Send Orders for Reprints to [email protected] 53 Drug Metabolism 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 Pharmacology and Toxicology, 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. Cytochrome P450 (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-glutamine, 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.