Powerful molecule

Substance found in the human body is the basis of biomaterials for treating lesions in the skin and hypertension

MIGUEL BOYAYANFlexible plastic film for covering stentsMIGUEL BOYAYAN

One of the smallest and most versatile molecules produced by the organism, nitric oxide (NO), is the raw material for new materials intended for treating high blood pressure, arteriosclerosis, burns and skin lesions. He synthesis and formulation of biomaterials that release in a controlled manner this molecule of only two atoms, one of oxygen and one of nitrogen, have yielded for the State University of Campinas (Unicamp) the request for registering six patents in Brazil. The researches started in 1995 at Unicamp’s Chemistry Institute, under the coordination of Professor Marcelo Ganzarolli de Oliveira. They have resulted in the preparation of three non-poisonous compounds, with antithrombotic, anti-inflammatory and antiproliferative (active in preventing cell growth) properties, which were added to an aqueous gel and to solid and liquid polymers.

The products have potential for replacing, with advantage, the nitroglycerin and sodium nitroprusside based medicines used for controlling crises of hypertension and angina pectoris. And also for use in angioplasty, a technique that consists of unblocking coronary arteries in a process of arteriosclerosis. The polymer containing nitric oxide, in the form of a very fine film, covers the stent, a small metallic mesh of some 2 centimeters in length used a mechanical support to prevent the artery from closing up again. The function of the substance in this case is to avoid the proliferation of the muscle cells of the wall of the artery, which even with the presence of the stent, can lead to a further obstruction of the vessel. Another very promising application is the use of the gel for treating wounds caused by burns and skin diseases, such as psoriasis (a non-contagious ailment that causes red, scaly, lesions) and cutaneous leishmaniasis, a disease that is endemic in tropical countries.

This line of research with nitric oxide began to be developed by Professor Ganzarolli when he returned to Brazil, after finishing his postdoctoral studies at the University of Southampton, in the United Kingdom. There, he was studying the photochemical aspect of sodium nitroprusside, a complex that donates NO and it is used in controlling blood pressure during operations and treatment for high blood pressure. But it was not known exactly how this medicine acted on the organism, until, in 1987, it was discovered that nitric acid was produced in the endothelium, a layer of cells of the wall of the blood vessels, and that it was responsible for controlling blood pressure in human beings and other mammals. After the American scientists Robert Furchgott, Ferid Murad and Louis Ignarro were awarded the Nobel Prize in Medicine and Physiology in 1998 for discovering the role of nitric oxide in the organism, researches relating to the molecule have multiplied. Accordingly, besides controlling blood pressure, it was discovered that it had several other important physiological functions.

Nitric oxide is produced in the cells of the brain and acts as a special neurotransmitter, as a consequence of its very rapid diffusion through the cell membranes. Furthermore, it is a key part in the organism’s system of defense against infections. “The invading agents are bombarded by the defense cells with a lethal mixture of NO and superoxide, a powerful oxidizing gent, forming other products that have a strong bactericidal and tumor combating action”, Ganzarolli says. The most popular use arising from the research with nitric oxide are the medicines that combat sexual impotence. They inhibit an enzyme (phosphodiesterase type 5), which leads to an increase in the production of NO in the muscle tissue of the cavernous body of the penis. The nitric oxide causes this tissue to relax, which allows blood to pass, an essential condition for an erection.In his studies into sodium nitroprusside, Ganzarolli found that the medicine released nitric oxide, but that is also showed a risk of intoxication by cyanide. When he returned to Brazil, he already came with the idea of creating a way of releasing the NO into the organism in a controlled fashion, but without the cyanides. At the time, he asked FAPESP for a research grant, which allowed him to set up the infrastructure for beginning the studies.

Natural transporter
Other discoveries, made at the time that the researcher was still in the United Kingdom, showed that the nitric oxide is transported in our organism by a class of molecules called nitrosothiols. These substances carry and preserve the NO in the transportation between the cells, because on its own it would not survive for long in the bloodstream. It reacts rapidly with oxygen and may interact with other radicals and transform itself into other nitrogenous products.Works at the time also indicated that the nitrosothiols had a vasodilative action. “I came back eager to synthesize these substances found inside the human body, as an alternative to the medicines in current use”, says Ganzarolli. “We managed to set up an innovative system for synthesizing and it worked, and we started to produce these nitrosothiols. As they are unstable in an aqueous solution, I tried another strategy, which is to incorporate these substances into a polymeric liquid matrix, to allow their controlled release and to stabilize them.”

To save time, the choice of the matrix fell upon non-poisonous polymers that were already used in other medicines. Formulations of this polymer, a viscous liquid similar in consistency to glycerin, were prepared with the incorporated drug. “We found that it was very stable inside this matrix, making it possible for it to be kept in a fridge for a very long time without it decomposing”, the researcher reports. “This was one effect that we discovered. The matrices bring about a much greater stability for the drug.” The discoveries also yielded three patent requests, referring to the method for synthesis and to the formulations, which were given honorable mentions in the 2001 and 2002 Governor of the State awards.

The vasodilative activity of the synthesized compounds was confirmed in in vivo tests with rats, carried out by a research group from Unicamp’s Biology Institute, which is working in partnership with Professor Ganzarolli. “Furthermore, we found that the vasodilative action was more potent than with sodium nitroprusside and lasted longer, without having the drawback of toxicity”, he reports. The results earned them a cover story of the Nitric Oxide magazine, which is a landmark for scholars of the theme.

Besides the liquid polymer, the researchers began to explore other kinds of materials. “We managed to incorporate these biomaterials in aqueous gels (hydrogels) and in solid polymeric films, a flexible plastic film that can be used both for applications on the skin and for covering intracoronary implant devices”, Ganzarolli describes. The hydrogels were tested in March and April of 2003 by student for a doctorate Amedea Barozzi Seabra, who has scholarship from FAPESP, in collaboration with Richard Weller, a dermatologist from the University of Edinburgh, in Scotland. In the course of two months in Scottish territory, Amedea tested the gel on the skin of healthy volunteers to assess the vasodilative action measured by the blood circulation. To do so, a laser-based technique was used.

The light passes through the skin and is reflected by the erythrocytes (the red blood cells responsible for transporting oxygen), which flow inside the blood vessels under the skin. The greater the speed of the erythrocytes, the greater to flow of blood. “The effect is the same that radar uses to detect cars that are speeding”, the researcher likens. When the gel was spread over the arm of the volunteers, a redness immediately appeared, as a consequence of the increased circulation. “This shows that it releases nitric oxide through the skin and has a potential for being applied in lesions from leishmaniasis and psoriasis and for treating burns.”

Controlled release
Other materials are being developed at the Chemistry Institute with the same potentialities as hydrogel. According to the coordinator of the research, this gel has a very interesting property, called reverse thermal gelification. In the fridge, it becomes liquid, and in the temperature range between 30º and 40ºC it is transformed into gel. This makes it possible to inject the solution subcutaneously. When it comes into contact with the skin, it turns into a gel and forms a deposit, from which the nitric oxide is released that is going to penetrate the systemic circulation (of the whole body). Depending on the behavior of the formulation, the release takes place in a slower or faster manner, and it may serve for different functions, like controlling blood pressure or having a cytotoxic action to kill off the protozoon that causes leishmaniasis.

The biomaterials that donate nitric oxide also have a good prospect for application for arteriosclerosis, an imunoinflamatory process that originates in the space right below the endothelium, the first layer of cells that covers the inside of the blood vessels. This is the place where the plaques of arteriosclerosis (atheromas) develop. When the plaque breaks up, it sets off the formation of a clot that blocks the flow of blood. If this happens in one of the coronary arteries, a region of the cardiac muscle that ceases to be irrigated, and the resulting complications may lead to death. One of the ways of unblocking the coronary arteries is angioplasty, a technique that consists of dilating the vessel by inflating a balloon, covered by the stent, which is introduced by a catheter at the place of the occlusion.

The balloon is removed, but the stent remains. However, as this procedure causes a lesion in the artery, the organism reacts and tries to heal it, and this may lead to the formation of a thrombus that blocks the artery (acute reocclusion), which occurs in 5 to 9% of the cases. Another possible consequence is that in this response to the trauma, the muscle tissue of the artery starts to proliferate, by cell division, leading to a thickening of the wall at the place of the lesion. As a result of this new layer of cells that forms, the vessel undergoes a remodeling and the cells pass through the metallic mesh of the stent, once again closing the vessel. This reocclusion, called restenosis, is one of the main problems of angioplasty and occurs in about 30 to 50% of the cases. As about 2 million stents a year are implanted in the world, there is a great demand for technologies that reduce the problem.

The studies with the stent coated with biomaterials enjoy the collaboration of a research group from the Heart Institute (InCor) of the Hospital and Clinics of the University of São Paulo (USP). “We are doing the chemical part of recovering and characterizing the release of nitric oxide in vitro”, says Ganzarolli. “As soon as we select the best system, we are going to hand the stents over to InCor, for them to be tested in animal models.” The preliminary tests were done at the Chemistry Institute with a very thin polymeric film, ideal for covering over irregularities in the metallic mesh. “We found that the polymer releases nitric oxide and has potential for use for the antiproliferative and antithrombotic properties of the NO”, reports the coordinator of the research..

The first tests for incorporating other drugs in the stents started to be carried out about eight years ago by several research groups scattered all over the world. Of these studies, the one that showed best results was the one that covered the stents with rapamycin, which has an antiproliferative action and has been used in treatment against rejection in kidney transplants. In April this year, Johnson&Johnson put onto the market a stent covered with this drug, the only one there is in clinical use at the moment. By the end of October, over 450,000 units of the product had already been sold. However, at the same time, the American agency that controls medicines, the Food and Drug Administration (FDA) published a warning that it had received about 300 reports advising the occurrence of thrombosis and reactions of hypersensitivity associated with the use of stents coated with rapamycin, with over 60 deaths associated. Although these numbers are small in the light of the success of the product, for Ganzarolli, this shows that there is room for putting onto the market stents coated with other drugs. Besides reducing the risks of toxicity, the biomaterials that donate nitric oxide permit localized application, with prolonged periods of release, as shown by the results achieved so far. The researcher intends to advance in his studies and points out that the technologies already developed can be transferred by the university to companies interested in producing the new materials industrially.

The project
Photochemistry of Metallic Complexes in Polymeric Matrices and Organized Systems (nº 95/02344-9); Modality Regular Line of Research Grants; Coordinator Marcelo Ganzarolli de Oliveira – Unicamp; Investment R$ 2,200.00 and US$ 20,342.00