Marcella Barbarino

Novel camptothecin analogue (gimatecan)-containing liposomes prepared by the ethanol injection method.

Small‐sized liposomes have several advantages as drug delivery systems, and the ethanol injection method is a suitable technique to obtain the spontaneous formation of liposomes having a small average radius. In this paper, we show that liposomal drug formulations can be prepared in situ, by simply injecting a drug‐containing lipid(s) organic solution into an aqueous solution. Several parameters should be optimized in order to obtain a final suitable formulation, and this paper is devoted to such an investigation. Firstly, we study the liposome size distributions determined by dynamic light scattering (DLS), as function of the lipid concentration and composition, as well as the organic and aqueous phases content. This was carried out, firstly, by focusing on POPC (1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphocholine) then on the novel L‐carnitine derivative PUCE (palmitoyl‐(R)‐carnitine undecyl ester chloride), showing that it is possible to obtain monomodal size distributions of rather small vesicles. In particular, depending on the conditions, it was possible to achieve a population of liposomes with a mean size of 100 nm, when a 50 mM POPC ethanol solution was injected in pure water; in the case of 50 mM PUCE the mean size was around 30 nm, when injected in saline (0.9% NaCl). The novel anticancer drug Gimatecan, a camptothecin derivative, was used as an example of lipophilic drug loading by the injection method. Conditions could be found, under which the resultant liposome size distributions were not affected by the presence of Gimatecan, in the case of POPC as well as in the case of PUCE. To increase the overall camptothecin concentration in the final liposomal dispersion, the novel technique of “multiple injection method” was used, and up to a final 5 times larger amount of liposomal drug could be reached by maintaining approximately the same size distribution. Once prepared, the physical and chemical stability of the liposome formulations was satisfactory within 24, as judged by DLS analysis and HPLC quantitation of lipids and drug. The Gimatecan‐containing liposomes formulations were also tested for in vitro and in vivo activity, against the human nonsmall cell lung carcinoma NCI‐H460 and a murine Lewis lung carcinoma 3 LL cell lines. In the in vitro tests, we did not observe any improvement or reduction of the Gimatecan pharmacological effect by the liposomal delivery system. More interestingly, in the in vivo Lewis lung carcinoma model, the intravenously administration of liposomal Gimatecan formulation showed a mild but significant increase of Tumor Volume Inhibition with respect to the oral no‐liposomal formulation (92% vs. 86 %, respectively; p < 0.05). Finally, our study showed that the liposomal formulation was able to realize a delivery system of a water‐insoluble drug, providing a Gimatecan formulation for intravenous administration with a preserved antitumoral activity.

Design, synthesis, and evaluation of biphenyl-4-yl-acrylohydroxamic acid derivatives as histone deacetylase (HDAC) inhibitors

A series of hydroxamic acid-based histone deacetylase (HDAC) inhibitors were designed on the basis of a model of the HDAC2 binding site and synthesized. They are characterized by a cinnamic spacer, capped with a substituted phenyl group. Modifications of the spacer are also reported. In an in vitro assay with the isoenzyme HDAC2, a good correlation of the activity with the docking energy was found. In human ovarian carcinoma IGROV-1 cells, selected compounds produced significant acetylation of p53 and alpha-tubulin. Most compounds showed an antiproliferative activity comparable to that of SAHA. At equitoxic concentrations, the tested compounds were more effective than SAHA in inducing apoptotic cell death. Compounds selected for in vivo evaluation exhibited a significant antitumor activity on three tumor models at well tolerated doses, thus suggesting a good therapeutic index.

Synthesis and Evaluation of New Hsp90 Inhibitors Based on a 1,4,5-Trisubstituted 1,2,3-Triazole Scaffold

Ruthenium catalyzed 1,3-cycloaddition (click chemistry) of an azido moiety installed on dihydroxycumene scaffold with differently substituted aryl propiolates gave a new family of 1,4,5-trisubstituted triazole carboxylic acid derivatives that showed high affinity toward Hsp90 associated with cell proliferation inhibition, both in nanomolar range. The 1,5 arrangement of the resorcinol, the aryl moieties, and the presence of an alkyl (secondary) amide in position 4 of the triazole ring were essential to get high activity. Docking simulations suggested that the triazoles penetrate the Hsp90 ATP binding site. Some 1,4,5-trisubstituted triazole carboxamides induced dramatic depletion of the examined client proteins and a very strong increase in the expression levels of the chaperone Hsp70. In vitro metabolic stability and in vivo preliminary studies on selected compounds have shown promising results comparable to the potent Hsp90 inhibitor NVP-AUY922. One of them, (compound 18, SST0287CL1) was selected for further investigation as the most promising drug candidate.

Novel 3,4-isoxazolediamides as potent inhibitors of chaperone heat shock protein 90.

A structural investigation on the isoxazole scaffold led to the discovery of 3,4-isoxazolediamide compounds endowed with potent Hsp90 inhibitory properties. We have found that compounds possessing a nitrogen atom directly attached to the C-4 heterocycle ring possess in vitro Hsp90 inhibitory properties at least comparable to those of the structurally related 4,5-diarylisoxazole derivatives. A group of compounds from this series of diamides combine potent binding affinity and cell growth inhibitory activity in both series of alkyl- and aryl- or heteroarylamides, with IC50 in the low nanomolar range. The 3,4-isoxazolediamides were also very effective in causing dramatic depletion of the examined client proteins and, as expected for the Hsp90 inhibitors, always induced a very strong increase in the expression levels of the chaperone Hsp70. In vivo studies against human epidermoid carcinoma A431 showed an antitumor effect of morpholine derivative 73 comparable to that induced by the reference compound 10.

Metabolic Approach to the Enhancement of Antitumor Effect of Chemotherapy: A Key Role of Acetyl-L-Carnitine

Purpose: Acetyl-l-carnitine (ALC) plays a relevant role in energy metabolism and stress response because of its function in the complex metabolic system regulating the acetyl-CoA levels that provide a source of acetyl groups for metabolic and acetylation-regulated processes. Because acetylation may influence p53 activity/stability and, therefore, the response to platinum compounds, this study was designed to investigate the effect of ALC in combination with platinum compounds. Experimental Design: The antiproliferative and antitumor activity studies were done in a panel of human tumor cell lines with functional or defective p53. The antimetastatic drug efficacy was investigated in the s.c. growing H460/M tumor subline, which is able to generate lung metastases. Results: ALC enhanced the sensitivity to cisplatin of tumor cells with functional p53. The sensitization by ALC was reflected in an improved in vivo antitumor efficacy of the combination over cisplatin alone in wild-type p53 lung tumors. ALC did not increase the cisplatin efficacy in the p53-mutant SW620 tumor. ALC exhibited a significant antimetastatic activity, and this effect was better exploited in combination with the histone deacetylase inhibitor, ST3595. The in vivo ALC/cisplatin combination caused the activation of p53, associated with protein acetylation and induction of target genes. Conclusions: ALC was effective in enhancing the antitumor potential of platinum compounds in wild-type p53 tumors. ALC, alone and in combination with a histone deacetylase inhibitor, exhibited an outstanding antimetastatic activity. Both effects, likely mediated by protein acetylation, may have implications for platinum-based therapy and combinations with histone deacetylase inhibitors. Clin Cancer Res; 16(15); 3944–53. ©2010 AACR.

ST7612AA1, a thioacetate-ω(γ-lactam carboxamide) derivative selected from a novel generation of oral HDAC inhibitors.

A systematic study of medicinal chemistry aimed at identifying a new generation of HDAC inhibitors, through the introduction of a thiol zinc-binding group (ZBG) and of an amide-lactam in the ω-position of the polyethylene chain of the vorinostat scaffold, allowed the selection of a new class of potent pan-HDAC inhibitors (pan-HDACis). Simple, highly versatile, and efficient synthetic approaches were used to synthesize a library of these new derivatives, which were then submitted to a screening for HDAC inhibition as well as to a preliminary in vitro assessment of their antiproliferative activity. Molecular docking into HDAC crystal structures suggested a binding mode for these thiol derivatives consistent with the stereoselectivity observed upon insertion of amide-lactam substituents in the ω-position. ST7612AA1 (117), selected as a drug candidate for further development, showed an in vitro activity in the nanomolar range associated with a remarkable in vivo antitumor activity, highly competitive with the most potent HDAC inhibitors, currently under clinical trials. A preliminary study of PK and metabolism is also illustrated.

Novel PARP-1 inhibitors based on a 2-propanoyl-3H-quinazolin-4-one scaffold.

Poly(ADP-ribose)polymerase-I (PARP-1) enzyme is involved in maintaining DNA integrity and programmed cell death. A virtual screening of commercial libraries led to the identification of five novel scaffolds with inhibitory profile in the low nanomolar range. A hit-to-lead optimization led to the identification of a group of new potent PARP-1 inhibitors, acyl-piperazinylamides of 3-(4-oxo-3,4-dihydro-quinazolin-2-yl)-propionic acid. Molecular modeling studies highlighted the preponderant role of the propanoyl side chain.

Efficient organic monoliths prepared by γ-radiation induced polymerization in the evaluation of histone deacetylase inhibitors by capillary(nano)-high performance liquid chromatography and ion trap mass spectrometry

New monolithic HPLC columns were prepared by γ-radiation-triggered polymerization of hexyl methacrylate and ethylene glycol dimethacrylate monomers in the presence of porogenic solvents. Polymerization was carried out directly within capillary (250-200 μm I.D.) and nano (100-75 μm I.D.) fused-silica tubes yielding highly efficient columns for cap(nano)-LC applications. The columns were applied in the complete separation of core (H2A, H2B, H3, and H4) and linker (H1) histones under gradient elution with UV and/or electrospray ionization (ESI) ion trap mass spectrometry (MS) detections. Large selectivity towards H1, H2A-1, H2A-2, H2B, H3-1, H3-2 and H4 histones and complete separation were obtained within 8 min time windows, using fast gradients and very high linear flow velocities, up to 11 mm/s for high throughput applications. The method developed was the basis of a simple and efficient protocol for the evaluation of post-translational modifications (PTMs) of histones from NCI-H460 human non-small-cell lung cancer (NSCLC) and HCT-116 human colorectal carcinoma cells. The study was extended to monitoring the level of histone acetylation after inhibition of Histone DeACetylase (HDAC) enzymes with suberoylanilide hydroxamic acid (SAHA), the first HDAC inhibitor approved by the FDA for cancer therapy. Attractive features of our cap(nano)-LC/MS approach are the short analysis time, the minute amount of sample required to complete the whole procedure and the stability of the polymethacrylate-based columns. A lab-made software package ClustMass was ad hoc developed and used to elaborate deconvoluted mass spectral data (aligning, averaging, clustering) and calculate the potency of HDAC inhibitors, expressed through a Relative half maximal Inhibitory Concentration parameter, namely R_IC(50) and an averaged acetylation degree.

Abstract 5556: Mice models of three human mesothelioma histotypes derived from primary patient tumors

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Mesothelioma, sometimes referred to as asbestos cancer, is known to be among the most aggressive and difficult to treat tumours. The disease attacks the mesothelium, a protective two-layered membrane that covers the internal organs of the body including the lungs, heart and abdominal organs. Mesothelioma can affect any of these layers, but is usually seen in the pleural (lung) or peritoneal (abdomen) mesothelium. The most commonly diagnosed form of this cancer is pleural mesothelioma, caused primarily by the inhalation of asbestos fibers. The prevalence of this cancer is estimated at less than 1% of all cancers, however its incidence is increasing, with an expected peak in the next 10-20 years. Mesotheliomas are tumours that have a poor prognosis due to limited treatment options. The most aggressive form is the sarcomatoid histotype, followed by the biphasic and the epithelioid histotypes.In order to setup in vivo models of human mesothelioma, translational related to human pathology in terms of histology, antigen expression and pharmacological response to therapy, we have established three mesothelioma in vivo models using primary cells derived from patients having different mesothelioma histotypes.We stably transfected MM-432 (sarcomatoid), MM-473 (epithelioid) and MM-487 (biphasic) mesothelioma cell lines with a luciferase expression vector. The selected high luc-espressing clones were inoculated intrapleurally in immunodeficient nude mice, and they successfully invaded and proliferated within the murine host. In particular, the epithelioid histotype presented tumour growth 100% of the time, after a short latency period. The biphasic histotype failed to present tumour growth in 30% of the cases, however was far more aggressive than the epithelial histotype upon establishment of tumour growth. Moreover, the ex-vivo Immunocytochemistry (i.e for CEA, EMA, Mesothelin, Podoplanin, calretinin) and biochemical characterization (i.e.EGFR, VEGF, Top1, TKs pathways) of the three histotypes, revealed histotype-related differences. Finally, the cytotoxic responses to a panel of antitumor drugs (i.e . Doxorubicin , Topotecan , Ciclofosfamide, Dacarbazina, Gemcitabine, Temozolomide, Bortezomib, 5-azacitidina, Paclitaxel, Etoposide, 5-FU, Cisplatin, and AZD-2281) revealed that the three original cell lines, compared to the corresponding luc-expressing clones, had similar sensitivities to drugs depending upon the tumor histotypes. Further, in vivo characterizations focused on chemotherapeutic responses of these models, are currently in progress. Citation Format: Claudio Pisano, Alison G. Cole, Marcella Barbarino, Erminia Bianchino, Mario Guglielmi, Carmela Melito, Grazia Mercadante, Alfonsina Porciello, Assunta Riccio, Ilaria La Porta, Sara Orecchia, Roberta Libener, Laura Mazzucco, Simonetta Andrea Licandro, Pasquale De Luca. Mice models of three human mesothelioma histotypes derived from primary patient tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5556. doi:10.1158/1538-7445.AM2014-5556

Abstract 1010: New thiol-ω(γ-lactam amide) SAHA analogues as potent histone deacetylase (HDAC) inhibitors..

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Histone deacetylase inhibitors (HDACi) are one of the last frontiers in pharmaceutical research. Two HDACi are already part of armamentarium of the anticancer drugs of the oncologists: Vorinostat, Zolinza® (SAHA) and Romidepsin, Istodax® (depsipeptide; FK-228), both licensed by the FDA for the treatment of cutaneous T-cell lymphoma (CTCL). Beyond them, more than twenty new drugs are currently under pre-clinical and clinical investigation as single agent and in combination therapies against different cancers and in other several novel therapeutic indications. Mainly they are hydroxamic acids and benzamide derivatives; however none has emerged superior to SAHA in terms of potency. In our search, we have selected a new generation of HDACi: thiol-based SAHA bearing a lactam amide in ω-position. This new generation was highly competitive compared to competing drugs, displaying sub-micromolar to low nanomolar inhibitor activity on HDACs, being especially powerful on HDAC6 isoform. They exhibited higher anti-proliferative activity than SAHA on different human cell lines. In vivo they were orally administered and showed a higher potency than SAHA, with a negligible toxicity. The overall profile of this new class of HDAC inhibitors, including synthesis and a comprehensive pharmacological characterization, will be presented. These encouraging results prompted us to select a drug candidate which is currently in a phase of pre-clinical evaluation. Citation Format: Giuseppe Giannini, Gianfranco Battistuzzi, Davide Vignola, Loredana Vesci, Ferdinando Maria Milazzo, Mario Berardino Guglielmi, Marcella Barbarino, Claudio Pisano, Walter Cabri. New thiol-ω(γ-lactam amide) SAHA analogues as potent histone deacetylase (HDAC) inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1010. doi:10.1158/1538-7445.AM2013-1010