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`1521-009X/12/4003-625–634$25.00
`DRUG METABOLISM AND DISPOSITION
`Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
`DMD 40:625–634, 2012
`
`Vol. 40, No. 3
`42770/3752997
`
`Deuterium Isotope Effects on Drug Pharmacokinetics. I. System-
`Dependent Effects of Specific Deuteration with Aldehyde Oxidase
`Cleared Drugs
`
`Raman Sharma, Timothy J. Strelevitz, Hongying Gao, Alan J. Clark, Klaas Schildknegt,
`R. Scott Obach, Sharon L. Ripp, Douglas K. Spracklin, Larry M. Tremaine, and Alfin D. N. Vaz
`Departments of Pharmacokinetics Dynamics and Metabolism (R.S., T.J.S., H.G., A.J.C., R.S.O., S.L.R., D.K.S., L.M.T.,
`A.D.N.V.) and Pharmaceutical Sciences (K.S.), Pfizer Global Research and Development, Groton, Connecticut
`
`Received September 14, 2011; accepted December 15, 2011
`
`ABSTRACT:
`
`The pharmacokinetic properties of drugs may be altered by kinetic
`deuterium isotope effects. With specifically deuterated model sub-
`strates and drugs metabolized by aldehyde oxidase, we demon-
`strate how knowledge of the enzyme’s reaction mechanism, spe-
`cies differences in the role played by other enzymes in a drug’s
`metabolic clearance, and differences in systemic clearance mech-
`anisms are critically important for the pharmacokinetic application
`of deuterium isotope effects. Ex vivo methods to project the in vivo
`outcome using deuterated carbazeran and zoniporide with hepatic
`systems demonstrate the importance of establishing the extent to
`which other metabolic enzymes contribute to the metabolic clear-
`
`ance mechanism. Differences in pharmacokinetic outcomes in
`guinea pig and rat, with the same metabolic clearance mechanism,
`show how species differences in the systemic clearance mecha-
`nism can affect the in vivo outcome. Overall, to gain from the
`application of deuteration as a strategy to alter drug pharmacoki-
`netics, these studies demonstrate the importance of understand-
`ing the systemic clearance mechanism and knowing the identity of
`the metabolic enzymes involved, the extent to which they contrib-
`ute to metabolic clearance, and the extent to which metabolism
`contributes to the systemic clearance.
`
`Introduction
`Deuteration of drugs to enhance their pharmacokinetic, pharmaco-
`dynamic, or toxicological properties has gained momentum as judged
`by a search of the SciFinder database with the search term “deuterated
`drugs.” Of 179 registries retrieved, 151 are since 2005 with an
`exponential growth since 2006. These include deuterated versions of
`patented and off-patent drugs with claims of increased efficacy, de-
`creased toxicity, reduced interpatient variability, and decreased drug
`dose or dosing frequency. Belleau et al. (1961) were among the first
`to demonstrate the pharmacodynamic effect of deuteration with ␣␣-
`dideuterated p-tyramine. The effect was attributed to decreased me-
`tabolism of p-tyramine by monoamine oxidases. Several reports that
`have examined the effect of deuteration on the pharmacokinetic and
`pharmacodynamic properties of drugs reveal results that include little
`to no effect (Tanabe et al., 1970; Farmer et al., 1979; Taylor et al.,
`1983; Burm et al., 1988; Dunsaed et al., 1995); increased systemic
`exposure, a pharmacodynamic effect, and receptor selectivity (Dyck
`et al., 1988; Schneider et al., 2006, 2007); and decreased toxicity
`(Najjar et al., 1978). However, in these studies the mechanisms
`underlying the observed effects or lack thereof were not examined.
`With the use of formyl-deuterated N-methylformamide, the hepato-
`
`Article, publication date, and citation information can be found at
`http://dmd.aspetjournals.org.
`http://dx.doi.org/10.1124/dmd.111.042770.
`
`toxicity was shown to be due to oxidative metabolism at the formyl
`carbon (Threadgill et al., 1989). Pohl and Gillette (1984 –1985) out-
`lined the kinetic basis for use of deuterated compounds to determine
`toxic metabolic pathways, and, in addition, Nelson and Trager (2003)
`have reviewed distinctions between “intrinsic KDIEs” and “observed
`KDIEs” in enzyme reaction mechanisms with particular emphasis on
`cytochrome P450 reactions. Foster (1984) and Kushner et al. (1999)
`have also discussed the application of deuterated drugs to drug phar-
`macokinetics, pharmacodynamics, and toxicity.
`A KDIE on the intrinsic metabolic clearance (CLint or Vmax/Km) is
`fundamental to the application of a deuteration strategy to alter drug
`pharmacokinetics. Multiple factors mute the magnitude of this isotope
`effect. These include substantial contribution to the metabolic clear-
`ance by conjugating enzymes (UDP-glucuronosyltransferases, sulfo-
`transferases, and glutathione transferases) and heteroatom oxidizing
`enzymes (flavin monooxygenases), in which carbon-hydrogen bonds
`are not broken; aspects of enzyme reaction mechanisms such as
`“metabolic switching” due to deuterium substitution, particularly im-
`portant with cytochrome P450 cleared molecules (Miwa and Lu,
`1987; Nelson and Trager, 2003); rate-limiting product release from
`enzymes, which mask intrinsic KDIEs (Ling and Hanzlik, 1989; Hall
`and Hanzlik, 1990; Bell-Parikh and Guengerich, 1999); and other
`biological processes such as organ blood flow-limited clearance, renal
`and/or biliary clearance by passive or active transport
`involving
`uptake or efflux pumps and enterohepatic recycling. Consequently, a
`
`ABBREVIATIONS: KDIE, kinetic deuterium isotope effect; LC, liquid chromatography; MS, mass spectrometry; QC, quality control; MRM, multiple
`reaction monitoring; AO, aldehyde oxidase; AUC, area under the curve.
`
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`SCHEME 1. Sequence of synthetic steps used to deuterate compounds used in this
`study.
`
`5.79 mmol, 3 Eq), and KI (20 mg, 0.12 mmol, 0.06 Eq). The resulting
`suspension was heated at 110°C for 4 h and then cooled to room temperature.
`The reaction was diluted with 20 ml of H2O and extracted with 20 ml of
`EtOAc. The EtOAc layer was washed two times with 10 ml of brine, dried over
`anhydrous Na2SO4, and concentrated under reduced pressure to afford 680 mg
`(89% yield) of b3 as a white solid. A suspension of b3 (500 mg, 1.27 mmol)
`in 35 ml of EtOD and 2 ml of tetraethylammonium was purged with N2 gas.
`To the suspension was added 30 mg of 10% Pd/C, and after purging with D2
`gas the reaction was stirred under a balloon of D2 gas for 3 h at room
`temperature. The reaction was then purged with N2 gas and filtered, and the
`filtrate was concentrated under reduced pressure. The residue was dissolved in
`30 ml of EtOAc, washed with 30 ml of H2O followed by 10 ml of brine, and
`dried over Na2SO4. The solvent was evaporated under reduced pressure, and
`the resulting residue was purified by flash column chromatography (ISCO
`silica gel cartridge eluting with 100% CH2Cl2 to 90% CH2Cl2/10% MeOH) to
`afford 360 mg (79% yield) of 1-[2H]carbazeran as a tan solid. LC-MS: m/z 362
`(MH⫹); monodeuterium isotopic content ⬎99%.
`2-[2H]Zoniporide. 2-[2H]zoniporide (CAS Registry Number 241800-98-6)
`was synthesized as shown in Scheme 1c. A suspension of quinoline carboxylic
`acid (c1, 4.00 g, 14.32 mmol) in 70 ml of EtOH and 1 ml of H2SO4 was heated
`at reflux for 18 h. An additional 1.5 ml of H2SO4 was added to the reaction,
`and reflux was continued for 20 h. The resulting reaction solution was cooled
`to room temperature and concentrated to approximately one-third of its volume
`by rotary evaporation. The residual solution was diluted with 150 ml of Et2O
`and washed two times with 50 ml of saturated aqueous NaHCO3 followed by
`50 ml of brine. The organic layer was dried over Na2SO4 and concentrated
`under reduced pressure to afford 3.85 g (88% yield) of the ethyl ester as a
`brown oil. The ester (2.35 g, 7.65 mmol) and 3-chloroperoxybenzoic acid (2.32
`g; 13.44 mmol, 1.8 Eq) in 70 ml of CHCl3 were stirred at room temperature for
`16 h. The reaction solution was diluted with 30 ml of CHCl3 and washed with
`50 ml of saturated aqueous NaHCO3, 50 ml of saturated aqueous NaHSO3, and
`finally 50 ml of brine. The organic layer was dried over Na2SO4 and concen-
`trated under reduced pressure to afford 2.74 g of crude c3 as a light brown oil.
`A suspension of c3 (3.30 g, 10.21 mmol) in 70 ml of D2O (99.9% D) was
`treated with 2 ml of 50% NaOD in D2O, and the resulting solution was heated
`at 100°C for 3 h (Kawazoe and Onishi, 1967). The reaction was cooled to room
`temperature, and the pH of the solution was adjusted to 4.0 by dropwise
`addition of D2SO4. The resulting suspension was extracted three times with
`100 ml of CHCl3. The combined organic layers were dried over Na2SO4 and
`concentrated under reduced pressure. The crude product residue was purified
`by flash column chromatography (ISCO silica gel cartridge eluting with 97.4%
`CH2Cl2/2.5% MeOH/0.1% AcOH to 94.9% CH2Cl2/5% MeOH/0.1% AcOH)
`to afford 2.20 g (73% yield) of c4 as a light yellow solid. LC-MS: m/z 297
`(MH⫹); monodeuterium isotopic content ⬎98%.
`
`KDIE on the intrinsic metabolic clearance alone may not translate into
`an alteration of the overall pharmacokinetics of a drug.
`Drug design strategies have successfully decreased the impact of
`cytochrome P450 enzymes in metabolic clearance, partly by increased
`use of nitrogen heteroaromatics in drug substructures. As a conse-
`quence, aldehyde oxidase is increasingly observed as an alternate
`metabolic pathway for clearance because of its ability to oxidize
`nitrogen heteroaromatic ring systems. The broad differential tissue
`distribution of this enzyme results in an inability to correlate in vitro
`intrinsic clearance to in vivo clearance, and failure of allometric
`scaling of clearance due to interspecies differences in this enzyme
`have resulted in design strategies to avoid its role in metabolic
`clearance (Pryde et al., 2010). An alternate approach to decrease
`clearance when this enzyme is involved may be the use of KDIEs with
`specifically deuterated substrates.
`In this study, we focused on aldehyde oxidase as the metabolic
`clearance enzyme using specifically deuterated substrates to establish
`mechanistic consistency across species and two Pfizer drugs, carba-
`zeran and zoniporide, for which some preclinical and clinical infor-
`mation was available, to demonstrate the importance of knowing the
`enzyme(s) involved in the metabolic clearance, their reaction mech-
`anisms, the species differences in metabolic pathways, and the use of
`in vitro methods to assess the probability for in vivo success. We have
`determined 1) in vitro intra- and intermolecular KDIEs for several
`aldehyde oxidase substrates, 2) in vitro KDIE on the intrinsic clear-
`ances and metabolic profiles in hepatocytes and hepatic subcellular
`fractions, and 3) the in vivo KDIEs on pharmacokinetic parameters
`for carbazeran and zoniporide administered orally and intrave-
`nously. Carbazeran was a phosphodiesterase-2 inhibitor for the
`treatment of chronic heart failure, discontinued because of low oral
`bioavailability (⬍5%) and short half-life in humans (Kaye et al.,
`1984), and zoniporide was a Na⫹/H⫹ exchanger-1 inhibitor for
`treatment of perioperative myocardial ischemic injury after sur-
`gery, for which high clearance was observed in humans and rats
`(Dalvie et al., 2010).
`
`Materials and Methods
`Unless otherwise stated, all reagents used in chemical syntheses and bio-
`chemical and biological studies were of reagent grade and used as such without
`further purification.
`Deuterated Substrates. Scheme 1 shows the synthetic approaches used to
`deuterate the substrates used in this study. The palladium-catalyzed reductive
`deuteration of ␣-chloro-heterocycles and the base-catalyzed deuterium ex-
`change of the ␣-hydrogen in heterocycle-N-oxides are well established meth-
`ods for the specific introduction of deuterium into selected sites of nitrogen
`heterocycles (Kawazoe and Onishi, 1967; Rylander, 1985).
`2-[2H]Quinoxaline, 1-[2H]phthalazine, and 2-[2H]quinoline. These were
`synthesized from their corresponding chloro derivatives by palladium-cata-
`lyzed reduction (Scheme 1a) (Rylander, 1985) and described for 2-[2H]qui-
`noxaline. A solution of 2-chloroquinoxaline (0.30 g, 1.82 mmol) in 10 ml of
`EtOD and 0.3 ml of tetraethylammonium was purged with N2 gas, and 20 mg
`of 20% Pd(OH)2/C was added. The suspension was then purged with D2
`(⬎99% isotopic purity) gas followed by stirring at room temperature for 4 h
`under a balloon of D2 gas. Before filtering, the reaction was purged with N2
`gas, and the filtrate was concentrated under reduced pressure. The residue was
`dissolved in 30 ml of EtOAc, washed with 30 ml of H2O, and concentrated
`under reduced pressure. Flash column chromatography of the residue on an
`ISCO silica gel cartridge eluting with 20% EtOAc/80% hexanes afforded 23
`mg (10% yield) of quinoxaline-d as an off-white solid. LC-MS: m/z 132
`(MH⫹); monodeuterium isotopic content ⬎99%.
`1-[2H]Carbazeran. 1-[2H]carbazeran (CAS Registry Number 70724-25-3)
`was synthesized as shown in Scheme 1b. A solution of 1,4-dichloro-6,7-
`dimethoxyphthalazine b1 (500 mg, 1.93 mmol) in 5 ml of dimethylformamide
`was treated with piperidine b2 (432 mg, 1.93 mmol, 1.3 Eq), K2CO3 (800 mg,
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`DEUTERIUM ISOTOPE EFFECTS ON DRUG PHARMACOKINETICS
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`A solution of c4 (0.72 g, 2.42 mmol) in 18 ml of MeOD was purged with
`N2 gas and treated with 126 mg of 10% Pd/C followed by ammonium formate
`(0.72 g, 11.42 mmol, 4.7 Eq). The resulting suspension was heated at 45°C for
`1 h and then cooled to room temperature and filtered. The filtrate was
`concentrated, and the resulting residue was diluted with 50 ml of H2O and
`extracted into 50 ml of CHCl3 containing 0.5 ml of AcOH. The organic layer
`was dried over Na2SO4 and concentrated under reduced pressure to afford 570
`mg (84% yield) of c5 as an off-white solid.
`A solution of c5 (0.35 g, 1.25 mmol) in 8 ml of SOCl2 was heated at reflux
`for 1 h. The solution was then cooled to room temperature and concentrated to
`a yellow solid by rotary evaporation. The solid was treated with 5 ml of toluene
`and again evaporated under reduced pressure to a solid. The solid was then
`suspended in 9 ml of tetrahydrofuran, and this suspension was added to a
`solution of guanidine-HCl (0.74 g, 7.75 mmol, 6.2 Eq) in 14 ml of 1 M aqueous
`NaOH and 7 ml of tetrahydrofuran. The reaction was heated at 45°C for 1 h
`and then cooled to room temperature to afford a biphasic solution. The organic
`layer was partially concentrated, and the resulting liquid was extracted with 25
`ml of 5:1 CHCl3-isopropyl alcohol. The organic layer was dried over Na2SO4
`and dried under vacuum. The resulting crude product was slurried in 5 ml of
`ice-cold EtOAc and filtered to afford 150 mg (38% yield) of 2-[2H]zoniporide
`as an off-white solid. LC-MS: m/z 322.2 (MH⫹); monodeuterium isotopic
`content ⬎98%.
`Biological Reagents. Human and rat liver cytosols were purchased from
`BD Gentest (Woburn, MA). Guinea pig liver S-9 and cytosol were purchased
`from XenoTech, LLC (Lenexa, KS). Pooled and cryopreserved hepatocytes
`from either human or rat livers were obtained from Celsis In Vitro Technol-
`ogies (Baltimore, MD).
`Human aldehyde oxidase was partially purified from pooled liver cytosol by
`ammonium sulfate precipitation as follows. To a liter of stirred human liver
`cytosol preparation (obtained as a recovery fraction from the preparation of
`liver microsomes) maintained in an ice-water bath, buffered with 100 mM
`potassium phosphate buffer, pH 7.4, and constantly monitored for pH, was
`added solid ammonium sulfate (in small amounts at a time) to four weight to
`volume cuts of 5, 15, 20, and 25% ammonium sulfate. The pH was constantly
`adjusted with a 1 Msolution of potassium mono-hydrogen phosphate
`(K2HPO4) to maintain it between 7.0 and 7.4. After each weight to volume
`addition of ammonium sulfate, the suspension was stirred for 30 min to
`equilibrate, then centrifuged at 9000g for 20 min to pellet precipitated protein.
`The protein pellets were redissolved in 100 ml of 10 mM potassium phosphate
`buffer, pH 7.4, and assayed for aldehyde oxidase activity, as measured by the
`oxidation of phenanthridine to phenanthridone. The aldehyde oxidase activity
`was recovered in the 20 and 25% w/v ammonium sulfate protein pellets. The
`oxidase activity of this preparation of aldehyde oxidase was stable at ⫺40°C
`for more than 1 year.
`Bioanalytical Procedures. A 1 atomic mass unit difference between the
`proto and deutero forms of the substrates used in these studies required an
`accurate correction of the mass spectral contribution from the natural abun-
`dance from 13C in the proto forms of the compounds to the base mass of the
`deutero forms. The difference in the contribution determined empirically from
`standard curves to that calculated from molecular formulas is less than 1%.
`Sample Preparation. All standards, QC samples, and samples were pre-
`pared using the Hamilton MicroLab STAR (Reno, NV) robotic sample prep-
`aration station. A working solution of 4 ␮g/ml zoniporide or deuterated
`zoniporide was prepared separately by diluting 1 mg/ml stock solution in 1:1
`dimethyl sulfoxide-acetonitrile. Sequential dilution of the working solution in
`blank Sprague-Dawley plasma yielded standard solutions of 0.1, 0.2, 0.5, 1, 5,
`10, 50, 100, and 200 ng/ml and QC samples of 0.4, 8, and 80 ng/ml in plasma.
`A working solution of 10 ␮g/ml carbazeran or deuterated carbazeran was
`prepared separately by diluting 1 mg/ml stock solution in 1:1 dimethyl sul-
`foxide-acetonitrile. Sequential dilution of the working solution in blank guinea
`pig plasma yielded standard solutions of 0.1, 0.2, 0.5, 1, 2, 5, 10, 50, 100, 200,
`and 500 ng/ml and QC samples of 0.8, 8, 80, and 400 ng/ml in plasma. In the
`cassette-dosed studies of zoniporide the early time points between 0.16 and
`0.75 h were diluted 10- and 5-fold in blank Sprague-Dawley plasma. For the
`carbazeran cassette-dosed study, samples at time points between 10 and 30 min
`were diluted 10-fold in blank guinea pig plasma with the exception of the
`10-min time point in the intravenous study that was diluted 20-fold. To each
`50-␮l standard or sample solution from the zoniporide study was added 200 ␮l
`
`of acetonitrile containing 10 ng/ml internal standard for protein precipitation.
`The solutions were mixed and centrifuged at 3000g for 20 min. Then 120 ␮l
`of the supernatant from standard and sample mixtures was transferred to a
`96-deep well plate and diluted 1:1 with 0.1% formic acid in water. To 120 ␮l
`of standard and sample solutions in the carbazeran study was added 480 ␮l of
`0.1% formic acid in 1:1 acetonitrile-water. After mixing, the solutions were
`analyzed by LC-tandem mass spectrometry as follows. An API 4000 mass
`spectrometer (Applied Biosystems/MDS Sciex, Foster City, CA) equipped
`with Turbo V sources and a TurboIonSpray interface integrated with a Prom-
`inence LC-AD20 binary pump (Shimadzu, Columbia, MD) and an autosampler
`(PAL; CTC Analytics AG, Zwingen, Switzerland) with a cool stack temper-
`ature controlled at 4 – 8°C was used for analysis. All instruments were con-
`trolled and synchronized by Analyst software from Applied Biosystems/MDS
`Sciex.
`Ten-microliter aliquots of the zoniporide samples were injected onto a C-18
`reversed phase column (Luna C18-2, 5-␮m, 2.0 ⫻ 30 mm; Phenomenex,
`Torrance, CA) equilibrated with 5% solvent B (0.1% formic acid in acetoni-
`trile) in solvent A (0.1% formic acid in water) and maintained for 0.6 min after
`injection followed by a linear gradient to 98% solvent B over 0.65 min and
`held for 0.55 min and then returned to the original conditions in 0.4 min for a
`cycle time of 3.0 min. The flow rate for the zoniporide analysis was 0.5
`ml/min. The column was equilibrated at 5% solvent B for 0.8 min before
`reinjection. For the analysis of carbazeran, the flow rate was 0.6 ml/min. The
`gradient was maintained at 5% solvent B for 0.6 min, followed by a linear
`increase to 98% solvent B in 0.65 min, and kept at 98% solvent B for 0.95 min,
`followed by a linear decrease to 5% in 0.2 min. The column was equilibrated
`at 5% B for 0.6 min before reinjection.
`The ionization parameters for the compounds were optimized by direct
`infusion of undiluted standards in 50% aqueous acetonitrile containing 0.1%
`formic acid. The multiple reaction monitoring (MRM) transitions for carbaz-
`eran and 1-[2H]carbazeran were m/z 361.2 to m/z 272.2 and m/z 362.2 to m/z
`273.2, respectively. For zoniporide and 2-[2H]zoniporide, the MRM transitions
`used were m/z 321.2 to m/z 262.1 and m/z 322.1 to m/z 263.1, respectively. A
`proprietary Pfizer compound was used as internal standard, transitions for
`which were m/z 364.3 to m/z 228.2. The dwell time of each MRM transition is
`50 ms.
`Response Correction and Data Processing for Pharmacokinetic Param-
`eters. Data were processed using Applied Biosystems/MDS SCIEX Analyst
`software, Excel, and Watson LIMS (version 7.2; Thermo Fisher Scientific,
`Waltham, MA). Analyte peaks were integrated using Analyst 1.4.2 for quan-
`titation and then exported to Excel for response correction. Because the
`difference between the proto and deutero isotopomers is 1 atomic mass unit, a
`correction for the deutero compound was necessary because of the contribution
`from the 13C natural abundance in the proto compound. The response correc-
`tion factor was calculated from the proto standards in the linear range of the
`instrument response; the sample response of the deuterated compound was
`then corrected by subtracting the interference from the proto compound. The
`response correction factor of deuteron zoniporide was determined from
`the proto zoniporide standards in the linear range of the instrument response.
`The responses for deutero zoniporide were corrected by subtracting the con-
`tribution from proto zoniporide. The corrected responses were uploaded to
`Watson LIMS for linear regression and calculations for sample concentrations
`and pharmacokinetic parameters.
`In Vitro KDIEs. The intramolecular deuterium isotope effects for
`1-[2H]phthalazine and 2-[2H]quinoxaline were determined from the ratio of the
`m/z 148 and m/z 147 mass spectrometric responses in their 1-phthalazone and
`2-quinoxalone products. Intermolecular isotope effects on the rate constants for
`substrate depletion were determined in cytosol, hepatocytes, S-9, or partially
`purified human aldehyde oxidase using a 1:1 mixture of the deutero and proto
`forms of quinoline, carbazeran, and zoniporide at 1.0 ␮M.
`Michaelis-Menten kinetic parameters for quinoline and 2-[2H]quinoline
`were determined with guinea pig liver cytosolic aldehyde oxidase with eight
`substrate concentrations of quinoline (spanning ⫾5 ⫻ Km) after the linear
`dependence on time and protein concentration were established. 2-Quinolone
`was quantitated by UV absorbance at 250 nm for the peak matched with m/z
`146. Kinetic parameters were determined from v versus [S] plots using XL-Fit
`version 4.0.
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`MH+ = 148 amu
`
`MH+ = 147 amu
`
`H
`
`NN
`
`N
`
`N
`
`NHNH
`
`O
`
`D
`H
`
`kH
`
`kD
`
`NN
`
`H
`
`D
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`substrate to internal standard versus time. Half-lives were calculated from the
`equation t1/2 ⫽ 0.693/first-order rate constant. Metabolites were identified by
`LC-MS from reactions used to determine half-lives in hepatocytes or S-9
`supplemented with cofactors or from reactions conducted at 10 ␮M concen-
`trations of the appropriate substrates.
`Kinetic Deuterium Isotope Effects on the Pharmacokinetics of Carba-
`zeran and Zoniporide in Guinea Pig and Rat. All procedures, including
`dosing methods, are within the guidelines approved by the Pfizer Institutional
`Animal Care and Use Committee. Male Hartley guinea pigs (325–350 g) and
`male Sprague-Dawley rats (250 –300 g) were used in all pharmacokinetic
`studies. Carbazeran and zoniporide were dosed as 1:1 mixtures of deutero and
`proto forms orally (in water) and intravenously as a bolus dose (in saline) via
`the jugular vein. Carbazeran was dosed at approximately 10 mg/kg b.wt. (5
`mg/kg each isotopic form) for both routes of administration, and zoniporide
`was administered orally at approximately 5 mg/kg b.wt. (2.5 mg/kg each
`isotopomer) and intravenously at approximately 2 mg/kg b.wt. (1 mg/kg each
`isotopomer) in saline. Blood samples (0.5 ml) were taken via the carotid artery
`at appropriate time intervals (between 10 min and 6 h). All samples were kept
`frozen at ⫺20°C until analysis. Pharmacokinetic parameters were determined
`only from the experimentally acquired data sets using Watson LIMS.
`
`Results
`Synthesis. NMR and mass spectrometric analysis of the deuterated
`compounds were consistent with their assigned specifically monodeu-
`terated structures as shown in Scheme 1.
`In Vitro Kinetic Deuterium Isotope Effects. To establish that
`interspecies differences observed for metabolism by aldehyde oxidase
`is not due to species-specific reaction mechanisms, the KDIE was
`determined for the metabolism of several substrates with liver cyto-
`solic aldehyde oxidase from human, rat, and guinea pig. The isotope
`effects determined were 1) intramolecular KDIE for 1-[2H]phthala-
`zine and 2-[2H]quinoxaline, 2) intermolecular KDIEs on the first-
`order rate constants for the oxidations of quinoline/2-[2H]quinoline,
`carbazeran/1-[2H]carbazeran, and zoniporide/2-[2H]zoniporide; and
`3) KDIE on the steady-state kinetic parameters for quinoline and
`2-[2H]quinoline with guinea pig liver cytosolic aldehyde oxidase. The
`aldehyde oxidase-susceptible carbon– hydrogen bonds adjacent to the
`aromatic nitrogens of phthalazine and quinoxaline are equivalent due
`to symmetry (Scheme 2). Replacement of either carbon– hydrogen
`bond by a carbon– deuterium bond provides a direct measure of the
`intrinsic KDIE from the ratio of the m/z 148 and m/z 147 ion current
`intensities in their respective lactam products (Nelson and Trager,
`2003). Table 1 shows the results for intra- and intermolecular KDIEs
`for oxidation of 1-[2H]phthalazine, 2-[2H]and quinoxaline, quinoline/
`2-[2H]quinoline,
`carbazeran/1-[2H]carbazeran,
`and zoniporide/2-
`[2H]zoniporide by aldehyde oxidase from human, rat, and guinea pig
`liver. Across species, the intramolecular KDIE was found to be
`between 4.7 and 5.1 for 1-[2H]phthalazine and 2-[2H]quinoxaline,
`
`kH/kD = m/z148 / m/z147
`
`O
`
`O
`MH+ = 148 amu
`D
`
`H
`MH+ = 147 amu
`O
`
`HN
`
`NN
`
`N
`
`NH
`
`kH
`
`kDD
`
`
`
`DH
`
`NN
`
`b
`
`kH/kD = m/z148 / m/z147
`SCHEME 2. Intramolecular deuterium isotope effect determined from the mass
`spectra of the 2-[2H]quinoxaline and 1-[2H]phthalazine metabolites formed by
`aldehyde oxidase.
`
`In general, reactions with hepatocytes and S-9 were conducted in a 5-ml
`volume (0.75 ⫻ 106 cells/ml for hepatocytes and 1 mg/ml protein for S-9) in
`Williams’ E medium (hepatocytes) or 50 mM potassium phosphate buffer, pH
`7.4 (liver S-9), whereas reactions with liver cytosol or partially purified human
`aldehyde oxidase were conducted in a 1.0-ml reaction volume in 50 mM
`potassium phosphate buffer, pH 7.4. Reactions were initiated by addition of
`substrate to the reaction mixtures that were preincubated at 37°C for approx-
`imately 5 min. Over a period of 60 min for cytosol and S-9 reactions and 90
`min for hepatocyte reactions, eight 100-␮l aliquots were removed and
`quenched in 100 ␮l of acetonitrile containing 1% formic acid. A 100-␮l aliquot
`of an internal standard solution (0.5 ␮M) was added; after mixing, the samples
`were filtered through a protein-binding filter membrane in a 96-well format. A
`25- to 50-␮l aliquot was analyzed by MRM of the proto and deutero substrates
`using substrate-specific transitions. Correction for the peak area of the 2H
`substrate was done by subtracting the appropriate percentage contribution due
`to the natural abundance 13C contribution from the 1H substrate. First-order
`rate constants were determined from the semilogarithmic plots of the ratio of
`
`TABLE 1
`KDIEs for the oxidation of quinoxaline, phthalazine, quinoline, carbazeran, and zoniporide by liver cytosolic aldehyde oxidase from human, rat, and guinea pig,
`hepatocytes from human and rat, and guinea pig S-9 supplemented with cofactors
`
`Substrate
`
`Human
`
`Hk/Dk
`
`Rat
`
`Guinea Pig
`
`Cytosol
`
`Hepatocytes
`
`Cytosol
`
`Hepatocytes
`
`Cytosol
`
`S-9 Supplemented
`
`2-[2H]Quinoxalinea
`1-[2H]Phthalazinea
`Quinolineb
`Carbazeranb
`Zoniporideb
`
`5.0
`5.1
`5.5
`4.8
`5.8
`
`N.D.c
`N.D.
`N.D.
`1.5
`1.9
`
`5.1
`5.0
`6.1
`5.0
`3.6
`
`N.D.
`N.D.
`N.D.
`4.6
`2.7
`
`4.7
`4.9
`6.0
`6.0
`4.8
`
`N.D.
`N.D.
`N.D.
`5.0
`1.5
`
`N.D., not determined.
`a Determined from the ratio of the peak area for m/z 148 (Hk, corrected for the M ⫹ 1 contribution from the m/z 147 ion) to the m/z 147 ion (Dk).
`b Determined from the ratio of the rate constants for disappearance of 2-[1H]quinoline (Hk) and 2-[2H]quinoline (Dk), 1-[1H]carbazeran (Hk) and 1-[2H]carbazeran (Dk), and 2-[1H]zoniporide (Hk)
`and 2-[2H]zoniporide (Dk).
`
`Auspex Exhibit 2004
`Apotex v. Auspex
`IPR2021-01507
`Page 4
`
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` at ASPET Journals on April 30, 2021
`
`DEUTERIUM ISOTOPE EFFECTS ON DRUG PHARMACOKINETICS
`
`629
`
`respectively. The intermolecular KDIEs on the first-order rate con-
`stants for metabolism of quinoline/2-[2H]quinoline, carbazeran/1-
`[2H]carbazeran, and zoniporide/2-[2H]zoniporide are between 3.6 and
`6.1 across species. The KDIEs on the steady-state kinetic constants for
`quinoline and 2-[2H]quinoline with guinea pig liver cytosolic alde-
`hyde oxidase show that the KDIE is primarily on Vmax (5.2) with a
`small effect on Km (1.1) resulting in a KDIE of 6.0 on the intrinsic
`clearance (Table 2). These results are consistent with a common
`reaction mechanism for aldehyde oxidases from human, rat, and
`guinea pig liver, where C–H bond cleavage occurs in the rate-limiting
`step and the KDIE is expressed on the intrinsic clearance in all
`species.
`Metabolically active hepatocytes have the complete complement of
`drug-metabolizing enzymes and consequently serve as the closest in
`vitro surrogate for in vivo hepatic metabolism (Fabre et al., 1990). The
`extent to which aldehyde oxidase contributes to the overall hepatic
`metabolic transformation of a drug may then be established by exam-
`ining the KDIE on the intrinsic clearance of the drug in hepatocytes.
`Guinea pig hepatocytes are not commercially available; therefore, the
`guinea pig liver S-9 fraction supplemented with NADPH and UDP-
`glucuronic acid cofactors and alamethicin (to permeate the micro-
`somal membrane) (Fisher et al., 2000) was used to mimic the hepa-
`tocyte system as closely as possible (Dalvie et al., 2009). The KDIEs
`for carbazeran in rat hepatocytes and guinea pig S-9 are 4.6 and 5.0,
`respectively (Table 1). These values are comparable to the KDIEs
`with cytosolic aldehyde oxidase of the three species examined (Table
`1) and suggest that in the guinea pig and rat aldehyde oxidase is
`probably the primary route of drug metabolic clearance. In contrast, in
`human hepatocytes the KDIE is significantly decreased (1.5) (Table
`1), suggesting that in humans other metabolic pathways contribute a
`greater extent of carbazeran’s hepatic metabolic clearance. Consistent
`with this interpretation, the glucuronide of carbazeran was identified
`as the major metabolite in human hepatocyte reactions with the
`aldehyde oxidase product secondary (Fig. 1A). Although the carbaz-
`eran glucuronide metabolite was also detected in the guinea pig S-9
`and rat hepatocyte reactions (Fig. 1, B and C, respectively), the levels
`were negligible in comparison with that for the aldehyde oxidase
`metabolite.
`The KDIEs for zoniporide with cytosolic aldehyde oxidase from
`human and guinea pig are similar (5.8 and 4.8) (Table 1). However,
`with human hepatocytes and guinea pig S-9 supplemented with co-
`factors, the KDIE is substantially reduced (1.9 and 1.5, respectively)
`(Table 1). This result suggests that the aldehyde oxidase pathway is
`not a major metabolic clearance mechanism for zoniporide in the
`human and guinea pig liver. With rat cytosol the KDIE is somewhat
`smaller (3.5) (Table 1) than would be expected if aldehyde oxidase
`were the only enzyme responsible for metabolism and is further
`decreased in rat hepatocytes (2.7) (Table 1). The decreased KDIE in
`rat cytosol and hepatocytes is accounted for by other metabolic
`pathways that are evident from the metabolic profile shown in Fig. 1D
`for rat hepatocytes. Hydrolysis of the acyl guanidine function to the
`carboxylic acid (M1) contributes approximately 10%, and other me-
`tabolites (M2, M3, M5, M7, M8, and M10) derived from oxidations
`by cytochromes P450 co