By David N. Leff
Being sent to bed without supper is a classic childhood punishment, but one in 10,000 babies born in the U.S. is on a life sentence of going without milk, cheese, meat, fish, bread and nuts ¿ forever.
These are among the high-protein foods that are rich in phenylalanine, an essential amino acid. Those ill-fated infants are born with an inherited metabolic disease called phenylketonuria, or PKU. Their livers lack the enzyme phenylalanine hydroxylase (PAH), which degrades phenylalanine.
The resulting amino-acid buildup has devastating neurological effects. Without treatment, PKU children incur irreversible mental retardation, with intelligence quotients around 50. They are extremely hyperactive, and many older victims suffer from epilepsy and psychoses. Their hair, skin and eyes are lighter than those of non-affected family members.
PKU is a recessive genetic disorder, inherited from both parents who are carriers of the mutated gene for the missing liver enzyme. In the U.S., it strikes one in every 10,000 live births, with an estimated 450 new cases entering the population each year.
Since the 1950s, all newborns in the industrial world are routinely screened for PKU, and promptly started on a lifelong course of treatment that many sufferers, and their families, find worse than the disease. Besides a total ban on consuming those high-protein foods, PKU patients must swallow a daily dose of a foul-smelling synthetic nutritional replacement product. Only low-protein fruits, vegetables and certain cereals are permitted.
Youngsters who comply with this dietary regimen usually grow up escaping the worst physical manifestations of PKU, but with obvious psychosocial problems derived from their dietary lifestyle. Moreover, observed pediatrician and molecular biologist Charles Scriver, ¿At the end of the first generation of treatment experience, fine-tuning of evaluation finds that there are important deficits in neuropsychological function, with the therapeutic modalities that were available. These make patient compliance difficult.¿
Scriver is professor of pediatrics and biology at McGill University, in Montreal, and at its affiliated Children¿s Hospital. Surveying the disheartening PKU scene, in which stricken youngsters rebel against their repellant and life-straitening diets, Scriver decided to seek a better way.
The title of his interim report in the current Proceedings of the National Academy of Sciences (PNAS), dated March 2, 1999, tells the story so far: ¿A different approach to treatment of phenylketonuria: Phenylalanine degradation with recombinant phenylalanine ammonia lyase [PAL].¿
When A Patient Needs A PAL
PAL is a recombinant enzyme that substitutes for liver PAH in mice that model the symptoms of PKU. To create PAL, Scriver teamed up with Montreal-based IBEX Technologies Inc., whose president and chief operating officer, Robert Heft, is a co-author of the PNAS paper.
¿We¿ve cloned that gene and expressed it in E. coli,¿ Heft told BioWorld Today, ¿and have developed purification methodology. That was the first step. [In] the second step, we have been developing various formulations for the enzyme, which is to be administered orally. If it is not carefully formulated, you would digest the enzyme just as any other protein. And, in conjunction with Scriver, we¿re designing the preclinical pharmacology experiments, which have actually been conducted by his group on this very special mouse model.¿
Scriver took the story from there.
¿These mice are a true counterpart of human PKU,¿ he said. ¿We started with two strains, mild and severe. Then my graduate student, Christineh Sarkissian, who is first author of the paper, decided to cross both strains to produce a hybrid, for two reasons. Three-fourths of human PKU patients are hybrids; that is, they have two different mutations in the PAH gene. So, to have a mouse that is a heteroallelic genotype is more like the human story. Secondly, the mouse allows us to study things we can¿t do in the human.
¿At the moment,¿ Scriver went on, ¿Christineh is measuring the cognitive and behavioral phenotype of these PKU-mimicking mice. They do show impaired learning and memory. This makes them a perfect model for studying our enzyme-substitution approach, using PAL instead of PAH.¿
The team¿s first experiment, he recounted, ¿was to give the mice a dose of PAL. We injected the naked enzyme intraperitoneally. That brought phenylalanine down to zero. But it¿s not the way we¿re going to treat the humans. We¿re not going to inject a naked enzyme into the abdominal cavity of patients every day. And even if we did, it would produce an immune response. What we¿re looking for now is an approach where we can give the enzyme by mouth. That requires protecting it from digestion. We¿ve gone a partial way along that route. We were able to get preliminary proof-of-principle results. Now we have work to do to redefine the activity of the enzyme, its stability, protectability and optimum dosage.¿
He compares the present stage of his PAL approach for treating PKU ¿to the equivalent early stage after insulin was discovered to treat diabetes. The IBEX people were highly encouraged to know that the scientific world thinks this approach is an important thing. Therefore, that will re-instigate confidence in financing of this project, which has been difficult because it seems to be such a long shot. At present, we have pretty well finished the description of the mouse without PAL treatment. So, next, we will be able to give them long-term dosage and study its effects.¿
¿We have found a way,¿ Scriver said, ¿to prepare a mouse so we can inject this stuff into its intestine without disturbing the animal, and do it day after day. We have a catheter installed surgically under the skin, coming out behind the neck, so the mouse can¿t eat its own catheter. Then, by the appropriate surgery we can inject the current preparation of the enzyme directly into the intestine.¿
Mice Prep Stage For People Trial By Year¿s End
The team has prepared a dozen mice, and they¿ve all survived that preliminary procedure. Scriver expects the initial preclinical regimen to continue for the next three months. ¿We need to know that we can get a long-term effect, and titrate dosage,¿ he said. ¿Then, we¿ll proceed to treating the mice long-term. The third stage will be to go for the first in-man clinical trials. We would like to use the enzyme in people, and some of our PKU patients are anxious to be part of that study.
¿Of course,¿ he said, ¿we have to have evidence of non-toxicity, and pass the whole proposal to do in-man studies past our institutional review board. We¿re aiming for doing the first in-man studies this year. The interface there is with Bob Heft of IBEX, as to whether they can have a suitable prep of the enzyme with activity we think will do the job.¿
Heft observed that there are ¿very substantial problems remaining to be solved as to the cost and logistics of this as a potential therapeutic. One is the amount of enzyme actually required to be therapeutically efficacious. The second problem is that the optimal pH [acid/alkaline value] of the enzyme is somewhere around 8.5. But the environment in the gastrointestinal tract is one log lower.
¿This is not something,¿ he concluded, ¿where there is a clear path yet to an ultimate therapeutic.¿ n