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Department of Drug Research and Medical Biotechnology, Fraunhofer Institute of Toxicology and Experimental Medicine, Hannover, Germany
(Received January 15, 2005; accepted May 6, 2005)
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Abstract |
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). Current data suggest that metabolism of drugs and other xenobiotics is thought to be associated with mainly six different cell types: type I and type II alveolar epithelial cells, alveolar macrophages, capillary endothelial cells, and ciliated and nonciliated (Clara cells) bronchiolar epithelial cells (Baron and Voigt, 1990; Anttila et al., 1997; Willey et al., 1997). Based on animal studies, the primary site of pulmonary cytochrome P450 (P450) monooxygenase activity resides in Clara cells and type II alveolar epithelial cells (AECs) (Bend et al., 1985; Voigt et al., 1990). In particular, AECs serve two major functions, both of which are essential for the maintenance of normal lung physiology. First, AECs synthesize and secrete pulmonary surfactant (Kasper and Singh, 1995), and second, they function as progenitors of newly formed pulmonary alveolar epithelium after lung injury and during normal lung cell turnover (Uhal, 1997). Additional functions include detoxification of airborne xenobiotics (Adamson et al., 1977), with the lung being the primary site for entry into systemic circulation. For elimination, certain xenobiotics undergo oxidation by phase I enzymes and additional reactions to foster excretion from the body (Guengerich and Shimada, 1991). Although most of these oxidations are beneficial, some may lead to reactive metabolites and cellular damage (Guengerich, 2000). In addition to drug delivery designed to treat pulmonary disease, administration of therapeutic agents by the inhalation route has been reported for the immunosuppressive agent cyclosporin (Keenan et al., 1992), interferons (Halme et al., 1994), and 
1-antitrypsin (McElvaney et al., 1991), among others.
We investigated the metabolism and pharmacokinetics of verapamil, a synthetic papaverin derivative, with L-type-calcium channel blocking activity and therapeutic use as an antianginal, antihypertensive, and antiarrhythmic agent (Woosley and Roden, 1983; Padrini et al., 1985; Piovan et al., 1995). Unfortunately, this drug suffers from extensive hepatic first pass metabolism. Because of its extensive metabolism, verapamil becomes rapidly inactivated, and this necessitates frequent dosage of this drug. This has stimulated research into new release forms and alternative routes for drug application. The objective of the present study was therefore to evaluate the plasma clearance and metabolism of verapamil after drug administration by inhalation. Specifically, rats were given single doses of verapamil by inhalation and intravenous routes, and the plasma and lung tissue concentrations of verapamil and its metabolites were measured at several time points using a validated and previously published high-performance liquid chromatography (HPLC)-MS method (Walles et al., 2002). Furthermore, we investigated the metabolism of verapamil in cultures of AECs, e.g., the site of drug entry into systemic circulation. Thus, the work in this manuscript explored the use of drug delivery by inhalation to avoid extensive first pass metabolism.
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Materials and Methods |
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) and was used to coat 12-well culture plates (Biochrom).
Cell Isolation and Culture. AECs were isolated as previously described by Richards et al. (1987) with slight modifications. Male Sprague-Dawley rats weighing approximately 120 g were anesthetized with ketamine (100 mg kg-1)/xylazine (Rompun) (10 mg kg-1) by intraperitoneal injection. After tracheotomy, the trachea was cannulated for artificial ventilation of the lung, and blood was removed from the lung by perfusion with ice-cold PBS via the pulmonary artery. The perfused lung plus trachea were then explanted and placed in a 50-ml plastic tube containing 30 ml of ice-cold PBS. The lungs were lavaged three times with 6 ml of PBS, and subsequently, 6 ml of 0.25% trypsin-EDTA solution was instilled into the lung via the trachea. The lung was incubated with trypsin for a total of 45 min at 37°C, and every 15 min, 4 ml of 0.25% trypsin-EDTA solution was added via the trachea. Trachea and bronchi were removed, and the lung tissue was chopped into 1- to 2-mm pieces. Ten milliliters of DNase (250 µg/ml) (Invitrogen GmbH, Karlsruhe, Germany) was added to the suspension, incubated for 10 min at 37°C in a shaking waterbath (60 rpm), and then filtered through a nylon mesh with 100- and 60-µm pore size. The resulting cell suspension was layered on a discontinuous Percoll gradient (heavy density 1.089 and low density 1.040) that was centrifuged at 250 g at 4°C for 30 min. The cell fraction at the interface between heavy- and low-density gradient was removed and mixed with PBS containing 50 µg/ml DNase. The cell suspension was again centrifuged at 140 g at 4°C for 6 min. The resulting cell pellet was resuspended in culture medium and subsequently cultured for 1 h on plastic to remove rapidly adhering cells, including lung macrophages. Subsequently, the cell suspensions were cultivated on rat tail collagen-coated 12-well plates. Approximately 4 x 106 cells were seeded per well in 1-ml culture medium and cultured for up to 7 days.
Histochemistry. Cytospin preparations of freshly isolated cells on days 3 and 6 in culture were prepared at an approximate density of 1 x 106 cells per slide and fixed in acetone/methanol (1:1) at -20°C for 8 min. Thereafter, the preparations were dried at room temperature (10–30 min). These cells were characterized by staining for alkaline phosphatase as a marker for epithelial cells by the method of Edelson et al. (1988) and by tannic acid polychrome stain for lamellar bodies, as described by Mason et al. (1985). The expression of surfactant protein C was analyzed by immunoperoxidase staining using the DAKO LSAB+ Kit (DAKO, Hamburg, Germany) in conjunction with a polyclonal rabbit anti-human surfactant protein C antibody (Byk Gulden, Konstanz, Germany).
Testosterone Hydroxylation Assay. Testosterone served as a substrate to probe for activity of P450 monooxygenases in cultured AECs (Arlotto et al., 1991). Therefore, AECs of day 3 in culture were exposed to culture medium containing 100 µM testosterone (Sigma, Taufkirchen, Germany) for 2, 4, 8, 12, and 24 h. Testosterone and its metabolites 2--hydroxytestosterone, 6--hydroxytestosterone, 6-ß-hydroxytestosterone, 16--hydroxytestosterone, and androstendione (Sigma) were analyzed by HPLC according to Arlotto et al. (1991) with slight modifications. 11--Hydroxyprogesterone (Sigma) was used as an internal standard for the quantitative determination of testosterone and its metabolites using the following procedures:
One microgram of 11--hydroxyprogesterone was added to 1 ml of cell culture supernatant. Following the addition of 100 µl of isopropanol, the samples were extracted with 5 ml of ethyl acetate by gentle shaking for 20 min. Extracts were evaporated to dryness, and the residues were reconstituted in 100 µl of the mobile phase (water/methanol/acetonitril, 60:25:15, v/v/v), and 80 µl of the sample was injected onto the HPLC system (HPLC series 1100; Hewlett Packard, Hamburg, Germany). The mobile phase was delivered at a flow rate of 1 ml/min using the HP 1100 Quaternary Pump. Chromatographic separation of metabolites was achieved on a C18 Nucleosil column with the dimensions 250 x 4 mm and a particle size of 5 µm (Macherey-Nagel, Düren, Germany) at a temperature of 30°C. The mobile phase consisted of water (solvent A), methanol (solvent B), and acetonitril (solvent C), and analysis was initiated with an isocratic elution of 60% A, 25% B, and 15% C for 12 min, followed by 45% A, 40% B, and 15% C for 3 min, and finally 45% A, 45% B, and 10% C thereafter. The total run time was 30 min per sample. Testosterone and metabolites were detected by UV absorption at 238 nm by comparison of retention times of individual reference standards.
P450 Induction in Cultured AECs. At day 3 in culture, AECs were treated for 48 h with 10 µM Aroclor 1254, a well known inducer of P450 monooxygenases (Borlak et al., 1996; Borlak and Thum, 2001).
Verapamil Metabolism. At day 3 in culture AECs were treated with 300 nM verapamil for 12 h. This concentration (300 nM verapamil) is based on therapeutic plasma values (Hamann et al., 1984). Culture supernatant was removed and frozen at -80° to await analysis.
Preparation of Lung Microsomes. Lungs were cut into small pieces and homogenized with an ultraturrax (Ika, Staufen, Germany) in KCl solution (0.15 M, pH 7.4) and thereafter centrifuged for 30 min at 11,000g and 4°C. The resulting supernatant was decanted and once again centrifuged at 110,000g and 4°C for 60 min. The resultant pellet was washed and resuspended in KCl solution (0.15 M, pH 7.4) and centrifuged for 40 min at 110,000g and 4°C. Then, the microsomal fraction was transferred into Tris-sucrose buffer (0.25 M sucrose, 20 mM Tris, and 5 mM EDTA, pH 7.4). The protein content was determined according to Smith et al. (1985). Microsomal solutions were frozen in liquid nitrogen and stored at -80°C until further use.
Lung Microsomal Metabolism Assays. A 1-ml reaction mixture containing 500 µg of microsomal protein in 0.1 M Tris buffer pH 7.4 + 1 mM NADPH (Sigma) was incubated with 300 nM verapamil for up to 60 min at 37°C. After incubation, the samples were frozen in liquid nitrogen and stored at -80°C until analysis.
Gene Expression Profiling. RNA was isolated from cultured AECs from days 3 to 7 using the Nucleo Spin RNAII Kit (Macherey-Nagel, Düren, Germany) according to the manufacturer's recommendation. For reverse transcription, 1 µg of RNA was denaturated for 5 min at 65°C, and reverse transcription was initiated by Omniscript Reverse Transcriptase (QIAGEN, Hilden, Germany). This reaction was carried out for 60 min at 37°C and stopped by heating to 95°C for 5 min. The resulting cDNA was frozen at -20°C until further use.
For PCR amplification of cDNA, a 25-µl reaction mixture was prepared containing 10x polymerase reaction buffer, 3 mM MgCl2, 0.4 nM dNTPs (Invitrogen), 400 nM concentration of the 3' and 5' specific primer (synthesized by Invitrogen, Hilden, Germany) (Table 1), 1 U Hot Star Taq polymerase (QIAGEN), and 1 µl of cDNA. PCR reactions were carried out in a T3 thermocycler (Biometra, Göttingen, Germany) using the following melting, annealing, and extension cycling conditions: denaturation for 45 s at 94°C, annealing for 60 s at 55°C, and extension for 60 s at 72°C for GAPDH (30 cycles), CYP2C11 and CYP4A1 (32 cycles), and CYP3A1, CYP2E1, CYP2J3, and CYP1B1 (36 cycles). PCR conditions for CYP1A1 (34 cycles), CYP1A2 (38 cycles), CYP2B1/2 (36 cycles), and CYP3A2 (38 cycles) were denaturation for 45 s at 94°C, annealing for 60 s at 57°C, and extension for 60 s at 72°C. Each PCR started with a 15-min denaturation step at 95°C and ended with a 10-min elongation step. Primer sequences are given in Table 1. PCR reactions were done within the linear range of amplification, after having established the chosen cycles to be in the mid-exponential phase for every primer set used. Amplification products were separated on a 1.8% agarose gel and were visualized by ethidium bromide under UV transillumination.
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Quantification of PCR-products was done with the program NIH Image V.1.62 and the software of the Kodak Image Station version ID 3.5 (Eastman Kodak, Rochester, NY). Data are expressed as the ratio of gene of interest (nominator) to the housekeeping GAPDH.
Statistical Analysis. Each analysis was based on n = 4 independent isolations. Means and standard deviations were calculated with the Microsoft Excel software package (version 2000; Redmond, WA). The Student's t test (unpaired) was used to detect statistical differences (p 
0.05).
Inhalation Studies. For 14 days and prior to exposure, animals were acclimated to external procedures to avoid any unphysiological breathing pattern. Verapamil was administered as dry aerosol by spray drying 0.8% (v/w) aqueous solution. Cascade impactor measurements of the aerosol size distribution revealed a mass median aerodynamic diameter of 1.4 µm and a geometric standard deviation of 1.5. A total of 21 male Sprague-Dawley rats weighing approximately 250 to 300 g were exposed to 50 mg/m3 verapamil by nose-only inhalation for 10, 20, 30, and 40 (three animals each time period), and 60 min (nine animals). After the 10-, 20-, 30-, and 40-min time point, three animals were killed immediately. In the case of the 60-min time point, three animals were killed immediately after the end of inhalation, whereas a further three animals were killed 60 min after the end of inhalation, and a further three animals were killed 180 min after the end of inhalation to enable direct comparison with the intravenous treatment group. The total inhaled dose after 60 min of exposure was calculated from the respiratory minute volume and the aerosol concentration to be 1.56 mg/kg. Based on an empirical correlation established by Stahl (1967), a value of 0.52 l/min was chosen for the respiratory minute volume in this calculation. From the inhaled dose, only the fraction deposited in the lung can become bioavailable. This fraction is estimated to be 30%, so that the relevant body dose of verapamil after 60 min of inhalation is approximately 0.5 mg/kg. In the case of intravenous application, 21 animals were given an intravenous dose of exactly 0.5 mg/kg body weight verapamil via the tail vein. After intravenous injection, three animals were killed after 10, 20, 30, 40, 60, 120, and 240 min. The blood was collected and centrifuged at 4000 rpm for 15 min at 4°C. Resultant plasma was frozen at -20°C. Furthermore, lungs were carefully explanted and frozen at -80°C. Blank plasma and tissue samples from untreated rats were obtained as well.
Bioanalytical Assay for Verapamil and Its Metabolites. Verapamil and its major metabolites were extracted by a column-switching technique with a restricted access material and by a matrix solid-phase dispersion method (MSPD) to analyze lung tissue samples as reported previously (Walles et al., 2002). Identification of the metabolites was achieved by liquid chromatography-MS as described elsewhere (Walles et al., 2002). The abundance of the metabolites was calculated using the peak ratio of the metabolites relative to that of verapamil. Only metabolites observed with a single-to-noise ratio (s/n) > 3 were quantified.
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Results |
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Cell Characterization. Table 2 summarizes the results obtained from cytospin preparations of rat AECs. Over 60% of freshly purified AECs and over 70% of AECs (day 3 in culture) were positive for surfactant protein C and tannic acid and alkaline phosphatase staining (Fig. 1, b–d, Table 2). We therefore provide evidence for the expression of markers of alveolar epithelial type II cells. Notably, at day 6 in culture, a significant decrease in staining for alkaline phosphatase (59%), lamellar bodies (70%), and surfactant protein C (63%) was observed (Table 2). Furthermore, transcript level of surfactant proteins A and B remained stable during culture, but the expression of genes coding for surfactant protein C and surfactant protein D decreased progressively. Figure 2 depicts a representative ethidium bromide stained gel of surfactant protein coding genes at days 3 to 7 in culture.
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We also investigated expression of P450 monooxygenases in cultures of AECs. With the exception of CYP1A2, CYP2C11, CYP3A1, and CYP4A1, which were below the level of detection, expression of transcripts of P450 isoforms CYP1A1, CYP1B1, CYP2B1/2, CYP2E1, CYP2J3, and CYP3A2 was similar during cell culture. Furthermore, CYP1A1, CYP1B1, and CYP2E1 mRNA expression was increased 6-, 2-, and 3-fold, respectively, after treatment of AECs with Aroclor 1254, a known P450 monooxygenases inducer (Fig. 3). The mRNA expression of genes coding for CYP2B1/2, CYP3A2, and CYP2J3 was unchanged after treatment of the AECs with Aroclor 1254 (Fig. 3). Furthermore, treatment of AECs with verapamil had no influence on P450 gene expression (data not shown).
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-hydroxytestosterone, 16--hydroxytestosterone, and 2--hydroxytestosterone (see Fig. 4). Approximately 5% of the testosterone was metabolically converted in a 24-h assay period.
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(integration was done mathematically using the mathematical expressions of the fitted curves.) There is a less than 5% difference between the two AUC values for the plasma and less then 11% for the lung tissue, therefore indicating that the experimental time period covers most of the kinetics of verapamil. Furthermore, the AUC after of intravenous and inhalation compare very well, the value for inhalation being 10% higher. Since the nominal doses after administration by inhalation and intravenous routes were nearly equal, we conclude that the bioavailability of verapamil after inhalation is essentially the same as after intravenous administration.
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). With cultures of AECs and with microsomal lung fractions, metabolism of verapamil proceeded with the production of metabolite D617, carbinolamin, and norverapamil (Fig. 7). Additionally, with microsomal fractions of rat lung, metabolite 24 was observed (Fig. 7).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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AECs have been reported to express P450 monooxygenases (Baron and Voigt, 1990) and are likely to play an important role in the pulmonary metabolism of drugs. To investigate the metabolic competence of cultured AECs, lung tissue was explanted, and cells were isolated as described previously by Richards et al. (1987). AECs and other lung cells overlap in cell density (w/v) (Bingle et al., 1990) and cell diameter (Haies et al., 1981; Crapo et al., 1982), which hampers efficient separation from other pulmonary cells. Characterization of AEC in culture should involve several cellular markers (Dobbs, 1990). We investigated AEC cultures by phase contrast microscopy and by studying lamellar bodies in the cytosol of cultured AECs and by investigating surfactant protein C protein expression. We show that isolated and cultivated AECs retained certain differentiation markers during culture. The lamellar bodies contained surfactant, a morphological hallmark of AECs. Laminar bodies are the main cellular storage compartments of pulmonary surfactant, and AECs produce surfactant protein A, B, C, and D (Kasper and Singh, 1995). In our study, gene expression of all four surfactant proteins was confirmed during 7 days of culture, albeit at varying intensities. Noteworthy, cells other than AECs also express surfactant proteins. For example, surfactant protein A is expressed in alveolar macrophages and Clara cells, as determined by immunocytochemistry (Walker et al., 1986; Williams et al., 1988). Nonetheless, surfactant protein C expression is restricted to AECs (Kasper and Singh, 1995), and therefore, surfactant protein C is a valuable marker for the characterization of this particular cell type. Indeed, surfactant protein C expression remained constant, when freshly isolated and day 6 cultured cells are compared (Table 2).
Additionally, alkaline phosphatase was proposed as marker for AECs. Although cells other than AECs express alkaline phosphatase activity as well, alveolar macrophages lack this phosphatase activity (Cohn and Weiner, 1963). A histochemical stain for alkaline phosphatase can therefore be used to distinguish AECs from alveolar macrophages (Edelson et al., 1988). In our study, 74% of freshly isolated AECs were positive for alkaline phosphatase staining.
Although AECs retained some of their differentiation markers, alkaline phosphatase activity, tannic acid polychrome staining, and surfactant protein C expression declined with time, e.g., between days 3 and 6 in culture (Table 2). We therefore chose 3-day-old cultures for studying the metabolism of testosterone and verapamil.
The metabolic competence of the cultured AECs was studied by measuring the mRNA expression levels of P450 monooxygenases and production of stereo- and site-specific hydroxylation products of testosterone, and reasonable correlations were found. We further show treatment of cultures of AECs with Aroclor 1254 to induce expression of certain P450 monooxygenases, i.e., CYP1A1, CYP1B1, and CYP2E1 and, therefore, provide evidence for cultured AECs to respond to P450 monooxygenase inducing agents.
We link the metabolic competence of cultured AECs to the metabolism of verapamil and compare findings with results from microsomal incubation assays. With lung microsomal fractions and with AECs, a total of four metabolites were observed, with norverapamil being the major metabolite. In strong contrast, the hepatic metabolism of verapamil resulted in 25 phase I and 14 phase II metabolites as recently reported by us (Walles et al., 2003). In human liver abundant expression of P450 enzymes resulted in extensive metabolism of verapamil, and its metabolism is primarily catalyzed by CYP3A4, CYP3A5, CYP2C8, CYP2C18, CYP2D6, and CYP2E1 (Tracy et al., 1999). Although the expression of an array of different P450 isoforms was detectable in cultured AECs of rat lung, only the rat ortholog to CYP3A5 is likely be a major contributor to pulmonary oxidation of verapamil. Hitherto, we show a good correlation between the limited expression of P450 isoforms relevant to verapamil metabolism and its limited metabolism in the alveolar epithelium.
We also investigated the metabolism of verapamil in vivo after administration by intravenous and inhalation routes. The level of verapamil in lung tissue, as well as in plasma, increased with inhalation time. This suggests fast transfer of verapamil via intracellular or paracellular transport from epithelium into lung tissue and blood. An important finding of our study was the similar elimination of verapamil from lung tissue and plasma after administration by intravenous and inhalation routes. The limited metabolism of verapamil in cultured AECs and in lung tissue after administration by inhalation is in good agreement, and we demonstrate that therapeutic plasma levels can be achieved. With lung tissue, we observed norverapamil as major metabolite, but the amounts were too small for proper quantification. Among many other metabolites, this metabolite is known to be a major breakdown product of verapamil. We found rat hepatocytes to produce large amounts of norverapamil (40% peak ratio in the MS mode relative to verapamil) as well (Walles et al., 2003). Obviously, inhalation of verapamil abrogated extensive hepatic metabolism, and we observed a kinetic of plasma elimination similar to intravenous administration. Notably, norverapamil was not detected in rat plasma after administration by inhalation, although three additional metabolites (carbinolamin and metabolites 9 and 19; see Fig. 5) were identified after intravenous application.
Our study clearly demonstrates the advantage of a drug application by inhalation for verapamil to achieve therapeutic plasma levels without suffering the consequences of extensive hepatic first pass metabolism. The need for frequent drug usage may therefore be overcome. In conclusion, drugs with pharmacokinetic defects, such as verapamil, may benefit from drug administration by inhalation to enter into systemic drug circulation.
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Acknowledgments |
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Footnotes |
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ABBREVIATIONS: P450, cytochrome P450; AECs, alveolar epithelial type II cells; MS, mass spectrometry; HPLC, high-performance liquid chromatography; MSPD, matrix solid-phase dispersion; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; AUC, area under the curve; s/n, single-to-noise.
Address correspondence to: Prof. Dr. Jürgen Borlak, Department of Drug Research and Medical Biotechnology, Fraunhofer Institute of Toxicology and Experimental Medicine, Nikolai-Fuchs-Str. 1, D-30625 Hannover. E-mail: borlak{at}item.fraunhofer.de
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1-antitrypsin treatment for cystic fibrosis. Lancet 337: 392-394.[CrossRef][Medline]
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