Print version ISSN 0074-0276
Mem. Inst. Oswaldo Cruz vol.92 s.1 Rio de Janeiro Nov. 1997
IMMUNOLOGICAL PROFILE OF TRYPANOSOMA CRUZI-INFECTED MICE SUBMITTED TO CHEMOTHERAPY WITH BENZNIDAZOLE
Olivieri, B.P., De Souza, A.P., Cotta de Almeida, V., Araújo-Jorge, T.C. & De Castro, S.L.
Lab. de Biologia Celular - DUBC- Instituto Oswaldo Cruz/FIOCRUZ, Av. Brasil 4365, Rio de Janeiro RJ 21045-900
There is a permanent search of alternative drugs for Chagas'disease chemotherapy, being most of the work performed only in vitro and concentrating the in vivo studies mainly in the acute phase, focusing only parasitaemia and mortality levels, without investigating the impact of the treatment on the parasite-specific immune responses, as well as on the host tissues, direct or indirect targets of this response.
Eight-week Swiss male mice were infected with 104 bloodstream forms of T. cruzi (Y strain) and treated orally with 100 mg/kg benznidazole/day/0-8 dpi (day post-infection) (group Bz). Non-infected (NI) and infected non-treated (INT) groups were used as controls.
|Peak Paras |
At 7 dpi, the hematocrite significantly decreased in the order: NI>Bz> INT, while the distribution of blood white cells was similar between the three groups. The body weight for Bz was similar to NI, while for INT at 14 and 20 dpi it was significantly lower.
At different times, animals of each group were sacrificed, analysed by the above parameters and organs were collected for (a) hystological and immunocytochemical determination of the inflammatory process at heart and liver, (b) quantitative determination of different cells of spleen and thymus. Splenomegaly and thymic atrophy were higher in INT than in Bz. The spleens of INT showed a relative increase in B cells at the parasitaemia peak, followed latter by an increase in T cells, specially in CD8+. The increase on this subset is apparently reverted in Bz, showing an increase in the ratio CD4+/CD8+. Comparing the absolute cell numbers, B and T expansion occurr in both groups, being observed latter in Bz than in INT. A possible association between this expansion of B and T cells with an anti-T.cruzi activity in Bz will be investigated. This is a preliminary report and the aim of this project is start with the immunological profile of infected mice treated with benznidazole, as a model to further comparison with other experimental agents.
Supported by PAPES/FIOCRUZ and CNPq.
HOW DO TRYPANOSOMA CRUZI INFECTED MICE RESPOND TO TREATMENT WITH CYCLOPHOSPHAMIDE AND CICLOSPORIN A.
Calabrese, K.S., Zaverucha do Valle, T., Paradela, A.S.R.C. & Gonçalves da Costa, S.C.
Lab. de Imunomodulação, Dep. de Protozoologia, Instituto Oswaldo Cruz, FIOCRUZ-Av. Brasil 4365, Manguinhos, CEP 21045-900, RJ.
Cyclophosphamide (CY) is an alkylating agent used in cancer therapy and once used as an immunosupressor in transplanted pacients, since it inhibits DNA replication. Ciclosporine A (CsA) is a drug extracted from a fungi called Tolipocladium inflatum that acts in DNA inhibiting the production of IL-2, working, though, as an immunosupressor agent too.
In the first one we gave an 200mg/kg dose of CY or CsA intraperitoneally (ip) 2 days before infection. In the second one the same dose was given 5 days after infection. In the last one the mice received a therapeutic dosis of 3mg/kg of CY or 10mg/kg of CsA ip every other day (eod) starting the day after infection in a total of 5 dosis. Control groups were done including treated non infected, only infected and normal mice.
The effect of CY and CsA treatment on spleen and thymus weight was evaluated. The infected groups treated with 200mg/kg (2 days before infection) as well as 3mg/kg (eod) of CY show a spleen index peak on day 12 after infection. The same occured with the infected non treated mice The spleen of group treated 5 days after infection with 200 mg/kg presented a hypotrophy during the first 12 days of infection.
Both groups treated with 200mg/kg of CY, 2 days before and 5 days after infection, demonstrated a reduced thymus weight on day 8. This reducing was more severe in the second group. Thymus weight remains stable when therapeutic dosis were used.
In all infected groups treated with CsA the spleen reached three times the weight of normal ones on day 12 after infection. On the other hand, the thymus index of all infected groups treated or not with CsA were smaller than normal ones.
Among the animals treated with CY the highest parasitaemia and mortality was observed in groups treated with CY 200mg/kg 5 days after infection. Whereas in CsA treated ones, the group receiving therapeutic dosis had the most severe parasitaemia and mortality.
Histopathological aspects of heart show a bipolar model in which inflammatory reaction without parasites was observed in mice treated with CY 200mg/kg 2 days before infection. The oposite occurs in animals treated 5 days after infection. This polarity is not seem in CsA treated mice in which inflammatory reaction and parasite number are equivalent.
Analisis of cells in the miocardium has been done by immunocytochemistry.
ACTIVITY OF PHENOLIC COMPOUNDS ISOLATED FROM BRAZILIAN PROPOLIS AGAINST T. CRUZI
Marcucci, M.C.*, Dantas, A.P.**, Ferreres, F.§, Bankova, V.S.#, and De Castro, S.L.**
*Faculdade de Engenharia e Ciências Tecnológicas, Universidade Metropolitana de Santos (UNIMES), **Laboratório de Biologia Celular, DUBC, Instituto Oswaldo Cruz, FIOCRUZ, CP 926, 21045-900, Rio de Janeiro, RJ, Brazil, §Laboratorio de Fitoquímica, CEBAS (CSIC) Murcia, Spain, #Institute of Organic Chemistry with Centre of Phytochemistry, Sofia, Bulgaria
Propolis is a resin collect by honeybees from plant exudates and used to protect the hive. At least 200 compounds were identified in different samples and most of the biological activities of propolis (such as microbicidal, antiviral, and antitumoral) are associated with its phenolic components (Marcucci, Apidologie, 26, 83, 1995). Previous work showed that in vitro ethanolic extracts of commercial resin were active against the three forms of T.cruzi (Higashi & De Castro J. Ethnopharmacol., 43, 149, 1994), but with no efficacy in the treatment of T. cruzi-infected mice, even by oral administration of doses up to 5g resin/kg/day (De Castro & Higashi J. Ethnopharmacol., 46, 55, 1995). These results led us to attempted its fractionation, selection and purification of active(s) principle(s).
Brazilian propolis is extensively used, specially in Japan, in foods and beverages aiming to improve human health, but there are only few reports about its composition and biological activity. We are investigating the phenolic composition of Brazilian propolis and its possible trypanocidal activity. We report the isolation and elucidation of two phenolic compounds - 3,5-diprenyl-4-hydroxycinnamic acid (I) and 2,2 dimethyl-6-carboxyethenyl-2H-l-benzopyrane (II) - from Brazilian propolis (Mandirituba, Paraná State sample #97). Their activity against bloodstream forms of T. cruzi (Y strain) was assayed and untreated and crystal violet (CV)-treated parasites were used as controls. Cell counts were performed after 24 h/4°C and the calculated ED50values were: I: 1.205±0.083 mg/ml, II: 0.770±0.057 mg/ml CV: 0.187±0.021 mg/ml. Compound II was considered a likely candidate to be one of most important components in Brazilian propolis with antibacterial activity (Aga et al., Biosci.Biothech.Biochem. 58, 945, 1994). In our case, both compounds presented lower activity than CV. Since propolis is a complex mixture, its biological activities, in several instances, are due not just to one compound, but to a synergistic action of several components. Other compounds are being chemically characterised for further assays against T.cruzi aiming a more complete analysis of the trypanocidal activity of phenolics isolated from Brazilian propolis.
Supported by PAPES/FIOCRUZ, CNPq and FAPESP.
SCREENING PLANT EXTRACTS AGAINST TRYPANOSOMA CRUZI TRYPOMASTIGOTES IN CULTURE
bFernandes-Ferreira, E., bSantos, R.R., cSant'Ana, A.E.G., eGrandi, T.S.M., dBrandão, M., aRibeiro, A., aAlves, T.M.A. & aZani, C.L.
aCentro de Pesquisas René Rachou, Cx Postal 1743, 30.190-002, Belo Horizonte, MG. E-mail: email@example.com; bLaboratório de Farmacologia Aplicada, FIOCRUZ, Rio de Janeiro, RJ; cDep. de Química, UFAL, Maceió, AL; dEPAMIG, Belo Horizonte, MG; eFundação Zoo-Botânica, Belo Horizonte, MG.
In a continuing effort to discover new trypanocidal substances for the development of drugs to prevent or treat Chagas'disease, we screened 72 extracts and fractions obtained from Brazilian plants. Different parts of 21 species belonging to 13 families were collected in the "Mata Atlântica" and "Cerrado". The powdered plants were divided in two parts: one extracted in a Soxhlet apparatus with methylenedichloride and methanol while the other was macerated with water at room temperature. The organic solvents were removed by evaporation under reduced pressure and water by freeze-drying. Some of the extracts obtained were further fractionated by solvent partitioning to afford semi-purified fractions. Each extract or fraction (25 mg) was dissolved in saline (1 mL) by sonicating at 37 °C during 1 h. At 2.5 mg/mL none of them lysed sheep's red blood cells after 48 h contact at 4° C. The trypomastigotes used in this screening were obtained from a T. cruzi infected MK2 cell culture. The parasites were harvested from the cell culture and the trypomastigote concentration adjusted with RPMI to 5 x 105 cells/mL. This suspension (100 µL) was added to each well of a microtitre plate followed by extracts/fractions solutions to attain a final concentration of 2.5 mg/mL. After 24 and 48 h the number of viable trypomastigotes was evaluated by microscopy and by 3H-thymidine incorporation. Controls without drugs and with gentian violet (50 µg/mL) were performed in parallel. All experiments were run in triplicate. Under these conditions 42 extracts (58 %) were active, eliminating all parasites in 24 h. Three (4 %) were partially active, eliminating 40-80% of the parasites after 48 h contact. The other 27 extracts (38 %) were completely inactive. The active extracts detected in this faster and more safe model will now be tested against trypomastigotes in murine blood obtained from mice experimentally infected with T. cruzi. The extracts active in this latter model will be fractionated using a bioassay-guided protocol to isolate and identify their trypanocidal component(s).
Financial support: PRONEX, FAPEMIG, FIOCRUZ, CNPq.
3-EPI-URSOLIC ACID, A TRYPANOSOMICIDAL TRITERPENE FROM ARRABIDAEA TRIPLINERVEA
Leite, J. P. V.1, Lombardi, J. A.2, Chiari, E.3 & Oliveira, A. B.1
1. Faculdade de Farmácia, UFMG, Belo Horizonte, MG, Brazil - 2. Departamento de Botânica, ICB, UFMG, Belo Horizonte - 3. Departamento de Parasitologia, ICB, UFMG, Belo Horizonte
In the search for new trypanosomicidal compounds that could be used as chemoprophylatic agents for preventing the transmission of Trypanosoma cruzi by blood transfusion we have carried on a screening of about 100 plant extracts by in vitro assays against T. cruzi trypomastigotes (Zani et. al. Phytomed., 1995, 2: 47; Chiari et. al. Phytother. Res. 1996, 10: 636; Oliveira et al. Phytother. Res. 1996, 10: 292). A. triplinervea was one of the active plant extracts. Bioguided fractionation of the deffatted leaves ethanolic extract was undertaken by chromatography on a silica gel column that was eluted with mixtures of increasing polarity of hexane, dicloromethane, ethtyl acetate and methanol. The fraction eluted with dicloromethane - ethyl acetate (1:1) was shown to be active and three main components were detected by TLC. Rechromatography of this fraction on a silica gel column afforded the major component which was identified as 3-epi-ursolic acid, by spectrometric analyses (IR, H1NMR and C13NMR). This triterpene was shown to be trypanosomicidal in the dose of 1.0mg/mL. As far as we are concerned, this is the first report on the trypanosomicidal effect of a non quinonoid triterpene. Tingenone, a quinonoid triterpene, was the only trypanosomicidal triterpene previously known (Sepulveda-Boza and Cassels, Planta Med. 1996, 62: 98).
Financial support: FAPEMIG, CNPq.
EFFECT OF TERPENES IN CULTURES OF PLASMODIUM FALCIPARUM.
Planchart Rojas A,. Nuñez C.V.1, Franca Roque N.1, Kimura E.A., Katzin A. M.
Departamento de Parasitologia ICB-USP; 1Departamento de Química Fundamental IQ-USP.
Plasmodium falciparum is the predominant species which causes malaria, with 120,000,000 new cases and up to 1,000,000 deaths per year globally. In Brazil it is responsible for 45% of cases. The increasing prevalence of P. falciparum strains displaying resistance to many anti-malaria drugs has necessitated the development of new compounds.
We have demonstrated the presence of N-linked glycoproteins in the ring and young trophozoites stages, and established their relationship in the differentiation of intraerythrocytic stages in P. falciparum. N-linked glycoproteins in young trophozoites seem to be essential for development to the schizont stage, since young trophozoites in tunicamicyn-treated cultures do not develop into schizont stage. Presence of N-glycoproteins suggest the presence of dolichol, and the intermediates of this biochemical pathway. Inhibition of dolichol biosynthesis or competition with similar molecules, as terpenes, could identify a new targets for development the anti-malaria drugs.
We evaluated the effect of terpenes in P. falciparum culture. Dried branch and ground of Mikania sp (Dr. F.G. Cruz, UFBa) were extract with dichloromethane. Subsequently a partition with hexane/methanol:water (95:5) was performed, the more polar phase was chromatographed on Silica, the fraction eluted with dichloromethane:methanol (95:5) was applied on Sephadex LH20 column (80 cm x 2 cm). Several fractions were tested in P. falciparum cultures synchronous at the ring stage, a fraction eluted after 120 ml of methanol (F-VII.10) was active on parasite. Treatment P. falciparum with 5 µg F-VII.10 for 48 h caused a 81% inhibition in development of the trophozoite to schizont stage without pigment formation and the parasites died, after 72 h of treatment inhibition of development of parasites increased to 95%. Other compound with know structure, as b-selinene and HO-sesquiterpenes were tested in cultures of P. falciparum. Treatment P. falciparum with 20 µg b-selinene for 48 h caused a 95% inhibition in development of the trophozoite to schizont stage without pigment formation and the parasites died, treatment of parasites with HO-sesquiterpenes inhibits 50% development of parasites after 72 h of treatment.
Biochemical studies to determine if biosynthesis of dolichol, glycoproteins and/or ubiquinones is modified or inhibited by treating P. falciparum with the compounds above mentioned are being developed in our laboratory.
Supported by FAPESP, CNPq.
PHOTOSENSITIZED INACTIVATION OF TRYPANOSOMA CRUZI, PLASMODIUM FALCIPARUM AND BABESIA DIVERGENS BY LIPOPHILIC PHEOPHORBIDES FOR BLOOD TRANSFUSION
Grellier, P1, Santus, R2, Santana, J3, Lauria-Pires, L3, Teixeira, ARL3, Dagan A4, Gatt, S4 & Schrével, J1.
1: Lab. de Biologie Parasitaire et Chimiothérapie, Muséum National d'Histoire Naturelle, 61 rue Buffon 75231 Paris Cedex 05, France; 2: Lab. de Photobiologie, MNHN, Paris, France; 3: Lab. Multidisciplinar de Pesquisa em Doença de Chagas, Universidade de Brasilia; 4: Dept. of Biochemistry, Hebrew University-Hadassah School of Medicine, Jerusalem, Israel.
Blood transfusion can transmit parasitic infections, such as those caused by Trypanosoma cruzi (Chagas' disease), Plasmodium (malaria) and Babesia (babesiosis). A higher degree of blood transfusion would be reached if methods were available for inactivating such parasites. We evaluated the effectiveness of photosensitization using lipophilic pheophorbides and red light illumination to eradicate red blood cells (RBC) infected with Plasmodium falciparum and with Babesia divergens, in whole blood. Studies by fluorescence microscopy and conventional fluorometry demonstrated the specific accumulation of pheophorbides in the infected RBC, compared with the uninfected RBC. The effectiveness of different derivatives in eradicating infected RBC was first estimated on parasite cultures. The best photosensitizer was the N-(4-butanol) pheophorbamide derivative (Ph4-OH) at 0.2 µM concentration and 5-min illumination. In whole blood, the eradication of B. divergens- and P. falciparum-infected RBC was obtained with 2 µM Ph4-OH and 10 min and 20 min illumination, respectively. Under these conditions of photosensitization, low levels of RBC hemolysis was noted only after 2 weeks of storage at 4°C and a subsequent 48 h incubation at 37°C. Furthermore, no reduction of negative charges of treated RBC was observed as well as no increase in the methemoglobin content. The study of the partition of Ph4-OH in the blood fractions has shown that Ph4-OH is mainly transported in plasma by high density lipoproteins (HDL) (Grellier P., et al. 1997. Vox Sang, 72: 211-220). This high affinity for HDL may explain the selective accumulation of lipophilic pheophorbide derivatives into the intracellular parasites as it was observed for HDL lipids (Grellier P., et al. 1991. J. Cell. Biol., 112: 267-277). Ph4-OH was also the most efficient pheophorbide derivative to eradicate T. cruzi trypomastigotes in vitro, in serum : 5 log10 killing was obtained with 5 µM Ph4-OH and 30 min illumination. Photosensitization with pheophorbide derivatives may be a promising approach to inactivation of transfusion-transmissible parasites in blood bank units.
STUDY OF THE INTERACTION BETWEEN TRIPANOCIDE 3-NITRO BENZALDEYDE GUANYL HYDRAZONE AND BOVINE SERUM ALBUMIN BY EQUILIBRIUM DIALYSIS AND NUCLEAR MAGNETIC RESONANCE.
Tinoco, L. W. and Figueroa-Villar, J. D.*
Departamento de Química, Instituto Militar de Engenharia, Praça General Tibúrcio 80, Rio de Janeiro, 22290-270, RJ, Brasil.
Our previous studies with guanyl hydrazones showed that these compounds are active against the trypomastigote forms of Trypanosoma cruzi in infected blood.1 One of the members of this family, 3-nitrobenzaldeyde guanyl hydrazone (3NBGH, ID50 182.6 mM) showed to be three times more active than gentian violet (ID50 536 mM). The use of this family of compounds to treat contaminated blood depends on their ability to act against the parasite without affecting the blood components. Preliminary results showed that the greater anti-trypanosome activity of the guanyl hydrazones is not associated with greater toxicity for the red cells.1 In order to study the possible interaction of these drugs with blood proteins, we have carried out nuclear magnetic resonance (NMR) and equilibrium dialysis studies of the interaction of 3NBGH with bovine serum albumin (BSA).
The equilibrium dialysis results show that the interaction of 3NBGH with BSA is weak and that the changes between the free and bounds states of the drug are fast. In this way, it was possible to determine the number of binding sites per molecule of BSA and the binding constant between the drug and the protein. The NMR relaxation times studies (T1 and T2) made possible to determine that the binding of 3NBGH with BSA is mainly electrostatic, and that it occurs mostly through the cationic end of the drug. It was also determined that the nitro group is a secondary site for interaction. Our results indicate that 3NBGH should be more appropriate than gentian violet to treat infected blood in blood banks. (CNPq-PADCT, CAPES, FINEP)
1- J.C. Messeder, L.W. Tinoco, E. M. Souza, R. Santa Rita, S. L. de Castro and J. D. Figueroa Villar, Bioorg. Med. Chem. Let., 5 (24), 3079-3084, 1995.
IN VIVO ACTIVITY OF SCH 56592 AGAINST DIFFERENT TRYPANOSOMA CRUZI STRAINS IN THE MURINE MODEL
Molina, J. T.1.3; Araújo, M.S.S.1;Urbina, J.A.2 & Brener, Z11.
Centro de Pesquisas René Rachou, FIOCRUZ, Belo Horizonte, MG, Brasil; 2. Biological Chemistry, IVIC, Caracas, Venezuela; 3. Instituto de Zoología, Universidad Central de Venezuela, Caracas, Venezuela.
CH56592 is a novel triazole antifungal agent that is a hidroxilated derivated of SCH51048 (Saksena et al., 1995). SCH56592 (SCH) was found to be 30-100 time more potent in vitro than ketoconazole or DO870 as antiproliferative agent and sterol biosynthesis inhibitor against epimastigotes and amastigotes of T. cruzi (Sugar & Liu, 1996). We evaluated in vivo the activity of SCH56592 and benznidazole (BZ) in murine model of acute Chagas disease on different T. cruzi strains (Y, SC-28, Colombiana, VL-10 and CL). Groups of 10 adults Swiss albino female were inoculated intraperitoneally with 104 blood trypomastigotes, the treatment was initiated 4 days after infection and given daily for 28 days, followed by a 7 days rest and another 14 days of treatment. The drug was suspended in 2% methylcellulose containing 0,5% Tween 80 and given by oral route. The groups controls received this suspension without the drug as a placebo. The methods of cure were assessed by hemoculture and xenodiagnosis, 30-60 days after the end of treatment. The percentage of cure is showed in table:
|T. cruzi strains % cure|
|SCH (5) ||80||70||50||50||100|
The results clearly indicate that SCH56592 is more active against resistant strains ( SC-28, Colombiana and VL-10), partially resistant (Y) and susceptible (CL) that BZ., currently used in the treatment and indicated that it should be further evaluated in clinical trials for the treatment of patients with Chagas disease.
ACTIVITY OF D0870-LOADED PLA-PEG NANOPARTICLES AGAINST A MURINE MODEL OF TRYPANOSOMA CRUZI INFECTION
Rodrigues Jr. J.M.1, Molina J. T.2 Gref R.3, Urbina J.A.4 and Brener, Z2
1. Depto. Produtos Farmacêuticos, Fac. de Farmácia da UFMG. Av. Olegário Maciel 2360, 30.180-112, Belo Horizonte (MG). 2. Centro de Pesquisas René Rachou. Belo Horizonte, MG, Brasil. 3. Laboratoire de Chimie-Physique Macromoléculaire (CNRS - URA 494), Nancy, France. 4. Biological Chemistry Lab., Instituto Venezolano de Investigaciones, Ap. 21827, Caracas (Venezuela)
Poly lactide (PLA) and poly ethileneglycol (PEG) biodegradable nanoparticles has shown their ability to avoid the opsonization and uptake by the phagocytes. The long plasma circulating characteristic of these systems offers the possibility of improving drug biovailability. The aim of this work was the incorporation of a sterol biosynthesis inhibitor, D0870, into stealth nanoparticles and evaluate their activity against Trypanosoma cruzi. Drug-loaded nanoparticles were obtained by the emulsion-method and showed average diameter comprised between 150 and 200 nm. The in vivo evaluation was carried out using an acute mouse model. Swiss albino mice, 10-15 by group, were inoculated with 104 blood stream trypomastigotes of CL strain. Four days after infection, mice were treated with drug-loaded nanoparticles by intravenous route during 30 consecutive days at doses of 0.75 to 3.0 mg/kg. D 0870 administered by oral route at the dose of 5 mg/kg was used as control. Untreated control received saline only. The cure rate was evaluated by hemoculture in liver-infusion tryptose (LIT) and xenodiagnostics with second instar Triatoma infestans nymphis 30 days after the end of treatment. Mice treated with D0870-loaded nanospheres had 100 % of blood parasite elimination after the first four injections in all doses. 50% of the untreated group died before the end of the experiment. No mortality was observed in treated groups. Although the smallest dose of drug-loaded nanoparticles showed not able to cure mice (only 30% of cure rate), mice which received the doses of 1.5 and 3.0 mg/kg had 100% of cure in both methods. The same cure rate was observed for oral treated group. Unloaded nanoparticles had no activity against T.cruzi.
Suported by CNPq, CONICIT, FAPEMIG
EFFECT OF NITRO GROUP ON THE TRYPANOCIDAL ACTIVITY OF NEW HETEROCYCLICS SYNTHESIZED FROM QUINONES ISOLATED FROM TABEBUIA S.P.
Dantas, A.P.1, Emery, F.S.2, De Moura, K,C,G.2, Santana, L.A.2, Pinto, M.C.F.R.2, Neves Pinto, C.2 , & Pinto, A.V.2 & de Castro, S.L.1
1Depto de Ultra-estrutura e Biol. Celular, IOC/FIOCRUZ,; 2Núcleo de Pesquisas em Prod. Naturais, UFRJ, RJ, Brazil
In a previous report, naphthopyrane skeletons have been synthesized and assayed as trypanocidal agents (Pinto et al., Arzneinm.Forschung 47I:74, 1997). These compounds are easily prepared from the reaction of aldehydes, ammoniating agents and quinones extracted from Tabebuia s.p., typical species of the Brazilian flora. Since the nitro group in several microbicides is a strong enzymophorus center for reductases, including nifurtimox and benznidazole, this characteristic led us to incorporate this group in naphthopyranimidazoles (I-IV).From the reaction of b-lapachone with o-, m- and p-nitrobenzaldehyde and ammonium acetate we obtained the corresponding imidazolic compounds (Ib,Ic,Id). The trypanocidal activity was assayed against bloodstream trypomastigotes of T. cruzi (Y strain) through the parameter ID50/24 h/4° C and compared with crystal violet (CV).
The activity of Ib, Ic and Id when compared with the parent compound Ia (R1=H; ID50 »14x<CV) showed that introduction of the nitro group on the benzene linked to the imidazole ring did not enhance the trypanocidal effect. However, this group on the para position (Ib) showed an ID50 value bellow CV. In relation to its meta and ortho analogues, the greater activity of Ib, where the inductive (electron withdrawing) effect upon the imidazole ring is higher, suggests that the para position presents a direct correlation with trypanocidal activity. The 21 compounds analysed so far, 12 with ID50£CV, a significative point in terms of a biological screening, present the following order of activity: IV>III>(I,II). These results lead us to the synthesis of new imidazoles of the type IV and oxazoles with an open chain (III). It is interesting to point out that imidazole rings are present in several known antiparasitic drugs.
Supported by CNPq, PAPES/FIOCRUZ, UFRJ
EFFECT OF THE ALKYLLYSOPHOSPHOLIPIDS ON TRYPANOSOMA CRUZI AND ON ITS INTERACTION WITH HEART CELLS
Santa-Rita, R.M., Jesus, A.M.C., Rodrigues, R.M., Barbosa, H.S., Meirelles, M.N.L. & De Castro, S.L.
Dept. Ultra-estrutura e Biologia Celular, Instituto Oswaldo Cruz/FIOCRUZ, 21045-900, RJ, Brazil
As part of a programme about the antileishmanial and antitrypanosomal activities of alkyllysophospholipids (ALPs), we investigate the effect of edelfosine (octadecyl-methyl-glycero-phosphocholine - Ed) on T. cruzi (Y strain) and on its interaction with heart muscle cells (HMC). ALPs were designed as potential immunomodulators and antimetabolites of phospholipid (PL) metabolism and later have been studied in tumour models, reaching some of them anticancer clinical trials.
The activity of Ed against trypomastigotes (TRY) increases with the temperature and decreases in the presence of blood. This decrease - measured by ID50/24h - was also observed at 4°C with ilmofosine (63.8±8.2 X 188.1±15.8mM) and with mitelfosine (47.2±7.0 X 166.9±11.0 mM). Ed was also assayed on the proliferation of amastigotes (AMA) and epimastigotes (EPI). After 24h of TRY-HMC interaction, the cultures were washed and treated with Ed, that caused both a dose- and a time-dependent inhibition on the infection process. After 3 days of treatment (dt), we detected both intact ans severely damage parasites and 40% of cells harboured differentiated TRY both in Ed7.5mM and control, corresponding, respectively to 1.5% and 22%of infected HMC.
EPI treated with 5µM Ed for 24h showed as initial alterations cell swelling and disorganization of the inner mitochondrion membrane, and with 10µM Ed, an extensive vacuolisation, presence of autophagic vacuoles, and disintegration of organelles. Cell swelling being one of the initial alterations observed suggests that Ed may induce permeability changes on the parasite's membrane. The antileishmanial effect of ALPs seems to involve interaction with membrane components, specially enzymes of PL metabolism (Croft et al., J.Antimicrob.Chemother. 38:1041,1996; Achterberg & Gercken, Mol.Biochem.Parasitol. 23:117,1987). Preliminary results showed that EPI treated with 30 mM Ed/4h (ID50/4h= 64.0±16.6 mM) after incubation with 32P presented alteration on the pattern of radioactivity incorporation in the different classes of PL. The next step is to investigate if Ed, a known inhibitor of mammalian PI-PLase C and PKC, causes a similar effect on T. cruzi.
Supported by EC (INCO-DC IC18-CT96-0084), CNPq and FIOCRUZ
TRYCICLIC DRUGS MODIFY EXPERIMENTAL CHAGAS DISEASE EVOLUTION IN TWO EXPERIMENTAL MODELS
Rivarola H.W..; Fernândez, A.R.* ; Enders, J.E. ; Paglini-Oliva, P. C t. Física Biom,dica, Inst. Fisiología, Fac. Cs. Médicas (U.N.Cba.). Santa Rosa 1085, 5000 Córdoba, Argentina - Support : SECyT , CONICOR -
*Established Investigator CONICET. Argentina.
Thioridazine (THI) and Clomipramine (CLO) are tricyclic drugs used in clinic therapeutics as neuroleptic drugs .Both have letal effects upon epimastigotes and trypomastigotes of T. cruzi, Tulahu,n strain ,in vitro. In order to analize the use of these trypanocidal drugs as therapy of T. cruzi infected mice we used two experimental models:1) Albino Swiss mice inoculated with 7x104 trypomastigotes/ animal, 2) inoculated with 50
parasites/ mouse. The infected animals were divided in the following groups: a) without treatment (control) b) treated with THI 80 mg / Kg for 3 days oral way c) treated with CLO 40 mg / Kg injected intraperitoneally 1 hour and 7 days post infection (pi) . Parasitemias, survival and histopathologic analysis of heart were done.
Non treated mice from model 1 survived 12 ñ 2 days pi reaching parasitemias of 6x105 parasites/ml. and from model 2 , 70% survived acute phase with a parasitemia of 3.25 x105 parasites/ml at 45 days pi and reached chronic phase without detectable parasites(135 days pi). Hearts from these groups presented morphological alterations typical of chagasic miocarditis .
THI treatment modified parasitemias levels turning them negative in both experimental models by day 20pi.Survival of both groups was 9 months and the histopathologic studies of heart showed mononuclear infiltrates without amastigotes nests during acute phase. Only isolated focus were detected at 75 pi. None structural alterations were detected at the end of the experiment.
CLO treatment also modified parasitemias levels in both models. We could not detect trypomastigotes in blood samples and the survival of these animals were similar to the treated with THI.Histopathological studies showed mild infiltrates .
The present results show that both tricyclic drugs have a direct effect upon parasitemia independently of the T. cruzi amount received by the host , significantly improved survival of treated mice and modified the evolution
of the experimental Chagas'disease.
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COUMARINS: POTENTIAL LEAD COMPOUNDS AGAINST CHAGAS DISEASE
Monteiro, M. R. 1; Vieira, P. C. 1, Fernandes, J. B.; Silva, M. F. G. F. 1; Pupo, M. T. 2; Pavão, F. 2, Oliva, G. 2, Albuquerque, S. 3
1 Laboratório de Produtos Naturais, DQ, UFSCar, C.P.676, CEP13565-905, São Carlos, SP, Brazil.
2 Laboratório de Cristalografia de Proteínas e Biologia Estrutural, IFSC, USP, C.P.369, CEP13560-970, São Carlos, SP, Brazil.
3 Departamento de Ciências da Saúde, FCFRP, USP, Ribeirão Preto, SP, Brazil.
Natural products have been extensively used as an important source of medicinal agents, particularly those from superior plants, which have provided most of the active compounds nowadays used as drugs or that served as lead compounds for further development: natural products (or their derivatives and analogues) represent 50% of all the drugs in clinical use. In the search for new compounds with trypanocidal activity, crude extracts, fractionated mixtures and purified compounds from plants of the families Rutaceae, Meliaceae and Myrcinaceae were evaluated using an in vivo bioassay against the trypomastigote form of T.cruzi. Through this bio-monitored approach, we were able to isolate from Cabralea canjerana (Meliaceae) the coumarin escopoletin, which showed LC50 of 59 mg/ml. Prenylated coumarins like osthol and phebalosin have also shown activity. Preliminary modeling studies using the program DOCK3.5, using as a putative target the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase from T.cruzi (GAPDH), have shown that these coumarins could have favorable energetic and topologic complementarity to the cofactor (NAD) binding site of the enzyme. Subsequent inhibitory assays of these coumarins against purified recombinant T.cruzi GAPDH, have shown that scopoletin, osthol and phebalosin present IC50 of 260 mM, 210 mM and 190 mM, respectively. Crystallographic studies of GAPDH complexed with coumarins are currently being sought.
Supported by FAPESP, PADCT-SBIO, CNPq, FINEP, WHO and the Howard Hughes Medical Institute.
IN THE SEARCH OF LEAD COMPOUNDS AGAINST CHAGAS DISEASE: SCREENING OF NATURAL PRODUCTS AGAINST THE ENZYME GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE (GAPDH) FROM TRYPANOSOMA CRUZI.
Pupo, M. T. 1; Oliva, G. 1 and Vieira, P. C.2
1 Laboratório de Cristalografia de Proteínas e Biologia Estrutural, Instituto de Física de São Carlos, USP, C.P.369, CEP13560-970, São Carlos, SP, Brazil.
2 Laboratório de Produtos Naturais, Departamento de Química, UFSCar, C.P.676, CEP13565-905, São Carlos, SP, Brazil.
The plant kingdom is a potential reservoir of new active molecules to be discovered that can act as leads to the design of new drugs. Medicinal plants have played a crucial role in the treatment of parasitic diseases. In the attempt to find active compounds against Chagas disease, we developed a screening test using the glycolitic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Trypanosoma cruzi as target The infectious form of T. cruzi depends on the glycolitic pathway as its energetic source; thus compounds blocking glycolysis could be used as potential drugs against this illness. The expression, purification, crystallization and the structure determination to medium resolution of T.cruzi GAPDH was already obtained and potential lead compounds are now being sought. Crude extracts, fractionated mixtures and pure compounds that showed in vitro activity against the trypamastigote form of T. cruzi were tested against the enzyme GAPDH. Species from genus Rapanea (Myrsinaceae) showed promising behavior, having extracts that inhibited 80-95% of the enzymatic activity in concentrations of 5mg/mL. Species from Meliaceae (Azadirachta indica and Trichilia claussenii), also presented inhibitory properties. The phytochemical studies of these extracts are being guided by this assay. We also tested a variety of pure compounds such as triterpenoids, steroids, sesquiterpenoids, antraquinones and coumarins.The latter class of compounds showed the most promising group of substances as potential GAPDH inhibitors (Table).
|1- seselin; 2- angustifolin; 3- phebalosin; 4- osthol; 5- scopoletin; 6- bergapten; 7- umbelliferone|
The structural studies of complexes of the inhibitory compounds with the target enzyme is an essential step for the understanding of the molecular mechanisms of inhibition and further optimization by structure-based design. For this purpose, extensive soaking and co-crystallization experiments are being conducted.
Supported by FAPESP, PADCT-SBIO, CNPq, FINEP, WHO and the Howard Hughes Medical Institute.
MODELLING OF A SPECIFIC INHIBITOR FOR IRON SUPEROXIDE DISMUTASE FROM LEISHMANIA DONOVANI CHAGASI
Silva # * , C. H. T. de P. and Garratt * , R. C.
# Instituto de Química de São Carlos, Universidade de São Paulo, Av. Dr. Carlos Botelho, 1465, São Carlos, 13560-250 SP, Brasil. * Instituto de Física de São Carlos, Universidade de São Paulo, Av. Dr. Carlos Botelho, 1465, São Carlos, 13560-250 SP, Brasil.
By virtue of their ability to dismutate O2 - . into H2O2 and O2, Superoxide dismutases (SODs) have been considered virulence factors in a number of intracellular pathogens as they represent a mechanism for evasion of the hosts immune attack.
Recently ( Silva et al., Mem. Inst. Oswaldo Cruz, 91 ( suppl.): 286, 1996 ), molecular modelling by structural homology was performed for the Leishmania Fe SOD, and the model obtained revealed differences in the proximities of the active site, when compaired with the human SOD crystal structure.
In this work, a screening of a database containing more than 100000 compounds was performed using the programs DOCK and LUDI . A limited number of compounds was selected using criteria such as synthetic viability and the existence of few conformers. In these compounds, structural bioisosteric modifications were designed in order to confer specificity and membrane penetrability, which can be evaluated calculating the lipophilicity of the compound based on its structure.
The results suggest that the new compounds should possess specificity, synthetic viability and the ability to cross membranes and may be of use in the chemotherapic treatment of Leishmaniasis.
PHENOTHIAZINE DERIVATIVES INHIBIT THE ACTIVITY OF ENERGY-DEPENDENT EFFLUX TRANSPORTERS IN LEISHMANIA PROMASTIGOTES
1Frézard, F., 2Essodaigui, M., 3Moreira, E.S.A., 3Anacleto, C., 2Garnier-Suillerot, A.
2Laboratoire de Physicochimie Biomoléculaire et Cellulaire, (CNRS - 2056), UFR Biomédicale, Université Paris Nord, 74 rue Marcel Cachin, Bobigny cedex 93017, France. 1Depart. de Fisiologia e Biofísica, 3Depart. de Microbiologia, ICB, UFMG, C.P.486, Belo Horizonte, 30161-910, MG, Brasil.
The phenothiazine neuroleptics, of which chlorpromazine is the prototype, are widely used for their antipsychotic, antianxiety, and antiemetic effect. Recently, their antileishmanial activity have been reported. However, their mechanism of action was not investigated. In other reports, these compounds were shown to inhibit the activity of the mammalian multidrug resistance (MDR) transporter P-glycoprotein. In view of these data, we investigated the possibility that phenothiazine might kill Leishmania by inhibiting an essential membrane transporter analog to P-glycoprotein. The present work have been divided in two parts. As a first part, we investigated in intact Leishmania promastigotes the presence of energy-dependent pump(s), using two fluorescent probes of the mammalian MDR transporters, including calcein acetoxy-methyl ester (Calc-AM). The conclusion of this first part was that energy-dependent efflux pump(s), capable of transporting Calc-AM and calcein, does exist in Leishmania promastigotes. As a second part, we studied the effect of five phenothiazine derivatives on the the accumulation of Calc-AM in L. guyanensis promastigotes, and on the efflux of calcein, product of the hydrolysis of Calc-AM by intracellular esterases. All tested phenothiazine derivatives, chlorpromazine (CLPO), triflupromazine (TFPO), trifluoperazine (TFP), prochlorperazine (PCLP), and thioridazine (TRD), did inhibit the efflux of Calc-AM and calcein from promastigotes. TFP, PCLP and TRD were more efficient than CLPO and TFPO. TFP at a concentration of 5 micromolar was sufficient to impair the function of the membrane transporter.
This work was supported by Université Paris Nord (France) and FAPEMIG (Brazil).
SYNTHESIS OF NEW 2-PROPENE-1-AMINE DERIVATIVES, TRYPANOCIDE ACTIVITIES AND THEIR SQUALENE EPOXIDASE ACTIVITY INHIBITION.
Oliveira, D.A.* , De Souza, A.O.* , Fernandes, A.M.P.*, Durán, M.*, Darcio G. Pereira*, Rodriguez, J.A& , Melo, P.S.&, De Castro, S.L.#, Souza-Brito, A.R.M.# , Haun, M.&, De Conti, R.*, Esposito, E.& , & Durán, N.*
Inst. Química, Biol. Chem. Lab.*, Inst.Biologia, Physiol. Depart.# , Inst. Biol. Culture Cell Laboratory& UNICAMP, Campinas, CEP 13083-970, S.P., Brazil , Ultrastruct. Mol. Biol.Depart., FIOCRUZ, R.J., Brazil.# and Fund.Tropical& , Campinas, S.P. Brazil.
A series of 3-(4'-bromo[1,1'-biphenyl]-4-yl)-3-(X)-N,N-dimethyl-2-propen-1-amine 2-propen-1-amines were synthetized (De Conti et al. Microbios 85, 83 (1996)) and a structure-activity relationship was previously discussed (De Conti et al. Eur.J. Med. Chem. 31, 915 (1996)). All of these compounds exhibited a high trypanocide activities in vitro on the three Trypanosoma cruzi forms (Oliveira et al. Mem. Inst. Oswaldo Cruz, 91, 320 (1996)). It was previously shown that 2-propene-1-amines act on ergosterol synthetase of T. cruzi epimastigote form (De Conti et al. XXIII Reunião Anual da SBBq, Maio 1994, Abstr. O1 (1993)). In order to study the structural-activity reltionship new trypanocides were synthetized. A new 2-propen-1-amine (X=2-furanyl) showed a trypanocidal activity of ID50 for trypamastigote 35. 1 ± 1.2, meanswhile X= phenyl showed a 174.8 ± 7.8 value.The logP for the furanyl and phenyl are 1.34 and 1.56, respectively, meaning that the liposolubility in this region does not affect the trypanocide activity. However, the lower resonance energy of furanyl (24 kcal/mol) compared to phenyl group (41 kcal/mol) could be important in this high activity of the furanyl against phenyl group in that position. The unsubstituted 2-propen-1-amine (X =phenyl) showed a trypanocidal selectivity for the cis-isomer (3 fold more effective than trans-isomer). Similar results with the bromo-derivative (X=Br-phenyl)(3.5 fold more effective than trans-isomer). Then, the cis-isomer of 2-propen-1-amines is the most active for trypanocide activity. The isomer mixture (cis/trans 1:1) of unsubstituted 2-propen-1-amine (X=phenyl) acting on T. rubrum (cepa 5507 CCT, from UFPE Collection, Recife, PE) was studied. The profile and MIC of the antifungal activity of unsubstituted 2-propen-1-amine (X=H) were determined by serial dilution with Sabouraud's dextrose broth (pH 6.5) in Petri plates. The results at three different concentrations (10-120 mM) showed that in 10th day only the control showed growth. After fungal sterol composition analyses by TLC, GLC anf HPLC a predominance of squalene in the treated fungus compared with untreated one was observed. This is an indication that this involves the inhibition of ergosterol biosynthesis at the point of squalene epoxidase. A systematic enzymatic inhibition study by the isomers and the new 2-propen-amines is in progress.
FURTHER STUDIES ON THE IN VIVO EFFECTS OF THE BROMO N,N-DIMETHYL-2-PROPEN-1-AMINE DERIVATIVE
Pereira D.G.*, de Castro S.L.** & Durán N.*
*Laboratório de Química Biológica, Instituto de Química, UNICAMP, SP;
**Depto. de Ultra-Estrutura e Biologia Celular, IOC/FIOCRUZ,RJ, Brazil.
It has been previously shown that the oral administration of the bromo derivative of 3-(4'-bromo-[1,1'-biphenyl]-4yl)-3-(4-X-phenyl)-N,N-dimethyl-2-propen-1-amine (B) displayed a strong suppressive effect on the parasitemia (Par) and cumulative mortality (%CM) of Swiss albino male mice infected with T.cruzi (104/mice, Y strain), while the unsubstituted analogue (H) was ineffective (Pereira et al., Mem.Inst. Oswaldo Cruz, 91, suppl., 313, 1996). In vitro studies showed that both derivatives displayed high trypanocidal activity and low cytotoxicity to bacteria and mammalian cells (de Conti et. al., Microbios 85: 83, 1996).
Giving continuity to this study, we measured the serum creatine kinase (CK) levels, and observed that, following the animals individually, control and treated groups showed significantly higher CK levels on 7 dpi (days post-infection) than the day before inoculation. Moreover, comparing CK values on 7 dpi, the group B20 (20mg B/kg/0-9dpi; Par=0, %CM=0) showed significantly lower levels of the enzyme than control (p<0.05) and H-treated groups.
We also assayed other schemes of treatment with B (5 mg/kg/0-9dpi and 200 mg/kg/5dpi) and compared with benznidazol (Bz 100mg/kg/0-9dpi):
|Par (104 p/ml)||226||12.4*||3.3*||3.3*|
|* p<0.01 t-Student test|
The results showed consistent parasitemic suppressions combined with prolonged time survivals, under all treatment. However, both bromo derivative treatments did not produce curative effect. The decrease of CK levels in the group B20 on the parasitemia peak (7 dpi) may be indicative of a reduced muscle damage due to this treatment. However, further experiments are needed in order to investigate if CK is a reliable parameter to monitor the course of infection.
Supported by: PADCT/FINEP, FAPESP (grant no. 96/1330-7) and FIOCRUZ.
PRELIMINARY STRUCTURE - ACTIVITY RELATIONSHIPS OF THE SOME N-ARYL-SYDNONES IN LEISHMANIA AMAZONENSIS PROMASTIGOTES
Torres. M.A.1,Echevarria, A.2 , Canto-Cavalheiro, M. M.1, and Leon, L.L.1
1 Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Depto. de Imunologia, 21045-900, Rio de Janeiro, RJ, Brasil
2 Universidade Federal Rural de Rio de Janeiro. Depto. de Química, 23851-970, Itaguaí, RJ, Brasil
Mesoionic systems have provided numerous compounds with useful and wide-ranging biological activities. These compounds have well separated regions of positive and negative charges. The association of this with the polyhetero-atomic system suggested a high probability of strong interactions with biomolecules. Searching for new anti-Leishmania drugs we chose and tested some mesoionic derivatives of the N-aryl-sydnones class, never used before on trypanosomatids. In this work we report the in vitro anti-Leishmania activity of four N-aryl-sydnones. Possible structure-activity relationships were investigated using computational tecniques. Leishmania amazonensis (MHOM/BR/77/LTB0016 strain) promastigotes were grown at 25 °C in Schneider's Drosophila medium supplemented with 10 % (v/v) heat-inactivated fetal calf serun and harvested from the medium in the late-log phase. Parasites were resuspended in fresh medium, counted in Neubauer chamber, adjusted to a concentration of 4x106 parasites/mL, and assayed with/without the drug, using pentamidine isethionate as reference drug. Theoretical calculations based on the semi-empirical AM1 method were performed with all compounds. Their most stable conformations were deduced from calculations. The resulting geometries were then employed for the determination of theoretical parameters like HOMO and LUMO energies, charges and dipole moments for each molecule. The preliminary results were promissing showing significant in vitro anti-Leishmania activity of the cyclohexylamine and chloro N-aryl-sydnones derivatives (LD50 < 30 mM). These results suggest that, in this small mumber of compounds, the variation of the para substituents of the benzene ring within the series appear to further modulate affinity so over a relatively wide range. On the other hand, the analysis of theoretical calculations indicate that the most relevant property in the activity is the dipole moment, and that higher in vitro anti-Leishmania activity values, are associated with higher dipole moments. Therefore, this indicates that the dipole is influential at the binding site suggesting that a shift of negative charge in the molecule favors high activity by electrostatic interactions with biomolecules. Nevertheless, the para substituents differ not only in their electronic character but also wiht respect to, for example, their lipophilic and steric contribution. Other than our preliminary studies, and analysis of the relationships between chemistry structure and anti-Leishmania activity has not been previously reported and, obviously, a detailed analysis of such effect is required.
Supported by CNPq/FIOCRUZ
IgG- NITROBENZOIC ACID CONJUGATES KILLS TRYPANOSOMA CRUZI
Carvalhaes, M. S* .; Santana, J. M**. & Teixeira, A. R. L**.
**Laboratório Multidisciplinar de Pesquisa em Doença de Chagas, UnB; * Laboratório de Imunoquímica, Universidade Federal de Goiás, Goiânia-GO, CP 131, CEP 74001-970
Hybrid molecules were constructed by direct linkage of a small M.W. drug, 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB), to IgG antibodies against T. cruzi antigens. Hybrid molecules were also constructed using succinimidyl-piridylthio-propionate (SPDP). Biological activity of hybrid molecules were assayed mixing 100ml (100mg) of each kind of molecule to 100ml of epimastigote suspension of T. cruzi (2 x 106 parasites/ml), and after 72h of incubation at 28oC, the number of free swimming parasites were determined. The normal rabbit IgG alone or linked to DTNB, with or without SPDP, immobilized only 10% ± 3,2 of the parasites. Rabbit immune IgG (RIIgG) immobilized 10% ± 4,3 of the cells; whereas, RIIgG linked to IgG in presence or absence of SPDP, immobilized 30% ± 5,3 and 45%± 5,7 of the parasites, respectively. We know by absorciometry, that two molecules of DTNB are linked to each IgG. These results indicated that immunoconjugates containing more drug are promising trypanocidal molecules.
IMMUNOCONJUGATE IgG - CLB KILLS TRYPANOSOMA CRUZI IN VITRO AND IN VIVO
* Santos, W.; * Silva, C. F.; * Rodriques, A. M.; · Santana , J. M; ·Teixeira, A. R. L; * Carvalhaes, M. S.
* Laboratório de Imunoquímica, IPTSP, Universidade Federal de Goiás, Goiânia-Goiás, Caixa Postal 131, CEP 74001-970
·Laboratório Multidisciplinar de Pesquisas em Doenças de Chagas, Universidade de Brasília, Brasília-DF, CEP 70910-900, Brazil.
We postulated that anti-Trypanosoma cruzi antibodies derivatized with low molecular weight trypanocidal drugs, may not be visible by the phagocitic system and therefore, would remain in host's circulation, and could direct the toxic drugs to the parasite. IgG molecules purified from rabbit serum by affinity chromatografy in sepharose-4B- protein A, were linked to chlorambucil spontaneously in 0,1M NaHCO3, pH 8,5, at room temperature for 4h ( molar ratio 1:8 ). Mobility of epimastigote forms (Y strain) was partially lost in vitro after incubation with immune IgG linked to CLB (IIgG-CLB). Death and growth inhibition of T. cruzi began at 48h and was maximum at 96h (% of death = 85,92 ; % of growth inhibition = 62,09). In control experiments, 500 mg/mL of normal IgG (NIgG), immune IgG (I IgG) , normal IgG linked to CLB (NIgG- CLB), in PBS or medium, did not kill and inhibit the growth of T. cruzi epimastigotes forms(1O 6 / mL). The endovenous administration of IIgG-CLB (200 mL of solutions 3 and 6 mM ) at 3, 5, 7 and 10 days after infection of BALB /c mice with 103 trypomastigotes forms of T. cruzi, Y strain, showed 22,7 - fold reduction in levels of parasitemia ( 1.456 ± 834,62) , when compared with those seen in animals treated with the some doses of NIgG , IIgG, NIgG- CLB or PBS ( 33.170 ± 17.606). These results show that treatment of T. cruzi with immunoconjugates constructed by hybridization of specific IgG antibodies with CLB can lead to death of the parasite.
GLUCANTIME ENTRAPPED IN LIPOSOMES: OPTIMIZATION OF ENCAPSULATION, EVALUATION AGAINST EXPERIMENTAL KALA-AZAR
1Frézard, F., 2Demicheli, C., 3Soares, C.F., 3De Souza, L.F., 3Michalick, M.S.M.
1Depart. de Fisiologia-Biofísica, ICB, UFMG, C.P. 486, 2Depart. de Química, ICEX, UFMG, C.P. 702, 3Depart. de Parasitologia, ICB, UFMG, C.P.486 - Belo Horizonte, 30161-910, MG, Brasil
Since the forties, the first-line treatment for leishmaniasis has been based on daily injections of pentavalent antimonials (Glucantimeâ and Pentostamâ). Unfortunately, these drugs are toxic and difficult to administrate because of their long-term treatment and high cost. Moreover, antimony unresponsiveness in visceral leishmaniasis (kala-azar) has become a serious clinical problem. Twenty years ago, the encapsulation of antimonials in liposomes was shown to improve strongly their effectiveness against experimental kala-azar. This effect was attributed to the fact that liposomes are rapidly cleared from blood circulation by liver, spleen and bone marrow macrophages, in which reside and proliferate Leishmania parasites. However, the low encapsulation efficiency of antimonials in liposomes has been until now one of the main obstacle to the development of a pharmaceutical product. In the present work, we evaluated different methods for the encapsulation of Glucantimeâ in liposomes. Using phosphatidylcholine (PC) and cholesterol (CHOL) as membrane components, we found the encapsulation efficiency to be higher in DRV (dehydration-rehydration vesicles) (10-20%) than in FATMLV (frozen and thawed multilamellar vesicles) (<10%) or REV (reverse-phase evaporation vesicles) (<10%). The inclusion of dicetylphosphate (DCP) in the DRV membrane increased the encapsulation efficiency to 50%. In order to evaluate the antileishmanial activity of the latter preparation, 4 groups of 10 hamsters were infected with a suspension of L. chagasi amastigotes and, 17 days after infection, were treated intraperitoneally with: (1) Glucantimeâ entrapped in PC/CHOL/DCP DRV at 60mg Sb/kg, (2) free Glucantimeâ at 100mg Sb/kg, (3) empty liposomes, (4) non-treated. After 45 days, animals were sacrified, and liver and spleen parasites were counted. A marked decrease of parasite count (at least 50-fold) was observed only in group (1), in which some animals did not show any parasite.
(supported by FAPEMIG, CNPq, PRPq-UFMG).
COMPARATIVE STUDY OF TWO ANTIMONIAL THERAPY SCHEDULES FOR TREATING CUTANEOUS LEISHMANIASIS
Azeredo Coutinho, RB & Mendonça, SCF.
Instituto Oswaldo Cruz, Fiocruz, Caixa Postal 926, Rio de Janeiro, 21045-900, RJ, Brasil.
The Brazilian Ministry of Health´s latest recommendation for the antimonial therapy of cutaneous leishmaniasis (CL) is: 10 to 20mg of Sb/[Kg.day] for 20 days, repeating this regimen if complete healing of lesions is not obtained within two weeks after its end. Before the publication of this recommendation, the most widely used antimonial regimen for treating cutaneous leishmaniasis in Brazil consisted of similar daily doses given in cycles with rest intervals between. The objective of this study was to compare these two therapeutic regimens with regard to adherence to therapy, proportion of therapeutic failure (need of additional treatment), side effects and mean time period for healing of lesions. Patients from an area where CL is due exclusively to Leishmania braziliensis were allocated into two groups with similar composition with regard to age, sex, number of lesions and period of disease progression before the onset of treatment. A group of 59 patients received the continuous schedule of 20 days and the other group of 33 patients received the intermittent treatment with three ten day cycles with ten day intervals between. In both groups the daily doses of antimonial (Meglumina antimoniato, provided by the Brazilian Ministery of Health) were given by deep IM injections. All patients were evaluated weekly by the same medical personnel at oupatient units of the Municipality of Rio de Janeiro´s Health Secretariat. In the group receiving the continuous schedule, 18,6% of the patients spontaneously interrupted the therapy, therapeutic failure occurred in 21% and side effects were observed in 28% of the cases. In the group undergoing the intermittent regimen only one patient spontaneously interrupted the treatment (3%), there was 9% of therapeutic failure and side effects were observed in 15,2% of the subjects. There was a statistically significant difference between the groups with regard to adherence to therapy (p<0,05, Fisher´s exact test). The mean time for the complete healing of lesions was similar: 97,4 ± 94,2 days for the continuous and 110,8 ± 61,1 days for the intermittent schedule. Our results show that, in our experience, the adherence to therapy is better with the intermittent schedule than with the continuous schedule of antimonial therapy.
Financial support: CNPq.
CHEMICAL ISOLATION OF AN APOLAR ANTILEISHMANIAL AND LYMPHOCYTE-SUPPRESSIVE SUBSTANCE PRESENT IN THE PLANT KALANCHOE PINNATA.
*Da-Silva S.A.G.; *Pinheiro, R.O.; Almeida, A.P.; Costa S.S.; Pimenta, F.P. & *Rossi-Bergmann, B.
*Instituto de Biofísica and Núcleo de Pesquisas de Produtos Naturais - UFRJ.
Our previous studies have shown that the aqueous extract of Kalanchoe pinnata protect BALB/c mice infected with Leishmania amazonensis when treated by oral route, and that protectiveness may be related to its immunosuppressive as well to its capacity to enhance macrophage microbicidal mechanisms. In order to identify the active substances present in the extract, chemical fractionation of the crude extract of K. pinnata was guided by both lymphocyte-suppressive and antileishmanial activities. The suppressive activity was assessed by inhibition of concanavalin A-induced proliferation of normal spleen cells, whereas antileishmanial activity was assessed by the decreased numbers of intracellular amastigotes after 48-hour culture in the presence of the fractions. Methanol extraction produced several fractions, of which the most apolar fraction 12 was the most active, suppressing about 80% of both lymphocyte proliferation and intracellular amastigote growth at 50 and 100 mg/mL, respectively. Further fractionation in dichloromethane, yelding the active 12S fraction, followed by ethanol:dichloromethane chromatography yelded the fraction F12S16-27 which was the most active, suppressing 60% of lymphoproliferation at 10 mg/mL. The 12S fraction was analysed by 1H RMN, indicating that it may be a long chain alcohol or terpene. These results suggest that the protective effect of K. pinnata may be due to apolar substances present in that plant.
ANTILEISHMANIAL ACTIVITY OF PESCHIERA AUSTRALIS EXTRACTS ON LEISHMANIA AMAZONENSIS IN VITRO
DELORENZI, JCMOB@; REZENDE, C#; PINTO, AC#; PADILHA, MRS*; SARAIVA, EMB@
@ Laboratório de Imunobiologia das Leishmanioses, Departamento de Imunologia, Instituto de Microbiologia; # Laboratório de Química de Produtos Naturais, Departamento de Química Orgânica, Instituto de Química e * Laboratório de Anatomia Vegetal e Fitoquímica Farmacêutica, Instituto de Biologia - UFRJ
Leishmaniasis is a major world wide health problem. Over 3 million people are infected over the world and 400 thousand new cases appear each year. Therapeutic failure in human leishmaniasis is a relevant problem in several coutries, mainly due to the emergence of drug resistent parasites. Clinical intervention is presently limited to antimonials, and few other choices, but disadvantages such as costs, long duration and side effects are reported for this treatment.
To search new drugs with antileishmanial activity we assayed stem extracts of P. australis on L. amazonensis promastigote growth in axenic medium and amastigote proliferation in infected mouse peritoneal macrophages. The different fractions were obtained by extracting with chloroform and hexane the crude extract ressuspended in acidified water (pH 2,5). These organic fractions were evaporated, ressuspended in dimetilsulfoxide and their activity tested. The effect on promastigote forms was assessed by cultivating parasites in the presence of each fraction and daily counting viable/motile forms. An inhibition of 100% was observed with 10 µg/ml of the chloroform on the 4th day of culture. When promastigotes were treated for oly 1hr with 100 µg/ml of the chloroform fraction, we observed a inhibition of 99% on their growth after 24hr.
The activity on amastigotes was tested by infecting mouse peritoneal macrophages with promastigotes (1:6 ratio, during 1hr, 35oC) and counting the number of intracellular parasites after 72hr. Two treatment protocols were evaluated for each fraction: a) addition of only one dose 1hr after the infection and b) dose addition once a day during three days after infection. A decrease of 90% in the survival was observed using 10 µg/ml of chloroform fraction in treatment b. Macrophages cultered in the presence of P. australis fractions shows no significant alteration in morphology, spreading and adherence to glass surface.
Gas chromatography and mass spectrometry analysis of the chloroform fraction suggest the presence of two indole alkaloids, coronaridine and voacangine. These alkaloids are under purification to better study their leishmanicidal activity.
Supported by: WHO, PRONEX, CAPES, CNPq.
EVALUATION OF "CRAVINHO" [POROPHYLLUM RUDERALE (JACQ.) CASS.] CRUDE EXTRACTS' ON LEISHMANIA (VIANNIA) BRAZILIENSIS AND LEISHMANIA (LEISHMANIA) AMAZONENSIS PROMASTIGOTES FORMS.
Jorge, A.S.; Silveira, T.G.V.; Arraes, S.M.A.A.; Zanzarini, P.D.; *Mello, J.C.P.; Bertolini, D.A.
Departamento de Análises Clínicas e *Departamento de Farmácia e Farmacologia. Universidade Estadual de Maringá - Av. Colombo, 5790 - 87.020-900 - Maringá - Paraná - Brasil.
The treatment for leishmaniasis in Brazil has been done basically with pentavalent antimonials, which have prolonged therapy and side effects. In this aspect, new chemotherapeutic agents have been tested. Efforts have been made in order to obtain natural products for the treatment of leishmaniasis. Thus, extract from plants of popular use in the treatment of leishmaniasis have been evaluated in relation to activity against Leishmania. The lyophilized crude extracts of "Cravinho" [Porophyllum ruderale (Jacq.) Cass.], with and without chlorophyll, were evaluated for anti-leishmanial activity. Promastigotes forms of Leishmania (Viannia) braziliensis strain MHOM/BR/87/M11272, and Leishmania (Leishmania) amazonensis strain MHOM/BR/89/M12766 grown in 199 (Difco) and LIT medium, respectively, at 25ºC in log stage were tested with different concentrations of extracts. The parasites were counted in a Neubauer's Chamber to evaluate inhibition of growth in comparison to the controls. For Leishmania (V.) braziliensis the studies with the extract without chlorophyll are in course. With chlorophyll the better inhibition happened in the interval between 0.75 mg/ml and 1.75 mg/ml, but in 0.75 mg/ml was observed 30.4%, 34.4% and 53.2% of the inhibition at 24, 48 and 72 hours of incubation, respectively. For Leishmania (L.) amazonensis in the concentration at 2.5 mg/ml of crude extract was observed growth inhibition of 45.5%, 85.2% and 91.1%, without chlorophyll, 46.3%, 64.1% and 87.6%, with chlorophyll, at the same incubation intervals. These results show that the crude extracts in use have anti-leishmanial activity "in vitro" mainly for the promastigotes forms of Leishmania (L.) amazonensis. In the sequence of this work we planned to assess the activity of the extracts on infected hamsters.
Supported by CNPq, UEM.
POTENCIAL EFFECTS OF DIARYLHEPTANOIDS EXTRACTED FROM CURCUMA SP. IN LEISHMANIA AMAZONENSIS PROMASTIGOTES
Araujo, C.A.C (1)., Alegrio, L.V(2)., Lima, M.E.F.(2), Castro, D.(2) and Leon, L.L.(1)
(1) Departamento de Imunologia, Instituto Oswaldo Cruz
(2) Departamento de Quimica, Universidade Federal Rural do Rio de Janeiro
Several classes of compounds with biological activities have been extracted from Curcuma sp, a zingiberaceous plant, and among them are the phenolic and non-phenolic diarylheptanoids. Current traditional Indian claims the use of some of these compounds against hepatic and biliary disorders, anorexia, diabetic wounds, etc. In previous work we described two phenolic diarylheptanoids, extracted and purified from Centrolobium sp, with a good "in vitro" activity on Leishmania amazonensis promastigotes. Considering our interest in these class of compounds, we are now studying the effects of some curcumin derivatives, which are also phenolic diarylheptanoids, obtained from Curcuma sp., on the same parasite. The following compounds were tested: 1) curcumin:1,7-bis(4-hydroxy-5-methoxyphenyl)-1,6-hepten-3,5-dione; 2) reduced curcumin:1,7-bis(4-hydroxy-5-methoxyphenyl)-heptan-3,5 -dione; 3) methylated curcumin: 1,7-bis(4,5-dimethoxyphenyl)-1,6-hepten-3,5-dione; 4) diol curcumin: 1,7-bis(4-hydroxy-5-methoxyphenyl)-hepten-3,5-diol and 5) reduced diol curcumin: 1,7-bis(4-hydroxy-5-methoxyphenyl)-heptan-3,5-diol. Leishmania amazonensis promastigotes were grown in LIT medium supplemented with 10% of fetal calf serum and the compounds solubilized in DMSO (the highest concentration used was 1.4%, v/v) were added to the culture, in a concentration range from 160 to 5mg/ml. After 24h incubation in a temperature of 26°C, the remaining parasites were counted in a Neubauer chamber and the results showed that the curcumin (1) and the methylated derivative (3) had the highest activity with LDs 50 of 23nM and 8.8nM, respectively. Further experiments are been done in order to clarify the role of the substituents radicals, such as the methyl group.
Supported by CNPq/FIOCRUZ
EVALUATION OF DRUG RESISTANCE IN LEISHMANIA SP.
Campos Y., Romero, P., Ponte-Sucre, A.I., and Mendoza-León, A. Laboratory of Biochemical and Molecular Biology of Parasites, Laboratory of Biophysics, I.B.E. and Laboratory of Molecular Physiology, I.M.E., Universidad Central de Venezuela, Apartado 47577, Caracas 1041-A, Fax: 58.2.753.5897.
The frequency observed in the lack of response of Leishmania sp to various drugs, including to those used for the treatment of the disease, is related to the development of resistance and emphazises the need for the comprehension of the biochemical and molecular mecanisms involved in this process. In previous studies we demonstrated a susceptibility of Leishmania to K+ transport blockers as glibenclamide (GLIB) with an LD50 of 7 µM. Later on we developed a Leishmania strain L. (Leishmania) NR resistant to GLIB (Gr). In this work we have studied the viability of Gr cells in the presence of high GLIB concentrations, as well as in the presence of drugs as colchicine and vincristine. The development of resistance to these two drugs is mediated by the multidrug resistance gene (mdr). GLIB resistance at low drug concentrations -30 µM- enhances the tolerance of the parasite to high GLIB concentrations (200 µM). Therefore, the growth of Gr cells is not impaired at 200 µM GLIB. Colchicine 250 µM decrease the growth of sensitive (Gs) cells by 36 % and by 54% in the presence of 50µM GLIB. A 24 % of decrease was observed in Gr. Vincrisine 7.5 µM decreases the growth of Gs by 26 % but Gr is insensitive to it. These results suggest that GLIB resistance developmentt includes a broad spectrum resistance to high GLIB concentrations as well as to drugs not directly related. The potentiatied effect observed in the presence of colchicine and GLIB suggest the existence of different resistance mechanism for each drug since sensitive strains are less sensitive to colchicine than vincristine.
Partially supported by the Venezuelan grants CDCH-UCV, 03.10.3750.96, 09.33.0018.94, PC.09.010.94, O9.103515.95, 09.33.3514.95 CONICIT RPIV-110034, S1-2674.
ENHANCED ANTI-LEISHMANIAL EFFECTIVENESS OF A PLANT-DERIVED CHALCONE BY ENTRAPMENT IN POLYMERIC NANOSPHERES.
Torres-Santos, E.C1.; Rodrigues, J.M.2; Pinto, E.F.1; Raposo, D.B.; Moreira, D.L.3; Kaplan, M.A.C.3 and Rossi-Bergmann, B1.
1Instituto de Biofísica Carlos Chagas Filho, UFRJ; 2Faculdade de Farmácia, UFMG; 3NPPN, UFRJ.
We have proposed to isolate and identify antileishmanial substances from Brazilian plants that could serve as new leads in the development of novel drugs against leishmaniasis. The extracts of several plant families criteriously selected according to the presence of certain chemical groups were screened against promastigotes and amastigotes of Leishmania amazonensis. We found that the inflorescences of the piperacean Piper aduncum contained a 2',6'-dihydroxy-4'-methoxichalcone extractable with dichloromethane which at 100 mg/ml inhibited DNA replication of both promastigote and amastigote forms of the parasite by 92% and 98%, respectively, while maintaining normal macrophage functioning and morphology. As an attempt to further increment its effectiveness, the chalcone was entrapped in poly (D,L-lactide) nanospheres. Entrapped chalcone was 25 % more potent in inhibiting amastigotes than its free form, at concentrations as low as 1mg/ml. In vivo studies showed that treatment of infected BALB/c mice with a total of 240 mg entrapped chalcone produced lesions 55 % smaller than treatment with empty nanospheres. Indeed, entrapped chalcone was superior to equivalent doses of Glucantime, which produced lesions 30% smaller than controls. These results show that the antileishmanial properties of the P. aduncum chalcone can be further potentiated by entrapment in biodegradable nanoparticles.
EFFECT OF HEAT SHOCK ON GLUCANTIME-RESISTANT LEISHMANIA (V.) GUYANENSIS CELL LINES
Rodrigues, P. H.; Moreira, E.S.A.; Petrillo-peixoto, M. L.
Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais - Av. Antônio Carlos 6627, Belo Horizonte, MG - CEP 31270-901
A wild type strain of Leishmania (V) guyanensis (IUMB/BR/85/M9945) and two glucantime-resistant cell lines named R14 and R21 were studied comparatively, in order to determine the effect of heat treatment on survival, viability and protein synthesis. All cell lines were kept cryopreserved and, for the experiments, they were maintained at 25oC in axenic liquid complex medium, in the absence of the drug, by limited number of passages at logarithmic phase. Survival of the parasites was determined by counting the number of cells immediately after heat exposure. Viability of cells which did not undergo lysis was estimated after incubation at 25oC, by counting the number of intact cells at 24 hours intervals. The generation time of control cultures specific of each cell line (15, 25 and 22 hours for the wild type, R14 and R21 respectively), was increased only after a longer exposure to 37ºC (240 minutes for the resistant cell lines and 120 minutes for the wild type). When cells were pre-treated at 120 minutes at 34ºC for induction of thermotolerance, no difference in parasite survival (28%, 78%, 38% for wild type, R14 and R21, respectively) was observed between pre-treated cells and control groups for all lines. Cell viability, however, was different: generation time of the pre-treated cells of the wild type was reduced, while no difference was observed with cells of R14 and R21 lines. Changes in protein synthesis for all cell lines subjected to heat shock were analyzed by SDS-PAGE. The most evident difference observed between wild type and resistant lines was the increase in the synthesis of a protein of 67 kDa, after heat treatment (120 minutes at 34ºC). However, no change was observed on the synthesis of this protein among the three lines, after the challenge of exposure to 180 minutes at 37ºC. Further characterization of the expression of HSP genes may explain the difference in thermotolerance existing among wild type and resistant lines, and enable the establishment of relationships of parasites heat shock response and drug resistance.