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Permeability of porcine nasal mucosa correlated with human nasal absorption
European Journal of Pharmaceutical Sciences 18 (2003) 47–53 www.elsevier.com / locate / ejps
Permeability of porcine nasal mucosa correlated with human nasal absorption ¨ b , Ola Camber c Cecilia Wadell a,b , *, Erik Bjork b
a ¨ ¨ , SE-151 85 Sodertalje ¨ ¨ , Sweden AstraZeneca R& D Sodertalje Department of Pharmacy, Uppsala University, SE-751 23 Uppsala, Sweden c InDex Pharmaceuticals AB, SE-171 77 Stockholm, Sweden
Received 13 June 2002; received in revised form 30 October 2002; accepted 30 October 2002
Abstract The Ussing chamber diffusion system was used as a model to study the apparent permeability across porcine nasal mucosa of eight drugs and molecules with different physicochemical characteristics, namely insulin, lidocaine, nicotine, PEG 4000, propranolol, sumatriptan, melagatran and an amino diether. A weak correlation was found between the apparent permeability coefficients and the corresponding literature data on the fraction absorbed after nasal administration in humans. In the case of passively transported drugs, a closer correlation was found than for the substances where other mechanisms such as carrier-mediated transport or possible efflux were involved. Factors influencing the correlation between in vitro and in vivo data are discussed and the importance of electrophysiological control of the viability status of the excised mucosa is emphasised. Although caution has to be exercised in view of the limitations of the in vitro system, it seems to be a useful tool when evaluating different factors influencing permeability of nasal mucosa. 2002 Elsevier Science B.V. All rights reserved. Keywords: Nasal mucosa; Pig; Ussing chamber; Electrophysiology; Permeability; Human
1. Introduction During the last decade several in vitro models using excised nasal mucosal segments and also monolayers of epithelial cells have been established, providing an opportunity to predict nasal absorption in vivo while avoiding problems associated with more complex whole animals (Schmidt et al., 1998; Werner and Kissel, 1996). These in vitro systems are regarded as convenient when investigating mechanisms of transport, metabolism and formulationrelated issues at an early stage in the drug development process, although factors such as clearance and efflux may influence the degree of correlation with in vivo absorption (Lee et al., 1997). With regard to oral administration, a variety of approaches for predicting absorption from permeability of intestinal cell lines or intestinal segments have been reported in the literature. The Caco-2 cell lines and diffusion chamber studies using rat intestinal segments are *Corresponding author. Tel.: 146-8-553-24947; fax: 146-8-55325080. E-mail address: [email protected] (C. Wadell).
currently used in the drug development process (Artursson et al., 2001; Lee et al., 1997; Stewart et al., 1997; Ungell, 1997). Moreover, various studies have been presented of predictions based on a single physicochemical parameter, e.g., lipophilicity, solubility and pKa , although generally no single method is capable in every respect of predicting drug absorption, especially where complex molecules are involved (Behl et al., 1998). An attempt to combine different parameters led to the implementation of the ‘rule of five’ as a tool for estimating solubility and permeability early on in the discovery process (Lipinski et al., 1997). An alternative approach is theoretical models predicting absorption, including, for instance, the biopharmaceutical drug classification whereby in vitro drug product dissolution and gastrointestinal permeability are correlated with in vivo bioavailability (Amidon et al., 1995) (Winiwarter et al., 1998). Another example is where the dynamic polar van der Waals’ surface area of b-adrenoreceptor antagonists was inversely correlated with permeability in Caco-2 cells and excised rat intestinal segments (Palm et al., 1996). It is of the utmost importance to consider the aim of a nasal absorption study before selecting the experimental
0928-0987 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0928-0987( 02 )00240-3
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model (Gizurarson, 1990; Illum, 1996). Accordingly, comparative screening studies on candidate drugs and enhancers are different from studies designed to give correlations with human data. In in vitro diffusion chamber studies with excised nasal mucosa from laboratory animals, the most reported and popular model seems to be rabbits (Bechgaard et al., 1993; Carstens et al., 1993; Gizurarson et al., 1991; Suzuki et al., 1999). Moreover, studies using mucosa from animals destined for slaughter, i.e., bovine, ovine or porcine mucosa, are seen in the literature (Hosoya et al., 1994; Reardon et al., 1993; Schmidt et al., 2000; Wheatley et al., 1988). The morphological similarities between porcine and human mucosa, combined with ethical considerations, are major reasons for our group to continue using the pig model. A number of studies where in vivo nasal absorption in various animal models is correlated with human data have recently been presented (Lindhardt et al., 2001a; Marttin et al., 1998; Merkus et al., 1991). However, as far as the authors are aware, no studies have been reported on the correlation between in vitro permeability of excised nasal mucosal segments and the corresponding nasal absorption in humans. The aim of the present study was to investigate the correlation between the permeability of porcine nasal mucosa mounted in diffusion chambers to different drug substances and the literature data on the corresponding fraction of drug absorbed after nasal administration in humans.
2. Materials and methods
pyruvate (4.9 mM) (Ungell, 1997). The KBR buffer was filtered through a 0.45-mm filter. The buffer had a pH of 7.4 and the osmolality was 300610 mOsmol / kg. All the chemicals in the KBR buffer were purchased from KEBO, ˚ Spanga (Sweden), or Sigma–Aldrich. Ultima Gold姠 Liquid scintillation cocktail and Soluene-350 were purchased ¨ Sweden). from Chemical Instruments (Lidingo,
2.2. Diffusion chamber set-up The diffusion chamber model used was a Costar vertical diffusion chamber system consisting of 9-mm low-volume chambers, an area available for diffusion of 0.64 cm 2 and a volume of 1.5 ml for each half cell (Precision Instrument Design, Tahoe City, CA, USA), as previously described (Wadell et al., 1999).
2.3. Tissue preparation Porcine nasal cavity mucosa was obtained from Pigham pigs from SQM Beef (Uppsala, Sweden). The cavity mucosa was carefully removed, stored on ice during transportation to the laboratory and thereafter put into a gassed KBR buffer (95% O 2 and 5% CO 2 ) and finally cut into appropriately sized pieces. The mucosal specimens were mounted in the chambers and prewarmed gassed KBR buffer was added, as previously described (Wadell et al., 1999). Throughout the studies the KBR and all other solutions were maintained at 37 8C and stirred using a gas system (95% O 2 and 5% CO 2 ) to provide both oxygenation and mixing of the chamber solution.
2.1. Materials 2.4. Electrophysiological measurements [N-methyl- 3 H]Nicotine (74.8 Ci / mmol, radiochemical purity 95%), nicotine, recombinant human insulin and propranolol hydrochloride were purchased from Sigma– Aldrich Sweden (Stockholm, Sweden). [ 14 C]Polyethylene glycol 4000 (44.8 Ci / mol, radiochemical purity 99.2%), 3 D-[2- H]glucose (16.4 Ci / mmol, radiochemical purity .99.7%) and [ 3 H]sumatriptan (82 Ci / mmol, radiochemical purity 98.8%) were purchased from Amersham–Pharmacia Biotech (Uppsala, Sweden). [ 3 H]Propranolol (21 Ci / mmol, radiochemical purity .97%) and D-[1- 14 C]mannitol (45–60 Ci / mol, radiochemical purity 97%) were purchased from Du Medical Scandinavia (Sollentuna, Sweden). [ 3 H]Melagatran (23.3 mCi / mmol, radiochemical purity 97%), [ 14 C]amino-diether (56.8 Ci / mol, radiochemical purity 98.1%) and [ 14 C]lidocaine (27 Ci / mol, radiochemical purity 92–94%) were obtained from ¨ ¨ ¨ AstraZeneca R&D (Molndal and Sodertalje, Sweden). Kreb’s bicarbonate Ringer’s solution (KBR) was prepared from CaCl 2 (1.25 mM), KCl (4.7 mM), KH 2 PO 4 (0.6 mM), MgSO 4 ?7H 2 O (1.2 mM), NaCl (108 mM), NaHCO 3 (16 mM), Na 2 HPO 4 (1.8 mM), D-glucose (11.5 mM), fumarate (5.4 mM), glutamate (4.9 mM) and
Electrophysiological measurements (potential difference (PD), transmucosal electrical resistance (R m ) and shortcircuit current (Isc )) were made to assess the integrity and viability of the mucosa. A four-electrode system, consisting of a pair of platinum electrodes, was used to pass the current across the membrane and a pair of Ag /AgCl electrodes connected to the agar bridges was used to measure the voltage over the nasal tissue. The computer program used for continuously monitoring and analysing electrophysiological data was developed by AstraZeneca. The voltage response for each pulse (five different current pulses from 230 to 30 mA, duration 235 ms) was measured 200 ms after the current was sent through the segment. In addition, 14 C-labelled mannitol and 3 H-labelled glucose were used as markers as a further control of tissue integrity.
2.5. Permeability studies After approximately 30 min of equilibration, the buffer on both sides of the tissue was replaced with 1.5 ml
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Table 1 Physico-chemical data and apparent permeability coefficients for the substances studied and corresponding data from the literature on the fraction of the dose absorbed following nasal administration to humans Substance
Mw (g / mol)
Amino diether Insulin Lidocaine Melagatran Nicotine Polyethylene glycol Propranolol Sumatriptan
251 5808 236 430 162 4000 259 295
f
log D
1.84 1.63 21.3 1.62 / 0.4 25.1 1.54 log P51.2
pKa
Conc. on the donor side of the chambers (mM)
Fraction absorbed (%)
Papp (310 6 ) (mean6S.D.)
9.3
0.5 18 1 0.25 0.025 0.02 0.1 12
58 a ,1 b 26 c 19 d 56 e 0.5–5 f 109 g 16 h
4964.4 0.0360.01 5268.3 6.262.7 128642 13615 2068 1463.3
7.86 2.0 / 7.0 / 11.5 6.16 / 10.96 – 9.5
a b c d e ¨ ¨ ¨ AstraZeneca R&D Sodertalje, Sweden; Moses et al., 1983; Scavone et al., 1989; Johansson et al., 1991; AstraZeneca R&D Molndal, Sweden; Donovan and Huang, 1998; g Hussain et al., 1980; h Duquesnoy et al., 1998.
prewarmed gassed KBR buffer. In the donor solutions, the concentration of insulin was 1 mg / ml, while the concentration of the radiolabelled substances was 1.0 mCi / ml. The total concentration of each substance in the chambers, including also cold substance for some of the substances, is shown in Table 1. Samples from the donor (20 ml) and the acceptor side (100 ml) of the chambers were withdrawn at various intervals up to 150 min (the acceptor samples were replaced with prewarmed gassed KBR buffer). The withdrawn radiolabelled samples were mixed with 10 ml of scintillation cocktail and the concentration of the substances was determined by liquid scintillation counting ¨ (LSC) using a Packard Tricarb 2100TR (CIAB, Lidingo, Sweden). Insulin was analysed by ELISA using a highly sensitive ELISA kit for insulin from Peninsula Laboratories (EIAH 7303, KeLab, Nacka Strand, Sweden). The apparent permeability (Papp ) between 15 and 150 min after addition of the substance was calculated using the equation: Papp 5 dQ / dt ? 1 /AC0 where dQ / dt is the steady-state appearance rate of the compound on the serosal side, C0 the initial concentration of the compound on the mucosal side of the membranes, and A the surface area of the membrane exposed to the compound. Following the permeability studies on the radiolabelled substances, mass balance studies were performed on random tissue specimens by dissolving the tissue in Soluene (1 ml) at 50 8C, followed by the addition of scintillation cocktail (10 ml). The concentration of the radiolabelled substances in the samples was determined by LSC and the mass balance was calculated using the following equation: Q rec 5 (Cs ?Vdonor 1 Q acceptor ) /(Co ?Vdonor ) 3 100% where Q rec is the total amount of substance recovered at the end of the experiment, Cs is the donor concentration at
the end of the experiment, Co is the initial donor concentration, Vdonor is the donor volume and Q acceptor is the total amount of substance recovered at the acceptor side.
2.6. Statistical analysis All permeability results are expressed as mean values6standard deviations (S.D.). The statistical analysis of the data concerning linear regression was performed using SAS v 8.01, SAS Institute (Cary, NC, USA).
3. Results and discussion In previous studies on permeability of Caco-2 cells and rat intestinal segments, a large number of compounds with different physicochemical data, e.g., lipophilicity, molecular weight and charge, were used and a close correlation between passive drug permeability and oral absorption in humans was observed (Artursson and Karlsson, 1991; ¨ et al., 1997; Yee, 1997). Artursson et al., 1993; Lennernas In the present study, various marketed compounds designed for systemic delivery were chosen, along with compounds investigated in nasal drug delivery research, provided that absorption data in humans were available. Drugs with different fraction absorbed were chosen, and those with an available radiochemical form were preferred. Since the oral route is often the first choice of drug delivery, the number of drugs administered by this route far exceeds the number of nasal administered drugs. The physicochemical properties and the structural diversity of the drugs suitable for nasal administration are also more limited than those of drugs intended for delivery to the gastrointestinal tract. With regard to the properties controlling absorption from the nasal cavity, correlations with nasal absorption have been found for the charge, lipophilicity and molecular weight of the molecules studied (McMartin et al., 1987). The pathways for absorption across the nasal mucosa are
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similar to other epithelia in the body. Of the four main routes available, transcellular passive diffusion is the dominant one, although the paracellular route is preferred for large or ionised molecules (Kimura et al., 1991). Diffusion chamber studies are regarded as useful tools for discriminating between passive and active transport processes (Schmidt et al., 1998). Passive diffusion is indicated by low energy dependency and equal flux rates in either direction, while active transepithelial transport is indicated by saturation kinetics and direction specificity. Briefly, the substances studied were two local anaesthetics: an amino diether and lidocaine, both considered to be transported passively transcellularly. In addition, insulin, a substance transported paracellularly used for diabetes, was studied, followed by nicotine, a diacidic base transported transcellularly by a carrier-mediated transport mechanism (Sayani and Chien, 1996; Schneider et al., 1996). Polyethylene glycol 4000 is commonly used as a non-absorbable marker, indicating damaged tissue (Donovan et al., 1990). It is a highly water-soluble compound, transported passively paracellularly (Donovan and Huang, 1998). Propranolol is a weak base used in the treatment of angina pectoris (b-blocker) and is absorbed rapidly and completely after nasal administration, which thus seems to be as effective as the intravenous route (Hussain et al., 1980). The highly plasma-bound substance is reported to be transported passively transcellularly (Artursson, 1990; Walgren and Walle, 1999), but is shown to be subject to intestinal efflux based on results in Caco-2 models (Chiou et al., 2001). The thrombin inhibitor melagatran, probably transported by the paracellular route due to its peptide-like characteristics, and sumatriptan, a 5HT 1 agonist used in the treatment of migraine and cluster headache, were also used (Duquesnoy et al., 1998; Gustafsson et al., 2001). Hence, substances with different mechanisms of transport were used in the studies. The tissue specimens included in the mean values of the results fulfilled the criteria on electrophysiological data set in earlier studies (Wadell et al., 1999). Briefly, the viability criteria involve the definition of non-viable tissue followed by discarding tissue specimens with a PD higher than (2)3 mV or a short circuit current below 60 mA / cm 2 just before the test solution was added. Physicochemical data on the substances used, literature data on fraction absorbed in humans and the permeability of porcine nasal mucosa to the substances are shown in Table 1. A general approach for choosing the substance concentration in the chambers when running Ussing chamber studies on excised intestinal mucosa—which also seems to be applicable to nasal mucosa—is to relate the substance concentration to oral human doses and compensate for differences in exposure time as well as exposed tissue area. For some substances, no literature data on human doses were found, in which case only radiolabelled substance was used in the Ussing chamber studies, which explains the low concentrations studied (Table 1).
Mass balance calculations showed that no radiolabelled substance was lost during the permeation studies for any of the substances. The correlation between the results for permeability of porcine nasal mucosa mounted in Ussing chambers to the substances and the corresponding literature data for the fraction of drug absorbed after nasal administration in humans is shown in Fig. 1. The correlation coefficient obtained when including propranolol was only 0.42, i.e., no correlation was found at the 95% confidence level. In view of the discussion of possible explanations to the discrepancy between in vivo and in vitro data on propranolol elsewhere in this publication, the substance was excluded from the graph, resulting in a correlation coefficient of 0.81 at the 95% confidence level. It is expected that the straight line will show a downward trend as it progresses, since a level will probably be reached where an increased permeability in vitro will not correspond to an increased absolute bioavailability in vivo, according to the sigmoid curve presented in earlier studies on the Caco-2 cells (Artursson and Karlsson, 1991). However, it was not possible to extrapolate from the straight line using these data. The results indicated that the best correlation was found for passively transported drugs compared to substances such as propranolol and nicotine, where other mechanisms seem to be involved in the transport. The high permeability achieved for polyethylene glycol 4000 combined with the large standard deviation (range: 0.7–35.1310 26 (mean 12.8, Table 1)) was unexpected. Since the apparent permeability of the permeability markers used (glucose and mannitol) and the electrophysiological measurements were unaffected, indicating a viable tissue, one explanation might be that small fractions of the radioactively labelled molecule have
Fig. 1. Literature data on fraction of drugs absorbed (%) (listed in Table 1) after human nasal administration versus apparent permeability data from Ussing chamber studies on porcine nasal mucosa.
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permeated the membrane. A more distinct and useful curve showing the correlation between the permeability of nasal tissue to drugs and their absorption in vivo may be possible, if future studies include some of the drugs such as hydroxycobolamine, midazolam, diazepam, estradiol and morphine which have recently been investigated for nasal administration in humans (Dooley et al., 2001; Illum et al., 2002; Knoester et al., 2002; Lindhardt et al., 2001b; Lonterman et al., 2000; Slot et al., 1997). In the case of propranolol, complete human absorption has been reported and accordingly the permeability achieved in vitro was lower than expected. Nevertheless, the result was comparable to permeability data in rabbit nasal mucosa (Kubo et al., 1994). In addition, large variability is seen on permeability data from intestinal cell lines and intestinal segments reported for this substance by different research groups (unpublished data). Moreover, as already mentioned, propranolol has been shown to be subject to efflux mechanisms in Caco-2 cell studies (Chiou et al., 2001). Chiou et al. also showed that the in vivo oral absorption of propranolol did not seem to be impeded by efflux and that a discrepancy might therefore exist between in vitro and in vivo results. On the other hand, propranolol is a highly permeable substance, i.e., the discrepancy is probably less significant than for drugs of low or moderate permeability. The relevance for nasal studies of these results on intestinal absorption is unclear. Nevertheless, provided that it is the p-glycoprotein that is involved in the efflux, a few studies have demonstrated the presence of this efflux protein in human nasal mucosa, although no comparison between the level of expression between nasal and intestinal mucosa has been made (Henriksson et al., 1997; Wioland et al., 2000). The (to some extent) deviating result achieved for nicotine might be explained by the fact that the substance is transported by a carrier-mediated mechanism. Possible explanations for a weak correlation for actively transported substances include some limitations for the in vitro system, i.e., a lower supply of co-factors to the transport proteins, a diminished concentration gradient across the excised mucosa and species differences in the expression of transport ¨ et al., 1997). proteins (Lennernas A disadvantage of the Ussing technique in particular and the use of in vitro techniques in general is the unphysiological diffusion pathways. Due to the lack of vascular supply, the molecules are forced to diffuse through the lamina propria (Ungell, 1997). Differences between permeability in vitro and bioavailability in humans might also be due to the fact that the sink conditions may be absent or functioning less well in the in vitro model and the possible difficulties with the unstirred water layers, which may be a potential diffusion barrier ¨ et al., especially for lipophilic drugs in vitro (Lennernas 1997). Contradictory results between in vitro studies and in vivo human studies may also be explained by decreased
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viability of the tissue in the in vitro study, with increased ¨ 1994). Accordpermeability as a consequence (Lennernas, ing to earlier discussion, the viability and integrity of the segments used was monitored by electrophysiological measurements and segments that did not fulfil the preset requirements were excluded (Wadell et al., 1999). Continuous measurement of electrophysiological data controlled and monitored in a computer program seems to be a reliable tool for keeping the viability status of the porcine mucosa under control. The automated electrophysiological data control system offers several advantages compared to the voltage clamp technique often used, i.e., less harmful to the tissue, more reliable and enabling continuous measurement and monitoring throughout the permeability studies (Bechgaard et al., 1992; Reardon et al., 1993).
4. Conclusions This is the first study comparing the apparent permeability of excised porcine mucosa with data from nasal absorption studies in humans. A weak correlation was found (a correlation coefficient of 0.81 at the 95% confidence level), probably mainly as a result of the limited number of substances available for comparative studies. In the case of passively transported drugs, a closer correlation was found compared to the substances where other mechanisms such as carrier-mediated transport or possible efflux were involved. Additional studies are needed, involving other compounds, in order to further investigate the influence of different mechanisms of transport on the correlation coefficient. In spite of the general limitations of an in vitro system, this model seems to be a useful tool when evaluating different factors influencing permeability of nasal mucosa.
Acknowledgements The authors would like to thank Bert Lindberg, SQM Beef, Uppsala, Sweden, Kristina Karlsson and Johanna Lindgren for excellent technical assistance. The authors would also like to thank Anna-Lena Ungell, Astra Zeneca ¨ R&D Molndal, Sweden for valuable scientific discussions.
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C. Wadell et al. / European Journal of Pharmaceutical Sciences 18 (2003) 47–53 Schmidt, M.C., Simmen, D., Hilbe, M., Boderke, P., Ditzinger, G., Sandow, J., Lang, S.R., Rubas, W., Merkle, H.P., 2000. Validation of excised bovine nasal mucosa as in vitro model to study drug transport and metabolic pathways in nasal epithelium. J. Pharm. Sci. 89 (3), 396–407. ¨ Schneider, N.G., Lunell, E., Olmstead, R.E., Fagerstrom, K.-O., 1996. Clinical pharmacokinetics of nasal nicotine delivery. A review and comparison to other nicotine systems. Clin. Pharmacokinet. 31 (1), 56–80. Slot, W.B., Merkus, F.W.H.M., Deventer, S.J.H.v., Tytgat, G.N.J., 1997. Normalization of plasma vitamin B12 concentration by intranasal hydroxycobolamin in vitamin B12-deficient patients. Gastroenterology 113, 430–433. Stewart, B.H., Chan, O.H., Jezyk, N., Fleisher, D., 1997. Discrimination between drug candidates using models for evaluation of intestinal absorption. Adv. Drug Del. Rev. 23, 27–45. Suzuki, K., Kawahara, K., Terada, N., Nomura, T., Konno, A., Fukuda, Y., 1999. Effect of ion transport inhibitors and methacholine on short-circuit current of isolated guinea pig nasal epithelium. Jpn. J. Physiol. 49, 99–106. Ungell, A.-L., 1997. In vitro absorption studies and their relevance to absorption from the GI tract. Drug Dev. Ind. Pharm. 23 (9), 879–892. ¨ Wadell, C., Bjork, E., Camber, O., 1999. Nasal drug delivery-evaluation
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of an in vitro method using porcine nasal mucosa. Eur. J. Pharm. Sci. 7, 197–206. Walgren, R.A., Walle, T., 1999. The influence of plasma binding on absorption / exsorption in the Caco-2 model of human intestinal absorption. J. Pharm. Pharmacol. 51, 1037–1040. Werner, U., Kissel, T., 1996. In-vitro cell culture models of the nasal epithelium: a comparative histochemical investigation of their suitability for drug transport studies. Pharm. Res. 13 (7), 978–988. Wheatley, M.A., Dent, J., Wheeldon, E.B., Smith, P.L., 1988. Nasal drug delivery: an in vitro characterization of transepithelial electrical properties and fluxes in the presence or absence of enhancers. J. Control. Release 8, 167–177. ¨ H., Winiwarter, S., Bonham, N.M., Ax, F., Hallberg, A., Lennernas, ´ A., 1998. Correlation of human jejunal permeability (in vivo) Karlen, of drugs with experimentally and theoretically derived parameters. A multivariate data analysis approach. J. Med. Chem. 41, 4939–4949. Wioland, M.-A., Fleury-Feith, J., Corlieu, P., Commo, F., Monceaux, G., Lacau-St-Guily, J., Bernaudin, J.-F., 2000. CFTR, MDR1 and MRP1 immunolocalization in normal human nasal respiratory mucosa. J. Histochem. Cytochem. 48 (9), 1215–1222. Yee, S., 1997. In vitro permeability across caco-2 cells (colonic) can predict in vivo (small intestinal) absorption in man-fact or myth. Pharm. Res. 14 (6), 763–766.
Permeability of porcine nasal mucosa correlated with human nasal absorption ¨ b , Ola Camber c Cecilia Wadell a,b , *, Erik Bjork b
a ¨ ¨ , SE-151 85 Sodertalje ¨ ¨ , Sweden AstraZeneca R& D Sodertalje Department of Pharmacy, Uppsala University, SE-751 23 Uppsala, Sweden c InDex Pharmaceuticals AB, SE-171 77 Stockholm, Sweden
Received 13 June 2002; received in revised form 30 October 2002; accepted 30 October 2002
Abstract The Ussing chamber diffusion system was used as a model to study the apparent permeability across porcine nasal mucosa of eight drugs and molecules with different physicochemical characteristics, namely insulin, lidocaine, nicotine, PEG 4000, propranolol, sumatriptan, melagatran and an amino diether. A weak correlation was found between the apparent permeability coefficients and the corresponding literature data on the fraction absorbed after nasal administration in humans. In the case of passively transported drugs, a closer correlation was found than for the substances where other mechanisms such as carrier-mediated transport or possible efflux were involved. Factors influencing the correlation between in vitro and in vivo data are discussed and the importance of electrophysiological control of the viability status of the excised mucosa is emphasised. Although caution has to be exercised in view of the limitations of the in vitro system, it seems to be a useful tool when evaluating different factors influencing permeability of nasal mucosa. 2002 Elsevier Science B.V. All rights reserved. Keywords: Nasal mucosa; Pig; Ussing chamber; Electrophysiology; Permeability; Human
1. Introduction During the last decade several in vitro models using excised nasal mucosal segments and also monolayers of epithelial cells have been established, providing an opportunity to predict nasal absorption in vivo while avoiding problems associated with more complex whole animals (Schmidt et al., 1998; Werner and Kissel, 1996). These in vitro systems are regarded as convenient when investigating mechanisms of transport, metabolism and formulationrelated issues at an early stage in the drug development process, although factors such as clearance and efflux may influence the degree of correlation with in vivo absorption (Lee et al., 1997). With regard to oral administration, a variety of approaches for predicting absorption from permeability of intestinal cell lines or intestinal segments have been reported in the literature. The Caco-2 cell lines and diffusion chamber studies using rat intestinal segments are *Corresponding author. Tel.: 146-8-553-24947; fax: 146-8-55325080. E-mail address: [email protected] (C. Wadell).
currently used in the drug development process (Artursson et al., 2001; Lee et al., 1997; Stewart et al., 1997; Ungell, 1997). Moreover, various studies have been presented of predictions based on a single physicochemical parameter, e.g., lipophilicity, solubility and pKa , although generally no single method is capable in every respect of predicting drug absorption, especially where complex molecules are involved (Behl et al., 1998). An attempt to combine different parameters led to the implementation of the ‘rule of five’ as a tool for estimating solubility and permeability early on in the discovery process (Lipinski et al., 1997). An alternative approach is theoretical models predicting absorption, including, for instance, the biopharmaceutical drug classification whereby in vitro drug product dissolution and gastrointestinal permeability are correlated with in vivo bioavailability (Amidon et al., 1995) (Winiwarter et al., 1998). Another example is where the dynamic polar van der Waals’ surface area of b-adrenoreceptor antagonists was inversely correlated with permeability in Caco-2 cells and excised rat intestinal segments (Palm et al., 1996). It is of the utmost importance to consider the aim of a nasal absorption study before selecting the experimental
0928-0987 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0928-0987( 02 )00240-3
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model (Gizurarson, 1990; Illum, 1996). Accordingly, comparative screening studies on candidate drugs and enhancers are different from studies designed to give correlations with human data. In in vitro diffusion chamber studies with excised nasal mucosa from laboratory animals, the most reported and popular model seems to be rabbits (Bechgaard et al., 1993; Carstens et al., 1993; Gizurarson et al., 1991; Suzuki et al., 1999). Moreover, studies using mucosa from animals destined for slaughter, i.e., bovine, ovine or porcine mucosa, are seen in the literature (Hosoya et al., 1994; Reardon et al., 1993; Schmidt et al., 2000; Wheatley et al., 1988). The morphological similarities between porcine and human mucosa, combined with ethical considerations, are major reasons for our group to continue using the pig model. A number of studies where in vivo nasal absorption in various animal models is correlated with human data have recently been presented (Lindhardt et al., 2001a; Marttin et al., 1998; Merkus et al., 1991). However, as far as the authors are aware, no studies have been reported on the correlation between in vitro permeability of excised nasal mucosal segments and the corresponding nasal absorption in humans. The aim of the present study was to investigate the correlation between the permeability of porcine nasal mucosa mounted in diffusion chambers to different drug substances and the literature data on the corresponding fraction of drug absorbed after nasal administration in humans.
2. Materials and methods
pyruvate (4.9 mM) (Ungell, 1997). The KBR buffer was filtered through a 0.45-mm filter. The buffer had a pH of 7.4 and the osmolality was 300610 mOsmol / kg. All the chemicals in the KBR buffer were purchased from KEBO, ˚ Spanga (Sweden), or Sigma–Aldrich. Ultima Gold姠 Liquid scintillation cocktail and Soluene-350 were purchased ¨ Sweden). from Chemical Instruments (Lidingo,
2.2. Diffusion chamber set-up The diffusion chamber model used was a Costar vertical diffusion chamber system consisting of 9-mm low-volume chambers, an area available for diffusion of 0.64 cm 2 and a volume of 1.5 ml for each half cell (Precision Instrument Design, Tahoe City, CA, USA), as previously described (Wadell et al., 1999).
2.3. Tissue preparation Porcine nasal cavity mucosa was obtained from Pigham pigs from SQM Beef (Uppsala, Sweden). The cavity mucosa was carefully removed, stored on ice during transportation to the laboratory and thereafter put into a gassed KBR buffer (95% O 2 and 5% CO 2 ) and finally cut into appropriately sized pieces. The mucosal specimens were mounted in the chambers and prewarmed gassed KBR buffer was added, as previously described (Wadell et al., 1999). Throughout the studies the KBR and all other solutions were maintained at 37 8C and stirred using a gas system (95% O 2 and 5% CO 2 ) to provide both oxygenation and mixing of the chamber solution.
2.1. Materials 2.4. Electrophysiological measurements [N-methyl- 3 H]Nicotine (74.8 Ci / mmol, radiochemical purity 95%), nicotine, recombinant human insulin and propranolol hydrochloride were purchased from Sigma– Aldrich Sweden (Stockholm, Sweden). [ 14 C]Polyethylene glycol 4000 (44.8 Ci / mol, radiochemical purity 99.2%), 3 D-[2- H]glucose (16.4 Ci / mmol, radiochemical purity .99.7%) and [ 3 H]sumatriptan (82 Ci / mmol, radiochemical purity 98.8%) were purchased from Amersham–Pharmacia Biotech (Uppsala, Sweden). [ 3 H]Propranolol (21 Ci / mmol, radiochemical purity .97%) and D-[1- 14 C]mannitol (45–60 Ci / mol, radiochemical purity 97%) were purchased from Du Medical Scandinavia (Sollentuna, Sweden). [ 3 H]Melagatran (23.3 mCi / mmol, radiochemical purity 97%), [ 14 C]amino-diether (56.8 Ci / mol, radiochemical purity 98.1%) and [ 14 C]lidocaine (27 Ci / mol, radiochemical purity 92–94%) were obtained from ¨ ¨ ¨ AstraZeneca R&D (Molndal and Sodertalje, Sweden). Kreb’s bicarbonate Ringer’s solution (KBR) was prepared from CaCl 2 (1.25 mM), KCl (4.7 mM), KH 2 PO 4 (0.6 mM), MgSO 4 ?7H 2 O (1.2 mM), NaCl (108 mM), NaHCO 3 (16 mM), Na 2 HPO 4 (1.8 mM), D-glucose (11.5 mM), fumarate (5.4 mM), glutamate (4.9 mM) and
Electrophysiological measurements (potential difference (PD), transmucosal electrical resistance (R m ) and shortcircuit current (Isc )) were made to assess the integrity and viability of the mucosa. A four-electrode system, consisting of a pair of platinum electrodes, was used to pass the current across the membrane and a pair of Ag /AgCl electrodes connected to the agar bridges was used to measure the voltage over the nasal tissue. The computer program used for continuously monitoring and analysing electrophysiological data was developed by AstraZeneca. The voltage response for each pulse (five different current pulses from 230 to 30 mA, duration 235 ms) was measured 200 ms after the current was sent through the segment. In addition, 14 C-labelled mannitol and 3 H-labelled glucose were used as markers as a further control of tissue integrity.
2.5. Permeability studies After approximately 30 min of equilibration, the buffer on both sides of the tissue was replaced with 1.5 ml
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Table 1 Physico-chemical data and apparent permeability coefficients for the substances studied and corresponding data from the literature on the fraction of the dose absorbed following nasal administration to humans Substance
Mw (g / mol)
Amino diether Insulin Lidocaine Melagatran Nicotine Polyethylene glycol Propranolol Sumatriptan
251 5808 236 430 162 4000 259 295
f
log D
1.84 1.63 21.3 1.62 / 0.4 25.1 1.54 log P51.2
pKa
Conc. on the donor side of the chambers (mM)
Fraction absorbed (%)
Papp (310 6 ) (mean6S.D.)
9.3
0.5 18 1 0.25 0.025 0.02 0.1 12
58 a ,1 b 26 c 19 d 56 e 0.5–5 f 109 g 16 h
4964.4 0.0360.01 5268.3 6.262.7 128642 13615 2068 1463.3
7.86 2.0 / 7.0 / 11.5 6.16 / 10.96 – 9.5
a b c d e ¨ ¨ ¨ AstraZeneca R&D Sodertalje, Sweden; Moses et al., 1983; Scavone et al., 1989; Johansson et al., 1991; AstraZeneca R&D Molndal, Sweden; Donovan and Huang, 1998; g Hussain et al., 1980; h Duquesnoy et al., 1998.
prewarmed gassed KBR buffer. In the donor solutions, the concentration of insulin was 1 mg / ml, while the concentration of the radiolabelled substances was 1.0 mCi / ml. The total concentration of each substance in the chambers, including also cold substance for some of the substances, is shown in Table 1. Samples from the donor (20 ml) and the acceptor side (100 ml) of the chambers were withdrawn at various intervals up to 150 min (the acceptor samples were replaced with prewarmed gassed KBR buffer). The withdrawn radiolabelled samples were mixed with 10 ml of scintillation cocktail and the concentration of the substances was determined by liquid scintillation counting ¨ (LSC) using a Packard Tricarb 2100TR (CIAB, Lidingo, Sweden). Insulin was analysed by ELISA using a highly sensitive ELISA kit for insulin from Peninsula Laboratories (EIAH 7303, KeLab, Nacka Strand, Sweden). The apparent permeability (Papp ) between 15 and 150 min after addition of the substance was calculated using the equation: Papp 5 dQ / dt ? 1 /AC0 where dQ / dt is the steady-state appearance rate of the compound on the serosal side, C0 the initial concentration of the compound on the mucosal side of the membranes, and A the surface area of the membrane exposed to the compound. Following the permeability studies on the radiolabelled substances, mass balance studies were performed on random tissue specimens by dissolving the tissue in Soluene (1 ml) at 50 8C, followed by the addition of scintillation cocktail (10 ml). The concentration of the radiolabelled substances in the samples was determined by LSC and the mass balance was calculated using the following equation: Q rec 5 (Cs ?Vdonor 1 Q acceptor ) /(Co ?Vdonor ) 3 100% where Q rec is the total amount of substance recovered at the end of the experiment, Cs is the donor concentration at
the end of the experiment, Co is the initial donor concentration, Vdonor is the donor volume and Q acceptor is the total amount of substance recovered at the acceptor side.
2.6. Statistical analysis All permeability results are expressed as mean values6standard deviations (S.D.). The statistical analysis of the data concerning linear regression was performed using SAS v 8.01, SAS Institute (Cary, NC, USA).
3. Results and discussion In previous studies on permeability of Caco-2 cells and rat intestinal segments, a large number of compounds with different physicochemical data, e.g., lipophilicity, molecular weight and charge, were used and a close correlation between passive drug permeability and oral absorption in humans was observed (Artursson and Karlsson, 1991; ¨ et al., 1997; Yee, 1997). Artursson et al., 1993; Lennernas In the present study, various marketed compounds designed for systemic delivery were chosen, along with compounds investigated in nasal drug delivery research, provided that absorption data in humans were available. Drugs with different fraction absorbed were chosen, and those with an available radiochemical form were preferred. Since the oral route is often the first choice of drug delivery, the number of drugs administered by this route far exceeds the number of nasal administered drugs. The physicochemical properties and the structural diversity of the drugs suitable for nasal administration are also more limited than those of drugs intended for delivery to the gastrointestinal tract. With regard to the properties controlling absorption from the nasal cavity, correlations with nasal absorption have been found for the charge, lipophilicity and molecular weight of the molecules studied (McMartin et al., 1987). The pathways for absorption across the nasal mucosa are
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similar to other epithelia in the body. Of the four main routes available, transcellular passive diffusion is the dominant one, although the paracellular route is preferred for large or ionised molecules (Kimura et al., 1991). Diffusion chamber studies are regarded as useful tools for discriminating between passive and active transport processes (Schmidt et al., 1998). Passive diffusion is indicated by low energy dependency and equal flux rates in either direction, while active transepithelial transport is indicated by saturation kinetics and direction specificity. Briefly, the substances studied were two local anaesthetics: an amino diether and lidocaine, both considered to be transported passively transcellularly. In addition, insulin, a substance transported paracellularly used for diabetes, was studied, followed by nicotine, a diacidic base transported transcellularly by a carrier-mediated transport mechanism (Sayani and Chien, 1996; Schneider et al., 1996). Polyethylene glycol 4000 is commonly used as a non-absorbable marker, indicating damaged tissue (Donovan et al., 1990). It is a highly water-soluble compound, transported passively paracellularly (Donovan and Huang, 1998). Propranolol is a weak base used in the treatment of angina pectoris (b-blocker) and is absorbed rapidly and completely after nasal administration, which thus seems to be as effective as the intravenous route (Hussain et al., 1980). The highly plasma-bound substance is reported to be transported passively transcellularly (Artursson, 1990; Walgren and Walle, 1999), but is shown to be subject to intestinal efflux based on results in Caco-2 models (Chiou et al., 2001). The thrombin inhibitor melagatran, probably transported by the paracellular route due to its peptide-like characteristics, and sumatriptan, a 5HT 1 agonist used in the treatment of migraine and cluster headache, were also used (Duquesnoy et al., 1998; Gustafsson et al., 2001). Hence, substances with different mechanisms of transport were used in the studies. The tissue specimens included in the mean values of the results fulfilled the criteria on electrophysiological data set in earlier studies (Wadell et al., 1999). Briefly, the viability criteria involve the definition of non-viable tissue followed by discarding tissue specimens with a PD higher than (2)3 mV or a short circuit current below 60 mA / cm 2 just before the test solution was added. Physicochemical data on the substances used, literature data on fraction absorbed in humans and the permeability of porcine nasal mucosa to the substances are shown in Table 1. A general approach for choosing the substance concentration in the chambers when running Ussing chamber studies on excised intestinal mucosa—which also seems to be applicable to nasal mucosa—is to relate the substance concentration to oral human doses and compensate for differences in exposure time as well as exposed tissue area. For some substances, no literature data on human doses were found, in which case only radiolabelled substance was used in the Ussing chamber studies, which explains the low concentrations studied (Table 1).
Mass balance calculations showed that no radiolabelled substance was lost during the permeation studies for any of the substances. The correlation between the results for permeability of porcine nasal mucosa mounted in Ussing chambers to the substances and the corresponding literature data for the fraction of drug absorbed after nasal administration in humans is shown in Fig. 1. The correlation coefficient obtained when including propranolol was only 0.42, i.e., no correlation was found at the 95% confidence level. In view of the discussion of possible explanations to the discrepancy between in vivo and in vitro data on propranolol elsewhere in this publication, the substance was excluded from the graph, resulting in a correlation coefficient of 0.81 at the 95% confidence level. It is expected that the straight line will show a downward trend as it progresses, since a level will probably be reached where an increased permeability in vitro will not correspond to an increased absolute bioavailability in vivo, according to the sigmoid curve presented in earlier studies on the Caco-2 cells (Artursson and Karlsson, 1991). However, it was not possible to extrapolate from the straight line using these data. The results indicated that the best correlation was found for passively transported drugs compared to substances such as propranolol and nicotine, where other mechanisms seem to be involved in the transport. The high permeability achieved for polyethylene glycol 4000 combined with the large standard deviation (range: 0.7–35.1310 26 (mean 12.8, Table 1)) was unexpected. Since the apparent permeability of the permeability markers used (glucose and mannitol) and the electrophysiological measurements were unaffected, indicating a viable tissue, one explanation might be that small fractions of the radioactively labelled molecule have
Fig. 1. Literature data on fraction of drugs absorbed (%) (listed in Table 1) after human nasal administration versus apparent permeability data from Ussing chamber studies on porcine nasal mucosa.
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permeated the membrane. A more distinct and useful curve showing the correlation between the permeability of nasal tissue to drugs and their absorption in vivo may be possible, if future studies include some of the drugs such as hydroxycobolamine, midazolam, diazepam, estradiol and morphine which have recently been investigated for nasal administration in humans (Dooley et al., 2001; Illum et al., 2002; Knoester et al., 2002; Lindhardt et al., 2001b; Lonterman et al., 2000; Slot et al., 1997). In the case of propranolol, complete human absorption has been reported and accordingly the permeability achieved in vitro was lower than expected. Nevertheless, the result was comparable to permeability data in rabbit nasal mucosa (Kubo et al., 1994). In addition, large variability is seen on permeability data from intestinal cell lines and intestinal segments reported for this substance by different research groups (unpublished data). Moreover, as already mentioned, propranolol has been shown to be subject to efflux mechanisms in Caco-2 cell studies (Chiou et al., 2001). Chiou et al. also showed that the in vivo oral absorption of propranolol did not seem to be impeded by efflux and that a discrepancy might therefore exist between in vitro and in vivo results. On the other hand, propranolol is a highly permeable substance, i.e., the discrepancy is probably less significant than for drugs of low or moderate permeability. The relevance for nasal studies of these results on intestinal absorption is unclear. Nevertheless, provided that it is the p-glycoprotein that is involved in the efflux, a few studies have demonstrated the presence of this efflux protein in human nasal mucosa, although no comparison between the level of expression between nasal and intestinal mucosa has been made (Henriksson et al., 1997; Wioland et al., 2000). The (to some extent) deviating result achieved for nicotine might be explained by the fact that the substance is transported by a carrier-mediated mechanism. Possible explanations for a weak correlation for actively transported substances include some limitations for the in vitro system, i.e., a lower supply of co-factors to the transport proteins, a diminished concentration gradient across the excised mucosa and species differences in the expression of transport ¨ et al., 1997). proteins (Lennernas A disadvantage of the Ussing technique in particular and the use of in vitro techniques in general is the unphysiological diffusion pathways. Due to the lack of vascular supply, the molecules are forced to diffuse through the lamina propria (Ungell, 1997). Differences between permeability in vitro and bioavailability in humans might also be due to the fact that the sink conditions may be absent or functioning less well in the in vitro model and the possible difficulties with the unstirred water layers, which may be a potential diffusion barrier ¨ et al., especially for lipophilic drugs in vitro (Lennernas 1997). Contradictory results between in vitro studies and in vivo human studies may also be explained by decreased
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viability of the tissue in the in vitro study, with increased ¨ 1994). Accordpermeability as a consequence (Lennernas, ing to earlier discussion, the viability and integrity of the segments used was monitored by electrophysiological measurements and segments that did not fulfil the preset requirements were excluded (Wadell et al., 1999). Continuous measurement of electrophysiological data controlled and monitored in a computer program seems to be a reliable tool for keeping the viability status of the porcine mucosa under control. The automated electrophysiological data control system offers several advantages compared to the voltage clamp technique often used, i.e., less harmful to the tissue, more reliable and enabling continuous measurement and monitoring throughout the permeability studies (Bechgaard et al., 1992; Reardon et al., 1993).
4. Conclusions This is the first study comparing the apparent permeability of excised porcine mucosa with data from nasal absorption studies in humans. A weak correlation was found (a correlation coefficient of 0.81 at the 95% confidence level), probably mainly as a result of the limited number of substances available for comparative studies. In the case of passively transported drugs, a closer correlation was found compared to the substances where other mechanisms such as carrier-mediated transport or possible efflux were involved. Additional studies are needed, involving other compounds, in order to further investigate the influence of different mechanisms of transport on the correlation coefficient. In spite of the general limitations of an in vitro system, this model seems to be a useful tool when evaluating different factors influencing permeability of nasal mucosa.
Acknowledgements The authors would like to thank Bert Lindberg, SQM Beef, Uppsala, Sweden, Kristina Karlsson and Johanna Lindgren for excellent technical assistance. The authors would also like to thank Anna-Lena Ungell, Astra Zeneca ¨ R&D Molndal, Sweden for valuable scientific discussions.
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