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The artemisinin therapy

A medicine against malaria derived from the Chinese plant will be produced in Brazil

MIGUEL BOYAYANThe start of the artemisinin purification processMIGUEL BOYAYAN

High fever and shivering are the most common symptoms of malaria, a disease caused by an organism of only a single cell, the protozoon called Plasmodium, and transmitted to man by the bite of the mosquito of genre Anopheles. The Brazilian Amazon concentrates almost the total number of Latin American cases, with an average registration of around 450,000 per year. The picture forecast for this year is not very encouraging. It is estimated that the number will reach more than 600,000 cases, with around 200,000 new cases in the state of Amazonas, half of them alone in the city of Manaus.

Since there is no vaccine to combat the illness, one of the treatments recommended by the World Health Organization (WHO) is performed using medicines that are derivatives of artemisinin, the main active ingredient extracted from sweet wormwood (Artemisia annua), a shrub that occurs naturally in China and Vietnam where it has been used for centuries by the population, in the form of tea, for the treatment of malaria fever.

Although the illness in endemic in Brazil, it is only now, with the research results from the Pluri-Disciplinary Center of Chemical, Biological and Agricultural Research (CPQBA), of the State University of Campinas (Unicamp), that a medicine made from the wormwood  leaves will be totally produced in the country by the company Labogen, from Indaiatuba, in the state of São Paulo. In 2006, the plant produced in Brazil will be processed and transformed into an anti-malaria medicine. Currently the raw material for producing the medicine used for malaria treatment is imported from China and Vietnam. “The major problem is that the imported material presents major variations in its level of purity, resulting in a non-standardized product”, says the researcher Mary Ann Foglio, the university’s research coordinator. “This is without taking into consideration that it’s important that the country be self-sufficient in the production of such a necessary medicine.”

The study of wormwood at the Unicamp research center began in 1988 when an exchange program was established between the CPQBA and the Chinese government. In scientific research carried out in China during the 70’s, the plant showed itself to have anti-malaria activity. At that time, various species of the Artemisia genre were evaluated in order to see which of them combated malaria fever. In this study it was verified that only two in fact presented activity. One of them was the Artemisia annua, which grows there with ease and has high levels of artemisinin, which can reach 1.2% of the plant’s dry weight. Based on these results, the Brazilian researchers decided to bring the plant to Brazil. To this end they managed to obtain seeds from China, from Vietnam and from other regions of Asia.

Impasse solved
The first step consisted in adapting the wormwood to the climatic conditions of Southeast Brazil, since the plant originated in a temperate climate, with rigorous winters and a summer with high luminosity, but not as warm as this region. “When it was attempted to cultivate this species in Brazil, the plant grew rapidly, flowered even more quickly, but produced almost nothing of the substance of interest”, says Mary Ann. The initial results were disappointing. The varieties with a high level of artemisinin had little biomass, while those with a lot of biomass had a low level of this substance.

This impasse was only solved when hybrids were developed that were capable of resisting the Southeast climate, results of the study carried out by Pedro Magalhães’ research team, who is the head of the Agrotechnology Division of the CPBQA. Through this process, the flowering of the plant was retarded so that there would be time for the chemical substance of interest to remain accumulated in the leaves. Today these hybrids reach approximately 1% of artemisinin, results very close to those obtained from the Chinese plants. “This took time, but we?ve advanced a lot”, says Mary Ann.

When the work was initiated, the first species had presented levels below 0.01%. This was an extremely low index, difficult to manage to isolate and economically unviable. At the same time as the work on acclimatizing the plant was being carried out, the researchers began to study the extraction process for artemisinin and to develop efficient analytical methodologies. Only then would it be possible to monitor the quantity of substance in the plant and in the extract and how much is lost during the process.

In 1998, ten years after the start of the studies with Artemisia, Unicamp requested a patent for the process of obtaining extracts for isolating pure artemisinin. “In the patent we guaranteed 98% purity of the substance”, related coordinator Mary Ann.

When this stage had been successfully completed, the researchers began to observe that a large quantity of residue produced during the process of isolating the substance of interest was discarded. In order to extract the artemisinin from the dry leaves an organic solvent was used. The initial result is a thick soup, which looks like a highly concentrated  green tea. But as the purification process advances a white crystal was obtained, which is the pure form of the artemisinin.

As the leaves contain only 1% of this substance, it is significant that the other 99% are compounds of various substances, such as chlorophylls, greases and other compounds that are eliminated. “We found it interesting to study these discarded components in order to evaluate a possible pharmacological interest for this material”, explains Mary Ann. “And in truth we verified that it’s very rich in substances that demonstrate pharmacological activity.” These substances have already been tested in rats for treating lesions coming from induced gastric ulcers, with positive results. Furthermore, it was verified within in vitro models that they possess anti-proliferate activity in eight strains of human tumor cells. These studies are still on going.

After having managed to define the parameters for cultivating the plant and for extracting and isolating the artemisinin, it was necessary to have a valid analytical methodology to guarantee the product’s quality when it would be placed on the market. This indeed is one of the demands by the National Health Surveillance Agency (Anvisa). To this end, the researchers developed an analytical methodology validated through liquid chromatography of high efficiency. For this technique, ultraviolet light allows the separate visualization of all of the chemical substances extracted from the plant. As the artemisinin molecule is not visible with ultraviolet, a universal detector of refractive index was used, which generates an intense electric signal for a determined quantity of sample. The research that resulted in the validation methodology, which guarantees reproduction, exactitude and precision analysis, began in 2002 and was funded by FAPESP.

With all of the stages completed, the only thing left was to confirm the production on an industrial scale. This became possible with the signing of a contract, in June of this year, for the transfer of the technology to the company Labogen by Unicamp’s Innovation Agency (Inova). “We’re passing on to the company all of the technology already established – from obtaining seeds, extraction of the vegetal material and production processes, with the quality control support that we’ve developed”, says Mary Ann. “We believe that by the end of 2006 we” ll have done all of the tests necessary for launching the product”, says José Machado de Campos Neto, the company’s executive director. “In the first phase we’ll produce only the main active ingredient, which will be sold to pharmaceutical laboratories that already have the medicine registered.” One of these laboratories that has already produced the anti-malaria medicine with the imported raw material is the Oswaldo Cruz Foundation (Fiocruz) of Rio de Janeiro.

In September, seedlings of the plant, of around 12 centimeters, were taken to the company’s farm. By the end of January they will have reached 2 meters and will then be ready for processing. During the first phase, around 100 kilograms of dry plant, in the second 1.5 tons and in the third year some 3 tons of the plant will be processed. “Only one kilogram of the pure substance is sufficient to treat the needs of serious malaria patients in Brazil”, says Mary Ann. This quantity could already be obtained during the project’s first phase, with the processing of 100 kilograms of the dry plant.

The excess that would be produced by the company in the subsequent phases could be exported to countries such as those in Africa, which register more than 100 million cases per year, the majority brought about by the protozoon Plasmodium falciparum, the most serious form of the disease. For this form, the WHO recommends that the treatment be carried out always with artemisinin associated with other medicines such as mefloquine. “This association is a recent guideline that has very high appeal”, says the medical infectologist Marcos Boulos, a professor of Infectious Diseases and Parasites at the Medical School of the University of São Paulo.

More than one hundred plasmodia exist, but in man only four species produce an illness. As well as the falciparum, the vivax, the malariae and the ovale. The protozoa are transmitted from one person to another through the bite of the mosquito Anopheles. In Brazil, malaria is caused by two species of plasmodium, by the vivax, which is the milder form, and by the falciparum, the serious form. The tea used for centuries in Southeast Asian countries to treat malaria fever only has an effect on the milder form of the disease, which in some cases, even without treatment, regresses naturally. For the more serious form, one can inclusively create resistance to the protozoon, as has occurred with derivatives of quinine, also originally extracted from the bark of the quinine tree (Cinchona pubescens) and frequently used since 1908 in Brazil.

Less pollutant
As the artemisinin is not soluble in water not in oil, it needs to pass through a chemical transformation so that it will be soluble in a solvent that can be administered to man. Two artemisinin derivatives, artemeter and sodium artesunate, soluble in oils and water, represent efficient alternatives in the treatment of the illness and allow for its application in the intravenous and intramuscular forms. It was up to the researcher Vera Rehder, from the Organic Chemistry and Pharmaceuticals Division of CPQBA, to improve this semi-synthetic process. “The total synthesis of artemisinin, which means the total production of the principal active ingredient in the laboratory, is possible, but it’s much more economically viable to obtain an extract starting from the plant’s leaves and to transform them in two chemical reactions”, argues Mary Ann. The latter process is faster, cheaper and less polluting.

The medicine currently used in Brazil for the more serious form of the illness is applied via an injection of three doses. Today, other forms of administration, such as oral, are being researched at the Technology in Medicines Institute – Far-Manguinhos, a laboratory linked to the Fiocruz Institute, which works with imported raw material in order to produce the anti-malaria. The major obstacle up until now for this type of compound manufacture is that the artemisinin dissolves in the stomach. New compounds point to dissolving in the intestine, where it will enter into the blood circulation in a more effective form. When overcoming this barrier, the Amazon region could benefit from the raw material produced here in Brazil, at all of the stages.

An ancient illness presence on the planet

Malaria is a parasitic disease that makes up part of humanity’s history. Data obtained from fossils indicate that the disease originated in Africa and that the protozoon that produces it has been present on the face of the Earth as long as man himself, or even longer. For the World Health Organization (WHO), malaria is a tropical and parasitical disease responsible for the second highest number of deaths in the world, losing out only to AIDS. It is estimated that more than 1 million children die each year in the African continent because of the disease or complications from it, such as anemia and renal insufficiency. Around 40% of the world’s population, which corresponds to approximately 2.4 billion people, live in risk areas for malaria transmission, which happens in more than one hundred countries.

The same person can catch the illness dozens of times. The mosquito contaminates when biting a sick person and then biting another to pass on the illness. Contamination can also occur, more rarelyr, by the use of infected syringes, blood transfusion or from the mother to her baby, at the moment of birth. After contamination, the symptoms appear between 9 and 40 days afterwards, depending upon the species of plasmodium. In Brazil, malaria has been registered since 1587. From the decade of the 1870s, with the start of rubber exploration, it became a major public health problem. In the decade of the 1940s around 6 million cases per year occurred in Brazil, reduced by way of campaigns to combat the disease, to 52,000 cases in 1970. Shortly afterwards, with the occupation of the Amazon, the cases began to increase in the region.

The projects
1. Implementation of the process to obtain an anti-malaria drug starting from Artemisia annua; Modality Public bidding MCT/MS/Finep – Bio-products; Coordinator Mary Ann Foglio – Unicamp; Investment R$ 490,000.00 (Finep)
2. High efficiency liquid chromatography in the analysis of seasonal variations of sesquiterpenic lactones present in Artemisia annua (nº 02/03004-2); Modality Regular line of Research Assistance; Coordinator Mary Ann Foglio – Unicamp; Investment R$ 117,120.00 and US$ 11,161.00 (FAPESP)