The kinase-activating mutation G2019S in leucine-rich repeat kinase 2 (LRRK2) is one of the most common genetic causes of Parkinson’s disease (PD) and has spurred development of LRRK2 inhibitors. Preclinical studies have raised concerns about the safety of LRRK2 inhibitors due to histopathological changes in the lungs of nonhuman primates treated with two of these compounds. Here, we investigated whether these lung effects represented on-target pharmacology and whether they were reversible after drug withdrawal in macaques. We also examined whether treatment was associated with pulmonary function deficits. We conducted a 2-week repeat-dose toxicology study in macaques comparing three different LRRK2 inhibitors: GNE-7915 (30 mg/kg, twice daily as a positive control), MLi-2 (15 and 50 mg/kg, once daily), and PFE-360 (3 and 6 mg/kg, once daily). Subsets of animals dosed with GNE-7915 or MLi-2 were evaluated 2 weeks after drug withdrawal for lung function. All compounds induced mild cytoplasmic vacuolation of type II lung pneumocytes without signs of lung degeneration, implicating on-target pharmacology. At low doses of PFE-360 or MLi-2, there was ~50 or 100% LRRK2 inhibition in brain tissue, respectively, but histopathological lung changes were either absent or minimal. The lung effect was reversible after dosing ceased. Lung function tests demonstrated that the histological changes in lung tissue induced by MLi-2 and GNE-7915 did not result in pulmonary deficits. Our results suggest that the observed lung effects in nonhuman primates in response to LRRK2 inhibitors should not preclude clinical testing of these compounds for PD.

Embryonic development is highly sensitive to xenobiotic toxicity and in utero exposure to environmental toxins affects physiological responses of the progeny. In the US, the prevalace of allergic asthma is inexplicably rising and in utero exposure to cigarette smoke (CS) increases the risk of allergic asthma (AA) and bronchopulmonary dysplasia (BPD) in children and animal models. We reported that gestational exposure to sidestream (secondhand) CS (SS) promoted nicotinic acetylcholine receptor-dependent exacerbation of AA and BPD in mice. Recently, perinatal nicotine injections in rats were reported to induce peroxisome proliferator-activated receptor gamma (PPARγ)-dependent transgenerational transmission of asthma. Herein, we show that F1 and F2 progeny from gestationally SS-exposed mice exhibit exacerbated AA and BPD that is not dependent on the decrease in PPARγ levels. Lungs from these mice show strong eosinophilic infiltration, excessive Th2 polarization, marked airway hyperresponsiveness, alveolar simplification, decreased lung compliance, and decreased lung angiogenesis. At the molecular level, these changes are associated with increased RUNX3 expression, alveolar cell apoptosis, and the antiangiogenic factor GAX, and decreased expression of HIF-1α and pro-angiogenic factors NF-κB and VEGFR2 in the 7-day F1 and F2 lungs. Moreover, the lungs from these mice exhibit lower levels of micro-RNA (miR)-130a and increased levels of miR-16 and miR-221. These miRs regulate HIF-1α-regulated apoptotic, angiogenic, and immune pathways. Thus the intergenerational effects of gestational SS involve epigenetic regulation of HIF-1α through specific miRs contributing to increased incidence of AA and BPD in the progenies.

The type II cell plays an important role in the response of the alveolar epithelium after lung injury through its synthesis and secretion of pulmonary surfactant, and by acting as the stem cell for the replacement of damaged type I epithelial cells. The nonciliated bronchiolar epithelial (Clara) cell is thought to play a similar role during repair of the bronchiolar epithelium. Recent evidence has suggested that epithelial cells may participate in aspects of the inflammatory response and regulation of fibroblast growth during pulmonary fibrosis through the production of and response to specific growth factors and cytokines. The cellular and molecular responses of epithelial cells and how they lead to the progression of events that defines the pulmonary parenchymal response to a class of particles is unclear. We used particles differing in size, chemical composition, and fibrogenicity in vivo and in vitro to elucidate early changes in proinflammatory and profibrotic cytokine and antioxidant gene expression in lung cells. Early increases in mRNA and protein for the proinflammatory cytokines interleukin (IL)-1 beta, IL-6, and tumor necrosis factor alpha have been observed in epithelial cells following exposure. These are accompanied by changes in specific epithelial genes including surfactant protein C and Clara cell secretory protein. The data indicate that effects on the epithelium are due to direct interactions with particles, not a result of macrophage-derived mediators, and suggest a more significant role in the overall pulmonary response than previously suspected. These results suggest that type II cell growth factor production may be significant in the pathogenesis of pulmonary fibrosis.

Idiopathic pulmonary fibrosis is a progressive and lethal disease and while there are now two approved drugs (Esbriet and Ofev) additional effective treatments are still needed. Recently, prostacyclin analogs such as iloprost and treprostinil (TRE) have been shown to exert some protection against bleomycin-induced pulmonary fibrosis in mice when administered in a prophylactic regimen. In this study, we evaluated the effect of the inhaled treprostinil prodrug hexadecyl-treprostinil (C16TR) formulated in a lipid nanoparticle (INS1009) administered therapeutically in a fibrotic rat model. Male Fischer 344 rats challenged with intra-tracheal saline instillation were then treated with daily inhaled phosphate buffered saline (PBS) while rats challenged with bleomycin sulfate (3.5-4.0 mg/kg) instillation were treated with either daily inhaled PBS, daily inhaled INS1009 (10, 30, or 100 μg/kg), or twice-daily orally with the anti-fibrotic compound pirfenidone (100 mg/kg). Dosing started on day 10 post-bleomycin challenge and continued until day 27 after bleomycin. Lungs were harvested 24 h after the last dose of treatment for evaluation of lung hydroxyproline content and pulmonary histology. Lung hydroxyproline content increased from 421 μg/lung lobe in saline challenged and PBS treated animals to 673 μg/lung lobe in bleomycin challenged and PBS treated rats. Treatment of bleomycin challenged rats with 10, 30, or 100 μg/kg INS1009 dose-dependently reduced lung hydroxyproline content to 563, 501, and 451 μg/lung lobe, respectively, and pirfenidone decreased hydroxyproline content to 522 μg/lung lobe. Histologically, both INS1009 (100 μg/kg) and pirfenidone (100 mg/kg) reduced the severity of subepithelial fibrosis. Single dose pharmacokinetic (PK) studies of inhaled INS1009 in bleomycin challenged rats showed dose-dependent increases in lung C16TR concentration and plasma TRE on day 10 post-bleomycin challenge. Multiple dose PK studies of inhaled INS1009 showed dose-dependent increases only in lung C16TR concentration on day 27 post-bleomycin challenge. We also investigated the effects of TRE on the cytokine transforming growth factor-β (TGF-β)-stimulated collagen gene and protein expressions in cultured human lung fibroblasts, assessed by real-time PCR and Sirius Red staining, respectively. In human fibroblasts, TRE (0.001-10 μM) inhibited TGF-β (20 ng/mL)-induced expression of collagen mRNA and protein in a concentration-dependent manner. These results demonstrated that inhaled INS1009, administered in a therapeutic dosing paradigm, dose-dependently (10-100 μg/kg) inhibited bleomycin-induced pulmonary fibrosis in rats. This effect may involve direct actions of TRE in suppressing collagen expression in lung fibroblasts.

Putative gain-of-function mutations in leucine-rich repeat kinase 2 (LRRK2), resulting in increased kinase activity and cellular toxicity, are a leading genetic cause of Parkinson's disease (PD). Hence, there is strong interest in developing LRRK2 kinase inhibitors as a disease-modifying therapy. Published reports that repeat dosing with two LRRK2 kinase inhibitors (GNE-7915 and GNE-0877) induce histopathological changes in the lung of non-human primates Fuji et al. 2015 (1) raised concerns about potential safety liability of LRRK2 kinase inhibitors. In the present study, we sought to determine whether previously observed effects in the lung: (a) represent on-target pharmacology, but with the potential for margin of safety, (b) are reversible upon drug withdrawal, and (c) are associated with pulmonary function deficits. To this end, we evaluated the histopathological effects, toxicokinetics and target inhibition of three structurally diverse 3 LRRK2 kinase inhibitors, GNE-7915 (30 mg/kg, BID, as a positive control), MLi-2 (15 and 50 mg/kg, QD) and PFE-360 (3 and 6 mg/kg, QD) following 2 weeks of dosing in non-human primates. Subsets of animals dosed with GNE-7915 or MLi-2 were evaluated after 2-week dose-free periods. All three LRRK2 kinase inhibitors induced mild cytoplasmic vacuolation of type II pneumocytes, as reported previously, confirming an on-target effect of these compounds. Interestingly, despite lower doses of both PFE-360 and MLi-2 producing nearly complete inhibition of LRRK2 kinase activity in the brain as assessed by levels of pS935-LRRK2, histopathological changes in lung were absent in animals treated with low-dose PFE-360 and observed only sporadically in the lowdose MLi-2 group. The lung effect was fully reversible at 2 weeks post-dosing of GNE-7915. In a second study of identical dosing with MLi-2 and GNE-7915, no deficits were observed in a battery of translational pulmonary functional tests. In aggregate, these results do not preclude the development of LRRK2 kinase inhibitors for clinical investigation in Parkinson's disease. IntroductionParkinson's disease (PD) is the second most common progressive and agerelated neurodegenerative disorder characterized by the loss of nigrostriatal dopamine neurons and accumulation of intracellular protein aggregates termed Lewy bodies (2).With an aging population, the prevalence of PD worldwide and the associated health care burden is expected to increase exponentially (3). Currently available drugs primarily augment dopaminergic neurotransmission to treat motor symptoms of PD, but do not affect the progression of the disease. Thus, there remains an urgent need to develop disease-modifying therapies for PD. 4Genetic studies have provided unprecedented insights into the pathogenesis of PD and suggested novel drug targets (4). One such target is leucine-rich repeat kinase 2 (LRRK2), which has attracted significant interest from the pharmaceutical industry for the following reasons: (a) point mutations in the LRRK2 gene represent the most common causes of ...