Postinfection A77-1726 treatment improves cardiopulmonary function in H1N1 influenza-infected mice

Abstract

Acute respiratory disease is associated with significant morbidity and mortality in influenza. Because antiviral drugs are only effective early in infection, new agents are needed to treat nonvaccinated patients presenting with late-stage disease, particularly those who develop acute respiratory distress syndrome. We found previously that the de novo pyrimidine synthesis inhibitor A77-1726 reversed the influenza-induced impairment of alveolar fluid clearance. Patients with acute respiratory distress syndrome and intact alveolar fluid clearance demonstrate lower mortality than those with compromised fluid clearance. We therefore investigated the effects of treatment with nebulized A77-1726 (67.5 mg/kg) on indices of cardiopulmonary function relevant to the diagnosis of acute respiratory distress syndrome. BALB/cAnNCr mice (8-12 wk old) were inoculated intranasally with 10,000 plaque-forming units/mouse influenza A/WSN/33 (H1N1). Pulse oximetry was performed daily. Alveolar fluid clearance, lung water, and lung mechanics were measured at 2 and 6 days after inoculation in live, ventilated mice by BSA instillation, magnetic resonance imaging, and forced-oscillation techniques, respectively. A77-1726 treatment at 1 day after inoculation delayed mortality. Treatment on Days 1 or 5 reduced viral replication on Day 6, and improved alveolar fluid clearance, peripheral oxygenation, and cardiac function. Nebulized A77-1726 also reversed influenza-induced increases in lung water content and volume, improved pulmonary mechanics, reduced bronchoalveolar lavage fluid ATP and neutrophil content, and increased IL-6 concentrations. The ability of A77-1726 to improve cardiopulmonary function in influenza-infected mice and to reduce the severity of ongoing acute respiratory distress syndrome late in infection suggests that pyrimidine synthesis inhibitors are promising therapeutic candidates for the management of severe influenza.

Figure 1.

A77 ameliorates nucleotide-mediated inhibition of alveolar fluid clearance. (A) Effect of treatment (Tx) with nebulized A77 (67.5 mg/kg) at 1 day postinoculation (DPI) (A77-Tx Day 1) on baseline alveolar fluid clearance rate (AFC) at 2 DPI (n = 7) and 6 DPI (n = 15), or A77 treatment at 5 DPI (A77-Tx Day 5) on AFC rate at 6 DPI (n = 6), compared with untreated animals (n = 12 per time point). (B) Effect of treatment with nebulized A77 (67.5 mg/kg) at 1 DPI (A77-Tx Day 1) on bronchoalveolar lavage fluid (BALF) ATP content at 2 DPI (n = 5) and 6 DPI (n = 9), or A77 treatment at 5 DPI (A77-Tx Day 5) on AFC at 6 DPI (n = 10), compared with untreated animals (n = 5 per time point). (C) Effect of treatment with nebulized A77 at 1 DPI (A77-Tx Day 1) on Day 2 AFC in the presence of 1.5 mM amiloride (AMIL) or 100 μM cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor–172 (CF-inh), compared with untreated animals (n = 7–9 per group). (D) Effect of treatment with nebulized A77 at 1 DPI (A77-Tx Day 1) on Day 2 AFC in Cl⁻-free (Na⁺ gluconate) BSA (n = 7 per group). Dotted lines indicate mean values for each parameter in uninfected mice. *P < 0.05 and ***P < 0.0005, versus uninfected mice. ^†P < 0.1, ^†P < 0.05, and ^#P < 0.0005, versus infected, untreated mice (UNTx) at the same postinfection time point.

Figure 2.

A77 prolongs survival, despite increasing postinfection weight loss and only limited antiviral effects. Effect of treatment of influenza-infected mice with nebulized A77 (67.5 mg/kg) at 1 DPI (A77-Tx DAY 1) or 5 DPI (A77-Tx DAY 5) on (A) mortality (n ≥ 10 per group), (B) body weight (BWT; percent change from baseline; n > 15 per group, including data from mice that died before 6 DPI), and (C) influenza virus replication (log titer in whole-lung homogenates; n ≥ 5 per group). ***P < 0.0005, versus untreated mice (UNTx). ^†P < 0.05 and ^‡P < 0.005, versus untreated mice at the same postinfection time point. FFU, focus-forming units.

Figure 3.

A77 improves cardiopulmonary function in conscious mice. Effects of treatment of influenza-infected mice with nebulized A77 (67.5 mg/kg) at 1 DPI (A77-Tx DAY 1) or 5 DPI (A77-Tx DAY 5) on (A) carotid arterial O₂ saturation (Sa_O2; percentage), (B) respiratory rate (RR; breaths/minute), (C) heart rate (HR; beats/minute), and (D) pulse distension (PD; μm) (n > 10 per group). Dotted lines indicate mean values for each parameter in uninfected mice. *P < 0.05, **P < 0.005, and ***P < 0.0005, versus uninfected mice. ^†P < 0.05 and ^#P < 0.0005, versus untreated mice (UNTx) at the same postinfection time point.

Figure 4.

A77 ameliorates bilateral airspace disease. Effects of treatment of influenza-infected mice with nebulized A77 (67.5 mg/kg) at 1 DPI (A77-Tx DAY 1) on (A) lung water content (percentage of total lung volume) at 2 DPI (n = 7) and 6 DPI (n = 4), compared with untreated animals (n = 6 per time point); < 0.1% T2-weighted (edema) signal was detected in uninfected mice (n = 8), and (B) total lung volume (cm³) in the same experimental groups. Dotted line indicates mean lung volume in uninfected mice. (C) Representative Day 6 midthoracic magnetic resonance image from an untreated, influenza-infected mouse. T1-weighted images (body tissues) are pseudocolored red. T2-weighted images (edema) are pseudocolored green. LV, left ventricular lumen. (D) Representative Day 6 midthoracic magnetic resonance image from an influenza-infected mouse treated with nebulized A77 at 1 DPI. All mice were anesthetized with isoflurane and imaged using an 11.7T BioSpec high-field small-animal magnetic resonance imaging system with respiratory and cardiac gating. Images were adjusted in brightness, contrast, and color balance for a more uniform appearance. These adjustments do not obscure, eliminate, or misrepresent any information presented in the original scans. ***P < 0.0005, compared with uninfected mice. ^†P < 0.05, versus untreated mice (UNTx) at the same postinfection time point.

Figure 5.

A77 attenuates the development of pulmonary edema. Effect of treatment of influenza-infected mice with nebulized A77 (67.5 mg/kg) at 1 DPI (A77-Tx DAY 1) or 5 DPI (A77-Tx DAY 5) on (A) lung water content (wet/dry weight ratio; n = 6–9 per group), (B) BALF protein content (μg/ml; n = 6–10 per group), and (C) BALF lactate dehydrogenase (LDH) content (mU/ml; n = 5–7 per group). Dotted lines indicate mean values for each parameter in uninfected mice. *P < 0.05, **P < 0.005, and ***P < 0.0005, versus uninfected mice. ^†P < 0.05 and ^#P < 0.0005, versus untreated mice (UNTx) at the same postinfection time point.

Figure 6.

A77 improves lung mechanics. Effects of treatment of influenza-infected mice with nebulized A77 (67.5 mg/kg) at 1 DPI (A77-Tx DAY 1) or 5 DPI (A77-Tx DAY 5) on (A) baseline total lung resistance (R_BASAL; cm H₂O · second/ml), (B) maximal airway resistance after the nebulization of 50 mg/ml methacholine (R_MAX; cm H₂O · second /ml), (C) dynamic lung compliance (C_DYN; ml/cm H₂O × 10), and (D) static lung compliance (C_ST; ml/cm H₂O × 10). All mice were anesthetized with valium/ketamine and mechanically ventilated on a FlexiVent computer-controlled piston ventilator (Scireq Respiratory Equipment, Montreal, PQ, Canada), with 8 ml/kg tidal volume, at a frequency of 150 breaths/minute, against 3 cm H₂O positive end-expiratory pressure (n = 5–7 per group). Dotted lines indicate mean values for each parameter in uninfected mice. **P < 0.005 and ***P < 0.0005, compared with uninfected mice. ^‡P < 0.005 and ^#P < 0.0005, versus untreated mice (UNTx) at the same postinfection time point.

Figure 7.

A77 reduces BALF neutrophil concentrations in influenza-infected mice. Effects of treatment of influenza-infected mice with nebulized A77 (67.5 mg/kg) at 1 DPI (A77-Tx DAY 1) or 5 DPI (A77-Tx DAY 5) on BALF numbers of (A) total cells, (B) neutrophils (PMNs), (C) alveolar macrophages (AMs), and (D) lymphocytes (Lymphs) (n ≥ 10 per group). All cell counts are expressed as × 10⁶/ml. Dotted lines indicate mean values for total cells and alveolar macrophage counts in uninfected mice (not shown for neutrophils and lymphocytes because counts for both cell types were less than 10⁴/ml in uninfected mice). *P < 0.05, **P < 0.005, and ***P < 0.0005, versus uninfected mice. ^‡P < 0.005 and ^#P < 0.0005, versus untreated mice (UNTx) at the same postinfection time point.