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Bovine gene yields medications

Studies on the fibroblast growth factor (FGF) suggest therapeutic potential

A piece of research by the Butantan Institute points to therapeutic possibilities for FGF (fibroblast growth factor) compound, produced from a bovine gene. The therapeutic functions range from the treatment of second degree burns to recovery from injury to spinal cord. The compound has also been used in a trunk-cell culture medium for the production of cell types with potential clinical use.

The biologist Paulo Lee Ho, coordinator of the research, has been studying FGF applications since 1989. The Fibroblast Growth Factor-2 project (FGF-2): Humanization, Expression and Possible Clinical Application, financed by FAPESP, is the result of a consultation made to him by the University of São Paulo’s (USP) Instituto do Coração (Heart Institute) (Incor).

Ho relates that FGF “was discovered in the 1970s by a Brazilian”, the professor at USP’s Chemistry Institute Hugo Aguirre Armelin, and that the corresponding bovine gene had been expressed in a bacterium – Escherichia coli – by another professor at that institute, Ângelo Geraldo Gambarini.

Part of a family of structurally similar growth factors, the FGF is multifunctional  – it can provoke different responses, depending on the type of cell with which it comes into contact. In the Butantan laboratories, the effects of one of the members of this family, FGF-2, have been tested. “We have tested some important actions, precisely to explore clinical cases: skin tissue healing, second degree burns, the reconstitution of the blood vessels of the myocardium, and the functional recovery of the nervous system”, says Ho.

In the case of tissue healing, the ability of FGF-2 to induce the proliferation of cells – mitogenic action – a response by cells that helps in healing. Another trial assessed the ability to induce the formation of blood vessels – angiogenetic action, precisely what aroused Incor’s interest in the research in the first place. The third trial sought to discover whether FGF-2 could induce the differentiation of certain cells in neurons, or maintain the viability of already differentiated neurons – neurotropic action.

Novel method
The possibility of using FGF-2 clinically was already known. But obtaining it based on a “humanized” bovine gene is the project’s innovation, which benefited from the work of the postdoctoral student Maria Leonor Sarno de Oliveira.

The team observed that the difference between human and bovine FGF was only two amino acids, quite close to one another in the molecular chain. To get to the humanized FGF, they started from the bovine cDNA. DNA is deoxyribonucleic acid, containing the genetic code present in all cells, while cDNA is supplementary DNA obtained from the messenger RNA – messenger ribonucleic acid that transmits the data of the genetic code – of the bovine FGF-2. To do this, they made two mutations at one time, “humanizing” the bovine FGF: the process consisted of modifying the bovine cDNA so that it was able to produce the same FGF as the human, switching the two different amino acids. With the bovine cDNA humanized, they could express it in a bacterium, the Escherichia coli.

The tests for assessing the mitogenic action of the FGF-2 were carried out in a fibroblast culture – cells characteristic of the connective tissue – maintained with fetal serum, the function of which is to induce cell growth. In the test, the researchers withdrew the serum from the culture, so that the cells would stop growing and then they added the purified FGF. The result was the induction of mitosis – the process of growth by cell division – with response varying according to the size of dose given.

To test the neurotropic action, they used cells with the ability to differentiate between neurons and they put them into contact with the FGF-2. Then they split into neurons – cells making up the nervous system.

In the third and fourth test, done jointly with Joaquim Coutinho Netto, of USP at Ribeirão Preto, the ability to form scar tissue and restore blood vessels was evidenced. The formation of scar tissue and the angiogenetic effect of FGF-2. The ear of the rabbit was punctured as far as the cartilage and a tampon with FGF-2 was placed there. The presence of the compound induced the replacement of the removed tissue and the formation of new blood vessels. It was noted, however, that the exclusive use of the FGF-2 was not enough to complete the process of forming the scar tissue. The wound caused by the puncture remained, even after the reconstitution of the tissue: the FGF-2 alone was unable efficiently to promote the growth of the epithelial cells that make up the skin. To solve the problem, the team tried to associate the FGF-2’s action with that of another compound, the keratinocyte growth factor.

In another trial, the FGF-2 was placed in the cornea of rabbits, a tissue that naturally has no blood vessels, but its presence induced the formation of blood vessels there too.

The laboratory tests have ended. The next stage of the project depends on the construction of a pilot plant enabling FGF to be produced on a larger scale, around 80 liters, under GMP (The acronym for Good Manufacturing Practice) conditions – appropriate for use in humans, according to the legislation. These conditions are designed, first, to assure quality. In addition, as they use recombinant DNA – genetically modified – the production process is conducted in such a way that these bacteria do not spread, even in the case of the E. coli, which is not foreign to man.

The team intends to patent the humanized FGF-2 made from a bovine gene. The same FGF produced in a similar way using recombinant DNA technology sells for an average of US$ 100 per 10 microgram. Production is expected to begin by midyear.

The conclusion of another project, undertaken in partnership with the Oswaldo Cruz Foundation (Fiocruz) of Rio de Janeiro also depends on the pilot plant: in it the group is working on developing an anti-helminthic vaccine – against schistosomiasis, an endemic disease in Brazil. Caused by the worm Schistossoma mansoni, it is contracted by the larva breaking into the skin when a person is exposed to contaminated water. The project is also at an advanced stage.

The vaccine will be produced from a protein also present on the surface of the worm, the Sm-14. The team of Paulo Lee Ho and de Ana Lúcia T. Oller do Nascimento, of the Butantan Molecular Biotechnology Laboratory, focused on improving the techniques of obtaining the Sm-14 by genetic engineering.

So far, Fiocruz has tested the vaccine on mice and rabbits and has achieved excellent results. According to the World Health Organization (WHO), only antigens that reach protection rate higher than 40% in tests on animals can be tried out on human beings. In mice vaccinated with the active form of the disease, a protection rate of around 50% was accomplished and, in rabbits, 100%. These animals, however, are not the best examples for protection tests: they are not natural targets since they do not normally enter water.

“We always use an animal for testing vaccines for human use, but the chosen model does not always give the same response as in a human: we have to carry out tests in humans to see whether it works or not”, says Ho.

The initial tests aim to discover whether the antigen is harmless to man. To carry them out, the group needs to produce the vaccine under proper conditions, which requires a working pilot plant. Ho believes that, once started, the tests on humans will be completed within five to ten years. The importance of the vaccine, in his view, lies in the ability to use it both for treatment and prevention of the disease.

The Projects
1. Fibroblast Growth Factor-2 (FGF-2): Humanization, Expression, and Possible Clinical Application (nº 99/08600-8); Type Support for research project; Coordinator Paulo Lee Ho – Butantan Institute of the State Health Secretariat; Investments R$ 5,000 and US$ 6,000
 2. Development of an Anti-helminthic Vaccine and the Challenge of Scaling it up to GMP Conditions for Phase I/II Clinical Testing in Humans (nº 98/14961-0); Type Support for research project; Coordinator Paulo Lee Ho – Butantan Institute of the State Health Secretariat; Investments R$ 83600 and US$ 95,785.86