La fibrose radio-induite est une complication sévère et tardive de la radiothérapie. Plusieurs études cliniques ont montré que la combinaison pentoxifylline-vitamine E est un traitement sûr et efficace contre la fibrose. Cependant, les mécanismes moléculaires de son efficacité restent inexplorés. Nous avons montré l’efficacité de la combinaison pentoxifylline-vitamine E dans l’entéropathie radique dans une faisabilité clinique. En parallèle, en utilisant un modèle unique, in vitro, de cellules musculaires lisses intestinales primaires isolées des personnes atteintes de l’entéropathie radique, nous avons montré une synergie entre la pentoxifylline et l’analogue hydrophile de vitamine E (trolox) qui permet à l’association d’inhiber l’expression de TGF-β1 au niveau de l’ARN messager et de la protéine. Cette action inhibitrice intervient au niveau transcriptionnel et conduit à une inhibition conséquente des cibles de la voie de signalisation TGF-β1/Smad (Col Iα1, FN1, PAI-1, CTGF), alors qu’elle semble sans effet sur la voie de signalisation Rho/ROCK. Pour la première fois, dans ces cellules issues de l’entéropathie radique, nous avons montré une surexpression de miR-210 ; microRNA induit par l’hypoxie. L’association pentoxifylline-trolox inverse la surexpression de miR-210 aussi bien dans les conditions normoxique que dans les conditions hypoxiques. L’implication de miR-210 dans l’entéropathie radique n’a pas été préalablement étudiée, néanmoins nous avons montré qu’un inhibiteur de miR-210 diminue l’expression de Col Iα1 dans ce modèle. L’effet anti-fibrosant exercé par l’association pentoxifylline-vitamine E est partiellement induit par l’inhibition de la cascade TGF-β1. L’inhibition de miR-210 est un deuxième mécanisme potentiel nécessitant d’autres investigations. Cette étude renforce les essais clinique antérieurs en montrant in vitro une synergie entre pentoxifylline et vitamine E et permettant de proposer cette association en première ligne thérapeutique dans la fibrose radio-induite. De plus, miR-210 est proposé comme une possible cible thérapeutique pour traiter la fibrose radio-induite. / Radiation-induced fibrosis is a serious late complication of radiotherapy. Pentoxifylline-vitamin E has proven effective and safe in clinical trials as treatment of fibrosis, while the molecular mechanism of its activity is yet unexplored. We showed efficacy of Pentoxifylline-vitamin E combination in radiation-induced enteropathy in a small clinical study. In parallel, using a unique in vitro model of primary smooth muscle cells isolated from intestinal samples isolated from humans with radiation enteropathy we showed that pentoxifylline and the hydrophilic analogous of vitamin E (trolox) synergize to inhibit TGF-β1 protein and mRNA expression. This inhibitory action is mediated at the transcriptional level and leads to subsequent inhibition of TGF-β1/Smad targets (Col Iα1, FN1, PAI-1, CTGF), while it has no effect on the Rho/Rock pathway. We have also demonstrated, for the first time, an overexpression of the hypoxia-induced microRNA miR-210 in the fibrotic cells. Pentoxifylline-trolox combination could reverse this miR-210 overexpression in normoxic and hypoxic conditions. While miR-210 has not been previously shown to be involved in radiation-induced enteropathy, we showed that miR-210 inhibitor could reduce mRNA expression of Col Iα1. The anti-fibrotic effect of combined pentoxifylline-vitamin E is at least in part mediated by inhibition of the TGF-β1 cascade. MiR-210 inhibition is another mechanism which needs further investigations. This study strengthens previous clinical data showing pentoxifylline-vitamin E synergy and supports its use as a first-line treatment of radiation-induced fibrosis. Also, it suggests miR-210 as a new potential therapeutic target for the treatment of this complication.

Thesis (PhD)--Stellenbosch University, 2000. / ENGLISH ABSTRACT: Pentoxifylline enhances the toxicity of radiation and has emerged as an effective modulator of the radiation response of tumour cells. The molecular mechanisms involved in the enhancement of radiotoxicity by pentoxifylline have not yet been elucidated. Cell cycle blocks, DNA repair and programmed cell death (apoptosis) are all pert of the cellular response to DNA damage and as such must be considered as targets of the drug. In this study, the influence of pentoxifylline on radiosensitisation, G2 block abrogation, DNA repair inhibition and the induction of apoptosis have been investigated in 8e11 and MeWo melanoma and 4197 and 4451 squamous cell carcinoma (SCC) cell lines. The influence of pentoxifylline on radiation-induced apoptosis in Jurkat J5 T-lymphocytic leukemia cells has also been assessed. Hela cervical carcinoma cells were used to investigate the molecular events involved in the abrogation of the G2 block by pentoxifylline. It is shown that pentoxifylline preferentially sensitises the TP53 mutant MeWo and 4451 cell lines and enhances radiotoxicity by factors of up to 14.5. In the MeWo melanoma, but not in the 4451 SCC cell line, radiosensitisation is accompanied by inhibition of DNA repair. No significant enhancement of radiation-induced apoptosis was observed in MeWo melanoma and 4451 SCC cells. However, Jurkat J5 cells showed an increase in apoptosis after irradiation in the presence of the drug. In irradiated Hela cervical carcinoma cells, pentoxifylline affects the expression of the two components of the mitosis promoting factor (MPF), namely cyclin 81 and p34cdC2, and rapidly restores cyclin 81/p34cdC2 ratios to control levels. Analysis of cyclin 81 expression in whole cells and isolated nuclei furthermore reveals an influence of the drug on the subcellular translocation of the MPF. It is concluded that G2 block abrogation is not the only mechanism involved in the radiosensitisation of tumour cells by pentoxifylline, but that DNA repair inhibition plays a role in certain cell types. Although pentoxifylline induces apoptosis in Jurkat J5 thymocytes, radiation-induced apoptosis plays no role in the radiosensitisation of the two TP53 mutant melanoma and sec cell lines. Abrogation of the G2 block by pentoxifylline, which sensitises tumour cells to a second irradiation or chemotherapeutic challenge, involves a modulation of the levels of cyclin 81 and p34cdC2, and the subcellular location of the MPF. These results are of utmost importance for the clinical potential of pentoxifylline as a dose modifier in cancer therapy. / AFRIKAANSE OPSOMMING: Pentoxifylline verhoog die toksisiteit van bestraling en het dus na vore getree as 'n effektiewe modulator van die sellulêre stralingsrespons in kankerselle. Die molekulêre meganismes betrokke by die verhoging van stralingstoksisiteit deur pentoxifylline is egter nog nie duidelik nie. Blokkering van die selsiklus, die herstel van ONS skade en geprogrammeerde seldood (apoptose) vorm almal deel van die sellulêre respons ná bestraling en word as sulks beskou as potensiële teikens van die middel. In hierdie studie is die invloed van pentoxifylline op stralings-sensitiwiteit, G2 blok verwydering, die vertraging van ONS herstel en die indusering van apoptose ondersoek in die Be11 en MeWo melanoom en 4197 en 4451 plaveisel-sel karsinoom sellyne. Die invloed van pentoxifylline op stralings-geïnduseerde apoptose in Jurkat J5 T-limfosiete is ook bestudeer. Hela servikale karsinoom selle is gebruik om die molekulêre gebeurtenisse rondom die verwydering van die G2 blok deur pentoxifylline te ondersoek. Dit word aangetoon dat pentoxifylline by voorkeur die radiosensitiwiteit van die TP53 mutante MeWo en 4451 sellyne verhoog, en stralingstoksisiteits verhogingsfaktore van tot 14.5 genereer. Hierdie effek gaan gepaard met die vertraging van ONS herstel in die MeWo melanoom, maar nie in die 4451 plaveisel-sel karsinoom sellyn nie. Die meting van apoptose toon geen noemenswaardige verhoging van stralings-geïnduseerde apoptose in MeWo melanoom óf in 4451 plaveisel-sel karsinoom selle nie. Jurkat J5 T-limfosiete toon egter wel 'n verhoging in apoptose wanneer bestraling in die teenwoordigheid van pentoxifylline gedoen word. In Hela servikale karsinoom selle affekteer pentoxifylline die uitdrukking van die twee komponente van die mitose promoverings faktor (MPF), naamlik siklien B1 en p34CdC2 , en restoreer die siklien 81/p34cdC2 verhoudings vinnig na normale vlakke. Ontleding van die siklien 81 uitdrukking in heel selle en in geïsoleerde selkerne toon verder dat die middelook die sub-sellulêre ligging van die MPF affekteer. Die gevolgtrekking word gemaak dat G2 blok verwydering nie die enigste meganisme is waardeur pentoxifylline radiosensitiwiteit verhoog nie, maar dat die vertraging van ONS herstel in sommige seltipes 'n rol speel. Alhoewel pentoxifylline apoptose verhoog in T-limfosiete, speel dit nie 'n rol in die verhoogde radiotoksisiteit wat waargeneem is in die TP53 mutante melanoom en plaveisel-sel karsinoom sellyne nie. Verwydering van die G2 blok deur pentoxifylline, wat selle meer sensitief kan maak vir 'n tweede stralings- of chemoterapie aanslag, behels die modulasie van siklien 81 en p34cdc2 vlakke en die sub-sellulêre ligging van die MPF. Hierdie resultate is van uiterste belang vir die kliniese aanwending van pentoxifylline as 'n dosis-modifiseerder in kankerterapie.

Despite recent advances, prognosis in metastatic prostate cancer remains poor. As with other cancers, tumor heterogeneity is an increasingly evident contributor in prostate tumorigenesis and developed resistance. Using in vitro and in vivo model systems, we examined novel diagnostic and therapeutic strategies in prostate cancer. In these studies, combination treatment with amuvatinib, a receptor tyrosine kinase inhibitor, and erlotinib, an epidermal growth factor inhibitor, was assessed for its ability to differentially modulate growth signaling in pathway diverse LNCaP (PTEN⁻) and DU-145 (PTEN⁺) human prostate cancer cell and mouse xenograft models. Our results suggest both individual mechanistic signaling activities, as well as benefits of the combination therapy though modulations of MAPK (pERK) and 4EBP1/cyclin D1 in growth signaling divergent PTEN+ and PTEN- prostate cancer cells. Additionally, despite the importance preanalytical tissue preservation on downstream diagnostic assays, exact protocols are not well defined and highly variable clinically and, as such, critical diagnostic information is lost. We show that a novel 2+2 fixation method induces target- and cell-specific alterations in immunostain intensity and efficacy. Importantly, cyclin D1 is increasingly utilized for as a clinical prognostic/diagnostic marker and demonstrated improved immunohistochemical staining efficacy with 2+2 fixation compared with treatment-matched xenograft protein alterations as assessed by western analysis. Finally, pentoxifylline (PTX) is a clinically utilized and well tolerated PDE inhibitor that has shown promise as a radio-/chemo-sensitization and anti-cancer agent against a variety of cancers. In these studies, we demonstrate that PTX induces cell and tumor growth inhibition in LNCaP prostate cancer cells. Mechanistically, PTX induces transient cellular signaling modulations of both the AMPK metabolic and AKT/mTOR growth pathways, while inducing autophagy. Also, PTX sensitizes LNCaP prostate cancer to cytotoxicity induced by first line chemotherapy docetaxel, inducing significant cellular apoptosis and reducing effective docetaxel concentrations by >10 fold for equivalent toxicity in viability assays. These findings nominate PTX as an adjunct therapy for the treatment of prostate cancer. In summary, these studies characterize the targeted signaling modulation by combination erlotinib and amuvatinib therapy, as well as pentoxifylline, for their use as therapies for prostate cancer. A novel fixation protocol was also assessed for improved diagnostic tissue preservation of critical signaling proteins. Further understanding in these areas will aid and expand the development of effective diagnostics, as well as emphasize the benefits of these and similar therapeutics for the treatment of prostate cancer.

Master of Science / Department of Kinesiology / Timothy I. Musch / Both cardiac and peripheral vasculature dysfunction likely contribute, in part, to elevations in TNF-[alpha] and exercise intolerance in chronic heart failure (CHF). The pharmaceutical TNF-[alpha] synthesis suppressor pentoxifylline (PTX) reduces plasma [TNF-[alpha]] and improves left ventricular (LV) function in CHF rats, but the effects of PTX on skeletal muscle blood flow (BF) and vascular conductance (VC) during exercise are unknown. We tested the hypothesis that PTX would elevate skeletal muscle BF and VC at rest and during submaximal treadmill exercise in CHF rats (coronary artery ligation). CHF rats received i.p. injections of 30 mg·kg[superscript]-[superscript]1·day[superscript]-[superscript]1 of PTX (CHF+PTX, n=13) or saline (CHF, n=8) for 21 days. Mean arterial pressure (MAP) and BF (radiolabeled microsphere infusions) were measured at rest and during treadmill exercise (20 m/min, 5% grade). Myocardial infarct (MI) size was not different between groups (CHF: 37±4, CHF+PTX: 37±3% of LV wall; p>0.05). Resting and exercising MAP was greater in CHF+PTX compared to CHF (p<0.05 for both). At rest, total hindlimb skeletal muscle BF and VC were not different between groups (p>0.05). However, during exercise PTX increased total hindlimb BF (CHF: 83±9, CHF+PTX: 114±8 ml·min[superscript]-[superscript]1·100g[superscript]-[superscript]1, p<0.05) and VC (CHF: 0.75±0.08, CHF+PTX: 0.88±0.06 ml·min[superscript]-[superscript]1·100g[superscript]-[superscript]1·mmHg[superscript]-[superscript]1, p<0.05). Furthermore, exercising BF was increased in 21, and VC in 11, of the 28 individual hindlimb muscles or muscle parts with no apparent fiber-type specificity. Thus, PTX administration augments skeletal muscle BF and VC during locomotory exercise in CHF rats, which carries important therapeutic implications for CHF patients.

Isquemia e reperfusão (I/R) de vasos mesentéricos que acarretam insuficiência vascular aguda são acompanhadas de insuficiência em múltiplos órgãos e estão associados a altas morbidade e mortalidade. Os benefícios da solução hipertônica e da pentoxifilina foram testados isoladamente e em conjunto para melhorar o fluxo sanguíneo e o estado de oxigenação dos tecidos com redução da reação inflamatória e de apoptose, tanto no intestino quanto no pulmão enquanto órgão alvo. Foram utilizados 24 ratos Wistar machos pesando entre 200 e 250g distribuídos em 4 grupos (n=6). Foram submetidos à laparotomia e período de isquemia por clampeamento dos vasos mesentéricos por 40 minutos. O período de reperfusão foi de 80 minutos. A cada 40 minutos foram realizadas coletas de sangue arterial para gasometria. Os animais foram alocados nos grupos: grupo I/R (IR) onde I/R foi realizada e os animais receberam 4ml/kg de solução salina isotônica. O grupo solução salina hipertônica (SH) recebeu 4ml/kg de solução hipertônica a 7,5% e o grupo pentoxifilina (PTX) recebeu 30mg/kg de pentoxifilina diluída em NaCl 0,9% em um volume total de 4ml/kg ao final da isquemia intestinal. O grupo solução salina hipertônica+pentoxifilina (SH+PTX) recebeu ambas as soluções com volume total de 4m/kg no mesmo momento. As biopsias de intestino e pulmão foram colhidas ao final do experimento e foram submetidas à coloração em HE e à imuno-histoquímica para COX 2, caspase-3 clivada e Bcl-2. Os valores de sO2 revelaram diferença estatisticamente significante aos 40 (p=0,0099) e 80 (p=0,0074) minutos de reperfusão na comparação do grupo IR com os grupos SH e SH+PTX. Os valores de lactato foram estatisticamente significantes aos 40 minutos (p=0,0069) e 80 minutos (p=0,0098) de reperfusão, entre os grupos IR e os grupos SH e SH+PTX. A avaliação histológica em HE no tecido intestinal evidenciou diferença estatisticamente significantes na comparação dos grupos SH (p=0,0200), PTX (p=0,0200) e HS+PTX (p=0,0412) com o grupo IR, assim como no tecido pulmonar, que evidenciou diferenças estatisticamente significantes na comparação de IR com SH (p=0,006), PTX (p=0,0433) e SH+PTX (p=0,0040). A marcação citoplasmática de COX 2 revelou diferença estatisticamente significante ao comparar o grupo IR com o grupo SH+PTX (p=0,0015), Na avaliação do tecido pulmonar foi observado o grupo IR estabelecendo diferenças estatisticamente significantes na comparação com os grupos SH (p=0,0455), PTX (0,0143) e SH+PTX (p=0,0455). A avaliação de apoptose por imunohistoquímica para caspase 3 clivada evidenciou diferença com significância estatística ente os grupos IR e SH (p=0,0085) e os grupos PTX e SH (p=0,0120) em tecido intestinal. Em tecido pulmonar, entre IR e PTX (p=0,0090) e SH e PTX (p=0.0412). A marcação citoplasmática de Bcl-2 evidenciou que somente entres os grupos IR e SH+PTX (p=0,0012) se estabeleceu significância estatística em tecido intestinal No pulmão a diferença foi evidenciada em relação ao grupo IR e os grupos SH (p=0,0128), PTX (p=0,0085) e SH+PTX (p=0,0066). Conclui-se que o uso associado de pentoxifilina e solução salina hipertônica oferece os melhores resultados dos pontos de vista metabólico, inflamatório e da inibição da apoptose / Ischemia-reperfusion (I/R) of the mesenteric vessels cause acute vascular insufficiency and is followed by multiple organs failure and high morbidity and mortality. The benefits of hypertonic saline (NaCl 7,5%) and pentoxifylline were tested to improve metabolic and oxygen status of tissues with reduced inflammatory reaction and apoptosis, both in the gut as in the lung as target organ. 24 male Wistar rats weighing 200 to 250g. They underwent laparotomy and ischemia by clamping the mesenteric vessels for 40 minutes. The reperfusion period was 80-minute long. Every 40 minutes, blood was collected for arterial gas analysis. The animals were set into 4 groups (n=6): I/R (IR) received isotonic saline just before reperfusion (4ml/kg). Hypertonic saline group (HS) received 7.5% hypertonic saline solution (4ml/kg) and Pentoxifylline group (PTX) received pentoxifylline (30mg/kg)diluted in NaCl 0.9% at the end of the mesenteric ischemia. Hypertonic saline+pentoxifylline group (SH-PTX) received both solutions at the same doses. Biopsies of intestine and lung were collected at the end of the experiment and were subjected to HE staining and immunohistochemistry staining to COX 2, cleaved caspase-3 and Bcl-2. About the values of sO2, a statistically significant difference was established when groups were compared at 40 (p=0.0099) minutes and 80 (p=0.0074) of reperfusion. The lactate values were statistically significant after 40 (p=0.0069) and 80 (p=0.098) minutes of reperfusion as well. Histological evaluation with HE revealed a statistically significant difference in the comparison of SH (p=0.0200), PTX (p=0.0200) and HS + PTX (p=0.0412) groups compared to IR group in intestine tissue and SH (p=0.0006), PTX (p=0.0433) and HS + PTX (p=0.040) in lung tissue. Cytoplasmic staining of COX 2 revealed a statistically significant difference when comparing IR (p=0.0015) to HS+PTX in intestinal tissue. The evaluation of the lung tissue for COX 2 showed IR group setting statistically significance when compared to HS 9p=0,045), PTX (0.0143) and HS+PTX (p=0.0455) groups. The evaluation of apoptosis by immunohistochemistry for cleaved caspase 3 revealed that there was a significant difference between IR versus SH (p=0.0085) and PTX versus HS (p= 0.0120) in intestinal tissue and IR versus PTX (p=0,0090) and PTX versus SH (p=0.0412) in lung tissue. The cytoplasmic Bcl-2 expression shows that only between groups IR versus HS+PTX (p = 0.0012) statistical significance was established in intestinal tissue. Lung tissue showed the difference was evidenced in relation to IR versus SH (p=0.0128), PTX (p=0.0085) and HS+PTX (p=0.0066). It may be concluded that the combined use of pentoxifylline and hypertonic saline offers best results on metabolic, inflammatory and apoptosis inhibitory aspects

Submitted by Jean Medeiros (jeanletras@uepb.edu.br) on 2016-03-02T17:03:56Z No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) PDF - Airlla Laana de Medeiros Cavalcanti.pdf: 2086060 bytes, checksum: b74e6eb69ebe6e0583efc0e2d23bedac (MD5) / Approved for entry into archive by Secta BC (secta.csu.bc@uepb.edu.br) on 2016-06-13T20:33:03Z (GMT) No. of bitstreams: 2 PDF - Airlla Laana de Medeiros Cavalcanti.pdf: 2086060 bytes, checksum: b74e6eb69ebe6e0583efc0e2d23bedac (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2016-06-13T20:33:10Z (GMT). No. of bitstreams: 2 PDF - Airlla Laana de Medeiros Cavalcanti.pdf: 2086060 bytes, checksum: b74e6eb69ebe6e0583efc0e2d23bedac (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-02-13 / Some inflammatory skin diseases are treated with drugs carried on conventional dosage forms that often fail to achieve adequate concentrations in the tissue to generate maximum pharmacological effect. Pentoxifylline (PTX) is one of these drugs widely studied due to its anti-inflammatory activity by inhibiting the production of TNFα and other proinflammatory cytokines. The use of PTX incorporated into microemulsions (ME) would be a novel alternative for the treatment of inflammatory skin disorders. This new drug delivery system can be used topically and can be able to increase the permeation through skin and the effectiveness of several drugs compared to conventional treatments. The aim of this work was to develop a microemulsion containing PTX (PTX-ME) for topical use. The formulation obtained from the pseudoternary phase diagrams was characterized and evaluated using methods such as polarized light microscopy (MLP), differential scanning calorimetry (DSC) and transmission electron microscopy (TEM). The in vitro release profile was determined using the model of Franz cells and antiedematogenic activity in vivo was determined using the technique of paw edema induced by carrageenan. The ME was composed of distilled water (5%), acid–capric/caprylic triglycerides (51%), Tween 80 (39.6%) and Brij TM 52 (4.4%). Analyzes of MLP, DSC, and TEM were able to confirm that the ME obtained was water-in-oil (W/O) type. The formulation was thermodynamically stable against thermic stress. Beside this, it had physicalchemical characteristics that allow its topical use. In vitro release of PTX-ME followed the Higuchi kinetic model. Additionally, it showed significant anti-inflammatory activity in paw edema induced by carrageenan in all stages of the assay. Consequently, the PTX-ME showed an interesting alternative for treatment of dermatological disorders. / Algumas doenças dermatológicas inflamatórias são tratadas com fármacos veiculados em formas farmacêuticas convencionais, que muitas vezes não atingem concentrações teciduais adequadas para gerar o efeito farmacológico máximo. A pentoxifilina (PTX) é um desses fármacos e tem sido amplamente estudada com relação a sua atividade anti-inflamatória por inibir a produção de TNF-α e outras citocinas pró-inflamatórias. O uso da PTX incorporada a uma microemulsão (ME) seria uma alternativa inédita para o tratamento de afecções dermatológicas inflamatórias. Esse novo sistema transportador de fármaco pode ser utilizado topicamente e é capaz de aumentar a permeação cutânea e a eficácia de vários fármacos em relação aos tratamentos convencionais. O objetivo deste trabalho foi desenvolver um sistema microemulsionado contendo PTX para aplicação tópica. A formulação obtida a partir de um diagrama de fase pseudoternário foi caracterizada e avaliada utilizando métodos como microscopia de luz polarizada (MLP), calorimetria exploratória diferencial (DSC) e microscopia eletrônica de transmissão (MET). O perfil de liberação in vitro foi determinado utilizando o modelo de células de Franz e a atividade antiedematogênica in vivo foi determinada através da técnica de edema de pata induzido por carragenina. A ME desenvolvida foi constituída por 5% de água destilada, 51% de triglicerídeos do ácido cáprico e caprílico, 39,6% de Tween ® 80 e 4,4% Brij ® 52. Através das análises de MLP, DSC e MET foi possível confirmar a estruturação do sistema como ME do tipo água em óleo (A/O). A formulação apresentou-se estável frente a estresses térmicos, além de possuir características físico-químicas que possibilitam seu uso por via tópica. A liberação in vitro da ME-PTX obedeceu ao modelo cinético de Higuchi e apresentou atividade anti-inflamatória significativa em edema de pata induzido por carragenina em todas as etapas do ensaio. Portanto, pôde-se concluir, que a veiculação da PTX através de um sistema microemulsionado mostrou-se uma alternativa interessante e inédita para o tratamento de afecções dermatológicas.

Orientador: James Venturini / Resumo: Paracoccidioidomicose (PCM) é uma micose sistêmica causada por fungos do gênero Paracoccidioides; suas principais formas clínicas são aguda/subaguda, crônica e residual. A PCM é uma doença restrita a países da América Latina com maior incidência no Brasil, especialmente entre os trabalhadores rurais. A maioria dos pacientes com a forma crônica da doença, mesmo após tratamento eficaz, apresentam sequelas, incluindo fibrose pulmonar e adrenal. Os problemas sociais, econômicos e psicológicos desencadeados pela fibrose pulmonar são subestimados; além disso, a fibrose na PCM permanece negligenciada, uma vez que não há tratamento. Dessa forma, o estudo teve por objetivo investigar a influência de drogas com potencial antifibrótico (pentoxifilina - PTX, azitromicina - AZT e talidomida - Thal) associadas aos tratamentos antifúngicos com itraconazol - ITC e cotrimoxazol - CMX em modelo murino de PCM pulmonar. Para tanto, camundongos BALB/c machos foram inoculados com leveduras do isolado 326 de P. brasiliensis e após 8 semanas de infecção foi dado início aos esquemas terapêuticos: PTX/ITC, PTX/CMX, AZT/ITC, AZT/CMX, Thal/ITC e Thal/CMX. Após 8 semanas de tratamento, os animais foram eutanasiados a fim de se avaliar a deposição de fibras colágenas, produção de hidroxiprolina, recuperação de fungos viáveis e a porcentagem das áreas com lesão nos pulmões e peso corporal. Visando identificar os mecanismos envolvidos foi avaliada a produção de TGF-β1, CCL3, IFN-γ, TNF-α, IL-10, VEGF, IL-6,... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Paracoccidioidomycosis (PCM) is a systemic mycosis caused by fungi of Paracoccidioides genus; the main clinical forms are acute/subacute, chronic and residual. PCM is a disease restricted to Latin American countries with a higher incidence in Brazil, especially among rural workers. Most patients with the chronic form, even after effective treatment, present sequelae, including pulmonary and adrenal fibrosis. The social, economic and psychological problems triggered by pulmonary sequels are underestimated. In addition, fibrosis in PCM remains neglected, since there is no treatment. The aim of this study was to investigate the influence of antifibrotic drugs (pentoxifylline - PTX, azithromycin - AZT and thalidomide - Thal) associated with antifungal treatments with itraconazole - ITC and cotrimoxazole - CMX in a murine model of pulmonary PCM. Male BALB/c mice were inoculated with P. brasiliensis “isolated 326” and after 8 weeks of infection the treatment were started: PTX/ITC, PTX/CMX, AZT/ITC, AZT/CMX, Thal/ITC and Thal/CMX. After 8 weeks of treatment, the mice were euthanized in order to evaluate the deposition of collagen fibers, hydroxyproline production, recovery of viable fungi and the percentage of areas with injury in lung and body weight. In order to identify the mechanisms involved, the production of TGF-β1, CCL3, IFN-γ, TNF-α, IL-10, VEGF, IL-6, IL-1β, IL-17 and IL-2 in the lung homogenate was evaluated. Our findings revealed that infected mice treated with PTX/ITC s... (Complete abstract click electronic access below) / Mestre

Graduation date: 1996

Pharmaceutical cocrystals use principles of crystal engineering for the design of crystalline forms of drugs and can improve their solubility, bioavailability, stability and other important properties without changing the efficacy of the drug. Herein reported are pharmaceutical cocrystals of two API's, caffeine and Pentoxifylline. Research has indicated that caffeine has the ability to reverse AB; plaque deposition in the brain (believed to be the primary cause of Alzheimer's pathogenesis) and thus revert memory and improve cognitive impairment. But owing to the fast absorption rate and short half life, a controlled release formulation of caffeine would be clinically beneficial. Thus, novel cocrystals of caffeine are presented with varying solubilities with respect to caffeine. The pharmaceutical cocrystals of caffeine used herein include: caffeine.cyanuric acid monohydrate, caffeine.syringic acid tetrahydrate, caffeine.chlorogenic acid and caffeine.catechin hydrate. Three caffeine cocrystals were prepared in our lab previously which include caffeine.ferulic acid, caffeine.ethyl gallate dihydrate and caffeine.caffeic acid. In addition, six caffeine cocrystal forms were reproduced from the literature and included in the solubility study: caffeine.quercetin, caffeine.salicylic acid, caffeine.1-hydroxy-2-napthoic acid, caffeine.gallic acid hemihydrate, caffeine.ellagic acid monohydrate and caffeine.coumaric acid. Dissolution studies were performed in aqueous media at room temperature. All of the cocrystals decreased the solubility of caffeine with the highest being a 278 fold decrease in the solubility of caffeine. Analysis of melting point, crystal packing efficiency and solubility of cocrystal former with solubility was also done to determine if they influenced the solubility. Presented herein are the results of the analyses. It was seen that solubility of the cocrystal former had no effect on the decrease in cocrystal solubility. Moreover melting point and solubility of the cocrystal could not be correlated probably due to the variability in the cocrystal formers. Crystal packing efficiency though did not show a high correlation with solubility but it was seen that highest solubility achieved by pure caffeine achieved the lowest crystal packing efficiency and vice versa suggesting its role in cocrystal solubility. Pentoxifylline is contraindicated for its use in autism. But owing to high solubility of the drug, a less soluble form of the drug would help in decreasing the half life and thereby help in forming a sustained form of the drug by modifying the inherent solubility of the API. Here, novel cocrystals of Pentoxifylline are presented with varying solubilities with respect to the API. The pharmaceutical cocrystals used herein include: pentoxifylline.benzoic acid, pentoxifylline.1-hydroxy-2-napthoic acid, pentoxifylline.salicylic acid, pentoxifylline.gallic acid, pentoxifylline. salicylamide, pentoxifylline.coumaric acid, pentoxifylline.caffeic acid and pentoxifylline.catechin hydrate. Dissolution studies were also performed in aqueous media at room temperature. All of the cocrystals decreased the solubility of Pentoxifylline with the highest being a 99 fold decrease in the solubility with pentoxifylline.coumaric acid. On analyzing melting point, crystal packing efficiency and relation of solubility of cocrystal former with solubility of cocrystal, as was done in the case of caffeine, the parameters showed no effect on solubility of the cocrystal.

Cyclin D1, a proto-oncogene, is required for progression from the G1 phase into the S phase of the cell cycle. Over-expression of cyclin D1 causes an increase in cell cycle progression and cell proliferation, implicating it in a variety of cancers including renal cell carcinoma (RCC). The rodent RCC cell model, QTRRE, and human RCC cell models, ACHN, 786-O and Caki-2, exhibit elevated levels of cyclin D1. Pentoxifylline (PTX), a non-specific phosphodiesterase inhibitor, is an FDA-approved hemorheologic agent used to treat intermittent claudication, stemming from peripheral vascular diseases, as well as other diseases involving defective locoregional blood flow. Treatment of QTRRE, ACHN, 786-O and Caki-2 with PTX caused a time- (0-24 hrs) and dose- (0-1.0 mg/mL) dependent decrease of cyclin D1 protein and p-Rb levels in whole cell lysate as well as cytosolic and nuclear fractions, albeit, to different extents within the models. Concomitant with cyclin D1 and p-Rb decrease, enhanced G1 phase cell cycle arrest was observed in the RCC models. Mechanistic studies in these RCC cell models were carried out to determine PTXs mechanism of action with regard to cyclin D1 protein level decrease. RT-PCR analysis showed no significant changes in cyclin D1 mRNA copy number in time- (0-24 hrs) and dose- (0-1.0 mg/mL) dependent PTX treatments. However, such treatments caused decrease in p-4EBP1 (Ser65), p-4EBP1 (Thr70), and p-4EBP1 (Thr37/46). Because PTX's ability to decrease cyclin D1 protein was prevented in the presence of the proteasome inhibitor, MG-132, studies were performed to determine whether cyclin D1 stability was decreased during PTX treatment. Cyclin D1 degradation is initiated by phosphorylation of residue Thr286 by GSK-3β. Inhibition of GSK-3β with LiCl or knockdown via siRNA in the presence of PTX failed to block cyclin D1 decrease. Moreover, PTX treatment in the presence of MG-132 revealed no significant increase in cyclin D1 p-Thr286 compared to control. Finally, using the protein synthesis inhibitor, CHX, PTX caused no significant decrease in cyclin D1 t₁/₂ (wt-HA and T286A-HA) compared to control. Sorafenib, a broad-spectrum (cRAF, bRAF, KIT, FLT-3, VEGFR-2, VEGFR-3, and PDGFR-β) kinase inhibitor, is FDA-approved for the treatment of RCC. Studies with sorafenib and PTX in the ACHN cell model were carried out to determine PTXs possible adjuvant role in inhibiting cell growth via cyclin D1 decrease and G1 phase arrest. MTS data showed PTX potentiates the anti-proliferative effects of sorafenib. PTX pre-treatment for 24 hrs was also lowered the effective dose of sorafenib from 50 μM to 5 μM. Further, ACHN xenograft tumor volumes from mice treated with PTX and sorafenib displayed significantly higher tumor growth inhibition compared to either drug treatment alone or vehicle. Finally, drug treated ACHN xenograft tissue displayed significantly lower cyclin D1, p-RB and p-4EBP1 levels. These results demonstrate a novel anti-cancer property of PTX and suggest its use as a possible adjuvant therapy in RCC treatment should be further explored.