y
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`1 of 27
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`Small Molecule
`Therapy for
`Genetic Disease
`
`Jess G. Thoene
`
`LUPIN EX. 1003
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`LUPIN EX. 1003
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`SMALL MOLECULE THERAPY FOR GENETIC DISEASE
`
`This book summarizes the substantial work that has been accomplished
`with simple molecules in the treatment of inborn errors of metabolism.
`These agents are discrete, often of natural origin, and provide pre(cid:173)
`dictable therapeutic responses. As such, they avoid many of the practi(cid:173)
`cal difficulties associated with gene and protein therapies.
`This book will enable interested clinician/scientists and others to
`rapidly survey the field, thus ascertaining what has been done as well
`as future directions for therapeutic research. Its important introductory
`chapters discuss the infrastructure of the field. These chapters focus
`on an introduction to pharmacokinetics and pharmacodynamics, a
`description of the FDA Office of Orphan Products, and a summary of
`the operation of the National Institutes of Health Office of Rare Dis(cid:173)
`eases Research. The remainder of the book is devoted to a review of
`small molecule therapy for genetic diseases. The book closely analyzes
`the cofactors used to augment the function of defective enzymes and
`the compounds that are able to use an alternative pathway to avoid
`the consequences of the metabolic block present in the patient. Among
`other therapies, the authors discuss the use of zinc and tetrathiomolyb(cid:173)
`date to treat Wilson disease and the use of cysteamine to treat nephro(cid:173)
`pathic cystinosis.
`
`Dr. Jess G. Thoene is currently Director of the Biochemical Genetics
`Laboratory in the Division of Pediatric Genetics at the University of
`Michigan in Ann Arbor and an Active Emeritus Professor of Pediatrics.
`He has held positions in numerous organizations, including Director of
`the Hayward Center for Human Genetics at Tulane University Health
`Sciences Center; Fellow and Medical Director of the Joseph P. Kennedy
`Jr. Foundation; member of the Board of Directors of Copley Pharmaceu(cid:173)
`ticals; and Chairman of the Board of Directors of the National Organi(cid:173)
`zation for Rare Disorders. He has authored numerous articles on inborn
`errors of metabolism, holds three U.S. patents, and is certified in pedi(cid:173)
`atrics and clinical biochemical genetics.
`
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`Small Molecule Therapy
`for Genetic Disease
`
`Edited by
`
`Jess G. Thoene
`University of Michigan
`
`eCAMBRIDGE
`V UNIVERSITY PRESS
`
`LUPIN EX. 1003
`3 of 27
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`-.J •
`
`~~~~~~~~~~~~- --~~~~~~~~~~~~-~~~~~~~~~~~- -
`
`CAMBRIDGE UNIVERSITY PRESS
`Cambridge, New York, Melbourne, Madrid, Cape Town, Singapore,
`Silo Paulo, Delhi, Dubai, Tokyo, Mexico City
`
`Cambridge University Press
`32 Avenue of the Americas, New York, NY 10013-2473, USA
`
`www.cambridge.org
`Information on this title: www.cambridge.org/9780521517812
`
`© Cambridge University Press 2010
`
`This publication is in copyright. Subject to statutory exception
`and to the provisions of relevant collective licensing agreements,
`no reproduction of any part may take place without the written
`permission of Cambridge University Press.
`
`First published 2010
`
`Printed in the United States of America
`
`A catalog record for this p11blicatio11 is available from the British Library.
`
`Library of Congress Cataloging in P11blicatio11 data
`
`Small molecule therapy for genetic disease I edited by Jess G. Thoene.
`p.;cm.
`Includes bibliographical references and index.
`ISBN 978-0-521-51781-2 (hardback)
`1. Metabolism, Inborn errors of - Chemotherapy - Handbooks, manuals, etc. 2. Metabolism,
`Inborn errors of - Gene therapy - Handbooks, manuals, etc. 3. Genetic disorders -
`Chemotherapy - Handbooks, manuals, etc.
`I. Thoene, Jess G.
`II. Title.
`[DNLM: 1. Genetic Diseases, Inborn - drug therapy. 2. Orphan Drug Production. 3. Rare
`Diseases - drug therapy. 4. Small Molecule Libraries - therapeutic use. QZ SO S635 2010]
`RC627.8.SSS 2010
`616.3'9042-dc22
`
`2010002899
`
`ISBN 978-0-521-51781-2 Hardback
`
`Cambridge University Press has no responsibility for the persistence or accuracy of URLs for
`external or third-party Internet Web sites referred to in this publication and does not guarantee
`that any content on such Web sites is, or will remain, accurate or appropriate.
`
`Every effort has been made in preparing this book to provide accurate and up-to-date
`information that is in accord with accepted standards and practice at the time of publication.
`Although case histories are drawn from actual cases, every effort has been made to disguise the
`identities of the individuals involved. Nevertheless, the authors, editors, and publishers can
`make no warranties that the information contained herein is totally free from error, not least
`because clinical standards are constantly changing through research and regulation. The
`authors, editors, and publishers therefore disclaim all liability for direct or consequential
`damages resulting from the use of material contained in this book. Readers are strongly advised
`to pay careful attention to information provided by the manufacturer of any drugs or
`equipment that they plan to use.
`
`5
`
`5l
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`Contents
`
`Contributors
`
`Preface
`
`SECTION I: INFRASTRUCTURE
`The U.S. Food and Drug Administration and the regulation of small
`molecules for orphan diseases
`Marlene E. Haffner and Tan T. Nguyen
`2 The Office of Rare Diseases Research: Serving a coordinating function
`at the National Institutes of Health
`Stephen c. Groft
`Introduction to pharmacokinetics and pharmacodynamics
`Juan J. L. Lertora and Konstantina M. Vanevski
`
`3
`
`SECTION II: COFACTORS
`4 Biotin and biotin-responsive disorders
`Kirit Pindolia and Barry Wolf
`5 Cobalamin treatment of methylmalonic acidemias
`Hans C. Andersson
`6 Sapropterin treatment of phenylketonuria
`Barbara K Burton
`7 L-camitine therapy in primary and secondary camitine
`deficiency disorders
`Susan C. Winter, Brian Schreiber, and Neil R. M. Buist
`
`SECTION Ill: UTILIZATION OF ALTERNATIVE PATHWAYS
`8 Cysteamine treatment of nephropathic cystinosis
`Jess G. Thoene
`
`9 Nitisinone use in hereditary tyrosinemia and alkaptonuria
`Wendy J. lntrone, Kevin J. O'Brien, and William A. Gahl
`1 O Alternative waste nitrogen disposal agents for urea cycle disorders
`Gregory M. Enns
`
`page vii
`xi
`
`3
`
`1 9
`
`35
`
`57
`
`68
`
`76
`
`86
`
`101
`
`114
`
`135
`
`v
`
`!ism,
`
`la re
`
`for
`ran tee
`
`tion.
`se the
`an
`least
`
`dvised
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`LUPIN EX. 1003
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`

`vi
`
`Contents
`
`11 POMP-based glucosylceramide synthesis inhibitors for Gaucher and
`Fabry diseases
`James A. Shayman
`12 Betaine treatment for the homocystinurias
`Amy Lawson-Yuen and Harvey L. Levy
`
`SECTION IV: METAL ION THERAPY
`1 3 Zinc and tetrathiomolybdate for the treatment of Wilson disease
`George J. Brewer
`1 4 Small copper complexes for treatment of acquired and inherited
`copper deficiency syndromes
`Stephen G. Kaler
`
`Index
`
`Color plates follow page 1 30.
`
`153
`
`1 73
`
`185
`
`202
`
`213
`
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`153
`
`173
`
`185
`
`202
`
`213
`
`Contributors
`
`Hans C. Andersson, MD, FACMG
`Director, Hayward Genetics
`Center
`Karen Gore Professor of Human
`Genetics
`Tulane University Medical Center
`New Orleans, LA
`
`George]. Brewer, MD
`Morton S. and Henrietta K. Sellner
`Emeritus Professor of Human
`Genetics
`Emeritus Professor of Internal
`Medicine
`Departments of Human Genetics and
`Internal Medicine
`University of Michigan School of
`Medicine
`Ann Arbor, MI
`
`Neil R. M. Buist, MB, ChB, FRCPE
`Professor Emeritus
`Departments of Pediatrics and Medical
`Genetics
`Oregon Health & Science University
`Portland, OR
`
`Barbara K. Burton, MD
`Professor of Pediatrics
`Northwestern University Feinberg
`School of Medicine
`Division of Genetics, Birth Defects,
`and Metabolism
`
`Children's Memorial Hospital
`Chicago, IL
`
`Gregory M. Enns, MB, ChB
`Associate Professor of Pediatrics
`Director, Biochemical Genetics
`Program
`Division of Medical Genetics
`Stanford University
`Stanford, CA
`
`William A. Gahl, MD, PhD
`Clinical Director, National Human
`Genome Research Institute
`National Institutes of Health
`Bethesda, MD
`
`Stephen C. Groft, PharmD
`Director, Office of Rare Diseases
`Research
`Department of Health and Human
`Services
`National Institutes of Health
`Bethesda, MD
`
`Marlene E. Haffner, MD, MPH
`Haffner Associates LLC
`Rockville, MD
`Former Director, Office of Orphan
`Products Development
`U.S. Food and Drug Administration
`Adjunct Professor, Departments of
`Medicine and Preventive Medicine
`and Biometrics
`
`vii
`
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`

`viii
`
`Contributors
`
`F. Edward Hebert School of Medicine
`and Biometrics
`Uniformed Services University of the
`Health Sciences
`Bethesda, MD
`
`F. Edward Hebert School of Medicine
`and Biometrics
`Uniformed Services University of the
`Health Sciences
`Bethesda, MD
`
`Wendy J. Introne, MD
`Staff Clinician, Office of the Clinical
`Director
`National Human Genome Research
`Institute
`National Institutes of Health
`Bethesda, MD
`
`Stephen G. Kaler, MD
`Head, Unit on Human Copper
`Metabolism; Program in Molecular
`Medicine
`Eunice Kennedy Shriver National
`Institute of Child Health and
`Human Development
`National Institutes of Health
`Bethesda, MD
`
`Amy Lawson-Yuen, MD, PhD
`Physician in Clinical Genetics and
`Pediatrics
`Woodcreek Healthcare
`Puyallup, WA
`
`Juan]. L. Lertora, MD, PhD
`Director, Clinical Pharmacology
`Program
`NIH Clinical Center
`National Institutes of Health
`Bethesda, MD
`
`Harvey L. Levy, MD
`Senior Physician in Medicine
`Children's Hospital Boston
`Professor of Pediatrics
`Harvard Medical School
`Boston, MA
`
`Tan T. Nguyen, MD, PhD
`Associate Professor of Pathology
`Department of Pathology
`
`Kevin J. O'Brien, RN, MS-CRNP
`Staff Clinician, Office of the Clinical
`Director
`National Human Genome Research
`Institute
`National Institutes of Health
`Bethesda, MD
`
`Kirit Pindolia, PhD
`Department of Medical Genetics
`Henry Ford Hospital
`Center for Molecular Medicine and
`Genetics
`Wayne State University School of
`Medicine
`Detroit, MI
`
`Brian Schreiber, MD
`Assistant Professor, Department of
`Medicine, Division of Nephrology
`Medical College of Wisconsin
`Milwaukee, WI
`Vice President, Medical Affairs
`Sigma Tau Pharmaceuticals
`Gaithersburg, MD
`
`James A. Shayman, MD
`Professor of Internal Medicine and
`Pharmacology
`Associate Vice President for Research,
`Health Sciences
`University of Michigan School of
`Medicine
`Ann Arbor, MI
`
`Jess G. Thoene, MD
`Director, Biochemical Genetics
`Laboratory
`Active Professor Emeritus of Pediatrics
`University of Michigan School of
`Medicine
`Ann Arbor, MI
`
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`Contributors
`
`ix
`
`Konstantina M. Vanevski, MD
`Special Volunteer Fellow
`Clinical Pharmacology Program
`NIH Clinical Center
`National Institutes of Health
`Bethesda, MD
`
`Susan C. Winter, MD
`Clinical Professor of Pediatrics
`University of California, San
`Francisco
`Medical Director, Medical Genetics
`
`Children's Hospital Central California
`Madera, CA
`
`Barry Wolf, MD, PhD
`Chair, Department of Medical
`Genetics
`Henry Ford Hospital
`Professor, Center for Molecular
`Medicine and Genetics
`Wayne State University School of
`Medicine
`Detroit, MI
`
`dicine
`
`of the
`
`IP
`inical
`
`arch
`
`cs
`
`and
`
`of
`
`tt of
`ology
`
`and
`
`·search,
`
`. of
`
`s
`
`~diatrics
`l of
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`1 O Alternative waste nitrogen disposal agents
`for urea cycle disorders
`
`Gregory M. Enns
`
`NATURAL HISTORY OF UREA CYCLE DISORDERS
`
`PATHOPHYSIOLOGY
`
`INHERITANCE
`
`EARLY EFFORTS AT HYPERAMMONEMIA THERAPY
`
`ALTERNATIVE PATHWAY MEDICATIONS
`
`MECHANISM OF ACTION
`
`Urea cycle intermediates
`Amino acylation products
`Pharmacokinetics of Ammonul
`
`DOSAGE
`
`SIDE EFFECTS
`
`U.S. FOOD AND DRUG ADMINISTRATION STATUS
`--
`- - - -
`- -- - -· · • --
`-
`-
`-
`-
`RES UL TS OF THERAPY
`
`FUTURE DEVELOPMENTS
`
`REFERENCES
`
`135
`
`136
`
`139
`
`140
`
`140
`
`140
`141
`142
`144
`
`145
`
`146
`
`147
`
`147
`
`149
`
`150
`
`NATURAL HISTORY OF UREA CYCLE DISORDERS
`
`Urea cycle disorders (UCDs) are inborn errors of metabolism characterized by
`episodic, life-threatening hyperammonemia secondary to partial or complete
`inactivity of enzymes responsible for eliminating nitrogenous waste. The urea
`cycle was initially elucidated by Krebs and Henseleit in 1932. 1 In 1958, argini(cid:173)
`nosuccinic acid lyase deficiency became the first enzymatic defect of the urea
`cycle to be identified, and reports of all others, except N-acetylglutamate syn(cid:173)
`thetase (NAGS) deficiency, followed in the 1960s.2 Ornithine transcarbamylase
`(OTC) deficiency is the most common UCD, followed by argininosuccinate syn(cid:173)
`thetase (AS) deficiency (citrullinemia), carbamoyl phosphate synthetase (CPS)
`deficiency, and argininosuccinate lyase (AL) deficiency. NAGS deficiency was first
`described in 1981 and has been documented in only a few patients.·i Estimates of
`overall incidence of UCDs in the United States have ranged from approximately
`1 in 25,000 to 1 in 8,200 births. 4•5
`
`135
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`

`136
`
`Gregory M. Enns
`
`Historically, mortality and morbidity have been high, with survivors com(cid:173)
`monly showing devastating neurological sequelae.2 UCDs are the most common
`cause of neonatal hyperammonemia and typically present with symptoms of poor
`feeding, lethargy, hypotonia, irritability, seizures, respiratory distress, grunting,
`and hyperventilation . Other disorders common in neonates, such as sepsis, car(cid:173)
`diac failure, and intracranial hemorrhage, are also in the differential diagnosis
`because similar clinical findings may occur in these conditions. Therefore, in
`all neonates presenting with nonspecific symptoms of distress, a plasma ammo(cid:173)
`nium level should be obtained. If the level of ammonium is elevated, diagnostic
`evaluations and treatment should be started immediately.
`Although presentation in the neonatal period has been well-documented,
`patients who have a partial enzyme deficiency typically manifest after the neona(cid:173)
`tal period. UCDs may strike at any age. Indeed, approximately two thirds of
`cases initially present after the neonatal period. 5 Clinical features may be subtle
`in such late-onset cases, leading to delays in diagnosis. In addition to acutely
`altered mental status, later-onset patients may have episodic ataxia, psychiatric
`and behavioral symptoms, psychomotor delay, and gastrointestinal complaints,
`such as Joss of appetite and episodic emesis. 6
`Many survivors of the initial hyperammonemic episode undergo recurrent
`attacks of hyperammonemia requiring hospitalization. Such episodes are typi(cid:173)
`cally preceded by an illness, especially a viral syndrome. Other events that con(cid:173)
`tribute to hyperammonemic episodes include dietary or medication noncompli(cid:173)
`ance and major life events, such as surgery, gastrointestinal bleeding, accidents,
`school stress, or parturition. Catabolic stress from viral illnesses appears to be a
`more significant risk factor than is increased intake of dietary nitrogen for causing
`7
`hyperammonemia. 5
`•
`A study of 260 UCO patients showed that onset of symptoms in the neonatal
`period results in the worst outcome (35%> survival approximately 11 years after
`the start of the study period), and patients who presented initially in late infancy
`have the best outcome (871X> survival; Figure 10-1). Percent survival to the final
`follow-up time point was highest for patients with AS deficiency (78% ), followed
`by girls with OTC deficiency (74%), and CPS-I deficiency (61%).5 Boys with OTC
`deficiency have the lowest survival rate over time (53%), as well as the lowest
`survival rate following hyperammonemic crises (71 %) .5 ·8
`Although alternative pathway therapy and other therapies, especially hemo(cid:173)
`dialysis, for UCDs has led to improved patient survival, cognitive impairment
`remains a common finding, especially in patients who have neonatal-onset dis(cid:173)
`ease.9
`
`PATHOPHYSlOLOGY
`
`Ammonia is present in all body fluids and exists mainly as ammonium ion at
`physiologic pH . Hyperammonemia is defined as a blood ammonia concentration
`greater than approximately 100 µmol / L in neonates or 50 µmol/L in children
`
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`

`lm-
`1on
`oor
`ng,
`
`~ar­
`)Sis
`in
`
`[10-
`;tic
`
`ed,
`aa-
`of
`•tie
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`ric
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`•nt
`pi(cid:173)
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`
`·n
`
`Alternative waste nitrogen disposal agents for urea cycle disorders
`
`137
`
`100
`90
`80
`70
`60
`50
`40
`30
`20
`10
`0
`
`Cl
`c:
`">
`·:;
`:;
`en
`c <ll
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`
`-·-·····--,
`·-- ··- -·· -···- ·· ·- ···· ·
`- - - - - - - - - - -:.. - :.._ ________________________ r····-· · .. ·····-·-··--·--··--····--··---··-··--·
`1----- - --- - -------- -
`
`100
`90
`80
`70
`60
`50
`40
`30
`20
`10
`0
`
`Cl
`
`c: ·:;
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`c Ql
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`
`0
`
`500
`
`1000
`
`1500
`
`2000
`Days
`
`2500
`
`3000
`
`3500
`
`4000
`
`Age Groups
`
`0-30 days
`- - - - - - - - - · >2- 16 years
`
`······················· 31 days-2 years
`
`Figure 10-1: Kaplan-Meier survival by age at first episode of hyperammonemia. Survival time
`was calculated as the amount of time between the discharge date of the last episode and the
`admission date from the first episode. Only 350/o of patients who presented with the first hyper(cid:173)
`ammonemic episode during the neonatal period were still alive at the final follow-up time point
`(approximately 11 years after the start of the study period). 5 Reproduced with permission from
`Acta Paediatrica .
`
`and adults (precise cutoffs vary depending on individual laboratory normative
`ranges). A five- to tenfold increase in blood ammonium levels is usually toxic
`to the nervous system. 10 Acute hyperammonemia causes astrocyte swelling and
`global cerebral edema, which affects brain white matter selectively. Changes
`involving the deep insular and perirolandic sulci may be reversible.11 In cases
`of severe hyperammonemia, however, permanent changes occur. Neuropatho(cid:173)
`logical findings in patients who have neonatal-onset proximal UCDs consist of
`gross cerebral atrophy, ventriculomegaly, delayed myelination, the appearance
`of Alzheimer type II astrocytes, ulegyria, and spongiform degeneration of the
`cortex, gray-white matter junction, and deep gray nuclei, including the basal
`ganglia and thalamus.11
`Hyperammonemia also causes increased cerebral cortical glutamine content,
`activation of astrocytic glutamine synthetase, and astrocyte swelling. Ammo(cid:173)
`nia diffuses freely across the blood-brain barrier and is rapidly incorporated
`into glutamine via glutamine synthetase. Glutamine synthetase, a cytosolic
`enzyme primarily localized to the astrocyte in the brain, catalyzes the following
`reaction :
`
`NHj + L-Glutamate +ATP__, L-Glutamine +ADP+ Pi .
`
`This reaction, therefore, represents a short-term means of buffering excess plasma
`ammonium. In theory, glutamine also may be an organic osmolyte that increases
`intracellular osmolarity. Such an increase in osmolarity would lead to increased
`cellular volume, as water enters the astrocyte, and subsequent cytotoxic cerebral
`edema.9· 12
`
`. , - - - - - - -- - - -·-
`
`:--- ~ I
`
`I
`
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`138
`
`Gregory M. Enns
`
`Although the "glutamine hypothesis" has been a leading explanation for
`the development of cerebral edema, other research has focused on glutamine(cid:173)
`independent mechanisms to explain the pathogenesis of hyperammonemic
`encephalopathy. In particular, the role of impaired brain oxidative metabolism
`in causing cerebral dysfunction associated with hyperammonemia is an area
`of active investigation. Acute hyperammonemia causes a decrease in brain
`metabolic rate and high-energy phosphate concentration and increased produc(cid:173)
`tion of toxic reactive oxygen species (ROS) by brain mitochondria. I.! Glutamine
`also enters mitochondria through a histidine-sensitive carrier. This process is
`potentiated by ammonia. Phosphate-activated glutaminase is located in the inner
`mitochondrial membrane and cleaves glutamine into glutamate and ammonia.
`Because of this localized production of ammonia, intramitochondrial ammonia
`levels have the potential to become high, leading to induction of mitochondrial
`permeability transition (MPT), increased oxidative and nitrosative stress, and
`astroglial dysfunction. The production of ROS and reactive nitrogen species and
`the induction of MPT have been hypothesized to initiate a cascade of events that
`includes activation of mitogen-activated protein kinases (MAPKs) and resultant
`failure of astrocytes to regulate their intracellular volume. 1 ~
`In addition, ammonium ions have a multitude of effects on mammalian neu(cid:173)
`rotransmitters, including systems involving cholinergic, serotonergic, and gluta(cid:173)
`matergic neurotransmission. The increased seizure predisposition in some UCO
`patients may be explained, in part, by increased brain concentrations of the
`excitatory amino acid neurotransmitters glutamate and aspartate. Increased lev(cid:173)
`els of these amino acids are present in the sparse-fur (spf) mouse, a model of
`OTC deficiency.15 Tryptophan, a precursor of serotonin, and quinolinic acid,
`an N-methyl-D-aspartate (NMDA) receptor agonist known to produce selec(cid:173)
`tive striatal cell loss, are similarly increased in sp( mice and in rats follow(cid:173)
`ing portacaval anastomosis. Ammonia also inhibits high-affinity transport of
`glutamate in astrocytes. This inhibition results in increased extracellular con(cid:173)
`centration of glutamate.16 These biochemical and pathological findings sug(cid:173)
`gest that NMDA-mediated excitotoxic brain injury may be occurring in UCD
`patients. 17 Ammonium ions also depress postsynaptic o:-amino-3-hydroxy-5-
`methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated currents. 16 AMPA
`receptors mediate fast synaptic transmission and are involved with learning and
`memory.
`Chronic hyperammonemia activates the L-system carrier, which results in a
`loss of NMDA receptor densities and increased uptake of tryptophan into the
`brain. 1(' Serotoninergic symptoms, such as anorexia, altered sleep patterns, and
`disorders of motor coordination, may be related to the increased brain turnover
`of serotonin observed in hyperammonemic states. 11 The adaptive changes in
`NMDA receptors that occur in chronic hyperammonemia result in a decrease in
`excitatory neurotransmission and impaired production of nitric oxide and cyclic
`guanosine monophosphate (cGMP) . Decreased cGMP production may inhibit
`long-term potentiation (LTP) in the hippocampus. Because L TP is a Jong-lasting
`
`I
`......
`
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`

`-
`
`for
`ine(cid:173)
`mic
`ism
`1rea
`·ain
`luc-
`1ine
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`ner
`'.lia.
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`rial
`md
`md
`hat
`ant
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`of
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`of
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`nd
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`nd
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`in
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`Jit
`ng
`
`Alternative waste nitrogen disposal agents for urea cycle disorders
`
`139
`
`Table 10-1. Molecular genetics of the UCDs
`
`Disorder
`
`Cellular
`compartment
`
`Gene
`
`Chromosomal Molecular
`location
`characteristics
`
`NAGS deficiency Mitochondrial matrix NAGS
`
`17q21.31
`
`CPS deficiency
`
`Mitochondrial matrix CPSl
`
`2q35
`
`OTC deficiency
`
`Mitochondrial matrix OTC
`
`Xp21.l
`
`AS deficiency
`(citrullinemia)
`AL deficiency
`
`Arginase
`deficiency
`
`Cytosol
`
`Cytosol
`
`Cytosol
`
`ASSl
`
`9q34.1
`
`ASL
`
`7cen-ql 1.2
`
`ARG
`
`6q23
`
`7 exons, spans ~ 5 Kb, ORF*
`~ 1.6 Kb, 534 amino acids
`38 exons, spans - 201 Kb, ORF
`~5.8 Kb, 1,500 amino acids
`10 exons, spans ~ 94 Kb, ORF
`~ 1.7 Kb, 354 amino acids
`15 exons, spans ~ 57 Kb, ORF
`~ 1.9 Kb, 412 amino acids
`1 6 exons, spans "'79 Kb, ORF
`~ 1.9 Kb, 464 amino acids
`8 exons, spans - 37 Kb, ORF
`~ 1.4 Kb, 322 amino acids
`
`' ORF: open reading frame.
`Note: Molecular characteristics of the UCDs are shown. Data were obtained
`the National Center for
`from
`Biotechnology Information (NCBI) Web sites (www.ncbi.nlm.nil1.gov/IEB/Research/Acembly/index.html !Ace View);
`www.ncbi.nlm.nih.gov/nuccore IEntrez Nucleotrde); www.ncbi.nlm.nig.gov/omim IOMIMJ).
`
`enhancement of synaptic transmission efficacy, considered to be the basis for
`some forms of learning and memory, this effect of hyperammonemia may
`be related to the abnormal cognitive function observed in patients who have
`UCDs. 18 Abnormal axonal growth, accompanied by decreased creatine and phos(cid:173)
`phocreatine levels (creatine is essential for axonal elongation) and alteration of
`brain cytoskeletal elements, are also observed in hyperammonemia. Glial fib(cid:173)
`rillary acidic protein (GFAP) is an important astrocytic protein involved in a
`multitude of cellular functions. GFAP is reduced, and microtubule-associated
`protein-2 (MAP-2) and neurofilament protein (NF-M) exhibit decreased phospho(cid:173)
`rylation, possibly through abnormal MAPK function caused by hyperammone(cid:173)
`mia.19 Although the precise interrelationship between these proposed patho(cid:173)
`genetic mechanisms is unclear, it is reasonable to suspect that these processes
`play at least some role in the mental impairment observed in UCD patients.
`
`INHERITANCE
`
`Urea cycle defects, with the exception of OTC deficiency, are inherited as autoso(cid:173)
`mal recessive traits. OTC deficiency is X-linked and, therefore, typically manifests
`more severely in males. Approximately fifteen percent of females with OTC defi(cid:173)
`ciency display symptoms, such as protein intolerance, cyclical vomiting, behav(cid:173)
`ioral and neurologic abnormalities, and even hyperammonemic coma, and are
`termed "manifesting heterozygotes." The severity of symptoms in such females
`is related to random X-inactivation and allelic heterogeneity, as well as to degree
`of environmental stress. Further details regarding the molecular genetics of the
`UCDs are provided in Table 10-1.
`
`LUPIN EX. 1003
`14 of 27
`
`

`

`1 40
`
`Gregory M. Enns
`
`EARLY EFFORTS AT HYPERAMMONEMIA THERAPY
`
`A number of different therapies aimed at removing accumulated ammonia in
`cases of hyperammonemic encephalopathy have been attempted, including lac(cid:173)
`tulose (reduces the production or absorption of the end products of bacterial
`nitrogen metabolism in the colon), exchange transfusion, peritoneal dialysi s
`(PD), hemodialysis, and supplementation with nitrogen-free analogues of essen(cid:173)
`tial amino acids. Although children treated with a-keto amino acid analogues
`showed some clinical improvement, such as improved seizure control attention
`span and weight gain, death in infancy was still common. Exchange transfusions
`are ineffective in managing hyperammonemia. PD has shown variable efficacy
`in treating hyperammonemia but, in general, is far inferior to hemodialysis. The
`early use of these treatments prolonged survival in some cases, but overall efficacy
`has been disappointing.20
`
`ALTERNATIVE PATHWAY MEDICATIONS
`
`In 1914, Lewis demonstrated that sodium benzoate could divert urea nitrogen
`to hippurate (HIP) nitrogen in two normal subjects. After ingestion of sodium
`benzoate, blood urea nitrogen and ammonia levels fell and urine HIP excretion
`rose markedly, with little change in total urine nitrogen excretion .21 Shiple and
`Sherwin later showed that oral administration of phenylacetate results in sub(cid:173)
`stitution of phenylacetylglutamine (PAGN) nitrogen for urea nitrogen in urine .
`Coadministration of benzoate and phenylacetate resulted in as much as 601l1i
`of urine nitrogen being excreted as HIP and PAGN. 22 Subsequently, the enzymes
`responsible for these reactions (acyl-coenzyme A [CoA] :glycine and acyl-CoA glu(cid:173)
`tamine N-acyltransferases) were identified and localized to both kidney and liver
`in humans and primates. Synthesis of HIP (from conjugation of glycine with ben(cid:173)
`zoate) and PAGN (from conjugation of glutamine with phenylacetate) requires
`adenosine triphosphate (ATP) and CoA. Pharmacogenetic factors partly deter(cid:173)
`mine the activity of enzymes responsible for formation of HIP and PAGN and,
`1
`therefore, play a role in determining the individual rate of nitrogen removal. 2
`·
`
`MECHANISM OF ACTION
`
`In 1979, Brusilow and colleagues suggested that the use of endogenous biosyn(cid:173)
`thetic pathways of non-urea waste nitrogen excretion could substitute for defec(cid:173)
`tive urea synthesis in patients who have UCDs. By promoting the synthesis of
`non-urea nitrogen-containing metabolites (the excretion rates of which are high
`or may be augmented), in theory, total body nitrogen load could be decreased
`despite abnormal urea cycle function. 24 The two classes of alternative pathway
`metabolites are (1) urea cycle intermediates (citrulline and argininosuccinate)
`and (2) amino acid acylation products (I llP and PAGNJ .
`
`LUPIN EX. 1003
`15 of 27
`
`

`

`Alternative waste nitrogen disposal agents for urea cycle disorders
`
`141
`
`Ornithine
`
`i
`/..
`/
`I Citrulline I·-"··
`
`.
`i
`i
`i
`
`~~"rt'~/
`
`I Argininosuccinate f .. -· .. :
`
`Supplemented_ ..... ·:Y
`Arginine
`.. -····
`
`Arginine
`
`Fumarate
`
`Figure 10-2: The urea cycle and alternative pathway therapy. Nitrogen flux through the urea
`cycle may be decreased by excretion of HIP and PAGN, molecules which contain one and two
`waste nitrogen atoms, respectively. In addition, citrulline (which contains one waste nitrogen
`atom) or argininosuccinate (which contains two waste nitrogen atoms) can serve as a vehicle
`of waste nitrogen excretion, depending on the location within t11e urea cycle of the deficient
`enzyme. For example, in AL deficiency, there is a block in argininosuccinate conversion to arginine.
`Supplementing AL deficiency patients with arginine increases production of argininosuccinate,
`which is then excreted in the urine along with its waste nitrogen. ARG: arginase. Reproduced with
`permission from NeoReviews, Vol. 7, page e490, Copyright © 2006 by the AAP.
`
`Urea cycle intermediates
`
`In AL deficiency, argininosuccinate accumulates and is excreted in the urine.
`Because argininosuccinate contains two waste nitrogen atoms, production of this
`metabolite can be exploited to excrete waste nitrogen in AL deficiency, provided
`that an adequate amount of ornithine is present to supply the necessary carbon
`skeletons for argininosuccinate biosynthesis. By administering pharmacologic
`doses of arginine, ornithine is synthesized by the action of arginase. Citrulline
`and argininosuccinate are then produced in turn by the sequential action of OTC
`and AS. In AL deficiency, argininosuccinate cannot be further metabolized and
`is excreted in the urine, along with waste nitrogen (Figure 10-2).-1
`Similarly, as long as sufficient arginine is supplied, citrulline can serve as
`a vehicle for waste nitrogen excretion in AS deficiency (citrullinemia; Figure
`10-2). When compared to argininosuccinate, however, citrulline has two major
`
`n
`c(cid:173)
`al
`is
`1-
`:s
`
`:y
`
`·n
`n
`·n
`d
`
`)(cid:173)
`e.
`Yc,
`:s
`1-
`:r
`
`l-
`:s
`r(cid:173)
`j,
`
`l-
`
`Jf
`h
`d
`y
`:)
`
`LUPIN EX. 1003
`16 of 27
`
`

`

`1 42
`
`Gregory M. Enns
`
`"AMMONIA-SCAVENGING" MEDICATIONS
`
`[PHENYLACETATE [
`
`CH -COO·
`
`CoA
`
`_·
`
`· GLYCINE
`
`Y GLUTAM/NE ©'
`~OATEl coo ©
`0 1
`0 y, CoA
`'
`Boowyl-CoA ~ Phooy"'"Yl-Col\
`co11 •[>iW2TEJ © • c~~o
`
`© "H-CH,-coo CH,-co-NH-r(CH,),-CONH,
`
`~_!"i_ENYLACETYLGLUTAMINE ]
`
`Figure 10-3: Mechanism of nitrogen scavenging by sodium benzoate and sodium pl1enylacetate:
`HIP and PAGN are formed by conjugation of benzoate witl1 glycine and pl1enylacetate witil glu(cid:173)
`tamine, respectively. Tl1ese reactions are performed by specific liver and kidney N-acyltransferases.
`HIP contains one waste nitrogen atom, and pl1enylacetylglutamine contains two waste nitrogen
`atoms. Botil HIP and PAGN are excreted in tile urine, effectively decreasing nitrogen flux t11rougil
`t11e urea cycle. ''Nitrogen atoms excreted. Reproduced witil permission from NeoReviews, Vol. 7,
`Page e490, Copyrigl1t C' 2006 by t11e AAP.
`
`disadvantages: (1) It contains only one waste nitrogen atom, and (2) a high per(cid:173)
`centage of filtered citrulline is reabsorbed, so urine excretion is relatively poor. 4 •23
`
`Amino acylation products
`
`Because of high renal clearance (five times the glomerular filtration rate), HIP is
`easily excreted by the kidneys. HIP biosynthesis, by conjugation of benzoate with
`glycine, is accomplished by the action of mitochondrial matrix enzymes (ben(cid:173)
`zoyl thiokinase and a glycine-specific N-acyltransferase; Figure 10- 3). Similarly,
`PAGN is formed by sequential action of phenylacetyl thiokinase and a glutamine(cid:173)
`specific N-acyltransferase. Because phenylacetate has t