FUNCTIONALIZED AMINO ACID DERIVATIVES-
`POTENT NEW AGENTS FOR THE TREATMENT OF EPILEPSY:
`SYNTHESIS, AND SPECTF|§$%C|DCé|;I_§_)lél\Sl\|D PHARMACOLOGICAL
`
`A Dissertation
`
`Presented to
`
`the Faculty of the Department of Chemistry
`
`University of Houston-University Park
`
`In Partial Fulfillment
`
`of the Requirements for the Degree
`
`Doctor of Philosophy
`
`By
`
`Judith D. Conley
`
`May, 1986
`
`Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
`
`EXHIBIT
`ACTAVIS, AMNEAL,
`AUROBINDO,
`BRECKENRIDGE,
`VENNOOT, SANDOZ,
`SUN
`
`|PR2014-01126-1018 p.1
`
`IPR2014-01126- Exhibit 1018 p. 1
`
`

`
`FUNCTIONALIZED AMINO ACID DERIVATIVES-
`
`POTENT NEW AGENTS FOR THE TREATMENT OF EPILEPSY:
`
`SYNTHESIS, AND SPECTROSCOPIC AND PHARMACOLOGICAL
`
`PROPERTIES
`
`/15552047. %/2%
`
`ith D. Conley
`
`J
`
`APPROVED:
`
`4 IM1 I4?
`
`Dr. Harold L. Koh , Chairman
`
`
`
`'
`/IA/\
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`
`I
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`.
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`
`I
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`Dr. Roy Vveinstein, Dean, College of Natural Science and Mathematics
`I9
`
`‘.'
`
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`
`'
`
`.
`
`Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
`|PR2014-01126- Exhibit 1018 p. 2
`
`IPR2014-01126- Exhibit 1018 p. 2
`
`

`
`COPYRIGHTED BY
`
`Judith D. Conley
`
`May, 1986
`
`Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
`|PR2014-01126- Exhibit 1013 p. 3
`
`IPR2014-01126- Exhibit 1018 p. 3
`
`

`
`ACKNOWLEDGEMENTS.
`
`The author would like to thank the University of Houston Department of
`
`Chemistry and the American Association of University Women for their financial
`
`support during the preparation of this dissertation through teaching assistant-
`
`ships and grants. Without the financial assistance of these groups, the comple-
`
`tion of this work would have not been possible.
`
`I would also like to acknowledge the faculty and staff of the Department of
`
`Chemistry at the University of Houston for their expertise and friendship. A
`
`special note of thanks is extended to Randy Wilkin, Ed Ezell, and Beatriz Fitz.
`
`Also, I would like to thank Drs. Douglas F. Dyckes. Thomas L. Lemke, Gary E.
`
`Martin, and Randolph P. Thummel for their additional comments on this work.
`
`A special acknowledgement goes to Dr. Darrell Watson whose initial
`
`findings made this project worthwhile to continue. Also, Iwould like to recognize
`
`Lisa Albee and Andrew McCallum for their technical assistance.
`
`I would also
`
`like to thank the past and present members of Dr. Kohn's research group who
`
`have been so very helpful along the way.
`
`I am grateful to my family for their love, encouragement, and patience.
`
`Thank you, Jody, Mike, Rand, Jennifer, and Tony.
`
`1 am indebted to my husband, Mark, for his encouragement and love which
`
`has been a constant throughout this work.
`
`I would like to thank him for keeping
`
`our daily lives short of chaos and always being there with aword of kindness
`
`Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
`|PR2014-01126- Exhibit 1018 p. 4
`
`IPR2014-01126- Exhibit 1018 p. 4
`
`

`
`when I needed it the most. There is no one else who could have put up with me
`
`during this time.
`
`Finally, I extend my deepest gratitude to Dr. Harold Kohn. His knowledge,
`
`wisdom, patience, sense of humor, understanding, enthusiasm, and encourage-
`
`ment directed me every inch of the way. His belief in me as a person and a
`
`chemist never waivered, even when my abilities were questioned by myself and
`
`others. Thank you just is not enough.
`
`Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
`|PR2014-01126- Exhibit 1018 p. 5
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`IPR2014-01126- Exhibit 1018 p. 5
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`

`
`DE DICATION .
`
`This dissertation is dedicated to my mother, Shirley, who has shown me the
`
`strength and love thatI possess inside myself. With that strength and love, Ican
`
`achieve any goal.
`
`Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
`|PR2014-01126- Exhibit 1013 p. 6
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`IPR2014-01126- Exhibit 1018 p. 6
`
`

`
`FUNCTIONALIZED AMINO ACID DERIVATIVES-
`POTENT NEW AGENTS FOR THE TREATMENT OF EPILEPSY:
`SYNTHESIS, AND SPECTI;%%(I3D%I;I%é\glD PHARMACOLOGICAL
`
`An Abstract of a Dissertation
`
`Presented to
`
`the Faculty of the Department of Chemistry
`
`University of Houston-University Park
`
`In Partial Fulfillment
`
`of the Requirements for the Degree
`
`Doctor of Philosophy
`
`BY
`
`Judith D. Conley
`
`May, 1986
`
`Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
`|PR2014-01126- Exhibit 1018 p. 7
`
`IPR2014-01126- Exhibit 1018 p. 7
`
`

`
`ABSTRACT.
`
`Inspection of known chemotherapeutic agents possessing depressant and
`
`anticonvulsant activity reveals a major structural pattern. Three functionalities are
`
`prevalent in many of these substrates: (1) a vicinal diamine linkage; (2) an
`
`oxygen atom on the ethylene chain bridging the two amino groups; and (3) an
`
`aromatic ring one carbon atom removed from an amino residue. Functionaiized
`
`amino acid derivatives satisfy these structural requirements. The parent
`
`compound in this study, N-acetyl-DL-alanine-N-benzylamide, has recently been
`
`shown to have high anticonvulsant activity. Structural analogs of the parent
`
`compound were synthesized where the acetyl group, the substituents on the
`
`on-carbon atom, and the N-benzylamide moiety were selectively modified. The
`
`syntheses of these adducts were achieved by known methods of peptide
`
`chemistry and their spectral properties were analyzed. The pharmacological
`
`activity of these adducts were evaluated at the National Institute of Neurological
`
`and Communicative Disorders and Stroke (ASP Project) at the National Insti-
`
`tutes of Health. The stereoisomers of those substrates possessing high anticon-
`
`vulsant activities were also synthesized and evaluated for their biological activity.
`
`Assessment of the pharmacological behavior of the functionalized amino
`
`acid derivatives revealed several significant findings. First, the biological activity
`
`of these substrates established a new class of antiepileptic agents which differ
`
`significantly in their mode of action from conventionally used drugs. Second,
`
`N-acetyI-DL-alanine-N-3-fluorobenzylamide
`
`and N-acetyl-DL-phenylglycine-N-
`
`vii
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`

`
`benzylamide were equally or more potent than the parent compound in the phar-
`
`macological testing. Third, selective substitution at the acyl group, the on-carbon
`
`atom, and the amido moiety of the functionalized amino acid derivatives
`
`governed the activities of the substrates in the MES, sc Met, and toxicity tests.
`
`Fourth, most of the compounds evaluated in this study were only active in the
`
`MES test. N-Acetyl-N‘-benzylurea, N-trimethylacetyl-DL-alanine-N-benzylamide,
`
`and N-acetyl-D-alanine-N-benzylamide were exceptions in that these substrates
`
`were also active in the sc Met test. Fifth, the pharmacological evaluation of the
`
`stereoisomers of N-acetylalanine-N-benzylamide and N-acetylphenylglycine-N-
`
`benzylamide demonstrated that the anticonvulsant activity displayed by the
`
`D-enantiomer was equal to or greater than that observed in the racemate, while
`
`the L-isomer exhibited significantly lower levels of anticonvulsant activity than the
`
`racemic substance. Sixth, the potent anticonvulsant activity of N-acety|-N'-ben-
`
`zylurea was uncovered.
`
`viii
`
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`

`
`TABLE OF CONTENTS.
`
`Bags.
`
`Acknowledgements.
`
`................................................................................................
`
`Dedication.
`
`................................................................................................................
`
`iii
`
`v
`
`Abstract.
`
`...................................................................................................................... vi
`
`ListofTab|es.
`
`.......................................................................................................... ..
`
`x
`
`List of Schemes.
`
`..................................................................................................... .. xiii
`
`l_ist of Figures.
`
`.......................................................................................................... xiv
`
`General Introduction.
`
`............................................................................................ ..
`
`1
`
`....................................... .. 19
`Chapter I. Functionalized Amino Acid Derivatives.
`A. Introduction.
`.............................................................................. .. 19
`B. Results and Discussion.
`..
`......................................... .. 28
`1. Synthesis.
`............. ... ....
`. ........ . . .. 28
`2. Spectral Evaluation.
`.................................. 40
`3. Pharmacological Evaluation.
`.................................... .. 64
`C. Experimental Section.
`.............................................................. 78
`
`
`
`..................................... .. 106
`Chapter II. Chiral Recognition in Epileptic Drugs.
`A. Introduction.
`........................................................................... .. 106
`B. Results and Discussion.
`....................................................... .. 111
`1. Synthesis.
`.................................................................. .. 111
`2. Spectral Evaluation.
`................................................ .. 113
`3. Pharmacological Evaluation.
`................................. .. 127
`C. Experimental Section.
`.......................................................... .. 135
`
`ChapterIH. General Conclusions and Future Considerations.
`
`.............. ..
`
`141
`
`References.
`
`......................................................................................................... .. 145
`
`ix
`
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`

`
`LIST OF TABLES.
`
`Lalzb.
`
`Basia.
`
`1. The International Classification of Epileptic Seizures.
`
`.................... .. 2
`
`2. The Revised Classification of Epileptic Seizures.
`
`............................. .. 3
`
`3. Antiepileptic Drugs Marketed in the United States.
`
`.......................... .. 16
`
`4. Functionalized Amino Acid Derivatives.
`
`............................................. .. 27
`
`5. Selected Physical and Spectral Data of DL-Amino Acid-N-
`Substituted Amides and Their Analogs (51).
`.................................... .. 32
`
`6. Selected Physical and Spectral Data of N-Acyl-DL-Amino
`Acid-N-Substituted Amides and Their Analogs (53) (Method A).
`
`33
`
`7. Selected Physical and Spectral Data of N~Acetyl-DL-Amino
`Acids and Their Analogs (53).
`.............................................................. .. 36
`
`8. Selected Physical and Spectral Data of N-Acyl-DL-Amino
`Acid-N-Substituted Amides and Their Analogs (5_3) (Method B).
`
`38
`
`9. Selected Infrared Spectral Data of DL-Amino Acid-N-
`Substituted Amides and Their Analogs (51).
`....................................... 41
`
`10. Selected Infrared Spectral Data of N-Acyl-DL-Amino
`Acid-N-Substituted Amides and Their Analogs (53).
`
`...................... .. 42
`
`11. Selected ‘H NMR Spectral Properties of DL-Amino Acid
`Methyl Ester Hydrochlorides (55).
`........................................................ 44
`
`12. Selected 1H NMR Spectral Properties of DL-Amino Acid-N-
`Substituted Amides and Their Analogs (51).
`.................................... .. 45
`
`13. Selected ‘H NMR Spectral Properties of N-Acetyl-DL-Amino
`Acids and Their Analogs (53).
`...........................................
`................ .. 47
`
`14. Selected ‘H NMR Spectral Properties of N-Acyl-DL-Amino
`Acid-N-Substituted Amides and Their Analogs (53).
`...................... .. 49
`
`15. Selected 130 NMR Spectral Properties of DL-Amino Acid
`Methyl Ester Hydrochlorides (55).
`........................................................ 55 .
`
`x
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`

`
`E1119.
`
`Raga
`
`16. Selected 13C Ni‘./lFl Spectral Properties of DL-Amino Acid-N-
`Substituted Amides and Their Analogs (51).
`................................... .. 56
`
`17. Selected 130 NMR Spectral Properties of N-Acetyl-DL-Amino
`Acids and Their Analogs (53).
`.............................................................. 57
`
`18. Selected 130 NMR Spectral Properties of N-Acyl-DL-Amino
`Acid-N-Substituted Amides and Their Analogs (53).
`..................... .. 59
`
`19. Phase I Pharmacological Evaluation of Racemic Substrates.
`
`..... .. 66
`
`20. Phase I Pharmacological Evaluation of Racemic Substrates.
`
`68
`
`21. Phase I Pharmacological Evaluation of Flacemic Substrates.
`
`..... .. 70
`
`22. Phase I Pharmacological Evaluation of Racemic Substrates.
`
`..... ..
`
`71
`
`23. Phase H Pharmacological Evaluation of Racemic Substrates.
`
`24. Phase IV Pharmacological Evaluation of Racemic Substrates.
`
`73
`
`74
`
`25. Phase V Pharmacological Evaluation of Racemic Substrates.
`
`....... 76
`
`26. Selected Physical and Spectral Data of D-, L-, and DL-Amino
`Acid-N-benzylamides (51).
`.................................................................. 112
`
`27. Selected Physical and Spectral Data of N-Acetyl-D-, L-, and
`DL-Amino Acid-N-benzylamides.
`.......................................................
`
`1 14
`
`28. Selected Infrared Spectral Data of D-, L-, and DL-Amino
`Acid-N-benzylamides (51).
`.................................................................. 116
`
`29. Selected Infrared Spectral Data of N-Acetyl-D-, L-, and
`DL-Amino Acid-N-benzylamides.
`.......................................................
`
`1 17
`
`30. Selected 1H NMR Properties of D-, L-, and DL-Amino Methyl
`Ester Hydrochlorides (55).
`.................................................................... 1 19
`
`31. Selected ‘H NMR Properties or D-, L-, and DL-Amino Acid-N-
`benzylamides (51).
`................................................................................. 120
`
`32. Selected 1H NMR Properties of N-Acetyl-D-, L-, and DL-Amino
`Acid-N- benzylamides.
`.......................................................................... 121
`
`xi
`
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`

`
`Iab_Le.
`
`Race
`
`33. Selected 130 NMR Properties of o-, L-, and DL-Amino Methyl
`Ester Hydrochlorides (55).
`................................................................. .. 122
`
`34. Selected 130 NMR Properties of D-, L-. and DL-Amino Acid-N-
`benzylamides (51).
`.............................................................................. .. 123
`
`35. Selected 130 NMR Properties of N-Acetyl-D-, L-, and DL—Amino
`Acid-N- benzylamides.
`....................................................................... .. 125
`
`36. Phase I Pharmacological Evaluation of Chiral Substrates
`and Their Corresponding Ftacemates.
`............................................. .. 128
`
`37. Phase 11 Pharmacological Evaluation of Chiral Substrates
`and Their Corresponding Racemates.
`............................................. .. 130
`
`38. Phase IV Pharmacological Evaluation of Chiral Substrates
`and Their Corresponding Racemates.
`............................................. .. 131
`
`39. Phase V Pharmacological Evaluation of Chiral Substrates
`and Their Corresponding Racemates.
`............................................. .. 133
`
`xii
`
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`IPR2014-01126- Exhibit 1018 p. 13
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`

`
`LIST OF SCHEMES.
`
`Scheme.
`
`Base.
`
`1.
`
`2.
`3.
`
`4.
`
`5.
`
`Synthesis of Functionalized Amino Acid Derivatives by
`Method A.
`............................................................................................. .. 30
`
`Proposed Mechanism forthe Esteritication of Amino Acids.
`...... .. 31
`Synthesis of N-Acetyl-N’-benzylurea(5;1g).
`.................................... 34
`
`Synthesis of Functionalized Amino Acid Derivatives by
`Method B.
`............................................................................................. .. 35
`
`Synthesis of N-Acetyldiphenylglycine-N-benzylamide (fia).
`
`.... ..
`
`39
`
`xiii
`
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`IPR2014-01126- Exhibit 1018 p. 14
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`

`
`LIST OF FIGURES.
`
`figure.
`
`we
`
`1.
`
`Hydrogen-bonded Arrangement of_N-Acety|-DL-3-amino-
`2-methy|-N-benzylpropanamide (5311).
`........................................... .. 53
`
`xiv
`
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`

`
`GENERAL INTRODUCTION.
`
`Epilepsy is a disease where the major symptom is seizure of the patient.
`
`The nature of the epileptic seizure led to its early discovery. The Babylonian civil
`
`code of Hammurabi (2080 B.C.), as well as early Hebrew scripts, mentioned the
`
`affliction. Hippocrates wrote the first monograph ("On the Sacred Disease," gs;
`
`400 B.C.) dealing with the full clinical aspects of epilepsy. A major seizure is
`
`also described in the New Testament (Mark IX: 17).1
`
`In the seventeenth century, Charles Le Pois first stated that all epilepsies
`
`originated in the brain. More than a century later, John Hughlings Jackson intro-
`
`duced the concept of a discharging epileptic focus in the brain (1870). William
`
`Gowers in 1885 modified this focal concept by classifying the disease into two
`
`classes. Partial seizures arose from a specific area of the brain, while general-
`
`ized seizures occurred from an unknown origin.-2
`
`Classification of epilepsies. Epilepsy has been described as being
`
`either idiopathic or symptopathic. Idiopathic implies that the cause of the epilep-
`
`sy is unknown and the seizure is the only sign or symptom of the disease. Symp-
`
`topathic epilepsy has a known cause (trauma, infection, neoplasm. or emotion).1
`
`A satisfactory classification of epilepsy has not been completely successful,
`
`primarily due to the limited knowledge of the pathological processes of the brain.
`
`The classifications that are used are based on the type of seizure-and not the
`
`type of epilepsy. The International Classification of Epileptic Seizures (Table 1)
`
`is the most widely accepted, although a revised form has been proposed (Table
`
`2). The first is based on clinical seizure type. electroencephalographic features
`
`(ictal and interictal), anatomic features, etiology. and age. The main feature of
`
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`

`
`2
`
`Iable 1. The International Classification of Epileptic Seizures.2
`
`1. Partial seizures (seizures beginning locally)
`A. Partial seizures with elementary symptomatology (generally without im-
`pairment of consciousness)
`1. With motor symptoms (includes Jacksonian seizures)
`2. With special sensory or somatosensory symptoms
`3. With autonomic symptoms
`4. Compound forms
`B. Partial seizures with complex symptomatology (generally with impairment
`of consciousness) (temporal lobe or psychomotor seizures)
`1. With impairment of consciousness only
`2. With cognitive symptomatology
`3. With affective symptomatology
`4. With "psychosensory" symptomatology
`5. With "psychomotor" symptomatology (automatisms)
`6. Compound torms
`C. Generalized seizures secondarily generalized
`
`II. Generalized seizures (bilaterally symmetrical and without local onset)
`A. Absence (petit mal)
`_
`B. Bilateral massive epileptic myoclonus
`C. Infantile spasms
`D. Clonic features
`E. Tonic features
`F. Tonic-clonic seizures (grand mal)
`G. Atonic seizures
`H. Akinetic seizures
`
`III. Unilateral seizures (or predominately)
`
`IV. Unclassified epileptic seizures (due to incomplete data)
`
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`
`3
`
`IaQ|e_2_, The Revised Classification of Epileptic Seizures.2
`
`1. Simple partial seizures (consciousness not impaired)
`A. With motor signs
`1. Focal motor without march
`2. Focal motor with march (Jacksonian)
`3. Versive
`4. Postural
`5. Phonatory (vocalization or arrest of speech)
`B. With somatosensory or special-sensory symptoms (simple hallucinations,
`e.g., tingling, light flashes, buzzing)
`1. Somatosensory
`2. Visual
`3. Auditory
`4. Olfactory
`5. Gustatory
`6. Vertiginous
`C. With autonomic symptoms or signs
`D. With psychic symptoms (disturbance of higher cortical function)
`1. ' Dysphasic
`2. Dysnesmic (e.g., deja vu)
`3. Cognitive (e.g., forced thinking)
`4. Affective (e.g., fear, anger)
`5. Illusions (e.g., macropsia)
`6. Structured hallucinations (e.g., music, scenes)
`II. Complex partial seizures (generally with impairment of consciousness; may
`sometimes begin with simple symptomatology)
`A. Simple partial onset followed by impairment of consciousness
`1. With simple partial features (A-D) and impaired consciousness
`2. With automatisms
`B. With impairment of consciousness at onset
`1. With impairment of consciousness only
`2. With automatisms
`III. Partial seizures evolving to generalized tonic-clonic (GTC) seizures (GTC
`with partial or focal onset)
`A. Simple partial seizures (1) evolving to GTC
`B. Complex partial seizures (II) evolving to GTC
`C. gi_iI'_nple partial seizures evolving to complex partial seizures evolving to
`C
`IV. Generalized seizures (bilaterally symmetrical and without local onset)
`A. Absence (petit mal)
`B. Bilateral massive epileptic myoclonus
`C. Infantile spasms
`D. Clonic features
`E. Tonic features
`F. Tonic-clonic seizures (grand mal)
`G. Atonic seizures
`V. Unilateral seizures (or predominately)
`VI. Unclassified epileptic seizures (due to incomplete data)
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`

`
`this classification is the distinction between seizures that are generalized from the
`
`onset and those that are partial or focal from the beginning and then later be-
`
`come generalized?
`
`The revised classification of epileptic seizures emphasizes factors based on
`
`direct observation. Only clinical seizure type and electroencephalographic (EEG)
`
`features are retained. A major difference of the revised classification from the
`
`former one is the separation of partial seizures into simple and complex. Partial
`
`seizures are classified primarily according to the state of patient consciousness
`
`during the attack. Consciousness is not impaired during a simple partial seizure,
`
`while impaired consciousness is characteristic of a complex partial seizure.2
`
`A simplified classification of seizures is used for the purpose of illustrating
`
`the specific action of antiepileptic drugs. Seizure types are divided into seven ca-
`
`tegories: grand mal, petit mal (absences), psychomotor, myoclonic, infantile
`
`spasms, febrile, and Jacksonian seizures.1
`
`Grand mal is characterized by the occurrence of maximal seizures asso-
`
`ciated with generalized tonic-clonic convulsions, loss of consciousness, and
`
`autonomic hyperactivity. The electroencephalograph (EEG) during the attack
`
`shows high voltage-fast activity. The essential feature of grand mal is the abnor-
`
`mal ease of generalized spread of an initial discharge to other areas of the brain.
`
`Grand mal is the most common single form of epilepsy and the most disrupting in
`
`the life of the patient.
`
`Petit mal is restricted to brief, frequent (5-1 00 per day) attacks of impaired
`
`consciousness associated with staring and eye movements, and, occassionally
`
`loss of posture and arm jerks. The EEG consists of a regular three per second
`
`spike and wave pattern. No specific etiology is known for petit mal and occurs
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`

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`most commonly before puberty.
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`Psychomotor seizures are characterized by confusion and purposeless
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`movements or behavior patterns. The EEG displays high voltage six per second
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`and flat four per second waves in widely separated areas of the brain.
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`Psychomotor epilepsy may originate in an anterior temporal region of the brain
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`and is often complicated by psychiatric illness.
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`Myoclonic seizures are distinguished by sudden sharp jerks of the head,
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`limbs, or trunk, lasting about one second and occurring in bursts of four or five in
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`three to six second intervals. The EEG may be normal during’ the attack, sugges-
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`ting an extrapyramidal origin. Myoclonic epilepsy is frequently accompanied by
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`grand mal seizures.
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`Infantile spasms occur in children from birth to an age of four and are asso-
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`ciated with jerking movements of the eyes, body, and arms. The EEG shows high
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`voltage slow waves and spikes. Febrile seizures occur only during fever in chil-
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`dren from the age of three to ten.
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`Jacksonian seizures consist of a sensory or motor march which may start as
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`a twitch in the thumb and may spread to other muscles. The EEG indicates that
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`the point of origin may be in the temporal or occipital areas of the brain. These
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`seizures often fade before puberty.
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`Model Assays: Analog Testing. The development of experimental mo-
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`dels for the testing of anticonvulsant drugs has been instrumental in the discovery
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`of effective antiepileptic agents.
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`In 1875, J. Crichton Browne described the con-
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`vulsive effects induced in animals by the administration of picrotoxin (1). At that
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`time, the convulsive actions of strychnine (g) were well known and had been ex-
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`tensively studied.3 Using faradic stimulation of the cerebral cortex, Albertoni
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`elicited electroshock seizures in dogs in 1882, but little was done with this proce-
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`, dure during the next fifty years.4 Pentylenetetrazole (Metrazol) (,3) was synthe-
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`’ sized in 1924 and its convulsant action in mice was demonstrated by Hillebrandt
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`in 1926.3
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`%
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`I-A
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`N
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`93
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`in 1937, Putnam and Merritt described a technique in which seizures were
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`electrically induced in cats by an interrupted (80 per sec) direct current delivered
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`to the brain for ten seconds through mouth-occipital electrodes. Using this new
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`electroshock procedure for creating convulsions in animals, these investigators
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`uncovered the pronounced anticonvulsant activity of 5,5-diphenylhydantoin (§_a)
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`(phenytoin, Dilantin).5 Phenytoin increased convulsive thresholds to such an ex-
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`tent that a "fit" could not be induced with current intensities four times the control
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`level. Significantly, this was the first example of experimental evaluation of
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`promising anticonvulsant agents prior to clinical use and paved the way for
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`intensified anticonvulsant research.
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`In 1944, Goodman and his associates began an intensive study of the phy-
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`siology and therapy of experimental convulsive disorders.6 Over the following
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`ten years, the Goodman group standardized the maximal electroshock seizure
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`test (MES), the minimal electroshock seizure test (EST), and the subcutaneous
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`pentylenetetrazole (Metrazol) seizure threshold test (so Met).7 In addition, they
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`developed the psychomotor electroshock seizure test (PsM)3 and the hypona-
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`tremic electroshock seizure threshold test (HET).9
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`In the maximal electroshock seizure test (MES), animals are stimulated by
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`a 60-cycle alternating current applied through corneal electrodes for 0.2 or 0.3
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`seconds. The characteristics of electroshock seizures are a tonic limb flexion of
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`one to two seconds, followed by a tonic limb extension of ten to twelve seconds,
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`and finally, generalized clonic movements for twelve seconds, creating a total
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`seizure lasting up to twenty-five seconds. Only abolition of the hind limb tonic-
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`extensor spasm is recorded as the measure of anticonvulsant activity. Drugs with
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`marked activity by this test are thought to prevent seizure spread and are likely to
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`be effective in grand mal and psychomotor seizures.1°
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`The minimal electroshock seizure threshold test (EST) consists of the appli-
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`cation of an alternating current one-seventh the strength used for the MES test
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`through corneal electrodes. The pattern for this type of seizure consists of facial,
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`lower jaw or foreleg clonus without loss of upright position, lasting 7 to 12 sec-
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`onds. Prevention of all seizure activity by the drug candidate is considered a
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`measure of anticonvulsant activity.1 0
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`The psychomotor electroshock seizure test (PsM) produces seizures which
`are characterized by abnormal behavior, stunning, and automatisms and is a test V
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`for normal minimal threshold. An unidirectional high voltage current (50-100 V) is
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`delivered for six seconds at a frequency of six impulses per seconds. The stun-
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`ning seizures seen in mice resemble those in psychomotor seizures and effective
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`drugs allow the animals to walk away immediately aftentvard.1°
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`The hyponatremic electroshock seizure threshold test (HET) lowers the
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`electroshock seizure threshold of the test animal by 50% by decreasing the
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`extracellular sodium concentration. Hyponatremia is achieved by the intraperlto-
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`neal injection of an isomolar glucose solution (5.5%). The effectiveness of the
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`active anticonvulsant agent is judged by its ability to raise the reduced hypona-
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`tremic seizure threshold.1°
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`The Metrazol (pentylenetetrazole) seizure threshold test (so Met) measures
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`the ability of the anticonvulsant drug to protect against seizures induced by a
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`subcutaneous injection of pentylenetetrazole. The dose used is the amount of
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`convulsant required to cause seizures in 97% of the animals tested (CD97: mice:
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`85 mg/kg; rats: 70mg/kg). Drugs with marked activity by this test are thought to
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`elevate seizure threshold and are likely to be effective against petit mal sei-
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`zures.1°
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`Seizures have been induced in certain strains of mice and rats by auditory
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`stimulation. The DBA (Dilute Brown Agouti coat colors) strain of the house
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`mouse has been known to be susceptible to seizures when exposed to a loud
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`mixed-frequency sound (12-16 kHz, 90-120 db) such as a doorbell.“ Seizures
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`are characterized by an initial phase of wild running and then followed by clonic
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`convulsions and tonic extension.”
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`Another model of epilepsy is the kindling phenomenon. Kindling is the pro-
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`gressively increased excitation of the brain induced by periodic electrical stimula-
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`tion of certain areas of the brain. Goddard13 chronically implanted stimulation
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`electrodes into mice, rats, and cats and daily delivered biphasic impulses (62.5
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`pulses per second for a duration of one second) to the amygdala region. Clonic
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`convulsions were observed about two weeks after the beginning of stimulation.
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`Photostimulation of the baboon Papio papio elicits myoclonic seizures that
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`are similar to those seen in humans.” After exposure to intermittent light
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`(twenty-five Hz), the animal experiences clonic movements in the eyelids which
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`spread to the face, neck, and the upper body. This phase of the seizure may
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`develop into general clonus.
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`Seizures have also been induced experimentally in different animal spe-
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`cies by hyperthermia, implantation of irritant substances, and other methods.
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`These experimental models of epilepsy have been reviewed.3
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`Development of the Various Classes of Antiepileptic Agents.
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`Drug development for the treatment of epilepsy began in the nineteenth century.
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`In 1857, Charles Locock introduced potassium bromide as the first anticonvul-
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`sant agent. Ten years later, the optimal dose and the reduction