Identification and validation of microtubule depolymerizing agent, CYT997, as a potential drug candidate for hepatocellular carcinoma.

BACKGROUND AND AIMS: Hepatocellular carcinoma (HCC) is a typically fatal malignancy with limited treatment options and poor survival rates, despite recent FDA approvals of newer treatment options. We aim to address this unmet need by using a proprietary computational drug discovery platform that identifies drug candidates with the potential to advance rapidly and successfully through preclinical studies. METHODS: We generated an in silico model of HCC biology to identify the top 10 small molecules with predicted efficacy. The most promising candidate, CYT997, was tested for its in vitro effects on cell viability and cell death, colony formation, cell cycle changes, and cell migration/invasion in HCC cells. We used an HCC patient-derived xenograft (PDX) mouse model to assess its in vivo efficacy. RESULTS: CYT997 was significantly more cytotoxic against HCC cells than against primary human hepatocytes, and sensitized HCC cells to sorafenib. It arrested cell cycle at the G2/M phase with associated up-regulations of p21, p-MEK1/2, p-ERK, and down-regulation of cyclin B1. Cell apoptosis and senescence-like morphology were also observed. CYT997 inhibited HCC cell migration and invasion, and down-regulated the expressions of acetylated tubulins, ß-tubulin, glypican-3 (GPC3), ß-catenin, and c-Myc. In vivo, CYT997 (20 mg/kg, three times weekly by oral gavage) significantly inhibited PDX growth, while being non-toxic to mice. Immunohistochemistry confirmed the down-regulation of GPC3, c-Myc, and Ki-67, supporting its anti-proliferative effect. CONCLUSION: CYT997 is a potentially efficacious and non-toxic drug candidate for HCC therapy. Its ability to down-regulate GPC3, ß-catenin, and c-Myc highlights a novel mechanism of action.

Uncultured adipose-derived regenerative cells (ADRCs) seeded in collagen scaffold improves dermal regeneration, enhancing early vascularization and structural organization following thermal burns.

OBJECTIVE: Advances in tissue engineering have yielded a range of both natural and synthetic skin substitutes for burn wound healing application. Long-term viability of tissue-engineered skin substitutes requires the formation and maturation of neo-vessels to optimize survival and biointegration after implantation. A number of studies have demonstrated the capacity of Adipose Derived Regenerative Cells (ADRCs) to promote angiogenesis and modulate inflammation. On this basis, it was hypothesized that adding ADRCs to a collagen-based matrix (CBM) (i.e. Integra) would enhance formation and maturation of well-organized wound tissue in the setting of acute thermal burns. The purpose of this study was to evaluate whether seeding uncultured ADRCs onto CBM would improve matrix properties and enhance healing of the grafted wound. METHODS: Full thickness thermal burns were created on the backs of 8 Gottingen mini-swine. Two days post-injury wounds underwent fascial excision and animals were randomized to receive either Integra seeded with either uncultured ADRCs or control vehicle. Wound healing assessment was performed by digital wound imaging, histopathological and immunohistochemical analyses. RESULTS: In vitro analysis demonstrated that freshly isolated ADRCs adhered and propagated on the CBM. Histological scoring revealed accelerated maturation of wound bed tissue in wounds receiving ADRCs-loaded CBM compared to vehicle-loaded CBM. This was associated with a significant increase in depth of the wound bed tissue and collagen deposition (p<0.05). Blood vessel density in the wound bed was 50% to 69.6% greater in wounds receiving ADRCs-loaded CBM compared to vehicle-loaded CBM (p=0.05) at day 14 and 21. In addition, ADRCs delivered with CBM showed increased blood vessel lumen area and blood vessel maturation at day 21(p=0.05). Interestingly, vascularity and overall cellularity within the CBM were 50% and 45% greater in animals receiving ADRC loaded scaffolds compared to CBM alone (p<0.05). CONCLUSIONS: These data demonstrate that seeding uncultured ADRCs onto CBM dermal substitute enhances wound angiogenesis, blood vessel maturation and matrix remodeling.