Send Orders for Reprints to [email protected] 53 Letters, 2019, 13, 53-63

RESEARCH ARTICLE

ISSN: 1872-3128 eISSN: 1874-0758

Comparison of Rat and Human Pulmonary Metabolism Using Precision-cut Lung Slices (PCLS)

Yildiz Yilmaz1,*, Gareth Williams1, Markus Walles1, Nenad Manevski1, Stephan Krähenbühl2 and Gian Camenisch1

1Pharmacokinetic Sciences, Novartis Institutes for Biomedical Research, Basel, Switzerland; 2Clinical and , University Hospital, Basel, Switzerland

Abstract: Background: Although the liver is the primary organ of drug metabolism, the lungs also contain drug-metabolizing enzymes and may, therefore, contribute to the elimination of drugs. In this investigation, the Precision-cut Lung Slice (PCLS) technique was standardized with the aims of charac- terizing and comparing rat and human pulmonary drug metabolizing activity. Method: Due to the limited availability of human lung tissue, standardization of the PCLS method was performed with rat lung tissue. Pulmonary enzymatic activity was found to vary significantly with rat A R T I C L E H I S T O R Y age and rat strain. The Dynamic Organ Culture (DOC) system was superior to well-plates for tissue in- cubations, while oxygen supply appeared to have a limited impact within the 4h incubation period used

Received: August 03, 2018 here. Revised: September 28, 2018 Accepted: October 08, 2018 Results: The metabolism of a range of phase I and phase II probe substrates was assessed in rat and

DOI: human lung preparations. (CYP) activity was relatively low in both species, whereas 10.2174/1872312812666181022114622 phase II activity appeared to be more significant.

Conclusion: PCLS is a promising tool for the investigation of pulmonary drug metabolism. The data indicates that pulmonary CYP activity is relatively low and that there are significant differences in en- zyme activity between rat and human lung. Keywords: Precision-cut lung slices (PCLS), rat lung metabolism, human lung metabolism, rat and human pulmonary metabo- lism activity, pulmonary disposition of phase I and phase II drugs, dynamic organ culture system, AFQ056.

1. INTRODUCTION and reduced systemic exposure and adverse reactions. Com- pounds absorbed via the inhaled route appear rapidly in the Although the primary function of the lung is gas ex- systemic circulation, bypassing any extensive hepatic first change, it also performs several important non-respiratory past effects [2]. The lung is a complex organ containing over functions. Through breathing, the lung is frequently exposed 40 different types of cells with varied functions and architec- to environmental toxicants and carcinogens and plays an ture [3]. The location of the pulmonary metabolizing en- important role in their detoxification. The same enzymes that zymes has been previously discussed [4]. Bronchial and play a role in the detoxification of these environmental com- bronchiolar epithelia, Clara cells, type II pneumocytes and ponents may also contribute to the metabolism and elimina- alveolar macrophages all express Cytochrome P450 (CYP) tion of inhaled, orally and intravenously administered drugs. enzymes [5, 6]. However, type I pneumocytes and endothe- The understanding of pulmonary diseases such as lung can- lial cells, which are most directly exposed to blood flow, cer, bronchial asthma and Chronic Obstructive Pulmonary contain only small amounts of CYPs [7]. Phase II enzymes Disease (COPD) has improved during the last twenty years such as UGT1A (glucuronyl transferase) and GST-P1 (glu- [1]. Consequently, the inhaled route of drug delivery has tathione S-transferase), which are important detoxifying en- gained more importance with the aim to treat these diseases zymes, are also found in lung cells [8, 9]. Due to this diver- directly. Inhaled drug delivery has several benefits including sity of cell types and variable metabolic activity, the study of a small drug load with high local concentrations, immediate pulmonary disposition is challenging. Therefore, suitable in delivery of the drug to the target with a rapid onset of action vitro tools are required to fully understand the potential role

of pulmonary metabolism in drug disposition. *Address correspondence to this author at the Novartis Institute for Bio- Pulmonary subcellular fractions such as microsomes and medical Research, Pharmacokinetic Sciences, Fabrikstrasse 14-1, Novartis Pharma AG, CH-4002 Basel, Switzerland; S9 are available from human and preclinical animal species. E-mail: [email protected] Although these in vitro systems require the addition of ap-

1874-0758/19 $58.00+.00 © 2019 Bentham Science Publishers Drug Metabolism Letters 54 Drug Metabolism Letters, 2019, Vol. 13, No. 1 Yilmaz et al. propriate cofactors such as Nicotinamide adenine dinucleo- not adversely affect CYP1A1 activity. The pulmonary me- tide phosphate (NADPH) and Uridine-diphosphate- tabolism of AFQ056 is sensitive regarding the conditions glucuronic acid trisodium salt (UDPGA), they provide a used and was therefore considered to be suitable for the op- convenient means for studying lung metabolism. Proteolytic timization of the PCLS conditions. Finally, the optimized enzymes are required for the preparation of subcellular frac- and standardized PCLS parameters were used to compare rat tions such as lung microsomes [10]. However, these subcel- and human pulmonary enzyme activity. Incubations were lular fractions lack the architecture of the intact cells and performed with a range of substrates for enzymes including may therefore not fully reflect the capacity of pulmonary CYPs, Flavin-containing Monooxygenases (FMOs), UDP- metabolism. Precision-cut Lung Slices (PCLS) are an alter- glucuronosyltransferases (UGTs) and Sulfotransferases native tool for studying lung metabolism and have several (SULTs). The substrates were selected based on literature important advantages over subcellular fractions as they pos- data and the availability of reference standards for parent sess the full range of cell types, intact tissue architecture and compound and metabolite quantification. cofactors at physiologically relevant concentrations [11, 12]. As a result, phase I and phase II metabolic processes are 2. MATERIALS AND METHODS fully represented by tissue slices [10]. The use of rat PCLS for drug metabolism investigations was first reported in 1977 2.1. Reagents, Chemicals, and Materials [13]. As an example, in vitro human pulmonary metabolism of ciclesonide, an inhaled corticosteroid, was investigated AFQ056 (mavoglurant) and NVP-CBJ474 (AFQ056-M3) using PCLS [14]. Furthermore, PCLS have recently been were synthesized at Novartis (Basel, Switzerland). Minimal used to study the metabolism of mavoglurant (AFQ056) Essential Medium (MEM, without L-, HEPES, and [15]. phenol red) and agarose UltraPure™ low melting point were purchased from Life Technologies (Carlsbad, CA). 4- One drawback of the PCLS model is that it generally re- Methylumbelliferone, 4-methylumbelliferyl glucuronide, 4- quires fresh tissue. Although this is routinely available for methylumbelliferyl sulfate, benzydamine, ramipril, triclosan, rodent species, its availability is limited for human and other carbazeran, diclofenac, diazepam, amodiaquine, sumatriptan, species such as dog and monkey. Nevertheless, the use of P-toluidine and 4-methylacetanilide were purchased from PCLS shows great promise as the methodology can be easily Sigma Aldrich. Benzydamine N-oxide, ramiprilat, triclosan applied to different organs such as liver, kidney and brain glucuronide, triclosan sulfate, 4-hydroxycarbazeran, 4- and may also be used to identify potential differences in drug hydroxydiclofenac, diclofenac glucuronide, midazolam, 1- metabolism between human and animals used for toxicologi- hydroxymidazolam, nordiazepam and N-desethylamo- cal studies [16]. Lung slices can be produced from limited diaquine were purchased from Toronto research chemicals. amounts of tissue and therefore, surgical waste material may be suitable for this purpose [17]. The preparation of subcel- 2.2. Standardization of the PCLS Incubation Conditions lular fractions from similar amounts of tissue would typically result in poor yields of protein. Although lung microsomes Rat lung tissue slices were prepared as previously de- and S9 are commercially available and relatively inexpen- scribed [15]. The impact of animal age was investigated by sive, their preparation time is usually longer than that of tis- performing incubations with lung slices from Sprague sue slices, potentially leading to loss of metabolic activity. Dawley (SD) rats aged between 6 and 12 weeks. In order to Furthermore, enzymatic activity will be adversely affected evaluate differences in enzyme activity due to rat strain, lung by repeated freeze-thaw cycles and this should be avoided tissue was also obtained from 6 week old Wistar Han (WH) when using microsomes and S9. In recent years, the use of rats. PCLS incubations were performed using titanium (type tissue cryopreservation has been investigated to reduce the C) roller inserts (Vitron Inc., Tucson, AZ). Three lung tissue dependence of the PCLS model on fresh material [18]. How- slices (average weight 0.024 g/slice) were positioned onto ever, robust methodology for tissue cryopreservation has not the roller inserts and then placed in glass vials (Fig. 1). The been developed so far and therefore, has not been used in the glass vials were prefilled with a modified dynamic organ current investigations. culture medium composed of MEM, HEPES (25 mM) and glucose (25 mM) [24, 25]. During the optimization process, Although lung slicing technology is commonly used in the DOC incubation system was compared with well-plates metabolism studies, the protocols used vary between labora- (Fig. 2) to determine if the pulmonary activity was depend- tories and there is still an opportunity to improve and stan- ent upon the incubation device. As the primary role of the dardize the technique [19]. Therefore, in this study, some of lung is gas exchange, the impact of the oxygen content in the the key experimental factors used in the PCLS procedure incubation system on drug metabolism was investigated. For were optimized with the aim of reducing tissue damage and that, metabolic activities were compared using an oxygen retaining lung metabolic activity. The PCLS incubation con- rich atmosphere (75% O , 5% CO ) and an atmosphere com- ditions were partly optimized and fully standardized using 2 2 posed of 5% CO and normal air (21% O content). the CYP1A1 substrate, AFQ056. CYP1A1 has previously 2 2 been shown to efficiently catalyse the oxidation of AFQ056, leading to the formation of the metabolite, CBJ474 [20]. 2.3. Preparation and Incubation of Lung Tissue Slices CYP1A1 is widely recognised as an extrahepatic enzyme Animal experiments were conducted with the approval of that is expressed in organs including kidney, intestine and the Cantonal Veterinary Authority of Basel City, Switzerland lung [21-23]. CYP1A1 mRNA is known to have a short half- (Protocol BS-2723). Male SD and WH rats, with an average life of approximately 2.4 h (Lekas et al., 2000) and it is age of 6 - 12 weeks and body weight of 200 - 250 g, were therefore critical that the experimental conditions used do used in this study (Charles River Germany). Fresh rat lungs Comparison of Rat and Human Pulmonary Drug Metabolism Drug Metabolism Letters, 2019, Vol. 13, No. 1 55

Fig. (1). Dynamic organ culture incubation system. For PCLS in- cubations, rat lung tissue slices (n=3, average weight 0.024 g/slice) were positioned onto the titanium (type C) roller inserts and then placed into glass vials. The glass vials were filled with a modified medium composed of MEM, HEPES (25 mM), glucose (25 mM) and test substrate (1 µM). Each time point was carried out in dupli- cate comprising 3 slices for each time point, and each incubation was carried out in triplicate. were obtained with the trachea and immediately put on ice after surgery until processing (Table. 1). The use of human lung tissue was approved by the local ethics committee of Basel, Switzerland (EKNZ 2015-355) and the studies were performed in accordance with the Dec- laration of Helsinki (1964 and subsequent revisions). In- Fig. (2). Precision cut lung slices incubation using a well-plate formed consent in writing was obtained from the study par- system. For PCLS incubations, 12 well-plates were filled with 2 mL ticipants. Tumour-free tissue samples from different regions of medium composed of MEM, HEPES (25 mM), glucose (25 mM) of the lung were collected post-surgery from six patients at and test substrate (1 µM). Rat lung tissue slices (n=3, average the Department of Thoracic Surgery, University Hospital weight 0.024 g/slice) were positioned on each plate, and incubated Basel. Basic demographic information is presented in Table for 4h. 2. The excised lung samples were immediately submerged in cold MEM medium. Samples were transported to our labora- Pre-warmed lung tissue (37°C for 5 min) was inflated tories at 4°C within 60 min of the time of surgical excision. with a 37°C solution of agarose (low melting point, 2.5%

Table 1. Important parameters for the tissue slice preparation.

Factors Subfactors Comments

Animals Strain Sprague Dawley & Wistar Han

Age 6-12 weeks

Fed/fasted Fed

Anesthesia Drug 3-5% Isofluran

Duration 3 min

Slice preparation Agarose gel Low melting point (2.5% v/v)

Slice thickness /weight ~416 µm / ~24 mg

Buffer MEM, HEPES, Glucose

Medium Volume 2 mL

Rotating frequency 4 rpm

Incubation conditions Temperature 37 °C

Duration of incubation 0-4 h

Incubation system Dynamic organ culture system & well-plate

These factors should be considered to minimize tissue damage in order to obtain an accurate and reproducible system for the determination of pulmonary drug metabolism. 56 Drug Metabolism Letters, 2019, Vol. 13, No. 1 Yilmaz et al.

Table 2. Demographic information of human lung donors.

Donor Gender Ethnicity Primary Lung Disease

1 Male Caucasian Bronchiectasis

2 Female Caucasian Lung cancer

3 Male Caucasian Lung cancer

4 Male Caucasian Lung cancer

5 Male Caucasian Lung cancer

6 Male Caucasian Lung cancer

Macroscopically healthy lung tissue surrounding the tumor or lesion was collected post-surgically from subjects aged 45 - 79 years.

Table 3. Probe substrates and corresponding metabolites used to investigate pulmonary metabolism.

Compound Metabolic Reaction Metabolite Responsible Enzyme

Triclosan Glucuronidation Triclosan glucuronide UGTs

Triclosan Sulfation Triclosan sulfate SULTs

4-MU Glucuronidation 4-MUG UGTs

4-MU Sulfation 4-MU-Sulfate SULTs

P-Toluidine N-Acetylation 4'-Methylacetanilide NATs

Midazolam Hydroxylation 1-Hydroxymidazolam CYP3A4

Amodiaquine N-deethylation N-Desethylamodiaquine CYP2C8

Diclofenac Hydroxylation 4-Hydroxydiclofenac CYP2C9

Diazepam N-demethylation Nordiazepam CYP2C19/3A

AFQ056 4-hydroxylation CBJ474 CYP1A1

Benzydamine N-Oxygenation Benzydamine-N-Oxide FMOs

Carbazeran Hydroxylation 4-Hydroxycarbazeran AO

Ramipril Hydrolysis Ramiprilat CES1

The probe substrates used in this study, their respective metabolites and responsible enzymes are shown. v/v) in MEM medium. The rat lungs were inflated via the a modified dynamic organ culture medium composed of trachea, whereas human lung tissue was inflated by injecting MEM, HEPES (25 mM) and glucose (25 mM). MEM buffer airways using a syringe and needle. This process of filling was pre-oxygenated prior to use by passing oxygen via a the lung with agarose allows for inflation of alveoli with the teflon tube, into the bottle. Oxygen pressure was regulated same embedding material and prevents the alveoli from col- by using a swagelock ventil. The tissue slices were pre- lapsing during the cutting process. The cutting process is incubated for 30 min at 37°C in a CO2 incubator (Hereaus, also easier and results in less surface damage to the tissue. Zurich, Switzerland) in an atmosphere of 75% O2 and 5% The slice thickness was also an important consideration. The CO2. The reactions were initiated by the addition of 1 µM of cut surface of the slice contains mainly damaged cells and if test compound and stopped after 240 min by adding 3mL of the slice thickness decreases then the overall metabolic ca- acetonitrile containing internal standard. The samples were pacity of the slice can be affected. Furthermore, if the slice vortex mixed and stored at -80°C until further processing. thickness increases this can lead to suboptimal metabolism A selection of phase I and phase II enzyme substrates due to reduced substrate delivery and gas diffusion [19]. (listed in Table 3) were used to compare enzyme activity in Slice thickness ( was chosen in this case based on ~416 µm) human and rat lungs. These compounds were incubated the previous findings [13, 19, 26, 27]. with tissue slices and the concentrations of substrate and For the incubations, three lung tissue slices (average corresponding metabolites were measured. The monitored weight 0.024 g/slice) were positioned onto titanium (type C) metabolites are also shown in Table 3. Probe substrates for roller inserts (Vitron Inc., Tucson, AZ). The inserts were drug-metabolizing enzymes were selected based on litera- then placed in glass vials that had been prefilled with 2mL of ture reports of the specific biotransformation pathways and Comparison of Rat and Human Pulmonary Drug Metabolism Drug Metabolism Letters, 2019, Vol. 13, No. 1 57 enzymes involved and availability of synthetic standards catalyze amodiaquine N-deethylation, diazepam N- enabling quantification of formed metabolites. 4- demethylation and diclofenac 4-hydroxylation in rats. De- Methylumbelliferone and triclosan have been described as spite this, amodiaquine, diazepam and diclofenac were still broad substrates for human UGTs and mammalian SULTs assumed to be suitable substrates to assess pulmonary [28, 29]. P-Toluidine was reported to be a substrate for CYP2C activity in this investigation. Benzydamine is re- human N-acetyltransferases present in human skin [30, 31]. ported to be a high-turnover substrate for human and rat Midazolam hydroxylation to 1’-hydroxymidazolam is FMOs [38, 39] and carbazeran hydroxylation to 4- catalysed by human and rat CYP3A [32, 33]. It was as- hydroxycarbazeran is catalysed by human and rodent Alde- sumed that 1- hydroxymidazolam formation would be suffi- hyde Oxidase (AO) [40-42]. Rampril hydrolysis to ramipri- cient to assess pulmonary CYP3A activity and therefore, lat was reported to be catalysed by human and rodent Car- quantification of 4-hydroxymidazolam was not performed in boxyl Esterase 1 (CES1) [43, 44]. this investigation. Amodiaquine N-deethylation is catalysed by human CYP2C8 leading to the formation of N- 2.4. Analytical Methods desethylamodiaquine [34]. N-demethylation of diazepam to form nordiazepam is predominantly catalysed by human Samples were quantitatively analysed using a Triple CYP2C19 and CYP3A [35]. Diclofenac hydroxylation to Quadrupole 6500 LC-MS/MS system (AB Sciex, Ontario, form 4’-hydroxydiclofenac is catalysed by human CYP2C9 CA) coupled to a UPLC system (Waters Acquity). Separa- [36]. The CYP2C subfamily is very complex with several tions were achieved using various reverse phase stationary different isoforms expressed in human and animal species phases. The analytical columns and mobile phase composi- [37]. The major isoforms expressed in human are CYP2C8, tions were developed for the simultaneous quantification of -2C9, 2C18 and 2C19. Although these isoforms are absent substrate and metabolite and are summarised in Table 4. The in rats, several other isoforms including CYP2C6, -2C7, - UPLC columns were operated at 40°C, with a mobile phase 2C11, -2C12, -2C13, -2C22 and -2C23 are expressed [37]. flow rate of 400 µL/min. The presence of metabolites was There is limited information regarding which isoenzymes confirmed by comparison with reference standards.

Table 4. LC-MS/MS methods for the determination of probe substrates and metabolites.

Gradient Gradient Ionization Analyte Column SRM Transition Gradient A B Profile Mode

AFQ056 Kinetex C8, 2.6 µm, 296/221 0.1% FA in H2O ACN A ESI + 2.1x50 mm CBJ474 312/237

4-MU 175/133 20 mM Phenomenex Luna Phenyl- 4-MU glucuronide 351/175 NH4HCO2 (pH MeOH B ESI - hexyl, 3 µm, 2.0x150 mm 4.2) in H2O 4-MU sulfate 255/175

Benzydamine 310/265 20 mM Supelco Ascentis C18, 3 µm, NH4HCO2 (pH ACN A ESI + 2.1x50 mm Benzydamine-N-Oxide 326/102 4.2) in H2O

Ramipril 417/234 20 mM Supelco Ascentis C18, 3 µm, NH4HCO2 (pH ACN A ESI + 2.1x50 mm Ramiprilat 389/156 4.2) in H2O

289/289 Triclosan 287/287 Supelco Ascentis C18, 3 µm, 0.1% FA in H2O ACN B ESI - Triclosan glucuronide 2.1x50 mm 463/287

Triclosan sulfate 367--> 175, 287

Carbazeran Supelco Ascentis C18, 3 µm, 361/272 0.1% FA in H2O ACN A ESI + 4-Hydroxycarbazeran 2.1x50 mm 377/288

Diclofenac 294 --> 250,214, 296/252

4-Hydroxydiclofenac HALO C18, 3 µm, 310/266, 312 --> 268, 232 0.1% FA in H2O ACN A ESI - 2.1x100 mm + guard 470 --> 193,250, 175, Diclofenac glucuronide 472/193 (Table 4) Contd… 58 Drug Metabolism Letters, 2019, Vol. 13, No. 1 Yilmaz et al.

Gradient Gradient Ionization Analyte Column SRM Transition Gradient A B Profile Mode

Midazolam 326/291 20 mM Supelco Ascentis C18, 3 µm, NH4HCO2 (pH ACN A ESI + 2.1x50 mm 1-Hydroxymidazolam 342/324 4.2) in H2O

Diazepam 285/193 20 mM Supelco Ascentis C18, 3 µm, NH4HCO2 (pH ACN A ESI + 2.1x50 mm Nordiazepam 271/208 4.2) in H2O

Amodiaquine Supelco Ascentis C18, 3 µm, 356/283 0.1% FA in H2O ACN C ESI + N-Desethylamodiaquine 2.1x50 mm 328/283

P-Toluidine Phenomenex Luna Phenyl- 108/93 0.1% FA in H2O ACN A ESI + 4'-Methylacetanilide hexyl, 3 µm, 2.0x150 mm 150/108

Estradiol Glucuronide (IS) 447/271

Gradient elution profiles A= 0-1 min 5% B, 5 min 50% B, 10-11 min 95% B, 12-15 min 5% B B= 0-1 min 5% B, 5 min 30% B, 6-12 min 95% B, 12.5-15 min 5% B C= 0-1 min 5% B, 6 min 80% B, 10-13 min 95% B, 13.5-15 min 5% B

Prior to analysis, samples were prepared as follows. The samples were thawed and sonicated for 5 min and 200 µL aliquots were transferred to a new tube containing 400 µL acetonitrile with internal standard, estradiol glucuronide. The diluted samples were vortex mixed and sonicated for 2 min and then centrifuged for 15 min at 15000 g. The supernatants were transferred to a new tube and evaporated to dryness under a stream of nitrogen. Finally, the samples were recon- stituted in 150 µL 95/5 (v/v) water/acetonitrile containing 0.1 % formic acid and analysed by LC-MS/MS. The LLOQ for the metabolites was 0.001 µM.

3. RESULTS 3.1. Standardization of the PCLS Assay Conditions The initial aim of this investigation was to standardize the PCLS incubation conditions. As mentioned earlier, some of the important incubation conditions were optimized using the CYP1A1 substrate, AFQ056. Furthermore, due to the limited availability of fresh human lung tissue, the optimiza- tion process was carried out using rat tissue with the assump- tion that rat lung is a suitable surrogate for human lung. Fig. (3). Age and strain dependent lung metabolism of AFQ056 in rats. AFQ056 (1 µM) was incubated for 4 h with lung tissue slices The PCLS results indicated that pulmonary CYP1A1 ac- obtained from 6 and 12 week old SD rats and 6 week old WH rats. tivity varied with rat age and rat strain (Fig. 3). AFQ056 The amount of metabolite, CBJ474, formed after 4 h was quantified turnover was approximately 1.5-fold higher in 6 week than and expressed as a percentage of initial AFQ056 concentration. in 12 week old SD rats. Similarly, pulmonary CYP1A1 ac- Data are presented as mean values ± S.D. (n=3). Each time point tivity in 6 week old SD rats was approximately 1.8-fold was carried out in duplicate comprising 3 slices for each time point, higher than in WH rats of the same age (Fig. 3). and each incubation was carried out in triplicate. The kinetic ex- periments showed linear formation of CBJ474 with time as shown The choice of the incubation vessel was also found to be in the previous investigations [15] (Fig. 4). An unpaired t-test was an important consideration when performing PCLS incuba- applied to the data and the level of statistical significance was set at tions. As shown in Fig. (4), greater CBJ474 formation was p<0.05. Significant differences in CYP1A1 activity (determined by observed with the DOC incubation system than with the well CBJ474 formation) were observed in 6 week old SD rats compared plates. Although the use of the rollers was more cumbersome to 12 week old SD rats. The same statistical test is also used to than a simple well plate, it is evident that they played a cru- compare the CYP1A1 activity (determined by CBJ474 formation) cial role in obtaining optimum enzymatic activity. in 6 week old SD and 6 week old WH rats. CYP1A1 activity was significantly higher in 6 week old SD rats than 12 week old SD (p- Given that in vivo, the lungs are directly exposed to the value <0.0413) and 6 week old SD and 6 week old WH (p value atmosphere, the influence of the oxygen content in the incu- <0.0186). bation buffer was investigated. The results indicated that Comparison of Rat and Human Pulmonary Drug Metabolism Drug Metabolism Letters, 2019, Vol. 13, No. 1 59

CYP1A1 activity was not influenced by atmospheric oxygen content during the 4h incubation period used here (Fig. 4). This insensitivity to the oxygen content may have been due to the fact that the incubation buffer was routinely saturated with oxygen prior to starting the incubation. The addition of this amount of oxygen may have been sufficient to support enzyme activity during the 4 h incubation period.

3.2. Comparison of Pulmonary Enzyme Activity in Rats and Humans The pulmonary metabolism of a selection of phase I and II substrates in rat and human lung tissue was compared us- ing the optimized conditions discussed above. The results suggested that there were considerable species differences with respect to the metabolism of the probe substrates tested (Fig. 5). Although CYP activity was generally low in both species, activity in human lung appeared to be remarkably lower than in rat. Furthermore, with the exception of CBJ474, it was not possible to detect the CYP mediated me- tabolites in human lung incubations. The formation of CBJ474 is mediated by CYP1A1 [15], an isoform that is Fig. (5). Comparison of rat and human pulmonary drug metaboliz- thought to be expressed in extrahepatic tissues including the ing activity. Substrates (1 µM) were incubated in the presence of rat lung. The data shown here demonstrates the presence of and human lung PCLS for 4 h. The metabolites were quantified and CYP1A1 in lung tissue albeit to a lesser extent in human are displayed as a percentage of initial substrate concentration. Data than in rat. are presented as mean values ± S.D. (n=3). Each time point was carried out in duplicate comprising 3 slices for each time point, and each incubation was carried out in triplicate. (Nomenclature is used for human enzymes).

served suggested that pulmonary aldehyde oxidase activity is low in both species. In contrast to the generally low oxidative metabolism ob- served here, pulmonary phase II processes appeared to be prominent. Triclosan and 4-MU underwent sulfation in rat and human lung with comparable turnover displayed in both species. Furthermore, toluidine N-acetylation also occurred in both species to a similar extent. Although glucuronidation of the UGT substrates, triclosan and 4-MU was observed in both rat and human lung, the velocity was significantly higher in rat than in human lung. CES1 mediated hydrolysis of ramipiril was comparable in rat and human lung slices.

4. DISCUSSION Although the liver has the highest metabolic capacity of all organs, the lung could also contribute to the total body clearance of drugs. Indeed, some inhaled drugs such as theo- Fig. (4). The impact of the incubation system on AFQ056 pulmo- phylline, salmeterol, isoprenaline, budesonide, and cicleson- nary metabolism. AFQ056 (1 µM) was incubated for 4 h with lung ide have been found to undergo significant metabolism in the tissue slices obtained from 6 week old SD rats. The impact of oxy- lung [45]. The lungs are a well perfused organ that receive gen supply on CBJ474 formation was investigated. Dynamic organ the entire cardiac output. Consequently, every compound culture (DOC) and well plate incubation systems were also com- given intravenously, intramuscularly, subcutaneously or pared. The metabolite, CBJ474, formed after 4 h was quantified and topically circulates through the lung before reaching the expressed as a percentage of initial AFQ056. Data are presented as liver. Circulating compounds will be exposed to pulmonary mean values ± S.D. (n=3). Each time point was carried out in dupli- enzymes and therefore, pulmonary first pass could be sig- cate comprising 3 slices for each time point, and each incubation nificant for drugs administered via these routes. was carried out in triplicate. The PCLS model allows for in vitro investigation of Similarly, benzydamine N-oxygenation was observed in pulmonary drug metabolism using fresh lung tissue. Since rat lung, whereas activity in human lung was negligible the slices contain all relevant cell and metabolite compo- (equivalent to approximately 1% of initial parent concentra- nents, the results obtained in vitro could predict the in vivo tion during 4 h). The limited carbazeran hydroxylation ob- pulmonary metabolism of specific drugs. Although the suc- 60 Drug Metabolism Letters, 2019, Vol. 13, No. 1 Yilmaz et al. cessful use of PCLS for drug metabolism studies has previ- to be made to the methodology. Rat lungs could easily be ously been demonstrated [14], there is a still a need to evalu- inflated with agarose via the intact trachea in order to pre- ate and improve the protocols used with the aim of optimiz- vent the alveoli from collapsing during the cutting process. ing this technique. Herein, the impact of several key factors However, human lung tissue was obtained as surgical waste, including rat age and rat strain, incubation vessels and oxy- and airways were generally not visible. Human tissue was gen supply were evaluated (Table 1). therefore carefully inflated via a series of small injections of agarose gel using a fine needle. Care was taken to avoid ap- As shown in Fig. (3), differences in CBJ474 formation plying excessive pressure that could have caused the alveoli were observed between 6 and 12 week old SD rats. These to burst, possibly leading to loss of metabolic activity. It is observations are consistent with the fact that in the rat, age- therefore possible that the lower enzyme activities observed related changes in anatomy, morphology, biochemistry, and physiology are known to result in corresponding changes in in in human lung is due to the quality of the lung slices and differences in enzyme expression. drug metabolizing activity [46-51]. The age of a rat can be defined in three specific life phases: infants (<3 week), juve- We were fortunate to obtain fresh human lung tissue niles (3-8 weeks), and adults (from 8 weeks up to 1.5 years). from the local University Hospital. Although the availability The data suggests that juvenile rats have higher pulmonary of human tissue was limited, we aimed to reduce experimen- CYP1A1 activities than adult rats. Similarly, differences in tal bias by processing the human tissue samples as carefully CYP1A1 mRNA expression and metabolic activity among and rapidly as possible. It can be assumed that the health rat strains have been reported previously [52]. The impact of status of the subjects had only a limited impact on the results this strain-dependent CYP1A1 activity can be seen in Fig. as the tissue collected was not tumorous. The PCLS tech- (3), where CBJ474 formation was shown to be greater in 6 nique is dependent upon the availability of fresh tissue and, week old SD than in WH rats of the same age. Given differ- therefore, samples were transported from the hospital to our ences in metabolic activity observed here, it is important that laboratory within one hour of isolation. Tissue slices were age and strain of animals used in this type of metabolic study then quickly prepared within 90 minutes to minimise any are standardized to obtain comparable results with low vari- loss of metabolic activity. ability. We therefore recommend that 6 week old juvenile Although several studies have been published in recent SD rats are used wherever possible. years concerning the characterization, expression levels and The choice of incubation vessel was also found to be im- localization of xenobiotic metabolizing enzymes in the lung, portant as higher CBJ474 turnover was seen with the DOC information about the functional aspects of these enzymes is system than with multi-well plates. The continuous rotation rare [4, 60, 61]. Possible reasons for this information gap of the titanium rollers in the DOC system may result in more may be that the activity of drug metabolizing enzymes in efficient oxygenation and optimal substrate and nutrient de- lung tissue is low (in particular regarding phase I metabolic livery to the tissue slices, thereby mimicking the in vivo enzymes) and that suitable systems for long-term measure- situation more closely [11, 53-56]. The primary drawback of ment of enzyme activities have not been available. PCLS the DOC system is that the number of incubations that can may close this gap. The data obtained in this investigation be performed in parallel is limited and that a specific incuba- shows that phase II activity in rat and human lung is rea- tor has to be purchased. Incubations could be more effi- sonably comparable and that phase II activity is considerably ciently performed using multi-well plates, which would al- higher than that of phase I enzymes (Fig. 5). This is consis- low for the scaling up of the number of incubations much tent with previously published data that summarised the ex- more easily. However, as shown in this study, the metabolic pression and activity of drug-metabolizing enzymes in hu- activity is compromised when well-plates are used (Fig. 4). man lung [9]. Although the influence of oxygen supply on lung slice vi- Pulmonary UGT activity was assessed using the non- ability has been reported previously [57, 58], our data selective UGT substrates, triclosan and 4-MU. Although suggests that oxygen supply has only a limited impact when UGT activity appeared to be higher in rat than in human incubation times are short (4h) and the buffer is pre- lung, this finding has to be interpreted with caution, since the oxygenated. Based on these results, subsequent incubations metabolism of triclosan and 4-MU depends not on the activ- were performed using buffer with pre-oxygenation and the ity of one specific UGT, but on the interplay of different DOC system with 75% of oxygen. UGT isoforms present in the lungs of these two species [62, The optimization process discussed here was performed 63]. with a single compound, AFQ056, a CYP1A1 substrate. While pulmonary CYP450 activity was low in both spe- While this may be considered to be a limitation, the well cies, the activity in human lung appeared to be markedly documented fragility of CYP1A1 [59] requires additional lower than in rat lung. The low pulmonary CYP450 activity care to maintain the activity when performing PCLS experi- observed here is in clear contrast to the role of this enzyme ments. The data shown here are consistent with previous family in the liver. Regarding Flavin mono-oxygenases, re- results [15], reflecting the reproducibility of our procedure. markably greater benzydamine N-oxygenation activity was We therefore consider AFQ056 to be a suitable compound observed in rat than in human lung. The pronounced N-oxide for monitoring the performance of the PCLS procedure. formation observed here in rat is consistent with previously The use of human tissue in this study turned out to be dif- obtained data [39]. FMOs are known to display complex ficult but nevertheless yielded valuable results. The prepara- species, tissue and gender dependent expression, which may tion of human tissue slices was technically more challenging account for the species difference observed in this investiga- than rat slices and therefore, appropriate modifications had tion [64-67]. FMO expression in human lung is low and the Comparison of Rat and Human Pulmonary Drug Metabolism Drug Metabolism Letters, 2019, Vol. 13, No. 1 61 major pulmonary isoform, FMO2 is catalytically inactive in UPLC = Ultra Performance Liquid Chromatography Caucasians [68, 69]. As this study only included lung tissue WH = Wistar Han from Caucasian donors, it is not surprising that limited N- oxide formation was observed. It would be interesting to 4-MU = 4-Methylumbelliferone explore pulmonary FMO activity in other populations, such as native Africans, where active FMO2 is expressed in lung ETHICS APPROVAL AND CONSENT TO PARTICI- [69]. Aldehyde oxidase mediated oxidation of carbazeran PATE was observed to a limited extent in both species. Aldehyde oxidase has been shown to be expressed in the bronchial Animal experiments were conducted with the approval of epithelium of rats [70]. The role of aldehyde oxidase in ex- the Cantonal Veterinary Authority of Basel City, Switzerland trahepatic metabolism has so far not been fully explored and (Protocol BS-2723). further investigations would be of interest. Human lung tissue was approved by the local ethics committee of Basel, Switzerland (EKNZ 2015-355). CONCLUSION Key experimental parameters were evaluated during our HUMAN AND ANIMAL RIGHTS optimization of the PCLS technique. The study demonstrates The studies were performed in accordance with the Dec- the importance of optimization and standardization of PCLS laration of Helsinki (1964 and subsequent revisions). and the conditions, which eventually resulted in a more robust reported experiments on animals were in accordance with the method to determine pulmonary metabolic activity. The rea- standards set forth in the 8th Edition of Guide for the Care son for our particular interest in the comparison of pulmo- and Use of Laboratory Animals (http:// grants.nih.gov/ nary enzymatic activity in rats and humans is that animal grants/olaw/Guide-for-thecare-and-use-of-laboratory- models are commonly used in the preclinical development of animals.pdf) published by the National Academy of Sci- new drugs. The results show that there are remarkable differ- ences. ences in pulmonary metabolic activity between rats and hu- mans, reflecting species and tissue dependent expression of CONSENT FOR PUBLICATION drug metabolizing enzymes. Care should therefore be taken when extrapolating metabolism data from animal models to Informed consent in writing was obtained from the study humans. PCLS turned out to be a valuable tool for the inves- participants. Tumour-free tissue samples from different re- tigation of pulmonary drug metabolism and should be devel- gions of the lung were collected post-surgery from six pa- oped further for assessing enzyme and metabolic activities tients at the Department of Thoracic Surgery, University not only in lungs but also in other organs. Hospital Basel, Switzerland.

LIST OF ABBREVIATIONS CONFLICT OF INTEREST AO = Aldehyde Oxidase The author declares no conflict of interest, financial or otherwise. CES1 = Carboxylesterase 1 COPD = Chronic Obstructive Pulmonary Disease ACKNOWLEDGEMENTS CYP = Cytochrome P450 The study was conducted with the great support from the PK Sciences group of Novartis institutes for biomedical re- DOC = Dynamic Organ Culture search. The authors would like to acknowledge Arnold De- FMOs = Flavin-containing Monooxygenase mailly for providing support in the generation of data. Prof. Dr. Didier Lardinois and Dr. Mark Nikolaj Wiese from Basel GST = S-transferase University Hospital are acknowledged for post-surgical col- LLQ = Lower Limit of Quantitation lection of discarded human lung samples. MEM = Minimal Essential Medium REFERENCES NADPH = Nicotinamide Adenine Dinucleotide Phos- [1] Del Donno, M.; Verduri, A.; Olivieri, D. Air pollution and reversi- phate ble chronic respiratory diseases. Monaldi Arch. Chest Dis., 2002, 57, 164-166. PCLS = Precision-cut Lung Slices [2] Rau, J.L. The inhalation of drugs: advantages and problems. Respir. Care Clin. N. Am., 2005, 50, 367-382. SD = Sprague Dawley [3] Stone, K.C.; Mercer, R.R.; Gehr, P.; Stockstill, B.; Crapo, J.D. Allometric relationships of cell numbers and size in the mammalian S.D. = Standard Deviation lung. Am. J. Respir. Cell Mol. Biol., 1992, 6, 235-243. [4] Zhang, J.Y.; Wang, Y.; Prakash, C. Xenobiotic-metabolizing en- SULT = Sulfotransferase zymes in human lung. Curr. Drug Metab., 2006, 7, 939-948. [5] Devereux, T.R.; Domin, B.A.; Philpot, R.M. Xenobiotic metabo- UDPGA = Uridine-diphosphate-glucuronic Acid Triso- lism by isolated pulmonary cells. Pharmacol. Ther., 1989, 41, 243- dium Salt 256. [6] Raunio, H.; Hakkola, J.; Hukkanen, J.; Lassila, A.; Päivärinta, K.; UGT = UDP-glucuronosyltransferase Pelkonen, O.; Anttila, S.; Piipari, R.; Boobis, A.; Edwards, R.J.

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