By David N. Leff

From sled dogs, oxen, camels and horses to locomotives, ships, automobiles and airplanes, every mode of human traction and transport is fueled by elemental oxygen - O₂. That goes double, of course, for us humans, not to mention all the other mammals on earth.

Deprive us of O₂ for only a few minutes and we die - of heart failure, stroke, drowning, strangulation. But as with an excess of other useful blessings, too much oxygen is more than enough. Whence, the mixed blessing of superoxide oxygen radicals - oxygen dragging one additional electron.

Whenever a mammal recycles food into energy, its cells generate highly reactive superoxide radicals. In many disease states - for example the inflammatory effect of arthritis, the reperfusion backlash of cerebral or myocardial ischemia, perhaps cancer and AIDS - those radicals turn against their own tissues, and damage or destroy their oxygen-radicalized cells. (See BioWorld Today, Sept. 29, 1999, p. 1.)

To be sure, the body has armed forces in reserve to put down such uprisings. These antioxidant bodyguards consist of enzymes called superoxide dismutase (SOD). Their munitions center on one of three metabolic metals - manganese (Mn), copper (Cu) or iron (Fe). Mn rides shotgun on the SOD recruited by the cell's mitochondrial organelle. The other two metals operate elsewhere in the cell or on its outside surface.

At first, in animal models at least, SOD looked good as an antioxidant therapeutic. But it fell short in human patients. "The main problem," said research pharmacologist Daniela Salvemini, "was the fact that the native SOD enzyme was of bovine origin - a bovine copper-zinc product. It was sold in Europe under the name of Orgatein, used with promising results in osteoarthritis and the side effects of cancer chemo- and radiation therapy. Unfortunately, what happened was the production of immunogenic side effects, because the product was of bovine origin. This was a not-unexpected finding. So unfortunately they had to stop Orgatein."

Salvemini is director of biology at MetaPhore Pharmaceuticals Inc., in St. Louis (the company's subtitle is "Metals in Medicine"). She and the firm's vice president of research, chemist Dennis Riley, set out to fashion a synthetic analog of SOD - a synzyme - to overcome that immunogenic hang-up and other SOD shortcomings.

Salvemini is lead author, and Riley senior author, of a progress report in today's Science, dated Oct. 8, 1999. Its title: "A nonpeptidylic [non-protein] mimic of superoxide dismutase with therapeutic activity in rats."

Synzyme Nine Years A-Borning

"In designing the mimetic," she told BioWorld Today, "the chemistry leader was Riley, and I was the pharmacology leader. He started this mimetic about nine years ago," Salvemini noted. "In a nutshell, the first step was to pick a metal, and we had a number of choices - copper, iron and manganese. We decided to go with the Mn form, because it's the least toxic of the three metals."

"The team's next step," she said, "was to go to a series of normal structure-function relationships. After that, our effort was to identify an appropriate ligand, to keep the Mn in its place, for enzyme catalysis. Then we had to decide: Which ligand do we pick? That took a lot of chemistry and computer modeling to come up with our SOD synzyme - M40403. To do it, we screened over a hundred molecules.

"The net result," Salvemini continued, "was a small mimetic enzyme, with catalytic activity approaching that of the native SOD enzyme, and of very high in vivo stability - meaning no Mn loss in vivo. The molecule really stayed intact when we put it into animals. And it showed very high efficacy in rat models of inflammation and reperfusion injury.

"The difference between our synzyme and the SOD molecule," she added, "is that the native enzyme's molecular weight is 32,000 kiloDaltons, whereas our mimetic is about 500. That's a 64-fold difference. A huge molecule compared to a very small one."

The co-authors put their mini-molecule to work in rats, testing its efficacy at reversing or abolishing the cellular devastation of free oxygen radicals in arthritic-like inflammation and ischemia-reperfusion tissue injuries. In both in vivo studies, animals treated with M40403 did better than animals who got SOD.

To simulate reperfusion, they clamped shut the splanchnic (visceral) arteries of rats for 45 minutes - long enough to induce ischemia. When blood flow to their intestines was allowed to resume, the circulatory shock killed most animal models within two hours, and all of them within four. But 90 percent of rats treated with M40403 survived.

For inducing intense local inflammation, a classical method is to inject a polysaccharide called carrageenan into the footpad of a rodent. Its paw swells up to a measurable maximum in three to six hours. As reported in Science, the co-authors gave six rats intravenous shots of M40403 half an hour before the carrageen treatment. This drastically reduced the edema and tissue damage at the paw, as well as releasing metabolic hallmarks of inflammation, notably white blood cells and immune-regulating cytokines.

Neutrophils, which are phagocytic (debris-scavenging) white blood cells, were present in force. "Superoxide," Salvemini observed, "can also up-regulate adhesion molecules, and as such elicit neutrophil recruitment and infiltration at sites of injury. In turn, neutrophils can get turned on, activated, and cause tissue injury.

"A very important role for our M40403, which we reported in Science," Salvemini pointed out, "is the fact that superoxide seems to be involved with the release of pro-inflammatory cytokines, such as tumor necrosis factor and interleukin-1. This is the first time that a SOD mimic has been shown to inhibit cytokine production. Let me emphasize," she said, "that we do not understand the mechanism by which superoxide is involved there, but it's something I intend to go in and explore."

Phase I Trials Foreseen In Second Half Of 2000

The Science paper concluded: "Understanding the signal transduction mechanisms used by free radicals to modify the course of disease will undoubtedly elucidate important molecular targets for future pharmacological intervention. SOD mimics such as M40403 can serve as tools to dissect these mechanisms."

In a press statement dated today, MetaPhore said that "The company intends to initiate Phase I trials for its lead SOD compound in the second half of 2000." (See BioWorld Today, Sept.13, 1999, p.1)