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Attack on the parasite

Genetic discoveries and new drugs fight the Trypanosome, which causes Chagas' disease

SIRIO J. B. CANÇADO Molecular structure of a compound of cubebine: O-dimethylaminocubebineSIRIO J. B. CANÇADO

Two novelties are promising to open up new fronts in the fight against Chagas’s diseases, an ailment that occurs only in the American continent, from the south of the United States to Argentina, and affects some 16 million victims, of which 6 million in Brazil. The first good news comes from Franca University (Unifran), in the region of Ribeirão Preto (SP), where researchers are betting on a future medicine based on cubebin, a substance extracted from the dry seed of the cubeb berries (Piper cubeba).

The other novelty came out of the laboratories of the Paraná Molecular Biology Institute (IBMP), a ramification of the Oswaldo Cruz Foundation (Fiocruz) set up in Curitiba (PR). The researchers from Paraná have discovered genetic mechanisms in the protozoon that causes the disease that favor the adaptation of the parasite to the different conditions of the organisms in which it installs itself. This same expedient means that it escapes more easily from the medicines used to contain the advance of Chagas.

New medicines against Chagas’s disease are always welcome. Today, the one most used is benznidazole, the active principle of Rochagan, a medicine produced by Roche, a pharmaceutical company headquartered in Switzerland. It retards the effects of the disease, but does not bring about a cure. Cubebin, to start with, is not going to modify this therapeutic picture, but it should make for treatments with fewer collateral effects, according to tests carried out in vitro (on cells) and in vivo (on mice), and compared with benznidazole.

The coordinator of the research, pharmacist Márcio Luis Andrade e Silva, from Unifran, a private institution from São Paulo, believes that a drug may be derived from at least one of the three compounds obtained using cubebin: methylpluviatolide, hinokinin, O-dmethylethylamine 6,6′-dinitroinokinin and nitrohinokinin. They have proved 100% effective in destroying one of the forms of the causer of the disease, the Trypanosoma cruzi protozoon, called trypomastigote, which circulates through the bloodstream, before it reaches the tissues of the heart or stomach. It may be that one of these new compounds may manage to destroy not only the circulating forms of Trypanosoma, but also those that lodge themselves in the tissues. The result would be the realization of a dream: the cure of Chagas’s disease.

Andrade e Silva knows that the path is a long one: there are at least another five years of work before arriving at a medicine that can be adopted by the public health system. The research, which enjoyed finance from FAPESP under the Young Researcher Support Program, has already yielded a patent registration request of the anti-Chagas activity of the cubebin derivatives with the National Institute for Intellectual Property (INPI). It has also awoken the interest of the Teuto Brasileiro laboratory, which is interested in taking part in the next stages of the research, which includes the complete synthesis of the most active derivatives, the study of other compounds, and testing toxicity and effectiveness in human beings.

The researchers intend to obtain and to test another ten or 15 derivatives of cubebin, in joint work with parasitologist Sérgio Albuquerque and pharmacist Jairo Knupp Bastos, both from the Ribeirão Preto Faculty of Pharmaceutical Sciences, of the University of São Paulo (USP). They also want to produce in the laboratory derivatives of cubebin, using intermediary organic molecules, this time in a joint work with Paulo Marcos Donate and Rosângela Silva, both from USP. Another challenge is to produce the molecule on a large scale, since the extraction of cubebin from pepper does not guarantee sufficient quantities of derivatives for going ahead with tests on human beings.

Of the four derivatives of cubebin already tested, two had not yet been described chemically: one is O-dimethylaminocubenin, which shows an amine radical (with one atom of nitrogen and two of hydrogen), and the other, nitrohinokinin, with a nitro group (one nitrogen and two oxygens) bound to an aromatic ring (a closed structure with six atoms of carbon and six hydrogen) of cubebin, both obtained from modifications of the original molecule. Cubenin, the basis for the two new compounds and for the other one, already known, hinokinin, can be extracted both from the dry seed of Piper cubeba and from the leaves of a shrub called in Brazil, bitch’s tit (Zanthoxylum naranjillo), although the berry has a concentration that is ten times greater. Up until now, the most active compound has been another one already known, methylpluviatolide, found in the leaves of the shrub and only now tested against Chagas’s disease.

The results obtained indicate that the compounds extracted from the Asiatic berry, in particular hinokinin and methylpluviatolide, “do not show any significant toxic effect, which is not the case with benznidazole”, says Andrade e Silva. The medical manuals warn that prolonged treatment with this substance causes alterations to the workings of the bone marrow and skin problems, for example.

Low toxicity
According to Márcio, the infected mice that were given the cubebin derivatives remained alive for up to three times longer than infected animals that received benznidazole. “Compared with cubenin, the four derivatives do not show much toxicity, and they have a very high lethal dose, over 2 grams”, the researcher avers. Even with continuous treatment, hinokinin did not damage the cells of the liver. And methylpluviatolide, according to him, kept the animal alive for 60 days more than benznidazole did. Cubenin began to be studied in Brazil at the end of the 80’s.

In 1988, researchers from USP’s Faculty of Pharmaceutical Sciences in Ribeirão Preto, oriented by chemist Otto Richard Gottlieb, isolated the substance from the leaves of the Zanthoxylum naranjillo. Jairo Knupp Bastos, who is now collaborating with Andrade e Silva, used to be part of this pioneering team that went on to confirm the low toxicity and the analgesic and anti-inflammatory activities of cubenin. The teams from Unifran and USP have now found that hinokinin, one of the derivatives of cubenin, besides its anti-Chagas activity, shows an analgesic and anti-inflammatory action close to that of indomethacin, one of the anti-inflammatories most in use today.

Thermal shock
A new medicine is also in the plans of the researchers from Curitiba. Certainly, it is not for the short term. What they are doing is to understand the protozoon’s genetic meanders. The IBMP’s most important discovery is the mechanisms used by Trypanosoma cruzi to adapt itself to the various environments where it manages to lodge itself. When it leaves the kissing bug, the insect that serves as its host, and infects an intermediary mammal, a cat, for example, which acts as a receptacle before man, the protozoon undergoes a thermal shock (the temperature goes from 28º to 37º Celsius), besides finding an environment rich in antibodies, proteins that take part in the destruction of invading microorganisms.

As the team from Paraná succeeded in revealing in some recently concluded work, the parasite’s resistance occurs when it gains time for the counterattack, stocking up on intermediary molecules – to be specific, messenger RNA, a type of ribonucleic acid (RNA). These molecules shorten the way, at the time of producing proteins with which the parasite manages to resist the change in temperature and the attack from the antibodies, which would kill it, were it not so agile. “It is the first time for this mechanism to be observed in a species of protozoon”, comments biologist Samuel Goldenberg, a director of the institute.

The discoveries may lead to new clues that help to hold back Chagas’s disease, by means of more effective medicines than those used nowadays. Difficult to diagnose, for remaining in silence for decades (in 60% of the cases, people do not show any signs of the symptoms of the infection), Chagas’s disease can bring about alterations to the workings of the heart and inflammations in the stomach, esophagus and intestines – this is when the therapeutic approach is changed and, instead of fighting the parasite, the doctors deal with the symptoms, sometimes putting in a pacemaker to prevent the infected individuals from dying.

Strategic stocks
The IBMP’s studies are a result of the simultaneous analysis of 4,200 genes deposited on a glass slide smaller than a box of matches – a biochip, probably the first with trypanosome genes done in Brazil. The biochip makes possible an overall analysis of the genes involved in the production of proteins. This process starts in the nucleus of the cell, where the DNA is, and finishes in the cytoplasm, a gelatinous sac that holds the nucleus.

Using the biochip, the researchers discovered that saving time in the production of defense proteins is not the protozoon’s only expedient for survival. An analysis of the genes also revealed that the Trypanosoma modifies the profile of the genes activated by the infected cells of the host mammals. Comparisons between cells of infected and healthy mice indicated that at least 370 genes are activated, and another 120 put into action during infection, resulting in damage to the heart, the stomach and the esophagus.

The project
Investigation into the Analgesic, Anti-inflammatory and Trypanocide Activities of Some Derivatives of Cubebine Obtained by Partial Synthesis (nº 99/13031-2); Modality Young Researcher Support Program;
Coordinator Márcio Luis Andrade e Silva – Unifran; Investment R$ 133,825.71