Background: Silica is the common name for silicon dioxide (SiO2) materials and exists in both crystalline and amorphous forms. While crystalline silica is known for its severe health effects, amorphous silica has been considered safe and applied in many areas. However, some recent studies have showed evidence of their toxicity, raising concerns about its use as nanomaterial for biomedical applications. When nanomaterials enter the body, they are enveloped in biological fluids rich in biomolecules, which compete for binding to the nanomaterial. Such effect could alter their surface chemistry and therefore affect their bio-distribution and interaction with cells. Aim and objectives: As part of the EU-funded NANODRUG network programme, the aim of this project was the in vitro toxicity assessment of commercially-sourced fumed and colloidal amorphous silica particles in relation to their physico-chemical properties and potential application as carriers for drug delivery. The objectives were 1) characterization of silica particles hydrodynamic (Hd) size and dispersity in different cell culture media; 2) in vitro toxicological assessment of silica particles in human skin cells; 3) delineation of toxicity mechanisms in relation to their size; 4) assessment of the influence of Foetal Bovine Serum (FBS) on particle Hd size and toxicity; and 5) contributing to the overall objective of the NANODRUG programme - development of safe nanodrugs for skin application - through collaborations with different partners.

Na engenharia de tecidos os biomateriais são usados tanto para induzir a formação óssea no tecido adjacente quanto agir como matriz temporária de células e outros agentes. Compósitos biodegradáveis a base de hidroxiapatita, colágeno e quitosana foram preparados como arcabouços para permitir a regeneração óssea. Este estudo teve como objetivo avaliar a toxicidade celular in vitro de novos compósitos de substituição óssea e o seu comportamento biológico após implantação subcutânea no processo de remodelação e cicatrização tecidual. Foram confeccionadas mantas a base de hidroxiapatita (HA), obtida por desproteinização e calcinação do osso bovino, colágeno (Col), obtido por tratamento alcalino do tendão bovino e quitosana (Qui), extraída do gládio de lula, nas seguintes proporções: B1) HA (mistura de partículas <0,2 mm e entre 0,2 e 1,18 mm, proporção 1:1) + Blenda (Col 1% + Qui 0,5%, proporção 1:1), proporção HA:Blenda 1:5; B2) HA (mistura de partículas <0,2 mm e entre 0,2 e 1,18 mm, na proporção 1:1) + Col 1%, proporção HA:Col 1:5; B3) HA (partículas <0,2 mm) + Col 1%, na proporção 1:5. O teste de citotoxicidade in vitro foi realizado pelo método de difusão de extrato em solução (brometo de tetrazolio - MTT). Os biomateriais descritos acima foram avaliados juntamente com o controle positivo (látex), sendo que todos foram submetidos a procedimentos de extração de acordo as normas da ISO10993-5. O meio de cultura foi utilizado como controle negativo. Foram utilizadas duas linhagens celulares: células tumorais de laringe humana HEp (ATCC-CCL-23) e células normais de rim de macaco verde africano (Cercopithecus aethiops) VERO (n=3). A citotoxicidade foi avaliada em um espectrofotômetro (absorbância de 570nm). No teste de biocompatibilidade as amostras foram implantadas no subcutâneo da região dorsal de 51 ratos Wistar. Histomorfometria de espessura e qualidade da cápsula fibrosa, interface implante-tecido, infiltrado inflamatório e crescimento celular interno foram realizados aos 3, 7 e 28 dias após cirurgia (n=5). Imunohistoquímica foi realizada para caracterizações adicionais do infiltrado de macrófagos. No teste de citotoxicidade as linhagens celulares mostraram resultados semelhantes. Não houve diferença entre os compósitos e o controle negativo. Foi demonstrado que os compósitos avaliados não apresentaram efeitos tóxicos em comparação ao controle positivo. No teste subcutâneo a qualidade e a espessura da cápsula não apresentaram diferenças significantes entre os biomateriais e os períodos de implantação. A resposta celular inflamatória aos 7 dias foi mais intensa no biomaterial B1, seguido pelo biomaterial B2 e B3. Uma baixa reação de corpo estranho foi observada em todos os biomateriais, caracterizada pela presença de poucos macrófagos. Ocorreu um intenso crescimento celular no interior do biomaterial B3 aos 28 dias. A análise histológica sugere que esses biomateriais são bem tolerados com baixa reação tecidual inflamatória. Concluímos que os três biomateriais avaliados não apresentaram evidências de citotoxicidade in vitro e, in vivo, mostraram uma boa biocompatibilidade. Esses materiais mostraram-se bons candidatos a matrizes para engenharia de tecidos e enxertos para regeneração óssea. / The biodegradable hydroxyapatite / chitosan / collagen composites of similar composition of the normal human bone were prepared as a biomimetic scaffold for bone replacement and regeneration. The aim of the present study was to evaluate the in vitro cytotoxicity of three new bone grafts and the influence of these synthetic grafts on the inflammatory response and remodeling of connective tissue during wound-healing process. Three biomaterials were developed: B1 = hydroxyapatite (particles size <0,2 mm + between 0,2 and 1,18 mm) / collagen 1% / chitosan 0.5%; B2 = hydroxyapatite (particles size <0,2 mm + between 0,2 and 1,18 mm) / collagen 1% and B3 = hydroxyapatite (particles size <0,2 mm) / collagen 1%. These biomaterials were investigated using a tetrazolium-based calorimetric assay (MTT assay). Latex was used as positive control and the culture medium as a negative control. Two cell lines were used to evaluate the effect of cell type on the toxicity results: human tumor cells HEp-2 (ATCC-CCL23) and monkey normal cells (VERO). Extracts from the biomaterials were obtained according to ISO 10993-12 standards. The cytotoxicity was assessed in a spectrophotometer (the absorbance was read at 570 nm). There were two independent experiments in triplicates. All values were expressed as mean values \'+ OU -\' standard deviation. In the biocompatibility test the samples were implanted subcutaneously in the dorsal lumbar region of 51 Wistar rats. Histomorphometric evaluation of capsule thickness, capsule quality, implant-tissue interface, infiltrate / inflammation and cellular growth within implant was performed 3, 7 and 28 days post-operatively (n=5). Immunohistochemistry was performed for further characterization of the macrophages infiltrate. In cytotoxicity test the cell lines showed similar results. There is no difference in values between the composites and negative control. The values of composites were significant different than the positive control. It was showed that none of the tested composites presents toxic effects for the cell lines evaluated in comparison with positive control. In the subcutaneous test the capsule thickness and capsule quality didn\'t show significant differences between the biomaterials and time of implantation. The biomaterial B1 showed significant difference in inflammatory cellular response than B2 and B3 at 7 days. A low ongoing foreign body reaction was observed in all biomaterials characterized by infiltration of few macrophages. A significant cellular growth within the B3 was observed at 28 days. Histological analysis suggested that the biomaterials were well tolerated with low inflammatory response. According to this study, the three bone grafts evaluated showed no evidence of cytotoxicity and good biocompatibility which makes them suitable candidates to design of bone replacement graft material.

Na engenharia de tecidos os biomateriais são usados tanto para induzir a formação óssea no tecido adjacente quanto agir como matriz temporária de células e outros agentes. Compósitos biodegradáveis a base de hidroxiapatita, colágeno e quitosana foram preparados como arcabouços para permitir a regeneração óssea. Este estudo teve como objetivo avaliar a toxicidade celular in vitro de novos compósitos de substituição óssea e o seu comportamento biológico após implantação subcutânea no processo de remodelação e cicatrização tecidual. Foram confeccionadas mantas a base de hidroxiapatita (HA), obtida por desproteinização e calcinação do osso bovino, colágeno (Col), obtido por tratamento alcalino do tendão bovino e quitosana (Qui), extraída do gládio de lula, nas seguintes proporções: B1) HA (mistura de partículas <0,2 mm e entre 0,2 e 1,18 mm, proporção 1:1) + Blenda (Col 1% + Qui 0,5%, proporção 1:1), proporção HA:Blenda 1:5; B2) HA (mistura de partículas <0,2 mm e entre 0,2 e 1,18 mm, na proporção 1:1) + Col 1%, proporção HA:Col 1:5; B3) HA (partículas <0,2 mm) + Col 1%, na proporção 1:5. O teste de citotoxicidade in vitro foi realizado pelo método de difusão de extrato em solução (brometo de tetrazolio - MTT). Os biomateriais descritos acima foram avaliados juntamente com o controle positivo (látex), sendo que todos foram submetidos a procedimentos de extração de acordo as normas da ISO10993-5. O meio de cultura foi utilizado como controle negativo. Foram utilizadas duas linhagens celulares: células tumorais de laringe humana HEp (ATCC-CCL-23) e células normais de rim de macaco verde africano (Cercopithecus aethiops) VERO (n=3). A citotoxicidade foi avaliada em um espectrofotômetro (absorbância de 570nm). No teste de biocompatibilidade as amostras foram implantadas no subcutâneo da região dorsal de 51 ratos Wistar. Histomorfometria de espessura e qualidade da cápsula fibrosa, interface implante-tecido, infiltrado inflamatório e crescimento celular interno foram realizados aos 3, 7 e 28 dias após cirurgia (n=5). Imunohistoquímica foi realizada para caracterizações adicionais do infiltrado de macrófagos. No teste de citotoxicidade as linhagens celulares mostraram resultados semelhantes. Não houve diferença entre os compósitos e o controle negativo. Foi demonstrado que os compósitos avaliados não apresentaram efeitos tóxicos em comparação ao controle positivo. No teste subcutâneo a qualidade e a espessura da cápsula não apresentaram diferenças significantes entre os biomateriais e os períodos de implantação. A resposta celular inflamatória aos 7 dias foi mais intensa no biomaterial B1, seguido pelo biomaterial B2 e B3. Uma baixa reação de corpo estranho foi observada em todos os biomateriais, caracterizada pela presença de poucos macrófagos. Ocorreu um intenso crescimento celular no interior do biomaterial B3 aos 28 dias. A análise histológica sugere que esses biomateriais são bem tolerados com baixa reação tecidual inflamatória. Concluímos que os três biomateriais avaliados não apresentaram evidências de citotoxicidade in vitro e, in vivo, mostraram uma boa biocompatibilidade. Esses materiais mostraram-se bons candidatos a matrizes para engenharia de tecidos e enxertos para regeneração óssea. / The biodegradable hydroxyapatite / chitosan / collagen composites of similar composition of the normal human bone were prepared as a biomimetic scaffold for bone replacement and regeneration. The aim of the present study was to evaluate the in vitro cytotoxicity of three new bone grafts and the influence of these synthetic grafts on the inflammatory response and remodeling of connective tissue during wound-healing process. Three biomaterials were developed: B1 = hydroxyapatite (particles size <0,2 mm + between 0,2 and 1,18 mm) / collagen 1% / chitosan 0.5%; B2 = hydroxyapatite (particles size <0,2 mm + between 0,2 and 1,18 mm) / collagen 1% and B3 = hydroxyapatite (particles size <0,2 mm) / collagen 1%. These biomaterials were investigated using a tetrazolium-based calorimetric assay (MTT assay). Latex was used as positive control and the culture medium as a negative control. Two cell lines were used to evaluate the effect of cell type on the toxicity results: human tumor cells HEp-2 (ATCC-CCL23) and monkey normal cells (VERO). Extracts from the biomaterials were obtained according to ISO 10993-12 standards. The cytotoxicity was assessed in a spectrophotometer (the absorbance was read at 570 nm). There were two independent experiments in triplicates. All values were expressed as mean values \'+ OU -\' standard deviation. In the biocompatibility test the samples were implanted subcutaneously in the dorsal lumbar region of 51 Wistar rats. Histomorphometric evaluation of capsule thickness, capsule quality, implant-tissue interface, infiltrate / inflammation and cellular growth within implant was performed 3, 7 and 28 days post-operatively (n=5). Immunohistochemistry was performed for further characterization of the macrophages infiltrate. In cytotoxicity test the cell lines showed similar results. There is no difference in values between the composites and negative control. The values of composites were significant different than the positive control. It was showed that none of the tested composites presents toxic effects for the cell lines evaluated in comparison with positive control. In the subcutaneous test the capsule thickness and capsule quality didn\'t show significant differences between the biomaterials and time of implantation. The biomaterial B1 showed significant difference in inflammatory cellular response than B2 and B3 at 7 days. A low ongoing foreign body reaction was observed in all biomaterials characterized by infiltration of few macrophages. A significant cellular growth within the B3 was observed at 28 days. Histological analysis suggested that the biomaterials were well tolerated with low inflammatory response. According to this study, the three bone grafts evaluated showed no evidence of cytotoxicity and good biocompatibility which makes them suitable candidates to design of bone replacement graft material.

The environmental bioavailability of contaminants, rather than their total concentrations in the soil compartment play a decisive role for the risks associated with contaminated sites. Various soil constituents and abiotic conditions have strong influence on bioavailability, which may vary substantially between different locations. It is therefore necessary to site-specifically use tools that reflect the fractions of contaminants that are available to biota and pose the highest potential environmental risks. Bioassays provide integrated toxic responses which include effects from unknown contaminants or combinatory toxic effects from mixtures of contaminants. Thus, biological effect data greatly contribute to establish more realistic exposure and risk-scenarios at contaminated sites. The work underlying this thesis presents possible techniques for high capacity screening for site-specific hazards at contaminated areas. By combining rapid water extractions and cell-based in vitro designs measures of the toxic potential in soils was obtained. Toxicologically bioavailable fractions of mixed metal pollution, including arsenic, were primarily investigated in this thesis. In two of the studies, environmental availability and toxicological bioavailability of arsenic was explored in CCA-contaminated soils. Application of cell-based in vitro screening techniques was also conducted at a metal contaminated industrial site to obtain spatial distribution of toxicity. Multivariate association techniques were employed in the interpretation of environmental exposure and cytotoxicity data. It was shown that cell-based in vitro systems for both basal cytotoxicity and specific end-points targeting arsenic could assess the toxic potential from extracts obtained by several water-based extraction techniques including Pressurised Liquid Extraction (PLE). The cell-based in vitro systems were found to add important information on the site-specific differences in arsenics genotoxic potential from CCA-contaminated soils. The results highlight the importance of taking speciation and toxicological bioavailability into account in the risk analysis, rather than to base risk estimates on total load of contaminants. The presented screening approach was successfully applied at a metal polluted industrial site where spatial distribution of toxicity was obtained. PLE extraction also provided means for combined toxicological and chemical screening of explosives in soils from live-fire training ranges. Multivariate association techniques highly facilitated the interpretation of complex environmental data. The PLE was found to be a rapid extraction technique that has sufficient environmental relevance to be used in environmental impact analyses. It was also concluded that other cell-based in vitro systems that target specific toxic effects have large potential for being used in screening for a variety of environmental chemicals. Keywords: Environmental availability, Environmental bioavailability, Toxicological bioavailability, mixture toxicity, hazard screening, contaminated soils, heavy metals, arsenic, CCA, explosives, soil extraction, water extracts, cell-based in vitro tests, cytotoxicity, genotoxicity, PLE, MVDA, PCA, PLS. / Föroreningars biotillgänglighet snarare än deras totala koncentration i markmiljön styr den risk som kan förknippas med förorenade områden. Biotillgängligheten är ofta långt från 100% p.g.a. en rad olika bindningsytor och processer i jorden som reducerar biotillgängligheten. Således kan biotillgängligheten variera kraftigt mellan olika förorenade platser och även inom samma plats till följd av de specifika förhållanden som råder på respektive plats. Tillämpning av biologiska indikatorer som ger ett mått på den samlade giftigheten från biotillgängliga föroreningar är därför viktiga verktyg i platsspecifika exponerings- och farobedömningar. Många biologiska tester är ofta laborativt intensiva och dyra och lämpar sig mindre väl i testning av ett stort antal prover vilket är önskvärt om en tillräcklig geografisk täckning ska uppnås över ett förenat område. Testsystem som har kapacitet att hantera många prover till en rimlig kostnad är därför mycket användbart för screening i ett inledande skede av en miljöriskanalys av ett förorenat område. Föreliggande avhandlingsarbete presenterar möjliga lösningar i att kombinera snabb vattenextraktionsmetodik med cellbaserade in vitro system för platsspecifik toxikologisk faroscreening av metallförorenade områden. Metodiken erbjuder hög kapacitet för många jordprover. Tillämpning av metodiken har gjorts mot huvudsakligen metallföroreningar, inklusive arsenik. I två delarbeten studerades två modelljordar från CCA-förorenade fastigheter avseende tillgänglighet och giftighet av framför allt arsenik. Vidare studerades om det med applicerad metodik gick att illustrera geografisk utbredning av toxicitet, mätt i cellbaserade in vitro system, som biotillgängliga föroreningar uppvisar på ett metallförorenat industriområde. Slutligen studerades lämpligheten i att använda PLE för kombinerad kemisk och toxikologisk screening av jordar från militära skjutfält som var förorenade med explosivämnen. Cellbaserade in vitro system för mätning av både generell toxicitet och mer specifika effektmarkörer för arsenik visade sig användbara vid mätning från flera vattenbaserade extraktionsmetoder, inklusive PLE (trycksatt vätskeextraktion). Resultaten visade på PLEs tillämplighet som en snabb extraktionsmetod med bibehållen relevans för miljöanalyser. Applikation av cellbaserade in vitro system på vattenextrakt från förorenad jord gav värdefull information bl.a. om platsspecifik genotoxisk potential där specieringen av arsenik hade avgörande betydelse i en fallstudie med CCA-förorenade jordar. Vattenextraktion av jordprover kombinerat med cellbaserade in vitro system kunde också ge en geografisk bild av den omedelbara faran från biotillgängliga metallföroreningar inom ett industriområde. Vattenextraktion med PLE visade sig även användbart för screening av explosivämnen där extrakten direkt kunde användas för såväl kemisk karakterisering som för toxikologisk analys. Även andra typer av in vitro system än de som användes i detta arbete har stor framtida potential för tillämpning i faroscreening av ett stort antal olika typer av miljöföroreningar.

Η σύγχρονη παραγωγή φαρμακευτικών και άλλων χημικών ουσιών και ο αναγκαίος τοξικολογικός τους έλεγχος επιφέρει την χρήση ενός μεγάλου αριθμού πειραματοζώων με αποτέλεσμα την αύξηση του κόστους αλλά και την έγερση ζητημάτων που σχετίζονται με την ασφάλεια και την βιοηθική. Στην βάση αυτού του προβληματισμού η ανάπτυξη νέων in vitro δοκιμασιών κυτταρο-τοξικότητας με δυνατότητα ακριβέστερης πρόβλεψης των αρχικών δόσεων οξείας από του στόματος τοξικότητας, προβάλλει ως αναγκαιότητα στις μέρες. Μέχρι τώρα έχουν χρησιμοποιηθεί σε in vitro δοκιμασίες μετασχηματισμένα, αθανατοποιημένα ή πρωτογενή κύτταρα καθώς και εμβρυικά βλαστικά κύτταρα (hESCs). Επίσης πρόσφατα χρησιμοποιήθηκαν μεσεγχυματικά βλαστικά κύτταρα από τον μυελό των οστών (BM-hMSCs). Τα κύτταρα αυτά ωστόσο παρουσιάζουν μειονεκτήματα που σχετίζονται με την δυσκολία απομόνωσης τους, την ετερογένεια τους, αλλά και τον πρόωρο φαινότυπο γήρανσης κατά την καλλιέργεια τους. Σε αυτή την μελέτη διερευνάται για πρώτη φορά η χρήση των βλαστικών κυττάρων της γέλης του Wharton (WJSCs) του ομφαλίου λώρου σε in vitro δοκιμασία κυτταροτοξικότητας. Τα κύτταρα αυτά παρουσιάζουν σημαντικά πλεονεκτήματα σε σχέση με άλλα μεσενχυματικά κύτταρα καθώς: απομονώνονται εύκολα, καλλιεργούνται εκτεταμένα με διατήρηση των βλαστικών τους ιδιοτήτων, δεν εγείρουν ηθικά ζητήματα για τη χρήσή τους, παρουσιάζουν γενετική και φαινοτυπική σταθερότητα και ήπιο ανοσολογικό προφίλ. Στην παρούσα μελέτη εξετάστηκαν παράλληλα και συγκριτικά με τα κύτταρα της γέλης του Wharton, οι κυτταρικές σειρές HepG2 (ηπατικού καρκινώματος) και NIH 3T3 (ινοβλάστες ποντικού) αλλά και τα μεσεγχυματικά κύτταρα λίπους AD-hSCs. Όπως προτείνεται από το πρωτόκολλο της ICCVAM δοκιμάστηκαν 12 ουσίες αναφοράς ενώ η μέτρηση της επιβίωσης των κυττάρων έγινε με την δοκιμασία MTS και NRU. Τα αποτελέσματα μας δείχνουν ότι τα κύτταρα της γέλης του Wharton μπορούν να αποτελέσουν αξιόπιστο και ελπιδοφόρο μοντέλο για δοκιμασίες in vitro τοξικότητας. Το μοντέλο αυτό μπορεί να λειτουργήσει συμπληρωματικά, ή ακόμα και να ξεπεράσει, ήδη επικυρωμένα μοντέλα κυτταροτοξικότητας . / The modern production of pharmaceuticals, other chemicals and their required toxicological controls results in the use of a large number of laboratory animals leading in increased costs as well as raising questions considering safety and bioethics. Alternatively, in vitro cytotoxicity assays are highlighted with the ability of a more accurate prediction of the starting dose of oral acute toxicity. Occasionally several cell lines have been used including transformed and immortalized cells or primary cells and embryonic stem cells (hESCs). For the same purpose adult mesenchymal stem cells derived from the bone marrow (BM-hMSCs) have been recently used but they exhibit difficulties in their isolation, heterogeneity, and premature senescence phenotype during their sub-cultivation. In this study for the first time we investigated the use of mesenchymal stem cells (WJSCs) isolated from fetal umbilical cord, in particular from the Wharton’s Jelly. These cells exhibit the advantage of easily being isolated and cultured in large quantities without ethical issues, genetic and phenotypic stability and subimmunological profile. Two different cell lines HepG2 (liver carcinoma) and NIH 3T3 (mouse fibroblasts) and mesenchymal adipose-derived stem cells AD-hSCs have been used and compared with the WJSCs in parallel. 12 substances have been tested for their cytotoxicity effect on cell survival using the MTS assay as suggested by ICCVAM. Our results indicate that this model is a reliable and promising approach for in vitro cytotoxicity tests on human cells and it can complement or even overpass validated cytotoxicity models.

Deux complexes de gallium solubles dans l'eau de formule [Ga(III)LCl], où L est la forme déprotonée de dérivés d'acide N-2-hydroxybenzyl-aspartique ont été synthétisés et caractérisés par RMN 1H et 13C, FT-IR, spectrométrie de masse et analyse élémentaire. Les données analytiques obtenues sont cohérentes avec une structure mononucléaire dans laquelle le cation gallium (III) est ligandé par l'un des deux groupes acide carboxylique, l'oxygène phénolique et l’atome d'azote du groupe 2-hydroxybenzylamino. Dans une telle structure, le ligand tridendate assure la liaison de l'ion métallique tandis que l'appendice carboxylique fournit la solubilité dans l'eau. La cytotoxicité du complexe gallium de l'acide (R)-2-(5-chloro-2-hydroxybenzylamino)succinique (GS2) a été évaluée contre les lignées cellulaires de cancer du sein humain MDA-MB231 et de fibrosarcome HT-1080. Nous avons établi que GS2 est plus cytotoxique que le dérivé dépourvu de chlore aromatique et que le chlorure de gallium. GS2 est capable d'induire l'apoptose par la régulation négative de la phosphorylation de l'AKT et l’arrêt du cycle cellulaire en G2M via l’activation de la voie des caspases 3/7. Bien que de nombreux effets moléculaires et cellulaires du Ga aient été décrits, y compris l'inhibition du protéasome et les activités ostéoclastiques, le complexe GS2 apparaît comme le premier complexe de gallium capable de réduire la phosphorylation d'AKT dans les cellules cancéreuses. L'activité de GS2 sur l'invasion cellulaire et sur l'expression et l'activité des métalloprotéinases matricielles (MMP) a été étudiée en utilisant une chambre de Boyden revêtue de collagène de type I. Nous avons analysé l'activité sur les MMPs par zymographie et dosage enzymatique en utilisant des substrats fluorogènes à haute affinité. Une inhibition sélective de MMP-14 a été observée pour bloquer la migration et l'invasion de cellules tumorales. L'expression de l'ARNm des MMP a été analysée par qRT-PCR. GS2 induit une diminution de l'invasion cellulaire. Un effet d'inhibition dose-dépendante a été observé sur les activités de MMP-2, MMP-9 et MMP-14. Une diminution de l'expression de l'ARNm de MMP-14 a été observée dans les deux lignées cellulaires, tandis que l'expression des ARNm de MMP-2 et de MMP-9 ne décroit que dans les cellules tumorales MDA-MB231. Les données obtenues sur l'expression de l'ARNm de MMP-14 ont été confirmées par analyse Western Blot. GS2 semble être une molécule capable de réduire l'activité de MMP-14 dans des maladies métastatiques cancéreuses présentant un niveau élevé d'expression et d'activité de MMP-14. En conclusion, ces données montrent que le complexe GS2, en combinaison avec une chimiothérapie cytotoxique, est un composé prometteur pour la thérapie anti-invasive et anticancéreuse. / Two water soluble gallium complexes with formula [Ga(III)LCl], where L is the deprotonated form of N-2-hydroxybenzyl aspartic acid derivatives were synthesized and characterized by 1H NMR, 13C NMR, FT-IR, mass spectrometry and elemental analysis. The analytical data are consistent with a mononuclear structure in which the gallium (III) cation is liganded by one of the two carboxylic acid groups, the phenol oxygen and the nitrogen atom of the 2-hydroxybenzylamino group. In such a structure, the tridendate ligand secures the binding of the metal ion whereas the carboxylic appendage provides the water solubility. The cytotoxicity of the gallium complex of (R)-2-(5-chloro-2-hydroxybenzylamino) succinic acid (GS2) was evaluated against human breast carcinoma MDA-MB231 and fibrosarcoma HT-1080 cell lines. The 5-chloro derivative GS2 was found to be more cytotoxic than the unsubstituted derivative and GaCl3. GS2 induces apoptosis through down-regulation of AKT phosphorylation, G2M arrest in cell cycle via activation of the caspase3/7 pathway. Although, many molecular and cell effects of Ga have been described, including proteasome inhibition and osteoclastic activities, GS2 appears as the first Ga compounds able to decrease AKT phosphorylation in cancer cells. The activity of GS2 on cell invasion and on the expression and activity of Matrix Metalloproteinases (MMPs) have been investigated using modified Boyden chamber coated with type I collagen. We analyzed the activity on MMPs by zymography and enzymatic assay using high affinity fluorogenic substrates. A selective inhibition of MMP-14 has been reported to blocks tumor cell migration and invasion. The expression of MMPs mRNA was analyzed by qRT-PCR. GS2 induces a decrease in cell invasion. A dose dependent inhibition effect was observed on MMP-2, MMP-9 and MMP-14 activities. A decrease in MMP-14 mRNA expression was observed in both cell lines, whereas MMP-2 and MMP-9 mRNA expression was decreased only in MDA-MB231 cells. Thus, we propose that GS2 compound may be a potential candidate to decrease the MMP-14 activity in cancer metastatic diseases presenting high level of MMP-14 expression and activity. Taken together, these data show that GS2, in combination with cytotoxic chemotherapy a is a promising compound for anti-invasive and anticancer therapy.

O carbonato de lodenafila é um inibidor da fosfodiesterase tipo 5, desenvolvido no Brasil, o qual é um dímero formado por duas moléculas de lodenafila ligadas por uma ponte carbonato. Encontra-se disponível sob a forma de comprimidos. Não existe monografia disponível para este fármaco em nenhum código oficial. Assim, o objetivo do presente estudo foi desenvolver métodos analíticos para análise qualitativa e quantitativa do carbonato de lodenafila em comprimidos, avaliação da estabilidade frente à degradação forçada, contemplando a cinética de fotodegradação, identificação e estudo de citotoxicidade in vitro dos produtos de degradação majoritários e desenvolvimento do teste de dissolução baseado em dados in vivo. A caracterização da substância química de referência foi realizada através da faixa de fusão por calorimetria exploratória diferencial, métodos espectrofotométricos na região do infravermelho (IV) e do ultravioleta (UV), assim como espectroscopia de ressonância magnética nuclear de 1H e 13C. Os métodos por cromatografia em camada delgada (CCD), espectrofotometria no UV, cromatografia líquida de alta eficiência (CLAE) e eletroforese capilar (EC) foram utilizados para identificar o fármaco na forma farmacêutica. Os seguintes métodos de análise quantitativa do fármaco em comprimidos foram desenvolvidos e validados: método indicativo da estabilidade por CLAE, espectrofotometria na região do UV e EC. Todos cumpriram com os parâmetros descritos pelas guias de validação e não apresentaram diferença significativa na determinação do fármaco. A cinética de fotodegradação do carbonato de lodenafila apresentou cinética de primeira ordem. Dois produtos majoritários encontrados na fotodegradação foram identificados por cromatografia líquida de ultra eficiência acoplada à espectrometria de massas (CLUE-MS/MS) como ácido 4-etoxi-3-(1-metil-7-oxo-3-propil-6,7-diidro- 1H-pirazol [4,3-d] pirimidina-5-il) benzenossulfônico e ácido 4-etoxi-3-(1-metil-7-oxo- 3-propil-6,7-diidro-1H-pirazol [4,3-d] pirimidina-5-il) benzenossulfínico. A avaliação da citotoxicidade in vitro indicou que a amostra de carbonato de lodenafila degradada não apresentou toxicidade. O teste de dissolução foi desenvolvido e validado utilizando como meio de dissolução 900 mL de HCl 0,1 M + lauril sulfato de sódio 1,5% (p/v), a 37 ± 0,5°C, pás a 25 rpm e quantificação por espectrofotometria no UV. O perfil de dissolução apresentou cinética de primeira ordem. De acordo com os resultados obtidos, todos os métodos propostos podem ser utilizados no controle de qualidade do carbonato de lodenafila. / Lodenafil carbonate is a PDE5 inhibitor developed in Brazil, which is a dimer formed by two lodenafil molecules linked by a carbonate bridge. It is currently available in tablets. There is no monograph available for this drug in any official code. Thus, the purpose of the present study was the development of analytical methods for qualitative and quantitative analysis of LC in tablets, evaluation of drug stability in forced degradation, comprising the determination of photodegradation kinetic, identification and in vitro cytotoxicity study of two major degradation products and develop a dissolution test based on in vivo data. The characterization of the reference chemical substance was performed by: melting range by differencial scanning calorimetry, infrared (IR) and ultraviolet (UV) spectrophotometry methods, as well as the 1H and 13C nuclear magnetic resonance spectroscopy. The methods by thin-layer chromatography (TLC), UV spectrophotometry, high-performance liquid chromatography (LC) and capillary electrophoresis (CE) were used to identify the drug in pharmaceutical formulations. The following methods, applied to the assay of the drug in tablets were developed and validated: a stability-indicating HPLC method, UV spectrophotometry and CE. All of them met the criteria described by the validation guidelines and showed no significant statistically difference in the drug determination. The photodegradation kinetics of lodenafil carbonate showed firstorder kinetics. Two degradation products found by photodegradation were identified by ultra performance liquid chromatography-tandem mass spectrometry (UPLCMS/ MS) as 4-ethoxy-3-(1-methyl-7-oxo-3-propyl-6,7-dihydro-1H-pyrazolo [4,3- d]pyrimidin-5-yl)-benzenesulfonic acid and 4-ethoxy-3-(1-methyl-7-oxo-3-propyl-6,7- dihydro-1H-pyrazolo [4,3-d]pyrimidin-5-yl) benzenesulfinic acid. The in vitro cytotoxicity evaluation indicated that the lodenafil carbonate degraded sample did not show toxicity. A dissolution test was developed and validated using 900 mL of 0.1 M HCl + 1.5% sodium lauryl sulfate (w/v), at 37 ± 0.5 °C as dissolution medium, paddle at 25 rpm and quantitation by UV spectrophotometry. The dissolution profile showed first order kinetics. According to the obtained results, all proposed methods can be used in the quality control of lodenafil carbonate.

O carbonato de lodenafila é um inibidor da fosfodiesterase tipo 5, desenvolvido no Brasil, o qual é um dímero formado por duas moléculas de lodenafila ligadas por uma ponte carbonato. Encontra-se disponível sob a forma de comprimidos. Não existe monografia disponível para este fármaco em nenhum código oficial. Assim, o objetivo do presente estudo foi desenvolver métodos analíticos para análise qualitativa e quantitativa do carbonato de lodenafila em comprimidos, avaliação da estabilidade frente à degradação forçada, contemplando a cinética de fotodegradação, identificação e estudo de citotoxicidade in vitro dos produtos de degradação majoritários e desenvolvimento do teste de dissolução baseado em dados in vivo. A caracterização da substância química de referência foi realizada através da faixa de fusão por calorimetria exploratória diferencial, métodos espectrofotométricos na região do infravermelho (IV) e do ultravioleta (UV), assim como espectroscopia de ressonância magnética nuclear de 1H e 13C. Os métodos por cromatografia em camada delgada (CCD), espectrofotometria no UV, cromatografia líquida de alta eficiência (CLAE) e eletroforese capilar (EC) foram utilizados para identificar o fármaco na forma farmacêutica. Os seguintes métodos de análise quantitativa do fármaco em comprimidos foram desenvolvidos e validados: método indicativo da estabilidade por CLAE, espectrofotometria na região do UV e EC. Todos cumpriram com os parâmetros descritos pelas guias de validação e não apresentaram diferença significativa na determinação do fármaco. A cinética de fotodegradação do carbonato de lodenafila apresentou cinética de primeira ordem. Dois produtos majoritários encontrados na fotodegradação foram identificados por cromatografia líquida de ultra eficiência acoplada à espectrometria de massas (CLUE-MS/MS) como ácido 4-etoxi-3-(1-metil-7-oxo-3-propil-6,7-diidro- 1H-pirazol [4,3-d] pirimidina-5-il) benzenossulfônico e ácido 4-etoxi-3-(1-metil-7-oxo- 3-propil-6,7-diidro-1H-pirazol [4,3-d] pirimidina-5-il) benzenossulfínico. A avaliação da citotoxicidade in vitro indicou que a amostra de carbonato de lodenafila degradada não apresentou toxicidade. O teste de dissolução foi desenvolvido e validado utilizando como meio de dissolução 900 mL de HCl 0,1 M + lauril sulfato de sódio 1,5% (p/v), a 37 ± 0,5°C, pás a 25 rpm e quantificação por espectrofotometria no UV. O perfil de dissolução apresentou cinética de primeira ordem. De acordo com os resultados obtidos, todos os métodos propostos podem ser utilizados no controle de qualidade do carbonato de lodenafila. / Lodenafil carbonate is a PDE5 inhibitor developed in Brazil, which is a dimer formed by two lodenafil molecules linked by a carbonate bridge. It is currently available in tablets. There is no monograph available for this drug in any official code. Thus, the purpose of the present study was the development of analytical methods for qualitative and quantitative analysis of LC in tablets, evaluation of drug stability in forced degradation, comprising the determination of photodegradation kinetic, identification and in vitro cytotoxicity study of two major degradation products and develop a dissolution test based on in vivo data. The characterization of the reference chemical substance was performed by: melting range by differencial scanning calorimetry, infrared (IR) and ultraviolet (UV) spectrophotometry methods, as well as the 1H and 13C nuclear magnetic resonance spectroscopy. The methods by thin-layer chromatography (TLC), UV spectrophotometry, high-performance liquid chromatography (LC) and capillary electrophoresis (CE) were used to identify the drug in pharmaceutical formulations. The following methods, applied to the assay of the drug in tablets were developed and validated: a stability-indicating HPLC method, UV spectrophotometry and CE. All of them met the criteria described by the validation guidelines and showed no significant statistically difference in the drug determination. The photodegradation kinetics of lodenafil carbonate showed firstorder kinetics. Two degradation products found by photodegradation were identified by ultra performance liquid chromatography-tandem mass spectrometry (UPLCMS/ MS) as 4-ethoxy-3-(1-methyl-7-oxo-3-propyl-6,7-dihydro-1H-pyrazolo [4,3- d]pyrimidin-5-yl)-benzenesulfonic acid and 4-ethoxy-3-(1-methyl-7-oxo-3-propyl-6,7- dihydro-1H-pyrazolo [4,3-d]pyrimidin-5-yl) benzenesulfinic acid. The in vitro cytotoxicity evaluation indicated that the lodenafil carbonate degraded sample did not show toxicity. A dissolution test was developed and validated using 900 mL of 0.1 M HCl + 1.5% sodium lauryl sulfate (w/v), at 37 ± 0.5 °C as dissolution medium, paddle at 25 rpm and quantitation by UV spectrophotometry. The dissolution profile showed first order kinetics. According to the obtained results, all proposed methods can be used in the quality control of lodenafil carbonate.

Made available in DSpace on 2014-12-17T14:16:31Z (GMT). No. of bitstreams: 1 ReginaCE_DISSERT.pdf: 2319971 bytes, checksum: 40c841a1d50cf6f734095c894d9d920d (MD5) Previous issue date: 2013-04-25 / Homeopathic medicines have been used for over two hundred years without the examination of their effects on in vivo and in vitro assays, due to the peculiarity of homeopathic preparations, the high dilution, which creates a challenge for the use of usual analytical techniques of quality control of medicine.Although there is scarcity of literature and variety of experiments, recently there have been some studies with few in vitro assays which have shown positive responses when evaluating the mechanism of action of homeopathic medicines which are able to act on a specific system.The present study aims to evaluate the efficacy of homeopathic products containing Momordica charantia through bioassays.Homeopathic products were tested by the MTT to assess cytotoxicity in RAW 264.7 (macrophage-like cells) and in tumor cells HeLa (human cervical adenocarcinoma cells), CHO K1 (Chinese hamster ovary cells), PANC-1 (human pancreas cancer cells) and PC-3 (human prostate cancer cells), dosage of inflammatory mediators NO, TNF-? and IL-6 released by RAW 264.7 cells, analysis of the death process and cell cycle changes of PC-3 by flow cytometry. The data demonstrate that homeopathic products of Momordica charantia did not show cytotoxicity to RAW 264.7, increased the production of inflammatory mediators by RAW 264.7 synergistically with LPS, showed cytotoxicity to PC-3 with change in its cell cycle inhibiting its proliferation, being the 30CH the most potent sample. Correlation studies were conducted in order to evaluate the possible in vitro applicable models to the quality control of homeopathic products with Momordica charantia. The data showed that the best applicable models in assessing the quality are the MTT to assess cytotoxicity in RAW 264.7 and PC-3 in 24 hours for Momordica charantia fruit products and dosage of NO production by RAW 264.7 with and without LPS / Os medicamentos homeop?ticos t?m sido utilizados por mais de duzentos anos, sem a verifica??o dos seus efeitos em ensaios in vivo e in vitro. Devido ? peculiaridade das prepara??es homeop?ticas, sua alta dilui??o, que cria um desafio para a utiliza??o das t?cnicas anal?ticas usuais do controle de qualidade de medicamentos. Embora exista escassez de literatura e heterogeneidade de experimentos, recentemente existem alguns estudos com poucos ensaios in vitro que t?m apresentado respostas positivas quando se avalia o mecanismo de a??o dos medicamentos homeop?ticos que s?o capazes de atuar em um sistema espec?fico. O presente estudo visa ? avalia??o da efic?cia dos produtos homeop?ticos contendo Momordica charantia atrav?s de bioensaios. Os produtos homeop?ticos foram avaliados pelo teste do MTT para verifica??o da citotoxicidade em RAW 264.7(macr?fago murino) e em c?lulas tumorais HeLa (Adenocarcinoma cervical uterino humano), CHO K1 (C?lulas de ov?rio de Hamster chin?s), PANC-1 (Carcinoma de p?ncreas humano) e PC-3 (Adenocarcinoma de pr?stata humano); pela dosagem de mediadores da inflama??o NO, TNF-? e IL-6 liberados por c?lulas RAW 264.7 e pela an?lise do processo de morte e da altera??o do ciclo celular de PC-3 por citometria de fluxo. Os dados demonstraram que os produtos homeop?ticos de Momordica charantia n?o apresentaram citotoxicidade para RAW 264.7, aumentaram a produ??o dos mediadores da inflama??o por RAW 264.7 sinergicamente com LPS, mostraram citotoxicidade para PC-3 com altera??o no seu ciclo celular inibindo sua prolifera??o, sendo 30CH a amostra mais potente. Foram realizados estudos de correla??o visando avaliar os poss?veis modelos in vitro aplic?veis ao controle de qualidade de produtos homeop?ticos contendo Momordica charantia. Os dados demonstraram que os melhores modelos aplic?veis na avalia??o da qualidade s?o o teste do MTT para avaliar citotoxicidade em RAW 264.7 e PC-3 em 24 horas para os produtos de Momordica charantia (fr) e dosagem da produ??o de NO por RAW 264.7 com e sem LPS

O carbonato de lodenafila é um inibidor da fosfodiesterase tipo 5, desenvolvido no Brasil, o qual é um dímero formado por duas moléculas de lodenafila ligadas por uma ponte carbonato. Encontra-se disponível sob a forma de comprimidos. Não existe monografia disponível para este fármaco em nenhum código oficial. Assim, o objetivo do presente estudo foi desenvolver métodos analíticos para análise qualitativa e quantitativa do carbonato de lodenafila em comprimidos, avaliação da estabilidade frente à degradação forçada, contemplando a cinética de fotodegradação, identificação e estudo de citotoxicidade in vitro dos produtos de degradação majoritários e desenvolvimento do teste de dissolução baseado em dados in vivo. A caracterização da substância química de referência foi realizada através da faixa de fusão por calorimetria exploratória diferencial, métodos espectrofotométricos na região do infravermelho (IV) e do ultravioleta (UV), assim como espectroscopia de ressonância magnética nuclear de 1H e 13C. Os métodos por cromatografia em camada delgada (CCD), espectrofotometria no UV, cromatografia líquida de alta eficiência (CLAE) e eletroforese capilar (EC) foram utilizados para identificar o fármaco na forma farmacêutica. Os seguintes métodos de análise quantitativa do fármaco em comprimidos foram desenvolvidos e validados: método indicativo da estabilidade por CLAE, espectrofotometria na região do UV e EC. Todos cumpriram com os parâmetros descritos pelas guias de validação e não apresentaram diferença significativa na determinação do fármaco. A cinética de fotodegradação do carbonato de lodenafila apresentou cinética de primeira ordem. Dois produtos majoritários encontrados na fotodegradação foram identificados por cromatografia líquida de ultra eficiência acoplada à espectrometria de massas (CLUE-MS/MS) como ácido 4-etoxi-3-(1-metil-7-oxo-3-propil-6,7-diidro- 1H-pirazol [4,3-d] pirimidina-5-il) benzenossulfônico e ácido 4-etoxi-3-(1-metil-7-oxo- 3-propil-6,7-diidro-1H-pirazol [4,3-d] pirimidina-5-il) benzenossulfínico. A avaliação da citotoxicidade in vitro indicou que a amostra de carbonato de lodenafila degradada não apresentou toxicidade. O teste de dissolução foi desenvolvido e validado utilizando como meio de dissolução 900 mL de HCl 0,1 M + lauril sulfato de sódio 1,5% (p/v), a 37 ± 0,5°C, pás a 25 rpm e quantificação por espectrofotometria no UV. O perfil de dissolução apresentou cinética de primeira ordem. De acordo com os resultados obtidos, todos os métodos propostos podem ser utilizados no controle de qualidade do carbonato de lodenafila. / Lodenafil carbonate is a PDE5 inhibitor developed in Brazil, which is a dimer formed by two lodenafil molecules linked by a carbonate bridge. It is currently available in tablets. There is no monograph available for this drug in any official code. Thus, the purpose of the present study was the development of analytical methods for qualitative and quantitative analysis of LC in tablets, evaluation of drug stability in forced degradation, comprising the determination of photodegradation kinetic, identification and in vitro cytotoxicity study of two major degradation products and develop a dissolution test based on in vivo data. The characterization of the reference chemical substance was performed by: melting range by differencial scanning calorimetry, infrared (IR) and ultraviolet (UV) spectrophotometry methods, as well as the 1H and 13C nuclear magnetic resonance spectroscopy. The methods by thin-layer chromatography (TLC), UV spectrophotometry, high-performance liquid chromatography (LC) and capillary electrophoresis (CE) were used to identify the drug in pharmaceutical formulations. The following methods, applied to the assay of the drug in tablets were developed and validated: a stability-indicating HPLC method, UV spectrophotometry and CE. All of them met the criteria described by the validation guidelines and showed no significant statistically difference in the drug determination. The photodegradation kinetics of lodenafil carbonate showed firstorder kinetics. Two degradation products found by photodegradation were identified by ultra performance liquid chromatography-tandem mass spectrometry (UPLCMS/ MS) as 4-ethoxy-3-(1-methyl-7-oxo-3-propyl-6,7-dihydro-1H-pyrazolo [4,3- d]pyrimidin-5-yl)-benzenesulfonic acid and 4-ethoxy-3-(1-methyl-7-oxo-3-propyl-6,7- dihydro-1H-pyrazolo [4,3-d]pyrimidin-5-yl) benzenesulfinic acid. The in vitro cytotoxicity evaluation indicated that the lodenafil carbonate degraded sample did not show toxicity. A dissolution test was developed and validated using 900 mL of 0.1 M HCl + 1.5% sodium lauryl sulfate (w/v), at 37 ± 0.5 °C as dissolution medium, paddle at 25 rpm and quantitation by UV spectrophotometry. The dissolution profile showed first order kinetics. According to the obtained results, all proposed methods can be used in the quality control of lodenafil carbonate.