UUUU—OSSflr'lerjltll-IU 19320.00
`Draco Mr'L-unu mt .wo DismH'IIoM
`Copyright EC.- 2004 by 'l'hc .-’\1‘r.tcr|ca1l Soctcty for Pharmacology and Experimental 'l'hcrapcutlcs
`DMD 32:“) 1‘). 200d
`
`I
`Vol. 32. No.
`litil-"l l] “87
`Printed in USA.
`
`NEW SECONDARY METABOLITES 0F 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, AND Henri Brunengraber
`
`Departments of Nutrition {T.K.. L.L.B.. 8.0., MAR, K.J.. K.T., F.D., ELK. HE.) and Pediatrics (BK. Wil. D.K.), Case Western
`Reserve University. Cleveland, Ohio; and Children ’s Hospitai (S. W.J and Department of Pediatrics (LN), University of
`Pennsylvania. Philadelphia. Pennsylvania
`
`[Received May 14, 2003; accepted September 2. 2003}
`
`This article is available online at http:ir’dmdaspetjoumalsorg
`
`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 which is unex-
`plained. The known metabolites of phenylbutyrate are phenylacetate,
`phenylacetylglu’camine, 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 fl-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 phenylbutyratc (PB'} is a highly effective drug for the
`treatment of patients with hyperammonemia resulting from inborn
`errors of urea synthesis (Batshaw et at. 1981. 2001; Brusilow. 1991).
`These patients excrete nitrogen as phcuylaccty‘lglutaminc (PAGN)
`(Balshaw et al.. 1981). the latter is also fonned when the patients are
`treated with phenylacctatc {PA}. However, PB is preferred as a
`prodnig of PA because it does not have the foul smell ol'the latter. in
`addition, PB shows promise for the treatment of cystic fibrosis be-
`cause it increases irons—membrane chloride conductance (ltubcnslcin
`and Zeitlin. 2000: Zeitlin et al.. 2002). Also. PB is used in clinical
`trials for the treatment ofsickIc—ccll anemia and thalasscrnia because
`
`l994:
`induces the Formation of fetal hemoglobin (Dover et at,
`it
`[loppe at al._. I999). Lastly. PB is used in clinical trials as a cytostatic
`antineoplastie agent. beeause it inhibits his-tone deacetylases and po—
`tcntiatcs the effect of cytotoxic agents on tumors (Samid et al.,
`I997":
`Gilbert ct al.. 2001).
`
`This work was supported by the National Institutes of Health [Research Grants
`DK58‘i EB. GATEMQE, and DK53?51 . and Training Grant DKDT319} and the Cleare—
`land Mt. Sinai Health Care Foundation.
`' Abbreviations used are: PB. 4—phenylbutyrate; PAGN, phenylacetylglu—
`tamine; PA, phenylacetate: PEIGN, 4-phenyIbLIIyrylglLrtamine: PHB. 3-hydroxy-rl—
`phenylbutyrate; PIC. 4—phenyI-trans-crotmate; PKB, 4-phenyl-3-ltetobutyrate;
`TMS.
`trimethylsilyl'. GC—MS. gas chromatography—mass spectrometry: SW.
`sweep width: TD. data points; COSY. correlation spectroscopy; HSDC. hetero
`nuclear single-quantum coherence; AUG, area under the curve.
`
`Address correspondence to: Henri anengraber, Depaflment of Nutrition,
`Room 280, Case Westem Reserve Universrty, 11000 Cedar Rd. Cleveland OH
`441. 06-?139. E-mail: hxoflflcwmcdu
`
`The clinical effectiveness of PE in some of these situations is
`
`limited by occasional incidences of toxicity at high doses (Carducci el
`al.. 2001; Gore et at. 2003). Coneems have been raised by clinical
`investigators who treat patients with large doses of PB as a sodiiun
`salt. First. the total amount of PB and its known metabolites excreted
`in urine (PA. PAGX) 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 sodium load
`of the treatment is potentially dangerous for patients with impaired
`cardiac andior renal Function. Third. the causticity of sodium PB can
`result in csophagal andjor gastric distress. even when it Is adminis—
`tered as a powder suspended in water (this has been extensively
`debated on the lnlemel discussion group Melub-L at littpzr’r'listslran-
`kcn.clcr‘mai|manr'listinfoimctab-L}. Neither the biochemical mecha-
`nismls) of PB toxicity nor the identity at the missing metabolites of
`PB is known. Also, it is not clear whether the metabolism of PB (21
`modified fatty acid) interferes with, or is influenced by. the metabo—
`lism of fatty acids and carbohydrates present in l‘oodstuITS. Lastly. it
`is 1101 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 it noninvasive probe of the
`HC- or
`'JC-Iabeling pattem of citric acid cycle intermediates in
`human liver [Magnusson et at. 1991; Yang et at, 1996). As part of
`these investigations, we recently identified pheuyIbutyrylglutamine
`(PBGN) as a new metabolite of PB [Comte at at. 2002). PBGN is
`presumably t‘onncd from the reaction of PB—CoA With glutaminc. by
`analogy with the lonnation of PAGN from the reaction between
`PA—(ToA and glutaminc [Webster ct at, I976). in normal adult sub—
`
`10
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`Horizon Exhibit 2018
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`NEW PH ENYLBUTYFIATE METABOLITES
`
`11
`
`o
`
`0
`
`o
`
`M-PI'IB
`
`C,
`
`-———b
`
`o o
`
`O
`
`1
`
`0.. “Eu—EH
`
`E0“.
`reflux
`
`”a:I
`
`o
`
`\/
`
`0
`
`o
`
`D D D
`
`D
`
`D
`
`0“ 0
`a.s-|2.z.t.4.4-1Hsi—rnn
`
`OH hm” mom—p m
`
`21-an
`SIHLI
`
`m" c
`
`nu;
`
`t-J-DH‘4CITM
`
`0H
`
`Pmrlmlone
`
`Nafltl'lJ
`
`0
`
`R-PI-IB
`
`l-FhenyI-Z-u-Jlllpropannl
`FIG. 1. Sc‘tteiuelfot' the synthesis thrrttuheted and denigrated standards.
`
`jccts who ingested a fairly low dose of PB (5 g/75 kg). we found tltat
`the total excretion of PB + PA + PAGN + PBGN accounted for only
`halt‘ the ingested PB (1031.: (Comte et al.. 2002]. The missing fraction
`of the dose may be disposed of either in urine as unknown metabo—
`lites, or in feces as ttnabsorbed PB and/or PB metabolites.
`In tlte present Study. we report
`the identification of additional
`metabolites of PB in humans. i.c.. R— and S~3—hydroxy4—phcnylbu—
`tyrate (PHB), phenylacctonc, and l—plienyl—E—propano]. as well as PA
`and PB glnenronides. We studied the mechanism of P113 fomtation
`t'rtmt PR in perfused rat livers and identified two additional tttelabtt—
`lites, 4-phcnyl-ri‘uns-ctotonatc (PIC) and 4-phcnyI-3-ketobutyrate
`(PKB). Lastly. we prepared sodium—tree esters ol‘ PB and investigated
`their bioavailability in rats. Glycerol-PB esters appear promising for
`the administration of large amounts of PB without the corresponding
`sodium load.
`
`Materials and Methods
`
`Materials. All chemicals used in syntheses. general chemicals. and solvents
`were obtained from Sigma-Ndrich (St. Louis. MO). All organic solvents were
`dried and distilled immediately before use. [Hhfl‘henylacetic acid @926) and
`[it-{dbenzcnc were purchased fi-orn lsnrec lnc. [Miamisburg Oil}. The derivati-
`zation agent .-'\-'~tnethy1—N—(trimethylsilylitrifluoroacctamidc was supplied by Regis
`Technologies. Inc. [Morton Grove. IL}. All aqueous solutions were made with
`water plu‘ificd with the Millin system (Milliporc Corporation. Bcdford. MA}.
`Preparation of lfnlabelcd and neutcratcd Standards. S—Z—Phenylbntyryl
`chloride was prepared by reacting S-Z-phcnylbutyric acid with 800:. and was
`vacuum—distilled and stored at 4°C as a 11.5 M solution in henrene. [3H5JPH
`was prepared by aluminum chloride-catalyzed condensation of 'y—bulyrolnc-
`tone with [:l‘lfi]hcnzene as previously described (Comte et at, 1002). y—Phe-
`nyl-rr'wis—crotonie acid was prepared by a FrideLCrafis reaction of benzene
`with ethyl y—bromo-trw.=.t-crotonate. followed by acid hydrolysis of ethyl
`y-phenyIvtt'mn—crotonotc (Loffler ct 511.. mm. The trans configuration of the
`product was continued by 'H NMR. [II1.]Phcnylacctylglycine was synthe-
`sized by reacting [El-lsjphcnylacetyl chloride with glycine. as described previ~
`
`£0771.
`and Tanaka.
`(Ramsdcll
`analog
`unlabeled
`the
`for
`ously
`[1H,]Phenylacctyl chloride was prepared by activation of [2[17]phcnylacctic
`acid with freshly distilled dichloromelhyl methyl ether and used immediately
`alter evaporation of excess dichlonomcthyl
`[methyl ether and of the methyl
`chlorot'ormate byproduct. The synthetic protocol for preparation ot'l-‘l-IB. PKB.
`[II'ISJPIIE phenylacctuuc. and I-plictiyl-Z-[2-2H]prupauol is outlined in Fig. l.
`Ethyl“ 4Phony!-3—kc.robt.rrymm (2: Fig.
`I) was synthesized by a method
`adapted from Capozzi et a]. t'l993‘t. The commercial isopropylidcne malonatc
`(2.2—dimelhy1—4.6ndiketo-l.3-dioxane. also called Meldrurn’s acid: Aldrich
`Chemical Co.. Milwaukee. WI} was reacted with phenylacetyl chloride in the
`presence of dry pyridine in anhydrous methylene chloride. The crude pheny-
`laccrylated Meldfltm‘s acid [compound I Fig l: was refluxed in absolute
`ethanol until evolution of C03 ceased (about. h). After evaporation of the
`solvent. ethyl 4-pheny 1-.-kctobutyrate was purified on a silica gel column.
`4-Pttem’t-3—l'retohurt-Tic acid (PKB). A [0% molar excess of l N NaOII
`solution was added slowly to ice-cooled ethyl 4-phenyl-3-ketobutyratc and
`stirred at room temperature until the organic phase- disappeared (approximately
`11 hi. The solution was acidified with l N HL‘l to pH 2 and extracted three
`times with 3 volumes ofethyl other. Alter drying uvcrNa:501. the solvent was
`evaporated to give the white solid product (yield 94%. mp. 70"(11.
`'l'l NMR
`(300 MHZ. 5. C [23(13): keto. 3.33 (s. 2H. CI-IQCOO). 3.73 (5. 2H. C'I'IzPh).
`It i 133 (in. 5H. Ph): cnol. 432 ts.
`ll-l. (Ill). l2.l3 ts. 111.0“): ketotenol =
`7.8:}.
`'JC NMR [TS MHz. 6. CD03): 48.36 {CHQCOOL 49.87 {CIIzPhL
`l2?.4l [(3—4 Phl.
`|2S.?3 (ES-3. [)5 Ph}. 129.50 (CG. (:6 Phi. 133.14 (C—ipso.
`Ph). 169.26 ((00). 201.57 (CO).
`1 reduction of4~phcny|—
`The identity of the product was also confirmed by l
`3-ketohnryrate with NaBHi. 2': extraction of tits-PUB with ethyl acetate. and
`3} TMS derivatization and Nth—positive chemical
`ionization [EC—MS. The
`mass spectrum of the derivative was identical to that of PIIB synthesized as
`described below. The 4—phenyl—3—kctobuty1’ic acid was stored at “80"C‘ to
`prevent decomposition. Just before use in liver perfusion experiments. the acid
`was dissolved in water and titrated to pH *— T .40.
`R. S ?- hfvdmrv 4-poetriltonrt'tic acid (PHB) Lthyl 4~phenyl—3ketohutyratc
`(2.06 g. 10 mmol) was mixed with 10 m] ofIIJO and a calculated amount of
`NaOI'lr'l-{30 was added dt'opwise with cooling to give 0.5M solution of [Ol-l‘].
`
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`i2
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`KASUMOV er AL.
`
`NaBH. {0.323, 10 mmol} was added and the mixture stirred for 24 h. After the
`reaction mixture was cooled to 0°C. the pH was brought to 7.0 with ”Cl {on}
`and more than half of water removed by Iyophiiizer. The pit was brought to 2
`by I-ICI it”: N} and the solution extracted 3 times with 3 volumes of ethyl ether.
`After drying the combined ether extract. the ether was evaporated giving a
`white solid product. Yield ?9%. Purity of product was assayed by (EC—MS after
`derivatization with TMS.
`R—3-Hvdru.r‘_r—nit-pherrr'ir‘rrrrr'r'we iR-PI-TB] was prepared by reducing. the cor-
`responding 4-phenyl-3-ketobutyric acid with ib'-clilorodiisopinocampheylbo
`lane [I “l-DiP-Cl] {Wang cl «1]..
`I999). The yield was 89% from PKB:
`cnantiomeric excess was 9?% [GIT-MS of the methyl .S'-2-phcnylbutyryl de-
`rivative (see below). and purity was confirmed by NM R].
`4—Phe—
`a.5—3anya:-o.t-_v-4—piiem.-igz.3.3.4.4.-‘H_.yimnu-aie
`ran—fuming).
`nyl-l’r-ketobutyric acid 0126':I g. LS rnmol) was suspended in 3 ml of 2[“120
`(99.9%] to which 0.36 ml cranes of Na0°H (3.6 mmol} in 2H30 was slowly
`added at 0 --5°C. This procedure exchanges III for 2H atoms on the methylene
`groups adjacent to the carbonyl. The solution was stirred at room temperature
`overnight and then lyophilized. The residue was dissolved in 3 ml of 311:0 and
`stirred for another 5 h at room temperature. The solution was cooled on ice.
`treated with Nali°I[.._ tea mg. 1.5 1111noll. and stirred overnight at room
`temperature [Des Rosiers ct al.. 1988}. After acidification to pH l
`to 2 with
`IICl (6 N). the solution was saturated with NaC'l and extracted three times with
`3 volumes of diethyl ether. Solvent evaporation yielded R.5‘-[°l-[_.]Pl{B (yield
`74%. purity 99% by NMR. MS isotopic enrichment 05% by GCvMS of the
`TMS derivative). The free acid was titrated to pH 8 with VaOl-I and stored
`frozen as a 0.5 M solution until use.
`”maniac-emitter was prepared by decarboxylating phenylkelobutyrate in acid
`at I00°C. It was reduced to 1-phcnyl-2-[2-°II]pmpanol with INK-1133114.
`Esters of Phenylbutyrate. Dihydroxyacetone-di-PB. glycerol-tri-PB. ri-
`hose—tetra~Pl3. giucose—penta-PB. and sorhitol—hexa—PB were prepared by re—
`acting the polyoli'sugar with excess phenylbntyryl chloride in the presence of
`pyridine and catalytic amounts of .-‘\-'.i\’~di111ethylatui11opy1'idine. Products were
`purified by flash column chromatography on silica. To prepare glycerol—mono—
`PB. isopnopylidcne glycerol was reacted with phenylbutyryi chloride as above.
`and the isopropylidcne group was. removed by mild acidic hydrolysis in water.
`The structure and purity of all products were confirmed by 'H and ”C NMR.
`The structure of glycerol-niono-PB was confirmed by acetylation with acetic
`anlrydride. fol lowed by GCLMS analysis of the derivative.
`Sample Preparation. For the determination of the [tee acids concentration
`(PA. PHB. PKB, PIC. and PE) in perfilsate. samples [0.1 ml} were spiked with
`0.1? pmol of [ll-17113.61. [1H5JPB. and R.S—[3H,]I’HB before deproteinizarinn
`with 20 pd of saturated sulfosalicylic acid. The slurries were saturated with
`NaCl. acidified with one drop of 6 M IICl. and extracted three times with 5 ml
`ofdiethyl ether. For the assay of conjugates of PI) and PA. 0.1—1’01 aliquots of
`final liver perfusate or of human urine were spiked with internal standards and
`treated with [.0 ml NC] (6 \l) at 90°C overnight to hydrolyze the conjugates.
`Glucuronides of PB and PA were identified by the amount ofthese compounds
`released after incubation of perfizsatc and urine samples with |G-glucurmridasc
`in 0.2 M ammonium acetate buffer. pH 5.0. overnight at VT.
`Bilc samples were analyzed for Ercc and total {conjugated + lice) PB and
`its metabolites. In the first series ofassays. 0.05-ml samples of'l'rile were spiked
`with internal standards. acidi tied to pll 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 NrIOH to .\l) at 90°C for 3 h before
`acidification and extraction.
`
`Phenylacetylglycine was extracted in acid and derivatized with rnethanolr'
`ll('l. For the assay of phciiylacetonc and l-phcnyI-Z-propanol. urine and
`perfusate samples were spiked with the structural analog l—phenyl—
`[2115]ethanol and then treated with NaBZIL to reduce phenylacetone to mono-
`dcuterntcd l-phcny—2~propanol. The labeled and unlabeled l—pbeiiyl—"‘~
`ptopanol were assayed as TMS derivatives.
`For the assays in urine. 0.l—rnl samples were spiked with 0.l 5 pmol
`[°H_.]PHB. acidified to pH i to 2 with HE]. saturated with Natl. and extracted
`three times with 3 ml of diethyl ether. The combined extracts were dried with
`Nazso4 and evaporated before reacting the residues with 'i'0 pl of This at
`60”C for 20 min.
`For the chiral assay of PHB cnantiomers (Powers et al.. 1994). 0.l-rnl
`samples were spiked with RS-[ZHSJPI-IB. and oidicr deproteinizcd with 50 p]
`
`to
`of saturated sulfosaiicylic acid (if containing proteins} or acidified to pH 1
`2 with MC] [for urine). Then. the slurries or solutions were saturated with NaCl
`and extracted three times with 3 ml of diethyl ether. The combined extracts
`Were dried with Nazism and evaporated before reacting the residues with 0.15
`mi of Incthanolr'l‘ltfl for l h at 65%]. to derivatize the carboxyl groups of the
`PHB enantiomers. After cooling.
`1 ml of water was added to the mixture and
`the liydroxyacid methyl ester was extracted with diethyl ether (three times in
`3 ml). Alter complete evaporation of the combined ether extract. 5-2-
`phenylbutyryl chloride benzene solution t_0.l ml, 0.5 M) and 0.05 ml of
`aqueous 12 N NaOH were added. After vorlcxing. the mixture was incubated
`for l h on a slow shaker at room temperature. The derivatives were extracted
`with ether t three times in 3 ml} and 1 ml of water. The combined ether phase
`was dried with Nazst.)fl and evaporated completely. 't'lie resrduc was dissolved
`in 0.] 1111 of ethyl acetate. and l 5.1.] was injected into the GC'-MS.
`(EC—MS Methods. All ofthe metabolites. except phenylacetylglycine. were
`analyzed as their TMS derivatives on a I'Iewlctt-Packard 5090 gas chromato-
`graph equipped with a 38-5 capillary column tot] m X 0.25 mm i.d.. 0.5 mm
`film thickness; Hewlett Packard. Palo Alto. CA) and coupled to a 5989A mass
`selective detector. Samples t0.2—l
`lpill were injected with a split ratio 20 to
`50: l. The carrier gas was helium (I mlr'min) and nominal initial pressure was
`20.01 psi. The injector port temperature was at 220°C. the transfer line at
`305°C. the source temperature at 200°C and quadrupole at 150°C. The column
`temperature program was: start at [00°C. hold for l min. increase by 8°C‘Imin
`to 2361:. increase by 35"(Tr'min to 310°C. t3 min at 3|0”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 msrion. at m3: 22w233 {PNEHdPA}. 254mg {PBrEHaPB}. 3251330
`[PllBr‘[°lls]Pll.B). and 252 (PFC). Note that PKB and phenylacctone had been
`reduced with NaB°li4 to monodeuterated R,3-3-hydroxy-4-phenylhutyrate
`(monitored at irix'z 3263330) and |~pheny|—2—pl'opanol (monitored at inf: 2001’
`Eli) andt'or 21152271 Also. since l-plrenyl-Z-pr'opanol was assayed with a
`standard of l~plrenyl[°l[.lethanol. ions monitored for this assay were 2tl0r'209
`or 2] 20226.
`For the analysis of chiral PIIB derivatives. the GC injector temperature was
`set at 380°C. The column {DB-S. 60 rn X 0.25 mm id. 0.5 mmfilm thickness;
`Hewlett Packard) program was modified to the initial 50°C for 2 min,
`increased by 15°Ci’min to 230°C. 25 min at 230°C.
`increased by 35°C to
`290°C; and held 10 min. Ions monitored were i) 358 WI
`-|
`18.
`i.e.. M -|
`hit-14+} for analytes and 2) 363 that + is + 5,
`i.e.. M + 5+NH4+1 for
`[IH5]PHB. The mass spectrometer was operated under ammonia- positive
`chemical ionization and was tuned aurormirically.
`Phcnylacctylglycinc was analyzed as its methyl ester derivative using an
`(JV-225 column ['29 m X 0 3.3 mm id,
`1 pin film thickness: QuadI-ex
`Corporation. Woodbridge. CT). This column yielded better resolution of
`N—phenylaeetylglycine methyl ester with no peak tailing. Samples [0.2—]
`lpal}
`wcre injected with a split ratio 20:1.Thc carrier gas was helium t constant flow
`l2 mli‘min). The injector port temperature was at 220°C, the transfer line at
`240°C. the source tcrnperaturc at 200°C. and quadrupole at 106°C. The column
`temperance program was: start at 90°C. hoid for l min. increase by |0'°C3’min
`to 240°C. 15 min at 240°C. Ai’tcr automatic calibration. the mass spectrometer
`was operated under ammonia-positive ioni7arion mode {pressure adjusted to
`optimize pcak areas]. ions monitored wcrc H208 1M + I. i.c.. M + l-li'I and
`225 {M +18.i.e.,M + I‘ll-1f) forthe analyte and 2] 215 [M + 2 + l. M +
`T + [‘I 'l and 232 (M + 'i + l3. M + i + N114" ) for N—L-‘tITJPA-glycine with
`a dwell time of 25 Instion.
`
`Areas under each chromatogram were determined by interactive computer
`integration. and corrected for narnrally occurring heavy isotopes and light
`isotopic irnptu‘itics in the synthesized labeled internal standards.
`NMR Spectroscopy. Proton NMR spectroscopy was performed at 400
`Mliz on a Bruker Avnncc (Bruiser. Newark. DE} [)MX 400 wide-bore spcc—
`tronteter using a 5—mm inverse probe. Full—strength urine samples were ob—
`tained by Iyophilizing 5 ml ofurine to dryness. The residue was dissolved in
`0.5 ml oszQO. and the solution was introduced into a 5—mm NMR tube. An
`external standard made of a sealed capillary containing a solution of trimeth—
`ylsilylpropionic acid in °H;O was introduced into the NMR tube and used as
`chemical shift rcfcrericc. Standard acquisition conditions were as follows for
`one-dimensional spectra: 45° pulse. S-s repetition time. water saturation during
`the relaxation delay. sweep width (SW: 6775 1-12, out data points (TD). and
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`NEW PH EN YLBUTYFIATE METABOLITES
`
`13
`
`32 scans of data collection. Two—dimensional correlation spectroscopy
`iCOSYl spectra were obtained with the following conditions for the second
`dimension: SW 3500 Hz. TD 2K. [6 scans. and for the first dimension. 512
`increments of 2T3 us zerofillcd lo lK. A uonshiflcd siuebell window was
`applied in both dimensions, and magmlude spectra were calculated. Two—
`dirncnsional
`‘[-lf'"C correlations via double insensitive nuclei enhanced by
`polarization transfer (HSQC} were performed in the phase—sensitive mode
`(TF‘Pl;
`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 117.. TU 2K. 128
`scans. and in the first dimension: SW l2 kHz. 256 increments of 20.? ps
`zei'ofillcd to 512. A shifted sincbell window was applied in both dimensions.
`Proton—decoupled carbon spectra of the concentrated urine samples were
`obtained at “30.62 MIIz in a 5-mm dual probe. Acquisition conditions were as
`follows: 20° pulse. repetition time [.3 5. SW 25 kHz. Tl) 64K. 40,000 scans.
`The free induction decays were zerolilled to 128K. and a Lorentz to Gauss
`transformation (LB = —1 Hz. GB = 0.1} was applied before Fourier trans—
`t‘orrnation.
`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 athte illness. Women had a negative pregnancy Last
`and were not breastfeeding. Seven subjects t_lh.ree men. four women: 3].? t
`5.0 years: I'll .3 I 3.4 cm: 79.5 I 5.9 kg} received detailed information on the
`purpose ofthc investigation and signed an informed consent form. After an
`overnight Fast. the subjects were admitted to the Clinical Research Center at
`7:30 AM. The); remained fasting until
`the completion of the study. An
`intravenous line was installed in the forearm with a saline infusion (20 rrilt'h}
`and a short blood sampling catheter was inserted into a superficial vein of the
`contralatcral hand. The hand was placed in a heating box at 601') for sampling
`of arterialized venous blood. At 8:00 AM, after baseline blood and urine
`sampling. each subject ingested 0.36 mnioli'kg (5 3135 kg] Na-PB. This Close
`corresponds to II
`to l?% of the doses commonly used in the treatment of
`patients with inborn errors of urea synthesis :04 0.6 g - kg" - day' 'j. Water
`intake was adjuster] to induce a diuresis of at least
`it‘ll) m|.-’30 min. Urine
`samples were collected at Martin intervals for the first 3 h after PB ingestion.
`and then every hour until 8 h. Urine samples were quickly frozen and stored
`at —80°C until analysis.
`Organ Perfusion Experiments. Livers from fed male Spraguedjawley rats
`kept on standard rat chow (200- 230 g) were perfused (Brunengraber et 21]..
`l‘Ti'S] with recirculating Krebs—Ringer~bicarbouate buffer containing 4% ho—
`vine serum albumin [fraction V. fatty acid poor; Intcrgcn. Purchase. NY} and
`10 mM glucose. The bile duct was cannulated with PF.
`I0 tubing IBD
`Bioacicnccs. San Jose. CA} for bile Collection. Throughout the 12-h experiment.
`sodium taurocholate £38 .Ll-I'I'tolr'hl was infused irtto the perfiJsion reservoir to
`stimulate bile [low {Robins and ancugraber. 1982}. After 30 min of equil-
`ibration. a calculated amount of either PB. R..S'-Pl'll3. or PKB was added to the
`perfusato to set an initial concentration ofS IuM. The perfusion continued until
`120 min. The pH of the pert-usate was monitored and kept at 7,3 to 2.4 by
`adding 0.3 \t[ Pistol-I. Samples of bile and perfusate were collected at regular
`intervals. For the assay of PKB, perfusate samples :2 ml) were treated imme
`diately with 0.31m of (Li M NaBSI-l4 in {Lt mM NaOH to convert unstable
`PKB to stable monodeuterated R..5‘~Pl'lB. Bile samples were collected every 30
`min. At the end of the experiment. the livers were quick—frozen with aluminum
`tongs prccoolcd in liquid nitrogen.
`Rat in Vivo Experiments. We tested the hioavailability tiltwti PB esters as
`a means to deliver large amounts of PB without the corresponding sodium
`load. Overnight—fasted rats [330 4400 g] were divided into six groups (5—? rats
`per group} for the testing of three different PB preparations: Na-PB. glycerol-
`mono—I’E. glycerol—tri—PB.
`ribose—tetra—PB. glucosepeuta—I’B. and sorbitol—
`hexa-PB. Each rat received one stomach gavage of the sodium salt or ester in
`an amount that delivered 2.15 mmol PBi‘kg. The weighed dose for each rat was
`mixed with 3 Llll of Tween and administered to the rats through a stomach
`gavage needle.
`Whole blood samples [100 200 pelt wen: taken at ‘-5. [5. 30. I50. [50. 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 all
`was transferred to an l-lppendorf‘ tube and quick frozen.
`
`Results
`
`Human Study. In our previous study, we had identified PBGN in
`the plasma and urine ofuormal adults who had ingested a small dose
`of PB. We now report the data of additional analyses conducted on the
`some samples of human urine. First. we subjected to NMR analysis
`two samples of urine produced by each subject before and 2 h after
`ingestion of PB. The NMR spectrum of the second sample, but not of
`the first, was highly suggestive of the presence of a product of
`hydroxylaticn ofthc side chain of PB. In the COSY spectrum ofthc
`lyophilized urine dissolved in D20 (alter PB ingestion), we identified
`a proton at 4.25 ppm coupled with two (ii-12‘s. The first Cl—l2 has
`protons at 2.8? and 2.70 ppm: the second has nearly identical protons
`at 2.4 ppm. A chemical shift at 4.25 ppm is likely corresponding to a
`proton coupled to the OH group. Therefore,
`the COSY spectrum
`revealed the presence of a metabolite having ~Cllz—CIIOII—Cllz—
`moiety. In the HSQC‘ spectrum thcsc proton signals con-elated with
`the following carbons: CH at 7‘0 ppm overlapping with other CH
`carbohydrate carbons, CH2 at 44.6 ppm and CH3 at 42.5 ppm. Lastly.
`in the aromatic region. signals at [29.6. 128.7. and 126.6 ppm corre-
`sponded to a monosubstihited phcriyl group having the same intensity
`per carbon as the signals of the wCHE—CHOHiTl-lg— group. We
`therefore concluded that B-hydroxy-PB (Pl-1B) was present in the
`urine of patients treated with PB. PllB would be a very likely
`metabolite since it would be formed via partial B-oxidation of PB to
`Pl-lB—CoA (presumably the S—cnautiomer), which would be hydro—
`lyzed to free PHB.
`'l'o further conform the identity of the urinary metabolite detected by
`NMR. we synthesized [unlabeled and R.S[31-1_.]Pl--lB. The di-TMS
`derivative of synthetic R.S—PHB was analyzed by GC—MS irt parallel
`with an extract of human urine (after PB ingestion) that had been
`rcactcd with TMS. 1n the sample derived from urine, we found a peak
`at the same retention time and with the same mass spectra (electron
`ionization and NHS—pusilivt: chemical
`ionization) as lht: slalltlnrtl
`tll‘
`Its-PHB. in addition. the NMR spectrum of synthetic FHB had the
`same chemical shifts as the material identified in human urine. This
`
`confimred the identity of P113 in human urine but did not yield
`information about its chirality. The ehimlity of excreted PllB yields
`infomiation on the mechanism of its formation (see below).
`For the chromatographic separation of PHB cnantiorncis from the
`synthetic raocmatc. we tried various chiral hydroxyl dcrivatization
`reagents before selecting the combination of l) methylation of the
`carboxyl group. and 2) reaction of the hydroxyl group with 8—2—
`phcnylbutyryl chloride (Powers ct al..l994l. The cxpcctccl derivatives
`of R— and SJ-‘HB were well separated. and their order ot‘elution was
`confimied using a sample of R-PltB that we had synthesized. R-Pl-lB
`clutcs ahead of the S—cICi-ivativc (Fig. 2).
`Chiral GC—MS analysis of thc hutnan urine samples (after PB
`ingestion) revealed that PHB is present as an cnantiomeric mixture
`with 10% R-PltB and 90% S—Pl-lB (Fig. 2). Figure 3 shows the time
`profile of(R+S)—PIIB excretion in urine after an oral bolus oFNa-PB.
`The excretion of (R+S)—Pl-lB peaked at 120 to 240 min. Eight hours
`alter ingestion of PB.
`the cumulative excretion of Iijl-Sl—PHB
`(1.35 t 0.13 mmol) amounted to 4.4 I 0.56% of the PB dose.
`Small amounts of phenylacetonc and l-phenyl-Z-propauol were
`identified in the urine samples (Table 1). Treatment of urine samples
`with fi—glucuronidase increased their PB -l- PA content. The total
`amount of PB + PA released by t‘l—glucuronidase amounted to 2.4 t
`0.3% ofthe PB dose.
`
`Pcrl'used Rat Liver Study. The metabolism of PB was studied in
`perfused rat livers by the addition of 5 mM PB to the recirculating
`perfusatc. the time profile of the PB concentration was curvilinear
`
`Page 4 of 10
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`4of10
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`4 of 10
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`Page 4 of 10
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`

`

`KASUMOV El AL.
`
`TABLE I
`
`Recnvmj- rgI'PB and its metabolites in lunnrm m'im.’ (it — x”) (“for the mini
`ingestion ”(1136 mumffkg Nat-PB and ;,I:'ridttr.'t'iwi (if-PR uret‘ubw‘itm by mr liver
`(H ‘- 5) perfused It't'fl.‘ 5 ”til-I PB
`“'3. of PB Uptake
`Mmuhulilt‘
`
`Humans
`Rats
`0.97 L 0.23
`|.29 L 0.33
`0.26 “' 0.06
`
`21.52 L 4.32
`7.64 "' 0.8?
`71.68 + 0.84
`5.25 L 0.88
`
`Fret: PB
`PB figlueumnide
`Free PA
`I‘A-glyeinc
`PA-fi-glueuttmidc
`PAGN“
`PBGN’
`[5.71 “' 1.63
`P1113
`4.54 1‘.’ 0.2L)
`PKB
`5.15 i 0.59
`PIC
`3.6? *7 0.23
`0.] 1 + 0.00?
`Phenylaeetoite
`Traec amounts
`0.01 1' 0.00l
`l—f'henyl—Z-propanol
`2.9? L (lt‘il
`Total bile met
`Total '14.]? L 5.58 62.4- '_ 2.I
`
`
`H] L 0J9
`32.6 '
`1.9
`21.5 ’ 2.4
`4.4 “' 0.36
`
`
`
`“ Previously identified tnelflbulitcr: {(‘tulttc id ml , Emil.
`
`14
`
`10
`
`it
`
`a 2 1 a
`
`Abundancex10‘
`
`EE
`
`.EE
`
`.
`
`£-
`
`E E3f
`
`l
`g
`
`39.64
`
`sass
`
`39.12
`
`39.76
`
`39.60
`
`39.34
`
`'I'lme (min.
`
`FIG, 2. Chiral CC—fl-fl' qut' rgf'R— and 5'—PH3 excreted in one .t'zmipt‘e' of'lnwtwi
`wine otter om! ingestion otflfié mmot-itg Nit—PB their! trace).
`The sample was spiked with HR [3H5]I'HB. [he enantitrrnur profile or which is
`shown by the thin trace.
`
`0.3
`
`0.2
`
`I}
`II
`
`:3:E.
`
`a E
`
`In
`0
`
`I
`
`II
`
`.-
`
`-womtmiwmmmmm
`
`Tim [Inln]
`
`Flt}. 3. Time wmwe of't'R-Sft—PHB urinary excretion in normal humans alter
`oral ingestion of {136 mmolfkg Nil-1’13 {mean 1' S.E.M.: U = I").
`
`(Fig. 4A). Plotting ol' the data under semilogaritlunie coordinates
`yielded a linear relationship compatible with a first order kinetic
`process for PR uptake. The total uptake anR amounted to 3 ID 1’ l6
`pmol ' ('90 min‘ I)t ' lit-'cr— '.
`Figure 4B shows the time accumulation of metabolites derived
`from PB and released into the perfusate. Phenylaeetylglyeine is a
`
`El PB-giueur.
`
`O PA-giucur.
`
`9 [R.S)-PHB
`
`0 [SJ-PHB
`
`* PA
`2'.) PA-gtydne
`
`3|]
`
`50
`
`TI]
`
`90
`
`1 1 D
`
`1 30
`
`1 50
`
`Time (min)
`
`.‘lrfetuholt'sm ot'PB in perfimed rat Event.
`Flo. 4.
`A. uplalrt: of PB from [he recirculating perfustlte. B, uucurnululiun of PB
`metaboliles in the perfiIsalc tn — 5 for all compounds except for PKB. where H —
`4-).
`
`kummt metabolite of PA in rats and dogs [Knoop, 1904; Ambrose and
`Sherwin.
`|933z James el al._. IWE). Ahtittl
`Ion/t. or the uptake ol' PB
`was accounted for by the production ofR— and S—l‘llB. of which about
`90% is the S—enantiomer. We also identified PIC. PKB. phenylae—
`etone. and l-phenyl-Z-propanol. which.
`to our best review of the
`literanti'e. have not been previously described as metabolites of PB.
`The identity of these metabolites was continued by GC—MS using
`standards we synthesized. Incubation of liver perfitsate samples with
`
`Page