safety and efficacy of 22 weeks of treatment with sapropterin dihydrochloride in patients with...

� 2008 Wiley-Liss, Inc. American Journal of Medical Genetics Part A 146A:2851–2859 (2008)

Safety and Efficacy of 22 Weeks of TreatmentWith Sapropterin Dihydrochloride in Patients

With Phenylketonuria

Phillip Lee,1 Eileen P. Treacy,2* Eric Crombez,3 Melissa Wasserstein,4 Lewis Waber,5

Jon Wolff,6 Udo Wendel,7 Alex Dorenbaum,8 Judith Bebchuk,9 Heidi Christ-Schmidt,9

Margretta Seashore,10 Marcello Giovannini,11 Barbara K. Burton,12

Andrew A. Morris13 and the Sapropterin Research Group1National Hospital for Neurology & Neurosurgery, Charles Dent Metabolic Unit, Queen Square, London, UK

2National Center for Inherited Metabolic Disorders, The Children’s University Hospital, Dublin, Ireland3Division of Medical Genetics, Department of Pediatrics, David Geffen School of Medicine at UCLA, Los Angeles, California

4Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York, New York5Children’s Medical Center of Dallas, Dallas, Texas

6Biomedical Genetics Program, Waisman Center, University of Wisconsin, Madison, Wisconsin7University Children’s Hospital, Heinrich-Heine University Dusseldorf, Dusseldorf, Germany

8BioMarin Pharmaceutical Inc., Novato, California9Statistics Collaborative Inc., Washington, District of Columbia

10Yale Department of Genetics, New Haven, Connecticut11Department of Pediatrics, San Paolo Hospital, University of Milan, Milan, Italy

12Division of Genetics, Children’s Memorial Hospital, Chicago, Illinois13Willink Biochemical Genetics Unit, Royal Manchester Children’s Hospital, Pendlebury, Manchester, UK

Received 10 March 2008; Accepted 18 September 2008

Phenylketonuria (PKU) is an inherited metabolic diseasecharacterized by phenylalanine (Phe) accumulation, whichcan lead to neurocognitive and neuromotor impairment.Sapropterin dihydrochloride, an FDA-approved syntheticformulation of tetrahydrobiopterin (6R-BH4, herein referredto as sapropterin) is effective in reducing plasma Pheconcentrations in patients with hyperphenylalaninemia dueto tetrahydrobiopterin (BH4)-responsive PKU, offering poten-tial for improved metabolic control. Eighty patients, �8 yearsold, who had participated in a 6-week, randomized, placebo-controlled study of sapropterin, were enrolled in this 22-week,multicenter, open-label extension study comprising a 6-weekforced dose-titration phase (5, 20, and 10 mg/kg/day of studydrug consecutively for 2 weeks each), a 4-week dose-analysisphase (10 mg/kg/day), and a 12-week fixed-dose phase(patients received doses of 5, 10, or 20 mg/kg/day based ontheir plasma Phe concentrations during the dose titration).Dose-dependent reductions in plasma Phe concentrations

were observed in the forced dose-titration phase. Mean (SD)plasma Phe concentration decreased from 844.0 (398.0) mmol/L (week 0) to 645.2 (393.4) mmol/L (week 10); the mean wasmaintained at this level during the study’s final 12 weeks(652.2 [382.5] mmol/L at week 22). Sixty-eight (85%) patientshad at least one adverse event (AE). All AEs, except one,were mild or moderate in severity. Neither the severe AEnor any of the three serious AEs was considered related tosapropterin. No AE led to treatment discontinuation. Saprop-terin is effective in reducing plasma Phe concentrationsin a dose-dependent manner and is well tolerated at doses of5–20 mg/kg/day over 22 weeks in BH4-responsive patientswith PKU. � 2008 Wiley-Liss, Inc.

Key words: clinical trial; phenylalanine; phenylketonuria;6R-BH4; sapropterin; tetrahydrobiopterin

How to cite this article: Lee P, Treacy EP, Crombez E, Wasserstein M, Waber L, Wolff J, Wendel U, Dorenbaum A,Bebchuk J, Christ-Schmidt H, Seashore M, Giovannini M, Burton BK, Morris AA, the Sapropterin ResearchGroup. 2008. Safety and efficacy of 22 weeks of treatment with sapropterin dihydrochloride in patients

with phenylketonuria. Am J Med Genet Part A 146A:2851–2859.

INTRODUCTION

Phenylketonuria (PKU) is a rare, autosomal reces-sive, inherited metabolic disease resulting frommutations in the gene encoding phenylalanine

*Correspondence to: Eileen P. Treacy, National Centre for InheritedMetabolic Disorders, The Children’s University Hospital, Temple Street,Dublin 1, Ireland. E-mail: [email protected]

Published online 16 October 2008 in Wiley InterScience(www.interscience.wiley.com)

DOI 10.1002/ajmg.a.32562

hydroxylase (PAH), the enzyme that converts pheny-lalanine (Phe) to tyrosine [Scriver and Kaufman,2001]. Phe is an essential amino acid which isingested in adequate amounts in a normal diet tomeet daily requirements for protein synthesis andothermetabolic processes. PAH is a required enzymein the metabolism of Phe; therefore, functionalmutations in PAH lead to an accumulation of Phein the blood and other tissues. As a result, PKU ischaracterized by elevated plasma Phe concentra-tions, which are toxic to the brain and can lead toneurocognitive and neuromotor impairment [Krauseet al., 1986; Moyle et al., 2007].

The current management of PKU involves limitingPhe intake through dietary restriction of wholeprotein and the use of Phe-free protein supplementsto meet daily dietary protein requirements. Althoughthese interventions can significantly reduce plasmaPhe concentrations and prevent the severe neuro-logical manifestations of the disease, compliancebecomes increasingly difficult with age because ofsocial constraints, the need for severe proteinrestriction [National Institutes of Health ConsensusDevelopment Panel, 2001], and the poor palatabilityof the Phe-free protein supplements [Rohr et al.,2001].

Several reports indicate that the majority of olderchildren, adolescents, and adults with PKU do nothave adequately controlled plasma Phe concentra-tions during dietary therapy [Azen et al., 1996; Walteret al., 2002]. Lack of adequate control of plasmaPhe concentration after early childhood has beenassociated with impairment of executive function,including deficits in working memory [White et al.,2001, 2002], impairment of frontal lobe functions[Leuzzi et al., 2004], decreased learning ability [Whiteet al., 2001, 2002], and reduced performance speed[Feldmann et al., 2005]. In addition, individuals withinadequately controlled plasma Phe concentrationsin infancy and childhood show increased behavioraland social integration problems [Azen et al., 1996;Smith and Knowles, 2000; Bhat et al., 2005; Gassioet al., 2005], higher risks for depression and anxietydisorders [Smith and Knowles, 2000], increased useof medication for attention deficit hyperactivitydisorder [Arnold et al., 2004], and lower socio-economic status and marriage rates [Bhat et al.,2005]. As a result, consensus treatment guidelinesrecommend that patients with PKU should maintainadequate control of plasma Phe concentrationsthroughout their lives [National Institutes of HealthConsensus Development Panel, 2001].

6R-tetrahydrobiopterin (6R-BH4) is a necessaryco-factor for PAH, the exogenous administration ofwhich has been shown to decrease plasma Pheconcentrations in BH4-responsive patients with PKU[Muntau et al., 2002]. In somepatients, treatmentwithBH4 can reduce the need for dietary restriction ofprotein and the use of Phe-free protein supplements

altogether [Shintaku et al., 2004; Belanger-Quintanaet al., 2005; Hennermann et al., 2005; Lambruschiniet al., 2005]. Sapropterin dihydrochloride is asynthetic formulation of tetrahydrobiopterin or 6R-BH4, herein referred to as sapropterin, which is FDA-approved to reduce blood Phe concentrations inpatients with hyperphenylalaninemia due to tetrahy-drobiopterin (BH4)-responsive PKU. In a random-ized, double-blind, placebo-controlled, Phase IIItrial [Levy et al., 2007], treatment with sapropterin,10 mg/kg/day (sapropterin dihydrochloride), for6 weeks was found both to be effective in reducingplasma Phe concentrations and to offer a favorablesafety profile in patients with PKU and hyper-phenylalanemia who had responded previously toan 8-day course of sapropterin. We report resultsfrom a 22-week, Phase III, open-label extensionstudy, which evaluated the safety and efficacy ofsapropterin in the treatment of PKU.

METHODS

The trial was an open-label, extension studyconducted at 26 centers in North America (Canadaand the USA) and Europe (France, Germany, Ireland,Italy, Poland, and the United Kingdom). The conductof the protocol at individual sites was approvedby the local Institutional Review Boards or EthicsCommittees, and the study was performed accordingto the principles of the International Conferenceon Harmonization Guidelines for Good ClinicalPractice, and the Declaration of Helsinki.

The study protocol was drafted and developed bythe study sponsor, BioMarin Pharmaceutical Inc.(Novato, CA). Representatives or employees of thesponsor were responsible for the administrationand monitoring of the study. Data managementwas performed by Pacific Data Designs Inc.(San Francisco, CA), and statistical analyses byStatistics Collaborative Inc. (Washington, DC).

Patients

An enriched population sample was used consist-ing of patients (�8 years of age) with PKU andhyperphenylalanemia who had been enrolled in therandomized, placebo-controlled, 6-week study [Levyet al., 2007]. The Levy et al. study required a bloodPhe level of �600 or 450 mmol/L after a protocolamendment at screening, after achieving �30%reduction in plasma Phe concentration during aprevious 8-day treatment course with sapropterin[Burton et al., 2007]. This selection method wasexpected to increase the probability of detecting atrue response to treatment.

Patients could participate provided they hadreceived at least 80% of the scheduled doses in theprevious randomized, placebo-controlled study[Burton et al., 2007]. Female patients of child-bearing

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American Journal of Medical Genetics Part A

potential were required to have a negative urinepregnancy testwithin 24hrprior to enrollment and tobe using acceptable measures of contraception. Allpatients were required to be willing to continue withtheir current diet during the study. Patients wereineligible for inclusion if they discontinued theprevious study for any reason other than withdrawalbecause of highplasmaPhe concentrations, or if theywere expected to require any investigational productor vaccine prior to completion of the study. Otherexclusion criteria were: pregnancy (or intendedpregnancy) or lactation; concurrent medical con-ditions or diseases that would interfere with theconduct of the study; the use of dihydrofolatereductase inhibitors [Ponzone et al., 2004], levodopa,or other medications that could influence the studyresults. Other medications could be given at theinvestigators’ discretion.

All adult patients provided informed, writtenconsent before inclusion in the study. In the case ofchildren, written informed consent was obtainedfrom their parents or guardians, and if applicable, thechild provided his or her assent.

Study Design

The study comprised three parts: a 6-week forceddose-titration phase, followed by a 4-week dose-analysis phase, and a 12-week fixed-dose phase.

Sapropterin was given once daily in the morning. Itwas provided as tablets, each containing 100 mgsapropterin dihydrochloride (each 100 mg sapro-pterin dihydrochloride contains 77 mg sapropterinbase), which were dissolved in 120–240 ml water,orange juice or apple juice. To allow the use ofwhole tablets, doses were calculated by multiplyingthe patient’s weight in kilograms (at week 0) by theassigned dose (5, 10, or 20 mg/kg/day) and roundingup to the next 100 mg unit dose.

Dosesof 5, 10, or 20mg/kg/daywere selectedbasedon the results of previous studies of efficacy and safetyduring extended treatment with BH4. In patientswith PKU, doses in this range have been shownto substantially reduce blood Phe levels [Ceroneet al., 2004; Shintaku et al., 2004; Steinfeld et al., 2004;Lambruschini et al., 2005; Trefz et al., 2005].

During the forced dose-titration phase, allpatients received doses of 5, 20, and 10 mg/kg/day(sapropterin dihydrochloride) for 2 weeks eachconsecutively, and during the dose-analysis phaseall patients received 10 mg/kg/day (sapropterindihydrochloride). The dose received during thefixed-dose phase was determined by the patient’splasma Phe concentrations at weeks 2 and 6 duringthe dose-titration phase. The dosage regimen overthe 22-week study period is shown in Table I.

Blood samples for the measurement of plasma Pheconcentrations were obtained 2.5–5 hr after a meal(preferably breakfast) at weeks 0, 2, 4, 6, 10, 12, 16,20, and 22. Plasma Phe concentrations were measur-ed by a validated ion-exchange technique atMayo Reference Services, Rochester, MN, USA(North American sites), or at the Laboratorium furKlinische Forschung GmbH, Raisdorf, Germany(European sites). The inter-assay coefficient ofvariation was 2.5% at a plasma Phe concentrationof 250 mmol/L (4.2 mg/dl) and 4.2% at a plasma Pheconcentration of 56 mmol/L (0.9 mg/dl). Plasma Pheconcentration was assessed by a central laboratoryunder blinded conditions. As this was an open-labelstudy, blinding to treatment was not required.

The efficacy variable of primary interest was theplasma Phe concentration. Long-term persistence ofthe response to sapropterin was assessed using theplasma Phe concentrations measured at the week 12,16, 20, and 22 visits. Plasma Phe measurementsfor each patient at the week 2, 4, and 6 visits,which correspond to measurements taken after each2-week dosing period (5, 20, and 10 mg/kg/day),were used to estimate the effect of dose on plasmaPhe concentrations for the three dose levels.

Safety was assessed by medical history, monitoringof adverse events (AEs; coded using the MedicalDictionary for Regulatory Activities coding diction-ary [MedDRA], version 8.1), and by physical exami-nation, measurement of vital signs, and clinicallaboratory evaluation (hematology, clinical chemis-try, and urinalysis). AEs were categorized accordingto their severity, relationship to study drug, andseriousness. The severity of AEs was assessed bythe investigator and categorized according to theimpact on usual activities: mild¼no limitation;

TABLE I. Dosage Regimen of Study Drug Over the 22-Week Study Period

Dose period Dose received

Forced dose-titration period (6 weeks) All patients received 5, 20, and 10 mg/kg/day consecutively for 2 weeks eachDose-analysis period (4 weeks) All patients received 10 mg/kg/dayFixed-dose period (12 weeks) Blood Phe concentration during forced dose-titration period Dose during fixed-dose

period<600 mmol/L (10.1 mg/dl) at week 2 and <240 mmol/L (4.0 mg/dl)

at week 65 mg/kg/day

�600 mmol/L at week 2 and �240 mmol/L at week 6 OR �240 mmol/Land <600 mmol/L at week 6

10 mg/kg/day

�600 mmol/L at week 6 20 mg/kg/day

Phe, phenylalanine.

SAPROPTERIN IN PATIENTS WITH PKU 2853


moderate¼ some limitation; and severe¼ inabilityto carry out usual activities. Serious AEs were definedas any AE that resulted in death, was life-threatening,required hospitalization or prolonged existinghospitalization, resulted in persistent or significantdisability or incapacity, was a congenital anomaly orbirth defect (in child or fetus of study subject exposedto study drug prior to conception or duringpregnancy), or could have jeopardized the patientor required medical or surgical intervention toprevent any of the other outcomes listed.

Statistics

The efficacy analyses included data for all patientsenrolled in the study, from all phases of the study.The primary efficacy endpoints included assess-ments of mean plasma Phe concentrations over timeand mean changes from week 0 in plasma Pheconcentrations over time. Ninety-five percent con-fidence intervals of the mean values for each timepoint were constructed. No formal statistical test wasperformed for the primary efficacy analysis. PlasmaPhe concentrations obtained at the week 2, 4 and6 visits, which each correspond to measurementstaken after each 2-week forced dose-titration period(5, 20, and 10 mg/kg/day), were analyzed using alongitudinal repeated-measures model. Each patientserved as their own control. SAS1 (version 9.1.3)software (SAS Institute Inc., Cary, NC) was used forall analyses. No imputation for missing data wasperformed for either the primary or the secondaryefficacy analyses.

The safety analyses included data for all patientswho had received at least one dose of sapropterin.

No formal sample-size calculation was performed.

RESULTS

A total of 80 patients (47 males, 33 females) wereenrolled, of whom 39 had received sapropterinand 41 had received placebo in the randomized,placebo-controlled study [Levy et al., 2007]. Of the 39patients previously treated with sapropterin, 7 (18%)transitioned directly into the current study with notreatment gap; the remaining 32 patients had amean (standard deviation; SD) gap in treatment of63.2 (23) days (range: 14–133 days) between thetwo studies. For the 41 patients who had receivedplacebo in the previous study and who did notenroll directly into the current study, the mean (SD)gap in treatment was 70.5� 26 days (range: 14–133 days).

All 80 patients were included in the intention-to-treat analysis. A total of 79 patients com-pleted week 22 of the study: 1 patient withdrewat week 16 due to non-compliance with studyprocedures. Demographic characteristics of thepatients are summarized in Table II.

Protocol deviations were reported for 39 (49%)patients. Of these, 32 (40%) had deviations related todosing and 19 (24%) haddeviations related to diet; allof these deviations were considered to be minor. Inaddition, one patient was required by her employerto have a vaccination for mumps. This patientwas exempted from the exclusion criteria that novaccination or investigational product be adminis-tered during the study.

Compliance with treatment (assessed by patientself-report) was generally good. Overall, 48 (60%)patients reported taking all doses correctly, 14 (18%)patients missed at least one dose and took noincorrect doses, 7 (9%) patients took at least onedose incorrectly and did not miss a dose, and11 (14%) patients took at least one dose incorrectlyand missed at least one dose. No patient took anydose higher than that prescribed.

During the dose-titration period, the mean (SD)amount of study drug received by patients during the5, 20, and 10mg/kg/daydosingperiodswas 5.7 (0.6),20.8 (0.7), and 10.7 (0.6) mg/kg/day, respectively. Inthe 12-week fixed-dose period, the mean (SD)amount of study drug received by patients in the 5,10, and 20 mg/kg/day dose groups was 6.6 (0.6),10.7 (0.6), and 20.5 (0.9) mg/kg/day, respectively.

A total of 19 patients reported changes in their dietduring the study: 4 patients reported a decrease inPhe intake for a period of more than 3 days, and12 patients reported a total of 15 incidences ofincreases in Phe intake lasting longer than 3 days.

Efficacy

Forced dose-titration and dose-analysisphases. During the forced dose-titration anddose-analysis phases of the study, the mean (SD)plasma Phe concentration decreased from 844.0(398.0)mmol/L (14.1 [6.6] mg/dl) at baseline (week 0)to 743.9 (384.4) mmol/L (12.4 [6.4] mg/dl) at week 2,580.8 (398.8) mmol/L (9.7 [6.7] mg/dl) at week 4,639.9 (381.8)mmol/L (10.7 [6.4] mg/dl) at week 6, and

TABLE II. Patient Demographic Characteristics (n¼ 80)

Demographic

Sex, n (%)Male 47 (59)Female 33 (41)

Ethnic origin, n (%)White 78 (98)Asian-Pacific Islander 1 (1)Other: White/Black/Arab 1 (1)

Age, years, mean (SD) 20.4 (9.6)Range (min, max) 8, 49

Height, cm, mean (SD) 165 (13)Range (min, max) 126, 191

Weight, kg, mean (SD) 67.3 (22)Range (min, max) 28, 144

SD, standard deviation.

2854 LEE ET AL.


645.2 (393.4) mmol/L (10.8 [6.6] mg/dl) at week 10(Fig. 1).

The three doses of sapropterin studied were allassociated with decreases in plasma Phe concen-trations compared with week 0 (Fig. 2). There wasevidence of a dose–response relationship (Fig. 1).During the dose-titration phase, patients receivingsapropterin, 10 or 20 mg/kg/day, showed signifi-cantly greater mean decreases in plasma Pheconcentration compared with week 0 than thosereceiving 5 mg/kg/day. The difference in themean (standard error) of the change from week 0in plasma Phe concentration between patients re-ceiving 5 and 10 mg/kg/day was 104 (22.2) mmol/L(1.7 [0.4] mg/dl) (P< 0.0001); the correspondingdifferences from week 0 between patients receiv-ing 5 and 20 mg/kg/day, and those receiving10 and 20 mg/kg/day, were 163 (22.2) mmol/L

(2.7 [0.4] mg/dl) (P< 0.0001) and 59 (22.2) mmol/L(1.0 [0.4] mg/dl) (P¼ 0.0085), respectively.

The proportions of patients achieving a decrease inplasma Phe concentration of at least 30% from week0 after 2 weeks of treatmentwith 5, 10, and 20mg/kg/day, were 25%, 46%, and 55%, respectively. Atthe end of the dose-analysis phase, during whichall patients received sapropterin, 10 mg/kg/day,37 patients (46%) showed a decrease in plasma Pheconcentrationof at least 30%, compared withweek 0.

Fixed-dose phase. During the fixed-dosephase, each patient’s sapropterin dose was deter-mined based on their week 2 and week 6 plasma Pheconcentrations. Of the 80 patients, 6 patients (8%)received 5 mg/kg/day, and 37 (46%) each received10 and 20 mg/kg/day; although the protocol allowedfor an increase in dose from 5 to 10 mg/kg/day, nopatient had a change in dose during this period.On average, patients maintained low mean (SD)plasma Phe concentrations, ranging between619.8 (371.0) mmol/L (10.3 [6.2] mg/dl) at week12 and 652.2 (382.5) mmol/L (10.9 [6.4] mg/dl)at week 22 (Fig. 1). Overall, the mean changefrom week 0 in plasma Phe concentration was�190.5 (355.7) mmol/L (3.2 [5.9] mg/dl) at week 22.The corresponding mean (SD) plasma Phe concen-trations at the end of the study in patients receiving 5,10, or 20 mg/kg/day were 437.8 (260.5) mmol/L(7.3 [4.4] mg/dl), 449.9 (193.1) mmol/L (7.5 [3.2] mg/dl),and 895.7 (407.2) mmol/L (14.9 [6.8] mg/dl), respec-tively (Fig. 2). As expected from the design of thestudy, patients with the highest week 0 plasmaPhe concentrations received the highest dose ofsapropterin, and the mean plasma Phe reductionsachieved in this group during the fixed-dosephase were comparable with those seen duringthe dose-titration phase. Similarly, patients receivinga fixed dose of 10 mg/kg/day showed plasmaPhe concentrations comparable with those achievedduring the corresponding period of the dose-titration phase. Patients receiving a fixed dose of5 mg/kg/day showed lower and more variableplasma Phe concentration reductions comparedwith the other groups; this observation could beexplained in part by the smaller number of patients inthis group.

Among the 6 subjects receiving 5 mg/kg/day, thepercent with a �30% reduction from baseline inblood Phe during the Fixed-Dose Phase rangedfrom 67% to 83%; for the 37 subjects receiving10 mg/kg/day, the percent ranged from 43% to 57%,and for the 37 subjects receiving 20 mg/kg/day, thepercent ranged from 38% to 42%. At the end of thefixed-dose period (week 22), 36 (46%) patientsachieved a decrease in plasma Phe concentration ofat least 30%, compared with week 0; the correspond-ing numbers within patients receiving 5, 10, or20 mg/kg/day were 3 (50%), 18 (49%), and 15 (42%),respectively.

FIG. 2. Mean plasma phenylalanine concentrations over time for all patientsand for patients receiving 5, 10, or 20 mg/kg/day sapropterin dihydrochlorideduring the fixed-dose period, identifying three cohorts (a cohort of6 patients who received the fixed dose of 5 mg/kg/day, 37 patients whoreceived the dose of 10 mg/kg/day and 37 patients who received the fixed doseof 20 mg/kg/day). Error bars indicate 95% confidence intervals; numbersattached to the bars indicate the number of patients included in the summaries.

FIG. 1. Mean plasma phenylalanine concentrations over time. Error barsindicate 95% confidence intervals; numbers attached to the bars indicate thenumber of patients included in the summaries.



Safety

A total of 260 AEs were reported by 68 (85%)patients. All except one of these events were mild ormoderate in severity, and no patient withdrew fromthe study because of an AE. The one AE ratedas severe was a tooth abscess, which was notconsidered serious and was not judged by theinvestigator to be related to sapropterin. The AEsreported for 5% or more of patients are summarizedin Table III.

A total of 82 (32%) AEs in 31 (39%) patients wereconsidered to be possibly or probably related tosapropterin. AEs thatwere considered tobeprobablyrelated to sapropterin were upper abdominal pain(1 patient), nausea (2 patients), headache (1 patient),dizziness (1 patient), and increased alanine amino-transferase (1 patient); with the exception of oneepisode of moderate nausea, all of these were mildin severity. AEs that were considered to be possiblyrelated to sapropterin and were reported by morethan one patient included: urinary tract (2 patients)or streptococcal infections (2 patients), vomiting(4 patients), diarrhea (2 patients), abdominal pain(2 patients), headache (8 patients), migraine(4 patients), pharyngolaryngeal pain (3 patients),cough (2 patients), decreased neutrophil counts(2 patients), and rash (2 patients). Thirty-one AEspossibly related to sapropterin were reported by1 patient each.

Three patients each experienced one serious AEduring the study. Two of these events, urinary tractinfection and spinal cord injury, occurred during thefixed-dose phase of the study. The third event, tibiafracture, occurred after the week-22 visit. The spinalcord injury and tibial fractures were accidental,sports-related injuries and the urinary tract infectionoccurred in a 13-year-old female with duplex kidneyand a history of prior infection.

Two patients had plasma Phe concentrations thatreached 2,000 mmol/L (33.3 mg/dl) or higher duringthe course of the study. One patient had plasma Pheconcentrations >2,000 mmol/L at week 6, which

decreased to 1,819 mmol/L (30.3 mg/dl) by week 10and increased again to �2,000 mmol/L at all visitsbetween week 12 and week 22: this patient receivedthe 20 mg/kg/day dose during the fixed-dose phase.The second patient had plasma Phe concentration>2,000 mmol/L at week 0, which decreased to766 mmol/L (12.8 mg/dl) by week 2: this patient alsoreceived 20 mg/kg/day during the fixed-dose phase,during which time plasma Phe concentrationsranged between 476 mmol/L (7.9 mg/dl) and873 mmol/L (14.6 mg/dl).

Apart from the reported AEs, no change in physicalexamination, vital signs or clinical laboratory evalua-tions requiring medical intervention occurred duringthe study.

DISCUSSION

This open-label extension study shows thatsapropterin at doses of 5–20mg/kg/day (sapropterindihydrochloride) produces a maintained decreasein plasma Phe concentration in BH4-responsivepatients with PKU. These results are consistent withthose of previous studies [Trefz et al., 2001; Shintakuet al., 2004; Steinfeld et al., 2004; Hennermann et al.,2005; Lambruschini et al., 2005],which show that oraltreatment with BH4 at doses of 5–20 mg/kg/day hasan acceptable safety profile and produces durableand significant reductions in plasma Phe concen-trations in BH4-responsive patients with PKU. In aprevious study by Burton et al. [2007], the proportionof patients with PKU who were responders tosapropterin was 20% (96/485); these patientsachieved a mean (SD) reduction in blood Phe levelof �392 (185) mmol/L between day 1 and day 8,representing a 50% mean reduction in overall bloodPhe level.

The forced dose-titration phase provided evidenceof a dose–response relationship, in that greaterdecreases in plasma Phe concentrations wereachieved with the two higher doses of sapropterincompared with the lowest dose. In line with theseresults, two previous studies have reported higherrates of response in patients receiving higherdoses of BH4. In the first study, 20 patients received10 mg/kg/day for 7 days, followed by a wash-outperiodof 1week and then 20mg/kg/day for 7 days. Areduction in plasma Phe concentration of more than30% was seen in 50% and 70% of patients receiving10 and 20 mg/kg/day, respectively [Matalon et al.,2005]. In the second study, higher rates of responseto 6R-BH4wereobservedwithdosesof 18–23mg/kgcompared with <18 mg/kg. In light of these data,the dose of sapropterin should be adjusted in eachindividual patient, according to their responsesto treatment, target plasma Phe concentration, andother treatment goals. If a patient’s blood Pheconcentration increases significantly, for exampleduring an illness, the decrease of plasma Phe evident

TABLE III. Adverse Events Reported Most Commonly*

Adverse event Number of patients (%)

Headache 16 (20)Pharyngo-laryngeal pain 12 (15)Nasopharyngitis 11 (14)Vomiting 10 (13)Diarrhea 8 (10)Upper respiratory tract infection 8 (10)Cough 7 (9)Dysmenorrheaa 3 (9)Migraine 6 (8)Back pain 4 (5)Gastroenteritis 4 (5)Influenza 4 (5)

*Adverse events reported by 5% or more of patients.aOnly female patients were included in the denominator (n¼ 33).

2856 LEE ET AL.


may change during the period of illness. Therefore,vigilant monitoring of such patients should beundertaken, including a focus on the importance ofadherence to a strict dietary regimen until thepatient’s blood Phe concentration returns to theirnormal level.

Standard forced-titration protocols use sequentialperiods of increasing dosage to identify a maximaltherapeutic effect or significant safety events. Suchdesigns may not distinguish between an increasedresponse related to higher dose and that related toincreasing duration of therapy and, thus, may lead tothe choice of doses that are higher than that requiredfor therapeutic benefit [Federal Register, 1994]. In thisstudy, to reduce the confounding effect of durationof therapy, we used a modified, forced dose-titrationphase in which doses did not increase sequentially.Instead, patients received three consecutive 2-weekcourses of sapropterin at doses of 5, 20, and 10 mg/kg/day (sapropterin dihydrochloride).One potentiallimitation of such a design is that there may be carry-over effects between doses. However, previousstudies have shown that although the beneficialresponse to a single dose (10 mg/kg) of 6R-BH4persists for at least 24 hr after treatment [Matalonet al., 2005], the half-life is estimated to be 3.3–5.1 hr[Fiege et al., 2004], thus the duration of each titrationperiod should be sufficient to limit any carry-overeffects without the need for wash-out periods.

During the fixed-dose phase (weeks 11–22), mostpatients were assigned to receive dosages of 10 or20 mg/kg/day, according to pre-defined plasma Pheconcentration criteria. On average, patients main-tained low plasma Phe concentrations suggestive ofthe persistence of sapropterin effects for at least10 weeks. During the fixed-dose phase, the patientsreceiving the highest dose of sapropterin (20 mg/kg/day) showed the lowest rate of response (proportionof patients with �30% reduction in plasma Pheconcentration) despite having the largest mean (SD)reduction in plasma Phe concentration at the end ofthe fixed-dose period (week 22; �209 [437] mmol/L).This is perhaps not surprising, as the patientsassigned to the 20 mg/kg/day dose were also thosewith the highest plasma Phe concentrations at theend of the dose-titration phase, as per the dosingalgorithm. The extent to which patients respond tosapropterin, therefore, appears to depend on Phetolerance (as measured in this study by their initiallevels of Phe), with those patients who had a lowerbaseline level of Phe showing a greater response.Previous studies have shown considerable inter-patient variability in BH4 responsiveness [Muntauet al., 2002] that may be governed by multiple factors.It is recognized that the emergent phenotype(Phe levels) is a complex trait with residual hydrox-ylation (PAH genotype) being a major determinant.Differences in Phe disposal have been identifiedbetween individuals of identical PAH genotype

[Scriver, 1998; Treacy et al., 1997] reflecting not onlyvariation in Phe hydroxylation but also differences inprotein synthesis rates and other genes involved inPhe homeostasis. Those patients assigned to the20 mg/kg/day dose may possibly show moderateresponsiveness.

The patients enrolled in this study had high plasmaPhe concentrations (�600 mmol/L or �450 mmol/Lafter protocol amendment) at entry to a previousrandomized, placebo-controlled study [Levy et al.,2007], and were advised to maintain their usual diets.Hence, they had variable dietary therapy and a widerange of baseline plasma Phe concentrations abovetheir target concentrations, as might be expected inclinical practice. These data indicate that, despitewide variations in baseline plasma Phe concentra-tions and dietary intake of Phe, treatment withsapropterin can help to maintain low plasma Pheconcentrations in an important subset of patientswith PKU, and may increase Phe tolerance, reducethe need for Phe-free protein supplements and allowincreased intake of dietary protein, enhancing theclinical management of many people with thisdisorder. The relaxation of strict dietary controlmay also improve overall compliance and adherenceto the treatment guidelines.

Although all patients entering this study hadachieved �30% reduction in plasma Phe concen-tration during a previous 8-day trial [Burton et al.,2007], the response rate at the end of the doseanalysis phase was 46%. It may be that evaluation ofresponsiveness over the previous 8-day treatmentcourse with sapropterin yielded false positive andnegative results, and that a response evaluationperiod of 1 month with a dose of 10 mg/kg/dayfollowed by 1 month at a dose of 20 mg/kg/day mayhave better enriched this study population.

The favorable safety profile of sapropterinobserved in this study is consistent with ourexperience in the previous study [Levy et al., 2007],and with studies with BH4 [Trefz et al., 2001;Shintaku et al., 2004; Steinfeld et al., 2004; Henner-mannet al., 2005; Lambruschini et al., 2005].MostAEswere mild or moderate in severity and resolvedspontaneously without discontinuation of treatment.No patient withdrew from the study because of AEs.

In conclusion, this study indicates that sapropterinis effective in reducing plasma Phe concentrations ina dose-dependent manner in BH4-responsivepatients with PKU, and is well tolerated at doses of5–20 mg/kg/day over 22 weeks.

ACKNOWLEDGMENTS

The authors would like to thank their fellowinvestigators of the Sapropterin Research Group.Study investigators: Canada: L. Clarke, ProvincialMedical Genetics Program, UBC Medical GeneticsResearch, Vancouver, BC; A. Feigenbaum, Hospital



for Sick Children, Toronto, ON; France: V. Abadie,Hopital Necker—Enfants Malades, Paris; D. Dobbe-laere, CHRU de Lille Hopital Jeanne de Flandres,Lille; F. Feillet, Hopital d’enfants—CHU BraboisService de Medecine Infantile, Vandoeuvre lesNancy; Germany: J. Hennermann, Charite CampusVirchow Klinikum, Otto-Heubner-Centrum furKinder-und Jugendmedizin, Berlin; F. Trefz, Klinikfur Kinder-und Jugendmedizin Reutlingen, Reutlin-gen; Italy: C. Meli, Azienda Ospedaliera Universita-ria, Catania; Poland: A. Milanowski, Instytut Matki iDziecka Apteka, Warszawa; United Kingdom: A.Chakrapani, Birmingham Children’s Hospital, Bir-mingham; M. Cleary, Great Ormond Street Hospital,London; United States of America: J. Baker, KaiserPermanente Medical Center, Oakland, CA; J. Bergof-fen, Kaiser Permanente, Genetics Department, SanJose, CA; D. Grange, St Louis Children’s Hospital,Division of Medical Genetics, Washington School ofMedicine, St Louis, MO; C. Harding,Oregon Health&Science University, Portland, OR; P. Harmatz, Child-ren’s Hospital Oakland, Oakland, CA; R. Koch,Children’s Hospital Los Angeles, Los Angeles, CA;H. Levy, Children’s Hospital of Boston MetabolismResearch, Boston, MA; L. Lipson, Kaiser Point WestMedical Office, Sacramento, CA; N. Longo, Univer-sity of Utah, Medical Genetics and Pediatrics, SaltLake City, UT; L. Randolph, Children’s Hospital LosAngeles, Los Angeles, CA; G. Vockley, Children’sHospital of Pittsburgh, Division of Medical Genetics,Pittsburgh, PA; C. Whitley, Fairview UniversityMedical Center, Pharmaceutical Services, Minneap-olis, MN. The authors would like to acknowledge theparticipation of the Yale Center for Clinical Research,supported in part by the CTSA Program Grant #1 UL1RR024139 and by the GCRC Program Grant #M01RR000125, from the National Center for ResearchResources, National Institutes of Health. This studywas sponsored by BioMarin Pharmaceutical Inc.,which had a significant role in study design,collection, analysis and interpretation of data, andthewritingof themanuscript. The studyprotocolwasdrafted and developed by the study sponsor.

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