Insulin from the liver

Doctors and researchers implant cells and create an alternative to pancreas transplants

MARI CLEIDE SOGAYAR / USPThe pancreatic islets (in red): selection and purification in eight hoursMARI CLEIDE SOGAYAR / USP

A 45-year-old business manager from the state of Bahia, Telma Rosário de Almeida, saw her life change overnight at the beginning of last December, when she was submitted to the first transplant of cells that produce insulin, the hormone that regulates the level of sugar in the blood, which over the next few years may benefit other diabetics like her. Telma’s diabetes, detected 26 years ago, worsened and ran out of control in 1998, when the daily injections of insulin became insufficient to stabilize the level of sugar, which started to oscillate frequently, as if on a roller-coaster; it would reach double the normal level, only to plummet less than an hour later to one tenth of the amount that it had reached.

These variations would cause constant discomfort and fainting fits that prevented her from working. For cases like this, the only solution available in Brazil is a transplant of the pancreas, the organ that produces insulin, which has been carried out regularly in the country since the 90s.

But Telma was lucky. She did not need to wait years on the waiting list for a donation and a pancreas transplant, an operation that takes up to nine hours. On the night of November 29th, 2002, a Friday, she received a phone call that made her take a plane from Salvador to São Paulo on the following day. Then, on Sunday, she was to be the first person in Brazil to receive an implant of the cells that produce insulin, the pancreatic islets, which took 35 minutes to be inserted in her organism.

Contrary to what could be expected, the cells that were to replace the hormone that her body had ceased to make were not lodged in the pancreas – this gland, some 20 centimeters in length and shaped like a cob of corn, located behind the stomach, is extremely delicate, and every effort is made to avoid meddling with it, because of the risk of severe inflammations.

It was in the portal vein, which irrigates the liver, through a 2- millimeter incision on the right side of the abdomen and a 30-centimeter needle, that the eight doctors under the coordination of endocrinologist Freddy Goldberg Eliaschewitz, from the Albert Einstein Hospital, injected some 250,000 pancreatic islets, a set of four different kinds of hormone-producing cells – insulin comes from one specific kind, the beta cells.

One of the peculiarities of this kind of implant, which nowadays is being carried out in 50 centers around the world, is precisely to make the liver, an organ that produces dozens of substances that are essential for the functioning of the organism, develop something it was not cut out to do: insulin. The liver was chosen for this task for its high capacity for regeneration, for its tolerance to the incorporation of matter that is foreign to it, and for consuming half the body’s insulin.

Financed by FAPESP, the National Council for Scientific and Technological Development (CNPq) and by the Financier of Studies and Projects (Finep), this project led to the mastery of the technique for obtaining and purifying the pancreatic islets. Implanted, in Brazil, by biologist Mari Cleide Sogayar, from the Chemistry Institute (IQ) at the University of São Paulo (USP), the method prizes nimbleness: only eight hours lapse between the moment when the pancreas is taken out of the donor with brain death and the researchers are separating the islets and leaving them ready for the transplant.

Function recovered
Three days after receiving the islets, Telma no longer felt any giddiness or discomfort. She started measuring the level of sugar in her blood only seven times a day, and no longer 25, as she did before the implant, and to inject two doses of insulin, instead of six. It was a sign that the recently implanted pancreatic islets were beginning to work and to replace the function of the original islets, destroyed by the defense system of the body itself, one of the characteristics of diabetes melittus 1.

They are heartening results, but are far from meaning an end to treatment. In the next six months, this business manager will have to undergo at least another two similar implants in the liver, in order to restore the normal quantity of insulin producing cells in the pancreas (about 1 million). In one year, the hope is hoped that the liver will take this extra work on board and fully replace this function of the pancreas. From this moment onwards, Telma will abandon the hormone applications, although without giving up the immunosuppressive medicines that prevent the rejection of the implant but increase the risk of infection and of cancer.

Before this kind of implant becomes available for a greater portion of the 500,000 victims of diabetes mellitus type 1 in Brazil, a few barriers will need to be overcome. One of them is to prove scientifically that the procedure is safe and effective. To do so, the Ministry of Health has authorized it to be carried out in another 17 diabetics over the next three months. Only then will the ministry assess the results and the possibility of including the implant on the list of procedures paid for by the National Public Health System.

Completing the experimental stage, though, does not seem so simple: the total cost of the implants needed to free a patient from the uncomfortable insulin injections is roughly US$ 100,000 – or US$ 20,000, should a donation of the immunosuppressive drugs be achieved -, an amount ten times higher than the cost of a pancreas transplant. Almost US$ 2 million would be needed, therefore, to comply with the minimum requirements for the safety and reliability of this new medical technique. “Even with business support and development agencies, it will probably be difficult to carry out all the necessary 17 transplants in three years”, Eliaschewitz recognizes.

It was he who decided to attempt in Brazil the transplant of the pancreatic islets, when the greater part of the foreign groups had still not found more encouraging solutions: only 11% of the patients were not taking insulin one year after the operation. In 1994, Eliaschewitz went to see Mari, at USP’s Chemistry Institute; she was studying the control of tumor cells and had experience in the cultivation of cells in the laboratory. When proposing that they begin the studies for isolating the pancreatic islets, he felt the resistance that he would have to overcome. “When I told her what I intended to do, Mari hesitated”, Eliaschewitz recalls. “But I said that I would only leave the room if she agreed to start the project”. Mari explains: “I had bad experience working with doctors before”. But she herself recognizes: “Today, we are a Jew and an Arab working in peace”.

Technique perfected
Over six years, a team of 17 doctors, biologists and biochemists sought to master the most complicated stage of the process: isolating the islets, an agglomeration of up to one thousand cells of four different kinds (alpha, beta, gamma and delta) which makes up only 1% of the pancreas. The majority of times, they would not achieve much success: the degree of purity of the isolated material would be no higher than 40%. In other words, only 40 out of each 100 cells separated were islets – the rest were cells that produce the digestive enzymes that the pancreas discharges into the small intestine.

The results only got better two years ago, after the team led by James Shapiro, from the University of Alberta, in Canada, perfected the technique for getting the islets. The modifications were small, but essential, and the researchers started to implant twice these sets of cells in the liver, the organ that best lends itself to housing this material. In an article published in 2000 on the New England Journal of Medicine, Shapiro reported the successful implant of pancreatic islets in seven persons, who one year after the operation ceased to depend on insulin – nowadays, the success rate of the procedure is close to 85%, a level similar to the one achieved with pancreas transplants.

Two months after the publication of the article, physician Carlos Aita, who is studying for a doctorate under Mari, was already to be found in the laboratory of Jonathan Lakey, from Shapiro’s team, to get to know details of the new technique for isolating the pancreatic islets. “On his return to Brazil, even without having at his disposal all the equipment used abroad, the results in redoing the isolation were much better”” explains Aita. But the researchers’ work was really given a push by the construction of a contamination free room at the Human Islets Transplant Unit at the Chemistry Institute. Concluded in 2001, at a cost of roughly US$ 500,000, financed by the institute itself and by FAPESP, the room made it possible for the researchers to extract islets with a degree of purity of up to 90%.

It is there, in an environment that is more sterile than a surgery room, that the researchers carry out the most sensitive stage of the process for getting the pancreatic islets: the digestion of the pancreas removed from the donor with brain death, done with enzymes like collagenase, capable of breaking down only the fibers that join the cells of the pancreas together, without, however, destroying the islets. The result is a pasty material, which is next centrifuged sothat the islets that one wants to implant in diabetics can be obtained, bythe differences in density. “The purer the suspension of islets, the lower the volume to be implanted in the patient will be, and accordingly the lower the risk of clogging the branches of the portal vein”, Mari observes.

It is also in this room that the researchers are working to overcome another challenge for the implant to become a viable alternative: producing the islets in the laboratory, thereby avoiding the need for so many donors – at the moment, each treatment has to consume two or three pancreases until the use of insulin is dispensed with.

“We are cultivating the islets in the laboratory, and, in future, we hope to use them successfully in transplants”, the researcher observes. In partnership with a company called Biomm, a division of Biobrás, Mari is developing microcapsules of a material compatible with the organism that can coat wrap up the islets and protect them from the attacks of the immune system. The microcapsules could be a way of reducing or even eliminating the need for immunosuppressive drugs, indispensable even in the case of a totally successful implant.

The Project
Isolation, Preservation, Cryopreservation and Microencapsulation of Pancreatic islets for Transplanting in Diabetic Patients (nº 96/12865-9); Modality Regular research benefit line; Coordinator Mari Cleide Sogayar – Chemistry Institute at USP; Investment R$ 233,185.03