`
`Contents lists available at ScienceDirect
`
`Molecular Genetics and Metabolism
`
`j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / y m g m e
`
`Phase 2 comparison of a novel ammonia scavenging agent with sodium
`phenylbutyrate in patients with urea cycle disorders: Safety, pharmacokinetics
`and ammonia control
`Brendan Lee a,b,*, William Rhead c, George A. Diaz d, Bruce F. Scharschmidt e, Asad Mian a,
`Oleg Shchelochkov a, J.F. Marier f, Martin Beliveau f, Joseph Mauney g, Klara Dickinson e, Antonia Martinez e,
`Sharron Gargosky e, Masoud Mokhtarani e, Susan A. Berry h
`a Baylor College of Medicine, One Baylor Plaza Rm R814, Houston, TX, United States
`b Howard Hughes Medical Institute, TX, United States
`c Pediatrics, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI, United States
`d Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY, United States
`e Hyperion Therapeutics, Inc., 601 Gateway Blvd., Ste. 200, South San Francisco, CA, United States
`f Pharsight Corp., Montreal, 2000 Peel St., Suite 570, Quebec, Canada
`g Chiltern, 2520 Independence Blvd., Ste. 202, Wilmington, NC, United States
`h Division of Genetics and Metabolism, University of Minnesota, 420 Delaware Str., SE, Minneapolis, MN, United States
`
`a r t i c l e
`
`i n f o
`
`a b s t r a c t
`
`Article history:
`Received 11 February 2010
`Received in revised form 18 March 2010
`Accepted 18 March 2010
`Available online 23 March 2010
`
`Keywords:
`Ammonia
`Clinical trial
`Phenylacetylglutamine
`Phenylbutyrate
`Urea cycle disorders
`
`Glycerol phenylbutyrate (glyceryl tri (4-phenylbutyrate)) (GPB) is being studied as an alternative to
`sodium phenylbutyrate (NaPBA) for the treatment of urea cycle disorders (UCDs). This phase 2 study
`explored the hypothesis that GPB offers similar safety and ammonia control as NaPBA, which is currently
`approved as adjunctive therapy in the chronic management of UCDs, and examined correlates of 24-h
`blood ammonia.
`Methods: An open-label, fixed sequence switch-over study was conducted in adult UCD patients taking
`maintenance NaPBA. Blood ammonia and blood and urine metabolites were compared after 7 days (steady
`state) of TID dosing on either drug, both dosed to deliver the same amount of phenylbutyric acid (PBA).
`Results: Ten subjects completed the study. Adverse events were comparable for the two drugs; 2 subjects
`experienced hyperammonemic events on NaPBA while none occurred on GPB. Ammonia values on GPB
`were 30% lower than on NaPBA (time-normalized AUC = 26.2 vs. 38.4 lmol/L; Cmax = 56.3 vs.
`79.1 lmol/L; not statistically significant), and GPB achieved non-inferiority to NaPBA with respect to
`ammonia (time-normalized AUC) by post hoc analysis. Systemic exposure (AUC0–24) to PBA on GPB was
`27% lower than on NaPBA (540 vs. 739 lg h/mL), whereas exposure to phenylacetic acid (PAA) (575 vs.
`596 lg h/mL) and phenylacetylglutamine (PAGN) (1098 vs. 1133 lg h/mL) were similar. Urinary PAGN
`excretion accounted for 54% of PBA administered for both NaPBA and GPB; other metabolites accounted
`for <1%. Intact GPB was generally undetectable in blood and urine. Blood ammonia correlated strongly and
`inversely with urinary PAGN (r = 0.82; p < 0.0001) but weakly or not at all with blood metabolite levels.
`Conclusions: Safety and ammonia control with GPB appear at least equal to NaPBA. Urinary PAGN, which is
`stoichiometrically related to nitrogen scavenging, may be a useful biomarker for both dose selection and
`adjustment for optimal control of venous ammonia.
`
`Ó 2010 Elsevier Inc. All rights reserved.
`
`Abbreviations: ASS, arginosuccinate synthetase deficiency; AUC0–24, 24-h area
`under the curve; Glycerol phenylbutyrate, generic name for glyceryl tri (4-
`phenylbutyrate); GPB, glycerol phenylbutyrate; HHH, ornithine translocase defi-
`ciency; NaPBA, sodium phenylbutyrate; PAA, phenylacetic acid; PAG, phenylacetyl
`glycine; PAGN, phenylacetylglutamine; PBA, phenylbutyric acid; PBG, phenylbuty-
`ryl glycine; PBGN, phenylbutyryl glutamine; PK, pharmacokinetic; TNAUC, time-
`normalized area under the curve; UCD, urea cycle disorder; ULN, upper limit of
`normal.
`* Corresponding author at: Department of Molecular and Human Genetics, Baylor
`College of Medicine, One Baylor Plaza Rm R814, Houston, TX 77030, United States.
`Fax: +1 713 798 5168.
`E-mail address: blee@bcm.tmc.edu (B. Lee).
`
`1096-7192/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved.
`doi:10.1016/j.ymgme.2010.03.014
`
`Introduction
`
`Urea cycle disorders (UCDs) comprise several inherited defi-
`ciencies of enzymes or transporters necessary for the synthesis of
`urea from ammonia [1–3]. UCDs result in the accumulation of toxic
`levels of ammonia in the blood and brain of affected patients and
`can present in the neonatal period or later in life depending on
`the severity and type of defect. UCD incidence is estimated to be
`1:8200 live births [1]. Hyperammonemia is the major cause of
`morbidity and mortality in UCD patients, and outcome during
`
`
`
`222
`
`B. Lee et al. / Molecular Genetics and Metabolism 100 (2010) 221–228
`
`hyperammonemic crises correlates with blood ammonia levels [4].
`Control of blood ammonia levels is the main objective of both acute
`and chronic management of UCD patients.
`Sodium phenylbutyrate (NaPBA) (US trade name: BUPHENYLÒ,
`EU: AMMONAPSÒ) is approved for the chronic adjunctive treatment
`of certain UCDs and lowers ammonia by enhancing excretion of
`waste nitrogen. It is a pro-drug that undergoes rapid beta-oxida-
`tion to phenylacetate, (PAA), a metabolically active compound that
`conjugates with glutamine via acetylation to form phenylacetyl-
`glutamine (PAGN) which is then excreted in the urine. PAGN, like
`urea, contains two molecules of nitrogen and therefore represents
`an alternate to urea for excretion of waste nitrogen [5]. The maxi-
`mum approved dose of 20 NaPBA grams per day (40 tablets per
`day) contains approximately 2363 mg of sodium, and current ‘‘Die-
`tary Guidelines for Americans 2005” recommends a sodium intake
`of 2300 mg/day for the general population and 1500 mg/day for
`individuals with hypertension and selected groups at risk for
`hypertension [6]. Some UCD patients may be at increased risk for
`hypertension, and a sodium-free oral treatment option would be
`especially beneficial for these patients [7,8].
`Glycerol phenylbutyrate (GPB) is an investigational agent being
`studied as an alternative therapy to NaPBA in UCD patients. It con-
`sists of a glycerol backbone with three molecules of PBA joined via
`ester linkage and is a pale yellow nearly odorless and tasteless oil.
`17.4 mL of GPB [1 tsp TID] delivers an amount of PBA equivalent
`to 20 g of NaPBA [40 tablets]).
`The safety and pharmacokinetic (PK) characteristics of GPB
`have been evaluated in pre-clinical models and in two prior clini-
`cal studies, including a randomized, crossover, open-label study in
`24 healthy male subjects administered a single oral dose of NaPBA
`and GPB (equivalent to 3 g/m2 of PBA), and an open-label study in
`32 adults, including 8 healthy adults and 24 adults with Child-
`Pugh grade A, B, or C cirrhosis (8 each), each of whom received
`a single 100 mg/kg dose of GPB followed by 1 week of BID
`dosing at 100 mg/kg per dose [9]. Collectively, these prior studies
`suggest that GPB exhibits satisfactory safety, achieves steady
`state within 4 days or less, and exhibits slow release characteris-
`tics. The present phase 2 study, the first in UCD patients, was de-
`signed to compare safety, PK and ammonia control of GPB with
`NaPBA.
`
`Materials and methods
`
`Study design and treatments
`
`This was a phase 2, open-label, fixed sequence, switch-over
`study in patients being treated with NaPBA for a UCD (confirmed
`via enzymatic, biochemical or genetic testing). Subjects 18 years
`old or older who had been treated with NaPBA for P2 weeks were
`eligible. Liver transplant, hypersensitivity to PBA, PAA or PAGN,
`clinically significant laboratory abnormalities or ECG findings, or
`any condition such as infection or medications that could affect
`ammonia levels were major exclusion criteria.
`After enrollment, subjects received NaPBA for at least 7 days,
`TID with meals at the dose level prescribed by the investigator.
`On the last day of NaPBA treatment they were admitted to an inpa-
`tient research unit for 24-h PK and ammonia monitoring. Depend-
`ing on dose, subjects were then either switched directly to 100%
`GPB, or GPB was introduced in step-wise weekly increments equiv-
`alent to 650 mg/kg/day of NaPBA, with the remainder of the PBA
`equivalent dose administered as corresponding weekly decre-
`ments in the dose of NaPBA. Initiation or increases in GPB dosing
`were done under observation in an appropriately monitored set-
`ting, and subjects were discharged after they were deemed clini-
`cally stable and after at least 48 h of observation. After at least
`7 days on 100% GPB administered TID at a dose equivalent to their
`
`prescribed dose of NaPBA in terms of PBA delivered, subjects were
`re-admitted to the research unit for 24-h PK and ammonia assess-
`ment, after which they were switched back to NaPBA.
`Subjects remained on their prescribed amount of dietary pro-
`tein throughout the study, received dietary counseling, were in-
`structed to record their diet for at least 3 days prior to each visit
`and were queried at the end of the study with respect to their pref-
`erence for NaPBA or GPB. Compliance was assessed by monitoring
`drug accountability records and inspection of the returned bottles
`and vials. Safety was assessed through standard safety laboratory
`tests, physical exams, serial triplicate ECG, and collection of ad-
`verse events. Efficacy was assessed by serial measurement of ve-
`nous ammonia. An independent Data and Safety Monitoring
`Board (DSMB) was chartered to oversee the conduct of the study
`and an interim analysis of safety, ammonia, and PK data was
`planned after 3 subjects completed the study.
`
`Pharmacokinetic and ammonia sampling
`
`Blood samples for analysis of intact GPB, for NaPBA and GPB
`metabolites including PBA, PAA, PAGN, phenylacetyl glycine
`(PAG), phenylbutyryl glycine (PBG) and phenylbutyryl glutamine
`(PBGN), as well as for venous ammonia were collected on the last
`day of dosing with either NaPBA or GPB at the following time
`points: at pre-first dose and at 30 min and 1, 2, 4, 5, 6, 8, 10, 12,
`and 24 h post-first dose. Urine was collected and analyzed for these
`same drug metabolites and collected in aliquots of 0–6 h (begin-
`ning with the time of the first dose of the day), 6–12 h and 12–24 h.
`
`Pharmacokinetic, pharmacodynamic and statistical analyses
`
`PK parameters for PBA, PAA, and PAGN in plasma, PAGN in ur-
`ine, as well as pharmacodynamic parameters for venous ammonia
`were calculated with non-compartmental methods using a vali-
`dated version of WinNonlin Enterprise (version 5.2). Individual
`plasma concentrations, urinary amounts and volumes were sum-
`marized with descriptive statistics (e.g. number of patients [n],
`mean, standard deviation [SD], median, minimum, and maximum).
`The following plasma PK parameters were calculated for PBA,
`PAA and PAGN using actual time–concentration profiles for each
`subject: area under the concentration versus time curve from time
`0 (pre-dose) to 24 h, calculated using the linear trapezoid rule
`steady state
`(AUC0–24), maximum plasma concentration at
`(Cmaxss), minimum plasma concentration at steady state (Cminss),
`time maximum plasma concentration at steady state (Tmaxss), and
`apparent clearance at steady state (CLss/F) (calculated as Dose/
`AUC0–24). The terminal elimination half-life of PBA and PAA could
`not be calculated due to the limited number of samples available
`after the last dose of GPB and NaPBA. The amount of PAGN
`excreted in urine over 24 h was calculated from urinary concentra-
`tion (by multiplying the urinary volume with urinary concentra-
`tions). The time-normalized area under the curve (TNAUC) and
`Cmaxss were calculated for venous ammonia, a pharmacodynamic
`marker. TNAUC was calculated as the AUC divided by the time
`spanned by the actual sampling period.
`Ammonia TNAUC and urinary excretion of PAGN were assessed
`using an ANOVA model with 90% CI for the difference in the means.
`The 90% CI were constructed from the analysis of variance in the
`logarithmic scale and back-transformed to the original scale. In-
`tra-patient coefficient of variability for PK and PD parameters were
`derived from the ANOVA model. Statistical analyses were per-
`formed using the LinMix module in WinNonlin Enterprise (version
`5.2). Correlates of blood ammonia were determined using Spear-
`man rank-order correlations. Measurement of total urinary nitro-
`gen (TUN) was performed by Elementar Rapid NIII Analyzer
`(Mayo Medical Laboratories, Rochester, MN) applying Dumas
`
`
`
`B. Lee et al. / Molecular Genetics and Metabolism 100 (2010) 221–228
`
`method of combustion [10] on frozen 24-h urine samples obtained
`after 7 days of treatment with NaPBA and GPB.
`
`Results
`
`Patient demographics and disposition
`
`A total of 13 subjects with a mean age of 37 (range 21–73) en-
`rolled in the study and 10 subjects (4 males and 6 females) com-
`pleted all the protocol defined study procedures (Table 1). One
`subject had an episode of hyperammonemia before switching to
`GPB. This subject was withdrawn from the study until stable and
`later re-entered and ultimately completed the study. One subject
`withdrew consent before transitioning to GPB and two other sub-
`jects were discontinued at the discretion of the investigators before
`receiving either study drug. One subject each had argininosucci-
`nate synthetase (ASS), and ornithine translocase (HHH) deficiency;
`the remaining subjects had ornithine transcarbamylase (OTC) defi-
`ciencies. Three subjects had neonatal or infantile onset, and all oth-
`ers had either childhood or adult onset UCD. Among the 10
`subjects who completed the study, NaPBA had been prescribed
`for an average (SD) of 9.04 (7.96) years at an average (SD) dose
`to 7.54 g/m2
`(1.65)
`of 191 (44.6) mg/kg/day, equivalent
`(range = 4.47–9.10 g/m2, 2 subjects were taking 20 g/day). Eight
`of the 10 subjects who completed the study were being prescribed
`NaPBA at doses below the recommended range of 9.9–13 g/m2
`(BUPHENYL PI). All but 1 subject switched from 100% NaPBA to
`
`Table 1
`Patients demographics.
`
`Gender [n (%)]
`Male
`Female
`
`Age (years) at screening
`Mean (SD)
`
`Height (cm)
`Mean (SD)
`
`Weight (kg)
`Mean (SD)
`
`UCD Diagnosis [n (%)]
`OTC Deficiencya
`ASS Deficiencyb
`HHH Syndromec
`
`Patients completing
`the study (N = 10)
`
`4 (40.0)
`6 (60.0)
`
`38.2 (17.85)
`
`165.6 (7.88)
`
`80.41 (31.647)
`
`223
`100% GPB in a single step, and 1 subject received 25% less GPB
`than the PBA molar equivalent of NaPBA due to dose calculation er-
`ror. Compliance with treatment was excellent; 99% of all sched-
`uled doses of either NaPBA or GPB were in fact taken based on
`monitoring of vials and bottles.
`
`Safety and tolerability
`
`A total of 21 AEs were reported for 7 subjects during 100% NaP-
`BA dosing as compared with 15 AEs for 5 subjects during 100% GPB
`dosing. Most AEs were categorized as mild (19/21 AEs during 100%
`NaPBA treatment and 13/15 AEs during 100% GPB treatment) (Ta-
`ble 2). During 100% NaPBA treatment, one AE (mental status
`
`Table 2
`Summary of treatment–emergent adverse eventsa.
`
`Adverse event term
`
`NaPBA
`N = 13
`
`Glycerol
`Phenylbutyrate
`N = 10
`
`All
`
`Related All
`
`Related
`
`Any AE (number of subjects)
`
`21 (7)
`
`Gastrointestinal disorders
`Nausea
`Dyspepsia
`Abdominal pain
`Gastro-oesophageal reflux disease
`Abdominal distension
`Abnormal faeces
`Constipation
`Diarrhoea
`Dry mouth
`Flatulence
`
`Metabolism and nutrition disorders
`Increased appetite
`Hyperammonaemia
`Dehydration
`
`Nervous system disorders
`Clonus
`Dizziness
`Dysgeusia
`Encephalopathy
`Nystagmus
`Tremor
`
`General disorders and
`administration site conditions
`Chills
`Hunger
`
`7 (3)
`2
`1
`2
`1
`0
`0
`0
`1
`0
`0
`
`3 (2)
`1
`1
`1
`
`6 (3)
`1
`1
`1
`1
`1
`1
`
`1 (1)
`
`1
`0
`
`6 (5)
`
`2 (2)
`0
`1
`0
`1
`0
`0
`0
`0
`0
`0
`
`1 (1)
`1
`0
`0
`
`2 (2)
`0
`1
`1
`0
`0
`0
`
`1 (1)
`
`1
`0
`
`15 (5)
`
`11 (5)
`
`5 (2)
`0
`0
`0
`0
`1
`1
`1
`0
`1
`1
`
`3 (3)
`3
`0
`0
`
`0
`0
`0
`0
`0
`0
`0
`
`5 (2)
`0
`0
`0
`0
`1
`1
`1
`0
`1
`1
`
`3 (3)
`3
`0
`0
`
`0
`0
`0
`0
`0
`0
`0
`
`1 (1)
`
`1 (1)
`
`0
`1
`
`0
`1
`
`8 (80.0)
`1 (10.0)
`1 (10.0)
`
`1 (10.0)
`2 (20.0)
`7 (70.0)
`
`9.04 (7.966)
`8.50
`0.0, 25.0
`
`3 (30.0)
`7 (70.0)
`
`190.79 (44.641)
`187.50
`144.0, 298.0
`
`0.55 (0.146)
`0.60
`0.3, 0.8
`6 (60%)
`
`UCD Onset [n (%)]
`Neonatal (0–630 days)
`Infantile (>30 days–62 years)
`Childhood or adult onset (>2 years)
`
`Duration of NaPBA Treatment (years)
`Mean (SD)
`Median
`Min, Max
`
`Type of NaPBA [n (%)]
`Powder
`Tablets
`
`NaPBA daily dose (mg/kg/day)
`Mean (SD)
`Median
`Min, max
`
`Average Protein intake during study (mg/kg/day)
`Mean (SD)
`Median
`Min, max
`Percentage of patients treated with L-citrolline
`
`a Ornithine transcarbamylase deficiency.
`b Arginosuccinate synthetase deficiency.
`c Ornithine translocase deficiency.
`
`Infections and infestations
`Herpes simplex
`
`Psychiatric disorders
`Food aversion
`Mental status change
`
`Respiratory, thoracic
`and mediastinal disorders
`Pharynogolaryngeal pain
`Cough
`Rhinorrhoeas
`
`Skin and subcutaneous
`tissue disorders
`Skin odour abnormal
`
`Investigations
`Weight increased
`
`Musculoskeletal and
`connective tissue disorders
`Back pain
`
`0
`0
`
`2 (2)
`1
`1
`
`0
`
`0
`0
`0
`
`1 (1)
`
`1
`
`0
`0
`
`1 (1)
`
`1
`
`0
`0
`
`0
`0
`0
`
`0
`
`0
`0
`0
`
`0
`
`0
`
`0
`0
`
`0
`
`0
`
`1 (1)
`1
`
`0
`0
`0
`
`0
`0
`
`0
`0
`0
`
`4 (2)
`
`1 (1)
`
`2
`1
`1
`
`0
`
`0
`
`1
`0
`0
`
`0
`
`0
`
`1 (1)
`1
`
`1 (1)
`1
`
`0
`
`0
`
`0
`
`0
`
`Source: UP 1204–003 Summary Tables 14.3.1 and 14.3.3.
`a Table reflects number of events and events reported during 7 days of NaPBA
`(sodium phenylbutyrate) prior to transition to glycerol phenylbutyrate, and 7 days
`of sole glycerol phenylbutyrate treatment after completion of transition from
`NaPBA treatment.
`
`
`
`224
`
`B. Lee et al. / Molecular Genetics and Metabolism 100 (2010) 221–228
`
`change) was considered moderate. During 100% GPB treatment,
`one subject with history of irritable bowel disease reported an
`AE (abdominal distension) that was considered severe and one
`AE (flatulence) that was considered moderate; both resolved with-
`out specific treatment. Two subjects experienced SAEs of hyperam-
`monemia while receiving NaPBA, one occurred before the subject
`began receiving GPB and one occurred 21 days after the subject
`had completed dosing with GPB and had switched back to NaPBA.
`Both were categorized as severe. There were no episodes of hyper-
`ammonemia on GPB.
`
`Pharmacokinetic and pharmacodynamic analyses
`
`All 10 patients who completed the study were considered
`evaluable for the PK analyses. Plasma PK parameters of PBA, PAA
`and PAGN and urinary PK parameters of PAGN are summarized
`in Table 3 and the 24-h concentration profiles are depicted in
`Fig. 1. Systemic exposure (AUC0–24) to PBA following GPB adminis-
`tration was 27% lower than that observed with NaPBA (540 vs.
`739 lg h/mL, respectively), whereas exposure levels of PAA (575
`vs. 596 lg h/mL, respectively) and PAGN (1098 vs. 1133 lg h/mL,
`respectively) were similar. PAG, PBG, and PBGN were not detect-
`able in plasma for either drug.
`The total amount of PAGN excreted in urine over 24 h following
`GPB treatment was slightly lower than that observed for NaPBA,
`but PAGN accounted for 54% of PBA delivered by both drugs (Ta-
`
`Table 3
`PK parameters and ammonia following NaPBA and glycerol phenylbutyrate
`administration.
`
`PK/PD parameters
`
`Arithmetic mean (CV%)
`
`PBA in plasma
`AUC0–24 (lg h/mL)
`Cmaxss (lg/mL)
`Cminss (lg/mL)
`PAA in plasma
`AUC0–24 (lg h/mL)
`Cmaxss (lg/mL)
`Cminss (lg/mL)
`PAGN in plasma
`AUC0–24 (lg h/mL)
`Cmaxss (lg/mL)
`Cminss (lg/mL)
`PAGN in urine
`Total excreted 0–24 h (lg)***
`0–6 h (lg)
`6–12 h (lg)
`12–24 h (lg)***
`Recovery of PBA as PAGN (%)
`
`Total urinary nitrogen in 24 h
`Mean (SD) g
`
`Ammonia
`TNAUC (lmol/L)
`Cmaxss (lmol/L)
`% normal ammonia values+
`
`Glycerol
`phenylbutyrate
`(n = 10)
`
`NaPBA (n = 10)
`
`540 (60.2)*
`70.1 (64.7)
`2.87 (265)
`
`575 (169)*
`40.5 (147)
`7.06 (310)
`
`740 (49.1)*
`141 (44.3)
`0.588 (255)
`
`596 (124)*
`53.0 (94.7)
`3.56 (194)
`
`1098 (44.2) *
`71.9 (55.9)
`12.1 (134)
`
`1133 (31.0)***
`83.3 (25.8)
`16.8 (86.3)
`
`10 784 747 (25.9)
`2381371 (61.3)
`3027310 (44.9)
`5433033 (50.4)
`54 (15)
`
`12 153 473 (48.2)
`2452838 (41.6)
`4859121 (54.7)
`4645447 (59.8)
`54 (16)
`
`9.0 (3.0)**
`
`9.6 (3.9)**
`
`26.2 (38.9)
`56.3 (49.5)
`59.5 (34.04)
`
`38.4 (51.0)
`79.1 (50.6)
`73.1 (27.04)
`
`Mean ammonia ratio
`(Glycerol phenylbutyrate /NaPBA)
`95% CI of ratio
`
`0.71
`0.44–1.14
`
`AUC0–24, area under the concentration from time 0 (pre-dose) to 24 h; Cmaxss,
`maximum plasma concentration at steady state; Cminss, minimum plasma con-
`centration at steady state; TNAUC, time-normalized area under the curve.
`+ % Normal ammonia values are presented as mean (SD).
`* n = 8.
`** n = 7.
`*** n = 9.
`
`ble 3). Peak urinary PAGN excretion for NaPBA occurred from 6–
`12 h after the first dose of the day as compared with 12–24 h for
`glycerol phenylbutyrate. Urinary PBA, PAA, PAG, PBG and PBGN
`each accounted for less than 1% of PBA administered. Total 24-h
`creatinine excretion after treatment with NaPBA or glycerol phen-
`ylbutyrate was similar with means (SD) of 1.08 (0.43) grams and
`1.03 (0.38) grams, respectively. The mean (SD) total urinary nitro-
`gen after treatment with NaPBA and GPB was similar, 9.6 (3.9) g
`and 9.0 (3.0) g, respectively.
`Mean (SD) glutamine levels (lmol/dL) in the 8 patients for
`whom measurements on both drugs were available were some-
`what higher on NaPBA as compared with GPB [739(294) vs.
`653(313)]; mean decrease = 86.6 (122); (p > 0.05).
`Blood ammonia values among all patients varied widely on both
`NaPBA (range 2–150 lmol/L; n = 101 values total) and on GPB
`(range 2–106 lmol/L, n = 99 total values) and also varied widely
`for any given patient on a single day (2.4- to 54-fold variation on
`NaPBA; average = 10.4-fold; 2.4- to 12.3-fold variation on GPB;
`average = 5.4-fold). Mean ammonia values were lower on GPB than
`on NaPBA when assessed as TNAUC (32% lower: 26.2 vs. 38.4 lmol/
`L, respectively) and Cmaxss (29% lower: 56.3 vs. 79.1 lmol/L,
`respectively) (Table 3). Mean ammonia TNAUC values for individ-
`ual subjects are depicted in Fig. 4; 27.0% of the ammonia values ob-
`tained while on GPB were above the upper limit of normal for
`ammonia at their respective study site (upper limit of normal ran-
`ged from 26–35 lmol/L at the four sites), as compared with 39.6%
`while on NaPBA (Fig. 3). These differences were attributable to
`lower ‘overnight’ ammonia levels (12–24 h) and did not reach sta-
`tistical significance (Fig. 2). A post hoc analysis indicated non-infe-
`riority of GPB in controlling ammonia compared to NaPBA with
`respect to TNAUC using standard non-inferiority methodology
`and the conventional 1.25 upper boundary for the 95% CI. The ratio
`of the least square geometric means (GPB/NaPBA) was 0.71 with a
`95% CI of 0.44–1.14.
`
`Correlates of blood ammonia
`
`Blood ammonia assessed as TNAUC correlated strongly and in-
`versely with 24-h urinary PAGN (r = 0.80; p < 0.0001) following
`administration of both NaPBA and GPB (Table 4 and Fig. 4); corre-
`lation with urinary PAGN output from 12–24 h was also significant
`(r = 0.75; p = 0.001). Blood ammonia did not correlate with AUC0-
`24 for either plasma PBA or PAA levels in blood for either drug and
`correlated weakly with plasma PAGN (r = 0.52; p = 0.04) (Table
`4). Urinary PAGN excretion (r = 0.71; p = 0.001) and venous ammo-
`nia (r = 0.55; p = 0.02) were also significantly correlated with the
`dose administered.
`
`Discussion
`
`GPB was well tolerated and no clinically important safety issues
`were identified. Hyperammonemic events requiring hospitaliza-
`tion and recorded as serious adverse events occurred in 2 subjects
`receiving NaPBA and were determined by the investigators to be
`due to non-compliance with medication.
`The PK characteristics of NaPBA and GPB in plasma were gener-
`ally similar, with the exception of PBA. The lower plasma levels of
`PBA in subjects on GPB treatment as compared to NaPBA may re-
`flect differences in the fractional conversion of PBA to PAA and
`PAGN for the two drugs prior to reaching the systemic circulation.
`This would be consistent with the 60% slower absorption of PBA
`when delivered as GPB vs. NaPBA, presumably because PBA is grad-
`ually released from GPB by pancreatic lipases as it passes through
`the gastrointestinal tract, which would allow more time for intra-
`
`
`
`B. Lee et al. / Molecular Genetics and Metabolism 100 (2010) 221–228
`
`225
`
`1 Week GPB (Visit 11-1) Day 1
`Last Day of NaPBA (Visit 2-1) Day 1
`LOQ = 1 μg/mL
`
`0
`
`2
`
`4
`
`6
`
`8
`
`10
`
`12
`
`14
`
`16
`
`18
`
`20
`
`22
`
`24
`
`Nominal Time (h)
`
`1 Week GPB (Visit 11-1) Day 1
`Last Day of NaPBA (Visit 2-1) Day 1
`LOQ = 1 μg/mL
`
`0
`
`2
`
`4
`
`6
`
`8
`
`10
`
`12
`
`14
`
`16
`
`18
`
`20
`
`22
`
`24
`
`Nominal Time (h)
`
`1 Week GPB (Visit 11-1) Day 1
`Last Day of NaPBA (Visit 2-1) Day 1
`LOQ = 1 μg/mL
`
`A
`
`200
`
`150
`
`100
`
`50
`
`0
`
`Mean (SD) Plasma PBA Concentration (μg/mL)
`
`B
`
`120
`
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`140
`
`120
`
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`Mean (SD) Plasma PAA Concentration (μg/mL)
`
`C
`
`Mean (SD) Plasma PAGN Concentration (μg/mL)
`
`0
`
`2
`
`4
`
`6
`
`8
`
`10
`
`12
`
`14
`
`16
`
`18
`
`20
`
`22
`
`24
`
`Nominal Time (h)
`
`Fig. 1. (A) Plasma phenylbutyric acid (PBA), (B) phenylacetic acid (PAA) and (C) phenylacetylglutamine (PAGN) were measured for 24 h following one week of dosing
`with either sodium phenylbutyrate (NaPBA) or glycerol phenylbutyrate (GPB) and are displayed as means ± SD. Times 0 and 24 h correspond to just prior to dosing and
`breakfast.
`
`hepatic / first pass conversion. PAG, PBG and PBGN were not mea-
`surable in blood.
`
`PAGN was the major urinary metabolite, with negligible
`amounts of PAA, PBA, PAG, PBG and PBGN (<1% of PBA dose for
`
`
`
`226
`
`B. Lee et al. / Molecular Genetics and Metabolism 100 (2010) 221–228
`
`1 Week GPB (Visit 11-1) Day 1
`Last Day of NaPBA (Visit 2-1) Day 1
`LOQ = 1 μmol/L
`
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`Mean (SD) Plasma Ammonia Concentration (μmol/L)
`
`0
`
`2
`
`4
`
`6
`
`8
`
`10
`12
`14
`Nominal Time (h)
`
`16
`
`18
`
`20
`
`22
`
`24
`
`Fig. 2. Venous ammonia was measured for 24 h following one week of dosing with either sodium phenylbutyrate (NaPBA) or glycerol phenylbutyrate (GPB) and is displayed
`as mean ± SD. Times 0 and 24 h correspond to just prior to dosing and breakfast.
`
`NH3
`
` NH3
`
`70
`60
`50
`40
`30
`20
`10
`0
`
`TNAUC NH3
`
`
`2978370
` U-PAGN
`
`9012846
`9750522
`7660091
`55473485921398
`9894241
`10757468
`12904931
`10041086
`12012321
`11512426
`13972797
`13745999
`11583483
`7539260
`6332433
`
`Fig. 4. Relationship between blood ammonia and urinary output of phenylacetyl-
`glutamine (PAGN). Blood ammonia assessed as time-normalized area under the
`curve (Y-axis) correlated inversely (r = 0.80; p < 0.001) with urinary PAGN output
`(X-axis). One subject was excluded from this post hoc analysis since TNAUC was
`calculable for only 6 h during treatment with GPB.
`
`nia values on NaPBA exceeded mean upper limit of normal for the
`study site laboratories and correlated inversely with NaPBA dose. A
`correlation with dose would not be expected in optimally managed
`patients if the target of management is normal ammonia values. It
`is interesting in this regard that 8 of 10 patients were being pre-
`scribed lower NaPBA doses than currently recommended in the ap-
`proved product labeling. Increasing dose in these subjects to
`within the labeled range (BUPHENYL package insert) might im-
`prove ammonia control and, considering the high proportion of
`UCD patients with self-reported neurological disability, potentially
`improve neurological outcome [3].
`Because of
`its clinical
`importance, additional correlates of
`ammonia control were sought with particular attention to clini-
`cally useful biomarkers. As compared with plasma PBA, PAA, or
`PAGN assessed at 24-h AUC, with which ammonia showed absent
`or weak correlation, blood ammonia measured as TNAUC corre-
`lated strongly and inversely with UPAGN (Table 4). This is consis-
`tent with the fact that urinary PAGN output is stoichiometrically
`related to waste nitrogen excretion and suggests that urinary
`PAGN may be useful in dose selection and adjustment.
`In his pioneering studies, Brusilow outlined the theoretical basis
`for the relationship between dietary protein intake and PAGN
`excretion [5]. He pointed out that 18 g of PAA, if completely con-
`verted to PAGN, should mediate excretion of 3.23 g of waste nitro-
`
` Ammonia (umol/L)
`80
`
`70
`
`60
`
`50
`
`40
`
`30
`
`20
`
`10
`
`0
`
`Mean
`
`Average Upper Limit of Normal
`
`ABPaN
`
`BPG
`
`Fig. 3. Venous ammonia in individual subjects following one week of dosing with
`either sodium phenylbutyrate (NaPBA; left) or glycerol phenylbutyrate (GPB; right).
`The values shown represent time-normalized area under the curve and are
`displayed as means ± SD. Times 0 and 24 h correspond to just prior to dosing and
`breakfast.
`
`each) excreted in urine. 24-h PAGN output was similar after NaPBA
`and GPB administration and accounted for 54% of the adminis-
`tered PBA dose for both drugs.
`Blood ammonia levels (TNAUC) were lower on GPB as compared
`with NaPBA. Although these differences did not achieve statistical
`significance, a post hoc analysis indicated non-inferiority of GPB as
`compared with NaPBA with respect to venous ammonia assessed
`as TNAUC, a preliminary finding that needs to be confirmed in a
`larger number of patients. This difference in ammonia was largely
`attributable to lower values between 12 and 24 h, a finding consis-
`tent with the delayed peak in urinary PAGN output following glyc-
`erol phenylbutyrate (12–24 h) as compared with NaPBA (6–12 h),
`which presumably reflects the delayed release characteristics of
`GPB. As for ammonia, glutamine levels, which have been shown
`to correlate with clinical symptoms [3], also tended to be higher
`on NaPBA than on GPB, although this difference did not reach sta-
`tistical significance. Collectively, these preliminary findings sug-
`gest that GPB is at least equivalent to NaPBA with respect to
`clearance of waste nitrogen and control of blood ammonia.
`Blood ammonia values varied widely both between patients
`and for the same patient on a given day. The average of all ammo-
`
`
`
`B. Lee et al. / Molecular Genetics and Metabolism 100 (2010) 221–228
`
`227
`
`Table 4
`Correlation between ammonia and plasma PAA, PBA and PAGN, and urinary PAGN (UPAGN)a,b.
`
`Plasma PAA
`
`Plasma PBA
`
`Plasma PAGN
`
`N
`R
`p
`
`15
` 0.23
`NS
`
`15
`0.08
`NS
`
`16
` 0.52
`0.04
`
`UPAGN
`
`18
` 0.80
`<0.0001
`
`Dose
`
`18
` 0.55
`0.02
`
`U-PAGN 12–24
`
`16
` 0.75
`<0.001
`
`Data from both NaPBA and glycerol phenylbutyrate were included in the analysis.
`Data from one subject with more than 50% missing data on glycerol phenylbutyrate were excluded.
`NS = not significant at a = 0.05.
`a Ammonia was measured as time-normalized area under the curve (TNAUC).
`b Spearman rank–order correlation.
`
`gen, an amount sufficient to completely replace urea nitrogen as a
`vehicle for waste nitrogen excretion in subjects receiving a low
`protein diet [1]. In support of this prediction, Brusilow, in two sep-
`arate studies, administered NaPBA or sodium phenylacetate to a
`7½-year-old male with carbamyl phosphate synthetase deficiency
`and a 38 year old male with ornithine transcarbamylase deficiency
`and reported that 80–90% and 92%, respectively, of the PAA admin-
`istered was excreted as urinary PAGN [5,11]. Furthermore, the
`FDA-approved label for NaPBA, currently marketed as BUPHENYLÒ
`(sodium phenylbutyrate), states that ‘‘A majority of the adminis-
`tered compound (approximately 80–100%) was excreted by the
`kidneys within 24 h as the conjugation product, phenylacetylgluta-
`mine. . .[corresponding to]. . . 0.12–0.15 g of phenylactylglutamine
`nitrogen. . .” [12].
`In the current study 54% of administered PBA was excreted as
`PAGN following the administration of either NaPBA or GPB. This
`lower fractional conversion to PAGN corresponds to a lower ‘cover-
`age’ of dietary protein per gram of NaPBA. Specifically, 1 g of PBA
`would be expected to mediate excretion of waste nitrogen derived
`from 2.4 g of dietary protein if completely converted to PAGN,
`but only 1.4 g of dietary protein at 60% conversion, assuming that
`nitrogen comprises 16% of dietary protein and that 47% of die-
`tary protein is excreted as waste nitrogen [5]. It is known that sec-
`ondary metabolites can be excreted after NaPBA treatment
`including glucuronides and phenylbutyrate beta-oxidation side
`products [13].
`The results further suggest that urinary PAGN output may be
`useful for dose adjustment. Individual ammonia values varied on
`average more than 7-fold over 24 h, even in the context of a con-
`trolled clinical study, and 24 h monitoring of ammonia (TNAUC)
`as performed in the present study are clinically impractical. Urine
`collections, by contrast, are routinely performed. Across both treat-
`ment periods, 24-h PAGN excretion was less than 10 g, which cor-
`responds to a NaPBA dose of 12 g assuming 54% conversion, 9
`times. In seven of these instances (77.8%), ammonia TN-AUC ex-
`ceeded 30 lmol/L (approximate average upper limit of normal
`amo