Open-label, Randomized, 2-way Crossover, Monocentric, Controlled Study to Evaluate the Effect on Daily PHE Fluctuation of PKU GOLIKE Versus Standard of Care in Patients With Phenylketonuria
Phase: NA
Conditions: Phenylketonurias
Sponsor: APR Applied Pharma Research s.a.

 
GLK-IT-2023  
  
 Ver.3.0 – 13.02.2024 
 
Page 1 of 10 
 
 
1 
PROTOCOL SUMMARY 
1.1 
Synopsis 
Title of study: 
Open-label, randomized, 2-way crossover, monocentric, controlled study to 
evaluate the effect on daily PHE fluctuation of PKU GOLIKE versus standard 
of care in patients with phenylketonuria 
Protocol number: 
GLK-IT-2023 V3.0 – 13.02.2024 
Sponsor: 
Applied Pharma Research S.A. 
Phase of 
development: 
Post-market 
Investigational site: 
ASST Santi Paolo e Carlo, Presidio Ospedale San Paolo, Via Antonio di Rudinì 
8 – 20142, Milano (Italy) – Clinical Department of Pediatrics. 
Aim of the study 
To document whether PKU Golike prolonged-release amino-acids 
formulation can achieve more stable phenylalanine blood levels with 
respect to the standard of care. 
Study description: 
This is an open-label, randomized, 2-way crossover, monocentric 
controlled study in patients (> 16 years old) with phenylketonuria (PKU). 
The comparison will be between the test product (PKU GOLIKE, a 
prolonged-release amino-acids (AAs) mixture) and standard of care.  
The study will consist of a screening visit (V1), two treatment periods with 
two test days each and a final visit planned at the end of the second 
treatment period (V2). Test days will occur on two consecutive preferably 
non-working/school days (T1-T2 and T3-T4,) preferably over two 
consecutive weeks. Treatment days in the two periods will have to be the 
same days of the week. T1 should be within two weeks after V1. The study 
products will be self-administered at home.  
Following informed consent and verification of eligibility criteria, 20 
patients with PKU will be randomized in a 1:1 ratio to one of the following 
two sequences: AB or BA where A=standard of care and B=PKU GOLIKE. In 
details:  
- 
AB: patients will receive 3 daily self-administrations of their usual 
standard of care for 2 consecutive days in the first period followed by 3 
daily self-administrations of PKU GOLIKE for 2 consecutive days in the 
second period.  
- 
BA: patients will receive 3 daily self-administrations of PKU GOLIKE for 
2 consecutive days in the first period followed by 3 daily self-
administrations of their usual standard of care for 2 consecutive days in 
the second period.  
No change in the randomization sequence will be allowed.  
Each patient will follow the same diet in terms of food, calories and 
nutrients ranges during the first days (T1 and T3) of each period and the 
same diet during the second days (T2 and T4) of each period, according to 

Product: PKU GOLIKE 
 
Version 3.0 
Clinical Study Protocol: GLK-IT-2023 
 
13 Feb 2024 
 
GLK-IT-2023  
  
 Ver.3.0 – 13.02.2024 
 
 
the age and body weight of the patient. The days before the test days each 
patient will follow her/his standard diet.  
PKU GOLIKE /standard of care will be the only protein substitute allowed 
on each test day, and no sports activities will be allowed on sampling days. 
Five blood spots will be collected on the second test day of each period.  
Timing of self-administrations, meals and blood spots will be standardized 
for each test day. On the test days, no food will be allowed outside of the 
defined time windows. The first self-administration of each test day will be 
performed after an overnight fasting (10 -12 h) and before any food intake. 
A patient’s e-diary will be used to collect information on patient compliance, 
24-hour blood spot collections, diet, daily activities, adverse events and 
event-related concomitant medications.  
Adverse events will be continuously monitored during the study, starting 
from informed consent. Adverse events will be collected by the patients (or 
by a parent/guardian) in the e-diary and during the telephone calls made 
by 
the 
Investigator 
to 
the 
patients. 
Moreover, 
patients 
(or 
parents/guardians) will be instructed to promptly report adverse events 
occurring during the study to the Investigator. 
The end of study (V2) will be performed (remotely, by phone) at the end of 
the second treatment period, within 2 weeks after the last test day. Patients 
prematurely discontinued from the study will be asked to attend (remotely, 
by phone) a discontinuation visit possibly taking place within 2 weeks from 
the last test day. At the final/discontinuation visit, the investigator will 
organize collection of blood spots test and residual PKU GOLIKE product 
from the patient’s domicile through a dedicated courier service. The 
account of the e-diary will be inactivated. 
Patients will receive a reimburse for the travel expenses, if required. 
Objectives and 
endpoints: 
Objectives 
Endpoints 
Primary efficacy objective 
Primary efficacy endpoint 
To compare the impact of GOLIKE 
versus 
standard 
of 
care 
on 
phenylalanine (Phe) blood levels  
Blood levels of Phe at pre-defined time 
points over 24 hours of each second 
test day.  
Secondary efficacy objectives 
Secondary efficacy endpoints 
To compare the impact of GOLIKE 
versus standard of care on tyrosine 
(Tyr) blood levels. 
Blood levels of Tyr at pre-defined time 
points over 24 hours of each second 
test day.  
Exploratory objective 
Exploratory endpoint 
To compare the impact of GOLIKE 
versus standard of care on branched-
chained 
amino 
acids 
(leucine, 
isoleucine, valine) blood levels.  
Blood levels of branched-chained 
amino 
acids 
(leucine, 
isoleucine, 
valine) at pre-defined time points over 
24 hours of each second test day.  
Study population: 
The study will enroll patients >16 years of age with PKU diagnosed at 
newborn screening and under a low Phe diet since diagnosis. 
Inclusion Criteria 
1. Signed, informed consent obtained by the patient prior to being 
enrolled into the study and prior to starting any data collection. For 

Product: PKU GOLIKE 
 
Version 3.0 
Clinical Study Protocol: GLK-IT-2023 
 
13 Feb 2024 
 
GLK-IT-2023  
  
 Ver.3.0 – 13.02.2024 
 
 
legally minor patients, signed written consent shall be obtained also 
by the parents/legal guardian.   
2. Male or female, aged ≥16 years.  
3. Patients with a registered diagnosis of PKU  
4. Ability and willingness to comply with all study procedures and 
availability for the duration of the study. 
5. Patients with mean value of blood Phe >360 µM in the previous 12 
months (calculated on at least 3 samples during the previous 12 
months; the last sample should be preferably obtained in the 30 
days preceding inclusion in the study). 
6. Patient taking free-AA and/or GMP as usual amino-acids 
supplementation. 
Exclusion Criteria 
1. Known 
or 
suspected 
hypersensitivity 
to 
any 
excipients/components of PKU GOLIKE. 
2. Treatment with any drug therapy for PKU  
3. Patient taking PKU GOLIKE as usual amino-acids supplementation 
4. Patient taking LNAA as usual amino-acids supplementation 
5. Any moderate to severe medical condition, which in the opinion of 
the Investigator would interfere with the study procedures or study 
outcome (reason to be provided) 
6. Any current participation in another clinical trial involving 
investigational or marketed products in the 3 months prior to the 
inclusion in this study. 
7. Pregnancy or lactation.  
Study intervention: 
Test product: PKU GOLIKE, a prolonged-release amino-acids (AAs) 
mixture. PKU GOLIKE is a FSMP for the dietary management of PKU.  
The product will be supplied in two formulations: PKU GOLIKE PLUS 16+ 
(granules) and PKU GOLIKE KRUNCH (tablets).  
For the test product, dosing will be adjusted by body weight in order to 
provide a PE total daily dose equal to the one in use by each patient in his 
standard of care (X g/kg). The prescribed formulation will be the granules 
but, at discretion of the physician, a part of the dose administered with the 
lunch on the first test day can be replaced with the tablets.  
Reference product: standard of care 
Test and reference products will be self-administered by patients at home 
and will be taken with food or liquid, according to the product leaflet. 
Timing of self-
administrations, 
meals and blood 
spots: 
First test day with 3 self-administrations of either standard of care or PKU 
GOLIKE: 
- h. 8:00: 1st product self-administration with breakfast 
- h 12:00: 2nd product self-administration with lunch 
- h 16:00: afternoon snack 
- h 20:00: 3rd product self-administration with dinner 
Second test days with 3 self-administrations of either standard of care or 
PKU GOLIKE:  
- h 8:00 (±15 min): 1st blood spot 

Product: PKU GOLIKE 
 
Version 3.0 
Clinical Study Protocol: GLK-IT-2023 
 
13 Feb 2024 
 
GLK-IT-2023  
  
 Ver.3.0 – 13.02.2024 
 
 
- 1st product self-administration (right after the blood spot) with 
breakfast 
- h 12:00 (±15 min): 2nd blood spot 
- 2nd product self-administration (right after the blood spot) with 
lunch 
- h 16:00 (±15 min): 3rd blood spot 
- right after the blood spot afternoon snack 
- h 20:00 (±15 min): 4th blood spot 
- 3rd product self-administration (right after the blood spot) with 
dinner 
- h 8:00 (±15 min) (following morning): 5th blood spot (in fasting 
conditions, before breakfast) 
On the test days, no food will be allowed outside of the defined time 
windows. 
Sample size 
determination: 
Considering the nature of the study [crossover, randomized, controlled with 
standard care (SC), efficacy of PKU GOLIKE (PG) in reducing the variation 
of Phe in comparison to SC], the testing hypothesis is to observe a major 
effect of the PG versus SC. 20 patients are adequate to demonstrate a 
difference of 30 micromoles/L with 40 micromoles/L of standard deviation 
between treatments at any blood spots with 80% power and 5% for two-
sided test. 
Patients that are not compliant or need to be excluded for other clinical 
reasons from the treatment schedule will be replaced in order to ensure 20 
subjects completing both treatment sequences. 
Statistical methods: 
General statistical methods 
Descriptive statistics (mean with standard deviation, minimum and 
maximum, median with the first and third quartile and 95% confidence 
interval) will be calculated for quantitative variables.  For qualitative 
variables, counts and percentages will be provided together with the 95% 
confidence interval. In calculation of percentages, patients with missing 
data will not be considered, unless otherwise specified.  
Analysis of the primary and secondary efficacy endpoints: 
The primary endpoint will be the comparison of the Phe at each time point 
by means of ANOVA for repeated measure (Wallenstein design) and post-
hoc Dunnett’s t test for multiple comparisons. 
The secondary endpoints time profile of Tyr, Leu, isoLeu and Val will be 
evaluated in blood spots with ANOVA for repeated measure (Wallenstein) 
and tested at each time points with post-hoc Dunnett’s t. 
Duration of study: 
Patients will be followed until the final visit/discontinuation visit, which 
will take place at the latest within 2 weeks from the last test day.  
Protocol 
version/date: 
V 3.0 dated Feb 13, 2024 
 

Product: PKU GOLIKE 
 
Version 3.0 
Clinical Study Protocol: GLK-IT-2023 
 
13 Feb 2024 
 
GLK-IT-2023  
  
 Ver.3.0 – 13.02.2024 
 
Page 5 of 10 
 
1.2 
Study Design 
 

Product: PKU GOLIKE 
 
Version 3.0 
Clinical Study Protocol: GLK-IT-2023 
 
13 Feb 2024 
 
GLK-IT-2023  
  
 Ver.3.0 – 13.02.2024 
 
Page 6 of 10 
 
1.3 
Schedule of Events (SoE) 
 
 
1st period  
2nd period  
 
Study period 
Screening Visit 
(V1)  
Test Day 
1  
(T1)   
Test Day 
2  
(T2)  
Test Day 
3  
(T3) * 
Test Day 
4  
(T4)   
Final / Discontinuation 
Visit 
(V2) § 
Informed Consent signature 
 
 
 
 
 
 
Inclusion/Exclusion Criteria 
 
 
 
 
 
 
Demographics 1 
 
 
 
 
 
 
Disease history 2 
 
 
 
 
 
 
Relevant Medical history & Prior medications 
 
 
 
 
 
 
Anthropometric measures 3 
 
 
 
 
 
 
Urine pregnancy test (if applicable) 
 
 
 
 
 
 
Diet preferences & Nutritional counseling 4 
 
 
 
 
 
 
Information on dosage/way of administration of SoC 

 
 
 
 
 
GOLIKE PKU dispensing 
 
 
 
 
 
 
Randomization 5  

 
 
 
 
 
Product self-administration according to 
randomization sequence  6 
 
 
 
 
 
 
Blood spots 7 
 
 
 
 
 
 
Blood spots collection by designated courier 8 
 
 
 
 
 
 
Adverse events 9 
 
Continuously through a patient’s diary 
  
Concomitant medications 10 
 
Continuously through a patient’s diary 
  
Diet 11 
 
On each test day through a patient’s diary 
  

Product: PKU GOLIKE 
 
Version 3.0 
Clinical Study Protocol: GLK-IT-2023 
 
13 Feb 2024 
 
GLK-IT-2023  
  
 Ver.3.0 – 13.02.2024 
 
Page 7 of 10 
 
Daily activities 12 
 
On each test day through a patient’s diary 
  
Compliance 13 
 
On each test day through a patient’s diary 
  
Information on 24-hour blood spots collection 14 
 
On each test day through a patient’s diary 
  
* Test days will occur on two consecutive preferably non-working/school days (T1-T2 and T3-T4) preferably over two consecutive weeks. Treatment days 
in the two periods will have to be the same days of the week. T1 should be within two weeks after V1. The second period is preferably to be done the week 
after the first period; nevertheless, one week of delay is admitted. 
§ The end of study will be performed (remotely, by phone) at the end of the second treatment period, within 2 weeks from the last test day. Patients 
prematurely discontinued from the study will be asked to attend (remotely, by phone) a discontinuation visit possibly taking place within 2 weeks from 
the last test day.  
1 Demographics information (age and sex). 
2 Disease history (year of phenylketonuria (PKU) diagnosis, date of start of low phenylalanine (Phe) diet, prior/concomitant treatments/diet for PKU in 
the last 3 months, disease severity according to classification described in the article at https://doi.org/10.1002/edm2.396; current standard of care to 
be documented). 
3 Anthropometric parameters (weight, height, and body mass index (BMI)). 
4 Information on recommended dietary patterns (food, calories and nutrient content) and diet preferences will be collected. Moreover, each patient will 
receive nutritional counselling for diet standardization to be followed on each test day. Diet instructions will be provided to the patient.  
5 Randomization in a 1:1 ratio to one of the following two randomization sequences: AB or BA where A=standard of care and B=PKU GOLIKE. 6 Products 
will be self-administered by the patients at home. For each period, patients will receive 3 daily self-administrations for 2 consecutive days in accordance 
to the assigned randomized sequence. The first self-administration of each test day will be performed after an overnight fasting (10-12 h) and before any 
food intake.  
7 Five blood spots to be collected by the patients on the second test day of each period at home, following the instructions received by the Investigator. 
Blood spots will be collected at 8:00 (±15 min) right before product self-administration and before any food intake, at 12:00 (±15 min) right before product 
self-administration and before food intake, at 16:00 (±15 min) before food intake, at 20:00 (±15 min) right before product self-administration and before 
food intake, and at 8:00 (±15 min) on the following morning, before any food intake.  
8 At the final/discontinuation visit, the investigator will organize collection of blood spots test at the patient’s home by a dedicated courier. 
9 Patients (or parent(s)/guardian) will be instructed by the Investigator to record any signs, symptoms or medical events occurring during the study until 
the final/discontinuation visit on a patient’s diary. The participant (or parent(s)/guardian) will be instructed to record the events starting on the day they 
happened and indicate in the diary when the events ended. Moreover, patients (or parent(s)/guardian) will be instructed to report adverse events timely 

Product: PKU GOLIKE 
 
Version 3.0 
Clinical Study Protocol: GLK-IT-2023 
 
13 Feb 2024 
 
GLK-IT-2023  
  
 Ver.3.0 – 13.02.2024 
 
Page 8 of 10 
 
to the Investigator. All adverse events (AEs) are to be reported by the Investigator starting from he informed consent until 30 days after the final dose of 
the study product. After this period, only serious AEs suspected of being related to the study product are to be reported. 
10 Any new (not already reported in the medical history as a current therapy) concomitant medication (including any supplements) taken during the study 
will be recorded on a patient’s diary. 
11Each patient will follow the same diet (= food ingested over 24 hours) in terms of food, calories and nutrients ranges during the first days (T1 and T3) of 
each period and the same diet during the second days (T2 and T4) of each period, according to the age and body weight of the patient. The days before the 
test days each patient will follow his standard diet. On the test days, no food will be allowed outside of the defined time windows.  
12 Information on the daily activities of each patient will be collected in the patient’s diary. No sport activities are allowed on sampling days  
13 Information on compliance to the study product will be collected in the patient’s diary on each test day. This information will include but will not be 
limited to information on the amount of product weighed and on whether the suspension/solution is ingested. 
14 Information on 24-hour blood spots will be collected in the patient’s diary on the second test day of each period. This information will include but will 
not be limited to the time of sample collection and information on whether some blood spots are not collected. 
 
 

Product: PKU GOLIKE 
 
Version 3.0 
Clinical Study Protocol: GLK-IT-2023 
 
13 Feb 2024 
 
GLK-IT-2023  
  
 Ver.3.0 – 13.02.2024 
 
Page 9 of 10 
 
1.4 
Details of test days  
 
 
First test days 
Second test days 
 
h 8:00 
h 12:00 
h 16:00 
h 20:00 
h 8:00  
(±15 
min) 
Right 
after the 
blood 
spot 
h 12:00 
(±15 
min) 
Right 
after the 
blood 
spot 
h 16:00 
(±15 
min) 
Right 
after the 
third 
blood 
spot 
h 20:00 
(±15 
min) 
Right 
after the 
blood 
spot 
h 8:00  
(±15 
min) 
Blood spots * 
 
 
 
 
 
 
 
 
 
 
 
 
 
Product 
self-
administration 
 
 
 
 
 
 
 
 
 
 
 
 
 
Meal § 
 
 
 
 
 
 
 
 
 
 
 
 
 
Reporting 
of 
information in 
the 
patient’s 
diary # 
 
Following the Instructions received by the Investigator 
* Blood spots must be collected before product self-administration and before food intake. 
§ No food is allowed outside of the defined time windows. 
# Information on patient’s compliance, 24-hour blood spots collection, diet, daily activities, adverse events, and concomitant medications. 
 

Product: PKU GOLIKE 
 
Version 2.0Clinical Study Protocol: GLK-IT-2023
 
 
09 May 2023 
GLK-IT-2023  
  
 Ver.3.0 – 13.02.2024 
 
Page 10 of 10