Faculty of Pharmacy, Reykjavik, Iceland2The Royal Danish School of Pharmacy, Department of Pharmacy, Copenhagen, Denmark3Ferring Pharmaceuticals, Copenhagen, DenmarkSummaryMinor changes in the administration technique used for intranasal instillation of clonazepam, have beenfound to influence the results significantly. A simple study was performed, where rabbits received 0.5 mgclonazepam intranasally. One group received the drug while fixed in a sitting position, where the othergroup was fixed in a supine position. The results show that both techniques where able to provide a rapidabsorption with a tmax around 3-4 min. The Cmax and AUC, however, were very different. The Cmax was foundto be 40 ng/ml and 86 ng/ml, respectively, and the AUC was found to be 891 and 2249 (ng/ml/min), respec-tively, for the sitting and the supine position. The relative bioavailability for sitting/supine was found to be38%. These results show that the administration technique is very important and should not be underesti-mated. *Correspondence: Sveinbjörn Gizurarson,University of Iceland, Faculty of PharmacyHofsvallagata 53, 107 Reykjavík, IcelandTel: +354 525 5807Fax: +354 525 4071E-mail: sveinbj@hi.isZJD 78 Two Intranasal  21/03/06  11:11  Side 35Scand. J. Lab. Anim. Sci. 2006 Vol. 33 No. 136analytical grade.The clonazepam was dissolved in a solution con-taining 5% GF in 4EG, by the means of an ultra-sonic bath (5 mg/ml). The formulation was pre-pared fresh prior the study.Sixteen New Zealand White rabbits, weighingabout 3 kg (Novo Nordisk A/S, Bagsværd,Denmark), were divided into two groups. Each rab-bit received 50 µl into each nostril, using anEppendorph pipette.The first group was manually fixed in a supine posi-tion during and for about 1 min after the adminis-tration.The second group was fixed in a sitting positionduring the administration. Blood samples were collected by venepuncture ofthe marginal ear vein prior to dosing and at 5, 10,20, 30 and 60 min. after dosing. Plasma was sepa-rated by centrifugation and stored at -20°C untilanalysed on HPLC.The plasma concentration of clonazepam wasanalysed by the HPLC method described byBechgaard et al. (1997) and calculated on the basisof the peak height, relative to an internal desmethyl-diazepam standard.Standard statistical methods (t-test) were usedthroughout. All pharmacokinetic values were cor-rected, relative to the dose/weight relationship. Thearea under the plasma concentration curve (AUC)was calculated, using the trapezoidal method.Results and DiscussionThe results from the experiments are shown inTable 1 and Fig. 1. The tables show that both tech-niques were equally rapid, relative to the rate ofabsorption (tmax was 2.75 to 4.00 min), but theamount absorbed was greatly affected by the tech-niques. The relative bioavailability (Frel(sitting/supine)) was only 37.7% and the Cmax wasfound to be 36.9 ± 20.3 ng/ml and 85.9 ± 21.8ng/ml for the sitting and supine position, respec-tively. The results were significant (p<0.05) andshow that the right choice of technique has muchmore influence than expected. Various factors may affect the systemic absorptionof the drugs from the nasal cavity such as the anato-my, the mucociliary clearance and the epithelialbarrier in different regions of the nasal cavity.Intranasal administration of a dye showed that thedistribution inside the nose was not the same forthese two techniques (unpublished results), but ithas been described that the location of the druginside the nasal cavity is of importance (Johanssonet al., 1991). Necropsy of rabbits receiving instilla-tion of a dye in a sitting position, show that the dyeis solely found on the septum and the nasal floor.Rabbits, however, fixed in a supine position, hadthe dye distributed all over the middle nasal con-chae as well as the septum and the nasal floor. Theconches are the most vascularised area inside thenasal cavity, which may explain the outcome of theresults. Other factors such as the viscosity, the vol-Table 1.Kinetic parameters for clonazepam in rabbits after single 0.5 mg intranasal dose, using two techniques(n=8).Sitting Supine % differenceposition position (supine/sitting)Dose (µg/kg) 165 ± 10 172 ± 11 4Cmax (ng/ml) 37 ± 20 86 ± 22 233tmax (min) 4,0 ± 4,6 2,8 ± 1,4 69kabs (ng/min) 15 ± 12 38 ± 17 250ke (ug/min) 11 ± 6 15 ± 4 137t0,5 (min) 62 ± 20 45 ± 9 73AUC (ng/ml/min) 891 ± 523 2249 ± 320 253JD 78 Two Intranasal  21/03/06  11:11  Side 36Scand. J. Lab. Anim. Sci. 2006 Vol. 33 No. 1ume of the vehicle, the bend from the nostrils intothe nasal cavity, the droplet size and the spray char-acteristics may also affect where and how far intothe nasal cavity the drug may travel (Harris et al.,1998) and may also affect the results. It is alsoimportant to keep the volume adequate so the drugis inside the nose and not lost in the interior or tothe nasopharynx: too many work has been pub-lished where up to 100 µL have been administered“intranasally” to mice, without checking the totalnasal volume of the animal (which is only 22 µL).In some animal species, it is almost impossible toadminister the formulations intranasally, unless theanimal is kept in a supine position, such as in mice(Egino et al., 1999). Due to the narrow nostrils, thesmall volume administered and the risk of failureduring administration, it is important to keep theanimal in a supine position and allow the animal toassist in receiving the formulation by using eachinhalation to deliver the drug into the nose. A study in humans, using 100-750 µL in one nos-tril, administered either as drops in a supine posi-tion or by spraying (over 2-3 sec with steady pres-sure and gently inhalation by the volunteer) in thesitting position supports these findings (Aoki &Crawley, 1976). Two formulations were tested, 3 or30% HSA, labelled with 99mTc (the 30% solutioncould not be sprayed). Although the results couldnot be translated into recommendations forintranasal dosing, there was an indication thatdroplets administered in a supine position resultedin good distribution for all volumes tested.Surprisingly, in this study, all spray resulted in poordistribution, which may indicate that the techniquewas not optimal. For spraying, the authors used aJencon Repette-injector designed to produce finespray (droplet size not indicated), where the tip washeld horizontally, resting just inside the nasal ori-fice, which may indicate that the majority of thespray did not reach the cavity of the nose. 37Figure 1Plasma concentrations after intranasal administration of 50 µl clonazepam solution (5 mg/ml) into eachnostril. (<) supine position; (®) sitting position.0,0020,0040,0060,0080,00100,00120,000 10 20 30 40 50 60 70Time (min)Plasma conc. (ng/ml) SittingSupineJD 78 Two Intranasal  21/03/06  11:11  Side 37Scand. J. Lab. Anim. Sci. 2006 Vol. 33 No. 138In conclusion, it has been shown that the techniqueused for instilling a drug into the nasal cavity hasmore influence on the outcome than expected. Asmall and simple change in the administration tech-nique may result in higher bioavailability. A factorof 2.5 fold increase in bioavailability was observedin this study.AcknowledgementsThe authors thank Novo Nordisk A/S (Bagsværd,Denmark) for sponsoring this study. Mr. Henrik M.Højrup and Mrs. Marianne Ravn are thanked fortheir skilful technical assistance. ReferencesAoki FY & JCW Crawley: Distribution and removalof human serum albumin-technetium 99minstilled intranasally. Br. J. Clin. Pharmac.1976, 3, 869-878Bechgaard E, S Gizurarson & RK Hjortkjær:Pharmacokinetic and pharmacodynamic respon-se after intranasal administration of diazepam torabbits. J. Pharm. Pharmacol. 1997, 49, 747-750Egino  K, R Lemus & M Karol M: The importanceof the diluent for airway transport of toluenediisocyanate following intranasal dosing ofmice. Inh. Toxicol. 1999, 11, 171-185Gizurarson S: Animal models for intranasal drugdelivery studies. Acta Pharm. Nord 1990, 2,105-122Gizurarson S. The relevance of nasal physiology tothe design of drug absorption studies. Adv.Drug Deliv. Rev. 1993, 11, 329-347Harris AS, E Svensson, ZG Wagner, S Lethagen &IM Nilsson: Effect of viscosity on particle size,deposition and clearance of nasal delivery sys-tems containing desmopressin. J. Pharm. Sci.1988, 77, 405-408Johansson C-J, P Olsson, M Bende, T Carlsson &PO Gunnarsson: Absolute bioavailability ofnicotine applied to different nasal regions. Eur.J. Clin Pharmacol. 1991, 41, 585-588JD 78 Two Intranasal  21/03/06  11:11  Side 38

Two Intranasal Administration Techniques Give Two Different Pharmacokinetic Results

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Two Intranasal Administration Techniques Give Two Different Pharmacokinetic Results

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