Collaborative research augments therapy for visceral leishmaniasis

Conducted by researchers at the Adolfo Lutz Institute in São Paulo and King’s College London, the study sought to reduce the toxicity of treatments for visceral leishmaniasis.

By Samuel Antenor

Agência FAPESP – A study performed jointly by researchers from the Adolfo Lutz Institute in São Paulo and from King’s College London in the United Kingdom has enabled the development of new therapeutic strategies to diminish the toxicity of treatments for visceral leishmaniasis, including the use of drug compounds already available on the market.

The project, “From trypanosomes to Leishmania: novel drug candidates for the treatment of neglected parasitic diseases,” was conducted between 2011 and 2013 by André Gustavo Tempone, coordinator of the Applied Toxinology Laboratory of the Department of Parasitology at the Adolfo Lutz Institute in São Paulo, and by Gerd Wagner of King’s College in London, under an agreement between the two institutions.

Caused by a protozoan of the genus Leishmania and transmitted by the sandfly (Phlebotomus papatasi), visceral leishmaniasis affects humans and animals such as dogs and cats, and this disease can cause damage to the liver, spleen and bone marrow. Left untreated, visceral leishmaniasis can cause death in a matter of months. The disease occurs throughout Brazil and in other countries with warm climates, such as India, Nepal, Sudan and Bangladesh, but can also occur in countries with temperate climates.

The drugs used in treatment – N-methylglucamine antimoniate (Glucantime) and amphotericin B – are highly toxic and can cause serious side effects. In addition, there are several reports of drug resistance, mainly in India, where the incidence of the disease is highest.

“In terms of treatment for leishmaniasis, we have looked at what has already been done to combat malaria, such as lowering drug toxicity in the body. In order for a therapeutic combination to weaken parasite resistance, we need to find synergistic combinations that can reduce the dose of the drugs used,” Tempone told Agência FAPESP.

The group of Brazilian and British researchers studied drug substances already in use to treat this and other illnesses as a way to reduce research costs and time.

Within the strategy of drug repositioning, the researchers tested nearly 200 substances out of the 2,750 drugs already approved by the US Food and Drug Administration (FDA), the agency that regulates the use of medicines in the United States.

King’s College London made its chemolibrary, containing nearly 400 synthesized compounds, available for tests of antiparasitic activity. “The joint work was essential,” Tempone noted.

According to Wagner, the research collaboration with the Adolfo Lutz Institute has been successful and has allowed the team at King’s College to conduct tests and studies using the expertise of the São Paulo laboratory.

“Collaboration with Tempone’s team has allowed us to test molecules in several antiparasitic assays and has included a visit to São Paulo by doctoral candidates from King’s College to test different compounds. The research led to the identification of various molecules that act against Leishmaniasis with potential for development,” said Wagner.

Over the course of the two-year project, the British team worked on the organic chemistry of the medicines, while the team from Adolfo Lutz worked on the aspects related to the parasitology of the disease. The focus of the research was to investigate the action of the repositioned drugs in fighting leishmaniasis. According to Wagner, the repositioning of drugs already used to treat other diseases eliminates the need for toxicity tests.

Nearly 1% of the substances offered potential action against the disease in the animal model (hamster), among which was chelating iron, used to treat conditions such as excess iron in the blood (secondary hemosiderosis).

One of the main features of the research, according to Tempone, was the use of nanotechnology in the form of liposomes – artificial spherical vessels used to carry medicines and biological substances – which significantly increased the potential action of substances in the animal model by directing the drugs to the target cell, or the host cell of the parasite.

Nanoliposomes were used for the encapsulation, delivery and controlled release of the potential medicines to treat infected cells. “The idea is to produce doses for intravenous use in order to reach the target cell, the macrophage,” said Tempone. Using this technique, the medicines began to act directly on the infected sites, increasing the chances of treatment success.

In 2013, the article “Conjugation to 4-aminoquinoline improves the anti-trypanosomal activity of Deferiprone-type iron chelators,” which reported the research on therapeutic repositioning, was published in the journal Bioorganic and Medicinal Chemistry by the researchers from the two institutions.

The group is now testing the medicines in an animal model to determine whether the compounds obtained in vitro can be used in humans. The ultimate goal is to make the production of new drugs viable in Brazil by sharing the research results with public laboratories.

Still a neglected disease

In Brazil, 3,000 to 4,000 cases of visceral leishmaniasis caused by Leishmania infantum, also known as Leishmania chagasi, are reported each year. The incidence of death from what the World Health Organization (WHO) classifies as a neglected disease is close to 10%.

Brazil has also reported the incidence of Leishmania braziliensis and Leishmania amazonensis, the latter of which is particularly frequent in the northern region. Both species are responsible for the occurrence of tegumentary leishmaniasis, a disease that causes infections in the skin and mucous membranes, resulting in disfigurement.

The dog is the vector most often used by the protozoa, which are transmitted to humans when the sandfly first bites an infected animal and then a person.

According to Tempone, the number of cases of visceral leishmaniasis in humans is growing in inland São Paulo because of the increased number of cases of canine visceral leishmaniasis. “These cases often occur where there had been no previous reports of human visceral leishmaniasis,” he said, adding that the incidence of the disease in dogs is also relatively high in Campo Grande (MS).

The Ministry of Health does not authorize the treatment of animals that carry the disease and calls for the euthanasia of infected canines. According to Tempone, the medicines used to combat human visceral leishmaniasis are not suitable for animals.

Throughout the world, tegumentary leishmaniasis and visceral leishmaniasis together affect nearly 12 million people, with cases reported in 98 countries, including countries in Europe and North America.

International partnerships

The next steps in the partnership with King’s College London include publishing data on in vitro studies conducted on the parasite that causes Chagas disease (Trypanosoma cruzi) and planning a compilation of new derivatives.

Currently, Tempone’s group is conducting a study with support from the British laboratory GlaxoSmithKline. The study’s preclinical tests on animals involve the drug liposomal buparvaquone, which is used by veterinary clinics to treat parasitosis. “We expect to continue animal testing for three more years, until we determine if possible clinical tests can be performed,” said the researcher.

The discovery of the possible use of liposomal buparvaquone in treating leishmaniasis occurred during the FAPESP-funded research, supported by a regular line of research grants.

Another study funded by FAPESP, conducted jointly by the Adolfo Lutz Institute and the Institute of Chemical, Pharmaceutical and Environmental Sciences of the Federal University of São Paulo (Unifesp), Diadema campus, has identified a new chemical prototype from an Atlantic Forest plant that shows a significant capacity to fight leishmaniasis.

The compound is being synthesized in partnership with Ohio State University (US), where Tempone coordinates projects in the area of new drugs for leishmaniasis, including studies on the mechanism of action and the synthesis of derivatives of the new anti-Leishmania substance isolated from the Brazilian plant.