Preclinical and Toxicology Assessment of ALW-II-41-27, an Inhibitor of the Eph Receptor A2 (EphA2)
Background and Objective:
The EphA2 receptor inhibitor ALW-II-41-27 has demonstrated promising results as an in vitro antagonist of Pneumocystis β-glucan-induced proinflammatory signaling. Given its effectiveness in this context, ALW-II-41-27 may hold potential as a candidate for further testing as an anti-inflammatory therapeutic in the rodent model of Pneumocystis pneumonia (PCP). This study aimed to assess its pharmacokinetics (PK), safety, and efficacy in vivo to determine its suitability for future anti-inflammatory drug testing in the context of PCP.
Methods:
In the initial phase, single-dose intraperitoneal (IP) injections of ALW-II-41-27 were administered at concentrations of 0, 10, 15, 20, and 30 mg/kg over a 24-hour treatment period to evaluate its pharmacokinetics in plasma, bronchoalveolar lavage fluid (BALF), and epithelial lining fluid (ELF). Based on these assessments, the optimal dosing of 20 mg/kg was selected for subsequent testing. Following this, mice were treated daily with either vehicle or 20 mg/kg of ALW-II-41-27 via IP injections for 10 days, with body weight recorded daily. On day 11, mice were weighed, euthanized, and tissues from the lungs, liver, and kidneys were harvested for histological analysis through H&E staining and pathology scoring. Lung samples were further analyzed for proinflammatory cytokines using enzyme-linked immunosorbent assay (ELISA) and for extracellular matrix production via quantitative PCR (qPCR). Postmortem blood samples were collected for complete blood count (CBC) and blood chemistry analysis. Additionally, ALW-II-41-27 was administered prior to a fungal β-glucan challenge to evaluate its in vivo effects on lung inflammation.
Results:
The pharmacokinetic assessment of ALW-II-41-27 following IP administration in C57BL/6 mice showed no significant changes in the body weight of the mice during the treatment period. ELISA analysis of proinflammatory cytokines in lung tissues revealed no major differences between the treatment and control groups. qPCR analysis of extracellular matrix-related transcripts also showed no significant differences in gene expression. Histological examination and pathology scoring of lung, liver, and kidney tissues did not indicate any significant toxicity in the animals. Further, blood chemistry and CBC analyses showed no major abnormalities, supporting the general safety of ALW-II-41-27 at the tested dose. When administered prior to intratracheal inoculation of fungal β-glucans, which is known to trigger a strong proinflammatory response in the lungs, ALW-II-41-27 significantly reduced lung tissue IL-1β levels, indicating its potential to modulate inflammation in vivo.
Conclusions:
In the initial safety and toxicology assessments, ALW-II-41-27 did not present any significant safety concerns across the measured parameters, including body weight, histology, blood chemistry, and cytokine levels. The results from this study suggest that ALW-II-41-27 may be a promising therapeutic candidate for further in vivo testing, particularly as an adjunct therapy to mitigate the harmful proinflammatory immune responses associated with Pneumocystis infection. These findings support the continued exploration of ALW-II-41-27 for its potential as a treatment option for managing inflammation in PCP. ALW II-41-27