The goal of this escalating single-dose phase I research study was to determine the safety, tolerability, pharmacokinetics, pharmacodynamics as well as in vitro metabolism and plasma protein binding of MS-275, a novel histone deacetylase inhibitor, in patients with solid tumors and lymphomas. A validated LC/MS assay was developed to quantitate MS-275 in plasma, human liver microsomes and urine. The pharmacokinetic (PK) evaluation was done using a non-compartmental approach. In-vitro plasma protein binding profile of MS-275 was characterized by a validated micro-equilibrium dialysis method. In vitro phase I and phase II hepatic metabolism of MS-275 were evaluated using human liver microsomes. A correlative covariate analysis was performed in an effort to explain the wide inter-individual variability among patients.Results from the study demonstrate that the validated LC-MS assay is specific, accurate, precise and sensitive. MS-275 demonstrates a substantial inter-individual PK variability in systemic exposure and clearance; exposures increase in near-proportion, while peak concentrations increase more than-proportionally with an increase in dose. Mean apparent oral clearance (CL/F) is independent of dose and exhibits apparent dose-independent PK behavior over the studied dose range. Oral absorption is highly variable. MS-275 has a 50-fold longer half-life in humans compared to pre-clinical species. PK/PD analysis showed significant correlation between occurrence of DLT and higher systemic exposures. Although there was an increase in the acetylation of histone H3 and H4 over time, preliminary analysis showed no significant correlation between PK parameters and change in % histone acetylation after 24 hours. MS-275 is moderately bound to plasma proteins. Hepatic phase I and II metabolic pathways are only minor routes of elimination, and MS-275 is neither a substrate for liver-specific organic anion transporting proteins, OATP1B1 and OATP1B3, nor a substrate for gastrointestinal efflux transporters ABCB1 (P-gp) or ABCG2. No significant correlation was found between CL/F and demographic, body measures and other clinical covariates, and inter-patient variability in CL/F remained similar in magnitude even after correcting dose for body surface area (BSA) or other body measures. BSA is not a significant predictor of MS-275 PK, and flat-fixed dosing can be used in the future.

Protooncogene Crk was found to be upregulated in tumours with aggressive and invasive potential. The adaptor protein Crk has an important role in cell signaling: it integrates signals from activated integrins and growth factors receptors via its SH2 domain and transmits the signal to its SH3 domain binding partners that activate the small GTPases Rac1, Rap1 and Ras. This leads to regulation of cell migration, proliferation and survival. The aim of this thesis project was to inhibit the Crk dependent signaling by a competitive inhibition of the Crk SH3 domain, using a high affinity CrkSH3 binding peptoid. Binding of the inhibitor to the Crk SH3 domain prevents binding of cellular Crk SH3 interaction partners and the corresponding signal transmission is impaired. In this thesis project the effect of the Crk SH3 inhibition on the invasiveness of cancer cells was analyzed. The observed inhibitory effect on cell invasion as well as on anchorage independent growth provides a proof of therapeutical relevance of targeting CrkSH3N domain by peptoide-based inhibitors. Powered by TCPDF (www.tcpdf.org)

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Cancers represent a group of unprecedented heterogeneous diseases and currently available anti-cancer therapies provide highly variable efficacy with unsatisfactory cure rates. A wide range of proteomic technologies are being used in quest for newer approaches which could significantly contribute to the discovery and development of selective and specific cancer biomarkers for monitoring the disease state and anti-cancer therapy success. Taking into consideration the above aspects, this research was undertaken to study cancer cell proteomes and their changes after anti-cancer treatment with specific focus on: (a) response to conventional anthracycline/anthracenedione drugs with respect to their different clinical efficacy and (b) identification of novel targets for therapy in cancer cells resistant to biological drugs such as inhibitors of (b1) cyclin-dependent kinases and (b2) Aurora kinases. This study identified several interesting key aspects related to the effects of daunorubicin, doxorubicin and mitoxantrone. With the main focus on early time intervals when the influence of apoptosis is minimised, changes common for all three drugs belonging mainly to metabolic and cellular processes were observed. More importantly, significant changes in proteins involved in the generation of precursor...

This diploma thesis brings new data about design, synthesis, physico-chemical characterisation and biological efficacy of the novel star-like HPMA-based conjugates intended for treatment of solid tumors. Recently, many different water-soluble drug delivery systems based on N-(2- hydroxypropyl)methacrylamide (HPMA) copolymers have been described. Here, we report synthesis and physico-chemical characterisation of high molecular weight star-like HPMA- based polymer carriers with low polydispersity prepared by controlled grafting of HPMA copolymers onto PAMAM dendrimer core. With the aim to keep the polydispersity of drug delivery system as low as possible, reversible Addition-Fragmentation Chain Transfer (RAFT) polymerisation was used for HPMA-based polymer precursor preparation. The end groups of the polymer presursors was afterwards used for grafting using carbodidimide condensation reaction or copper free click chemistry on polyamidoamine (PAMAM) dendrimers resulting in a formation of star-like high-molecular-weight (HMW) drug carriers. Described synthetic procedure provided preparation of star-like HMW drug carriers with Mw between 1.105 - 3.105 g/mol and narrow distribution of Mw. The model drug, doxorubicin (Dox), was attached to the hydrazide group containing polymer cariers by pH- sensitive...

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Broad distribution and activity limit the utility of anti-cancer compounds by causing unacceptable systemic toxicity and narrow therapeutic indices. To improve tumour accumulation, drug-loaded macromolecular assemblies have been designed to replace conventional surfactant-based formulations. Their nanoscale size enhances tumour accumulation via hyperpermeable vasculature and reduced lymphatic drainage. Incorporating targeting ligands introduces cell specificity through receptor-specific binding and uptake, enabling drugs to reach intracellular targets. In this work, the targeting properties of polymer nanoparticle micelles of poly(2-methyl-2-carboxytrimethylene carbonate-co-D,L-lactide)-graft-poly(ethylene glycol)-furan (poly(TMCC-co-LA)-g-PEG) were verified using in vitro and in vivo models of breast cancer. To select a relevant mouse model, the vascular and lymphovascular properties of two tumour xenograft models were compared. Greater accumulation of a model nanocarrier was observed in orthotopic mammary fat pad (MFP) tumours than size matched ectopic subcutaneous tumours, suggesting that the organ environment influenced the underlying pathophysiology. Immunostaining revealed greater vascular thickness, density and size, and thinner basement membranes in MFP tumours, likely contributing to greater blood perfusion and vascular permeability. Based on these observations, MFP tumour-bearing mice were used to characterize the pharmacokinetics and biodistribution of a taxol drug, docetaxel, encapsulated in poly(TMCC-co-LA)-g-PEG nanoparticles. The nanoparticle formulation demonstrated longer docetaxel circulation in plasma compared to the conventional surfactant-based formulation. As a result, greater docetaxel retention was uniquely measured in tumour tissue, extending exposure of tumour cells to the active compound and suggesting potential for increased anti-cancer efficacy. Furthermore, active targeting of antibody-modified nanoparticles to live cells was shown to be selective and receptor-specific. Binding isotherms were used to quantify the impact of antibody density on binding strength. The equilibrium binding constant increased linearly with the average number of antibodies per particle, which is consistent with a single antibody-antigen interaction per particle. This mechanistic understanding enables binding behaviour to be adjusted in a predictive manner and guides rational nanoparticle design. These studies validate poly(TMCC-co-LA)-g-PEG nanoparticles as a platform for targeted delivery to cancer on both a tissue and cellular level, forming a compelling justification for further pre-clinical evaluation of this system for safety and efficacy in vivo.

Broad distribution and activity limit the utility of anti-cancer compounds by causing unacceptable systemic toxicity and narrow therapeutic indices. To improve tumour accumulation, drug-loaded macromolecular assemblies have been designed to replace conventional surfactant-based formulations. Their nanoscale size enhances tumour accumulation via hyperpermeable vasculature and reduced lymphatic drainage. Incorporating targeting ligands introduces cell specificity through receptor-specific binding and uptake, enabling drugs to reach intracellular targets. In this work, the targeting properties of polymer nanoparticle micelles of poly(2-methyl-2-carboxytrimethylene carbonate-co-D,L-lactide)-graft-poly(ethylene glycol)-furan (poly(TMCC-co-LA)-g-PEG) were verified using in vitro and in vivo models of breast cancer. To select a relevant mouse model, the vascular and lymphovascular properties of two tumour xenograft models were compared. Greater accumulation of a model nanocarrier was observed in orthotopic mammary fat pad (MFP) tumours than size matched ectopic subcutaneous tumours, suggesting that the organ environment influenced the underlying pathophysiology. Immunostaining revealed greater vascular thickness, density and size, and thinner basement membranes in MFP tumours, likely contributing to greater blood perfusion and vascular permeability. Based on these observations, MFP tumour-bearing mice were used to characterize the pharmacokinetics and biodistribution of a taxol drug, docetaxel, encapsulated in poly(TMCC-co-LA)-g-PEG nanoparticles. The nanoparticle formulation demonstrated longer docetaxel circulation in plasma compared to the conventional surfactant-based formulation. As a result, greater docetaxel retention was uniquely measured in tumour tissue, extending exposure of tumour cells to the active compound and suggesting potential for increased anti-cancer efficacy. Furthermore, active targeting of antibody-modified nanoparticles to live cells was shown to be selective and receptor-specific. Binding isotherms were used to quantify the impact of antibody density on binding strength. The equilibrium binding constant increased linearly with the average number of antibodies per particle, which is consistent with a single antibody-antigen interaction per particle. This mechanistic understanding enables binding behaviour to be adjusted in a predictive manner and guides rational nanoparticle design. These studies validate poly(TMCC-co-LA)-g-PEG nanoparticles as a platform for targeted delivery to cancer on both a tissue and cellular level, forming a compelling justification for further pre-clinical evaluation of this system for safety and efficacy in vivo.

La recherche contre le cancer est aujourd’hui tournée vers les thérapies ciblées. Dans ce contexte, la nucléoline et la nucléophosmine sont fortement impliquées dans la croissance tumorale et l’angiogenèse associée et surexprimées dans les cellules tumorales et endothéliales activées. Elles apparaissent donc comme des cibles de choix. Le pseudopeptide HB-19 lie la nucléoline de surface, inhibe la croissance cellulaire de nombreuses lignées de cellules tumorales et induit la mort de ces cellules tumorales par apoptose. D’autre part, il inhibe, in vitro et in vivo, plusieurs étapes de l’angiogenèse tumorale. Ces deux activités mènent, in vivo, à l’inhibition de la croissance tumorale dans de nombreux modèles de croissance tumorale chez la souris. Dans le but d’améliorer les activités observées avec HB- 19, des pseudopeptides dérivés de ce dernier ont été synthétisés. Ainsi le NucAnt 6L (N6L) montre une activité 5 à 10 fois supérieure à celle de HB-19 selon les modèles. Son activité anti-métastatique a également été démontrée. L’étude du mécanisme d’action des pseudopeptides a permis d’identifier deux nouveaux récepteurs: les héparanes sulfates et la nucléophosmine. L’importance du TIMP-3 dans son activité anti-métastatique a également été soulignée. Enfin, aucune toxicité n’a été observée chez les souris aux doses employées et la synthèse de N6L peut être effectuée à l’échelle industrielle. N6L apparaît donc comme un composé prometteur pour une thérapie anti-cancéreuse / The cancer research is nowadays interested in targeting therapies. In this context, nucleolin and nucleophosmin are proteins highly involved in tumor growth and angiogenesis and over-expressed in activated endothelial and tumor cells. So, they appear as very promising targets. The pseudopeptide HB-19 binds to cell surface-expressed nucleolin, inhibits different tumor cell growth and induces cell death by apoptosis. Furthermore, it inhibits, in vitro and in vivo, several steps of angiogenesis. These two activities lead, in vivo, to the suppression of tumor growth and angiogenesis in several mice models. In order to improve the activities observed with HB-19, new compounds derived from HB-19 were synthesized. So, NucAnt 6L (N6L) show 5 to 10 fold stronger anti-tumoral activity than HB- 19 depending of the model. Study of their action mechanism allowed us to identify two new receptors: nucleophosmin and heparan sulfates. The importance of TIMP-3 in anti-metastatic activity has also been highlighted. Finally, no toxicity has been observed in mice treated with N6L which can easily industrially be synthesized. N6L appears to be a promising compound for anti-cancer therapies

TRAIL (TNF-related apoptosis inducing ligand) est une protéine anti-tumorale capable de se lier spécifiquement aux récepteurs agonistes de mort (TRAIL-Rl ou DR4 et TRAIL-R2 ou DR5) des cellules cancéreuses et d'induire leur apoptose sans être toxique pour les cellules saines. Grâce à leurs propriétés exceptionnelles, notamment leur biocompatibilité, les nanotubes de carbone et surtout les SWCNTSs sont utilisés dans un large éventail d'applications et sont considérés très prometteurs pour révolutionner la thérapie anticancéreuse en nanomédecine. Les SWCNTSs sont connus par leur diffusion rapide dans un milieu aqueux tel que le sang, ouvrant la voie de développement de nouveaux nanovecteurs de médicaments. L'objectif principal de nos travaux de thèse a consisté à fonctionnaliser TRAIL sur des SWCNTSs pour mimer sa fonction membranaire en induisant une forte agrégation des récepteurs et déclencher l'apoptose (mort cellulaire programmée). Dans un premier temps, la fonctionnalisation des SWCNTSs avec TRAIL a été réalisée: adsorption non covalente des molécules de PSE sur les nanotubes via 1t-1t stacking, puis greffage du TRAIL au complexe SWCNTS-PSE pour former le nanovecteur (nommé NPT). Ensuite, nous avons caractérisé notre NPT par différentes méthodes (RAMAN, XPS, IR, MET, STEM ... ) afin d'estimer le taux de greffage du TRAIL sur le NPT, qui était environ de 80%. Dans un deuxième temps, nous avons étudié les paramètres thermodynamiques tels que le pH et la température du NPT en comparaison avec TRAIL seul par une approche chromatographique d'affinité (CHLP). Les résultats obtenus montrent une meilleure affinité du nanovecteur par rapport à TRAIL seul avec le récepteur TRAIL-R2 immobilisé sur la colonne chromatographique. En outre, des calculs de docking ont montré également que le complexe NPT couplé aux homotrimères de TRAIL est le plus stable une fois docké au récepteur TRAIL-R2. Ainsi, nous avons montré que les interactions de type Van der Waals et des liaisons hydrogène régissent l'association NPT-DR5 pour un pH supérieur à 7,4 (comme pour TRAIL seul). Enfin, notre nanovecteur s'est avéré plus efficace que TRAIL seul dans des différents tests menés in vitro sur des plusieurs types de lignées tumorales. Le NPT a permis une augmentation du potentiel pro­apoptotique de TRAIL avec un gain de fonction apoptotique estimé entre 10-20 fois par rapport à celui obtenu avec TRAIL seul. Dans ce travail, nous fournissons ainsi une preuve de concept que les nanovecteurs basés sur la fonctionnalisation du TRAIL avec les SWCNTSs peuvent être utiles pour les futurs traitements anti-cancéreux en nanomédecine. / TRAIL (TNF-related apoptosis inducing ligand) is a protein involved in immune anti-tumor surveillance. This cytokine is able to bound specifically to agonist death receptors (TRAIL-Rl or DR4 and TRAIL-R2 or DR5) of cancer cells, inducing apoptosis without being taxie to healthy cells. Thanks to their exceptional properties such as biocompatibility, carbon nanotubes and especially single-walled carbon nanotubes (SWCNTSs) are used in a wide range of applications and are considered to be very promising for cancer therapy in nanomedicine. The SWCNTSs are known to rapidly diffuse in aqueous media such as blood, opening the way for the development of new drug nanovectors or nanocarriers. The main purpose of this work is to functionalize SWCNTSs with TRAIL to mimic the membrane function of TRAIL by inducing a strong aggregation of death receptors and then induce apoptosis. First of all, the choice of SWCNTS functionalization with TRAIL was considered the first key in this thesis: non-covalent adsorption of PSE molecules on the nanotubes via 1t-1t stacking and TRAIL was next attached to a SWCNTS-PSE to form our nanovector, called NPT. Then, the NPT was characterized by various methods (Raman, XPS, IR, TEM, STEM, ... ) in order to estimate the grafted degree of TRAIL on the NPT surface (about 80%). Secondly, we investigated the ef:fects of the thermodynamic parameters such as pH and temperature on NPT versus TRAIL by a chromatographie approach (HPLC). The results showed a better affinity for NPT compared to TRAIL alone with the TRAIL-R2 receptor immobilized on the chromatographie colurnn. In addition, docking calculations have also shown that the NPT complex coupled to TRAIL homotrimers is the most stable when docked to DR5. Thus, we have demonstrated that Van der Waals interactions and hydrogen bonds govem the NPT-DR5 association for pH > 7.4 (as for TRAIL). Finally, our TRAIL-based SWCNTSs nanovectors (NPT) proved to be more efficient than TRAIL alone towards death receptors in triggering cancer cell killing in vitro. These NPTs increased the pro-apoptotic potential of TRAIL by nearly 10 to 20-fold in different Human tumor cell lines tested including colorectal, non-small cell lung cancer, or hepatocarcinomas. We provide in this work a proof of concept that nanovectors based on SWCNTS functionalization with TRAIL may be useful for future cancer treatments in nanomedicine.