Dissertations / Theses on the topic 'Diminazeno'

This paper aimed to describe some lab and histological findings from an experimental infection of Trypanosoma (Trypanozoon) evansi Steel, 1885 (Balbiani, 1888) in rats (Rattus novergicus) belonging to the Wistar strain, testing the effectiveness of three medications. These animals were divided in 4 groups of 10 each and treated to three different chemical treatments after the detection of more than eight tripomastigotes for visual side of the blood smears at the light microscope with 1000 increase. Was tested injectable oxytetracycline 10%, benzonidazol per oss and injectable diminazene 7% while one infected and untreated group remained as control. Evaluation of treatment efficacy was performed through daily blood smears stained by Panotico Rápido® for 60 days. The rats were necropsied aiming histological examination. Treatment with diminazene was the most efficient since no parasites having significant difference (p < 0.05) between males and females of that group, which presented survive after the treatment of 14,4 days more than the males. Histological alterations findings were not specific.
Este trabalho objetivou verificar os achados laboratoriais e histológicos da infecção experimental por Trypanosoma (Trypanozoon) evansi (Steel, 1885) Balbiani, 1888, em ratos (Rattus norvegicus) da linhagem Wistar, testando a eficácia de três medicamentos. Foram utilizados 40 ratos, divididos em quatro grupos de 10 cada, sendo cada grupo composto por 5 machos e cinco fêmeas, os quais foram tratados com três quimioterápicos distintos após a detecção de parasitemia superior a oito tripomastigotas por campo visual do esfregaço sangüíneo ao microscópio de luz (1000 vezes). Testou-se oxitetraciclina 10% injetável, benzonidazol comprimidos de 100mg e aceturato de diminazeno 7% injetável comparados com um grupo controle, experimentalmente infectado, mas não tratado. As avaliações foram feitas por contagens diárias da presença de hemoparasitas em esfregaço sangüíneo, corado por Panótico Rápido®, por 60 dias. Os ratos foram necropsiados e avaliados histologicamente à procura de alterações microscópicas dos órgãos afetados. O tratamento feito com aceturato de diminazeno foi o mais eficaz no controle da parasitemia, havendo diferença significativa (p < 0.05) entre machos e fêmeas desse grupo, as quais apresentaram período de sobrevida após o tratamento de 14,4 dias superior aos machos. Histologicamente as alterações encontradas nos órgãos afetados foram inespecíficas.

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Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG
Activation of the Angiotensin Converting Enzyme 2 (ACE2)-Angiotensin-(1-7) [Ang(1-7)]-Mas Receptor axis results in protective effects in the cardiovascular system. ACE 2 is an important component of Renin-Angiotensin System, because it is able to convert Angiotensin II in Ang-(1-7). Recents studies have shown that diminazene aceturate (DIZE) act as an activator of ACE 2. The objective of this study was to evaluate the cardiovascular effects of chronic treatment with DIZE in pressureoverloaded rats and the possible mechanisms involved in these effects. Male Wistar rats (200-350 g) were divided in four groups: (1) Sham; (2) Coarcted (abdominal aortic banding, CAA); (3) CAA + DIZE (1 mg/kg, gavage); e (4) CAA + DIZE (1 mg/kg, gavage) + A-779 (120 µg/day, osmotic mini-pumps). Twenty one days after surgery procedure, the blood pressure was recorded, the hearts were isolated and perfused according to Langendorff method. Vascular reactivity was measured by isolated aortic ring preparation. In order to evaluate the cardiac hypertrophy, the left ventricular mass index (VMI) was calculated through the ratio of the left ventricular wet weight to tibia length. The cross-sectional area of cardiomyocytes (CSA) was also evaluated. The mRNA levels for ANP, BNP e TGF-β were also evaluated by qRT-PCR. The expression of ACE-2 and ERK1/2, AKT, mTOR, GATA-4, catalase and SOD proteins involved in hypertrophic pathways was analyzed by Western Blot technique. The results are presented as means ± SEM. One-way ANOVA followed by the Newman-Keuls post-test was used to analyze the blood pressure, cardiac morphometric parameters, isolated heart, qRT-PCR and Western Blot experiments. Two-way ANOVA followed by the Bonferroni post-test was used for aortic rings preparation protocols. All statistical analyses were considered significant at P<0.05. Isolated hearts from coarcted rats presented a significant decrease in the left ventricular end systolic pressure (128.1 ± 9.0 vs. 79.1 ± 12.8 mmHg in CAA, P<0.05), left ventricular developed pressure (118.1 ± 8.9 vs. 69.0 ± 12.7 mmHg in CAA, P<0.05), +dP/dt (2295.0 ± 161.8 vs. 1406.0 ± 246.5 mmHg/s in CAA, P<0.05) and dP/dt (1787.0 ± 166.0 vs. 1066.0 ± 181.9 mmHg/s in CAA, P<0.05). The DIZE treatment attenuated all of these effects induced by CAA. Moreover, DIZE treatment increased the coronary flow in hypertrophic hearts (CAA: 11.6 ± 0.8 vs. CAA+DIZE: 15.8 ± 0.6 mL/min, P<0.05). This effect was blocked by A-779. Pressure–overloaded hearts showed a significant increase in VMI (0.17 ± 0.01 vs. 0.21 ± 0.01 g/cm in CAA, P<0.05) and CSA (8.98 ± 0.54 vs. 10.72 ± 0.27 µm in CAA, P<0.05). The chronic treatment with DIZE prevented the heart hypertrophy (10.72 ± 0.27 in CAA vs. 9.25 ± 0.23 µm in CAA+DIZE, P<0.05). Indeed, treatment with A-779 attenuated the antihypertrophic effect induced by DIZE treatment. The coarcted rats presented a increase in mRNA expression of ANP, BNP and TGF-β and the DIZE treatment reverted this effect. The pressure overload decreased the acetylcholine-induced relaxation in aortic rings from coarcted rats and treatment with DIZE was not able to improve this effect. The coarctation decreased the phosphorylation of the AKT, which was not changed by DIZE treatment. The expression of ACE 2, total and phosphorylated ERK1/2, total AKT, mTOR, SOD and catalase was not changed by coarctation or by ACE 2 activation. These results show that the chronic treatment with DIZE was efficient in preventing the left ventricular hypertrophy and cardiac dysfunction induced by pressure overload. These effects were independent of changes in the expression of ACE 2, ERK1/2, AKT, mTOR, SOD and catalase. Thus, DIZE has important therapeutic potential for cardiovascular diseases.
A ativação do eixo Enzima Conversora de Angiotensina 2 (ECA2)-Angiotensina-(1-7) [Ang-(1-7)]-Receptor Mas resulta em importantes efeitos protetores no sistema cardiovascular..A ECA 2 é um importante componente do Sistema ReninaAngiotensina, pois hidrolisa a Angiotensina II em Ang-(1-7). Recentes estudos tem demonstrado que o aceturato de diminazeno (DIZE) apresenta capacidade de aumentar a atividade da ECA 2. Sendo assim, o objetivo deste estudo foi avaliar os efeitos cardiovasculares do DIZE nas mudanças induzidas por sobrecarga pressórica e possíveis mecanismos intracelulares envolvidos nestes efeitos. Foram utilizados ratos Wistar (200-350 g), divididos em quatro grupos: (1) Sham; (2) Coarctados (coarctação da aorta abdominal, CAA), (3) CAA + DIZE (1 mg/kg, gavagem); e (4) CAA + DIZE (1 mg/kg, gavagem) + A-779 (120 µg/dia, mini-bombas osmóticas). Decorridos 21 dias da coarctação, a pressão arterial dos animais foi registrada, os corações foram isolados e perfundidos pelo método de Langendorff com pressão constante. A reatividade vascular foi avaliada por preparação de anéis de aorta isolada. Para avaliar a hipertrofia cardíaca, o peso dos ventrículos esquerdos foi normalizado pelo comprimento das tíbias e expresso como índice de massa ventricular (IMV), além da área de secção transversa dos cardiomiócitos (AST) ser também medida. Os níveis de mRNA para ANP, BNP e TGF-β também foram avaliados por qRT-PCR. A expressão de ECA 2 e das proteínas ERK1/2, AKT, mTOR, GATA-4, SOD e catalase, envolvidas em vias pró-hipertróficas, foi analisada através da técnica de Western Blot. Os resultados foram apresentados como média ± erro padrão da média. Para as análises de pressão arterial média, coração isolado e parâmetros morfométricos, qRT-PCR e Western Blot, foi utilizado o teste ANOVA One Way seguido pelo pós-teste de Newman-Keuls. Para a reparação de anéis de aorta isolada, foi usado ANOVA Two Way seguido pelo teste de Bonferroni. As diferenças foram consideradas significativas com P<0,05. Os corações isolados dos ratos coarctados apresentaram diminuição significativa da pressão ventricular esquerda ao final da sístole (128,1 ± 9,0 vs. 79,1 ± 12,8 mmHg em CAA, P<0,05), pressão desenvolvida pelo ventrículo esquerdo (118,1 ± 8,9 vs. 69,0 ± 12,7 mmHgem CAA, P<0,05), +dP/dt (2295,0 ± 161,8 vs. 1406,0 ± 246,5 mmHg/s em CAA, P<0,05) e -dP/dt (1787,0 ± 166,0 vs. 1066,0 ± 181,9 mmHg/s em CAA, P<0,05). A ativação da ECA 2 atenuou a disfunção ventricular esquerda induzida pela coarctação. O tratamento com DIZE aumentou o fluxo coronariano dos corações hipertrofiados (CAA: 11,6 ± 0,8 vs. CAA+DIZE: 15,8 ± 0,6 mL/min, P<0,05). Este efeito foi loqueado pelo A-779. Os corações submetidos a sobrecarga pressórica mostraram um umento significativo do IMV (0,17 ± 0,01 vs. 0,21 ± 0,01 g/cm em CAA, P<0,05) e AST (9,37 ± 0,55 vs. 10,72 ± 0,27 µm em CAA, P<0,05). A ativação da ECA 2 preveniu a hipertrofia cardíaca (AST: 10,72 ± 0,27 vs. 9,25 ± 0,23 µm em CAA + DIZE, P<0,05). O tratamento com A-779 atenuou o efeito anti-hipertrófico produzido pelo DIZE nos corações coarctados. A coarctação também promoveu aumento da expressão de mRNA de ANP, BNP e TGF-β e o tratamento com DIZE reverteu esse efeito. A sobrecarga pressórica diminuiu o relaxamento induzido por acetilcolina em anéis de aorta isolada e o tratamento com o ativador da ECA 2 não foi capaz de alterar esse efeito. A coarctação diminuiu a fosforilação da AKT e o tratamento com DIZE não foi capaz de alterá-la. Não foram encontradas alterações na expressão das proteínas ECA 2, ERK1/2 total e fosforilada, AKT total, mTOR, GATA-4, SOD e catalase. Tais resultados mostram que o tratamento crônico com DIZE apresenta efeitos cardioprotetores contra a disfunção cardíaca induzida pela sobrecarga pressórica através da diminuição da hipertrofia ventricular esquerda, sem mudanças na expressão de ECA 2, ERK1/2, AKT, mTOR, GATA-4, SOD e catalase. Portanto, o DIZE possui importante potencial terapêutico frente a doenças cardiovasculares.

Conselho Nacional de Desenvolvimento Científico e Tecnológico
The aim of this study was to evaluate the utilization of a standard treatment with diminazene aceturate against the infection caused by T. evansi, associated to sodium selenite and vitamin E. In vitro tests showed trypanocidal effect related to the treatment with diminazene aceturate and sodium selenite, but vitamin E had no harmful effect on the trypanosomes. In vivo experiments utilized a total of 72 adult outbreed females rats, separated into 9 groups (A, B, C, D, E, F, G, H and I), 8 animals each. Group A was the uninfected group; groups B to I were infected with 0.2 mL of blood containing 106 trypanosomes. Parasitemia was estimated daily by microscopic examination of blood smears. Group B served as positive control; group C was treated with diminazene aceturate; group D with sodium selenite; group E with vitamin E; group F received an association of diminazene aceturate and sodium selenite; group G received an association of diminazene aceturate and vitamin E; group H received an association of diminazene aceturate, sodium selenite and vitamin E, and group I received an association of sodium selenite and vitamin E. Diminazene aceturate was administrated in a single dose on the 3rd day post infection (PI). Sodium selenite and vitamin E were administered at the 3rd and 23rd day PI. In vivo tests showed increase of longevity in groups treated with diminazene aceturate associated with sodium selenite (groups F and H). No difference was found between groups C and E, thus the vitamin E did not increase the efficacy of treatment against T. evansi when associated to diminazene aceturate. The curative efficacy of treatments was 37.5, 87.7, 37.7 and 75% to the groups C, F, G and H, respectively. Other treatments showed no efficacy. The sodium selenite when combined with chemotherapy may represent an alternative in the treatment of trypanosomosis.
O objetivo deste estudo foi avaliar a utilização de um tratamento padrão contra a infecção causada pelo T. evansi, baseado na utilização do aceturato de diminazeno associado ao selenito de sódio e a vitamina E. Os testes in vitro mostraram um efeito tripanocida relacionados ao tratamento com aceturato de diminazeno e selenito de sódio; contudo a vitamina E não gerou nenhum efeito nocivo sobre o tripanossomas. Experimentos in vivo utilizaram um total de 72 fêmeas adultas de ratos, separados em 9 grupos (A, B, C, D, E, F, G, H e I), com 8 animais cada grupo. O grupo A serviu como grupo não infectado; grupos de B a I foram infectados com 0,2 mL de sangue contendo 106 tripanossomas. A parasitemia foi estimada diariamente por exame microscópico de esfregaços sanguíneo. O grupo B serviu como controle positivo; grupo C, tratado com aceturato de diminazeno; grupo D, com selenito de sódio; grupo E, com vitamina E; grupo F, recebeu uma associação de aceturato de diminazeno e selenito de sódio; grupo G, associação de aceturato de diminazeno e vitamina E; grupo H, associação de aceturato de diminazeno, selenito de sódio e vitamina E; e por fim o grupo I o qual recebeu uma associação de selenito de sódio e vitamina E. O aceturato de diminazeno foi administrado em dose única no 3º dia pós-infecção (PI). Selenito de sódio e vitamina E foram administradas no 3º e 23º dias PI. Os testes in vivo mostraram aumento da longevidade nos grupos tratados com aceturato de diminazeno associado ao selenito de sódio (grupos F e H). Não foi encontrada diferença entre os grupos C e E, portanto, a vitamina E não aumentou a eficácia do tratamento contra T. evansi quando associado ao aceturato de diminazeno. A eficácia curativa dos tratamentos foi de 37.5, 87.7, 37.7 e 75% para os grupos C, F, G e H, respectivamente. Os demais tratamentos não mostraram eficácia. Assim, podemos sugerir que o selenito de sódio, quando combinado com a quimioterapia pode representar uma alternativa no tratamento da tripanossomose.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
The aim of this study was to develop and to evaluate the therapeutic efficacy of liposomes diminazene aceturate and in vitro and by using mice experimentally infected with Trypanosoma evansi. In vitro tests were performed in culture medium at concentrations of 0.25, 0.5, 1, 2 and 3 mg/ml of diminazene aceturate convetional (CDMZ) and liposomal (L-DMZ). A total of 114 rats (Rattus norvegicus) were used of in vivo test. These rats were divided into 6 groups (A, B, C, D, E and F). Group A served as a negative control (uninfected and untreated). Rats in Group B served as a positive control and were infected with T. evansi. Animals in Group C were infected and treated with L-DMZ (single dose, 3.5 mg/kg-1), Group D was composed with infected and treated with C-DMZ animals (single dose, 3.5 mg/kg-1), Group E infected treated with L-DMZ animals for 5 consecutive days (3.5 mg/kg-1/dia) and Group F infected treated with C-DMZ animals for 5 consecutive days (3.5 mg/kg-1/dia). In vitro, a dose-dependent trypanocidal effect of L-DMZ was observed against the parasite. In vivo, the efficacy of L-DMZ and C-DMZ in different therapeutic protocols was similar. The analysis of biochemical and histological parameters on the 7th and 40th post-treatment revealed alterations in liver and kidney enzymes, and histological alterations in the structure of organs, especially in the animals treated with L-DMZ at 5 consecutive days. The results of this study showed that liposomal formulations may be a new alternative for the treatment of tripanosomoses, but future research could be undertaken to improve the conduction of liposomes and direction for greater efficiency.
Este estudo teve como objetivo desenvolver e testar lipossomas de aceturato de diminazeno em testes in vitro e in vivo visando o controle de Trypanosoma evansi. O teste in vitro foi realizado em meio de cultura nas concentrações de 0,25, 0,5, 1, 2 e 3 μg/mL de aceturato de diminazeno convencional (C-DMZ) e lipossomal (L-DMZ). Para os testes in vivo foram utilizados 114 ratos (Rattus norvegicus) divididos em seis grupos (A, B, C, D, E e F) em dois experimentos, um para avaliar a eficácia e outro para analisar os parâmetros bioquímicos e histológicos. O grupo A foi utilizado como controle (animais sadios), B (animais infectados e não tratados), C (animais infectados e tratados com aceturato de diminazeno lipossomal com dose única 3,5 mg/kg-1), D (animais infectados e tratados com aceturato de diminazeno convencional com dose única 3,5 mg/kg-1), E (animais infectados e tratados com aceturato lipossomal por 5 dias consecutivos com a dose de 3,5 mg/kg-1/dia) e grupo F (animais e infectados tratados com aceturato convencional por 5 dias consecutivos com a dose de 3,5 mg/kg-1/dia). O teste in vitro com o lipossoma de aceturato de diminazeno mostrou uma maior mortalidade dose-dependente do T. evansi em meio de cultura se comparado ao medicamento comercial e os parasitos morreram mais rapidamente que nos grupos de aceturato de diminazeno convencional e controle. Nos resultados dos testes in vivo, a eficácia do aceturato de diminazeno lipossomal e convencional nos diferentes protocolos terapêuticos foram similares. A análise dos parâmetros bioquímicos e histológicos realizados no 7º e 40º pós-tratamento revelaram alterações nas enzimas hepáticas e renais, além de modificações na estrutura dos órgãos, principalmente nos animais tratados com lipossomas na maior dosagem. Os resultados obtidos neste estudo demonstraram que as formulações lipossomais podem ser uma nova alternativa para o tratamento das tripanossomoses. Futuras pesquisas poderiam ser realizadas para melhorar o carreamento e a direção dos lipossomas para uma maior eficácia.

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O Aceturato de Diminazeno (DIZE) é utilizado como medicamento anti- trypanosomal de uso veterinário que é capaz de aumentar a velocidade catalítica da enzima conversora de angiotensina- 2 (ECA-2). Portanto, o objetivo do nosso trabalho foi investigar os efeitos do tratamento com DIZE no remodelamento cardíaco após infarto agudo do miocárdio (IAM). Além disso, a proposta foi comparar os dados do tratamento com DIZE com modelo experimental tratado com Losartan (Los). Ratos Wistar Macho foram submetidos ao procedimento cirúrgico de oclusão permanente da artéria coronária esquerda para produzir IAM, os animais controles foram submetidos ao mesmo procedimento cirúrgico, porém sem a oclusão arterial. Após a cirurgia os animais foram distribuídos em cinco grupos: (1) Ratos Controles + Placebo (Con-Pla; n= 8); (2) Ratos Controles + DIZE (Con-DIZE; n= 6); (3) Ratos Infartados + Placebo (IM-Pla; n= 7); (4) Ratos Infartados + DIZE (IM-DIZE; n= 6); (5) Ratos Infartados + Losartan (IM-Los; n= 7), foram administrados diariamente injeção subcutânea de 15 mg/kg (DIZE e/ou Los) e placebo (NaCl 0.9%), durante 28 dias. Após 28 dias os animais foram anestesiados para obter as variáveis hemodinâmicas intracardíaca. Após registro hemodinâmico o coração foi parado em diástole com uma injeção intravenosa de KC1 (1M). Um cateter de duplo lúmen foi inserido dentro da cavidade do ventrículo esquerdo (VE) para obter os dados da curva pressão volume. Os Corações foram fixados em paraformol e processados para análise histológica de hipertrofia dos miócitos e conteúdo de colágeno. O grupo IM-Pla apresentou aumento significativo da pressão diastólica final do ventrículo esquerdo (26±3,3 mmHg) e redução na dp/dt+ (3014 ± 161 mmHg/s) e dpdt- ( -2333±91 mmHg/s) e pressão sistólica do VE (101±3,1 mmHg), quando comparados com os grupos tratados com DIZE (PDFVE: 15±1,6 mmHg/s; dp/dt+ 3884±104 mmHg/s; dpdt-: -2798±120 e PSVE: 110±0,7 mmHg) e IM-Los (PDFVE: 16±2,9; dp/dt+: 4146±131 mmHg/s; dpdt-: 2823±136 mmHg/s e PSVE: 111±3,5 mmHg). A hemodinâmica do ventrículo direito (VD) mostrou aumento de pressão do VD no grupo IM-Pla (40±0,6 mmHg) e redução da pressão no grupo IM-DIZE (37±2 mmHg). Os grupos de IM-DIZE (0,33±0,03 mmHg/mL e 0,64±0,01 mmHg/mL) e IM-Los (0,36±0,03 mmHg/mL e 0,65±0,04 mmHg/mL) apresentaram menor dilatação e rigidez no ventrículo esquerdo, quando comparados com com o grupo infartados sobre Pla (0,39±0,03 mmHg/mL e 0,78±0,02 mmHg/mL). O aumento da fração volumétrica de colágeno no VE foi parcialmente prevenido pelo tratamendo com DIZE ou Los. A hipertrofia dos miocitos pós-infarto permaneceu inalterado pelo tratamento com DIZE. Como esperado o tratamento com losartan atenuou o crescimento hipertrófico no ventriclo esquerdo quando comparado aos grupos controles. Em conclusão, nosso estudo apresentou que o DIZE foi parcialmente eficaz para prevenção de mudanças hemodinâmicas induzidas por infarto em ratos semelhante à observação em ratos infartados tratados com Los. As duas drogas também previnem o aumento da deposição de colágeno no miocárdio do ventrículo esquerdo. Em relação a hipertrofia dos miocitos, porém, apenas Los apresentou um efeito preventivo. Palavras Chaves Infarto do miocárdio, ECA-2, Diminazeno, Losartan, Insuficiência Cardíaca

Diminazene is the therapy of choice for canine babesiosis in South Africa. Differences in the dosage described for diminazene usage and the occurrence of mortality at doses equal to or close to the recommended treatment dose for the treatment of canine babesiosis have been described. This has necessitated the need to more fully understand the absorption and disposition of diminazene in dogs. An intravenous (i.v.) as well as an intramuscular (i.m.) pharmacokinetic study was conducted to determine the pharmacokinetics of diminazine in healthy dogs as well as to describe the binding characteristics of diminazine (in the blood) in vivo and in vitro. Diminazene pharmacokinetics showed a large inter-individual variation after i.m. administration at 4.2 mg/kg (% CV 37 – 163) with a rapid absorption (K01-Hl - 6.6 + 10.8 min resulting in a Cmax of 1849 + 268.7 ng/ml at Tmax of 20 min. There was a rapid distribution phase (T½a 21.6 + 11.4 min) with the distribution into the peripheral compartment being more rapid than the distribution back in to the central compartment. A mean elimination half-life (T½b 5.31 + 3.89 h) was derived. At 1 h after i.m. injection, 75 % of the diminazene in whole blood was in the plasma fraction. Compartmental analysis of the i.v. data after diminazene administration at 2 mg/kg revealed a Cmax of 3725 + 1672.8 ng/ml with a rapid distribution phase (T½a 7.0 + 6.2 min) with a long elimination half-life (T½b - 32.0 ± 28.8 h). The distribution into the peripheral compartment was more rapid than the distribution back into the central compartment as measured by K12 and K21 (K12 - 8.78 + 8.71; K21 0.32 + 0.25). The i.v. pharmacokinetic results were very variable between the dogs with a % CV of 55.5 – 137.2. We hypothesize that the rapid distribution phase is a result of diminazene being sequestered into the liver, followed by a slow terminal phase were diminazene is both redistributed to the peripheral tissues and renally excreted. The T½b of 32.0 ± 28.8 h in the i.v. study is considerably longer than the elimination half-life (T½b - 5.31 + 3.89 h) found in the i.m. study. This is most likely due the 25 ng/ml limit of detection of the HPLC, detecting the i.v. tail but not the i.m. tail. This is not surprising as the Cmax levels following i.v administration were more than 2 times higher than after i.m. administration. Further pharmacokinetic studies with diminazene in dogs should take account of the rapid absorption of diminazene after i.m. administration and the low levels of diminazene in the terminal phases. The initial sequestration of diminazene in the liver and distribution to the peripheral compartment needs further clarification. With the knowledge gained of the pharmacokinetics of diminazene in healthy dogs, a population pharmacokinetic study in dogs with babesiosis is recommended. This will allow us to more fully appreciate alterations in the pharmacokinetics of diminazene in diseased populations and the potential covariants exerting an effect. It is our current recommendation that diminazene given i.m. at 4.2 mg/kg not be repeated within a 21 day period.
Dissertation (MMedVet (Med))--University of Pretoria, 2003.
Companion Animal Clinical Studies
unrestricted

African trypanosomiasis remains a major health problem to both humans and animals due to lack of effective treatment or vaccine to control the disease. Animal trypanosomiasis is considered one of the most important diseases affecting livestock production and agricultural development in sub-Saharan Africa. Although the use of trypanocides remain the most important method for controlling the disease in animals, the mechanisms of action of these compounds are not completely known. The overall aim of this thesis is to decipher the molecular mechanisms involved in Trypanosoma congolense-induced cytokine production and how this is modulated by the trypanocide, Diminazene aceturate (Berenil). First, I investigated the molecular and biochemical mechanisms of action of Berenil to determine whether in addition to trypanolytic effect, it exerts a modulatory effect on the host immune system. Although it is known that T. congolense infection in mice is associated with increased production of pro-inflammatory cytokines by macrophages, the intracellular signaling pathways leading to the production of these cytokines remain unknown. Therefore, I investigated the innate receptors and intracellular signaling pathways that are involved in T. congolense-induced pro-inflammatory cytokine production in macrophages. Next I further determined whether the inhibitory effect of Berenil on proinflammatory cytokine production in macrophages is specific to T. congolense. I found that Berenil treatment significantly reduced the immune activation and proinflammatory cytokine production in infected mice suggesting that in addition to its direct trypanolytic effect, Berenil also modulates the host immune response to the parasite. Next, I show that T. congolense induced pro-inflammatory cytokine production in macrophages is dependent on phosphorylation of mitogen-activated protein kinase (MAPK) and signal transducer and activation of transcription (STAT) proteins in a TLR2-dependent manner. I further show that Berenil treatment downregulates T. congolense as well as LPS induced cytokine production by affecting the phosphorylation of MAPK and STAT proteins. Collectively, the results from this thesis provide novel insights into T. congolense-induced activation of the innate immune system and modulation of host immune response by Berenil. These findings are significant and could help in developing newer and better therapeutic strategies against the disease, in particular, and inflammatory responses in general.
October 2016

Bovine trypanosomosis is a serious constraint to livestock development in large parts of Mozambique. In most areas where tsetse flies are present, the disease in livestock is controlled using curative and prophylactic trypanocidal drugs. Those drugs have been used for many years and new drugs are unlikely to become available in the near future. As a result, trypanosomes have developed resistance against the currently available trypanocidal compounds. Drug resistance has been detected in various African countries and is a serious impediment to the control of livestock trypanosomosis. A study was initiated to determine whether drug resistant trypanosome strains are present in Zambézia Province of Mozambique. The aim of this study was to determine the sensitivity of Trypanosoma congolense isolates from Chinde, Nicoadala and Maganja da Costa Districts to diminazene aceturate, isometamidium chloride and homidium chloride. To assess the effect of the farming system and the intensity of drug regimens on the development of drug resistance, trypanosome isolates were collected from cattle from subsistence, semisubsistence and commercial livestock production systems. Drug-use practices in each of the production systems were determined using a questionnaire. The methodology used to assess the level of drugs resistance in the trypanosome isolates was the standardized method described by Eisler et al. (2001). Seven isolates were selected for resistance testing. For each of the seven isolates, five different doses varying between 0.01-20 mg/kg body weight for isometamidium chloride, 0.01-10 mg/kg body weight for homidium chloride and 1-30 mg/kg body weight for diminazene aceturate were used. For each dose rate six mice were treated intraperitoneally with the appropriate quantity of the drug dissolved in 0.2 ml of sterile distilled water 24 hours after the inoculation of the blood containing the trypanosomes. The control mice (six mice per trypanocidal drug) received the same amount of water without the drug. In four of the seven isolates high levels of multiple drug resistance (diminazene aceturate and isometamidium chloride) were detected. One isolate had a low level of multiple (diminazene aceturate and isometamidium chloride) drug resistance. Two isolates were susceptible to both diminazene aceturate and isometamidium chloride. One of those was highly susceptible to isometamidium chloride even at the lowest dose rate. The observed levels of drug resistance could in most cases be correlated to the drug-use practices in the particular livestock production system. The results obtained from homidium chloride treatment are not conclusive, because most the mice cured after receiving 10 mg/kg body weight of the drug. Hence more research is required to establish the homidium threshold in mice. The results of this study should be useful to define the strategy of disease control in places where resistance of trypanocide were been reported.
Dissertation (MSc (Veterinary Science))--University of Pretoria, 2005.
Veterinary Tropical Diseases
unrestricted

Cette thèse porte sur le développement d’une formulation colloïdale de diminazène (DMZ) à l’aide de nanoparticules polysaccharidiques cationiques (NP+) pour le traitement de la Trypanosomiase Africaine (TA).Nous avons étudié dans un premier temps le procédé de chargement des NP+ en DMZ base. Nous avons constaté que l’ajout de phospholipides dans la matrice des NP+ est nécessaire à l’association de DMZ. La quantité de phospholipide est d’ailleurs le facteur limitant de l’indice de saturation des NP+ en DMZ. Afin de ne pas dégrader le principe actif, lors de son chargement, le procédé choisi est le « post-loading » qui correspond à un mode opératoire en conditions douces : ajout d’une solution de DMZ dans une suspension de NP+ à cœur huileux. Nos résultats montrent que cette formulation reste stable durant 6 mois à 4°C ne libérant pas de DMZ et le protégeant de l’oxydation. Dans un second temps, nous avons évalué l’efficacité thérapeutique du DMZ formulé. Les tests in vitro sur Trypanosoma brucei brucei montrent une amélioration de l’activité trypanocide du DMZ. Les tests réalisés sur un modèle aigu de TA, ont mis en évidence que la dose efficace est équivalente au DMZ libre (3 mg/kg)
This thesis focuses on the development of a colloidal formulation of diminazene (DMZ) using cationic polysaccharide nanoparticles (NP+) for the treatment of African Trypanosomiasis. We first studied the process of DMZ loading in NP+. The addition of phospholipids in the matrix of the NP+ appeared to be necessary for the DMZ association. So, the amount of phospholipids is the limiting factor of the saturation index of NP+ with DMZ. To avoid the drug degradation during its formulation, we choose the \\\"post-loading\\\" technique which corresponds to a procedure with mild conditions: adding a DMZ solution in a suspension of NP+ containing an oily core. DMZ loaded into 70DGNP+ was found to be protected against oxidation and was stable for at least 6 months at 4°C. In a second step, we evaluated the therapeutic efficacy of formulated DMZ. In vitro tests on Trypanosoma brucei brucei showed an improvement of the DMZ trypanocidal activity. Tests on an acute model of Trypanosomiasis showed that the effective dose is equivalent to the free DMZ (3 mg / kg)

The aim of this thesis was to provide a picture of the trypanosomosis and drug resistance prevalence in Eastern Province of Zambia, to understand the underlying factors of drug resistance (drug use habits), to improve the diagnosis of trypanosomosis in livestock and finally, to improve the diagnosis of isometamidium resistance in T.congolense. After an introductory part where available trypanosomosis and trypanocide resistance diagnostic methods are described and discussed, the body of the thesis is divided in two main sections. In the first section are presented the results of a cross-sectional and a longitudinal epidemiological survey describing the geographical distribution of trypanosomosis cases, of resistant isolates and of cattle treated with isometamidium chloride. The results of the monitoring of unsupervised treatments of cattle with isometamidium by farmers and veterinary assistants with the Isometamidium-ELISA technique are also presented. The second section describes the development of two new diagnostic methods, the first one allowing the diagnosis of trypanosome infections with high sensitivity and specificity through semi-nested polymerase chain reaction and restriction fragment length polymorphism. This is the first report of a pan-trypanosome PCR test (a single PCR test for the diagnosis of all important pathogenic trypanosomes of cattle). The second new method that was developed allows the diagnosis of isometamidium resistant T.congolense strains by PCR-RFLP. This is the first report of a PCR based diagnostic test of trypanocide resistance in T. congolense.

Doctorat en sciences, Spécialisation biologie moléculaire
info:eu-repo/semantics/nonPublished

碩士
國立中興大學
獸醫學系暨研究所
105
Canine babesiosis which is a protozoal, tickborne, hemolytic disease with worldwide distribution and global significance and specially caused by Babesia gibsoni is a big challenge and critical issue in the treatment until right now. It seems not to be complete eradicated by a traditional single drug or some new combination treatments such as azithromycin with atovaquone, and clindamycin, diminazene, with imidocarb, and enrofloxacin, doxycycline with metronidazole. After treatment, canine babesiosis is easily to relapse again or have drug-resistant problems which have been studied recently. The combination treatment protocol is more efficiency than the single drug administrated to canine babesiosis in most studies. It is the life-threaten and emergency disease which needs the appropriate treatment in dogs. The aim of this study is to compare the efficacy of a combination of clindamycin, doxycycline, and metronidazole (CDM) to that of diminazene and clindamycin-doxycycline-metronidazole (DCDM) for the treatment of natural B. gibsoni infections, 16 client-owned dogs were random collected in the study. Seven dogs were treated with CDM combination and 9 dogs were treated with DCDM. Seven of the 7 dogs in the CDM group and 8 of the 9 dogs in the DCDM one completely recovered, but one of the DCDM group was died in acute IMHA after received the diminazene injection. The statistical differences in the numeric variables (HCT, HGB, and PLT) between CDM and DCDM groups were noticed only at the 7th day (p=0.013; p=0.019; p=0.037, respectively) after assessed by Wilcoxon’s rank-sum test. The changes of the serum biochemisty after treatment were shown that the ALT, and ALKP levels were elevated higher than first day (4/7 and 2/8, respectively). There is one dog individual in each group with highly GGT after treatment, but the TBIL of all dogs were normal in the treatment period. The conclusion is that it is good efficacy and no significate difference in both groups but diminaneze is not available in many countries in order to CDM will be the choice of treatment for B. gibsoni. Although the CDM treatment protocol might elevate the serum liver enzyme levels, it is not appeared serious clinical signs with vomit or dropped appetite. After combine with ursodiol and silymarin treatment, the serum live enzyme levels return back to the normal range which may means CDM treatment should be used with caution in animal with liver dysfunction.

Les micelles polyioniques ont émergé comme des systèmes prometteurs de relargage de médicaments hydrophiles ioniques. Le but de cette étude était le développement des micelles polyioniques à base de dextrane pour la relargage de médicaments hydrophiles cationiques utilisant une nouvelle famille de copolymères bloc carboxymethyldextran-poly(éthylène glycol) (CMD-PEG). Quatre copolymères CMD-PEG ont été préparés dont deux copolymères identiques en termes de longueurs des blocs de CMD et de PEG mais différent en termes de densité de charges du bloc CMD; et deux autres copolymères dans lesquels les blocs chargés sont les mêmes mais dont les blocs de PEG sont différents. Les propriétés d’encapsulation des micelles CMD-PEG ont été évaluées avec différentes molécules cationiques: le diminazène (DIM), un médicament cationique modèle, le chlorhydrate de minocycline (MH), un analogue semi-synthétique de la tétracycline avec des propriétés neuro-protectives prometteuses et différents antibiotiques aminoglycosidiques. La cytotoxicité des copolymères CMD-PEG a été évaluée sur différentes lignées cellulaires en utilisant le test MTT et le test du Bleu Alamar. La formation de micelles des copolymères de CMD-PEG a été caractérisée par différentes techniques telles que la spectroscopie RMN 1H, la diffusion de la lumière dynamique (DLS) et la titration calorimétrique isotherme (ITC). Le taux de relargage des médicaments et l’activité pharmacologique des micelles contenant des médicaments ont aussi été évalués. Les copolymères CMD-PEG n'ont induit aucune cytotoxicité dans les hépatocytes humains et dans les cellules microgliales murines (N9) après 24 h incubation pour des concentrations allant jusqu’à 15 mg/mL. Les interactions électrostatiques entre les copolymères de CMD-PEG et les différentes drogues cationiques ont amorcé la formation de micelles polyioniques avec un coeur composé du complexe CMD-médicaments cationiques et une couronne composée de PEG. Les propriétés des micelles DIM/CMDPEG ont été fortement dépendantes du degré de carboxyméthylation du bloc CMD. Les micelles de CMD-PEG de degré de carboxyméthylation du bloc CMD ≥ 60 %, ont incorporé jusqu'à 64 % en poids de DIM et ont résisté à la désintégration induite par les sels et ceci jusqu'à 400 mM NaCl. Par contre, les micelles de CMD-PEG de degré de carboxyméthylation ~ 30% avaient une plus faible teneur en médicament (~ 40 % en poids de DIM) et se désagrégeaient à des concentrations en sel inférieures (∼ 100 mM NaCl). Le copolymère de CMD-PEG qui a montré les propriétés micellaires les plus satisfaisantes a été sélectionné comme système de livraison potentiel de chlorhydrate de minocycline (MH) et d’antibiotiques aminoglycosidiques. Les micelles CMD-PEG encapsulantes de MH ou d’aminoglycosides ont une petite taille (< 200 nm de diamètre), une forte capacité de chargement (≥ 50% en poids de médicaments) et une plus longue période de relargage de médicament. Ces micelles furent stables en solution aqueuse pendant un mois; après lyophilisation et en présence d'albumine sérique bovine. De plus, les micelles ont protégé MH contre sa dégradation en solutions aqueuses. Les micelles encapsulant les drogues ont maintenu les activités pharmacologiques de ces dernières. En outre, les micelles MH réduisent l’inflammation induite par les lipopolysaccharides dans les cellules microgliales murines (N9). Les micelles aminoglycosides ont été quant à elles capable de tuer une culture bactérienne test. Toutefois les micelles aminoglycosides/CMDPEG furent instables dans les conditions physiologiques. Les propriétés des micelles ont été considérablement améliorées par des modifications hydrophobiques de CMD-PEG. Ainsi, les micelles aminoglycosides/dodecyl-CMD-PEG ont montré une taille plus petite et une meilleure stabilité aux conditions physiologiques. Les résultats obtenus dans le cadre de cette étude montrent que CMD-PEG copolymères sont des systèmes prometteurs de relargage de médicaments cationiques.
Polyion complex (PIC) micelles have emerged as promising delivery systems of ionic hydrophilic drugs. It was the aim of this study to develop dextran-based PIC micelles for the delivery of hydrophilic cationic drugs using a new family of carboxymethyldextranblock- poly(ethylene glycol) (CMD-PEG) copolymers. Four CMD-PEG copolymers were prepared: (i) two copolymers identical in terms of the length of CMD and PEG blocks, but different in terms of the charge density of the CMD block; and (ii) two copolymers in which the charged block is the same, but the PEG block is of different molecular weight. The micellization of CMD-PEG copolymers and drug delivery aspects of the resulting micelles were evaluated using different cationic drugs: diminazene (DIM), a model cationic drug, minocycline hydrochloride (MH), a semisynthetic tetracycline antibiotic with promising neuroprotective properties and different aminoglycoside antibiotics. The cytotoxicity of CMD-PEG copolymers was evaluated in different cell lines using MTT and Alamar blue assays. CMD-PEG micelles encapsulating different drugs were characterized using different techniques, such as 1H NMR spectroscopy, dynamic light scattering (DLS), and isothermal titration calorimetry (ITC). The pattern of drug release and pharmacological activity of micelles-encapsulated drugs were also evaluated. The CMD-PEG copolymers did not induce cytotoxicity in human hepatocytes and murine microglia (N9) in concentrations as high as 15 mg/mL after incubation for 24 h. Electrostatic interactions between CMD-PEG copolymers and different cationic drugs triggered the formation of PIC micelles with a CMD/drug core and a PEG corona. The properties of DIM/CMD-PEG micelles were strongly dependent on the degree of carboxymethylation of the CMD block. Micelles of CMD-PEG copolymers having degree of carboxymethylation ≥ 60%, incorporated up to 64 wt% DIM, resisted salt-induced disintegration in solutions up to 400 mM NaCl and sustained DIM release under physiological conditions (pH 7.4, 150 mM NaCl). In contrast, micelles of CMD-PEG of degree of carboxymethylation ~ 30% had lower drug content (~ 40 wt% DIM) and disintegrated at lower salt concentration (∼ 100 mM NaCl). The CMD-PEG copolymer that showed the most satisfactory micellar properties, in terms of high drug loading capacity, sustained drug release and micelles stability was selected as a potential delivery system of minocycline hydrochloride (MH) and different aminoglycosides. CMD-PEG micelles encapsulating either MH or aminoglycosides had small size (< 200 nm in diameter), high drug loading capacity (≥ 50 wt% drug) and sustained drug release. These micelles were stable in aqueous solution for up to one month, after freeze drying and in the presence of bovine serum albumin. Furthermore, the micelles protected MH against degradation in aqueous solutions. Micelles-encapsulated drugs maintained their pharmacological activity where MH micelles reduced lipopolysaccharides-induced inflammation in murine microglia (N9) cells. And aminoglycosides micelles were able to kill a test micro-organism (E. coli X-1 blue strain) in culture. Aminoglycosides/CMD-PEG micelles were unstable under physiological conditions. Micelle properties were greatly enhanced by hydrophobic modification of CMD-PEG. Thus, aminoglycosides/dodecyl-CMD-PEG micelles showed smaller size and better stability under physiological conditions. The results obtained in this study show that CMD-PEG copolymers are promising delivery systems for cationic hydrophilic drugs.

Trypanocidal Drug Resistance is presently recognized as an important constraint for trypanosomosis control efficiency in the endemic areas. No recent and up to date report is available for the Matutuíne District, which is preventing the establishment of an appropriate control strategy in this endemic area. The present study was carried out in order to assess and map the diminazene resistance in T. congolense populations circulating in bovines in the Matutuíne District. Between 2009 and 2011, a longitudinal survey was performed, allowing an updated vision of the disease prevalence and of the drug resistance situation. A total of 2427 bovine blood samples were collected on filter paper in all five administrative areas in Matutuine (Belavista, Zitundo, Catuane, Katembe and Machangulo). The parasitological (buffy coat) prevalence was 17.3% while using the molecular test 18S PCR, a prevalence of 46% was recorded. Four trypanosome species were detected: the pathogenic T. congolense (savannah and Kilifi type), T. vivax, T. b. brucei and the non-pathogenic T. theileri. The T. congolense savannah type was the predominant species with a parasitological prevalence of 12.7% (309) (although including the Kilifi type which was not possible to distinguish by buffy coat) and using the molecular test was 22.3% (540) as single infection; 1.6% (149) as mixed infection with T. theileri. These results confirm the Matutuíne District as a trypanosomosis-endemic area and even indicate stable disease status, since the prevalence is almost identical to that reported 13 years before. Additionally, it indicates that the T. congolense savannah type remains the main etiologic agent of bovine trypanosomosis in the region. Concerning the diminazene resistance in Matutuíne District, the 689 T. congolense savannah 18S PCR positive samples were submitted to the Ade2 PCR followed by the DpnII-PCR-RFLP. Only 21% (143) of the samples were amplified on the Ade2 PCR, confirming the low sensitivity of this PCR tool compared to the 18S PCR.. Ade2 positive PCR s were from four administrative areas in Matutuíne s i.e. Belavista (55.9%), Zitundo (25.2%), Catuane (15.4%) and Katembe (2.1%). Machangulo was not evaluated. The DpnII-PCR-RFLP revealed the occurrence of all three possible profiles of the TcoAT1/TcoNT10 gene: sensitive homozygous (17.5%), sensitive heterozygous or mixed (44.8%), and resistant (37.8%). Such TcoAT1/TcoNT10 gene allele distributions have been reported in areas without or with moderate trypanocidal drug usage. Therefore we can assume that this is the status of the Matutuíne District and also that, diminazene resistance is not a major problem in the region, allowing the use of the sanative pair strategy for trypanosomosis control in the area. For the evaluation of the correlation between the molecular DpnII-PCR-RFLP and the in vivo standardized mouse test for diminazene resistance diagnosis, 24 T. congolense savannah type isolates were evaluated by both tests. All of them (24/24) were sensitive to the drug at the dose of 10 mg/kg and 20 mg/kg bw in the in vivo drug sensitivity mouse test with the relapses being checked only by parasitological methods. The molecular test revealed 5/24 homozygous sensitive, 8/24 heterozygous sensitive and 11/24 homozygous resistant isolates.. Based on these data, the K statistic revealed a slight agreement between both tests (K= 0.15), supporting the idea of possibly misleading information in the drug sensitivity mouse test when the relapses are detected by parasitological methods. The molecular tool for detection of resistance to diminazene should not be used at the individual level but rather considering the distribution of the resistance alleles as indicator of drug use intensity. This correlation should be further explored. However, the result of the mouse test also indicates that diminazene resistance in this region has not yet been established.
Dissertation (MSc)--University of Pretoria, 2014.
tm2016
Veterinary Tropical Diseases
MSc
Unrestricted