Defense Date
2026
Document Type
Thesis
Degree Name
Master of Science
Department
Pharmaceutical Sciences
First Advisor
Masahiro Sakagami
Abstract
Lung fibrosis (LF) is a progressive, irreversible pathological condition in the lungs that is ultimately fatal. Current drug treatments (nintedanib, pirfenidone, and nerandomilast) slow disease progression but cause significant systemic adverse effects due to oral administration. Transient receptor potential vanilloid 4 (TRPV4), a mechanosensitive ion channel, may play a role in LF progression and represent a novel anti-LF target. Ginkgetin (GKT), a natural biflavonoid, is a potent TRPV4 inhibitor with anti-inflammatory and antioxidative properties. Local lung delivery could achieve high local lung concentrations while minimizing systemic exposure. Accordingly, this project evaluates GKT's therapeutic intervention efficacy in LF via local lung delivery through the following three specific aims: (1) to demonstrate GKT inhibition of TGF-β1-induced fibroblast activation in vitro; (2) to assess therapeutic intervention efficacy in a bleomycin (BLM)-induced LF rat model; (3) to evaluate whether GKT inhibits calcium-dependent signaling; and (4) to compare pharmacokinetics (PK) of GKT following local lung versus systemic delivery.
Human LF-derived lung fibroblasts (LL29 cells) were treated with GKT (0-10 μM) following TGF-β1 stimulation. Cell proliferation (MTT assay) and differentiation to myofibroblasts (alpha smooth muscle actin, α-SMA expression via Western blot) were measured. Male Sprague-Dawley rats received BLM (2.5 mg/kg on day 1 and 1.25 mg/kg on day 4), followed by GKT (0.5 mg/kg) treatment via orotracheal (OT) spray or subcutaneous (SC) injection 5 days weekly over a two-week period from day 7 to day 21. Outcome measures included histological airspace fibrosis assessment, lung collagen and hydroxyproline levels, treadmill endurance, myofibroblast and profibrotic markers (α-SMA and transforming growth factor beta, TGF-β), calcium-dependent signaling (phosphorylated calcium/calmodulin-dependent protein kinase II, p-CaMKII), oxidative stress (malondialdehyde, MDA), and neutrophil infiltration (myeloperoxidase, MPO). GKT PK in plasma and lung homogenates were also determined using a validated high performance liquid chromatography with ultraviolet (HPLC-UV) method following both routes of administration.
GKT at 10 μM inhibited TGF-β1-induced LL29 cell proliferation by 103% and differentiation to myofibroblasts by 85%. Local OT delivery of GKT significantly attenuated BLM-induced LF across all outcome measures: fibrotic formation remained substantially reduced; collagen and hydroxyproline levels were reduced by 100% and 83%, respectively; treadmill endurance was longer (20 vs. 6 minutes); α-SMA and TGF-β levels were decreased by 100% and 94%, respectively; p-CaMKII, MPO, and MDA levels were also reduced by 93%, 78%, and 93%. In contrast, SC delivery was less effective, supporting the superiority of the local lung delivery. PK analysis revealed quantifiable GKT remaining in the lungs only following OT delivery (12 μg at 1 hour and 8.2 μg at 3 hours), whereas systemic exposure remained below the lower limit of quantification (LLOQ). No detectable GKT was observed in either lungs or plasma following SC injection, consistent with its lower therapeutic efficacy.
This thesis demonstrates that GKT exerts potent anti-LF activity in both cellular and animal models and may therefore be a promising therapeutic candidate for LF via local lung delivery. The reduction in lung p-CaMKII expression supports the hypothesis that its therapeutic effects may involve TRPV4 inhibition, a unique activity previously reported for GKT. Local lung delivery achieved higher lung GKT concentrations while maintaining negligible systemic exposure, potentially offering an advantage over current oral therapies, which are associated with dose-limiting systemic and gastrointestinal toxicities. Taken together, these findings suggest that local lung TRPV4 inhibition may represent a mechanistically distinct therapeutic approach for LF and that TRPV4 inhibitors or antagonists warrant further investigation as potential therapeutic candidates.
Rights
© Cimone Richardson
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
7-28-2026