Molecular Genetics and Metabolism 103 (2011) 323–329
`
`Contents lists available at ScienceDirect
`
`Molecular Genetics and Metabolism
`
`j ou r n a l h o m e pa g e : ww w. e l s ev i e r. c o m/ l o c a t e / y mg m e
`
`Ammonia control in children with urea cycle disorders (UCDs); Phase 2 comparison
`of sodium phenylbutyrate and glycerol phenylbutyrate☆
`Uta Lichter-Konecki a,⁎, G.A. Diaz b, J.L. Merritt II c, A. Feigenbaum d, C. Jomphe e, J.F. Marier e, M. Beliveau e,
`J. Mauney f, K. Dickinson g, A. Martinez g, M. Mokhtarani g, B. Scharschmidt g, W. Rhead h
`a Children's National Medical Center, 111 Michigan Ave NW, Washington, DC, 20010, USA
`b Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY, USA
`c Seattle Children's Hospital, 4800 Sand Point Way NE, Seattle, WA, USA
`d The Hospital for Sick Children, 555 University Avenue, Toronto Ontario, Canada
`e Pharsight, Montreal, 2000 Peel St., Suite 570, Quebec, Canada
`f Chiltern, 2520 Independence Blvd., Ste. 202, Wilmington NC, USA
`g Hyperion Therapeutics, Inc., 601 Gateway Blvd., Ste. 200, South San Francisco, CA, USA
`h Pediatrics, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI, USA
`
`a r t i c l e
`
`i n f o
`
`a b s t r a c t
`
`Article history:
`Received 17 February 2011
`Received in revised form 21 April 2011
`Accepted 21 April 2011
`Available online 5 May 2011
`
`Keywords:
`Urea cycle disorders
`Ammonia scavengers
`Hyperammonemia
`Phenylacetate
`Sodium phenylbutyrate
`Glycerol phenylbutyrate
`
`Twenty four hour ammonia profiles and correlates of drug effect were examined in a phase 2 comparison of
`sodium phenylbutyrate (NaPBA) and glycerol phenylbutyrate (GPB or HPN-100), an investigational drug
`being developed for urea cycle disorders (UCDs).
`Study Design: Protocol HPN-100-005 involved open label fixed-sequence switch-over from the prescribed
`NaPBA dose to a PBA-equimolar GPB dose with controlled diet. After 7 days on NaPBA or GPB, subjects
`underwent 24-hour blood sampling for ammonia and drug metabolite levels as well as measurement of
`24-hour urinary phenyacetylglutamine (PAGN). Adverse events (AEs), safety labs and triplicate ECGs
`were monitored.
`Results: Eleven subjects (9 OTC, 1 ASS, 1 ASL) enrolled and completed the switch-over from NaPBA (mean
`dose=12.4 g/d or 322 mg/kg/d; range=198–476 mg/kg/d) to GPB (mean dose=10.8 mL or 0.284 mL/kg/d
`or 313 mg/kg/d; range = 192–449 mg/kg/d). Possibly-related AEs were reported in 2 subjects on NaPBA
`and 4 subjects on GPB. All were mild, except for one moderate AE of vomiting on GPB related to an
`intercurrent illness. No clinically significant laboratory or ECG changes were observed. Ammonia was
`lowest after overnight fast, peaked postprandially in the afternoon to early evening and varied widely over
`24 h with occasional valuesN 100 μmol/L without symptoms. Ammonia values were ~25% lower on GPB vs.
`NaPBA (p≥ 0.1 for ITT and p b 0.05 for per protocol population). The upper 95% confidence interval for the
`difference between ammonia on GPB vs. NaPBA in the ITT population (95% CI 0.575, 1.061; p = 0.102) was
`less than the predefined non-inferiority margin of 1.25 and less than 1.0 in the pre-defined per-protocol
`population (95% CI 0.516, 0.958; p b0.05). No statistically significant differences were observed in plasma
`phenylacetic acid and PAGN exposure during dosing with GPB vs. NaPBA, and the percentage of orally
`administered PBA excreted as PAGN (66% for GPB vs. 69% for NaPBA) was very similar. GPB and NaPBA dose
`correlated best with urinary-PAGN.
`Conclusions: These findings suggest that GPB is at least equivalent to NaPBA in terms of ammonia control,
`has potential utility in pediatric UCD patients and that U-PAGN is a clinically useful biomarker for dose
`selection and monitoring.
`
`© 2011 Elsevier Inc. All rights reserved.
`
`Abbreviations: ASL, argininosuccinate lyase deficiency; ASS, argininosuccinate synthetase deficiency; AUC0–24, 24 h area under the curve; CV%, coefficient of variation; DSMB,
`Data Safety and Monitoring Board; GPB, glycerol phenylbutyrate (generic name for glyceryl tri (4-phenylbutyrate), also referred to as HPN-100); ITT, intention to treat; NaPBA,
`sodium phenylbutyrate; NH324-hour AUC, ammonia 24-hour area under the curve; OTC, ornithine transcarbamylase deficiency; PAA, phenylacetic acid; PAGN, phenylacetylglutamine;
`PBA, phenylbutyric acid; PK, pharmacokinetic; UCD, urea cycle disorder; ULN, upper limit of normal; U-PAGN24-hour Excr, PAGN excreted in urine over 24 h.
`☆ ClinicalTrials.gov identifier: NCT00947544.
`⁎ Corresponding author at: Department of Pediatrics, George Washington Univ. Medical Center, Division of Genetics and Metabolism, Children's National Medical Center,
`111 Michigan Ave NW, Washington, DC 20010, USA.
`E-mail address: ulichter@cnmc.org (U. Lichter-Konecki).
`
`1096-7192/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
`doi:10.1016/j.ymgme.2011.04.013
`
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`1. Introduction
`
`U. Lichter-Konecki et al. / Molecular Genetics and Metabolism 103 (2011) 323–329
`
`Urea cycle disorders (UCDs), which comprise several inherited
`enzyme and transporter deficiencies, result in the accumulation of toxic
`levels of ammonia in the blood and brain and can present in the neonatal
`period or later in life depending on the severity and type of defect [1–3].
`Control of hyperammonemia, the major cause of morbidity and
`mortality in UCD patients, is a major objective of treatment [4,5].
`Batshaw, Brusilow and coworkers introduced the concept of
`alternative pathway pharmacological therapy for UCDs [4,6,7]. These
`pioneering studies led to the availability of sodium phenylbutyrate
`(NaPBA), which is approved in the US (trade name: BUPHENYL®)
`(sodium phenylbutyrate) Powder and Tablets) and Europe (trade
`name: AMMONAPS®) for the chronic treatment of UCDs involving
`deficiencies of carbamylphosphate synthetase (CPS), ornithine trans-
`carbamylase (OTC), or argininosuccinic acid synthetase (AS) and
`lowers ammonia by enhancing excretion of waste nitrogen in the
`form of phenylacetylglutamine (PAGN). Despite the fact that
`alternative pathway therapy with NaPBA has been utilized for over
`two decades, there are little systematically collected data available
`pertaining to fasting and postprandial ammonia levels or correlates of
`drug effect in pediatric patients [8]. GPB is an investigational agent
`being developed for UCDs. Like NaPBA, it contains phenylbutyric acid
`(PBA), a pro-drug that is converted via β-oxidation to the active
`moiety, phenylacetic acid (PAA), which combines with glutamine to
`form PAGN that is excreted in the urine. However, unlike NaPBA
`which is a salt, GPB is a pre-pro-drug that contains no sodium
`and consists of three molecules of PBA joined to glycerol in ester
`linkage. It is hydrolyzed in the small intestine by pancreatic lipases to
`release PBA and glycerol, PBA is absorbed more slowly than when
`administered as NaPBA, and the glycerol is presumably digested
`like dietary glycerol consumed in the form of long chain triglycerides
`[8–10].
`
`2. Materials and methods
`
`2.1. Study design and treatments
`
`This phase 2, open-label, fixed sequence, switch-over study enrolled
`pediatric patients ages 6 or above being treated with NaPBA for a UCD
`(confirmed via enzymatic, biochemical or genetic testing). Major
`exclusion criteria included liver transplant, hypersensitivity to PBA,
`PAA or PAGN, clinically significant laboratory abnormalities or ECG
`findings, or conditions or medications that could affect ammonia levels.
`Enrolled subjects received NaPBA for at least 7 days, three times
`daily with meals at the dose level prescribed by the investigator. On the
`last day of NaPBA treatment they were admitted to a study unit and
`underwent 24-hour blood sampling for pharmacokinetic (PK) and
`ammonia measurements. Subjects were then switched to GPB at the
`PBA molar equivalent of their prescribed NaPBA dose. Initiation of GPB
`dosing was done under observation in an appropriately monitored
`setting and subjects were discharged after they were deemed clinically
`stable. After 7 days of treatment with GPB subjects were re-admitted to
`the research unit for 24-hour PK and ammonia monitoring, after which
`they were offered enrollment into a long-term extension study. Only the
`results of the switch-over part of the protocol are reported here.
`Subjects received dietary counseling and remained on their
`prescribed amount of dietary protein throughout the study. Diet
`was carefully monitored on study days 7 and 14 during the 24-hour
`blood sampling as well as for at least 3 days prior to each visit via
`diaries. Subjects also were queried at the end of the study with respect
`to their preference for NaPBA or GPB. Compliance with study drug was
`assessed by daily recording of missed doses by the subject and
`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 adverse events. Efficacy was assessed by serial measurement of
`venous 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 conducted after
`6 subjects completed the study.
`
`2.2. Biochemical analyses
`
`NaPBA and GPB metabolites including PBA, PAA, and PAGN were
`measured by validated liquid chromatography tandem mass spec-
`trometry methods at the bioanalytical
`laboratory, Quest Pharma
`Services. Venous ammonia was measured by the accredited hospital
`laboratory at each site; plasma amino acids were measured by Baylor
`Medical Genetics Laboratories.
`
`2.3. Pharmacokinetic and ammonia sampling
`
`Blood samples for analysis of venous ammonia, intact GPB, and
`NaPBA and GPB metabolites were collected on the last day of dosing
`with either NaPBA or GPB at time zero (pre dose and pre-breakfast)
`and 4, 8, 12, 16, 20 and 24 h post-first dose. Lunch and dinner typically
`were eaten after the 4 and 8 h collections, respectively. Plasma amino
`acids were collected at time 0 (fasting) on days 7 and 14. Urine was
`collected in aliquots of 0–12 h (beginning with the first dose of the
`day) and 12–24 h.
`
`2.4. Pharmacokinetic analyses
`
`Pharmacokinetic parameters of PBA, PAA and PAGN in plasma and
`urine were calculated using a validated version of WinNonlin®
`Enterprise (Version 5.2). Statistical analyses were performed using
`WinNonlin v.5.2 (LinMix Module). Plasma PK parameters, including
`mean and coefficient of variation (standard deviation [SD], expressed
`as a percentage of the mean), were calculated using actual time-
`concentration profiles for each subject and included the following:
`Area under the concentration vs. time curve from time 0 (pre-dose) to
`24 h (AUC0–24), calculated using the linear trapezoid rule, maximum
`plasma concentration at steady state (Cmaxss), minimum plasma
`concentration at steady state (Cminss), time of maximum plasma
`concentration at steady state (Tmaxss), and apparent clearance at
`steady state (CLss/F) (calculated as Dose/AUC0–24 over the bioavailable
`fraction [F]). The amount of PAGN excreted in urine over 24 h was
`calculated by multiplying urine volume with urinary concentrations.
`Summary tables and figures were generated using WinNonlin®
`AutoPilot™ (Version 1.1.1), a configurable software application that
`works with WinNonlin®, and third-party reporting tools, including
`SigmaPlot® versions 9.01 and 10.1 and Microsoft® Office Word and
`Excel 2003 and 2007.
`
`2.5. Ammonia analyses
`
`The primary efficacy endpoint for the switch over phase of the
`study was 24-hour ammonia AUC (NH324-hour AUC), calculated based
`on the sequence of ammonia concentrations outlined above. An
`imputation algorithm was prespecified in the statistical analysis plan
`to allow for calculation of NH324-hour AUC for subjects with missing
`ammonia values.
`
`2.6. Efficacy endpoints and statistical analyses
`
`The primary efficacy endpoint was predefined as comparison of
`NH324-hour AUC on the last day of NaPBA treatment with the last day of
`GPB treatment. Secondary efficacy endpoints included the maximum
`ammonia concentrations and percentage of abnormal ammonia
`values on the last day of treatment with NaPBA vs. GPB. All subjects
`who received any amount of both study medications were included in
`
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`U. Lichter-Konecki et al. / Molecular Genetics and Metabolism 103 (2011) 323–329
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`325
`
`Table 1
`Patient demographics.
`
`Patients completing the study
`(N=11)
`
`the intention-to-treat (ITT) population, which was the primary
`population for analysis of efficacy and pharmacokinetic parameters.
`A per-protocol population was also prospectively defined as all
`subjects from the ITT population who (a) exhibited ≥80% compliance
`with study medication during inpatient stays (days 7 and 14), (b) did
`not use sodium benzoate within 48 h prior to days 7 and 14,
`(c) differed with respect to protein intake on study days 7 and 14
`by ≤50%, and (d) had calculable ammonia AUC (as defined above) for
`both days 7 and 14.
`Non-inferiority analysis of GPB to NaPBA with respect to ammonia
`control was prospectively defined. An analysis of variance (ANOVA)
`model for the natural log-transformed NH324-hour AUC was constructed
`with factors for treatment as a fixed effect and subject as a random
`effect. The 90% CI for the difference between GPB and NaPBA means
`(GPB minus NaPBA) on the natural log scale was constructed using the
`least square means from the ANOVA model. The difference and lower
`and upper confidence interval of NH324-hour AUC values were
`exponentiated to express the results as geometric means, ratio of
`geometric means, and corresponding CI on the original scale. Non-
`inferiority was to be concluded if the upper bound of the 90% CI was
`less than or equal to 1.25. Correlates of drug dose were determined for
`both NaPBA and GPB using Spearman rank-order correlations. A
`superiority analysis was predefined for the per-protocol population.
`
`3. Results
`
`3.1. Patient demographics, disposition and compliance
`
`Eleven subjects (age range 6–17; 1 male and 10 females) enrolled
`and all 11 subjects completed the protocol defined study procedures.
`As defined by the DSMB Charter, enrollment was temporarily paused
`after six subjects completed the switchover portion of the study
`pending DSMB approval to complete enrollment. Subject demo-
`graphics are summarized in Table 1. One subject each had arginino-
`succinate synthetase (ASS) and arginosuccinate lyase (ASL)
`deficiency; the remaining 9 subjects had ornithine transcarbamylase
`(OTC) deficiencies. Six subjects had neonatal or infantile onset and the
`remainder had later onset UCD. NaPBA had been prescribed for an
`average (SD) of 74.7 (48.2) months at an average (SD) NaPBA dose of
`12.4 (4.4) g/d (equivalent to an average of 322 mg/kg/d or 10.2 g/m2)
`and received the PBA equivalent dose of GPB (average (SD)=10.8 mL,
`equivalent to an average dose of 0.284 mL/kg/d or 313 mg/kg/d). Five
`subjects received NaPBA tablets and 6 received NaPBA powder during
`the study, and 2 subjects who received NaPBA via an NG tube took
`GPB orally during the study. Compliance with treatment was
`excellent; N98% of all scheduled doses of either NaPBA or GPB were
`apparently taken based on monitoring of vials and bottles.
`Dietary protein prescription at baseline among the 11 subjects
`(mean, SD) was 0.75±0.29 g/kg/d (27.7±9.48 g/day). Compliance
`on study with diet was less uniform than compliance with respect to
`study drug. While two subjects showed N50% variance in protein
`intake on days 7 vs. 14 and were excluded from the per protocol
`analysis, overall total mean protein intake was very similar on NaPBA
`and GPB (0.64±0.35 g/kg/d or 24.35 g/d and 0.61±0.16 g/kg/d or
`23.98 g/d on days 7 and 14 respectively; Table 4), as was the
`distribution of dietary protein throughout the day with both GPB and
`NaPBA at breakfast (4.96±2.497 and 5.46±2.943 g, respectively),
`lunch (6.41±4.759 and 5.98±5.332 g, respectively), and dinner
`(6.74±4.653 and 5.71±5.197 g, respectively).
`
`3.2. Safety and tolerability
`
`During the switch over period of the study all reported AEs were
`categorized as mild except one episode of vomiting graded as
`moderate on GPB, which resolved and was attributed to intercurrent
`illness and deemed unrelated to study drug (Table 2). There were no
`
`1 (9.1%)
`10 (90.9%)
`
`10.2 (3.95)
`
`133.66 (16.900)
`
`41.79 (20.135)
`
`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
`ASL deficiencyc
`UCD onset [n (%)]
`Neonatal (0 – b=30 days)
`Infantile (N30 days – b=2 years)
`Childhood or adult onset (N2 years)
`Duration of NaPBA treatment (months)
`Mean (SD)
`Median
`Min, Max
`Type of NaPBA [n (%)]
`Powderd
`Tablets
`NaPBA daily dose (mg/kg/day)
`12.41 (4.392)
`Mean (SD)
`10.50
`Median
`8.0–20.0
`Min, Max
`2 (18.2%)
`Subjects with a G-tube
`Average prescribed protein intake during study (g/kg/day)
`Mean (SD)
`0.75 ±0.29
`Patients treated with L-Citrulline (%)
`9 (81.8%)
`
`9 (81.8%)
`1 (9.1%)
`1 (9.1%)
`
`3 (27.3%)
`3 (27.3%)
`5 (45.5%)
`
`74.68 (48.220)
`76.00
`0.5 - 162.0
`
`7 (63.6%)
`4 (36.4%)
`
`a Ornithine transcarbamylase deficiency.
`b Arginosuccinate synthetase deficiency.
`c Arginosuccinate lyase deficiency.
`d One patient was switched from powder to tablets for the study.
`
`episodes of hyperammonemic crisis, predefined as blood ammonia
`exceeding 100 µmol/mL plus signs or symptoms of hyperammonemia
`on either NaPBA or GPB. Each patient was asked by the investigator or
`representative to evaluate his or her drug preference on day 14 of the
`study; all 11 subjects stated a preference for GPB.
`
`3.3. Pharmacokinetic analyses
`
`All 11 patients were considered evaluable for the PK analyses, as
`were all measurable concentration values. Values below the lower limit
`of quantification were treated as zero. Individual plasma metabolite
`
`Table 2
`Adverse events 1.
`
`Preferred term
`
`NaPBA (N=11)
`
`GPB (N=11)
`
`Number of subjects with at least one AE
`Grade 1
`Grade 2
`
`Lymphadenopathy (Grade 1)
`Abdominal pain upper (Grade 1)
`Vomiting (Grade 2)
`Decreased appetite (Grade 1)
`Ear infection (Grade 1)
`Upper respiratory tract infection (Grade 1)
`Cardiac murmur (Grade 1)
`Dermatitis contact (Grade 1)
`
`2 (18.2%)
`2 (18.2%)
`0 (0.0%)
`
`1 (9.1%)
`0 (0.0%)
`0 (0.0%)
`1 (9.1%)
`0 (0.0%)
`0 (0.0%)
`1 (9.1%)
`0 (0.0%)
`
`4 (36.4%)
`3 (27.3%)
`1 (9.1%)
`
`0 (0.0%)
`2 (18.2%)
`1 (9.1%)
`0 (0.0%)
`1 (9.1%)
`1 (9.1%)
`0 (0.0%)
`1 (9.1%)
`
`1 Table reflects number of adverse events reported during 7 days of dosing with sodium
`phenylbutyrate (NaPBA) and 7 days of dosing with glycerol phenylbutyrate (GPB).
`
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`326
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`U. Lichter-Konecki et al. / Molecular Genetics and Metabolism 103 (2011) 323–329
`
`Table 3
`Sodium phenylbutyrate (NaPBA) and glycerol phenylbutyrate (GPB) plasma metabolite concentrations (ug/mL) *.
`
`Time
`
`PBA
`
`NaPBA
`
`0.843 ±1.28
`b1–3.48
`
`Trough (24 h)
`Mean ±SD
`Range
`Peak (12 h)
`Mean ±SD
`36.8±38.2
`2.28–109
`Range
`Range of single measurements (0–24 h)
`Range
`b1–109
`
`GPB
`
`6.43±16.0
`b1–54.5
`
`74.8±52.6
`4.14–161
`
`b1–161
`
`PAA
`
`NaPBA
`
`0.817 ±1.03
`b1–2.84
`
`68.8 ±47.4
`3.43–148
`
`b1–148
`
`* PBA = phenylbutyric acid; PAA = phenylacetic acid; and PAGN = phenylacetylglutamine.
`
`GPB
`
`9.13 ±18.6
`b1–62.9
`
`78.6 ±65.2
`12.1–244
`
`b1–244
`
`PAGN
`
`NaPBA
`
`4.71 ±3.08
`b1–7.94
`
`72.2 ±28.5
`30.8–113
`
`b1–116
`
`GPB
`
`27.7 ±39.2
`2.63–137
`
`88.8 ±33.8
`32.0–136
`
`2.63–153
`
`values varied widely for both NaPBA and GPB whether drawn at peak
`(12 h), trough (24 h; selected over time zero because of monitored
`dosing) or throughout the course of the day (0–24 h) (Table 3). Intact
`GPB was not detectable in plasma.
`Plasma PK parameters of PBA, PAA and PAGN and urinary PK
`parameters of PAGN are summarized in Table 4 and the 24-hour
`concentration profiles are depicted in Fig. 1. Mean systemic PBA exposure
`(AUC0–24) following GPB administration was ~2.7-fold higher (pb0.01)
`than that observed with NaPBA. However, when compared with NaPBA
`administration, there was less variability in plasma PBA levels during
`dosing with GPB, as reflected by the coefficient of variation for AUC, Cmax
`and Cmin (Table 4). Mean (AUC0–24) systemic PAA and PAGN exposure
`during dosing with GPB as compared with NaPBA did not differ
`significantly, albeit also with directionally greater values on GPB as
`compared with NaPBA. Minimum (Cmin) values of PAA were significantly
`greater during GPB as compared with NaPBA dosing.
`
`Table 4
`PK parameters and ammonia at steady state dosing with sodium phenylbutyrate and
`glycerol phenylbutyrate (Study days 7 and 14).
`
`PK/PD parameters
`
`PBA in plasma mean (CV%)
`AUC0–24 (μg·h/mL)
`Cmaxss (μg/mL)
`Cminss (μg/mL)
`PAA in plasma mean (CV%)
`AUC0–24 (μg·h/mL)
`Cmaxss (μg/mL)
`Cminss (μg/mL)
`PAGN in plasma mean (CV%)
`AUC0–24 (μg·h/mL)
`Cmaxss (μg/mL)
`Cminss (μg/mL)
`PAGN in urine mean (CV%)
`Total excreted 0–24 h (g)
`Recovery of PBA as PAGN (%) Fe0–24 (%)
`Ammonia mean (SD)
`AUC (μmol/L)
`Cmaxss (μmol/L)
`Total number (%) of ammonia values
`above ULN⁎
`Diet
`Actual protein intake (g/d) Mean (SD)
`Actual protein intake (g/kg/d) Mean (SD)
`Judged compliant with diet (days 7, 14)
`Difference in ammonia between GPB and NaPBA
`Mean difference of AUC (μmol/L) (SD)
`Ratio of Geometric means
`90% Confidence interval
`95% Confidence interval
`
`Glycerol
`phenylbutyrate
`(n =11)
`
`NaPBA
`(n=11)
`
`631 (44.9)
`95.6 (42.0)
`1.50 (99.8)
`
`236 (105.2)
`37.4 (101.6)
`0.366 (171.3)
`
`964 (63.6)
`90.5 (69.1)
`2.99 (122.1)
`
`773 (73.3)
`75.1 (64.4)
`0.674 (130.5)
`
`1378 (40.2)
`105 (33.5)
`13.1 (64.9)
`
`1015 (44.7)
`74.8 (37.3)
`4.63 (66.4)
`
`12.5 (56.9)
`66.4 (23.9)
`
`12.5 (51.3)
`69.0 (23.9)
`
`603.8 (187.92)
`47.77 (12.800)
`24 (31.6)
`
`814.6 (322.36)
`55.66 (21.607)
`14 (18.4)
`
`23.98 (9.891)
`0.61 (0.16)
`8/11 (72.7%)
`
`24.35 (12.445)
`0.64 (0.35)
`9/11 (81.8%)
`
`-210.8 (310.89)
`0.781
`(0.609, 1.002)
`(0.575, 1.061)
`
`AUC0–24: Area under the concentration from time 0 (pre-dose) to 24 h, Cmaxss: Maximum
`plasma concentration at steady state, Cminss: Minimum plasma concentration at steady
`state, CV% — coefficient of variation.
`⁎ % abnormal ammonia values presented as mean (SD); the denominator is the total
`number of ammonia values at all time points (76).
`
`Similar to plasma, all urines were judged analyzable. One subject
`had no urine sample for the first interval and another did not void
`during the 12–24 h post-dose collecting interval, therefore their total
`PAGN amount excreted over 24 h (U-PAGN24-hour Excr) was adjusted as
`prospectively detailed in pharmacokinetic analysis plan. 24-hour
`PAGN excretion following GPB treatment was very similar to that
`observed for NaPBA (66% vs. 69% recovery of PBA as urinary PAGN),
`although peak urinary PAGN occurred later in the day during GPB
`treatment as compared with NaPBA treatment (percentage of total
`output from 0 to 12 and 12 to 24 h approximately 45% and 55% for GPB
`vs. 57% and 43% for NaPBA).
`
`3.4. Plasma amino acids
`
`Three patients had glutamine values above the normal range
`(266–746 umol/L) on GPB as compared with 6 patients on NaPBA.
`Mean [SD] plasma glutamine levels were non-statistically significant-
`ly lower on GPB (650.3 [187.3] umol/L) vs. NaPBA (725.1 [204.2]
`umol/L). Mean [SD] plasma branched chain amino acid levels were
`similar with NaPBA vs. GPB (isoleucine=34.1 [20.2] vs. 38.6 [9.2],
`leucine=64.0 [38.3] vs. 68.0 [16.9], and valine=134.5 [65.36] vs.
`112.0 [36.36] on NaPBA and GPB, respectively).
`
`3.5. Blood ammonia
`
`Ammonia levels varied widely, increased several fold during the day,
`peaking at around 8–12 h, and in four samples (3 during NaPBA and 1
`during GPB treatment, respectively) exceeded 100 umol/L in the absence
`of clinical symptoms. Only 2 of 154 blood ammonia values were missing;
`all NH324-hour AUC values were calculable and no imputation was
`required. Average ammonia values tended to be lower on GPB than on
`NaPBA assessed as NH324-hour AUC, average blood ammonia, average
`Cmaxss or the percentage of values above the upper limit of normal (as
`per normal values at the respective study site; range 29–54 μmol/L)
`(Table 4, Figs. 2 and 3), although these differences did not achieve
`statistical significance. GPB was determined to be non-inferior to NaPBA
`with respect to ammonia control based on the pre-specified analysis, as
`the upper boundaries of both the 90% (0.609, 1.002) and 95% (0.575,
`1.061) confidence intervals fell below 1.25. Analysis of ammonia control
`in the pre-specified per-protocol population demonstrated significantly
`lower ammonia (623.1 vs. 897.2), assessed as NH324-hour AUC on GPB as
`vs. NaPBA (pb0.05). This per-protocol analysis on 9 of 11 subjects
`excluded the two with a variance of over 50% in dietary protein intake on
`days 7 and 14.
`
`3.6. Effect of age on PK and ammonia during GPB dosing
`
`PK parameters during steady state dosing with GPB analyzed
`separately for subjects ages 6–11 (n=7) vs. 12–17 (n=4) were similar,
`as were mean (SD) ammonia values assessed as NH324-hour AUC (Table 5).
`
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`
`327
`
`GPB Day 14
`NaPBA Day 7
`LOQ = 1 µg/mL
`
`Fig. 2. Twenty Four Hour Ammonia Values on Sodium Phenylbutyrate and Glycerol
`Phenylbutyrate. Venous ammonia was measured for 24 h following one week of dosing
`with either sodium phenylbutyrate (NaPBA; continuous line) or glycerol phenylbutyrate
`(GPB; dotted line) in 11 pediatric subjects. The top panel depicts mean (SE) ammonia
`concentrations over 24 h. The bottom panel depicts overall ammonia values, where the
`bottom and top of the ‘box’ represent the 25th and 75th percentile of all values, the
`horizontal line within the box represents the mean, the open diamond within the box
`represents the median, and the top and bottom of the lines correspond to the maximum
`and minimum observed values, respectively. The open circles above the box for ammonia
`(24-hours AUC) on GPB represent outliers above the 75th percentile.
`
`4. Discussion
`
`No clinically important safety issues were identified during GPB
`dosing and tolerability was satisfactory. No hyperammonemic crises
`occurred during either NaPBA or GPB treatment. Plasma PBA exposure
`was ~2.7 times greater (p b0.01) during dosing with GPB as compared
`with NaPBA, whereas plasma PAA and PAGN exposure differed by
`
`100
`
`10
`
`1
`
`0.1
`
`100
`
`10
`
`1
`
`0.1
`
`100
`
`(µg/mL)
`
` (µg/mL)
`
`10
`
`(µg/mL)
`
`Mean (SD) Plasma PBA Concentration
`
`Median (SD) Plasma PAA Concentration
`
`Median (SD) Plasma PAGN Concentration
`
`0
`
`2
`
`4
`
`6
`
`8
`
`10
`12
`14
`Nominal Time (h)
`
`16
`
`18
`
`20
`
`22
`
`24
`
`GPB Day 14
`NaPBA Day 7
`LOQ = 1 µg/mL
`
`0
`
`2
`
`4
`
`6
`
`8
`
`10
`12
`14
`Nominal Time (h)
`
`16
`
`18
`
`20
`
`22
`
`24
`
`GPB Day 14
`NaPBA Day 7
`LOQ = 1 µg/mL
`
`1
`
`0
`
`2
`
`4
`
`6
`
`8
`
`10
`12
`14
`Nominal Time (h)
`
`16
`
`18
`
`20
`
`22
`
`24
`
`Fig. 1. Plasma phenylbutyric acid (PBA; top panel), phenylacetic acid (PAA; middle
`panel) and phenylacetylglutamine (PAGN; bottom panel) were measured for 24 h
`following one week of dosing with either sodium phenylbutyrate (NaPBA) or glycerol
`phenylbutyrate (GPB) and are displayed as median ±SD. Times 0 and 24 h correspond
`to just prior to dosing and breakfast.
`
`3.7. Correlates of drug dose and ammonia
`
`Drug dose correlated directly and most consistently and strongly
`with urinary PAGN for both NaPBA and GPB treatments (r=0.866;
`p b0.0001 for both NaPBA and GPB combined) (Table 6). Drug dose
`did not correlate with plasma PBA levels assessed as AUC0–24.
`Drug dose did correlate, albeit less strongly and consistently, with
`plasma PAGN and PAA, assessed as AUC0–24. Ammonia assessed as
`NH324-hour AUC correlated neither with blood levels of PBA, PAA or
`PAGN nor with urinary PAGN output. Blood ammonia Cmax correlated
`positively with glutamine, although the correlation was modest
`(r=0.25) and did not achieve significance in this small sample size.
`
`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
`mean ±SD. Times 0 and 24 h correspond to just prior to dosing and breakfast.
`
`
`Page 5 of 7
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`328
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`U. Lichter-Konecki et al. / Molecular Genetics and Metabolism 103 (2011) 323–329
`
`Table 5
`PK parameters and ammonia at steady state dosing with glycerol phenylbutyrate
`(Study day 14) by age range.
`
`PK/PD
`parameters
`
`All subjects
`(n =11)
`
`Ages 6–11
`(n =7)
`
`Ages 12–17
`(n =4)
`
`(Day 14; 23.98±9.891 g/d or 0.61±0.16 g/kg/d) and NaPBA (Day 7;
`24.35±12.445 g/d or 0.64±0.35 g/kg/d).
`One possible explanation for the directionally lower ammonia
`values is the generally higher plasma metabolite levels observed
`during GPB dosing. As summarized in Table 4, GPB treatment resulted
`in higher plasma PBA levels overall, including higher peak and trough
`levels, and similar differences in favor of higher plasma levels for PAA
`and PAGN. Plasma PAA levels were better sustained and directionally
`higher over night, as manifested by higher trough levels and this could
`be causally related to the directionally lower ammonia levels during
`GPB dosing.
`The PAA levels are of interest in that neurological toxicity has been
`reported in cancer subjects administered high doses of PAA
`intravenously; this toxicity was associated with PAA blood levels
`ranging from ~499 to 1285 μg/mL [11,12]. However, PAA blood levels
`observed during steady state dosing with both NaPBA and GPB were
`well below the levels reported by Thibault to be associated with
`neurological symptoms (Table 4).
`Another possible explanation for and contributing factor to the
`ammonia findings may be slower gastrointestinal absorption of PBA
`when delivered as GPB. This is supported by prior PK/PD modeling
`(10) and, in the present study, by the proportion of PAGN excreted
`during the 0–12 and 12–24 h time periods (45% and 55% for GPB vs.
`57% and 43% for NaPBA). A slower gastrointestinal absorption of PBA
`is also indicated by lesser variation in PBA concentrations during GPB
`dosing in this study (Table 4).
`While the number of subjects is small, ammonia and PK findings
`appeared to be generally similar during steady state GPB among
`subjects ages 6–11 and 12–17, suggesting that GPB metabolism and
`effect is not dependent on age, per se, within this range (Table 5).
`The present study underscores the difficulty clinicians face in making
`decisions regarding drug dosing based on blood ammonia. Even under
`the controlled conditions of the present trial,
`including excellent
`compliance with diet and study drug in patients whose ammonia
`values were viewed as well controlled by their physicians prior to
`enrollment, ammonia values on average varied more than 10-fold over
`the course of the day. This observation suggests that random values for
`blood ammonia are of limited utility and that ammonia levels should be
`drawn at a constant time in relation to meals and medication for
`monitoring of treatment. Blood levels of metabolites are also problem-
`atic with respect to therapeutic monitoring. As summarized in Tables 3
`and 4 and depicted in Fig. 1, blood levels of PBA, PAA and PAGN all varied
`widely over the course of the day. Levels at individual time points and
`even peak or trough levels varied considerably among these patients
`(Table 3), suggesting that therapeutic metabolite blood levels when
`collected randomly or at peak or trough time points are difficult if not
`impossible to define. Although PAA and/or PAGN exposure assessed as
`24 h area under the curve and measured with 8 samples over 24 h
`did correlate with dose, the correlation was weak and inconsistent and
`24-hour sampling is impractical in routine practice.
`Urinary output of PAGN,
`in contrast to blood levels of drug
`metabolites, shows promise as a biomarker of dose selection and
`compliance monitoring in pediatric UCD patients and is practical to
`perform in routine clinical practice. This makes theoretical sense,
`since PAGN output is stoichiometrically related to waste nitrogen
`scavenging, and PAGN output, measured over 24 h, correlated
`strongly and positively with drug dose (R= 0.909; p b0.001 for GBP
`and R=0.753; p=0.007 for NaPBA).
`Many metabolic specialists use glutamine levels to monitor treatment
`and adjust drug doses. Among the subjects in this study, who were
`viewed as well-controlled under the supervision of a metabolic specialist
`at entry, glutamine levels decreased, although not statistically signifi-
`cantly, after switching from NaPBA to GBP. Glutamine levels also
`correlated modestly and positively with ammonia. However, the
`correlation did not achieve statistical significance and needs evaluation
`in a larg