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Iron against tuberculosis

In experiments on animals, a metallic compound kills Koch’s bacillus more rapidly and with fewer side effects

MIGUEL BOYAYANShantytown in São Paulo: the poor account for 98% of the deaths from tuberculosis in the worldMIGUEL BOYAYAN

In the endless battle that humanity is waging against one of the greatest killers of all times, Mycobacterium tuberculosis, the main bacterium that causes tuberculosis, a group of Brazilian researchers hopes shortly to provide a new arsenal of chemical weapons to beef up the combat against the microscopic enemy, which every year scythes the lives of 2 to 3 million persons, the absolute majority in poor countries: synthetic drugs especially designed to exploit the pathogen’s weak points in order to  kill it in the quickest and least toxic way for man The goal is to reduce the time of treatment and its side effects. A molecule with an apparent potential for meeting this set of objectives was presented, discretely, in a two-page article in the January issue of the British scientific magazine Chemical Communications, of the Royal Society of Chemistry, of the United Kingdom. Baptized informally as IQG 607, the metallic compound – a ferrocyanide associated to a molecule of Isoniazid, (INH), a drug that for 50 years has been the flagship in treatment against tuberculosis – has already been patented in Brazil and is at the final stage of pre-clinical tests with mice. The molecule is one more promising result from a group of scientists connected with the Brazilian Network for Researching and Combating Tuberculosis ( Rede TB-TB Network). Created 3 years ago, it is a venture that groups about 170 scientists from 47 research institutes from several states in the country who are studying this disease, which is neglected by the major pharmaceutical laboratories for being a health problem typical of the poor.

Although there is still a journey of some years ahead, until the candidate antituberculosis drug proves really safe and efficient, the initial results of the experiments with the new molecule are heartening.  “In animals, it not only kills the common variety of M. tuberculosis, but also the main mutant strains of the bacterium that have become resistant to the medicines”, explains biochemist Diógenes Santiago Santos, from the Pontifical Catholic University of Rio Grande do Sul (PUC-RS), the coordinator of the studies with IQG 607 and other compounds against the causal agent of tuberculosis, also known as Koch’s bacillus. “As it has proved to be less toxic than the drugs that are available on the market today, it may perhaps be used in higher dosages than those prescribed for isoniazid, and thereby speed up the process of curing the patient.”  To show that the new molecule tends to cause fewer side effects, Santos comes up with data from an experiment carried out in his laboratory. Rodents that were given daily a high quantity of the new drug (1 gram of the molecule per kilo of weight) took 20 days to die, whereas those who took 250 milligrams of isoniazid, a dose four times smaller, died in just four hours.

Selective and refined drugs, which attack the bacillus more than man, may lead to a considerable reduction in the time needed to treat tuberculosis Nowadays, the treatment lasts six months and four different medicines are given to the patients.The treatment takes a long time, because the medicines available are very toxic and have to be given in small doses. Accordingly, with the succession of drugs given to the sick over half a year, the doctors manage to kill or sterilize the various strains of bacteria that may be infecting the patients, without causing them any major discomfort. Even so, for one reason or another, like the undesired side effects of chemotherapy (nausea, vomiting, and jaundice), a significant portion of the patients do not complete the long course of treatment, recommended by the World Health Organization (WHO). “About 15% of the people who seek our services have abandoned the treatment”, says Reynaldo Dietze, the director of the Infectious Diseases Nucleus of the Federal University of Espírito Santo (Ufes).  These people, plus the patients who were not completely cured, due to errors in the therapeutic procedures, are candidates for having relapses and for developing multiresistant tuberculosis, which is difficult and expensive to treat.  In Brazil, this more aggressive form of the disease – characterized by its resistance to at least two anti-TB drugs, usually isoniazid and rifampicin – is still rare, accounting for about 1% of the 120,000 new cases of the illness a year. But in other parts of the world, such as in the former Soviet republics, it now means a serious new obstacle to controlling the disease.

After having shown signs that it has been under control for some decades, thanks to a cocktail of cheap drugs capable of curing 95% of the patients and to the massive adoption of a preventive vaccine (BCG), this ancient illness has gained new robustness with the emergence of Aids, in the 1980s. “In Brazil and abroad, a lot of people thought that tuberculosis was under control and that it was no longer necessary to invest in research, but this idea was a mistake”, explains Afranio Kritski, from the Federal University of Rio de Janeiro (UFRJ), another researcher from the TB Network.  Present today in one third of humanity, M. tuberculosis, which is transmitted in the air and causes infections chiefly in the lungs, has formed a malignant pair with the HIV virus. Usually, 90% of the people who are carrying the bacillus in their organism are never going to develop tuberculosis, nor will they even be capable of transmitting it through the air. But weakened individuals, with their immune defense system debilitated, like Aids sufferers, are easy prey for the infections caused by the bacterium.  The large refugee camps and the recent (forced) migratory movements of large contingents of human beings around the globe are also pointed out as being causes of the recrudescence of tuberculosis.

In 1993, the World Health Organization (WHO) declared that the situation of the disease was one of an emergency in the world and instituted the date of March 24 as World Tuberculosis Day, a way of calling attention to the problem. “It is clear that the emergence of Aids and mass migrations have helped to increase the number of cases of tuberculosis in the world”, explains Antonio Ruffino Netto, from the Ribeirão Preto School of Medicine of the University of São Paulo (FMUSP/RP), the coordinator of the epidemiological studies of the TB Network.  “But we cannot forget that the presence of the bacillus in the organism is necessary, but not sufficient to cause the disease. The real cause of the disease is social inequality.” Closed, small and cramped places, such as refugee camps, prisons, and even overpopulated shantytowns and slums, mean environments that favor the dissemination of the bacillus, which infects one person a second in some part of the planet, 8 million individuals a year.  If, at the end of the 19th century, the disease went so far as to be romanticized and even described as a disease that hits even the highest strata of society, tuberculosis is seen today essentially as a disease that is almost restricted to the more humble layers of the population (see the text on page 36, with a brief history of the disease).  It is estimated that 95% of the cases and 98% of the deaths occur in poor countries. Brazil is the only country of the Americas to figure on the list of the 22 nations that concentrate 80% of the occurrences of tuberculosis in the world. Officially, the disease kills from 5,000 to 6,000 people a year in Brazil, but specialists estimate that the number of deaths must reach 10,000.

The idea of altering the formula for isoniazid – and thus of creating a molecule against tuberculosis – took shape after science discovered the mechanism of action of this drug, at the end of the 1990s. The drug interferes with the synthesis of mycolic acids, one of the chemical ways responsible for the production of the cell walls of Koch’s bacillus. By doing so, it prevents the full formation of this hard structure, essential for protecting the cells from the pathogen. Accordingly, the drug does not allow the bacterium to multiply, which finally leads to its death. Talking like this, in shorthand, the mechanism of action of isoniazid – the oldest and most important one of the medicines that patients take in the course of treatment – even seems simple.  But, in fact, it is a complex process, which unleashes a series of chemical reactions, not only in the pathogen that it is supposed to kill, but in its host as well. “The isoniazid is metabolized in the human liver, forming compounds like hydrazine, which are toxic for this organ and for the central nervous system”, Santos says.  Hence the nausea, the vomiting, and other side effects that trouble the patients during the treatment against tuberculosis. Besides being intricate, the mechanism of action of the drug on the bacillus is indirect: to have some therapeutic effect, the isoniazid has to be activated by the catalase-peroxidase enzyme, produced by a gene present in the tuberculosis bacterium, the KatG. Unless this happens, the drug will not work.

But, after all, how has all this knowledge about the mechanism of action of isoniazid assisted the Brazilian researchers in making a new potential drug?  The clue that this drug, by twisted  ways, was capable of killing the bacterium, because it acted on the metabolic way linked to the synthesis of the mycolic acids, responsible for the formation of the pathogen’s cell wall, provided input for the scientists to design compounds capable of reaching precisely the same target – only by a more direct path. By a chemical shortcut that could be more efficient in besieging the bacillus and cause less side effects in man. “Our objective was to create molecules that would act in this metabolic way, one of the bacterium’s weak points, but that would not need to be activated by the enzyme produced by the KatG gene, nor by the human hepatic system”, explains chemist Ícaro de Souza Moreira, vice-rector of the Federal University of Ceará (UFCE), who conceived the new ferrous compound, whose chemical formula is [Fe11(CN)5(INH)]3-. This prerequisite is justified: KatG is one of the five genes of Koch’s bacillus in which mutations have already been detected that make isoniazid ineffective against tuberculosis. Changes in the composition of the KatG seem to be linked to half the cases in which the tuberculosis bacterium shows greater resistance to the action of isoniazid. Accordingly, designing an anti-TB  molecule with an a action independent of this gene seems to be a way developing a drug with a potential for treating the common cases – and the more serious ones as well – of tuberculosis.

The strategy of designing totally new chemical complexes by joining together metallic compounds with isoniazid was put in place when the researches began, about three years ago. “The metals combine more easily in the cellular medium”, Santos comments. With this approach, Moreira, who comes from Ceará and is the group’s inorganic chemist, developed ten different molecules. Some of them were complexes that interacted with ruthenium, others with cobalt, and yet others with iron, as is the case of the IQG 607 molecule. Iron has been given priority, for being a metal that less dear than the others, since the goal is to develop cheap and effective drugs against tuberculosis. That is why the studies with IQG 607, a ferrous compound, are at a more advanced stage. “But, besides the IQG 607, we saw that another five molecules kill Koch’s bacillus”, says Moreira.

To find the ideal molecule, the one that suits best the most vulnerable metabolic ways of the bacillus, the researchers resorted to sophisticated resources. In the Biomolecular Systems Laboratory of the São Paulo State University (Unesp) in São José do Rio Preto, physicist Walter Filgueira de Azevedo Jr. simulated, in three dimensions, how the fit would be of the metallic compounds – not just of the IQG 607, but of the other nine candidate drugs – with enoyl-reductase, the target enzyme of the chemical way to be attacked.  “This way, even before any in vitro test being done, we already had information about which compounds best fitted the active site of the enzyme”, Azevedo Jr explains. The IQG 607 was one of them.

The molecule and the other metallic compounds under development are not the only hope offered by the TB Network. In the laboratory, about a thousand plant extracts have now been tested, of which 30 have displayed some interesting result. Groups from all the regions of the country are trying to develop more effective examinations for the detection of the disease, and vaccines and medicines against the disease. In the quest for this common objective, some ventures sometimes resort to very singular resources, in a diversity of approaches that instead of stimulating competition, fosters complementary work in the efforts made in the ambit of the TB Network.  A group of researchers headed up by biochemist Célio Lopes Silva, from FMUSP/RP, has, for example, developed a gene vaccine, using the DNA of the M. tuberculosis, which proved capable of preventing and curing the disease, in experiments with animals (see Pesquisa FAPESP 81). Although it was conceived for people with tuberculosis, the vaccine is now a target for clinical tests on human beings who have an advanced form of cancer of the head and neck and are not responding to any other kind of treatment. “We should have the first results of the experiments in six months”, claims Silva, who is also the general coordinator of the TB Network.

Expensive, time-consuming, fiddly, and with an unpredictable result, the tests on human beings are the final acid test which all and every new medicine or vaccine has to pass, in order to have their marketing approved.  First, their toxicity and side effects are assessed. Next, what the ideal dose is. Then, their effectiveness. With the potential drugs against tuberculosis, it is no different.  Santos estimates that all the stages of clinical tests with the IQG 607 molecule should consume a quantity equivalent to US$ 40 million, in the course of two or three years of experiments with at least 2,00 persons. It is a amount that, if it is never obtained, will mean that the possible remedy against tuberculosis will have the same end as so many other promising molecules: interesting information that has never left the pages of a scientific article.  “The majority of the major international laboratories do not invest in drugs for the diseases of the poor”, Santos points out.  “And the Brazilian pharmaceutical industry has neither the habit nor the necessary funds for financing clinical researches.”

He does not, however, get discouraged, and presently is trying to convince a British laboratory to foot the bill for the clinical tests with the IQG 607. Another way out is to battle for finance from governments and philanthropic entities. Last month, the Bill & Melinda Gates Foundation, created by the billionaire owner of Microsoft, the world’s largest software company, donated some US$ 83 million to an American non-profit institution that is researching into new vaccines against tuberculosis, the Aeras Global TB Vaccine Foundation. This entity has just started the tests on human beings of a new, genetically modified version of BCG.

Famous victims of the bacillus

Probably described for the first time in old Indian texts, pulmonary tuberculosis was called tisic by the ancient Greeks, since the days of Hippocrates, the father of Medicine, over 2,000 years ago. The word means weakening or wear, a clear allusion to one of the most visible symptoms of the disease, in an age when there was still no effective treatment: the progressive consumption of the health of its victims. Since then, tuberculosis was given other names – white plague, consumption, and phthisis.  The World Health Organization (WHO) forecasts that, unless the current escalation of the disease is not checked, another 100 million individuals will perish in the next two decades – the majority of them poor people, of a productive age (between 15 and 50), who, to a large extent, could be cured with the medicines already available.

A particular and paradoxical trait of tuberculosis – the contemporary name for the ailment is derived from the kind of lesion that the Mycobacterium tuberculosis bacterium causes in the lungs, characterized by nodules or tubercles of dead gray matter – is its frequent romanticization, sometimes glamorization, in certain epochs.  The principal muse of the Renaissance, immortalized in famous paintings by the Florentine Sandro Botticelli, like the Birth of Venus, and in a posthumous portrait done by Piero de Cosimo, Simonetta Vespucci died phthisical, 22 years old, in 1476. Her paleness and delicateness, probably caused by the action of the bacillus, were seen as ideal traits of feminine beauty. Tuberculosis seems to have exerted a morbid fascination amongst artists and intellectuals, especially amongst writers, who when they did not succumb to the disease would make their personages suffer from it.

In spite of the cause of tuberculosis having been determined in 1882 by German bacteriologist Robert Koch, medicine took decades to find more effective treatments than to prescribe spells of clean fresh air in the sanatoriums. Amongst the patients who were withering away in those days, there was a levy of artists, whose financial condition was not always one of the best.  Between 1848 and 1855, the novelist sisters Charlotte, Emily and Anne Brontë – the first less than 40 years old, and the last two at around 30 – died of tuberculosis. In music, one of the most famous victims was the Polish composer Frédéric Chopin, who died in 1849, before completing his fortieth year.  Koch’s bacillus also put an early full stop to the literary career of Franz Kafka: the Czech writer departed a littler over the age of 40, in 1924.  In an age in which there were now drugs against the disease, British actress Vivien Leigh died of tuberculosis in 1967, 53 years old.  In Brazil, there is no lack of examples of illustrious fatal victims of tuberculosis: Carlos Alves the poet died at the age of 24 in 1871 and musician Noel Rosa, at 26, in 1937.  Not to mention Álvares de Azevedo, Augusto dos Anjos, José de Alencar…

The Project
New Strategies for Controlling Tuberculosis in Brazil; Coordinator
Diógenes Santiago Santos –  PUC-RS; Investment R$ 2,500,000.00 (CNPq, Ministry of Health and Unesco)