Pulmonary deposition and metabolite formation of ML3403, a second generation p38 MAPK inhibitor, in the isolated perfused and ventilated rat lung

S Ammon-Treiber1,2, M Schwab1,2, G Ganchev1, U Delabar2, S Laufer3, TE Mürdter1. 1Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart and University of Tübingen, 70376, Germany, 2University Hospital Tübingen, Department of Clinical Pharmacology, 72076, Germany, 3Institute of Pharmacy, University of Tübingen, Department of Pharmaceutical and Medical Chemistry, 72076, Germany

Mitogen activated protein kinases (MAPK) play a key role in cell regulation. Among small molecular anti-cytokine agents and drug candidates for the treatment of chronic inflammatory lung diseases, ML3403 belongs to the second generation of diarylimidazole-type p38 MAP kinase inhibitors. The aim of the present study was to determine pulmonary deposition of the p38 MAPK inhibitor ML3403 as well as potential lung specific formation of its known active metabolite ML3603 using the ex vivo model of the isolated perfused and ventilated rat lung (IPL). In addition, Cytochrome P450 mediated oxidation of ML3403 was investigated in human liver microsomes as well as in human lung microsomes.

Isolated lungs from male WISTAR rats (weight 282 ± 27g, mean ± SD, n = 4-5/group) were placed in a 37°C perfusion chamber, ventilated via a tracheal cannula and perfused for 30min, 60min or 120min with ML3403 (total amount 109.2µg) in an albumin containing Krebs-Henseleit perfusion buffer via the pulmonary artery in a recirculating manner. Concentrations of ML3403 and ML3603 in perfusate and lung tissue were determined by LC-MS/MS.

We chose the ML3403 dose to reach approx. 1μM peak concentrations in the recirculating perfusion buffer, because ML3403 has been shown to inhibit p38α in the low micromolar range (IC50 = 0.38μM) (Laufer et al., 2003).

Peak perfusate concentrations of ML3403 were observed after 5min within the three different perfusion series (mean cmax 984 ± 74 nM, 857 ± 107 nM, 978 ± 100 nM, mean ± SEM, respectively). ML3403 rapidly accumulated in lung tissue and reached tissue concentrations >20µM which did not differ significantly between 30min, 60min and 120min, suggesting an early saturation of lung tissue with ML3403. Perfusate concentrations of ML3603 were low in total but steadily increased over time: 8.22 ± 1.18 nM at 30min, 12.1 ± 1.22nM at 60min and 34.8 ± 3.14 nM at 120min (mean ± SEM, p < 0.001 120min vs 30min and 120min vs. 60min). Stable lung mechanics such as tidal volume, lung compliance and airway resistance were observed during perfusion with ML3403, reflecting good local tolerability. Formation of ML3603 in human liver microsomes followed Michaelis-Menten kinetics (KM: 2.8±0.4 µM; Vmax: 600±26 pmol/min/mg protein). In lung microsomes formation rates were much smaller compared to liver microsomes and KM was increased which is in agreement to the known low expression levels of CYPs in lung.

It can be concluded that ML3403 rapidly accumulates in lung tissue and that the lung is capable to metabolize ML3403 to its sulfoxide metabolite ML3603.

This study was supported by IZEPHA, Tübingen, the Robert Bosch Foundation, Stuttgart, and BMBF (# 0315280C), Berlin, Germany

References:

Laufer SA, Wagner GK, Kotschenreuther DA, Albrecht W (2003) Novel substituted pyridinyl imidazoles as potent anticytokine agents with low activity against hepatic cytochrome P450 enzymes. J Med Chem 46(15):3230-44.