Centers for disease control and prevention (CDC)Trypanosoma cruzi under a microscope: fighting it with nitric oxide and rutheniumCenters for disease control and prevention (CDC)
New hope has appeared for the treatment of Chagas disease. At the end of 2007, researchers at the University of São Paulo (USP) and in São Carlos and Ribeirão Preto completed successful test tube and clinical trials with chemical compounds based on nitric oxide and ruthenium, from the platinum group of metals, which killed trypanosome cruzi, the parasite responsible for the disease. Considered by the World Health Organization (WHO) as a disease that has been overlooked – one of those that pharmaceutical companies do not believe has a great enough profit potential to make it worth their while developing drugs to fight them. The disease affects 16 to 18 million of the 100 million people in Latin America who form the population at risk (normally in the lower economic classes). Of those who are infected, around 50 thousand die every year in the region. According to the Ministry of Health, in Brazil there are 6 million infected people, of which 300 thousand are in the state of São Paulo. Although the formula developed by USP is still a long way from becoming a drug (between 5 to 10 years of research would be necessary), studies indicate the possibility of a new treatment for the disease.
At the end of last year, the university signed a confidentiality agreement with a local (unnamed) laboratory to continue the research which produced two registered patents and an article in the British Journal of Pharmacology. The importance of this also lies in the fact that in decades no new drug has been launched to treat the illness.
To prove the efficiency of the ruthenium compounds in the treatment of infected mice, the São Paulo scientists had to face a long path. The research started more than ten years ago and was financed by two FAPESP projects. The objective was to understand the synthesis, reactivity and the chemical base of compounds capable of absorbing or liberating nitric oxide, a colorless gas that is part of several of our bodily biological processes. Apart from being a potent vasodilator, nitric oxide is part of the process which helps the functioning of our kidneys, penile erections, the contraction of the uterus at the time of birth and the destruction of microorganisms harmful to man. As explained by Douglas Wagner Franco, a professor at the Institute of Chemistry at USP Sao Carlos, “right from the start our intention was to understand the chemistry of these compounds in order to use them as transporters of nitric oxide.” “We needed formulas with low toxicity, capable of dissolving in water, stable in the presence of oxygen in a soluble or solid state and capable of releasing nitric oxide in a controlled manner.”
Some years ago laboratory trials on mice showed that the compounds developed by the team of professor Franco and professor Elia Tfouni from the Department of Chemistry at the School of Philosophy, Sciences and Letters at Riberão Pires, were involved in vasodilator processes. At this stage of the work, they also had the participation of researchers Marta Helena Krieger and Dora Maria Grassi-Kassisse, from the Institute of Biology at the State University of Campinas (Unicamp). The interest in learning the potential use of these formulas to fight infections, principally Chagas, is the theme of the doctorate pursued at IQSC by Jean Jerley Nogueira da Silva. He took advantage of the fact that infections generate a lack of oxygen in the tissues of the body. As a result, in exactly these situations they create an ideal setting for ruthenium compounds to be activated. To help with this work, Jean Silva had the help of the immunologist João Santana da Silva of FMRP, the School of Medicine at USP’s Ribeirão Preto campus.
The initial results confirmed that nitric oxide blocks the reproduction of T. cruzi in its epimastigote form – one of three forms that the parasite assumes throughout its life – characterized by having a flagellum, a frontal appendix used for locomotion, beside the protozoan nucleus. In order to understand the course of the study one must know how Chagas disease is transmitted. The disease is so called because it was discovered by the Brazilian public health expert Carlos Chagas in 1909. It is caused by a beetle known as the barbeiro (Triatoma infestans). They are infected by sucking the blood of contaminated organisms (skunks, opossums, dogs, cats, rodents or humans). In the bug, the T. cruzi is found in the form of epimastigotes which can multiply, but not infect. To arrive at the final portion of the insect’s gastrointestinal system, T. cruzi assumes another form called trypomastigote, which is when the flagellum migrates to the rear part, which is infectious but incapable of multiplying. The transition happens when the insect bites someone. As it sucks the blood, it defecates on the area, contaminating its victim with protozoans. On entering the bloodstream of the host, the trypomasigote invades the red blood cells or muscular tissue and assumes its third and final form, called the amastigote, with no flagellum, and has a great capacity for multiplication among the cells.
Toxic effect
Jean Silva said “we still do not know the exact nature of the action of our formulas on the parasite , but we know that apart from blocking the proliferation of the different types of epimastigotes, they can kill the trypomastigotes and amastigotes even in mice.” Throughout the studies, dozens of compounds created by IQSC were tested and 13 produced more promising results. Of these, the researchers selected the two most active and less toxic ones to continue their research. The names of the compounds are unpronounceable: hexafluorophosphate (or tetrafluoroborate) of transnitrosyltetraaminoisonicotinamide of ruthenium (II) and hexafluorophosphate (or tetrafluoroborate) of transnitrosyltetraaminoimidazole of ruthenium (II). These went on to be tested at FMRP. Franco commented that “some worked better than benznidazole, a classic drug used to treat people who have Chagas disease, with no toxic effects in the doses used, which were up to 900 times less than the dose of benznidazole.”
Mice were used in the live trials and, once infected, were treated for 15 days with one or the other form of ruthenium. The control group was made up of infected mice that were treated or not with benznidazole at the same dosage. “After two weeks of treatment we saw that the level of parasitaemia (the quantity of parasites per ml of blood) was 60% less in the control group compared to those treated with benznidazole. As a result of this reduction we observed survival of up to 100% of the animals” said João Silva. “Apart from that we saw that the compounds are capable of eliminating the parasite in the muscular part of the heart during the acute phase of the disease, something which benznidazole does not do efficiently.” The live trials began in mid 2006 and were concluded at the end of last year.
The risk of blood transfusions contaminated by T. cruzi is another serious problem related to the Chagas disease. In the United States it is estimated that 100 thousand people are infected due to blood transfusions and transplants originating from people in endemic areas. In this case the compounds also produced better results when compared to the antiseptic agent gentian violet (or methylrosaniline chloride), recommended by the WHO for the treatment of infected blood. “Treatment with this drug takes 24 hours whereas our compounds become active in the first hour. This fast elimination of protozoans is important because urgent transfusions become safer,” said Jean Silva.
According to Franco, the next stage of the research is to understand the action mechanism of the formulas. “We don’t know in which enzymes our compounds act or whether they work inside or outside the parasite. Another relevant point is to find out whether the compounds are also efficient in the chronic phase of the illness, as the current available treatment is only effective for the acute phase of the disease, which goes from the second to the fourth month after the bite. During this period a red and swollen lesion can develop at the site of infection. Other possible symptoms are fever, anorexia, lumps on the body and swelling of the liver and spleen. Swelling of the heart can occur in more serious cases. About 30% of the people contaminated by the protozoans become chronic cases, but may show no symptoms for 10 to 20 years. In this period the parasite continues reproducing, causing irreversible damage to the heart and nervous system.
The group of researchers expects that thanks to the agreement signed with the local laboratory, they will be able to continue to pursue this research. João Silva of FMRP states that “we’re ready to run pharmacological and pre-clinical trials which precede trials on humans.”
The Project
Thermal and photochemical reactivity of nitrosyl, ruthenium complexes, knowledge and control of the reactivity of nitric oxide
Type
Thematic Project
Coordinator
Douglas Wagner Franco – USP
Investment
R$ 515.060,30 and US$ 541.346,06 (FAPESP)
