Abstract
The pulmonary and hepatic expression and catalytic activities of phase I and II drug-metabolizing enzymes were compared using human lung and liver tissue, and lung parenchymal cells (LPCs) and cryopreserved hepatocytes. Cytochrome P450 gene expression was generally lower in lung than in liver and CYP3A4 expression in lung was negligible. Esterase gene expression was similar in lung and liver. Expression of all sulfotransferase isoforms in lung was similar to or higher than that in liver. Lung tissue expressed low levels of UGT. However, the expression of UGT2A1 in lung was higher than that in liver. There was a range of catalytic activities in LPCs, including cytochrome P450, esterase, and sulfation pathways. Phase I activities were generally less than 10% of those determined in hepatocytes. Rates of ester hydrolysis and sulfation in LPCs were similar to those in hepatocytes. When measurable, glucuronidation in LPCs was present at very low levels, reflecting the gene expression data. The metabolism of salbutamol, formoterol, and budesonide was also investigated. Production of salbutamol-4-O-sulfate and budesonide oleate was observed in LPCs from at least two of three donor preparations studied. Formoterol sulfate and low levels of formoterol glucuronide were detected in one of three donors. In general, drug-metabolizing capability of LPCs is low compared with liver, although some evidence for substantial sulfation and deesterification capacity was observed. Therefore, these data support the use of this cell-based system for the investigation of key routes of xenobiotic metabolism in human lung parenchyma.
Inhaled drugs are among the frontline therapies for respiratory diseases including asthma and chronic obstructive pulmonary disease (Barnes, 2006). The overall contribution of pulmonary drug metabolism to the disposition of inhaled drugs is often assumed to be minor compared with that of the liver (Ding and Kaminsky, 2003). However, detailed, systematic investigations of the expression and catalytic activity of drug-metabolizing capacity of pulmonary cells are limited.
The lung is a complex organ consisting of approximately 40 different cell types (Nemery and Hoet, 1993). This complex architecture reflects the physiological role of the lung as the gas exchange interface between the blood and the external environment. As a frontline barrier, the lung requires protection from a wide range of gaseous and particulate xenobiotic substances that may be inhaled. The lung has therefore developed a wide range of protective detoxification systems. There are numerous publications reporting the presence of xenobiotic enzymes in the lung, from cytochromes P450 (P450s) normally found in liver, such as CYP1A, CYP2A, CYP2B, CYP2C, CYP2D, and CYP3A, to a number of reportedly lung-specific P450s such as CYP2S, CYP2F, and CYP4B1 and non-P450 systems such as epoxide hydrolase (Pacifici et al., 1988; Ding and Kaminsky, 2003; Castell et al., 2005; Bernauer et al., 2006; Zhang et al., 2006). In addition to phase I enzymes, phase II enzymes such as UGT, SULT, glutathione S-transferase, and peptidase activity have also been reported (Lorenz et al., 1984; Sidorowicz et al., 1984; King et al., 2000; Glatt et al., 2001; Zhang et al., 2006). Due to the low level of drug-metabolizing activity in the lung, many published studies are based on detection of mRNA rather than direct measurement of catalytic activity, although a number of studies have reported catalytic activity in both animal and human lung tissue preparations. However, some debate still exists between the specific location of drug-metabolizing capability in the lung and its relevance to inhaled therapy. For example, inhaled medicines will first come into contact with the bronchial epithelial cells of the lung. It therefore seems logical that the lung epithelium will have most relevant drug-metabolizing capability. Indeed, it has been reported that the majority of the drug-metabolizing capacity of rodent lung is present within epithelial Clara cells (Ding and Kaminsky, 2003). However, the same cells in human are reported to contain less endoplasmic reticulum and, therefore, reduced activity of drug-metabolizing capability (Castell et al., 2005). The actual location and importance of the drug-metabolizing enzymes within the human lung therefore remains unclear. However, it is evident that the metabolic fate of compounds administered to the lung will be influenced by the method of delivery of the drug, the corresponding residence time of the drug within the lung tissue, and the pulmonary drug elimination processes, of which metabolism will be a contributory factor.
Oral pulmonary therapy is an established route of administration, and there is an increasing interest in the use of this route for systemic drug delivery (Gonda, 2006). However, the location and relevance of human pulmonary drug metabolism for inhaled medicines is unclear at present; therefore, the aim of the current study was to investigate the drug-metabolizing capability of the human lung. In particular, demonstration of both phase I and II pathways as well as non-P450-mediated metabolism was considered essential, as these pathways are reportedly involved in the metabolism of inhaled therapies such as salbutamol, beclomethasone dipropionate (BDP), budesonide, and formoterol (Jonsson et al., 1995; Pacifici et al., 1996; Tunek et al., 1997; Joyce et al., 1998; Daley-Yates et al., 2001; Zhang et al., 2002). Freshly isolated mixed lung parenchymal cell (LPC) preparations derived from human lung were used to develop an in vitro model of human pulmonary metabolism. Metabolism data obtained in human lung cells were compared with those obtained in cryopreserved human hepatocytes.
Materials and Methods
Reagents. 7-Ethoxycoumarin (7-EC), coumarin, 7-hydroxycoumarin (7-HC), ethoxyresorufin (ER), resorufin, 4-hydroxy diclofenac, testosterone, 4-methylumbelliferone (4-MU), androstenedione, 1-naphthyl β-d-glucuronide, sodium bicarbonate, acetaminophen glucuronide, William's medium E, and l-glutamine were supplied by Sigma-Aldrich (Poole, Dorset, UK). S-Mephenytoin, 4-hydroxy mephenytoin, bufuralol, 1-hydroxy bufuralol, 1-hydroxy midazolam, 4-hydroxy midazolam, 7-HC sulfate, 7-HC glucuronide, acetaminophen sulfate, midazolam, and 6β-hydroxy testosterone were supplied by SAFC Corp. (St. Louis, MO). Hydroxy bupropion was supplied by Toronto Research Chemicals Inc. (Toronto, ON, Canada). α-Naphthyl sulfate potassium salt was supplied by Research Organics Inc. (Cleveland, OH). Acetaminophen, diclofenac, budesonide, bupropion, astemizole, BDP, beclomethasone monopropionate (BMP), beclomethasone, salbutamol, salbutamol 4-O-sulfate, 1-naphthol, and formoterol were greater than 99% pure and were supplied by GlaxoSmithKline Research and Development (Stevenage, UK). Cryopreserved human hepatocytes (n = 10 donors), hepatocyte thawing medium, and antibiotics were supplied by In Vitro Technologies (Baltimore, MD). Trypan blue was supplied by MP Biomedicals Inc. (Irvine, CA). AmpliTaq Gold DNA polymerase, AmpErase uracil-N-glycosylase, and MGB probes were supplied by Applied Biosystems (Warrington, UK). Acetylated bovine serum albumin and M-MLV Reverse Transcriptase were supplied by Promega (Southampton, UK). DNase I was supplied by Invitrogen (Paisley, UK). deoxynucleoside-5′-triphosphates were supplied by BioGene Limited (Kimbolton, UK). Primers and TaqMan probes were supplied by Sigma-Genosys (Haverhill, UK). All other chemicals and reagents were of analytical grade or higher.
Human Tissue. All human lung samples were procured with the informed consent of the donor or donor's next of kin, and with the approval of the appropriate local ethics committee. Liver pieces were obtained from a number of “ethically approved sources” and Local Research Ethics Committee approval was granted. Microsomes were prepared from this tissue in-house using a standard differential centrifugation method. Details of the donors are shown in Table 1.
Determination and Quantification of mRNA Present in Human Lung Parenchyma and Human Liver. Total RNA was extracted from lung and liver parenchyma using a TRIzol-based method and was DNase-treated before quantitative real-time, reverse transcriptase-polymerase chain reaction (PCR). Gene-specific primer probe sets (Table 2) were designed at Asterand and quantitative real-time, reverse transcriptase-PCR analysis was performed as described previously (Bowen et al., 2000). Total RNA samples were annealed to optimized concentrations of specific primer probe sets that exhibit no significant homology to any other mRNA as determined by BLAST searches, and reverse-transcribed using M-MLV Reverse Transcriptase. Quantitative sequence detection was performed using an ABI 7900 (Applied Biosystems). Expression of the target genes and GAPDH was quantified using a proprietary multiplexing protocol. Successful reverse transcriptions and PCR were confirmed by the coamplification of GADPH mRNA. Gene expression data were expressed as copy number/100 ng of total RNA based on Asterand's global standard curve.
Preparation of Human Isolated Lung Parenchymal Cells. LPCs were isolated from macroscopically normal sections of human lung parenchyma by trypsin digestion. In brief, lung parenchyma was washed thoroughly with phosphate-buffered saline to remove any blood. The tissue was then minced with scissors and digested in an appropriate volume of digestion medium (minimal essential medium supplemented with 2 mM l-glutamine, 20 mM HEPES, 0.25 mg/ml DNase I, and 2 mg/ml trypsin) twice for 30 min each on a shaking platform at 37°C. The resulting cell suspensions were filtered through 150-μm gauze into tubes containing fetal bovine serum to inactivate the trypsin. The cells were then washed twice by pelleting at 300g for 10 min at 4°C and resuspending in William's medium E (phenol red-free) supplemented with 2 mM l-glutamine. Cell viability was determined by trypan blue exclusion and the initial viability of all lung cell preparations was greater than 90%.
Freshly Isolated Lung Parenchymal Cell Incubations. Probe substrate solutions were prepared in dimethyl sulfoxide, diluted in William's medium E containing l-glutamine, and added to preparations of human LPCs in suspension at final cell concentrations of either 1.5 × 106 cells/ml (donors 1-4) or 1 × 106 cells/ml (donors 5-8). The final incubation volume was 2 ml. The final concentration of dimethyl sulfoxide in the incubations was <0.2%.
Probe substrates were incubated at 2 μM for determination of rates of metabolism and at least 10-fold higher for the investigation of metabolic routes. Substrate concentrations for the rate analysis were chosen to represent a concentration lower than hepatic Km but high enough to be easily detected by LC-MS/MS. Route substrate concentrations were chosen to correlate with approximate hepatic Km for P450 probe substrates. For phase II probe substrates and inhaled drugs, a nominal substrate concentration of 25 μM was used.
Incubations designed to investigate routes of metabolism were carried out at concentrations approximating the hepatic Km. Table 3 details the probe substrates used to characterize drug metabolism in both freshly isolated human LPCs and cryopreserved hepatocytes, together with their final incubation concentrations. Incubations were performed at 37°C and samples were removed at 0, 5, 15, 30, 45, 60, 90, 120, and 240 min for rate determination and at 0, 2, and 12 h for route determination.
Cryopreserved Hepatocyte Incubations. Hepatocytes were thawed as described by the supplier and incubated at 1 × 106 cells/ml in William's medium E containing l-glutamine. Probe substrates were incubated at 1 μM for rate incubations and at equivalent substrate concentrations to the lung cell incubations for route investigation (Table 3). Data were generated in three independent experiments performed with the same batch of pooled cryopreserved hepatocytes. For incubations with both human LPCs and cryopreserved hepatocytes, the rate of depletion of probe substrates and/or the rate of production of known major, or enzyme-specific, metabolites were measured by LC-MS/MS.
Sample Analysis. Samples for the determination of the rates and routes of metabolism of all probe substrates were analyzed using sensitive and specific LC-MS/MS methods. The LC-MS/MS system comprised an Agilent 1100 binary pump (Agilent, Waldbronn, Germany) and an HTS PAL CTC autosampler (CTC Analytics, Zwingen, Switzerland) coupled to a Sciex 4000-QTrap mass spectrometer (MDS Sciex, Concord, ON, Canada) fitted with a pneumatically assisted electrospray ion source. Separations were achieved using various reversed phase stationary phases including Luna C18(2) 5 μm, Synergi Max RP 4 μm, and Synergi Hydro 4 μm (Phenomenex, Torrance, CA), depending upon individual substrates and metabolites. All high-performance liquid chromatography columns were 50 × 2 mm i.d., operated at 40°C, and eluted with a mobile phase flow rate of 0.8 ml/min. As with the stationary phase, the mobile phase composition was chosen depending upon substrate and metabolite(s). Formic acid (0.1% v/v in water/acetonitrile), ammonium formate (10 mM in water/methanol), and ammonium acetate (10 mM in water/methanol) were all used. In each case, gradient elution was used. The gradient elution profiles were adjusted to give sufficient retention and separation of substrate and metabolite(s) while keeping the chromatographic run to within 5 min. Individual substrate and metabolite(s) were analyzed within the same analytical run in all but two cases. Chromatographic resolution of acetaminophen and its sulfate and glucuronide conjugates was required to avoid overestimation of acetaminophen due to in-source degradation of the cochromatographing conjugated metabolites. Because simultaneous resolution of both metabolites from parent was not achieved, acetaminophen was analyzed separately from its metabolites. Budesonide and its fatty acid ester metabolite, budesonide oleate, could not be analyzed using the same high-performance liquid chromatography method because of the extreme hydrophobicity of budesonide oleate compared with budesonide.
Detection was achieved by selected reaction monitoring (SRM) for specific product ions derived from collision-induced dissociation of the analyte precursor ion. The optimum mode of analysis, positive or negative ion, was used for each individual analyte based on highest sensitivity. Protonated (MH+) or deprotonated (MH-) molecules were used as precursor ions in all cases, with the exception of budesonide oleate, where, in negative ion mode, an acetate adduct (M + CH3COO-) was used as the precursor ion (Tunek et al., 1997).
The presence of metabolites was confirmed by cochromatography with reference standards. Where reference material was unavailable, methods were developed based upon the product ion spectrum of the parent molecule and the substrate/putative metabolite molecular weight offset, with the theoretical SRM transitions being monitored within the analytical run. Retention time reference samples for the metabolites of formoterol were generated by incubating formoterol with fresh rat hepatocytes. The resulting sample was used for method development, monitoring SRM mass transitions based on published product ion spectra (Rosenborg et al., 1999). Additional SRM transitions were included as qualifiers wherever possible to increase the degree of confidence in identification of metabolites. The methods used for the analysis of probe substrates and their metabolites in hepatocyte and LPC incubation samples are summarized in Table 4.
Sample preparation consisted of centrifugation and dilution of supernatant with an equal volume of water before direct injection onto the LC-MS/MS system. In the case of formoterol and salbutamol samples, a dilution factor of 3 was used to prevent peak splitting.
Analysis of the rates of metabolism was performed by monitoring depletion of the parent compound over the incubation time profile. Peak area ratios to internal standard were used for all substrates with the exception of 7-hydroxy coumarin, 1-naphthol, acetaminophen, and formoterol; for these compounds, parent peak area only was used.
Data Analyses. Rates of depletion of parent compound were fitted to a model of exponential decay. The resulting elimination rate constant (kel) was converted to an in vitro clearance (CLin vitro) using the following equation: CLin vitro = kel · V.
Results
The expression of drug-metabolizing enzymes in human lung parenchyma, expressed as copy number per 100 ng of total RNA, compared with that in human liver is presented in Figs. 1 and 2 respectively. Of the genes investigated, CYP1A1, CYP1A2, CYP3A4, EPHX2, SULT2A1, UGT1A1, UGT1A4, and UGT2B11 were expressed at low levels in human lung parenchyma with an average copy number of <100 per 100 ng of total RNA. All other genes were expressed at levels >100 copies per 100 ng of total RNA.
The expression of most of the phase I drug-metabolizing enzymes studied in human lung parenchyma was significantly lower than that in liver (p < 0.01). Relatively higher expression of epoxide hydrolase (EPHX1) and esterase was measured, approximately 20% of hepatic levels. However, this level of expression was also significantly lower than that in liver (p < 0.05). The expression of the SULT isoforms SULT1A1, 1A2, and 1A3/4 was not significantly different in the lung compared with that in liver. SULT2B1/2 expression was approximately 500-fold greater in lung compared with that in liver, a statistically significant difference (p < 0.01). The expression of UGT in lung parenchyma was significantly lower (p < 0.01) and less than 5% of that expressed in liver. However, the mean expression of one UGT isoform in lung, UGT1A6*2, was not significantly different from that in liver, whereas the mean expression of UGT2A1 was 5-fold and significantly (p < 0.01) higher in lung compared with liver.
These differences in expression were reflected in the catalytic activities of LPCs (Table 5). The catalytic turnover of the phase I metabolic probes investigated was <10% that observed in hepatocytes, whereas some phase II and esterase probes showed relatively higher activities (Table 6).
The selected metabolites of the probe substrates investigated in LPCs and hepatocytes are shown in Table 7. For most of the probe substrates investigated in hepatocytes, similar metabolites were produced in pulmonary tissue. However some differences were also observed. For the CYP3A4 substrates testosterone and midazolam, a smaller range of metabolites was observed in LPCs compared with hepatocytes, with low levels of 6β-hydroxy testosterone and 1-hydroxy midazolam produced. No glucuronide metabolites of umbelliferone, 4-MU, or acetaminophen were detected, although sulfation of both umbelliferone and 4-MU was observed in both hepatocytes and LPCs. Trace levels of 1-naphthol glucuronide were detected in LPCs from one of three donors.
The metabolism of the marketed inhaled compounds salbutamol, formoterol, and budesonide was also investigated (Tables 5 and 6). LPCs produced the sulfate metabolite of salbutamol in two of three donors, although in one of these donors only trace amounts of metabolite were observed. Sulfate and trace levels of glucuronide metabolites of formoterol and budesonide oleate were observed in LPCs from one of three donors.
Discussion
The current studies have shown that freshly isolated human LPCs have the capability to metabolize drugs, albeit at considerably reduced rates compared with hepatic preparations. The expression of a range of drug-metabolizing enzymes including P450, carboxylesterase, epoxide hydrolase, and sulfotransferase observed in these studies is consistent with previously reported data (Lorenz et al., 1984; Pacifici et al., 1988, Petruzzelli et al., 1988; Toussaint et al., 1993; Zhang et al., 2006). We did not determine the uptake of the individual substrates into the LPCs, although the assumption was that if the substrates entered hepatocytes, they would also enter LPCs. Uptake could be investigated further in separate experiments to determine whether any lack of metabolism was due to low or no uptake of the compounds.
The previously observed low expression of CYP3A4 in lung tissue (Toussaint et al., 1993; Anttila et al., 1997; Castell et al., 2005; Raunio et al., 2005) is supported by these studies. First, there was no evidence for the formation of hydroxy budesonide, a metabolic pathway predominantly mediated by CYP3A4 (Jonsson et al., 1995). Second, the poor expression of CYP3A4 in lung parenchyma was indicated by the metabolism of testosterone, which resulted primarily in the production of androstenedione without substantial production of the CYP3A-mediated 6β-hydroxy testosterone metabolite. The conversion of testosterone to androstenedione occurs predominantly via a dehydrogenase pathway (Sanderson and van den Berg, 2003) or P450s other than CYP3A4 (Yamazaki and Shimada, 1997). The involvement of CYP2C19 in the oxidation of testosterone to androstenedione has been reported previously (Yamazaki et al., 1997); however, low levels of CYP2C activity were also observed in the current studies. In contrast to CYP3A4, CYP3A5 was expressed at low levels in lung parenchyma and may therefore play a role in the pulmonary metabolism of midazolam and testosterone (Patki et al., 2003), as some 6β-hydroxy testosterone and 1-hydroxy midazolam production was observed, albeit at low levels.
The presence or absence of CYP2C activity in the lung is currently poorly understood (Zhang et al., 2006). In the current studies, little or no metabolism of diclofenac or S-mephenytoin was observed in LPCs, indicating poor CYP2C catalytic activity in these cells. These data are in keeping with previously published data (Toussaint et al., 1993). The results from our studies may also support the hypothesis that CYP2C expression in the lung is low and limited to specific cell types, such as the serous cells of bronchial glands (Zhang et al., 2006).
The hydrolysis and sulfation of some of the probe substrates and inhaled medicines in lung tissue observed in these studies has been previously documented. Hydrolysis of BDP to BMP and beclomethasone has been reported in lung homogenates in vitro (Foe et al., 2000) and after administration to human (Daley-Yates et al., 2001). The sulfation of salbutamol has been described previously in lung homogenate in vitro (Pacifici et al., 1996) although this particular pathway was shown not to occur after administration of salbutamol to human (Ward et al., 2000). Interestingly, phase II metabolites of formoterol, a compound known to be sulfated and glucuronidated in human (Rosenborg et al., 1999; Zhang et al., 2002) were not consistently observed in these studies. In contrast, the oleate metabolite of budesonide was observed, indicating the presence of an active fatty acid pathway. The fatty acid conjugate of budesonide has been reported to be responsible for the duration of action of budesonide in human (Tunek et al., 1997).
Of particular interest in these studies is the apparent and consistent ability of LPCs to hydrolyze substrates such as BDP, and to produce sulfate conjugates of a number of probe substrates while lacking extensive expression of UGT or the ability to glucuronidate specific probe substrates. Previously reported Western blot analysis (King et al., 2000; Turgeon et al., 2001) and catalytic studies have indicated the presence of UGT protein and UGT activity in the human lung. Glucuronidation of 1-naphthol or 4-nitrophenol has been reported in human pulmonary microsomes (Conner et al., 1997; Ghosheh and Hawes, 2002). Other catalytic studies have shown resorufin glucuronidation activity in isolated porcine lung epithelial cells (Blickwede and Borlak, 2005), and perfused rat lung studies have demonstrated the glucuronidation of 4-MU (Aitio et al., 1976). In-house studies have also shown low level formation of naphthyl glucuronide in alamethicin-treated human lung microsomes, albeit at less than 10% of hepatic activity (unpublished data). In contrast, other studies have indicated little or no UGT activity in human lung tissue (Toussaint et al., 1993; Zheng et al., 2002). In the current studies, the absence of UGT catalytic activity for the UGT probe substrates studied, namely, 1-naphthol, acetaminophen, and 4-MU, is consistent with the low expression of UGT isoforms observed. This indicates either a reduced inherent UGT activity in LPCs, the expression of an isoform different from those investigated in these studies, or a lack of suitable cofactors (uridine diphosphoglucuronic acid) in these lung cell preparations. Another possibility is that UGT activity in the pulmonary tissue is concentrated in cell types not represented in high concentrations in these studies, such as pulmonary epithelial cells as demonstrated in animal species (Yamashiki et al., 2002; Blickwede and Borlak, 2005) or more highly expressed in the tissues of the upper respiratory tract of human subjects (Zheng et al., 2002).
The data from the current studies indicate that unless an inhaled drug has specific sites susceptible to metabolism, such as hydrolysis or sulfation, or is particularly metabolically labile, then inhaled medicines are unlikely to have reduced efficacy due to metabolic clearance in the lung. However, at least one strategy for extending duration of action of inhaled drug candidates is to increase the retention of the compound within the lung. The longer the compound remains in the lung, assuming that the compound remains in the same compartment as the drug-metabolizing enzymes, there is the possibility that drug metabolism could contribute to the retention, efficacy, or toxicity of the drug. With regard to the potential for toxicity, there are few reported cases of clinical pulmonary toxicity due to the metabolic activation of an inhaled topical medicine. Inhaled medicines are generally potent compounds administered at very low doses and such low doses may mitigate the possibility of toxicity. Conversely, there is some evidence of toxicity arising due to lung-specific metabolism of some agents in animal species (Higenbottam et al., 2004).
It has been reported that specific metabolic capabilities can be localized in some pulmonary cell types. For example, Clara cells from preclinical species contain a relatively high drug-metabolizing capacity (Ding and Kaminsky, 2003), and human macrophages exhibit epoxide hydrolase and glutathione transferase activities (Petruzzelli et al., 1988). In addition, drug transporters, such as the multidrug resistance, multidrug resistance protein, peptide transporter, and lung resistance protein systems have also been shown to be expressed in human lung tissue (Hamilton et al., 2002; Groneberg et al., 2004). Both localized metabolism and transport could affect the disposition of an inhaled drug in vivo. Although the current studies show that LPCs are capable of drug metabolism, it is possible that epithelial cells lining the respiratory tract may influence the disposition of an inhaled therapy to a greater extent than parenchymal cells, which will only have access to the distributed portion of either an inhaled or oral dose.
The current experiments used a mixed population of human parenchymal cells as a pragmatic solution to developing a method to investigate drug-metabolizing capability of the lung. There has been no correction for the location or identity of the predominant cell types represented in the mixed cell population. This, together with the lack of a suitable scaling factor, currently rules out attempting to scale the metabolic processes observed to predict lung clearance as a proportion of plasma clearance. Further work in this area to characterize the population of cells recovered compared with intact tissue would be required before quantitative assessments of lung tissue stability could be obtained.
In summary, an in vitro system for the investigation of particular enzyme activities in human lung has been investigated. At present, the model can be used to rank potential drug candidates for metabolic stability in the lung. Further studies will focus on continued optimization of the in vitro approach, including investigation of metabolism in specific cell types within the lung, metabolism capability in different disease states (for example, asthma and chronic obstructive pulmonary disease), and characterizing the impact of drug transporters in the lung.
Acknowledgments
We thank Florence Athersmith, Lisa Gould, and Gabriela Cruz for technical assistance.
Footnotes
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doi:10.1124/dmd.107.015966.
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ABBREVIATIONS: SULT, sulfotransferase; BDP, beclomethasone dipropionate; BMP, beclomethasone monopropionate; P450, cytochrome P450; LPC, lung parenchymal cell; 7-EC, 7-ethoxycoumarin; EPHX, epoxide hydrolase; ER, ethoxyresorufin; 7-HC, 7-hydroxycoumarin; LC-MS/MS, liquid chromatography-tandem mass spectrometry; 4-MU, 4-methylumbelliferone; PCR, polymerase chain reaction; SRM, selected reaction monitoring; UGT, uridine glucuronosyltransferase.
- Received March 26, 2007.
- Accepted July 9, 2007.
- The American Society for Pharmacology and Experimental Therapeutics