The Effectiveness of Kuvan in Amish PKU Patients

Phenylketonuria (PKU) is caused by a deficiency of the enzyme phenylalanine hydroxylase
(PAH). PAH is responsible for the rate limiting step in phenylalanine catabolism. This
reaction is dependent on tetrahydrobiopterin (BH4). BH4 is a catalytic cofactor for the
enzyme PAH. Supplementation with synthetic BH4 (saproterin dihydrochloride or KuvanTm) has
been shown to activate residual PAH activity and lower Phe levels in certain patients. It may
also have a chaperone effect by binding abnormal PAH protein and decreasing its degradation.
Saproterin is an approved oral drug developed to reduce blood Phenylalanine (Phe) levels and
increase Phe tolerance in BH4 responsive patients with hyperphenylalaninemia.

Certain genotypes have been found to be good predictors of Saproterin responsiveness;
however, these correlations are imperfect. Therefore the standard of care is to perform a
trial of Saproterin therapy to assess responsiveness in every PAH patient regardless of
genotype. Currently this recommendation does not include patients with two null mutations.

The purpose of this study is to determine if Amish patients with PKU show responsiveness
after a high dose, prolonged Saproterin trial. The population of interest has a high
frequency of a specific splice site mutation, the 1066-11G>A mutation. This splice site
mutation leads to protein conformational changes and abrogation of function. Previous studies
of this genotype have indicated <1% residual activity of the PAH enzyme and an insignificant
responsiveness to Saproterin. However, in this specific study Phe levels were evaluated only
over 24 hours after a single-dose BH4 challenge at the standard dose of 20mg/kg.

Recently, observation in several Amish patients suggests some promise that these patients may
be responsive to Saproterin after a longer trial of the medication (Personal comment - Samuel
Yang, MD). In addition, studies evaluating PAH enzyme activity in these patients, have found
evidence that this genotype may need higher cofactor concentrations for optimal enzyme
activity and therefore may require a higher dose of Saproterin then the currently recommended
20mg/kg (9). To date, no formal clinical trial has been done.

To answer the question of whether this genotype is responsive to Saproterin at higher doses,
over a prolonged period, the investigators propose a randomized clinical trial comparing
blood Phe levels in patients treated with standard-dose Saproterin to those treated with
high-dose Saproterin. The results of this study could reveal a new population of Saproterin
responsive PKU patients. This could call into question other null mutations of the PAH gene
and their responsiveness to Saproterin. This may lead to the investigation of genotypes
previously thought to be unresponsive to Saproterin. Further, diet treatment alone has proven
to be difficult for the Amish population for cultural and economic reasons.

This study will include patients from three local centers with the potential of a fourth, out
of state, center participating. All patients will be enrolled through University Hospitals
Case Medical Center. Consent will be obtained over the phone or in person.

- Amish patients from University Hospitals Case Western Reserve/Rainbow Babies and
Children's Hospital/Center for Human Genetics

- Amish patients from Akron Children's hospital in Akron, Ohio

- Amish patients from The Center for Children with Special Needs (DDC clinic),
Middlefield, Ohio

- Patients from Shodair Children's Hospital in Helena, Montana. These patients may be
recruited if more patients are required to meet our enrollment goal.

Study patients will present to their preferred clinic for 5 office visits. Initial
testing/Part 1: length 4 weeks

Recruited patients will present to clinic for part 1 of the study. After informed consent is
obtained, molecular genetic testing will be performed. Testing will include sequence analysis
of the PAH gene. This testing is necessary to identify the specific genotypes present in each
patient. The investigators expect the majority of this population to be homozygous or
heterozygous for the above described splice site mutation. Any patients found to have one (or
no) identifiable mutations after sequencing will not be included in the study. Genotypes will
be grouped and analyzed separately at the conclusion of the study. Patients who do not wish
to undergo genetic testing will not be included in the study.

Each patient will also have an initial blood Phe level drawn in the form of a dried blood
spot on filter paper card that will be supplied by the testing company. All Phe testing will
be performed in a Clinical Laboratory Improvement Amendment (CLIA) certified laboratory at
The Clinic for Special Children in Strasburg, PA.

Baseline assessment tools (BRIEF, PKU-QOL) will also be performed at this initial visit.
Patients will be given a unique study number by our investigational drug pharmacy and will
begin part 1 of the study as described below.

In part 1 of the study, patients will not receive any medication. Each patient will be
treated with diet alone. Patients will maintain a stable, Phe restricted diet (including
formula) that is consistent with their diet at the time of enrollment. This will be monitored
by food diaries kept for 3 days of each week. Specifically, patients will be expected to keep
diaries for the 3 consecutive days leading up to blood draw. Based on these diaries, average
weekly Phe intake and Phe tolerance will be calculated and recorded. Patients will also
complete a food frequency tool, which will be reviewed at part 1, 2 and 3's initial visit. We
will use all of the tools above, plus allow patients regular access to our dietician in order
to encourage the maintenance of a stable diet.

Blood Phe concentration will be measured weekly (each Monday) by blood spot cards. Patients
will not require weekly clinic visits to have Phe levels drawn. Blood spot cards will be
performed at their home and then mailed to the reference laboratory. In order to teach and
enable patients to become comfortable performing their own blood spots at home, nursing staff
(from the DDC clinic) will travel to each patient's house weekly for part 1 of the study
(total of 3 home blood spots). For parts 2 and 3 of the study, those patients and families
who are able, will perform their own blood spots at home. Those families who remain
uncomfortable performing their own blood spots will continue to have their blood spots
collected by home nursing.

Initial visit:

- Venous blood draw for PAH gene sequencing

- Baseline Phe level by dried blood spot card in office

- Executive functioning and quality of life assessment tools (BRIEF, PKU-QOL) given

- Baseline diet information collected and recorded. Average weekly Phe calculated, Phe
tolerance calculated.

- Food frequency tool completed and reviewed.

Blood Phe values and Phe tolerance from part 1 of the study (on diet treatment alone) will be
compared to blood Phe values and Phe tolerance from parts 2 and 3, on low-dose and high-dose
treatment.

Part 2: length - 4 weeks

Study numbers will be randomized into "standard-dose Saproterin" or "high-dose Saproterin"
groups (treatment group A or treatment group B). The investigational drug pharmacy will
provide study patients with their medication in kits, at the beginning of each treatment
period. Goal high-dose Saproterin dosing will be 40mg/kg (rounded up to the nearest 500mg),
provided in the form of pre-packaged 500mg packets of powder. Labeled dosing on these packets
will be covered by the investigational drug pharmacy and unidentifiable to patients.
Standard-dose Saproterin will be 20mg/kg (rounded to the nearest 100mg) provided in the form
of 100mg tablets. Dosing of the tablets will be unidentifiable to patients. Medication will
be given orally once daily.

Initial part 2 visit: after 4 week diet only period

- Food diaries collected for the previous 4 weeks (3 per week). Average weekly Phe
calculated, Phe tolerance calculated.

- Food frequency tool completed and reviewed.

- Baseline Phe level drawn by dried blood spot card in office

- Executive functioning and quality of life assessment tools (BRIEF, PKU-QOL) given

- Provided with medication kits (high or standard-dose, 4 week supply)

Final part 2 visit: after 4 weeks of treatment

- Food diaries collected for the previous 4 weeks (3 per week). Average weekly Phe
calculated, Phe tolerance calculated.

- Executive functioning and quality of life assessment tools (BRIEF, PKU-QOL) given

- Empty medication bottles/packet kits collected from each patient to assess compliance

- Review of any potential adverse events

This is the end of part 2. There will then be a 2 week "wash out period" where patients
receive no treatment. The length of this washout period was determined based on the mean
elimination half-life of saproterin and previous clinical trials. The mean elimination
half-life of saproterin ranges from 3.9 to 17 hours. In previous trials, washout periods have
consisted of 7 and 30 days. No efficacy assessments have been performed to differentiate or
support one washout period over the other (BioMarin Pharmaceuticals, Inc. Data on file).
Based on the elimination range above, we determined that a two week washout period is
sufficient. Patients should maintain their steady diet during this time.

Part 3: length - 4 weeks The cross-over will occur between treatment groups: A to B and B to
A.

Initial part 3 visit: after 2 week washout period

- Food diaries collected for the previous 2 weeks (3 per week). Average weekly Phe
calculated, Phe tolerance calculated.

- Food frequency tool completed and reviewed.

- Baseline Phe level performed by dried blood spot card in office

- Executive functioning and quality of life assessment tools (BRIEF, PKU-QOL) given

- Provided with medication kits (high or standard-dose, 4 week supply)

- Review of any potential adverse events

Final part 3 visit: after 4 weeks of treatment

- Food diaries collected for the previous 4 weeks (3 per week)

- Executive functioning and quality of life assessment tools (BRIEF, PKU-QOL) given

- Empty medication bottles/ kits collected from each patient to assess compliance

- Review of any potential adverse events

During all parts, patients will maintain a stable, Phe restricted diet including formula
(same diet as in part 1 of the study). All Phe levels will be blinded and researchers will
not have access to them until the conclusion of the study. At the conclusion of the study,
after all data have been analyzed, a complete list of blood Phe levels obtained during the
study (both on high and low-dose treatment) will be sent to each patient's primary metabolic
physician. Physicians can then discuss long term Saproterin treatment with their patients
based on their Phe levels.

The study will conclude after all parts are complete and groups A and B have both received a
trial of high and standard-dose Saproterin. All medication will be discontinued at this time.

Inclusion Criteria:

- Current diagnosis of PKU with the following:

- Age of at least 2 years or older

- Baseline Phe level of > 360 umol/L

- Willing to maintain a stable diet

- Patient or guardian are willing and able to provide written, signed informed consent
after the nature of the study has been explained and prior to any research-related
procedures

- Are willing to comply with all study procedures

- Two identifiable mutations found on PAH gene sequencing

- Any patients already taking Saproterin (or have taken in the past), must have a
treatment end date at least 14 days prior to Day 1 of the study.

Exclusion Criteria:

- Any patient currently taking Saproterin who has taken the medication at any point in
the 14 days prior to Day 1 of the study

- Under 2 years of age

- Unwilling to maintain a stable diet

- Patients with baseline Phe levels < 360 umol/L

- Patients unable to comply with all study procedures

- Patients unable to provide written, signed informed consent

- One (or no) identifiable mutations found on PAH gene sequencing