A Publicatioll of The American~ Society
`for Pharmacology and Experimental Therapeutics
`Founded by John J. Abel-1909
`
`)
`
`-
`
`H
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`Edited for the Society by S. J. Enna
`
`e Published by Williams & Wilkins
`
`Roxane Labs., Inc.
`Exhibit 1006
`Page 001
`
`

`
`COPYRIGHT© 1998 BY THE AMERICAN SOCIETY FOR PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
`
`Roxane Labs., Inc.
`Exhibit 1006
`Page 002
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`

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`VOLUME 286
`SEPTEMBER 1998
`NUMBER 3
`
`-The Journal of
`PHARMACOLOGY
`
`AND EXPERIMENTAL THERAPEUTICS
`
`Contents
`
`Role of 5-HT 4 Receptors in the Mouse Passive A voidance Test
`
`Inhibition of Cholesterol Biosynthesis in Primary Cultured Rat
`Hepatocytes by Artichoke (Cynara scolymus L.) Extracts
`Pharmacological Characterization of Human ml Muscarinic
`Acetylcholine Receptors with Double Mutations at the
`Junction of TM VI and the Third Extracellular Domain
`Substance P Induction of Itch-Associated Response Mediated
`by Cutaneous NK1 Tachykinin Receptors in Mice
`Evidence against Anandamide as the Hyperpolarizing Factor
`Mediating the Nitric Oxide-Independent Coronary Vasodilator
`Effect of Bradykinin in the Rat
`Concentration-Effect Relationship of [-Propranolol and
`Metoprolol in Spontaneous Hypertensive Rats after Exercise(cid:173)
`Induced Tachycardia
`The Role of Dopaminergic Systems in the Perinatal Sensitivity
`to 3,4-Methylenedioxymethamphetamine-Induced
`Neurotoxicity in Rats
`Transport of L-Valine-Acyclovir Via the Oligopeptide
`Transporter In The Human Intestinal Cell Line, Caco-2
`Calcium-Mediated Second Messengers Modulate the
`Expression of Behavioral Sensitization to Cocaine
`Multiple Actions of Methohexital on Hippocampal CAl and
`(\ Cortical Neurons of Rat Brain Slices
`Chronic Administration of Taurine to Aged Rats Improves the
`Electrical and Contractile Properties of Skeletal Muscle Fibers
`
`CVT-124, a Novel Adenosine A1 Receptor Antagonist with
`Unique Diuretic Activity
`
`Comparing the Subjective, Psychomotor and Physiological
`Effects of Intravenous Pentazocine and Morphine in Normal
`Volunteers
`Hydrogen Peroxide-Induced Stimulation of L-Type Calcium
`Current in Guinea Pig Ventricular Myocytes and Its Inhibition
`by Adenosine Al Receptor Activation
`Effect of KATP Channel Blocker U37883A on Renal Function
`in Experimental Diabetes Mellitus in Rats
`Inhibitory Effects of Nitric Oxide Donors on Nitric Oxide
`Synthesis in Rat Gastric Myenteric Plexus
`Characterization of the Effects of the Partial Dopamine
`Agonist Terguride on Cocaine Self-Administration in the Rat
`Full Agonistic Properties of Bay x 3702 on Presynaptic and
`Postsynaptic 5-HT lA Receptors: Electrophysiological Studies in
`the Rat Hippocampus and Dorsal Raphe
`Dopamine D2 Receptors Mediate Glomerular Hyperfiltration
`Due To Amino Acids
`
`Nicoletta Galeotti, Carla Gh~lardini and
`Alessandro Bartolini
`Rolf Gebhardt
`
`X.-P. Huang, F. E. Williams,
`S. M. Peseckis and W. S. Messer, Jr.
`
`Tsugunobu Andoh, Tetsuro Nagasawa,
`Masamichi Satoh and Y asushi Kuraishi
`David Fulton and John Quilley
`
`Lena Brynne, Mats 0. Karlsson and
`Lennart K. Paalzow
`
`Norberto Aguirre, Meritxell Barrionuevo,
`Berta Lasheras and Joaquin Del Rio
`
`Remco L. A. de Vrueh, Philip L. Smith and
`Chao-Pin Lee
`R. C. Pierce, E. A. Quick, D. C. Reeder,
`Z. R. Morgan and P. W. Kalivas
`Liang Zhang, Yu Zhang and
`Richard Wennberg
`Sabata Piemo, Annamaria De Luca,
`Claudia Camerino, Ryan J. Huxtable and
`Diana Conte Camerino
`Miklos Gellai, George F. Schreiner,
`Robert R. Ruffolo, Jr., Tracey Fletcher,
`Robin De Wolf and David P. Brooks
`James P. Zacny, Joanna L. Hill,
`Matthew L. Black and Parvine Sadeghi
`
`George P. Thomas, Stephen M. Sims,
`Michael A. Cook and Morris Karmazyn
`
`Volker Vall on, Margitta Albin us and
`Doreen Blach
`Kenji Hosoda, Toku Takahashi,
`Masayuki A. Fujino and Chung Owyang
`Luigi Pulvirenti, Claudia Balducci,
`Marla Piercy and George F. Koob
`Jianming Dong, Claude de Montigny and
`Pierre Blier
`
`Gerd Luippold and Bernd Muhlbauer
`
`1115. I'
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`1122
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`1129
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`1140
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`1146
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`1152
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`1159
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`1166
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`1171
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`1177
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`1183
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`1191
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`1197
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`1208
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`1215
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`1222
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`1231
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`1239
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`1248
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`Contents continued on iv
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`Roxane Labs., Inc.
`Exhibit 1006
`Page 003
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`Contents continued from iii
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`Further Characterization of the Expression in Liver and
`Catalytic Activity of CYP2B6
`
`Effects of N-Methyl-D-Aspartate Receptor Antagonists on
`Discriminative Stimulus Effects of Naloxone in Morphine(cid:173)
`Dependent Rats Using theY-Maze Drug Discrimination
`Paradigm
`Potentiation and Inhibition of Nicotinic Acetylcholine
`Receptors by Spermine in the TE671 Human Muscle Cell Line
`
`Antiplatelet Efficacy of XV 459, A Novel Nonpeptide Platelet
`GPIIb/IIIa Antagonist: Comparative Platelet Binding Profiles
`with c7E3
`
`Application of Liquid Chromatography/Mass Spectrometry in
`Accelerating the Identification of Human Liver Cytochrome
`P450 Isoforms Involved in the Metabolism of Iloperidone
`A Study on the Metabolism of Etoposide and Possible
`Interactions with Antitumor or Supporting Agents by Human
`Liver Microsomes
`
`Differential Blockade of the Antinociceptive Effects of
`Centrally Administered Cannabinoids by SR141716A
`Vasoregulatory Prostanoid Generation Proceeds Via
`Cyclooxygenase-2 in Noninflamed Rat Lungs
`Role of Vasopressin on Adrenergic Neurotransmission in
`Human Penile Blood Vessels
`
`Modifications by Superoxide-Generating Agent,
`Neurotransmitters and Neuromodulators of Nitroxidergic
`Nerve Function in Monkey Cerebral Arteries
`Apparent Insensitivity of the Hotplate Latency Test for
`Detection of Antinociception Following Intraperitoneal,
`Intravenous or Intracerebroventricular M6G Administration
`to Rats
`Interaction of 2' ,2' -Difluorodeoxycytidine (Gemcitabine) and
`Formycin B with the Na+ -Dependent and -Independent
`Nucleoside Transporters of Ehrlich Ascites Tumor Cells
`S 16924 ((R)-2-{1-[2-(2,3-Dihydro-Benzo[1,4] Dioxin-5-Yloxy)(cid:173)
`Ethyl]-Pyrrolidin-3yl}-1-( 4-Flu oro-Phenyl)-Ethanone ), a Novel,
`Potential Antipsychotic with Marked Serotonin (5-HT)lA
`Agonist Properties: I. Receptorial and Neurochemical Profile
`in Comparison with Clozapine and Haloperidol
`S 16924 ((R)-2-{1-[2-(2,3-Dihydro-Benzo[1,4] Dioxin-5-Yloxy)(cid:173)
`Ethyl]-Pyrrolidin-3yl}-1-( 4-Fluoro-Phenyl)-Ethanone ), a Novel,
`Potential Antipsychotic with Marked Serotonin (5-HT)lA
`Agonist Properties: II. Functional Pvofile in Comparison to
`Clozapine and Haloperidol
`
`Potentiation of CD95L-Induced Apoptosis of Human
`Malignant Glioma Cells by Topotecan Involves Inhibition of
`RNA Synthesis but Not Changes in CD95 or CD95L Protein
`Expression
`Role of Cyclooxygenase-2 in the Healing of Gastric Ulcers in
`Rats
`
`1253
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`1260
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`1269
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`1277
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`1285
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`1294
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`1301
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`1309
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`1315 jfl~
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`1321
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`1326
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`1333
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`1341 •
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`-~
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`1356 'II
`
`(I
`
`Sean Ekins, Mark V andenBranden,
`Barbara J. Ring, Jennifer S. Gillespie,
`Tian J. Yang, Harry V. Gelboin and
`Steven A. Wrighton
`Ivan 0. Medvedev, Olga A. Dravolina and
`Anton Y. , Bespalov
`
`Zuoyi Shao, Ian R. Mellor,
`Matthew J. Brierley, John Harris and
`Peter N. R. U sherwood
`Shaker A. Mousa, Jeffrey M. Bozarth,
`William Lorelli, Mark S. Forsythe,
`Martin J. M. C. Thoolen, Thomas M. Reilly and
`Paul A. Friedman
`A. E. Mutlib and J. T. Klein
`
`Takashi Kawashiro, Kouwa Yamashita,
`Xue-Jun Zhao, Eriko Koyama,
`Masayoshi Tani, Kan Chiba and
`Takashi Ishizaki
`
`Sandra P. Welch, John W. Huffman and
`John Lowe
`L. Ermert, M. Ermert, A. Althoff,
`M. Merkle, F. Grimminger and W. Seeger
`
`Gloria Segarra, Pascual Medina,
`Cristina Domenech, Jose M. Vila,
`Juan B. Martinez-Leon, Martin Aldasoro and
`Salvador Lluch
`
`Tomio Okamura, Hideyuki Fujioka,
`Kazuhide Ayajiki and Noboru Toda
`
`Samantha M. South and Maree T. Smith
`
`Trisha Burke, Stephanie Lee,
`Peter J. Ferguson and James R. Hammond
`
`Mark J. Millan, Alain Gobert,
`Adrian Newman-Tancredi, Valerie Audinot,
`Frans;oise Lejeune, Jean-Michel Rivet,
`Didier Cussac, Jean-Paul Nicolas,
`Olivier Muller and Gilbert Lavielle
`Mark J. Millan, Rudy Schreiber,
`Anne Dekeyne, Jean-Michel Rivet,
`Karin Bervoets, Michaelis Mavridis,
`Claude Sebban, Sophie Maurel-Remy,
`Adrian Newman-Tancredi,
`Michael Spedding, Olivier Muller,
`Gilbert Lavielle and Mauricette Brocco
`
`Stephan Winter and Michael Weller
`
`1374
`
`Jun-ichi Shigeta, Satoru Takahashi and
`Susumu Okabe
`
`1383
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`fJ1 1f
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`Contents continued on v
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`Roxane Labs., Inc.
`Exhibit 1006
`Page 004
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`Contents continued from iv
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`Hepatic Sinusoidal Membrane Transport of Anionic Drugs
`Mediated by Anion Transporter Nptl
`
`The Effects of Stress on Homeostasis in JCR-LA-cp Rats: The
`Role of Nitric Oxide
`
`Purification of Two Rat Hepatic Proteins with A-Esterase
`Activity Toward Chlorpyrifos-Oxon and Paraoxon
`Modulation of Epileptiform Activity by Adenosine A1
`Receptor-Mediated Mechanisms in the Juvenile Rat
`Hippocampus
`EP iEP 2 Receptor-Specific Prostaglandin E 2 Regulation of
`lnterleukin-6 Generation by Human HSB.2 Early T Cells
`Protective Effect of Bismuth Nitrate Against Injury to the
`Bone Marrow by y-lrradiation in Mice: Possible Involvement
`of Induction of Metallothionein Synthesis
`Dissociation of Angiotensin II -Stimulated Activation of
`Mitogen-Activated Protein Kinase Kinase from Vascular
`Contraction
`Saquinavir, an HIV Protease Inhibitor, Is Transported by
`P-Glycoprotein
`M2 Muscarinic Autoreceptors Modulate Acetylcholine Release
`in the Medial Pontine Reticular Formation
`HMR 1883, a Novel Cardioselective Inhibitor of the ATP(cid:173)
`Sensitive Potassium Channel. Part 1: Effects on
`Cardiomyocytes, Coronary Flow and Pancreatic P-Cells
`HMR 1883, a Novel Cardioselective Inhibitor of the ATP(cid:173)
`Sensitive Potassium Channel. Part II: Effects on Susceptibility
`to Ventricular Fibrillation Induced by Myocardial Ischemia in
`Conscious Dogs
`Prenatal Exposure to Fluoxetine (Prozac) Produces Site(cid:173)
`Specific and Age-Dependent Alterations in Brain Serotonin
`Transporters in Rat Progeny: Evidence from
`Autoradiographic Studies
`Serotonin-Mediated Palmitoylation and Depalmitoylation of G
`Alpha Proteins in Rat Brain Cortical Membranes
`Identification of New Human CYP2C19 Alleles (CYP2C19*6
`and CYP2Cl9*2B) in a Caucasian Poor Metabolizer of
`Mephenytoin
`
`ERRATUM
`
`2 ERRATUM
`
`Index, Volume 286, July-September, 1998
`
`Hikaru Y abuuchi, lkumi Tarnai,
`Kyoko Morita, Tomoko Kouda,
`Ken-ichi Miyamoto, Eiji Takeda and
`Akira Tsuji
`Juan C. Leza, Eduardo Salas,
`Grzegorz Sawicki, James C. Russell and
`Marek W. Radomski
`Amber L. Pond, Howard W. Chambers,
`Cody P. Coyne and Janice E. Chambers
`Virginia Tancredi, Margherita D' Antuono,
`Astrid N ehlig and Massimo A voli
`
`Li Zeng, Songzhu An and
`Edward J. Goetzl
`
`Nobuhiko Miura, Masahiko Satoh,
`Nobumasa Imura and Akira Naganuma
`
`Stephanie W. Watts, Jennifer A. Florian and
`Kimberly M. Monroe
`
`Annice E. Kim, Jay M. Din taman,
`DavidS. Waddell and Jeffrey A. Silverman
`
`H. A. Baghdoyan, R. Lydic and
`M. A. Fleegal
`
`Heinz Gogelein, Jens Hartung,
`Heinrich C. Englert and
`Bernward A. SchOlkens
`
`George E. Billman, Heinrich C. Englert and
`Bernward A. Scholkens
`
`Theresa M. Cabrera-Vera and
`George Battaglia
`
`Shubhada Bhamre, Hoau-Y an Wang and
`Eitan Friedman
`Gordon C. lbeanu, Joyce A. Goldstein,
`Urs Meyer, Simone Benhamou,
`Christine Bouchardy, Pierre Dayer,
`Burhan I. Ghanayem and Joyce Blaisdell
`
`1391
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`1397 4
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`•
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`1404
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`1412
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`1420
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`1427
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`1431
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`1439
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`1446
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`1453
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`1465
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`1474
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`1482
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`1490
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`1495
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`1496
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`1497
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`0022-3565/98/2863-1285$03.00/0
`THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
`Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics
`JPET 286:1285-1293, 1998
`
`Vol. 286, No. 3
`Printed in U.S.A.
`
`App-lication of Liquid Chromatography/Mass Spectrometry in
`Accelerating the Identification of Human Liver Cytochrome
`P450 lsoforms Involved in the Metabolism of lloperidone
`
`A. E. MUTLIB and J. T. KLEIN
`Department of Chemical Research, Neuroscience Therapeutic Area, Hoechst Marion Rousse! Inc., Bridgewater, New Jersey
`Accepted for publication May 11, i 998
`This paper is available online at http://www.jpet.org
`
`ABSTRACT
`[1-[4-[3-[4-(6-fluoro-1 ,2-benzisoxazol-3-yl)-1-piper(cid:173)
`lloperidone,
`idinyl]propoxy]-3-methoxyphenyl]ethanone, 1, is currently under(cid:173)
`going clinical trials as a potential antipsychotic agen~. The metab(cid:173)
`olism of iloperidone was studied in human liver microsomes to
`define the metabolic pathways and to identify the cytochrome
`P450 (CYP) isoforms responsible for the formation of major ilo(cid:173)
`peridone metabolites. lloperidone was extensively metabolized in
`vitro via hydroxylation, reduction and 0-demethylation to produce
`1-[4-[3-[4-(6-fluoro-1 ,2-benzisoxazol-3-yl)-1-piperidinyl]propoxy]-
`3-methoxyphenyl]-2-hydroxyethanone, 4; 4-[3-[4-(6-fluoro-1 ,2-
`benzisoxazol-3-yl)-1-piperidinyl]propoxy]-3-methoxy-a-methyl(cid:173)
`benzene methanol, 3, and 1-[4-[3-[4-(6-fluoro-1 ,2-benzisoxazol-
`3-yl)-1-piperidinyl]propoxy]-3-hydroxyphenyl]ethanone, 2, re(cid:173)
`spectively, in decreasing order of abundance. The major in vitro
`metabolite, 4, present in trace quantities ir.1 urine, was postulated
`to be either eliminated in bile as a conjugate or further metabolized
`to a phenol, 4-[3-[4-(6-fluoro-1 ,2-benzoisoxazol-3-yl)-piperidin-
`1-yl]propoxy]-3-methoxyphenol, 5. The formation of the three ma(cid:173)
`jor in vitro metabolites 2, 3 and 4 was NADPH dependent. The
`major circulating and urinary metabolite in humans dosed with 1
`was metabolite 3. The mean apparent Km and V max for formation
`
`of 2 by human liver microsomes was 7.4 ± 3.0 pM and 0.0343 ±
`0.0134 nmol min- 1 mg- 1
`, respectively. The mean apparent Km
`and V max for 3 was 101.2 ± 34.7 pM and 0.·1414 ± 0.0346 nmol
`min- 1 mg- 1
`, respectively. The mean apparent Km and Vmax for 4
`was 39.7 ± 10.8 J-LM and 0.1372 ± 0.056 nmol min- 1 mg-1,
`respectively. The CYP isoenzymes responsible for the formation
`of metabolites 2, 3 and 4 were determined by using selective
`chemical inhibitors and by correlation studies. Metabolites 2 and 4
`were formed by CYP3A4 and by the polymorphic CYP2D6 re(cid:173)
`spectively. Metabolite 3 is postulated to be produced mainly by a
`cytosolic enzyme(s), although CYP3A, CYP1A2 and CYP2E1
`isozymes were shown to be involved in its formation as well. The
`power of liquid chromatography/mass spectrometry in greatly
`accelerating the process of identifying the human liver CYP iso(cid:173)
`forms involved in the metabolism of iloperidone was demon(cid:173)
`strated in this study. Liquid chromatography/mass spectrometry
`was used in the initial studies to confirm the identities of the
`metabolites. This was followed by accurate and reliable quantita(cid:173)
`tion of individual metabolites present in biological extracts by
`operating· the mass spectrometer in th~ selected ion monitoring
`mode.
`
`Iloperidone, 1- [ 4-[3- [ 4-( 6-fluoro-1,2-benzisoxazol-3-yl)-1-pi(cid:173)
`peridinyl]propoxy]-3-methoxyphenyl]ethanone, is a dopa(cid:173)
`mine-Diserotonin-5-HT2 antagonist that has been demon(cid:173)
`strated recently from completed phase II clinical trials, as a
`potential atypical antipsychotic with a low propensity to
`cause extrapyramidal side effects (Strupczewski et al., 1990,
`1995; Corbett et al., 1993; Szewczak et al., 1995; Szczepanik
`'"'€t al., 1996). Iloperidone was found to be extensively metab(cid:173)
`olized by 0-dealkylation, N-dealkylation, reduction and hy(cid:173)
`droxylation in rats, dogs and humans (Mutlib et al., 1995a).
`It was shown that iloperidone was metabolized via 0-deal(cid:173)
`kylation to yield 6-fluoro-3-[1-(3-hydroxypropyl)-4-piperidi(cid:173)
`nyl]-1,2-benzisoxazole and 1-[4-[3-[4-(6-fluoro-1,2-benzisox(cid:173)
`azol-3-yl)-1-piperidinyl]propoxy] -3-hydroxyphenyl] ethanone.
`
`Received for publication February 16, 1998.
`
`Oxidative N-dealkylation led to the formation of 6-fluoro-3-
`(4-piperidinyl)-1,2-benzisoxazol and a secondary metabolite,
`3-[( 4-acetyl-2-methoxy )phenoxy] propionic acid. Iloperidone
`was reduced to 4-[3-[4-(6-fluoro-1,2-benzisoxazol-3-yl)-1-pip(cid:173)
`eridinyl]propo:xy]-3-metho:xy-a-methylbenzene methanol which
`was identified as the major circulating metabolite in humans and
`rats. Hydroxylation ofiloperidone produced 1-[4-[3-[4-(6-fluoro-1,2-
`benzisoxazol-3-yl)-1-piperidinyl]propo:xy]-2-hydroxy-5-methoxy(cid:173)
`phenyl]ethanone and 1-[4-[3-[4-( 6-fluoro-1,2-benzisoxazol-3-yl] -1-
`piperidinyl]-3-methoxyphenyl]-2-hydro:xyethanone, the later of
`which was found to be the principal circulating metabolite in dogs.
`The identities of all these metabolites were established by compar(cid:173)
`ing the LC/MS retention times and mass spectral data with syn(cid:173)
`thetic standards (Mutlib et al., 1995a).
`Metabolism studies with human liver microsomes indicated
`that the major metabolite produced in vitro was 1-[4-[3-[4-(6-
`
`ABBREVIATIONS: GYP, cytochrome P450; LC/MS, liquid chromatography/mass spectrometry; NMR, nuclear magnetic resonance; HPLC, high
`performance liquid chromatography; SIM, selected ion monitoring.
`
`1285
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`1286
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`Mutlib and Klein
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`Vol. 286
`
`fluoro-1,2-benzisoxazol-3-yl)-1-piperidinyl] propoxy-3-methoxy(cid:173)
`phenyl]-2-hydroxyethanone, 4. However, this metabolite was
`present in very small quantities in human plasma and urine
`indicating further metabolism or excretion of this compound in
`bile. The reduced product, 4-[3-[4-(6-fluoro-1,2-benzisoxazol-3-
`yl)-1-piperidinyl]propoxy]-3-methoxy-a-methylbenzene metha(cid:173)
`nol, 3, was found as the next major metabolite in the in vitro
`extracts. This was the major circulating metabolite in human
`plasma. The 0-demethylated metabolite, 1-[4-[3-[4-(6-fluoro-
`1 ,2-benzisoxazol-3-yl)-1-pi peridinyl] propoxy] -3-hydroxyphe(cid:173)
`nyl] ethanone, 2, was the third most abundant microsomal me(cid:173)
`tabolite.
`The particular isoenzymes responsible for the formation of
`the three major metabolites 2, 3 and 4 (fig. 1) by human liver
`microsomes was determined using specific chemical inhibi(cid:173)
`tors and by correlation analyses. The use of these two tech(cid:173)
`niques in characterizing the CYP450 involved in the metab(cid:173)
`olism of xenobiotics has been described previously (Inaba et
`al., 1985; Miners et al., 1988, 1995; Murray et al., 1990;
`Veronese et al., 1991; Andersson et al., 1993; Tassaneeyakul
`et al., 1993a, 1993b; Thummel et al., 1993; Wrighton et al.,
`1993; Chang et al., 1994; Halpert et al., 1994). To confirm the
`involvement of polymorphic CYP2D6 in the formation of the
`major in vitro metabolite 4, metahQlism studies were carried
`out with eDNA-expressed CYP2D.6. The kinetics (V max and
`Krn) for the formation of metabolites 2, 3 and 4 were also
`evaluated using microsomes from four different human livers
`and the average intrinsic clearance of each metabolite was
`obtained from the calculated V max and Krn values.
`
`Methods
`Materials and reagents. Acetonitrile, methanol, formic acid and
`· ethyl acetate (all HPLC grade solvents, EM Scienc~, Gibbstown, NJ),
`ammonium hydroxide, glacial acetic acid, a-phosphoric acid, 85% and
`am;monium formate (Fisher Scientific, Fair Lawn, NJ), Bond-Elut
`Certify, 130 mg/~0 ml cartridges (Varian, Harbor City, CA), were
`purchased from their respective suppliers. Other reagents were of
`either HPLC or analytical grade and were used without further
`purification.
`Human liver microsomes were obtained from International Insti(cid:173)
`tute for the Advancement of Medicine (Exton, P A). Microsomes pre-"'··
`pared from cells transfected with cDNAs encoding human CYP2D6,
`CYP2E1, CYP2A6 and CYP3A4 were obtained from Gentest Corpo(cid:173)
`ration (Woburn, MA).{3-Glucuronidase (Helix Pomatia, Type 1), glu(cid:173)
`cose-6-phosphate, NADP, NADPH and glucose-6-phosphate dehy(cid:173)
`drogenase were obtained from Sigma Chemical (St. Louis, MO).
`The chemical inhibitors were obtained from the following sources:
`furafylline and sulfaphenazole, Research Biochemicals International
`(Natick, MA); troleandomycin and diethyldithiocarbamate, Sigma
`Chemical; and quinidine, Aldrich Chemical (Milwaukee, WI).
`Synthesis ofmetabolic standards. The synthesis ofilopepdone, 1-[4-
`[3-[4-( 6-fluoro-1,2-benzisoxazol-3-yl)-1-piperidinyl]propoxy-3~methoxyphe­
`nyl]ethanone (1), 1-[4-[3-[4-(6-fluoro-1,2-benzisoxazol-3-yl)-1-piperidinyl](cid:173)
`propoxy] -3-hydroxyphenyl] ethanone (2), 4-[3-[ 4-( 6-fluoro-1,2-benzisoxa(cid:173)
`zol-3-yl)-1-piperidinyl]propoxy]-3-methoxy-a-methylbenzene methanol (3),
`1-[4- [3-[ 4-( 6-fluoro-1,2-benzisoxazol-3-yl)-1-piperidinyl]propoxy] -3-me(cid:173)
`thoxyphenyl]-2-hydroxyethanone ( 4) and 1-[4-[3-[4-(6-fluoro-1,2-benzisox(cid:173)
`azol-3-yl)-1-piperidinyl]propoxy-3-'ethoxyphenyl]ethanone (internal stan(cid:173)
`dard) have been described previously (Strupczewski et a(., 1995).
`Metabolite 5 was synthesized as described below.
`Acetic acid 4-(3-chloropropoxy)-3-methoxyphenyl ester: 3-Chlo(cid:173)
`roperoxybenzoic acid (50-60%, 3 g) was added to a solution of 1-[4-
`
`2
`
`0-Demethylation~
`
`3
`
`Reduction (cytosolic and
`microsomal enzymes)
`
`iloperidone, 1
`
`Hydroxylation (CYP2D6)
`
`Fig. 1. Structures of iloperidone (1) and metabo(cid:173)
`lites 2, 3, 4 and 5. The isozyme(s) responsible for the
`formation of metabolites 2 and 4 are shown.
`
`5
`
`Roxane Labs., Inc.
`Exhibit 1006
`Page 007
`
`

`
`1998
`
`(3-chloropropoxy)-3-methoxyphenyl]ethanone (2.0 g, 8.2 mmol) in 50
`ml of chloroform. The resultant solution was allowed to stand at
`ambient temperature for 24 hr, then was washed with saturated
`aqueous NaHC0 3 solution (50 ml), and then dried (anhy. MgS04 ),
`filtered and concentrated in vacuo to 1.5 g of an oil. Flash chroma(cid:173)
`tography (silica gel, 20 mm X 100 mm, 50% heptane/dichlorometh(cid:173)
`ane) afforded 1.3 g (61 %) of a pale yellow oil. An analytical sample
`(0.5 g) was purified by bulb-to-bulb distillation via Kugelrohr oven
`(220°C/0.2 mm) to afford 0.39 g of a pale yellow oil. IR (neat): 2970,
`2940, 1765, 1610, 1520, 1210, 1030 cm- 1
`1H-NMR (CDC13 ): 8 2.17-
`;
`2.31 (m, 2H), 2.27 (s, 3H), 3.72-3.79 (t, 2H, J = 6.4 Hz), 3.83 (s, 3H),
`4.11-4.17 (t, 2H, J = 5.9 Hz), 6.59-6.64 (m, 2H), 6.86-6.90 (m, 1H)
`and MS: m/e (rei. intensity) 258 (M+, 50), 216 (100), 140 (66), 125
`(16), 111(14). Calculated for C12H15Cl04 , 55.71% C, 5.84% H, 13.70%
`Cl, 24.74% 0; found: 55.58% C, 5.83% H, 13.86% Cl and 24.39% 0.
`4- [3- [ 4-( 6-Fluoro-1 ,2-benzisoxazol-3-yl)-pi peri din -1-yl] propoxy] -3-
`methoxyphenol (5): A mixture of 6-fluoro-3-(4-piperidinyl)-1,2-
`benzisoxazole (Strupczewski et al., 1995) (4.0 g, 18.2 mmol), acetic
`acid 4-(3-chloropropoxy)-3-methoxyphenyl ester (5.0 g, 19.3 mmol)
`and K2C03 (milled, 8.0 g, 57.9 mmol) in 50 ml of 1-methyl-2-pyrro(cid:173)
`lidinone was heated with stirring at 95-100°C for 1.5 hr. After
`cooling, the mixture was diluted with ethyl acetate (500 ml), washed
`with water (2 X 300 ml), then brine, and then dried (anhy. MgS04 ),
`filtered and concentrated in vacuo to 11 g of a dark oil. Gradient flash
`chromatography (silica, 40 mm X 120 mm, dichloromethane, fol(cid:173)
`lowed by 1%, 2%, 3%, 4%, and then 5% methanol in dichloromethane,
`afforded 1.4 g (19.2%) of a brown solid. Recrystallization from ace(cid:173)
`tonitrile (15 ml) afforded 1.0 g (13.7%) of light tan crystals, mp
`160-161 oc. IR (KBr): 3460, 3080, 2940, 1615, 1520, 1450, 1210,
`1120, 1035, 950, 825 em-\ 1H-NMR (DMSO-d6 ): 8 1.80-3.32 (m,
`13H), 3.71 (s, 3H), 3.84-3.92 (t, 2H, J = 6.4 Hz), 6.21-6.27 (dd, 1H,
`J = 2.7, 8.6 Hz), 6.40 (d,1H, J = 2.7 Hz), 6.75 (d, 1H, J = 8.6 Hz),
`7.21-7.31 (dt, 1H, J = 2.2, 9.1 Hz), 7.63-7.69 (dd, 1H, J = 2.2, 9.1
`Hz), 7.95-8.02 (dd, 1H, J = 5.3, 8.8 Hz), 8.94 (bs, 1H, exchangeable
`with deuterium oxide) and MS: m/e (rei. intensity) 400 (M+, 100),
`262 (28), 233 (33), 96 (31). Calculated for C22H 25FN20 4 , 65.99% C,
`6.29% H, 7.00% N; found 65.87% C, 6.22% H, 7.06% N.
`General instrumentation. Melting points were determined on a
`Thomas-Hoover capillary apparatus (uncorrected). IR spectra were
`obtained with either a Perkin-Elmer 547 or Nicolet 205 FT-IR spec(cid:173)
`trometer. NMR spectra for the synthetic compounds were obtained
`using Varian Gemini 200 spectrometer. Chemical shifts are reported
`relative ·to tetramethylsilane. Mass spectra of synthetic standards
`were obtained on a Finnigan 4500 MS equipped with a Super IN COS
`data system. Elemental analysis were performed by Robertson Mi(cid:173)
`crolit Labbratories (Madison, NJ).
`LC/MS. A Spectra Physics SP 8800 ternary pump and a Waters
`autosampler (Model 717) were used for all LC/MS analysis. Chroma(cid:173)
`tography was carried out at ambient temperature using a base
`deactivated 100 X 4.6 mm, 5 11-m, Hypersil C18 analytical column
`(Keystone Scientific, Bellefonte, PA). No guard columns were used
`during the assays. The mobile phase delivered at 0.55 ml/min con(cid:173)
`sisted of a 70:30 v/v mixture of acetonitrile and 2.5 mM ammonium
`formate adjusted to pH 3.5 with 96% formic acid. The mobile phase
`was filtered through a 0.45 11-m nylon filter (Rainin Instruments,
`. ., Woburn, MA) and degassed for 5 min under vacuum. A splitter was
`set up in the electrospray interface that led to ~20% of the HPLC
`effluent to be sprayed in the source of the mass spectrometer. Post(cid:173)
`column plumbing consisted of capillary tubings obtained from
`Polymicro Technologies (North Phoenix, AZ). The internal diameters
`of these capillaries were 50 and 100 11-m. The samples were intro(cid:173)
`duced onto the HPLC column by the autoinjector set at a run time of
`11 min.
`Mass spectrometric detection was carried out using a PE-SCIEX
`API 111· triple-stage quadrupole instrument (PE-SCIEX, Thornhill,
`Ontario, Canada) using the electrospray interface, modified (pneu(cid:173)
`matically assisted) by introduction of zero grade air at 40 psi co-axial
`to the direction of liquid flow. The electrospray needle was main-
`
`CYP450 Metabolism of lloperidone
`
`1287
`
`tained at 4500-5000 volts. The nitrogen curtain gas was set at 0.9
`liter/min. The orifice potential and electron multiplier settings were
`+ 55 V and -3.8 kV, respectively. The interface plate was heated to
`65°C to prevent condensation of liquids. To quantitate the analytes
`present in the incubation mixtures, the mass spectrometer was op(cid:173)
`erated in the selected ion monitoring mode (SIM) with a dwell time
`of 266 msec. The eluant from HPLC was introduced into the source
`via the electrospray interface, generating the positively charged
`pseudomolecular ions (MH+) of2, 3, 4 and 5 at m/z 413, 429,443 and
`401, respectively. The ions were monitored using the Q3 quadru(cid:173)
`poles. For quantitative studies peak-area ratios of analytes (either
`mlz 413, 429 or 443) to the internal standard (mlz 441) were used for
`the construction of calibration curves. For qualitative studies, the
`mass spectrometer was operated in the positive mode with a scan
`range of 100 to 1000 amu. The output signal from the mass spec(cid:173)
`trometer was interfaced to a Quadra 950 Macintosh computer oper(cid:173)
`ating RAD and MacQuan Softwares (PE-SCIEX) for data collection,
`peak integration and analysis.
`Standard solutions. Separate standard solutions of1, 2, 3, 4 and
`5 were prepared in 0.1% aqueous acetic acid or distilled water using
`volumetric flasks to give concentrations of ~ 1 mg/ml of each. The
`stock solutions were diluted with distilled water in series to prepare
`solutions ofknown concentrations (100, 10, 1, 0.1 and O.Ofng/~-tD of
`1, 2, 3 and 4 for quantitative studies. A working standard solution of
`10 ng/11-l of internal standard was used during the entire assay. The
`stock solutions were kept refrigerated and discarded after one
`month. The working standards were prepared fresh daily.
`
`In Vitro Incubation with Human Liver Microsomes
`General. Unless otherwise stated, the in vitro incubations were
`done with the addition of either NADPH (2 mM) or the NADPH
`regenerating system {NADP (1 mM), glucose-6-phosphate (10 mM),
`glucose-6-phosphate dehydrogenase (2 IU)} to a mixture consisting of
`magnesium chloride (5 mM) and human microsomal protein (0.3
`mg). The metabolic reactions were initiated by the addition of ilo(cid:173)
`peridone (0.4-40 11-M). All kinetic and CYP inhibition studies were
`done using NADPH instead of the NADPH regenerating system. The
`volume was adjusted to 1 ml with 0.1 M phosphate buffer (pH 7.4)
`and the incubation mixtures were shaken gently in a water bath held
`at 37°C. Unless otherwise stated the incupations were carried out for
`20 min. Using these conditions ~16% ofiloperidone was found to be
`converted to metabolites. In vitro incubations with eDNA-expressed
`CYP isozymes (CYP2D6, CYP3A4, CYP2E1 and CYP2A6) were car(cid:173)
`ried out using identical conditions as described for microsomal incu(cid:173)
`·bations except 0.5 mg of eDNA expressed proteins were used.
`Time, protein and substrate linearity. To determine if the
`production of metabolites 2, 3 and 4 from 1 was linear with time, the
`substrate and protein concentrations were kept at 10 11-g/ml (23 11-M)
`and 0.5 mg/ml, respectively. A set of 1 ml incubations were carried
`out and the reaction terminated by the addition of 2 ml of 0.083 M
`phosphoric acid at 5, 10, 15, 20, 30, 45 and 60 min. Incubations were
`done in duplicate at each time point. Internal standard (200 ng of 5)
`was added to each sample before being extracted on the solid phase
`cartridges as described below. For quantitation, metabolite stan(cid:173)
`dards 2, 3 and 4 were added (10-2000 ng/rnJ) to boiled microsomes,
`the internal standard was added and samples extracted as described
`below. The levels of each of the metabolites 2, 3 and 4 were deter(cid:173)
`mined from the linear standard curves. The levels of metabolite
`obtained at each time point were expressed as nmole/mg protein.
`After the initial experiments showed that the production ~f all
`three metabolites were linear up to 30 min, a study was conducted
`whereby the human liver microsomal protein concentration was
`varied (0.1, 0.2, 0.3, 0.5 and 1.0 mg/ml) in the incubation mixture.
`The incubation conditions were same as described above except the
`substrate concentration was 10 11-g/ml (23 11-M) and incubation time
`was 30 min. Standard curves of each metabolite were constructed in
`the range of 10-2000 ng/ml of incubation mixture. Samples, includ-
`
`Roxane Labs., Inc.
`Exhibit 1006
`Page 008
`
`

`
`1288
`
`Mutlib and Klein
`
`Vol. 286
`
`ing the calibration standards were extracted on solid phase car(cid:173)
`tridges as described below.
`A study was conducted to determine the linearity of formation of
`metabolites 2, 3 and 4 as nmollmin/mg of protein versus the added
`iloperidone concentrations. Using a protein concentration of 0.5
`mg/ml and 30 min incubation time, the production of metabolites at
`substrate concentrations of2.3, 4.7, 11.7, 23.4, 46.8, 70.3 and 117 ILM
`was' determined. The incubation conditions and extraction methods
`were same as described above. The concentrations of metabolites 2,
`3 and 4 obtained at each substrate level were calculated from stan(cid:173)
`dard curves prepared in the range 20-2000 ng/ml of each compound.
`Kinetic studies: V max and K,. values for four different hu(cid:173)
`man liver microsomes. Human liver microsomes were initially
`phenotyped by International Institute for the Advancement of Med(cid:173)
`icine (Exton, PA). The kinetics of 0-demethylation, hydroxylation
`and reduction of iloperidone by human liver microsomes were deter(cid:173)
`mined over a concentration range of 4.8 to 468 ILM using livers
`HL-051, HL-052, HL-056 and HL-060. Incubations were carried out
`as described above (see In Vitro Incubation with Human Liver Mi(cid:173)
`crosomes) in duplicates, with protein concentration of 0.3 mg/ml and
`20 min incubation time. The reaction was started by the addition of
`NADP. The concentrations of each metabolite was determined from
`standard curves prepared in the concentration range of 20 ng to 2500
`ng/ml. After obtaining the concentration of each metabolite at differ(cid:173)
`ent substrate concentrations for the four liver samples, the V max and
`Km values were obtained from the graphical analyses of Lineweaver(cid:173)
`Burke plots using the Pharmacologic Calculation System, version 4.2
`(a), software (see table 1).
`CYP inhibitor studies. To confirm the isoenzymes responsible
`for the production of metabolites 2, 3, and 4, microsomal incubations
`were carried out in the presence of specific chemical inhibitors: 2.5
`and 25 ILM of quinidine (CYP2D6), 2.5 and 25 ILM of sulfaphenazole
`(CYP2C9), 5 and 50 ILM of diethyldithiocarbam