In vitro, ex vivo and in vivo examination of buccal absorption of metoprolol with varying pH in TR146 cell culture, porcine buccal mucosa and Göttingen minipigs

In vitro, ex vivo and in vivo examination of buccal absorption of metoprolol with varying pH in TR146 cell culture, porcine buccal mucosa and Göttingen minipigs

European Journal of Pharmaceutical Sciences 49 (2013) 117–124 Contents lists available at SciVerse ScienceDirect European Journal of Pharmaceutical ...

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European Journal of Pharmaceutical Sciences 49 (2013) 117–124

Contents lists available at SciVerse ScienceDirect

European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps

In vitro, ex vivo and in vivo examination of buccal absorption of metoprolol with varying pH in TR146 cell culture, porcine buccal mucosa and Göttingen minipigs René Holm a,b,⇑, Emil Meng-Lund b, Morten B. Andersen a,b, Mads L. Jespersen a,b, Jens-Jacob Karlsson c, Mats Garmer c, Erling B. Jørgensen a, Jette Jacobsen b a

Biologics and Pharmaceutical Science, H. Lundbeck A/S, Ottiliavej 9, DK-2500 Valby, Denmark Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark c Department of Discovery DMPK, H. Lundbeck A/S, Ottiliavej 9, DK-2500 Valby, Denmark b

a r t i c l e

i n f o

Article history: Received 29 December 2012 Received in revised form 21 February 2013 Accepted 24 February 2013 Available online 14 March 2013 Keywords: Metoprolol Buccal absorption IVIVC Mini-pigs pH TR146 cell culture

a b s t r a c t This work studied the buccal absorption of metoprolol in vitro, ex vivo and in vivo as a function of buffered pH at 7.4, 8.5, 9.0 and 9.5. Permeability studies showed a correlation (r2 = 0.92) between in vitro TR146 cell culture and ex vivo porcine buccal mucosa in a modified Ussing chamber. A higher apparent permeability was observed at higher pH values, i.e. the more compound that was unionised the higher the permeability. In vivo studies were conducted in anaesthetised Göttingen mini-pigs. A clear influence of pH on the absorption was seen and a significant higher absolute bioavailability was obtained after buccal dosing (58–107%) compared to oral (3%) administration, ranging 58–107% and 3%, respectively. Macroscopically, no local toxic effects were observed by visual inspection of mini-pig cheeks. A very clear level C in vitro in vivo correlation (r2 = 0.98) was obtained between the observed in vitro permeabilities and the bioavailability observed in vivo, suggesting that the two in vitro models have good predictive power for drug delivery, which could be a useful tool for future formulation developments intended for buccal delivery. Ó 2013 Elsevier B.V. All rights reserved.

1. Introduction The pharmaceutical industry constantly faces the challenge of providing efficient treatment delivery options that are acceptable to patients and physicians. The high patient compliance associated with oral administration makes this the primary route of drug delivery, so a novel drug delivery system must offer better therapeutic outcomes compared to e.g., to oral administration. Oral drug administration of drugs may be limited by hepatic first pass metabolism and the risk of acid or enzymatic degradation in the gastrointestinal tract (GI) (Devries et al., 1991; Pathan et al., 2008). The use of buccal drug delivery, i.e., drug administration through the mucosal membrane lining the cheeks is a way to bypass these problems. Drugs are immediately drained into the reticulated vein, which directly accesses the systemic circulation (Pathan et al., 2008), thereby bypassing hepatic first pass metabolism and avoiding presystemic degradation in the GI tract (Devries et al., 1991; Mao et al., 2009; Pathan et al., 2008). Buccal delivery can be used for both systemic and local treatment.

⇑ Corresponding author at: Biologics and Pharmaceutical Science, H. Lundbeck A/S, Ottiliavej 9, DK-2500 Valby, Denmark. Tel.: +45 3643 3596; fax: +45 3643 8242. E-mail address: [email protected] (R. Holm). 0928-0987/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejps.2013.02.024

A key issue for buccal absorption is the permeability of the drugs through the buccal mucosa. A basic assumption is that the permeability and the local environment adjacent to the mucosa can be changed to enhance drug permeation (Gibaldi and Kanig, 1965; Pathan et al., 2008). Most drugs delivered successfully via the buccal route are small dose molecules with a log P ranging from 1.6 to 3.3 whereas large hydrophilic molecules are generally poorly absorbed (Smart, 2005). Log P is by definition the logarithmic ratio for the oil:water partition ratio of the unionised form of the molecule. Many compounds are ionised at physiological pH, which is why a more suitable way of evaluating the ionisation of compounds is by using their log D values. When determining log D the oil:aqueous buffer partitioning is done at a defined pH, i.e. a proportion of the compound may be ionised depending on the pKa and pH. Consequently, the degree of ionisation of a compound, consequently, influences the relative lipophilicity, which is an important parameter for oral (Shore et al., 1957) and buccal absorption (Shojaei et al., 1998). The influence of the degree of ionisation on the permeability of a drug has been tested in numerous in vitro studies (Consuelo et al., 2005; Coutel-Egros et al., 1992; Deneer et al., 2002; Tavakoli-Saberi and Audus, 1989; Wang et al., 2009). Common to these studies, buccal absorption depends on the degree of ionisation of the drug. The in vitro TR146 cell line has been proposed as a model of the human

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buccal epithelium (Jacobsen et al., 1995, 1996; Nielsen and Rassing, 1999, 2000a,b). The TR146 cells originate from a human buccal carcinoma (Rupniak et al., 1985), and when cultured it forms an epithelium resembling that of the buccal mucosa (Jacobsen et al., 1995), with the appropriate differentiation patterns seen in human non-keratinized epithelium (Jacobsen et al., 1999). However, the TR146 cell culture model is less of a barrier compared to human and porcine buccal epithelium, as demonstrated by a significantly greater permeability to tritiated water, mannitol, testosterone, dextrans, and nicotine (Nielsen and Rassing, 1999, 2000b, 2002; Nielsen et al., 1999), which may be due to the cancerous nature of the original cells. The ability of the TR146 cell line to differentiate between formulation systems as a predictor of the in vivo absorption relative to an ex vivo porcine model lacks investigation. In vivo studies in dogs and humans (Gul et al., 2007; Streisand et al., 1995; Zhang et al., 2002) have also confirmed that buccal absorption of drugs depends on the degree of ionisation. However, contradictory results have been published (Wang et al., 2010; Zhang et al., 1989) and generally the studies in vivo are affected by the experimental difficulties in conducting such studies. When conducting buccal bioavailability studies in animals, it is essential that the animal and the human oromucosa are similar. Since the morphology and composition of porcine oromucosa have been reported to be similar to man (Patel et al., 2012; Shojaei, 1998; Squier and Hall, 1985), pigs have been selected as the species used in the present study. To our knowledge, drug bioavailability in minipigs upon buccal administration as a function of the ionisation degree has, to our knowledge, not been evaluated nor has the correlation between the in vitro buccal permeability and in vivo bioavailability been addressed. The Food and Drug Administration (FDA) uses metoprolol to define high solubility and high permeability in oral administration (Dahan et al., 2010), while a similar definition does not exist for buccal drug delivery. There are several reasons why metoprolol is potentially a good model compound for buccal delivery; (i) a potent drug, i.e. low dose; (ii) high apparent aqueous solubility (Birudaraj et al., 2005; Patel and Vavia, 2009) enables a small dosage volume, which is needed for the available administration area of the cheek; (iii) first pass metabolism (Borg et al., 1975a) after oral administration, hence a higher bioavailability after buccal delivery is expected and (iv) suitable for pH-dependent absorption studies as metoprolol is a secondary amine, with a pKa value around 9.5 (Avdeef and Berger, 2001; Caron et al., 1999; Schoenwald and Huang, 1983). The aim of the present study, was to study the bioavailability of metoprolol when administered in vehicles with different pH values as a means to enhance buccal absorption in vitro and in vivo. Furthermore, the relative correlation between the investigated methods was evaluated.

2. Materials and methods 2.1. Materials Metoprolol tartrate, bisoprolol hemifumerate, methyl cellulose, HEPES H4034, and methanol of HPLC grade were purchased from Sigma–Aldrich (St. Louis, MO, USA). Sodium phosphate dibasic dodecahydrate and sodium phosphate monobasic monohydrate was obtained from Merck (Darmstadt, Germany) and sodium tetraborate dibasic decahydrate from Bie & Berntsen (Rødovre, Denmark). Fetal Bovine Serum (FBS), Hank’s Balanced Salt Solution (HBSS)  10, Mg2+, Ca2+ and Dulbecco’s Modified Eagle Medium (DMEM)-high glucose was obtained from GibcoÒ (Paisley, UK). ZoletilÒ for anaesthesia was obtained from Virbac (Carros cedex, France) and Neutral eye gel from Actavis (Gentofte, Denmark). Ultima Gold™ Scintillations fluid, D-[1-14C]mannitol (spec. act.

58.8 mCi/mmol) and [ring-3H]metoprolol (spec. act. 28.9 Ci/mmol) were obtained from Perkin Elmer (Waltham, MA, USA), BD Falcon™ cell culture inserts (transparent polyethylene terephthalate, 0.4 lm pore diameter, 2.0 ± 0.2  106 pores/cm2 with a nominal porosity of 0.25% [value supplied by manufacturer], and a growth area 0.9 cm2) were purchased from BD Biosciences (Erembodegem, Belgium). All reagents were of analytical or HPLC grade and used without further purification. Deionised water was obtained from a Millipore Milli-Q Ultrapure water purification system (Billerica, MA, USA). 2.2. TR146 permeability and sensitivity study The TR146 cell line was provided by Imperial Cancer Research Technology (London, UK) and cultured as previously described (Jacobsen et al., 1995). Briefly, the culture was maintained in cell culture flasks (25 cm2) (Nunc, Roskilde, DK) at 37 °C in 5% CO2/ 95% air at 98% humidity. The culture medium consisted of DMEM supplemented with 10% FBS, 100 IU/ml penicillin, and 0.1 mg/ml streptomycin. Subcultivation was performed using a mixture of 0.5% (w/v) trypsin and 0.2% (w/v) EDTA. Three to 14 passages were used. For the permeability and subsequent viability study, TR146 cells were seeded on BD Falcon™ cell culture inserts, at a plating density of approximately 2.4  104 cells/cm2, cultivated for 27–29 days changing the culture medium 3 times weekly. On the day of the study, the inserts were gently rinsed twice with 25 mM HEPES HBSS (hHBSS) and the initial transepithelial electrical resistance (TEER) was measured at room temperature (Endohm and voltmeter EVOM from World Precision Instruments (Sarasota, FL, USA)). The donor media consisted of 25 mM phosphate (pH 7.4) or 25 mM borate (pH 8.5, 9.0 and 9.5) in HBSS, with the addition of 0.1 mM metoprolol as the test compound and 0.1 mM mannitol as a marker for paracellular transport. The final activity of radiolabeled metoprolol and mannitol was 0.4 lCi/ml. The receptor media consisted of 25 mM phosphate (pH 7.4) and the volume applied to the basolateral and apical side was 2.0 and 0.8 ml, respectively. The study was carried out at 37 °C using a thermostatic horizontally shaker (125 rpm). The pH of the receptor medium was measured after the permeability study using a pH meter (Radiometer Analytical, Lyon, France) with a microelectrode (Methrom, Herisau, Switzerland). During the permeability study (0–120 min), 100 ll samples were taken from the basolateral side and replenished with an equal volume of fresh buffer. Also, donor samples were taken at t = 0 and t = 120 min. Each sample was added to a counting vial together with 2 ml of scintillation liquid, which was then shaken and counted (Packard, Canberra, Australia). After the permeability experiment, the inserts were emptied and the MTS-PMS viability test was performed as previously described (Jacobsen et al., 1996). The inserts were incubated for 2 h with hHBSS (n = 6) or 0.2% SDS (n = 2) as positive or negative controls, respectively. A solution of MTS, 240 lg/ ml and PMS, 2.4 lg/ml in HBSS was added to the apical side and pure HBSS was added to the basolateral side. The plate was incubated at 37 °C horizontally shaken and protected from light. After 2 h the absorbance was measured at 492 nm using a microplate reader. The viability was calculated from the following equation:

viability ¼

Atest  Aneg Apos  Aneg

ð1Þ

where Atest, Aneg, Apos are the absorbance of test, negative control, and positive control, respectively. 2.3. Ex vivo porcine permeability study Buccal mucosa was obtained from domestic pigs (UCR, Denmark). The skin and the major part of the submucosa were trimmed away with surgical scissors within 3 h after sacrificing the pig and

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before freezing to 20 °C. On the day of the ex vivo study, the mucosa was thawed at room temperature, and a tissue slicer (Thomas Scientific, Swedesboro, NJ, USA) was used to isolate the mucosal permeability barriers, i.e. the epithelium, the basal lamina, and a minimum of submucosa. The tissue was sliced to a thickness of 610 ± 130 lm (mean ± SD, n = 22). Tissue was mounted in a modified Ussing chamber (Physiologic Instruments Inc., San Diego, CA, USA) with an area of 0.40 cm2, adding 2.0 ml transport medium in both donor and receptor chambers. The temperature was kept at 36 ± 1 °C during the permeability study and stirring was maintained by a gas lift supplying 2 bubbles of atmospheric air per second. All buffers were adjusted to isoosmosis (290 mOsm/l) by adding sodium chloride, and pH was adjusted with diluted sodium hydroxide. The receptor medium was 100 mM phosphate buffer pH 7.4. The donor media consisted of 100 mM phosphate (pH 7.4) or 100 mM borate (pH 8.5, 9.0 and 9.5) in HBSS, with the addition of 0.1 mM metoprolol as the test compound and 0.1 mM mannitol as a marker for paracellular transport. The final activity of the radiolabeled metoprolol and mannitol was 0.4 lCi/ml. During the permeability study (0–240 min), samples were collected and treated as described in the cell culture section. After the permeability study, the tissues were rinsed with HBSS and dissolved in 1.0 ml of concentrated sulphuric acid by heating to 70 °C and subsequently quantified by liquid scintigraphy. 2.4. Data analysis of permeability The steady state flux of metoprolol and mannitol was calculated from the accumulated amount of drug permeating the epithelium as a function of time at the linear portion of the curve. The apparent permeability (Papp, cm/s) was calculated from the steady state flux (Eq. 2):

Papp ¼

dQ dt

 ð2Þ

A  C0

where dQ/dt is the steady state flux (dpm/s), A is the area of diffusion area (cm2) and C0 is initial donor concentration (dpm). 2.5. Metoprolol formulations for in vivo study The oral liquid and the solution for intravenous injection were prepared by dissolving metoprolol tartrate (50 and 20 mg/ml, respectively) in 100 mM phosphate buffer (pH 7.4) and adjusting pH to 7.4 with 1 M NaOH. The solutions were then passed through a sterile 0.20 lm filter (Millex-FG, Millipore Corporation, Billerica, MA, USA). The content of metoprolol was analysed by high performance liquid chromatography with UV detection (HPLC-UV). The buccal gels were prepared under constant magnetic stirring by mixing 1% (w/v) methylcellulose and buffer in a glass bottle with a Teflon lid, heated to 60 °C on a water bath, cooled to room temperature, and finally dissolving 50 mg/mL metoprolol in the gels at pH 7.4–9.0, and 25 mg/ml metoprolol in a gel at pH 9.5. For the gel at pH 7.4, a 100 mM phosphate buffer was used, whereas for gels at pH 8.5–9.5, a 25 mM borate buffer was added

Table 1 Overview of buccal gels administered to mini-pigs. pH of gel

Concentration of metoprolol (mg/mL)

Osmolality (mOsm/kg) (mean ± SD, n = 3)

7.4 8.5 9.0 9.5

50 50 50 25

432 ± 6.0 407 ± 3.1 413 ± 3.6 284 ± 6.6

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to get similar osmolalities of the buffers with different pH values (Table 1). The gel at pH 9.5 was administered at double volume due to the solubility profile of metolpolol. Saliva is a hypotonic solution with 150–200 mOsm/kg (Giuseppina et al., 2006). For this reason, osmolality was kept low for this formulation to avoid additional flux of water into the gel. The content of metoprolol was analysed by HPLC-UV. 2.6. In vivo mini-pig study The protocol for the in vivo study was approved by the Animal Welfare Committee, appointed by the Danish Ministry of Justice. All animal procedures were carried out in compliance with EC Directive 86/609/EEC and with the Danish law regulating experiments with animals and the NIH guidelines on animal welfare. Male Göttinge minipigs (16.2–20 kg on the experimental days) were obtained from Ellegaard Göttingen Minipigs A/S (Dalmose, Denmark) and acclimatised for 14 days before initiation of the study. The pigs were examined weekly by a veterinarian and observed closely after each experimental day. Before entering the experiment, the mini-pigs were fasted for 18–20 h with free access to water. Buccal gels and intravenous injection were tested in four mini-pigs in a non-randomised cross-over study, with a washout period of 7 days between each treatment. The animals were anesthetised with 0.1 mg/kg ZoletilÒ and additional injections of ZoletilÒ were given during the course of the experiment. The eyes were treated with Neutral eye gel (Actavis, Gentofte, Denmark) to avoid dryness. The mini-pigs were placed in dorsal recumbence on a temperature controlled table (37 °C) and covered with a thermo blanket. The mini-pigs were intubated to ensure free respiratory passages during anaesthesia and to comply with a possible emergency. Prior to and 4 h after administration of the formulations, i.e. buccal gel, intravenous injection or oral solution, the pH at the upper buccal right site of the cheek was measured with a flat pH electrode (InlabÒSurface, Mettler-Toledo, Glostrup, Denmark) conneted to a portable pH-meter Cyberscan 200 (Eutech Instruments, Nijkerk, Netherlands). The mini-pigs received the buccal gels in the upper right buccal site of the cheek. Blood samples (3.0 mL) were obtained by individual vein puncture of the vena jugularis. The oral solution was administered by gavage and the solution for intravenous injection in the vena jugularis. Blood samples were collected at – 5 min and 5, 15, 30, 45, 60, 90, 120, 180, and 240 min after administration. The blood samples were collected in EDTA coated tubes and immediately centrifuged for 10 min at 4 °C, 2765g (Centrifuge Multifuge 1 S-R, Heraeus, Hanau, Germany). Plasma was harvested and stored at 20 °C until analysis. 2.7. Analysis of metoprolol Quantitative analysis of the buccal gels, oral liquid and solution for intravenous injection was performed by reverse phase HPLCUV: detection at 274 nm, flow rate of 1 ml/min and a column temperature of 35 °C. The Merck HPLC system used consisted of an L-7300 column oven, an L-7400 UV detector with an L-7200 autosampler, an L-7110 pump and a D-7000 interface (Merck, Darmstadt, Germany). Separations were achieved using a C18 XBridge column (3.5 lm, 4.6  150 mm) (Waters Corporation, Milford, MA, USA). The HPLC mobile phase consisted of methanol (50%, v/v)/phosphate buffer (50 mM, pH 7.4) (50%, v/v). The samples were passed through a 0.20 lm filter (Millex-FG, Millipore Corporation, Billerica, MA, USA). The level of detection by this method was 60 ng/mL, level of quantification was 0.2 lg/mL and repeatability 1.7%. The method was shown to be linear to 16 lg/ mL. Prior to analysis, samples were diluted with mobile phase to

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an appropriate concentration of metoprolol, within the linear range of the standard curve. Analysis of the plasma samples was performed by ultra performance liquid chromatography (UPLC) connected to a tandem mass spectrometer (MS/MS): Waters Acquity Ultra Performance Liquid Chromatograph system, (Waters Corp., Milford, MA, USA) equipped with a binary solvent delivery system (pump), a sample manager module with autosampler, and a column compartment/heater. A switch valve connected the UPLC to the mass spectrometer. The mass chromatogram was performed using a MDS SCIEX 4000 LC-MS/MS system with a Turbo Ion SprayÒ (ESI) as an interface (Applied Biosystems, Carlsbad, CA, USA), operating in the positive ion electrospray mode. Operational settings: metoprolol and the internal standard, bisoprolol, were detected at a precursor > product ion mass to charge ratio (m/z) of 268.10 > 116.10 and 326.10 > 116.10 using a depolarisation potential of 71/66 and a collision cell exit potential of 8/10, respectively. Collision gas (CAD) 7.00, temperature (TEM) 600 °C, IS 4500 V, Entrance potential (EP) 10.0 V and collision energy (CE) 27.0 V. Nitrogen was used for the nebulizer and collision gases. Analyst 1.4.2 software was used for controlling the UPLC-MS/MS as well as acquiring and analysing the results. For the UPLC analysis, chromatographic separation was performed on a 50 mm  2.1 mm Waters ACQUITY BEH C8 1.7 lm column. The mobile phases contained 0.1% ammonium hydroxide in water (A) and acetonitrile with 0.1% ammonium hydroxide (B). The total run time was 3.0 min and a gradient system was used. The gradient conditions were 98% A and 2.0% B from initial time to 5.0% A and 95.0% B at 1.50 min, 5.0% A and 95.0% B from 1.50 to 2.00 min, 98% A and 2.0% B at 2.20 min and finally 98% A and 2.0% B from 2.20 to 3.0 min. The flow rate was 0.60 mL/min. The column temperature was maintained at 40 °C, the injection volume was 10 lL and the needle wash solution contained acetonitrile (25%, w/w)/isopropanol (25%, w/w)/water (24.9%, w/w)/methanol (25%, w/w)/formic acid (0.1%, w/w). Plasma samples was prepared by adding 150 lL of a 1.0 ng/mL bisoprolol solution (internal standard) dissolved in acetonitrile with 0.1% aqueous ammonium hydroxide into 25 lL plasma, making the sample alkaline and precipitating the plasma proteins. The solution was subsequent centrifuged at 6200 rpm at 4 °C for 20 min. To 100 lL of the supernatant, 100 lL of an aqueous 0.1% ammonium hydroxide solution was added, followed by centrifugation at 6200 rpm at 4 °C for 5 min. The supernatant was thereafter analysed with the UPLC-MS/MS as described above. The sample preparation was done with Beckman Coulter Biomek NXp (SelectScience Ltd., Bath, UK). Each time a new series was analysed, a fresh standard curve was prepared with a robot Tecan Genesis RSP 200 (Tecan Group Ltd., Männedorf, Switzerland). The peak area correlated linearly with the plasma concentration of the metoprolol in the range of 0.33–1000 ng/mL plasma. If the plasma sample drug concentration was above 1000 ng/ml, the sample was diluted appropriately in blank plasma before analysis.

2.8. Pharmacokinetic data analysis Pharmacokinetic parameters were calculated using WinNonlin Professional version 5.2 (Pharsight Corporation, Mountain View, CA, USA). The plasma concentration–time profiles of metoprolol after intravenous dosing were fitted to a two compartment model, while a non-compartmental model was used to analyse the buccal data. The area under the curve (AUC) was determined using the linear trapezoidal method and extrapolation of the last measured plasma concentration to infinity for the animals dosed intravenously. AUC0–4h for metoprolol after oral and buccal administration was calculated using the linear trapezoidal rule from time zero to the last measured plasma concentration at 4 h post-dose. The total

bioavailability (F) of metoprolol was calculated for the individual animal using the following equation:

Fa ¼

    AUCbuccal DoseIV  AUCIV Dosebuccal

ð3Þ

AUCIV is the area under the curve following intravenous administration of metoprolol, AUCbuccal following buccal administration. DoseIV and Dosebuccal is the dose administered intravenous and buccally, respectively. 2.9. Statistical analysis Sigma Stat for Windows software, version 3.0.1 from SPSS Inc. (Chicago, IL, USA) was used for the statistical calculations. The statistical comparisons used one way analysis of variance (ANOVA) followed by a Student–Neuman–Keuls test. Two sided p-values below 5% (p < 0.05) were considered statistically significant. 3. Results and discussion Buccal administration offers several opportunities, e.g. it can be advantageous for patients who are unable to swallow, have gastric disorders or limited access to water at the administration time. In addition, buccal absorption bypasses hepatic first-pass metabolism, which is why this administration route can be used for compounds with significant first-pass metabolism, or to reduce the dose requires, thereby lowering the potential side effects of the compound and/or its metabolites (Mao et al., 2009). Disadvantages of buccal drug delivery, includes (i) high requirements with respect to organoleptic properties, (ii) the patient may not eat or drink while taking the medication (Patel et al., 2011), (iii) a limited absorption area, and (iv) limited amount of liquid available for dissolution of a solid dose when compared to the gastrointestinal tract (Devries et al., 1991). Previous studies have found the pKa of metoprolol lies between 9.24 and 9.63 and log P from 1.88 to 1.96 (Avdeef and Berger, 2001; Caron et al., 1999; Schoenwald and Huang, 1983). This is in accordance with our values of 9.47 for the pKa and 1.86 for log P (data not shown). Metoprolol has an aqueous solubility greater than 50 mg/mL at pH 7.4, which is why it is an excellent candidate for the evaluation of the ionisation effects on absorption following buccal administration. 3.1. In vitro permeability studies in TR146 cell culture and ex vivo porcine buccal mucosa Metoprolol had a pH dependent apparent permeability coefficient (Papp) in both in vitro models (Fig. 1), when the pH value was increased from pH 7.4 to 8.5, 9.0 and 9.5. The apparent permeability obtained in the two models was similar over the pH range, as presented in Fig. 1, with the corresponding permeability values shown in Table 2. The apparent permeability in Caco-2 cells (derived from human epithelial colorectal adenocarcinoma) at pH 7.4 at both the apical and basolateral sides have previously been reported to be 116  106 s/cm (Neuhoff et al., 2003). Both the TR146 and the ex vivo porcine buccal mucosa model had a lower metoprolol permeability than the Caco-2 cells, probably reflecting the differences in the two tissues. A correlation between the two models was observed, see Fig. 2, which may explain why the higher permeability previously seen in the TR146 cell line when compared to the porcine ex vivo model (Nielsen and Rassing, 1999, 2000b, 2002; Nielsen et al., 1999) does not appear to influence the predictability of the in vivo performance for metoprolol. The lag-time of 76 ± 22 min (mean ± SD, n = 22) for metoprolol transport across

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Fig. 1. Apparent permeability coefficient (Papp) for metoprolol at different pH values in ex vivo porcine buccal mucosa model (d) and filter-grown TR146 cell culture (N). Mean ± SD (n = 4–8 and 4 for the Ussing chamber model and cell culture, respectively).

Table 2 Apparent permeability coefficient (Papp) for metoprolol at different pH values in an ex vivo porcine buccal mucosa model and filter-grown cell culture TR146. Mean ± SD for the Ussinf chamber model (n = 4–8) and cell culture (n = 4). pH

Apparent permeability coefficient (Papp) Porcine buccal model

TR146 cell line

7.4 8.0 8.5 9.5

8.0  106 ± 8.6  107 3.5  105 ± 5.7  106 5.0  105 ± 1.5  105 7.3  105 ± 1.1  105

1.09  105 ± 5.2  107 3.6  105 ± 1.2  106 5.3  105 ± 9.1  107 6.2  105 ± 2.7  106

ex vivo porcine buccal mucosa showed no pH dependency. No significant lag-time was observed in the cell culture model. In order to study the integrity of the epithelial barrier, the permeation of [14C]mannitol was measured. The cell culture showed a small increase in mannitol permeability at higher pH values [118 ± 4.6% (pH 8.5), 130 ± 7.2% (pH 9.0) and 129 ± 4.3% (pH 9.5)] compared to pH 7.4 (100 ± 3.3%). Mannitol permeability in ex vivo porcine buccal mucosa increased almost 10-fold when raising the pH from 7.4 to 9.5. No significant decrease was observed in the TEER value (136 ± 17.1 X cm2, mean ± SD, n = 24) from the start to the end of the study in the cell culture model. For unclear

Fig. 2. Correlation between apparent permeability coefficient (Papp) for metoprolol at different pH values in an ex vivo porcine buccal mucosa model and filter-grown TR146 cell culture. The solid line represents the linear least squares regression line, R2 = 0.96. Mean ± SD (n = 4–8 and n = 4 for the Using chamber model and cell culture, respectively).

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reasons, the resistance increased from pH 9.0 (207 ± 10 X cm2, mean ± SD, n = 4) to pH 9.5 (253 ± 18 X cm2, mean ± SD, n = 4). The MTS-PMS assay performed immediately after the permeability study in the TR146 cell culture model showed a decrease in function with increased pH. Cell viability (mean ± SD, n = 4) relative to the positive control hHBSS (100%, n = 6, pH 7.4), was 95.0 ± 4.7% (pH 8.5), 90.9 ± 2.1% (pH 9.0) and 78.1 ± 5.9% (pH 9.5). The decrease at pH 9.5 was not a matter of concern, as this pH is >2 pH units above the pH of saliva. The present results indicate a parallel relation between increased Papp of mannitol and decreased viability/sensitivity. The pH of the donor media was measured after the permeability study in both in vitro models. In cell culture model, the pH slightly decreased over time from 7.40 to 7.25 ± 0.04, 8.50 to 8.00 ± 0.02, 9.00 to 8.56 ± 0.04 and 9.50 to 8.99 ± 0.02 (means ± SD, n = 4). This could be explained by (i) release of hydrogen ions due to metabolism and (ii) transporters capable of transporting hydrogen ions resulting in a net transfer with the concentration gradient. Argument (iii) can be supported by the fact that the decrease in pH is significantly lower when having an identical pH value (7.40) at the apical and basolateral side. The ex vivo model in porcine buccal mucosa showed no changes in donor pH during the 4 h of the transport study. The recovery was 102.3 ± 5.4% for metoprolol and 100.0 ± 0.62% for mannitol in the ex vivo model (mean ± SD, n = 21), respectively. The porcine tissue showed an accumulation of 4.2 ± 0.2% metoprolol across the investigated pH values. There was no relationship between the accumulated amount of metoprolol and the thickness of the excised porcine buccal mucosa. This accumulation could be a reflection of the buccal mucosa architecture, as it is composed of 20–40 layers of squamous epithelial cells with many prominent ridges. Accordingly, the excised mucosa was made up of a variable number of epithelial cell layers imbedded in the submucosa. The recovery was 88.8 ± 6.4% for metoprolol and 106.2 ± 5.3% for mannitol in the cell culture model (mean ± SD, n = 16). 3.2. Pharmacokinetic profile following intravenous administration The plasma concentration–time profile of metoprolol after intravenous injection of 2 mg/kg metoprolol in anesthetised mini-pigs is presented in Fig. 3. The profile could be described by a bi-exponential equation:

C pl ¼ 552  e4:4t þ 143  e0:42t

ð4Þ

where Cpl is the concentration of metoprolol (ng/ml) in plasma and t is time (in minutes). The AUC0? was 32,231 ± 6537 ng min/ml,

Fig. 3. Plasma concentration–time profile of metoprolol after intravenous administration of 2 mg/kg to anesthetised mini-pigs. Plasma concentration (d, mean ± SEM, n = 4). The solid line is the profile based upon the two compartmental model in equation 3.

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AUC0?last was 29,049 ± 3775 ng min/ml, clearance 72.6 ± 10.2 ml/ min/kg and volume of distribution 7732 ± 672 L/kg. The results were used for the calculation of the absolute bioavailability after oral and buccal administration. 3.3. Oral and buccal administration of metoprolol to mini-pigs The plasma concentration–time profile of metoprolol after buccal administration of gels with pH values ranging 7.4–9.5 and an oral liquid pH 7.4 to mini-pigs are presented in Fig. 4 and the corresponding pharmacokinetic parameters in Table 3. The oral cavity of the mini-pigs was examined for reddening or other signs of irritation before and after the experiment and on the following days. No signs of irritation were observed for any of the formulations. This is consistent with the reported resilient properties of the buccal epithelium and its short recovery time after stress or damage (Devries et al., 1991; Patel et al., 2011). The pH at the surface of the buccal mucosa was measured as an indicator of potential changes or tissue damage (Table 4). Some variations of the surface pH values were observed. The anaesthesia and the intubation of the mini-pigs during the 240 min study period may have contributed to decreased salivary flow and subsequent dryness of the oromucosal surface. This may have affected the mucosal surface pH. Otherwise no clear physiological meaningful trend could be observed between the groups. A fast absorption was seen following buccal and oral absorption of metoprolol, and there were no statistical differences between Tmax values for these two routes of administration, i.e. 35 min and 53 min, respectively (Table 3). This is expected to be a reflection of the high solubility and good permeability of metoprolol. Cmax was significantly lower for animals dosed orally, whereas only a trend towards a higher Cmax with a higher pH could be observed for the bucally dosed animals, which did not reach statistical significance. This is most likely caused by the difference in the fraction of unionised metoprolol absorbed from buccal gels with different pH. Generally, orally dosed drugs undergo hepatic first pass metabolism, whereas buccally delivered drugs bypass it (Devries et al., 1991; Pathan et al., 2008). A significantly lower absolute

bioavailability of metoprolol was observed in the animals orally dosed animals (3) compared bu bucally dosed animals (58– 107%). The absolute bioavailability of metoprolol after oral administration to rats has been reported to be dose dependent, varying between 4% and 60% (Borg et al., 1975b; Yoon et al., 2011) compared to 38–60% in humans (Tanabe et al., 2007). The present study showed that the mini-pigs metabolised metoprolol at a higher level than rat and humans. Furthermore, the lower bioavailability obtained following oral drug administration indicates that bioavailability measured after buccal administration is a result of buccal absorption and not swallowed metoprolol. There is a significant difference between the bioavailability of buccally administered gels at pH of 7.4 versus 9.0 and 9.5. Increased AUC and F% as a function of increasing pH was observed (Table 3), suggesting that the bioavailability of metoprolol increased as the degree of ionisation decreased. Gels at pH 7.4 and 8.5 were not significantly different, though a tendency towards higher bioavailability was seen at pH 8.5 where a higher fraction of the compound was unionised (Fig. 4). This study therefore indicated that pH adjustment of a buccal gel formulation is an effective way to increase the bioavailability of an ionisable drug administered buccally. To our knowledge, studies of pH-dependent buccal delivery in minipigs have to our knowledge not previously been described in the literature, but similar tendencies have been suggested by other in vivo studies of other species, such as man and dogs (Adrian et al., 2006; Gul et al., 2007; Henry et al., 1980; Streisand et al., 1995). Contradictory results to the present study have been reported for other beta-blockers (Wang et al., 2010; Zhang et al., 1989), though primarily based upon in vitro investigations and in vivo evaluation at only one pH in both studies. Wang and co-workers used a concept around pHmax, defined as the pH value with the maximal aqueous solubility for a given drug. Adjusting the formulation to this pH was suggested to lead to a maximum potential absorption (Wang et al., 2010). The results obtained in the present work do not support this hypothesis; however, in the present study a compound with sufficient solubility was investigated. Increased aqueous solubility was therefore unnecessary compared with effects on the permeability. Finding an optimal formulation is clearly a

Fig. 4. Plasma concentration–time profile of metoprolol after buccal and oral drug administration of 5 mg/kg metoprolol to anesthetised mini-pigs. Buccal gels; pH 7.4(N), pH 8.5(j), pH 9.0(), pH 9.5(d), insert oral liquid. Mean ± SEM (n = 4).

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Table 3 Noncompartmental pharmacokinetic values obtained after oral and buccal administration of metoprolol (5 mg/kg) at different pH-values to anaesthetised mini-pigs (mean ± SEM, n = 4).

Amount drug un-ionised (%)c Cmax (ng/mL) Tmax (min) AUC0–4h (ng min/mL) Absolute bioavailability (%) a b c

Oral Liquid

Buccal gel (pH 7.4)

Buccal gel (pH 8.5)

Buccal gel (pH 9.0)

Buccal gel (pH 9.5)

– 21 ± 4a 53 ± 23 2453 ± 166 3.3 ± 0.2a

0.8 401 ± 79 39 ± 4 41,567 ± 5641 58 ± 8b

9.7 519 ± 88 35 ± 4 57,638 ± 4916 81 ± 7

25 747 ± 58 36 ± 11 69,705 ± 3103 96 ± 6

52 853 ± 202 34 ± 11 77,090 ± 10,080 107 ± 14

Significantly different from all the buccal formulations. Significantly different from buccal gels with pH 9.0 and 9.5. Calculated from the measured pKa value.

Table 4 pH on the surface of buccal mucosa in mini-pigs before and after administration of metoprolol formulations (mean ± SD, n = 4). pH at the porcine buccal mucosal surface Treatment IV

Before administration (time = 0 min) After administration (time = 240 min)

7.3 ± 0.3 6.9 ± 0.1

compromise between solubility and permeability and should be adjusted for the specific compound and its biopharmaceutical and physic-chemical parameters. The dependency of absorption on pH was in accordance with the results observed in vitro with the TR146 cells and porcine buccal mucosa in Ussing chambers. When evaluating the ex vivo porcine buccal permeability as a function of in vivo bioavailability in mini-pigs, a positive correlation (r2 = 0.9752) was obtained (Fig. 5), equal to a level C in vitro in vivo correlation (IVIVC). Very few attempts have been made to obtain IVIVC for buccal delivery, and additional compounds and variations in formulation should be tested to confirm the validity of this correlation. The ex vivo porcine buccal model could potentially be used as a screening tool. Kapil et al. (2012) have recently reported a level A correlation between drug release measured in Franz diffusion cells using a dialysis membrane compared to pharmacokinetic data obtained from a rabbit in vivo study of a prolonged release buccoadhesive film containing rivastigmine. Though Kapil and co-workers only used three rabbits in their study, these findings are highly interesting from a

Oral

Buccal gel

7.6 ± 0.2 7.3 ± 0.5

pH 8.5

pH 9.0

pH 9.5

7.2 ± 0.1 6.6 ± 0.1

7.4 ± 0.1 6.5 ± 0.2

7.1 ± 0.1 7.3 ± 0.1

drug delivery point of view as they, together with the data obtained in this study, provide positive indications towards developing predictive in vitro methods for buccal drug delivery, which could provide an important formulation tool for this administration route. 4. Conclusion In summary, the present study has shown a correlation between in vitro predictions and in vivo bioavailability of metoprolol administered buccally to mini-pigs. Metoprolol was well absorbed in vitro in TR146 cells and porcine buccal mucosa in Using chambers and a higher permeability was found when the pH increased, i.e. when the fraction of unionised compound increased. The oral bioavailability of metoprolol in mini-pigs was very low. The absorbed metoprolol following buccal delivery was therefore mostly a reflection of compound absorbed through the buccal cavity. Therefore, this study suggests good predictive power from in vitro studies for the evaluation of buccal administered drug candidates, although further work is needed to validate this level C IVIVC correlation and to identify the important physical chemical parameters for buccal absorption. Acknowledgments The Drug Research Academy is acknowledged for material grants for the in vitro study. The personnel in the animal facilities at H. Lundbeck A/S are thanked for their skilful and professional handling of the animals, which makes studies like this possible. David John Simpson at H. Lundbeck A/S is highly acknowledge for linguistic help. References

Fig. 5. Correlation between apparent permeability (Papp) for metoprolol in the ex vivo porcine buccal mucosa model and in vivo absolute bioavailability of metoprolol obtained from mini-pigs administered buccally with metoprolol gels with varying pH values. The solid line represents the linear least squares regression line, R2 = 0.9752. Data presented as mean ± SEM, n = 4–8 for in vitro data and n = 4 for in vivo.

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