Neuroimaging and Neurocognitive Assessment and Response to Sapropterin Dihydrochloride Treatment in Phenylketonuria

We plan to enroll 36 subjects with PKU. They will all be counseled to follow the PKU diet.
They will be randomized to receive Kuvan. We will plan to enroll 12 in the diet alone group
and 24 in the Kuvan group with the hope of 12 being responders (as defined by a 30% drop in
blood phe levels). Those who do not have a blood response with decreased Phe will still be
evaluated for a neurological response as measured by neurocognitive testing and neuroimaging
as outlined in this protocol. All 24 in the Kuvan group will receive a 1 month trial of
Kuvan and then the responders will have their insurance cover the drug and for the non
responders Biomarin will supply Kuvan.

Aim 1: To test the hypothesis that stable patients with PKU who are not currently on diet
have specific brain biomarkers quantifiable by DTI, fMRI and MRS and that the levels of
these biomarkers correlate with the clinical severity and outcome. Our pilot study and the
research of others have suggested the following as potential biomarkers for neural injury in
PKU:

- Metabolic abnormalities: elevated Phe concentrations as measured by MRS.

- White matter microstructure: measured by apparent diffusion coefficient, (ADC), using
DTI.

- Working memory and attention/executive function: measured by fMRI, Near infrared
spectroscopy (NIRS) and neuropsychological testing.

We will study these potential biomarkers in a group of well characterized adults with PKU
who are not currently on diet or a strict diet as evidenced by elevated Phe, but who were
identified by the newborn screen and may have been on diet during childhood, with varying
severity of clinical involvement.

Aim 2: To measure changes in biomarkers validated in Aim 1 after return to diet +/- Kuvan
(sapropterin dihydrochloride; Kuvan)

Based on our preliminary data and that of others, Aim 1 should provide us with a number of
candidate biomarkers for brain injury in PKU. We expect that these will include three types
of markers that can be correlated: metabolic, structural and cognitive. We will use these
biomarkers to study subjects during return to diet and/or initiation of Kuvan.

Aim 3: To compare cognitive performance in our subjects prior to and after return to diet
+/- Kuvan. using the following tests:

Comprehensive Trail making test (CTMT) -Parts A and B Stroop WASI (IQ, digit span) BRIEF
Edinburgh Handedness Inventory Hand Preference Demonstration Test

Outcome variables The primary endpoints in this study are 1) an estimate of the white matter
damage (macroscopic and microscopic) in participants with PKU versus controls at baseline
and after return to diet +/- Kuvan; 2) determination of a profile of brain biochemical
abnormalities in participants with PKU versus controls at baseline and after return to
diet/Kuvan, 3) assessment of cognitive and motor systems abnormalities (by fMRI and
cognitive testing) in the participant cohort as compared to age matched typically developing
non disease controls at baseline and after return to diet/Kuvan; 4) coefficient of
variability of scoring on traditional neurocognitive.

After the conclusion of this study, future areas of research would include expansion to
study long term effects of Kuvan, study of more impaired populations and response to Kuvan,
and using optical imaging methods we are currently piloting in OTCD.

Method to investigate biomarkers of disease We plan to use 1HMRS to identify markers of
disease severity such as elevations of Phe and decreases in NAA. This will be compared in
participants and normal age matched controls. Additionally, diffusion tensor imaging will
allow us to probe white matter fiber tracts that may be especially vulnerable in this
disorder selectively. Lastly, fMRI may identify regions of differential activation in the
brain that are different in participants versus age matched controls at rest and with a
cognitive task probing working memory and attention.

Inclusion Criteria:

1. Patients with PKU identified on Newborn screening and with phe concentration >12mg/dl
on the newborn screen

2. Baseline phe level at study enrollment > 20 mg/dl (this is the level required for
inclusion in the study, regardless of the level used to make diagnosis)

3. Age range: 18-45 years

4. Able to comply with neuroimaging without requiring sedation (typically requires IQ
over 65). The IQ will be checked with the WASI (Weschler Adult scales of
intelligence) before determining eligibility

5. Able to undergo neuroimaging safely (i.e. without presence of ferromagnetic devices)

6. Subject has ability to follow instructions in English

7. Female of childbearing age who is sexually active agrees to urine pregnancy test

8. Availability to come to Washington, DC to participate in this study

Exclusion Criteria:

1. Age range <18 or >45 years

2. Inability to comply with neuroimaging without the use of sedation (low IQ or
claustrophobic)

3. Presence of ferromagnetic device(s) that preclude safe imaging including cardiac
pacemaker, neural pacemaker, surgical clips in the brain or blood vessels,
surgically implanted metal plates, screws or pins, cochlear implants or
metal objects in their body

4. Pregnant female or breastfeeding at screening or planning to become pregnant at any
time during the study.

5. Baseline phe < 20 mg/dl

6. Currently on Kuvan

7. History of substance abuse