Christopher Royer

Evaluation of inhaled carbon monoxide as an anti-inflammatory therapy in a nonhuman primate model of lung inflammation

Carbon monoxide (CO) confers anti-inflammatory protection in rodent models of lung injury when applied at low concentration. Translation of these findings to clinical therapies for pulmonary inflammation requires validation in higher mammals. We have evaluated the efficacy of inhaled CO in reducing LPS-induced lung inflammation in cynomolgus macaques. LPS inhalation resulted in profound neutrophil influx and moderate increases in airway lymphocytes, which returned to baseline levels within 2 wk following exposure. CO exposure (500 ppm, 6 h) following LPS inhalation decreased TNF-α release in bronchoalveolar lavage fluid but did not affect IL-6 or IL-8 release. Lower concentrations of CO (250 ppm, 6 h) did not reduce pulmonary neutrophilia. Pretreatment with budesonide, a currently used inhaled corticosteroid, decreased LPS-induced expression of TNF-α, IL-6, and IL-8, and reduced LPS-induced neutrophilia by ∼84%. In comparison, CO inhalation (500 ppm, for 6 h after LPS exposure) reduced neutrophilia by ∼67%. Thus, inhaled CO was nearly as efficacious as pretreatment with an inhaled corticosteroid at reducing airway neutrophil influx in cynomolgus macaques. However, the therapeutic efficacy of CO required relatively high doses (500 ppm) that resulted in high carboxyhemoglobin (COHb) levels (>30%). Lower CO concentrations (250 ppm), associated with anti-inflammatory protection in rodents, were ineffective in cynomolgus macaques and also yielded relatively high COHb levels. These studies highlight the complexity of interspecies variation of dose-response relationships of CO to COHb levels and to the anti-inflammatory functions of CO. The findings of this study warrant further investigations for assessing the therapeutic application of CO in nonhuman primate models of tissue injury and in human diseases. The study also suggests that akin to many new therapies in human diseases, the translation of CO therapy to human disease will require additional extensive and rigorous proof-of-concept studies in humans in the future.

A novel nonhuman primate model of cigarette smoke-induced airway disease.

Small animal models of chronic obstructive pulmonary disease (COPD) have several limitations for identifying new therapeutic targets and biomarkers for human COPD. These include a pulmonary anatomy that differs from humans, the limited airway pathologies and lymphoid aggregates that develop in smoke-exposed mice, and the challenges associated with serial biological sampling. Thus, we assessed the utility of cigarette smoke (CS)-exposed cynomolgus macaque as a nonhuman primate (NHP) large animal model of COPD. Twenty-eight NHPs were exposed to air or CS 5 days per week for up to 12 weeks. Bronchoalveolar lavage and pulmonary function tests were performed at intervals. After 12 weeks, we measured airway pathologies, pulmonary inflammation, and airspace enlargement. CS-exposed NHPs developed robust mucus metaplasia, submucosal gland hypertrophy and hyperplasia, airway inflammation, peribronchial fibrosis, and increases in bronchial lymphoid aggregates. Although CS-exposed NHPs did not develop emphysema over the study time, they exhibited pathologies that precede emphysema development, including increases in the following: i) matrix metalloproteinase-9 and proinflammatory mediator levels in bronchoalveolar lavage fluid, ii) lung parenchymal leukocyte counts and lymphoid aggregates, iii) lung oxidative stress levels, and iv) alveolar septal cell apoptosis. CS-exposed NHPs can be used as a model of airway disease occurring in COPD patients. Unlike rodents, NHPs can safely undergo longitudinal sampling, which could be useful for assessing novel biomarkers or therapeutics for COPD.

Highly selective inhibition of myosin motors provides the basis of potential therapeutic application.

Significance Defects in myosin function are linked to a number of widespread and debilitating diseases, including asthma, chronic obstructive pulmonary disease, and hypertrophic cardiomyopathy. We report here the discovery of an allosteric site that modulates myosin motor function with high specificity that opens the path toward new therapeutic solutions. Identification of specific antimyosin drugs that significantly alter a motor’s function is an imperative first step toward the development of targeted and effective treatments for such diseases. Highly specific drugs against different members of the superfamily would also provide exquisite tools to investigate in cells their functional role. Additionally, detailed, high-resolution studies of the interaction of drugs with their myosin targets provide insights into the molecular mechanism of motor function. Direct inhibition of smooth muscle myosin (SMM) is a potential means to treat hypercontractile smooth muscle diseases. The selective inhibitor CK-2018571 prevents strong binding to actin and promotes muscle relaxation in vitro and in vivo. The crystal structure of the SMM/drug complex reveals that CK-2018571 binds to a novel allosteric pocket that opens up during the “recovery stroke” transition necessary to reprime the motor. Trapped in an intermediate of this fast transition, SMM is inhibited with high selectivity compared with skeletal muscle myosin (IC50 = 9 nM and 11,300 nM, respectively), although all of the binding site residues are identical in these motors. This structure provides a starting point from which to design highly specific myosin modulators to treat several human diseases. It further illustrates the potential of targeting transition intermediates of molecular machines to develop exquisitely selective pharmacological agents.

The in vivo efficacy and side effect pharmacology of GS-5759, a novel bifunctional phosphodiesterase 4 inhibitor and long-acting β2-adrenoceptor agonist in preclinical animal species

Bronchodilators are a central therapy for symptom relief in respiratory diseases such as chronic obstructive pulmonary disease (COPD) and asthma, with inhaled β2‐adrenoceptor agonists and anticholinergics being the primary treatments available. The present studies evaluated the in vivo pharmacology of (R)‐6‐[[3‐[[4‐[5‐[[2‐Hydroxy‐2‐(8‐hydroxy‐2‐oxo‐1,2‐dihydroquinolin‐5‐yl)ethyl]amino]pent‐1‐ynyl]phenyl]carbamoyl]phenyl]sulfonyl]‐4‐[(3‐methoxyphenyl)amino]‐8‐methylquinoline‐3‐carboxamide (GS‐5759), a novel bifunctional compound with both phosphodiesterase 4 (PDE4) inhibitor and long‐acting β2‐adrenoceptor agonist (LABA) activity, which has been optimized for inhalation delivery. GS‐5759 dose‐dependently inhibited pulmonary neutrophilia in a lipopolysaccharide (LPS) aerosol challenge model of inflammation in rats with an ED50 ≤ 10 μg/kg. GS‐5759 was also a potent bronchodilator with an ED50 of 0.09 μg/kg in guinea pigs and 3.4 μg/kg in dogs after methylcholine (MCh) and ragweed challenges respectively. In cynomolgus monkeys, GS‐5759 was dosed as a fine‐particle dry powder and was efficacious in the same dose range in both MCh and LPS challenge models, with an ED50 = 70 μg/kg for bronchodilation and ED50 = 4.9 μg/kg for inhibition of LPS‐induced pulmonary neutrophilia. In models to determine therapeutic index (T.I.), efficacy for bronchodilation was evaluated against increased heart rate and GS‐5759 had a T.I. of 700 in guinea pigs and >31 in dogs. In a ferret model of emesis, no emesis was seen at doses several orders of magnitude greater than the ED50 observed in the rat LPS inflammation model. GS‐5759 is a bifunctional molecule developed for the treatment of COPD, which has both bronchodilator and anti‐inflammatory activity and has the potential for combination as a triple therapy with a second compound, within a single inhalation device.

The neonatal susceptibility window for inhalant allergen sensitization in the atopically predisposed canine asthma model.

Allergic asthma often begins in early life and, although many risk factors have been enumerated, the specific factors that initiate disease progression in an individual remain unclear. Using our dog model of early life allergen inhalation, we tested the hypothesis that the atopically biased neonatal immune system would exhibit tolerance to ragweed if allowed to mature normally before exposure or artificially through innate immune stimulation with early life exposure. Dogs were subjected to a series of inhalational ragweed exposures from 1 to 20 weeks old, with or without inhalation of a Toll‐like receptor 4 (TLR4) agonist (CRX‐527), or from 13 to 31 weeks old. Serum allergen‐specific antibody response was assessed at 4, 8 and 20 weeks after the last sensitizing exposure. At 24 or 35 weeks old, airway hyper‐responsiveness to methacholine and ragweed challenges and pulmonary inflammation by bronchoalveolar lavage were tested 1 and 4 days after ragweed challenge at 28 or 39 weeks old. Allergen‐free immune maturation resulted in no airway hyper‐responsiveness and very little ragweed‐specific IgE relative to the control group, but eosinophilia developed upon ragweed challenge. TLR4 agonism yielded no airway hyper‐responsiveness, but a strong airway neutrophilia developed upon ragweed challenge. Our data indicate that an atopic predisposition creates a critical window in which allergen exposure can lead to an asthmatic phenotype. Allergen‐free immune maturation may lead to allergen tolerance. TLR4 agonism before early life allergen exposure may abrogate the development of allergen‐specific bronchonconstriction, but allergen‐specific pulmonary inflammation remains a strong concern.

Myocyte repolarization modulates myocardial function in aging dogs

Studies of myocardial aging are complex and the mechanisms involved in the deterioration of ventricular performance and decreased functional reserve of the old heart remain to be properly defined. We have studied a colony of beagle dogs from 3 to 14 yr of age kept under a highly regulated environment to define the effects of aging on the myocardium. Ventricular, myocardial, and myocyte function, together with anatomical and structural properties of the organ and cardiomyocytes, were evaluated. Ventricular hypertrophy was not observed with aging and the structural composition of the myocardium was modestly affected. Alterations in the myocyte compartment were identified in aged dogs, and these factors negatively interfere with the contractile reserve typical of the young heart. The duration of the action potential is prolonged in old cardiomyocytes contributing to the slower electrical recovery of the myocardium. Also, the remodeled repolarization of cardiomyocytes with aging provides inotropic support to the senescent muscle but compromises its contractile reserve, rendering the old heart ineffective under conditions of high hemodynamic demand. The defects in the electrical and mechanical properties of cardiomyocytes with aging suggest that this cell population is an important determinant of the cardiac senescent phenotype. Collectively, the delayed electrical repolarization of aging cardiomyocytes may be viewed as a critical variable of the aging myopathy and its propensity to evolve into ventricular decompensation under stressful conditions.

Connective Tissue Growth Factor Promotes Pulmonary Epithelial Cell Senescence and Is Associated with COPD Severity.

ABSTRACT The purpose of this study was to determine whether expression of connective tissue growth factor (CTGF) protein in chronic obstructive pulmonary disease (COPD) is consistent in humans and animal models of COPD and to investigate the role of this protein in lung epithelial cells. CTGF in lung epithelial cells of ex-smokers with COPD was compared with ex-smokers without COPD by immunofluorescence. A total of twenty C57Bl/6 mice and sixteen non-human primates (NHPs) were exposed to cigarette smoke (CS) for 4 weeks. Ten mice of these CS-exposed mice and eight of the CS-exposed NHPs were infected with H3N2 influenza A virus (IAV), while the remaining ten mice and eight NHPs were mock-infected with vehicle as control. Both mRNA and protein expression of CTGF in lung epithelial cells of mice and NHPs were determined. The effects of CTGF overexpression on cell proliferation, p16 protein, and senescence-associated β-galactosidase (SA-β-gal) activity were examined in cultured human bronchial epithelial cells (HBECs). In humans, CTGF expression increased with increasing COPD severity. We found that protein expression of CTGF was upregulated in lung epithelial cells in both mice and NHPs exposed to CS and infected with IAV compared to those exposed to CS only. When overexpressed in HBECs, CTGF accelerated cellular senescence accompanied by p16 accumulation. Both CTGF and p16 protein expression in lung epithelia are positively associated with the severity of COPD in ex-smokers. These findings show that CTGF is consistently expressed in epithelial cells of COPD lungs. By accelerating lung epithelial senescence, CTGF may block regeneration relative to epithelial cell loss and lead to emphysema.

Cigarette smoke and HIV synergistically affect lung pathology in cynomolgus macaques

In the era of combined antiretroviral therapy (cART), lung diseases such as chronic bronchitis (CB) and chronic obstructive pulmonary disease (COPD) are common among persons living with HIV (PLWH), particularly smokers. Although smoking is highly prevalent among PLWH, HIV may be an independent risk factor for lung diseases; however, the role of HIV and cigarette smoke (CS) and their potential interaction in the development of chronic lung diseases among PLWH has not been delineated. To investigate this interaction, cynomolgus macaques were exposed to CS and/or simian-adapted human immunodeficiency virus (SHIV) and treated with cART. The development of CB and the lung functions were evaluated following CS±SHIV treatment. The results showed that in the lung, SHIV was a strong independent risk factor for goblet cell metaplasia/hyperplasia and mucus formation, MUC5AC synthesis, loss of tight junction proteins, and increased expression of Th2 cytokines/transcription factors. In addition, SHIV and CS synergistically reduced lung function and increased extrathoracic tracheal ring thickness. Interestingly, SHIV infection generated significant numbers of HIV-gp120+ epithelial cells (HGECs) in small airways and alveoli, and their numbers doubled in CS+SHIV-infected lungs. We conclude that even with cART, SHIV independently induces CB and pro-COPD changes in the lung, and the effects are exacerbated by CS.

VTX-1463, a novel TLR-8 agonist, attenuates nasal congestion after ragweed challenge in sensitized beagle dogs.

VTX‐1463 is a selective toll‐like receptor (TLR) 8 agonist that activates a subset of innate immune cells to produce a unique cytokine profile. Delivery of VTX‐1463 via nasal spray may modulate the nasal response in allergic rhinitis. The aim of this study was to determine the effects of VTX‐1463 on the nasal response in a dog model of allergic rhinitis. Ragweed (RW)‐sensitized dogs were pretreated with increasing doses of VTX‐1463 1 day prior to RW challenge or with two doses (4 or 8 days and 1 day prior to RW). Changes in nasal cavity volume (NV) were determined by acoustic rhinometry and nasal lavage fluid was assessed for histamine, lipid mediators, and cellular infiltrates at sequential times following RW challenge. VTX‐1463 pretreatment significantly preserved NV during the acute response to RW challenge for all doses tested. The area under the curve (AUC) for NV over the 1.5 h assessment period in RW challenged vehicle controls averaged 51.5% (SEM: ±2.12%) of the baseline NV over all studies. A single 100 µg dose of VTX‐1463 given 1 day prior to RW yielded an AUC for NV of 69.3% (±6.59%) of baseline, while a 1000 µg dose administered twice (8 days and 1 day prior to RW) resulted in an AUC for NV of 85.4% (±4.74%, P < 0.05) of baseline. For a single 1000 µg VTX‐1463 dose 1 day prior to RW, multiple mediators produced by mast cells, including histamine, PGE2, PGD2, and cysteinyl LTs, were significantly reduced relative to the vehicle control. The selective TLR8 agonist, VTX‐1463, preserved NV in a dose‐dependent manner in the acute phase of a nasal allergic response. The therapeutic effect appears to result from attenuated mast cell mediator release. Modulating the local cytokine response via TLR8 agonism appears to have a therapeutic effect on the acute allergic nasal response.

LRRK2 kinase inhibitors induce a reversible effect in the lungs of non-human primates with no measurable pulmonary deficits

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 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 low-dose 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.