Molecular Genetics and Metabolism 100 (2010) 221-228
`
`Contents lists available at ScienceDirect
`
`Molecular Genetics and Metabolism
`
`s
`Molecular Gene1ic~
`und Metabolism
`
`ELSEVIER
`
`journal homepage: www.elsevier.com/locate/ymgme
`
`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 ct. Bruce F. Scharschmidt e. As ad Mian a.
`Oleg Shchelochkov a. ].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
`'Pediatrics. Medical College of Wisconsin. 8701 Watertown Plank Rd .• Milwaukee. WI. United States
`• 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
`r Pharsight Corp .. Montreal. 2000 Peel St.. Suite 570. Quebec. Canada
`g Chiltem. 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
`
`ARTICLE INFO
`
`ABSTRACT
`
`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
`Phenylacetylgl utamine
`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 NaPEA. 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 NaPEA. Blood ammonia and blood and urine metabolites were compared after 7 days (steady
`state) ofTID 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 NaPEA while none occurred on GPB. Ammonia values on GPB
`were ~30% lower than on NaPEA (time-normalized AUC = 26.2 vs. 38.4!lmoljL; Cmax = 56.3 vs.
`79.1 llmoi/L; not statistically significant). and GPB achieved non-inferiority to NaPEA with respect to
`ammonia (time-normalized AUC) by post hoc analysis. Systemic exposure (AUC0 _24 ) to PBA on GPB was
`27% lower than on NaPEA (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 NaPEA and GPB; other metabolites accounted
`for <1%.1ntact 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 NaPEA. 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.
`
`© 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(cid:173)
`ciency; NaPEA, sodium phenylbutyrate; PAA, phenylacetic acid; PAG, phenylacetyl
`glycine; PAGN, phenylacetylglutamine; PBA, phenylbutyric acid; PBG, phenylbuty(cid:173)
`ryl glycine; PBGN, phenylbutyryl glutamine; PK, pharmacokinetic; TNAUC, time(cid:173)
`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 © 2010 Elsevier Inc. All rights reserved.
`doi: 1 0.1016/j.ymgme.201 0.03.014
`
`Introduction
`
`Urea cycle disorders (UCDs) comprise several inherited defi(cid:173)
`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
`
`Par Pharmaceutical, Inc. Ex. 1004
`Par v. Horizon, IPR of Patent No. 9,561,197
`Page 1 of 8
`
`

`

`222
`
`B. Lee et al.fMolecular 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 t rade name: BUPHENYL•,
`EU: AMMO NAPS•) 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(cid:173)
`tion to phenylacetate, (PAA), a metabolically active compound that
`conjugates with glutamine via acetylation to form phenylacetyl(cid:173)
`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(cid:173)
`mum approved dose of 20 NaPBA grams per day ( 40 tablets per
`day) contains approximately 2363 mg of sodium, and current "Die(cid:173)
`tary Guidelines for Americans 2005" recommends a sodium intake
`of 2300 mgjday for the general population and 1500 mgjday 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(cid:173)
`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 (PI<) characteristics of GPB
`have been evaluated in pre-clinical models and in two prior clini(cid:173)
`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 gjm 2 of PBA), and an open-label study in
`32 adults, including 8 healthy adults and 24 adults with Child(cid:173)
`Pugh grade A, B, or C cirrhosis (8 each), each of whom received
`a single 100 mgjkg dose of GPB followed by 1 week of BID
`dosing at 100 mgjkg 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 (cid:173)
`tics. The present phase 2 study, the first in UCD patients, was de(cid:173)
`signed to compare safety, PI< 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 :;,2 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(cid:173)
`tient research unit for 24-h PI< and ammonia monitoring. Depend(cid:173)
`ing on dose, subjects were then either switched directly to 100%
`GPB, or GPB was introduced in step-wise weekly increments equiv(cid:173)
`alent to ~50 mgjkgjday of NaPBA, with the remainder of the PBA
`equivalent dose administered as corresponding weekly decre(cid:173)
`ments in the dose of NaPBA. Initiation or increases in GPB dosing
`were done under observation in an appropriately monitored set(cid:173)
`ting, and subjects were discharged after they were deemed clini(cid:173)
`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 PI< and ammonia assess(cid:173)
`ment, after which they were switched back to NaPBA.
`Subjects remained on their prescribed amount of dietary pro(cid:173)
`tein throughout the study, received dietary counseling, were in(cid:173)
`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(cid:173)
`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(cid:173)
`verse events. Efficacy was assessed by serial measurement of ve(cid:173)
`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 PI< 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(cid:173)
`ning with the time of the first dose of the day), 6- 12 hand 12- 24 h.
`
`Pharmacokinetic, pharmacodynamic and statistical analyses
`
`PI< parameters for PBA, PAA, and PAGN in plasma, PAGN in ur(cid:173)
`ine, as well as pharmacodynamic parameters for venous ammonia
`were calculated with non-compartmental methods using a vali(cid:173)
`dated version of WinNonlin Enterprise (version 5.2). Individual
`plasma concentrations, urinary amounts and volumes were sum(cid:173)
`marized with descriptive statistics (e.g. number of patients [n],
`mean, standard deviation [SD], median, minimum, and maximum).
`The following plasma PI< 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
`(AUC0 _ 24), maximum plasma concentration at steady state
`(Cmax55 ), minimum plasma concentration at steady state (Cmin55 ),
`time maximum plasma concentration at steady state (Tmax55 ), and
`apparent clearance at steady state (CL55/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(cid:173)
`tion (by multiplying the urinary volume with urinary concentra(cid:173)
`tions). The time-normalized area under the curve (TNAUC) and
`Cmax55 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 AN OVA 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(cid:173)
`tra-patient coefficient of variability for PI< and PD parameters were
`derived from the ANOVA model. Statistical analyses were per(cid:173)
`formed using the LinMix module in WinNonlin Enterprise (version
`5.2). Correlates of blood ammonia were determined using Spear(cid:173)
`man rank-order correlations. Measurement of total urinary nitro(cid:173)
`gen (TUN) was performed by Elementar Rapid NIII Analyzer
`(Mayo Medical Laboratories, Rochester, MN) applying Dumas
`
`Par Pharmaceutical, Inc. Ex. 1004
`Par v. Horizon, IPR of Patent No. 9,561,197
`Page 2 of 8
`
`

`

`B. Lee et al.fMolecular Genetics and Metabolism 100 (2010) 221-228
`
`223
`
`method of combustion [ 1 0] 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(cid:173)
`rolled in the study and 10 subjects ( 4 males and 6 females) com(cid:173)
`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(cid:173)
`jects were discontinued at the discretion of the investigators before
`receiving either study drug. One subject each had argininosucci(cid:173)
`nate synthetase (ASS), and ornithine translocase (HHH) deficiency;
`the remaining subjects had ornithine transcarbamylase (OTC) defi(cid:173)
`ciencies. Three subjects had neonatal or infantile onset, and all oth(cid:173)
`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 gjm2
`of 191
`(44.6) mgjkgjday, equivalent
`(1.65)
`(range = 4.47- 9.10 gjm2
`, 2 subjects were taking 20 gjday). Eight
`of the 10 subjects who completed the study were being prescribed
`NaPBA at doses below the recommended range of 9.9- 13 gjm 2
`(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 (em)
`Mean (SD)
`
`Weight (kg)
`Mean (SD)
`
`Patients completing
`the study (N = 10)
`
`4 (40.0)
`6 (60.0)
`
`38.2 (17.85)
`
`165.6 (7.88)
`
`80.41 (31.647)
`
`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(cid:173)
`ror. Compliance with treatment was excellent; ~99% of all sched(cid:173)
`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(cid:173)
`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(cid:173)
`ble 2). During 100% NaPBA treatment, one AE (mental status
`
`Table 2
`Summary of treatment- emergent adverse events•_
`
`Adverse event term
`
`NaPEA
`N=13
`
`Glycerol
`Phenylbutyrate
`N = 10
`
`All
`
`Related All
`
`Related
`
`Any AE (number of subjects)
`
`21 (7)
`
`6 (5)
`
`15 (5)
`
`11 (5)
`
`Gastrointestinal disorders
`Nausea
`Dyspepsia
`Abdominal pain
`Castro-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
`
`2 (2)
`0
`
`2
`1
`0
`0
`0
`1
`0
`0
`
`3 (2)
`1
`
`6 (3)
`1
`1
`
`0
`1
`0
`0
`0
`0
`0
`0
`
`1 (1)
`1
`0
`0
`
`2 (2)
`0
`1
`1
`0
`0
`0
`
`5 (2)
`0
`0
`0
`0
`1
`
`5 (2)
`0
`0
`0
`0
`1
`
`0
`
`0
`
`3 (3)
`3
`0
`0
`
`3 (3)
`3
`0
`0
`
`0
`0
`0
`0
`0
`0
`0
`
`0
`0
`0
`0
`0
`0
`0
`
`1 (1)
`
`1 (1)
`
`1 (1)
`
`1 (1)
`
`0
`
`0
`1
`
`UCD Diagnosis [n (%)]
`OTC Deficiency•
`ASS Deficiencyb
`HHH Syndrome'
`
`UCD Onset [n (%)]
`Neonatal (0-~30 days)
`Infantile (>30 days-~2 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 (mgfkgfday)
`Mean (SD)
`Median
`Min, max
`
`Average Protein intake during study (mgfkgfday)
`Mean (SD)
`Median
`Min, max
`Percentage of patients treated with L-citrolline
`
`a Ornithine transcarbamylase deficiency.
`b Arginosuccinate synthetase deficiency.
`' Ornithine translocase deficiency.
`
`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
`03, 0.8
`6 (60%)
`
`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
`
`1
`0
`
`0
`0
`
`2 (2)
`1
`
`0
`
`0
`0
`0
`
`1 (1)
`
`0
`0
`
`1 (1)
`
`1
`0
`
`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
`
`0
`
`0
`
`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.
`• Table reflects number of events and events reported during 7 days of NaPEA
`(sodium phenylbutyrate) prior to transition to glycerol phenylbutyrate, and 7 days
`of sole glycerol phenylbutyrate treatment after completion of transition from
`NaPEA treatment
`
`Par Pharmaceutical, Inc. Ex. 1004
`Par v. Horizon, IPR of Patent No. 9,561,197
`Page 3 of 8
`
`

`

`224
`
`B. Lee et al.fMolecular 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(cid:173)
`out specific treatment. Two subjects experienced SAEs of hyperam(cid:173)
`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(cid:173)
`ammonemia on GPB.
`
`Pharmacokinetic and pharmacodynamic analyses
`
`All 10 patients who completed the study were considered
`evaluable for the PI< analyses. Plasma PI< parameters of PBA, PAA
`and PAGN and urinary PI< 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(cid:173)
`tration was 27% lower than that observed with NaPBA (540 vs.
`739 11g h/mL, respectively), whereas exposure levels of PAA (575
`vs. 596 11g h/mL, respectively) and PAGN (1 098 vs. 1133 11g h/mL,
`respectively) were similar. PAG, PBG, and PBGN were not detect(cid:173)
`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
`AUCo-24 (J.lg hjml)
`Cmax" (J.lgjml)
`Cmin" (J.lg/ml)
`
`PAA in plasma
`AUC0 _24 (J.lg hjml)
`Cmax" (J.lg/mL)
`Cmin" (J.lg/ml)
`
`PAGN in plasma
`AUC0 _24 (J.lg hjmL)
`Cmax" (J.lg/ml)
`Cmin" (J.lgjml)
`
`PAGN in urine
`Total excreted 0-24 h (J.lg)"'
`0-6 h (J.lg)
`6-12 h (J.lg)
`12-24 h (J.lg)"'
`Recovery of PBA as PAGN (%)
`
`Total urinary nitrogen in 24 h
`Mean (SD) g
`
`Ammonia
`TNAUC (J.lmoljl)
`Cmax" (J.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 phenylburyrate jNaPBA)
`95% CI ofratio
`
`0.71
`0.44-1.14
`
`AUC0 _24, area under the concentration from time 0 (pre-dose) to 24 h; Cmax",
`maximum plasma concentration at steady state; Cmin". minimum plasma con(cid:173)
`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(cid:173)
`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(cid:173)
`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 (~-tmol/dL) in the 8 patients for
`whom measurements on both drugs were available were some(cid:173)
`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 ~-tmoljL; n = 101 values total) and on GPB
`(range 2-106 ~-tmol/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 ~-tmol/
`L, respectively) and Cmax55 (29% lower: 56.3 vs. 79.1 ~-tmol/L,
`respectively) (Table 3 ). Mean ammonia TNAUC values for individ(cid:173)
`ual subjects are depicted in Fig. 4 ; 27.0% ofthe ammonia values ob(cid:173)
`tained while on GPB were above the upper limit of normal for
`ammonia at their respective study site (upper limit of normal ran(cid:173)
`ged from 26-35 ~-tmol/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(cid:173)
`tistical significance (Fig. 2). A post hoc analysis indicated non-infe(cid:173)
`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(cid:173)
`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(cid:173)
`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(cid:173)
`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(cid:173)
`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 PI< characteristics of NaPBA and GPB in plasma were gener(cid:173)
`ally similar, with the exception of PBA. The lower plasma levels of
`PBA in subjects on GPB treatment as compared to NaPBA may re(cid:173)
`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(cid:173)
`ually released from GPB by pancreatic lipases as it passes through
`the gastrointestinal tract, which would allow more time for intra-
`
`Par Pharmaceutical, Inc. Ex. 1004
`Par v. Horizon, IPR of Patent No. 9,561,197
`Page 4 of 8
`
`

`

`B. Lee et al.fMolecular Genetics and Metabolism 100 (2010) 221-228
`
`225
`
`1 Week GPB (Visit 11-1) Day 1
`-
`-o- Last Day ofNaPBA (Visit 2-1) Day 1
`LOQ= 1 f.lgimL
`
`0
`
`2
`
`4
`
`6
`
`10
`
`12
`
`14
`
`16
`
`18
`
`20
`
`22
`
`24
`
`Nominal Time (h)
`
`1 Week GPB (Visit 11- 1) Day 1
`-
`-o- Last Day ofNaPBA (Visit 2-1) Day 1
`LOQ= 1 flg/mL
`
`0
`
`2
`
`4
`
`6
`
`10
`
`12
`
`14
`
`16
`
`18
`
`20
`
`22
`
`24
`
`Nominal Time (h)
`
`1 Week GPB (Visit 11- 1) Day 1
`-
`-o- Last Day of NaPBA (Visit 2-1) Day 1
`LOQ= 1 f.lgimL
`
`A 200
`
`0
`
`B 120
`
`0
`
`c 140
`
`3
`~ 120
`6
`
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`0
`
`2
`
`4
`
`6
`
`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 (NaPEA) or glycerol phenylbutyrate (GPB) and are displayed as means± SD. Times 0 and 24 h correspond to just prior to dosing and
`breakfast.
`
`hepatic I first pass conversion. PAG, PBG and PBGN were not mea(cid:173)
`surable in blood.
`
`PAGN was the major urinary metabolite, with negligible
`amounts of PAA, PBA, PAG, PBG and PBGN (<1% of PBA dose for
`
`Par Pharmaceutical, Inc. Ex. 1004
`Par v. Horizon, IPR of Patent No. 9,561,197
`Page 5 of 8
`
`

`

`226
`
`B. Lee et al.fMolecular Genetics and Metabolism 100 (2010) 221 - 228
`
`1 Week GPB (Visit 11-1) Day 1
`-
`--o-- Last DayofNaPBA (Visit2-l) Day 1
`LOQ= 1 fllllOIJL
`
`100
`
`~
`~
`" 80
`
`.9 i 0 " 60
`
`0 u
`"' ·a
`0 s s 40
`<
`"' s
`i(j
`0::
`0
`~
`,;
`" ::>:
`
`20
`
`0
`
`0
`
`2
`
`4
`
`6
`
`14
`12
`10
`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 (NaPEA) or glycerol phenylbutyrate (GPB) and is displayed
`as mean± SD. Times 0 and 24 h correspond to just prior to dosing and breakfast.
`
`Ammonia (umoi/L)
`80,--------------------------------------------
`
`-........ ·· ..
`
`70 +-----------~~~-----------------------------------
`
`-~ ... ... __
`60 +-----------~~~,,~----------------------------
`, ,, ,,
`50r-----~~~-,--~-~_-=-------------
`' ',
`40r--~~~~~~~~~=:-------
`& Mean
`'·
`-- ...... -.!:':" · ....
`30 ~-~-~-~-~-~-~--~-~-~-~-~-~-~-~-~-~-~-~-~-~-~-~-~--~-~-~-~-~~~-~-~~~~-~-~-~-~- ~--~-~-~-~----_-_-_-_
`Average Upper Limit of Normal
`• •. _ • ·, -~
`20t-----~~~~~~~~------
`
`NH3
`
`"- "-- ~
`
`~
`
`1\
`I \
`"""-'\ ~ I
`\
`v
`"J
`~
`
`I---NH3I
`
`"'
`\. ....
`
`I
`...
`
`--.
`
`70
`60
`£ 50
`
`z 40
`
`0
`~ 30
`~ 20
`10
`0
`
`10+-----------------------------~~----------
`
`0+-------------------,-------------------.
`NaPBA
`GPB
`
`Fig. 4. Relationship between blood ammonia and urinary output of phenylacetyl(cid:173)
`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.
`
`Fig. 3. Venous ammonia in individual subjects following one week of dosing with
`either sodium phenylbutyrate (NaPEA; 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 (cid:173)
`tered PBA dose for both drugs.
`Blood ammonia levels (TN AU C) 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(cid:173)
`tent with the delayed peak in urinary PAGN output following glyc(cid:173)
`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(cid:173)
`tistical significance. Collectively, these preliminary findings sug(cid:173)
`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-
`
`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(cid:173)
`scribed lower NaPBA doses than currently recommended in the ap(cid:173)
`proved product labeling. Increasing dose in these subjects to
`within the labeled range (BUPHENYL package insert) might im(cid:173)
`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(cid:173)
`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(cid:173)
`lated strongly and inversely with UPAGN (Table 4). This is consis(cid:173)
`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(cid:173)
`verted to PAGN, should mediate excretion of 3.23 g of waste nitro-
`
`Par Pharmaceutical, Inc. Ex. 1004
`Par v. Horizon, IPR of Patent No. 9,561,197
`Page 6 of 8
`
`

`

`B. Lee et al.fMolecular Genetics and Metabolism 100 (2010) 221-228
`
`227
`
`Table 4
`Correlation between ammonia and plasma PAA, PBA and PAGN, and urinary PAGN (UPAGN)"·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 NaPEA and glycerol phenylbutyrate were included in the analysis.
`Data from one subject with more than 50% missing data on glycerol phenylbutyrate were excluded.
`NS ~ n