Fibroblasts are key effector cells involved in fibrotic remodelling. They are not only the main source of extracellular matrix deposition in fibrotic tissues but may also modulate inflammatory processes and vascular remodelling.
Numerous studies show that hedgehog signalling is implicated in fibrotic tissue remodelling in a variety of fibrotic disorders, including SSc. Hedgehog signalling is essential to organ development. During homeostasis, the activity of this signalling pathway is very low in the majority of cells and tissues, except for stem cells, in which hedgehog signalling has an important role in the regulation of behaviour and function. Nevertheless, during fibrotic tissue remodelling, hedgehog signalling is activated and has pathogenic effects. The expression of sonic hedgehog (SHH, which is a key hedgehog ligand in the context of skin fibrosis) and of GLI1 and GLI2 (hedgehog signalling-associated transcription factors) are upregulated in the skin of patients with SSc. In addition, the SHH levels in the serum of patients with SSc are increased compared with healthy individuals and correlate with fibrotic burden. Hedgehog signalling is highly interlinked with other pathways that are implicated in fibrotic tissue remodelling; for example, the activation of hedgehog signalling in SSc is caused, in part, by TGFβ, which not only induces the expression of SHH but also activates the promoter of GLI2 to upregulate GLI2 expression in fibroblasts. Hedgehog signalling also stimulates fibroblasts to differentiate into myofibroblasts and induces skin fibrosis. Pharmacological or genetic inactivation of hedgehog signalling ameliorates fibrosis in a wide variety of mouse models of fibrosis and in many different organs.
The inhibition of hedgehog signalling is currently being evaluated for its anti-fibrotic effects in pulmonary fibrosis. Current pharmacological efforts to target hedgehog signalling focus on small molecule inhibitors of Smoothend (SMO). SMO is a G protein coupled receptor that activates GLI transcription factors in response to hedgehog ligand-receptor binding (these ligands include SHH, Indian hedgehog and desert hedgehog). Two SMO inhibitors, sonidegib and vismodegib, have been approved by the FDA for the treatment of basal cell carcinoma. In a phase IIa RCT of patients with idiopathic pulmonary fibrosis (IPF), individuals receiving the SMO inhibitor ENV-101 (200 mg daily) were found to have a significantly higher predicted FVC mean change from baseline after 12 weeks than those receiving placebo (1.9% increase in the ENV-101 arm versus 1.3% decrease in the placebo arm). Although further studies with more patients and longer follow-up are required to confirm these results, the increase in FVC upon ENV-101 treatment might be indicative of anti-fibrotic remodelling induced by SMO inhibition. Indeed, preclinical findings demonstrate that inhibition of hedgehog signalling can deactivate myofibroblasts and induce re-differentiation into resting fibroblasts. A confirmatory phase IIb trial of ENV-101 in IPF is currently recruiting (NCT06422884).
Fipaxalparant is a small molecule inhibitor that inhibits lysophosphatidic acid receptor 1 (LPAR1). This inhibitor blocks the effects of phospholipid lysophosphatidic acid (LPA). LPA is a small molecule mediator that is released during enzymatic breakdown of lipid membranes by the enzyme autotaxin. LPA is part of the tissue injury response that can promote inflammation and scarring and evidence suggests that LPA is a mediator of bleomycin-induced lung fibrosis. LPA signals through a family of receptors (LPAR1-LPAR6), which are expressed on a variety of cells that contribute to the pathogenesis of SSc. LPAR1 is considered the dominant LPA receptor in the pathogenesis of fibrotic tissue remodelling. Clinical trials of LPAR antagonists in IPF have shown positive results. A small phase IIa trial of an LPAR1 antagonist in diffuse cutaneous SSc (dcSSc) demonstrated that treatment with this antagonist attenuated LPA-regulated target genes in vivo and showed a beneficial trend in modified Rodnan Skin Score (mRSS) over 12 weeks compared with placebo. This trial led to a larger randomized controlled phase IIb study (BEACON), which did not reach its primary end point of changes in FVC (NCT04781543).
Other approaches that target LPA signalling, such as autotaxin inhibition, have also been investigated. Ziritaxestat, an autotaxin inhibitor, was evaluated in IPF and in SSc with positive results for mRSS in a small phase IIa study. However, parallel trials in IPF reported safety concerns and a lack of efficacy, and increased mortality was reported in the group receiving the highest dose in the ISABELA trials, which led to the clinical development of ziritaxestat being discontinued. Based on these results, targeting LPA signalling in SSc is currently considered challenging.
The regulation of tissue repair and development pathways by members of the TGFβ family is well established. TGFβ has three major isoforms (TGFβ1, TGFβ2 and TGFβ3) that signal through the same receptor complex. Although the downstream canonical and non-canonical signalling pathways are shared, these isoforms differ in their bioavailability, accessory protein binding and cell and tissue-specific expression patterns. TGFβ ligands are generally complexed to latent TGFβ binding proteins and are released from sequestered, inactive forms bound to matricellular proteins in response to conformational changes of integrins. All three TGFβ isoforms seem to be profibrotic in preclinical models and findings suggest that attenuating TGFβ signalling can prevent or even reverse fibrosis in preclinical models. Ample evidence indicates that TGFβ pathways are upregulated in many fibrotic diseases, including SSc. The first study of TGFβ inhibition in SSc evaluated the monoclonal antibody CAT-192 (metelimumab), which binds to TGFβ1 (ref. ). Although this phase I-II trial showed some positive dose-dependent changes in mRSS at 6 months, these changes did not reach statistical significance. In addition, biomarker studies in this trial were limited and it remains unclear if in vivo antagonism of TGFβ signalling pathways occurred.
Other approaches involve targeting TGFβ ligands. An uncontrolled trial of fresolimumab in SSc showed encouraging molecular benefits; the target genes COMP and TSP1 were downregulated and a two-gene score, proposed as a biomarker for the progression of skin fibrosis, improved. However, there were concerns about toxicity including gastrointestinal tract effects, vascular lesions and a possible increase in the incidence of proliferative lesions of the skin (keratoacanthomas). The TGFβ1/3 ligand trap, AVID200, has been evaluated in SSc and in myelofibrosis; however, these studies were too small and/or lacked placebo arms to definitively test the hypothesis of blocking TGFβ ligands as an anti-fibrotic approach in SSc. Determining lack of efficacy or unacceptable toxicity is crucial as the TGFβ pathway remains one of the most relevant targets for SSc owing to extensive preclinical evidence. The emergence of the activin signalling inhibitor, sotatercept, as an approved therapy for pulmonary arterial hypertension (PAH), including PAH associated with connective tissue disease, with remarkable benefit in clinical trials, confirms the feasibility of targeting TGFβ superfamily members and serves as a reminder that these pathways are closely interdependent and so targeting one member might affect signalling by others.
Currently, there is an ongoing phase Ib dose ranging trial of RG-6315 (RO-7303509), a human monoclonal antibody that targets TGFβ3, in SSc (NCT05462522). RG-6315 is administered via subcutaneous or intravenous routes and is a novel molecular entity. It is hypothesized that TGFβ3 might be an important profibrotic mediator and that neutralization could have fewer adverse effects than targeting all TGFβ ligands, TGFβ1 or TGFβ2 (ref. ). Conversely, historical literature supports TGFβ3 having anti-fibrotic effects based on evidence from non-scarring fetal wound healing in animal models. This finding was not substantiated in clinical trials and thus clinical development of recombinant TGFβ3 as a potential anti-fibrotic therapy has ceased.