Will Tetrahydrobiopterin Have A Role In PKU Treatment?

By Virginia Schuett

From the Spring/Summer 2003 issue of National PKU News

The story of tetrahydrobiopterin (BH4) and its possible role in PKU treatment is a rapidly evolving one. We first presented the idea of "BH4-responsive PKU" in the Spring/Summer 2002 issue. The following story is based on a presentation at the international PKU meeting in Denmark (June 2002) by Dr. Rueben Matalon of the University of Texas Medical Branch; it is augmented with additional research results available since then.

PKU is caused by a defect in activity of the phenylalanine hydroxylase (PAH) enzyme. In order for the PAH enzyme to function properly it requires a cofactor. All enzymes in the human body require cofactors, many of which are vitamins. The cofactor for PAH is a "vitamin-like" substance called tetrahydrobiopterin, or BH4 for short. Without BH4, the PAH enzyme cannot function. Our bodies make BH4, so we do not have to take it as a supple-mental vitamin. When production of BH4 is defective, the resulting disorder can be more serious than PKU. The symptoms are seizures, mental retardation, and neurological changes. But that disorder is not the subject of this discussion.

"BH4-Responsive PKU"

Despite normal production of the cofactor in PKU, there is a new concept regarding treating PKU with BH4. This concept was put forward in a 1999 paper from Japan where three children with very mild PKU were given an oral BH4 load at a dose of 20mg/kg of body weight. Surprisingly, their blood phe levels dropped sharply after the BH4 load. BH4 production was confirmed to be normal for these children and yet the extra BH4 mysteriously caused a drop in blood phe. Needless to say, this publication caused quite a stir in the PKU research community. Subsequently, PKU centers in Europe and elsewhere gave BH4 loads to some of their patients with mild PKU. The publications that followed all dealt with mild PKU. The prediction based on this limited early research was that about 10-20% of mild PKU patients might respond to BH4 loading. Given their normal BH4 production, what explanation is there for lowered blood phe levels after a BH4 load?

One of the many possibilities is that in patients with mutations resulting in some residual PAH enzyme activity, the additional BH4 somehow improves the enzyme activity even more, causing a drop in blood phenylalanine levels.

In fact, Dr. Seymour Kaufman from the National Institutes of Health first suggested this idea more than 25 years ago. In an article published in the Journal of Biological Chemistry, he reported on his experiments adding methyl-BH4 to rat liver. Surprisingly, PAH activity increased in the livers. He suggested that patients with PKU might benefit from methyl-BH4. However, since the methyl-BH4 compound is not exactly the same as plain BH4, that idea was ignored for 25 years-until the Japanese publication.

Design of the U.S. Pilot Study

So far, they have studied 30 patients. They will continue studying an extra 10 by the end of the project, including patients with classical PKU as well as milder forms.

Dr. Flemming Güttler at the J. F. Kennedy Institute in Denmark determined which PKU genetic mutations each of the study subjects have. They then sent all mutations to Dr. Raymond Stevens and his colleagues at the Scripps Research Institute in La Jolla, California. This group used information about each mutation to assess its effect on the enzyme according to the exact position of the mutation in the enzyme's structure (see Winter 2003 issue for a visual depiction of the PAH enzyme structure). These are important studies. We know there are more than 500 different genetic mutations that lead to PKU. Knowledge of the effect of specific mutations on the enzyme can be very helpful generally in the treatment of PKU patients-and now it can be useful for predicting those who may respond to BH4.

The U.S. pilot study involved a one-time loading dose of BH4. Because BH4 is very costly, the study was based on a single loading dose. Also, because of the cost, they used just 10mg/kg instead of the 20 mg/kg used by other investigators. They gave BH4 in a 10 mg/kg dose orally after an overnight fast, then sampled blood for phe and tyrosine at intervals over a 24-hour period.

They have submitted a paper for publication that deals with 25 study patients. Of these, 13 have classical PKU (defined as blood phe levels over 20 mg/dl or 1200 µmol/L on a normal diet). The remaining 12 have "atypical PKU" or "mild PKU (defined as blood phe levels between 10 and 20 mg/dl or 600 and 1200 µmol/L on a normal diet); or "very mild hyperphe" (defined as blood phe levels less than 10 mg/dl or 600 µmol/L on a normal diet).

To-date, the study has included 30 patients with PKU (an additional 5 patients since they submitted the paper). The age range is 6 months to 24 years. They kept all patients on their normal diet regimen since they did not want to interfere with good diet compliance. During the study, all but four patients were on some form of diet treatment.

Study Results

Last year in Dublin, Dr. Matalon and Dr. Koch presented a preliminary report of this study at a meeting of The Society for the Study of Inborn Errors of Metabolism. What surprised them as well as people at the conference was that, in addition to the expected response of those with more mild forms of PKU, there was a favorable response in some patients with what they designated "classical" PKU. In fact, about 50% of the group of 13 patients with classical PKU had a considerable response to a BH4 load-up to a 50% reduction in their blood phe level at 24 hours. However, no one knows if this would be a lasting effect for these patients, or for any others who have a response to a single BH4 load.

Contrary to popular belief, in even the most classical forms of PKU, there is typically some small residual enzyme activity (with just a few exceptions). Even though no one else has reported it in classical PKU, in the U.S. pilot study group, BH4 must have enhanced in some way the small remaining enzyme activity in these patients with classical PKU-at least for the 24 hour study period. In Figure 1 you can see an example of this. In this patient, blood phe level dropped from 6 mg/dl (360 µmol/L) to below 2mg/dl (120 µmol/L) 24 hours after the BH4 load.

All patients in the study had normal pterin profiles, and normal dihydropteridine reductase enzyme levels. This shows that they did not have defects other than PKU that would cause elevated blood phe levels.

Overall, 64% of the total patient group in the pilot study responded to the BH4 load by at least a 40-50% drop in blood phe levels at 24 hours. Blood tyrosine showed an upward trend, but this was not statistically significant.

Other Recent BH4 Research

Findings of the U.S. pilot study are encouraging, but somewhat unique. For example, in a series of 31 patients from Germany, Muntau and collegues showed a favorable response among 87% patients (27 out of the 31) with mild PKU and mild hyperphe as described in a recently published paper (Tetrahydrobiopterin as an alternative treatment for mild phenylketonuria, N. Engl. J. Med. 347:26, pp: 2122-2132, Dec. 2002). These patients were loaded with phe, then with BH4. Seven mutations were "probably" and six were "potentially" associated with response to BH4 after a load and 15 hours of follow-up. The study had only 7 patients with classical PKU, none of whom responded to the BH4 load. The U.S. pilot study has twice as many patients with classical PKU, and that population has more heterogeneity in the mutations represented; this may explain why the U.S. group saw a response in classical PKU patients when others have not.

The German researchers were able to select out a group of 5 children with mild PKU (ages 4-14) to try longer-term treatment with BH4. They used daily doses of 7.1-10.7 mg/kg body weight over a period of 166-263 days. This is one of the only longer-term studies of BH4. The BH4 treatment led to an increase in dietary phe tolerance: from a range of 8.5-30 mg/kg body weight before BH4, to 17.9-90 mg/kg during treatment. Average blood phe levels remained the same, confirming greater tolerance for dietary phe.

In a study by another German group (Weglage, et. al., J. Inher. Metab. Dis. 25, pp: 321-322, 2002), only 3 patients out of 87 showed a temporary decrease in blood phe levels with BH4 loading (the other 84 were not at all responsive). It is unclear why, but even though these three patients had a positive response to BH4 during loading, their blood phe levels increased over time despite continued BH4 therapy (10 mg/kg). BH4 did not prove an effective therapy in the long term and was discontinued.

Need for More Research

Additionally, we need information on potential toxicity during long-term usage before BH4 can be deemed an acceptable alternative or adjunct therapy in PKU treatment. Worldwide, more than 350 patients with true defects in BH4 production have been treated with the cofactor over the past two decades. Dr. Nenad Blau of Switzerland reports no side effects in over 200 patients tested using BH4 from Schirk's Laboratory in Switzerland, the major producer of BH4 for patient use. However, these are not double-blinded studies. There is a Japanese product that has a slightly different chemical composition that also has been used; out of 318 BH4-deficient patients, adverse reactions reported include sleep disorders, increased frequency of urination, and loose stools.

Remember that the patients who have been studied over a period of years are BH4 deficient, unlike PKU patients who have normal BH4 levels. We cannot assume that giving BH4 to people with normal BH4 levels will necessarily be the same as giving it to people who are BH4-deficient.

Whether long-term BH4-responsive patients will be able to stop the diet or will need to continue some restriction also must be determined. Dr. Koch and Dr. Matalon hope to extend their study and look at these and other treatment issues over a lengthy period of time, if funding becomes available.

Obtaining BH4 Tablets

Cost of BH4 Treatment

Even if BH4 treatment is found to be effective, cost is an issue. For a 10 mg/kg dose, it would cost more than $7,000 yearly for a 1-3 year old child; for a school-age child, more than $14,000; for a teenager/adult, $35-40,000 or more per year. This does not include any formula that may still be necessary. Possibly the optimal dose will be less than 10 mg/kg (but it may be more). Also, with increased demand, and possible production of BH4 in the U.S., the cost might decrease somewhat.

Conclusion

In some cases of mild PKU, and possibly even in some cases of classical PKU, treatment with BH4 ultimately may have a role in PKU management. But much more work is needed to resolve the many scientific questions and practical problems that remain before BH4 can be viewed as a treatment option. There are ongoing studies in Europe and several in the U.S. We will continue to keep you informed as more research results become available.