North American and Canadian businessmen have created a pharmaceutical company to produce and sell a Brazilian invention: the LDE (low density emulsion) particles, artificial lipoproteins which will serve as the vehicle for medicines used in combating cancer and in the reduction of the toxicity of these drugs. The creation is of Dr. Raul Maranhão, professor at the Pharmaceutical Sciences School of the University of São Paulo (USP), Director of the Lipids Laboratory of the Heart Institute (Incor) of USP and now a member of the Board of the company, iCell Therapeutics Corp.
Incor and its financing organ, the Zerbini Foundation, signed an agreement with iCell through which they cede the patent, and in return, become shareholders with participation and representation on the Board of iCell, which is looking for resources to establish itself. José Antônio Ramires, the Director of Incor, clarifies that the patent has not been sold. “We deposited the patent as our part of the capital and we signed a contract which at its the end, in the case the company does not sell the product, we can reclaim the patent”. The agreement, which will result in the installation of the Center of Technological Development of iCell within Incor itself, allows the production and world commercialization of LDE.
When the LDE began to be developed, with a similar behavior to bad cholesterol or LDL (low density lipoprotein), the objective was only to create preventative examinations for atherosclerosis – the accumulation of fatty material in the arteries. The studies began in the 80s, with the target being the metabolism of kilomicron (Km), another lipoprotein linked to atherosclerosis. Contrary to the fractions of cholesterol LDL and HDL (high density lipoprotein), the metabolism of Km is not measured in the blood: its concentration varies with the quantity and quality of consumed fat, plus by the velocity of its absorption by the intestine. Conclusion: to study it, it would be necessary to follow its path through the organism.
Affinity with receptors
Consequently, Dr. Maranhão developed an artificial kilomicron. Injected with a radioactive marker, following it allowed for a better understanding of the obstruction of the arteries and showed that people who have coronary diseases have greater difficulty to metabolize and take out of the bloodstream the lipoprotein particles swallowed in food.
Dr. Maranhão says that there has were attempts of similar studies with LDL, but little advance had been made: “We are not allowed to inject blood derivatives of one person into another for research purposes, due to the risk of transmission of illnesses such as Aids and hepatitis. Also, the injection of LDL of the patient himself, and afterwards collection and preparation of the material, demands a large available source of volunteers, which would make the research something complex and impractical”. Hence the idea of an artificial particle which could be a tool for the identification of individuals with slower metabolisms and to stimulate prevention.
The first experiments with LDE on mice brought a new piece of data which dramatically changed the direction of the research: the artificial particle was capable of linking itself to the receptors of the cells, and even with greater affinity than some of the natural particles. Dr. Maranhão explains that the structure of LDE is very similar to that of natural LDL: a nucleus concentrated of cholesterol and an ester of cholesterol, and a surface formed from only one layer of phospholipids.
Therefore, it does not have apoproteins, the links between the lipid particle and the cellular receptors – especially the ApoB-100, which covers the natural LDL. Surprisingly, its lack doesn’t make a difference. Also, on entering into circulation, the LDE began to attract and incorporate apoproteins, especially the ApoE which, therefore, linked itself to the receptor. “It is as if the bond is made in a cooperative manner, in the case of ApoB only one molecule links the particle to the receptor, while with the LDE between two and four molecules of ApoE make the link.”
Another vocation for LDE had been discovered, that would make the specialist inlipids and physiology make his way towards the field of oncology. He already knew that the tumor cells have up to 100 times more LDL receptors than normal cells, and thus this lipoprotein is removed much more quickly from the blood circulation. That is why the majority of cancer patients have a significant reduction in the level of blood cholesterol. For Dr. Maranhão, this probably occurs because the accelerated proliferation of the neoplastic (cancerous) cell requires greater quantities of cholesterol and of other lipids, necessary for its multiplication and its survival.
Beginning with the discovery that LDE bonds so easily to the cellular receptors, the researcher worked on the hypothesis that it could serve as a vehicle to transport chemotherapies and selectively hit the neoplastic cells like a “Trojan horse”. “This has always been the ideal for any therapist a mechanism which would select the sick cells, preserving the healthy ones.”
There is no lack of attempts. One of the techniques most studied is that of the liposomes, which tend to concentrate themselves in more vascular cells – typical of tumors. Preparations with liposomes have already transported anti-fungus reagents in the treatment of infectious complications, above all, Aids. However, in relation to tumors, for Dr. Maranhão, the liposome is still an ” uncontrolled air balloon.”
He has already tested LDE on patients. Hisstudy, published in 1994 in the magazine Cancer Research involved14 people with breast cancer and 22 with ovary tumors (13 malignant and 9 benign). “We injected the emulsion with radioactive marking before the surgery of the removal of the tumor. Afterwards, we compared the normal ovarian tissue with the neoplastic tissue to measure the level of radiation.”While the benign tumor of the ovary captured the same quantity of active particles to that of the normal tissue, the neoplastic (cancerous) had ten times more particles than the healthy cells. In the breast cancer cases, the concentration of LDE was four times more.
Since the lipid emulsion united selectively to cancerous cells, it is presumed that the healthy ones remain protected from the emulsion chemotherapies. Tests on mice were carried out with the chemotherapy BCNU (carmustine). Afterwards, 42 patients at the Hospital das Clínicas and 20 at the Sírio-Libanês Hospital in São Paulo, with the help of the oncologist Dr. Antônio Carlos Buzaid, were evaluated. The volunteers had tumors of the breast, kidney, colon, bones and prostrate.
A triple dose
It was verified that with the LDE that the patients could tolerate doses of BCNU three times more. “The maximum dose that is normally used”, says Maranhão, “is between 150 and 200 milligrams per square meter (mg/m²) of body surface, with the known collateral effects – nausea, loss of hair and other more drastic such as depression of the bone marrow, which leads to disturbances of coagulation and of the immunology system. We verified that up to 400 mg/m² the toxicity is minimum, or that is to say, no significant collateral effects were verified. We increased the dose to 600 mg/m² and even then there was good tolerance”. Nevertheless, he believes it is too early to determine if the efficiency of the drug changes or not when conducted by the LDE, since, for example, the majority of the patients studied went through other treatments. “It is difficult to evaluate and document the responses to a treatment.”
The next stage includes the incorporation of two chemotherapies on LDE: paclitaxel (taxol) and etoposide. The preliminary tests have been promising. “For now, the two drugs were tested on mice. The taxol proved to be ten times less toxic and with the etoposide the toxicity was 16 times less.” (see table) The thematic project that Dr. Maranhão will be developing until 2003 will have two groups: one of cardiac transplants, and another of the systemic lupus erythematosus – a self-immune illness that deregulates the defense system. It has already been verified that transplants and patients with lupus show a great tendency to develop precocious atherosclerosis. The obstruction of the coronary arteries normally occurs after the first year of the transplant and is the greatest cause of death of transplants, which appears linked to the post-surgery immunological reactions. In the lupus patients, the risk of coronary obstruction is 50 times greater than that of the general population.
In a project which finished in 1995, Dr. Maranhão verified grave metabolic disturbance of quilomicron in patients with the lupus erythematosus system and cardiac transplants, results which were published last year in the magazines Arthritis and Rheumatism and Transplantation. In the present project, he intends to see if there are alterations of removal of LDL, both in the patients of lupus (in collaboration with Eloísa Bonfá) and in those with transplants.
Some encouraging inciting results came from other tests. With the collaboration of Dr. Carmen Cristiano, of her team, of professor Carlos Eduardo Negrão, of the Physical Education Institute of USP, Dr. Maranhão compared athletes and inactive people with normal levels of LDL and equivalent. “The athletes removed LDE from circulation twice as fast. Itwas also verified that the velocity of removal of the particle is proportional to the so called maximum VO2, the index of the consumption of oxygen which evaluates lung capacity.”
He hopes that, in the future, any person might benefit from a preventative examination with LDE, to detect precocious metabolic disturbances which do not appear in a simple blood examination. “Today, the cholesterol examination is like a photograph. It states how many milligrams of cholesterol there are in 100 milliliters of blood at a specific moment. Although the total cholesterol is a reflection of what enters into circulation, made by the liver, and of what comes out, entering into the cells via the receptors. It is this entire process that we can’t visualize with the LDE, just like a film.”
Ten years of fighting for the patent
Dr. Raul Maranhão didn’t lose his patience when he went through the trails of bureaucracy in order to obtain the patent of his invention, without which he could not advance. “It was a crusade which began in 1991, when we made the first consultations at INPI (National Institute of Industrial Property)”. At that time, the INPI could do very little: there was not a Brazilian law about patents which could regulate the production of medicines, which would only be approved in May of 1996.
As well, in the United States things weren’t easy. The patent, requested in 1992, took four years to come out, after a long back and forth journey of documents. In his point of view, “in spite of being competent and with a good level of knowledge in the areas of biomedicine”, the patent office workers in New York didn’t have much knowledge in the area in question. The major difficulty was to prove that the invention was not similar to anything already patented. “An emulsion could be an infinity of products, even a cream for face wrinkles.”
With the patent in his pocket, Dr. Maranhão contacted some national companies, but the conversations didn’t go anywhere. “The national industry still needs incentives to create a tradition in the development of pharmaceutical products”, he states.
The partnership with iCell solved the problem, but Dr. Maranhão has begun other battles. One of them is the clinical studies which still have to be done, this time outside of Brazil, so that the LDE will be accepted abroad. The production techniques will also have to be reviewed. Today, at Incor, the quantity of LDE sufficient for a patient absorbs three to four days of work “. At full speed, we might be able to attend to ten patients in a week”, says Renato Barboza, a laboratory technician. It is enough for the research but not for industrial production.
Artificial Lipoproteins in the Investigation of Dislipidemias and in the Treatment of Cancer (nº 99/01229-2); Modality Thematic project; Coordinator Dr. Raul Cavalcante Maranhão – Heart Institute (Incor) of USP; Investment US$ 364,678.00 and R$ 150,627.00