A Macromolecule Linked Prodrug of Rapamycin Shows Potent Growth Inhibitory Effects Against Activated Fibroblasts Mimicking Fibroproliferative Transformation in Pulmonary Hypertension

Introduction: Pulmonary hypertension (PH) is a life-threatening disease characterized by a progressive loss of vascular cell homeostasis with an irreversible shift toward the activated proliferative phenotype. New drug delivery approaches with improved efficacy are urgently needed for better clinical management of the disease. The present studies evaluated a novel prodrug-based strategy designed to durably suppress PH through enhanced delivery of rapamycin. We hypothesized that a long-circulating rapamycin prodrug constructed with a bioeliminable polyethylene glycol (PEG) scaffold would show efficacy as a new anti-PH drug candidate in cell culture and animal model studies. Methods: A new carrier-linked prodrug was synthesized by reversibly attaching rapamycin to 4-arm PEG (PEG-[RAPA] 4 , Mn = 40 kDa). Cell growth inhibitory effects of the construct were studied in vitro with proliferative fetal lung fibroblasts (HEL299) and transformed fibroblast-type cells (SK-LMS1), and in vivo using SK-LMS1 xenografts established in athymic nude mice. The results were analyzed by comparing the growth curve slopes (one-way ANOVA with the Tukey post hoc test). Results: Rapamycin (1-5 μM) potently inhibited proliferation of both fetal and transformed fibroblasts (75-95% inhibition 7 days post-treatment) in PDGF-BB-stimulated growth conditions (40 ng/ml, 2% FBS). Serum-stimulated (10% FBS) growth of both cell lines was also suppressed with remarkable potency at all tested concentrations (≥50% and ≥65% inhibition for HEL299 and SK-LMS1, respectively). In animal model studies, PEG-[RAPA] 4 given 2х/week strongly mitigated disease progression throughout the 4-week treatment period, whereas all animals in ‘no treatment’ and ‘blank PEG’ control groups were eliminated after reaching the end point. Statistical analysis confirmed markedly delayed growth of SK-LMS1 xenografts in the prodrug group (p = 0.032). Conclusions: Polymeric carrier-linked prodrug delivery of rapamycin effectively inhibits growth of activated fibroblasts in vitro and in a preclinical model recapitulating the altered vascular cell phenotype and proliferative character of the human disease. PEG-[RAPA] 4 is a promising candidate for further evaluation as a potential therapy for PH.