No description available.

No description available.

No description available.

Diamidines are small molecules that generally possess antiparasitic properties and bind preferentially to the minor groove of AT rich DNA. With the goal of getting a better understanding of the thermodynamic driving forces and binding affinities, a series of pentamidine analogs were investigated with various AT rich DNA by ITC, UV-Vis and fluorescence spectroscopic methods. Findings suggest that the substitution of the linker oxygen of pentamidine to a nitrogen slightly improves the binding affinity. All of the investigated compounds are entropically driven at 25 oC with non-alternating AT DNA. Additionally, the increased fluorescence of the nitrogen and sulfur linked analogs will enable future work to be done with fluorescence microscopy to help determine if and where these compounds accumulate in the target organism.

Myotonic dystrophy (DM) is an inherited disease characterized by myotonia, insulin resistance, cardiomyopathy, and cognitive deficiencies. DM is a triplet repeat disorder, meaning that affected individuals carry anywhere between 50 and thousands of CTG/CCTG repeats in their genetic makeup. When transcribed into RNA, these repeats become “toxic” in the sense that they serve to bind and sequester important RNA binding proteins. One such family of proteins, the Muscleblind-like (MBNL) family, is important in the regulation of alternative mRNA splicing, and thus the sequestration of MBNL proteins leads to a number of mis-splicing events. Many of these events are directly correlated to DM symptoms. While there is no known cure for DM, the use of small molecules to treat symptoms is a well-characterized therapeutic tactic with immense promise. Pentamidine is a small molecule that was found to reverse mis-splicing in both DM cell and mouse models. Mechanistically, this molecule is particularly unique because unlike many small molecules, which physically displace MBNL from the toxic CUG RNA, pentamidine reduces CUG RNA levels, possibly through inhibition of CTG transcription. Chapter I summarizes alternative splicing mechanisms and regulation, defines MBNL protein structure and function, describes DM pathophysiology and molecular mechanism, and finally provides an overview of pentamidine characterization as a small molecule therapeutic. Chapter II reports the development of an in vitro T7 transcription assay, which allowed us to compare the relative efficacy by which pentamidine is able to inhibit the transcription of various repeat and non-repeat DNA sequences. This chapter further reports the characterization of a series of methylene linker analogues of pentamidine, which were also characterized through the T7 transcription assay. Chapter III details our thorough structure-activity relationship investigation of bisbenzamidine analogues of pentamidine, both in in vivo and in vitro models. Chapter IV describes our characterization of actinomycin D, a known transcription inhibitor and chemotherapeutic, within the DM disease framework. Chapter V summarizes these data, which ultimately serve as a proof of concept for the potential of CTG transcription inhibition in therapeutic contexts and broadly describe their application in other repeat diseases. This dissertation contains previously published and unpublished co-authored material. / 10000-01-01

Pentamidine is used to treat Pneumocystis carinii pneumonia. The incidence of this infection in pregnancy has paralleled the increasing incidence of acquired immunodeficiency syndrome in pregnancy. Using the in vitro bidirectionally perfused human placenta, we studied the transfer of pentamidine across the placenta. Pentamidine was added to the maternal circulation at therapeutic concentrations (2 wg/ml). No transfer of pentamidine was detectable with a newly devised high-performance liquid chromatography method sensitive to 0.05 wg/ml of pentamidine. Increasing the pentamidine concentration tenfold produced a low level of transfer to the fetal circuit. Fetal concentrations were far below maternal perfusate concentrations. Placental tissue levels were higher than media levels. These data are suggestive of minimal drug transfer to the fetus and significant concentration of the drug in placental tissue. (Am J Obstet Gynecol 1989;160:759-61.)

Pouco se sabe sobre o mecanismo de ação da pentamidina, um composto utilizado no tratamento das leishmanioses. Para uma melhor compreensão do mecanismo de ação assim como o mecanismo de resistência à pentamidina, foi isolado um gene que codifica um membro da família de transportadores ABC, chamado PRP1 capaz de conferir resistência à pentamidina em formas promastigotas e amastigotas de Leishmania spp. O tratamento dos transfectantes que superexpressam a PRP1 com pentamidina em presença de concentrações não tóxicas de verapamil, um inibidor de transportadores ABC, foi capaz de reverter a resistência mediada por esse transportador. Foram ainda isolados nesse estudo duas linhagens de L. amazonensis resistentes à pentamidina. A análise molecular dos parasitos indicou que esses mutantes não apresentaram nenhuma amplificação de DNA, inclusive do gene PRP1 que também não se mostrou superexpresso em ambas as linhagens. As duas linhagens resistentes à pentamidina tiveram sua resistência revertida quando tratadas com verapamil, indicando que o mecanismo de resistência nesses mutantes pode estar associado a um transportador ABC. Os resultados obtidos nesse estudo fornecem dados para uma melhor compreensão do mecanismo de resistência à pentamidina e sugerem um provável potencial da associação pentamidina e verapamil no tratamento da doença. / Little it is known about the mechanism of action of pentamidine, an compound used for leishmaniases chemotherapy. To understand the mechanism of action and resistance of pentamidine, it was isolated a gene that codifies a member of ABC transporter family, named as PRP1 able to confer pentamidine resistance in promastigotes and amastigotes of Leishmania spp. Treatment of transfectants overexpressing PRP1 with pentamidine in presence of non toxic concentration of verapamil, an inhibitor of ABC transporters, was able to reverse the drug resistance mediated by this transporter. Two lines of L. amazonensis resistant to pentamidine were selected. Molecular analysis of parasites indicated that these mutants do not contain amplified DNA, including the PRP1 gene either not associated with overexpression in both lines. The two lines resistant to pentamidine had their resistance reversed when treated with verapamil, indicating that the mechanism of resistance may be associated to an ABC transporter. The results of this work lead to new insights for a better understanding of the mechanism of of resistance suggesting a probably potencial of pentamidine and verapamil association in the chemotherapy.

Pouco se sabe sobre o mecanismo de ação da pentamidina, um composto utilizado no tratamento das leishmanioses. Para uma melhor compreensão do mecanismo de ação assim como o mecanismo de resistência à pentamidina, foi isolado um gene que codifica um membro da família de transportadores ABC, chamado PRP1 capaz de conferir resistência à pentamidina em formas promastigotas e amastigotas de Leishmania spp. O tratamento dos transfectantes que superexpressam a PRP1 com pentamidina em presença de concentrações não tóxicas de verapamil, um inibidor de transportadores ABC, foi capaz de reverter a resistência mediada por esse transportador. Foram ainda isolados nesse estudo duas linhagens de L. amazonensis resistentes à pentamidina. A análise molecular dos parasitos indicou que esses mutantes não apresentaram nenhuma amplificação de DNA, inclusive do gene PRP1 que também não se mostrou superexpresso em ambas as linhagens. As duas linhagens resistentes à pentamidina tiveram sua resistência revertida quando tratadas com verapamil, indicando que o mecanismo de resistência nesses mutantes pode estar associado a um transportador ABC. Os resultados obtidos nesse estudo fornecem dados para uma melhor compreensão do mecanismo de resistência à pentamidina e sugerem um provável potencial da associação pentamidina e verapamil no tratamento da doença. / Little it is known about the mechanism of action of pentamidine, an compound used for leishmaniases chemotherapy. To understand the mechanism of action and resistance of pentamidine, it was isolated a gene that codifies a member of ABC transporter family, named as PRP1 able to confer pentamidine resistance in promastigotes and amastigotes of Leishmania spp. Treatment of transfectants overexpressing PRP1 with pentamidine in presence of non toxic concentration of verapamil, an inhibitor of ABC transporters, was able to reverse the drug resistance mediated by this transporter. Two lines of L. amazonensis resistant to pentamidine were selected. Molecular analysis of parasites indicated that these mutants do not contain amplified DNA, including the PRP1 gene either not associated with overexpression in both lines. The two lines resistant to pentamidine had their resistance reversed when treated with verapamil, indicating that the mechanism of resistance may be associated to an ABC transporter. The results of this work lead to new insights for a better understanding of the mechanism of of resistance suggesting a probably potencial of pentamidine and verapamil association in the chemotherapy.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2014-05-21T13:01:57Z No. of bitstreams: 1 Gilmara de Souza Sampaio.Metilglucamina...2013.pdf: 742977 bytes, checksum: d3a3b5d150f8150fa303862cd610b04f (MD5) / Made available in DSpace on 2014-05-21T13:01:57Z (GMT). No. of bitstreams: 1 Gilmara de Souza Sampaio.Metilglucamina...2013.pdf: 742977 bytes, checksum: d3a3b5d150f8150fa303862cd610b04f (MD5) Previous issue date: 2013 / Fundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, Brasil / O prognóstico da leishmaniose cutânea (LC) com o uso dos antimoniais pentavalentes (Sb+5) de um modo geral é considerado bom, embora alguns casos tornem-se refratários à terapêutica tradicional. Infelizmente, não existem marcadores de gravidade da doença ou marcadores de resposta terapêutica, limitando a utilização de formas de tratamento mais efetivas. Em alguns casos, porém, existe a necessidade de utilizar outras drogas, como a anfotericina B (desoxocolato) e as pentamidinas (isotionato e mesilato), consideradas como drogas de 2ª escolha no tratamento das leishmanioses, sendo de fundamental importância à busca de novos esquemas terapêuticos. O objetivo do estudo foi comparar a eficácia entre o antimoniato-N-metilglucamina (Glucantime®) e o isotionato de pentamidina (Pentacarinat®) no tratamento da forma cutânea da leishmaniose tegumentar (LT). Trata-se de um estudo experimental, do tipo ensaio clínico, randomizado, aberto e controlado, com controles ativos (pacientes submetidos a tratamento com antimonial pentavalente). Foram cadastrados 173 pacientes com a forma cutânea da LT em atividade. O paciente com suspeita de LC foi submetido à avaliação clínica e posteriormente submetido a exames laboratoriais específicos (IDRM, exame histopatológico da lesão da pele). Foram avaliados os critérios de inclusão e, em seguida, alocados em um dos grupos para o tratamento proposto G1: Isotionato de Pentamidina (Pentacarinat®) na dose de 4mg/kg/peso de 2 em 2 dias por 5 doses com aplicação intramuscular 1x ao dia; e G2: Antimoniato-N-metilglucamina (Glucantime®), dose de 15mg/Sb+5/kg/dia durante 20 dias com aplicação endovenosa 1x ao dia. Os pacientes foram acompanhados por um período de três meses após o final do tratamento, para determinação dos desfechos principais: Cura cínica ou falha terapêutica. O acompanhamento evolutivo após o ensaio terapêutico foi de 12 meses, para avaliar a possibilidade de recidiva de lesão(ões). A duração total do estudo foi de 18 meses. A amostra do estudo foi composta, em sua maioria, por indivíduos do sexo masculino (58,3%), adultos jovens, com faixa etária entre 20-30 (34,1 %), que trabalhavam na área rural (85,1%), portadores de lesões ulceradas únicas e ou múltiplas (64,6%), geralmente em membros inferiores. A incidência de cura clínica foi maior nos pacientes tratados com Glucantime®, com 55(61,8%) dos pacientes curados, enquanto que os dos pacientes tratados com isotionato de pentamidina (Pentacarinat®), 26(34,2%) evoluíram com cura clínica. Os resultados descritos neste estudo mostram que o isotionato de pentamidina (Pentacarinat®), medicamento considerado de 2ª escolha dentro das alternativas oferecidas pelo Ministério da Saúde (MS), não foi uma droga com melhor resposta terapêutica, quando comparada à droga de 1a escolha (Glucantime®). A relação risco versus benefício, portanto, não justifica a substituição do antimoniato-N-metilglucamina (Glucantime®) pelo isotianato de pentamidina (Pentacarinat®) nas doses usadas pelos pacientes em nosso estudo. / The prognosis of cutaneous leishmaniasis (CL) with the use of pentavalents antimonials (Sb+5) is generally considered good, although in some cases have become refractory to conventional therapy. Unfortunately there are no markers of disease severity or markers of therapeutic response, limiting the use of more effective forms of treatment. In some cases, however, there is a need for other drugs such as amphotericin B (desoxycholate) and pentamidine (isethionate and mesylate), which are considered as the second choice in the treatment of leishmaniasis, since few studies with reduced doses of these drugs demonstrated encouraging results in tegumentary leishmaniasis (TL), which is paramount in the search for new therapeutic regimens using proven antileishmanial drugs. We have compared the effectiveness between the N-methylglucamine antimoniate (Glucantime®) and pentamidine isethionate (Pentacarinat®) in the treatment of CL in an endemic area of tegumentary leishmaniasis (TL), Jiquiriçá Valley, Bahia, Brazil. This is an experimental study of the clinical trial kind, randomized, open and uncontrolled, with active controls (patients treated with Sb+5). The choice of an open trial was mainly due to the medications studied here have different ways of administration. 173 patients with active TL (cutaneous form) were enrolled, the follow up, after the therapeutic trial was 12 months long, in order of the possibility of relapse. All patients with suspected lesion underwent clinical evaluation and subsequently made the specific laboratory tests (Montenegro Reaction /MR, biopsy of borders of the lesion for histopathological examination). Once with immunological and parasitological diagnosis, all inclusion criterias were then evaluated, after that were allocated to one of the groups of the proposed treatment, the G1 group: Pentamidine isethionate (Pentacarinat®) at a dosage of 4mg/kg of weight at every 2 days with 5 intramuscular doses, once a day, and the G2 group: N-methylglucamine antimoniate (Glucantime®) 15mg/Sb+5/kg/day dosage for 20 days with intravenous injection 1x a day. Patients were followed for three months after the end of treatment, the parameter for determination of cure was essentially clinical. The study lasted for 18 months. The sample was composed mostly by males (58,3%), young adults (34,1%), peasants, patients with ulcerated lesions, usually in the legs. The incidence of clinical cure was higher in patients treated with Glucantime®, with 55 (61,8%) than patients treated with pentamidine isethionate (Pentacarinat®), 26 (34,2%) healed. The results described in this study showed that pentamidine isethionate (Pentacarinat®), drug of 2nd choice within the alternatives offered by Health Ministry, is not the drug with the better therapeutic response, compared to the drug of the 1st choice, so the risk/benefit balance does not justify the replacement of N-methylglucamine Antimoniate (Glucantime®) by pentamidine isethionate (Pentacarinat®).

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-05-08T17:25:12Z No. of bitstreams: 1 elgteelminborgesdepaula.pdf: 3070841 bytes, checksum: 4405f18ee4db96eba54abf3c61692232 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-05-17T14:03:07Z (GMT) No. of bitstreams: 1 elgteelminborgesdepaula.pdf: 3070841 bytes, checksum: 4405f18ee4db96eba54abf3c61692232 (MD5) / Made available in DSpace on 2017-05-17T14:03:07Z (GMT). No. of bitstreams: 1 elgteelminborgesdepaula.pdf: 3070841 bytes, checksum: 4405f18ee4db96eba54abf3c61692232 (MD5) Previous issue date: 2013-08-08 / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / Este trabalho compreende um estudo de sistemas supramoleculares estabilizados por interações não covalentes, formados entre substâncias utilizadas no tratamento de doenças negligenciadas, como as leishmanioses e a malária, com β-ciclodextrina (βCD). Investigou-se a associação do antileishmanico Isotionato de Pentamidina (PNT), do antimalárico Artesunato (AS) e do potencial antimalárico 1-(7-cloroquin-4-il)-tiossemicarbazida (CQT) com βCD. Os compostos envolvendo βCD e PNT foram obtidos pelo método de liofilização, ao passo que os compostos envolvendo AS e CQT foram obtidos por secagem por nebulização. Os compostos foram caracterizados por técnicas no estado sólido por espectroscopia vibracional da região do infravermelho (IV), análise térmica (TG/DTA) e microscopia eletrônica de varredura (MEV). Em solução, utilizou-se espectrometria de massas com ionização por eletrospray (IES-EM), ressonância magnética nuclear uni e bidimensional (1H e ROESY) e titulação calorimétrica isotérmica (ITC) (apenas o sistema envolvendo PNT). Além disso, os compostos tiveram sua atividade biológica investigada. Combinando técnicas termodinâmicas e estruturais como ITC, IES-EM e RMN, o processo de inclusão e os parâmetros termodinâmicos foram identificados. Os dados experimentais obtidos por ITC e IES-EM sugerem a formação simultânea de diferentes complexos supramoleculares em solução. Além disso, os dados de RMN sugerem uma inclusão profunda de PNT na cavidade de βCD. Os testes biológicos realizados in vivo, evidenciaram a atividade por via oral do composto de inclusão estudado (βCD:PNT)1:1, em relação ao fármaco livre, o qual é inativo. Tais resultados foram promissores no que se refere à investigação de uma alternativa terapêutica para o tratamento da Leishmaniose. Para o sistema formado entre AS e βCD, as caracterizações indicaram a formação de um sistema supramolecular envolvendo as duas espécies. Experimentos do tipo DOSY, indicaram o mesmo coeficiente de difusão para fármaco e macromolécula, evidenciando assim que os precursores evoluem juntos. Para CQT, verificou-se que o composto de inclusão (βCD:CQT) não foi formado. Deste modo, preparou-se o composto (αCD:CQT)1:1, o qual foi devidamente caracterizado. Os resultados obtidos por ROESY indicaram interações entre os hidrogênios benzênicos, H5, H6 e H8, com os hidrogênios H-3 e H-5 situados na cavidade da molécula hospedeira, envidenciando a formação do composto de inclusão com αCD. Os ensaios biológicos para os antimaláricos AS e CQT foram realizados in vivo. A toxicidade aguda do composto de inclusão (βCD:AS)1:1 não apresentou toxicidade aos camundongos após 5 dias de administração. Adicionalmente, realizou-se ensaios para avaliar a atividade citotóxica de AS, (βCD:AS)1:1 e CQT. Os resultados indicaram que as moléculas não apresentaram toxicidade in vitro para células renais e hepáticas. A atividade in vivo de CQT foi testada contra P. berghei, constatando-se o potencial antimalárico foi ativo no quinto e sétimo dias, reduzindo aproximadamente 66 % da parasitemia. / This work includes a study of supramolecular systems stabilized by non-covalent interactions, formed between the substances used in the treatment of neglected diseases such leishmaniasis and malaria, with β-cyclodextrin (βCD). We investigated the association of antileishmanial pentamidine isethionate (PNT), the antimalarial artesunate (AS) and potential antimalarial 1,7-chloroquinolinyl-thiosemicarbazide (CQT) with βCD. The compounds involving PNT were obtained by lyophilization, while compounds involving AS and CQT were obtained by spray drying. The compounds were characterized by standard solid state methods such as vibrational spectroscopy in the infrared regiond (IR), thermal analysis (TG/DTA) and scanning electron microscopy (SEM). In solution, we used mass spectrometry with electrospray ionization (ESI-MS), nuclear magnetic resonance (1H and ROESY) and isothermal titration calorimetry (ITC) (only the system involving PNT). Furthermore, the compounds have biological activity investigated. Combining thermodynamic and structural techniques such as ITC, ESI-MS and NMR, the inclusion process and the thermodynamic parameters were identified. The experimental data obtained by ESI-MS and ITC suggest the simultaneous formation of different supramolecular complexes in solution. Also, NMR data suggest a deep inclusion of PNT in βCD cavity. The biological tests performed in vivo showed the oral activity of inclusion compound studied (βCD:PNT)1:1, compared to free drug which is inactive. These results are promising with regard to the investigation of alternative therapeutic alternatives for leishmaniasis treatment. For the system formed between AS e βCD the characterizations indicated the formation of a supramolecular system involving two species. DOSY experiments indicated the same diffusion coefficient for the drug and the macromolecule, showing the formation of inclusion compound (βCD:AS)1:1. It was verified that (βCD:CQT) inclusion compound was not formed. Thus the compound (αCD:CQT)1:1 was prepared, which was properly characterized. The ROESY results indicated interactions between the aromatic hydrogens, H5, H6 and H8 with H-3 and H-5 located in the cavity of host molecule, showing the formation of inclusion compound with αCD. The biological tests for antimalarials AS and CQT were performed in vivo. Acute toxicity of the inclusion compound (βCD:AS)1:1 showed no toxicity to mice after 5 days of administration. Additionally, tests were conducted to evaluate the cytotoxic activity of AS, (βCD:AS)1:1 and CQT. The results indicated that the molecules showed no toxicity in vitro in renal and hepatic cells. The in vivo activity of CQT was tested against P. Berghei, noting the potential antimalarial was active in the fifth and seventh days, reducing approximately 66% parasitemia.