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Body with more gas

Compounds containing ruthenium, which liberate or capture nitric oxide, could serve as a basis for future medications

New collection of osteology and myology, of Jacques Gamelin, 1779

“We could present ourselves as projectionists of made to measure chemical compounds that donate or capture nitric oxide.” On imagining this possibility, Elia Tfouni does not intend to give up modesty, but is merely summarizing the results of ten years of work with Douglas Franco – both are chemists and professors at the University of Sao Paulo (USP). In conjunction with researchers from the Federal University of Sao Carlos (UFSCar), professors Tfouni, Franco and their respective teams, have developed close to fifty (50) compounds which in initial laboratory studies show themselves to be capable of absorbing or releasing nitric oxide, a colorless gas essential to the organism that appears and disappears all the time: its half life, when half of the total number of molecules break down, is only five seconds.

Produced, consumed and reproduced all the time, nitric oxide facilitates blood circulation, kidney function, the destruction of harmful microorganisms, penis erection and the contraction of the uterus at the time of giving birth, as well as serving as a chemical messenger between the neurons of the brain. Few molecules are as versatile and omnipresent, although this chemical combination of one atom of nitrogen with one atom of oxygen had been viewed over decades mainly as a residue liberated at the exhaust pipe of motorcars – until three American pharmacologists demonstrated its importance for human beings and won the Nobel Prize for Medicine in 1998.

At times it may well be good to reduce the quantity of nitric oxide in circulation within the organism; at other times, the best thing is to increase the supply of the molecule linked to life and death, to pleasure and pain. In a septic shock, as is named the brutal drop in blood pressure that comes about from a bacteria infection, there is excessive production of nitric oxide – associated as well with schizophrenia, Alzheimer’s disease, diabetes and asthma. In these cases its abundance is not desirable and medicines that could reduce its concentration in the organism would be very welcome. At other times, what is needed is the opposite effect, that is to keep the maximum possible quantity of nitric oxide in circulation, when taking into consideration its property of promoting the dilation of veins and arteries: medication such as Viagra are in fact based on this effect, by way of which the blood circulates more generously to the penis. The reverse situation, the shortage of nitric oxide makes the blood vessels contract and turns the possibility of a heart attack more imminent.

Franco and Tfouni enjoy immensely adding that they have in their hands something new that avoids heart attacks and at the same time is a national Viagra. But no so. Their studies have been purely chemical and they are only just beginning tests on animals, the first stage of a long journey towards safe applications in human beings. The compound that is in the most advanced stage of research is colloquially called RuNO, the abbreviation of an almost pronounceable name, trans-nitrosyltetraammin(triethylphosphite)ruthenium hexafluorophosphate. In the laboratory, RuNo shows itself to be efficient at reducing the blood pressure of rats with hypertension, according to a study made in conjunction with Marta Krieger, from the State University of Campinas (Unicamp), and published in 2002 in the magazine Nitric Oxide: Biology and Chemistry.

Alessander Acácio Ferro, an ex-doctoral student of professor Tfouni, has developed other apparently promising compounds. One is called hexafluorophosphate of ruthenium (II) trans-nitrosilchloro (1,4,8,11-tetra azacyclotetradecane) – or simply, cyclam – and it exhibits an action twenty times slower when compared with sodium nitroprussiate, a compound used for treating heart attacks, which rapidly replaces the nitric oxide. Even though the work has just begun, according to professor Franco, the result suggests that cyclam could be used to maintain the blood pressure stable, more than for resolving emergency situations as is done with nitroprussiate or nitroglycerine, which also liberates nitric oxide, as well as being a powerful explosive.

In a less advanced stage are the studies of Jean Jerley Nogueira da Silva, one of professor Franco’s students, on the potential use of these compounds in combating infections. In collaboration with João Santana, from the Medical School of USP in Ribeirao Preto, Jean has verified that nitric oxide blocks the reproduction of the protozoa Trypanosoma cruzi, which causes Chagas’s Disease, against which for decades now new medicines have not come forward. Jean has selected three compounds with high destructive power: within an hour, each one of the three killed between 60% to 92% of the parasites grown on a culture.

Preliminary tests
This research group reached their results by looking at the ionic properties – electrically charged particles – of the chemical element named ruthenium. In its electrically neutral form, ruthenium is a white metal used in the production of non-corrosive alloys and in jewelry as a substitute for platinum. With the loss of two electrons, it becomes Ru2+ and an experimental model for the development of new medications, as it combines easily with nitric oxide and forms compounds that are only slightly reactive. “This is the simplest way of carrying out chemical synthesis that we have discovered”, says Franco, whose team is an integral part of the Chemistry Institute of Sao Carlos, whilst Tfouni and his students work out of the laboratories at USP’s Philosophy, Sciences and Arts School at Ribeirao Preto.

The ruthenium compounds are still treated with care, because of their toxicity. Even at that, in controlled doses, they have been tested throughout the entire world against some illnesses, making up an alternative for today’s adopted strategies that control the stock of gas acting upon enzymes that produce nitric oxide or breaking them down after having complied with its task. In a revision article published last year in the Current Topics in Medicinal Chemistry,  Celine Marmion and her team from the Royal College of Surgeons, in Dublin, Ireland, in conjunction with researchers from the Canadian company AnorMED, plotted out a rich future for the donors and captors of nitric oxide based on ruthenium compounds when describing the results of the new compounds tested on rats, pigs, cats and rabbits against wide ranging problems, such as hypertension, cancer, heart attack and inflammation.

The first clinical tests have also been done on human beings. In a study also published last year in Clinical Cancer Research, researchers from the National Cancer Institute of Holland reported the discovery of the best dose of a ruthenium based compound that is apt to avoid the spreading of tumors, after having treated thirty four patients with different doses. In parallel, specialists at the University of Trieste, In Italy, under the direction of Enzo Alessio, have found a formula given the name NAMI-A, which was tested on twenty- four people and which impedes the proliferation of cancerous cells. “The ruthenium compound that we’ve developed and tested is a lot less toxic than the anti-tumor compounds with platinum, but it’s clear that not all of ruthenium’s derivatives are of low toxicity”, comments  Enzo Alessio, the study’s director, during 2004 in the magazine Current Topics in Medicinal Chemistry. In Bulgaria, a group has found a ruthenium derivative that combats leukemia in human cells.

Each research group adopts a basic chemical structure, from which they derive all of their compounds, much in the same manner as starting from a single chassis, models of simple or more luxurious cars are manufactured. In the compounds formulated by the USP team, whose properties have been described in around forty (40) scientific articles published over the last ten years, ruthenium occupies the center of an imaginary eight-faced solid, in the form of two pyramidal units each with a square base. It was inside this atomic platform that RuNO was formed, up until now the example of the most generous of donors of nitric oxide, which reacts 10,000 faster than another, for now the most parsimonious of the oxide delivered, cyclamNO (this is the same cyclam as that immense name, but now without the nitric oxide or NO).

“We could create intermediate compounds that retard or accelerate the liberation of nitric oxide by varying the chemical ligands”, says  Tfouni. Ligands are the molecules that form the external skeleton of this eight-faced solid. The four vertices of the common base of the two pyramids are formed by relatively complex molecules, made up of groups of between ten to forty atoms, such as multi-pyridines, tetraamines, aminopolycarboxylates or salen, in accordance with the chemical class to which they pertain. On the top one of the pyramids there are simpler ligands – sulfite, phosphite, chloride or simply water – that are decisive in the liberation of nitric oxide, situated in another extreme of this complex structure.

Runaway electron
In collaboration with researchers from the University of Arizona, the University of California and the National Health Institutes (NIH), of the United States, the USP chemists constructed their substances in the laboratory in such a way that the nitric oxide will be released it receives a light beam of sufficient energy or when it meets a solitary electron, the elementary particle of negative charge that orbits the atomic nucleus. Next, according to professor Tfouni, the position that the nitric oxide had occupied becomes vacant and is taken over by another molecule – generally water. This same structure, now with a water molecule occupying the nitric oxide’s position, but with one less electron, performs the reverse function and turns itself into a nitric oxide captor.

This is a perfect union between an atomic structure with one electron less and a gas made up with an electron that easily escapes. By losing an electron the nitric oxide becomes more reactive – is hungry for an electron, shall we say – and rapidly adheres to other molecules of the organism, an example being the iron of hemoglobin, the protein that distributes oxygen to the body’s cells. Franco and Tfouni have made use of this instability of nitric oxide to synthesize their compounds that function like magnets of greater or lesser intensity: the nitric oxide comes close, allows its runaway electron to escape and latches on, be it in a more intense manner, be it in a more tenuous manner, to the ruthenium ion of this structure, which is at that moment waiting for an electron – and thus this nitric oxide goes out of circulation, at least temporarily. “The nitric oxide can escape from some structures but not from others”, comments Tfouni.

In order to arrive at these results, this USP team of chemists on average work a week in preparing the reactions between the initial reactant compounds, which are colored red, blue, green or yellow, but which lose their color when they combine with the oxygen and the nitrogen of nitric oxide. Only in the end are the powders produced, generally light yellow or reddish-brown, which liberate or capture nitric oxide within a few seconds. But things are not yet finished. Another week of tests and analysis are run through until finally they arrive at the certainty that the reaction products are exactly those expected. “If we were to discover that the compound was impure”, says Tfouni, “we would have to go back to the beginning and start again.” If one is dealing with new arrangements of the compounds, then the work takes even longer.

But what are the real chances of these compounds moving ahead, passing through all of the stages and turning themselves effectively into medications? Sixty-year-old Franco, and sixty-one-year-old Tfouni know that they are dealing with a long journey, in view of the difficulty of passing on academic research results to the Brazilian sector of the chemical-pharmaceutical industry, which is not conspicuous by its familiarity with scientific research, which could help them to depend less on importations. In 2004, according to a survey produced at the start of March, the imbalance in the commercial balance of the pharmaceutical industry was accentuated, with exports at US$ 351 million (25% more than the previous year) and imports running at U$ 1.8 billion (17% more). The Brazilian Federation of Pharmaceutical Industry (Febrafarma) itself, responsible for this survey, recognizes that this difference is only going to close when they themselves widen their investments in research and development.

Even at that, the work continues. At the end of last year, Patrícia Zanichelli, from professor Franco’s team, obtained the first samples of macromolecules named dendrimers, containing twenty seven repeating ruthenium, ligands and nitric oxides, and also of silica – or pure sand – embedded in the donors and in the captors of nitric oxide, whose distribution in the organism would then be more controlled and directed only to some organs of the human body. With these special pure sands, developed by Fabio Gorzoni Doro and Kleber Queiroz Ferreira, two of professor Tfouni’s doctorate students,  it is intended to cover the surface of stainless steel and thus increase the efficiency of the devices known as stents, a type of spring that maintains the arteries open and helps to avoid a heart attack – each of which costs around US$ 2,000.00. Stents already exist that have been covered with antibiotics, liberated during one or two weeks, but not donors of nitric oxide of long duration, as the USP chemists would like. “We’d like these compounds to last as long as the very steel of the stent itself”, said professor Tfouni. “It’s very difficult, but getting close is already very good.” He knows that a good stainless steel lasts at least twenty years.

The Project
Thermal and photochemical reactivity of nitrosil complexes of ruthenium, knowledge and control of the reactivity of coordinated nitric oxide (nº 99/07109-9); Modality Thematic Project; Coordinator Douglas Wagner Franco – IQSC/USP; Investment
R$ 1,787,508.80 (FAPESP)