Multivariate-based selection of drugs and implications of aerosol presentation for inhalation toxicology in rats
Creators: name: Paul, Graham
Publisher: name: F1000 Research Limited
Year: 2019

description: Many inhaled drugs have been developed as crystalline dry powder formulations for prolonged stability and to optimise clinical drug delivery. Inhalation exposure of animals requires large masses of drug, relative to other dose routes in pre-clinical development. Surrogate formulations or administration routes are sometimes proposed to expedite early clinical evaluation. Such strategies defer discharging of liabilities such as altered pharmacokinetics-pharmacodynamics (PK/PD) or tolerability of the final formulation until later in development. Using three compounds of common pharmacology but differing physicochemical properties, the aim of this work was to determine if altered presentation of an inhaled drug affected exposure or toxicology and hence if such shortcuts are prudent.<br><br>Rats (n=6/group) were administered GSK‑899, GSK‑677 or GSK‑361 for up to 28 days by snout-only inhalation exposure (one hour daily) at target doses of ≤15 mg/kg/day. The compounds were administered to rats as aerosols generated from dry powder formulations of micronised crystals (all compounds) or amorphous powder (GSK‑899 only) blended with lactose, or as nebulised aqueous solutions. Rats were euthanized after 28 days of treatment and the lungs were fixed in 10% buffered formalin, embedded in paraffin wax, sectioned and stained with haematoxylin and eosin for examination by light microscopy. Additional rats (n=3/group/timepoint) were exposed and the lungs taken following a single dose or 28 days of treatment for homogenisation and HPLC‑MS/MS analysis of the parent compounds. Macrophages were also harvested from satellite animals (one rat per compound and aerosol form) by bronchoalveolar lavage using 0.9% (w/v) aqueous sodium chloride. The cells were fixed in 4% formaldehyde and 1% glutaraldehyde (v/v/v) in phosphate buffer, embedded in Agar 100 resin, sectioned and stained with uranyl acetate and lead citrate for examination by transmission electron microscopy.<br><br><i>In vitro</i> dissolution rates in simulated lung fluid were relatively fast for amorphous GSK-899 and crystalline GSK‑677, and relatively slow for crystalline GSK‑899 and GSK‑361. <i>In vivo</i>, crystalline GSK‑899 (low solubility) was most persistent in rat lung tissue, with no appreciable decrease in drug-homogenate concentration at 24 hours and a 7-fold increase from Days 1 to 28. Post dose drug-lung concentrations for amorphous GSK‑899, GSK‑677 (crystalline and nebulised aerosols) and crystalline GSK‑361 were similar and reduced at 24 hours. However, GSK‑677 (most soluble) increased from Days 1 to 28 indicating a degree of accumulation. Conversely, crystalline GSK‑361 was least persistent in lung tissue with no evidence of accumulation Days 1 to 28; drug-lung homogenate concentrations were lowest for nebulised GSK‑361 (≤1 mg/kg/day evaluated) and non quantifiable at 24 hours. This correlated with no GSK‑361-related lung pathology. A non-adverse dose dependent increase in the severity and incidence of multifocal foamy macrophage aggregates was evident for nebulised and dry powder formulations of GSK‑899 and GSK‑677. Administration of GSK‑899 at 15 mg/kg/day was also associated with adverse inflammatory changes characterised by interstitial mononuclear inflammatory cell infiltration, which was marked for the crystalline form. Transmission electron microscopy confirmed the presence of particles in macrophages harvested from rats administered amorphous or crystalline GSK‑899 only.<br><br>Crystalline GSK‑361, which was of low solubility but high membrane permeability <i>in vitro</i>, showed no drug-lung accumulation <i>in vivo</i> or pulmonary toxicity. Differences in dissolution rate for amorphous and crystalline GSK‑899 correlated with drug-lung concentrations and a shift in the severity of lung pathology suggesting that the physical state of a drug can influence observed toxicity. However, inflammatory changes at 15 mg/kg/day were nevertheless adverse for both dry powder aerosol forms of GSK‑899 and the safety margin between the no observed adverse effect level (NOAEL) and the hypothetical clinical exposure was thus unaffected. This suggests that membrane permeability may be a greater driver than <i>in vitro</i> solubility for mitigating toxicopathology when designing inhaled drugs.