Dissertations / Theses on the topic 'New Drug Application'

A medicina clínica apresentou a partir dos anos 1960 uma certa inflexão em seus mecanismos internos de produzir conhecimento, assim como em sua forma de aplicar esses conhecimentos na prática. A principal diferença em relação ao passado foi que, a partir dessa época, os problemas de natureza clínica, como diagnóstico diferencial, terapêutica e estimativa de prognóstico, passaram a ser processados predominantemente por instrumentos de análise padronizados e, principalmente, submetidos ao escrutínio de uma razão empírico-matemática. A entidade paciente deixou de ser um ente emissor de sintomas e sinais que são processados por um médico, para ser uma nova entidade em que esses signos, previamente estabelecidos e validados pelos estudos clínicos e pela razão matemática, são encaixados pelos médicos nas manifestações dos pacientes. Na mesma época em que esse processo ocorreu o mundo ocidental vivia um período de crise que se caracterizava por um baixo crescimento econômico e por um questionamento por parte da sociedade dos princípios normativos, tanto éticos quanto morais, que regiam sua forma de viver. Nos Estados Unidos, a partir de 1962 passou a ser uma exigência legal que toda droga nova, antes de ser comercializada, deveria provar, através de testes científicos, que ela tinha eficácia terapêutica, que de fato funcionava na patologia que se propunha tratar. A metodologia para promover esse tipo de demonstração foi construída a partir dessa exigência. As regras de prova científica de eficácia foram construídas a partir dessa demanda legal, e são chamadas genericamente de Epidemiologia Clínica. Vários agentes participaram ativamente do processo de definição das regras do método científico que foram então, a partir dessa data, implementadas e sedimentadas. Destacam-se nesse debate, a comunidade acadêmica, a sociedade civil, os economistas, os advogados e juizes, os agentes do governo, e, finalmente, a indústria farmacêutica. Essa última assumiu uma posição de destaque, secretariando, e, de certa forma impondo uma agenda de discussão. O motivo por detrás dessa atitude foi uma profunda crise de legitimidade das regras de operação do negócio farmacêutico em dois de seus principais componentes: os desenvolvimentos tecnológicos, responsáveis pelas inovações na área terapêutica, e, a garantia de um mercado historicamente monopolístico, legitimada pela instituição secular da lei de patentes. Ambos esses institutos passaram, nas décadas de 1960 e 1970, por um conturbado processo de rediscussão de seus fundamentos. Defende-se que essa função de secretariar a discussão por parte da indústria farmacêutica teve um papel de destaque na construção das regras de cientificidade que passaram, desde então, a regular o ato médico. A implementação, aceitação e sedimentação como princípio normativo de prova de verdade dessa metodologia científica, sobretudo na comunidade médica, ocorreu de uma forma muito particular. Discute-se nesse trabalho a história dessa implementação sob duas perspectivas: a história oficial conforme descrita pelo Departamento de História do Food and Drug Administration (FDA, Agência Federal Norte-americana), e, alternativamente, através de uma análise dos discursos proferidos por personagens que direta e efetivamente participaram dessa discussão. Médicos, farmacologistas, advogados, legisladores, economistas, profissionais da indústria farmacêutica e agentes do governo emitiram e discutiram opiniões, e estas foram registradas e publicadas. Esse material compõe a matéria prima em cima da qual trabalha-se no sentido de compor uma história que acaba por ter alguns pontos diferenciais em relação à versão oficial. Em torno desses pontos procura-se produzir um discurso sobre a relação entre ciência, lei, economia, e, prática médica. A dimensão científica dessa história se mistura intensamente com outros aspectos igualmente importantes como: debates legislativos, interesses econômicos de segmentos privados, papel do Estado na regulação econômica, confiabilidade na neutralidade das publicações científicas, participação do mundo acadêmico no desenvolvimento tecnológico de um país, etc.. Defendese que a fusão desses discursos em um formato consensual construiu uma situação em que a referida dimensão técnico-científica passou a ter uma importância relativamente maior do que as outras como instrumento de legitimação da verdade em medicina na sociedade, e que esse fato teve algumas importantes conseqüências práticas, destacando-se: um processo progressivo de desumanização do atendimento médico e a produção de um sistema de barreiras que dificultam o exercício de uma critica eficiente e positiva por parte dos profissionais ligados à pratica médica. Por último, em torno do conceito de humanismo, abordado particularmente dentro da tradição filosófica ocidental, discute-se o quanto essa hipertrofia da dimensão técnica e suas conseqüências práticas, podem ser questionadas em seus fundamentos epistemológicos visando a reconstrução de uma prática médica mais humana e emancipadora.<br>Since 1960 Clinical Medicine suffered a kind of inflection in its internal mechanisms of producing theoretical knowledge, as well as in the way that this knowledge is applied in practical life. The most important difference in relation to the past was that problems of clinical nature like, differential diagnosis, therapeutical decisions, and prognosis estimation, started to be predominantly processed by standardized analytical instruments, and most important, they were always previously submitted to an empirical-mathematical reasoning. Individual patients were no longer a being that reported signs and symptoms that were processed by a physician, they started to be a new entity in which these signs, previously established and validated by clinical studies have to be necessarily engrafted by the physicians in patients manifestations. At the same time that this process occurred, western world was living an important critical period, characterized by a very slow economic growth, and by a reevaluation of its ethical and moral values. After 1962, in the United States, it became obligatory to prove, through empirical scientific evidence that a new drug was effective, before marketing and sales authorization was issued to a company who wanted to launch the drug. The scientific method designed to prove efficacy of a drug was actually developed after this legal demand. This method is called generically Clinical Epidemiology. Several actors participated in the discussion of the rules of this method. Medical Schools, government representatives, pharmaceutical industry can be cited; the latter played a very special role, since it acted as a secretary of the whole process. The reason behind this was that, at those times, the two major pillars of the pharmaceutical business, innovation capacity and patent law, were being severely criticized, and proposals for changing the way this things were being conducted in American society were about to become a reality. We defend the position that the attitude of the pharmaceutical business representatives, were crucial for the establishment of the scientific rules that were considered consensual, and that these rules, for many reasons, started to be the paradigm of medical reasoning and individual decision in medical problems. Implementation, acceptance, and maintenance of this new clinical scientific method that was born after the legal demand for prove of efficacy of a new drug, particularly in medical community, occurred in a very particular way. We discuss the history of this process under two separate perspectives: the official history, as described by History Department of the Food and Drug Administration, and alternatively, through an analysis of the speeches of persons who actually participated directly in this discussion. Physicians, pharmacologists, lawyers, legislators, economists, pharmaceutical industry representatives, government members and politicians, all these groups, emitted their opinions and these were registered and published. This is the row material that was used to composite a new story of the whole process, and the result of this work is somehow different from the official history reported before. The scientific dimension of this story is mixed up with other important aspects like: legislative debates, private economical interests, the role of the State in regulating the economy, academic participation in decisions related to economic growth of a country, etcWe try to prove that the intersection of all these interests in a consensual framework built up a situation in which the previously referred technical-scientific dimension started to have a relatively bigger importance in relation to the other aspects as an instrument to legitimate what is truth (or what is false) in clinical medicine, to the whole body of the society. This fact brought two important practical consequences: a progressive reduction in other human aspects of clinical medicine apart from technology, and, the development of a system of barriers that jeopardize the possibility of a critical attitude towards the scientific method from those who practice medicine. Around the concept of humanism, studied particularly inside western philosophical tradition, we discuss how much this so called hypertrophy of the technical-scientific dimension and its practical consequences can be scrutinized and questioned in its epistemological foundations in order to rebuild a new medicine more human and critical.

In recent years, a number of reports have focused on the use of polyesters in drug delivery due to their intrinsic biocompatibility and biodegradability. In this thesis, aliphatic polyesters were synthesized by polycondensation reaction and ring opening polymerization reactions. The properties of the polymers and drug delivery potential of the resultant materials were evaluated. In the polycondensation reactions, a series of aliphatic polyesters of similar molecular weight were synthesized by reacting 1,10-decanediol with different ratios of succinic acid/phenylsuccinic acid and the effects of phenyl group side-chain substitution on polymer properties was investigated. A solvent-free melt polycondensation method using scandium (III) triflate as catalyst at an industrially relevant temperature (120 °C) was used. As the phenyl content increased, the polymers changed from semicrystalline to amorphous in state. The loading capability of polymers was checked by formulating nanoparticles containing coumarin 6 as a fluorescent dye analogue of active drugs. A polymer with a 70/30 ratio of succinic acid and phenylsuccinic acid showed the highest dye loading among the set of materials synthesised. This polymer was found to be degradable over time under selected experimental conditions. Amphiphilic block co-polymers from the PluronicTM class were used to stabilize, in PBS, nanoparticles formed from these polyesters by nanoprecipitation routes. The metabolic activity, cell membrane integrity and lysosomal functions of C3A cells dosed with the polymers were determined to observe the cytocompatibility of the highest dye-loaded nanoparticles. Activity relative to undosed C3A cells was retained at more than 80% in the all of the assays. Imaging of Pluronic coated and uncoated nanoparticles in C3A cells suggested that both types of the nanoparticles were endocytosed in the early stage of the study (within 10 min). The internalization of nanoparticles was increased progressively over the study time. These results indicated the possible utility of the selected polymers in diagnostic and delivery applications. Ring opening polymerization (ROP) reactions were used for the synthesis of a diblock (mPEG-b-PεDL) and a triblock (PεDL-b-PEG-b-PεDL) copolymer from a seven membered ε-decalactone (ε-DL) monomer obtained from renewable sources. A diblock (mPEG-b-PεDL) copolymer was compared with structurally similar mPEG-b-PCL copolymer synthesized via ROP of ε-caprolactone (ε-CL) monomer, which can be considered as a non-renewable monomer. A six membered δ-decalactone (δ-DL) was also used for the synthesis of a diblock copolymer (mPEG-b-PδDL) to compare the reaction kinetics and properties of the copolymers. The copolymers were prepared via bulk polymerisation using 1,5,7-Triazabicyclo[4.4.0]dec-5-ene (TBD) as a metal-free catalyst to replace the conventionally used stannous octoate [Sn(Oct)2]. A higher polymerization efficiency was achived with TBD compared to Sn(Oct)2 catalyst. However, a notable difference in the reaction temperature required for ε-DL and δ-DL polymerization was observed. The comparison with a structural analogue, i.e. ε-CL, demonstrated that the ε-DL polymerization was inhibited due to the presence of the alkyl chain of ε-DL monomer. However, a higher reaction time (12 h for TBD and 24 h for Sn(Oct)2) in CROP of ε-DL was addressed by using microwave based ring opening polymerization (MROP) reaction. The MROP was adopted as a ‘green’ and cheap heating method alternative to conventional heating (CROP) for the synthesis of mPEG-b-PεDL diblock copolymers using TBD as a catalyst. All the reactions were conducted in bulk. The MROP was designed based on the dielectric properties of all the reacting materials, as it was found that ε-DL monomers showed good absorption of MW radiation (tanδ>0.5). Accordingly, MROP resulted in a higher rate of ε-DL polymerization compared to CROP but comparison of the synthesis of mPEG-b-PCL copolymer by MROP indicated that the presence of the alkyl chain in ε-DL monomer significantly reduced the rate of polymerization. The synthesized mPEG-b-PεDL copolymer was investigated as a potential drug delivery vehicle for solubilization and controlled delivery of indomethacin. The indomethacin loading and release from mPEG-b-PεDL micelles (amorphous core) was compared against well-established mPEG-b-PCL micelles (semicrystalline core). The drug-polymer compatibility was also determined through a predictive computational approach to access the drug solubilisation (or drug loading) into hydrated micelles. The micelles were prepared by solvent evaporation method and characterized for size, morphology, indomethacin (IND) loading and release. Both of the micelle formulations showed a uniform distribution of spherical micelles with size <60 nm. However, a significantly higher size of empty mPEG-b- PεDL micelle was observed compared to mPEG-b-PCL micelles. A higher compatibility of the drug was predicted with PCL core as determined by modified Flory-Huggins interaction parameters (sp) using the Hanson solubility parameter (HSP) approach. The compatibility of the drug was determined for both of the segments (hydrophilic and hydrophobic) of the copolymers and found to be in the order of sp (PεDL)> sp (mPEG)> sp (PCL). The predictions suggested that more IND should encapsulate within the micelles with PCL core compared to PDL core, but the IND loading experiments revealed an overall higher loading in PεDL core (6.55 wt%) compared to PCL core (5.39 wt%) (P < 0.05, unpaired student’s t-test). However, consideration of the IND loading per unit volume of the micelles revealed that the PCL cored micelles was able to load 1.5 times more compared to the PεDL cored micelles. This result illustrated the higher compatibility of the IND with PCL core in accordance with the solubility parameter calculations. These data also suggested that the overall higher IND loading in PεDL core was attributable to the amorphous nature of the core which increased the core volume by 1.81 times compared to the PCL core. Drug release studies showed the sustained release pattern from both of the micelle systems although the semicrystalline PCL core (80% drug release in 110 h) was able to release the drug for a longer period compared to PεDL core (80% drug release in 72 h). Cell viability tests demonstrated the cytocompatibility of the mPEG-b-PεDL polymer. The micelles were internalized effectively in the early stages of the study and progressively increased with time. The results of the present thesis suggested that novel aliphatic polyester can be good candidates for the drug delivery applications and further studies can explore the possible applications of these polymers in the biomedical field.

Thesis (S.M.)--Massachusetts Institute of Technology, Engineering Systems Division, Technology and Policy Program, 2007.<br>Includes bibliographical references (p. 83-86).<br>In this thesis, the control of hypertension provides the backdrop for my effort to investigate how clinical trial design has evolved for antihypertensive drug submissions reviewed and approved by the Food and Drug Administration between 1988 and 2001. To do this, I have constructed and undertaken a preliminary analysis of a number of quantitative surrogate measures of complexity and scale, such as trial design, numbers of patients, treatment lengths, active drug comparators, number of indications pursued, number of indications approved, and approval times. In addition, I review how practice guidelines for the treatment of hypertension have changed with advancing clinical and biological knowledge. I attempt to investigate whether a link exists between the changing characteristics of clinical trials for antihypertensive therapies and the evolving guidelines for treating hypertension, promulgated by the Joint National Committee Report on the Detection, Evaluation, and Treatment of High Blood Pressure, (JNC), a committee assembled by the National Heart, Lung, and Blood Institute.<br>(cont.) Although the number of New Drug Applications (NDAs) examined in the antihypertensive class is too small to permit rigorous statistical analyses, I am nonetheless able to observe a number of apparent trends within the set of NDA submissions for antihypertensives approved by the FDA. Specific trends I observe in support of increasing trial complexity include: 1) trial sizes increase over time as measured by patient enrollments per trial (p-value = 0.003); 2) clinical trial designs over time have included greater numbers of arms per trial (p-value = 0.022); and 3) the number of drug-drug interaction studies in antihypertensive NDAs has increased with time (p-value = 0.027). These trends offer preliminary support for the hypothesis that clinical trials associated with NDA applications for antihypertensives have become more complex over the last two decades. The mechanisms responsible for the observed increase in complexity are less clear. Based on available information, I cannot determine if FDA guidance documents or informal correspondence were responsible for making antihypertensive clinical trials more complicated, or whether pharmaceutical companies introduced greater complexity into the trial design for commercial reasons.<br>(cont.) Furthermore, while I observe that FDA guidelines did not precisely track changes in JNC guidelines for treating hypertension, it is not clear whether the discrepancies are meaningful. Future research might attempt to identify more precisely the causes of increasing clinical trial complexity, and attempt to relate trial complexity to the cost of drug development more generally.<br>by John Simon Howe MacNeil.<br>S.M.

In this contribution the design and development on new approaches for targeted or controlled drug delivery is proposed. The selected starting material for the synthesis of these drug delivery systems (DDSs) was pullulan. Pullulan is a natural and linear polysaccharide that possesses excellent water solubility, biocompatibility and biodegradability. In virtues of the presence of multiple anchoring points along its chain, pullulan represents also an ideal carrier to build up colloidal drug delivery systems. Initially, pullulan oxidation was performed and deeply investigated as a convenient method for its functionalization. This strategy allowed the synthesis of a polymer therapeutic suitable for the treatment of the hepatocellular carcinoma (HCC). According to statistics, HCC is the third cause of cancer related death and the problems regarding its treatment are mainly caused by the considerable consequences of the first-pass effect and the lack of selectivity towards cancer cells. Thus, we aimed at developing a pullulan based DDS with higher selectivity for liver cancer cells. In literature a peptide sequence called preS121-47, portion of a protein located in the envelope of the Hepatitis B virus, was reported to be responsible for hepatocytes recognition, interaction and virus internalization. Moreover, the proposed receptor for this peptide, named SERPINB3, has been found overexpressed in HCC, thus representing a favourable target. Pullulan was modified with the introduction of seven peptide preS121-47 molecules and the selectivity towards cells overexpressing SERPINB3 (HepG2/SERPINB3) was proved to be 2 times higher when compared with both control cells not overexpressing SERPINB3 (HepG2/EMPTY) and control polymer without targeting agent. Then, the introduction of doxorubicin, linked to pullulan through an hydrazone pH-sensitive cleavable bond, lead to the formation of a complete polymer therapeutic which showed improved efficacy on hepatocellular carcinoma cells. Intending to explore other protocols for pullulan functionalization, its activation with p-toluenesulfonyl chloride and subsequent substitution with propargylammine was carried out. The display of alkyl groups along the polysaccharide chain permits the introduction of linking groups through copper-catalyzed azide-alkyne click chemistry reaction (CuAAC). Two rhodamine labelled polymers containing labile linking groups, i.e. hydrazone and ester, were synthesized. The stability of these conjugates in response to the pH was tested in comparison with a control polymers including only covalent bonds. Pullulan functionalized with the ester bond showed less stability that what expected, while sufficient retention of rhodamine, used as fluorescent drug model, was observed for the hydrazone bond. The potential use of these products as carrier for the sustained drug release in the vitreous of the eye was then evaluated. The flexible and highly biocompatible pullulan-based products demonstrated ex vivo to move slowly into the vitreous of an intact porcine eye. In vitro tests confirmed the safeness and high internalization levels of such bioconjugates on retina epithelium cells (ARPE-19). Furthermore, considering the peculiar features of the anatomy of the eye, we hypothesized to investigate the potential use of cell penetrating enhancer (CPE) decorated liposomes for improved entrance to the retinal cells after the administration through intravitreal injection. Taking advantage of the expertize on polysaccharide functionalization, the synthesis of an innovative CPE molecule starting from a maltotriose core was accomplished in our laboratory. This non-peptidic and polycationic TAT-like system was used in the present work for the decoration of liposomes. Different combinations of TAT and PEG2kDa-DSPE were evaluated and tested, but the formulation containing equal amount of these two decorating agents showed the most favourable characteristic for ophthalmic applications. Indeed, the diffusion coefficient calculation performed ex vivo on porcine eyes highlighted the possibility to tune liposomes mobility in vitreous by tuning their superficial properties. The surface decoration of liposomes with PEG2kDa-DSPE endowed liposomes with slipping properties, improving their vitreous mobility, while the presence of TAT molecule conferred enhanced uptake and penetration ability towards retina cells. Indeed, in vitro cell studies on ARPE-19 confirmed the low toxic effects and excellent level of uptake achieved with the co-decoration with PEG and TAT.<br>La tecnologia farmaceutica negli ultimi decenni si è indirizzata in maniera sempre più consistente verso lo sviluppo di nuovi sistemi in grado di ovviare ai limiti delle convenzionali terapie. La maggior parte delle molecole ad azione terapeutica possiede, infatti, alcuni ostacoli all’ampio utilizzo, come la scarsa solubilità in acqua, un profilo farmacocinetico inadeguato e la mancata selettività per l’organo bersaglio. Pertanto, lo sviluppo di metodologie che consentano un direzionamento sito-specifico e controllato di farmaci può essere di fondamentale aiuto non solo per l’ottimizzazione della terapia ma anche per il miglioramento della qualità della vita del paziente. In quest’ottica, il presente progetto di ricerca si è focalizzato nello sviluppo di sistemi a base polisaccaridica per il rilascio direzionato e controllato di farmaci. I polisaccaridi costituiscono infatti una piattaforma ideale per la sintesi di sistemi di drug delivery grazie alla loro notevole solubilità in acqua, biocompatibilità e biodegradabilità. Inoltre, possiedono diversi punti di ancoraggio per il legame con farmaci, agenti direzionanti o sonde fluorescenti. Il pullulano è stato scelto come polisaccaride modello per lo studio delle metodiche di funzionalizzazione e lo sviluppo di carrier polimerici. L’unità ripetitiva del pullulano è il maltotriosio, formato da tre molecole di glucosio legate da legami α(1-4) glicosidici. Ciascuna unità di maltotriosio è invece legata alla successiva attraverso un legame α(1-6) glicosidico. Questa peculiare copresenza di legami α(1-4) e α(1-6) conferisce al pullulano una struttura lineare e flessibile, adatta alla funzionalizzazione. Inizialmente il pullulano è stato ossidato utilizzando il periodato di sodio. Questo ossidante è selettivo per i dioli vicinali e permette l’apertura dell’anello piranosico e la formazione di due gruppi aldeidici. Questi gruppi aldeidici possono successivamente reagire con un gruppo amminico attraverso la reazione di amminazione riduttiva. Si ottiene in questo modo una base di Schiff che può essere ridotta con l’utilizzo di agenti riducenti quali cianoboroidruro di sodio e il boroidruro di sodio. Quello che si forma nel passaggio finale è pertanto un legame amminico stabile. L’ossidazione del pullulano è stata inizialmente sfruttata per il legame con una sonda fluorescente scelta come modello tra una libreria di composti sintetizzati presso il laboratorio della Prof.ssa Maria Grazia Ferlin dell’Università di Padova (Dip. di Scienze del Farmaco). La sintesi del coniugato fluorescente Pull-38 è stata di valido aiuto nello studio delle proprietà spettrofotometriche e spettrofluorimetriche della sonda. Infatti, attraverso tecniche spettrofluorimetriche, microscopia confocale e studi di tossicità e uptake in vitro, è stato possibile valutare il potenziale di tali composti nell’ambito della biologia cellulare. Successivamente, la stessa tecnica di coniugazione ha permesso la sintesi di un coniugato per il direzionamento sito-selettivo al carcinoma epatocellulare (HCC). In questo studio il pullulano è stato funzionalizzato con l’agente di targeting preS121-47 e con la rodamina come agente fluorescente. E’ riportato in letteratura che il peptide preS121-47, porzione della proteina preS1 presente nel capside del virus dell’epatite B, possiede un ruolo fondamentale nell’interazione del virus con la cellula epatica e nella sua internalizzazione. Il recettore di tale peptide è indicato con il nome di SERPINB3. La caratterizzazione del coniugato ha evidenziato il legame di sette molecole di peptide per catena di pullulano, con conseguente formazione di strutture a grappolo della dimensione di circa 150 nm. In collaborazione con il gruppo della Prof.ssa Patrizia Pontisso dell’Università di Padova (Dip. di Medicina) si è testata l’effettiva selettività del coniugato per le cellule sovra-esprimenti SERPINB3. L’uptake in tali cellule è risultato 2.5 volte maggiore rispetto a quello ottenuto nei normali epatociti. Il sistema è stato poi implementato con l’introduzione di doxorubicina, farmaco scelto come modello. E stato introdotto un legame idrazonico acido labile tra il backbone polimerico e il farmaco per garantire il rilascio di doxorubicina all’interno dell’endosoma. Si è dimostrato con le analisi al DLS e le immagini al TEM che i coniugati contenenti doxorubicina auto-assemblano a costituire strutture sferiche della dimensione di circa 170 nm. L’efficacia di tale coniugato si è rivelata doppia sia rispetto al pullulano controllo privo di preS121-47 che alle cellule controllo non esprimenti SERPINB3. In una seconda fase del progetto il pullulano è stato attivato con p-toluene cloruro di solfonile e successivamente sostituito con propargilammina. L’introduzione del propargile ha permesso quindi la funzionalizzazione del carrier attraverso la reazione di click chemistry tra un gruppo azidico e un alchino. Sono stati sintetizzati in questo modo tre diversi coniugati di pullulano marcato con rodamina. La rodamina è stata legata al polimero attraverso un legame idrazonico e un legame estereo. E’ stata poi valutata la stabilità di tali legami alla variazione di pH, ponendoli a confronto con un coniugato controllo pullulano-rodamina contenente solo legami covalenti. Tali coniugati polisaccaridici sono quindi stati testati in collaborazione con il gruppo del Prof. Arto Urtti dell’Università di Helsinki durante i sei mesi trascorsi presso il suo laboratorio (Centre for Drug Research, Faculty of Pharmacy, Helsinki). L’obiettivo di tali polimeri è quello, attraverso la somministrazione intravitreo, di garantire un rilascio sostenuto di farmaco per il trattamento di patologie della parte posteriore dell’occhio, quali la degenerazione della macula o la retinopatia diabetica. La mobilità di tali coniugati è stata valutata ex vivo nell’occhio intatto di maiale attraverso microscopia confocale dimostrando che essi vengono trattenuti nel reticolo del vitreo grazie alla natura polisaccaridica e flessibile del pullulano. I test di tossicità realizzati in vitro su cellule di epitelio di retina hanno confermato l’alta tollerabilità dei coniugati sintetizzati. Infine, gli studi di uptake condotti con l’utilizzo del citofluorimetro e del microscopio confocale hanno rivelato l’alto livello di internalizzazione dei prodotti, confermando l’effettiva possibilità di sviluppo di futuri carrier a base di pullulano per l’applicazione oculare. Infine, l’esperienza nella modifica di polisaccaridi maturata nel nostro laboratorio di ricerca ha permesso lo sviluppo di un innovativo cell penetrating enhancer (CPE) a partire da un core maltotriosidico. Questa nuova molecola di natura non peptidica e poli-argininica (TAT) è stata utilizzata per la decorazione di liposomi utilizzabili nel delivery oculare. Diverse formulazioni di liposomi decorati superficialmente con PEG2kDa-DSPE e TAT sono state prese in considerazione e studiate presso l’Università di Helsinki durante i mesi trascorsi nel laboratorio del Prof. Arto Urtti. E’ stato verificato attraverso studi ex vivo che liposomi cationici (TL) decorati in superficie con solo TAT sono incapaci di muoversi nel vitreo e rimangono intrappolati a causa delle interazioni elettrostatiche che si sviluppano con i componenti anionici dell’umor vitreo, come l’acido ialuronico e il solfato di condroitina. Al contrario, l’aggiunta di PEG aumenta la mobilità dei liposomi conferendogli la capacità di “scivolare” e penetrare attraverso le maglie dell’umor vitreo. I test di tossicità condotti in vitro con l’utilizzo delle cellule ARPE-19 hanno rivelato che nonostante i liposomi cationici siano tossici per le cellule, tale tossicità sia notevolmente ridotta in presenza PEG. I liposomi sono stati poi caricati con calceina per mimare la presenza di un farmaco idrofobico e valutarne l’uptake. Si è misurato un eccezionale livello di internalizzazione nelle ARPE-19 per i liposomi decorati con il TAT o con TAT e PEG rispetto ai liposomi privi di cell penetrating enhancer. Pertanto, i liposomi sia PEGylati che decorati in superficie con il TAT si sono dimostrati la miglior formulazione testata per l’adeguata mobilità nel vitreo, la ridotta tossicità e l’elevato uptake sulle cellule di retina.

2014 - 2015<br>The major technological challenge encountered when formulating new dosage forms is to find a way to adapt drug release kinetics to specific clinical requirements, with the aim to enhance the therapeutic action of both new and old drugs. Research in chronopharmacology has demonstrated the importance of biological rhythms in drug therapy and this has led to a new approach to the development of drug delivery systems which are required to enable the drug release timing and control. This approach has a strong impact on the treatment of chronic inflammatory-based diseases which symptoms are circadian-dependent such as the so-called early morning pathologies (EMPs_e.g. arthritis, arthrosis). The conventional treatment of these pathologies consists of daily administrations of anti-inflammatory medicines (NSAIDs or SAIDs), which are often unable to coordinate drug release with clinical symptoms onset resulting in inefficient therapy and poor patient compliance. In the case of individuals who suffer from rheumatoid arthritis and related painful joint disorders, the NSAIDS may be more effective when the drug is administered at least 4 to 6 hours before the pain reaches its peak in the early morning. In this context, my PhD project focused on the design and development of chronotherapeutic drug delivery systems characterized by time-specific and prolonged release of model anti-inflammatory drugs able to fit with the specific therapeutic needs of EMPs. In particular the PhD program involved the design and development of: chronotherapeutic systems loaded with short half life (Ketoprofen and Ketoprofen lysine salt) or with long half life NSAIDs (Piroxicam); and chronotherapeutic systems loaded with SAIDs (Prednisolone). These formulations were designed as monolayer, core/shell and floating microparticles tested as self-consistent formulation or into specific capsules as final dosage form. Formulations were produced by prilling/ionotropic gelation technique using natural (alginate, pectin), semi-synthetic (hydroxypropilmethylcellulose) or synthetic (methacrylic acid - methyl methacrylate copolymer) polymeric carriers as release modifiers. These formulations were characterized in terms of drug content and encapsulation efficiency (UV), morphology (SEM, SEM-EDX), solid state (DSC, FT-IR) swelling behavior and drug release profile (USP apparati II and IV). The in vivo anti-inflammatory effectiveness of the formulations showing the better in vitro technological features was studied using a modified protocol of carrageenan induced edema in rats. The optimized drug delivery systems showing a delayed and time-specific drug release in vitro as well as a delayed and prolonged in vivo anti-inflammatory effect compared to pure drugs, could be considered as potential chronotherapeutic agents to be taken at bedtime and able to act in the early morning. [edited by author]<br>XV n.s.(XXIX ciclo)

The subject of this thesis is multicolour bioluminescence analysis and how it can provide new tools for drug discovery and development.The mechanism of color tuning in bioluminescent reactions is not fully understood yet but it is object of intense research and several hypothesis have been generated. In the past decade key residues of the active site of the enzyme or in the surface surrounding the active site have been identified as responsible of different color emission. Anyway since bioluminescence reaction is strictly dependent from the interaction between the enzyme and its substrate D-luciferin, modification of the substrate can lead to a different emission spectrum too. In the recent years firefly luciferase and other luciferases underwent mutagenesis in order to obtain mutants with different emission characteristics. Thanks to these new discoveries in the bioluminescence field multicolour luciferases can be nowadays employed in bioanalysis for assay developments and imaging purposes. The use of multicolor bioluminescent enzymes expanded the potential of a range of application in vitro and in vivo. Multiple analysis and more information can be obtained from the same analytical session saving cost and time. This thesis focuses on several application of multicolour bioluminescence for high-throughput screening and in vivo imaging. Multicolor luciferases can be employed as new tools for drug discovery and developments and some examples are provided in the different chapters. New red codon optimized luciferase have been demonstrated to be improved tools for bioluminescence imaging in small animal and the possibility to combine red and green luciferases for BLI has been achieved even if some aspects of the methodology remain challenging and need further improvement. In vivo Bioluminescence imaging has known a rapid progress since its first application no more than 15 years ago. It is becoming an indispensable tool in pharmacological research. At the same time the development of more sensitive and implemented microscopes and low-light imager for a better visualization and quantification of multicolor signals would boost the research and the discoveries in life sciences in general and in drug discovery and development in particular.

The subject of this thesis is multicolour bioluminescence analysis and how it can provide new tools for drug discovery and development.The mechanism of color tuning in bioluminescent reactions is not fully understood yet but it is object of intense research and several hypothesis have been generated. In the past decade key residues of the active site of the enzyme or in the surface surrounding the active site have been identified as responsible of different color emission. Anyway since bioluminescence reaction is strictly dependent from the interaction between the enzyme and its substrate D-luciferin, modification of the substrate can lead to a different emission spectrum too. In the recent years firefly luciferase and other luciferases underwent mutagenesis in order to obtain mutants with different emission characteristics. Thanks to these new discoveries in the bioluminescence field multicolour luciferases can be nowadays employed in bioanalysis for assay developments and imaging purposes. The use of multicolor bioluminescent enzymes expanded the potential of a range of application in vitro and in vivo. Multiple analysis and more information can be obtained from the same analytical session saving cost and time. This thesis focuses on several application of multicolour bioluminescence for high-throughput screening and in vivo imaging. Multicolor luciferases can be employed as new tools for drug discovery and developments and some examples are provided in the different chapters. New red codon optimized luciferase have been demonstrated to be improved tools for bioluminescence imaging in small animal and the possibility to combine red and green luciferases for BLI has been achieved even if some aspects of the methodology remain challenging and need further improvement. In vivo Bioluminescence imaging has known a rapid progress since its first application no more than 15 years ago. It is becoming an indispensable tool in pharmacological research. At the same time the development of more sensitive and implemented microscopes and low-light imager for a better visualization and quantification of multicolor signals would boost the research and the discoveries in life sciences in general and in drug discovery and development in particular.

Monoclonal antibodies (mAbs) are extensively used in several therapeutic fields and in particular are used against cancer as therapeutic entity and/or targeting agents. The last application belongs to the several strategies to promote selective drug delivery in order to spare healthy tissues from unspecific toxicities of invasive therapies, as anticancer treatments, and to improve final clinical outcomes. Among all, targeted therapy includes antibody-drug conjugates (ADCs) approach, which exploit the synergic action of potent cytotoxic agents conjugated to a monoclonal antibody (mAb) coupled by specific linkers. The antibody acts as drug-carrier and targeting agent, and eventually also as a drug per se, in order to selectively kill cancer cells. As single therapeutic agents, both mAbs and highly cytotoxic drugs present critical issues in their clinical use. mAbs usually have to be used in combination therapy with other drugs because the majority of them show an insufficient, although specific, clinical response. On the other hand, chemotherapeutic drugs, especially the very potent ones, have a narrow therapeutic window and, therefore, their therapeutic doses are close to the maximum tolerated dose. Consequently, they present heavy side effects owing to the lack of tumour cells selectivity. The bright idea to combine mAbs and cytotoxic drugs in a unique entity offers the possibility to overcome their limitations. Traditionally, drugs have been coupled in a random way to lysine or cysteine residues, and the final product heterogeneity unfortunately has relevant impact on ADC activity, characterization and manufacturing, thus affecting the efficacy of the therapy. In an era in which product homogeneity and batch-to-batch reproducibility are essential pre-requisites for pharmaceutics, the development of new technologies for site-directed coupling have become a primary focus. New methodologies for site-specific conjugation are now widely investigated, and some example are the introduction of cysteine residues using site-directed mutagenesis, the use of enzymes, the insertion of un-natural amino acids and the conjugation to Fc N-Glycans. Nevertheless, such approaches require a specific development of each new ADC, thus offering few opportunities of know-how sharing between different projects, in fact these are often limited to the linker stability and the drug activity. In this work, a new construct for the delivery of active drugs has been proposed based on mAbs non-covalently interacting with a Fc-binding moiety (FcBM) that carries also the drugs. The aim is to achieve a higher degree of homogeneity and create a versatile and adaptable drug delivery system. Drug is not linked directly to the FcBM but through a linker constituted of a linear PEG chain. The drug-PEG-FcBM/antibody systems, from here called Antibody-Drug Systems (ADSs), present some advantages with respect to ADCs: they can be a versatile platform with which different mAbs can be used by simple mixing on demand with a drug-Fc binding module on the basis of the target tumour/disease to be treated. FcBM is the core of the system and must present unique binding properties for the Fc of antibodies. In this work, Protein G (22.8 kDa), a bacterial Fc binding receptor, and a goat Fab’ (≈ 50 kDa) against human Fc were tested as FcBM candidate, while a PEG chain was selected as linker for drug/dye attachment. Protein G was selectively PEGylated at the N-terminus with a PEG 20kDa, while Fab’ was mono-, bi- and tri-PEGylated with a PEG 5 kDa after the reduction of the sulfhydryl bridges at the hinge region of the progenitor F(ab’)2. Circular dichroism studies showed that the conjugates preserved protein secondary structure and isothermal titration calorimetry experiments determined that their affinity for Fc is about 107-108 M-1. After the complexation with a model mAb (Trastuzumab or Rituximab), NIR labelled ADSs were tested in vitro by cytometry analysis showing a high selectivity for antigen expressing cells. Furthermore, when a FcBM is conjugated to labelling molecule instead of a drug it would allow the switch from therapeutic application to diagnosis purposes. Tubulysin A (TubA), a potent inhibitor of tubulin polymerisation, was selected as drug model and tethered to the free end of PEG through a disulphide bond. TubA-PEG-Protein G/Trastuzumab ADS showed a preferential cytotoxic activity against the HER2+ cell line (SKBR-3), thus supporting the approach for both diagnostic and therapeutic purposes in cancer. A preliminary biodistribution study was performed in immunodeficient NSG mice, inoculated via subcutaneous injection with IGROV-1 (HER2-) and SKOV-3 (HER2+) tumour cells. Total body scanning showed a very rapid accumulation of Cy5-PEG-Protein G/Trastuzumab within 8 hours from ADS injection. Finally, uptake and intracellular trafficking of an ADS composed by a mouse IgG2a against ICAM-1 and Protein G as FcBM were evaluated in human umbilical vein endothelial cells (HUVECs). HUVECs were stimulated with TNFα to promote the expression of ICAM-1 receptor toward which the ADS was targeted. Fluorescence microscopy was used to follow ADS internalization by endothelial cells. The results disclosed that mechanism of uptake was based on CAM-mediate endocytosis, which relies in the binding between the receptor and anti ICAM-1 antibody. After internalization, the ADS followed the classical pathway toward the lysosomes to be degraded. This proves the possibility to achieve a selective intracellular delivery of active agents through ADS approach and open the way of new applications of this technology.<br>Gli anticorpi monoclonali (mAbs) sono largamente utilizzati in diversi campi terapeutici e in particolare nel contro il cancro, come entità terapeutica e/o agenti direzionanti. Quest'ultima applicazione rientra nell’insieme delle diverse strategie per promuovere il trasporto selettivo dei farmaci al sito d’azione, al fine di risparmiare i tessuti sani dalla tossicità aspecifiche propria di molte terapie invasive, quali la chemioterapia, e per migliorare i risultati clinici finali. Tra le terapie direzionate rientra l'uso degli anticorpi coniugati a farmaci, che sfruttano l'azione sinergica di potenti agenti citotossici coniugati ad un anticorpo monoclonale attraverso specifici linker. L'anticorpo agisce come agente di trasporto e agente direzionante, e anche come farmaco di per sé, al fine di colpire selettivamente le cellule cancerose. Come singoli agenti terapeutici, sia gli anticorpi monoclonali che i farmaci altamente citotossici presentano problemi critici nel loro impiego clinico. Gli anticorpi monoclonali di solito devono essere usati in terapia combinata con altri farmaci perché la maggior parte di essi mostra una risposta clinica insufficiente, sebbene specifica. D'altra parte, i farmaci chemioterapici, specialmente quelli molto potenti, hanno una ristretta finestra terapeutica e, pertanto, le dosi terapeutiche sono prossime alla dose massima tollerata. A causa della mancanza di selettività per le cellule tumorali, questi farmaci presentano pesanti effetti collaterali. La brillante idea di unire anticorpi monoclonali e farmaci citotossici in un'entità unica offre la possibilità di superare i limiti appena discussi. Tradizionalmente, i farmaci sono stati coniugati in modo aspecifico ai residui di lisina o cisteina; tuttavia, l'eterogeneità del prodotto finale ha un impatto rilevante sull'attività, sulla caratterizzazione e sulla produzione dei coniugati farmaco-anticorpo, influenzando così l'efficacia della terapia. In un'epoca in cui l'omogeneità del prodotto e la riproducibilità da lotto a lotto sono prerequisiti essenziali per lo sviluppo di una nuova forma farmaceutica, lo studio di nuove tecnologie per la coniugazione sito-specifica è diventato un obiettivo primario. Nuove metodologie per la coniugazione site-specifica sono ora ampiamente studiate e alcuni esempi sono: l'introduzione di residui di cisteina mediante mutagenesi sito-diretta, l'uso di enzimi, l'inserimento di aminoacidi non naturali e la coniugazione ai glicani della regione Fc. Tuttavia, tali approcci richiedono uno sviluppo mirato e a sé stante per ogni nuovo coniugato farmaco-anticorpo, offrendo quindi poche opportunità di adattamento delle metodologie tra diversi progetti, che hanno come elementi limitanti la stabilità del linker e l'attività del farmaco. In questo lavoro, è stato proposto un nuovo costrutto per la somministrazione di farmaci attivi basato su anticorpi monoclonali che interagiscono in modo non covalente con una molecola avente alta affinità per la regione Fc (detta FcBM) e che lega anche i farmaci. L'obiettivo è il raggiungimento di un grado più elevato di omogeneità e la creazione di un sistema di somministrazione di farmaci versatile e adattabile. Il farmaco non è legato direttamente all'FcBM ma è presente un linker costituito da una catena di PEG lineare. I sistemi anti-PEG-FcBM/anticorpo, da qui chiamati sistemi farmaco-anticorpo (ADS), presentano alcuni vantaggi rispetto i coniugati farmaco-anticorpo: costituiscono una piattaforma versatile con la quale si possono utilizzare diversi anticorpi semplicemente per aggiunta al modulo farmaco-FcBM in base al tumore bersaglio/malattia da trattare. L’FcBM è il cuore del sistema e deve presentare proprietà di legame uniche per la regione Fc degli anticorpi. In questo lavoro, la proteina G (22,8 kDa), un recettore batterico per l’Fc e un Fab' caprino (≈ 50 kDa) contro l’Fc umano sono stati testati come potenziali candidati per il ruolo di FcBM, mentre una catena PEG è stata selezionata come linker per l'attacco del farmaco/marcatore. La proteina G è stata selettivamente PEGhilata all'N-terminale con un PEG 20kDa, mentre il Fab' è stato mono-, bi- e tri-PEGhilato con un PEG 5 kDa dopo la riduzione dei ponti sulfidrilici nella regione di cerniera del F(ab')2 progenitore. Studi di dicroismo circolare hanno dimostrato che i coniugati hanno conservato la struttura secondaria della proteina, mentre gli esperimenti di calorimetria isotermica di titolazione hanno determinato che la loro affinità per l’Fc è circa 107-108 M-1. Dopo la complessazione con un anticorpo modello (Trastuzumab o Rituximab), gli ADS marcati con coloranti sono stati testati in vitro mediante analisi citometrica, che mostra un'elevata selettività per le cellule che esprimono l'antigene. Inoltre, il legame dell’FcBM a un marcatore anziché ad un farmaco consentirebbe il passaggio dal campo terapeutico al campo diagnostico. La Tubulisina A (TubA), un potente inibitore della polimerizzazione della tubulina, è stato selezionato come farmaco modello e coniugato all’estremità libera del PEG attraverso un legame disolfuro. Il sistema TubA-PEG-Protein G/Trastuzumab ha mostrato un'attività citotossica preferenziale contro la linea cellulare HER2 + (SKBR-3), dimostrando così la possibile applicazione sia per scopi diagnostici che terapeutici nel cancro. Uno studio preliminare di biodistribuzione è stato condotto su topi NSG immunodeficienti, inoculati mediante iniezione sottocutanea con cellule tumorali IGROV-1 (HER2-) e SKOV-3 (HER2 +). La scansione total body ha mostrato un accumulo molto rapido di Cy5-PEG-Protein G/Trastuzumab entro 8 ore dall'iniezione. Infine, l’internalizzazione e il traffico intracellulare di un ADS, composto da un anticorpo murino IgG2a contro l'ICAM-1 umano e la proteina G come FcBM, sono stati valutati in cellule endoteliali di vena ombelicale umana (HUVEC). Le HUVEC sono state stimolate con il TNFα per promuovere l'espressione del recettore ICAM-1 verso il quale l'ADS è direzionato. La microscopia di fluorescenza è stata utilizzata per seguire l'internalizzazione dell’ADS nelle cellule endoteliali. I risultati hanno rivelato che il meccanismo di internalizzazione è l'endocitosi mediata da CAM, che si basa sul legame tra il recettore e l'anticorpo anti-ICAM-1. Dopo l'internalizzazione, l'ADS segue il classico percorso verso i lisosomi per essere degradato. Ciò dimostra la possibilità di ottenere un direzionamento intracellulare selettivo di agenti attivi attraverso l'approccio ADS e apre la strada a nuove applicazioni per questa tecnologia.

The purpose of the present study was to formulate new vesicular carriers to optimize the topical delivery of anti-inflammatory drugs: diclofenac, quercetin and curcumin. In the first work concentrated and interconnected penetration enhancer containing vesicles (PEVs) are proposed as carriers for dermal delivery of diclofenac. PEVs were prepared by using a commercial phosphatidylcholine mixture (180 mg/m) and transcutol in different amounts. Conventional liposomes were also prepared and tested as control. All vesicles showed a mean size ranging from 75 to 253 nm with fairly narrow size distribution, negative zeta potential value, and drug loading capacity between 48 and 70%. SWAXS studies showed that composition affected vesicle structure and morphology: 10 and 30% transcutol PEVs were unilamellar while liposomes and 20% transcutol PEVs were multilamellar. Rheological studies demonstrated that control liposomes and 10 and 30% transcutol containing PEVs behaved as Newtonian fluids while 20% transcutol containing PEVs showed a plastic behavior. Ex vivo (trans)dermal delivery experiments showed an improved skin deposition of diclofenac when PEVs were used. Vesicle toxicity and uptake of fibroblasts, target of inflammation treatment, were evaluated by MTT test and fluorescence microscopy. Control liposomes and PEVs were both able to interact and being internalized by the 3T3 fibroblasts at all time exposure tested. Furthermore, PEVs showed to be able to reduce the in vitro drug toxicity. The aim of the second study was to improve the knowledge of drug–glycol–phospholipid-interactions and their effects in lamellar vesicle suitability as drug delivery systems. Liposomes were prepared using hydrogenated soy phosphatidylcholine (P90H, 60 mg/ml) and diclofenac sodium salt at two con-centrations (5–10 mg/ml). To obtain innovative vesicles two permeation enhancers with glycol group, diethyleneglycol monoethyl ether and propylene glycol, were added to the water phase at different ratios (5%, 10%, and 20%). Vesicle organization was deeply investigated by physico-chemical characterization, including differen-tial scanning calorimetry and small-angle diffraction signal analysis while macroscopic structure behavior was evaluated by rheological studies. Results evidenced that the presence of the penetration enhancer and diclofenac sodium salt led to structural rearrangements within and among vesicles forming a tridi-mensional and complex architecture in which vesicles were closely packed and interconnected. This new design allowed a change in the physical state of dispersions that became highly viscous liquid or soft-solid-like, thus forming an ideal system for topical application able of both adhering to the skin and delivering the drug. © 2013 Elsevier B.V. All rights reserved. In the last work biocompatible quercetin and curcumin nanovesicles were developed as a novel approach to prevent and restore skin tissue defects on chronic cutaneous pathologies. Stable and suitable quercetin- and curcumin-loaded phospholipid vesicles, namely liposomes and penetration enhancercontaining vesicles (PEVs), were prepared. Vesicles were made from a highly biocompatible mixture of phospholipids and alternatively a natural polyphenol, quercetin or curcumin. Liposomes were obtained by adding water, while PEVs by adding polyethylene glycol 400 and Oramix_CG110 to the water phase. Transmission electron microscopy, cryogenic-transmission electron microscopy and small- and wideangle X-ray scattering showed that vesicles were spherical, oligo- or multilamellar and small in size (112–220 nm). In vitro and in vivo tests underlined a good effectiveness of quercetin and curcumin nanovesicles in counteracting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induced lesions and inflammation. Myeloperoxydase activity, used to gauge inflammation, was markedly inhibited by quercetin liposomes (59%) and curcumin liposomes and polyethylene glycol (PEG)-PEVs (_68%). Histology showed that PEG-PEVs provided an extensive re-epithelization of the TPA-damaged skin, with multiple layers of thick epidermis. In conclusion, nanoentrapped polyphenols prevented the formation of skin lesions abrogating the various biochemical processes that cause epithelial loss and skin damage.

The first polysulfenamides were synthesized with S-N and N-S-N bonds along the backbone. We demonstrated that sulfenamides were stable in polar protic and aprotic solvents, but degraded rapidly when exposed to acidic conditions. Microparticles were fabricated from polysulfenamides with S-N bonds, their surfaces were readily functionalized, and they were internalized by cells allowing for intracellular delivery of their cargo. These microparticles were also stable at physiological pH, degraded under acidic conditions, and possessed minimal toxicity towards cells. This work demonstrated that polysulfenamides form the basis for a new set of polymers for drug delivery that greatly differ from prior work in this field. New biodegradable polymers with N-S-N bonds along the backbone were synthesized. These were the first polymers with these bonds and possessed many of the same characteristics as polymers synthesized with S-N bonds. The synthesis and characterization of comb block copolymers with arms composed of poly(lactic acid), poly(butyl acrylate), and poly(styrene-b-vinylpyridine) were described. The self-assembled morphologies in the solid state of comb tri- and tetrablock copolymers with poly(styrene) were also described. These assemblies demonstrated that well-ordered and complex morphologies were assembled from these polymers. The steric effect of substitutions on oxanorbornenes in ring opening metathesis polymerization (ROMP) was investigated. Oxanorbornenes substituted with methyls at the bridgehead positions showed limited reactivity with the Grubbs first and second generation catalysts and the Grubbs first generation methylidene catalyst.

The development of more efficient extraction methods is the key to improving access to natural products with therapeutic uses. Immobilised metal affinity chromatography (IMAC) has conventionally been used to purify proteins from biological mixtures. This thesis aimed to expand the applications of IMAC beyond protein purification by developing two new innovations of IMAC. First, multi-dimensional IMAC (MD-IMAC) was developed for the simultaneous purification of multiple non-protein-based bacterial secondary metabolites from a single culture. The MD-IMAC system, comprised of two in-series IMAC resins charged with different metal ions, was designed to exploit differences between the coordination chemistries of different compounds. A system was optimised for two clinical metabolites, desferrioxamine B (DFOB) and bleomycin (BLM). Retention of compounds was dependent on the metal ion used to charge the resin, the immobilised chelating ligand denticity, and column order. The technique was used successfully to resolve native DFOB and BLM from a culture of Streptomyces verticillus. Second, the utility of IMAC as a method of screening natural sources for metalloenzyme inhibitors was examined. Three microbial sources (Penicillium citreonigrum, Streptomyces incarnatus, Salinispora tropica) were processed on Ni(II)-, Cu(II), and Zn(II)-IMAC, followed by assay screens for inhibition against histone deacetylases (HDACs), 5-lipoxygenase and tyrosinase. Proof-of concept was established, with a putative HDAC inhibitor ([M + H+]+, m/z = 827.2800) identified from S. incarnatus. More material and further analyses are required to structurally characterise the bioactive species. This study is the first to demonstrate these applications of IMAC. As IMAC is an aqueous-compatible, green technology, MD-IMAC could be used to expedite pharmaceutics processing and reduce reliance on organic solvents. IMAC could also provide a new way of screening natural sources for metalloenzyme inhibitors.

A drug discovery project typically starts with a pharmacological hypothesis: that the modulation of a specific molecular biological mechanism would be beneficial in the treatment of the targeted disease. In a small-molecule project, the next step is to identify hits, i.e. molecules that can effect this modulation. These hits are subsequently expanded into hit series, which are optimised with respect to pharmacodynamic and pharmacokinetic properties, through medicinal chemistry. Finally, a drug candidate is clinically developed into a new drug. This thesis concerns the use of structure-based virtual screening in the hit identification phase of drug discovery. Structure-based virtual screening involves using the known 3D structure of a target protein to predict binders, through the process of docking and scoring. Docking is the prediction of potential binding poses, and scoring is the prediction of the free energy of binding from those poses. Two new methodologies, based on post-processing of scoring results, were developed and evaluated using model systems. Both methods significantly increased the enrichment of true positives. Furthermore, correlation was observed between scores and simple molecular properties, and identified as a source of false positives in structure-based virtual screening. Two target proteins, Mycobacterium tuberculosis ribose-5-phosphate isomerase, a potential drug target in tuberculosis, and Plasmodium falciparum spermidine synthase, a potential drug target in malaria, were subjected to docking and virtual screening. Docking of substrates and products of ribose-5-phosphate isomerase led to hypotheses on the role of individual residues in the active site. Additionally, virtual screening was used to predict 48 potential inhibitors, but none was confirmed as an inhibitor or binder to the target enzyme. For spermidine synthase, structure-based virtual screening was used to predict 32 potential active-site binders. Seven of these were confirmed to bind in the active site.

<p>Neuroblastoma is one of the most common solid cancers of early childhood. In Sweden, approximately 10-15 cases occur annually. The overall five-year neuroblastoma survival in Europe is approximately 45%. Since cancer treatment involves drugs with risks of side effects in the growing child, there is a need for more effective and less toxic drugs. One new approach in cancer treatment is inhibition of tumor angiogenesis, i.e., of new blood vessel growth into the tumor. An angiogenesis inhibitor may be combined with cytostatic drugs to enhance the efficacy. The aim of this study was to investigate how drugs could be used to inhibit angiogenesis and tumor growth in a xenograft model of human neuroblastoma in nude mice. </p><p>The tumors express the angiogenesis stimulator vascular endothelial growth factor (VEGF) on both protein and mRNA levels. The angiogenesis inhibitors SU5416 (an inhibitor of VEGF signalling) and TNP-470 (an inhibitor of endothelial cell proliferation) inhibited angiogenesis in our model. TNP-470, however, inhibited angiogenesis without significant reduction of the tumor growth, in contrast to SU5416. </p><p>We also discovered that the cytostatic drug CHS 828 could cause regression of neuroblastoma tumors in the model when given orally at a low daily dose, alone or in combination with the angiogenesis inhibitor SU5416 or TNP-470. </p><p>Furthermore, a new use of the cardiac glycoside digoxin was found. Digoxin inhibited FGF-2 -stimulated bovine capillary endothelial cell growth in vitro, and inhibited angiogenesis in vivo in the chick chorioallantoic membrane assay (CAM). It also inhibited neuroblastoma growth by approximately 50% in our neuroblastoma model. </p><p>In conclusion, CHS 828 and digoxin represent two classes of drugs with potent antitumor effects that may be valuable in treatment of neuroblastoma, either alone or in combination with angiogenesis inhibitors.</p>

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Civil and Environmental Engineering; and, (S.M.)--Massachusetts Institute of Technology, Engineering Systems Division, Technology and Policy Program, 2007.<br>Includes bibliographical references (p. 99-102).<br>Efforts in managing diabetes, including the medical advances in novel therapies and public health policies of disease control and prevention, have not reduced the disease prevalence since 1990s. I analyze this phenomenon from the technology and policy viewpoints underlying diabetes treatment and prevention, in order to control and manage the disease in a cost-benefit balanced manner. The innovative performance of the antidiabetic drug therapy is investigated by analyzing the fifteen New Drug Applications (NDAs) of the antidiabetics approved by the US Food and Drug Administration since the early 1990s. I examine the characteristics of the clinical trials supporting NDAs and observe how the complexity of clinical trials has changed over time. Nine out of the twenty-five selected indicators are found to exhibit an increasing trend of complexity. The trend is more pronounced in the oral antiglycemics group (seven indicators) than the subcutaneous group (two indicators). Interestingly, this trend in increasing complexity in clinical trials is generally consistent with that of the increasing R&D costs in the pharmaceutical industry, possibly account for the declining innovative performance of the industry over the time period under investigation. A system dynamics approach is applied to assess current public health policies in diabetes control and prevention.<br>(cont.) The benefit of system thinking is to avoid potential policy resistance by identifying the problematic characteristics of the system, such as time-delays, feedback, and structure of stocks and flows. For diabetes management, the public health system can be considered a "dynamic-complex" system in terms of current policy made by the National Diabetes Control and Prevention Program. Despite providing earlier and expanded screening as well as improved availability and accessibility of treatment for diabetes, the policy results in the increase of prevalence. More undiagnosed people are diagnosed, thus increasing the incidence, whereas people already diagnosed prolong their lifespan due to the better and more accessible medical care. A future successful chronic disease management program should systematically integrate the efforts from both the treatment and prevention perspectives.<br>by Hsiao-Lan Sharon Lin.<br>S.M.

En la present tesi doctoral es descriu un procediment general per a l'obtenció de nanopartícules mesoporoses de sílice (MSNs) regioselectivament bifuncionalizades de forma ortogonal amb diferents grups funcionals. L'estratègia sintètica consisteix en la preparació de MSNs mitjançant co-condensació, seguit d'una posterior funcionalització covalent, mentre el tensioactiu es troba encara present en l'estructura de les MSNs. Seguint aquesta metodologia, s'han sintetitzat les nanopartícules bifuncionalizades amina-azida (MSN-(NH2)i(N3)o), amina-isotiocianat (MSN-(NH2)i(NCS)o) i amina-aldehid (MSN-(NH2)i(CHO)o), per al seu ús en aplicacions biomèdiques. En primer lloc, s'han sintetitzat i caracteritzat de forma homogènia i reproduïble les nanopartícules de referència (MSN-NH2) que permetran les successives funcionalitzacions. Aquestes nanopartícules aminades s'han fet servir posteriorment per a la síntesi de sensors de naftalimida. S'ha aconseguit desenvolupar un procediment general per a la introducció de 4-amino-1,8 naftalimides. Aquestes naftalimides han estat provades com a sensor i portes lògiques per a la detecció de H+ i F-. D'altra banda, s'ha descrit un protocol per preparar amino-azida-MSNs de forma regioselectiva. Aquestes MSNs han estat funcionalitzades per primera vegada amb foldàmers catiònics i la seva capacitat per creuar membranes citoplasmàtiques i viabilitat ha estat estudiada, així com l'ús d'aquests sistemes per a l'alliberament intracel·lular de Doxorubicina (DOX) de forma controlada. També s'ha realitzat un nou protocol per preparar MSNs amb isotiocianat en la seva estructura. La metodologia sintètica és general i es pot aplicar, en principi, a qualsevol MSNs aminada. L'eficiència de la funcionalització és comparable a la cicloaddició de coure (CuAAC) evitant els protocols d'aïllament i d'eliminació del metall. Seguint aquesta metodologia, s'han preparat unes noves amino-isotiocianat-MSNs per al disseny d'un nano-contenidor capaç d'alliberar el fàrmac Ataluren de forma controlada, per el seu us en la distròfia muscular de Duchenne (DMD). S'han aconseguit sintetitzar amina-aldehid-MSNs. Aquestes MSNs s'han aplicat com una nanoplataforma simple i versàtil capaç d'alliberar de forma dual una barreja CPT/DOX per al tractament del càncer, mitjançant l'ús d'estímuls de pH. Mentre un fàrmac és absorbit dins de la superfície interior, l'altre està unit covalentment a la superfície externa, actuant així, a la vegada, com a fàrmac i com agent bloquejant de porus. Aquest sistema respon als estímuls de pH i tots dos fàrmacs són només alliberats en un medi àcid.<br>En la presente tesis doctoral se describe un procedimiento general para la obtención de nanopartículas mesoporosas de sílice (MSNs) regioselectivamente bifuncionalizadas de forma ortogonal con distintos grupos funcionales. La estrategia sintética consiste en la preparación de MSNs mediande co-condensación, seguido de una posterior funcionalización covalente, mientras el tensioactivo se encuentra todavía presente en la estructura de las MSNs. Siguiendo esta metodología, se han sintetizado las nanopartículas bifuncionalizadas amina-azida (MSN-(NH2)i(N3)o), amina-isotiocianato (MSN-(NH2)i(NCS)o) y amina-aldehído (MSN-(NH2)i(CHO)o), para su uso en aplicaciones biomédicas. En primer lugar, se han sintetizado y caracterizado de forma homogénea y reproducible las nanopartículas aminadas de referencia (MSN-NH2) que permitirán las sucesivas funcionalizaciones, con un tamaño de 50 nm y 100 nm aproximadamente. Estas nanopartículas aminadas se han usado posteriormente para la síntesis de sensores de naftalimida. Se ha conseguido desarrollar un procedimiento general para la introducción de 4-amino-1,8 naftalimidas. Estas naftalimidas han sido probadas como sensores y puertas lógicas para la detección de H + y F-. Por otra parte, se ha descrito un protocolo para preparar amino-azida-MSNs de forma regioselectiva. Estas MSNs han sido funcionalizadas por primera vez con foldámeros catiónicos y su capacidad para cruzar membranas citoplasmáticas y viabilidad ha sido estudiada, así como el uso de estos sistemas para la liberación intracelular de doxorubicina (DOX) de forma controlada. También se ha realizado un nuevo protocolo para preparar MSNs con isotiocianato en su estructura. La metodología sintética es general y puede aplicarse, en principio, a todo tipo de MSNs aminadas. La eficiencia de la funcionalización es comparable a la cicloadición de cobre (CuAAC) evitando los protocolos de aislamiento y de eliminación del metal. Siguiendo esta metodología, se han preparado unas nuevas amino-isotiocianato-MSNs para el diseño de un nano-contenedor capaz de liberar el fármaco Ataluren de forma controlada. Se ha logrado sintetizar amina-aldehído-MSN. Estas MSNs se han aplicado como una nanoplataforma simple y versátil capaz de liberar de forma dual una mezcla CPT/DOX para el tratamiento del cáncer, mediante el uso de estímulos de pH. Mientras un fármaco es absorbido dentro de la superficie interior, el otro está unido covalentemente a la superficie externa, actuando así como fármaco y como agente bloqueante de poro. Este sistema responde a los estímulos de pH y ambos fármacos son solamente liberados en un medio ácido.<br>In this PhD dissertation, a general procedure for the obtaining of different regioselective orthogonal bifunctionalized mesoporous silica nanoparticles (MSNs) has been carried out. The strategy consists of a covalent functionalization of co-condensed monodispersed and uniform aminated-MSNs, where tensioactive is still present in its structure. Three bifunctionalized MSNs, amine-azide (MSN-(NH2)i(N3)o), amine-isothiocyanate (MSN-(NH2)i(NCS)o) and amine-aldehyde (MSN-(NH2)i(CHO)o), with efficient “click” reactions, have been synthetized for its use in biomedical applications. First, a well characterized batch of precursor aminated-MSNs (MSN-(NH2)) has been prepared. The best conditions for the synthesis of homogenous and reproducible MSN-(NH2) with a size between 50-100 nm have been studied. These aminated-MSNs have been used for the synthesis of naphthalimide sensors where a general procedure for the introduction of 4-amine-1,8-naphthalimides has been developed. These naphthalimides have been tested as potential logic gates for the detection of H+ and F-. A straightforward protocol to prepare amine-azide MSNs has been described. These MSNs have been functionalized with quinolin cationic foldamers for the first time. The ability of these foldamer-MSNs to cross cytoplasmic membranes and its viability has been studied. The penetrating capacity of foldamer-MSNs have been used for intracellular delivery of Doxorubicin (DOX). A new protocol to prepare isothiocyanate functionalized MSNs is described. The synthetic methodology is general and can be applied, in principle, to all type of aminated MSNs. The efficiency of the functionalization is comparable to the copper cycloaddition (CuAAC) avoiding isolation and copper removal protocols. Following this methodology, new amino-isothiocyanate-MSNs have been prepared for the design of a nano-container able to release the drug Ataluren in a controlled manner, for the treatment of Duchenne muscular dystrophy (DMD). Regioselective bifunctionalized amine-aldehyde-MSNs have been synthetized. These MSNs have been applied as a versatile nanoplatform able to release dual synergistic CPT/DOX mixture for cancer treatment only by using pH stimuli. While CPT is absorbed at the inner surface, DOX is covalently linked to the external surface acting both as an active and a capping agent (pH=4).

Peptidomimetics can mimic hierarchical structures of peptides and proteins. Thus, they are extensively studied for therapeutic applications. To break the limitation of backbones and frameworks and expand the peptidomimetics family, a new class of peptidomimetics - “γ-AApeptides” was developed. Design of γ-AApeptides is based on the chiral peptide nucleic acids (PNAs) backbone. The World Health Organization estimates that one -third of all deaths in the world are on account of infectious diseases. AMPs are important because of their high activity against broad spectrum microbes, less susceptible to grow resistance and selectivity in binding to bacterial cells over human cells. γ-AApeptides as a new class of peptidomimetics have increased stability and enhanced chemical diversity. We have developed polymyxin mimic cyclic peptides, small linear molecules and hydantoin derivatives as potent antibiotic agents with γ-AApeptides. They have good bioactivity and selectivity. Combinatorial library is key technology for accelerating the discovery of novel therapeutic agents. One-bead-two-compound γ-AApeptides-based library was developed and screened against SMYD2 protein which is essential for tumor growing.

Con el fin de relacionar la estructura y la actividad de series de compuestos, es importante usar descriptores moleculares relevantes. Los descriptores GRIND y VolSurf pertenecen a una nueva familia de descriptores llamado libre de alineamiento. Es decir, que no necesitan alinear los compuestos con el fin de comparar sus campos de interacciones molecular. En este estudio se ha aplicado esos descriptores para la selección de reactivos químicos a partir de una amplia base de datos. La selección se ha echo mediante un protocolo que permite maximizar la diversidad de la muestra y así obtener unos compuestos muy informativos. También se ha desarrollado nuevos descriptores de forma que están basado en los cambios de curvatura de la superficie molecular. Los resultados obtenidos indican que los nuevos descriptores de forma se integran muy bien en los descriptores GRIND originales y que permiten identificar los efectos de forma tanto favorable como desfavorable. Además, se ha desarrollado nuevos descriptores libre de alineamiento llamado 'anchor-GRIND' que usan un átomo de cada molécula como punto de referencia para la comparación de los campos de interacciones molecular. Los descriptores 'anchor-GRIND' permiten una descripción mas precisa y mas sencilla que los descriptores GRIND lo que los hace mas relevante para el análisis de ciertas familias de compuestos.<br>In order to correlate the differences of structure with the differences of activity of series of compounds, it is important to use relevant molecular descriptors. The GRIND and VolSurf descriptors belong to the so-called alignment-free descriptors family. In other words, they do not require to align the compounds in order to compare its molecular interaction fields. In this study, we applied these descriptors to the selection of chemical reagent from a database of compounds. The selection has been done following a protocol which allows to maximize the diversity of the sample and so to obtain some compounds highly informative. In addition we developed new shape descriptors which are based on the changes of curvature of the molecular surface. The results obtained show that the new shape descriptors are well integrated in the original GRIND descriptors. Furthermore, we designed new alignment-free descriptors called 'anchor-GRIND' which use one atom of each molecule as a reference point for the comparison of the molecular interaction fields. The 'anchor-GRIND' descriptors allow a more precise and more simple description than the GRIND descriptors, which makes them more relevant for the analysis of some families of compounds.

[ES] La presente tesis doctoral titulada "Mesoporous silica and gold-based nanodevices: new controlled delivery platforms for biomedical applications" se centra en el diseño, síntesis, caracterización y evaluación de distintos nanodispositivos híbridos orgánico-inorgánicos. En concreto, se utilizan como soporte nanopartículas mesoporosas de sílice y nanopartículas de oro para su aplicación biomédica, en concreto en el campo del cáncer de mama. En el primer capítulo se introduce el marco general en el que se engloban los estudios realizados. Se presentan los conceptos relacionados con nanotecnología y nanomedicina, así como la interacción de las nanopartículas a nivel biológico con el organismo y las células. Finalmente, se introducen conceptos básicos del cáncer de mama y la aplicación de nanomateriales como terapia. A continuación, en el segundo capítulo, se exponen los objetivos de la presente tesis doctoral que son abordados en los siguientes capítulos experimentales. En el tercer capítulo se describe el primer nanomaterial para la liberación controlada de dos inhibidores (navitoclax y S63845) de las proteínas anti- apoptóticas de la familia Bcl-2. Este sistema se ha diseñado con el objetivo de superar la resistencia a navitoclax en un modelo celular de cáncer de mama triple negativo. En concreto, se han preparado nanopartículas mesoporosas de sílice cargadas con navitoclax y S63845, y funcionalizadas con un aptámero dirigido a la proteína de superficie MUC1, que actúa como puerta molecular. En este trabajo hemos demostrado que las nanopartículas diseñadas son internalizadas preferentemente por células tumorales de cáncer de mama. También hemos demostrado la capacidad de las nanopartículas de revertir la resistencia a navitoclax en un modelo celular de cáncer de mama triple negativo. Además, ponemos de manifiesto la disminución del principal efecto adverso (trombocitopenia) asociado a la administración del navitoclax en su formulación libre, gracias a la encapsulación en las nanopartículas. En el capítulo cuatro se presenta un sistema sensible a pH para la liberación controlada de un cargo fluorescente y la maquinaria de edición génica basada en el sistema CRISPR/Cas9, dirigido a la edición del gen codificante de la proteína fluorescente verde (GFP, del inglés gren fluorescent protein). El nanodispositivo está constituido por nanopartículas mesoporosas de sílice cargadas con rodamina B, funcionalizadas con polietilenimina y revestidas con el plásmido codificante del sistema CRISPR/Cas9. En este trabajo se ha demostrado el escape lisosomal de las nanopartículas, mediado por el efecto esponja de protones de la PEI. Asimismo, mostramos un nanodispositivo pionero en su campo, basado en nanopartículas mesoporosas de sílice, capaz de realizar la doble función de llevar a cabo la edición del gen codificante de GFP y la liberación exitosa del cargo fluorescente. En el quinto, y último, capítulo experimental se propone una nueva aproximación para realizar una terapia enzimática prodroga empleando nanopartículas de oro como transportadores enzimáticos. En este caso, se aborda la funcionalización de nanopartículas de oro con la enzima peroxidasa de rábano (HRP, del inglés horseradish peroxidase), capaz de transformar la prodroga inocua ácido indol-3-acético en especies radicales que resultan tóxicas para las células tumorales. En este capítulo se ha demostrado el efecto terapéutico del nanodispositivo en combinación con la prodroga en modelos celulares de cáncer de mama de los subtipos luminal A y triple negativo. Además, se ha confirmado la eficacia terapéutica del sistema en esferoides tumorales formados por células de cáncer de mama triple negativo. Por último, se presentan en el capítulo seis las conclusiones extraídas del desarrollo de esta tesis doctoral. Los resultados obtenidos en este trabajo contribuirán al desarrollo de nuevos nanomateriales inteligentes con aplicación en diversas áreas de la nanomedicina.<br>[CA] La present tesi doctoral titulada "Mesoporous silica and gold-based nanodevices: new controlled delivery platforms for biomedical applications" se centra en el disseny, síntesi, caracterització i avaluació de diferents nanodispositius híbrids orgànic-inorgànics. En concret, s'utilitzen com a suport nanopartícules mesoporoses de sílice i nanopartícules d'or per a la seua aplicació biomèdica, en concret en el camp del càncer de mama. En el primer capítol s'introdueix el marc general en el qual s'engloben els estudis realitzats. Es presenten els conceptes relacionats amb la nanotecnologia i nanomedicina, així com la interacció de les nanopartícules a nivell biològic amb l'organisme i les cèl·lules. Finalment, s'introdueixen conceptes bàsics del càncer de mama i l'aplicació de nanomaterials com a teràpia. A continuació, en el segon capítol, s'exposen els objectius de la present tesi doctoral que són abordats en els següents capítols experimentals. En el tercer capítol es descriu el primer nanomaterial utilitzat per a l'alliberament controlat de dos inhibidors (navitoclax i S63845) de les proteïnes anti-apoptòtiques de la família Bcl-2. Aquest sistema s'ha dissenyat amb l'objectiu de superar la resistència a navitoclax en un model cel·lular de càncer de mama triple negatiu. En concret, s'han preparat nanopartícules mesoporoses de sílice carregades amb navitoclax i S63845, i funcionalitzades amb un aptàmer dirigit a la proteïna de superfície MUC1, que actua com a porta molecular. En aquest treball hem demostrat que les nanopartícules dissenyades són internalitzades preferentment per cèl·lules tumorals de càncer de mama. També hem demostrat la capacitat de les nanopartícules de revertir la resistència a navitoclax en un model cel·lular de càncer de mama triple negatiu. A més, posem de manifest la disminució del principal efecte advers (trombocitopènia) associat a l'administració del navitoclax en la seua formulació lliure, gràcies a l'encapsulació en les nanopartícules. En el capítol quatre es presenta un sistema sensible a pH per a l'alliberament controlat d'una càrrega fluorescent i la maquinària d'edició gènica basada en el sistema CRISPR/Cas9, dirigit a l'edició gènica del gen codificant de la proteïna fluorescent verda (GFP, del anglés gren fluorescent protein). El nanodispositiu està constituït per nanopartícules mesoporoses de sílice carregades amb rodamina B, funcionalitzades amb polietilenimina i revestides amb el plàsmid codificant del sistema CRISPR/Cas9. En aquest treball s'ha demostrat la fuga lisosomal de les nanopartícules, mediat per l'efecte esponja de protons de la PEI. Així mateix, vam mostrar un nanodispositiu pioner en el seu camp, basat en nanopartícules mesoporoses de sílice, capaç de realitzar la doble funció de dur a terme l'edició del gen codificant de la GFP i l'alliberament exitós de la càrrega fluorescent. En el cinqué i últim capítol experimental es proposa una nova aproximació per a realitzar una teràpia enzimàtica prodroga emprant nanopartícules d'or com a transportadors enzimàtics. En aquest cas, s'aborda la funcionalització de nanopartícules d'or amb l'enzim peroxidasa de rave (HRP, del anglés horseradish peroxidase), capaç de transformar la prodroga innòcua àcid indol-3-acètic en espècies radicals que resulten tòxiques per a les cèl·lules tumorals. En aquest capítol s'ha demostrat l'efecte terapèutic del nanodispositiu en combinació amb la prodroga en models cel·lulars de càncer de mama dels subtipus luminal A i triple negatiu. A més, s'ha confirmat l'eficàcia terapèutica del sistema en esferoides tumorals formats per cèl·lules de càncer de mama triple negatiu. Finalment, en el capítol sis es presenten les conclusions extretes del desenvolupament d'aquesta tesi doctoral. Els resultats obtinguts en aquesta tesi contriburan al desenvolupament de nous nanomaterials intel·ligents amb aplicació en diverses àrees de la nanomedicina.<br>[EN] This Ph.D. thesis entitled "Mesoporous silica and gold-based nanodevices: new controlled delivery platforms for biomedical applications" is focused on the design, synthesis, characterisation, and evaluation of several hybrid organic-inorganic nanomaterials. We have developed mesoporous silica nanoparticles and gold nanoparticles for biomedical applications, specifically in the breast cancer area. The first chapter includes an overview of the concepts related to the research performed. Introductory notions about nanotechnology and biomedicine are presented, as well as the basis of the interactions of nanoparticles with biological systems. Finally, breast cancer disease and the application of nanomaterials as therapy are described. Next, in the second chapter, the objectives addressed in the following experimental chapters are displayed. In the third chapter, we present the first nanomaterial for the controlled delivery of two inhibitors (navitoclax and S63845) of the Bcl-2 anti-apoptotic proteins. This nanosystem has been designed to overcome navitoclax resistance in a triple-negative breast cancer cellular model. We have prepared mesoporous silica nanoparticles loaded with navitoclax and S63845 and functionalised with an aptamer targeting MUC1 surface protein as a molecular gate. In this work, the specific targeting of the nanodevice to breast cancer cells has been demonstrated. The ability to overcome navitoclax resistance has been shown in navitoclax-resistant triple-negative breast cancer cells. Furthermore, navitoclax encapsulation in the nanoparticles has proved to reduce the main adverse effect (thrombocytopenia) associated with free formulated drug administration. In the fourth chapter, we describe a pH-responsive nanosystem for the controlled co-delivery of a fluorescent cargo and the genome-editing machinery based on CRISPR/Cas9, which targets the green fluorescent protein (GFP) coding gene. The nanodevice consists of mesoporous silica nanoparticles loaded with rhodamine B, functionalised with polyethyleneimine, and capped with the CRISPR/Cas9 plasmid. In the present work, we have shown the lysosomal scape capacity of the nanodevice enhanced by the proton sponge effect of PEI. We have also demonstrated a pioneering mesoporous silica-based nanodevice efficient in the simultaneous genome editing of the GFP gene (as a model gene) and the successful release of a fluorescent cargo (as a model drug). In the fifth and last experimental chapter, we propose a new approximation to develop enzyme prodrug therapy using gold nanoparticles as enzyme carriers. In this case, we use gold nanoparticles functionalised with the enzyme horseradish peroxidase (HRP), which transforms the non-toxic prodrug indol-3-acetic acid into radical species toxic to tumour cells. In this chapter, the therapeutic effect of the nanodevice in combination with the prodrug has been demonstrated in two breast cancer cell subtypes (luminal A and triple-negative breast cancers). Also, the therapeutic effect of the material has been corroborated in multicellular tumour spheroid-like cultures formed by triple-negative breast cancer cells. Finally, in the sixth chapter, the conclusions derived from the presented studies and the general conclusions of this Ph.D. thesis are released. The obtained results will promote the development of new smart nanomaterials with diverse biomedical applications.<br>Gema Vivo-Llorca thanks the Generalitat Valenciana for her fellowship ACIF/2017/072. Vicente Candela-Noguera thanks the Spanish Government for his fellowship FPU15/02753. We would like to thank Servier for the workart used in the figures of this manuscript (Servier Medical Art https://smart.servier.com/). We thank the Spanish Government (project RTI2018-100910-B-C41 (MCUI/AEI/FEDER, UE); SAF2017-84689-R-B (MCUI/AEI/FEDER, UE)) and the Generalitat Valenciana (project PROMETEO/2018/024 and PROMETEO/2019/065) for support.<br>Vivo Llorca, G. (2021). Mesoporous silica and gold-based nanodevices: new controlled delivery platforms for biomedical applications [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/172713<br>TESIS

New imidazotetrazinones have been synthesised that possess features in their structures to release aziridinium ions upon ring opening. Unstable 2-aminoethylisocyanates were required in this preparation, which were synthesized with BOC-protection of the amino group to counteract the reactivity of the amine towards the isocyanate group in the case of aliphatic amines; in contrast, anilinoethylisocyanates were synthesized unprotected. Substituents with a range of electron-withdrawing and electron-releasing properties were introduced at the p-position of the aniline ring. A 13C-labelled study confirmed the release of the aziridinium ion by these imidazotetrazinones in neutral pH buffer solution. Furthermore the kinetics of the hydrolysis in neutral aqueous solution of some these new tetrazines were similar to temozolomide, in addition to useful acid stability. Other imidazotetrazinones were synthesised for the purpose of releasing alcohols and phenols. Their synthesis was performed with a one-carbon linker between the imidazotetrazinone 3-position and the alcohols or phenols to be released. The release of alcohol and phenol through the hydrolysis of the intermediate diazonium ions to the unstable hemiacetals that decomposed to the alcohol and phenol was confirmed by 1H NMR. The kinetics of the hydrolysis of these tetrazines in neutral aqueous solution showed a faster reaction rate compared with temozolomide (t1/2 = 0.53 and 0.36 h compared with temozolomide 1.4 h).<br>Full text was made available at the end of the embargo period, 1st Feb 2016.

New imidazotetrazinones have been synthesised that possess features in their structures to release aziridinium ions upon ring opening. Unstable 2-aminoethylisocyanates were required in this preparation, which were synthesized with BOC-protection of the amino group to counteract the reactivity of the amine towards the isocyanate group in the case of aliphatic amines; in contrast, anilinoethylisocyanates were synthesized unprotected. Substituents with a range of electron-withdrawing and electron-releasing properties were introduced at the p-position of the aniline ring. A 13C-labelled study confirmed the release of the aziridinium ion by these imidazotetrazinones in neutral pH buffer solution. Furthermore the kinetics of the hydrolysis in neutral aqueous solution of some these new tetrazines were similar to temozolomide, in addition to useful acid stability. Other imidazotetrazinones were synthesised for the purpose of releasing alcohols and phenols. Their synthesis was performed with a one-carbon linker between the imidazotetrazinone 3-position and the alcohols or phenols to be released. The release of alcohol and phenol through the hydrolysis of the intermediate diazonium ions to the unstable hemiacetals that decomposed to the alcohol and phenol was confirmed by 1H NMR. The kinetics of the hydrolysis of these tetrazines in neutral aqueous solution showed a faster reaction rate compared with temozolomide (t1/2 = 0.53 and 0.36 h compared with temozolomide 1.4 h).

La malaria es una enfermedad infecciosa producida por Plasmodium, protozoo transmitido a través de la picadura de mosquitos del género Anopheles. Aunque existen tratamientos y medidas de prevención, en los países tropicales sigue teniendo una alta incidencia debido a problemas como la aparición de resistencias (tanto a los fármacos como a los insecticidas), la inexistencia de una vacuna eficaz y la necesidad de herramientas de diagnóstico más baratas y sencillas para llevar a zonas rurales, entre otras cuestiones socioeconómicas. A nivel de investigación, las herramientas de nanotecnología se exploran para mejorar la eficacia de fármacos, como componentes de vacunas o fabricación de nuevos fármacos o insecticidas. Sin embargo, las herramientas de direccionamiento de los sistemas de tratamiento o las usadas en detección de antígenos para el diagnóstico aún no cumplen los requisitos de relación coste/efectividad para una enfermedad de los países en desarrollo. Este trabajo se ha enfocado en la exploración de interacciones ligando-diana de los compuestos glicosaminoglicanos sulfatados y el desarrollo de nuevos elementos direccionadores, los aptámeros, como nuevas herramientas con posible aplicación en la eliminación de la malaria. Esta investigación ha quedado compilada en tres artículos. Los dos primeros artículos resumen los resultados de la aplicación de heparina y sus derivados para bloquear el desarrollo del parásito, tanto en las fases de infección de mamíferos como en el mosquito. En el tercer artículo se describe la selección y caracterización de aptámeros con potencial aplicación al diagnóstico de la malaria. Estas herramientas de direccionamiento hacia el parásito son más baratas que los anticuerpos y se podrían aplicar en sistemas nanotecnológicos de transporte de fármacos, utilizar como adyuvantes de tratamientos o incluir en pruebas de diagnóstico.

L’objectif de ce travail a été de développer deux approches analytiques dédiées à l’analyse toxicologique des nouveaux produits de synthèse (NPS) dans différentes matrices biologiques (sang, urine et cheveux). La première est basée sur le criblage non ciblé par chimiluminescence sur biopuces et chromatographie liquide couplée à la spectrométrie de masse haute résolution (LC-HRMS) et la deuxième correspond à un criblage ciblé par spectrométrie de masse en tandem (LC-MS/MS). Ces deux approches ont ensuite été appliquées dans des études observationnelles pour évaluer la consommation de NPS dans des populations à risques de surdosage, de pharmacodépendance ou de soumission chimique dans un contexte clinique ou médico-judiciaire.La dernière partie a été consacrée au développement d’un nouvel outil analytique de traitement des données issues de la LC-HRMS qui a permis d’étudier le métabolisme de 9 NPS in vitro sur des cultures de microsomes du foie humain (HLM) et in vivo sur des échantillons biologiques d’usagers de ces drogues. Cette dernière approche a permis la création d’une bibliothèque de spectres de haute résolution composée de 228 métabolites dont certains ont été proposés comme marqueurs pertinents d’exposition aux NPS dont ils sont issus.Ce travail a été concrétisé par la rédaction de 10 publications scientifiques et a permis d’initier plusieurs collaborations pluridisciplinaires<br>The aim of the present work was to develop two analytical approaches dedicated to the analysis of new psychoactive substances in different biological matrices (blood, urine and hair). The first approach is based on untargeted screening by both biochip array technology chemiluminescence assay and liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS) and the second corresponds to a targeted screening by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). These two approaches were then applied in observational studies to assess the consumption of NPS in high risk populations (overdose, drug abuse, drug facilitated crimes) in clinical and forensic settings. The last part of the work was devoted to the development of a new analytical tool for LC-HRMS data processing which made it possible to study the metabolism of 9 NPS In vitro on human liver microsomes (HLM) and In vivo in biological samples from drug users. This approach has enabled the creation of HRMS spectral library containing 228 metabolites, some of which have been proposed as relevant markers of NPS exposure.This work has resulted on 10 scientific publications and allowed to initiate many multidisciplinary collaborations

L'environnement marin est doté d’une diversité fongique encore trop faiblement explorée puisqu’on estime qu’environ 10% des champignons marins ont fait l’objet d’une étude. Dans ce contexte, le projet de thèse décrit dans ce manuscrit est focalisé sur le potentiel biotechnologique des champignons marins isolés d’éponges marines. Ces champignons sont caractérisés par une importante biodiversité et chimiodiversité susceptible de conduire à de nouvelles molécules bioactives. Il s’agit d’un projet pluridisciplinaire qui joint la mycologie, la chimie, la biochimie et les biotechnologies. Il couvre la stratégie complète de découverte de nouveaux produits naturels avec l'isolement et l'identification des souches fongiques à l’extraction et l'isolement des molécules ainsi que l’évaluation des propriétés biologiques. Le manuscrit est divisé en trois parties principales : - La première partie est dédiée à l’isolement des communautés fongiques cultivables associées à quatre éponges de l’océan Atlantique et trois éponges de Méditerranée. Nous avons obtenu au total 129 taxons parmi lesquels 84,5% ont pu être identifiées jusqu’au niveau de l’espèce via une approche polyphasique basée sur des techniques morphologiques, moléculaires et phylogénétiques. Parmi ces derniers, nous avons décrit pour la première fois deux espèces : Thelebolus balaustiformis et Thelebolus spongiae. Nos travaux ont permis de souligner la spécificité des communautés fongiques hébergées par chaque éponge ce qui laisse à penser que les éponges sont capables de recruter leur propre mycobiote. - La seconde partie est consacrée à l'étude de la diversité chimique des champignons marins associés à l'éponge Grantia compressa en utilisant l'approche OSMAC (une souche – de nombreux composés). Les résultats obtenus ont révélé les difficultés à obtenir des conditions de culture optimales. De façon générale et pour tous les champignons, les milieux riches en nutriments favorisent à la fois le développement du mycélium et la production de métabolites secondaires. Parmi les champignons isolés, Eurotium chevalieri MUT 2316 produits de nombreux métabolites, comparativement aux autres champignons. Dans ce contexte, nous avons pu isoler et caractériser dix composés. - La troisième partie est dédiée à l’évaluation des propriétés biologiques (pharmacologiques et environnementales) des différentes molécules isolées. Six composés ont montré des propriétés antibactériennes notamment l'isodihydroauroglaucine qui s’est avérée active vis-à-vis de la plupart des bactéries à Gram-positif testées et pour laquelle une activité bactéricide a pu également être décelée. La dihydroauroglaucine et le physcion inhibent complètement la réplication du virus de la grippe A tandis que la neoechinuline inhibe le virus de l'herpès simplex 1. Enfin, les différentes molécules ont été évaluées pour leurs propriétés antifoulings susceptibles de rentrer dans la composition de peintures plus respectueuses de l’environnement. Les molécules inhibent à de très faibles concentrations l'adhésion et la croissance de bactéries et de microalgues impliquées dans le biofouling. Par ailleurs, la combinaison de molécules isolées d’E. chevalieri MUT 2316 inhibe la totalité des bactéries et microalgues testées. Les travaux menés ont permis de mettre en avant l’importante biodiversité et chimiodiversité de champignons marins hébergés par les éponges. Les molécules isolées d’E. chevalieri MUT 2316 sont susceptibles de valorisation dans différents domaines de recherche tels que le développement de nouveaux médicaments ou de peintures antifoulings plus respectueuses de l’environnement<br>Marine environment represents an untapped source of fungal diversity, where it has been estimated that about 10% of fungi have been explored until now. Due to the lack of knowledge on marine fungi and their incredible biotechnological potential, this Ph.D. thesis focuses on a highly promising group of fungi: those associated with marine sponges. These fungi are both characterized by high biodiversity and chemodiversity, being the most successful producers of new bioactive molecules. On these premises, the main goal of the research was to cover the firsts and fundamentals aspects of the natural products discovery pipeline: from the isolation and identification of fungi from sponges to the isolation of molecules and the evaluation of their biological activity. This resulted in a multidisciplinary Ph.D. project that enclosed mycology, chemistry, biochemistry and biotechnology. In a “funnel-like” perspective, using multidisciplinary experimental approaches three main parts were developed: - The first aim was to isolate the fungal communities associated with sponges using several isolation techniques to increase the number of cultivable fungi. Four and three sponges were respectively collected in the Atlantic Ocean and in the Mediterranean Sea. Overall, 129 taxa were obtained; thanks to a polyphasic approach based on morphological, molecular and phylogenetic techniques, 84.5% of them were identified at the species level. Two fungal species Thelebolus balaustiformis and Thelebolus spongiae were here first described, updating the knowledge on marine fungal diversity. This work underlined the specificity of the fungal community for each sponge, leading to think that these animals are able to recruit their own mycobiota. - The second part was based on the investigation of the chemical diversity of marine fungi associated with the sponge Grantia compressa, using the OSMAC approach (One Strain – Many Compounds). Not surprisingly, it has been difficult to define a condition that promotes both the development of the mycelium and the secondary metabolites production for all fungi; generally, rich nutrients media are the best candidates to achieve the above-mentioned results. Among the tested fungi, Eurotium chevalieri MUT 2316 produce more metabolites than any other fungus and ten pure compounds were isolated. - The third part of this Ph.D. project aimed to test the biological activity of the ten fungal molecules. Two main research fields, pharmaceutical and environmental, were chosen as potential targets. Six compounds showed antibacterial activity, with isodihydroauroglaucin active against most of the Grampositive bacteria tested also with bactericidal activity. Dihydroauroglaucin and physcion were able to completely inhibit the replication of Influenza A virus, while neoechinulin completely inhibited Herpes Simplex Virus 1. Finally, the last series of bioassays aimed to face the urgent need of environmentally friendly antifouling and highlighted several molecules already active at extremely low concentrations, inhibiting the adhesion and growth of both bacteria and microalgae. As result, a mix of few compounds produced by E. chevalieri MUT 2316 would inhibit all the bacteria and microalgae tested. In conclusion, this Ph.D. project highlighted the outstanding biodiversity and chemodiveristy of marine fungi inhabiting sponges. The molecules isolated from E. chevalieri MUT 2316 found applications in different research fields and represent promising candidates for the development of new drugs and antifouling paints

During the past few decades gene therapy has become a promising alternative for the treatment of many incurable diseases such as cancer. This extremely complex disease possesses characteristics that make most of the generally used treatments rather ineffective. The research work presented in this thesis attempts to take advantage of novel non-viral gene therapeutic approaches based on plasmidic DNA and nanoparticulated systems that might provide the foundation for the development of a novel therapeutic treatment from the production to a real application in the everyday life of cancer patients. Hence the production of an expression vector that encodes for a tumor suppressor, p53 was promoted in recombinant organisms. Subsequently the different plasmid DNA isoforms were isolated and the supercoiled isoform, the one biologically active and with enhanced transfection efficiency was purified by affinity chromatography. Following this purification and recovery the development of novel nanoparticle systems that could deliver the exogenous DNA into the malignant cells was promoted. Nanoparticulated systems produced with chitosan demonstrated small size ranges and suitable properties for the encapsulation of plasmidic DNA. Additionally, transfection of eukaryotic neoplastic cells revealed the suitability of the nanocarrier as a novel delivery system of p53 expression vectors to cancer.

碩士<br>國立中央大學<br>化學學系<br>102<br>The first part of my research is about annular rings with controllable sizes are successfully created on the surface of COOH-functionalized mesoporous silica (namely, XC-CAR-10, where X represents the number of carbons in the diamines used) for the adsorption of metal ions. Particularly, excellent adsorption ability of 3C-CAR-10 towards Er3+ (ca. 174 μg/mg) was achieved. Reversible phosphorylation of proteins is a common theme in the regulation of important cellular functions such as growth, metabolism, and differentiation. Although, the number of cellular phosphoproteins is relatively high, the phosphorylated residues themselves are generally of low abundance due to the sub-stoichiometric nature. Additionally, phosphopeptides with phosphate groups are able to form stable complexes with the lanthanide ions by the formation of precipitation. In this study, thus, we reported an annular mesoporous adapted with trivalent lanthanide ions Er3+ on for the applications of phosphopeptide enrichment and cleaning-up. The second part of my research is about a new style drug carrier with designed DNA as a cap to control drug release. We synthesized mesoporous silicas with an amine group on the surface, named AM-SBA15. Due to owning positive charge of AM-SBA15, it can be formation of positive-negative interaction between AM-SBA15 and DNA phosphate groups at neutral condition. Thus, we designed a short length DNA with 17 EcoRV cutting cites as caps on the surface of AM-SBA15 mesoporous silica. According to investigation results, DNA shows a good capability for covering pores on the surface of AM-SBA15 so as to not allow fluorescein released. Remarkably, the fluorescein will be released upon EcoRV added because restriction enzyme of EcoRV digests DNA into smaller fragments. Finally, we develop a novel platform as a drug carrier and release system. Drugs will be released when composites are triggered by specific restriction enzyme.

FDA decisions and late-stage clinical trial results regarding new pharmaceutical approvals can cause extreme moves in the share price of small biopharmaceutical companies. Throughout the clinical trial process, many potential investors are exposed to market-moving information before such information is made available to the investing public. An investor who wished to profit from advance knowledge about clinical trial results may use the publicly traded options markets in order to increase leverage and maximize profits. This research examined options data surrounding the public release of information pertaining to the efficacy of clinical trials and approval decisions made by the FDA. Events were identified for small pharmaceutical companies with fewer than three currently approved drugs in an attempt to isolate the effect of individual clinical trial and FDA-related events on the share price of the underlying company. Option data were analyzed using logistic regression models in an attempt to predict phase II and III clinical trial outcome results and FDA new drug approval decisions. Implied volatility, open interest, and option contract delta values were the primary independent variables used to predict positive or negative event outcomes. The dichotomized version of a predictor variable designed to estimate total investment exposure incorporating open interest, option contract delta values, and the underlying stock price was a significant predictor of negative pharmaceutical related events. However, none of ii the variables examined in this research were significant predictors of positive drug research related events. The estimated total investment exposure variable used in this research can be applied to the prediction of future clinical trial and FDA decision related events when this predictor variable shows a negative signal. Additional research would help confirm this finding by increasing the sample size of events that potentially follow the same pattern as those examined in this research.<br>text

博士<br>國防醫學院<br>生命科學研究所<br>102<br>Anti-tuberculosis drug-induced hepatotoxicity (ATDIH) is the most common adverse drug reaction caused by first-line anti-TB drugs, i.e. isoniazid (INH), pyrazinamide (PZA) and rifampin (RIF). Among them, INH is the most potent single agent against TB but is also the most frequent cause of ATDIH. Prior researches indicated that hepatotoxic free radicals and hydrazine, deriving from catalysis of INH by CYP2E1 and amidase, are the major contributions to INH induced liver injury. The hepatotoxic side effects should rationally be avoided through suitable modifications on CYP2E1 and amidase enzyme activity. In addition, it has been reported that certain genetic polymorphisms of the metabolic enzymes are also the risk factors contributed to ATDIH. Therefore, it is important to understand the correlations between the genetic variation of these metabolic enzymes and the clinical outcome in patients. The primary objectives of this study are: (1) To screen of effective novel amidase inhibitors from common pure compounds derived from food, natural products or pharmaceutical excipients, so that to inhibit amidase activities from human liver and intestines and to improve clinical benefits by avoiding unwanted side effects. (2) To investigate the genetic polymorphisms of metabolic enzymes and correlate them with the susceptibility of anti-TB drugs induced hepatotoxicity in TB patients, so as to select representative high risk genotypes in patients with the hope for applying the genetic information in routine rapid diagnosis. In the current study, we use INH as the model drug to establish an in vitro screening test for amidase inhibition. Among 94 Chinese herbal medicine, pure compounds and pharmaceutical excipients tested, 7 were found potent on inhibiting amidase activities by at least 50% or above. HUCHE033, a pure compound with 65.9% in vitro amidase inhibition, was chosen for subsequent animal study in vivo. Effect of protection from INH or INH/RIF induced hepatotoxicity has been demonstrated in mice animal model. Furthermore, it also showed in vivo inhibitory effects on the intrinsic amidase and CYP2E1 in pharmacokinetic studies, both in human and mice. On the other hand, we analyzed the allele frequency of 43 SNP genetic polymorphisms on INH related metabolic enzymes, including NAT2, CYP2E1 and CES1. Risks for developing ATDIH among different genotypes were also compared. Results showed that, 7 NAT2 SNPs and 4 CES1 SNPs were highly correlated to the incidence of ATDIH, while 7 CYP2E1 SNPs were shown correlated with the intensity (or severity) of ATDIH. Among the best predictive genotypes, patients with high risk genotypes had 3.8 to 14.0 folds higher odds to develop ATDIH, as defined according different levels of liver injury, as compared to those without the genotypes. In conclusion, the study successfully identified amidase inhibitors with competent potential for developing new anti-TB drug combinations with low hepatotoxic side effects. Several SNP genotypes from the drug metabolic enzyme NAT2, CYP2E1 and CES1 were also demonstrated predicting values on susceptibilities of ATDIH in patients. These results should help to provide novel pathways on development of new diagnosis or medications for caregivers to combat with TB.

碩士<br>國立臺北科技大學<br>生物科技研究所<br>99<br>Alzheimer’s disease is the most common cause of dementia characterized by progressive cognitive impairment in the elderly. It is a chronic, slowly progressive neurodegenerative disorder. The gradual loss of memory, decline in other cognitive functions, and decrease in functional capacity result in death approximately 8-10 years after the onset of the symptoms. It is accompanied by dysfunctions in the cholinergic neurotransmission of the central nervous system. Hence, most of the drugs approved for AD treatment are acetylcholinesterase inhibitors (AChEIs), which can enhance cholinergic neurotransmission by increasing acetylcholine availability in the synaptic cleft. In this study, molecular docking experiments combined with a consensus scoring function were conducted to predict the binding affinities of a total of 88 AChEIs, in which 68 and 20 compounds were used in the training and test sets, respectively, and to characterize the structural features of the catalytic gorge of acetylcholinesterase (AChE) toward binding. Our results yielded correlation coefficients R2 = 0.8439 and 0.9573 for the training and test sets, respectively, after partial least squares regression and leave-one-out cross-validation coefficient Q2 = 0.6291, indicating that the consensus scoring function developed here is applicable to bioactivity prediction and structural characterization for AChE inhibition. The identification of the protein-ligand interactions produces a list of those residues within the dual binding site of AChE, which make the most important hydrogen bond,

β-lactamase enzymes catalyze the irreversible hydrolysis of the four-membered cyclic amide ring characteristic of β-lactam antibiotics rendering them inactive and useless against pathogenic bacteria. Understanding structure activity relationships between β-lactam antibiotics and β-lactamases is important for designing novel β-lactams, β-lactamase inhibitors, and β-lactam-based fluorescent probes for rapid diagnosis of β-lactam antibiotic resistant infections. The first half of this study focuses on the class A β-lactamase BlaC from Mycobacterium tuberculosis (Mtb) and addresses intermolecular interactions between BlaC and substrates, inhibitors, and biosensors that influence their kinetic parameters with BlaC and activities against Mtb. The substrate structure activity relationship explained the molecular basis for differential innate resistance of Mtb to faropenem, biapenem, and tebipenem by showing the interactions between BlaC and the lactams that govern differential acyl-intermediate stability and affinity. The inhibitor structure activity relationship revealed features of the BlaC active site that can be exploited to enhance binding and inhibition of BlaC by benzoxaboroles, and demonstrates their utility as potentiators of β-lactam antibiotic activity against Mtb. BlaC-specific β-lactam based fluorescent probes were designed and optimized for Mtb detection. Their utility was demonstrated by detecting down to 10 colony forming units of bacillus Mycobacterium bovis Calmette–Guérin (BCG) in human sputum. The second half of this study focuses on the New Delhi Metallo-β-lactamase-1 (NDM-1), which is rapidly generating bacterial resistance to nearly all β-lactams. The NDM-1 gene encodes a class B1 metallo-β-lactamase enzyme. Purified recombinant NDM-1 was biochemically and biophysically characterized. The crystal structures of apo and monometalated NDM-1 provided structural insight into metal binding and the promiscuous enzymatic activity of NDM-1. Mechanistic details of the NMD-1 reaction were examined by comparing crystal structures of NDM-1 in complex with an unhydrolyzed β-lactam substrate and with hydrolyzed products. These structures were used for quantum mechanics / molecular mechanics simulations to estimate the free energy along the β-lactamase reaction coordinate. The results suggest that NDM-1 uses bulk water as the nucleophile that attacks the β-lactam ring, and a coordinated hydroxide ion or water molecule as the catalytic base depending on pH.

博士<br>國立中興大學<br>化學系<br>93<br>Aminoalcohols have been recognized as good ligands for catalyzing many asymmetric reactions, such as borane reduction of ketone and diethylzinc addition to aldehyde. However, aminothiols were relatively rare to be studied as catalysts for similar reactions. Recently, we found aminothiol with simple structure could actually be used as an excellent chiral catalyst for asymmetric addition of diethylzinc to aldehyde. The enantioselectivity can be as high as 99.0% e.e. when only 0.02mol% of aminothiol was used as to catalyze the addition process. These phenomena indicated that the aminothiol was extremely efficient to accelerate the addition process in asymmetric fashion. These aminothiols were easily prepared in large quantity from some naturally occurring aminoacids through a few simple steps. All of the properties described above have made these aminothiols become practically useful for industrial purpose. This disseration will discuss the stoichiometry of organozinc as well as the aminothiol ligands used in asymmetric reaction. Meanwhile, solvent effects and temperature effects of the title reaction will also be discussed.

Commonly prescribed small molecule drugs can have net-positive and well-understood safety profiles when prescribed individually, but unexpected consequences when taken at the same time. Detection of these drug-drug interactions (DDIs) continues to be a critical and unmet area of translational research. The Centers for Disease Control and Prevention (CDC) estimate that one third of Americans are concurrently taking two or more prescription drugs, and DDIs are estimated to be responsible for 17% of all drug adverse events. The consequences of DDIs can be relatively minor (headache, skin rash) or much more severe (bleeding, liver toxicity). At a cellular level, DDIs can occur as a result of both drugs competing for metabolism (known as pharmacokinetic interactions) or targeting the same protein target or biological pathway (pharmacodynamic interactions). Clinical trials typically focus on the effects of individual drugs, leaving DDIs to usually be discovered only after the drugs have been approved. One of the most carefully studied drug adverse events is long QT syndrome (LQTS), an unexpected change in the heart's electrical activity that can lead to a potentially fatal ventricular tachycardia known as torsades de pointes (TdP). Some patients have genetic mutations that lead to congenital forms of LQTS, while drug-induced LQTS typically occurs via block of the hERG potassium channel (KCNH2) responsible for ventricular repolarization. After a number of high profile drugs were withdrawn from the market due to discovered risk of TdP, the FDA issued guidelines so that pharmaceutical companies could anticipate and test for this side effect before a new drug is approved. These recommendations have helped prevent new QT-prolonging drugs from entering the market, but nonetheless over 180 approved drugs have been associated with drug-induced LQTS. While information on individual QT-prolonging drugs is thus readily available to clinicians, little has remained known about DDIs (QT-DDIs). There are many more commonly prescribed drugs that are safe when given individually but could increase TdP risk when administered together. This troubling situation is compounded by the fact that traditional post-market surveillance algorithms are poorly equipped to sensitively and specifically detect DDIs. Data science – the application of rigorous analytical methods to large datasets – offers an opportunity for predicting previously unknown QT-DDIs. Some biomedical datasets (such as drug-target binding affinities and experiments to determine protein-protein interactions) have been collected explicitly for research, while other valuable datasets (such as electronic health records) were initially recorded for billing purposes. Each data modality has its own important set of advantages and disadvantages, and integrative data science approaches can incorporate multiple types of data to help account for these limitations. In this thesis we develop new data sciences techniques that combine clinical, biological, chemical, and genetic data. These approaches are explicitly designed to be robust to biased and missing data. We apply these new methodologies to (1) predict new QT-DDIs, (2) validate them experimentally, and (3) investigate their molecular and genetic mechanisms. We exemplify this approach in the discovery of a previously unknown QT-DDI between ceftriaxone (cephalosporin antibiotic) and lansoprazole (proton pump inhibitor); importantly, both drugs have no cardiac indications and are safe when given individually. The clinical data mining, drug target prediction, biological network analysis, genetic ancestry prediction, and experimental validation methods described in this thesis form the basis for a comprehensive pipeline to predict QT-DDIs rapidly and robustly. They also provide an opportunity for further enriching our understanding of LQTS biology and ultimately enabling the design of safer drugs.

碩士<br>東吳大學<br>財務工程與精算數學系<br>105<br>The biotechnology industry is known as the star industry in the 21st century, and it contains unlimited business opportunities. However, the development of new drugs usually takes decades. During this time, facing a huge demand for funds, the uncertainty results of clinical trials, the potential competitors to join, and the unsure of future market demands…all these factors may raise the difficulties of evaluating the project value. If the traditional discounted cash flow method (DCF method) is used for evaluation, it fails to show the flexibility of the decision-making, and therefore it is less suitable for the new drug research and the development projects. In view of the above characteristics, this study considers a variety of uncertain factors that can influence the evaluation of R & D value of new drugs. The least-squares Monte Carlo simulation (LSM) is used as the main model to evaluate the value of the program. Also, in order to concern about the decision-making flexibilities that the decision makers have during the R & D process of new drugs, this study joins one of the real options—option to abandon in this model. In addition, this study will also take the possibilities of clinical trials adoption into the consideration of value assessment, in order to make the model closer to the reality. Finally, the analysis of sensitivity in each parameter of the model is used to summarize the important variables that affect the R & D value of the new drug, which can be used as the basis for the later research reference or improvement. Key words: discounted cash flow method, least-squares Monte Carlo simulation method, real options, option to abandon

The rational design of systems for controlled drug delivery is an important area of research for advancing new therapies for many diseases. Nanomaterial based controllable drug delivery platforms would overcome many of the major drawbacks in pharmacological therapy, because they would store the therapeutic molecules during transportation within the body and provide triggered and finely controlled release of the agent at the target site. In this context, lipid vesicles as liposomes attracted, since their discovery, growing interest for their potential applications as drug delivery vectors. They are now considered clinically established nanometer-scaled systems for the delivery of cytotoxic drugs or agents for biomedical applications. However, liposomes can be further engineered to improve their performance in terms of stability and controlled delivery, since the rapid degradation, due to the reticuloendothelial system (RES), and inability to achieve sustained drug delivery, over a prolonged period, limit their biological efficacy and use in pharmaceutics. Interesting results were obtained in terms of physical stability through the approach that provides the combination of different biomaterials within the same delivery lipid system. Following this concept, it was possibile to change the surface properties of the bilayer, by coating it with water-soluble polymers, like polyethylene glycol, leading to “stealth liposomes”. More recently, satisfying achievements resulted from the modification of the internal structure of liposomes, with the aim to convert the aqueous inner core into a soft and elastic hydrogel, obtaining structures able to retain the cargo for longer time, without unwanted burst release of the delivered compound. An ideal drug delivery platform should encompass not only the carrier stability, but also controllable timing, dosage and site specificity of drug release, and permit remote, noninvasive and reliable switching of the therapeutic agent, in order to prevent deleterious side effects of cytotoxic drugs toward normal and healthy tissue. Much of innovations in materials design, for drug delivery, manifest in producing “smart liposomes” that are able to respond to “smart triggers”, in order to realize on-demand processes, allowing for tailored release profiles with excellent temporal and dosage control. In principle, the ondemand drug delivery is becoming feasible through the design of stimuli-responsive systems that recognize their microenvironment and react in a dynamic way. Specifically, it is possible to engineere the liposomal structure making it capable of responding to physical, chemical or biological triggers. Among the endogenous or exsogenous stimuli that can be applied, magnetic stimulus represents a potential trigger for the remotely-on demand release, evaluating that normal biological tissues are essentially transparent to low-frequency magnetic fields. Through the encapsulation of superparamagnetic nanoparticles, giving rise to the Magneto-Liposomes (MLs), it is possibile to modulate the transmembranal drug diffusion by using an external magnetic field with intensity significantly lower that no heat generation, harmful to healthy tissues, is observed. Another challenging way to activate release from liposomes is the use of pulsed electric fields. In particular, since it was demonstrated that electric pulses of shorter duration (nanosecond) and higher intensity (in the order of MV/m) directly interact not only with cell membrane, but also with internal cell organelles of nanometer dimensions, nanosecond electric pulses are proposed as sufficient signals to generate an alteration of the liposomal transmembrane voltage, which is followed by the formation of temporary hydrophilic pores. Without implying the phenomenon of irreversible poration, the nanosecond pulses therefore can be considered useful external stimuli to trigger the simultaneous permabilization of liposomes and cells membranes, in order to let that the chemical load internalized in the vesicles to be released inside the cells. In this scenario is placed the main activity of this Ph.D. thesis, whose aim is to provide a multiscale and multidisciplinary approach to demonstrate the capability of liposomes to prove effective smart systems for the on-demand and modified drug delivery, able to minimize off-target effects and maximize programmability of therapy. Following a briefly overview of this Ph.D. thesis is given. In Chapter 3 is reported the study which highlights the utility in trapping MNPs within phospholipid vesicles, generating hybrid magneto-responsive constructs. Particularly, in this work the inclusion of hydrophilic Fe3O4 nanoparticles (MNPs) within phospholipid vesicles, characterized by different rigidity and stiffness, was investigated as novel strategy for improving stability and reactivity of these MNPs, since the integration in liposomes may prevent MNPs from aggregation and extend their potential use in the environmental remediation. The stability of these hybrid systems was indirectly investigated evaluating the ability of retaining a fluorescent marker in their structure, under both mechanical and thermal stress conditions. In particular, for the mechanical stress test, a low intensity nonthermal alternating magnetic field (AMF) was applyed to magnetic liposomes. The AMF could, in fact, cause a mechanical destabilization of the vesicle membrane, due to MNPs oscillation within the liposomes, which may induce the release of the dye. In Chapter 4, according to the results obtained in the previous work, shown in Chapter 3, is presented the research project which combines engineering skills, specifically focused on electromagnetic fields, with competences in synthesis and characterization of hybrid magnetic nanocarriers, to assess a remotely on-demand drug delivery. Specifically, here is refiled the possibility to trigger drug release from high-transition temperature magnetoliposomes (high-Tm MLs) entrapping MNPs, through a magneto-nanomechanical approach, where the mechanical actuation of the MNPs is used to enhance the membrane permeability, avoiding temperature rise. Since the AMF, as an external magnetic signal, found rare application in clinic, in this case, the ability of the non-thermal pulsed electromagnetic fields (PEMFs), that are already employed in theraphy, due to their antiinflammatory effects, was tested, in order to verify if, once applied to high-Tm MLs, PEMFs could be able to efficiently trigger the transmembranal drug diffusion. In the Chapter 5 and 6 is illustrated another sophisticated and innovative drug delivery strategy to activate an efficient on-demand release. Specifically, in Chapter 5 is reported the theoretical work and the experimental proof-of-concept of the possibility of applying ultrashort (ns) and intense (MV/m) external pulsed electric fields (nsPEFs), to remotely trigger the release from liposomes of nanometer-sizing. The nanoelectropermeabilization, that probably occurs with the formation of transient pores in the bilayer, because of the external pulsed electric field enforcement, was evaluated in relation to the diffusion across the bilayer of a probe, previously trapped in the core of liposomes. To support the experimental data, a numerical model of liposomes suspension, exposed to nsPEF by means a standard electroporation cuvette, was carried out according with the experimental conditions. In Chapter 6 was demonstrated, once again, the possibility of permeabilizing the liposomal membrane, applying the same type of pulses, described in Chapter 5, but, in this case, delivered to the nanometer-sized lipid vesicles by a coplanar exposure system. Furthermore, the electropermeabilization mechanism in liposomes membrane was investigated through the Raman Coherent anti-Stokes spectroscopy (CARS), highlighting, for the first time, the experimental proof of the role of water molecules of the interstitial phase in the electropermeabilization of vesicles bilayer. Finally, in Chapter 7 is described the project who leaded to the development of a novel hybrid lipid-polymer nanocostructs, designed to merge the beneficial properties of both polymeric drug delivery systems and liposomes in a single nanocarrier and at the same time take care of liposomes limitations, such as the physical and chemical stability issues. Starting from previous studies on the use of liposomes as template to create nanohydrogel, this research brought to the novel Gel-in-Liposome (GiL) systems, relying from the combination of lipid vesicles and the polymer polyethylene glycol-dimethacrylate (PEG-DMA) at two different molecular weight. These hybrid systems are characterized by the presence of a chemically crosslinked polymeric network within the aqueous compartment of liposomes. The effect of PEG-DMA, on the properties of the new lipid-polymer nanosystems and on the related changes of the membrane permeability and stability, against different stresses, were evaluated to understand GiL potential use as drug carriers in clinics.