Pre-clinical and clinical development of leukemia stem cell inhibitors in acute leukemias

In Leukemias, recent developments have demonstrated that the Hedgehog pathway plays a key-role in the peculiar ability of self renewal of leukemia stem cells. The aim of this research activity was to investigate, through a first in man, Phase I, open label, clinical trial, the role and the impact, mainly in terms of safety profile, adverse events and pharmacokinetics, of a Sonic Hedgehog inhibitor compound on a population of heavely pretreated patients affected by AML, CML, MF, or MDS, resistant or refractory to standard chemotherapy. Thirty-five patients have been enrolled. The drug was administered orally, in 28 days cycles, without rest periods. The compound showed a good safety profile. The half life was of 17-35 hours, justifying the daily administration. Significant signs of activity, in terms of reduction of bone marrow blast cell amount were seen in most of the patients enrolled. Interestingly, correlative biological studies demonstrated that, comparing the gene expression profyiling signature of separated CD34+ cells before and after one cycle of treatment, the most variably expressed genes were involved in the Hh pathway. Moreover, we observed that many genes involved in MDR (multidrug resistance)were significantly down regulated after treatment. These data might lead to future clinical trials based on combinatory approaches, including, for instance, Hh inhibitors and conventional chemotherapy.

Development of musculoskeletal models for the design and the pre-clinical validation of hip resurfacing prosthesis

Background. The surgical treatment of dysfunctional hips is a severe condition for the patient and a costly therapy for the public health. Hip resurfacing techniques seem to hold the promise of various advantages over traditional THR, with particular attention to young and active patients. Although the lesson provided in the past by many branches of engineering is that success in designing competitive products can be achieved only by predicting the possible scenario of failure, to date the understanding of the implant quality is poorly pre-clinically addressed. Thus revision is the only delayed and reliable end point for assessment. The aim of the present work was to model the musculoskeletal system so as to develop a protocol for predicting failure of hip resurfacing prosthesis. Methods. Preliminary studies validated the technique for the generation of subject specific finite element (FE) models of long bones from Computed Thomography data. The proposed protocol consisted in the numerical analysis of the prosthesis biomechanics by deterministic and statistic studies so as to assess the risk of biomechanical failure on the different operative conditions the implant might face in a population of interest during various activities of daily living. Physiological conditions were defined including the variability of the anatomy, bone densitometry, surgery uncertainties and published boundary conditions at the hip. The protocol was tested by analysing a successful design on the market and a new prototype of a resurfacing prosthesis. Results. The intrinsic accuracy of models on bone stress predictions (RMSE < 10%) was aligned to the current state of the art in this field. The accuracy of prediction on the bone-prosthesis contact mechanics was also excellent (< 0.001 mm). The sensitivity of models prediction to uncertainties on modelling parameter was found below 8.4%. The analysis of the successful design resulted in a very good agreement with published retrospective studies. The geometry optimisation of the new prototype lead to a final design with a low risk of failure. The statistical analysis confirmed the minimal risk of the optimised design over the entire population of interest. The performances of the optimised design showed a significant improvement with respect to the first prototype (+35%). Limitations. On the authors opinion the major limitation of this study is on boundary conditions. The muscular forces and the hip joint reaction were derived from the few data available in the literature, which can be considered significant but hardly representative of the entire variability of boundary conditions the implant might face over the patients population. This moved the focus of the research on modelling the musculoskeletal system; the ongoing activity is to develop subject-specific musculoskeletal models of the lower limb from medical images. Conclusions. The developed protocol was able to accurately predict known clinical outcomes when applied to a well-established device and, to support the design optimisation phase providing important information on critical characteristics of the patients when applied to a new prosthesis. The presented approach does have a relevant generality that would allow the extension of the protocol to a large set of orthopaedic scenarios with minor changes. Hence, a failure mode analysis criterion can be considered a suitable tool in developing new orthopaedic devices.

Conjugation of Toll Like Receptor 7 agonist through conjugation to immunogenic proteins. Synthesis, characterization, formulation and pre-clinical evaluation

The next generation of vaccine adjuvant are represented by a wide ranging set of molecules called Toll like agonists (TLR’s). Although many of these molecules are complex structures extracted from microorganisms, small molecule TLR agonists have also been identified. However, delivery systems have not been optimized to allow their effective delivery in conjunction with antigens. Here we describe a novel approach in which a small molecule TLR agonist has been conjugated directly to antigens to ensure effective co delivery. We describe the conjugation of a relevant protein, a recombinant protective antigen from S.pneumoniae (RrgB), which is linked to a TLR7 agonist. Following thorough characterization to ensure there was no aggregation, the conjugate was evaluated in a murine infection model. Results showed that the conjugate extended animals’ survival after lethal challenge with S.pneumoniae. Comparable results were obtained with a 10 fold lower dose than that of the native unconjugated antigen. Notably, the animals immunized with the same dose of unconjugated TLR7 agonist and antigen showed no adjuvant effect. The increased immunogenicity was likely a consequence of the co-localization of TLR7 agonist and antigen by chemical binding and is was more effective than simple co-administration. Likely, this approach can be adopted to reduce the dose of antigen required to induce protective immunity, and potentially increase the safety of a broad variety of vaccine candidates

Analisi preclinica di nuove strategie terapeutiche basate sull'inibizione di protein-chinasi nell'osteosarcoma.

Scopo: L’obiettivo del presente programma di studio è stato quello di identificare e validare nuovi possibili bersagli terapeutici per l’osteosarcoma (OS) partendo dall’analisi del chinoma umano. Risultati: L’analisi del profilo di espressione genica ottenuta su 21 campioni clinici di OS ad alto grado di malignità ha permesso di selezionare le seguenti chinasi di possibile rilevanza biologica per l’OS: AURK-A, AURK-B, CDK2, PIK3CA, PLK-1. Le chinasi selezionate sono state validate tramite RNA interference. Successivamente è stata valutata l’efficacia dei relativi inibitori specifici: VX-680 e ZM-447439 inibitori delle Aurora-chinasi, Roscovitina di CDK2 e NMS1 di PLK-1, già inclusi in studi clinici. In termini d’inibizione della crescita cellulare le linee sono risultate maggiomente sensibili ai farmaci VX-680 e NMS1. E’ stata osservata una minor sensibilità ai farmaci VX-680, ZM447439 e NMS1 nelle linee doxorubicina(DX)-resistenti (caratterizzate da elevati livelli di espressione di ABCB1), indicando questi farmaci come potenziali substrati di ABCB1. La Roscovitina, nonostante i valori di IC50 elevati, non sembrerebbe substrato di ABCB1. La validazione preclinica di VX-680 e ZM447439 è stata completata. La forte inibizione della crescita è causata da endoreduplicazione per mancata citodieresi con conseguente formazione di una popolazione iperploide e apoptosi. Inoltre, VX-680 inibisce la motilità e la capacità di formare colonie. Esperimenti di associazione farmacologica mostrano che VX-680 interagisce positivamente con tutti i chemioterapici convenzionali impiegati nel trattamento dell’OS. NMS-1 produce interazioni positive con la DX in linee cellulari DX-resistenti, probabilmente grazie all’effetto revertante esercitato su ABCB1. La Roscovitina produce interazioni positive con CDDP e DX nelle varianti resistenti, effetto probbilmente dovuto al ruolo di CDK2 nei meccanismi di riparo del DNA. Conclusioni: L’analisi in vitro dell’attività degli inibitori ha permesso di identificare VX-680 come nuovo farmaco di potenziale interesse clinico, soprattutto in virtù delle sue interazioni sinergiche con i chemioterapici di uso convenzionale nel trattamento dell’osteosarcoma.

Droga, alcol e lavoro: La prevenzione come promozione della salute

Lo scopo di questa dissertazione è quello di costruire un modello di promozione della salute nel contesto di lavoro in relazione al consumo di sostanze psicoattive fra lavoratori, attraverso il confronto tra la situazione italiana e inglese. L’ipotesi di fondo rimanda all’idea che i luoghi di lavoro possano rappresentare setting d’elezione per i progetti di prevenzione non solo perché alcuni studi dimostrano l’esistenza di fattori di rischio connessi alla mansione rispetto alle condotte relative allo stile di vita, ma anche perché il consumo di alcol e droghe è altamente diffuso tra i lavoratori e questo comporta rischi per la sicurezza e la salute personale nonché quella dei colleghi di lavoro. Si tratta quindi di indagare il rapporto tra contesto lavorativo e utilizzo di sostanze al fine di suggerire  alla luce degli studi internazionali in materia e delle riflessioni condotte dai soggetti coinvolti nella ricerca che si andrà a presentare  linee guida e indicazioni operative per la realizzazione di interventi di promozione alla salute nei contesti professionali. A tal fine, saranno analizzati gli esiti di 13 focus group che hanno coinvolto esperti italiani e 6 interviste somministrate a esperti inglesi volti a definire la situazione attuale in Italia e Gran Bretagna in materia di prevenzione del consumo di alcol e droghe nei luoghi di lavoro. In particolare, l’analisi verterà sulle seguenti aree: - Percezione circa la diffusione dei consumi nei luoghi di lavoro - Presentazione delle politiche adottate, in logica comparativa, tra i due paesi. - Analisi critica degli interventi e problematiche aperte. L’analisi del materiale empirico permette di delineare due modelli costruiti sulla base dei focus group e delle interviste: - in Italia si può affermare che prevalga il cd. modello della sicurezza: di recente trasformazione, questo sistema enfatizza la dimensione del controllo, tanto che si parla di sorveglianza sanitaria. É orientato alla sicurezza concepita quale rimozione dei fattori di rischio. Il consumo di sostanze (anche sporadico) è inteso quale espressione di una patologia che richiede l’intervento sanitario secondo modalità previste dal quadro normativo: una procedura che annulla la discrezionalità sia del datore di lavoro sia del medico competente. Si connota inoltre per contraddizioni interne e trasversali rispetto alle categorie lavorative (i controlli non si applicano alle professioni associate a maggiore prestigio sociale sebbene palesemente associate a rischio, come per esempio i medici) e alle sostanze (atteggiamento repressivo soprattutto verso le droghe illegali); - in Gran Bretagna, invece, il modello si configura come responsabilità bilaterale: secondo questo modello, se è vero che il datore di lavoro può decidere in merito all’attuazione di misure preventive in materia di alcol e droghe nei luoghi di lavoro, egli è ritenuto responsabile della mancata vigilanza. D’altro canto, il lavoratore che non rispetta quanto previsto nella politica scritta può essere soggetto a licenziamento per motivi disciplinari. Questo modello, particolarmente attento al consumo di tutte le sostanze psicoattive (legali e illegali), considera il consumo quale esito di una libera scelta individuale attraverso la quale il lavoratore decide di consumare alcol e droghe così come decide di dedicarsi ad altre condotte a rischio. Si propone di ri-orientare le strategie analizzate nei due paesi europei presi in esame attraverso la realizzazione di un modello della promozione della salute fondato su alcuni punti chiave: – coinvolgimento di tutti i lavoratori (e non solo coloro che svolgono mansioni a rischio per la sicurezza) al fine di promuovere benessere secondo un approccio olistico di salute, orientato ad intervenire non soltanto in materia di consumo di sostanze psicoattive (legali e illegali), ma più in generale sulle condotte a rischio; – compartecipazione nelle diverse fasi (programmazione, realizzazione e valutazione del progetto) del lavoratore, datore di lavoro e medico competente secondo una logica di flessibilità, responsabilizzazione condivisa fra i diversi attori, personalizzazione e co-gestione dell’intervento; – azione volta a promuovere i fattori di protezione agendo simultaneamente sul contrasto dei fattori di rischio (stress, alienazione, scarso riconoscimento del ruolo svolto), attraverso interventi che integrano diverse strategie operative alla luce delle evidenze scientifiche (Evidence-Based Prevention); – ricorso a strumenti di controllo (drug testing) subordinato all’esigenza di tutelare l’incolumità fisica del lavoratore e dei colleghi, da attuarsi sempre e comunque attraverso prassi che non violino la privacy e attraverso strumenti in grado di verificare l’effettivo stato di alterazione psico-fisica sul luogo di lavoro; – demedicalizzazione delle situazioni di consumo che non richiedono un intervento prettamente sanitario, ma che al contrario potrebbero essere affrontate attraverso azioni incentrate sul care anziché la cure; – messa a disposizione di servizi ad hoc con funzione di supporto, counselling, orientamento per i lavoratori, non stigmatizzanti e con operatori di formazione non solamente sanitaria, sull’esempio degli EAPs (Employee Assistence Programs) statunitensi. Si ritiene che questo modello possa trasformare i contesti di lavoro da agenzie di controllo orientate alla sicurezza a luoghi di intervento orientati al benessere attraverso un’azione sinergica e congiunta volta a promuovere i fattori di protezione a discapito di quelli di rischio in modo tale da intervenire non soltanto sul consumo di sostanze psicotrope, ma più in generale sullo stile di vita che influenza la salute complessiva.

Anticancer drug screening from images of zebrafish embryogenesis

During the previous 10 years, global R&D expenditure in the pharmaceuticals and biotechnology sector has steadily increased, without a corresponding increase in output of new medicines. To address this situation, the biopharmaceutical industry's greatest need is to predict the failures at the earliest possible stage of the drug development process. A major key to reducing failures in drug screenings is the development and use of preclinical models that are more predictive of efficacy and safety in clinical trials. Further, relevant animal models are needed to allow a wider testing of novel hypotheses. Key to this is the developing, refining, and validating of complex animal models that directly link therapeutic targets to the phenotype of disease, allowing earlier prediction of human response to medicines and identification of safety biomarkers. Morehover, well-designed animal studies are essential to bridge the gap between test in cell cultures and people. Zebrafish is emerging, complementary to other models, as a powerful system for cancer studies and drugs discovery. We aim to investigate this research area designing a new preclinical cancer model based on the in vivo imaging of zebrafish embryogenesis. Technological advances in imaging have made it feasible to acquire nondestructive in vivo images of fluorescently labeled structures, such as cell nuclei and membranes, throughout early Zebrafishsh embryogenesis. This In vivo image-based investigation provides measurements for a large number of features at cellular level and events including nuclei movements, cells counting, and mitosis detection, thereby enabling the estimation of more significant parameters such as proliferation rate, highly relevant for investigating anticancer drug effects. In this work, we designed a standardized procedure for accessing drug activity at the cellular level in live zebrafish embryos. The procedure includes methodologies and tools that combine imaging and fully automated measurements of embryonic cell proliferation rate. We achieved proliferation rate estimation through the automatic classification and density measurement of epithelial enveloping layer and deep layer cells. Automatic embryonic cells classification provides the bases to measure the variability of relevant parameters, such as cell density, in different classes of cells and is finalized to the estimation of efficacy and selectivity of anticancer drugs. Through these methodologies we were able to evaluate and to measure in vivo the therapeutic potential and overall toxicity of Dbait and Irinotecan anticancer molecules. Results achieved on these anticancer molecules are presented and discussed; furthermore, extensive accuracy measurements are provided to investigate the robustness of the proposed procedure. Altogether, these observations indicate that zebrafish embryo can be a useful and cost-effective alternative to some mammalian models for the preclinical test of anticancer drugs and it might also provides, in the near future, opportunities to accelerate the process of drug discovery.

Development of muliplexed analytical methods based on bioluminescent whole-cell biosensors

The subject of this Ph.D. research thesis is the development and application of multiplexed analytical methods based on bioluminescent whole-cell biosensors. One of the main goals of analytical chemistry is multianalyte testing in which two or more analytes are measured simultaneously in a single assay. The advantages of multianalyte testing are work simplification, high throughput, and reduction in the overall cost per test. The availability of multiplexed portable analytical systems is of particular interest for on-field analysis of clinical, environmental or food samples as well as for the drug discovery process. To allow highly sensitive and selective analysis, these devices should combine biospecific molecular recognition with ultrasensitive detection systems. To address the current need for rapid, highly sensitive and inexpensive devices for obtaining more data from each sample,genetically engineered whole-cell biosensors as biospecific recognition element were combined with ultrasensitive bioluminescence detection techniques. Genetically engineered cell-based sensing systems were obtained by introducing into bacterial, yeast or mammalian cells a vector expressing a reporter protein whose expression is controlled by regulatory proteins and promoter sequences. The regulatory protein is able to recognize the presence of the analyte (e.g., compounds with hormone-like activity, heavy metals…) and to consequently activate the expression of the reporter protein that can be readily measured and directly related to the analyte bioavailable concentration in the sample. Bioluminescence represents the ideal detection principle for miniaturized analytical devices and multiplexed assays thanks to high detectability in small sample volumes allowing an accurate signal localization and quantification. In the first chapter of this dissertation is discussed the obtainment of improved bioluminescent proteins emitting at different wavelenghts, in term of increased thermostability, enhanced emission decay kinetic and spectral resolution. The second chapter is mainly focused on the use of these proteins in the development of whole-cell based assay with improved analytical performance. In particular since the main drawback of whole-cell biosensors is the high variability of their analyte specific response mainly caused by variations in cell viability due to aspecific effects of the sample’s matrix, an additional bioluminescent reporter has been introduced to correct the analytical response thus increasing the robustness of the bioassays. The feasibility of using a combination of two or more bioluminescent proteins for obtaining biosensors with internal signal correction or for the simultaneous detection of multiple analytes has been demonstrated by developing a dual reporter yeast based biosensor for androgenic activity measurement and a triple reporter mammalian cell-based biosensor for the simultaneous monitoring of two CYP450 enzymes activation, involved in cholesterol degradation, with the use of two spectrally resolved intracellular luciferases and a secreted luciferase as a control for cells viability. In the third chapter is presented the development of a portable multianalyte detection system. In order to develop a portable system that can be used also outside the laboratory environment even by non skilled personnel, cells have been immobilized into a new biocompatible and transparent polymeric matrix within a modified clear bottom black 384 -well microtiter plate to obtain a bioluminescent cell array. The cell array was placed in contact with a portable charge-coupled device (CCD) light sensor able to localize and quantify the luminescent signal produced by different bioluminescent whole-cell biosensors. This multiplexed biosensing platform containing whole-cell biosensors was successfully used to measure the overall toxicity of a given sample as well as to obtain dose response curves for heavy metals and to detect hormonal activity in clinical samples (PCT/IB2010/050625: “Portable device based on immobilized cells for the detection of analytes.” Michelini E, Roda A, Dolci LS, Mezzanotte L, Cevenini L , 2010). At the end of the dissertation some future development steps are also discussed in order to develop a point of care (POCT) device that combine portability, minimum sample pre-treatment and highly sensitive multiplexed assays in a short assay time. In this POCT perspective, field-flow fractionation (FFF) techniques, in particular gravitational variant (GrFFF) that exploit the earth gravitational field to structure the separation, have been investigated for cells fractionation, characterization and isolation. Thanks to the simplicity of its equipment, amenable to miniaturization, the GrFFF techniques appears to be particularly suited for its implementation in POCT devices and may be used as pre-analytical integrated module to be applied directly to drive target analytes of raw samples to the modules where biospecifc recognition reactions based on ultrasensitive bioluminescence detection occurs, providing an increase in overall analytical output.