Οι κυκλοδεξτρίνες είναι κυκλικοί ολιγοσακχαρίτες αποτελούμενοι από μόρια α-D-γλυκοπυρανόζης που συνδέονται με α-1-4 γλυκοζιτικούς δεσμούς και παράγονται από το άμυλο. Διαφέρουν μεταξύ τους από τον αριθμό των μονάδων γλυκοπυρανόζης της δομής τους. Οι κυκλοδεξτρίνες διακρίνονται σε δύο ομάδες: τις φυσικές και τις τροποποιημένες. Οι πιο κοινές φυσικές κυκλοδεξτρίνες είναι οι α-κυκλοδεξτρίνη, β-κυκλοδεξτρίνη και γ-κυκλοδεξτρίνη, που αποτελούνται από 6, 7 και 8 μονάδες γλυκοπυρανόζης, αντίστοιχα. Επίσης, οι τροποποιημένες κυκλοδεξτρίνες εμφανίζουν σημαντικά πλεονεκτήματα σε σχέση με τις φυσικές. Η δομή των κυκλοδεξτρινών προσομοιάζει το σχήμα κόλουρου κώνου με μια υδρόφιλη εξωτερική επιφάνεια και μια λιπόφιλη εσωτερική κοιλότητα. Η υδρόφιλη εξωτερική επιφάνεια εξασφαλίζει καλή υδατοδιαλυτότητα για το μόριο της κυκλοδεξτρίνης, ενώ η υδρόφοβη εσωτερική κοιλότητα δημιουργεί το κατάλληλο περιβάλλον για τον εγκλωβισμό ολόκληρου του μορίου μιας βιοδραστικής ένωσης ή τμήματος αυτού. Οι κυκλοδεξτρίνες μπορούν να αλληλεπιδράσουν με μόρια κατάλληλου μεγέθους για το σχηματισμό συμπλόκων έγκλεισης. Ο σχηματισμός των συμπλόκων έγκλεισης των κυκλοδεξτρινών με τις βιοδραστικές ενώσεις συχνά έχει ως αποτέλεσμα τη τροποποίηση των φυσικών και χημικών ιδιοτήτων του εγκλωβιζόμενου μορίου. Η συμπλοκοποίηση των κυκλοδεξτρινών έχει χρησιμοποιηθεί επιτυχώς για την αύξηση της διαλυτότητας, του ρυθμού διαλυτοποίησης, της χημικής σταθερότητας και βιοδιαθεσιμότητας ελάχιστα διαλυτών και αδιάλυτων βιοδραστικών ενώσεων. Επίσης, οι κυκλοδεξτρίνες μπορούν να χρησιμοποιηθούν για τη μείωση ή κάλυψη ανεπιθύμητων οσμών και δυσάρεστων γεύσεων, την αντιμετώπιση των προβλημάτων ασυμβατότητας μεταξύ των βιοδραστικών ουσιών και μεταξύ των βιοδραστικών ουσιών και των εκδόχων και για τη κάλυψη των παρενεργειών των φαρμάκων. Η ιτρακοναζόλη είναι μία τριαζόλη με αντιμυκητιασική δράση ευρέως φάσματος, η οποία είναι πρακτικά αδιάλυτη στο νερό σε φυσιολογικές συνθήκες και περισσότερο διαλυτή σε πολύ όξινες συνθήκες, με αποτέλεσμα να παρουσιάζει πολύ μικρή βιοδιαθεσιμότητα μετά από του στόματος χορήγηση. Η ιτρακοναζόλη είναι μία ασθενής βάση με πολύ μικρή υδατοδιαλυτότητα. Η υδατοδιαλυτότητα της ιτρακοναζόλης σε ουδέτερο pH είναι ~ 1 ng/ml και σε pH 1 είναι ~ 4μg/ml. Οι κυκλοδεξτρίνες που μελετήθηκαν στη παρούσα εργασία είναι οι : υδροξυπρόπυλο-β-CD (ΗP-β-CD), μέθυλο-β-CD (Με-β-CD), υδροξυαίθυλο-β-CD (HE-β-CD) και υδροξυπρόπυλο-γ-CD (ΗP-γ-CD). Στόχοι της παρούσας εργαστηριακής άσκησης είναι:  ο προσδιορισμός της επίδρασης των παραπάνων κυκλοδεξτρινών στην διαλυτότητα της ιτρακοναζόλης σε όξινα υδατικά διαλύματα  ο χαρακτηρισμός των στερεών συμπλόκων της ιτρακοναζόλης με τις προς εξέταση κυκλοδεξτρίνες που παρασκευάσθηκαν με διαφορετικές τεχνικές. / Cyclodextrins are cyclic (α-1,4)-linked oligosaccharides of α-D-glucopyranose derived from starch. They differ from one another by the number of glucopyranose units in the structure. Cyclodextrins can be classified in two groups: natural and modified. The most common natural cyclodextrins are α-cyclodextrin, β–cyclodextrin andι γ-cyclodextrin which consist of six, seven and eight glucopyranose units respectively. Also, modified cyclodextrins offer significant advantages in comparison with natural cyclodextrins. The cyclodextrin structure provides a molecule shaped like a segment of a hollow cone with an exterior hydrophilic surface and interior hydrophobic cavity. The hydrophilic surface generates good water solubility for the cyclodextrin and the hydrophobic cavity provides a favorable environment in which the entire or part of the drug molecule is fitted. Cyclodextrins can interact with appropriately sized molecules to result in the formation of inclusion compounds. Inclusion complex formation between cyclodextrins and drugs often modifies the physical and chemical properties of the guest molecules. Cyclodextrin complexation has been successfully used to improve solubility, dissolution rate, chemical stability and bioavailability of sparingly soluble or insoluble drugs. Also, cyclodextrins can be used to reduce or eliminate unpleasant smell or tastes of drugs, prevent drug-drug or drug-additive interactions and reduce or prevent drug side effects. Itraconazole is an orally broad-spectrum triazole antifungal agent, which is practically insoluble in water at physiological conditions and more soluble only in extremely acidic media, leading to poor oral biovailability. ITZ is a very poorly water soluble, weak base. The aqueous solubility is estimated at ~ 1ng/ml at neutral pH and ~ 4μg/ml at pH = 1. [2] The chemical structure of ITZ is shown in Figure 1. The cyclodextrins tested in this work were : Hydroxypropyl-β-Cyclodextrin (HP-β-CD), Methyl-β-Cyclodextrin (Me-β-CD), Hydroxyethyl-β-Cyclodextrin (He-β-CD) and Hydroxypropyl-γ-Cyclodextrin (HP-γ-CD). The aims of this work are:  to evaluate the influence of above cyclodextrins on the solubility of ITZ at acified aqueous solutions  to characterize the solid complexes formed between cyclodextrins and ITZ prepared by different techniques.

Title of thesis: Polymeric stabilizers maintaining the saturation solubility of itraconazole nanocrystals after dissolution process Author: Jana Kubačková Department: Pharmaceutical Technology Supervisor: PharmDr. Ondřej Holas, Ph.D. Specialized supervisor: Assoc. Prof. Leena Peltonen, Ph.D. The increase of bioavailability of poorly water soluble drugs is still an issue. One of the techniques improving aqueous drug substance solubility, and consequently enhancing bioavailability, is formation of nanoparticles. However, the bioavailability is determined by the concentration of the dissolved drug achieved at the time of absorption. This fact emphasizes the importance of the maintenance of the high solubility until the absorption area is reached. Sufficiently stabilised nanocrystalline drugs offer a solution to this problem. In this thesis, the solid nanoparticle formations of an antifungal agent itraconazole (ITZ) are presented. Wet milling was employed to create the nanosuspension stabilised by binary mixture of stabilisers or by a single stabiliser. An aggregation inhibitor Poloxamer 407 (F127) in the combination with a polymeric precipitation inhibitor hydroxypropyl methylcellulose (HPMC) or polyvinyl pyrrolidone (PVP) at different ratios, or a single precipitation inhibitor, were utilised. The...

Considerando a importância do felino doméstico nos relatos zoonóticos da esporotricose, assim como os problemas relacionados a terapêutica da micose nessa espécie, o estudo objetiva avaliar a atividade in vitro da terbinafina e itraconazol frente ao S. schenckii; estudar a ação desses fármacos no tratamento da esporotricose sistêmica experimental; analisar as enzimas hepáticas e hemograma dos animais submetidos ao tratamento antifúngico; estudar as possíveis alterações macroscópicas nos animais inoculados com S. schenckii assim como realizar o retroisolamento do agente. Para o teste in vitro, foram utilizados 10 isolados de S. schenckii (seis felinos, três humanos e um de cão), os quais foram avaliados quanto a suscetibilidade frente aos fármacos através da técnica de microdiluição em caldo de acordo com o CLSI para as fases leveduriforme e filamentosa do agente. A leitura da CIM foi visual, correspondendo à menor concentração do antifúngico em que não houve crescimento visível do S. schenckii quando comparado ao controle positivo. O teste in vivo, foi realizado através da inoculação pelas vias intraperitonial e veia lateral da cauda de células fúngicas (2x103 cél/mL) de dois isolados de S. schenckii (felino e canino) em 120 ratos norvergicus, divididos em quatro grupos: tratado terbinafina (250mg/kg) e itraconazol (100mg/kg); diluente terbinafina (250mg/kg de solução de água destilada com 5% de DMSO e 1% de Tween 20) e itraconazol (100mg/kg de água destilada estéril), sendo administrados através de sondagem oral uma vez ao dia, durante 30 dias. Paralelamente foram realizados hemogramas e avaliação das enzimas hepáticas (ALT e FA), assim como o acompanhamento da evolução clínica e análise histopatológica de todos animais experimentais. No final do período experimental os animais foram necropsiados e realizado análise macroscópica com a coleta dos órgãos para a obtenção do retroisolamento do S. schenckii. A CIM para a terbinafina obtida entre todos os isolados na fase leveduriforme de esporotricose felina variaram de 0,055μg/mL a 0,109μg/mL, enquanto que para os isolados humanos e de cão foi de 0,055μg/ml. Para o itraconazol, a CIM frente aos isolados de esporotricose felina foi de 0,875μg/mL a 1,75μg/mL, já para os casos humanos foi de 0,219μg/mL e para o isolado de cão de 1,75μg/mL. Para a fase filamentosa do agente foi observada uma CIM para a terbinafina entre os isolados de felinos de 0,875 μg/mL, enquanto que os isolados humanos e de cão a CIM foi de 0,219μg/mL. Para o itraconazol os isolados felinos resultaram numa CIM de 3,5μg/mL, já nos isolados de esporotricose humana e em cão a CIM foi de 1,75μg/mL. Quanto ao estudo in vivo, os animais tratados com terbinafina e itraconazol resultaram em menor freqüência de alterações macroscópicas assim como na obtenção do retroisolamento do agente em relação aos grupos controle. Não foi detectada alterações nas enzimas hepáticas, hemograma e nas avaliações macro e histopatológicas dos animais submetidos ao tratamento antifúngico. Com esses resultados conclui-se que a terbinafina e itraconazol possuem atividade in vitro frente ao S. schenckii e que as doses estudadas dos fármacos são eficazes no tratamento da esporotricose experimental sistêmica, assim como não produzem alterações das enzimas hepáticas avaliadas e do hemograma. / Considering the importance of the domestic feline in the zoonotic reports of the esporotrichosis, as well as the related problems the therapeutics of the mycosis in that species, the study objective was evaluate the activity in vitro of terbinafine and itraconazole against to S. schenckii; study the activity of those drugs in the treatment of the experimental systemic esporotrichosis; analyze the hepatic enzymes and blood count of the animals submitted to the antifungal treatment; study the possible macroscopic alterations in the animals inoculated with S. schenckii as well as realized the agent retroisolament. For the test in vitro, 10 isolated of S. schenckii were used (six felines, three humans and one canine), which were performed as the susceptibility against to the drugs through the microdiluição technique in broth in accordance with the CLSI for the phases yeast and mycelial of the agent. The reading of CIM was visual, corresponding to smallest concentration of the antifungal in that there was not visible growth of S. schenckii when compared to the positive control. The test in vivo, was performed through the of the inoculation intraperitonial and lateral vein of the tail of fungal cells (2x103 cél/mL) of two isolated of S. schenckii (feline and canine) in 120 mice norvergicus, divided in four groups: treaty terbinafine (250mg/kg) and itraconazole (100mg/kg); diluent terbinafine (250mg/kg of solution of water distilled with 5% of DMSO and 1% of Tween 20) and itraconazol (100mg/kg of water distilled sterile), being administered by gavage once a day, for 30 days. Parallel blood counts and evaluation of the hepatic enzymes were accomplished (ALT and F.A), as well as the accompaniment of the clinical evolution and analysis histopathologic of all animal experimental. In the end of the experimental period the animals were autopsied and performed macroscopic analysis with the collection of the organs for the obtaining of the detected S. schenckii growth. CIM for the terbinafine obtained among all the isolated in the phase yeast of feline esporotrichosis oscillated between 0,055μg/mL the 0,109μg/mL, while for the isolated humans and canine was of 0,055μg/ml. For the itraconazole, the CIM against of the isolated of feline esporotrichosis was from 0,875μg/mL to 1,75μg/mL, for the human cases it went of 0,219μg/mL and for the isolated canine of 1,75μg/mL. For the mycelial phase a CIM was observed for the terbinafine among the isolated of felines of 0,875 μg/mL, while the isolated humans and canine CIM was of 0,219μg/mL. For the itraconazole the isolated felines resulted in a CIM 3,5μg/mL, in the isolated of human esporotrichosis and canine CIM was of 1,75μg/mL. As for the study in vivo, the animals treaties with terbinafine and itraconazole resulted in smaller frequency of macroscopic alterations as well as in the obtaining of the detected agent growth in relation to the groups control. It was not detected alterations in the hepatic enzymes, blood count and in the evaluations macro and histopathologic of the animals submitted to the antifungal treatment. With those results we are to report that the terbinafine and itraconazol apresent activity in vitro against to S. schenckii and that the studied doses of the drugs are effective in the treatment of the systemic experimental esporotrichosis, as well as they don't produce alterations of the appraised hepatic enzymes and blood count.

Considerando a importância do felino doméstico nos relatos zoonóticos da esporotricose, assim como os problemas relacionados a terapêutica da micose nessa espécie, o estudo objetiva avaliar a atividade in vitro da terbinafina e itraconazol frente ao S. schenckii; estudar a ação desses fármacos no tratamento da esporotricose sistêmica experimental; analisar as enzimas hepáticas e hemograma dos animais submetidos ao tratamento antifúngico; estudar as possíveis alterações macroscópicas nos animais inoculados com S. schenckii assim como realizar o retroisolamento do agente. Para o teste in vitro, foram utilizados 10 isolados de S. schenckii (seis felinos, três humanos e um de cão), os quais foram avaliados quanto a suscetibilidade frente aos fármacos através da técnica de microdiluição em caldo de acordo com o CLSI para as fases leveduriforme e filamentosa do agente. A leitura da CIM foi visual, correspondendo à menor concentração do antifúngico em que não houve crescimento visível do S. schenckii quando comparado ao controle positivo. O teste in vivo, foi realizado através da inoculação pelas vias intraperitonial e veia lateral da cauda de células fúngicas (2x103 cél/mL) de dois isolados de S. schenckii (felino e canino) em 120 ratos norvergicus, divididos em quatro grupos: tratado terbinafina (250mg/kg) e itraconazol (100mg/kg); diluente terbinafina (250mg/kg de solução de água destilada com 5% de DMSO e 1% de Tween 20) e itraconazol (100mg/kg de água destilada estéril), sendo administrados através de sondagem oral uma vez ao dia, durante 30 dias. Paralelamente foram realizados hemogramas e avaliação das enzimas hepáticas (ALT e FA), assim como o acompanhamento da evolução clínica e análise histopatológica de todos animais experimentais. No final do período experimental os animais foram necropsiados e realizado análise macroscópica com a coleta dos órgãos para a obtenção do retroisolamento do S. schenckii. A CIM para a terbinafina obtida entre todos os isolados na fase leveduriforme de esporotricose felina variaram de 0,055μg/mL a 0,109μg/mL, enquanto que para os isolados humanos e de cão foi de 0,055μg/ml. Para o itraconazol, a CIM frente aos isolados de esporotricose felina foi de 0,875μg/mL a 1,75μg/mL, já para os casos humanos foi de 0,219μg/mL e para o isolado de cão de 1,75μg/mL. Para a fase filamentosa do agente foi observada uma CIM para a terbinafina entre os isolados de felinos de 0,875 μg/mL, enquanto que os isolados humanos e de cão a CIM foi de 0,219μg/mL. Para o itraconazol os isolados felinos resultaram numa CIM de 3,5μg/mL, já nos isolados de esporotricose humana e em cão a CIM foi de 1,75μg/mL. Quanto ao estudo in vivo, os animais tratados com terbinafina e itraconazol resultaram em menor freqüência de alterações macroscópicas assim como na obtenção do retroisolamento do agente em relação aos grupos controle. Não foi detectada alterações nas enzimas hepáticas, hemograma e nas avaliações macro e histopatológicas dos animais submetidos ao tratamento antifúngico. Com esses resultados conclui-se que a terbinafina e itraconazol possuem atividade in vitro frente ao S. schenckii e que as doses estudadas dos fármacos são eficazes no tratamento da esporotricose experimental sistêmica, assim como não produzem alterações das enzimas hepáticas avaliadas e do hemograma. / Considering the importance of the domestic feline in the zoonotic reports of the esporotrichosis, as well as the related problems the therapeutics of the mycosis in that species, the study objective was evaluate the activity in vitro of terbinafine and itraconazole against to S. schenckii; study the activity of those drugs in the treatment of the experimental systemic esporotrichosis; analyze the hepatic enzymes and blood count of the animals submitted to the antifungal treatment; study the possible macroscopic alterations in the animals inoculated with S. schenckii as well as realized the agent retroisolament. For the test in vitro, 10 isolated of S. schenckii were used (six felines, three humans and one canine), which were performed as the susceptibility against to the drugs through the microdiluição technique in broth in accordance with the CLSI for the phases yeast and mycelial of the agent. The reading of CIM was visual, corresponding to smallest concentration of the antifungal in that there was not visible growth of S. schenckii when compared to the positive control. The test in vivo, was performed through the of the inoculation intraperitonial and lateral vein of the tail of fungal cells (2x103 cél/mL) of two isolated of S. schenckii (feline and canine) in 120 mice norvergicus, divided in four groups: treaty terbinafine (250mg/kg) and itraconazole (100mg/kg); diluent terbinafine (250mg/kg of solution of water distilled with 5% of DMSO and 1% of Tween 20) and itraconazol (100mg/kg of water distilled sterile), being administered by gavage once a day, for 30 days. Parallel blood counts and evaluation of the hepatic enzymes were accomplished (ALT and F.A), as well as the accompaniment of the clinical evolution and analysis histopathologic of all animal experimental. In the end of the experimental period the animals were autopsied and performed macroscopic analysis with the collection of the organs for the obtaining of the detected S. schenckii growth. CIM for the terbinafine obtained among all the isolated in the phase yeast of feline esporotrichosis oscillated between 0,055μg/mL the 0,109μg/mL, while for the isolated humans and canine was of 0,055μg/ml. For the itraconazole, the CIM against of the isolated of feline esporotrichosis was from 0,875μg/mL to 1,75μg/mL, for the human cases it went of 0,219μg/mL and for the isolated canine of 1,75μg/mL. For the mycelial phase a CIM was observed for the terbinafine among the isolated of felines of 0,875 μg/mL, while the isolated humans and canine CIM was of 0,219μg/mL. For the itraconazole the isolated felines resulted in a CIM 3,5μg/mL, in the isolated of human esporotrichosis and canine CIM was of 1,75μg/mL. As for the study in vivo, the animals treaties with terbinafine and itraconazole resulted in smaller frequency of macroscopic alterations as well as in the obtaining of the detected agent growth in relation to the groups control. It was not detected alterations in the hepatic enzymes, blood count and in the evaluations macro and histopathologic of the animals submitted to the antifungal treatment. With those results we are to report that the terbinafine and itraconazol apresent activity in vitro against to S. schenckii and that the studied doses of the drugs are effective in the treatment of the systemic experimental esporotrichosis, as well as they don't produce alterations of the appraised hepatic enzymes and blood count.

Considerando a importância do felino doméstico nos relatos zoonóticos da esporotricose, assim como os problemas relacionados a terapêutica da micose nessa espécie, o estudo objetiva avaliar a atividade in vitro da terbinafina e itraconazol frente ao S. schenckii; estudar a ação desses fármacos no tratamento da esporotricose sistêmica experimental; analisar as enzimas hepáticas e hemograma dos animais submetidos ao tratamento antifúngico; estudar as possíveis alterações macroscópicas nos animais inoculados com S. schenckii assim como realizar o retroisolamento do agente. Para o teste in vitro, foram utilizados 10 isolados de S. schenckii (seis felinos, três humanos e um de cão), os quais foram avaliados quanto a suscetibilidade frente aos fármacos através da técnica de microdiluição em caldo de acordo com o CLSI para as fases leveduriforme e filamentosa do agente. A leitura da CIM foi visual, correspondendo à menor concentração do antifúngico em que não houve crescimento visível do S. schenckii quando comparado ao controle positivo. O teste in vivo, foi realizado através da inoculação pelas vias intraperitonial e veia lateral da cauda de células fúngicas (2x103 cél/mL) de dois isolados de S. schenckii (felino e canino) em 120 ratos norvergicus, divididos em quatro grupos: tratado terbinafina (250mg/kg) e itraconazol (100mg/kg); diluente terbinafina (250mg/kg de solução de água destilada com 5% de DMSO e 1% de Tween 20) e itraconazol (100mg/kg de água destilada estéril), sendo administrados através de sondagem oral uma vez ao dia, durante 30 dias. Paralelamente foram realizados hemogramas e avaliação das enzimas hepáticas (ALT e FA), assim como o acompanhamento da evolução clínica e análise histopatológica de todos animais experimentais. No final do período experimental os animais foram necropsiados e realizado análise macroscópica com a coleta dos órgãos para a obtenção do retroisolamento do S. schenckii. A CIM para a terbinafina obtida entre todos os isolados na fase leveduriforme de esporotricose felina variaram de 0,055μg/mL a 0,109μg/mL, enquanto que para os isolados humanos e de cão foi de 0,055μg/ml. Para o itraconazol, a CIM frente aos isolados de esporotricose felina foi de 0,875μg/mL a 1,75μg/mL, já para os casos humanos foi de 0,219μg/mL e para o isolado de cão de 1,75μg/mL. Para a fase filamentosa do agente foi observada uma CIM para a terbinafina entre os isolados de felinos de 0,875 μg/mL, enquanto que os isolados humanos e de cão a CIM foi de 0,219μg/mL. Para o itraconazol os isolados felinos resultaram numa CIM de 3,5μg/mL, já nos isolados de esporotricose humana e em cão a CIM foi de 1,75μg/mL. Quanto ao estudo in vivo, os animais tratados com terbinafina e itraconazol resultaram em menor freqüência de alterações macroscópicas assim como na obtenção do retroisolamento do agente em relação aos grupos controle. Não foi detectada alterações nas enzimas hepáticas, hemograma e nas avaliações macro e histopatológicas dos animais submetidos ao tratamento antifúngico. Com esses resultados conclui-se que a terbinafina e itraconazol possuem atividade in vitro frente ao S. schenckii e que as doses estudadas dos fármacos são eficazes no tratamento da esporotricose experimental sistêmica, assim como não produzem alterações das enzimas hepáticas avaliadas e do hemograma. / Considering the importance of the domestic feline in the zoonotic reports of the esporotrichosis, as well as the related problems the therapeutics of the mycosis in that species, the study objective was evaluate the activity in vitro of terbinafine and itraconazole against to S. schenckii; study the activity of those drugs in the treatment of the experimental systemic esporotrichosis; analyze the hepatic enzymes and blood count of the animals submitted to the antifungal treatment; study the possible macroscopic alterations in the animals inoculated with S. schenckii as well as realized the agent retroisolament. For the test in vitro, 10 isolated of S. schenckii were used (six felines, three humans and one canine), which were performed as the susceptibility against to the drugs through the microdiluição technique in broth in accordance with the CLSI for the phases yeast and mycelial of the agent. The reading of CIM was visual, corresponding to smallest concentration of the antifungal in that there was not visible growth of S. schenckii when compared to the positive control. The test in vivo, was performed through the of the inoculation intraperitonial and lateral vein of the tail of fungal cells (2x103 cél/mL) of two isolated of S. schenckii (feline and canine) in 120 mice norvergicus, divided in four groups: treaty terbinafine (250mg/kg) and itraconazole (100mg/kg); diluent terbinafine (250mg/kg of solution of water distilled with 5% of DMSO and 1% of Tween 20) and itraconazol (100mg/kg of water distilled sterile), being administered by gavage once a day, for 30 days. Parallel blood counts and evaluation of the hepatic enzymes were accomplished (ALT and F.A), as well as the accompaniment of the clinical evolution and analysis histopathologic of all animal experimental. In the end of the experimental period the animals were autopsied and performed macroscopic analysis with the collection of the organs for the obtaining of the detected S. schenckii growth. CIM for the terbinafine obtained among all the isolated in the phase yeast of feline esporotrichosis oscillated between 0,055μg/mL the 0,109μg/mL, while for the isolated humans and canine was of 0,055μg/ml. For the itraconazole, the CIM against of the isolated of feline esporotrichosis was from 0,875μg/mL to 1,75μg/mL, for the human cases it went of 0,219μg/mL and for the isolated canine of 1,75μg/mL. For the mycelial phase a CIM was observed for the terbinafine among the isolated of felines of 0,875 μg/mL, while the isolated humans and canine CIM was of 0,219μg/mL. For the itraconazole the isolated felines resulted in a CIM 3,5μg/mL, in the isolated of human esporotrichosis and canine CIM was of 1,75μg/mL. As for the study in vivo, the animals treaties with terbinafine and itraconazole resulted in smaller frequency of macroscopic alterations as well as in the obtaining of the detected agent growth in relation to the groups control. It was not detected alterations in the hepatic enzymes, blood count and in the evaluations macro and histopathologic of the animals submitted to the antifungal treatment. With those results we are to report that the terbinafine and itraconazol apresent activity in vitro against to S. schenckii and that the studied doses of the drugs are effective in the treatment of the systemic experimental esporotrichosis, as well as they don't produce alterations of the appraised hepatic enzymes and blood count.

Die Aspergillose ist eine Erkrankung des Respirationstraktes bei Vögeln, vor allem in Gefangenschaft gehaltenen tropischen Papageien, Greifvögeln und Pinguinen. Itraconazol ist hierbei ein häufig eingesetztes Therapeutikum und wird in der Vogelmedizin per os verabreicht (JONES und OROSZ 2000; KELLER 2011). Es zeigten sich gute Wirksamkeiten und Resistenzlagen. Bei über 90 % der Aspergillus fumigatus Isolate aus Wild- und Hausvögeln konnte eine MHK von 0,5 µg/ml eruiert werden (BEERNAERT et al. 2009). Allerdings kam es nach oraler Gabe von Itraconazol zu einem verzögerten Wirkungseintritt sowie, vor allem bei Graupapageien, zu Nebenwirkungen (KRAUTWALD-JUNGHANNS 2011b; PSCHERER 1995). Um ausreichende Wirkspiegel in Lunge und Luftsäcken zu erreichen, ist die Verabreichung einer hohen oralen Dosis notwendig. Dies führt wiederum zu hohen systemischen Konzentrationen und somit zu hohen potenziellen Nebenwirkungen (LUMEIJ et al. 1995). Ein Ziel in der Verbesserung der Therapiemöglichkeiten der Aspergillose ist es daher, einen hohen lokalen Wirkspiegel mit gleichzeitig geringen systemischen Konzentrationen zu erreichen. Ein Ansatz besteht in der inhalativen Verabreichung von z. B. Nanosuspensionen, da hier das Medikament direkt in Lunge/Luftsäcke appliziert werden kann. Ziel dieser Arbeit war die Sammlung von ersten Daten über den Einsatz einer neuartigen Itraconazol-Nanosuspension zur Verabreichung per inhalationem an Tauben (Columba livia f. domestica). Hierzu wurden in einer ersten Studie pharmakokinetische Spiegel in Blut sowie Lungengewebe nach einmaliger Gabe zweier Konzentrationsstufen (1 % und 10 %) erfasst. In der zweiten Studie wurde die Verträglichkeit sowie die Pharmakokinetik nach 14 tägiger Inhalation der Suspension in den Dosierungsstufen 1 %, 4 % und 10 % evaluiert. In der ersten Studie wurden die Tauben in zwei Hauptgruppen sowie eine Placebogruppe randomisiert aufgeteilt und erhielten einmalig über 30 min die Itraconazol-Nanosuspension per inhalationem in den Dosierungsstufen 1 % und 10 % bzw. isotonische Natriumchlorid-Lösung. Keines der Tiere zeigte Nebenwirkungen, die in Zusammenhang mit der Medikation gestellt werden konnten. Zur Erstellung eines pharmakokinetischen Profils wurden Blutproben nach 1 h, 4 h, 24 h, 48 h, 72 h und 96 h sowie Lungengewebe von je vier Tauben nach der Euthanasie entnommen und darin die Itraconazol sowie die OH Itraconazol Konzentrationen bestimmt. Alle vorhandenen Werte zu einem gegebenen Zeitpunkt wurden gemittelt, woraus sich ein zusammengesetztes Profil ergab. Nach singulärer Verabreichung zeigten sich geringe systemische Spiegel mit einer Peak-Konzentration von 0,01 µg/ml Itraconazol 4 h post inhalationem bzw. 0,04 µg/ml OH-Itraconazol 24 h post inhalationem bei der 1%igen Nanosuspension und 0,065 µg/ml Itraconazol 24 h nach der Inhalation bzw. 0,365 µg/ml OH Itraconazol 24 h nach der Inhalation bei der 10%igen Dosierungsgruppe. In den Lungen konnten weitaus höhere Spiegel mit 9,1 µg/g Itraconazol und 0,223 µg/g OH Itraconazol 1 h post inhalationem bei der 1%igen Dosierungsgruppe bzw. 91,13 µg/g Itraconazol 1 h nach der Inhalation und 1,081 µg/g OH Itraconazol 4 h nach der Inhalation bei der 10%igen Dosierungsgruppe detektiert werden. Bei der zweiten Studie wurden die Tiere in drei Hauptgruppen sowie eine Placebogruppe randomisiert aufgeteilt und erhielten 14 Tage lang über je 30 min die Itraconazol-Nanosuspension per inhalationem in den Konzentrationen 1 %, 4 % und 10 % bzw. istotonische Natriumchlorid Lösung. Zur Abklärung der Verträglichkeit wurden die Tiere täglich adspektorisch, sowie alle sieben Tage ausführlich klinisch untersucht. Weiterhin wurden vor und nach der Inhalationsperiode detaillierte Blutuntersuchungen durchgeführt. Bei keiner der Untersuchungen konnten Unverträglichkeitsreaktionen auf das zu testende Medikament festgestellt werden. Auch nach 14 tägiger Gabe konnten im Plasma nur geringe Itraconazol-/OH Itraconazol-Konzentrationen gemessen werden. Aufgrund der teilweise sehr hohen Lungenspiegel (17,14 µg/g bei der 4%igen und 185 µg/g bei der 10%igen Nanosuspension je 24 h post inhalationem) und hohen Eliminationshalbwertszeiten (von über 40 h) sind jedoch hohe und langanhaltende Wirkspiegel am Infektionsort gegeben. Abschließend kann gesagt werden, dass die erlangten Daten über den Einsatz der neuartigen Itraconazol-Nanosuspension zur Verabreichung per inhalationem bei Tauben keine klinischen Nebenwirkungen erkennen ließen und sich ein effektives pharmakokinetisches Profil zeigte. Mit den hohen lokalen Lungenspiegeln nebst geringen systemischen Konzentrationen und somit geringen zu erwartende Nebenwirkungen erscheint das Medikament durchaus zum Einsatz gegen die Aspergillose bei Vögeln geeignet zu sein.

Die Aspergillose ist eine Erkrankung des Respirationstraktes bei Vögeln, vor allem in Gefangenschaft gehaltenen tropischen Papageien, Greifvögeln und Pinguinen. Itraconazol ist hierbei ein häufig eingesetztes Therapeutikum und wird in der Vogelmedizin per os verabreicht (JONES und OROSZ 2000; KELLER 2011). Es zeigten sich gute Wirksamkeiten und Resistenzlagen. Bei über 90 % der Aspergillus fumigatus Isolate aus Wild- und Hausvögeln konnte eine MHK von 0,5 µg/ml eruiert werden (BEERNAERT et al. 2009). Allerdings kam es nach oraler Gabe von Itraconazol zu einem verzögerten Wirkungseintritt sowie, vor allem bei Graupapageien, zu Nebenwirkungen (KRAUTWALD-JUNGHANNS 2011b; PSCHERER 1995). Um ausreichende Wirkspiegel in Lunge und Luftsäcken zu erreichen, ist die Verabreichung einer hohen oralen Dosis notwendig. Dies führt wiederum zu hohen systemischen Konzentrationen und somit zu hohen potenziellen Nebenwirkungen (LUMEIJ et al. 1995). Ein Ziel in der Verbesserung der Therapiemöglichkeiten der Aspergillose ist es daher, einen hohen lokalen Wirkspiegel mit gleichzeitig geringen systemischen Konzentrationen zu erreichen. Ein Ansatz besteht in der inhalativen Verabreichung von z. B. Nanosuspensionen, da hier das Medikament direkt in Lunge/Luftsäcke appliziert werden kann. Ziel dieser Arbeit war die Sammlung von ersten Daten über den Einsatz einer neuartigen Itraconazol-Nanosuspension zur Verabreichung per inhalationem an Tauben (Columba livia f. domestica). Hierzu wurden in einer ersten Studie pharmakokinetische Spiegel in Blut sowie Lungengewebe nach einmaliger Gabe zweier Konzentrationsstufen (1 % und 10 %) erfasst. In der zweiten Studie wurde die Verträglichkeit sowie die Pharmakokinetik nach 14 tägiger Inhalation der Suspension in den Dosierungsstufen 1 %, 4 % und 10 % evaluiert. In der ersten Studie wurden die Tauben in zwei Hauptgruppen sowie eine Placebogruppe randomisiert aufgeteilt und erhielten einmalig über 30 min die Itraconazol-Nanosuspension per inhalationem in den Dosierungsstufen 1 % und 10 % bzw. isotonische Natriumchlorid-Lösung. Keines der Tiere zeigte Nebenwirkungen, die in Zusammenhang mit der Medikation gestellt werden konnten. Zur Erstellung eines pharmakokinetischen Profils wurden Blutproben nach 1 h, 4 h, 24 h, 48 h, 72 h und 96 h sowie Lungengewebe von je vier Tauben nach der Euthanasie entnommen und darin die Itraconazol sowie die OH Itraconazol Konzentrationen bestimmt. Alle vorhandenen Werte zu einem gegebenen Zeitpunkt wurden gemittelt, woraus sich ein zusammengesetztes Profil ergab. Nach singulärer Verabreichung zeigten sich geringe systemische Spiegel mit einer Peak-Konzentration von 0,01 µg/ml Itraconazol 4 h post inhalationem bzw. 0,04 µg/ml OH-Itraconazol 24 h post inhalationem bei der 1%igen Nanosuspension und 0,065 µg/ml Itraconazol 24 h nach der Inhalation bzw. 0,365 µg/ml OH Itraconazol 24 h nach der Inhalation bei der 10%igen Dosierungsgruppe. In den Lungen konnten weitaus höhere Spiegel mit 9,1 µg/g Itraconazol und 0,223 µg/g OH Itraconazol 1 h post inhalationem bei der 1%igen Dosierungsgruppe bzw. 91,13 µg/g Itraconazol 1 h nach der Inhalation und 1,081 µg/g OH Itraconazol 4 h nach der Inhalation bei der 10%igen Dosierungsgruppe detektiert werden. Bei der zweiten Studie wurden die Tiere in drei Hauptgruppen sowie eine Placebogruppe randomisiert aufgeteilt und erhielten 14 Tage lang über je 30 min die Itraconazol-Nanosuspension per inhalationem in den Konzentrationen 1 %, 4 % und 10 % bzw. istotonische Natriumchlorid Lösung. Zur Abklärung der Verträglichkeit wurden die Tiere täglich adspektorisch, sowie alle sieben Tage ausführlich klinisch untersucht. Weiterhin wurden vor und nach der Inhalationsperiode detaillierte Blutuntersuchungen durchgeführt. Bei keiner der Untersuchungen konnten Unverträglichkeitsreaktionen auf das zu testende Medikament festgestellt werden. Auch nach 14 tägiger Gabe konnten im Plasma nur geringe Itraconazol-/OH Itraconazol-Konzentrationen gemessen werden. Aufgrund der teilweise sehr hohen Lungenspiegel (17,14 µg/g bei der 4%igen und 185 µg/g bei der 10%igen Nanosuspension je 24 h post inhalationem) und hohen Eliminationshalbwertszeiten (von über 40 h) sind jedoch hohe und langanhaltende Wirkspiegel am Infektionsort gegeben. Abschließend kann gesagt werden, dass die erlangten Daten über den Einsatz der neuartigen Itraconazol-Nanosuspension zur Verabreichung per inhalationem bei Tauben keine klinischen Nebenwirkungen erkennen ließen und sich ein effektives pharmakokinetisches Profil zeigte. Mit den hohen lokalen Lungenspiegeln nebst geringen systemischen Konzentrationen und somit geringen zu erwartende Nebenwirkungen erscheint das Medikament durchaus zum Einsatz gegen die Aspergillose bei Vögeln geeignet zu sein.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 114-127).

A esporotricose, micose subcutânea causada pelo fungo dimórfico Sporothrix schenckii, acomete o homem e várias espécies de animais, sendo os felinos domésticos a espécie mais comumente envolvida nos relatos zoonóticos da enfermidade. Considerando as dificuldades terapêuticas no tratamento da micose, incluindo toxicidade e o desenvolvimento de resistência aos antifúngicos disponíveis, o estudo objetivou avaliar a eficácia in vivo do imunomodulador (1-3) glucana e em associação ao Itraconazol no tratamento da esporotricose sistêmica experimental, além de avaliar a presença do S. schenckii na cavidade oral e a produção de óxido nítrico. Foram utilizados 96 ratos Wistar, machos, os quais foram divididos em seis grupos de 16 animais, sendo eles G1: 0,5mg de glucana previamente (três doses) e pós a inoculação; G2: 0,5 mg glucana pós inoculação; G3: grupo controle; G4: 0,5mg de glucana associada a 10mg/kg de itraconazol; G5: 10mg/kg de itraconazol; e grupo G6: duas doses de 0,5mg de glucana associado a 10mg/kg de itraconazol. Os animais foram inoculados com 0,1ml do inóculo fúngico pela via intraperitoneal e veia lateral da cauda, após uma semana iniciou-se o tratamento dos grupos, quando estes apresentavam sinais clínicos da enfermidade. Após acompanhamento clínico durante a cinco semanas de tratamento e isolamento do agente na cavidade oral dos animais, estes foram eutanasiados e necropsiados para a coleta de sangue, avaliação anatomopatológica dos órgãos, retroisolamento do agente, quantificação das unidades formadoras de colônias (UFC) do baço e quantificação de óxido nítrico. Na primeira avaliação foi observado no grupo G1, uma prevenção da enfermidade desencadeada pela glucana, uma vez que, os animais pertencentes a esse grupo não apresentaram sinais clínicos, lesões anatomopatológicas e obtiveram a menor quantificação de UFC (p<0,5). No entanto no final do período experimental o G4 foi significativamente melhor dentre os grupos de tratamento, pois, findou o período com 70% dos animais sem qualquer alteração clínica, lesões macroscópicas nos órgãos e retroisolamento do agente, além de demonstrar menor quantificação nas UFCs e não ocorrer nenhum óbito decorrente da enfermidade. Os grupos G2, G5 e G6 foram significativamente melhor que o grupo controle uma vez que este grupo apresentou uma mortalidade de 31,25%, além de que 100% dos animais permanceram com alterações clínicas, lesões em órgão como fígado, baço e testículo e demonstraram maior quantificação de UFC (p<0,5). Quanto á produção de óxido nítrico foi verificado maior quantidade nos grupos que receberam a glucana em detrimento aos demais grupos. Em vista dos resultados obtidos conclui-se que a glucana induz a produção de óxido nítrico e os cinco tratamentos demostraram resultados satisfatórios, no entanto, a associação do antifúngico e (1-3) glucana propiciou uma regressão mais precoce das lesões no tratamento da esportricose sistêmica experimental. / Sporotrichosis is a subcutaneous mycosis caused by the dimorphic fungus Sporothrix schenckii, which affects humans and a wide variety of animals, domestic cats involved in reports of zoonotic illness. Considering the difficulties in the therapeutic treatment of ringworm, including toxicity and the development of resistance to antifungals available, the study aimed to evaluate the efficacy of the immunomodulator in vivo (1-3) glucan and in combination with Itraconazole in the treatment of experimental systemic sporotrichosis, beyond to evaluate the presence of S. Schenckii in the oral cavity of experimental animals and the production of oxide nitric. Were used 96 male Wistar rats, which were divided into 6 groups of 16 animals, and they were G1: 0.5 mg of glucan in advance (three doses) and after inoculation G2: 0.5 mg glucan after inoculation; G3: control group; G4: 0.5 mg of glucan associated with 10mg/kg of itraconazole; G5: 10mg/kg of itraconazole, and G6: two doses of 0.5 mg of glucan associated 10mg/kg of the itraconazole. The animals were inoculated with 0.1 ml of fungal inoculum by intraperitoneal and lateral tail vein, after one week started treatment groups when they showed clinical signs of disease. After clinical monitoring during the five weeks of treatment and isolation of the agent in the oral cavity of animals, they were euthanized and necropsied to collect blood, anatomopathological evaluation of organs, organ cultures of the agent, and quantification of colony forming units (CFU) of spleen. In the first evaluation was observed in G1, a prevention of the disease triggered by glucan, since the animals from this group showed no clinical signs, anatomopathologicals lesions and obtained the smaller number of CFU (p <0.5). However at the end of experimental period the G4 was significantly better among the treatment groups, therefore, ended the period with 70% of animals without any clinical alteration, macroscopic lesions in organs and organ cultures of the agent, beyond to demonstrate smaller number in CFUs and not occur any deaths resulting from illnes .The groups G2, G5 and G6 were significantly better than the control group since this group had a mortality rate of 31.25%, besides that 100% of the animals remained with clinical changes, lesions in organs such as liver, spleen and testis and demonstrated greater quantification of CFU (p <0,5). As the production of nitric oxide has been found much in the groups receiving the glucan rather than to the other groups. In view of these obtained results it is concluded that glucan induces the production of nitric oxide and the five treatments demonstrated efficacy to the reference sporotrichosis experimental systemically, however, the combination of antifungal (1-3) glucan provided an early regression of the lesions.

Pythium insidiosum, classificado no Reino Stramenopila e Classe Oomycetes, é o agente etiológico da pitiose, uma doença diagnosticada principalmente em eqüinos, caninos e humanos. As atividades in vitro do voriconazol, itraconazol e terbinafina, sozinhos ou em combinação, foram estudadas empregando uma metodologia de macrodiluição baseada na técnica M 38-A (CLSI, 2002) contra 30 isolados clínicos de Pythium insidiosum, com o objetivo de avaliar novas medidas para tratar infecções causadas por este agente utilizando quimioterapia. As combinações (terbinafina e voriconazol; terbinafina e itraconazol) tiveram suas interações avaliadas utilizando a técnica de checkerboard seguindo o modelo da macrodiluição em caldo. Terbinafina foi ativa quando testada sozinha, mostrando uma concentração inibitória mínima (CIM) e concentração fungicida mínima ≤ 8,0 mg/L para todos os isolados. Voriconazol e itraconazol foram inativos contra os isolados testados quando usados sozinhos. A combinação de voriconazol com terbinafina foi sinérgica contra 17% das amostras e indiferente em 25 (83%) amostras. O uso concomitante de terbinafina e itraconazol foi sinérgico contra 17% e indiferente contra 83% das amostras. As interpretações de ambas as interações foram equivalentes para 26 isolados (87%): vinte e três foram indiferentes e três mostraram efeito sinérgico. Antagonismo não foi observado. A terapia combinada é uma alternativa à monoterapia, especialmente para pacientes com infecções invasivas que são difíceis de tratar. Estes resultados precisam ser correlacionados com relatos clínicos; e ensaios in vivo, usando modelos animais, podem preencher a lacuna entre avaliações clínicas e avaliações in vitro das drogas. / Pythium insidiosum is classified in the kingdom Stramenopila, class Oomycetes. It causes pythiosis, a disease mainly diagnosed in horses, dogs, and humans. In order to evaluate new approaches to treat infections caused by Pythium insidiosum using chemotherapy, the in vitro activities of voriconazole, itraconazole and terbinafine, alone or in combination, against 30 clinical isolates were studied employing a macrodilution methodology based on the M 38-A technique (CLSI, 2002). The combinations (terbinafine plus voriconazole and terbinafine plus itraconazole) had their interactions evaluated using the checkerboard technique following the broth macrodilution design. Terbinafine was active when used as a single drug, showing minimal inhibitory concentration (MIC) and minimal fungicidal concentration ≤ 8,0 mg/L for all the isolates. Voriconazole and itraconazole were inactive against the isolates tested when used alone. The combination of voriconazole with terbinafine was synergic against 17% of the strains and indifferent on 25 (83%) strains. The concomitant use of terbinafine and itraconazole was synergic against 17% and indifferent against 83% of the strains. The interpretations of both interactions were equivalent for 26 isolates (87%): twenty three were indifferent and three showed synergistic effect. Antagonism was not observed. Combination therapy provides an alternative to monotherapy, especially for patients with invasive infections that are difficult to treat. These results need to be correlated with clinical outcomes, and in vivo assays using experimental animal models could bridge the gap between in vitro and clinical evaluation of drugs.