Detection of hormones in surface and drinking water in Brazil by LC-ESI-MS/MS and ecotoxicological assessment with Daphnia magna

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Etablierung eines high content imaging-basierten in vitro Testsystems zur Evaluierung genotoxischer Substanzen

Zur Registrierung von Pharmazeutika ist eine umfassende Analyse ihres genotoxischen Potentials von Nöten. Aufgrund der Vielzahl genotoxischer Mechanismen und deren resultierenden Schäden wird ein gestaffeltes Testdesign durch die ICH-Richtlinie S2(R1) „Guidance on genotoxicity testing and data interpretation for pharmaceuticals intended for human use S2(R1)“ definiert, um alle genotoxischen Substanzen zu identifizieren. Die Standardtestbatterie ist in der frühen Phase der Arzneimittelentwicklung aufgrund des geringen Durchsatzes und des Mangels an verfügbarer Substanzmenge vermindert anwendbar. Darüber hinaus verfügen in vitro Genotoxizitätstests in Säugerzellen über eine relativ geringe Spezifität. Für eine vollständige Sicherheitsbeurteilung wird eine in vivo Testung auf Kanzerogenität benötigt. Allerdings sind diese Testsysteme kosten- und zeitintensiv. Aufgrund dessen zielen neue Forschungsansätze auf die Verbesserung der Prädiktivität und die Erfassung des genotoxischen Potentials bereits in der frühen Phase der Arzneimittelentwicklung ab. Die high content imaging (HCI)-Technologie offeriert einen Ansatz zur Verbesserung des Durchsatzes verglichen mit der Standardtestbatterie. Zusätzlich hat ein Zell-basiertes Modell den Vorteil Daten relativ schnell bei gleichzeitig geringem Bedarf an Substanzmenge zu generieren. Demzufolge ermöglichen HCI-basierte Testsysteme eine Prüfung in der frühen Phase der pharmazeutischen Arzneimittelentwicklung. Das Ziel dieser Studie ist die Entwicklung eines neuen, spezifischen und sensitiven HCI-basierten Testsytems für Genotoxine und Progenotoxine in vitro unter Verwendung von HepG2-Zellen gewesen. Aufgrund ihrer begrenzten metabolischen Kapazität wurde ein kombiniertes System bestehend aus HepG2-Zellen und einem metabolischen Aktivierungssystem zur Testung progenotoxischer Substanzen etabliert. Basierend auf einer vorherigen Genomexpressionsprofilierung (Boehme et al., 2011) und einer Literaturrecherche wurden die folgenden neun unterschiedlichen Proteine der DNA-Schadensantwort als putative Marker der Substanz-induzierten Genotoxizität ausgewählt: p-p53 (Ser15), p21, p-H2AX (Ser139), p-Chk1 (Ser345) p-ATM (Ser1981), p-ATR (Ser428), p-CDC2 (Thr14/Tyr15), GADD45A und p-Chk2 (Thr68). Die Expression bzw. Aktivierung dieser Proteine wurde 48 h nach Behandlung mit den (pro-) genotoxischen Substanzen (Cyclophosphamid, 7,12-Dimethylbenz[a]anthracen, Aflatoxin B1, 2-Acetylaminofluoren, Methylmethansulfonat, Actinomycin D, Etoposid) und den nicht-genotoxischen Substanzen (D-Mannitol, Phenforminhydrochlorid, Progesteron) unter Verwendung der HCI-Technologie ermittelt. Die beste Klassifizierung wurde bei Verwendung der folgenden fünf der ursprünglichen neun putativen Markerproteine erreicht: p-p53 (Ser15), p21, p-H2AX (Ser139), p-Chk1 (Ser345) und p-ATM (Ser1981). In einem zweiten Teil dieser Arbeit wurden die fünf ausgewählten Proteine mit Substanzen, welche von dem European Centre for the Validation of Alternative Methods (ECVAM) zur Beurteilung der Leistung neuer oder modifizierter in vitro Genotoxizitätstests empfohlen sind, getestet. Dieses neue Testsystem erzielte eine Sensitivität von 80 % und eine Spezifität von 86 %, was in einer Prädiktivität von 84 % resultierte. Der synergetische Effekt dieser fünf Proteine ermöglicht die Identifizierung von genotoxischen Substanzen, welche DNA-Schädigungen durch eine Vielzahl von unterschiedlichen Mechanismen induzieren, mit einem hohen Erfolg. Zusammenfassend konnte ein hochprädiktives Prüfungssystem mit metabolischer Aktivierung für ein breites Spektrum potenziell genotoxischer Substanzen generiert werden, welches sich aufgrund des hohen Durchsatzes, des geringen Zeitaufwandes und der geringen Menge benötigter Substanz zur Substanzpriorisierung und -selektion in der Phase der Leitstrukturoptimierung eignet und darüber hinaus mechanistische Hinweise auf die genotoxische Wirkung der Testsubstanz liefert.

Influence of traumatic impaction and pathological loading on knee menisci

Nearly half of the US population faces the risk of developing knee osteoarthritis (OA). Both in vitro and in vivo studies can aid in a better understanding of the etiology, progression, and advancement of this debilitating disorder. The knee menisci are fibrocartilagenous structures that aid in the distribution of load, attenuation of shock, alignment and lubrication of the knee. Little is known about the biochemical and morphological changes associated with knee menisci following altered loading and traumatic impaction, and investigations are needed to further elucidate how degradation of this soft tissue advances over time. The biochemical response of porcine meniscal explants was investigated following a single bout of dynamic compression with and without the treatment of the pharmaceutical drug, anakinra (IL-1RA). Dynamic loading led to a strain-dependent response in both anabolic and catabolic gene expression of meniscal explants. By inhibiting the Interleukin-1 pathway with IL-1RA, a marked decrease in several catabolic molecules was found. From these studies, future developments in OA treatments may be developed. The implementation of an in vivo animal model contributes to the understanding of how the knee joint behaves as a whole. A novel closed-joint in vivo model that induces anterior cruciate ligament (ACL) rupture has been developed to better understand how traumatic injury leads to OA. The menisci of knees from three different groups (healthy, ACL transected, and traumatically impacted) were characterized using histomorphometry. The acute and chronic changes within the knee following traumatic impaction were investigated. The works presented in this dissertation have focused on the characterization, implementation, and development of mechanically-induced changes to the knee menisci.

Doseamento microbiológico de neomicina e bacitracina - desenvolvimento e validação de método rápido em microplacas

Os ensaios microbiológicos são utilizados para avaliar a atividade antimicrobiana desde a descoberta do uso dos antibióticos. Apesar dos ensaios microbiológicos serem amplamente empregados para determinação da potência de antibióticos em formas farmacêuticas, uma vez que fornecem a medida da atividade biológica, apresentam limitações quanto a baixa reprodutibilidade e tempo de análise. O objetivo deste trabalho foi desenvolver, otimizar e validar ensaio colorimétrico rápido em microplaca para determinar a potência da neomicina e bacitracina em produtos farmacêuticos. Metodologias de análise fatorial e análise de superfície de resposta foram utilizadas no desenvolvimento e otimização da escolha do microrganismo, da composição do meio de cultura, da proporção de inóculo, e das concentrações de cloreto de trifeniltetrazólio e dos antibióticos. Os métodos otimizados foram validados pela avaliação da linearidade, precisão, exatidão e robustez. Análise estatística mostrou equivalência entre o método de difusão em ágar e o método colorimétrico rápido em microplaca para ambos antibióticos. Além disso, o ensaio em microplaca apresentou vantagens em relação à reprodutibilidade, sensibilidade, tempo de incubação e quantidades necessárias de meio de cultura e soluções para ambos os antibióticos.

Studies in asymmetric and heterocyclic synthesis: I. chiral ketones II. Quinolones III. Trifluoromethylated pyrones

This thesis is split into three sections based on three different areas of research. In the first section, investigations into the α-alkylation of ketones using a novel chiral auxiliary is reported. This chiral auxiliary was synthesised containing a pyrrolidine ring in the chiral arm and was applied in the preparation of α-alkylated ketones which were obtained in up to 92% ee and up to 63% yield over two steps. Both 3-pentanone and propiophenone based ketones were used in the investigation with a variety of both alkyl and benzyl based electrophiles. The novel chiral auxiliary was also successful when applied to Michael and aldol reactions. A diamine precursor en route to the chiral auxiliary was also applied as an organocatalyst in a Michael reaction, with the product obtained in excellent enantioselectivity. In the second section, investigations into potential anti-quorum sensing molecules are reported. The bacteria Pseudomonas aeruginosa is an antibiotic-resistant pathogen that demonstrates cooperative behaviours and communicates using small chemical molecules in a process termed quorum sensing. A variety of C-3 analogues of the quorum sensing molecules used by P. aeruginosa were synthesised. Expanding upon previous research within the group, investigations were carried out into alternative protecting group strategies of 2-heptyl-4-(1H)- quinolone with the aim of improving the yields of products of cross-coupling reactions. In the third section, investigations into fluorination and trifluoromethylation of 2-pyrones, pyridones and quinolones is reported. The incorporation of a fluorine atom or a trifluoromethyl group into a molecule is important in pharmaceutical drug discovery programmes as it can lead to increased lipophilicity and bioavailability, however late-stage incorporation is rarely reported. Both direct fluorination and trifluoromethylation were attempted. Eight trifluoromethylated 2-pyrones, five trifluoromethylated 2-pyridones and a trifluoromethylated 2-quinolone were obtained in a late-stage synthesis from their respective iodinated precursors using methyl fluorosulfonyldifluoroacetate as a trifluoromethylating reagent.

The high price of drug patents: Australia, patent law, pharmaceutical drugs and the Trans-Pacific Partnership

This week, the secrecy surrounding an independent Australian report on patent law and pharmaceutical drugs has been lifted, and the work has been published to great acclaim...

A dynamic mechanical method for determining the silicone elastomer solubility of drugs and pharmaceutical excipients in silicone intravaginal drug delivery rings

The silicone elastomer solubilities of a range of drugs and pharmaceutical excipients employed in the development of silicone intravaginal drug delivery rings (polyethylene glycols, norethisterone acetate, estradiol, triclosan, oleyl alcohol, oxybutynin) have been determined using dynamic mechanical analysis. The method involves measuring the concentration-dependent decrease in the storage modulus associated with the melting of the incorporated drug/excipient, and extrapolation to zero change in storage modulus. The study also demonstrates the effect of drug/excipient concentrations on the mechanical stiffness of the silicone devices at 37°C.

Co-melt fluidised bed granulation of pharmaceutical powders: Improvements in drug bioavailability

This study investigates the use of co-melt fluidised bed granulation for the agglomeration of model pharmaceutical powders, namely, lactose mono-hydrate, PEG 10000, poly-vinyl pyrolidone and ibuprofen as a model drug. Granulation within the co-melt system was found to follow a nucleationâ??steady growthâ??coating regime profile. Using high molecular weight PEG binder, the granulation mechanism and thus the extent of granulation was found to be significantly influenced by binder viscosity. The compression properties of the granulate within the hot fluidised bed were correlated using a novel high temperature experimental procedure. It was found that the fracture stress and fractural modulus of the materials under hot processing conditions were orders of magnitude lower than those measured under ambient conditions. A range of particle velocities within the granulator were considered based on theoretical models. After an initial period of nucleation, the Stokes deformation number analysis indicated that only velocities within the high shear region of the fluidised bed were sufficient to promote significant granule deformation and therefore, coalescence. The data also indicated that larger granules de-fluidised preventing agglomeration by coalescence. Furthermore, experimental data indicated that dissipation of the viscous molten binder to the surface was the most important factor in the latter stages of the granulation process. From a pharmaceutical perspective the inclusion of the model drug, ibuprofen, combined with PVP in the co-melt process proved to be highly significant. It was found that using DSC analysis on the formulations that the decrease in the heat of fusion associated with the melting of ibuprofen within the FHMG systems may be attributed to interaction between PVP and ibuprofen through inter-molecular hydrogen bonding. This interaction decreases the crystallinity of ibuprofen and facilitates solubilisation and bioavailability within the solid matrix.

Slow Release Drug Dissolution Profile Prediction in Pharmaceutical Manufacturing: a Multivariate and Machine Learning Approach

Slow release drugs must be manufactured to meet target specifications with respect to dissolution curve profiles. In this paper we consider the problem of identifying the drivers of dissolution curve variability of a drug from historical manufacturing data. Several data sources are considered: raw material parameters, coating data, loss on drying and pellet size statistics. The methodology employed is to develop predictive models using LASSO, a powerful machine learning algorithm for regression with high-dimensional datasets. LASSO provides sparse solutions facilitating the identification of the most important causes of variability in the drug fabrication process. The proposed methodology is illustrated using manufacturing data for a slow release drug.

Structure and viscoelastic behavior of pharmaceutical biocompatible anionic microemulsions containing the antitumoral drug compound doxorubicin

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Physical enhancement of transdermal drug application: is delivery technology keeping up with pharmaceutical development?

Advances in molecular biology have given us a wide range of protein and peptide-based drugs that are unsuitable for oral delivery because of their high degree of first-pass metabolism. Though parenteral delivery is the obvious answer, for the successful development of commercial chronic and self-administration usage formulations it is not the ideal choice. Transdermal delivery is emerging as the biggest application target for these agents, however, the skin is extremely efficient at keeping out such large molecular weight compounds and therapeutic levels are never going to be realistically achieved by passive absorption. Physical enhancement mechanisms including: iontophoresis, electroporation, ultrasound, photomechanical waves, microneedles and jet-propelled particles are emerging as solutions to this topical delivery dilemma. Adding proteins and peptides to the list of other large molecular weight drugs with insufficient passive transdermal fluxes to be therapeutically useful, we have a collection of pharmacological agents waiting for efficient delivery methods to be introduced. This article reviews the current state of physical transdermal delivery technology, assesses the pros and cons of each technique and summarises the evidence-base of their drug delivery capabilities.

Drug combination: pharmaceutical technologies applied to the manufacturing of combined dosage forms for oral administration

Lo scopo di questa ricerca di dottorato è stato lo studio di una forma di dosaggio flessibile e personalizzabile, indirizzata alle necessità individuali di ogni paziente, per il trattamento dell’iperplasia prostatica benigna. La terapia proposta prevede l’utilizzo di due farmaci, un alfa bloccante (farmaco A) e un inibitore delle 5- fosfodiesterasi (farmaco B) e, somministrati in una singola forma di dosaggio contenenti differenti dosi e combinazioni dei due farmaci. Lo sviluppo di un sistema di rilascio per la somministrazione orale di farmaco A e farmaco B è stato realizzato grazie alla tecnologia Dome Matrix. La tecnologia si basa sull’assemblaggio di moduli utilizzati come elementi di controllo del rilascio. L’assemblaggio dei moduli può essere ottenuto attraverso diverse configurazioni. Sono stati quindi realizzati sistemi assemblati in grado di galleggiare sul contenuto gastrico; la prolungata permanenza della forma farmaceutica nello stomaco favorisce la solubilizzazione dei due principi attivi che quindi potrebbero raggiungere il sito di assorbimento nel primo tratto intestinale già in dispersione molecolare, condizione ideale per essere assorbiti. La prima parte della ricerca è stata focalizzata sulla realizzazione di un sistema assemblato a rilascio modificato di farmaco A. Moduli contenenti diversi dosaggi di farmaco sono stati assemblati in varie configurazioni e dosi differenti per ottenere una forma di dosaggio flessibile, adattabile alle esigenze terapeutiche del paziente. La seconda parte del lavoro di tesi ha riguardato la realizzazione di un sistema assemblato, contenente entrambi i farmaci in associazione. L’ultima parte della ricerca è stata svolta presso la “University of Texas at Austin” sotto la supervisione del Professor Nicholas Peppas. Il lavoro svolto è stato focalizzato sullo studio delle caratteristiche di rigonfiamento dei singoli moduli di farmaco e dei loro sistemi assemblati; il comportamento di tali sistemi è stato investigato anche grazie all’utilizzo della tecnica di tomografia computerizzata a raggi X.