Chitin Derived Small Molecule AVR-48 Reprograms the Resting Macrophages to an Intermediate Phenotype and Decrease Pseudomonas aeruginosa Mouse Lung Infection
Published: 11 November 2022
AVR-48 is a structural derivative of chitin previously shown by our laboratory to significantly decrease lung injury parameters in LPS, hyperoxia and sepsis-induced rodent models. The current study objectives are to determine the cellular mechanism of action and demonstrate efficacy in a mouse bacterial lung infection model. For in vitro receptor binding and macrophage polarization studies, C57Bl/6J mouse derived spleens and human peripheral blood mononuclear cells (hPBMCs) were treated with AVR-48 ± LPS or biotin conjugated AVR-48. Different macrophage types were determined using flow cytometry and secreted cytokines were measured using ELISA. In vivo, a CD-1 mouse Pseudomonas aeruginosa lung infection was treated with AVR-48, assessing bacterial colony forming unit (CFU), IL-10 and IL-17A levels in lung and blood samples. AVR-48 binds to both the toll-like receptor 4 (TLR4) and the CD163 receptor on mouse monocytes. In hPBMCs, frequency of intermediate macrophages increased upon AVR-48 treatment for 72 h. Increased bacterial phagocytosis/intracellular killing were observed in THP-1 cells and reduction in CFU in CD-1 mouse lungs. Binding of AVR-48 to both TLR4 and CD163 receptors bring the macrophages to an intermediary stage, resulting in increased phagocytosis and decreased inflammation, altogether providing an optimal immune balance for treating lung injury and infection.
Keywords: chitin analog; immunomodulation; TLR4; CD163; lung injury; bacterial infection; phagocytosis; anti-inflammatory; intermediate macrophages
Chitin-Derived AVR-48 Prevents Experimental Bronchopulmonary Dysplasia (BPD) and BPD-Associated Pulmonary Hypertension in Newborn Mice
Published: 9 August 2021
Bronchopulmonary dysplasia (BPD) is the most common complication of prematurity and a key contributor to the large health care burden associated with prematurity, longer hospital stays, higher hospital costs, and frequent re-hospitalizations of affected patients through the first year of life and increased resource utilization throughout childhood. This disease is associated with abnormal pulmonary function that may lead to BPD-associated pulmonary hypertension (PH), a major contributor to neonatal mortality and morbidity. In the absence of any definitive treatment options, this life-threatening disease is associated with high resource utilization during and after neonatal intensive care unit (NICU) stay. The goal of this study was to test the safety and efficacy of a small molecule derivative of chitin, AVR-48, as prophylactic therapy for preventing experimental BPD in a mouse model. Two doses of AVR-48 were delivered either intranasally (0.11 mg/kg), intraperitoneally (10 mg/kg), or intravenously (IV) (10 mg/kg) to newborn mouse pups on postnatal day (P)2 and P4. The outcomes were assessed by measuring total inflammatory cells in the broncho alveolar lavage fluid (BALF), chord length, septal thickness, and radial alveolar counts of the alveoli, Fulton’s Index (for PH), cell proliferation and cell death by immunostaining, and markers of inflammation by Western blotting and ELISA. The bioavailability and safety of the drug were assessed by pharmacokinetic and toxicity studies in both neonatal mice and rat pups (P3-P5). Following AVR-48 treatment, alveolar simplification was improved, as evident from chord length, septal thickness, and radial alveolar counts; total inflammatory cells were decreased in the BALF; Fulton’s Index was decreased and lung inflammation and cell death were decreased, while angiogenesis and cell proliferation were increased. AVR-48 was found to be safe and the no-observed- adverse effect level (NOAEL) in rat pups was determined to be 100 mg/kg when delivered via IV dosing with a 20-fold safety margin. With no reported toxicity and with a shorter half-life, AVR-48 is able to reverse the worsening cardiopulmonary phenotype of experimental BPD and BPD-PH, compared to controls, thus positioning it as a future drug candidate.
Small Immunomodulatory Molecules as Potential Therapeutics in Experimental Murine Models of Acute Lung Injury (ALI)/Acute Respiratory Distress Syndrome (ARDS)
Acute lung injury (ALI) or its most advanced form, acute respiratory distress syndrome (ARDS) is a severe inflammatory pulmonary process triggered by a variety of insults including sepsis, viral or bacterial pneumonia, and mechanical ventilator-induced trauma. Currently, there are no effective therapies available for ARDS. We have recently reported that a novel small molecule AVR-25 derived from chitin molecule (a long-chain polymer of N-acetylglucosamine) showed anti-inflammatory effects in the lungs. The goal of this study was to determine the efficacy of two chitin-derived compounds, AVR-25 and AVR-48, in multiple mouse models of ALI/ARDS. We further determined the safety and pharmacokinetic (PK) profile of the lead compound AVR-48 in rats. ALI in mice was induced by intratracheal instillation of a single dose of lipopolysaccharide (LPS; 100 µg) for 24 h or exposed to hyperoxia (100% oxygen) for 48 h or undergoing cecal ligation and puncture (CLP) procedure and observation for 10 days. Both chitin derivatives, AVR-25 and AVR-48, showed decreased neutrophil recruitment and reduced inflammation in the lungs of ALI mice. Further, AVR-25 and AVR-48 mediated diminished lung inflammation was associated with reduced expression of lung adhesion molecules with improvement in pulmonary endothelial barrier function, pulmonary edema, and lung injury. Consistent with these results, CLP-induced sepsis mice treated with AVR-48 showed a significant increase in survival of the mice (80%) and improved lung histopathology in the treated CLP group. AVR-48, the lead chitin derivative compound, demonstrated a good safety profile. Both AVR-25 and AVR-48 demonstrate the potential to be developed as therapeutic agents to treat ALI/ARDS.
Chitin Analog AVR-25 Prevents Experimental Bronchopulmonary Dysplasia
Infants born extremely preterm are at a high risk of developing bronchopulmonary dysplasia (BPD) which is characterized by large, simplified alveoli, increased inflammation, disrupted and dysregulated vasculogenesis, decreased cell proliferation, and
increased cell death in the lungs. Due to lack of specific drug treatments to combat this condition, BPD and its long-term complications have taken a significant toll of healthcare resources. AVR-25, a novel immune modulator experimental compound,
was able to partially recover the pulmonary phenotype in the hyperoxia-induced experimental mouse model of BPD. We anticipate that AVR-25 will have therapeutic potential for managing human BPD.
Novel Chitohexaose Analog Protects Young and Aged mice from CLP Induced Polymicrobial Sepsis
In Gram-negative bacterial sepsis, production of excess pro-inflammatory cytokines results in hyperinflammation and tissue injury. Anti-inflammatory cytokines such as IL-10 inhibit inflammation and enhance tissue healing. Here, we report a novel approach to treat septicemia associated with intraabdominal infection in a murine model by delicately balancing pro- and anti-inflammatory cytokines. A novel oligosaccharide compound AVR-25 selectively binds to the TLR4 protein (IC50 = 0.15 μM) in human peripheral blood monocytes and stimulates IL-10 production. Following the cecal ligation and puncture (CLP) procedure, intravenous dosing of AVR-25 (10 mg/kg, 6–12 h post-CLP) alone and in combination with antibiotic imipenem protected both young adult (10–12 week old) and aged (16–18 month old) mice against polymicrobial infection, organ dysfunction, and death. Proinflammatory cytokines (TNF-α, MIP-1, i-NOS) were decreased significantly and restoration of tissue damage was observed in all organs. A decrease in serum C-reactive protein (CRP) and bacterial colony forming unit (CFU) confirmed improved bacterial clearance. Together, these findings demonstrate the therapeutic ability of AVR-25 to mitigate the storm of inflammation and minimize tissue injury with high potential for adjunctive therapy in intra-abdominal sepsis.
Chitohexaose Activates Macrophages by Alternate Pathway through TLR4 and Blocks Endotoxemia
Sepsis is a consequence of systemic bacterial infections leading to hyper activation of immune cells by bacterial products resulting in enhanced release of mediators of inflammation. Endotoxin (LPS) is a major component of the outer membrane of Gram negative bacteria and a critical factor in pathogenesis of sepsis. Development of antagonists that inhibit the storm of inflammatory molecules by blocking Toll like receptors (TLR) has been the main stay of research efforts. We report here that a filarial glycoprotein binds to murine macrophages and human monocytes through TLR4 and activates them through alternate pathway and in the process inhibits LPS mediated classical activation which leads to inflammation associated with endotoxemia. The active component of the nematode glycoprotein mediating alternate activation of macrophages was found to be a carbohydrate residue, Chitohexaose. Murine macrophages and human monocytes up regulated Arginase-1 and released high levels of IL-10 when incubated with chitohexaose. Macrophages of C3H/HeJ mice (non-responsive to LPS) failed to get activated by chitohexaose suggesting that a functional TLR4 is critical for alternate activation of macrophages also. Chitohexaose inhibited LPS induced production of inflammatory molecules TNF-a, IL-1b and IL-6 by macropahges in vitro and in vivo in mice. Intraperitoneal injection of chitohexaose completely protected mice against endotoxemia when challenged with a lethal dose of LPS. Furthermore, Chitohexaose was found to reverse LPS induced endotoxemia in mice even 6/24/48 hrs after its onset. Monocytes of subjects with active filarial infection displayed characteristic alternate activation markers and were refractory to LPS mediated inflammatory activation suggesting an interesting possibility of subjects with filarial infections being less prone to develop of endotoxemia. These observations that innate activation of alternate pathway of macrophages by chtx through TLR4 has offered novel opportunities to cell biologists to study two mutually exclusive activation pathways of macrophages being mediated through a single receptor.