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Neurological reagent

Butantan signs antibody patent licensing agreement with Millipore

BUTANTAN instituteRat nerve cells under the effect of the Nudel reagent. The nuclei of the neurons are shown in blue and the action of the Endo A protein in yellow or red.BUTANTAN institute

This month two small, 10 ml flasks containing polyclonal antibodies, produced by the Butantan Institute in São Paulo will be sent to Millipore in the USA, a company that specializes in the worldwide distribution of reagents for scientific use. The product is totally new and will be used for research in the neurology area, including tests for the development of future medication. The antibody selectively recognizes the enzyme, endooligopeptidase A, also known as Eopa, Endo A or Nudel, the acronym for nuclear distribution element-like. This molecule plays a part in intracellular transport in the central nervous system and in the arrangement of neurons during the formation of the embryo.

“In the central nervous system Endo A is found in the neurons and there is evidence that it plays an important role in schizophrenia, lissencephaly, also known as smooth brain disorder, and in neurodegenerative diseases,”  explains pharmacist Mirian Hayashi, a professor at the Pharmacology Department of the Federal University of São Paulo (Unifesp) an d coordinator of the FAPESP-funded patent project. A substantial part of the work was carried out at the Butantan Institute, where Mirian worked for more than ten years in the laboratories of the Center for Applied Toxinology (CAT), one of FAPESP?s Research, Innovation and Dissemination Centers (Cepid).

Endo A has two known functions. One is to bind with other proteins expressed in the neurons of the nervous system, enabling the intracellular transport and rearrangement of these neurons in the brain. Thus, the serious pathology known as smooth brain, characterized by the absence of convolutions in this organ, called cortical gyri, is associated with a dysfunction in the bonding of Endo A with another protein called Lis1, genetically related to this sickness. “The neurons do not distribute themselves over the surface of the brain and cortical gyri are not formed during the embryo’s formation,”  explains Mirian. The child, therefore, dies in the uterus or is born severely mentally retarded and generally does not live for more than three years. Researchers know that the bond of Endo A with another protein called DISC1, which is related to schizophrenia, also plays a fundamental role in this disorder’s molecular mechanism. Affected patients express a protein with a mutation that does not bind to Endo A. This non-binding is also directly related to the molecule’s second function, which is to break down peptides (protein fragments). Everything indicates that this activity is related to the physiopathology of neurological disorders, although to date it has been impossible to identify which peptides are destroyed in the brain of a living organism by Endo A.

Initial discovery
“Nudel may  also be involved in diseases such as Alzheimer’s and Parkinson’s, because it’s important for cell transport and may have an influence on the bond between neurons,”  says Professor Antonio Carlos Martins de Camargo, CAT coordinator. He was the researcher who discovered this enzyme in 1967. Though he was the first to describe Endo A and also Endo B, likewise identified for its capacity to degrade neuron-transmitters, and to write almost a hundred scientific articles, in addition to having been the advising tutor for dozens of theses relating to these proteins, the scientific community only really paid attention to this enzyme in 2000.  At this time,  US and Japanese researchers isolated the same protein and published a series of articles in Neuron magazine about their findings; they also renamed Endo A Nudel, and all without mentioning Camargo or his team. However, in 2002, after a looking at the literature involved, researchers from the English pharmaceutical company, Merck Sharp & Dohme (MSD), recognized the importance of the Brazilian group and asked a consultancy company to develop studies with Nudel.

The result of this work was jointly presented three years later in an article in the Proceedings of the National Academy of Sciences (PNAS), where it was proved that Endo A and Nudel were the same protein. The role this molecule plays was also demonstrated in the migration of neurons and its enzymatic action in breaking down the peptides and neuropeptides of this protein was proved. The only reason the studies in partnership with Merck did not continue was because the company closed down its research center in England due to the financial problems it faced after sales of its anti-inflammatory drug, Vioxx, were suspended.

With the identity and importance of Endo A proved, the preparation of good antibodies for this protein became extremely important, because it works like a tool for identifying and inhibiting the enzyme, two factors that are essential in laboratory studies for checking the causes and understanding the molecular mechanisms of some neurological diseases. The antibody as a product can be sold as a laboratory reagent and can serve as a means of study to enable the development of new drugs. This is why Millipore and its subsidiary, Upstate, which has a catalogue of products dedicated to the sale of antibodies, are going to receive 10 ml of antibodies for rat Nudel and 10 ml of antibodies for human Nudel. This material will be split up and repackaged by the company and sold in smaller doses of approximately 0.1 ml. “We’re going to see what demand is like and if necessary we’re going to produce more,”  says Mirian.

Special breed
For this task Mirian has the collaboration of Osvaldo Sant’Anna, also a researcher from the Butantan Institute, who offered his breed of mice that are specially selected for preparing highly specific antibodies. The production of 10 ml involves five or six mice, specially bred for this purpose. The Endo A injected into the mouse, which then develops the antibody (after the animal’s blood has been drained and purified), was produced in an agreed way. In this technique the genes of human and rat Nudel were cloned and multiplied using the  DNA technique, in which Escherichia coli bacteria are used to produce the enzyme.

The rights of the team in this process are guaranteed by an international patent, which, in addition to Mirian and Camargo, mentions the inventors as being researcher Fernanda Portaro and Ph.D. student Juliano Guerreiro. The licensing agreement was analyzed and negotiated by FAPESP’s Technology Patenting and Licensing Center (Nuplitec). The terms of the negotiations are confidential and the period covered by the agreement is ten years. “I believe that in the future it will be possible to diagnose neurological diseases using Nudel. Then it will also be possible to license the right to produce diagnosis kits, which have also been included in the registered patent and that are obviously a lot more profitable,”  says Camargo.

The Project
Endooligopeptidase A, patent request
PAPI (Intellectual Property Support Program)
Mirian Hayashi – Unifesp
R$34,261.67 and US$57,417.78 (FAPESP)