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Unexpected action from a distance

Neurons can transport painkillers and anti-inflammatory drugs

sergio_livro.tifreproduction from the book “The Diary of Frida Kahlo” Sérgio Henrique Ferreira has always been curious and persevering. He often surprises his family and friends by concocting dishes with exotic combinations of ingredients in his kitchen. He also combines ingredients in his laboratory at the University of São Paulo in Ribeirão Preto, where for the last 36 years he has investigated how the compounds that block pain act; pain is one of the most uncomfortable symptoms of inflammation. The combination of curiosity and perseverance led him, in 1972, to discover how acetyl salicylic acid – the active principle of aspirin – prevents inflammation and reduces the pain. Years later, he demonstrated how morphine, the oldest and most powerful painkiller, suppresses pain by acting directly on the nerves responsible for the sensitivity of the body’s organs and tissues – not only because morphine acts on the brain and other parts of the central nervous system, as had been previously believed. And now an unexpected discovery has emerged from the laboratory of this pharmacologist, a native of the city of Franca, in the State of São Paulo: the interaction of painkillers and anti-inflammatory drugs with a special group of cells known as the nociceptive neurons.

These neurons, which innervate the skin, muscles, bones, blood vessels, and viscera, are the entrance through which pain enters the organism. They are called nociceptive because they detect environmental stimuli that are harmful to the body, such as the heat coming from the flame of a lighted match, and conduct them to the central nervous system, where they are interpreted as pain.

Ferreira, in partnership with the team headed by pharmacologist Berenice Lorenzetti from the Federal University of Paraná, noticed that specific compounds able to directly or indirectly combat pain can travel relatively long distances inside these neurons – in human beings, this distance can be more than one meter long – without losing their painkilling or anti-inflammatory characteristics.

The transportation of drugs by these cells explains why, for example, injecting a painkiller such as diclofenac or morphine near a deep wound in the leg acts way beyond the site where the injection is applied and can be as efficient in blocking pain as if the painkiller had been injected in the lumbar region of the backbone – from where it easily reaches the spinal cord fluid and other organs of the central nervous system.

Becoming acquainted with this form of transportation also opens up the possibility for the future development of new strategies for dispensing painkillers and anti-inflammatory drugs with fewer side effects. Acute pain such as the kind provoked by advanced forms of cancer or major surgery is lessened nowadays by the application of painkillers and anti-inflammatory drugs close to the spinal cord, a procedure that generally requires medical supervision, because the pharmaceutical drugs can affect the brain nerves and centers that control breathing, and in some situations, lead to death.

“Maybe some day these cases will be treated less aggressively, by means of intramuscular injections, in view of the fact that muscles are innervated by these neurons,” says Ferreira. If this alternative is successful, it will close the door which provides the access for pain to enter the body. Ferreira explains the reasoning by comparing the human body to a building. “If you don’t allow the dog to go past the ground floor, it will not go up to the tenth floor,” he says. “Understanding how to block the entrance to the building will allow us to control the pain that does not originate in the central nervous system.”

“However, we still have to conduct tests to verify whether this strategy actually works,” says the pharmacologist, who began his scientific career nearly fifty years ago as an assistant to one of the most renowned Brazilian researchers – Rio de Janeiro doctor Maurício da Rocha e Silva, who passed away in 1983, and who had discovered bradykinin, a nonapeptide released by adding enzymes of the venom extracted from the jararaca, the Brazilian lancehead snake, and controls blood pressure.

It took Ferreira and his team 15 years to understand what the experiments were showing. The first clues that some pharmaceutical drugs could travel through the neurons emerged in the mid 1990’s, when he and Berenice applied compounds into the cerebrospinal fluid of rats. These compounds stimulate the nociceptive neurons and make them more sensitive to painful stimuli.

They did not expect that the application of the compound in the cerebrospinal fluid would affect regions of the body as distant as the paws, even though the spinal cord has neuron endings that innervate them. After being given the injection, however, the rats began to pull back their paw when submitted to an impact they had been indifferent to previously; this indicated that the rats had become as sensitive to the touch as a thumb after being hammered.

As Ferreira was not able to explain this result, he searched for more evidence that this was a real effect. He also wanted some of his ideas on this phenomenon to be set aside for a while to mature. Meanwhile, pharmacologists Mani Funez, a member of the team headed by Berenice, and Djane Duarte, a post-graduate student working at Ferreira’s laboratory, decided to investigate what would happen if, at the same time, the animals were given a dose of painkiller in their paws, far from the site where the painkilling pharmaceutical drug had been applied. In a second round of tests, they inverted the site of the injection’s application and verified that, even when the painkiller was injected far from the sensitizing compound, it still eliminated the pain.

In a third phase of the test, they added a third compound – called antagonist, because it inhibits the painkiller’s action – and applied it together with the sensitizing agent. In other words, when they injected the painkiller into the rat’s paw, they injected the sensitizing agent and the antagonist into the cerebrospinal fluid and vice-versa. Then they compared the results with the results of the tests on another group of animals that had been given two compounds with antagonistic action (one a painkiller and the other an agent that inhibits the effect of the painkiller) simultaneously in the same place – only in the paws or only in the spinal cord.

The antagonist pharmaceuticals applied in distant places should not interact and annul one other’s effects if they are not transported through the neurons. In other words, in the absence of that interaction, the antagonist should not eliminate the painkilling effect of the morphine, injected into the paw or into the cerebrospinal fluid. This is not what they observed.

Tabulae anatomicae (1741), by Pietro Berrettini da Cortona

national library of medicine Tabulae anatomicae (1741), by Pietro Berrettini da Cortonanational library of medicine

When the antagonistic compounds were applied into bodily regions that were a few centimeters apart – simultaneously into the paw and the spinal cord – the effects were mutually annulled, in a similar manner to what the researchers had observed in the tests in which both compounds were injected into the paw or into the spinal cord. Ferreira named this interaction between compounds applied at points far from each other telentagonism, a phenomenon that was not previously believed to occur in neurons. At that time, researchers were only familiar with some small molecules that were transported very slowly inside those cells.

That interaction was so unexpected that it took some time before the researchers realized that this was a new phenomenon. “We did not believe in the results,” says Mani, the first author of the article that describes the phenomenon in the December 9 issue of Proceedings of the National Academy of Sciences (PNAS). “We thought that if we injected the painkiller into the rat’s paw, the drug’s action would be restricted to the site of the injection.”

Apparently, that interaction does not occur with all the drugs that act on the neurons. Mani and Djane observed that teleantagonism, the consequence of the transportation of substances in the neuron, occurred with two classes of pharmaceutical drugs: painkillers from the morphine family and the anti-inflammatory drugs from the aspirin family. The painkillers block the chemical reactions associated with the transmission of the pain message inside the neurons. The aspirin group – the world’s bestselling drug – acts in different cells, including neurons, preventing the production of compounds that make them sensitive to painful stimuli.

“We spent ten years doing experiments, thinking and re-thinking the results. At one point we even thought that we might have committed some kind of methodological mistake,” says Ferreira, who describes the discovery as being serendipitous, which means someone being lucky enough to find something valuable purely by chance. “We feel that the time has come to announce our findings and see what other researchers have to say about this phenomenon,” says the pharmacologist, who coordinated the research work that resulted in the article published in the PNAS journal.

However, the description of teleantagonism only clarifies some of the doubts. Researchers still don’t know, for example, how these compounds are transported inside the neurons – are they carried by proteins, and therefore consume energy? Or do they passively spread out by means of diffusion, in the manner of table salt molecules in a glass of water? The injection of a morphine-antagonist drug marked with radioactive material allowed the researchers to record the duration of the trip inside the nociceptive neuron. When applied to one of the paws, the drug took 90 minutes to travel throughout the entire extension of the neuron to the spinal cord. “The knowledge we have of the neurons’ physiology and of the transportation of molecules is not enough to explain why the compounds are distributed so quickly,” says Mani.

Another pathway
Ferreira believes there is a third explanation for the transportation of the drugs. They might be transported by means of an intricate network of nanometric-sized tubes – the microtubules – that comprise the cell’s inner structure. “I wonder if on this scale the speed of the transportation would still follow the parameters we are used to?” Ferreira asks. “This hypothesis could be tested. From the mechanical point of view, it is possible to build microtubules and measure the transportation speed inside and on the outer surface of the microtubules.”

Having opened up all his ideas, Ferreira has started to design a broader and more integrated view of how certain painkillers and anti-inflammatory drugs act on the organism and has started to gain a better understanding of morphine’s action on the nociceptive neurons that he and Meire Nakamura identified in 1979. The key to explain why these drugs applied into the muscle are able to inhibit pain in a broader region of the body is not only in the transportation of these drugs in the neuron. The key is possibly related to the region of the cell in which they act.

Ferreira believes that the pain-blocking chemical reactions occur in an area of the neuron that is known as the cell body – and not on the neuron’s endings, called axons. The cell body contains the genetic material and the characteristics that maintain the cell alive. An anatomical characteristic seems to allow the neuron’s cell body, or soma, to act as a kind of pharmacological bridge between the periphery of the body and the central nervous system, according to the researchers’ article published in the December issue. The neurons’ cell bodies innervate arms, legs and viscera – that is, the entire body – and are grouped in ganglia, the tissue mass structures located a few centimeters away from the spinal cord. The ganglia are in contact with the cerebrospinal fluid that bathes the central nervous system. “This characteristic integrates the pharmacological effects observed in the peripheral nervous system with those of the central nervous system,” says Ferreira.

In view of this new understanding on how pharmaceutical drugs interact with neurons, Mani dares to imagine possible progress in relation to dealing with pain. “Who knows – perhaps some day we will create compounds that can be injected into the muscle to act directly on the points of therapeutic interest in the central nervous system, without causing the undesired effects observed when these pharmaceutical drugs are given orally or intravenously?” First, however, the researchers will have to confirm whether the phenomenon described in the rodents also occurs in human beings. As Ferreira tells his students, science demands curiosity and a certain dose of daring.

The Project
Inflammatory reaction: mediators involved in the genesis of pain, migration and activation of leucocytes and septicemia (nº 01/07838-2); Type Thematic Project; Coordinator Sérgio Henrique Ferreira – USP-RP; Investment R$ 2,535,897.80 (FAPESP)

Scientific articles
FUNEZ, M.I., et al. Teleantagonism: a pharmacodynamic property of the primary nociceptive neuron. PNAS, v. 105, no. 49, p. 19 038-19 043, 9 Dec. 2008.