Inhibition of ALK5 Signaling Induces Physeal Dysplasia in Rats

Abstract

TGF-|β|, and its type 1 (ALK5) receptor, are critical to the pathogenesis of fibrosis. In toxicologic studies of 4 or more days in 10-week-old Sprague-Dawley rats, using an ALK5 inhibitor (GW788388), expansion of hypertrophic and proliferation zones of femoral physes were noted. Subphyseal hyperostosis, chondrocyte hypertrophy/hyperplasia, and increased matrix were present. Physeal zones were laser microdissected from ALK5 inhibitor-treated and control rats sacrificed after 3 days of treatment. Transcripts for TGF-|β|1, TGF-|β|2, ALK5, IHH, VEGF, BMP-7, IGF-1, bFGF, and PTHrP were amplified by real-time PCR. IGF and IHH increased in all physis zones with treatment, but were most prominent in prehypertrophic zones. TGF-|β|2, bFGF and BMP7 expression increased in proliferative, pre-and hypertrophic zones. PTHrP expression was elevated in proliferative zones but decreased in hypertrophic zones. VEGF expression was increased after treatment in pre-and hypertrophic zones. ALK5 expression was elevated in prehypertrophic zones. Zymography demonstrated gelatinolytic activity was reduced after treatment. Apoptotic markers (TUNEL and caspase-3) were decreased in hypertrophic zones. Proliferation assessed by Topoisomerase II and Ki67 was increased in multiple zones. Movat stains demonstrated that proteoglycan deposition was altered. Physeal changes occurred at doses well above those resulting in fibrosis. Interactions of factors is important in producing the physeal dysplasia phenotype.

Nonclinical Safety Evaluation of a Transforming Growth Factor β Receptor I Kinase Inhibitor in Fischer 344 Rats and Beagle Dogs

“…LY2157299 resulted in changes to the bone similar to previous reports [12,13]. Reversibility of bone changes was assessed following the 1-month study in rats revealing a band of increased trabecular density that was separated from the normal physis by a zone of normal endochondral ossification.…”

“…Osseous changes included physeal and subphyseal changes in the femur, tibia, and sternum of rats that occurred after just 14 days of dosing and were similar to those previously described with TGF-β inhibition [12,13]. The zones of maturation and hypertrophy were expanded in the physes of the long bones and endplates of the sternabrae, and the primary and secondary spongiosa were denser with increased connectivity (Figure 8).…”

“…Other pharmaceutical companies have published similar valvular toxicities with inhibitors from their development efforts, indicating that the lesions described are likely an on target effect [12,13]. Many explanations have been considered to understand mechanism of this phenomenon; no definitive explanation has been provided at this time [12].…”

“…Inhibition of TGF-β signaling results in a physeal dysplasia that is observed in rats administered small molecule inhibitors of TGF-β RIK [12,13]. Rats administered repeated doses of small molecule TGF-β RIK inhibitors develop a valvulopathy that is characterized by inflammation, hemorrhage and stromal hyperplasia [12][13][14]. Valvulopathy in rats is likely a result of direct inhibition of the TGF-β signaling pathway, rather than an "off-target" effect, because multiple structurally diverse small molecule inhibitors have produced similar effects.…”

“…Within the last 5 years, a small number of publications have disclosed the effects of TGF-β inhibition in nonclinical toxicology species. Inhibition of TGF-β signaling results in a physeal dysplasia that is observed in rats administered small molecule inhibitors of TGF-β RIK [12,13]. Rats administered repeated doses of small molecule TGF-β RIK inhibitors develop a valvulopathy that is characterized by inflammation, hemorrhage and stromal hyperplasia [12][13][14].…”