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Large size growth hormone

PIPE company develops medication using recombinant DNA techniques and reaches agreement to manufacture the product more cheaply and replace the imported product for dwarfism (nanism)

Hospitals, healthcare posts and distributors of pharmaceutical products should receive the first bottles of human growth hormone produced in Brazil in the first quarter of 2002. At present, the product is imported and costs the country an outlay of millions of dollars. Better still, the hormone produced here will be around 30% cheaper that the imported product. The medication used in particular to treat children suffering from dwarfism brought about by hormone deficiency, that of the human growth hormone or hGH, from, Human Growth Hormone, is the result of research by the Genosys Biotecnológica company that has been financed by FAPESP under its Small Business Innovation Research (PIPE). In October 2000, Genosys found the right partner to produce and sell the hGH, the pharmaceutical company Braskap, also Brazilian owned.

Growth hormone deficiency affects one in every 15,000 children and the only treatment is hormone replacement. Diagnosing the deficiency in producing hGH should preferably be done between the ages of 3 end 5. The treatment continues until the cartilages stop growing at around 16 years of age. A child takes 0.1 IU (international units) a day per kilo of body weight. It involves about 12 years of treatment, costing in all R$ 360,000. The partnership between Genosys and Braskap will enable this sum to be brought down to around R$ 250,000.

In a normal individual, the release of the hormones in the blood reaches its peak during sleep and during adolescence. Between 20 and 25 years of age, the body begins to produce less, until it is almost zero at around 60 years of age. This growth hormone in itself has no activity. It is released by the pituitary gland (hypophysis), located at a base of the brain. It reaches the liver through the blood stream and induces the production of IGF3 and of IGF1 (Insulin-like Growth Factor), the substances that really promote growth. The use of the growth hormone in treating children began in the mid 60s, when the pituitary gland was obtained from corpses.

In the United States, the National Pituitary Agency was set up to collect the glands. In the 70s, the recombinant DNA technique emerged enabling the hormone’s coding gene to be cloned in genetically modified bacteria. At the same time, it was discovered that using a pituitary gland obtained from corpses was associated with Creutzfeldt-Jacob’s disease. This disease is the human version of “mad cow” disease, and is marked by physical and mental degeneration, and this led to a worldwide banon using the hormone obtained directly from the pituitary.

Academic product
Genosys produces the hormone using the recombinant DNA technique. Cloning the hormone’s coding gene was first done by professor Hamza Fahmi Ali El Dorry, of the Biochemistry Department of the Chemistry Institute of the University of São Paulo (USP). That year, he invited the biochemist Jaime Francisco Leyton, who had been his doctoral student and who had a postdoctorate degree at Chicago Medical School, in the United States. “He invited me to undertake the production of the hormone with him”, tells Leyton. “Genosys was founded based on incentive that PIPE offered, since we wanted to continue doing research while we looked for partners and finance”.

Partners wanted
With the project approved right after the PIPE issued its first invitation, Genosys received R$ 66,000 plus a further US$ 101,000 to develop the end product. The purchase of equipment was included in these sums. The search for commercial partners was not easy. “It was a pilgrimage. We consulted several companies, and we held various meetings, but pharmaceutical companies were either unwilling to invest in research and development, or they wanted a huge financial return from the association”, comments Leyton.

Genosys ended up consulting BNDESPar – the BNDES’s affiliate company specializing in providing capital for Brazilian companies by buying shares or convertible debentures – and the project was approved on merit, but the funds would only be released if there were a partner for selling the hormone. “We sat down with representatives of a pharmaceutical company and of BNDESPar, who would provide the money, but we could reach no agreement. We were providing the technology and obtaining the capital, but even so the pharmaceutical company wouldn’t close the deal”.

In mid-2000, Genosys and Braskap set out to reach an understanding, and discovered that they had various points in common, and the agreement was signed at the end of the year. The contract with Braskap is essentially a consortium: “The purpose is to produce and sell the hGH. Genosys’s main contribution is its production expertise, while the most valuable contribution that Braskap brings to the partnership is its experience in distribution and sales”, explains Leyton.

Today, after taking part in the 3rd edition of the Venture Forum (see Pesquisa Fapesp no. 64) , Leyton is negotiating the participation of risk capital investors in the company, thus ensuring better financial health for Genosys and more investment in developing new products.

Final trials
Before going on the market, the Genosys hormone still has to go through clinical trials. So far, the entire production process has been developed and validated, but the hormone has not been given to a human being; it has only been tested on animals and approved, with the help of the Toxicological Diagnostics Laboratory (Ladtox), under the coordination of professor Luiz Carlos de Sá Rocha.Even these acute and chronic toxicity tests, using 1.5 and 10 time the therapeutic dose in three species of animal will have to be done again using the hormone produced on Braskap’s premises, because of a legal requirement.

Trials in humans will be quick; the hormone just has to be given and its responsein the blood stream checked. Another factor facilitating the release of the hormone on the Brazilian market is its similarity to products manufactured abroad. Genosys’s greatest difficulty in developing the hormone was mastering the downstream process (purification), about which no company that has this mastery is willing give any solid clues. Until a few years ago, cloning the gene was the most complex stage.

Nowadays the technology is better known. Researchers obtain the gene that codes the hormone production from the pituitary and modify it to meet the needs of the bacterium Escherichia coli where the hGH will be produced. This DNA sequence is inserted into a DNA molecule of the bacterium (plasmid ). Once this plasmodium is in place within the bacterium, it will begin producing the hormone. The synthesis is activated by the presence in the culture of a chemical inducer.

Production capacity
The next stage is to promote the fermentation of the bacteria in a 200-liter fermenter. “Today, we can easily produce 12,000 4-I.U. ampoules a month,” says Leyton. A child suffering from dwarfism uses around 250 ampoules a year. Thus, the Genosys/Braskap consortium’s production capacity can supply up to 576 children. “But we have every chance of raising this to 40,000 ampoules a month”.

Fat and efficient
The bacteria are excellent hormone producers. They produce so much that the product has to be stored in the so-called inclusion corpuscles, which can easily be seen under a microscope. With the bacteria fattened up, the downstream process can begin. The first thing to do is to “break” the bacteria to release the corpuscles and to wash them. Then the hormone inside the corpuscles is made soluble and placed in its original arrangement.

Then, it is the turn of the chromatographic purification processes, in which a aqueous solution containing the hGH passes through a succession of processes such as ionic exchange and molecular sifting, among others, to remove the impurities from the hormone. Only about 20% to 28% of the hGH the bacteria have produced remain; the rest is lost. Leyton explains that this yield is economically feasible, and it is preferable to accept this loss in order to obtain a highly purified product.

In the following stages, a conserving agent is added and the desired concentration of the hormone is established. Finally, the solution will be lyophilized (freeze dried in a vacuum). Using this process, the powdered hGH will remain stable for up to two years. The degree of purity required in the production of the growth hormone is much higher than for other products also produced by genetic engineering, such as a vaccine, for example, explains Leyton. “When you are vaccinated and there is reaction, a slight fever, people regard this as normal.

But the vaccine ought not to produce any reaction at all. What happens in purifying the vaccine is that producers, for purely financial reasons, are satisfied with 95% or 96% purity.” This is possible as a person will take a only one single shot of vaccine and at most a re-enforcing shot some time later, andif some slight fever occurs, it will soonabate. But in the case of the hormone, the child will take it practically every day and no impurity can be tolerated in the vaccine. “The secret of hGH production is purifying it”.

Free supply
The main buyer of the hormone will be the public healthcare system, which supplies the medication to children free of charge and also to adults because, even after adolescence, patients use the hormone in decreasing doses for the rest of their lives. The Central Institute of the Hospital das Clínicas of USP’s School of Medicine supplies 320 people with their hGH needs, most of them children.

According to Berenice de Mendonça, head of the hospital’s Endocrinology Development Unit the treatment of children suffering from dwarfism because of hormone deficiency in the State of São Paulo can be considered good. The hGH used in the various hospitals in the State that treat this problem is supplied by the State Health Department and by the Ministry of Health. In addition to the 320 supplied by the Central Institute, more than 100 children receive hGH at the Children’s Institute, also associated with the hospital.

Growing and diversifying
Braskap has 20 years experience in the market to help it distribute the medication. Located in Sorocaba, the company’s main business is the manufacturing of gelatin capsules for medications (1 billion a year) for various pharmaceutical companies. There are 15 manufactures of these capsules in the world, three of them in Brazil: two foreign companies and Braskap, which is wholly Brazilian owned. In 1995, the company also set up its pharmaceutical division, which now has ten medications in production.

According to the company’s president, Augusto Mattos, Braskap’s investment in the partnership will be focused on the construction of a specific area for producing the hormone, equipment, clinical trials, and setting up a promotion and selling team. “We estimate the initial investment at US$ 2 million. We forecast that in the early years the hormone will account for 5% of the company’s production capacity.”

For Mattos, the pioneering nature of the two companies is a reason for optimism. “Brazil has an excellent community of researchers, which, with the support of the business community, will bring great development to the country in all sorts of areas”. The contract signed by Braskap and Genosys provides for the development of new products.

Uses approved and abuses denied
Laboratory-produced human growth hormone has so far, according to the prestigious US Food and Drug Administration (FDA), been approved for the following uses: dwarfism resulting from hormone deficiency and Turner’s syndrome, a congenital condition associated with a defect or deficiency of the X chromosome affecting girls, which, among other consequences, leads to short stature in 95% of those affected and sterility. It is prescribed for treating patients with an advanced stage of Aids, when people begin to lose muscles mass.

Other uses, including in Brazil, are in the case of renal transplants and adults with a deficiency of the hormone. In the United States, there is a movement in medical circles that is demanding FDA approval for the geriatric use of hGH. There is also research in progress in Brazil on geriatric use, in the treatment of people with serious burns and in reducing abdominal fat.

In the case of geriatric use, studies in recent years suggest there are benefits in combating osteoporosis, in lowering muscle mass and reducing localized fat deposits. The results are not yet conclusive. According to José Antonio Miguel Marcondes, president of the São Paulo Endocrinology and Metabolism Society, an article on the geriatric use of hGH was published in the New England Journal of Medicine four years ago.

“The benefits for the elderly seem uncontested, although the survey published was limited in terms of sample size”, says Marcondes. In the case of healthy people that attend health clubs who want to use hGH to gain body mass and look younger, the issue takes on a shade. “This type of use is at the very least improper, because of the lack of any detailed studies and the risks run, which range from diabetes to cancer”.

There are other serious risks for people who use hGH without proper medical guidance, and who do not suffer from any deficiency of the hormone: growth of the feet, hands, chin and the eye socket (under the eyebrows) and the cartilaginous extremities (the ear and nose), growth of existing tumors, cirrhosis, pain in the joints, swelling, and high blood pressure caused by liquid retention, as well as the emergence of coronary disease.

Classified as an anabolic steroid by the International Olympic Committee, hGH is subject to special control (sale against medical prescription in two copies, both to be retained by the drugstore) by the National Sanitary Surveillance Agency, according to a resolution issued in February 15, 2001.

The project
Production of the Human Growth Hormone using Recombinant DNA Technology (nº 97/07394-0); Type Small Companies’ Technological Innovation Program (PIPE); Coordinator Jaime Francisco Leyton – Genosys; Investment R$ 232,134.21