0090-9556/04/3201-10—19$20.00
`DeauG METABOLISM AND Disposition
`Copyright © 2004 by The American Society for Pharmacology and Experimental Therapeutics
`DMD 32:10-19, 2004
`
` LISL/LLILL&?7
`Printed in ULS.A,
`
`NEW SECONDARY METABOLITES OF PHENYLBUTYRATE IN HUMANS AND RATS
`
`Takhar Kasumov, Laura L. Brunengraber, Blandine Comte, Michelle A. Puchowicz,
`Kathryn Jobbins, Katherine Thomas, France David, Renee Kinman, Suzanne Wehrli,
`William Dahms, Douglas Kerr, Itzhak Nissim, Anp Henri Brunengraber
`
`Departments of Nutrition (T.K., L.L.B., B.C., M.A.P., K.J., K.T., F.D., R.iK., H.B.) and Pediatrics (R.K., W.D., D.K.), Case Western
`Reserve University, Cleveland, Ohio; and Children’s Hospital (S.W.) and Department of Pediatrics (I.N.), University of
`Pennsyivania, Philadelphia, Pennsylvania
`
`(Received May 14, 2003; accepted September 2, 2003)
`
`This article is available online at http://dmd.aspetjourals.org
`
`ABSTRACT:
`
`Phenylbutyrate is used to treat inborn errors of ureagenesis, ma-
`lignancies, cystic fibrosis, and thalassemia. High-dose phenylbu-
`tyrate therapy results in toxicity, the mechanism of whichis unex-
`plained. The known metabolites of phenylbutyrate are phenylacetate,
`phenylacetylglutamine, and phenylbutyrylglutamine. These are ex-
`creted in urine, accounting for a variable fraction of the dose. We
`identified new metabolites of phenylbutyrate in urine of normal
`humans and in perfused rat livers. These metabolites result from
`interference between the metabolism of phenylbutyrate and that
`of carbohydrates and lipids. The new metabolites fall into two
`
`
`categories, glucuronides and phenylbutyrate B-oxidation side
`products. Two questions are raised by these data. First, is the
`nitrogen-excreting potential of phenylbutyrate diminished by in-
`gestion of carbohydrates or lipids? Second, does competition
`between the metabolism of phenylbutyrate, carbohydrates, and
`lipids alter the profile of phenylbutyrate metabolites? Finally, we
`synthesized glycerol esters of phenylbutyrate. These are par-
`tially bioavailable in rats and could be used to administer large
`doses of phenylbutyrate in a sodium-free, noncaustic form.
`
`Sodium phenylbutyrate (PB') is a highly effective drug for the
`treatment of patients with hyperammonemia resulting from inborn
`errors of urea synthesis (Batshawet al, 1981, 2001; Brusilow, 1991).
`These patients excrete nitrogen as phenylacetylglutamine (PAGN)
`(Batshawct al., 1981). The latter is also formed whenthe patients are
`treated with phenylacetate (PA). However, PB is preferred as a
`prodrug of PA because it does not have the foul smell of the latter. In
`addition, PB shows promise Jor the treatment of cystic [ibrosis be-
`cause it increases frans-membrane chloride conductance (Rubenstein
`and Zeitlin, 2000; Zeitlin et al., 2002), Also, PB is used in clinical
`trials for the treatment of sickle-cell anemia and thalassemia because
`
`induces the formation offetal hemoglobin (Dover et al., 1994:
`it
`Hoppe et al., 1999), Lastly, PB is used in clinical trials as a cytostatic
`antineoplastic agent, because it inhibits histone deacetylases and po-
`tentiates the effect of cytotoxic agents on tumors (Samid etal., 1997;
`Gilbert et al., 2001).
`
`This work was supported by the NationalInstitutes of Health (Research Grants
`DK581 26, GA79495, and DK53761, and Training Grant DKO7319) and the Cleve-
`land Mt. Sinai Health Gare Foundation.
`' Abbreviations used are: PB, 4-phenyloutyrate; PAGN, phenylacetylglu-
`tamine; PA, phenylacetate; PRGN, 4-phenylbutyryigiutamine; PHB, 3-hydroxy-4-
`phenylbutyrate; PrC, 4-phenyl-trans-crotonate; PKB, 4-phenyl-3-ketobutyrate;
`TMS,
`trimethylsilyl; GC-MS, gas chromatography-mass spectrometry; SW,
`sweep width; TD, data points; COSY, correlation spectroscopy; HSQC, hetero
`nuclear single-quantum coherence; AUC, area under the curve.
`
`Address correspondence to: Henri Brunengraber, Department of Nutrition,
`Room 280, Case Wester Reserve University, 11000 Cedar Ad., Claveland OH
`44106-7139. E-mail: hxo8@cwru.edu
`
`The clinical effectiveness of PB in some ofthese situations is
`
`limited by occasional incidences of toxicity at high doses (Carducci et
`al., 2001; Gore et al., 2002). Concerns have been raised by clinical
`investigators who treat patients with large doses of PB as a sodium
`salt. First. the total amount of PB and its known metabolites exereted
`in urine (PA, PAGN) is less than the administered PB dose, some-
`times as low as 50%. Some of the unknown metabolites might
`contribute to PB toxicity at high doses. Second, the large sodiumload
`of the treatment is potentially dangerous for patients with impaired
`cardiac and/or renal function. Third, the causticity of sodium PB can
`result in esophagal and/or gastric distress, even when it
`is adminis-
`tered as a powder suspended in water (this has been extensively
`debated on the Internet discussion group Metab-L at http://lists.fran-
`ken.de/mailman/listinfo/metab-L). Neither the biochemical mecha-
`nism(s) of PB toxicity nor the identity of the missing metabolites of
`PB is known. Also. it is not clear whether the metabolism of PB (a
`modified fatty acid) interferes with, or is influenced by, the metabo-
`lism offatty acids and carbohydrates present in foodstuffs. Lastly, it
`is not known whether stimulation of lipolysis under stress conditions
`interferes with PB metabolism.
`
`interest in PB metabolism was related to it being a
`Our original
`precursor of PAGN, which can be used as a noninvasive probe of the
`"SC. or
`'C-labeling pattern ofcitric acid cycle intermediates in
`human liver (Magnusson et al.. 1991: Yang et al., 1996). As part of
`these investigations, we recently identified phenylbutyrylglutamine
`(PBGN) as a new metabolite of PB (Comte et al., 2002). PBGN is
`presumably formed from the reaction of PB-CoA with glutamine, by
`analogy with the formation of PAGN from the reaction between
`PA-CoA and glutamine (Websteret al., 1976). In normal adult sub-
`
`10
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`Horizon Exhibit 2018
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`Par v. Horizon
`Par v. Horizon
`IPR2017-01768
`IPR2017-01768
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`NEW PHENYLBUTYRATE METABOLITES
`
`cm
`
`©
`xX ,
`o
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`qa = Oo
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`OH )NaOD/D,0
`HC $
`2SNABD,
`
`R,S-|2,2,3,4,4-7H5|-PHB
`
`1) Na
`2) Hel
`
`PKB
`
`(-pip-ciIM
`
`OH
`
`“1006=
`
`ry
`
`Phenylacetone
`
`Need,
`
`D
`
`OH
`
`1-Phenyl-2-[2H propanol
`R-PHB
`Fic. 1. Scheme jor the svathesis of unlabeled and deuterated standards.
`
`1977).
`and Tanaka,
`(Ramsdell
`analog
`unlabeled
`the
`for
`ously
`jects who ingested a fairly low dose of PB (5 g/75 kg), we found that
`[711,]Phenylacety! chloride was prepared by activation of [7[,]phenylacetic
`the total excretion of PB + PA + PAGN + PBGNaccounted for only
`acid with freshlydistilled dichloromethy] methyl ether and used immediately
`half the ingested PB dose (Comte et al., 2002). The missing fraction
`after evaporation of excess dichloromethyl methyl ether and of the methyl
`of the dose may be disposed of either in urine as unknown metabo-
`chloroformate byproduct. The synthetic protocol for preparation of PHB, PKB,
`lites, or in feces as unabsorbed PB and/or PB metabolites.
`(H.JPHB,phenylacetone. and |-pheny!-2-[2-*H]propanolis outlined in Fig. 1.
`In the present study, we report
`the identification of additional
`Ethyl 4-Phenyl-3-ketobutyrate (2; Fig. 1) was synthesized by a method
`metabolites of PB in humans. i.e., R- and S-3-hydroxy-4-phenylbu-
`adapted from Capozzi et al. (1993). The commercial isopropylidene malonate
`tyrate (PHB), phenylacetone, and |-pheny|-2-propanol, as well as PA
`(2,2-dimethy]-4.6-diketo-|.3-dioxane, also called Meldrum’s acid; Aldrich
`and PB glucuronides. We studied the mechanism of PHB formation
`Chemical Co., Milwaukee, WI) was reacted with phenylacetyl chloride in the
`from PB in perfusedrat livers and identified two additional metabo-
`presence of dry pyridine in anhydrous methylene chloride. The crude pheny-
`lacetylated Meldrum’s acid (compound 1, Fig. 1) was refluxed in absolute
`lites, 4-phenyl-tais-crotonate (PYC) and 4-phenyl-3-ketobutyrate
`ethanol until evolution of CO, ceased (about 3 h). After evaporation of the
`(PKB), Lastly, we prepared sodium-tree esters of PB and investigated
`solvent, ethyl 4-phenyl-3-ketobutyrate was purified onasilica gel column.
`their bioavailability in rats. Glycerol-PB esters appear promising for
`4-Phenyl-3-ketobutvric acid (PRB). A 10% molar excess of | N NaOll
`the administration of large amounts of PB without the corresponding
`solution was added slowly to ice-cooled ethyl 4-phenyl-3-ketobutyrate and
`sodium load.
`stirred at room temperature until the organic phase disappeared (approximately
`12 h). The solution was acidified with | N HC] to pH 2 and extracted three
`limes with 3 volumes of ethyl ether. Afler drying over Na.SQ,, the solvent was
`evaporated to give the white solid product (yield 94%, m.p. 70°C).
`'H NMR
`(300 MHz, 6, CDCI): keto, 3.33 (s, 2H, CH,;COO), 3.78 (s, 2H, CH;Ph),
`7.7 7.33 (m, 5H, Ph); enol, 4.82 (s, 1H, CH), 12.13 (s, 1H, OH); keto/enol =
`7.8:1. °C NMR (75 MHz, 6, CDCI,): 48.36 (CH,COO), 49.87 (CH,Ph),
`127.41 (C-4 Ph), 128.73 (C-3, C-5 Ph), 129.50 (C-2, C-6 Ph), 133.14 (C-ipso,
`Ph), 169.26 (COQ), 201.57 (CO).
`The identity of the product wasalso confirmed by |) reduction of 4-pheny!-
`3-ketobutyrate with NaBH,, 2) extraction of &,5-PHB with ethyl] acetate, and
`3) TMS derivatization and NH,-positive chemical
`ionization GC-MS. The
`mass spectrum ofthe derivative was identical to that of PHB synthesized as
`described below. The 4-phenyl-3-ketobutyric acid was stored at —80°C to
`prevent decomposition. Just before use in liver perfusion experiments, the acid
`was dissolved in waler and Lilrated to pH = 7.40.
`R,S-3-Hydroxy-4-phenylbutyrie acid (PHB), Ethyl] 4-phenyl-3-ketobutyrate
`(2.06 g, 10 mmol) was mixed with 10 ml of H,O and a calculated amount of
`NaOH/H,0was added dropwise with cooling to give 0.5M solution of [OH].
`
`Materials. All chemicals used in syntheses, general chemicals, and solvents
`were obtained from Sigma-Aldrich (St. Louis, MO). All organic solvents were
`dried and distilled immediately before use. ["H;|Phenylacetic acid (99%) and
`(PH,Jbenzene were purchased from Isotee Inc. (Miamisburg, OH), The derivati-
`zation agent N-methyl-N-(trimethylsilyltrifluoroacctamide was supplied by Regis
`Technologies, Inc. (Morton Grove, IL}. All aqueous solutions were made with
`water purified with the Milli-Q system (Millipore Corporation, Bedford, MA).
`Preparation of Unlabeled and Deuterated Standards. §-2-Phenylbutyry!
`chloride was prepared by reacting 5-2-phenylbutyric acid with SOCI,, and was
`vacuum-distilled and stored at 4°C as a 0.5 M solution in benzene. 7H.JPB
`was prepared by aluminum chloride-catalyzed condensation of -y-butyrolac-
`tone with [H,Jhenzene as previously described (Comte et al., 2002). y-Phe-
`nyl-trans-crotonic acid was prepared by a Fridel-Crafts reaction of benzene
`with ethyl y-bromo-rrans-crotonate, followed by acid hydrolysis of ethyl
`y-phenyl-tans-crotonate (Loffler et al., 1970). The srans configuration of the
`product was confirmed by 'H NMR. [*H,]Phenylacetylglycine was synthe-
`sized by reacting [°H;]phenylacety] chloride with glycine, as described previ-
`
`Materials and Methods
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`12
`
`KASUMOVET AL.
`
`NaBH,(0.372, 10 mmol) was added and the mixture stirred for 24 h. After the
`reaction mixture was cooled w 0°C, the pH was brought to 7.0 with HC] (6N}
`and more than halfof water removed by lyophilizer. The pH was brought to 2
`by HCI (6 N) andthe solution extracted 3 times with 3 volumes of ethy] ether.
`After drying the combined ether extract. the ether was evaporated giving a
`white solid product. Yield 79%. Purity of product was assayed by GC-MSafter
`derivatization with TMS.
`R-3-Hydroxy-4-phenylfniyrate (R-PHB) was prepared by reducing the cor-
`responding 4-phenyl-3-ketobutyric acid with #-chlorodiisopinocamphey|lbo
`rane [(—)-DIP-Cl] (Wang et al., 1999). The yield was 89% from PKB;
`enantiomeric excess was 97% [GC-MS of the methy] S-2-phenylbutyry] de-
`rivative (see below). and purity was confirmed by NMR).
`R,S-3-Hydroxy-4-phenyl/2,2,3,4,4-H<Jbutyrate
`(R,S-[°HJPHB). 4-Phe-
`nyl-3-ketobutyrie acid (0.267 g, 1.5 mmol) was suspended in 3 ml of 7H,Q
`(99.9%) to which 0.36 ml of 40% of NaO*H (3.6 mmol) in 7H,O was slowly
`added at 0-S°C. This procedure exchanges 'H for 7H atoms on the methylene
`groups adjacentto the carbonyl. The solution was stirred at room temperature
`overnight and then lyophilized. The residue wasdissolved in 3 ml of 7H,Oand
`stirred for another 3 h at room temperature. The solution was cooled onice,
`treated with NaB7IL,
`(63 mg, 1.5 mmol}, and stirred overnight at room
`temperature (Des Rosiers et al., 1988). After acidification to pH |
`to 2 with
`TIC] (6 N}, the solution was saturated with NaCl and extracted three times with
`3 volumes ofdiethyl ether. Solvent evaporation yielded R,S-[°H,JPHB (yield
`74%, purity 99% by NMR, M5 isotopic enrichment 95% by GC-MSof the
`TMS derivative). The free acid was titrated to pH § with NaOHand stored
`frozen as a 0.5 M solution until use.
`Phenylacetone was prepared by decarboxylating phenylketobutyrate in acid
`at 100°C. It was reduced to 1-phenyl-2-[2-*I]}propanol with NaB*IL,.
`Esters of Phenylbutyrate. Dihydroxyacetone-d!-PB, glycerol-tn-PB, ri-
`bose-tetra-PB, glucose-penta-PB, and sorbitol-hexa-PB were prepared by re-
`acting the polyol/sugar with excess phenylbutyry! chloride in the presence of
`pyridine and catalytic amounts of N.N-dimethylaminopyridine. Products were
`purified by flash column chromatography on silica. To prepare glycerol-mono-
`PB, isopropylidene glycerol was reacted with phenylbutyry! chloride as above.
`and the isopropylidene group was removed bymild acidic hydrolysis in water.
`The structure and purity of all products were confirmed by 'H and ‘°C NMR.
`The structure of glycerol-mono-PB was confirmed byacetylation with acetic
`anhydride, followed by GC-MS analysis of the derivative.
`Sample Preparation. For the determination of the free acids concentration
`(PA, PHB, PEB, PrC, and PB) in perfusate, samples (0.1 ml) were spiked with
`0.17 pmol of ?H,JPA, [?H,]PB, and &,S-(7H,]PHB before deproteinization
`with 20 yl of saturated sulfosalicylic acid. The slurries were saturated with
`NaCl, acidified with one drop of 6 M HC], and extracted three times with 5 ml
`ofdiethy! ether, For the assay of conjugates of PB and PA, 0.1-ml aliquots of
`final liver perfusate or of human urine were spiked with internal standards and
`treated with 1.0 ml HCI (6 N) at 90°C overnight to hydrolyze the conjugates.
`Glucuronides of PB and PA were identified by the amount of these compounds
`released after incubation of perfusate and urine samples with B-glucuronidase
`in 0.2 M ammoniumacetate buffer, pH 5.0, overnight at 37°C.
`Bile samples were analyzed for free and total (conjugated + free) PB and
`its metabolites. Tn the first series of assays, 0.05-m1 samples af hile were spiked
`with internal standards. acidified to pH 2 to 2.5, and extracted three times with
`diethyl ether. In the second series of assays, samples spiked with internal
`standards were hydrolyzed with 0.3 ml of NaOH (6 N) at 90°Cfor 3 h before
`acidification and extraction.
`
`Phenylacetylglycine was extracted in acid and derivatized with methanol’
`HC]. For the assay of phenylacetone and 1|-pheny|-2-propanol, urine and
`perfusate samples were spiked with the structural analog 1-phenyl-
`[PH.Jethanol and then treated with NaB*H, to reduce phenylacetone to mono-
`deuterated 1-pheny-2-propanol. The labeled and unlabeled |-phenyl-2-
`propanal were assayed as TMS derivatives.
`For the assays in urine, 0.1-ml samples were spiked with 0.15 mol
`(H.JPHB.acidified to pH 1 to 2 with HCl, saturated with NaCl, andextracted
`three times with 3 ml of diethyl ether. The combined extracts were dried with
`Na,SO, and evaporated before reacting the residues with 70 jul of TMS at
`60°C for 20 min.
`For the chiral assay of PHB enantiomers (Powers et al., 1994), 0.1-ml
`samples were spiked with 2,5-PH.]PHB, and either deproteinized with 50 pl
`
`to
`ofsaturated sulfosalicylic acid (if containing proteins) or acidified to pH |
`2 with HCI (for urine}. Then,the slurries or solutions were saturated with NaC]
`and extracted three times with 3 ml of diethy! ether. The combined extracts
`were dried with Na,SO, and evaporated before reacting the residues with 0.15
`ml] of methanol/HCI for 1 h at 65°C, to derivatize the carboxy! groups ofthe
`PHB enantiomers. After cooling,
`| ml of water was added to the mixture and
`the hydroxyacid methyl ester was extracted with diethyl ether (three times in
`3 ml). After complete evaporation of the combined ether extract. 5-2-
`phenylbutyry! chloride benzene solution (0.1 ml, 0.5 M) and 0.05 ml of
`aqueous 12 N NaOH were added. After vortexing, the mixture was incubated
`for | h ona slowshaker at room temperature. The derivatives were extracted
`with ether (three times in 3 ml) and | ml of water. The combined ether phase
`was dried with Na,SO, and evaporated completely. The residue was dissolved
`in 0.1 ml of ethyl acetate, and 1 ysl was injected into the GC-MS.
`GC-MS Methods. All of the metabolites, except phenylacetylglycine, were
`analvzed as their TMS derivatives on a Hewlett-Packard 589) gas chromato-
`graph equipped with a ZB-5 capillary column (60 m * 0.23 mm id, 0.5 mm
`film thickness; Hewlett Packard. Palo Alto, CA) and coupled to a 5989A mass
`selective detector. Samples (0.2-1 jul) were injected with a split ratio 20 to
`50:1. The carrier gas was helium (1 ml/min) and nominal initial pressure was
`20.61 psi. The injector port temperature was at 270°C, the transfer line at
`305°C, the source temperature at 200°Cand quadrupole at 150°C. The column
`temperature program was: start at 100°C. hold for | min, increase by 8°C/min
`to 236°C, increase by 35°C/min to 310°C, 6 min at 310°C, After automatic
`calibration, the mass spectrometer was operated under ammonia-positive ion-
`ization mode. Appropriate ion sets were monitored with a dwell time of 25 to
`35 ms/ion, at m/z 226/233 (PA/?H,)PA), 254/259 (PB/PH.JPB), 325/330
`(PLB/PU.]PIB), and 252 (PrC). Note that PKB and phenylacetone had been
`reduced with NaB?H,
`to monodeuterated R,5-3-hydroxy-4-phenylbutyrate
`(monitored at m/z 326/330) and 1-phenyl-2-propanol (monitored at m/z 200/
`210 and/or 217/227). Also, since 1-phenyl-2-propanol was assayed with a
`standard of I-phenyl[*II,Jethanol, ions monitored forthis assay were 200/209
`or 217/226.
`Forthe analysis of chiral PIIB derivatives, the GC injector temperature was
`set at 280°C. The column (DB-5, 60 m X 0.25 mm i.d., 0.5 mm filmthickness;
`Hewlett Packard) program was modified to the initial 150°C for 2 min,
`increased by 15°C/min to 230°C, 25 min at 230°C,
`increased by 35°C to
`290°C, and held 10 min. Ions monitored were 1} 358 (M +
`18, i.e, M 4
`NH,*) for analytes and 2) 363 (M + 18 + 5, ie, M + S+NH,*) for
`(°H.JPHB. The mass spectrometer was operated under ammonia- positive
`chemical ionization and was tuned automatically.
`Phenylacetylglycine was analyzed as its methyl ester derivative using an
`OV-225 column (29 m X 0.32 mmid., 1
`yam film thickness; Quadrex
`Corporation, Woodbridge, CT). This column yielded better resolution of
`N-phenylacetylglycine methyl ester with no peak tailing. Samples (0.2—1 jl)
`were injected with a split ratio 20:1. The carrier gas was helium (constant flow:
`1.2 ml/min). The injector port temperature was at 220°C, the transfer line at
`240°C, the source temperature at 200°C, and quadrupole at 106°C. The column
`temperature program was: start at 90°C. hold for | min, increase by 10°C/min
`to 240°C, 15 min at 240°C. Aficr automatic calibration, the mass spectrometer
`was operated under ammonia-positive ionization made (pressure adjusted to
`optimize peak areas). lons monitored were 1) 208 (M + 1, ic. M + H™! and
`225 (M + 18, ie., M + NH,*) for the analyte and 2) 215 (M+7+1.M+
`7 + H*) and 232 (M + 7 + 18,M + 7 + NH,”*) for N-PH,]PA-glycine with
`a dwell time of 24 ms/ion.
`
`Areas under each chromatogram were determined by interactive computer
`integration, and corrected for naturally occurring heavy isotopes and light
`isotopic impurities in the synthesized labeled internal standards.
`NMR Spectroscopy. Proton NMR spectroscopy was performed at 400
`MHz on a Bruker Avance (Bruker, Newark. DE)DMX 400 wide-bore spec-
`trometer using a 5-mm inverse probe. Full-strength urine samples were ob-
`tained by lyophilizing 5 ml of urine to dryness. The residue was dissolved in
`0.5 ml of 7H,O, and the solution was introduced into a S-mm NMR tube. An
`external standard made of a sealed capillary containing a solution of trimeth-
`ylsilvlpropionic acid in 7H,O was introduced into the NMRtubeand used as
`chemical shift reference. Standard acquisition conditions were as follows for
`one-dimensional spectra: 45° pulse, 8-s repetition time, water saturation during
`the relaxation delay, sweep width (SW) 6775 Hz, 64K data points (TD), and
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`NEW PHENYLBUTYRATE METABOLITES
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`13
`
`32 scans of data collection. Two-dimensional correlation spectroscopy
`(COSY) spectra were obtained with the following conditions for the second
`dimension: SW 3500 Hz, TD 2K, 16 scans, and for the first dimension, 312
`increments of 278 jas zerofilled to 1K. A nonshified sinebell window was
`applied in both dimensions, and magnitude spectra were calculated. Two-
`dimensional
`'H/'*C correlations via double insensitive nuclei enhanced by
`polarization transfer (HSQC) were performed in the phase-sensitive mode
`(TPP; time-proportional receiver phase incrementation) using gradients for
`coherence selection and carbon decoupling during acquisition. The following
`conditions were used in the second dimension: SW 3200 Hz, TD 2K, 138
`scans, and in the first dimension: SW 12 kHz, 256 increments of 20.7 jus
`zerofilled to 512. A shifted sinebell window was applied in both dimensions.
`Proton-decoupled carbon spectra of the concentrated urine samples were
`obtained at 100.62 MHzin a 3-mm dual probe. Acquisition conditions were as
`follows: 20° pulse, repetition time 1.3 s. SW 25 kHz, TD 64K, 40,000 scans.
`The free induction decays were zerofilled to 128K, and a Lorentz to Gauss
`transformation (LB = —1 Hz, GB = 0.1) was applied before Fourier trans-
`formation.
`Clinical Investigation. The protocol was reviewed and approved by the
`Institutional Review Board of University Hospitals of Cleveland. All subjects
`were free of any chronic or acute illness. Women had a negative pregnancytest
`and were not breastfeeding. Seven subjects (three men, four women; 31.7 +
`3.0 vears; 171.3 = 3.4 cm: 79.5 = 3.9 kg) received detailed information on the
`purpose ofthe investigation and signed an informed consent form. After an
`overnight fast, the subjects were admitted to the Clinical Research Center at
`7:30 AM. They remained fasting until
`the completion of the study. An
`intravenous line was installed in the forearm with a saline infusion (20 ml/h)
`and a short blood sampling catheter was inserted into a superficial vein of the
`contralateral hand. The hand wasplaced in a heating box at 60°Cfor sampling
`of arterialized venous bload. At 8:00 AM, after baseline blood and urine
`sampling, each subject ingested 0.36 mmol/kg (3 g/75 kg) Na-PB. This dose
`corresponds to 11 to 17% of the doses commonly used in the treatment of
`patients with inborn errors ofurea synthesis (0.4—0.6 g-kg'+ day '). Water
`intake was adjusted to induce a diuresis of al least 100 ml/30 min. Urine
`samples were collected at 30-min intervals for the first 3 h after PB ingestion,
`and then every hour unt] 8 h. Urine samples were quickly frozen andstored
`at —80°C until analysis.
`Organ Perfusion Experiments. Livers from fed male Sprague-Dawleyrats
`kept on standard rat chow (200-230 g) were perfised (Brunengraberet al..
`1975) with recirculating Krebs-Ringer-bicarbonate buffer containing 4% bo-
`vine scrum albumin (fraction V, fatty acid poor; Intergen, Purchase, NY) and
`10 mM glucose. The bile duct was cannulated with PE 10 tubing (BD
`Biosciences, San Jose, CA) for bile collection. Throughout the 2-h experiment,
`sodium taurocholate (38 jumol/h) was infused into the perfusion reservoir to
`stimulate bile flow (Robins and Brunengraber, 1982). After 30 min of equil-
`ibration, a calculated amount of either PB, &,5-PHB, or PKB was added to the
`perfusate to set an initial concentration of 5 mM. The perfusion continued until
`120 min. The pHofthe perfusate was monitored and kept at 7.3 to 7.4 by
`adding 0.3 M NaOH, Samples of bile and perfusate were collected at regular
`intervals. For the assay of PKB, perfusate samples (2 m!) were treated imme-
`diately with 0.2 ml of 0.1 M NaB*H, in 0.1 mM NaOH to convert unstable
`PKB to stable monodeuterated &,$-PHB. Bile samples were collected every 30
`min. At the end ofthe experiment, the livers were quick-frozen with aluminum
`tongs precooled in liquid nitrogen.
`Rat in Vivo Experiments. We tested the bioavailability of two PR esters as
`a means to deliver large amounts of PB without the corresponding sodium
`load. Overnight-fasted rats (330—400 ¢) were divided into six groups (5—7 rats
`per group) forthe testing ofthree different PB preparations: Na-PB, glycerol-
`mono-PB, glycerol-tri-PB, ribose-tetra-PB, glucose-penta-PB, and sorbitol-
`hexa-PB. Each rat received one stomach gavage of the sodiumsalt orester in
`an amount that delivered 2.15 mmol PB/ke. The weighed dose for each rat was
`mixed with 3 ml of Tween and administered to the rats through a stomach
`gavage needle.
`Whole bloud samples (100-200 pal) were taken al —3, 15, 30, 60, 150, 240,
`330, 420, and 480 min from a small incision in a tail vein. Blood was collected
`in heparinized microcapillary tubes and centrifuged. The plasma (50-100 pl)
`was transferred to an Eppendorf tube and quick frozen.
`
`Results
`
`Human Study. In our previous study, we had identified PBGN in
`the plasma and urine of normal adults who had ingested a small dose
`of PB. We now report the data of additional analyses conducted on the
`same samples of humanurine. First, we subjected to NMR analysis
`two samples of urine produced by each subject before and 2 hafter
`ingestion of PB. The NMRspectrumof the second sample, but not of
`the first, was highly suggestive of the presence of a product of
`hydroxylation of the side chain of PB. In the COSY spectrumof the
`lyophilized urine dissolved in D,O (alter PB ingestion), we identified
`a proton at 4.25 ppm coupled with two CH,’s. The first CH, has
`protons al 2.87 and 2.70 ppm: the second has nearly identical protons
`at 2.4 ppm. A chemical shift at 4.25 ppmis likely corresponding to a
`proton coupled to the OH group. Therefore,
`the COSY spectrum
`revealed the presence of a metabolite having -CH,-CHOH-CH,—
`moiety. In the HSQC spectrum these proton signals correlated with
`the following carbons: CH at 70 ppm overlapping with other CH
`carbohydrate carbons, CH, at 44.6 ppm and CH, at 42.5 ppm. Lastly,
`in the aromatic region, signals at 129.6, 128.7, and 126.6 ppmcorre-
`sponded to a monosubstituted phenyl group having the sameintensity
`per carbon as the signals of the -CH,-CHOH-CH,— group. We
`therefore concluded that B-hydroxy-PB (PHB) was present in the
`urine of patients treated with PB. PHB would be a very likely
`metabolite since it would be formed via partial B-oxidation of PB to
`PHB-CoA (presumably the S-enantiomer), which would be lydro-
`lyzed to free PHB.
`‘Lo further confirm the identity of the urinary metabolite detected by
`NMR, we synthesized unlabeled and R,S[H.]PHB. The di-TMS
`derivative of synthetic R.S-PHB was analyzed by GC-MS inparallel
`with an extract of human urine (after PB ingestion) that had been
`reacted with TMS. In the sample derived from urine, we found a peak
`at the same retention time and with the same mass spectra (electron
`ionization and NH,-positive chemical
`ionization) as the standard of
`#S-PHB. In addition, the NMR spectrum of synthetic PHB had the
`same chemical shifts as the material identified in human urine. This
`
`confirmed the identity of PHB in human urine but did not yield
`information about its chirality. The chirality of excreted PHB vields
`information on the mechanismofits formation (see below).
`For the chromatographic separation of PHB enantiomers from the
`synthetic racemate, we tried various chiral hydroxyl derivatization
`reagents before selecting the combination of 1) methylation of the
`carboxyl group, and 2) reaction of the hydroxyl group with S-2-
`phenylbutyryl chloride (Powers ct al.,1994). The expected derivatives
`of A- and S-PHB were well separated, and their order of elution was
`confirmed using a sample of R-PHB that we had synthesized. R-PHB
`elutes ahead of the S-clerivative (Fig. 2).
`Chiral GC-MSanalysis of the human urine samples (after PB
`ingestion) revealed that PHB is present as an enantiomeric mixture
`with 10% R-PHB and 90% S-PHB (Fig. 2). Figure 3 shows the time
`profile of (R+8)-PHB excretion in urine after an oral holus of Na-PB.
`The excretion of (R+S)-PHB peaked at 120 to 240 min. Eight hours
`after ingestion of PB,
`the cumulative excretion of (R+5)-PHB
`(1.35 + 0.13 mmol) amounted to 4.4 = 0.56% ofthe PB dose.
`Small amounts of phenylacetone and 1-phenyl-2-propanol were
`identified in the urine samples (Table 1). Treatment of urine samples
`with B-glucuronidase increased their PB + PA content. The total
`amount of PB + PA released by B-glucuronidase amounted to 2.4 +
`0.3% of the PB dose.
`
`Perfused Rat Liver Study. The metabolism of PB was studied in
`perfused rat livers by the addition of 5 mM PBto the recirculating
`perfusate. ‘Ihe time profile of the PB concentration was curvilinear
`
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`

`

`KASUMOVET AL.
`
`TABLE 1
`
`Recavery af PB and its metabolites in human urine ( = 7) after the aral
`ingestion of0.36 mmalikg Na-P8 and production of PB metabolites hy rat liver
`(n= S) perfused with 5 mit PB
`%o of PB Uptake
`Metabolite
`Humans
`Rats
`
`Free PB
`0,97 + 0,23
`PB-#-glucuronide
`1.29 + 0.33
`Free PA
`0.26 + 0.06
`PA-glycine
`PA-§-glucuronide
`PAGN"®
`PBGK”
`15.71 + 1.63
`PHB
`4.54 + 0.29
`PKB
`5.15 + 0.59
`Pil
`3.67 + 0.23
`O.11 £ 0.007
`Phenylacetone
`Trace amounts
`0.01 + 0.001
`1-Phenyl-2-propanol
`2.97 + 0.61
`Total bile met
`Total 74,12 4 5.58 62.4 12.1
`
`
`L.11 + 0.19
`32.6 1 1.9
`21.5 © 2.4
`44> 0.56
`
`
`
`21,52 + 4.32
`7.64 = 0.87
`7.68 + 0.84
`5.25 + 0.88
`
`“ Previously identified metabolites (Comte ev af , 2002),
`
`14
`
`Abundancex10%
`
`es
`
`3 2 1 0
`
`Y (R)-PHE
`
`a () PB-giucur.
`
`# (R,S)-PHB
`© (S)-PHB
`
`* PA
`© PA-glycine
`@ PA-glucur.
`® Prc
`¥ PKB
`
`Wy Phenylac.
`
`=£a&
`
`=
`
`E =32
`
`g
`
`39.64
`
`39.68
`
`39.72
`
`39.76
`
`39.80
`
`39.04
`
`Time (min)
`
`Fic, 2. Chiral GC-MSassay of R- and $-PHB excreted in one sample of human
`urine after oral ingestion af 0.36 mmole Na-PB (hold trace).
`The sample was spiked with RS (7H; JPHB, the enantiomer profile of which is
`shown by the thin trace.
`
`0.3
`
`0.2
`
`a4
`
`a=a a£3 5
`
`30
`
`50
`
`70
`
`90
`
`110
`
`130
`
`150
`
`9
`-60
`
`o
`
`60
`
`120
`
`1800
`
`«2400
`
`300
`
`360s
`
`420
`
`480
`
`Time (min)
`
`Fic. 3, Time course of (R—S\-PHB urinary excretion in normal humansafter
`oral ingestion of 0.36 mmol/kg Na-PB (mean + S.E.M.; » = 7).
`
`(Fig. 4A). Plotting of the data under semilogarithmic coordinates
`yielded a linear relationship compatible with a first order kinetic
`process for PB uptake. The total uptake of PB amounted to 310 + 16
`pmol - (90 min~!) > liver” '.
`Figure 4B shows the time accumulation of metabolites derived
`from PB and released into the perfusate. Phenylacetylglycine is a
`
`Time (min)
`
`Fig. 4. Metabolism of PB in perfised rat livers.
`A, uptake of PB from the recirculating perfusate. B, accumulation of PB
`metabolites in the perfusate (n = 5 for all compounds except for PRB, where n =
`4).
`
`knownmetabolite of PA in rats and dogs (Knoop, 1904: Ambrose and
`Sherwin. 1933; James et al., 1972). About 16%of the uptake of PB
`was accounted for by the production of R- and S-PLIB. of which about
`90% is the S-enantiomer. We also identified PrC, PKB, phenylac-
`etone, and 1-phenyl-2-propanol, which,
`to our best review of the
`literature. have not been previously described as metabolites of PB.
`The identity of these metabolites was confirmed by GC-MS using
`standards we synthesized. Incubation ofliver perfusate samples with
`
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`

`, 0-30
`
` [ free [PHB] (mM)
`Mi
`total [PHB] (mM)
`
`|
`
`C1) free [PA] (mM)
`i ictal [PA] (mM)
`
`
`
`(0 free [PB] (mM)
`
`BH total [PB] (mM)
`
`2
`<=
`
`/
`
`== a
`
`,
`
`=
`
`Eao
`
`30-60
`
`60-90
`
`90-120
`
`time (min)
`
`Fic. 5. Biliary excretion of PB andits metabolites (R+5)-PHB and PA by
`perfused tat livers.
`The white bars showthe biliary concentrations of the free metabolites. The dark
`
`hars showthe total concent
`ns of metabolites after alkaline hydrolysis.
`
`B-glucuronidase revealed the formation of PB and PA glucuronides.
`which accounted for 22 and 5% of t