Effects of Kuvan on Melatonin Secretion

Background: Phenylketonuria (PKU) is a genetic condition due to missing one of the key
enzymes of phenylalanine (Phe) degradation. The missing enzyme is phenylalanine hydroxylase
(PAH). Because of the deficiency of the enzyme, plasma Phe is highly elevated compared with
other plasma amino acids. This abnormal ratio i.e., Phe vs. each of the other large neutral
amino acids (LNAA) which are transported into the brain as precursors of neurotransmitters
through the common transportater LAT1, is likely to contribute to neurotransmitter
deficiencies in individuals with PKU. Patients who do not restrict Phe intake develop mental
retardation and neuropsychological disorders. Treatment of PKU has been historically limited
to restriction of Phe intake, meaning protein restriction.

The FDA recently approved a new medication, Kuvan, a synthetic form of tetrahydrobiopterin
(BH4) manufactured by BioMarin Pharmacetical Inc., for patients with PKU. Kuvan is a
cofactor for PAH, tyrosine hydroxylase, and tryptophan hydroxylase. Patients with PKU
typically undergo a month-long trial of Kuvan to determine if they are responders to the
drug, meaning blood phenylalanine drops by more than 30 percent. Those who are not
responders do not usually continue Kuvan, however our recent study (HS 08-00147) showed that
classical PKU patients who are not Kuvan responders demonstrated improvement in their
maladaptive behaviors, suggesting some effects of Kuvan in the CNS system without reducing
blood phenylalanine concentrations (Moseley et al, manuscript under preparation). Seven
subjects who did not respond to Kuvan with lower blood Phe levels had significant increases
in blood tyrosine after 6 months (p=0.015), along with improvement in the Vineland
Internalizing and Overall Maladaptive Behavior Indexes at 12 months (p=0.032 and 0.049,
respectively). Kaufman reported that Kuvan at dose of 20 mg/kg/day increased CSF BH4 levels
(1). Based on these observations, it is likely that Kuvan has some effects on
neurotransmitter metabolism.

Supplementation of large neutral amino acids (LNAA) as a medical food has been used in
Europe for the past 25 years and it is believed to be effective in treating patients with
PKU. In the article by Shindeler et al. (2), 16 individuals (7 males and 9 females; age 11y
to 45 y) participated in the study which consisted 4 weeks wash-out and 4 two-weeks stages
(on phenylalanine restricted diet and with/without LNAA, and on no phenylalanine restricted
diet and with/without LNAA). No specific adverse effects are reported in the article. Large
neutral amino acid therapy for the treatment of PKU has been done in the past (3). LNAA is
used primarily for adolescents and adults who cannot adhere to the standard phe-restricted
diet. LNAA treatment has been approved for PKU patients just like other special formulas.
Our previous studies have demonstrated deficiency of serotonin in the brain, as evidenced by
low nocturnal melatonin secretion and reduced urine dopamine compared to controls, and that
supplementation with LNAA improves, but does not normalize these markers.

Rationale: Patients with PKU are thought to be deficient in tyrosine and tryptophan in the
brain tissue level because of the abnormal phe/tyrosine and phe/tryptophan ratios. Previous
studies showed low serotonin levels in cerebrospinal fluid in PKU individuals and some
improvement was observed after tryptophan supplementation (4). Our prior study suggested
deficiency of tryptophan in the brain tissue was demonstrated by low nocturnal melatonin
secretion and 6-sulfatoxymelatonin, which is a stable metabolite of melatonin (5). LNAA
supplementation resulted in some improvement in melatonin secretion, however, melatonin
secretion in patients with PKU on LNAA was still significantly lower than the control group.
Supplementation of Kuvan may activate the brain tyrosine and tryptophan hydroxylases (6),
resulting in improvement of nocturnal melatonin secretion and urine melatonin and dopamine
concentrations. By supplementing LNAA increased amount of neurotransmitter precursor amino
acids will be transported into the brain and activated brain tyrosine and tryptophan
hydroxylases by Kuvan will act on these precursors to produce neurotransmitters, i.e.,
serotonin and dopamine. These synergistic effects may increase melatonin and dopamine levels
to closer to the control levels.

Aims of the Study: To show scientific evidence of improvement in metabolism of
neurotransmitters including serotonin and dopamine in patients with PKU treated with LNAA
only, Kuvan only, and Kuvan and LNAA.

Objectives: This investigator initiated study has three objectives: (1) To evaluate if
tetrahydrobiopterin (Kuvan) supplementation has beneficial effects on melatonin secretion
and urine dopamine levels in individuals with PKU when they are and are not treated with
large neutral amino acids; (2) To demonstrate synergistic effects of LNAA supplementation
and Kuvan therapy in improvement of neurotransmitter metabolism in individuals with PKU; and
(3) To gather pilot information necessary to design a larger, multicenter trial of these
interventions, should results of these pilot studies indicate further study is warranted.

Study Methodology: This study will be conducted in adult patients with PKU in four 4-week
phases: phase I (large neutral amino acid/LNAA), phase II (Washout), phase III (Kuvan only)
and phase IV (Kuvan and LNAA). Kuvan supplementation will be adjusted to provide 20
mg/kg/day. LNAA therapy is adjusted to the manufacturer's instruction (total tablets of LNAA
is 1/2 X BW kg). On the last day of each phase, subjects will stay approximately 14 to 18
hours overnight at the CTU at USC University Hospital. Blood specimens will be collected
every 2 hours from 7:00 pm to 7:00 am. First void urine will be collected on the day of
discharge (patients will be asked to void urine at 11:00 pm and the first void urine will be
collected).

Blood will be collected in a red top tube (5 ml) through an IV line which is saline locked
during the study period. Avoid hemolysis by collecting blood through an IV line into a
reasonably large peripheral vein. Blood will be left to clot for 45 minutes at room
temperature (18-28 C) and protected from light (a dim flash light or a yellow light < 100
lux). Serum will be collected and stored at - 20 C until they are subjected to analysis for
plasma melatonin and plasma amino acids. Urine 20 ml first void sample will be collected
into two plain tubes (10 ml per each tube) and stored in a deep freezer (-20 C) until
subjected to be analyzed for dopamine and 6-sulfatoxymelatonin.

This is an open-labeled study. During all four 4-week phases, subjects will remain on their
protein-restricted diet. LNAA tablets are not considered a medicine, but are under the
category of medical foods; examples include low protein foods. LNAA tablets will be provided
by the LNAA manufacture (Applied Nutrition). Kuvan will be taken per orally with breakfast
and LNAA tablets will be taken with meals three times per day.

Inclusion Criteria:

- Adult English-speaking patients who have confirmed PKU, and have tolerated Kuvan
therapy at 20 mg/kg/day in the past, and are currently on the LNAA supplements will
be considered as candidates for the study.

- Subjects must be able to stop LNAA therapy for 4 weeks.

- This will be determined by the subjects themselves, based on their past personal
experiences.

Exclusion Criteria:

- Individuals who have never taken Kuvan, who have never been on LNAA therapy, who are
under the age of 18, or who do not speak English will be excluded.

- Subjects who cannot stop LNAA therapy for 4 weeks will be excluded.