Improvement of pyrazolo[3,4-d]pyrimidines pharmacokinetic properties: Nanosystem approaches for drug delivery

Pyrazolo[3,4-d]pyrimidines are a class of compounds with a good activity against several cancer cell lines. Despite the promising anticancer activity, these molecules showed a poor aqueous solubility. This issue could threat the future development of pyrazolo[3,4-d]pyrimidines as clinical drug candidates. With the aim of improving their solubility profile and consequently their pharmacokinetic properties, we have chosen four compounds (1-4) on the base of their anti-neuroblastoma activity and we have developed albumin nanoparticles and liposomes for the selected candidates. Albumin nanoparticles and liposomes were prepared and characterized regarding size and ? -potential distribution, polidispersity index, entrapment efficiency and activity against SH-SY5Y human neuroblastoma cell line. The most promising nanosystem, namely LP-2, was chosen to perform further studies: confocal microscopy, stability and drug release in physiological conditions, and biodistribution. Altogether, the obtained data strongly indicate that the encapsulation of pyrazolo[3,4-d]pyrimidines in liposomes represent an effective method to overcome the poor water solubility

Development of liposome-based drug delivery system to improve drug-like properties and anticancer activity of tyrosine kinase inhibitors with pyrazolo [3,4-d] pyrimidine structure.

Cancer is a multifactorial disease whose causes are still unknown. Frequently, mutations in several genes play an important role in cellular growth processes and, based on their specific roles, they are usually distinguished in proto-oncogenes and oncosuppressors. One of the most studied oncogenes is c-Src, encoding for a non-receptor tyrosine kinase protein that plays a multitude of roles in cell signalling. The activation of c-Src is involved in the control of many functions, including cell adhesion, growth, movement, and differentiation by a different set of cell surface receptors. c-Src is found to be over- expressed and mutationally activated in a wide variety of human cancers. Moreover, c- Src may have an influence on the development of the metastatic phenotype. Significant progresses in the understanding of cancer biology have prompted extensive research within novel classes of anticancer drugs. A number of tyrosine kinase inhibitors targeting the c-Src tyrosine kinase (as well as related tyrosine kinases) have been developed for therapeutic use. 4-aminopyrazolo [3,4-d] pyrimidines compounds were firstly used as c-Src family kinases (SFKs) inhibitors in 1996 but later demonstrated to inhibit all SFKs members with IC50 values in the low nanomolar range. These compounds have been extensively used for studying the biological pathways of SFKs and, even though they are a class of promising anti-tumor compounds because of their good activity against several cancer cell lines, these molecules showed a poor aqueous solubility, limiting them as clinical drug candidates. In the continuing effort to find new anticancer agents, our group conducted large studies on a series of new c-Src inhibitors with a pyrazolo [3,4-d] pyrimidine scaffold. The aim of this thesis was to improve the solubility profile and the pharmacokinetic properties of selected compounds, chosen on the base of their inhibitory activity on c- Src. To overcome the poor water solubility of these molecules, pyrazolo [3,4-d] pyrimidines compounds were encapsulated in liposomes formulations. Liposomes were prepared and characterized for size, ζ potential distribution and polydispersity index (PDI) by dynamic light scattering in order to obtain a homogeneous population of small unilamellar vesicles (SUV); subsequently, fluorescent liposomes were prepared to assess the ability of liposomes to interact with neuroblastoma (SH-SY5Y) and glioblastoma (U87) cells by confocal microscopy; finally, encapsulation efficiency and activity by cellular assays were studied to determine cytotoxicity, showing that liposomes loaded with pyrazolo [3,4-d] pyrimidine compounds determine a cytotoxic effect in SH-SY5Y (Neuroblastoma) or U87 (Glioblastoma) cell line equal to or higher than the free compound solubilised in DMSO. Moreover, biodistribution of liposomes loaded with pyrazolo [3,4-d] pyrimidine was evaluated in male Sprague Dawley rats after 24h of treatment. Altogether, the obtained data strongly indicate that the encapsulation of pyrazolo [3,4-d] pyrimidine compounds in liposomes represent an effective method to improve the biodistribution of pyrazolo [3,4-d] pyrimidines and attribute a therapeutic efficacy to this novel formulation. During my Ph.D. period, I also focused on the study of active targeting of liposomes against tumor areas that overexpress plasmin. The plasminogen/plasmin system plays a key role in tumor development and, in particular, in progression and metastasis. The aim of my project was to functionalize the surface of stealth liposomes encapsulating pyrazolo [3,4-d] pyrimidines with a peptide able to bind plasmin. Since plasmin is overexpressed in tumor areas, the bond between plasmin and the peptide in liposomes surface would lead to a destabilization of the liposomes and consequently to a greater drug release in the tumor site. The obtained liposomes were characterized after purification evaluating the size, the ζ potential distribution, the polydispersity index (PDI) and the encapsulation efficiency (EE). Furthermore, several tests to quantify the peptide bound to liposomes were performed, unfortunately with poor results. Moreover, peptide-bearing fluorescent liposomes were also prepared and tested on hepatocarcinoma cell line HepG2 to investigate any change in the cellular uptake. In addition, cytotoxicity assays on HepG2 cells were performed to test the efficacy of liposomes conjugated with the peptide with respect to non-conjugated liposomes, both encapsulating the selected compounds. Liposomes functionalized with the peptide resulted more stable and with better physicochemical properties than non-functionalized liposomes. Furthermore, confocal microscopy of fluorescent liposomes highlighted the ability of liposomes with the peptide to penetrate in hepatocarcinoma cells after 1h of treatment with respect to non-functionalized liposomes, not able to be internalized after the same time. Finally, cytotoxicity data showed that liposomes with the peptide have lower IC50 values than non-functionalized liposomes. In conclusion, data obtained showed that it was obtained a good functionalized liposomal formulation able to be recognized by hepatocarcinoma cells secreting plasmin. Further in vivo tests will be performed to study the ability of these liposomes to release the compound in tumor areas over-expressing plasmin

Molecular insights to the bioactive form of BV02, a reference inhibitor of 14-3-3σ protein-protein interactions

BV02 is a reference inhibitor of 14-3-3 protein-protein interactions, which is currently used as chemical biology tool to understand the role of 14-3-3 proteins in pathological contexts. Due to chemical instability in certain conditions, its bioactive form has remained unclear. Here, we use NMR spectroscopy to prove for the first time the direct interaction between the molecule and 14-3-3σ, and to depict its bioactive form, namely the phthalimide derivative 9. Our work provides molecular insights to the bioactive form of the 14-3-3 PPI inhibitor and facilitates further development as candidate therapeutic agent

Plasmin-Binding Tripeptide-Decorated Liposomes Loading Pyrazolo[3,4- d]pyrimidines for Targeting Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the most fatal cancer types worldwide. HCC cells were proved to overexpress c-Src and Sgk1, a tyrosine and a serine-threonine kinase, respectively, whose role is crucial for the development and progression of the tumor. Pyrazolo[3,4-d]pyrimidine derivatives are a class of tyrosine kinase inhibitors that have shown good activity against HepG2. HCC cells were also proved to overexpress plasmin, which is localized on the cell surface bound to its receptors. In this study, a tripeptide with sequence d-Ala-Phe-Lys, which binds a specific reactive site of plasmin, was synthesized and characterized. This tripeptide was used to decorate liposomes encapsulating three selected pyrazolo[3,4-d]pyrimidines. Liposomes bearing tripeptide have been characterized, not showing remarkable differences with respect to the corresponding tripeptide-free liposomes. In vitro HepG2 cell uptake profiles and cytotoxicities showed that the presence of the tripeptide on the liposomal membrane surface improves the cell-penetrating ability of liposomes and increases the activity of two of the three tested compounds

The FHP01 DDX3X Helicase Inhibitor Exerts Potent Anti-Tumor Activity In Vivo in Breast Cancer Pre-Clinical Models

Inhibition of DDX3X expression or activity reduces proliferation in cells from various tumor tissues, in particular in breast cancer, and its expression often correlates to tumor aggressiveness. This makes DDX3X a prominent candidate for the design of drugs for novel personalized therapeutic strategies. Starting from an in silico drug discovery approach, a group of molecules has been selected by molecular docking at the RNA binding site of DDX3X. Here, the most promising among them, FHP01, was evaluated in breast cancer preclinical models. Specifically, FHP01 exhibited very effective antiproliferative and killing activity against different breast cancer cell types, among which those from triple-negative breast cancer (TNBC). Interestingly, FHP01 also inhibited WNT signaling, a key tumorigenic pathway already correlated to DDX3X functions in breast cancer model cell lines. Ultimately, FHP01 also caused a significant reduction, in vivo, in the growth of MDA MB 231-derived TNBC xenograft models. Importantly, FHP01 showed good bioavailability and no toxicity on normal peripheral blood mononuclear cells in vitro and on several mouse tissues in vivo. Overall, our data suggest that the use of FHP01 and its related compounds may represent a novel therapeutic approach with high potential against breast cancer, including the triple-negative subtype usually correlated to the most unfavorable outcomes because of the lack of available targeted therapies

One drug for two targets: Biological evaluation of antiretroviral agents endowed with antiproliferative activity

AIDS-related cancer diseases are malignancies with low incidence on healthy people that affect mostly subjects already immunocompromised. The connection between HIV/AIDS and these cancers has not been established yet, but a weakened immune system is certainly the main cause. We envisaged the possibility to screen a small library of compounds synthesized in our laboratory against opportunistic tumors mainly due to HIV infection like Burkitt's Lymphoma. From cellular assays and gene expression analysis we identified two promising compounds. These derivatives have the dual action required inhibiting HIV replication in human TZM-bl cells infected with HIV-1 NL4.3 and showing cytotoxic activity on human colon HT-29 and breast adenocarcinoma MCF-7 cells. In addition, preclinical in vitro adsorption, distribution, metabolism, and excretion studies highlighted a satisfactory pharmacokinetic profile

Plasmin-Binding Tripeptide-Decorated Liposomes Loading Pyrazolo[3,4‑<i>d</i>]pyrimidines for Targeting Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the most fatal cancer types worldwide. HCC cells were proved to overexpress c-Src and Sgk1, a tyrosine and a serine-threonine kinase, respectively, whose role is crucial for the development and progression of the tumor. Pyrazolo­[3,4-<i>d</i>]­pyrimidine derivatives are a class of tyrosine kinase inhibitors that have shown good activity against HepG2. HCC cells were also proved to overexpress plasmin, which is localized on the cell surface bound to its receptors. In this study, a tripeptide with sequence d-Ala-Phe-Lys, which binds a specific reactive site of plasmin, was synthesized and characterized. This tripeptide was used to decorate liposomes encapsulating three selected pyrazolo­[3,4-<i>d</i>]­pyrimidines. Liposomes bearing tripeptide have been characterized, not showing remarkable differences with respect to the corresponding tripeptide-free liposomes. <i>In vitro</i> HepG2 cell uptake profiles and cytotoxicities showed that the presence of the tripeptide on the liposomal membrane surface improves the cell-penetrating ability of liposomes and increases the activity of two of the three tested compounds

2-Aminothiazolones as anti-hiv agents that act as gp120-cd4 inhibitors

We report here the synthesis of 2-aminothiazolones along with their biological properties as novel anti-HIV agents. Such compounds have proven to act through the inhibition of the gp120-CD4 protein-protein interaction that occurs at the very early stage of the HIV-1 entry process. No cytotoxicity was found for these compounds, and broad antiviral activities against laboratory strains and pseudotyped viruses were documented. Docking simulations have also been applied to predict the mechanism, at the molecular level, by which the inhibitors were able to interact within the Phe43 cavity of HIV-1 gp120. Furthermore, a preliminary absorption, distribution, metabolism, and excretion (ADME) evaluation was performed. Overall, this study led the basis for the development of more potent HIV entry inhibitors

Aptamer Functionalization of Nanosystems for Glioblastoma Targeting through the Blood–Brain Barrier

Polymeric nanoparticles (PNPs) may efficiently deliver in vivo therapeutics to tumors when conjugated to specific targeting agents. Gint4.T aptamer specifically recognizes platelet-derived growth factor receptor β and can cross the blood–brain barrier (BBB). We synthesized Gint4.T-conjugated PNPs able of high uptake into U87MG glioblastoma (GBM) cells and with astonishing EC₅₀ value (38 pM) when loaded with a PI3K-mTOR inhibitor. We also demonstrated in vivo BBB passage and tumor accumulation in a GBM orthotopic model

Prodrugs of Pyrazolo[3,4-d]pyrimidines: From Library Synthesis to Evaluation as Potential Anticancer Agents in an Orthotopic Glioblastoma Model

Pyrazolo[3,4-d]pyrimidines are potent protein kinase inhibitors with promising antitumor activity but suboptimal aqueous solubility, consequently worth being further optimized. Herein, we present the one-pot two-step procedure for the synthesis of a set of pyrazolo[3,4-d]pyrimidine prodrugs (1a-8a and 9a-e) with higher aqueous solubility and enhanced pharmacokinetic and therapeutic properties. ADME studies demonstrated for the most promising prodrugs a better aqueous solubility, a favorable hydrolysis in human and murine serum, and an increased ability to cross cell membranes with respect to the parental drugs, explaining their better 24 h in vitro cytotoxicity against human glioblastoma U87 cell line. Finally, the 4-4a couple of drug/prodrug was also evaluated in vivo, revealing a profitable pharmacokinetic profile of the prodrug associated with a good efficacy. The application of the prodrug approach demonstrated to be a successful strategy for improving aqueous solubility of the parental drugs, determining a positive impact also in their biological efficacy