By David N. Leff

Back near the beginning of life on earth, when the Animal Kingdom began to munch on the Vegetable Kingdom, plants in self-defense mounted chemical warfare against the herbivores. They evolved production of a variety of alkaloids — bitter, toxic proteins — in order to stop being eaten. Among these alkaloids was nicotine.

Whereupon, so the theory goes, evolution then endowed certain members of the Animal Kingdom with a series of enzymes to neutralize those poisonous plant proteins. Among these detoxicants is a cluster of proteins called cytochrome P450, which features three main enzymic families that deal with external toxins from the environment.

The second member of this cytochrome tribe is an enzyme acronymically named CYP2A6. "Actually," observed molecular pharmacologist Rachel Tyndale, at the University of Toronto, "CYP2A6 is mostly involved in ridding the human body of nicotine."

CYP2A6 breaks down 60 to 80 percent of inhaled nicotine — which drives the brain's pleasurable reaction to cigarette smoking — into a simpler molecule, cotinine, which is excreted.

Insurance companies have long tested for cotinine in the blood or urine of applicants, to verify their claims to being non-smokers. Tyndale, whose laboratory focuses on gene defects in the cytochrome P450 family, and how they affect drug dependency, questions the validity of this cotinine test.

"In our smoking population," she told BioWorld Today, "we have 10 percent that inactivate nicotine to cotinine at a very different rate than the other 90 percent, and reflects a different kind of smoking behavior."

Of the world's population, now edging 6 billion men, women and children, Tyndale noted, "one out of three over the age of 15 smokes."

Non-Addicts Inherited Mutant Enzyme

The gene encoding the CYP2A6 enzyme resides on the long arm of human chromosome 19. What that enzyme does in a healthy, non-smoking body is anybody's guess. "Its wild-type form has no known endogenous role at all," Tyndale observed.

"Two mutations occur in the structural, or coding, region of the CYP2A6 gene," Tyndale pointed out. "Both create a null, or completely defective, gene product — an inactive protein." A fortunate fraction of the population harbors these defective proteins.

"Within dependant smokers," Tyndale explained, "the active CYP2A6 enzyme removes nicotine more slowly, so they simply have to restock their nicotine levels at greater intervals. Hence, they smoke fewer cigarettes to do that.

"When people are learning to smoke — the other side of the coin — the levels of nicotine stick around for a longer time, but they have principally negative effects in a non-tolerant beginner. The nicotine causes dizziness and nausea for example. And we think that in the people who are slower at getting rid of these effects — because they harbor an inactive enzyme — that tips the balance toward them not wanting to smoke."

Tyndale is the principal author of a brief paper in today's issue of Nature, dated June 25, 1998, and titled: "Nicotine metabolism defect reduces smoking."

To reach this conclusion, she and her co-authors looked for defective CYP2A6 genes in the chromosomes of 428 present and former cigarette smokers:

* tobacco-addicted volunteers.

* tobacco-and-alcohol addicts.

* never-addicted controls, who had tried smoking and soon quit.

Among the two addicted cohorts, the team found 12.3 percent with defective CYP2A6 genes vs. 19.6 percent in the habit-avoiding controls.

Then they asked whether the 12.3 percent of addicts with impaired nicotine metabolism smoked fewer cigarettes per capita than the 87.7 percent with wild-type genes.

Those who had inherited a mutant allele from only one parent (heterozygotes) consumed 129 smokes a week, whereas those with two active, wild-type genes from both parents had a weekly cigarette usage of 159.

From these findings, Tyndale and her co-authors reasoned that the inhibitory effects on nicotine wrought by defective CYP2A6 genes might well decrease the carcinogenic effects of smoking.

"We believe that blocking the removal of nicotine from the body via this pathway," Tyndale said, "would work toward decreasing the number of cigarettes that dependant smokers need to smoke. And we would use this as a process for reducing the harm that's due to exposure to the carcinogens in cigarettes, and also as a route to smoking cessation.

"There's evidence to show," she went on, "that it would be easier to quit if you're smoking five cigarettes a day than if you were smoking 30 a day."

She thinks that "this is a good approach, both in smokers who are having a lot of trouble quitting, and also in smokers to whom we can just give an extra tool to help them quit."

Nicogen Inc. Start-Up To Develop Enzyme Inhibitor

Tyndale and her co-authors envisage an orally administered medication. She makes the point that "we have no other target except the liver. Drugs that come in through the stomach and the gastrointestinal tract go to the liver first. And they would be able to inhibit the enzymes right at the very beginning of their existence."

The university has filed for four patents worldwide covering the inventions involved in the CYP2A6 inhibition approach. "We're in the process," Tyndale observed. "I'm hoping that toward the end of this summer or in the fall, we'll have some data that we'll be able to make public. We're interested in moving quickly."

"When we started this project," she continued, "it was clear that if we just simply made it public without protecting the intellectual property, it would be very hard to develop this. A pharmaceutical company with which we sought a commercial collaboration would need protected proprietary information.

"So what we did was to pursue the patent route. And in fact we had some investment money come in from a limited partnership. So in July of last year we established a small company ourselves to do the beginning studies. After that we would hope to partner with a pharmaceutical company to actually do the larger-scale studies, and produce a proper oral medication."

Their new start-up firm, Nicogen Inc., is located in Montreal.

Over recent years, a number of genes have surfaced with the ability to free nicotine-dependant rodents from their addictions. Such research findings have buttressed the argument by tobacco companies that nicotine is not truly addictive.

"Many of those genes that have been identified," Tyndale pointed out, "have to do with the reward system in the brain, the dopamine system. And that's where the dopamine receptor variants and monoamine oxidase enzymes will probably be very important.

"The difference here," she added, "is that CYP2A6 is a gene that very specifically acts on nicotine itself. It provides even further support for the fact that nicotine is indeed an addictive drug, which smokers are very carefully replenishing on a daily basis." *