UNIVERSIDADE FEDERAL DE PERNAMBUCO CENTRO DE CIÊNCIAS BIOLÓGICAS PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS BIOLÓGICAS DOUTORADO EM CIÊNCIAS BIOLÓGICAS
JULIANA KELLE DE ANDRADE LEMOINE NEVES
IMIDAZOLÍDINAS ESQUISTOSSOMICIDAS: AVALIAÇÃO ULTRAESTRUTURAL, ATIVIDADE CITOTÓXICA E IMUNOMODULADORA
RECIFE – 2010
UNIVERSIDADE FEDERAL DE PERNAMBUCO CENTRO DE CIÊNCIAS BIOLÓGICAS PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS BIOLÓGICAS DOUTORADO EM CIÊNCIAS BIOLÓGICAS
IMIDAZOLÍDINAS ESQUISTOSSOMICIDAS: AVALIAÇÃO ULTRAESTRUTURAL, ATIVIDADE CITOTÓXICA E IMUNOMODULADORA
Tese apresentada ao Programa de Pós- Graduação em Ciências Biológicas – Universidade Federal de Pernambuco, como parte dos requisitos para a obtenção do Título de Doutor em Ciências Biológicas.
Orientadora: Dra. Suely Lins Galdino
Co-orientadora: Dra. Mônica Camelo P.A. Albuquerque
RECIFE – 2010
Neves, Juliana Kelle de Andrade Lemoine Imidazolidinas esquistomicidas: avaliação ultraestrutural, atividade citotóxica e imunomoduladora/ Juliana Kelle de Andrade Lemoine Neves. – Recife: O Autor, 2010.
166 folhas : il., fig., tab. Orientadora: Suely Lins Galdino Co-orientadora: Mônica Camelo P.A. Albuquerque Tese (doutorado) – Universidade Federal de Pernambuco. Centro de Ciências Biológicas. Ciências Biológicas, 2010. Inclui bibliografia e anexos.
1. Esquistossomose 2. Imidazolidinas 3. Citotoxicidade I. Título.
616.963 CDD (22.ed.) UFPE/CCB-2010-102
"Aquele que tentou e não conseguiu
é superior àquele que nada tentou”.
Arquimedes
Dedico a
Meu esposo e filho André Lemoine e Ângelo Lemoine
Minha mãe Maria José AGRADECIMENTOS
A Deus que me deu oportunidade de vida, pela força e coragem pela caminhada longa que foi e será;
A Professora Dra. Suely Lins Galdino, do Laboratório de Planejamento e Síntese de Fármacos, do Departamento de Antibióticos da UFPE, pela orientação e credibilidade;
A Professora Dra. Mônica Camelo Pessoa Azevedo de Albuquerque, do Departamento de Medicina Tropical da UFPE, pela orientação e credibilidade;
Ao Professor Dr. Ivan da Rocha Pitta, do Laboratório de Planejamento e Síntese de Fármacos, do Departamento de Antibióticos da UFPE, pela cooperação para realização deste trabalho e credibilidade;
A Professora Dra. Maria do Carmo Alves de Lima, do Laboratório de Planejamento e Síntese de Fármacos, do Departamento de Antibióticos da UFPE, pela orientação, incentivo, credibilidade e amizade e por todos momentos de alegria;
A Professora Dra. Paloma Lys de Medeiros do Departamento de Histologia e Embriologia da UFPE, pela cooperação e disponibilidade prestada;
A Professora Dra. Valéria Rêgo Alves Pereira do Departamento de Imunologia do Centro de Pesquisas Aggeu Magalhães pelos grandes incentivos, colaboração e pela atenção dispensadas no decorrer da reta final do trabalho e esclarecimentos prestados nos momentos de dúvidas;
A Dra. Cristiane Moutinho Lagos de Melo do Departamento de Imunologia do Centro de
Pesquisas Aggeu Magalhães, pela grande amizade, colaboração, dedicação e disponibildade prestada;
A Dra. Maira Galdino da Rocha Pitta do Departamento de Antibióticos pela colaboração para realização deste trabalho; A Dra. Christina Alves Peixoto do Laboratório de Ultraestrutura do Centro de Pesquisas
Aggeu Magalhães (FIOCRUZ) e toda sua equipe pela colaboração na realização da microscopia eletrônica de varredura.
A Sandra Botelho do Departamento de Antibióticos pela grande ajuda nas sínteses dos compostos;
A todos do Laboratório de Imunologia do LIKA, pela colaboração e agradável convívio durante o período de experimentação;
Aos colegas do Laboratório de Planejamento e Síntese de Fármacos (GPIT/UFPE);
Ao Programa de Pós-Graduação em Ciências Biológicas, pela oportunidade do Doutorado;
A Adenilda Eugênia de Lima, secretária do Curso de Pós-Graduação em Ciências Biológicas, pela competência e grande convívio;
E a minha família, por sempre acreditar em mim e superarem pacientemente os meus momentos de ausência.
LISTA DE ABREVIATURAS E SIGLAS
Cepa BH – Cepa oriunda de Belo Horizonte – MG
DMSO – Dimetilsulfóxido
ELISA - Enzyme-Linked Immunosorbent Assay
ESQ – Esquistossomose
FIOCRUZ – Fundação Oswaldo Cruz
FUNASA – Fundação Nacional de Saúde
GGVS – Gerência Geral de Vigilância à Saúde
GPIT – Grupo de Pesquisa e Inovação Terapêutica
IFN- Interferon gama
IL – Interleucina
LIKA – Laboratório de Imunopatologia Keizo Asami
LPSF – Laboratório de Planejamento e Síntese de Fármacos
MEC - Matriz Extracelular
MEV – Microscopia Eletrônica de Varredura
NO – Óxido nítrico
OMS – Organização Mundial de Saúde
PECE – Programa Especial de Controle da Esquistossomose
UFPE – Universidade Federal de Pernambuco
WHO - World Health Organization
LISTA DE FIGURAS Figura 1: Medicamentos desenvolvidos entre 1975 a 2004. Fonte: CHIRAC e 20 TORREELE, 2006. Figura 2: Theodor Bilharz . Fonte: www.yearofscience.org. 21 Figura 3: Vermes adultos de Schistosoma. (A) Macho e (B) Fêmea. Fonte: PICASA, 23 2009. Figura 4: Distribuição mundial da esquistossomose. Fonte: GRYSSELS et al, 2006. 24 Figura 5: Áreas endêmicas da esquistossomose mansônica no Brasil. Fonte: 25 AMARAL et al., 2006. Figura 6: Prevalência da esquistossomose dos municípios da Zona da Mata no 27 estado de Pernambuco. Fonte: BARBOSA et al., 2006. Figura 7: Caramujo do gênero Biomphalaria. Fonte: www.york.ac.uk, 2009. 29 Figura 8: Segmento anterior do corpo do macho (à esquerda) e da fêmea (à 31 direita), mostrando os principais órgãos: a-ventosa oral e boca, b-porção anterior do intestino, c-acetábulo ou ventosa ventral, d-vesícula seminal, e-canal deferente, f-testículos, g-porção média bifurcada do intestino, h- ceco, i-orifício genital feminino, j-útero contendo dois ovos, k-um ovo em processo de formação da casca no oótipo, l-oviduto, m- ovário, n- viteloduto, o-glândulas vitelinas. Fonte: REY, 2001. Figura 9: Fígado sem alterações (A); hepatomegalia com o lóbulo esquerdo 33 aumentado (B), baço com conformações normais (C); esplenomegalia mostrando um crescimento do órgão ultrapassando o ultimo arco costal na altura do umbigo (D). Fonte: medlineplus, 2009. Figura 10: Composição celular e de componentes da matri extraceular em 34 granulomas esquistossómticos hepáticos. (Modificado a partir de CARVALHO, et al 2008). Figura 11: Tipos de moléculas de adesão, fatores de crescimento e seus receptores 34 em granulomas esquistossomóticos. (Modificado a partir de CARVALHO, et al 2008). Figura 12: Esquema da reação granulomatosa em fígado de camundongo. Fonte: 35 HITARA e FUKUMA, 2003. Figura 13: Granuloma esquistossomótico. Fonte:38 PEARCE e MACDONALD, 2002. 36 Figura 14: Ciclo de vida do Schist45osoma mansoni. Fonte: GRYSSELS et al, 2006. 38 Figura 15: Desenvolvimento da resposta imune na infecção. Fonte: PEARCE e 43 MACDONALD, 2002. Figura 16: (I) 5,5-Difenil-imidazolidina-2,4-diona e (II) 5-(p-clorofenil)-5-metil- 45 imidazolidina-2,4-diona. Figura 17: (I) 5-Benzilideno-2-tioxo-imidazolidin-4-ona e (II) 2-amino-5- 46 benzilideno-imidazolidina-2,4-diona
LISTA DE TABELAS
Tabela 1: Grandes editais temáticos na área de doenças negligenciadas. 21 Tabela 2: As principais espécies que infectam seres humanos, as regiões onde são 22 encontradas e seus hospedeiros intermediários. Tabela 3: Número de infecções das cidades da Zona da Mata do estado de 28 Pernambuco, em destaque as cidades com maior número de infecções. Tabela 4: Principais fármacos esquistossomicidas desenvolvidos no século XX. 50 .
SUMÁRIO
LISTA DE ABREVIATURAS E SIGLAS
LISTA DE FIGURAS
LISTA DE TABELAS
RESUMO
ABSTRACT
1 INTRODUÇÃO 15
2 REVISÃO DA LITERATURA 18
2.1 Doenças negligenciadas 19
2.2 Esquistossomose 21
2.3 Distribuição geográfica e dados epidemiológicos 23
2.4 Hospedeiro Intermediário 29
2.5 Classificação e morfologia 30
2.6 Morbidade, fisiologia e imunopatologia 32
2.7 Ciclo biológico 37
2.8 Diagnóstico 38
2.9 Controle e prevenção quimioterapica 39
2.10 Resistência ao tratamento 40
2.11 Imunopatologia 41 2.12 Imidazolidinas bioativas 44
2.13 Fármacos esquistossomicidas 46
3-OBJETIVOS 53
3.1 Geral 54
3.2 Específicos 54
4 CONCLUSÃO 55
5 PERSPECTIVAS 57
6 REFERÊNCIAS 59
ANEXOS 74
Biological and immunological activity of new Imidazolidines front of adult worms of
Schistosoma mansoni
Immunological studies and in vitro schistosomicide action of new imidazolidine derivatives
Antischistosomal action of thioxo-imidazolidine compounds: an ultrastructural study
Comissão de Ética de Experimentação Animal
RESUMO
A esquistossomose é uma doença debilitante e endêmica, distribuída em 74 países, causada por parasitas do gênero Schistosoma, onde existem cinco espécies de interesse médico, apenas o Schistosoma mansoni é endêmico no Brasil. Existem aproximadamente 207 milhões de pessoas infectadas pela esquistossomose no mundo, no Brasil estima-se sete milhões de portadores de esquistossomose mansônica e é considerada uma endemia em franca expansão atingindo aproximadamente 19 estados brasileiros. O praziquantel é o único fármaco para o tratamento de todas as esquistossomoses no mundo. O objetivo do nosso trabalho foi a busca de novos compostos biologicamente ativos para o combate da esquistossomose, através da síntese de derivados imidazolidínicos e avaliação da atividade esquistossomicida. Os derivados imidazolidínicos das séries 5-benzilideno-3-benzil-4- tioxo-imidazolidin-2-ona e 5-arilazo-4-tioxo-imidazolidin-2-ona foram avaliados in vitro frente a vermes adultos de Schistosoma mansoni (Cepa BH). Todos derivados avaliados in vitro apresentaram atividade esquistossomicida, sendo que os compostos LPSF/PT05, PT10 e PT11 provocaram mortalidade de 100% em 24 horas nas concentrações de 320 e 200 M e o LPSF/PT09 mortalidade de 100% em 48 horas também na maior concentração 320 M. O LPSF/PT05 e LPSF/PT10 foram analisado em microscopia eletrônica de varredura. Os derivados também foram avaliados quanto a citotoxicidade e viabilidade celular, apresentando uma toxicidade inferior ao praziquantel e uma baixa mortalidade celular (apoptose e necrose) em relação ao padrão. Na dosagem de citocinas (IL-10 e IFN- ) nenhum dos compostos apresentou indução dessas proteínas, porém todos os compostos analisados apresentaram uma indução no óxido nítrico estatisticamente significativo.
Palavras-chave: Esquistossomose, imidazolidinadionas, Ensaios in vitro .
ABSTRACT
Schistosomiasis is a debilitating and endemic, distributed in 74 countries, caused by parasites of the genus Schistosoma, which has five species of medical interest, only the Schistosoma mansoni is endemic in Brazil. There are approximately 207 million people infected with schistosomiasis in the world, in Brazil an estimated seven million people with schistosomiasis and is considered an endemic disease on the rise amounting to about 19 states. The Praziquantel is the main drug for the treatment of schistosomiasis in all the world. The aim of our study was the search for new biologically active compounds for the fight against schistosomiasis, through the synthesis of imidazolidine. Derivatives imidazolidine series 5-benzylidene-3-benzyl-4-thioxo-imidazolidin-2-one and 5-arilazo-4- thioxo-imidazolidin-2-one were evaluated in vitro against the adult worms of Schistosoma mansoni (strain BH). Derivatives evaluated in vitro all showed schistosomicidal activity and the LPSF/PT05, PT10 and PT11 had 100% mortality within 24 hours in higher concentrations 320 and 200 M and LPSF/PT09 100% mortality within 48 hours also the highest concentration 320 M. The LPSF/PT05 e LPSF/PT10 analyzed in scanning electron microscopy. Derivatives were also evaluated for cytotoxicity and cell viability, showing a lower toxicity to praziquantel and a low mortality in relation to the cellular standard. The measurement of cytokines (IL-10 e IFN- ) none of the compounds showed induction, but all the compounds showed an induction analyzes nitric oxide statistically significant.
Keywords: Schistosomiasis, imidazolidinedione, Assay in vitro.
INTRODUÇÃO 1. Introdução
A Química Medicinal estuda as razões moleculares da ação dos fármacos de maneira a descrever a relação entre a estrutura química e a atividade farmacológica, hierarquizando as diferentes contribuições funcionais. No contexto inverso, inclui-se o planejamento e o desenho estrutural de novas substâncias que possuam propriedades farmacoterapêuticas úteis, capazes de representarem novas entidades químicas, candidatas a protótipos de novos fármacos de uso seguro. A química medicinal é uma disciplina híbrida que está no centro de um grande espaço inter e multidisciplinar constituído pela biofísica, biologia molecular, bioquímica, clínica médica, físico-química, fisiologia, patologia, química biológica, química inorgânica, química orgânica, entre outras. Tratando do planejamento racional das drogas, sua síntese, sua elucidação estrutural, o desenvolvimento de ensaios farmacológicos e estudos de relações estrutura-atividade farmacológica (WILLIAMS e LEMKE, 2002; IUPAC, 2010).
As doenças infecciosas parasitárias continuam sendo um obstáculo ao desenvolvimento social e econômico dos países mais pobres, afetando as populações pobres e marginalizadas. As alternativas quimioterápicas para o tratamento da maioria das doenças parasitárias são extremamente limitadas. A esquistossomose, doença debilitante causada por parasitas do gênero Schistosoma constitui um dos maiores problemas mundiais enfrentados na saúde pública, visto que está distribuída em várias regiões geográficas. E mundialmente só perde para malária em número de pessoas infectadas (CARVALHO et al, 2008).
O Schistosoma é endêmico, afetando, aproximadamente, 76 países tropicais e subtropicais, principalmente países da África, Ásia e América Latina, sendo que nas Américas, ocorre apenas a espécie S. mansoni. No Brasil, ocorre nas regiões Norte, Nordeste, e no Norte das regiões Sul e Sudeste (GREVELDING, 2004).
Atualmente, estima-se no mundo 207 milhões de pessoas infectadas com as esquistossomoses, com um total de 650 milhões expostos ao risco de contrair a doença. Há aproximadamente seis a oito milhões de portadores de esquistossomose mansoni no Brasil. No mundo o S. mansoni infecta cerca de 70 milhões de indivíduos (CHITSULO et al., 2000; CIMERMAN, 2001; WHO, 2008). O ciclo do Schistosoma é complexo, pois exige a presença de hospedeiro intermediário, representados por diferentes espécies de moluscos pertencentes ao gênero: Bullinus, Biomphalaria, Oncomelania e Neotricula (CARRERA, 1990; NEVES,2005), com as seguintes formas evolutivas do S. mansoni: verme adulto (macho e fêmea), ovo, miracídio, esporocisto, cercária e esquistossômulo.
Na dependência de elevadas cargas parasitárias e, conseqüentemente, do número de ovos com formações granulomatosas, instala-se, progressivamente, o regime de hipertensão portal, com o conseqüente aparecimento de esplenomegalia de caráter congestivo e de circulação colateral, inclusive de varizes de esôfago. As fêmeas liberam centenas de ovos por dia, um número substancial ficam presos ao intestino e fígado, principalmente. Tem-se, assim, a forma hepatoesplênica com hipertensão portal da esquistossomose, definida como descompensada quando houver sangramento digestivo alto (GRYSCHEK, 2001; HELMY et al., 2009).
Há relatos de resistência ao praziquantel em cepas de Schistosoma principalmente na África, aonde o fármaco vem sendo utilizado agressivamente por longos períodos. Com a possível resistência a este fármaco são utilizadas doses maiores para se obter um melhor resultado (LIANG, 2001; ISMAIL et al., 1996). Porém, faz-se necessário o desenvolvimento de vacinas e o planejamento e síntese de novos fármacos que constituem novas alternativas terapêuticas para a parasitose em questão.
As imidazolidinas tiveram no passado um representante na clínica médica como um fármaco de ação comprovada frente a vermes adultos de S. mansoni, o niridazol (CATTO; TRACY; WEBSTER, 1984). A propriedade esquistossomicida in vitro frente a vermes adultos de S. mansoni dos derivados imidazolidínicos vem sendo relatada por Oliveira e colaboradores (2004) e Albuquerque e colaboradores (2006).
Derivados imidazolidínicos sintetizados por nossa equipe no Laboratório de Síntese e Planejamento de Fármacos-LSPF, ressaltam a contribuição química do núcleo imidazolidínico. Os resultados já obtidos com derivados imidazolidinônicos-3,5- dissubstituídos revelam que estas moléculas são portadores de significante atividade esquistossomicida (OLIVEIRA et. al., 2004; SOARES, 2004; ALBUQUERQUE et. al., 2005; PITTA, 2005).
REVISÃO BIBLIOGRÁFICA 2 REVISÃO BIBLIOGRÁFICA
2.1 Doenças negligenciadas
Doenças negligenciadas são doenças que não só prevalecem em condições de pobreza, mas também contribuem para a manutenção do quadro de desigualdade, já que representam forte entrave ao desenvolvimento dos países. Como exemplos de doenças negligenciadas, podemos citar: dengue, doença de Chagas, esquistossomose, hanseníase, leishmaniose, malária, tuberculose, entre outras. Segundo dados da Organização Mundial de Saúde (OMS), mais de um bilhão de pessoas estão infectadas com uma ou mais doenças negligenciadas, o que representa um sexto da população mundial (BRASIL, 2010).
Essas doenças têm sido progressivamente marginalizadas por aqueles encarregados pelos programas de pesquisa tanto do setor público quanto do privado, essencialmente porque as pessoas que sofrem de doenças negligenciadas são pobres, e não oferecem um retorno lucrativo suficiente para que a indústria farmacêutica invista em pesquisa e desenvolvimento de novos medicamentos voltados para tais doenças. Fica claro, portanto, que a crise na falta de medicamentos para doenças negligenciadas não chegou às atuais proporções por falta de conhecimento científico, e nem somente pelo hiato entre a pesquisa básica e a pré-clínica. Esta crise é o resultado tanto das insuficientes políticas públicas voltadas para P&D de medicamentos de interesse nacional dos países em desenvolvimento, quanto da falha de mercado, provocada pelo baixo interesse econômico que esses pacientes representam para a indústria (CHIRAC e TORREELE, 2006; MJUR, 2007).
Entre 1975 e 2004, apenas 21 medicamentos (figura 01) foram registrados para doenças tropicais e tuberculose, ainda que estas doenças constituam mais de 11% da carga global de doenças. Durante o mesmo período, 1.535 medicamentos foram registrados para outras doenças (CHIRAC e TORREELE, 2006).
.
Figura 1: Medicamentos desenvolvidos entre 1975 a 2004. Fonte: CHIRAC e TORREELE, 2006.
Um estudo sobre o financiamento mundial de inovação terapêutica para doenças negligenciadas revelou que menos de 5% deste financiamento foram investidos no grupo das doenças negligenciadas, ou seja, doença do sono, leishmaniose visceral, esquistossomose e doença de Chagas, ainda que mais de 500 milhões de pessoas sejam ameaçadas por estas quatro doenças parasitárias (MORAN, 2009).
No intuito de agregar grupos de pesquisa e apoiar atividades de pesquisas científicas, tecnológicas e de inovação (tabela 1), em 2008, foram selecionados projetos para formação e consolidação de institutos nacionais de ciência e tecnologia (INCT), em uma iniciativa do MS, MCT, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Banco Nacional de Desenvolvimento Econômico e Social (BNDES) e das FAP do Amazonas, Minas Gerais, Pará, Rio de Janeiro, São Paulo e Santa Catarina. Entre os INCT que tiveram recursos do MS, existem alguns com grande potencial de contribuição para a temática de doenças negligenciadas e o combate às doenças: INCT em Tuberculose, INCT de Gestão da Inovação em Doenças Negligenciadas, INCT de Biotecnologia Estrutural e Química Medicinal em Doenças Infecciosas e INCT de Vacinas (BRASIL, 2010).
Tabela 1. Grandes editais temáticos na área de doenças negligenciadas.
ANO EDITAL RECURSOS 2003 Rede Tuberculose R$ 1,9 milhões 2004 Dengue R$ 945 mil 2005 Hanseníase R$ 2,5 milhões 2006 Doenças negligenciadasa R$ 17 milhões 2007 Doenças negligenciadas R$ 22 milhões 2008 Rede Malária R$ 15,4 milhões 2009 Rede Dengue R$ 22,7 milhões aDiferentemente do edital de 2008, o edital de doenças negligenciadas de 2006 não incluiu esquistossomose (BRASIL, 2010).
2.2 Esquistossomose
Foi Bilharz (figura 2), em 1852, quem descreveu um parasita intravascular durante uma necropsia em um rapaz, para qual deu o nome de Distomum haematobium e posteriormente em 1858 Weinland denominou o gênero deste helminto de Schistosoma.
A doença é popularmente conhecida como “xistose”, “barriga-d’água” ou “mal-do- caramujo” o qual foi trazida durante o tráfico de escravos e com os imigrantes orientais e asiáticos, entretanto, apenas o S. mansoni se fixou por encontrar bons hospedeiros intermediários e pelas condições ambientais semelhantes às da região de origem (figura 06) (REY, 2001; NEVES, 2005).
De acordo com a Organização Mundial da Saúde existem cinco espécies de Schistosoma que representam risco a saúde publica devido a sua capacidade de infectar humanos (tabela 2).
Figura 2: Theodor Bilharz. Fonte: www.yearofscience.org Tabela 2: Principais espécies que infectam seres humanos, as regiões onde são encontradas e seus hospedeiros intermediários.
Espécies de Schistosoma Regiões Endêmicas Gêneros de caramujo
Esquistossomose intestinal
S. mansoni África, Oriente Médio e América Biomphalaria do Sul
S. intercalatum África (raro, somente no oeste da Bulinus África)
S. japonicum Ásia (primeiramente encontrado
no Japão, hoje ocorre Oncomelania principalmente na China).
S. mekongi Ásia (próximo ao rio Mekongi) Neotricula
Esquistossomose urinária
S.haematobium África e Oriente Médio Bulinus
Fonte: WHO, 2009
As espécies do gênero Schistosoma, que têm importância epidemiológica em medicina humana, são o S. haematobium, S. japonicum, S. mekongi, S. intercalantum e o S. mansoni. Os vermes adultos vivem no sistema porta migrando para as veias mesentéricas causando lesões em vários órgãos (MALTA, 1994; NEVES, 2005). O Schistosoma é um trematódeo da família Schistosomatidae que compreende espécies completamente adaptadas ao parasitismo que se aloja nos vasos sanguíneos. Ao contrário de outros trematódeos, os causadores da esquistossomose não são hermafroditas, ou seja, possuem sexos separados, os machos são achatados e as fêmeas são finas e cilíndricas (figura 03). As espécies diferenciam por sua morfologia, forma de diagnóstico, gêneros do hospedeiro intermediário e localização do parasita no hospedeiro.
A
B
Figura 3: Vermes adultos de Schistosoma. (A) Macho e (B) Fêmea. Fonte: PICASA, 2009.
2.3 Distribuição geográfica e dados epidemiológicos
Apesar do sucesso dos programas de controle, estima-se no mundo 207 milhões de pessoas infectadas (BLANCHARD, 2004) com 120 milhões sintomáticas e 20 milhões com a doença em estado agravado, sendo um total de 650 milhões expostos ao risco de contrair a doença de uma das esquistossomoses (CHITSULO et al., 2000; WHO, 2009) (figura 4). Entre 207 milhões de pessoas com a doença, 85 % vivem na África, onde a grande maioria das fatalidades ocorre devido ao S. haematobium, porém o S. mansoni causa 130.000 mortes por ano, pela hipertensão portal (WHO, 2007). No mundo, existem aproximadamente 70 milhões de infectados pelo S. mansoni (CHITSULO et al., 2004; CIMERMAN, 2001).
Figura 4: Distribuição Mundial da Esquistossomose. Fonte: GRYSSELS et al, 2006.
No Brasil, antes da implantação do Programa Especial de Controle da Esquistossomose (PECE), a esquistossomose atingia entre dez e doze milhões de pessoas, sendo considerada uma das mais importantes áreas de ocorrência da doença (BINA e PRATA, 2003). Há cerca de 7,01 milhões de portadores de esquistossomose mansoni (CHITSULO et al., 2004; CIMERMAN, 2001) (figura 5).
No país, a área endêmica para esquistossomose abrange 19 estados com aproximadamente 26 milhões de habitantes expostos ao risco. Ocorre de forma endêmica e focal desde o estado do Maranhão até Minas Gerais (FUNASA, 2002).
Figura 5: Áreas endêmicas da esquistossomose mansônica no Brasil. Fonte: AMARAL et al., 2006.
A esquistossomose é primariamente uma endemia rural e periurbana que, em algumas cidades como Belo Horizonte, Salvador e Recife estão tornando-se urbana, devido à migração descontrolada e à falta de saneamento básico. No Nordeste vem sendo registrado na Região Metropolitana do Recife, imposta por uma mudança na indústria agro- açucareira que vem promovendo o deslocamento de trabalhadores para a periferia das grandes cidades, onde não há condições básicas de saneamento e moradia, favorecendo o surgimento de novos focos urbanos da doença como os registrados em São Lourenço da Mata e na Ilha de Itamaracá (IGLÉSIAS, 1997; BARBOSA et al. 2000; XIMENES et al., 2000; BERGQUIST, 2002). A esquistossomose é endêmica em 90 dos 185 municípios do Estado de Pernambuco. Com uma área total de 98.933 Km², o Estado possui 19.425 Km² de área esquistossomótica, sendo 16.693 Km² de área endêmica e área focal de 2.732 Km², os quais correspondem a um percentual de 19,63 % da superfície pernambucana. A prevalência média foi de 8,7 % e a média anual de internações, de 1999 a 2003, foi de 377, taxa de 5,12/10 mil internações. O número médio de óbitos, de 1998 a 2002, foi de 139,2 com taxa de mortalidade de 1,76/100 mil habitantes (GGVS/LABEND/ESQ, 2001; BRASIL, 2005). A figura 6 mostra a prevalência da esquistossomose nos municípios da Zona da Mata do estado de Pernambuco, na parte mais clara com prevalência moderada de 10 a 50% e na parte mais escura com prevalência baixa, menor que 10% (BARBOSA et al., 2006), na tabela 3 mostra o número de infecções das cidades da Zona da Mata, destacando-se os municípios de Aliança, Condado, Escada, Itambé, Itaquitinga, Jaqueira, Rio Fomoso e Tracunháem com prevalência alta de número de pessoas infectadas com Schistosoma mansoni.
10%-50% (prevalência moderada) < 10% (baixa prevalência)
Figura 6: Prevalência da esquistossomose dos municípios da Zona da Mata no estado de Pernambuco (BARBOSA et al., 2006).
Tabela 3: Número de infecções das cidades da Zona da Mata do estado de Pernambuco, em destaque as cidades com maior número de infecções.
Município Escolas Examinados %SM GM epg
(BARBOSA et al., 2006)
2.4 Hospedeiro intermediário
Em 1913, foi descrito o hospedeiro intermediário da doença: moluscos do gênero Biomphalaria (figura 7) com concha espiral plana, que podem medir de 10 a 40 mm. Miyaki e Suzuki, autores do feito inédito, demonstraram também que a cercária (larva do Schistosoma) transmite a doença ao homem quando penetra em sua pele.
Figura 7: Caramujo do gênero Biomphalaria. Fonte: www.york.ac.uk, 2009
Os caramujos transmissores da esquistossomose mansoni no Brasil pertencem ao:
Filo Mollusca
Classe Gastropoda
Subclasse Pulmonata
Ordem Basommatophora
Família Planorbidae
Gênero Biomphalaria
Já foram identificadas dez espécies pertencentes ao gênero Biomphalaria no Brasil. São elas: B. glabrata, B. tenagophila, B. straminea, B. amazonica, B. peregrina, B. occidentalis, B. intermedia, B. schrammi, B. oligoza e B. kuhniana. Destas, apenas as três primeiras foram encontradas eliminando cercárias na natureza, sendo, portanto, transmissoras da esquistossomose mansoni nas Américas (BARNES, 1996).
São moluscos de água doce, conhecidos como planorbídeos e, popularmente, como caramujos, têm a concha em espiral, com as voltas ou giros no mesmo plano e, por isso, recebem a denominação de planorbídeo. Os caramujos planorbídeos criam-se e vivem na água doce de corrégos, riachos, valas, alagados, brejos, açudes, represas ou outros locais onde haja pouca correnteza, quando jovens alimentam-se de vegetais em decomposição e de folhas verdes e põem ovos, dos quais, depois de alguns dias, nascem novos caramujos que crescem e tornam-se adultos. O hábitat de preferência do Biomphalaria para colonização é de microflora rica, bastante matéria orgânica, boa insolação, temperatura média da água entre 20°C e 26°C, pH neutro tendendo a alcalino, salinidade abaixo de 3 por 1.000, pouca turbidez e velocidade da água inferior a 30cm/s, com leito raso, lodoso ou rochoso e vegetação enraizada mais próxima das margens (NEVES, 2005).
O B. glabrata é o mais importante transmissor de S. mansoni na região neotropical, não só pelo alto grau de suscetibilidade de suas populações ao parasito como pela extensão de sua distribuição geográfica (CARVALHO, 2008).
2.5 Classificação e morfologia
Os parasitas transmissores da esquistossomose pertencem.
Reino Animalia
Sub-reino Metazoa
Filo Platyhelmintes
Classe Trematoda
Subclasse Digenea
Superfamília Schistosomatoidea
Família Schistosomatidae
Subfamília Schistosomatinae
Gênero Schistosoma (BARNES, 1996).
O S. mansoni macho mede entre 0,6 a 1,4 cm de comprimento por 0,11 cm de largura e sua cor é branca. É largo na posição mediana e afilado nas extremidades do corpo. Na extremidade anterior traz uma ventosa oral afunilada e logo após uma segunda ventosa, a ventral, pedunculada, também denominada de acetábulo e ainda um canal ginecóforo, onde fica alojada a fêmea. O aparelho genital masculino compreende de seis a oito massas testiculares pequenas, situadas dorso ventralmente, no início do segmento posterior. A fêmea tem o corpo cilíndrico comprido e fino medindo cerca de 1,2 a 1,6 cm de comprimento por 0,016 cm de largura, sua cor é acinzentada devido ao tubo digestivo conter um pigmento derivado da digestão sanguínea, hemozoína. Em sua região anterior, existem estruturas especializadas na fixação do verme ao hospedeiro, as ventosas, estando a ventosa acetabular e pedunculada muito perto da oral. O ovário é oblongo e lobado, ficando na metade anterior do corpo. Nos dois sexos, o tubo digestivo se inicia na extremidade anterior, no fundo da ventosa oral, compreendendo um esôfago sem espessamento muscular, o sistema excretor, tem início nos solenócitos, convergindo para dois canais longitudinais que desembocam em uma pequena vesícula excretora (BARNES et al., 1996; REY, 2001) (figura 8).
Figura 8: Segmento anterior do corpo do macho (à esquerda) e da fêmea (à direita), mostrando os principais órgãos: a-ventosa oral e boca, b-porção anterior do intestino, c- acetábulo ou ventosa ventral, d-vesícula seminal, e-canal deferente, f-testículos, g-porção média bifurcada do intestino, h-ceco, i-orifício genital feminino, j-útero contendo dois ovos, k-um ovo em processo de formação da casca no oótipo, l-oviduto, m-ovário, n- viteloduto, o-glândulas vitelinas (REY, 2001).
Após alguns dias de infecção no hospedeiro definitivo, os esquistossômulos chegam ao fígado e alimentam-se de sangue, na terceira semana começa a atração dos sexos por quimiotaxia, iniciando o acasalamento e completam seu desenvolvimento na quarta semana e migram para as vênulas, mesentério e plexo hemorroidário. A nutrição deles é feita através da ingestão de sangue venoso (REY, 2002; NEVES, 2005).
2.6 Morbidade, fisiopatologia e imunopatologia
Embora a parasitose possa resultar em morte, a sua natureza crônica reduz a capacidade das pessoas infectadas para o trabalho. Em crianças, pode causar anemia, nanismo e de uma reduzida capacidade de aprender (WHO, 2009).
A principal causa de mortalidade e morbidade na esquistossomose humana são as fibroses hepáticas, que envolve o espaço portal, devido a isso a resposta granulomatosa e a fibrose hepática vêm sendo intensamente estudadas na patogênese da resposta imune celular do S. mansoni. O granuloma tem um papel hepatoprotetor por sequestrar as toxinas secretadas pelos ovos (PHILLIPS e COLLEY, 1978).
A doença é causada principalmente pela deposição dos ovos dos vermes adultos nos vasos sanguíneos que rodeiam a bexiga ou intestinos. O sinal clássico da esquistossomose urinária é hematúria (sangue na urina). Fibrose ureteral e hidronefrose são achados comuns em casos avançados, e o câncer na bexiga também é uma possível complicação em estágio final. Na esquistossomose intestinal um quadro clínico de dor abdominal, diarréia, sangue nas fezes e fígado aumentado é comum em casos avançados e freqüentemente associada à ascite e outros sinais de aumento da pressão portal. Nesses casos, pode haver também esplenomegalia (LOPES et al., 2008; WHO, 2008).
Diante diso, a formação de granuloma associada a fibrose poderá conduzir a hipertensão portal, uma das maiores causas de mortalidade e morbidade da esquistossomose (LOPES et al., 2008).
As agressões que ocorrem no organismo parasitado pelo S. mansoni são decorrentes das lesões diretas do parasito e da resposta do hospedeiro a tais lesões (PRATA, 1996). A maioria das pessoas infectadas permanece assintomáticas. A sintomatologia clínica corresponde ao estágio de desenvolvimento do parasito no hospedeiro, podendo ser dividida em: a)Dermatite Cercariana, fase de penetração das cercárias através da pele. Varia desde assintomática até uma dermatite urticariforme, com erupção papular, eritema, edema e prurido, podendo durar até cinco dias após a infecção; b)Esquistossomose Aguda caracteriza-se após 3-7 semanas de exposição com febre, anorexia, dor abdominal e cefaléia. Com menor freqüência, o paciente pode apresentar diarréia, náuseas, vômitos, tosse seca. Também conhecida como febre de KATAYAMA (DOHERTY et al., 1996), a esquistossomose aguda é comum em áreas de alta taxa de transmissão, que são mediados por formação de imunocomplexos e na maioria do casos com a deposição de ovos nos tecidos do hospedeiro. Sintomas respiratórios também foram descritos, (70% dos casos) do que em pacientes infectados por S. haematobium (BETHLEM et al., 1997; COOKE et al., 1999). As manifestação inflamatória deve-se ao processo migratório do parasita, que passa pelos pulmões (pequena circulação), antes de atingir o sistema hepático (SCHWARTZ et al., 2000; SALANTRINI et al., 2002; ROSS et al., 2002). c)Esquistossomose Crônica, fase que se inicia a partir dos seis meses após a infecção, podendo durar vários anos. Podendo surgir os sinais de progressão da doença em diversos órgãos, atingindo graus extremos de severidade como hipertensão pulmonar e portal, ascite, ruptura de varizes do esôfago (figura 10) (REESE et al., 1989; REY, 2002; GRYSSELS et al, 2006).
Figura 9 - Fígado sem alterações (A); hepatomegalia com o lóbulo esquerdo aumentado (B), baço com conformações normais (C); esplenomegalia mostrando um crescimento do órgão ultrapassando o ultimo arco costal na altura do umbigo (D). Fonte:medlineplus, 2009.
A esquistossomose humana e experimental é uma doença sistêmica podendo comprometer vários órgãos, como fígado, baço, intestino e outros. Porém, a maioria dos estudos se concentra nas alterações hepáticas e intestinais. Três alterações patológicas merecem mais destaque: presença de granulomas, fibrose de Symmer e alterações vasculares, como hipertensão portal, varizes esofágicas (ANDRADE, 2004). Os granulomas provocados por ovos de Schistosoma mansoni expressam durante o seu desenvolvimento (maturação e involução) vários componentes de matriz extracelular (MEC) (figura 11) e várias moléculas de adesão (CAMs) (figura 12), os quais são especialmente distribuídos em seu interior e sofrem variações (modulações) temporais, em suas expressões. TIPOS DE CÉLULA
Monócitos/Macrófagos Células epitelióides Eosinófilos Neutrófilos Mastócitos Linfócitos (T, B, K) Fibroblastos
Miofibroblastos
COMPONENTES DA MATRIZ EXTRACELULAR
Colágenos Fibronectina Figura 10: Composição celularFibras e de elásticas componentes da matri extraceular em granulomas esquistossómticos hepáticos (ModificadoLaminina a partir de CARVALHO, et al 2008).
MOLÉCULAS DE FATORES DE CRESCIMENTO
ADESÃO
Fator estimulador de colônias (CSF) Integrinas Entropoetina Selectinas Leptina Imunoglobulinas Fator transformador de crescimento Caderina beta (TGF- ) Octudinas Interferon gama (IFN- ) Conexinas
Figura 11: Tipos de moléculas de adesão, fatores de crescimento e seus receptores em granulomas esquistossomóticos (Modificado a partir de CARVALHO, et al 2008). Com a maturação dos ovos, os neutrófilos começam a infiltra-se em torno deles com aproximadamente oito dias de implantação dos ovos. Com o surgimento de uma extensa necrose aparecem células como macrófagos, linfócitos, fibroblastos e eosinófilos entorno da lesão (10 dias). O aumento da reação do tecido chega ao máximo em duas semanas, com necrose dos hepatócitos, rodeados de componentes da matriz extracelular. Em quatro semanas a reposta Th1 diminui e a Th2 aumenta juntamente com as IL-4, IL-5, e IL-13 e com seis semanas a fibrose hepática (HIRATA e FUKUMA, 2003) (figura 13).
0 dia 8 dias 10 dias
2 semanas 4 semanas 6 semanas
Figura 12: Esquema da reação granulomatosa em fígado de camundongo. Fonte: HITARA e FUKUMA, 2003.
O granuloma é um nódulo de tecido inflamatório composto de agregado de macrófagos ativados e de linfócitos T, muitas vezes com associação de necrose e fibrose (figura 14). A inflamação granulomatosa é uma forma de reação de hipersensibilidade tardia (TH), frequentemente em resposta aos microrganismos persistentes. Ele é encontrado num grupo relativamente pequeno de doenças infecciosas e autoimunes, sendo definido como uma área focal de inflamação granulomatosa (WYNN e CHEEVER, 1995).
Hepatócitos
Ovo
Colágeno CD4+ T Eosinófilo Outras Cél Macrófago
Figura 13: Granuloma esquistossomótico. Fonte: PEARCE e MACDONALD, 2002.
O granuloma atinge o seu maior tamanho durante a fase aguda da infecção, diminuindo na fase crônica. Nesse processo são envolvidos vários mecanismos, incluindo alterações nos padrões de citocinas produzidas por células Th1 e Th2 (MONTESANO et al., 1997).
Consiste de um agregado microscópico de células epitelióides rodeadas por um colar de linfócitos e eventualmente alguns plasmócitos. Sua gênese está fortemente relacionada a processos imunes e suas células apresentam, nas preparações de rotina, um citoplasma rosa-pálido, com grânulos, que aparenta se fundir com o citoplasma das células vizinhas. O núcleo é oval ou alongado, menos denso que o de um linfócito e pode apresentar dobras na membrana nuclear. Granulomas de idade ”avançada” desenvolvem uma cápsula formada por fibroblastos e tecido conectivo (NEVES, 2005).
O elemento anatopatológico básico do processo esquistossomótico crônico e sua lesão típica é o granuloma que se forma do ovo do parasita. Os antígenos secretados no interior do ovo maduro atravessam os tecidos e se disseminam nas circunvizinhanças destes, que são chamados de antígenos solúveis do ovo (SEA) que são fundamentais para a formação da reação granulomatosa, ou seja da doença (REY, 2001; NEVES, 2005).
2.7 Ciclo Biológico
O ciclo evolutivo do S. mansoni é do tipo heteroxênico com dois hospedeiros, um intermediário (molusco) e um definitivo (vertebrado). Os ovos do S. mansoni são eliminados pelas fezes do hospedeiro definitivo (figura 9), homem. Na água, estes eclodem, liberando larvas microscópicas, ciliadas, chamadas de miracídio, que completam o seu ciclo vital em apenas 48 horas. Se não encontram um hospedeiro intermediário, essas larvas, ficam incapazes de infestar o homem e morrem. Mas se encontram os hospedeiros apropriados, nele penetram e aí continuam o seu ciclo evolutivo formando esporocistos dando origem a novas larvas que retornam ao meio líquido. Após 4-6 semanas os caramujos começam a liberar as cercarias, elas nadam através de movimentos vibratórios e circulares até encontrar um hospedeiro vertebrado, e penetrar ativamente por sua pele, onde perdem a cauda e transformam-se em esquistossômulos (BLANCHARD, 2004). Aqueles que não são destruídos pelo sistema imunológico do hospedeiro, seguem através da corrente sanguínea, passando pelo coração, pulmões e fígado. Nos vasos do sistema porta-hepático, eles alcançam a fase adulta, acasalam-se e migram para as vênulas da parede intestinal, onde o ciclo começa novamente (ROSS et al, 2002). Um verme adulto vive, em média, 3-5 anos, mas pode alcançar 30 anos. O potencial teórico de reprodução de um par de vermes é aproximadamente de 600 bilhões de esquistossomos (GRYSSELS et al, 2006).
Figura 14: Ciclo de vida do Schistosoma mansoni. Fonte: GRYSSELS et al, 2006.
2.8 Diagnóstico
No diagnóstico clínico, deve-se levar em conta a fase da doença e o histórico do paciente tais como origem, hábitos, contato com água, entre outros. Algumas formas de diagnósticos podem ser aplicadas: exame parasitológico das fezes, biópsia ou raspagem da mucosa retal, testes sorológicos, ultra-sonografia, palpação do fígado e baço. A esquistossomose pode ser confundida com diversas doenças, em função das diferentes manifestações que ocorrem durante sua evolução:
Dermatite cercariana – seu quadro clínico pode ser confundido com manifestações exantemáticas como sarampo, rubéola, escarlatina e dermatites causadas por outros tipos de cercárias de aves aquáticas. Esquistossomose aguda ou toxêmica – o diagnóstico diferencial deve ser feito com outras doenças infecciosas agudas, tais como febre tifóide, malária, hepatite viral anictérica, estrongiloidíase, amebíase, mononucleose, tuberculose miliar e ancilostomose aguda.
Esquistossomose crônica – nessa fase, a doença pode ser confundida com amebíase, estrongiloidíase, giardiase e demais parasitoses, além de outras afecções que cursam com hepatoesplenomegalia, tais como calazar, leucemia, linfoma, salmonelose prolongada, esplenomegalia tropical e cirroses. Para se chegar ao diagnóstico da esquistossomose é muito importante não apenas os resultados laboratoriais mas também os dados epidemiológicos, como história de banhos em águas com caramujos e procedência do doente. A esquistossomose aguda, por exemplo, é mais freqüente em pessoas que não vivem em áreas endêmicas (BRASIL, 2005).
No diagnóstico parasitológico ou exame de fezes pode ser feito utilizando o método de Hoffman, que é uma técnica qualitativa de sedimentação espontânea, e pelo o método de Kato-Katz que é uma técnica quantitativa que determina o número de ovos por grama de fezes indicando a carga parasitária (KATZ et al., 1972).
A biópsia ou raspagem da mucosa retal, apesar de não fazer parte dos exames de rotina, é recomendável em casos suspeitos com parasitológico negativo, além de permitir uma verificação mais rápida do efeito da quimioterapia, entretanto o resultado não significa necessariamente a cura, pois as fêmeas podem não terem sido mortas, mas somente cessado temporariamente a postura de ovos (NEVES, 2005). Os testes sorológicos não possuem sensibilidade/especificidade suficiente para sua aplicação. A ultra-sonografia é um dos diagnósticos mais importantes, principalmente na fase crônica da doença onde são observadas as alterações hepáticas e determinado com precisão o grau de fibrose (REY, 2001; FUNASA, 2003).
2.9 Controle e prevenção quimioterápica
O controle da esquistossomose é um desafio para muitos países endêmicos. Apesar dos esforços para controlar a doença ela continua sendo uma das infecções parasitárias mais prevalentes do mundo e um problema de saúde pública mundial, perdendo apenas para a malária nos países tropicais e subtropicais (SAVIOLI et al., 1997). A nova estratégia coordenada pela Organização Mundial de Saúde (OMS) sobre uso de drogas antihelmintica tornará possível o controle da esquistossomose em comunidades pobres e marginalizados, por conta do baixo custo e segurança dos medicamentos que agora estão amplamente disponíveis (WHO, 2008).
No combate à esquistossomose várias medidas devem ser adotadas como, eliminação sistemática dos hospedeiros intermediários no seu habitat natural, drenagem de lagos e córregos ou pelo combate biológico empregando peixes, a tilápia, que se alimenta desse molusco. No entanto não é uma medida correta vista o desequilíbrio ecológico. Outra medida seria identificar as áreas contaminadas e submetê-las a processos de saneamento. Evitando que os excrementos de pessoas portadoras da doença possam ser carregados pela água das chuvas. As medidas de profilaxia consistem basicamente em medidas preventivas, de fundamental educação sanitária das populações sobre o modo de transmissão da doença. Assim como o tratamento da população infectada (OLIVEIRA, 1997; REY, 2002).
A estratégia para o controle da esquistossomose tem como objetivo reduzir a mortalidade através do tratamento com praziquantel, que é a única droga disponível. Praziquantel tem sido utilizado com sucesso nos últimos 20 anos para a esquistossomose no Brasil, Camboja, China, Egito, Marrocos e Arábia Saudita. Tratamento pelo menos três vezes durante a infância para a prevenção da doença na idade adulta. O recente foco e consenso sobre doenças tropicais negligenciadas, sublinharam que algumas destas doenças podem ser tratadas em larga escala com medicamentos seguros e eficazes, e em intervalos regulares. Tal tratamento é feito individualmente, sem diagnóstico, assim que defini a área hiperendêmica. O Praziquantel é o fármaco utilizado para o tratamento de todas as esquistossomoses, no entanto está havendo falhas em estudos nos laboratórios e no campo (CIOLI e PICA-MATTOCCIA, 2004; RENGANATHAN e CIOLI, 1998).
2.10 Resistência ao Tratamento
A evolução da resistência dos helmintos é decorrente da inexistência de métodos para detectar o seu desenvolvimento. Então, muitos casos não são detectados em estágios iniciais, reduzindo ou mesmo anulando, a possibilidade do processo ser revertido. Para diminuir os níveis de resistência, recomenda-se prosseguir a terapia com cautela ou retirar totalmente o medicamento. Para os portadores da esquistossomose torna-se impossível, pois o Praziquantel representa o único medicamento capaz de combater a doença (BENNETT et al., 1997; CIOLI e PICA-MATTOCCIA, 2004).
Resistência a um determinado fármaco, significa que houve uma redução na sensibilidade após ter sido tratada por ele. Tolerância é uma insensibilidade ao fármaco inata, onde a população não tinha tido contato prévio com o mesmo (DOENHOFF et al., 2002).
O uso de quimioterápicos no controle de doenças infecciosas ao longo do tempo pode ser apontado como fator principal no desenvolvimento de resistências. O uso extensivo e doses exageradas de anti-helmínticos poderá resultar na resistência, afetado o controle das parasitoses. A resistência aos fármacos tem ocorrido em conseqüência do uso abusivo (COLES, 1999; SANGSTER e GILL, 1999).
Os primeiros relatos da resistência ao praziquantel registrados na literatura vieram de um foco no Norte do Senegal na África, onde o fármaco não teria sido eficaz em torno de 18-39 % dos casos (GRYSEELS et al., 1994; STELMA et al., 1995), o qual poderia ter sido devido a sua epidemiologia, ou seja, de acordo com a imunologia do paciente, a carga parasitária, o número de vermes imaturos e a reinfecção (CIOLI e PICA-MATTOCCIA, 2003). A diminuição da susceptibilidade dos esquistossômulos com a administração do Praziquantel foi repentina e a monitoração desse fenômeno deve ser ativa assim como o desenvolvimento de novos fármacos esquistossomicida, visto que ao longo do tempo as conseqüências podem se agravar (DOENHOFF et al., 2002).
2.11 Imunopatologia
A esquistossomose mansônica é basicamente uma doença que decorre da resposta inflamatória granulomatosa que ocorre em torno dos ovos vivos do parasito. Os antígenos são secretados pela membrana interna da casca do ovo maduro, chamada envelope de Von Lichtenberg, que apresenta toda maquinaria de síntese protéica, núcleo próprio, inúmeras mitocôndrias e uma extensa rede de retículo endoplasmático rugoso indicando alto nível de síntese protéica (NEVES, 2005). Os antígenos induzem a uma resposta imunológica humoral e celular que são elementos fundamentais na formação da reação granulomatosa e, portanto da doença. O organismo apresenta múltiplas camadas de defesa, pelas quais o organismo se defende contra a invasão de patógenos. As mesmas podem ser classificadas em: barreiras físicas – que compreendem a pele, a autolimpeza e a microbiota normal do indivíduo; imunidade inata e imunidade adaptativa. A pele é a primeira defesa do corpo humano contra a infecção armando uma barreira resistente e impenetrável de epitélio protegido por camadas de células queratinizadas (PARHAM, 2001).
Quando a barreira física é rompida e os patógenos penetram nos tecidos moles do corpo, entra em ação a imunidade inata, que utiliza mecanismos de reconhecimento molecular geral para detectar a presença de microrganismos e não leva à imunidade prolongada contra aquele patógeno em particular. A resposta imune adaptativa, em contraste, focaliza especificamente o invasor em questão gerando memória imunológica para o mesmo, através da seleção clonal de células imunes para esse patógeno. As células responsáveis por ambas as respostas imunes, inata e adaptativa, são principalmente os leucócitos e as células teciduais relacionadas a eles (BENJAMINE et al., 2002; PARHAM, 2001).
A infecção parasitária pode ser particularizada pela alta resistência dos parasitos às defesas imunes e pela cronicidade de sua ação. Os parasitos são capazes de sobreviver e de replicar-se nos seus hospedeiros, porque são bem adaptados para resistirem às defesas imunes. Resistem à fagocitose, multiplicam-se no interior de macrófagos e, em alguns casos, resistem a lise mediada pelo complemento. Os helmintos, de uma maneira geral, sobrevivem nos tecidos extracelulares e, com freqüência, sua eliminação é dependente de tipos especiais de resposta de anticorpo (ABBAS et al. 2003).
Na infecção pelo S. mansoni tem-se uma resposta Th1 e Th2, o que chamamos de uma dicotomia da resposta imune, antes da oviposição ocorre a resposta Th1 e depois da oviposição ocorre a resposta Th2 (GRYZCH et al., 1991; WYNN et al., 1993; KUBY, 2002; PEARCE e MACDONALD, 2002) (figura 15). E é na resposta Th2 que se tem uma reação granulomatosa de agregados de leucócitos, linfócitos, eosinófilos e macrófagos (PEARCE e MACDONALD, 2002; STADECKER et al., 2004).
Após infecção Aguda Crônica
cercária esquistosômulo vermes adultos ovos
Figura 15: Desenvolvimento da resposta imune na infecção pelo Schistosoma Fonte: PEARCE e MACDONALD, 2002. A resposta Th1 é definida pela produção de interferon gama (INF-γ) e está associada com a imunidade mediada por células, incluindo reações de hipersensibilidade tardia, recrutamento e ativação de macrófagos inflamatórios e leucócitos e respostas citotóxicas que levam a proteção contra microrganismos celulares. Os linfócitos Th1 respondem otimamente aos antígenos apresentados pelas células B, utilizando como co- estimulante o CD80 e produzem ainda IL-2, linfotoxinas e fator de necrose tumoral, não possuindo receptores para a IL-1 e TNF-β (CHTANOVA e MACKAY, 2001).
A resposta Th2 é caracterizada pela produção de IL-4 e IL-5 e está associada com a imunidade humoral, incluindo a ativação de eosinófilos e mastócitos levando a proteção contra microrganismos extracelulares. Os linfócitos Th2 respondem otimamente aos antígenos apresentados pelos macrófagos e células dendríticas mielóides, são especificamente ativados por sinais gerados pelo CD86 e mediante o estímulo do antígeno ou na presença de IL-1, citocina fundamental em seu processo de ativação, produzem IL-4, IL-5, IL-6, IL-10, IL-13 (YANG et al., 2005). O subtipo Th3 foi identificado posteriormente por produzir elevados níveis do fator de crescimento transformante (TGF- , além de IL-4 e IL-10 em quantidades variáveis. As células Th3 apresentam um fenótipo imunossupressor de tolerância oral e autoimunidade em modelos experimentais (SING et al., 1999). Além das células T CD4+, que deram origem ao paradigma Th1/Th2, outros tipos celulares como linfócitos T CD8+, células dendríticas, macrófagos e células NK também produzem citocinas de ambos os tipos, ou seja, tipos 1 e 2 (KIM et al., 2002). Isso levou a divisão do estudo das respostas imunológicas em resposta do tipo Th1 ou Th2 intrinsecamente relacionadas aos tipos de citocinas produzidas pelas diferentes populações celulares (KOURILSKY e TRUFFA-BACHI, 2001).
As interleucinas 5 e 10 desempenham papéis importantes na infecção, quando estão ausentes durante a infecção aguda a IL-5 impede a eosinofilia, mas não tem nenhum efeito sobre o tamanho e a fibrose do granuloma (SHER, 1990). Sugerindo que a IL-5 desempenha um papel secundário na patogênese. Contudo, a IL-10 parece desempenhar um papel na regulação do tamanho do granuloma (FLORES-VILLANUEVA et al., 1996). Este fato levanta indícios de que a IL-10 diminui a quantidade das populações de células Th1 inflamatórias, ou torna-as anérgicas, levando a um desenvolvimento de uma resposta Th2 estável (FLORES-VILLANUEVA et al., 1996; REISER e STADECKER 1996).
As células como os macrófagos possuem funções na imunidade inata e como células efetoras atuam na imunidade adquirida. Os macrófagos que são ativados na resposta Th1 secretam mediadores inflamatórios, como o óxido nítrico (NO) (JORENS et al., 1995; MACMICKING et al., 1997).
Na infecção pelo S. mansoni, a ativação dos macrófagos pelo IFN- e por outros co- fatores induz à produção de NO pela reação enzimática. O NO torna-se uma molécula efetora, causando a morte do verme (JAMES, 1995).
O maior aumento na produção de NO é na oviposição dos vermes adultos, no entanto, com o início de resposta Th2 por parte do hospedeiro, a produção de NO diminui, uma vez que a resposta Th1 foi inibida (BRUNET et al., 1999). Estudos em camundongos infectados deficientes em IL-4 demonstraram, através de uma polarização da resposta Th1, com alta produção de IFN- e NO após a oviposição, que a citocina IL-4 tem grande importância no controle do NO e na produção do granuloma, fazendo assim com que a sobrevida do camundongo aumente (PATTON et al., 2002).
2.12 Imidazolidinas Bioativas As imidazolidinas ou hidantoínas e seus derivados são uma classe de substâncias que despertam grande interesse por apresentarem importantes atividades farmacológicas, tais como anticonvulsivante, antiarrítmico e no tratamento de complicações diabéticas crônicas. Esses compostos também promovem agregação de plaquetas e inibição da aldose redutase. As imidazolidinas possuem um grupo metilênico muito reativo no carbono-5, o que permite a síntese de inúmeros derivados a partir da condensação com aldeídos aromáticos (ROSSI, 2000).
Luttermoser e Bond (1954) a partir de modificações estrutural no anel imidazolidínico, observaram que a 5,5-difenil-imidazolidina-2,4-diona e a 5-(p-clorofenil)- 5-metil-imidazolidina-2,4-diona obtidas (figura 12) apresentavam atividade frente a vermes adultos em S. mansoni de ratos infectados.
O H O H N N
N O N O H3C H H I II
Figura 16: (I) 5,5-Difenil-imidazolidina-2,4-diona e (II) 5-(p-clorofenil)-5-metil- imidazolidina-2,4-diona
A imidazolidina-2,4-diona foi descoberta em 1861 por Bayer durante um trabalho com ácido úrico. Em 1875 Bayer sintetizou a imidazolidina-2,4-diona pela primeira vez através do aquecimento do bromo-acetiluréia com amônia alcoólica, mais tarde Harries e Weiss em 1900 obtiveram a imidazolidina-2,4-diona a partir do éter etílico de glicina e cianato de potássio em presença do ácido clorídrico (ELDERFIELD et al., 1957; FINKBEINER, 1965).
Em 1899, Ruhemann e Cunnington obtiveram derivados do 5–benzilideno imidazolídínicos pelo aquecimento do fenil proprionato de etila com uréia em solução alcoólica na presença de etóxido de sódio, onde substituiu a uréia pela tioúreia e guanidina, obtendo o 5–benzilideno-2-tioxo-imidazolidin-2-ona e o 2-amino-5-benzilideno- imidazolidin-4-ona (figura 13) (JOHNSON et al., 1915). O H O H N N
CH N CH N S NH H H I II
Figura 17: (I) 5-Benzilideno-2-tioxo-imidazolidin-4-ona e (II) 2-amino-5-benzilideno- imidazolidina-2,4-diona
Derivados imidazolidínicos sintetizados por nossa equipe no Laboratório de Síntese e Planejamento de Fármacos ressaltam a contribuição química do núcleo imidazolidínico. Os resultados já obtidos com derivados imidazolidinônicos-3,5-dissubstituídos revelam que essas moléculas são portadores de significante atividade esquistossomicida (OLIVEIRA et. al., 2004; SOARES, 2004; ALBUQUERQUE et. al., 2005; PITTA, 2005). Alguns desses derivados já se encontram patenteados pelo INPI sob o numero de depósito 000362/03, em 02/10/2003, e vários em fase de desenvolvimento (PITTA et. al, 2003).
Atualmente observa-se o aparecimento rápido de cepas resistentes aos fármacos disponíveis Praziquantel e Oxamniquine, sendo este último não mais incluso dentre os medicamentos essenciais (WHO, 2003), porém ainda utilizado na clínica médica em Pernambuco.
Os resultados obtidos em nosso Laboratório de Planejamento e Síntese como os derivados imidazolidínicos frente aos vermes adultos de Schistosoma mansoni (OLIVEIRA et al., 2004; ALBUQUERQUE et al.,2005), nos impulsionou ao planejamento e a síntese de novos derivados 3-benzil-5-benzilideno-4-tioxo-imidazolidin-2-onas e a avaliação da atividade esquistossomicida in vitro e in vivo na busca de novos agentes esquistosomicidas.
2.13 Fármacos esquistossomicidas
Vários fármacos foram desenvolvidos para o combate do Schistosoma, desde do começo do século XX, estas apresentaram graves efeitos colaterais que tornaram a sua utilização terapêutica proibida. A história da quimioterapia esquistossomicida representou sempre um desafio no encontro de um tratamento eficaz e seguro desde o começo do século XX. Foram utilizados vários fármacos para o tratamento da esquistossomose, tais como: Tártaro emético, Niridazol, Lucantone, Hicantone, entre outros (COLES, 2002).
Foram usados na quimioterapia esquistossomicida há aproximadamente 50 anos, mas hoje obsoleto por boas razões. O tártaro emético (tabela 4) foi descoberto acidentalmente em 1918 por Christopherson, observando uma tribo Berbere que estavam sendo tratados com a droga para leishmaniose. Ele percebeu uma interrupção no parasita S. hematobium. A droga foi injetada por via intravenosa e com doses crescentes. As maiores preocupações foram os efeitos colaterais como, diarréias, cólicas, vômitos, rachaduras na pele e até mesmo problema cardiovasculares; sendo mais freqüentes com o uso da terapia e por ser uma droga de uso prolongado. Muitos médicos preferiram dar o antimônio somente aos pacientes hospitalizados. O tártaro emético é eficaz contra as três espécies de Schistosoma o S. haematobium, S. japonicum e S. mansoni com menor índice de eficácia para o S. japonicum (CIOLI et al., 1995).
A emetina (tabela 4) foi usada a partir de 1920 para o tratamento das infecções do S. japonicum e seu uso mais tarde foi estendido para o S. mansoni. Seu efeito era satisfatório, no entanto as doses eram próximas do limite da toxicidade (CIOLI et al., 1995). Logo após introduziu a 2,3 dehidroemetina (tabela 4), que tem as mesmas atividades da emetina, mas com metade da toxicidade e com efeitos colaterais modestos sendo necessário o acompanhamento dos pacientes. Mas a droga ainda é usada para o tratamento da amebíase (CIOLI et al., 1995).
O niridazol (tabela 4) é um derivado do nitrotiazol que foi introduzido em 1964 por Ciba-Geigy com o nome de Ambilhar . A droga recebeu atenção considerável, por ser uma das alternativas adiantadas dos antimoniais e por ter vantagens na administração oral (CIOLI et al., 1995). O composto não é muito eficaz e apresenta diversos inconvenientes sérios, tais como varias doses orais e ocorrência de efeitos colaterais como mutagenicidade, carcinogenicidade, embriotoxicidade, imunossupressão (FONTANILLES, 1969). A experiência clínica diz que o niridazol é mais eficaz em crianças do que em adultos (CIOLI et al., 1995). O metrifonato (tabela 4) é um composto organofosforado, 2,2,2-tricloro-1- hidroxietil dimetil fosfanato, que foi introduzido em 1955 com os nomes de DipterexO ou Dylox3 (LORENZ et al., 1955). Em 1962, um médico belga, Jaques Cerf observou a possibilidade de atividade anticolinesterásica em helmintos e mais tarde observou eficácia para o S. haematobium, no entanto comprovou que ele não tinha atividade frente ao S. mansoni. Hoje, ele não é mais usado contra esse parasita (CIOLI et al., 1995).
O nitrofurano, em 1960, foi relatado alguma atividade para as infecções do S. japonicum em animais e humanos. O furapromidium, derivado do nitrofurano foi usado largamente na China, no entanto verificou-se alta toxicidade o que conduziu o abandono dos nitrofuranos (tabela 4) (CIOLI et al., 1995). Robinson e colaboradores em 1970, demonstraram atividade esquistossomicida em trans-5-amino-3-[2-(5-nitro-2- furil)-vinil]- 1,2,4-oxadiazole, molécula análogas ao niridazol. Observou-se que a toxicidade aguda era baixa, porém mostrou mutações cancerígenas e a substância foi descontinuada para o tratamento das esquistossomíases humana (CIOLI et al., 1995).
Em 1938, descobriu-se um composto ativo chamado de Miracil, outros três compostos desta série foram sintetizados e testados o Miracil B, C e D. O Miracil D estava pronto para ser experimentado clinicamente, logo seus estudos foram interrompidos devido a segunda guerra mundial. Em 1945, foi introduzido na parte clínica com o nome de lucantona (tabela 4) ou 1-[2-(dietilamino)etilamino]-4-metil-9H-tioxant-9-one, onde foi eficaz frente ao S. mansoni e S. haematobium quando administrados oralmente. Devido aos seus efeitos colaterais como problemas no sistema cardiovascular, sistema nervoso central e gastrointestinais, não é mais utilizada (CIOLI et al., 1995).
O hicantona (tabela 4) é análogo do lucantona. O hicantona é ativa frente ao S. mansoni e S. haematobium, porém com baixa atividade nos vermes adultos fêmeas e nas formas imaturas, nos experimentos in vitro (CIOLI et al., 1995).
A atividade anti-helmíntica do amoscanato (tabela 4) ou 4-isotiocianato-4'- nitrodifenilamino foi relatado, em 1976, por Striebel. O fármaco é uma substância cristalina amarela, insolúvel em água e na maioria dos solventes orgânicos (STRIEBEL, 1976). Uma característica atrativa do amoscanato é por sua larga atividade para as doenças como esquistossomíase, filariose e até nematódeos gastrointestinais. Nos experimentos o amoscanato mostrou-se eficaz a todas as principais espécies de Schistosoma. Com apenas uma dose oral, os ratos infectados foram curados, a toxicidade era baixa (CIOLI et al., 1995). No entanto ele não consta na lista modelo de medicamentos essenciais da World Health Organization (WHO, 2003).
O oltipraz (tabela 4) é um derivado do ditiol-tiona, sintetizado em 1976 na indústria Rhone-Poulenc, na França (BARREAU et al., 1977). É um pó vermelho fino, insolúvel na água e solúvel em lipídeos. É ativo frente ao S. mansoni, S. haematobium e S. intercalatum e inativo ao S. japonicum. Os efeitos tóxicos em longo prazo não foram relatados. Os principais efeitos colaterais são náuseas, vômitos, dores abdominais, sonolência (CIOLI et al., 1995).
A tubericidina (tabela 4) ou 7-deazoadenosina é um análogo da purina que pode ser incorporado em ácidos nucléicos do Schistosoma. Seu uso como esquistossomicida foi em 1971, sendo eficaz na cura de ratos infectados com as principais espécies de Schistosoma, nãosendo relatado nenhuma toxicidade aparente (JAFFE et al., 1971; CIOLI et al., 1995).
A atividade antiparasitária da ciclosporina A (tabela 4) foi descoberta, em 1981, por Bueding e colaboradores ao tentar suprimir a formação de granuloma de ratos infectados por S. mansoni. Observaram que ratos tratados tiveram uma redução drástica no número dos vermes e que esta redução era mais pronunciadas ao tratar de infecções imaturas e os mais afetados eram os machos. A ciclosporina A além de atividade contra o Schistosoma tem atividade frente ao plasmódio, leishmania, cestodos, filaria entre outros (CIOLI et al., 1995).
A oxamniquine (tabela 4) e o praziquantel (tabela 4), ambas são administradas por via oral em dose única, que são relativamente bem toleradas, com baixa toxicidade, e apresentam bons percentuais de cura, em torno de 80 % para adultos e de 70 % para crianças abaixo de 15 anos (KATZ, 2004).
Estudos realizados em camundongos infectados experimentalmente por S. mansoni e tratados com o oxamniquine mostraram que os vermes machos são mais susceptíveis, o fármaco produz edema e necrose do tegumento, com posterior aparecimento de lesão em bolhas. O praziquantel, em estudos in vitro, provoca contrações tônicos-clônicas no parasito, com posterior parada na postura de ovos. O praziquantel é ativo contra todas as espécies de Schistosoma que parasitam o homem, ao contrário da oxamniquine, que só é ativa contra o S. mansoni. Sendo esta a razão principal para a retirada da oxamniquine da lista dos medicamentos essenciais da Organização Mundial de Saúde (CIMERMAN, 2001; WHO, 2003).
O praziquantel induz o influxo de cálcio através do tegumento e das células musculares do verme causando contração, demonstrando a presença de sítios sensíveis ao praziquantel. A eficácia do praziquantel foi constatada em estudos realizados na Zona da Mata do Estado de Pernambuco, em 52 povoados, onde a substituição da oxamniquine que foi usada durante dois anos pelo praziquantel usado durante um ano, em pessoas infectadas, resultou em uma diminuição dos índices de infecção. Verificando-se então a melhor eficiência do praziquantel (BECK et al., 2001).
Tabela 4: Principais fármacos esquistossomicidas desenvolvidos no século XX.
ANO NOME COMERCIAL ESTRUTURA
1918 Tártaro emético ou OH OK C O O C H H C O OH O C H Tártaro de antimônio e Sb O Sb H C O H O O C H potássio O H KO O C OH
1920 Emetina
1920 2,3 De-hidroemetina O
N O H H
H H CH2 O HN
O 1945 Lucatona
N 1945 Hicantona O HN
S O
1955 Metrifonato O O P O
OH Cl Cl Cl
1964 Ambilhar ou O 2N O H S N Niridazol N N
CH3 1964 Oxamniquine ou Mansil CH2NHCH CH3 NH
NO2 CH2OH 1970 Praziquantel ou Cestox ou Cisticid N O
N
O
1971 Tubericidina NH2 N
N N HO O
OH HO
1976 Amoscanato
1976 Oltipraz N N
S S H3C S
H3C H C C 1981 Ciclosporina A H C CH H 3 3 CH2 CH H3C CH3 H C CH3 CH H C CH CH2 H3C 3 CH 3 CH2 CH3 H3C N C CO N C C N C CO N C C N CH2 CO O H O H3C CO HCH2C CH H3C N CH H3C N O H O H H 3 O C CH N C C N C C NH C C N CO C H CH3 H CH3 O CH2 CH CH2 CH H3C CH3 CH CH H C CH H3C 3 3 3
OBJETIVOS 3. Objetivos
3.1. Geral
O presente trabalho pretendeu contribuir, no âmbito da Química Medicinal, no desenvolvimento de novos agentes esquistossomicidas 5-arilazo-3-benzil-4-tioxo- imidazolidin-2-onas (LPSF/PT) e 3-benzil-5-benzilideno-4-tioxo-imidazolidin-2-onas (LPSF/RZS).
3.2. Específicos
Síntezar moléculas das séries 5-arilazo-3-benzil-4-tioxo-imidazolidin-2-onas (LPSF/PT) e 3-benzil-5-benzilideno-4-tioxo-imidazolidin-2-onas (LPSF/RZS);
Elucidar e caracterizar as estruturas, através de métodos espectroscópicos convencionais (infravermelho, ressonância magnética nuclear de hidrogênio, ultravioleta e espectrometria de massas) dos compostos sintetizados;
Avaliar a suscetibilidade in vitro do Schistosoma mansoni (cepa BH) frente aos derivados imidazolidínicos;
Analisar o tegumento do parasita através da microscopia eletrônica de varredura;
Avaliar a citotoxicidade, viabilidade celular, óxido nítrico e dosagem de citocinas.
CONCLUSÃO 4 Conclusão
Os derivados imidazolidinicos testados tiveram resultados relevantes quanto a viabilidade dos vermes apresentando uma mortalidade máxima nas doses maiores e medianas, inibição da oviposição dos vermes, alterações tegumentares e descasalamentos. Também houve uma indução significativa do óxido nítrico em quatro compostos analisados LPSF/PT-9 e PT-10; LPSF/RZS-2 e RZS-5.
Na apoptose e na necrose celular os compostos imidazolidinicos apresentaram valores inferiores ao praziquantel, assim como a citotoxicidade dos derivados imidazolidinicos também foi inferior ao praziquantel.
Na microscopia eletrônica de varredura foi observado danos no tegumento dos vermes muito semelhante ao praziquantel, único fármaco de referência para a esquistossomose tendo então os compostos imidazolidinicos como possíveis candidatos a fármacos.
PERSPECTIVAS
5 Perspectivas
Diante de todos os resultados favoravéis: citotoxicidade, viabilidade celular, análise da microscopia eletrônica de varredura, fica claro que os derivados imidazolidínicos são fortes candidatos a fármacos esquistossomicidas, fazendo-se necessários estudos mais aprofundados no que diz respeito a elucidação do mecanismo de ação dos compostos.
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ANEXOS Parasitology Research
Dear Ms. Neves:
It is a pleasure to accept your manuscript entitled "Biological and immunological activity of new imidazolidines against adult worms of Schistosoma mansoni" in its current form for publication in the Parasitology Research.
Thank you for your fine contribution. On behalf of the Editors of the Parasitology Research, we look forward to your continued contributions to the Journal.
Sincerely, Prof. Bill Chobotar Managing Editor, Parasitology Research [email protected]
Biological and immunological activity of new Imidazolidines front of adult worms of Schistosoma mansoni
Juliana Kelle de Andrade Lemoine Neves1 , Sandra Sarinho1, Cristiane Moutinho Lagos de Melo2, Valéria Rêgo Alves Pereira2, Maria do Carmo Alves de Lima1, Ivan da Rocha Pitta1, Mônica Camelo Pessoa de Azevedo Albuquerque3,4, Suely Lins Galdino1 .
1Laboratório de Planejamento e Síntese de Fármacos (LPSF) do Departamento de Antibióticos-UFPE, Pernambuco, Brazil, 2Departamento de Imunologia do Centro de Pesquisas Aggeu Magalhães – CPqAM/FIOCRUZ-UFPE, Pernambuco, Brazil, 3Laboratório de Imunopatologia Keizo Asami LIKA- UFPE, 4 Departamento de Medicina Tropical-UFPE, Pernambuco, Brazil.
Correspondence Address: Juliana K A L Neves, Departamento de Antibióticos, Centro de Ciências Biológicas, Universidade Federal de Pernambuco. Av. Prof. Moraes Rego s/n, Cidade Universitária, 50670- 901 Recife, PE, Brazil. Phone: +55 81-2126-8347; Fax: +55 81 2126-8346. e-mail: [email protected]
ABSTRACT The only available drug for treating schistosomiasis is praziquantel (PZQ), however there are already reports of resistance to treatment, then it is necessary to search for the development of new compounds to combat schistosomiasis. We test new products and LPSF/RZS-2 Imidazolidines LPSF/RZS-5 against adult worms of Schistosoma mansoni. IC50, cytotoxicity, immune response and cell viability tests were also available for these Imidazolidines. In different concentrations ranging from 40-640 µM, the Imidazolidines produced motor abnormalities, unpaired inhibition oviposition and a mortality of 24 hours for the higher concentrations. Although no changes in IFN- and IL-10, LPSF/RZS-2 and LPSF/RZS-5 induced production of nitric oxide and showed similar behavior to praziquantel in the proof of cell death.
Keywords: imidazolidine, Schistosoma mansoni , in vitro, praziquantel
Abbreviations LPSF Laboratório de Planejamento de Síntese de Fármacos PZQ Praziquantel RZS 5-benzylidene-3-benzyl-4-thioxo-imidazolidin-2-one RZS-2 5-(4-chloro-benzylidene-3-(4-nitrebenzyl)-4-thioxo-imidazolidin-2-one RSZ-5 5-(4-fluoride-benzylidene-3-(4-nitrebenzyl)-4-thioxo-imidazolidin-2-one
Introduction
Medicinal Chemistry is an interdisciplinary science that allows rational planning, the synthesis and the structural elucidation of drugs. Aim also the development of pharmacological assays and studies of structure-biological activity. These studies describe the interaction between biological and chemical systems, which allow the development of new compounds and use the chemotherapy as a major alternative to cure various diseases (Monge et al., 2000).
Infectious parasitary diseases remain obstacles to socio-economic development of poor countries. On the other hand, chemotherapy alternatives for treatment of most parasitary disease are very extremely limited. Schistosomiasis, caused by parasites of Schistosoma genus, is an endemic disease affecting, approximately, 76 tropical and subtropical countries, especially Africa, Asian and Latin America. On America and Brazil only occurs S. mansoni specie (Grevelding, 2004, Chitsulo et al, 2000).
The schistosome life cycle is complexes and requires a snail intermediate host and a mammalian definitive host. The infection begins with cercarial penetration of human skin. Following entry into the circulatory system, parasites travel through the lungs and ultimately situate in the liver where they grow, differentiate, mate, and then migrate to the urogenital system or mesenteric veins of the hepatoportal system to commence egg production (Rai et al., 2009).
The reference drug for treatment of schistosomiasis is praziquantel (PZQ), but this drug is inefficient in immature forms and now has reports of resistance in some strains this has worried the world's public health organizations (Ismail et al., 1999, Pica-Mattoccia et al. and Cioli, 2004). Therefore many studies have been testing the effectiveness of new drugs against many schistosomiasis strains.
Imidazolidines and your derivates are substances class that had shown anti-convulsive and antiarrhythmic pharmacological activities (Rossi and Zelnik, 2000). These composites have been used as anti- schistosome agents in previous studies by our group (Oliveira et al., 2004, Albuquerque et al., 2005, Pitta et al., 2006). Here we investigated the action of new imidazolidines derivatives 5-(4-clorobenzilideno)-3-(4- nitrobenzil)-4-tioxo-imidazolidin-2-ona (LPSF/RZS-2) and 5-(4-fluorbenzilideno)-3-(4-nitrobenzil)-4-tioxo- imidazolidin-2-ona (LPSF/RZS-5) against Schistosoma mansoni adult worms. Cellular viability test, cytotoxicity and immunomodutory activity induced by these new composites on immune spleen cells were also available.
Materials and methods
Compounds The new imidazolidines composites of 5-benzylidene-3-benzyl-4-thioxo-imidazolidin-2-one (RZS) were obtained through the synthesis performed at the Laboratorio de Planejamento de Sintese de Farmacos (LPSF), Universidade Federal de Pernambuco - Brazil, which were duly identified by nuclear magnetic resonance of hydrogen, infrared and spectroscopy mass. The praziquantel was purchase from Sigma Chemical Co., (St Louis, MO, USA - lot. 044K1032).
Parasites and intermediary hosts The BH strain (BH - Belo Horizonte, MG, Brazil) of S. mansoni that has been maintained in the Laboratorio de Imunopatologia Keizo Asami-LIKA was used throughout this study. The strain was kept after it had passed through Biomphalaria glabrata molluscs provided by the Department of Tropical Medicine (Universidade Federal de Pernambuco).
Animals used like definitive hosts Were used mice Swiss females, average weight 20±2 at the, were obtained and maintained at the animal facilities of Laboratorio de Imunologia Keizo Asami (LIKA) of the Universidade Federal de Pernambuco, Recife, Brazil. The animals were infected by exposure to a S. mansoni cercarian suspension containing approximately 100 cercarian, using the tail immersion technique (Oliver and Stirewalt, 1952). Animals were housed in a controlled temperature and light environment, and were given water and commercial chow ad libitum. The experiments were approved by the Federal University of Pernambuco’s Animal Experiments Ethics Committee, Process nº.23076.016877/2004-76, in accordance with Law 9.605 Article 32 Decree 3179. Art 17.
Anti-S. mansoni assay For the in vitro test with S. mansoni, LPSF/RZS-2 and LPSF/RZS-5 imidazolidines derivatives were dissolved in 1.6% dimethyl sulphoxide (DMSO) and used in concentrations varying from 32 to 320 µM, which were added to the medium containing the worms after a period of 2 h of adaptation to the culture medium. Duplicates were carried out for each concentration used. The parasites were kept for 5 days and monitored every 24 h to evaluate their general condition: motor activity, alterations in the tegument, mortality rate. The control worms were treated with 1.6% of DMSO in an RPMI 1640 medium.
Animals used for immunological, cytotoxic and cellular viability assays Male BALB/c mice (6 to 8 weeks old) were raised at the animal facilities of the Oswaldo Cruz Foundation (Rio de Janeiro, Brazil) and maintained at the animal facilities of the Aggeu Magalhães Research Center of the Oswaldo Cruz Foundation in Recife, Brazil. All mice were sacrificed and your spleens were removed in accordance with the Oswaldo Cruz Foundation Commission for Experiments with Laboratory Animals (Ministry of Health, Brazil, 0266/05).
Obtaining of spleen cells Spleen cells were obtained according to previous protocol (Pereira et al.,2004). After killing the animal with
CO2 gas, the spleen of each mouse was removed aseptically and placed in Falcon tube containing RPMI 1640 with fetal calf serum (complete medium). In a vertical flow, each spleen was transferred to a Petri dish where they were soaked. The cell suspensions obtained were transferred to Falcon tubes containing approximately 10 mL of incomplete medium by spleen, centrifuged at 4°C, 200 x g for 5 minutes. After discarding the supernatant, distilled water was added to the sediment to promote lysis of red blood cells. The supernatant, containing no cellular debris was collected and centrifuged at 4 °C, 200 xg for 5 minutes. The sediment (containing cells) was resuspended in complete RPMI 1640. An aliquot of each cell suspension was separated, diluted in trypan blue to be quantified in a Neubauer chamber and the cell viability was determined.
In vitro cytotoxicity assay The cytotoxicity of the compounds was determined using BALB/c mice splenocytes (6x105 cells.well-1) cultured in 96-well plates in RPMI 1640 media (Sigma Chemical Co., St. Louis, MO) supplemented with 10% of fetal calf serum (FCS; Cultilab, Campinas, SP, Brazil) and 50 µg.mL-1 of gentamycin (Novafarma, Anápolis, GO, Brazil). Each imidazolidine was evaluated in six concentrations (1, 5, 10, 25, 50 and 100 μg.mL-1), in triplicate on two independent assays. Cultures were incubated in the presence of 3H-thymidine -1 (Amersham Biosciences) (1 μCi well ) for 24 h at 37º C and 5% CO2. After this period, the content of the plate was harvested to determine the 3H-thymidine ([3H]TdR) incorporation using a beta-radiation counter (β- matrix 9600, Packard). The toxicity of the compounds was determined by comparing the percentage of 3H- thymidine incorporation (as an indicator of cell viability) of imidazolidines-treated wells in relation to untreated wells. Non-cytotoxic concentrations were defined as those causing a reduction of 3H-thymidine incorporation below 30% in relation to untreated controls.
Measurement of cytokine levels in splenocytes supernatants Spleen cells were cultured in 24-well plates (TPP) at a density of 106 cells.well-1. Cytokines were quantified in 24 h, 48 h, 72 h and 6 day supernatants from cultures stimulated with ConA (2.5 µg.mL-1) and PHA (5.0 µg.mL-1) mitogens and LPSF/RZS-2 and LPSF/RZS-5 (1 µg.mL-1), or maintained only in culture medium (control). The levels of IL-10 and IFN were measured by sandwich ELISA, according to the manufacturer’s suggested protocols. The monoclonal antibodies used were from Kit OptEIA (BD Biosciences), being previously titled. Plates with 96 wells (Nalge Nunc International Corporation) were sensitized with specific anti-cytokine antibodies (according to the manufacturer’s instructions) and incubated “overnight” at 4 °C. Cytokine standards were added after serial dilution from their initial concentration (16000 pg.mL-1). After washes, 50 l of all samples and standards were added in duplicate and the plate incubated for 2 hours at room temperature. Subsequently, the specific antibodies were combined with biotin (according to the manufacturer’s instructions) and incubated for 1h30min at room temperature. Revealer solution was added containing 2.2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS). Reaction was blocked with 1 M sulphuric acid and the reading was carried out in a spectrophotometer (Bio-Rad 3550, Hercules, CA) at 415 nm. Sample concentrations were calculated in the linear region of the titration curve of cytokine standards, and final concentrations were expressed in pg/ml, using the Microplate Manager Version 4.0 software (Bio-Rad laboratories).
In vitro nitrite analysis Mice spleen cells were used to evaluate the concentration of nitrite, while treated with Con A (2,5 µg.mL-1), PHA (5.0 µg.mL-1), LPSF/RZS-2 and LPSF/RZS-5 (1, 10 and 100 µg.mL-1), and after 24 h, 48 h, 72 h and 6 days of incubation. Culture media were carefully collected for subsequent measurement by colorimetric Griess method (Ding et al., 1988). NO concentration was estimated by the standard curve (3.12 - 100 µmol.mL-1).
Analysis of cellular viability using Annexin V-FITC and Propidium iodide staining Immune spleen cells were treated with Praziquantel (PZQ) or LPSF/RZS-2 and or LPSF/RZS-5 at 1 µg.mL-1 concentration, respectively. These treated cells were maintained in culture in 24-well plates (TPP) for 24, 48 and 72 hours for analyze the cellular viability these cells after this 3 times of assay. Cells without treatment (control) were used like negative control. After this, lymphocytes were centrifuged at 4°C, 450 xg for 10 minutes. After discarding the supernatant, the sediment was added 1 mL of PBS 1X and were centrifuged at 4°C, 450 xg for 10 minutes. After discarding the supernatant, the pellet was resuspended in a binding buffer (10 mM HEPES (pH 7.4), 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, and 1.8 mM CaCl2) and was added annexin V conjugated with fluorescein isothiocyanate (FITC) (1:500) and propidium iodide (PI, 20 µg.mL-1; 106 cells) in each identified cytometer tube. Flow cytometry was performed on a FACSCalibur (Becton Dickinson Biosciences, Mountain View, Ca, USA) analyzed using Cell Quest Pro software (Becton Dickinson). Results analysis was performed on graphs by dot plot. Double positive (Annexin-FITC+/PI+) were considered spleen cells in the late stage of apoptosis, whereas only PI+ cells were necrotic cells. Annexin-FITC+/PI- were represented splenocytes in the early stage of apoptosis. Double negative was considered viable cells.
Statistical analysis Data were analyzed using non-parametric tests. To detect differences between groups, the Mann- Whitney U test and tukey t test were used. All results were expressed by mean values of groups standard deviation and were analyzed considering the value of p < 0.05 (statistically significant).
Results PZQ citotoxity was superior to imidazolidines derivatives and anti-S. mansoni Many techniques are utilized to elucidate cytotoxic mechanisms induced by drugs on different cells. The present study used Tritiate Timidine ([3H]TdR) assay, on immune spleen cells by BALB/c mice, given that the timidine acts through the incorporation of tritium into the DNA of cells and is the most commonly utilized technique today for cytotoxity detection in immune cells (Pechhold et al., 2002). Praziquantel was used like standard drug (positive control) and cells without treatment like negative control. Results did show that LPSF/RZS-2 and LPSF/RZS-5 presented nontoxic effects on 5 and 1 µg.mL-1 concentrations (Table 1). On the other hand praziquantel showed higher toxicity on splenocytes on all tested concentrations (Table 2). LPSF/RZS-2 and LPSF/RZS-5 also were tested against S. mansoni adult worms and results were expressed in terms of the IC50 (µM) values. The imidazolidines derivatives show activity against adult worms of S. mansoni. LPSF/RZS-2 induced 100% of mortality at 640 μM concentration on first 48 hours (Table 1). After 96 hours LPSF/RZS-2 did show similar behavior to Praziquantel and induced 100% of mortality on all concentrations (Table 2). LPSF/RZS-5 on first 24 hours induced 100% of mortality by adult worms for majority concentrations and after 48 hours did not observed any activity by worms (Table 1). Many physiological alterations were observed on adult worms exposure to imidazolidines and praziquantel drugs (see Tables 1 and 2). Oviposition by adult worms also did not was observed on any concentration of these imidazolidines and control group (worms without treatment) remained viable during all the time of observation.
LPSF/RZS-2 and LPSF/RZS-5 imidazolidines did not produced IFN- and IL-10 cytokines
For immunological assays we investigated IL-10 and IFN- cytokines and nitric oxide production on supernatants of cultures by splenocytes stimulated in vitro with LPSF/RZS-2 and LPSF/RZS-5 at 1 µg.mL-1 concentrations. Because yours immunological properties Phytohemagglutinin (PHA at 5.0 µg.mL-1) and Concanavalin A (Con A at 2.5 µg.mL-1) mitogens were used like positive controls and without stimulus (cells + medium) was used like negative control. Although higher and statistical values were produced for Con A and PHA mitogens on all experimental times of cultures (data not show), neither experimental imidazolidines tested were efficient on IL-10 and IFN- production on supernatants of cultures.
LPSF/RZS-2 and LPSF/RZS-5 induces higher values of nitrite production
The effects in the NO-induced production in murine spleen cells was performed on in vitro cultures treated with LPSF/RZS-2 and LPSF/RZS-5 at 100, 10 and 1 µg.mL-1 concentrations. At 24 hours LPSF/RZS- 2 and LPSF/RZS-5 on 10 and 1 µg.mL-1 concentrations did show higher and significant values in relation to control (Figure 1A). At this same time LPSF/RZS-5 at 1 µg.mL-1 concentration was statistically superior to LPSF/RZS-2 on 10 and 1 µg.mL-1 concentrations (Figure 1A). After 48 hours was observed that only LPSF/RZS-2 was statistically superior to control at 10 µg.mL-1 concentration (p < 0.04). LPSF/RZS-5 did not show significant values at this time. The higher values of nitrite production induced by imidazolidines composites were observed at 72 hours of treatment. LPSF/RZS-2 was statistically superior to control at 100 and 1 µg.mL-1 concentrations and LPSF/RZS-5 was also superior to control at 100, 10 and 1 µg.mL-1 concentrations (Figure 1B). The last time of culture, i.e., 6th day, LPSF/RZS-2 and LPSF/RZS-5 did show similar behavior and was statistically superior to control only at 10 µg.mL-1 concentration (p < 0.001).
Low cellular death induced by LPSF/RZS-2 and LPSF/RZS-5 imidazolidines Cellular viability was realized for measure cellular damage induced by imidazolidines derivatives and for all compounds were used 1 µg.mL-1 concentration. On 24 hours of assay, PZQ showed higher and statistically values in relation to LPSF/RZS-2 and LPSF/RZS-5 to cell death by apoptosis and was superior to LPSF/RZS-2 by necrosis. But, these values were not significant in relation to control. PZQ showed higher values of cellular death in relation to imidazolidines and control at 48 hours. At this same time LPSF/RZS-5 was statistically superior to LPSF/RZS-2 for both cellular death indices. At 72 hours LPSF/RZS-2 and LPSF/RZS-5 were superior to control on apoptosis. On necrosis PZQ and RZS-5 demonstrated higher values in relation to control and LPSF/RZS-5 was superior to LPSF/RZS-2. All results of cellular viability test were represented on Figure 2A and B.
Discussion LPSF/RZS-2 and LPSF/RZS-5 were less cytotoxic and induced superior mortality of adult worms in relation to Praziquantel. But in relation to absence of oviposition by adult worms and motor abnormalities imidazolidines were similar to PZQ. In fact, PZQ at low concentrations, in vitro, appears to impair the function of the worms’ suckers and at higher concentrations increases the contraction (irreversibly at very high concentrations) of the worm’s strobilla (chain of proglottids) (Xiao et al., 1985; Cioli and Pica- Mattoccia, 2003), causes irreversible focal vacuolization with subsequent cestodal disintegration at specific sites of the cestodal integument (Martin, 1997; Badreldin, 2006). These same physiological alterations were observed on adult worms treated with imidazolidines here and in others studies realized by our group (Albuquerque et al., 2002; Soares et al., 2004). Many drugs have been compared with praziquantel and its cytotoxicity and ability to kill adult worms of S. mansoni has been shown equivalent to reference drug (Dayan, 2003; Sayed, 2008). However, similar to praziquantel, the mechanism by which the imidazolidines exerts schistosomicide activity in vitro is still unclear. So far, the main activities assigned for imidazolidines are antibacterial, antiamebic, anti-T. cruzi and anti-schistosome activities (Kalyaham et al., 1992; Sharma and Khan, 2001; Pitta et al; 2006; Caterina, 2008). Imidazolidines also show activity on apoptotic cells with melanoma, your route was identified in S. mansoni (Dubois et al., 2009) and has been suggest a possible activity at the cholinergic level receptors, since the nervous system of S. mansoni, with unique pharmacological and physiological characteristics, can be used in the research of compounds for human and animal use. Many studies have shown Th1 and Th2 response balance on schistosomotic patients. Furthermore, these same studies strongly suggest that resistance to infection is multifactorial and that it can not be clearly correlated with a single immune mechanism. Here, imidazolidines derivatives did not induce significant production of IFN- neither IL-10, but the potent anti-schistosomotic agent, the nitric oxide, was strongly stimulated. Against parasitosis, there is ample direct and indirect evidence that NO can act as an antischistosomal and, more broadly, antiparasitic molecule (Brunet, 2001; Colasanti, 2002; Rivero, 2006). NO produced by human white cells has been shown to kill larval schistosome parasites (James and Glaven, 1989) and is a molecule which has been shown to kill a multitude of eukaryotic and prokaryotic organisms and against which no other defense mechanism has, to our knowledge, been reported (Brophy and Pritchard, 1992; Ahmed et al., 1992). In addition, studies have shown that compounds that induce nitric oxide release possess potential immunomodulatory properties (Curtin, 2002; Korhonen, 2008). Here, we observe that imidazolidines induced nitric oxide production and this behavior can be indicate the possible immunostimulant activity induced by imidazolidines derivatives. Many drugs have been shown damage on cell morphology on in vitro assays. (Malheiros et al, 2000) using trifluoperazine (TFP), dibucaine (DBC) and Praziquantel observed cytotoxic effects, as haemolysis and release of membrane lipids, on human erythrocyte membranes, in a dose-dependent mechanism. Here, on cellular death test, we observed that PZQ showed higher values on all the experimental times and on some results was superior to imidazolidines when tested on immune spleen cells. But the behavior among experimental composites was very similar indicating that it is possible the activation of similar routes in the induction of cell death among the compounds evaluated and PZQ drug reference. However, more assays are needed to answer this question. The chemical groups introduced on any substance are important because can be essential for manifestation and intensity of many biologic action, in consequence of effect induced by biofunctional groups (Korolkovas, 1974). Here, we observe structural similarity by imidazolidines synthesized with niridazole (Ambilhar®), chemically named 1-(5-nitro-tiofeno-3-il)-imidazolidin-2-ona. Imidazolidines shows the same pharmacoforic group of niridazole and present HC=C-N and C-N=N-C-N substitutions on position 5 of the imidazolidine ring Chand and cols. (2005) observed that C-N=N-C-N can be acts like kelant agents because the nitrogen of imidazolidine and the nitrogen of arilaze groupment acts like donor centers. The biofunctional groups of imidazolidines was by alkyl radicals, chlorine atoms (LPSF/RZS-2) and fluoride (LPSF/RZS-5). These radicals can be changed the basicity and or lipophilicity of drugs, inducing, changes in interactions with the receptor sites and in the absorption phenomena and transport through different cellular compartments. We believe that this characteristic may be responsible for better biological response of imidazolidine derivatives. LPSF/RZS-2 and LPSF/RZS-5 imidazolidines derivatives were less cytotoxicity that Praziquantel, showed relevant antischistosomal activity in vitro, induced higher nitric oxide production by immune spleen cells and showed similar behavior to praziquantel on cellular death test. Thus is possible that these new imidazolidines derivatives can be future candidates to schistosomotic drugs.
Conclusions The results obtained with derivatives LPSF/RZS-2 and LPSF/RZS-5 suggest that the best contribution of these compounds were due to the presence of the nitro group, which acts as a group parasitophorous. Another important contributor to activity schistosomicide are the halogens chlorine and fluorine, which have the maximum effect in a position para the aromatic ring. The sum of these effects were observed in derivatives with a mortality of 100% in most concentrations. Despite having produced no toxic effects and produce cytokines IFN and IL-10, they also showed production of nitric oxide statistically significant.
Acknowledgements This work was supported by grants from the Financiadora de Estudos e Projetos (FINEP), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).
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concentration (μM)
640 320 160 80 40 a Observation Cytotoxicity c time Mortality (%) IC50 (μM) (worms) (µg.mL-1)b 24 h 80 73 42 0 0 158 48 h 100 85 64 0 0 tegument morphology altered, 145 LPSF/RZS-2 72 h 100 85 100 74 0 not paired, no sucker 5 75 96 h 100 100 100 100 26 adherence, absence of eggs 65 120 h 100 100 100 100 38 65
concentration (μM)
640 320 160 80 40
Observation Cytotoxicity b time Mortality (%) IC50 (μM) (worms) (µg.mL-1)a 24 h 100 100 100 100 34 65 tegument morphology altered, 48 h 100 100 100 100 95 NDd not paired, no sucker LPSF/RZS-5 72 h 100 100 100 100 100 5 ND adherence, absence of eggs, 96 h 100 100 100 100 100 ND appearance of bubbles 120 h 100 100 100 100 100 ND a Control group paired adults worms without apparent morphological change, presence of eggs. b Expressed as the highest concentration tested that was not cytotoxic for the BALB/c mice splenocytes. Values in µM are showed in parentheses. c Calculated at five concentrations using data obtained from at least three independent experiments, with a SD less than 10% in all cases. d ND- no determined Table 2: In vitro effects of Praziquantel against Schistosoma mansoni adult worms
concentration (μM)
80 40 20 10
Observation a Cytotoxicity c time Mortality (%) IC50 (μM) (worms) (µg.mL-1)b 24 h 60 45 36 30 37,02 48 h 66 56 50 45 35,08 lower movements and contractions, PZQ 72 h 76 67 57 53 <1 33,91 absence of eggs 96 h 100 100 100 92 15,36 120 h 100 100 100 92 15,36 a Control group paired adults worms without apparent morphological change, presence of eggs. b Expressed as the highest concentration tested that was not cytotoxic for the BALB/c mice splenocytes. Values in lM are showed in parentheses. c Calculated at seven concentrations using data obtained from at least three independent experiments, with a SD less than 10% in all cases.
Figure 1: Nitrite production on immune spleen cell cultures of BALB/c mice comparing experimental imidazolidines. A – 24 hours of assay. LPSF/RZS-2 and LPSF/RZS-5 on 10 and 1 µg.mL-1 concentrations did show higher values in relation to control and LPSF/RZS-5 at 1 µg.mL-1 concentration was statistically superior to LPSF/RZS-2 on 10 and 1 µg.mL-1 concentrations. B – 72 hours of assay. LPSF/RZS-2 was statistically superior to control at 100 and 1 µg.mL-1 concentrations and LPSF/RZS-5 was also superior to control at 100, 10 and 1 µg.mL-1 concentrations. Horizontal bars represent the average of three independent experiments per group. p < 0.001.
Figure 2: Imidazolidines derivatives effect on immune spleen cells viability. A - apoptosis investigation utilizing annexin V at 24, 48 and 72 hours of assay. At 24 hours PZQ was superior to LPSF/RZS-2 and LPSF/RZS-5 on apoptosis and superior to LPSF/RZS-2 to necrosis, but not in relation to control. PZQ was statistical superior to control, LPSF/RZS-2 and LPSF/RZS-5 at 48 hours. LPSF/RZS-5 was statistically superior to LPSF/RZS-2 at same time. At 72 hours LPSF/RZS-2 and LPSF/RZS-5 were superior to control, but not among them. B - Necrosis assay using propidium iodide at 24, 48 and 72 hours of assay. PZQ demonstrated higher quantity necrotic cells in relation to control, LPSF/RZS-2 and LPSF/RZS-5 at 48 hours. LPSF/RZS-5 was also statistically superior to LPSF/RZS-2 at same time. At 72 hours PZQ and LPSF/RZS-5 were superior to control and LPSF/RZS-5 did show higher values in relation to LPSF/RZS-2. Horizontal bars represent the average of three independent experiments per group. p < 0.05. PARASITOLOGY RESEARCH
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Immunological studies and in vitro schistosomicide action of new
imidazolidine derivatives
Juliana Kelle de Andrade Lemoine Neves1, , Sandra Sarinho1, Cristiane
Moutinho Lagos de Melo2, Valéria Rêgo Alves Pereira2, Maria do Carmo Alves de Lima1, Ivan da Rocha Pitta1, Mônica Camelo Pessoa de Azevedo
Albuquerque3,4, Suely Lins Galdino1
1Laboratório de Planejamento e Síntese de Fármacos (LPSF) do
Departamento de Antibióticos-UFPE, Pernambuco, Brazil, 2Departamento de
Imunologia do Centro de Pesquisas Aggeu Magalhães – CPqAM/FIOCRUZ-
UFPE, Pernambuco, Brazil, 3Laboratório de Imunopatologia Keizo Asami LIKA-
UFPE, 4 Departamento de Medicina Tropical-UFPE, Pernambuco, Brazil.
Correspondence Address: Juliana K A L Neves, Departamento de
Antibióticos, Centro de Ciências Biológicas, Universidade Federal de
Pernambuco. Av. Prof. Moraes Rego s/n, Cidade Universitária, 50670-901
Recife, PE, Brazil. Phone: +55 81-2126-8347; Fax: +55 81 2126-8346. e-mail: [email protected]
ABSTRACT
Schistosomiasis remains a major public health problem with 207 million people infected and further 779 million at risk. The reference drug for treatment of schistosomiasis is praziquantel (PZQ), but this drug is inefficient in immature forms. Aim of this study was testing new imidazolidines derivatives LPSF/PT09 and LPSF/PT10 against adult worms of Shistosoma mansoni. IC50, cytotoxicity, immune response and cell viability assays were also available for these imidazolidines. At different concentrations ranging from 32 to 320 M, imidazolidines was promoted motor abnormalities in breeding and not paried of worms and a mortality of 24 hours for higher concentrations. Despite not having changes in IFN- and IL-10, LPSF/PT09 and LPSF/PT10 induced the nitric oxide production and showed similar behavior to praziquantel on cellular death test. Thus is possible that these new imidazolidines derivatives can be future candidates to schistosomotic drugs.
Key words: Shistosoma mansoni, in vitro, imidazolidine.
1.INTRODUCTION
Parasites comprise an important group of human pathogenic organisms affecting the lives of approximately 2 billion people mostly in developing countries in the tropics and sub-tropics [1]. Schistosomiasis remains a major public health problem with 207 million people infected and further 779 million at risk. The disability caused by this chronic helminthic infection was recently reassessed in order to correctly represent its high impact on public health, previously underestimated [2, 3, 4].
Immunological studies have been describes that in mice resistance to schistosome reinfection has been correlated mainly with a Th1 response while pathology correlates to a Th2 response [5]. Cytokines like IL-4, IL-13, TNF- and IFN- are closely related with schistosomiasis disease and are prevalent on schistosome-associated fibrosis [6]. IL-10 has been demonstrated to correlate with the control of morbidity in humans [7] and nitric oxide produced at higher levels by macrophages, has cytotoxic activity against parasites and exerts an important function in immune system modulation on parasitosis [8].
So far, the reference drug for treatment of schistosomiasis is praziquantel
(PZQ), but this drug is inefficient in immature forms and now has reports of resistance in some strains this has worried the world's public health organizations [9,10]. Therefore many studies have been testing the effectiveness of new drugs against many schistosomiasis strains [11,12] .
Imidazolidines and your derivates are substances class that had shown anti-convulsive and antiarrhythmic pharmacological activities. Furthermore, the imidazolidines have a methylenic group very reactive on carbone-5, that to allow the synthesis of many derivate through aromatic aldehyde condensation
[13].
Imidazolidines have been used as anti-schistosome agents in previous studies by our group [14,15,16]. Here we investigated the action of new imidazolidines derivatives 5-(4-cloro-arilazo)-3-(4-cloro-benzil)-4-tioxo- imidazolidin-2-ona (LPSF/PT-09) and 3-(4-cloro-arilazo)-5-(4-cloro-benzil)-4- tioxo-imidazolidin-2-ona (LPSF/PT-10) against Schistosoma mansoni adult worms. Cellular viability test, cytotoxicity and immunomodutory activity induced by these new composites on immune spleen cells were also available.
2. MATERIALS AND METHODS
2.1.Compounds
Derivatives imidazolidinicos 5 - (4-chloro-arilazo) -3 - (4-chloro-benzyl)-4- thioxo-imidazolidin-2-one (LPSF/PT-09) and 3 - (4-chloro-arilazo) -5 - (4-chloro- benzyl)-4-thioxo-imidazolidin-2-one (LPSF/PT-10) were obtained through the synthesis performed at the Laboratorio de Planejamento de Sintese de
Farmacos (LPSF), Universidade Federal de Pernambuco - Brazil, which were duly identified by nuclear magnetic resonance of hydrogen, infrared and spectroscopy mass. The praziquantel was purchase from Sigma Chemical Co.,
(St Louis, MO, USA - lot. 044K1032).
2.2. Parasites and intermediary hosts
The BH (BH - Belo Horizonte, MG, Brazil) strain of S. mansoni that has been maintained in the Laboratorio de Imunopatologia Keizo Asami-LIKA was used throughout this study. The strain was kept after it had passed through Biomphalaria glabrata molluscs provided by the Department of Tropical
Medicine (Universidade Federal de Pernambuco).
2.3. Animals used like definitive hosts
Were used mice Swiss females, average weight 20±2 at the, were obtained and maintained at the animal facilities of Laboratorio de Imunologia Keizo Asami
(LIKA) of the Universidade Federal de Pernambuco, Recife, Brazil. The animals were infected by exposure to a S. mansoni cercarian suspension containing approximately 100 cercarian, using the tail immersion technique [17].
Animals were housed in a controlled temperature and light environment, and were given water and commercial chow ad libitum. The experiments were approved by the Federal University of Pernambuco’s Animal Experiments
Ethics Committee, Process nº.007639/2007-12, in accordance with Law 9.605
Article 32 Decree 3179. Art 17.
2.4. Anti-S. mansoni assay
For the in vitro test worms were maintained in a RPMI-1640 medium (Sigma
Chemical Co., St. Louis, MO) buffered to pH 7.5, supplemented with HEPES
(20 mM), 10% foetal bovine serum, penicillin (100U.mL-1), and streptomycin
(100 µg.mL-1). Incubation was carried out at 37°C in a humid atmosphere containing CO2 5%. LPSF/PT-9 and LPSF/PT-10 imidazolidines derivatives were dissolved in 1.6% dimethyl sulphoxide (DMSO) and used in concentrations varying from 32 to 320 µM which were added to the medium containing the worms after a period of 2 h of adaptation to the culture medium.
Duplicates were carried out for each concentration used. The parasites were kept for 5 days and monitored every 24 h to evaluate their general condition: motor activity, alterations in the tegument, mortality rate. In the PZQ group used the same methodology, but the concentration ranged from 10 to 80 µM. The control worms were treated only DMSO in RPMI 1640 medium.
2.5. Animals used for immunological, cytotoxic and cellular viability assays
Male BALB/c mice (6 to 8 weeks old) were raised at the animal facilities of the
Oswaldo Cruz Foundation (Rio de Janeiro, Brazil) and maintained at the animal facilities of the Aggeu Magalhães Research Center of the Oswaldo Cruz
Foundation in Recife, Brazil. All mice were sacrificed and your spleens were removed in accordance with the Oswaldo Cruz Foundation Commission for
Experiments with Laboratory Animals (Ministry of Health, Brazil, 0266/05).
2.6. Obtaining of spleen cells
Spleen cells were obtained according to previous protocol [18]. After killing the animal with CO2 gas, the spleen of each mouse was removed aseptically and placed in Falcon tube containing RPMI 1640 with fetal calf serum (complete medium). In a vertical flow, each spleen was transferred to a Petri dish where they were soaked. The cell suspensions obtained were transferred to Falcon tubes containing approximately 10 mL of incomplete medium by spleen, centrifuged at 4°C, 200 x g for 5 minutes. After discarding the supernatant, distilled water was added to the sediment to promote lysis of red blood cells.
The supernatant, containing no cellular debris was collected and centrifuged at
4 °C, 200 x g for 5 minutes. The sediment (containing cells) was resuspended in complete RPMI 1640. An aliquot of each cell suspension was separated, diluted in trypan blue to be quantified in a Neubauer chamber and the cell viability was determined.
2.7. In vitro cytotoxicity assay
The cytotoxicity of the compounds was determined using BALB/c mice splenocytes (6 x 105 cells.well-1) cultured in 96-well plates in RPMI 1640 medium (Sigma Chemical Co., St. Louis, MO) supplemented with 10% of fetal calf serum (FCS; Cultilab, Campinas, SP, Brazil) and 50 µg.mL-1 of gentamycin
(Novafarma, Anápolis, GO, Brazil). Each imidazolidine was evaluated in six concentrations (1, 5, 10, 25, 50 and 100 μg.mL-1), in triplicate on two independent assays. Cultures were incubated in the presence of 3H-thymidine
-1 (Amersham Biosciences) (1 μCi.well ) for 24 h at 37º C and 5% CO2. After this period, the content of the plate was harvested to determine the 3H-thymidine
([3H]TdR) incorporation using a beta-radiation counter (β-matrix 9600, Packard).
The toxicity of the compounds was determined by comparing the percentage of
3H-thymidine incorporation (as an indicator of cell viability) of imidazolidines- treated wells in relation to untreated wells. Non-cytotoxic concentrations were defined as those causing a reduction of 3H-thymidine incorporation below 30% in relation to untreated controls. In praziquantel were also used six concentrations (1, 5, 10, 25, 50 and 100 μg.mL-1).
2.8. Measurement of cytokine levels in splenocytes supernatants
Spleen cells were cultured in 24-well plates (TPP) at a density of 106 cells.well-1.
Cytokines were quantified in 24 h, 48 h, 72 h and 6 day supernatants from cultures stimulated with Con A (2.5 µg.mL-1) and PHA (5 µg.mL-1) mitogens and
PT-9 and PT-10 (1 µg.mL-1), or maintained only in culture medium (control). The levels of IL-10 and IFN were measured by sandwich ELISA, according to the manufacturer’s suggested protocols. The monoclonal antibodies used were from Kit OptEIA (BD Biosciences), being previously titled. Plates with 96 wells
(Nalge Nunc International Corporation) were sensitized with specific anti- cytokine antibodies (according to the manufacturer’s instructions) and incubated
“overnight” at 4°C. Cytokine standards were added after serial dilution from their initial concentration (16000 pg.mL-1). After washes, 50 l of all samples and standards were added in duplicate and the plate incubated for 2 hours at room temperature. Subsequently, the specific antibodies were combined with biotin
(according to the manufacturer’s instructions) and incubated for 1h30min at room temperature. Revealer solution was added containing 2.2-azino-bis (3- ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS). Reaction was blocked with 1 M sulphuric acid and the reading was carried out in a spectrophotometer (Bio-Rad 3550, Hercules, CA) at 415 nm. Sample concentrations were calculated in the linear region of the titration curve of cytokine standards, and final concentrations were expressed in pg.mL-1, using the Microplate Manager Version 4.0 software (Bio-Rad laboratories).
2.9. In vitro nitrite analysis
Mice spleen cells were used to evaluate the concentration of nitrite, while treated with Con A (2,5 µg.mL-1), PHA (5 µg.mL-1), LPSF/PT-9 and LPSF/PT-10
(1, 10 and 100 µg.mL-1), and after 24 h, 48 h, 72 h and 6 days of incubation.
Culture media were carefully collected for subsequent measurement by colorimetric Griess method [19]. NO concentration was estimated by the standard curve (3.12 - 100 µmol.mL-1).
2.10. Analysis of cellular viability using Annexin V-FITC and Propidium iodide staining
Immune spleen cells were treated with Praziquantel (PZQ) or LPSF/PT-9 and or
LPSF/PT-10 at 1 µg.mL-1 concentration, respectively. These treated cells were maintained in culture in 24-well plates (TPP) for 24, 48 and 72 hours for analyze the cellular viability these cells after this 3 times of assay. Cells without treatment (control) were used like negative control. After this, lymphocytes were centrifuged at 4°C, 450 x g for 10 minutes. After discarding the supernatant, the sediment was added 1 mL of PBS 1X and were centrifuged at 4°C, 450 x g for
10 minutes. After discarding the supernatant, the pellet was resuspended in a binding buffer (10 mM HEPES (pH 7.4), 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, and 1.8 mM CaCl2) and was added annexin V conjugated with fluorescein isothiocyanate (FITC) (1:500) and propidium iodide (PI, 20 µg.mL-1; 106 cells) in each identified cytometer tube. Flow cytometry was performed on a
FACSCalibur (Becton Dickinson Biosciences, Mountain View, Ca, USA) analyzed using Cell Quest Pro software (Becton Dickinson). Results analysis was performed on graphs by dot plot. Double positive (Annexin-FITC+/PI+) were considered spleen cells in the late stage of apoptosis, whereas only PI+ cells were necrotic cells. Annexin-FITC+/PI- were represented splenocytes in the early stage of apoptosis. Double negative was considered viable cells.
2.11. Statistical analysis
Data were analyzed using non-parametric tests. To detect differences between groups, the Mann-Whitney U test and tukey t test were used. All results were expressed by mean values of groups standard deviation and were analyzed considering the value of p<0.05 (statistically significant).
3. RESULTS
3.1. PZQ citotoxity was superior to imidazolidines derivatives
For the purpose of determining selectivity, the action derivatives imidazolidines were performed against of spleen cells from BALB/c mice (a fine method which is effective for evaluation of specific T lymphocyte cytotoxicity), using praziquantel as standard drug. Cytotoxicity assays using immune spleen cells by mice showed that LPSF/PT-9 presented nontoxic effects on 10, 5 and 1
µg.mL-1 concentrations and LPSF/PT-10 not exhibit cytotoxicity at 5 and 1
µg.mL-1 concentrations (Table 1). On the other hand praziquantel showed higher toxicity on splenocytes on all tested concentrations (Table 2). LPSF/PT-9 and LPSF/PT-10 also were tested against S. mansoni adult worms and results were expressed in terms of the IC50 (µM) values. PZQ also was used like the reference schistosomicide drug. The in vitro mortality of adult worms was observed at different concentrations by imidazolidine derivatives. The imidazolidines derivatives did show activity against adult worms of S. mansoni.
LPSF/PT-09 induced 100% of mortality at 320 μM concentration on first 48 hours. On 72 hours this same behavior was present in 100 μM concentrations.
LPSF/PT-10 induced 100% of worms mortality on 24 hours at 320 μM concentration. On 72 hours LPSF/PT-10 induced this same mortality index for 200 and 100 μM concentrations. Oviposition by adult worms also did not was observed on any concentration of these imidazolidines. Control group remained viable during all the time of observation (Table 1).
3.2. LPSF/PT-9 and LPSF/PT-10 imidazolidines did not produced IFN- and
IL-10 cytokines
For immunological assays we investigated IL-10 and IFN- cytokines and nitric oxide production on supernatants of cultures by splenocytes stimulated in vitro with LPSF/PT-9 and LPSF/PT-10 at 1 µg.mL-1 concentrations. Because yours immunological properties Phytohemagglutinin (PHA at 5 µg.mL-1) and
Concanavalin A (Con A at 2.5 µg.mL-1) mitogens were used like positive controls and without stimulus (cells + medium) was used like negative control.
Although higher and statistical values were produced for Con A and PHA mitogens on all experimental times of cultures (data not show), neither experimental imidazolidines tested were efficient on IL-10 and IFN- production on supernatants of cultures.
3.3. LPSF/PT-9 and LPSF/PT-10 induces higher values of nitrite production
The effects in the NO-induced production in murine spleen cells was performed on in vitro cultures treated with LPSF/PT-9 and LPSF/PT-10 at 100,
10 and 1 µg.mL-1 concentrations. Higher and statistical values were observed for both LPSF/PT-9 and LPSF/PT-10 at 100 µg.mL-1 concentration. At 24 hours
LPSF/PT-9 on 100 and 1 µg.mL-1 concentrations showed higher values in relation to control and LPSF/PT-10 at 100 µg.mL-1 was superior to control. At this same time LPSF/PT-10 was statistically superior to LPSF/PT-9 at 100 µg.mL-1 concentration (Figure 1A). After 48 hours was observed that LPSF/PT-9 and LPSF/PT-10 100 µg.mL-1 was statistically superior to control and among itselves (Figure 1B). Although LPSF/PT-9 presented higher values, only
LPSF/PT-10 at 100 µg.mL-1 did show statistical difference in relation to control at 72 hours (Figure 1C). The last time of culture, LPSF/PT-9 and LPSF/PT-10 at
100 µg.mL-1 did show statistical difference in relation to control. At 6 days we also observe that LPSF/PT-9 at 100 µg.mL-1 was statistical superior to
LPSF/PT-10 at 10 µg.mL-1 (Figure 1D).
3.4. Low cellular death induced by LPSF/PT-9 and LPSF/PT-10 imidazolidines
Cellular viability was realized for measure cellular damage induced by imidazolidines derivatives and for all compounds were used 1 µg.mL-1 concentration. After 24 hours it observed that the number of apoptotic cells was higher in relation to cells on late apoptosis and necrotic cells. At this time, PZQ was statistically superior to LPSF/PT-9 and LPSF/PT-10 imidazolidines (Figure
2A), but did not show higher values in relation to control cells (cells without treatment). On late apoptosis we observed that LPSF/PT-10 was superior to
LPSF/PT-9 and PZQ, but not in relation to control. LPSF/PT-10 demonstrated higher quantity necrotic cells in relation to control, PZQ and LPSF/PT-9 (Figure
2B). On 48 hours we observed that PZQ showed higher values in relation to others compounds. PZQ was superior to LPSF/PT-9 (Figure 2A) and LPSF/PT-
10 at apoptosis, late apoptosis and necrosis, but only in necrosis these values were statistical superior to control (Figure 2B). 72 hours was the unique time that presented statistical difference in relation to control for all composites tested. On apoptosis PZQ, LPSF/PT-9 and LPSF/PT-10 were superior to control and PZQ was superior to LPSF/PT-10 (Figure 2A). PZQ and LPSF/PT-9 induced more late apoptosis in relation to control and both composites were statistical superior to LPSF/PT-10. This behavior was similar on necrosis assay, i.e., PZQ and LPSF/PT-9 were superior to control and LPSF/PT-10 (Figure 2B).
4. DISCUSSION
Imidazolidines derivatives have been studied actually. The main activities assigned for these compounds are antibacterial, antiamebic, anti-T. cruzi and anti-schistosome [20, 21, 16, 22]. Here we analyze the cytotoxicity and immunological properties of new imidazolidines derivatives named LPSF/PT-9 and LPSF/PT-10 against Schistosoma mansoni worms.
LPSF/PT-9 and LPSF/PT-10 were less cytotoxic and induced superior mortality of adult worms in relation to Praziquantel. But in relation to absence of oviposition by adult worms and motor abnormalities, imidazolidines were similar to PZQ. Many drugs have been compared with praziquantel and its cytotoxicity and ability to kill adult worms of S. mansoni has been shown equivalent to praziquantel [23,24]. However, similar to praziquantel, the mechanism by which the imidazolidines exerts schistosomicide activity in vitro is still unclear. Many studies have been demonstrated that PZQ at low concentrations, in vitro, appears to impair the function of the worms’ suckers and at higher concentrations increases the contraction (irreversibly at very high concentrations) of the worm’s strobilla (chain of proglottids) [25; 26], causes irreversible focal vacuolization with subsequent cestodal disintegration at specific sites of the cestodal integument [27,28]. Besides these same aspects [15,16], imidazolidines show activity on apoptotic cells with melanoma, your route was identified in S. mansoni [29] and should be investigated to its participation in the death of the parasites.
Many studies have shown Th1 and Th2 response balance on schistosomotic patients. Before of treatment, patients in the acute phase as well as with the severe hepatosplenic form of the disease, have high levels of pro- inflammatory cytokines in their serum (IL-1, IL-6 and IFN- ). For other hand, patients with the intestinal clinical form have higher levels of IL-10 [5, 30].
Furthermore, these same studies strongly suggest that resistance to infection is multifactorial and that it can not be clearly correlated with a single immune mechanism. Stephaniel [31] affirm that low concentrations of IL-10, IL-4, and
TGF- inhibited macrophage larvicidal activity, as well as oxide nitric (NO) production, in a synergistic mechanism. The induction of T-lymphocyte proliferation by mitogens involves both cell-cell interaction and molecular communication. One of the changes observed after stimulation of lymphocytes by mitogens like PHA or Con A are increased ion fluxes and transport of other substrate across the plasma membrane. The consequences of the increased
K+, for example, included increased intracellular levels of Na2+ and Ca2+ in addition to decreased Mg2+ and K+ in PHA stimulated lymphocytes. This fact shows the requirements for calcium in lymphocyte activation and its participation in the process [32]. The central event in the activity of PZQ on schistosomes may well be the phenomenon of Ca influx into the tegument that is observed within a couple of minutes after drug exposure. However, the mechanisms leading to this alteration in Ca homeostasis are not clear [33,34]. In our study, imidazolidines derivatives did not induce significant production of
IFN- neither IL-10.
On the other hand, the potent anti-schistosomotic agent, the nitric oxide, was strongly stimulated. Nitric oxide is a molecule that shows effects only to a target located very close to the NO source. This behavior is important for avoided toxicity to surrounding tissues and indicating that the larvicidal action of
NO does not require interaction with other effector cell products [31]. Against parasitosis, there is ample direct and indirect evidence that NO can act as an antischistosomal and, more broadly, antiparasitic molecule [35,36,37]. NO produced by human white cells has been shown to kill larval schistosome parasites [35] and is a molecule which has recently been shown to kill a multitude of eukaryotic and prokaryotic organisms and against which no other defense mechanism has, to our knowledge, been reported [38, 39]. In addition, studies have shown that compounds that induce nitric oxide release possess potential immunomodulatory properties [40]. Here, we observe that LPSF/PT-9 and LPSF/PT-10 imidazolidines stimulated NO production by immune spleen cells and this behavior may indicate possible immuno stimulant activity induced by imidazolidines derivatives.
The two way cellular death, apoptosis and necrosis, differ fundamentally on morphology, biochemistry and biological relevance. Apoptotic cells are generated in vast numbers in the central lymphoid organs such as the thymus and bone marrow and are daily removed by phagocytic cells for homeostasis maintenance of these tissues. On the other hand, cellular death by necrosis occurs, generally, on response to several injuries suffered by these cells that have ruptured the plasmatic membrane leading to homeostasis loss and constituent release that starts an inflammatory response [41]. However, the distinction among initial apoptosis, late apoptosis and necrosis is still being largely discussed.
Many drugs have been shown damage on cell morphology on in vitro assays. Malheiros [42] using trifluoperazine (TFP), dibucaine (DBC) and
Praziquantel observed cytotoxic effects, as haemolysis and release of membrane lipids, on human erythrocyte membranes, in a dose-dependent mechanism. Here, on cellular death test, we observed that PZQ showed higher values on all the experimental times and on some results was superior to imidazolidines when tested on immune spleen cells. But the behavior among experimental composites was very similar, especially at 72 hours of assay, indicating that it is possible the activation of similar routes in the induction of cell death among the compounds evaluated and PZQ drug reference. However, more assays are needed to answer this question.
LPSF/PT-9 and LPSF/PT-10 imidazolidines derivatives showed relevant antischistosomal activity in vitro, induces higher nitric oxide production by immune spleen cells and showed similar behavior to praziquantel on cellular death test. Thus is possible that these new imidazolidines derivatives can be future candidates to schistosomotic drugs, but further studies is needed to elucidate the mechanisms induced for this response. Understanding the mechanisms that mediate the effects that this compounds have on the immune system will provide information that will make these compounds as future candidates to schistosomotic drugs.
ACKNOWLEDGEMENTS
This work was supported by grants from the Financiadora de Estudos e
Projetos (FINEP), Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior (CAPES), and Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq).
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Table 1: In vitro effects of LPSF/PT-09 and LPSF/PT-10 against Schistosoma mansoni adult worms Concentration (μM)
320 200 100 50 32
Observation Cytotoxicity b time Mortality (%) IC (μM) (worms) (µg.mL-1)a 50
24 h 74 79 80 19 0 51,74 not paired, no sucker 48 h 100 80 83 44 0 59,19 LPSF/PT-09 adherence, absence of 72 h 100 100 100 63 0 10 49,52 eggs, tegument 96 h 100 100 100 88 6 45,32 morphology altered 120 h 100 100 100 88 18 43,06
Concentration (μM)
320 200 100 50 32
Observation Cytotoxicity b time Mortality (%) -1 a IC (μM) (worms) (µg.mL ) 50
24 h 100 65 54 0 0 not paired, no sucker 115,8 48 h 100 100 85 0 0 adherence, absence of 95,31 LPSF/PT-10 72 h 100 100 100 0 0 eggs, appearance of 5 76,82 96 h 100 100 100 0 0 bubbles, tegument 76,82 120 h 100 100 100 0 0 morphology altered 76,82 a Expressed as the highest concentration tested that was not cytotoxic for the BALB/c mice splenocytes. Values in µM are showed in parentheses. b Calculated at five concentrations using data obtained from at least three independent experiments, with a SD less than 10% in all cases.
Table 2: In vitro effects of Praziquantel against Schistosoma mansoni adult worms Concentration (μM)
80 40 20 10
a b Observation Cytotoxicity c time Mortality (%) -1 IC (μM) (worms) (µg.mL ) 50 lower movements and 24 h 60 45 36 30 contractions, absence of eggs, 37,02 48 h 66 56 50 45 tegument morphology altered 35,08 PZQ 72 h 76 67 57 53 <1 33,91 96 h 100 100 100 92 15,36 120 h 100 100 100 92 15,36 a Control group paired adults worms without apparent morphological change, presence of eggs. b Expressed as the highest concentration tested that was not cytotoxic for the BALB/c mice splenocytes. Values in lM are showed in parentheses. c Calculated at seven concentrations using data obtained from at least three independent experiments, with a SD less than 10% in all cases.
Figure 1: Nitrite production on immune spleen cell cultures of BALB/c mice comparing experimental imidazolidines. Control is referent to not stimulated culture. A and B – on 24 and 48 hours of assay, respectively. On these two experimental times, LPSF/PT-9 and LPSF/PT-10 showed higher values in relation to control and among itselves. C – 72 hours of assay. LPSF/PT-10 at 100 µg.mL-1 did show statistical difference in relation to control. D - LPSF/PT-9 and LPSF/PT-10 at 100 µg.mL-1 did show statistical difference in relation to control and among itselves at 6 days of assay. Horizontal bars represent the average of three independent experiments per group. p < 0.05.
24 h 48 h 72 h
A
B
Figure 2: Imidazolidines derivatives effect on immune spleen cells viability. A - apoptosis investigation utilizing annexin V at 24, 48 and 72 hours of assay. PZQ was statistical superior to PT-9 and PT-10 imidazolidines but not in relation to control at 24 hours. At 48 hours PZQ was superior to LPSF/PT-9 and LPSF/PT-10 imidazolidines, but not in relation to control. PZQ, LPSF/PT-9 and LPSF/PT-10 were superior to control and PZQ was superior to PT-10 at 72 hours of assay. B - Necrosis assay using propidium iodide at 24, 48 and 72 hours of assay. PT-10 demonstrated higher quantity necrotic cells in relation to control, PZQ and LPSF/PT-9 at 24 hours. PZQ was superior to LPSF/PT-9 and LPSF/PT-10 imidazolidines and in relation to control on 48 hours of assay. PZQ and LPSF/PT-9 was superior to control and LPSF/PT-10 at 72 hours. Horizontal bars represent the average of three independent experiments per group. p < 0.05.
Imidazolidinas Esquistossomicidas: avaliação ultraestrutural, atividade citotóxica e imunomoduladora
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Neves, Juliana K. A. L. 132 Imidazolidinas Esquistossomicidas: avaliação ultraestrutural, atividade citotóxica e imunomoduladora
Trabalho para publicação
Revista: International Parasitology
ISSN: 1383-5769
Antischistosomal action of thioxo-imidazolidine compounds: an ultrastructural study
Autors: aJuliana Kelle A. L. Neves, aMaria do Carmo Alves de Lima, bValéria Rego Alves Pereira, bCristiane Moutinho Lagos de Melo, cChristina Alves Peixoto, aIvan Rocha Pitta, dMônica Camelo Pessoa Azevedo Albuquerque, aSuely Lins Galdino .
aLaboratório de Planejamento e Síntese de Fármacos (LPSF), Departamento de Antibióticos, Centro de Ciências Biológicas, Universidade Federal de Pernambuco, Av. Prof. Moraes Rego s/n, Cidade Universitária, 50670-901 Recife, PE, Brazil. bLaboratório de Imunogenética, Departamento de Imunologia, Centro de pesquisas Aggeu Magalhães, Fundação Oswaldo Cruz (FIOCRUZ), 50670-420 Recife, PE, Brazil. cLaboratório de Ultraestrutura, Departamento de Entomologia, Centro de Pesquisas Aggeu Magalhães, Fundação Oswaldo Cruz (FIOCRUZ), 50670-420 Recife, PE, Brazil. dLaboratório de Imunopatologia Keizo Asami (LIKA), Universidade Federal de Pernambuco, Av. Prof. Moraes Rego s/n, Cidade Universitária, 50670-901 Recife, PE, Brazil.
Correspondence Address: Juliana K A L Neves,Departamento de Antibióticos, Centro de Ciências Biológicas, Universidade Federal de Pernambuco. Av. Prof. Moraes Rego s/n, Cidade Universitária, 50670-901 Recife, PE, Brazil. Phone: +55 81-2126-8347; Fax: +55 81 2126-8346. e-mail: [email protected]
Neves, Juliana K. A. L. 133 Imidazolidinas Esquistossomicidas: avaliação ultraestrutural, atividade citotóxica e imunomoduladora
Abstract: Schistosomiasis is a disease caused by helminthes of genus Schistosoma, which threats approximately 207 million people worldwide. Lately, strain of Schistosoma mansoni developed tolerance and resistance against Praziquantel, drug available in the market for the treatment of disease, justifying studies that seek alternatives for prevention, treatment and cure of this disease. This study aimed to evaluate the in vitro activity of new imidazolidine derivatives of the series 5-benzylidene-3- benzyl-4-tioxo-imidazolidine-2-one (LSFP/PT-5) and 5-arilazo-4-tioxo- imidazolidine-2-one (LSFP/PT-11) against the adult worms of Schistosoma mansoni. LPSF/PT-5 and LPSF/PT-10 imidazolidines derivatives showed relevant schistosomicide activity in vitro and induced significant ultrastructural alterations on worms and cellular death similarly to praziquantel. Thus is possible that these new imidazolidines derivatives can be future candidates to schistosomotic drugs, but further studies is needed to elucidate the mechanisms induced for this response.
Keywords Schistosomiasis, Schistosoma mansoni, in vitro, derivative imidazolidione
Neves, Juliana K. A. L. 134 Imidazolidinas Esquistossomicidas: avaliação ultraestrutural, atividade citotóxica e imunomoduladora
1. Introduction
Schistosomiasis mansoni infection, an endemic disease of worldwide importance in terms of public health, affects large geographical areas in several countries. This tropical disease affects approximately 207 million people worldwide and is responsible for almost 500,000 deaths each year [1,2]. In Brazil, there are
8–10 million peoples infected and about 30 million at risk of infection [3].
Praziquantel (PZQ) remains the standard drug for the treatment of schistosomiasis worldwide. PZQ is effective against all Schistosoma species infecting humans and has been extensively tested. But, this drug is inefficient in immature forms and now has reports of resistance in some strains this has worried the world's public health organizations [4,5]. The imidazolidines are a broad class of bioactive pentagonal heterocyclic compounds with diverse biological activity [6]. Imidazolidines have an antifugal, antimicrobial, anti-T. cruzi and schistosomicidal properties [7]. The schistosomicidal properties of imidazolidine derivatives have been demonstrated by in vitro studies with adult S. mansoni worms [8,9,10]. Here, we investigated two new imidazolidines compounds against S. mansoni adult worms. The 3-benzyl-5-
(4-chloro-arylazo)-4-thioxo-imidazolidin-2-one, named LPSF-PT05 (CAS Registry
Number 197504-87-3) and 5-(4-flúor-arilazo)-3-(4-cloro-benzil)- 4-tioxo- imidazolidin-2-one, named LPSF- PT-11 [11] were synthesized by the Laboratório de Planejamento e Síntese de Fármacos-LPSF (UFPE) using diazonium ions formed from a phenylamine that acts as an electrophylic reagent and engages with the active hydrogen in position 5 of 3-benzyl-4-thioxo-imidazolidine-2-one, yielding the arylazo imidazolidine 5-(4-fluor-arilazo)-3-(4-cloro-benzil)- 4-tioxo-imidazolidin-
Neves, Juliana K. A. L. 135 Imidazolidinas Esquistossomicidas: avaliação ultraestrutural, atividade citotóxica e imunomoduladora
2-ona. In addition to anti-S. mansoni test, we analyzed the cytotoxicity of this compounds and investigated the cellular death induction promoted by these new imidazolidines.
2. MATERIALS AND METHODS
2.1. Compounds
Imidazolidines derivatives 3-benzyl-5-(4-chloro-arylazo)-4-thioxo- imidazolidin-2-one (LPSF/PT-05) and 5-(4-fluor-arilazo)-3-(4-cloro-benzil)- 4-tioxo- imidazolidin-2-ona (LPSF/PT-11) were obtained through the synthesis performed at the Laboratorio de Planejamento de Sintese de Farmacos (LPSF), Universidade
Federal de Pernambuco - Brazil, which were duly identified by nuclear magnetic resonance of hydrogen, infrared and spectroscopy mass. The praziquantel was purchase from Sigma Chemical Co., (St Louis, MO, USA - lot. 044K1032).
2.2. Parasites and intermediary hosts
The BH (BH - Belo Horizonte, MG, Brazil) strain of S. mansoni that has been maintained in the Laboratorio de Imunopatologia Keizo Asami-LIKA was used throughout this study. The strain was kept after it had passed through Biomphalaria glabrata molluscs provided by the Department of Tropical Medicine (Universidade
Federal de Pernambuco).
2.3. Animals used like definitive hosts
Were used mice Swiss females (20±2 gram of weight), were obtained and maintained at the animal facilities of Laboratorio de Imunologia Keizo Asami (LIKA)
Neves, Juliana K. A. L. 136 Imidazolidinas Esquistossomicidas: avaliação ultraestrutural, atividade citotóxica e imunomoduladora of the Universidade Federal de Pernambuco, Recife, Brazil. The animals were infected by exposure to a S. mansoni cercarian suspension containing approximately 100 cercarian, using the tail immersion technique [12]. Animals were housed in a controlled temperature and light environment, and were given water and commercial chow ad libitum. The experiments were approved by the Federal
University of Pernambuco’s Animal Experiments Ethics Committee, Process nº.007639/2007-12, in accordance with Law 9.605 Article 32 Decree 3179. Art 17.
2.4. Anti-S. mansoni assay
For the in vitro test worms were maintained in a RPMI-1640 medium (Sigma
Chemical Co., St. Louis, MO) buffered to pH 7.5, supplemented with HEPES (20 mM), 10% fetal bovine serum, penicillin (100U.mL-1), and streptomycin (100 µg.mL-
1 ). Incubation was carried out at 37 °C in a humid atmosphere containing CO2 5%.
LPSF/PT-5 and LPSF/PT-11 imidazolidines derivatives were dissolved in 1.6% dimethyl sulphoxide (DMSO) and used in concentrations varying from 32 to 320
µM which were added to the medium containing the worms after a period of 2 h of adaptation to the culture medium. Duplicates were carried out for each concentration used. The parasites were kept for 5 days and monitored every 24 h to evaluate their general condition: motor activity, alterations in the tegument, mortality rate. In the PZQ group used the same methodology, but the concentration ranged from 10 to 80 µM. The control worms were treated only DMSO in RPMI
1640 medium.
2.5. Animals used for cytotoxic and cellular viability assays
Neves, Juliana K. A. L. 137 Imidazolidinas Esquistossomicidas: avaliação ultraestrutural, atividade citotóxica e imunomoduladora
Male BALB/c mice (6 to 8 weeks old) were raised at the animal facilities of the
Oswaldo Cruz Foundation (Rio de Janeiro, Brazil) and maintained at the animal facilities of the Aggeu Magalhães Research Center of the Oswaldo Cruz
Foundation in Recife, Brazil. All mice were sacrificed and your spleens were removed in accordance with the Oswaldo Cruz Foundation Commission for
Experiments with Laboratory Animals (Ministry of Health, Brazil, 0266/05).
2.6. Obtaining of spleen cells
Spleen cells were obtained according to previous protocol [13]. After killing the animal with CO2 gas, the spleen of each mouse was removed aseptically and placed in Falcon tube containing RPMI 1640 with fetal calf serum (complete medium). In a vertical flow, each spleen was transferred to a Petri dish where they were soaked. The cell suspensions obtained were transferred to Falcon tubes containing approximately 10 mL of incomplete medium by spleen, centrifuged at 4
°C, 200 x g for 5 minutes. After discarding the supernatant, distilled water was added to the sediment to promote lysis of red blood cells. The supernatant, containing no cellular debris was collected and centrifuged at 4 °C, 200 x g for 5 minutes. The sediment (containing cells) was resuspended in complete RPMI
1640. An aliquot of each cell suspension was separated, diluted in trypan blue to be quantified in a Neubauer chamber and the cell viability was determined.
2.7. In vitro cytotoxicity assay
Neves, Juliana K. A. L. 138 Imidazolidinas Esquistossomicidas: avaliação ultraestrutural, atividade citotóxica e imunomoduladora
The cytotoxicity of the compounds was determined using BALB/c mice splenocytes (6 x 105 cells.well-1) cultured in 96-well plates in RPMI 1640 medium
(Sigma Chemical Co., St. Louis, MO) supplemented with 10% of fetal calf serum
(FCS; Cultilab, Campinas, SP, Brazil) and 50 µg.mL-1 of gentamycin (Novafarma,
Anápolis, GO, Brazil). Each imidazolidine was evaluated in six concentrations (1, 5,
10, 25, 50 and 100 μg.mL-1), in triplicate on two independent assays. Cultures were incubated in the presence of 3H-thymidine (Amersham Biosciences) (1 μCi.well-1) for 24 h at 37º C and 5% CO2. After this period, the content of the plate was harvested to determine the 3H-thymidine ([3H]TdR) incorporation using a beta- radiation counter (β-matrix 9600, Packard). The toxicity of the compounds was determined by comparing the percentage of 3H-thymidine incorporation (as an indicator of cell viability) of imidazolidines-treated wells in relation to untreated wells. Non-cytotoxic concentrations were defined as those causing a reduction of
3H-thymidine incorporation below 30% in relation to untreated controls. In praziquantel were also used six concentrations (1, 5, 10, 25, 50 and 100 μg.mL-1).
2.10. Analysis of cellular viability using Annexin V-FITC and Propidium iodide staining
Immune spleen cells were treated with Praziquantel (PZQ) or LPSF/PT-5 and or
LPSF/PT-11 at 1 µg.mL-1 concentration, respectively. These treated cells were maintained in culture in 24-well plates (TPP) for 24, 48 and 72 hours for analyze the cellular viability these cells after this 3 times of assay. Cells without treatment
(control) were used like negative control. After this, lymphocytes were centrifuged at 4 °C, 450 x g for 10 minutes. After discarding the supernatant, the sediment was
Neves, Juliana K. A. L. 139 Imidazolidinas Esquistossomicidas: avaliação ultraestrutural, atividade citotóxica e imunomoduladora added 1 mL of PBS 1X and were centrifuged at 4 °C, 450 x g for 10 minutes. After discarding the supernatant, the pellet was resuspended in a binding buffer (10 mM
HEPES (pH 7.4), 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, and 1.8 mM CaCl2) and was added annexin V conjugated with fluorescein isothiocyanate (FITC) (1:500) and propidium iodide (PI, 20 µg.mL-1; 106 cells) in each identified cytometer tube.
Flow cytometry was performed on a FACSCalibur (Becton Dickinson Biosciences,
Mountain View, Ca, USA) analyzed using Cell Quest Pro software (Becton
Dickinson). Results analysis was performed on graphs by dot plot. Double negative
(Annexin-FITC-/PI-) was considered viable cells. Annexin-FITC+/PI- were represented splenocytes in the early stage of apoptosis. Double positive (Annexin-
FITC+/PI+) were considered spleen cells in the late stage of apoptosis and only PI+, cells were considered like necrotic.
2.11. Scanning Electronic Microscopy
At 1, 3, 6 e 12 h after treatment with 100 µM (LPSF/PT-5 and LPSF/PT-11 imidazolidines) or 80 µM (PZQ) the worms as were fixed in 2,5% glutaraldehyde in
0.1M phosphate buffer (pH 7.2) for 2 h at ambient temperature. Then, they were washed twice in the same buffer and post-fixed in 1% osmium tetroxide in phosphate buffer for 1 h ambient temperature. All worms were dehydrated in ethanol, washed several times in 100% ethanol, critical-point-dried in CO2, mounted on stubs, coated with gold, and examined under a JEOL 25SII scanning electronic microscope.
Neves, Juliana K. A. L. 140 Imidazolidinas Esquistossomicidas: avaliação ultraestrutural, atividade citotóxica e imunomoduladora
2.12. Statistical analysis
Data were analyzed using non-parametric tests. To detect differences between groups, the Mann-Whitney U test and tukey t test were used. All results were expressed by mean values of groups standard deviation and were analyzed considering the value of p<0.05 (statistically significant).
3. Results
PZQ citotoxity was superior to imidazolidines derivatives and showed anti-S. mansoni activity
For the purpose of determining selectivity the cytotoxicity induced by imidazolidines, we utilized the Tritiate Timidine ([3H]TdR) assay on immune spleen cells by BALB/c mice. This technique use Tritiate Timidine that acts through the incorporation of tritium into the DNA of cells and is the most commonly utilized technique today for cytotoxity detection in immune cells [14]. Praziquantel was used like standard drug (positive control) and cells without treatment like negative control. Results did show that LPSF/PT-5 and LPSF/PT-11 presented nontoxic effects on 5 and 1 µg.mL-1 concentrations (Table 1). On the other hand praziquantel showed higher toxicity on splenocytes on all tested concentrations
(Table 2). LPSF/PT-5 and LPSF/PT-11 also were tested against S. mansoni adult worms and results were expressed in terms of the IC50 (µM) values (Table 1). PZQ also was used like the reference schistosomicide drug (Table 2). The two imidazolidines derivatives induced 100% of mortality at 100 μM concentration on first 24 hours of worms exposition and this behavior was repeated on others times
Neves, Juliana K. A. L. 141 Imidazolidinas Esquistossomicidas: avaliação ultraestrutural, atividade citotóxica e imunomoduladora of exposition (Table 1). Physiological alterations were induced by all experimental drugs (imidazolidines and praziquantel) and motor abnormalities of worms were similar between drugs (Tables 1 and 2). Oviposition by adult worms also did not was observed on any concentration of these imidazolidines and control group
(worms without treatment) remained viable during all the time of observation.
LPSF/PT-5 and LPSF/PT-11 induced ultrastructural alterations on worms tegument
Parasites were exposure to 100 M of LPSF/PT-5 and LPSF/PT-11 or 80 M of
PZQ and were collected on 1, 3, 6 and 12 hours of interval for ultrastructural investigation. Male worms without treatment were utilized like negative control.
Serious damages induced by imidazolidines on worms were observed and were similar to PZQ. With the derived LPSF/PT-5, after 1 hour of incubation, imidazolidine promoted blistering and peeling of the tegument, and after 3 hours of contact there is an increasing number of bubbles between a trabeculae and changes in windy after 6 hours of incubation and disruption of the bubbles change after 12 hours of seed coat with an increasing peeling on tegument of worm
(Figures 1). After 1 hour of incubation with the compound LPSF/PT-11, we observed blisters and swelling of the integument and loss of a few thorns in the tubers; after 3 hours increased swelling and hardly see thorns; after 6 hours of contact we noted an elongation of worm when compared with the LPSF/PT-5 and
PZQ after 12 hours peeling the skin (Figures 2). Moderate damage on tegument surface was observed on male worms after 1 hour of incubation with PZQ at 80 M
Neves, Juliana K. A. L. 142 Imidazolidinas Esquistossomicidas: avaliação ultraestrutural, atividade citotóxica e imunomoduladora concentration. After 1 hour of contact with the PZQ, we observed contraction on parasite, changes in trabeculae, swelling, reduction and loss of spines on the tegument. We also observed a large number of bubbles around the trabeculae
(Figures 3). After 3 hours of incubation with PZQ, there was the increase of number of bubbles on the worm tegument (Figures 3). After 6 hours worms showed curve and retracted appearance and after 12h of exposure, we observed on worm tegument: trabeculae vacuolization, severe swelling and disruption of bubbles. Control shown the normal body surface with normal appearance of the tubers and a number of thorns (Figures 4).
PZQ was induced major cellular death than imidazolidines
To confirm the ultrastructural results some assays were performed to evaluation the presence of cellular death induction by imidazolidines derivate. This assay was done using BALB/c mice splenocytes exposure to LPSF/PT-5,
LPSF/PT-11 or PZQ at 1 M concentration and for measurement of cellular damage we investigated apoptosis and necrosis aspects. Results showed that at
24 hours PZQ induced higher apoptosis on splenocytes in relation to LPSF/PT-5 and LPSF/PT-11 imidazolidines and LPSF/PT-11 was superior to LPSF/PT-5
(Figure 5A), but these drugs did not show statistical significance in relation to control (cells without treatment). At this same time, it observed that LPSF/PT-5 was superior to LPSF/PT-11 on necrosis (Figure 5B), but also not in relation to control. PZQ induced most apoptosis than LPSF/PT-5 and LPSF/PT-11 imidazolidines on 48 hours of assay, but not in relation to control (Figure 5A).
Neves, Juliana K. A. L. 143 Imidazolidinas Esquistossomicidas: avaliação ultraestrutural, atividade citotóxica e imunomoduladora
However, PZQ induced higher and statistical necrosis on splenocytes of control,
LPSF/PT-5 and LPSF/PT-11 imidazolidines. LPSF/PT-11 was also superior to
LPSF/PT-5 at this same time (Figure 5B). At to last time of assay, 72 hours,
LPSF/PT-5 and LPSF/PT-11 induced higher apoptosis levels (Figure 5A) and PZQ and LPSF/PT-11 induced higher necrosis levels in relation to control cells (Figure
5B).
Discussion
Schistosomiasis is a negligence disease that has one drug of choice, praziquantel. Because of this, the Special Program for Research & Training in Tropical Diseases promote by WHO provides opportunities for studies in the planning of anti-parasitic and encourages worldwide synthesis of new compounds for these neglected diseases [15]. Indeed, many drugs have been compared with praziquantel and its cytotoxicity and ability to kill adult worms of S. mansoni has been shown equivalent to praziquantel [16,17]. Here,
LPSF/PT-5 and LPSF/PT-11 were less cytotoxic and induced superior mortality of adult worms in relation to Praziquantel. But in relation to absence of oviposition by adult worms and motor abnormalities imidazolidines were similar to reference drug.
PZQ at low concentrations, in vitro, appears to impair the function of the worms’ suckers and at higher concentrations increases the contraction (irreversibly at very high concentrations) of the worm’s strobilla (chain of proglottids) [18,19], causes irreversible focal vacuolization with subsequent cestodal disintegration at specific sites of the cestodal integument [20,21]. These same physiological alterations were
Neves, Juliana K. A. L. 144 Imidazolidinas Esquistossomicidas: avaliação ultraestrutural, atividade citotóxica e imunomoduladora observed on adult worms treated with imidazolidines here and in others studies realized by our group [9,22].
Actually, imidazolidines were being investigated as your anti-parasite action.
On schistosomiasis, imidazolidines composites did show action against adult worms [10] and for anti-T. cruzi assays these composites were promising against epimastigotes forms [7]. However, similar to praziquantel, the mechanism by which the imidazolidines exerts schistosomicide activity in vitro is still unclear. Many studies affirm that imidazolidines did show activity on apoptotic cells with melanoma, your route was identified in S. mansoni [23] and has been suggest a possible activity at the cholinergic level receptors, since the nervous system of S. mansoni, with unique pharmacological and physiological characteristics, can be used in the research of compounds for human and animal use [24].
The ultrastructural analysis observed on our results indicated that like praziquantel, imidazolidines induced serious damages on S. mansoni tegument.
Previous works also indicated that new compouds had been demonstrated anti-S. mansoni activity. Using artemether like treatment against schistosimiasis, Xiao [25] showed contraction and swelling of the integument with fusion of the tubers, blisters and a disorder in the arrangement of the spines. Soliman [26] also demonstrated that medroxyprogesterone acetate induced loss or reduction of some thorns in the tubers, rupture of some tubers, and tissue collapse of the damaged area, mild to moderate erosion and flaking of the coat. In addition, effects of artesunate were also studied in which male worms showed blisters in the oral sucker, swelling trabeculae, complete loss of spines emergence of bubbles
Neves, Juliana K. A. L. 145 Imidazolidinas Esquistossomicidas: avaliação ultraestrutural, atividade citotóxica e imunomoduladora between the trabeculae, accompanied by the outbreak of such an extensive peeling and erosion of the tegument of the worm [27, 28,29].
Many drugs have been shown damage on cell morphology on in vitro assays and Annexin V appears to be a potent discriminator between viable and apoptotic cells. Furthermore, propidium iodide may be added to specifically stain the cells which have compromised plasma membrane integrity [30]. Unlike ultrastructural damage observed on S. Mansoni tegument induced by imidazolidines derivatives LPSF/PT-5 and LPSF/PT-11, the cellular viability test demonstrated that new imidazolidines compounds did show significant splenocytes cell death only at 72 hours of treatment. Malheiros [31] using trifluoperazine (TFP), dibucaine (DBC) and Praziquantel observed cytotoxic effects, as haemolysis and release of membrane lipids, on human erythrocyte membranes, in a dose- dependent mechanism. Here, on cellular death test, we observed that PZQ showed higher values on all the experimental times and on some results was superior to imidazolidines. But, the behavior by PT-11 was very similar to PZQ, indicating that it is possible the activation of similar routes in the induction of cell death among the compounds evaluated and drug reference. However, more assays are needed to answer this question.
LPSF/PT-5 and LPSF/PT-10 imidazolidines derivatives showed relevant schistosomicide activity in vitro and induced significant ultrastructural alterations on worms and cellular death similarly to praziquantel. Thus is possible that these new imidazolidines derivatives can be future candidates to schistosomotic drugs, but further studies is needed to elucidate the mechanisms induced for this response.
Neves, Juliana K. A. L. 146 Imidazolidinas Esquistossomicidas: avaliação ultraestrutural, atividade citotóxica e imunomoduladora
Acknowledgment
This work was supported by grants from the Financiadora de Estudos e Projetos
(FINEP), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
(CAPES), and Conselho Nacional de Desenvolvimento Científico e Tecnológico
(CNPq).
References
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[4] Ismail, M., Botros, S., Metwally, A., William, S., Farghally, A., Tao, L.-F., Day, T.A., Bennett, J.L. Resistance to praziquantel: direct evidence from Schistosoma mansoni isolated from Egyptian villagers. Am. J. Trop. Med. Hyg. 1999; 60: 932– 935.
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[14] Pechhold K, Craighead N, Wesch D, Kabelitz D. 2002. Measurement of cellular proliferation. Met. Microb. 32: 77-97.
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[16] A.D. Dayan. Albendazole, mebendazole and praziquantel. Review of non- clinical toxicity and pharmacokinetics. Acta Tropica 86 (2003) 141-159.
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[18] Xiao SH, Catto BA, Webster LT (1985) Effects of praziquantel on different developmental stages of Schistosoma mansoni in vitro and in vivo. Journal of Infectious Disease 151: 1130–1137.
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[22] SOARES, A.L.M.; Síntese e avaliação da atividade biológica de novos derivados Arilazo-Imidazolidínicos e Arilideno-Tiazolidínicos frente a vermes adultos de Schistosoma mansoni (Cepa BH).2004.140p. Disser (Mestrado) – Departamento de Antibióticos, Universidade Federal de Pernambuco, Recife.
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[24] Thibaut, JPB, Monteiro, LM, Leite, LCC, Menezes, CMS, Lima, LM, Noel, F. (2009) The effects of 3-methylclonazepam on Schistosoma mansoni musculature are not mediated by benzodiazepine receptors. European Journal of Pharmacology. 606: 9–16.
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[26] Soliman MFM, Ibrahim MM. Antischistosomal action of atorvastatin alone and concurrently with medroxyprogesterone acetate on Schistosoma haematobium harboured in hamster: surface ultrastructure and parasitological study. Acta Tropica 93 (2005) 1–9
[27] Jiraungkoorskula W, Sahaphonga S, Sobhonc P, Riengrojpitaka S, Kangwanrangsan N. Effects of praziquantel and artesunate on the tegument of adult Schistosoma mekongi harboured in mice. Parasitology International 54 (2005) 177 – 183.
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[31] Sônia V.P. Malheiros, M. Alexandra Brito, Dora Brites, Nilce Correa Meirelles. Membrane effects of trifluoperazine, dibucaine and praziquantel on human erythrocytes. Chemico-Biological Interactions 126 (2000) 79–95.
Neves, Juliana K. A. L. 151 Imidazolidinas Esquistossomicidas: avaliação ultraestrutural, atividade citotóxica e imunomoduladora
Table 1: In vitro effects of LPSF/PT-05 and LPSF/PT-11 against Schistosoma mansoni adult worms
Concentration (μM)
320 200 100 50 32
Observation Cytotoxicity b time Mortality (%) -1 a IC (μM) (worms) (µg.mL ) 50
24 h 100 100 100 21 2 57,79 48 h 100 100 100 64 10 not paired, no sucker 47,44 LPSF/PT-05 72 h 100 100 100 64 12 adherence, absence of eggs, 5 48,59 96 h 100 100 100 71 25 tegument morphology altered 47,37 120 h 100 100 100 100 25 39,75
Concentration (μM)
320 200 100 50 32
Observation Cytotoxicity b time Mortality (%) -1 a IC (μM) (worms) (µg.mL ) 50
24 h 100 100 100 39 11 51,33 not paired, no sucker 48 h 100 100 100 56 16 49,58 adherence, absence of eggs, LPSF/PT-11 72 h 100 100 100 85 84 5 52,11 appearance of bubbles, c 96 h 100 100 100 100 89 ND tegument morphology altered 120 h 100 100 100 100 100 ND
a) Expressed as the highest concentration tested that was not cytotoxic for the BALB/c mice splenocytes. Values in µM are showed in parentheses. b) Calculated at five concentrations using data obtained from at least three independent experiments, with a SD less than 10% in all cases. c) Not determined.
Neves, Juliana K. A. L. 152 Imidazolidinas Esquistossomicidas: avaliação ultraestrutural, atividade citotóxica e imunomoduladora
Table 2: In vitro effects of Praziquantel against Schistosoma mansoni adult worms
Concentration (μM)
80 40 20 10
a b Observation Cytotoxicity c time Mortality (%) -1 IC (μM) (worms) (µg.mL ) 50
24 h 60 45 36 30 37,02 48 h 66 56 50 45 lower movements and 35,08 PZQ 72 h 76 67 57 53 contractions, absence of eggs, <1 33,91 96 h 100 100 100 92 tegument morphology altered 15,36 120 h 100 100 100 92 15,36 a) Control group paired adults worms without apparent morphological change, presence of eggs. b) Expressed as the highest concentration tested that was not cytotoxic for the BALB/c mice splenocytes. Values in lM are showed in parentheses. c) Calculated at seven concentrations using data obtained from at least three independent experiments, with a SD less than 10% in all cases.
Neves, Juliana K. A. L. 153 E F t
b d
G H t vv
I J
K L
d
d
Fig. 1: Scanning electron microscopy of the tegument of S. mansoni group LPSF/PT-5 in E (1000x) have observed the appearance of bubbles (b) in one hour incubation with the secondary F (3350X) also at a time, scaling (d) and wrinkling of the tegument , G (200x) in three hours of contact there is change in the ventral sucker (vv) of the parasite, H (1000x) in three hours an increase in the number of bubbles between the trabeculae (t), I (1000x) and J ( 3350X) in six hours of contact, the outbreak of blisters, K (1000x) and L (3350X) in twelve hours of incubation an increase in shedding of the tegument at different resolutions.
Imidazolidinas Esquistossomicidas: avaliação ultraestrutural, atividade citotóxica e imunomoduladora
M N
O P
Q R
S T
Fig. 2:Scanning electron microscopy of the tegument of S. mansoni group LPSF/PT-11 in M (1000x) large number of bubbles (b) and swelling of the seed coat in one hour incubation with the compound, N (3350X) also at a time, swelling and loss of spines of the trabeculae (t), O (3350X) in three contact hours, swelling and hardly see thorns, P (70x) in three hours and Q (50x) in six hours, there is an extension of the parasite, R (1000x) within six hours of contact, S (1000x) and T (3350X) in twelve hours of incubation changes the tegument.
Neves, Juliana K. A. L. 155 Imidazolidinas Esquistossomicidas: avaliação ultraestrutural, atividade citotóxica e imunomoduladora
U V
b
d
X
Fig. 3: SEM showing the changes of the tegument of the adult worm S. mansoni when exposed to PZQ in 1, 3, 6 and 12 hours respectively. U (3350X) blisters (b) and scaling (d) of the tegument in V (70x) and X (70x) contraction of the parasite and Y (3350X) outbreak of blisters, loss of spines and retraction of the seed coat.
Neves, Juliana K. A. L. 156 Imidazolidinas Esquistossomicidas: avaliação ultraestrutural, atividade citotóxica e imunomoduladora
vo A B vo vv
vv
C DD
t t
Fig. 4. Images of S. mansoni adult male control, A (70x) and B (200x) worms with normal and windy tegument in C (1000x) and D (3350X) showing a large number of trabeculae (t) with its thorns.
Neves, Juliana K. A. L. 157 Imidazolidinas Esquistossomicidas: avaliação ultraestrutural, atividade citotóxica e imunomoduladora
Figure 5: Imidazolidines derivatives effect on immune spleen cells viability. A - apoptosis investigation utilizing annexin V at 24, 48 and 72 hours of assay. PZQ was statistical superior to PT-5 and PT-11 at 24 and 48 hours. Only at 72 hours PT-5 induced higher apoptosis in relation to control and PT-11 was superior to PT-5 at 24 hours and control at 72 hours. B - Necrosis assay using propidium iodide at 24, 48 and 72 hours of assay. PZQ was superior to PT-5 and PT-11 imidazolidines and in relation to control at 48 hours of assay. PZQ was also superior to control at 72 hours of assay. PT-5 was only statistically superior to PT-11 at 24 hours and PT-11 induced higher necrosis levels at 48 and 72 hours in relation to control. Horizontal bars represent the average of three independent experiments per group. p < 0.05.
Neves, Juliana K. A. L. 158 Imidazolidinas Esquistossomicidas: avaliação ultraestrutural, atividade citotóxica e imunomoduladora
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Collate acknowledgements in a separate section at the end of the article before the references and do not, therefore, include them on the title page, as a footnote to the title or otherwise. List here those individuals who provided help during the research (e.g., providing language help, writing assistance or proof reading the article, etc.).
Units
Follow internationally accepted rules and conventions: use the international system of units (SI). If other units are mentioned, please give their equivalent in SI.
Nomenclature and units
Follow internationally accepted rules and conventions: use the international system of units (SI). If other quantities are mentioned, give their equivalent in SI. You are urged to consult IUPAC: Nomenclature of Organic Chemistry: http://www.iupac.org/ for further information.
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Math formulae
Present simple formulae in the line of normal text where possible and use the solidus (/) instead of a horizontal line for small fractional terms, e.g., X/Y. In principle, variables are to be presented in italics. Powers of e are often more conveniently denoted by exp. Number consecutively any equations that have to be displayed separately from the text (if referred to explicitly in the text).
Footnotes
Footnotes should be used sparingly. Number them consecutively throughout the article, using superscript Arabic numbers. Many wordprocessors build footnotes into the text, and this feature may be used. Should this not be the case, indicate the position of footnotes in the text and present the footnotes themselves separately at the end of the article. Do not include footnotes in the Reference list. Table footnotes Indicate each footnote in a table with a superscript lowercase letter.
Artwork
Electronic artwork General points • Make sure you use uniform lettering and sizing of your original artwork. • Save text in illustrations as "graphics" or enclose the font. • Only use the following fonts in your illustrations: Arial, Courier, Times, Symbol. • Number the illustrations according to their sequence in the text. • Use a logical naming convention for your artwork files. • Provide captions to illustrations separately. • Produce images near to the desired size of the printed version. • Submit each figure as a separate file.
A detailed guide on electronic artwork is available on our website: http://www.elsevier.com/artworkinstructions You are urged to visit this site; some excerpts from the detailed information are given here.
Formats Regardless of the application used, when your electronic artwork is finalised, please "save as" or convert the images to one of the following formats (note the resolution requirements for line drawings, halftones, and line/halftone combinations given below): EPS: Vector drawings. Embed the font or save the text as "graphics". TIFF: color or grayscale photographs (halftones): always use a minimum of 300 dpi. TIFF: Bitmapped line drawings: use a minimum of 1000 dpi. TIFF: Combinations bitmapped line/half-tone (color or grayscale): a minimum of 500 dpi is required. DOC, XLS or PPT: If your electronic artwork is created in any of these Microsoft Office applications please supply "as is".
Please do not:
• Supply embedded graphics in your wordprocessor (spreadsheet, presentation) document; • Supply files that are optimised for screen use (like GIF, BMP, PICT, WPG); the resolution is too low; • Supply files that are too low in resolution; • Submit graphics that are disproportionately large for the content.
Color artwork Please make sure that artwork files are in an acceptable format (TIFF, EPS or MS Office files) and with the correct resolution. If, together with your accepted article, you submit usable color figures then Elsevier will ensure, at no additional charge, that these figures will appear in color on the Web (e.g., ScienceDirect and other sites) regardless of whether or not these illustrations are reproduced in color in the printed version. For color reproduction in print, you will receive information regarding the costs from Elsevier after receipt of your accepted article. Please indicate your preference for color in print or on the Web only. For further information on the preparation of electronic artwork, please see http://www.elsevier.com/artworkinstructions.
Please note: Because of technical complications which can arise by converting color figures to "gray scale" (for the printed version should you not opt for color in print) please submit in addition usable black and white versions of all the color illustrations.
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Figure captions Ensure that each illustration has a caption. Supply captions separately, not attached to the figure. A caption should comprise a brief title (not on the figure itself) and a description of the illustration. Keep text in the illustrations themselves to a minimum but explain all symbols and abbreviations used.
Text graphics Present incidental graphics not suitable for mention as figures, plates or schemes at the end of the article and number them "Graphic 1", etc. Their precise position in the text can then be indicated. See further under Electronic artwork. If you are working with LaTeX and have such features embedded in the text, these can be left, but such embedding should not be done specifically for publishing purposes. Further, high-resolution graphics files must be provided separately.
Tables
Number tables consecutively in accordance with their appearance in the text. Place footnotes to tables below the table body and indicate them with superscript lowercase letters. Avoid vertical rules. Be sparing in the use of tables and ensure that the data presented in tables do not duplicate results described elsewhere in the article.
References
Citation in text Please ensure that every reference cited in the text is also present in the reference list (and vice versa). Any references cited in the abstract must be given in full. Unpublished results and personal communications are not recommended in the reference list, but may be mentioned in the text. If these references are included in the reference list they should follow the standard reference style of the journal and should include a substitution of the publication date with either "Unpublished results" or "Personal communication" Citation of a reference as "in press" implies that the item has been accepted for publication.
Web references As a minimum, the full URL should be given and the date when the reference was last accessed. Any further information, if known (DOI, author names, dates, reference to a source publication, etc.), should also be given. Web references can be listed separately (e.g., after the reference list) under a different heading if desired, or can be included in the reference list.
References in a special issue Please ensure that the words 'this issue' are added to any references in the list (and any citations in the text) to other articles in the same Special Issue.
Reference style Text: Indicate references by number(s) in square brackets in line with the text. The actual authors can be referred to, but the reference number(s) must always be given. Example: "..... as demonstrated [3,6]. Barnaby and Jones [8] obtained a different result ...." List: Number the references (numbers in square brackets) in the list in the order in which they appear in the text. Examples: Reference to a journal publication: [1] J. van der Geer, J.A.J. Hanraads, R.A. Lupton, The art of writing a scientific article, J. Sci. Commun. 163 (2000) 51–59. Reference to a book: [2] W. Strunk Jr., E.B. White, The Elements of Style, third ed., Macmillan, New York, 1979. Reference to a chapter in an edited book: [3] G.R. Mettam, L.B. Adams, How to prepare an electronic version of your article, in: B.S. Jones, R.Z. Smith (Eds.), Introduction to the Electronic Age, E-Publishing Inc., New York, 1999, pp. 281– 304.
Supplementary data
Elsevier accepts electronic supplementary material to support and enhance your scientific research. Supplementary files offer the author additional possibilities to publish supporting applications, high- resolution images, background datasets, sound clips and more. Supplementary files supplied will be published online alongside the electronic version of your article in Elsevier Web products, including ScienceDirect: http://www.sciencedirect.com. In order to ensure that your submitted material is directly usable, please provide the data in one of our recommended file formats. Authors should submit the material in electronic format together with the article and supply a concise and descriptive
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caption for each file. For more detailed instructions please visit our artwork instruction pages at http://www.elsevier.com/artworkinstructions.
Submission checklist
It is hoped that this list will be useful during the final checking of an article prior to sending it to the journal's Editor for review. Please consult this Guide for Authors for further details of any item. Ensure that the following items are present: One Author designated as corresponding Author: • E-mail address • Full postal address • Telephone and fax numbers All necessary files have been uploaded • Keywords • All figure captions • All tables (including title, description, footnotes) Further considerations • Manuscript has been "spellchecked" and "grammar-checked" • References are in the correct format for this journal • All references mentioned in the Reference list are cited in the text, and vice versa • Permission has been obtained for use of copyrighted material from other sources (including the Web) • Color figures are clearly marked as being intended for color reproduction on the Web (free of charge) and in print or to be reproduced in color on the Web (free of charge) and in black-and-white in print • If only color on the Web is required, black and white versions of the figures are also supplied for printing purposes For any further information please visit our customer support site at http://epsupport.elsevier.com.
Use of the Digital Object Identifier
The Digital Object Identifier (DOI) may be used to cite and link to electronic documents. The DOI consists of a unique alpha-numeric character string which is assigned to a document by the publisher upon the initial electronic publication. The assigned DOI never changes. Therefore, it is an ideal medium for citing a document, particularly 'Articles in press' because they have not yet received their full bibliographic information. The correct format for citing a DOI is shown as follows (example taken from a document in the journal Physics Letters B): doi:10.1016/j.physletb.2003.10.071 When you use the DOI to create URL hyperlinks to documents on the web, they are guaranteed never to change.
Proofs
One set of page proofs (as PDF files) will be sent by e-mail to the corresponding author (if we do not have an e-mail address then paper proofs will be sent by post) or, a link will be provided in the e-mail so that authors can download the files themselves. Elsevier now provides authors with PDF proofs which can be annotated; for this you will need to download Adobe Reader version 7 (or higher) available free from http://www.adobe.com/products/acrobat/readstep2.html. Instructions on how to annotate PDF files will accompany the proofs (also given online). The exact system requirements are given at the Adobe site: http://www.adobe.com/products/acrobat/acrrsystemreqs.html#70win. If you do not wish to use the PDF annotations function, you may list the corrections (including replies to the Query Form) and return them to Elsevier in an e-mail. Please list your corrections quoting line number. If, for any reason, this is not possible, then mark the corrections and any other comments (including replies to the Query Form) on a printout of your proof and return by fax, or scan the pages and e-mail, or by post. Please use this proof only for checking the typesetting, editing, completeness and correctness of the text, tables and figures. Significant changes to the article as accepted for publication will only be considered at this stage with permission from the Editor. We will do everything possible to get your article published quickly and accurately. Therefore, it is important to ensure that all of your corrections are sent back to us in one communication: please check carefully before replying, as inclusion of any subsequent corrections cannot be guaranteed. Proofreading is solely your responsibility. Note that Elsevier may proceed with the publication of your article if no response is received.
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Offprints
The corresponding author, at no cost, will be provided with a PDF file of the article via e-mail. For an extra charge, paper offprints can be ordered via the offprint order form which is sent once the article is accepted for publication. The PDF file is a watermarked version of the published article and includes a cover sheet with the journal cover image and a disclaimer outlining the terms and conditions of use.
For inquiries relating to the submission of articles (including electronic submission where available) please visit this journal's homepage. You can track accepted articles at http://www.elsevier.com/trackarticle and set up e-mail alerts to inform you of when an article's status has changed. Also accessible from here is information on copyright, frequently asked questions and more. Contact details for questions arising after acceptance of an article, especially those relating to proofs, will be provided by the publisher.
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