A New Anticancer Agent for the Hedgehog-dependent tumors: from Drug Discovery to Drug Delivery

This Ph.D. thesis focuses on two main topics: Part A. Identification of a natural compound capable of inhibiting the Hedgehog signalling pathway Hedgehog (Hh) signaling is essential for tissue development and stemness and its deregulation has been observed in many tumors. For this reason, the understanding of the poorly elucidated mechanism of Gli1-mediated transcription allows to identify novel molecules blocking the pathway at a downstream level, which represents a crucial goal in cancer therapy. Here, we clarify the structural requirements of the pathway effector Gli1 for binding to DNA and identify the natural isoflavone Glabrescione B (GlaB) as the first small molecule binding to Gli1 zinc-finger and impairing Gli1 activity by interfering with its interaction with DNA. Remarkably, as a consequence of its robust inhibitory effect on Gli1 activity, GlaB inhibited the growth of Hedgehog-dependent tumor cells such as medulloblastoma (MB) and basal cell carcinoma (BCC) both in vitro and in orthotopic xenograft mice and in allograft mice models, as well as the self-renewal ability and clonogenicity of tumor-derived stem cells. Moreover, since extraction methods developed allow to get very limited amounts of pure isoflavone, we provided the total synthesis of Glabrescione B which foresees just three steps route with a overall yield 15%. This synthetic strategy allowed us the preparation of five derivatives with the aim to elucidate the structure-activity relationships and to clarify the molecular mechanism behind the Hedgehog signalling modulation.Part B. Drug Delivery Strategies Nanoparticles are submicrometer-sized carriers designed to improve the biodistribution of systemically administered (chemo)therapeutic agents. By delivering pharmacologically active agents more effectively and more selectively to the target site, nanocarriers aim to improve the balance between the efficacy and the toxicity of systemic (chemo)therapeutic administrations. Indeed, nanomaterials with an intrinsic ability to be used for imaging purposes, such as iron oxide–based magnetic nanoparticles (MNPs), are increasingly being loaded with drugs or alone for combining disease diagnosis and therapy. In this study, non-ionic surfactant vesicles (niosomes) loaded with lipophilic and hydrophilic MNPs have been prepared. Vesicles have been characterized in terms of dimensions, ζ-potential, time stability, bilayer characteristics and overall iron content. The quantification of the effective diameter of the MNPs entrapped in some niosomes was deduced from magnetic force microscopy. The encouraging obtained results proved that such vesicles could be promising carriers for the delivery of hydrophilic and lipophilic MNPs, thereby prompting various opportunities for the development of suitable strategies of both diagnosis and therapy (“theranostics”). In part A it has been showed that GlaB is a drug able to inhibit Hh-dependent tumors. However, the major drawbacks in its clinical translation for cancer therapy are its poor water solubility, poor pharmacokinetics and limited bioavailability at the tumor site. In order to, on the one hand, enhance the bioavailability of GlaB and, on the other one, to target selectively the MB cancer cells, different niosomal formulations have been designed that could pass through the blood-brain barrier. The novelty of the performed study lies in the use of two different polisorbates (namely Tween 20 and Tween 80) as surfactants in the formulation. They have been used in vesicles preparation, to act as an anchor for apolipoprotein E (apo E) from blood plasma. The particles seem to mimic LDL and interact with the LDL receptor leading to their uptake by the endothelial cells. In an attempt to formulate niosomal vesicles of smallest size and narrow polydispersity index, different ratio of Tween 20 and Tween 80 were studied in combination with two different purification techniques. With the purpose of delivering high doses of the natural compound GlaB, to solid tumors after systemic administration, for therapeutic applications in vivo, long-circulating GlaB loaded oil- cored polymeric nanocapsules (NC-GlaB) were designed and formulated. Furthermore, we showed that NC-GlaB is effective at inhibiting the proliferation of different Hedgehog-dependent cell lines in vitro. Notably, however, both GlaB and NC-GlaB are remarkably more effectively against pure cancer stem cells lines than cancer cells (composed by normal cancer cells and a subpopulation of cancer stem cells). These studies provide early evidence of a nanoparticle-encapsulated agent that might be useful for chemotherapy in patients affected by a Hedgehog-dependent tumor.