# Targeting of C-ROS-1 Activity Using a Controlled Release Carrier to Treat Craniosynostosis in a Preclinical Model of Saethre-Chotzen Syndrome

## TL;DR

This study explores a drug-based treatment for craniosynostosis in a mouse model of Saethre-Chotzen Syndrome, aiming to avoid invasive surgery.

## Contribution

A novel pharmacological approach using crizotinib with controlled release carriers to treat craniosynostosis is proposed and tested.

## Key findings

- Crizotinib delivered via microdisks showed greater efficacy at lower concentrations in reducing bone formation at coronal sutures.
- The drug maintained suture patency without adverse effects on major organs or blood parameters.
- The bone inhibitory effects diminished after treatment cessation, suggesting a need for ongoing therapy.

## Abstract

Saethre-Chotzen syndrome (SCS) is one of the most prevalent craniosynostosis, caused by a loss-of-function mutation in the TWIST-1 gene, with current treatment options relying on major invasive transcranial surgery. TWIST-1 haploinsufficient osteogenic progenitor cells exhibit increased osteogenic differentiation potential due to an upregulation of the transmembrane tyrosine kinase receptor, C-ROS-1, a TWIST-1 target gene known to promote bone formation. The present study assessed the efficacy of suppressing C-ROS-1 activity using a known chemical inhibitor to C-ROS-1, crizotinib, to halt premature coronal suture fusion in a preclinical mouse model of SCS. Crizotinib (1 μM, 2 μM, or 4 μM) was administered locally over the calvaria of Twist‐1del/+ heterozygous mice prior to coronal suture fusion using either a nonresorbable collagen sponge (quick drug release) or a resorbable sodium carboxymethylcellulose microdisk (slow sustained release). Coronal suture fusion rates and bone parameters were determined by μCT imaging and histomorphometric analysis of calvaria postcoronal suture fusion. Results demonstrated a dose-dependent increase in the efficacy of crizotinib to maintain coronal suture patency, with no adverse effects to brain, kidney, liver, and spleen tissue, or blood cell parameters. Moreover, crizotinib delivered on microdisks resulted in a greater efficacy at a lower concentration to reduce bone formation at the coronal suture sites compared to sponges. However, the bone inhibitory effects were found to be diminished by over time following cessation of treatment. Our findings lay the foundation for the development of a pharmacological nonsurgical, targeted approach to temporarily maintain open coronal sutures in SCS patients. This study could potentially be used to develop similar therapeutic strategies to treat different syndromic craniosynostosis conditions caused by known genetic mutations.