003 1-3998/86/201 I-11 17$02.00/0
`PEDIATRIC RESEARCH
`Copyright © 1986 International Pediatric Research Foundation, Inc.
`
`Vol. 20, No. 11, 1986
`Printed in U.S.A
`
`Waste Nitrogen Excretion Via Amino Acid
`Acylation: Benzoate and Phenylacetate in
`Lysinuric Protein Intolerance
`
`OLLI SIMELL, ILKKA SIPILA, JUKKA RAJANTIE, DAVID L. VALLE, AND
`SAUL W. BRUSILOW
`
`Children's Hospital, University of Helsinki, SF-00290 Helsinki, Finland; and DepartmentofPediatrics, The
`Johns Hopkins University School ofMedicine, Baltimore, Maryland 21205
`
`ABSTRACT. Benzoate and phenylacetate improve prog-
`nosisin inherited urea cycle enzymedeficiencies by increas-
`ing waste nitrogen excretion as amino acid acylation prod-
`ucts. We studied metabolic changes caused by these sub-
`stances and their pharmacokinetics in a biochemically
`different urea cycle disorder, lysinuric protein intolerance
`(LPI), understrictly standardized induction of hyperam-
`monemia. Five patients with LPI received an intravenous
`infusion of 6.6 mmol/kg L-alanine alone and separately
`with 2.0 mmol/kg of benzoate or phenylacetate in 90 min.
`Blood for ammonia, serum urea and creatinine, plasma
`benzoate, hippurate, phenylacetate, phenylacetylgluta-
`mine, and amino acids was obtained at 0, 120, 180, and
`270 min. Urine was collected in four consecutive 6-h pe-
`riods. Alanine caused hyperammonemia: maximum in-
`crease 107, 28-411 uM (geometric mean, 95% confidence
`interval); ammonia increments were nearly identical after
`alanine + benzoate (60, 17-213 uM)and alanine + phen-
`ylacetate (79, 13-467 uM) (NS). Mean plasma benzoate
`was 6.0 mM when extrapolated to the end of alanine +
`benzoate infusions; phenylacetate was 4.9 mM at the end
`of alanine + phenylacetate. Transient toxicity (dizziness,
`nausea, vomiting) occurred in four patients at the end of
`combined infusions, and we suggest upper therapeutic
`plasma concentrations of 4.5 mM for benzoate and 3.5
`mM for phenylacetate. Benzoate and phenylacetate then
`decreased following first-order kinetics with tins of 273
`and 254 min, respectively. Maximal plasma hippurate
`(0.24, 0.14-0.40 mM) was lower than maximal phenylac-
`etylglutamine (0.48, 0.22-1.06 mM, p = 0.008). Orotic
`acid excretion was 5.62, 1.84—17.14 umol/kg per h after
`alanine, but only 1.07, 0.04—25.62 umol/kg per h after
`alanine + benzoate ( p < 0.151) and 2.74, 0.01-16.25 »mol/
`kg per h after alanine + phenylacetate (p < 0.016). Urea
`excretions were in the same rangeafter all loads. Urinary
`hippurate nitrogen after alanine + benzoate and phenyl-
`acetylglutamine nitrogen after alanine + phenylacetate ac-
`counted for an average of 12 and 22 of that in urea in the
`first 6 h. Of the benzoate and phenylacetate given, 65 and
`51%were excreted in 24 h as hippurate and phenylacetyl-
`
`less than 3.5% appeared un-
`glutamine, respectively;
`changedin urine. (Pediatr Res 20: 1117-1121, 1986)
`
`Abbreviations
`
`LPI, lysinuric protein intolerance
`iv, intravenous
`
`In 1914, Lewis (1) showed that benzoate modifies waste nitro-
`gen excretion by decreasing production of urea via excretion of
`waste nitrogen as the acylation product of glycine with benzoate,
`i.e. hippurate. A few years later (2) phenylacetate was noted to
`be anotherefficient acylating agent, combining with glutamine
`to form phenylacetylglutamine. Excretion of these nitrogenous
`products decreases the requirement for urea synthesis. Despite
`these observations, these alternative ways of waste nitrogen ex-
`cretion were not used clinically until the encouraging results in
`the treatment of inborn errors of the urea cycle were reported
`(3-9).
`In life-threatening hyperammonemia measurements of the
`effects and metabolism of benzoate and phenylacetate are diffi-
`cult if not impossible. We thus selected to test these substances
`in LPI, a disease with typical clinical picture (10, 11), well-
`characterized hyperammonemia, and defective transport of the
`diaminoacidsat the basolateral membraneofthe intestinal (12-
`14), renal (15, 16), and probably liver (17, 18) epithelial cells.
`Wegave the patients an iv alanine load, which leaves healthy
`subjects unaffected but causes moderate hyperammonemia in
`the patients, either alone or together with benzoate or phenyl-
`acetate. The results indicate that hippurate and phenylacetylglu-
`tamine nitrogen excretions accounted for over 10 and 20%,
`respectively, of the amount of nitrogen excreted in urea in the
`first 6 h. Neither one of these substances was able to abolish
`alanine-induced hyperammonemia in these patients, although
`orotic aciduria diminished.
`
`MATERIALS AND METHODS
`
`Received December 10, 1985; accepted June 6, 1986.
`Address for correspondence and reprint requests Dr. Olli Simell, Children’s
`Hospital, University of Helsinki, 00290 Helsinki, Finland.
`This study was supported by the Finnish Academy, by the Sigrid Jusélius
`Foundation and Foundation for Pediatric Research, Finland, and by Grants ROI-
`HD-11134, MOI-RR-00052, and KO7-NS-00342 from the National Institutes of
`Health, by the Randi Klavan Memorial Fund. and by the Kittering Family
`Foundation. O.S. was a Visiting Scientist at
`the McGill University-Montreal
`Children’s Hospital, sponsored by the Medical Research Council of Canada, during
`preparation of the manuscript. D.L.V, was an investigator in the Howard Hughes
`Medical Institute.
`
`1; a
`Patients. Five Finnish children (see the legend of Fig.
`sister-brother pair, otherwise no consanguinity between the pa-
`tients) with a mean age (range) of 8.8 (2.8-12.6) yr, weight for
`age of —1.6 [—0.9-(—3.2)] SD and height for age of —2.4 [—1.3-
`(—4.1)] SD and homozygous for LPI were studied after informed
`consent and acceptanceofthe study by the ethics committee of
`the hospital.
`The patients were of normalintelligence, had hepatospleno-
`megaly, hypotrophic muscles, osteoporosis, and aversion to pro-
`tein-rich food. They excreted massive amountsoflysine andless
`1117
`
`Par Pharmaceutical, Inc. Ex. 1007
`Par v. Horizon, IPR of Patent Nos. 9,254,278, 9,095,559, and 9,326,966
`Page 1 of 5
`
`

`

`1118
`
`SIMELL FT AL.
`
`500
`
`400
`
`(pmol/l) ALA + BA
`
`BLOODAMMONIA
`
`300
`
`200
`
`100
`
`ALA + PAA
`
`1. Blood ammonia responses to 6.6 mmol/kg loadsof alanine infused iv in 90 min either alone (ALA) or together with 2.0 mmol/kg of
`Fig.
`benzoate (ALA + BA) or phenylacetate (ALA + PAA) to five Finnish patients with lysinuric protein intolerance. For identification, the patients are
`named from A to E;Aisthesister of B.
`
`TIME (h)
`
`of arginine and ornithine. All except one patient with recent
`diagnosis had been oncitrulline supplementation, 0.5 mmol/g
`of daily dietary protein. Even on this regimen the protein intake
`had remained low (12, 19). The citrulline supplementation was
`stopped 48 h before the infusion studies.
`The infusions were started after an overnight fast at 0900 h,
`and no food was allowed during the first 6 h. L-Alanine, 6.6
`mmol/kg, was given iv as a 5% aqucous solution in 90 min,
`followed by 0.9% saline, 200 ml/m? of body surface area per h
`for additional 4.5 h. This load causes a rapid increase in serum
`urea in healthy controls but blood ammonia and urinary orotic
`acid excretion remain unchanged (19). A similar infusion of
`alanine was given to each patient together with 2.0 mmol/kg of
`sodium benzoate, and on a separate day, with 2.0 mmol/kg of
`sodium phenylacetate.
`Blood and urine samples. Blood for ammonia, serum urea and
`creatinine, plasma benzoate, hippurate, phenylacetate, phenyl-
`acetylglutamine, and amino acids was drawn at 0, 90, 120, 180,
`and 270 min. Urine was collected in four consecutive 6-h periods.
`Blood samples were immediately placed on ice and processed for
`measurement or storage at —20° C. Urine was collected on ice
`and frozen at the end ofeach collection.
`Measurements. Blood ammonia was measured with an am-
`moniaspecific electrode (Orion Research Inc., Cambridge, MA)
`(20). Plasma and urinary benzoic acid, hippuric acid, phenyla-
`cetic acid, and phenylacetylglutamine were measured by reverse
`phase liquid chromatography with the use of a Waters Cis
`column, with a 20% methanolsolution in 0.0! M acetate buffer,
`pH 3, as an eluant. Plasma and urinary amino acids were
`quantitated with a Beckman 121 M AminoAcid Analyzer using
`lithium buffers and norleucineas internal standard.
`Statistical analysis. Values are expressed, if not otherwise
`stated, as geometric means with 95% confidence limits. For
`statistical evaluation, Mann-Whitney’s U test was used.
`Urinary values are expressed normalized to body weight. Re-
`sults normalized to creatinine excretion gave little new infor-
`mation and were omitted.
`
`RESULTS
`
`Clinical symptoms. The youngest patient was pale and floppy
`for | h after alanine load, but others tolerated alanine-induced
`hyperammonemia without symptoms. Four patients, including
`the youngest one, had symptomsat the end ofloads containing
`
`benzoate or phenylacetate. Three complained of dizziness and
`two vomited at the end of alanine + benzoate loads, and three
`were pale and dizzyafter alanine + phenylacctate. The symptoms
`disappeared in 45—60 min.
`Blood ammonia. The fasting blood ammonia concentration
`was normal (70 »M)in 11 of the 15 measurements, and elevated
`in four (Fig.
`1). The three hyperammonemic values at the
`beginning of the alanine + phenylacetate infusions were in
`patients having just arrived at the hospital in an overnighttrain;
`their ammonia increments were well within the range of the
`other subjects. Ammonia values usually peaked at 120 min. The
`geometric mean of peak increments wasslightly higher after
`alanine (107, 28-411 4M) than after alanine + benzoate (60, 17-
`213 4M, NS) and alanine + phenylacetate (79, 13-467 uM, NS).
`The concentrations returned to basal values in all except one
`subject by 270 min.
`Serum urea and creatinine. The increase in the geometric
`mean values of serum urea after alanine (from 4.5 to 5.1 mM)
`and alanine + benzoate (from 6.6 to 8.3 mM) loads was caused
`by rising values in one patient. The values were stable in the
`others. Urea increased in this patient also after alanine + phen-
`ylacetate load.
`Serum creatinine fluctuated slightly and randomly in the in-
`dividual patients. The log mean values remained stable after
`alanine,
`increased after alaninac + benzoatc, and decreased
`slightly after alanine + phenylacetate.
`Plasma benzoate and hippurate. Plasma benzoate peaked 2 h
`after the start of the infusion and decreasedlinearly with a mean
`t,2 of 273 min (Fig. 2). By extrapolation, benzoate concentration
`at the end ofinfusion (at 90 min) was 6.01, 5.17-6.98 mM.
`Plasma hippurate levels peaked 120 min after start of the
`infusions (0.24, 0.14-0.40 mM) and remained nearly stable for
`the next 3 h (Fig. 2), implying that synthesis from benzoate,
`distribution between plasma and other compartments and elim-
`ination were balanced, despite continuous decrease in plasma
`benzoate concentration. At all time points, the hippurate con-
`centrations were less than 10% those of benzoate.
`Peak
`Plasma_ phenylacetate and phenylacetylglutamine.
`plasma concentration of phenylacetate was at 120 min after the
`alanine + phenylacetate infusion (Fig. 3) and wassignificantly
`(p = 0.008) below the concentration of plasma benzoate reached
`after the alanine + benzoate load. The disappearance curve of
`phenylacetate from plasma waslinear with a t,,2 of 254 min,
`similar to that of benzoate. By extrapolation, phenylacetate
`concentration was 4.77, 3.71-6.11 mM at the endofthe infusion.
`
`Par Pharmaceutical, Inc. Ex. 1007
`Par v. Horizon, IPR of Patent Nos. 9,254,278, 9,095,559, and 9,326,966
`Page 2 of 5
`
`

`

`BENZOATE AND PHENYLACETATE IN HYPERAMMONEMIA
`
`1119
`
`Plasma phenylacetylglutamine values were stable in [80- and
`270-min samples in two subjects butstill increasing in three at
`270 min (Fig. 3). Mean value thus peaked at 270 min; concen-
`tration peaks (0.48, 0.22~1.06 mM)were abovethose of hippur-
`ate after alanine + benzoate (p = 0.008) (Figs. 2 and 3). Phen-
`ylacetylglutamine/phenylacetate percentages (linear means +
`
`ALA + BA
`
`ALA +BA
`
`3
`
`
`
`TIME (h)
`
`Fig. 2. Plasma benzoate and hippurateafter alanine + benzoate loads
`in patients with lysinuric protein intolerance. For details see legend to
`Figure |.
`
`a
`2
`<
`<2
`sit
`ow>
`os
`<
`aE
`ase
`=
`w
`ae
`a
`
`Fig. 3. Plasma phenylacetate and phenylacetylglutamine concentrations after alanine + phenylacetate loads in patients with lysinuric protein
`intolerance. For details see legend to Figure 1.
`
`TIME (h)
`
`
`
`OROTICACIDEXCRETION
`
`(umol/kg/h)
`
`
`
`ALA+ BA
`
`ALA +PAA 12-18
` Do
`
`
`O-6
`
`12-18
`6-12
`
`18-24
`
`6-12
`COLLECTION HOURS
`
`18-24
`
`0-6
`
`$218
`6-12
`
`18-24
`
`Fig. 4. Urinary orotic acid excretions after alanine, alanine + benzoate, and alanine + phenylacetate infusions in patients with lysinuric protein
`intolerance. Urine wascollected for four consecutive 6-h periods. For details of the infusions see legend to Figure 1.
`
`Par Pharmaceutical, Inc. Ex. 1007
`Par v. Horizon, IPR of Patent Nos. 9,254,278, 9,095,559, and 9,326,966
`Page 3 of 5
`
`SD), 7+ 2, 12+ 4, and 19 + 10 at the three measurement points
`(Fig. 3), were clearly above the hippurate/benzoate percentages
`after alanine + benzoate infusions(Fig. 2).
`Plasma aminoacids. Plasma alanine concentrations at the
`four measurement times and peak increments (5.86, 3.56-9.67;
`6.39, 2.99-13.65 and 4.66, 1.05-20.70 mM)differed insignifi-
`cantly after the three loads. Infusion of alanine caused aninitial
`rise in plasma glycine concentration in all patients, whereas
`glycine increased in only three patients after alanine + benzoate
`and in four patients after alanine + phenylacetate. The peak
`incrementsin plasma glutamine + glutamic acid were 1.08, 0.25-
`4.70; 0.84, 0.26-2.71; and 0.22, 0.16-2.99 mM (NS)after the
`three loads, respectively. Plasma proline concentrations increased
`and plasmacitrulline rose slightly after alanine and alanine
`benzoate loads but decreased after alanine + phenylacetate.
`Arginine, ornithine, and lysine remained unchanged. Plasma
`concentrations of several other amino acids were slightly higher
`at 120 min as compared with the fasting value but responses
`were similarafter all three loads.
`Orotic acid excretion. The patients excreted 10 times more
`orotic acid during the first 6-h collection after the alanine load
`(5.62, 1.84-13.14 wM/kg per h) (Fig. 4) than they do in their
`daily urine (17). Addition of benzoate or phenylacetate to the
`infusion decreased this value to 19% (1.07, 0.04-25.6 umol/kg
`per h, p = 0.151) and 49% (2.74, 0.01-16.25 yzmol/kg per h, p
`= 0.016) respectively, of alanine alone.
`Urea excretion. Twenty-four-h urea excretion was 8.36, 4.51-
`15.49 mmol/kg after alanine, as compared with 9.30, 6.12-14.13
`mmol/kg after alanine + benzoate (NS) and 7.99, 4.22-15.15
`mmol/kg after alanine + phenylacetate (NS) with highest values
`in the first two 6-h collections (data not shown).
`
`Ww
`z
`=
`<
`5
`aon
`Sat SS.
`n> os
`<t
`E
`JOE
`aor
`a!
`>
`z
`Ww
`
`=a
`
`1.0
`
`ALA + PAA
`
`
`
`
`
` o—— 0 PLASMABENZOICACID(mmol/l)
`
`
` PLASMAHIPPURICACID(mmol/!)
`
`ALA + PAA
`
`

`

`1120
`
`SIMELL ET AL.
`
`Benzoate and hippurate excretion. Less than 2% of the admin-
`istered dose of benzoate appeared unchangedin the urine in 24
`h. Hippurate excretion peaked in the first 6-h collection, de-
`creased steadily and then wasin the range of basal excretion in
`the last collection (Fig. 5). The combined amountof benzoate
`and hippurate excreted in 24 h (1.33, 0.71-2.52 mmol/kg)
`accounted for 67, 35-126% ofthe benzoate dose given.
`Phenylacetate and phenylacetylglutamine excretion. The
`amount of phenylacetate excreted unchanged in 24 h was 61, 9-
`425 pmol/kg or 3, 0.4-21% of the dose infused. Excretion peaked
`in the first collection period, and approached zero values in the
`third and fourth collections.
`For unknown reasons, measured urinary phenylacetylgluta-
`mine excretion in one patient markedly exceeded the infused
`dose of phenylacetate (Fig. 5). When her data are deleted from
`the results, 809, 297-2200 umol or 40, 15-110% of infused
`phenylacetate was excreted as phenylacetylglutamine in 24 h.
`The major part of phenylacetylglutamine excretion occurred
`within 12 h but small amounts continued to be excreted even in
`the last collection.
`Molar excretions of the acylation products (hippurate and
`phenylacetylglutamine) by the patient group were almost iden-
`tical (Fig. 5) but “hippurate excretors” and “phenylacetylgluta-
`mine excretors” differed. Further, excretions of the acylation
`products did notcorrelate with orotic acid excretions, or peak or
`mean blood ammoniclevels in individual patients.
`Amino acid excretion. Mean 24-h alanine excretion was less
`than 2% of the administered dose and identical after the three
`
`
`
`HIPPURICACIDEXCRETION
`
`(mmol/kg/h)
`
`EXCRETION 0-6
`PHENYLACETYLGLUTAMINE
`
`D
`
`ALA+ PAA
`
`(mmol/kg/h)
`
`loads. Excretion of alanine and other amino acids, including
`ornithine, arginine, and lysine was increased in the first collec-
`tion, but returned to basal rates (data not shown).
`Relative nitrogen excretions. The major part of urinary nitro-
`gen output occurred as urea, but excretion as hippurate after
`alanine + benzoate and as phenylacetylglutamineafter alanine
`+ phenylacetate formed 11.5 and 22.1% ofthe urea nitrogen in
`the first collection period, respectively (Table 1). The excretion
`of nitrogen in other measured constituents of the urine, including
`orotic acid, was small after all loads.
`
`DISCUSSION
`
`Toxic dosage or plasma concentrations associated with acute
`toxicity after intravenous benzoate or phenylacetate are currently
`poorly known. Theclinical responses (dizziness, nausea, vomit-
`ing) in our patients after the infusions ofalanine + benzoate and
`alanine + phenylacetate suggest that the 2.0 mmol/kg dose is
`likely to cause symptomsin the majority of subjects. However,
`simultaneous hyperammonemia may have influenced the clini-
`cal symptoms. A dose between |1.5—1.8 mmol/kg, given iv in 90
`min during a hyperammonemiccrisis is probably safe, and the
`plasma concentrations reached should perhaps not exceed 4.0-
`4.5 mM for benzoate or 3.0-3.5 mM for phenylacetate (calcu-
`lated by extrapolating the mean plasma curves ofthese substances
`after the loads to the assumed peak concentration at the end of
`the infusion, minus 30%). The calculated iv dose is within the
`range now safely used in patients with urea cycle enzymedefi-
`ciencies (8). Plasma concentrations measured in such patients
`imply that somewhat higher peak phenylacetate values (exceed-
`ing 4 mM) are acceptable.
`Thety,2 values in plasma for both benzoate and phenylacctate
`were short and almost identical, suggesting fast metabolism and
`distribution into tissues. Our results clearly indicate that the
`main metabolic pathway of these substances is via conjugation
`to hippurate and phenylacetylglutamine, respectively. Compari-
`son of plasma benzoate and hippurate concentrations after the
`alanine + benzoate infusions (Fig. 2) suggests that benzoate
`concentration was notrate-limiting for acylation and lower con-
`centrations might be as efficient for hippurate production with-
`out causing side effects. Plasma phenylacetate values after the
`alanine + phenylacetate infusions also remained high for a long
`period, and excretion ofthe acylation product continued for even
`longer.
`The 6.6 mmol/kg dose of L-alanine iv has been experimentally
`found suitable for induction of mild hyperammonemiain LPI
`(10). In theory, addition of 2.0 mmol/kg of benzoate or phenyl-
`acetate to the load optimally leads to stoichiometric elimination
`of 2 and 4 mmol/kg of nitrogen as hippurate and phenylacetyl-
`glutamine, respectively. Because lower doses of alanine are tol-
`erated without hyperammonemia in LPI, we assumed that these
`benzoate and phenylacetate doses, despite of the apparent non-
`
`12-18
`6-12
`18-24
`COLLECTION HOURS
`
`0-6
`
`12-18
`18-24
`
`6-12
`
`Fig. 5. Urinary hippurate excretion after alanine + benzoate loads
`and phenylacetyiglutamine excretion after alanine + phenylacetate loads
`in patients with lysinuric protein intolerance. For details see the legend
`to Figure |.
`
`Table 1. Nitrogen excretion (umol/kg body wt perh, or percent ofnitrogen excreted in urea) in five patients with lysinuric protein
`intolerance in thefirst 6-h collections afier loads ofalanine, alanine + benzoate, and alanine + phenylacetate*
`Load
`
`Alanine -
`784; 364-1970
`
`_ %
`100
`
`Alanine + benzoate
`1100; 400-3020
`126; 29-542
`
`N excreted in
`Urea
`Hippurate
`Phenylacetylgluta-
`mine
`Orotic acid
`Alanine
`Glycine
`GJutamine + glutamic
`acid
`10.4; 1.3-85.0
`1.1
`8.7; 6.5-11.6
`Lysine
`14.6; 2.3-92.5
`1.1
`8.6; 10.8-26.1
`Creatinine
`* Values are log means; 95% confidence limits. Percentages are calculated for the means.
`
`11.2; 3.7-34.3
`11.8; 0.8-167.2
`3.6; 0.3-55.9
`$8.7; 3.5-101.1
`
`1.4
`1.5
`0.5
`2.4
`
`2.1; 0.1-51.2
`16.2; 2.0-129.2
`1.8; 0.1-43.3
`23.9; 1.3-92.5
`
`% _ _Alanine + phenylacetate
`100
`841; 396-3020
`11.5
`1.6; 0.2-11.1
`186; 28-1200
`
`0.2
`1.5
`0.2
`1.3
`
`1.0
`1.3
`
`5.5; 0.0-32.5
`14.2; 1.9-104.4
`1.9; 1.5-19.9
`4.5; 0.5-38.1
`
`14.4; 4.0-Si.1
`17.4; 8.4-35.9
`
`%
`100
`0.2
`22.1
`
`0.7
`Ld
`0.2
`0.5
`
`1.7
`2.1
`
`Par Pharmaceutical, Inc. Ex. 1007
`Par v. Horizon, IPR of Patent Nos. 9,254,278, 9,095,559, and 9,326,966
`Page 4 of 5
`
`

`

`BENZOATE AND PHENYLACETATE IN HYPERAMMONEMIA
`
`1121
`
`REFERENCES
`
`Acknowledgments. Theskillful technical assistance of Ms. Mar-
`jatta Viikari, Ellen H. Gordes, and Evelyn Bull is acknowledged.
`The authors thank Huguette Rizziéro and Lynne Prevost (McGill
`University-Montreal Children’s Hospital Research Institute) for
`preparing the typescript.
`
`stoichiometry, would prevent the induced hyperammonemia.
`Instead,in this study, blood ammonia values and urinary orotic
`acid excretion were only moderately diminished by addition of
`benzoate or phenylacetate to the alanine load, suggesting that
`hyperammonemia, carbamyl phosphate, and, subsequently, or-
`otic acid production occur rapidly after alanine infusion and are
`notefficiently abolished by simultaneous infusion of the acylat-
`ing agents. Induction of hyperammonemia by protein intake (5
`9, 21) probably leads to more even distribution of nitrogen in
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`J Biol Chem 18:225-231
`metabolic compartments and, consequently, may be associated
`2. Shiple GT, Sherwin CP 1922 Synthesis of amino acids in animal organisms.I.
`with better responses to treatment with the acylating agents.
`Synthesis of glycocoll and glutamine in the human organism. J Am Chem
`Interestingly, correlation of orotic acid excretion with blood
`Soc 44:618-624
`ammonia was good in these experiments as in previous studies
`3. Brusilow S, Valle DL, Batshaw ML 1979 New pathways of nitrogen excretion
`in inborn errors of urea synthesis. Lancet 1:452-454
`(22-23).
`4. Brusilow S, Tinker J, Batshaw ML 1980 Aminoacid acylation: A mechanism
`Our findings of the excretion rates of hippurate and pheny!-
`of nitrogen excretion in inborn errors of urea synthesis. Science 207:659-
`acetylglutamine after the loads differ somewhat from earlier
`661
`findings in healthy subjects. Lewis’s volunteer, receiving 10 g of
`5. Batshaw ML,Painter MJ, Sproul GT, Schafer IA, Thomas GH, Brusilow $
`1981 Therapy of urea cycle enzymopathies: Three case studies. Johns Hop-
`sodium benzoate orally, excreted 23% of urinary total nitrogen
`kins Med J 148:34-40
`as hippurate, but an equimolar decrease occurred in urea +
`6, Smith I 1981 The treatment ofinborn errors of the urea cycle. Nature 291:378-
`ammonia nitrogen excretions. We did not measuretotal nitrogen
`380
`7. Batshaw, ML, Brusilow S, Waber L,Blom W, Brubakk AM, Burton B, Cann
`Outputin our patients, but hippurate nitrogen excretion was 12%
`H. Kerr D, Mamunes P, Myerberg D. Schafer 1 1982. Treatment of inborn
`of urea nitrogen excreted in the first 6 h after the load. We also
`errors of urea synthesis. N Engl J Med 306:1387-1392
`found that 54% ofthe single phenylacetate dose was excreted as
`8. Brusilow SW, Danncy M, Waber LJ, Batshaw M, Burton B, Levisky L. Roth
`phenylacetylglutamine in 24hafter the load. Earlier Ambrose e¢
`K, McKeethren C, Ward J 1984 Treatment of episodic hyperammonemia
`al, (24), after having administered 5-7 g/day of phenylacetate to
`in children with inborn errors of urea synthesis. N Engl J Med 310:1630-
`1634
`a healthy man, found 98% ofthe dose in urine as phenylacetyl-
`9. Brusilow W 1984 Arginine, an indispensable amino acid for patients with
`glutamine. In another study (25), 91% of the 85 mg/kg dose was
`inborn errors of urea synthesis. J Clin Invest 74:2144-2148
`excreted in the urine as phenylacetylglutamine.
`10. Simeil O, Perheentupa J, Rapola J, Visakorpi JK, Eskelin LE 1975 Lysinuric
`In patients with urea cycle enzyme deficiency who have re-
`protein intolerance. Am J Med 59:229-240
`11. Rajantie J, Simell O. Rapola J. Perheentupa J |980 Lysinuric protein intoler-
`ceived benzoate or phenylacetate treatment with proper meas-
`ance: a two-yeartrial of dietary supplementation therapy with citrulline and
`urement of nitrogen excretion (3-5, 7-9), urea excretion re-
`lysine. J Pediatr 97:927-932
`mained unchanged even though the total nitrogen excretion
`12. Rajantie J. Simell O. Perheentupa J |980 Intestinal absorption in lysinuric
`increased by approximately the amount accountable for hippur-
`protein intolerance: impaired for diamino acids, normalfor citrulline. Gut
`21:519-524
`ate or phenylacetylglutamine, just as in our experiments. The
`13. Rajantie J, Simell O, Perheentupa J 1980 Basolateral membrane transport
`cause for the discrepancy in the urea excretions after administra-
`defect for lysine in lysinuric protein intolerance. Lancet 1:1219-1221
`tion of benzoate or phenylacetate in healthy subjects and in
`14. Desjeux J-F. Rajantie J, Simell O, Dumontier A-M., Perheentupa J 1980
`patients with urea cycle failure remains open. However,it sug-
`Lysine fluxes across the jejunal epithelium in lysinuric protein intolerance.
`J Clin Invest 64: 1382-1387
`gests that urea excretion in urea cycle diseases (often with a close
`15. Simell O, Perheentupa J 1974 Renal handling of diaminoacids in lysinuric
`to zero enzymeactivity of the cycle in vitro) cannot be easily
`protein intolerance. J Clin Invest 54:9-17
`changed and represents nitrogen coming from another pool than
`16. Simei! O, Perheentupa J 1974 Defective metabolic clearance of plasmaarginine
`the oneaffected in similar experiments in healthy subjects (8, 9).
`and ornithine in lysinuric protein intolerance. Metabolism 23:691-701
`17. Simell O 1975 Diamino acid transport into granulocytes and liverslices of
`After a single dose of benzoate or phenylacetate with alanine,
`patients with lysinuric protein intolerance. Pediatr Res 9:504-508
`the acylation products formed [2 and 22% ofthe urea nitrogen
`18. Rajantie J, Simell O, Perheentupa J 1983 “Basolateral” and mitochondrial
`in the urine immediately after the dose. During prolonged ad-
`membranetransport defect in the hepatocytes in lysinuric protein intoler-
`ance. Acta Paediatr Scand 72:65-70
`ministration the percentages are probably higher (3-5, 7-9). The
`19. Rajantie J 1981 Orotic aciduria in lysinuric protein intolerance: dependence
`fast disappearance from plasma of both benzoate and phenylac-
`on the urea cycle intermediates. Pediatr Res |5:115-119
`ctate suggests that frequent oral doses or continuousinfusion are
`20. Proelss HF, Wright BW 1973 Rapid determination of ammoniain a perchloric
`optimal ways for their administration.
`acid supernate from blood, by use of an ammonia-specific electrode. Clin
`Chem 19:1162-1169
`In summary,this study showsthat waste nitrogen excretion as
`21. Brusilow SW, Valle DL 1985 Identification of heterozygosity for ornithine
`hippurate or phenylacetylglutamine can be induced in patients
`transcarbamylase deficiency (OTCD). Pediatr Res 19:244A(abstr)
`with LPI by infusion of benzoate or phenylacetate. Neither one
`22. Kesner I 1965 The effect of ammonia administration on orotic acid excretion
`of these substances, when added to an iv alanineinfusion, is able
`in rats. J Biol Chem 240:1722-1724
`to abolish the resulting hyperammonemia andorotic aciduria in
`23. Milner JA, Visek WJ 1975 Urinary metabolites characteristic of urea-cycle
`aminoacid deficiency. Metabolism 24:643-651
`the patients. In hyperammonemiccrisis, addition of benzoate,
`24. Ambrose AM, Power FW, Shervin CP 1933 Further studics on the detoxifica-
`phenylacetate, or both to the iv ornithine or arginine or oral or
`tion of phenylacetic acid. J Biol Chem 101:669-675
`iv citrulline treatment of LPI (10,
`| 1) may prove helpful, just as
`25. James MO, Smith RL, Williams RT, Reidenberg M 1972 The conjugation of
`they are effective in hyperammonemic urea cycle enzyme defi-
`phenylacetate acid in man, sub-human primates and some none-primate
`species. Proc R Soc Lond [Biol] 182:25-35
`ciencies (3-9).
`
`Par Pharmaceutical, Inc. Ex. 1007
`Par v. Horizon, IPR of Patent Nos. 9,254,278, 9,095,559, and 9,326,966
`Page 50f5
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