By David N. Leff

Science Editor

Perched high above the rest of the human body, the brain shares one exclusive property with the lower-down testes. Both organs are immunologically privileged. The eyeball's cornea also belongs to this members-only club. No antibodies or killer T cells, hunting alien intruders, need apply to these guarded, gated premises.

In the brain, those gates consist of tight junctions between the endothelial cells surrounding that organ, and keeping out large blood-borne proteins, such as antibodies. This seamless cerebral chastity belt is well named the blood-brain barrier (BBB).

"But when there's too much activity in the brain," observed neuroscientist Matthew During, "when there's a disease process going on, such as a stroke or severe epilepsy seizure, then the tight junctions loosen up a bit, and enable antibodies to get across the BBB. And that's just the site we want," he pointed out. "The antibodies cross into the brain, and bind to its glutamate receptor, which is in part mediating the damage.

"Glutamate," During explained, "is the major excitatory neurotransmittter in the brain. How one brain neuron talks to the other, and communicates and excites it to action, is by releasing glutamate. This molecule acts only on the cell-surface proteins that are its receptors. They come in many different flavors and colors, and are typically classed as NMDA and non-NMDA receptors. NMDA stands for N-methyl-D-aspartate, which is a chemical analogue of glutamate and aspartate. The NMDA receptor, when it's activated, acts as an ion channel, and allows calcium to pour into the cell.

"That usually is a good thing under normal brain signaling," During went on, "but when there's an insult like this to the brain, too much calcium comes through these pores, and sets off a whole set of events that end in cell destruction."

During directs the Central Nervous System Gene Therapy Center at Jefferson Medical College in Philadelphia. He is corresponding author of a paper in today's Science, dated Feb. 25, 2000, titled: An oral vaccine against NMDAR1 with efficacy in experimental stroke and epilepsy." (See BioWorld Today, Oct. 26, 1998, p. 1.)

"With this single-shot oral vaccine," he told BioWorld Today, "we have demonstrated that we can generate autoantibodies to a brain protein - the NMDA receptor - that can be therapeutic in a prophylactic sense. In other words, that can potentially protect the brain from insults that might occur in individuals at risk of epilepsy, stroke, brain trauma, and perhaps neurodegenerative diseases as well."

Antibody Gate-Crashes Barrier On Demand

During and his co-authors base their strategy on the little-noticed phenomenon that when injured, the brain transiently relaxes the tight seal of its blood-brain barrier, and breaches its immune privilege. "That," he pointed out, "enables the immune system antibodies to rush into the brain, during this window of opportunity, and talk to its neurons."

Armed with this passkey, the Jefferson team constructed a gene transfer plasmid consisting of a recombinant adeno-associated virus (AAV) coupled to a complementary DNA sequence encoding an antigenic NMDA receptor. Then they raised a small army of epileptic rats, by injecting them with kainic acid.

"This chemical, kainic acid, isolated from seaweed," During noted, "is a very potent structural analogue of glutamate. It crosses the BBB, activates glutamate receptors, and doing so causes seizures, because it selectively goes to parts of the brain that generate epilepsy.

"Our first proof-of-principle study," he recounted, "was to use this good animal model of temporal lobe epilepsy - probably the most difficult form of human epilepsy to treat. "In an initial in vivo experiment," he continued, "we immunized a group of 20 or 30 rats with the NMDA plasmid, plus two control cohorts. We administered the vaccine by oral orogastric tube dosing of AAV expressing the specific NMDA gene. And gave placebos to similar numbers in the control groups. Then we injected epileptogenic kainic acid into nine of the NMDA-vaccinated animals.

"We subsequently showed in brain autopsies that we could get this humoral [B cell] antibody response. The antibodies came up with respectable titers at about one month, and at five months had increased to very high levels.

"Of the nine NMDA-immunized rats, seven were completely normal. They ran around, didn't even look as though they'd been given the drug, displayed excellent learning and memory, and their brains showed no injury. Of the two rats that did get seizures, one didn't have circulating antibody. The vaccine obviously didn't take in that animal, so it wasn't surprising that it had epilepsy. The other rat, which had prolonged seizures, revealed a brain free of all damage, which we found remarkable.

"So after the success of our epilepsy experiments," During continued, "we decided to move to a more demanding brain disease, something incredibly difficult to treat, namely ischemic stroke."

To create this animal model, the co-authors injected endothelin, a powerful blood vessel constrictor, into the brains of 38 rats, to produce a very large, unilateral subcortical stroke that blocked the brain's middle cerebral artery. "Using this endothelin stroke model," During recounted, "we did 10 NMDA rats and 18 controls. In the subsequently vaccinated cohort, the total stroke volume was decreased by a dramatic 70 percent."

Stroke, Seizure Trials Need Industry Support

The co-authors are now planning a Phase I clinical trial in people at imminent risk of stroke.

"Our first goal," During said, "is to move toward patients with subarachnoid hemorrhage, who have had a little aneurysm in the brain. Those patients run a very high risk - over 50, maybe 60 percent - of severe vasospasm and ischemic stroke, over the course of the next week or two.

"At that point our planned Phase I protocol is not to vaccinate them, but just to infuse the antibody itself, because it's a bit safer, and can be done on a shorter-term time frame. If we show in that situation that it's safe and effective, then we'll move back to patients with severe carotid stenosis who are not good candidates for surgery, and then people with severe hypertension.

"I'd like to be in the clinic within a year's time for both stroke and epilepsy Phase I trials," During said, but added, "We're going to need some sort of partnership with a pharmaceutical or biotech company to get these plans off the ground.".