Evaluating Lung Exposure and Clearance of an Inhaled Drug in Dogs by HPLC-MS/MS Analysis of Tissue or Lavage Fluid and MALDI-MSI of Lung Sections
Creators: name: Paul, Graham R
name: Marshall, Peter S
Publisher: name: F1000 Research Limited
Year: 2019

description: Non‑terminal sampling of bronchoalveolar lavage fluid (BALF) or lung biopsies may facilitate monitoring of drug-lung dose in animals with repeated administration and clinical translation. This study investigated lung accumulation and clearance of a drug of low membrane permeability (7 nm/sec) and solubility (37 µg/mL) in dogs using high performance liquid chromatography with mass spectrometry (HPLC‑MS/MS) or matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI‑MSI) and considered the utility of BALF and biopsy-sized samples to assess dosimetry.<br><br>Male dogs (n=2/group) inhaled a powdered drug once or for 14 days (1 hour; snout-only) at a clinically relevant dose of 16 µg/kg (equating to 800 µg in humans) or toxicologically relevant dose of 4000 µg/kg, and were sampled for plasma up to 24 hours post exposure on Days 1 and 14. Immediately after a single dose or 24 hours after the 14<sup>th</sup> dose, dogs were euthanized and sampled for BALF and lung. Additional dogs were euthanized and sampled for BALF and lung after an off-dose period of 14 days. Lung tissue (0.5-1.0g) taken proximally, centrally and distally from the main airway of lobes was homogenized for HPLC-MS/MS analysis of drug; a biopsy-sized sample (2‑5 mg; single dose; n=1/group) was taken from adjacent tissue. The intermediate lung lobe (n=1/group) was sectioned for MALDI-MSI and drug-homogenate analysis or flushed with saline for HPLC‑MS/MS analysis of drug in BALF.<br><br>Systemic exposure (plasma AUC<sub>0-t</sub>) in dogs at 4000 µg/kg was similar on Days 1 and 14. A paucity of quantifiable data precluded calculation of plasma AUC<sub>0-t</sub> at 16 µg/kg.<br><br>Drug-lung distribution was even across lobes, with accumulation more pronounced after 14 days at 4000 µg/kg. Mean lung concentrations 24 hours after the 14<sup>th</sup> dose were up to 3.3-fold higher than immediately after a single dose indicating a degree of lung clearance during the treatment period. Drug-lung concentrations in dogs inhaling 4000 µg/kg persisted after the 14-day off-dose period. After the off-dose period, drug concentration was 32% of the post single dose value and 65% of post repeat dose value indicating partial drug clearance during and on cessation of treatment.<br><br>At 4000 µg/kg, drug concentrations for biopsies were 2 to 3-fold higher than lung homogenate, indicating the focal samples overestimated drug-lung concentrations. Drug was not quantifiable in biopsy-sized samples of dogs inhaling 16 µg/kg.<br><br>Differences between groups for drug mass recovered in BALF were inconsistent with trends in lung homogenate and biopsy data, suggesting BALF was an unreliable surrogate for estimating lung dose.<br><br>Drug-lung distribution seen by MALDI-MSI was uniform throughout the examined lung sections. At 4000 µg/kg, image intensity for each dosing regimen indicated drug accumulation (with increased signal intensity associated with major airways) after repeated administration and evidence of clearance after the off-dose period. The spatial resolution (30μm) was insufficient for assessment of intracellular distribution.<br><br>In conclusion, drug-lung accumulation after repeated administration of 16 or 4000 µg/kg with partial clearance after an off-dose period reflected the low aqueous solubility and membrane permeability of the drug. Drug mass in BALF, versus lung tissue/biopsy samples, suggested lavage <i>per se</i> was an unsuitable sampling method for estimating lung dose. Biopsy samples overestimated drug-lung concentrations but showed a potential application for monitoring trends in lung dose with administration of toxicologically relevant doses. However, the limit of quantification was insufficient to quantify drug in biopsies at a clinically relevant dose.