By David N. Leff

Science Editor

From acne and arthritis to anthrax, cholera and plague, minocycline is a wide-spectrum antibiotic. It has a decades-long history of attacking both Gram-negative and Gram-positive bacteria. Now this drug, a semi-synthetic chemical derivative of tetracycline, is being sicced on Huntington's disease (HD) - an inherited, neurodegenerative disorder, with no apparent microbial target.

HD strikes five people in a million - men and women alike. The trouble is it usually strikes them only in their 40s and 50s. Until then they don't know if they have been spared by the genetic Russian roulette that may have cursed a parent or grandparent. While still in the prime of reproductive life, they may give birth to a new generation, uncertain in turn of their fate.

In recent years, discovery on human chromosome 4 of the mutated gene for HD, huntingtin by name, and the aberrant protein it expresses, huntingtin, has ushered in prenatal diagnosis. But HD remains completely untreatable, and invariably fatal. Neuroscientists are working hard to petition for clemency, or at least reprieve, from this pitiless verdict.

One among them is neurosurgeon Robert Friedlander, of Harvard-affiliated Brigham and Women's Hospital in Boston. He is senior author of a progress report in Nature Medicine for July 2000. Its title: "Minocycline inhibits caspase-1 and caspase-3 expression and delays mortality in a transgenic mouse model of Huntington's disease."

Caspases are enzymes in the body dedicated to promoting programmed cell death - apoptosis - normally of worn-out or defective cells and tissues. Abnormally, apoptosis can kill healthy cells, such as brain neurons, thereby bringing on neurodegenerative diseases. At least, that's the current hypothesis. Friedlander bolstered that notion last year by inhibiting caspases in a mouse model of HD, using a chemical called zVAD-fmk. It slowed progression of their disease. (See BioWorld Today, May 20, 1999, p. 1.)

Now, he and his co-authors have gone that effort several better.

"For one thing," he told BioWorld Today," that tripeptide caspase inhibitor we used last year to slow mouse mortality has never been used in humans, and probably can't be. But our new compound, minocycline, is a drug that's been known for 30 years, and is in the pharmacy now. That's the main difference between both agents. What's more," he added, "minocycline, unlike tetracycline, its chemical precursor, crosses the blood-brain barrier."

How To Create A True-To-Huntington's Mouse

"The HD transgenic mouse model," Friendlander observed, "is not an exact replica of the human condition, but it is a pretty good imitation. The way its creators made it," he recalled, "was that they found the mutation in the huntingtin gene, which expresses a very big protein - 350 kiloDaltons with 67 exons. They then made the HD mouse by taking exon 1, a very small part of the protein, but the one that has the expanded CAG repeats - the genomic hallmarks of the disease.

"They introduced that construct into the genome of a mouse embryo, which grew up with a disease pretty similar to the human HD. From the behavioral point of view, it had typical tremors and weight loss, plus seizures and diabetes. Human HD patients," Friedlander pointed out, "don't usually have seizures, or diabetes.

"From a cerebral viewpoint," he continued, "the brain atrophies. There's neuronal cell death. The HD mice form neuronal inclusions, with huntingtin protein fragments, which is similar to what happens in the human brain."

In the co-authors' in vivo experiments, Friedlander recounted, "We injected minocycline intraperitoneally into the HD mice every day, starting at 6 weeks of age. They were just at the late presymptomatic stage of the disease. Actually, at that point their brains already have a lot of pathology. Some of the neuronal inclusions have formed by that age, and neurotransmitter receptor levels are starting to change.

"We monitored progress of the disease two ways," he narrated. "One, by putting the animals on a Rotarod. This is a motor-driven cylinder, rotating at variable speeds per minute. We rated their stability by the amount of time they could stay on the rod at set speeds. Second, we looked at mortality - from a whole-mouse point of view."

Friedlander summed up the results: "The decline on the Rotarod was slower in mice treated with minocycline, and their life was prolonged 14 percent." He said, "We tested tetracycline as a control, and found that it did not have a significant effect. So it's not just that we were giving a systemic antibiotic, and that's how the mice lived longer.

"Then," he went on, "we looked at the activation of caspase-1, which is active in the brains of humans and mice with HD. The minocycline not only inhibited this activation of the enzyme but - probably more importantly - blocked its production approximately 50 percent.

"A new finding that we reported," he pointed out, "is that another very important caspase is caspase-3. The general consensus in the field is that caspase-3 is probably the apoptotic agent that at the end does the actual killing of the cell. And the production of caspase-3 was blocked almost 100 percent by minocycline. This is the first drug that's ever been shown to do that, in any model."

Besides curbing the caspases, their experiments showed, minocycline also cooled another enzyme - inducible nitric oxide synthase (iNOS), which generates harmful free oxygen radicals. "In our preclinical mouse experiments," Friedlander said, "minocycline also - remarkably - inhibited the production of iNOS. So we looked in autopsied human brains with HD compared to normals, and there was a very significantly elevated activation of iNOS in the human HD specimens."

Clinical Trials 'Hopefully This Fall'

On the down side, Friendlander cautioned, "The issues that we need to watch with minocycline are several: One is that the HD mice seem to have more side effects than the normal mice when given the antibiotic. At higher doses, it was toxic for the animals. But I think that's probably an artifact of this transgenic mouse, which for some reason has very bad problems with diabetes. That's not something that's the same with human HD patients.

"I think minocycline made the diabetes much worse," he continued, "and that's why they died. For that reason, it's important for us to conduct a Phase I/II safety trial with minocycline in symptomatic HD patients, which we're hopefully going to be doing in the fall of this year."