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NEUROPSYCHOPHARMACOLOGICAL INVESTIGATIONS WITH 4H-3-METHYLCARBOXAMIDE-3,4-BENZOXAZINE-Z-ONE: DEMONSTRATION OF A NOVEL SPECTRUM OF CENTRAL ACTIVITY BY John James Vrbanac, Jr. A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Pharmacology 1978 ABSTRACT NEUROPSYCHOPHARMACOLOGICAL INVESTIGATIONS WITH 4H-3-METHYLCARBOXAMIDE-3,4-BENZOXAZINE-2-ONE: DEMONSTRATION OF A NOVEL SPECTRUM OF CENTRAL ACTIVITY BY John James Vrbanac, Jr. Since the fortuitous discovery of the mood elavating effects of the monoamine oxidase inhibitor (MAOI) ipronia- zid and the tricyclic antidepressant (TCA) imipramine in the mid-1950's, there has been a persistent search for new and improved antidepressant agents lacking the bothersome side effects and toxicities assiciated with the MAOL and the TCA. A new and apparently efficacious antidepressant agent, caroxazone, was compared with various, MAOI and TCA for effectiveness anui potency to protect against reserpine- induced and a-methyltyrosine-induced depression of a learn- ed motor skill, to potentiate the psychomotor stimulant and toxic effects of concomitant administration of (L)3,4-di- hydroxyphenylalanine and a peripheral decarboxylase inhi- bitor, to inhibit brain type-A MAO activity in xitgg and for effects on the concentration of norepinephrine in the hypothalamus using rats as subjects. The results strongly suggest that the central pharmacodynamic profile of carox- azone is fundamentally different from that of either the TCA of the MAOI. ACKNOWLEDGEMENTS The author gratefully acknowledges Ms. Renate Lillie- fors for assisting in the preparation of this manuscript and Dr. Richard H. Rech for his valuable suggestions and for proof-reading the final draft. My gratitude is also extended to all committee members for extending to me their valuable time. ii PREFACE "As Bokonon invites us to sing along with him: If you wish to study a 'grandfalloon', Just remove the skin of a toy balloon." Kurt VOnnegut, Jr. iii TABLE OF CONTENTS '-PRELIMINARY PAGES" Acknowledgements ........................................... Preface .................................................... List of Tables ............................................. List of Figures o0.0000000000000000...00000000000000.0000... —TEXT PAGES- INTRODUCTION —— General Introduction for Thesis °°°°°°° SECTION I — Antagonism of Reserpine-induced Behavioral Depression: Comparison of Phenelzine, Isocarboxizid, Desi- pramine, Amitriptyline, Imipramine, Methylphenidate, grAm- phetamine’ Cocaine and Caroxazone o0.000000000000000oooooooo INTRODUCTION ooooooooooooooooooooooo METHODS Subjects, Compounds, etc. ..... Experimental Protocol o........ Stastical Analysis ---°------oo mfimgs..u.u.n.u.u.n.u.n.n DISCUSSION 00000000000000.0000oooooo SECTION II — Prevention of a-Methyltyrosine-induced Behavioral Depression: Comparison of Phenelzine and Car0xazone ooooooooooooooooooooooococo-00.000.00.00...g..... INTRODUCTION 00000000000000.0000...- METHODS Subjects, Compounds, etc. °°--- Experimental Protocol ooooooooo StaStical Analysis oooooooooooo .iv ii iii vi vii 1-13 14-56 14 16 17 20 21 49 57-67 57 S9 60 61 TABLE OF CONTENTS (continued) Resu1t8 and Discussion 00000000000000 SECTION III — Potentiation of L-DOPA-induced Behavioral Stimulation: Comparison of Phenelzine and Caroxazone ----'-° INTRODUCTION 000000000000000000000000 METHODS Subjects, Compounds, etc. 00-0-0 Experimental Protocol °°°°°°---- Stastical Analysis °°°°'°------- RESULTS 00000000000000000000000000000 DISCUSSION 00000000000000000000000000 SECTION IV — Comparison of Phenelzine, Desipramine, Amitriptyline and Caroxazone as Inhibitors of Cerebral Mono- amine oxidase: .23 Vitro Studies 0000000000000000000000000000 INTRODUCTION 000000000000000000000000 METHODS Subjects, Compounds, etc. '°°--- Experimental Protocol ---------- Stastical Analysis ---.......... mfimgg.u.n.u.u.u.u.u.u.n.. SECTION V — I3 Vivo Studies: Effect of Phenelzine and Caroxazone on the Concentration of NOrepinephrine in Rat Hypothalamus 000000000000000000000000000000000000000000000000 INTRODUCTION 000000000000000000000000 METHODS SUbjects, compounds, etc. 000000 Experimental Protocol 0000000000 StaStical Analysis 0000000000000 RESULTS 00000000000000000000000000000 IBIBLJCMSRAFWFI.............................................. APPENDAGE TO THE BIBLIOGRAPHY: Gen- eral References 00000000000000000000 V ,62 68-86 68 71 72 73 74 84 87-105 87 89 89 98 99 106-121 106 107 107 108 113 122-139 137 TAdfl.E LIST OF TABLES Test for acute antagonism of rotarod decrement 4 hours after reserpine, 2 mg/kg00000000000000000000000000 Test for long-term antagonism of rotarod decrement 24 hoUrs after reserpine00000000000000000000000000000000000 Test for acute prevention of rotarod decrement 2 hours after reserpine00000000000000000000000............ The effects of phenelzine and aMT given alone and in combination on rotarod performance0000000000000000000 The effects of caroxazone and GMT when given alone and in combination on rotarod performance"°"°'°°°°°°°° Caroxazone vs. phenelzine in potency to impart a lethal effect to the combination of 32 mg/kg L-DOPA and 75 mg/kg HMD0000000000000000000000000000000000000000 In Vitro Measurement of MAO activity toward d3—DA as a substrate000000000000000000000000000000000000000000 Effect of Caroxazone and Phenelzine on the Concen- tration of Norepinephrine in Rat Hypothalam °'°°°°°°--- Potency ratio for caroxazone and phenelzine in Specific test situations00000000000000000000000000000000 vi PAGE 27 28 30 63 64 82 102 114 120 FIGURE 1 10 11 12 13 LIST OF FIGURES PAGE Chemical structures of caroxazone and some mono- amine oxidase inhibitors (MAOI)--°----------°~0.°°--- 2 Chemical structure of some tricyclic antidepressants and of the phenothiazine nucleuS°°°°°°°-°°'°°°°'°'°°° 3 Biochemical pathways for synthesis and degreda- tion of dopamine0000000000000000000000000000000000000 7 Major metabolic pathways for norepinephrine.......... 8 Synthesis and metabolism of serotonin (5-HT)......... 9 Log dose-response curve for reserpine-induced 1083 Of rOtatOd performance00000000000000000000000000 23 Time course for reserpine impairment of rotarod perfomnceO00......0OOOOOOOOOOOOOOOOOOOOOOO0.00...O 25 Four-hour prevention log dose-response curves for isocarboxAzid, phenelzine and caroxazone ............ 32 Time course of prevention of reserpine depression of rotarod performance following various doses of isocarboxazid000000000000000000000000000000000000 35 Time course of prevention of reserpine depression of rotarod performance following various doses of phenelzine000000000000000000000000.00000000000000000 37 Time course of prevention of reserpine depression of rotarod performance following various doses Of caroxazone0.0......OOOOOOOOOOOOOOOOOI0.0.0.000... 39 Time course for acute antagonism of reserpine depression of rotarod performance following gfamphet- amine (1 mg/kg), methylphenidate (18 mg/kg) or Cocaine (18 lug/kg)0.00000000000000000000000000000000 42 Time course for long-term antagonism of reserpine depression of rotarod performance following geamphet- amine (1 mg/kg), mehtylphenidate (18 mg/kg) or cocaine (18 mg/kg)0000000000000000000000000000000000 vii 43 LIST OF FIGURES (continued) FIGURE 14 15 16 17 18 19 20 21 22 23 24 25 26 Log-spaced dose-response for acute and long-term antagonism of reserpine depression of rotarod per- formance following various doses of cocaine"'°"""° Time course of complete prevention of reserpine de- pression of rotarod performence following various doses of caroxazone or phenelzine""""°"°""""° Time course of complete prevention of reserpine de- pression of rotarod performance following various doses of isocarb0xazid00000000000000000000000000000000 Log-spaced dose—response (LDR) curves for phenelzine and caroxazone efficacy in preventing aMT-induced loss of rotarod performance in previously trained ratSOCOOCOOOOOOOOOOOO00.000.00.000...OOOOOOOOOOOOOOOOO Phenelzine interaction with L-DOPA following HMD pretreatment.0.0.0.0...OOOOOOOOOOOOOOOOOOOOOOOOIOOO... Caroxazone interaction with L-DOPA following HMD pretreatment000000000000000000000000000000000000000000 Accumulative motor activity COuntS00000000000000000000 Electron impact mass spectra of 3-methoxytyramine, 3-methoxytyramine-d3, dopamine and dopamine-d3 pentafluoropropionyl derivatives°°'°°°'°'°"'°°°'°°'°° Electron impact mass spectra of a mixture of dop- amine and d3-dopamine, pentafluoropropionic anhy- dride derivative00000000000000000000000000000000000000 Representative standard curve for d3-d0pamine quantitation by GC/MS mass fragmentography, 431/428 ion pair00000000000000000000000000000000000000 Disapperrance of 100 pg of d3-DA Over a 90-minute period000000000000000000000000000000000000000000000000 Estimation of the log dose-response curve for phenelzine, caroxazone, amitriptyline and desi- pramine 15 vitro inhibition of rat brain MAO acti- vity with dopamine as substrate00000000000000000000000 TIM scans for NE and d3-NE0000000000000000000000000000 viii PAGE 47 S3 55 65 76 78 81 93 95 97 101 104 110 LIST OF FIGURES (continued) FIGURE 27 28 Representative standard curve for norepinephrine quantitation by GC/MS mass fragmentography, 577/578 ion pair000000000000000000000000000000000000 0000 Hypothalamic norepinephrine concentration in rat brain following various doses of phenelzine or caroxazoneOOOOCOOOOOOO0......COO...OOOOIOOOOOOOOIOOOOOOO ix PAGE 112 115 INTRODUCTION General Introduction for Thesis INTRODUCTION Modern psychopharmacology came into being in 1950 with the syntheses of the phenothiazine neuroleptic chlorpro- mazine (Delay and Deniker, 1952). Psychotherapeutic agents for the treatment of affective disorders also became avail- able in the 1950's when the mood elevating effects of iproniazid were noticed in patients suffering from tuber- culosis. The inhibition of monoamine oxidase (MAO; mono- amine; 02 oxidoreductase; EC 1.4.3.4) by iproniazid was first described by Zeller et ai. (1952). Iproniazid was not introduced into general use in the treatment of de- pressive disorders until 1957 and many other monoamine oxidase inhibitors (MAOI) that are efficacious in the treat- ment of depressive syndromes have been described since then. Figure 1 shows the chemical structure of some once widely used MAOI antidepressants. A second class of antidepressants, the tricyclic antidepressants (TCA), also came into being in the 1950's as a result of molecular modifications of some of the earlier antihistaminic drugs. The antidepressant efficacy of this class of drugs was quickly recognized and their use rapidly became widespread. Figure 2 shows the chemical structures of some TCA and of the phenothiazine nucleus. CAROXA ZONE 4H-3-methy1carboxamide-1,3-benzoxazine-2-one QCHZ CH2 -NH-NH2 OCH-H2 .FH-NH2 PHENEL ZINE TRANYLCYPROMINE QCHZ 2Nmuac—fl—T' NO‘ / “CH 3 ISOCARBOXAZID Figure 1. Chemical structure of caroxazone and some monoamine oxidase inhibitors (MAOI). O O 3 O N CH2 CH2 CHZN (CH3) 2 Imipramine CH-CH2CH2N(CH3)2 Amitriptyline '0 | O =0 0 Doxepin (CH2)3NHCH3 Desipramine .0 CH(CH2)2NHCH3 O Nortriptyline 000 (4H2)3NHCH3 Protriptyline R2 Phenothiazine R1 Nucleus Figure 2. Chemical structures of some tricyclic antidepressants and of the phenothiazine nucleus. 4 The MAOI drugs are a rather heterogeneous group of compounds in that they exert many different effects and have various chemical structures. All of the MAOI that are clinically efficacious in the treatment of endogenous depression exert a prolonged suppression of monoamine oxidase activity after 12.2122 and EETXEEEE administra- tion (Neff and Goridis, 1972; Moore, 1971; Planz gt gt., 1972; Tipton, 1972; Eiduson, 1972; Youdim and Sandler, 1967; Youdim, 1972a, 1972b; Spector gt gt., 1963). In this respect these compounds constitute a relatively homogene- ous drug class in that only minor differences in the du- ration of drug effects exist. Monoamine oxidase is believed to exist in multiple forms (i.e., isoenzymes) in the mamma- lian central nervous system (CNS) and other tissues as well (Collins gt gt., 1970; Sandler and Youdim, 1972; Youdim, 1967, 1972a, 1974; Youdim gtht., 1974; Youdim and Sandler, 1967). The significance of multiple forms of cerebral MAO in clinical depression and its treatment with MAOI, even the existence of these forms, has been debated in much of the current literature (Jain, 1977; Fuller, 1972; Neff and Yang, 1974; Fuentes and Neff, 1975; Maitre gt gt., 1976; Planz, 1972; Sandler and Youdim, 1974; Housley gt gt., 1976; Neff and Goridis, 1972; Waldmeier gt gt., 1976; Waldmeier and Maitre, 1975). Differences in substrate affinities for different MAO types seen tg_ttttg may be of little significance to $3 2122 antidepressant activities. For example, there are negligible qualitative differences be- tween the MAOI in animal antidepressant screening tests 5 at doses that maximally suppress brain level of enzyme ac- tivity and bring about changes in the concentrations of brain monoamine neurotransmitters and their metabolites (Everett, 1967; Hill and Tedeshi, 1971; Iversen and Iversen, 1975; Moore, 1971; Rech, 1975; Maitre gt gt., 1976). MAOI pretreatment will prevent a number of behavioral effects due to reserpine (Blaschko and Chruschiel, 1960; Smith, 1962), potentiate the stimulant effects of indirect- acting central nervous system (CNS) stimulants such as amphetamine (Scheckel gt gt., 1969), potentiate the CNS stimulant effects of L-DOPA and L-tryptophan (Everett, 1967; Rech and Thut, 1976; Creveling gt gt., 1968; Everett, 1957, 1967, 1970; Spector, 1967; Thut, 1970; Grahame-Smith, 1974; Wiegland and Perry, 1961) and prevent the general behavioral depression associated with tyrosine hydroxylase inhibition by a—methyltyrosine (Moore and Rech, 1967). MAOI reverse the depressed affect in a certain prOportion of patients suffering from endogenous depression. But MAOI also lack specificity in perturbating brain monoamine sys- tems, many side effects and interactions resulting from enzyme inhibition in peripheral nerves and the liver. The complications associated with chronic suppression of MAO activity in hepatic and cardiovascular tissues are widely appreciated. MAOI-induced hepatotoxicity, impaired red- green color vision and neurologic damage are believed to be unrelated to MAO inhibition (Neff and Yang, 1976). Because of these serious side effects many clinicians view the routine use of this class of antidepressants to be a questionable practice. The clinician's choice of antidepressant therapy is presently limited to drugs that are classified as having either the MAOI or tricyclic antidepressant (TCA) types of activity, as defined by various laboratory procedures (Hill and Tedeschi, 1971; Askew, 1965; Iversen and Iversen, 1975; Rech, 1974, 1975; Sigg, 1959, 1965; Sulser, 1961a, 1962). The effects of MAOI on the concentration of 5-HT, NE and DA metabolites in the CNS is not different from what one would expect (Yang and Neff, 1974). Figures 3, 4 and 5 show synthetic and degradative pathways for 5-HT, NE and DA. Enzyme abbreviations are given in the figure legend. Dramatic increases in the 3-0-methylmetabolites of the two catecholamines occur following MAOI treatment. The de- creased brain levels of the carboxylic and alcoholic meta- bolites of DA, 5-HT and NE are also very pronounced. How- ever, the altered levels of these metabolites does not correlate well with the duration of MAOI bahavioral "anti- depressant" effects in laboratory animals. On the other hand, the time course of altered 3-0-methyl-catecholamine metabolite levels correlates well with the duration of "antidepressant" activity (Waldmeier gt gt., 1976; Maitre gt g]_.. , 1976) . TCA are clinically efficacious and exhibit fewer dele- terious side effects than MAOI, and currently are the drugs of choice (Honigfeld, 1973; Goodman and Gilman, 1975; Bielski and Friedel, 1978; Payson, 1971). Drugs classified as TCA are reported to inhibit the synaptic reuptake process COOH COOH / / CH2CHNH2 C 2CHNH2 TH HO OH OH L-Tyrosine L-DOPA CH2CH2NH2 D O CH3-O OH 3—Methoxytyramine MAO Norepinephrine V V HZCOOH CHZCOOH (EMT . ° 0 HO CH3O H OH 3,4-Dihydroxyphenyl- Homovanillic Acid acetic Acid Figure 3. Biochemical pathways for synthesis and degre- dation of dOpamine. Abbreviations: L-AAAD, L-aromatic amino acid decarboxylase; COMT, catechol-O-methyltransfer- ase; D-B-O, dopamine-B-oxidase; MAO, monoamine oxidase; TH, tyrosine hydroxylase. 8 |H HO NHZ Norepinephrine ‘5 t5 0H Q CH30 H0 H2 3,4-Dihydroxyphenylglycol Normetanephrine £5? C6 ‘0 0130 H0 3-Methoxy-4-hydroxyphenylglycol Figure 4. Major metabolic pathways for norepinephrine. Abbreviations: COMT, catechol-O-methy1transferase; MAO, monoamine oxidase; AlDH, alcohol dehydrogenase. NH2 / NH2 CH 2cacoon / / HO /CHZCHCOOH O > O N H H TIXPtOPhan 5—Hydroxytryptophan CHZCHZNHZ HO CHZCOOH O MAO &A0 0 N N H H 5-Hydroxyindole- Serotonin 3-acetic Acid Figure 5. Synthesis and metabolism of serotonin (5-HT). Abbreviations: TryH, tryptOphan hydroxylase; MAO, mono- amine oxidase; A0, aldehyde oxidase; L-AAAD, L-aromatic amino acid decarboxylase. 10 at serotonergic and noradrenergic synapses in the mammalian CNS (Carlsson gt gt., 1968, 1969a, 1969b, 1969c; Corrodi and Fuxe, 1968, 1969; Schildkraut gt gt., 1969; Schubert gt gl., 1970; Meek and Werdinius, 1970; Alpers and Himwich, 1972). Such studies suggested that those tricyclic anti- depressants which have tertiary amines in their side chains, such as amitriptyline or chlorimipramine, are more potent blockers of, and thus preferentially inhibit, the uptake mechanisms for central serotonergic neurons. In contrast, those antidepressants of the secondary amine type, such as desmethylimipramine, preferentially inhibit uptake mecha— nisms for central noradrenergic neurons (Carlsson gt El-r 1969a, 1969b, 1968; Fuxe and Ungerstedt, 1968; Shaskan and Snyder, 1970; Dubinsky gt gt., 1973). Chronic admi- nistration of those antidepressants effecting 5-HT uptake processes retards central 5-HT turnover and decreases brain levels of 5-HT and 5-HIAA (Corrodi and Fuxe, 1968; Schild- kraut, 1969c; Schubert gt gt., 1970; Meek and Werdinius, 1970). It has been postulated that these effects are medi- ated through some feed-back mechanism which reduces impulse flow in these 5-HT neurons (Corrodi and Fuxe, 1969; Schubert, 1970). The literature on secondary amine tricyclic antide- pressant effects on central catecholamine metabolism is less consistent. Although some investigators have reported decreases in the turnover of NE (Glowinski and Axelrod, 1966; Schanberg gt gt., 1967), others have failed to confirm this observatiOn (Corrodi and Fuxe, 1968; Schubert, 1970; Corrodi gt gt., 1967). However, secondary amine tricyclic 11 antidepressants do potentiate the effects of any drug which increases NE levels in the synaptic cleft (Scheel- Krueger, 1972). These observations are interesting since abnormalities in the release of one or more of the cate- cholamine and indolamine putative CNS neurotransmitters have been seriously implicated as possible disorders be- hind the clinical manifestations of affective disorders (Davis, 1970; Schildkraut, 1970). The emphasis of basic laboratory and clinical investigations into the psycho- pharmacology of antidepressants reflects this prejudice. MAOI offer an alternative treatment in patients not responsive to TCA and are effective in some of these re- fractory cases. There is considerable interest in finding new drugs with antidepressant efficacy that do not have the potential toxicities of the MAOI, but have a broader thera- peutic spectrum and less troublesome side effects than the TCA compounds (anticholinergic, antihistaminic, antisero- tonergic). A new series of drugs, the 1,3-benzoxazines, synthe- sized by Farmitalia Research Laboratories in Milan, Italy, have been screened for antidepressant prOperties (RJA. Carrano, personal communication; Suchowsky, 1969c, 1969d). Of the agents studied to date, 4H-3-methylcarboxamide-l,3- benzoxazine-Z-one (caroxazone) has shown the most promise. Pharmacological tests in a variety of animal species indi- cate that caroxazone exhibits many of the drug interactions associated with clinically useful antidepressants (Suchowsky gt gt., 1969a, 1969b; R.A. Carrano, personal communication). 12 Initial clinical trials also indicate that this drug shows considerable promise as an antidepressant. Caroxazone increases brain catecholamine and 5-hydro- xytryptamine levels at doses that are in the same range as those showing "antidepressant" activity in the functional screening tests in animals. This is also true for the classical MAOI. The potency of this drug is also in the same range as the more commonly used MAOI (phenelzine, tra- nylcypromine). Thus, ta gt!g_potency and Spectrum of acti- vity of caroxazone in these tests would suggest a mechanism similar to that of the MAOI. However, when caroxazone was administered 12.2122! analysis of MAO activity of brain homogenates did not show a reduction in enzyme activity (Suchowsky gt gt., 1969b). Repeated treatment with caro— xazone was found to increase S-HIAA levels (R.A. Carrano, personal communication). Caroxazone does not appear to have any antihistaminic, anticholinergic or antiserotonergic activity, and therefore would not be expected to cause some of the adverse side effects seen with the TCA (i.e., xero- stomia, constipation, blurred vision, etc.). Caroxazone also differes from a TCA in a lack of effect on norepine- phrine or S-hydroxytryptamine uptake (Suchowsky gt gt., 1969a). Caroxazone does not exhibit amphetamine-like CNS stimulatory activity (Suchowsky gt gt., 1969a, 1969c). This observation is important since g-amphetamine and ampheta- mine-like CNS stimulants also exhibit many of the drug inter— actions associated with clinically efficacious antidepressant 13 agents (i.e., anti-reserpine activity: Rech, 1964, 1975; Pirch, gt gt., 1967; Moore, 1971; Hill and TedesChi, 1971; Iversen and Iversen, 1975; McKearney, 1968; Rech and Stolk, 1970). The research eff‘rt presented here was designed to better characterize the pharmacodynamics of caroxazone as they relate to "antidepressant" activity. These studies have been organized into 5 sections. The first three sec- tions describe experiments which compared caroxazone with other clinically efficacious antidepressants for activity to elicite "antidepressant" activities as defined by three different experimental procedures. The fourth section describes experiments comparing some of these drugs for potency and effectiveness to inhibit rat brain MAO EEEEEEQ' usingthpamine as the enzyme substrate. The last section describes the results of experiments comparing phenelzine and caroxazone for activity to influence the concentration of norepinephrine in the hypothalamus of the rat. Each of the sections contains a brief introduction, description of experimental protocol in a methods section, a results section and a discussion section (or a combined results and discussion section). SECTION I Antagonism of Reserpine-Induced Behavioral Depression: Comparison of Phenelzine, Isocarboxazid, Desipramine, Amitriptyline, Imipramine, Methylphenidate, g-Amphetamine, Cocaine and Caroxazone SECTION I INTRODUCTION Reserpine treatment produces a wide variety of effects when administered to laboratory animals, most of which are easy to observe and quantitate (i.e., decreased spontaneous motor activity, hypothermia, impairment of various learned behaviors, etc.). One prOperty shared by MAOI and TCA is the ability to both reverse (i.e., the antidepressant drug administered after reserpine) and prevent (the antidepres- sant administered before reserpine) certain components of the reserpine depressant Spectrum. Caroxazone is reported to also exhibit anti-reserpine activity in a variety of species (Suchowsky gt gt., 1969a, 1969b). For example, caroxazone and imipramine are reported to be approximately equipotent in reversing reserpine-induced hypothermia, decreased spontaneous motor activity and ptosis, and, in general, caroxazone exhibits anti-reserpine activity that is very similar to that seen for wellknown antidepressants. Reserpine depletes central stores of norepinephrine (NE), dopamine (DA) and 5-HT (Alpers and Shore, 1967). Pretreat- ment with a MAOI will prevent the depletion, whereas TCA are without preventative effects in this particular situa- tion (Tipton, 1972; Christmas gt gt., 1972; Pirch, 1967; Rech, 1975; Iversen and Iversen, 1975; Moore, 1971). 14 15 Pretreatment with caroxazone will also prevent reserpine— induced depletion of cerebral biogenic amine stores (RJA. Carrano, personal communication; Suchowsky, 1969b). The following study examines the anti-reserpine effects of caroxazone, MAOI, TCA and some CNS stimulant drugs (i.e., methylphenidate, cocaine and g-amphetamine). These drugs were included in the study since g-amphetamine has been used in man, although without much success, as a short-term antidepressant (Payson, 1971). Cocaine effects in this experimental situation were not known and this was another consideration. The results contained in this section have been submitted for journal publication (Vrbanac gt gt., 1978a). METHODS Experimental methods generally follow previously published procedures for examining various drug interac- tions using rotarod performance in the rat (Rech gt gt., 1966; Moore and Rech, 1967). Subjects All subjects used in this study were female Sprague- Dawley rats weighing from 200 to 250 9. Subjects were purchased from Spartan Farms, Haslett, Michigan, and main- tained in laboratory animal facilities with controlled temperature (22°C), humidity (45 %) and diurnal lighting (i.e., 12 hour light cycle from 7 a.m. to 7 p.m.). Purina Rat Chow and water were available gg libitum. Drugs The following drugs were used for this study: reserpine alkaloid (S.P. Penick and Co., New York, N.Y.), 4-H-3- methylcarboxamide-l,3-benzoxazine-2-one (caroxazone, free base; Farmitalia Research Laboratories, S.P.A., Milan, Italy), phenelzine (sulfate salt; Warner-Lambert Research Institute, Morris Plains, N.J.), isocarboxazid (Hoffman-LaRoche, Inc., Nutley, N.J.), tranylcypromine (sulfate salt; Smith, Kline and French Laboratories, Philadelphia, Pa.), desipramine (hydrochloride salt; Merrell National Laboratories, Inc., 16 17 Cincinnati, Ohio), imipramine (hydrochloride salt; Geigy Pharmaceutical Division, Ardsly, N.Y.), amitriptyline (hydrochloride salt; Merck, Sharp and Dohme, West Point, Pa.), geamphetamine (hydrochloride salt; Sigma Chemical Co., St. Louis, Mo.), methylphenidate (hydrochloride salt; Geigy Pharmaceutical Division, Ardsly, N.Y.) and cocaine (hydrochloride salt; Mallinckrodt Chemical Works, St. Louis, Mo.). All drugs were dissolved in 0.9 % saline except for caroxazone and reserpine. Caroxazone was suspended in 0.5 % methylcellulose and reserpine was dissolved in glacial acetic acid and diluted with distilled water to a concentra- tion of 2.0 mg reserpine per m1 of dilute acetic acid (2-3 % v/v). Solutions used in the construction of the log-Spaced dose-response curve for reserpine-induced loss of rotarod performance were prepared by dilution of 4.0 mg reserpine/m1 3.0 % acetic acid stock solution (i.e., 0.32, 0.56, 1.0, 1.8, 2.0 and 3.2 mg reserpine/ml). TrainingeProcedure Subjects were trained to walk on a rotating cylinder, or "rotarod" (RR; 5 inches diameter, 9.5 revolutions per minute). An animal was considered to be trained when it remained on the cylinder for 180 continuous seconds. Ani- mals were placed on the cylinder with the head pointing in the direction of rotation and thus were required to learn to turn around, as well as walk forward, on the cylinder. Drug effects in these rats were examined one or two days later. The same procedures and performance criteria were 18 used during drug testing as for training. Thus, 100 % performance for any animal required that the subject turn around initially as well as remain on the cylinder for a period of 180 seconds. The same rotarod used to train subjects was used for drug testing. Dosing and Testigg Protocol Four procedures were carried out in the drug tests: acute antagonism, long-term antagonism, acute prevention, and long-term prevention. For the acute antagonism reser- pine (2 mg/kg) was administered 4 hours before testing on the rotarod (RR), and the test drugs were administered 2 hours before the RR determination. The protocol used to evaluate the effects of g-amphetamine, methylphenidate and cocaine was slightly different. Two hours following the reserpine treatment subjects were tested on the rotarod and scores recorded. Subjects were immediately treated with one of the three test drugs and the time of injection was re- corded for each subject. Only those subjects showing essen- tially complete loss of rotarod performance were treated (loss of rotarod performance arbitrarily defined as a score of 10 seconds or less on the rotarod). Subjects were tested 20 minutes after receiving the test drug and a second score was recorded. The time course of effects was determined for doses showing activity to antagonize the effects of reserpine treatment. For the long-term antagonism RR was measured at 2 and 24 hours after reserpine, the test drug was administered 19 immediately after the 24-hour measurement, and RR was again determined at 26 hours after reserpine. The protocol used to evaluate the effects of g-amphetamine, methylphe- nidate and cocaine differed from this procedure in exactly the same manner described previously for the acute preven- tion procedure. Therefore, the last RR measurement was taken 20 minutes after injection of one of the stimulant test doses. The acute prevention experiment required the injec- tion of the test drug at 4 hours before placing the subjects on the RR, and 2 hours before reserpine (2 mg/kg). The long-term prevention experiments were of 2 types. The first procedure called for the test drug to be injected 2 hours before reserpine (2 mg/kg), followed by RR determinations at 4, 6, 8, 10 and 24 hours after administering the test drug. However, if performance had completely deteriorated to the non-protection level by 8 or 10 hours, subsequent rotarod measurements were cancelled. The second experiment of the long-term prevention type involved the administra- tion of caroxazone, 32 mg/kg, 2 hours before reserpine (2 mg/kg). RR behavior was tested at 4 and 26 hours after caroxazone, but additional injections of 11 mg/kg of caro- xazone were given at 4, 8, 12, 16, 20 and 24 hours. These additional doses had been calculated to be a maintenance dose of caroxazone based on first-order elimination and a blood level half-life of 6 hours (R.A. Carrano, personal communication). A control group received 0.5 % 20 methylcellulose in place of caroxazone at these same times. The 32 mg/kg dose of caroxazone was chosen since this was maximally effective in preventing RR disruption at 2 hours after reserpine, as determined in pilot studies. Statistical Analysis All experimental designs contained the apprOpriate controls. The highest dose of each test drug was examined for possible effects On RR when given with the reserpine vehicle (dilute acetic acid). A reserpine dose-response pattern on RR was also determined at two and four hours, and the time course of effect following 2 mg/kg reserpine was examined at 2, 6, 8, 10 and 24 hours. Tests for statistical significance were the Mann- Whitney U test for differences between independent samples (one-tailed, P<.05) for the majority of the tests. The non— parametric multiple comparisons by simultaneous test proce- dures, an a posteriori test of samples with equal measures based on U, the Wilcoxin-Mann-Whitney U test for multiple comparisons, was applied to time-course studies (P<.05). The Wilcoxin signed-ranks test for differences between re- lated samples (two-tailed, P<.05) was applied to the long- term antagonism studies. RESULTS Reserpine Dose-Response and Time-Course for Rotarod Impairment Reserpine was administered in log-spaced doses (LDR) ranging from 0.32 to 4.0 mg/kg and effects on rotarod per- formance (RR) were established 2 hours thereafter. The results obtained at 2 hours are seen in Figure 6. The se- cond LDR was obtained using a RR that turned slightly faster (10.0 revolutions per min). Doses below 1.0 mg/kg (mean RR score at 1.0 mg/kg = 130 sec, on the slower RR, which was used in all subsequent experiments) did not sig— nificantly influence RR behavior. A dose of 1.8 mg/kg de- creased RR scores to a mean of 8 sec, less than 5 % of control. Thus, the dose response curve is very steep. A dose of 2 mg/kg was chosen for convenience, since earlier studies had been done with this dose and it represents a dose only slightly larger than that producing maximal im- pairment. Figure 7 shows the time-course for disruption of RR following a single 2 mg/kg injection of reserpine. The large plot demonstrates the onset of RR impairment as de- terminations made every 20 minutes for 2 hours. The insert plots a longer time-course, to 24 hours. The behavior was completely disrupted by 100 minutes after reserpine and remained maximally impaired for up to 12 hours. By 24 hours 21 Figure 6. 22 Log-spaced dose-response curve (LDR) for reserpine-induced loss of rotarod perfor- mance. Subjects were tested two hours after receiving an i.p. injection of reser- pine. Doses tested were 0.32, 0.56, 1.0, 1.8, 2.0, 3.2 and 4.0 mg/kg (n=4). Vehicle alone had no effect on subject performance (not shown). The circles show results ob- tained with the rotarod used in all sub- sequent drug tests (9.5 revolutions/min). The second LDR, indicated by squares, was obtained using a rotarod that turned slightly faster (10.0 revolutions/min). 23 Gszv mmo: 335m 53 N.m m4 at.” mm. mm. _ _ r . o / I HUNG! I I I O z E :8 m N G S 0 N “a 0 oomaaom mmzoqm I . W. M 99298 mmamfi .. I .. oma an... mzmmmmmmm . z ’ I I I I : 0 633m . l. 03 Figure 7. 24 Time course for reserpine impairment of rotarod performance. Results are expressed as the average time i S.E.M. in seconds (n=12) on rotarod as determined every 20 minutes for the initial 120 minutes (large plot) and at 4, 6, 8, 10, 12 and 24 hours (displayed in small insert) following 2 mg/kg of reserpine (i.p.). The Mann- Whit- ney test for multiple comparisons of groups with equal measures was used for statisti- cal analysis. Results for comparison of a) 20, 40, 60, 80, 100 and 120 minute measures and b) 0, 6, 12 and 24 hour mea- sures are displayed in the conventional manner (p<.05). Statistical difference between various means is also shown with broken connecting lines, in this and in Figures 3, 4 and 5. 25 Figure 7 180 'k. 180‘ -1oo / JV AK A’ O O I ‘. 30" . u I 120 \ 20f- ' I. \ ' , F10 / \ a z \ 1O ‘ | I’ L I I 5 \ ' / I \ 0-0-0-0-0.0 0 \ 0 I TTTTT I T v I I I l - \ o 12 24 60 Ordinate: Seconds and % of [D Control (left) \\\\\~ Abscissa: Hours .\ I 0 ‘ - C l T T I I T ’41 O 60 120 TIME FOLLOWING RESERPINE - MINUTES 26 there was only a slight recovery. These results confirm previous observations of reserpine effects on RR (Pirch EE.E1-r 1967), and they also parallel in time of onset the depletion of brain monoamines and induction of slow waves in the electrocorticogram. Acute Antagonism of Resetpine Effects by Antidepressants The results of the experiments to determine acute an— tagonism of reserpine effects are listed in Table 1. None of the drugs examined reversed the reserpine decrement in RR. The potential antagonists were also administered after the acetic acid vehicle; at doses listed in Table 1 they did not have a significant effect on RR, the animals per- forming as well as controls (data not included in Table 1). Long-term Antagonism of Reserpine Effects by Antidepressants Groups of 5-6 rats each were trained on the RR to criterion and injected with 2 mg/kg reserpine. The groups were again assessed for RR two hours following reserpine to assure the reserpine impairment (Table 2). At 24 hours after reserpine, the rats were tested for RR and injected immediately thereafter with 10 mg/kg of isocarboxazid, phe- nelzine, caroxazone, desipramine, or amitriptyline. They were once more tested for RR two hours later (26 hours after reserpine). Comparing the RR scores at 26 hours with those at 24 hours indicated that none of the antidepressant drugs was capable of antagonizing the RR impairment on the second day after reserpine. 27 TABLE 1 Test for acute antagonism of rotarod decrement 4 hours after reserpine, 2 mg/kg Treatment‘ Seconds on RR, n mean i SD 1. Acetic Acid vehicle 180.0i0.0 12 2. Reserpine Control2 (Initial observation) 5.05:2.02 12 3. Caroxazone 10 mg/kg 4.67il.97 6 Paired Control (reserpine alone + drug vehicle) 7.17i7.75 6 4. Caroxazone 20 mg/kg 4.17i2.93 6 Paired Control 3.40:0.55 5 5. Caroxazone 32 mg/kg 6.00:2.00 6 6. Isocarboxazid 10 mg/kg ll.33i5.15 6 Paired Control 5.67:1.75 6 7. Isocarboxazid 20 mg/kg 5.20:2.68 5 Paired Control 4.67tl.03 6 8. Tranylcypromine 20 mg/kg 3.20i0.20 5 Paired Control 5.50:4.23 6 9. Phenelzine 32 mg/kg 5.1011.67 6 10. Desipramine 32 mg/kg 10.50:4.81 6 11. .-Amitriptyline 32 mg/kg 5 . 32:2 .59 5 1None of the treatments listed significantly antago- nized the reserpine impairment of rotarod performance. 2Reserpine and paired controls all averaged at 2.8 % of untreated (acetic acid vehicle) performance. .mcflmuommu mo cowuomncfl Hmumo mosey m50flum> um ooumuow may ooxams Am no mncv mooum 20mm Dona mocoomm mo Hmnfisc .z.m.m some can we wsao> comma .ummu mm Hooslmm wnu up muommmm mcaonmmmn omuficommucm haucmoflwflcmflm mmsuo ummu can no mooz .pmmu mm Hconlvm mnu Hmwum MHODMHoQEEH mx\mE OH HO mmoo m CH commumflcflfiom mos unflaommucm Hafiucmuom commd o.oao.oma maaasnaauuaaa 28 N.¢H@.HH v.m H¢.w o.HNaN.hm N.Nao.m o.HHHm.mH N.m Hm.OH o.owo.oma OGHEMHQHMOQ w.mao.va m.m Hm.wH m.m Ho.oa o.oao.oma OGOmeOHmo h.NHN.m N.m H~.HN o.mmwh.mv o.oao.cmH mafiudmcmnm m.¢ah.ma H.v HN.©H m.m Hh.MH ~N.©Hm.m>a ownmxonHMOOmH Gm vm N . o flmono umoa mx\ma N .mcwouwmmn Houmm musom mcwmuomou Hmumm mason vm Dawsonooo commuou mo Emwcommucm EnoulmcoH now Once N Ems”. 29 Acute Prevention of Reserpine Effects by Antidepressants Listed in Table 3 are the RR scores of groups pretrea- ted with the various antidepressant drugs to assess preven— tion of reserpine impairment. When caroxazone, phenelzine, isocarboxazid, or tranylcypromine was administered 2 hours before reserpine and RR tested at 4 hours, the usual decre— ment due to reserpine was completely prevented. Gross ob— servations also clearly indicated that these animals showed no significant signs of having received reserpine. On the other hand, the groups treated with a tricyclic antide- pressant (TCA) and the reserpine showed much of the ptosis, hunching, and immobility of the reserpine controls. The larger doses (desipramine and amitriptyline) showed only a very slight protection against the RR disruption by reser- pine, and imipramine was ineffective in the dose used. A more complete dose-response pattern for acute anta- gonism of reserpine was established for isocarboxazid, phenelzine and caroxazone, as illustrated in Figure 8. Essentially complete protection against reserpine impairment was achieved by isocarboxazid in a dose of 3.2 mg/kg and by phenelzine and caroxazone in a dose of 5.6 mg/kg. Thus, the threshold doses for prevention of reserpine effects among the 3 antidepressants are quite close, although caroxazone is slightly less potent than the other two agents. The ca- roxazone protection is more variable over the range of higher dosage than in the case of isocarboxazid and phenel- zine. Because of this variability a larger number of sub- jects and doses were employed to determine the caroxazone 30 TABLE 3 Test for acute prevention of rotarod decrement 2 hours after reserpine Seconds on RR, T reament mean i S.E.M. n 1. vehicle (+ Reserpine 2 mg/kg) 5.20i0.6 20 2. Caroxazone 20 mg/kg 180 $0.01 5 Caroxazone 40 mg/kg 180 $0.0 6 3. Phenelzine 18 mg/kg 180 $0.0 8 Phenelzine 32 mg/kg 180 $0.0 8 4. Isocarboxazid 20 mg/kg 180 $0.0 6 Isocarboxazid 40 mg/kg 180 $0.0 5 5. Tranylcypromine 20 mg/kg 180 $0.0 6 6. Desipramine 32 mg/kg ll.60$4.02 8 7. Imipramine 18 mg/kg 5.50$1.7 6 a. Amitriptyline 32 mg/kg 18.40$6.22 12 1Treatments 2 through 5 were completely effective in preventing reserpine impairment. 2These secoes are significantly greater that reserpine controls by the Mann‘Whitney U Test, but the effect was just significant at p< 0.05. Figure 8. 31 Four-hour prevention log-spaced dose-res- ponse curves for isocarboxazid, phenelzine and caroxazone. Response is the average time in seconds that each group remained on the rotarod when tested 4 hours after antidepressant administration (i.p., and 2 hours after 2 mg/kg reserpine, i.p.). The data displayed for phenelzine and iso- carboxazid is the average for 8 subjects except for the 0.56 and 1.8 mg/kg doses of phenelzine (n=7) and the 0.32 and 0.56 mg/kg doses of isocarboxazid (n=6 and 7, respectively). A total of 12 doses of caroxazone was studied with n equal to 7-16 subjects per group. 8) H N C) AVERAGE TIME ON ROTAROD (N 180 0') O I 32 Figure 8 a???“ C?_ l I l 0.18 0.32 0.56 1.0 1.8 3.2 5.6 DOSE (HG/KG) ISOCARBOXAZID PHENELZINE CAROXAZONE l l l l 10 18 32 56 33 dose-response curve. Long-term Prevention of Reserpine Effects by Antidepressant The 3 antidepressant drugs isocarboxazid, phenelzine and caroxazone were compared for long-term protection against reserpine disruption of RR as depicted in Figures 9, 10 and 11. Each antidepressant drug was administered in varying doses two hours before reserpine. RR was measured just before reserpine injection and at 2, 4, 6, 8 and 22 hours after reserpine (4, 6, 8, 10 and 24 hours following antidepressant), or until the protection was completely lost. Figure 9 shows that doses of isocarboxazid of 3.2 mg/kg and greater afford a long-term protection against reserpine impairment of RR, being quite significant although not complete at 24 hours after reserpine. On the contrary, doses of 1.8 mg/kg and less of isocarboxazid exerted a weak and transient protection. Figure 10 indicates a similar pattern for phenelzine in interacting with reserpine. Doses of 5.6 mg/kg and greater protected against the reserpine disruption of RR to a significant degree for at least 24 hours. Phenelzine in doses of 3.2 mg/kg and less afforded partial protection only transiently, up to 4 hours after the depressant. Caroxazone interacted with reserpine in a very different pattern, as seen in Figure ll. The duration of the protective effect was dose-related between 5.6 and 56 mg/kg, but even at the largest dose the effect did not ex- tend beyond 8 hours after reserpine. Figure 9. 34 Time course of prevention of reserpine depression of rotarod performance following various doses of isocarboxazid. Response is the average time in seconds when tested 4 hours after isocarboxazid and 2 hours after 2 mg/kg of reserpine, with subsequent testing at 6, 8, 10 and 24 hours after isocarboxazid. Each group contained 8 sub- jects except for the lowest two doses (n=6 for 0.32 mg/kg and n=7 for 0.56 mg/kg). Statistical treatment of the data is de— scribed in the Methods section. Statistic- ally significant differences between vari- ous means are represented by broken lines (p<.05). Figure 10. 36 Time course of prevention of reserpine de- pression of rotarod performance following various doses of phenelzine. Response is the average time in seconds when tested 4 hours after phenelzine and 2 hours after 2 mg/kg reserpine, with subsequent testing at 6, 8, 10 and 24 hours after phenelzine. Each group contained 8 subjects except for the 0.56 and 1.8 mg/kg doses (n=7). Statistical treatment is described in the Methods section. Statistically signifi- cant differences between various means are represented by broken lines (p<.05). Figure 11. 38 Time course of prevention of reserpine de- pression of rotarod performance following various doses of caroxazone. Response is the average time in seconds, testing 4 hours after caroxazone and 2 hours after 2 mg/kg reserpine, with subsequent testing at 6, 8 and 10 hours after caroxazone. N=8 for the 1.0, 1.8, 3.2, 5.6 and 18 mg/kg groups. For all other groups n=7. Sta- tistical treatment is described in the Methods section. Statistically signifi- cant differences between various means are represented by broken lines (p<.05). SECONDS 0N ROTAROD 180 F '>K‘ —EJ EQEOXAzoyE E] . “CNN 7 22.3 ‘\ ‘\ \ \ CD 18.0 A 10.0 ‘\ \.\ \ i: 3: 120 - i \\ \ V \ v 1:8 H O\ A 1.0 ‘ \ \\ x CONTROL \\ .0 - l \ \A X “.Ix o \ Q\\l' C) "' \ \ \ D \“V I \\3 [I] 0 1 X\$ x>>§>¥ 0 2 4 6 8 10 TIME (HOURS) 40 To determine if caroxazone prevention of reserpine RR disruption could be extended by repeated administration, we carried out the next series of experiments. Caroxazone, 32 mg/kg, was injected 2 hours before reserpine, and RR was measured at 4 and 26 hours after this dose of the antide- pressant. In addition, doses of 11 mg/kg of caroxazone were injected at 4-hour intervals after the 32 mg/kg dose, up to 24 hours. A control group received the same treatment, except that caroxazone vehicle was injected in place of the drug. The caroxazone-treated group walked the RR for l74.8i3.l sec (mean i S.E.M.) at 4 hours and 108:14.8 sec at 26 hours. The control (reserpine-treated) rats walked the RR for 29.5:ll.7 sec at 4 hours and 24.4i9.3 sec at 26 hours. Therefore, maintenance of caroxazone levels over an extended period also extends the protection against the effects of reserpine and yields a pattern of interaction resembling that seen after a single dose of an MAOI. Acute and Long-term Antagonism of Reserpine Effects bngNS Stimulants The results obtained for the CNS stimulants are dis- played in Figures 12, 13 and 14 and will only be discussed briefly. Figures 12 and 13 show both g-amphetamine and methylphenidate to be effective antagonists of reserpine- induced depression of RR performance, as others have repor- ted and in keeping with the generally accepted profile of amphetamine-like activity (Smith, 1962; McKearney, 1968; Moore, 1971; Rech and Stolk, 1970; Stolk and Rech, 1968; 41 Figure 12. Time course for acute antagonism of reser- pine depression of rotarod performance following gfamphetamine (1 mg/kg), methyl- phenidate (18 mg/kg) and cocaine (18 mg/kg). Results are expressed as the mean time each group remained on the rotarod (n=8). Significant differences are shown with an asterisk (multiple comparisons by non- parametric simultaneous test procedure of data within each drug group, p<.05). 42 A352 :5 m2: 8H ofi. on r} _ _ o I L» Di. .t. 36*me 362868 .. AV Sims dmfiafimsgum I 0 Sims 3386823382 . D 4.. NH wnsmflm rmN flOHVlOH N0 SUNODBS 43 Figure 13. Time course for long-term antagonism of reserpine depression of rotarod performance following d-amphetamine (1 mg/kg), methyl- phenidate T18 mg/kg) and cocaine (18 mg/kg). Results are expressed as the mean time each group remained on the rotarod (n=8). Testing protocol is described in the Methods section. All median scores ob- tained after CNS stimulant administration are significantly different from the con- trol median score obtained just prior to treatment with stimulants. Significant differences are shown with an asterisk (multiple comparisons by nonparametric simultaneous test procedure of data within each drug group, p100). Traditionally, RR data has been expressed as the group mean score. Another common expression is the time to 50 % re- covery of depressed RR performance (50 % of n). The data diSplayed in Figures 9, 10 and 11 show caroxazone to be approximately equipotent with phenelzine and slightly less potent than isocarboxazid. Although caroxazone is shown to exert a less consistent reversal of the reserpine-induced loss of RR performance, the maximal effect appears to be approximately the same as the maximal effect seen with the MAOI, generally speaking. However, caroxazone is seen to exert preventative effects that are quite different if the data is replotted in a quantal fashion (i.e., the traditional LDR). Figures 15 and 16 show the data contained in Figures 9, 10 and 11 expressed as the % of the subjects in each group showing complete protection from reserpine effects (i.e., % of subjects remaining on the RR for 180 seconds). The data is expressed as a % since not all groups contained the same number of subjects. Caroxazone is seen to exert anti-reserpine activity that is less effective than that afforded by the two MAOI. The effect is completely lost between the 6 and 8 hour readings, even for the highest doses of caroxazone. This is in sharp contrast to the preventative action of phenelzine and,to a slightly lesser degree, that of isocarboxazid. The four hour values seen for caroxazone cannot be compared to the LDR seen in Figure 8 since this latter curve was constructed using a larger number of sub- jects at doses > 3.2 mg/kg. If, however, the data for Figure 15. 52 Time course of complete prevention of re- serpine depression of rotarod performance following various doses of caroxazone or phenelzine. Response is the % of subjects in each group remaining on the RR for the full 180 seconds, testing 4 hours after caroxazone or phenelzine and 2 hours after 2 mg/kg reserpine, with subsequent testing at 6, 8 and 10 hours after caroxazone or phenelzine. N=8 for the 3.2, 5.6 and 18 mg/kg caroxazone groups. For all other caroxazone groups n=7. For all phenelzine groups n=8. Data for repeated administra- tion of caroxazone 4-hour maintenance dose (calculated from Ke=0.001925) after initial 32 mg/kg treatment is also shown (0). 53 Hz” -— : ma a o.m HHH mz_~4mzmza N.m nu L7 NV a N.m Nmu mw “322%: mm 0 mzo~ a iw lal a... 314/ ma musmflm /% No Nom NOOH 33N0dsau z Figure 16. 54 Time course of complete prevention of re- serpine depression of rotarod performance following various doses of isocarboxazid. Response is the % of subjects in eaoh group remaining on the RR for the full 180 se- conds when tested 4 hours after isocarbo- xazid and 2 hours after 2 mg/kg of reser- pine, with subsequent testing at 6, 8, 10 and 24 hours after isocarboxazid. Each group contained 8 subjects. Open-faced circles show results obtained upon repeated administration of 4-hour caroxazone main- tenance doses (calculated for Ke=0.001925) after an initial 32 mg/kg treatment. 55 am LJ. .1 if Homumwmmmv ozoN mxoumu IV 0..— m H I Av m.m I = m m I.- o.oa ..AV o.ma..4V mx\mfi :H once 4 .///I/m V HmmOOIO mzme OH O O a N NI _ _ _ _ _ ~ OllI. 4.11.»le OH musmflm om OOH (1N3383d) 33N0d838 56 caroxazone displayed in Figure 8 was replotted as the % of subjects performing to training criteria, then the dose of 5.6 mg/kg would be seen as approximately 50 % effective at 4 hours. Both Figures 15 and 16 show the data obtained for chronic (24 hour) administration of caroxazone. The effect differs dramatically not only from the single treatment caroxazone data, but paradoxically, shows caroxazone to exert greater protection at 24 hours than all doses of either MAOI. Pharmacokinetic studies in man, dogs and rats have shown that the kinetics of elimination after repeated admin- istration Ixf caroxazone do not differ from single admin- istration kinetics, nor does the pattern of tissue distri- bution change upon repeated administration (R.A. Carrano, personal communication). Thus, it seems very unlikely that caroxazone accumulated in these subjects. Besides, the to- tal dose of caroxazone administered is only 98 mg/kg and not very different from the highest single dose used (56 mg/kg), on a log scale. The results Obtained for repeated admin- istration are consistent with the hypothesis that the anti- reserpine effects of caroxazone, and presumably its central effects in general, are completely reversible and are depen- dent upon blood levels of unbound caroxazone. SECTION II Prevention of aMethyltyrosine-induced Behavioral Depression: Comparison of Phenelzine and Caroxazone SECTION II INTRODUCTION The previous study suggested that caroxazone is funda- mentally different from the classical MAOI in the pattern of preventing the behavioral depressant effects of reserpine. This study attempts to further characterize the Spectrum of behavioral effects of caroxazone. The amino acid d-methyl- tyrosine (aMT) is a reversible inhibitor of tyrosine hydroxy- lase and thus interferes with the synthesis of NE and DA (Spector gt $1., 1965). Because dMT enters the brain (via amino acid transport systems) it has been used extensively as a pharmacological tool to study the functions of central catecholaminergic neurons. Doses of aMT sufficient to maximally inhibit brain tyrosine hydroxylase activity, elicite a behavioral depression, with the onset and magni- tude of this depression correlated with changes in catechol- amine levels, and aMT-induced decreases in Spontaneous motor activity, decrement of conditioned avoidance behavior and impaired performance of a learned motor skill have been re- ported (Moore, 1971; Moore and Rech, 1967; Rech 33 al., 1966). The effects of caroxazone on GMT-treated animals have not been reported. If administered to rats as a pretreatment, MAOI prevent aMT—induced behavioral depression as well as the aMT-induced reduction in NE and DA. TCA are without 57 58 effect in this test (Hill and Tedeshi, 1971; Iversen and Iversen, 1975; Moore, 1971; Moore and Rech, 1967; Rech, 1975). Based upon the results contained in Section I it was decided that phenelzine, which was nearly equipotent with caroxazone in preventing reserpine-induced depression of RR performance, would be compared with caroxazone for preventative effects against aMT-induced depression of the same learned motor skill. Repeated administration of aMT is necessary for this agent to consistently depress RR performance in rats to a level that is appropriate for evaluation of the preventa- tive effects of a second drug. It was obvious from the re- sults of Section I that repeated administration of caroxa- zone would be necessary and also appropriate. METHODS Subjects All subjects used in this study were essentially as described previously in Methods, Section I. Drugs The drugs used in this study were caroxazone (free base), phenelzine (sulfate salt) and DL-a-methyltyrosine methylester (aMT; hydrochloride salt, Aldrich Chemical, Milwaukee, Wisconsin). Phenelzine was dissolved in 0.9 % NaCl (phenelzine vehicle). The concentration of phenelzine was adjusted for each dose such that the volume of 0.9 % NaCl injected at each treatment was always equal to 1.0 ml/kg body weight. aMT was dissolved in distilled water (D.W., aMT vehicle). The concentration of aMT was always 10 mg/ml of D.W. and the volume of injection for all subjects was 10 ml/kg body weight (to minimize renal toxicity resulting from precipitation of aMT in the renal tubules, Hook and Moore, 1969; Mbore £5 31., 1967). Caroxazone was suspended in 0.5 % methylcellulose (caroxazone vehicle). The concen- tration of caroxazone was adjusted for each dose such that the volume of 0.5 % methylcellulose injected at each treat- ment was always equal to 1.0 ml/kg body weight. 59 60 Training Procedure The subjects were trained as described previously in the Methods portion of Section I. Dosing and Testing Protocol In the initial series of experiments subjects received a single i.p. injection of phenelzine in doses of either 0 (saline vehicle), 1.8, 5.6 or 18 mg/kg. There were 12 sub- jects receiving each dose of phenelzine or phenelzine vehicle. Half of the subjects in each of the four groups were treated 2, 5 and 8 hours later with 100 mg/kg aMT. The remaining subjects received aMT vehicle alone. Thus, there were a total of 8 treatment groups with 6 subjects in each group. Two hours following the last injection of aMT all subjects were tested for rotarod performance. The total time on the rotarod, up to 180 seconds, was recorded for each subject. In the next series of experiments subjects were admin- istered caroxazone in doses of 0 (0.5 % methylcellulose ve- hicle), 2, 4, 8, 10, 16, 32 and 64 mg/kg three hours before the first injection of aMT or aMT vehicle (total number of groups equal to 16 with 6 subjects per group). Caroxazone effects in the CNS are apparently completely reversible with elimination of the drug from the body (see Section I) and are thus directly related to the blood levels of the drug (R.A. Carrano, personal communication; Suchowsky g5 g1., l969a,b). Therefore, a maintenance dose of caroxazone was calculated (for Ke=0.001925 min-1) and administered along with each of the three aMT treatments. Three hours 61 following the third injection of dMT (or aMT vehicle) plus caroxazone maintenance dose (or caroxazone vehicle) subjects were tested for rotarod performance. The total time on the rotarod up to 180 seconds was recorded for each subject. Statistical Analysis Tests for statistical significance were the Mann-Whitney U test for differences between independent samples and the nonparametric multiple comparisons by simultaneous test procedure, an g pgsteriori test of samples with equal meaq sures based upon U, the Wilcoxin-Mann-Whitney statistic (Sokal and Rohlf, 1969). RESULTS AND DISCUSSION The results of the initial experiments with phenelzine are seen in Table 4. GMT treatment alone depressed rotarod performance to about the same extent seen in a previous study of aMT-induced depression of rotarod performance (Moore and Rech, 1967). MAOI (phenelzine) pretreatment almost come pletely prevented this effect, as expected. Doses of 5.6 and 18 mg/kg of phenelzine are seen to protect against the deficit while the 1.8 mg/kg dose was not effective (nonpara- metric simultaneous test procedure). The results of the caroxazone experiments are seen in Table 5. aMT treatment alone significantly depressed rota- rod performance (p<.01) to about the same extent as was seen in previous experiments. Doses of caroxazone in the range of 8-64 mg/kg were active in preventing the impaired rotarod behavior caused by aMT injections. All doses of caroxazone alone were seen to have no effect on rotarod performances. Figure 17 displays log-spaced dose-response (LDR) curves for phenelzine and caroxazone effectiveness in preventing dMT-induced loss of rotarod performance. ReSponse is plotted in the traditional manner as the % of the subjects responding (remaining on the rotarod for 180 seconds). The LDR curves show the drugs to be approximately equipotent in preventing GMT-induced behavioral depression. The minimum effective 62 63 .AOHumHumumID mmspHSSIscszcHxOOHHS map mswms mump owuuofimummIsoc How muscmooum umwu muomsmuH93Hm upmummfioo mum muooflosm Umummuu 925 on» mHao «H Ho.vm new masono HHM How mo.vw umv msomHHmmEoo mHnHmmom HHm mcmeE mo muHsmmH map mzonm msfluoomnmpcou .Hooououm wsHummu cam usmfiumouu How axon man on momma .muomnn5m O Umswmucoo msoum comm .mcsoomm OOH on as commuou men so mpsoowm :H mafia came on» ma msHm> scams OH m.m O..H 0.0 H OH o.m O.H O.o Iamx\mfiv OGHNHmsmsm mo mmoa wmummuu OHOH£m>I825 u muomnndm ooummuu 925 Nmmuoom cmfipmz mdouw :H mmocwHOOMHa DeGOHMHcmHm h.vmwm.mmH m.HNHo.h¢H m.H~HO.HO >.Oum.Ov IHHOO m x mx\ma OOHV muomnnzm pmummuu Bad o.o Ho.OOH o.o Ho.OOH m.m~Hh.va h.mum.vhH +Aumum3_pmHHHumHo mx\HE OHV aboum Hoaucoo OHOHnm> 626 mx\me OH mx\me O.m mx\me O.H mx\mfi o.o IOGHNHOGOEQ mo mmon Homz O m.o ca ODMMHDm msHunswcm Homz O m.o usmfiumouumum ”A.2.mwmwv GOHmuOm no mwcoomm SH OEHH sum: mosmenomumm commuou so soflpmsHono :H can mson sm>flm azamxswmsHNHmcmnm mo muommmm one v mqmdfi 64 .HHO.VOV dump OHHumfimummso: How amuscmooum ummu msowcmuHDEHm an mcomHHmmEoo mHmHuHsz n .HGHE OOO u may COHHMGHEHHO Hmpuo HmHHm How OOHMH50HOO mw0© mosmcmucHMSH .z.m.m H mnsoomm OOH on as counpou map so mEHu same on» mH msHm> sommA Nm O O N O NO O v N O Amx\mfiv wcHNHmsmnm mo omoo pmpmouu OHOHnm> 92 u pwummuu BS mmmuoom QMHUOE mfloum sH mmosoummmHv DGMOHMHQOHO N. OHHm. NOH O 0.0 H0.00H O 0.0H O.vO O. O H0.00H O 0.0 H0.00H O Om.m O.Nm N. ONHO. HmH O 0.0 H0.00H O OO.v 0.0H O. O HN.th O 0.0 H0.00H O mm.N 0.0H v. O HO. HbH O 0.0.H0.00H O «O.N 0.0 H. ONHO. OOH O 0.0 H0.00H O hH.H O.¢ O. O HN. wNH O N.NHHO.hOH O mm.O O.N O.h H0.00 OH h.m H0.0>H OH 0.0 0.0 8 x mi? 83 92a c mHofiEoo 6829 925 : msoum usmfiummua monmsmusHmE HMHHHGH Amx\mEV wmoo muonxoumu ~A.z.m.m HO mucoomm cH oonmuou so mEHu sums H mocmEHomumm ponuon co :oHumsHono CH cam mcon sw>Hm cmn3 928 can msoumxoumo mo muommmm one m mqmdm. Figure 17. 65 LOg-Spaced dose-response (LDR) curves for phenelzine and caroxazone efficacy in pre- venting aMT—induced loss of rotarod perfor- mance in previously trained rats. Response is the % of the subjects showing complete prevention of aMT effects (i.e., score is equal to control training criteria of 180 seconds). Open-faced characters are for paired MT vehicle controls (i.e., subjects treated with either phenelzine, caroxazone, phenelzine vehicle or caroxazone vehicle and also with distilled.water, thecxMT ve- hicle). Caroxazone and phenelzine are seen to be approximately equipotent in this test. The caroxazone LDR on the left takes into consideration the apparently completely reversible nature of the central effects of caroxazone (for Ke=0'001925)' = mean of 180 seconds) Z RESPONSE (100 % 66 Figure 17 100- A 0 AA AOA+AA A \ /’ A ’ \ V / \ I \ / \ O A. A C! 75" Minimum effective dose I *p< .05 I **p< .01 50‘ *1: A 4- A D.W. 9I_I~I_T_ O . — Phenelzine 25- A ‘ — Caroxazone U I — Phenelzine Veh. V v — Caroxazone Veh. OJ I I I I I 1.0 3.2 10 32 100 MG/KG OF CAROXAZONE OR PHENELZINE (LOG-SPACED) VEHICLE (0.0) 67 doses (MED) are shown (*) to be 5.6 mg/kg and 8 mg/kg for phenelzine and caroxazone, respectively. The results of the previous study showed that caroxazone has anti-reserpine activity but that this activity is rapidly lost in a few hours as caroxazone blood levels fall. It is very likely that a similar situation would also exist for caroxazone's preventive effects on aMT-induced behavioral depression. The central actions of caroxazone are apparently completely reversible and thus the magnitude of a given drug effect is directly related to the time following administration of the drug. Phenelzine, on the other hand, exerts a stable level of "antidepressant" activity for many hours or even days after a single dose, presumable due to the irreversible nature of the MAO inhibition that outlasts the presence of the drug in the body. Thus, it was apprOpriate to calculate a "corrected" MED for caroxazone (for Ke=0.001925 min'l). The corrected MED for caroxazone is 5.7 mg/kg. SECTION II I Potentiation of L-DOPA-induced Behavioral Stimulation: Comparison of Phenelzine and Caroxazone SECTION III INTRODUCTION L-DOPA (3,4-dihydroxyphenylalanine) penetrades the blood brain barrier freely despite the large hydroqen bond- ing capacity of this substituted amino acid. This is ap- parently due to its high affinity for the large neutral amino acid tranSport system (Oldendorf, 1974). Various effects are seen when L-DOPA is administered systemically to laboratory animals, including behavioral and electro- physiolocical changes. L-DOPA is an intermediate in the formation of dOpamine (DA) and norepinephrine (NE) from phenylalanine (Blaschko g5 g1., 1937; Gurin and Delluve, 1947) and L-DOPA by itself is considered to have little di- rect biological activity (Carlsson g3 g1., 1957; Blaschko and Chrusciel, 1960; Smith and Dews, 1962). Since L-DOPA is the natural precursor of DA and NE, various effects seen following administration of L-DOPA have usually been attri- buted to enhanced activity of brain catecholamine pathways (Moore and Rech, 1967; Kadzielawa and.Widy-Tyszkiewicz, 1970; Everett, 1961; Carlsson g5 g1., 1957). Formation of excess amounts of DA and NE is apparently dose-dependent since the enzyme tyrosine hydroxylase is the rate-limiting step in the formation of NE and DA. The roles played in eliciting the various effects (behavioral and electrOphysiological 68 69 observations) by NE and DA formed from exogenously-admin- istered L-DOPA have been the subject of much debate (Chan and Webster, 1971; Ernst, 1969; Everett, 1968; Van Rossum gt 31., 1964; Rech g;_g1., 1968; Taylor and Snyder, 1970; Rech and Thut, 1976). Other investigators have indicated that release of S-hydroxytryptamine “34“” from central stores or the formation of active metabolites such as 3-O-methyl- dOpa may contribute as well (Ng ggwgl., 1970; Neuburg and Thut, 1974; Scheckel gt 31., 1969). The DOPA-potentiation test devised by Everett (1967) and Everett EE.§l° (1964) was originally intended to screen for non-MAOI antidepressant activity. Subjects were pre- treated with a MAOI (pargyline) and subsequently administered a large dose of L-DOPA along with the compound being evalu- ated and the combination was compared with MAOI and L-DOPA along. The ability for test drugs to elicit peripheral adrenergic signs (sympathetic stimulation: salivation, pilo- erection, etc.) and central adrenergic activity (irritabi-' lity, increased spontaneous motor activity, etc.) was the measure for antidepressant activity. This procedure was subsequently modified to screen for MAOI-type CNS activity (Rech and Thut, 1976; Thut and Rech, 1972). When relatively low doses of L-DOPA are combined with a MAOI (with peri- pheral decarboxylase activity having been inhibited by pre- treatment with a third drug),the resultant effect on animal behavior is a very dramatic dose-dependent (doses of the MAOI) stimulation of spontaneous motor activity (along with the other signs indicative of general stimulation of the 70 catecholaminergic systems in the CNS). The procedure em- ployed in this particular study is essentially the same as the previously reported, except that the test compound (phenelzine or caroxazone) was administered along with L-DOPA, instead of before L-DOPA treatment (Rech and Thut, 1976; Thut and Rech, 1972). The following study compares caroxazone with phenelzine for effectiveness and potency to potentiate the psychomotor stimulant and toxic effects of concomitant administration of L-DOPA and a peripheral de- carboxylase inhibitor. The results contained in this study have been submitted for journal publication (Vrbanac gE‘gl., 1978c). METHODS Subjects Female Sprague-Dawley rats, 200-250 g, and male Sprague-Dawley rats, 300-500 g (Spartan Farms, Haslett, Michigan) served as experimental subjects. Experiments measuring drug-induced alterations in spontaneous loco- motor activity used only females as subjects. Males were used in experiments comparing phenelzine and caroxazone for potency to elicit a lethal effect to the combination of 32 mg/kg L-DOPA and 75 mg/kg HMD (see drugs below). Care of subjects was described earlier (Methods portion of Section I). Drugs The following drugs were used in this study: phenel- zine (sulfate salt).L-dihydroxyphenylalanine (L-DOPA; Nutritional Biochemicals Corporation, Cleveland, Ohio), 8-)3,4-dihydroxyphenyl)-a-hydrazine-a-methyl-DL-proprionic acid (HMD; Merck, Sharp and Dohme Research Laboratories, West Point, Pa.), caroxazone (free base), desipramine (hydrochloride salt) and amitriptyline. Phenelzine was dissolved in 0.9 % NaCl vehicle. All other drugs were suspended in 0.5 % methyl cellulose. Drugs were always administered i.p. in a volume of 1.0 ml/kg (first injection) 71 72 and 2.0 ml/kg (second injection). Dosing and Testing Protocol All subjects were pretreated with HMD, 75 mg/kg, to block peripheral decarboxylation of L-DOPA (HMD inhibits the enzyme L-aromatic amino acid decarboxylase and does not pass the blood brain barrier to any significant degree at this dose). Thirty minutes following HMD treatment half of the subjects received L-DOPA (32 mg/kg) plus either phenelzine or caroxazone (drug group) and the other half received L-DOPA or L-DOPA vehicle thirty minutes following HMD pre- treatment. Each drug group (n=8) was paired with a control group (n=8) for all doses of phenelzine and caroxazone ad- ministered. One hour following the second injection indi- vidual subjects were placed in plastic animal cages (31 cm x 36.5 cm x 17 cm, 1 x w x b). Each cage was placed upon an electromagnetic motor activity apparatus (two Stoelting Electronic Activity Monitors). Counts were totaled for 15 minutes. Motor activity was also determined for lS-minute periods starting at two and three hours after the second in- jection. For each group of control subjects or drugged sub- jects half of the animals were recorded on each counter. Counts were always determined using the same counter at l, 2 and 3 hours for any individual. The two counters were previously calibrated to give approximately the same number of counts for any given level of animal activity. Pairing of control subjects with drug subjects eliminated day-to-day variability as a factor since only results obtained on any 73 particular day were compared statistically. Testing for motor activity occurred only between 1300 and 1700 hours. Each subject was used only once. Doses of phenelzine used were 1.8, 3.2, 5.6, 10 and 18 mg/kg; caroxazone doses were 10, 18, 32, 56 and 100 mg/kg. Statistical Analysis The Mann-Whitney U test for differences between inde— pendent samples and the Wilcoxin ranked-sums test were used to determine significant differences (p<.05) between control and drug group median scores (both tests gave identical re- sults). Comparisons between control (n=8) and drug groups (n=8) were made for each of the three sets of data (at l, 2 and 3 hours) and for the three periods as accumulative motor ac tivi ty counts . RESULTS The results of the motor activity experiments are seen in Figures 18 and 19. The control data comparing HMD plus L-DOPA with HMD plus vehicle is not shown but was found to be not significantly different or show any trend toward a difference (p<.10). Figure 18 shows the one, two and three hour counts obtained after various doses of phenelzine were combined with 32 mg/kg of L-DOPA, with peripheral de- carboxylase activity inhibited by HMD (n=8). The control mean (0.0 dose) was calculated using control group counts for all 5 doses of phenelzine (i.e., n=40). Significantly different drug and control group medians occurred at doses of phenelzine of 3.2 mg/kg or greater. The 3.2 mg/kg data was not quite significant at 3 hours and just significant at l and 2 hours (.025 p<.05). The 5.6 mg/kg dose clearly potentiated L-DOPA, the effect being quite dramatic (p<.01). The lowest dose, 1.8 mg/kg, did not significantly increase the median motor activity compared to vehicle control. The 1.8 mg/kg dose produced a slight but not significant inter- action (.05 p<.10). Thus, 3.2 mg/kg is a threshold or mini- mally effective dose of phenelzine in-this particular expe- rimental situation. The maximally effective dose was clear- ly 10 mg/kg. Phenelzine at 18 mg/kg produced serious toxi- city. Two subjects died before the one hour reading and had 74 Figure 18. 75 Phenelzine interaction with L-DOPA following HMD pretreatment. Animals were treated with 75 mg/kg HMD and 30 minutes later with 32 mg/kg of L-DOPA and 0.0, 1.8, 3.2, 5.6, 10 or 18 mg/kg of phenelzine. The average counts for 8 subjects is shown for each dose at l,:2 and 3 hours after the last injection. The control average counts displayed are for all 40 control subjects. Each subject trea- ted with phenelzine was paired with one con- trol subject. Statistical significance was determined by the Mann-Whitney U test. A significant difference occurred at doses marked with an asterisk. 76 «no: m «so: N moo: H OH 3 O5. ~.mm.H ca 3 3 8m ~.m méoé 3 S O...“ «:24 o.o Omen O HO IHIIHI |I_.l I OOO I H .1 H H. .. I OOOH F1. 5:25sz EOOIITOENOOZOE .. IO OH musmHm. SanOD EinNIN SI SOVHBAV Figure 19. 77 Caroxazone interaction with L-DOPA follow- ing HMD pretreatment. Animals were treated with 75 mg/kg HMD and 30 minutes later with 32 mg/kg L-DOPA and 0.0, 10, 18, 32, 56 or 100 mg/kg of caroxazone. The average counts for 8 subjects is shown for each dose as measured at l, 2 and 3 hours after the last injection. The control average counts displayed are for all 40 control subjects. Each subject treated with caroxazone was paired with one control subject. Statisti- cal significance was determined by the Mann- Whitney U test as described in the Methods section (p<405). Significant differences occured at doses indicated (*). 78 «no: m «no: N «no: H OOH Om Nm OH OH 0.0 OOH Om Nm OH OH. 0.0 OOH Om Nm OH OH. 0.0 Human o IH. H. I HL _ IHL I 8N JL HI IAIQI .. 7 I 8: Lr * t J 22541233IEOOLIOZONSSHS LI I OOO Ir OH wusmHm * I SiNflOD SinNIN SI BOVHBAV 79 to be replaced. The other six subjects all lived but showed an overt behavior that was not consistent with the behavior seen with doses of 5.6 and 10 mg/kg. Instead of showing hyperreactivity to their environment, most of these subjects were relatively unresponsive to all types of environmental stimuli. Behavior was similar to the extreme stimulation seen shortly after a very high (lethal) dose of gfampheta- nune in rats. The caroxazone-L-DOPA interaction data is seen in Figure 19. The one obvious difference is an approximate lO-fold shift in the threshold dose needed for a significant, or almost significant increase in motor activity as compared to the dose-response pattern with phenelzine. Figure 20 plots the percent increase in accumulative counts (counts for l, 2 and 3 hour periods are summed) above control as a function of log dose. The potency to produce an effect is not the only difference noted. The maximal effects (maximal stimulation of motor activity counts) were also quite diffe- rent. Caroxazone maximal effect was only half that of the phenelzine response. Caroxazone by itself did not decrease spontaneous motor activity over controls at doses of 56 and 100 mg/kg in control. The results of the L-DOPA-interaction experiment were unexpected in the sense that 100 mg/kg of caroxazone did not appear to elicit a toxic interaction in this particular experimental situation, while 10 mg/kg of phenelzine appeared to be toxic and 18 mg/kg was lethal in 2 of 8 subjects tested. The dose-response curves for both phenelzine and caroxazone were extended to lethal effects. Figure 20. 80 Accumulative motor activity counts. Total counts over the three periods is expressed as % of paired control group mean accumu- lative counts and expressed as log-spaced dose—response.curves for phenelzine and caroxazone treatments (n=8). Threshold doses for significant increases in accumu- lative counts were 1.8 mg/kg of phenelzine and 18 mg/kg of caroxazone. The maximal effect seen with phenelzine is two times greater than the maximal effect seen with caroxazone. Z OF CONTROL ACCUMULATIVE MOTOR ACTIVITY COUNTS J: O O 1 300 - 200 - 100 1 81 Figure 20 O —PHENELZINE -CAROXAZONE * - P<.05 n - p<.01 “ .** O A\A Q I o A\A . ./ ./ 1,0 118 3:2 516 1b 18 3'2 5'6 1b0 LOG-SPACED DOSE (MG/KG) 82 TABLE 6 Caroxazone vs. phenelzine in potency to impart a lethal effect to the combination of 32 mg/kg L-DOPA and 75 mg/kg HMD Number of Subjects Dead (24 hours) Test Drug Dose (mg/kg) Phenelzine Caroxazone Female Male Female Male 10 0/8 --- 0/8 --- 18 2/8 --- 0/8 --- 32 --- 0/8 0/8 --- 56 ~-- 3/8 0/8 --- 100 --- 8/8 0/8 --- 180 --- 4/4 --- --- 320 --- --- 0/8 0/8 560 --- --- 2/4 0/8 1 2 MLD 18 56 560 --- 1 Minimum lethal dose observed for male and female subjects. 2The i.p. LD5 for caroxazone in rats has been re- ported to be 1532 mg/kg (R.A. Carrano, personal communication). 83 Because of limited supplies of caroxazone it was only possible to calculate a minimum lethal dose (MLD) using 8 subjects per dose. An LDlOO was estimated for phenel- zine. The results are seen in Table 6. Experimental pro- tocol is unchanged from before. The obvious conclusion is that the phenelzine-L-DOPA interaction is dramatically more toxic than the caroxazone-L-DOPA interaction, at least over a 24-hour time span. DISCUSSION Spontaneous motor activity peak effects for caroxazone were found to be only half that for phenelzine. Doses of caroxazone greater than 100 mg/kg were not valid to include since depression of spontaneous motor activity was seen for caroxazone treated subjects at a dose of 180 mg/kg (4 sub- jects). The resultant behavioral effects of higher doses of caroxazone in combination with L-DOPA are therefore complex and would complicate the comparison with phenelzine data or with the lower doses of caroxazone. The quantitative diffe- rences between caroxazone and phenelzine measured in the mo- tor activity experiments (potency and maximal effects) were no more dramatic than the qualitative differences observed in animal behavior and general appearance. These differences were so clear that caroxazone-DOPA and phenelzine-DOPA teated subjects were easily distinguished from one another on casual observation. The caroxazone combination resulted in far less overt hyperreactivity to environmental stimuli (such as hand clapping and handling), fewer and less pro- nounced signs of sympathetic stimulation and less fighting behavior. For example, subjects treated.with 3.2 and 5.6 mg/kg of phenelzine subjectively appeared to show a more dramatic interaction with L-DOPA than subjects treated with 56 and 100 mg/kg caroxazone and L-DOPA. The relative 84 85 mildness of effects of the caroxazone-DOPA combination come pared to the phenelzine-DOPA combination, even for the higher doses of caroxazone, is reflected in the MLD's of the two treatments (18 mg/kg vs. 560 mg/kg, for females). These findings add to the other evidence indicating basic pharma— codynamic differences between caroxazone and classical MAOI. Doses of caroxazone from 100 to 560 mg/kg should have been far more toxic than they were if caroxazone exerts central effects very similar to MAOI (i.e., inhibition of MAO acti- vity iglyiyg towards biologically active amines derived from L-DOPA). Likewise, the signs of CNS stimulation at the lower doses should have resembled more the phenelzine- DOPA treated subjects. The earlier data is worth discussing from another per- spective. Reserpine exerts central depressant effects long after most of the administered dose has left the body. Per- sumably this action is due to depletion of 5-HT, NE and DA from nerve terminals by interference with processes necessary for neurotransmitter uptake and storage in synaptic vesicles. Exactly how this is accomplished is not known. However, it is reasonable to assume that this process involves binding of reserpine to receptors on or inside synaptic vesicles. The data obtained for phenelzine and isocarboxazid at 24 hours (Section I) indicates that Ka>>Kd for this binding process and that the residual reserpine that remains after the bulk of the administered dose has been removed from the body is still biologically active. This has been suggested to be the case in the peripheral nervous system (Alpers and 85a Shore, 1967). Since the half-life for recovery of MAO activity following MAOI administration is in the order of many days, the loss of preventative effects at 24 hours in- dicates that some pharmacological action of the MAOI present at 10 hours has been lost, or diminished considerably, and that the loss of this drug action allows the residual re- serpine to impair neuronal function, even though NE, DA and 5-HT stores have been protected. The existence of a small, functionally important, rapidly-recovering pool of MAO would fit into this hypothetical scenario. The presence of such a pool could also explain the lack of significant MAO inhi- bition (as measured lg giggg) by caroxazone at lower anti- reserpine doses coupled.with a spectrum of "antidepressant" activity that suggests a close relationship to the MAOI in bioassay procedures, since with this model only a small selective portion of central MAO activity need be inhibited for the expression of antidepressant effects. This latter pr0posal may also explain inconsistencies in the duration of functional effects of MAOI as compared with their duration of enzyme inhibition (Waldmeier and Maitre, 1976; Maitre and Waldmeier, 1976). These investigators showed that L-DOPA potentiation by MAOI, as well as the accumulation of dOpamine and 3-methoxytyramine, was short-lived, less than 24 hours in half-life even with large doses of MAOI. Smaller doses of MAOI were observed to inhibit the enzyme (igflyiggg measure- ment) with a half-life of several days to over a week, in addition to provoking changes in homovanillic acid and 86 3,4-dihydroxyphenylacetic acid levels in striatum.over this same time period. Although the above speculations may seem attractive, it.must be admitted that the relationship of the antidepres- sent activity of the MAOI to inhibition of this enzyme is still very tenuous. SECTION IV Comparison of Phenelzine, Desipramine, Amitriptyline and Caroxazone as Inhibitors of Cerebral Monoamine Oxidase: £2 Vitro Studies SECTION IV INTRODUCTION The analytical techniques of gas-liquid chromatography (GC) and gas-liquid chromatography-mass spectrometry (GC/MS) mass-fragmentography have recently been very successfully applied to the qualitative and quantitative analysis of biogenic amine neurotransmitters and their metabolites. Most techniques develOped from volatile electrOphilic de- rivatives of these compounds. These electrophilic deriva- tives can be detected in picogram quantities by GC using electron capture detection (ECD). The GC/MS mass-fragmento- graphy technique also involves the formation of a stable volatile derivative of the compound under analysis as well as a known amount of a deuteriumrlabeled internal standard. The relative intersities of apprOpriate mass fragments for the compound being analyzed and its deuterated derivative can be used for quantitation. A very efficient analytical approach that combines the use of GC and GC/MS systems in- volves the use of the GC/MS system to positively identify retention time values of electrOphilic derivatives of the compound in question and apprOpriate internal standards of chemical similarity, and then the use of GC/ECD in routine quantitative analysis of biological samples. Various GC and GC/MS techniques for the qualitative and quantitative 87 88 detection of homovanillic acid (HVA, 3-methoxy-4-hydroxy- phenylacetic acid) (Sjoquist gt gt., 1973; Dziedzic and Git- low, 1973; Gordon gt gt., 1974; Sjoquist and Anggard, 1972), 3-methoxy-4-hydroxyphenethy1ene glycol (MHPG) (Dziedzic and Gitlow, 1973; Dekirmenjian and Maas, 1974), 3,4-dihydroxy- phenylacetic acid (DOPAC) (Pearson and Sharman, 1974), normetanephrine (NM) (Narasimhachari, 1974; Lhuguenot and Maume, 1974; Abramson, 1974; Capella and Horning, 1966), vanillyl mandelic acid (VMA) (Gordon gt gl., 1974; Nara- simhachari, 1974; Watson and Wilk, 1974), dopamine (Lhuguenot and Maume, 1974; Abramson, 1974; Capella and Horning, 1966; Kilts gt gt., 1976; Koslow gt gt., 1972), norepinephrine (Abramson, 1974; Capella and Horning, 1966; Koslow gt gt., 1972; Lhuguenot and Maume, 1974), S-Hydroxyindole acetic acid (S-HIAA) (Watson and Wilk, 1974; Bertilsson gt gt., 1972), serotonin (5-HT) (Capella and Horning, 1966; Abramson, 1974; Cattabeni gt gt., 1972) and 3-methoxytyramine (3-methyxy-4- hydroxyphenyl ethylamine) (Capella and Horning, 1966; Kilts gt gt., 1976; Abramson, 1974) have been reported in the lite- rature. This section describes experiments designed to assess some it ytttg neurochemical effects of caroxazone using GC/MS Mass-fragmentoqraphy. Caroxazone is compared with phenelzine, amitriptyline and desipramine for effectiveness to inhibit rat brain MAO i2.!i££2l using 2,5,6-trideuterod0pamine as substrate. METHODS MAO activity was determined by a slight variation of published procedures (Wurtman and Axelrod, 1964; Glowinski gt 31., 1966). Female Sprague-Dawley rats were decapitated and the brains removed. Forebrain was homogenized in 8 ml of ice-cold isotonic saline. To 1.0 ml of the homogenate were added 0.6 ml of pH 7.3 phosphate buffer and 8.4 ml of distilled water (D.W.) or D.W. containing various concen- trations of caroxazone, desipramine, amitriptyline, or phenelzine (all salts were normalized as moles of free base). All tissues and drug solutions were fresh for each deter- mination. The incubation medium was allowed to stabilize at 37°C for 15 minutes, with shaking, and then 100 pg of 3,4-dihydroxy-2,5,6-trideuter0phenylethylamine hydrochloride (d3-DA) was added as substrate. d3-DA was prepared by acid exchange of the aromatic hydrogens in deuterium oxide acidi- fied with deuterium chloride (Lindstroem gt g1., 1974; dopamine HCl was purchased from Aldrich Chemical Co., Mil- waukee, Wisconsin). The chemical and isotopic purity of the d3-DA used in this study was greater than 99 % (determined by GC/MS) . One hundred pl aliquots were placed in one dram vials at 0, 15 and 30 minutes following the addition of d3-DA. Kinetic parameters for this particular experimental situation 89 90 were studied in an earlier experiment. Samples were taken every 5 minutes in the initial experiments (n=3) and every 15 minutes later on (n=5). The decline in substrate was linear up to 45 minutes. Linear regression analysis using 0-, 15-, 30- and 45-minute data points showed a significant correlation coefficient (p<.01). The correlation coeffi- cient for regressionanalysis of the 0-, 15- and 30-minute data points was highly significant (p<.001). In all sub- sequent tp‘yitgg determinations of MAO activity samples were collected only at 0, 15 and 30 minutes. Enzyme acti- vity was stopped by addition of 0.3 ml of methanol and 0.2 ml of 0.1 N HCl to each 100 p1 sample of the incubation medium. Samples were heated in a water bath (60°C) under a stream of nitrogen until completely dry (10 to 20 minutes). To each sample, 30 p1 ethylacetate and 20 pl pentafluorOpro- pionic anhydride (PFPA) were added. Samples were dessicated and placed in a refrigerator until analysis could be per- formed. Standard curves were prepared each day by adding a fixed amount of dopamine HCl (264 pmol) and varying amounts of d3-DA (52 to 1299 pmol). Each standard curve consisted of at least 7 points along with one blank (internal standard alone). The protein concentration in each incubation medium was determined by the method of Lowry gt gt. (1951). Enzyme activity was expressed as nanomoles d3-DA metabolized/hour/ mg protein and is shown in the test as a percentage of the total MAO activity inhibited. Control reaction velocity de- termined under these conditions was shown in an earlier ex- periment to be a close approximation of Vmax (determined by 91 Lineweaver-Burke plot of first—order decay data). The re- action velocity for 100 % inhibition was arbitrarily set as the rate of decay seen when 2 x 10-3 M phenelzine was pre- sent in the incubation medium. Quantitation of DA in the samples was performed by multiple ion detection (MID, mass-fragmentography) using a Finnigan 3200E gas chromatography/mass spectrometry system interphased with a System Industries 150 data analysis unit. A 1.6 m x 2 mm (i.d.) silanized glass column packed with 3 % SP-2250 on 80.100 Supelc0port was used for separation. From 0.1 to 0.5 pl of each sample was injected into the system. Injection port temperature was 250°C and the column temperature was 155°C. ‘The He carrier gas flow rate was 10 ml/min. Fragmentation was accomplished by electron im- pact at 70 eV and 0.5 mA. Ion pairs selected for monitoring of DA were m/e+=431/428,284/281 and 268/265. This analyti- cal technique has been described in detail previously along with analysis of the fragmentation pattern of the penta- fluoroprOpionic anhydride derivative of DA by electron im- pact at 70 eV (Kilts gt gt., 1976). If the reader examines this reference the following corrections should be noted. With respect to the fragmentation pattern of the pentafluoro- prOpionic anhydride derivative of 3-methoxytyramine, it was stated that m/e=296 results from loss of OCOC2F5 from the 4 position on the aromatic ring. The m/e=296 results primarily from cleavage between the a-carbon and the nitrogen. Also, for both DA and 3-MT, the a-carbon-nitrogen cleavage involves loss of one hydrogen from the a-carbon to the leaving group. Figure 21. 92 Electron impact mass Spectra of 3-methoxy- tyramine, 3-methoxytyramine-d3, dOpamine and depamine-d3 pentafluorOprOprionyl de- rivatives. See test for instrument condi- tions. 93 0): 330 330 Doc Jen SON 3. r P D I! ? r d} T E o A _ H H lflN a 3. I... S» ..... a «Uh A a .on W C 1 o . m . m P?» w @339 [M a. a 2 .0 Saw .5. Ho 9.6250 min 3 8.6390 02 0): 8O 08 000 OOn I CON I coho I _ I 2 H A 3...: .8...- H. n... w M a 100 M a J I 0 O O O p.) O‘BI\m'\hu\~tobobn\u (M o a, I .. 3. ca“ 2: NI. 9262.03 main .5 no 695833 .33. OxE cow - own I 8.. I con 3.“ 3. “$7. 0! fl 1. cl \. IS a an mm A a on H g a. c a t «ASN.3»; ntuNAv um. . .N o A O. D I... “R g a 3:: OZZOECvZ «Cl ma 4.2 P5 m. ACO— o): 80 80 00v 00¢ 8N 8.0 .3... _ I. I... _ nu Hu 3 m M a 8 m mo. U K. emu wxsfu «.6 O oufu K o N.” i 2.. 3,... 2.52.8 «Eu 3 EA 2. Hm mpsmHm Figure 22. 94 Electron impact mass spectra of a mixture of dopamine and d -d0pamine, pentafluoro— prOpionic anhydri e derivative. Ordinate plots relative intensity and the abscissa plots m/e+. Scan range covers m/e+ = 250 to m/e+ = 450. 95 omv Dov 0mm OOM omN HOO\ONO \ 1.. __ Ox II HO SO33 omov HMSHOH ESEHSHZ .v O0.0H O0.0H Oh.mH OO.NH .HHO.vm umv sz SSS «SOQIH SSH: SoHHmSHn IEoo SH Sm>HO Sosz >HH>Huom HOHQE msomcmu Icomm SH ommmHOSH HSSOHHHSmHm m OSHHHOHHO wmop umoSOH ”momop m>Huommmm uSmHm>HSOm .m mm.o mm.o om.H mm.H .AHo.vS HMO uommmm m>Humuem> Imum HSMOHHHSOHm m OSHuHoHHm mmow umm30H "ODSmEHomHmm ponmuou mo SOHmmoummp 92 Ho S0HHSO>OHQ How mOOOU uSmHm>Hsvm .N hm.o m0.0 O0.0 OO.H .HHO.vm HMO uomwmm m>HumuSo> Imum HSSOHHHSmHm m OSHHHOHHO OOOU ummsoH “SUSSEHOHHOQ commuon mo SonmmHmmp OSHQ Iummmu mo SoHuSm>mHm How mmmop uSmHm>HSUm .H N mx\UmE mx\ma NOH\OOE mx\me mmoc cmuomuuoo mmow pmuomHHoOSD S OSHNHOSOSS oH mu mmo u .u p mSonxoumu mSOHumsuHm ummu OHHHommm SH mSHNHmSmSm pSm wSonxonmo How oHHMH NOSmuom m ”MAM/NB 121 about the normal function of these enzyme systems, we are not likely to have a clear understanding of drugs that alter their activity. The results of this study indicate that caroxazone differs at least to some extent in interacting with brain monoamine mechanisms from the TCA and the MAOI in rats. Probably the most significant finding of these studies was the relatively low toxicity of caroxazone in the L-DOPA potentiation experiments since this has direct cli- nical relevance. As a last thought, it should be kept in mind that it is always risky to extrapolate from animals to man in drug research. Because of the extreme complexity of the mamma- lian CNS and the uniqueness of the human experience, such extrapolations are often of questionable value in the disci- pline of psychOpharmacology. Therefore, clinical compari- sons of caroxazone with the traditional MAOI will be re- quired before very much more can be said about the psycho— dynamic effects of this new drug. BIBLIOGRAPHY BIBLIOGRAPHY ABRAMSON, F.P., MCCAMAM, M.W. AND MCCAMAN, R.E.: Femto- mole analysis of biogenic amines and aminoacids using functional group mass Spectrometry. Anal. Biochem. 51; 482-499, 1974. ALPERS, H.S. AND HIMWICH, H.E.: The effects of chronic imipramine administration on rat brain levels of serotonin, S-hydroxyindoleacetic acid, norepinephrine and dopamine. II.Pharmacol. Exp. Ther. ttg: 531-538, 1972. ALPERS, H.S3 AND SHORE, P.A.: Persistent binding of reser- pine-H in heart: Association with norepinephrine depletion. Pharmacologist g: 183, 1967. ALPER, R.H., VRBANAC, J.J., KILTS, C.D. AND RECH R.H.: Comparison of phenelzine and caroxazone as poten- tiators of L-DOPA. Fed. Proc. 31: 773, 1978. ASKEW, B.M.: A simple screening procedure or imipramine- like antidepressant agents. Life Sci. 3: 725-730, 1963 AXELROD, J.: Metabolism of epinephrine and other sympa- thomimetics. Physiol. Rev. 32; 751-776, 1963. AXELROD,CL,'WHITBY, L.G. AND HERTTING, 6.: Effects Of psychoactive drugs on the uptake of H -norepinephrine by tissue. Science 133: 383-384, 1961. BARNES, C.D. AND ELTHERINGTON, L.G.: "Drug Dosage In Lab- oratory Animals: A Handbook". University of Calif- ornia Press, Berkely and Los Angeles, Calif., 1965. BERTILSSON, L., ATKINSON, A.J., ALTHANUS, J.R., HARFAST, A., LINGREN, J.E. AND HOLMSTEAD, B.: Quantitative deter- mination of 5-hydroxy-3-indoleacetic acid in cerebro- Spinal fluid by gas chromatography-mass stectrometry. Anal. Chem. 43; 1434-1438, 1972. BIELSKI, R.J. AND FRIEDEL, R.O.: Prediction of tricyclic antidepressant responce. Arch. Gen. Psychiat. 3;: 1479-1489, 1976. 122 123 BLASCHKO,H. AND CHRUSCHIEL, T.L. : The decarboxylation of amino acids related to tyrosine and their awaken- ing action in reserpine-treated mice. J. Physiol. (Lond.) lit: 272-284, 1960. BLASCHKO, H., TICHTER, D. AND SCHLOSSMONN, H.: The oxi- dation of adrenaline and other amines. Biochem. J. 3;: 2187-2196, 1937. BLASCHKO, H.: The Specific action of L-DOPA decarboxylase. J. Physiol. (Lond.) gg: 50P-51P, 1939. BLATCHFORD, D.:, HOLZBUER, M. AND YOUDIM, M.B.H.: Sub- strate and strain dependent differences in the devel- opment of monoamine oxidase in the rat brain. Br. J. Pharmacol. g3: 251-252, 1975. BOPP, B. AND BIEL, J.H.: Antidepressant drugs. Life Sci. 415-423, 19740 BRAESTRUP, C., ANDERSENT, H. AND RANDRUP, A.: The mono- amine oxidase B inhibitor deprenyl potentiates phenyl- ethylamine behavior in rats without inhibition of catecholamine metabolite formation. Eur. J. Pharmacol. 33: 181-187, 1975. CAPELLA, P. AND HORNING, E.C.: Seperation and identifica- tion of biogenic amines by gas-liquid chromatography. Anal. Chem. gt: 316-321, 1966. CARLSSON, A., CORRODI, H., FUXE, K. AND HOKFELT, T.: Effect of antidepressant drugs on the depletion of intra- neuronal 5-hydroxytryptamine stores caused by 4-methyl- a-ethyl-meta—tyramine. Eur. J. Pharmacol. _5_: 357-366, l969a. CARLSSON, A., CORRODI, H., FUXE, K. AND HOKFELT, T.: Effect of some antidepressant drugs on the depletion of intra- neuronal brain catecholamine stores caused by 44,adi- methylmetatyramine. Eur. J. Pharmacol. _5_: 367-373, l969b. CARLSSON, Ab: Pharmacology of synaptic monoamine trans- mission. Prog. Brain Res. 2;: 53-59, 1969. CARLSSON, A: Effects of centrally acting drugs on catechol- amine biosynthesis. Neurosci. Res. Progr. Bull. 5: 59- 63, 1967. CARLSSON, A., FUXE, K. AND UNGERSTEDT, V.J.: The effect of imipramine on 5-hydroxytryptamine neurons. J. Pharm. Pharmacol. 29: 150-152, 1968. 124 CARLSSON, A., LINDQUIST, M. AND MAGNUSSON, T.: 3,4-di- hydroxyphenylalanine and S-hydroxytryptamine as reserpine antaginists. Nature (Lond.) 180: p1200, 1957a. CARLSSON, A., ROSERGREN, E., BERTLER, A. AND NELSSON, J.: Effect of reserpine on the metabolism of catechol- amines, tg: "PsychOtropic Drugs", Garattini, S. and Gheti, V., Eds., Elsevier, Amsterdam, 366-372, 1957b. CARLTON, P.L.: Potentiationcfifthe behavioral effects of amphetamine by imipramine. Psychopharmacologia t: 364-376, 1961. ICARRANO, R.A.: "Confidential Background Document: Carox- azone Investigational Use Only", Department of Pharma- cology/Toxicology, Adria Laboratories, Wilmington, Deleware, May 1976. CATABENI, F., KOLSOW, S.H. AND COSTA, E.: Gas chromato- graphic-mass spectrometric assay of four indole- alkylamines of rat pineal. Science 178: 166-168, 1972. CHAN, O.L. AND WEBSTER, R.A.: Importance of noradrenaline found in a functional pool in maintaining Spontaneous locomotor activity. Br. J. Pharmacol. it: 700-708, 1971. CHRISTMAS, A.J., COULSON, C.J., MAXWELL, D.R. AND RIDDELL , D.: A comparison of the pharmacological and biochem- ical properiies of substrate-selective monoamine oxi- dase inhibitors. Br. J. Pharmacol. 52: 490-503, 1972. COLLINS G.G.S., SANDLER, M.,.WILLIAMS, E.D. AND YOUDIM, M.B.H.: Multiple forms of human brain mitochondrial monoamine oxidase. Nature (Lond.) 225: 817-820, 1970. CORRODI, H. AND FUXE, K.: The effect of catecholamine pre- curssonr and monoamine oxidase inhibiton on the amine levels of central catecholamine neurons after reser- pine treatment or tyrosine hydroxylase inhibition. Life Sci. g: 1345-1350, 1967. 1This document contains selected internal research reports compiled and translated into English by Farmatilia, Milano, Italy, for use by Adria Laboratories and a general overview of preclinical studies to date. These reports represent those studies investigation the "anti- depressant" properties of caroxazone relative to tricyclic antide- pressants and monoamine oxidase inhibitors. Current address of Adria Laboratories is Columbus, Ohio. 125 CORRODI, H. AND FUXE, K.: The effect of imipramine on central monoamine neurons. CL Pharm. Pharmacol. g9: 230-231, 1968. CORRODI, H. AND FUXE, K.: Decreased turnover in central 5-HT nerve terminals induced by antidepressant drugs of the imipramine type. Eur. J. Pharmacol. 1: 56-59, 1969. CORRODI, H., FUXE, K. AND HOEKFELT, T.: The effect of some psychoactive drugs on central monoamine neurons. Eur. J. Pharmacol. t: 363-368, 1967. CORRODI, H. AND HANSSON, L.C.F.: Central effects of an inhibitor of tyrosine hydroxylation. Psychophar- macologia t9: 116-125, 1966. CREVELING, C.R., DALY, J., TOKUYAMA, J. AND WITKOP, B.: The combined use of a-methyltyrosine and threo-di- hydroxyphenylserine: Selective reduction of dOpamine levels in the central nervous system. Biochem. Pharmacol. l1: 65-70, 1968. DAVIS, J.M.: Theories of the biological etiology of af- fective disorders. Int. Rev. Neurobiol. t3: 145-155, 1970. DAVES, J.M.: The efficacy of the tranquilizing and anti- depressant drugs. Arch gen. Psychial. it: 552-572, 1965. DEKIRMERJAIN, H. AND MAAS, J.: 3-Methoxy-4-hydroxyphen- ethyleneglycol in plasma. Clin. Chim. Acta gg: 203- 210, 1974. DELAY, J. AND DENIKER, P.: Trente-huit cas de psychosis traitees par la cure prolongee et continue de 4560 RF. Le congres des A1. at Neurol. de Langue Fr. 22‘ "Compte rendu Congres." Masson et Cie, Paris, 1952. DUBINSKY, B., KARPOWICZ, J.K. AND GOLDBERG, M.E.: Effects of tricyclic antidepressants on attack elicited by hypothalamic stimulation: Relation to brain biogenic amines. J. Pharmacol. Exp. Ther. ttl: 550-557, 1973. DZIEDIZIC, S.W. AND GITLOW, S.E.: Cerebrospinal fluid, homovanillic acid and isohomovanillic acid. J. Neuro- chem. 3g, 333-335, 1974. EIDUSON, S.: Ontogenic development of monoamine oxidase, £2? "Advances in Biochemical PsychOpharmacology", Costa, E. and Sandler, M., Eds., Raven Press, New York, 271-287, 1972. 126 ERNST, A.: The role of biogenic amines in the extrapyra- midal system. Acta Physiol. Pharmacol. Neerl. t5: 141-154, 1969. EVERETT, G.M.: Some electrOphysiological and biochemical correlates of motor activity and aggressive behavior, In: "Neuro-Psycho-Pharmacology", VOlume 2, Rothlin, E., Ed., Elsevier, New York, p. 479-484, 1961. EVERETT, G.M.: The DOPA response potentiation test and its use in screening for antidepressant drugs, £3: "Antidepressant Drugs", Garattini, S. and Dukes, M.N.G., Eds., Excerpta Medica Foundation, Amsterdam, p. 164, 1967. EVERETT, G.M.: Role of dopamine in aggressive and motor responses in male mice. Pharmacologist lg: 181, 1968. EVERETT, G.MI.AND BORSCHERDING, J.W.: L-DOPA: Effect on concentrations of dopamine, norepinephrine, and sero- tonin in the brains of mice. Science 168: 849- 850, 1970. EVERETT, G.M., DAVIN, J.C. AND TOMAN, J.E.P.: Pharmacolo- gical studies of monoamine oxidase inhibitors. Fed. Proc. it: 388, 1959. FUENTES, J.A. AND NEFF, N.H.: Selective monoamine oxidase inhibitor drugs as aids in evaluating the role of type A and B enzymes. Neuropharmacol. t5: 819-825, 1975. FULLER, RJW.: Selective inhibition of monoamine oxidase. Adv. Biochem. PsychOpharm. 5: 339-354, 1972. FUXE, K. AND UNGERSTEDT, U.: Histochemical studies on the effect of (+)amphetamine, drugs of the imipramine group and tryptamine on central catecholamine and S-hydroxytryptamine neurons after intraventricular injeciton of catecholamines and 5-hydroxytryptamine. Eur. J. Pharmacol. 3: 235-244, 1968. GELPI, E., PERALTA, E. AND SEGURA, J.: Gas chromatography- mass spectrometry of catecholamines and tryptamines: Determination of gas chromatographic profiles of the amines, their precursors and their metabolites. J. Chromatogr. Sci. lg: 701-709, 1974. GLOWINSKI, J. AND AXELROD, J.: Effects Of drugs on the disposition of 3H-norepinephrine in the rat brain. Pharmacol. Rev. Lg: 775-778, 1966. 127 GLOWINSKI, J., AXELROD, J. AND IVERSEN, L.L.: Regional studies of catecholamine in the rat brain, 12‘ "Effects of Drugs on the Disposition of and Metabolism of 3H-norepinephrine and 3H-d0pamine". J. Pharmacol. Exp. Ther. igg: 30-41, 1966. GOLDSTEIN, A., ARONOW, L. AND KALMAN, S.M.: "Principles of Drug Action", Harper and Row, New York, pp. 280- 342, 1969. GOODMAN, L.S. AND GILMAN, A.: "The Pharmacological Basis of Therapeutics", MacMillan Co., New York. 1975. GORDON, E.K., OLIVER, J., BLACK, K. AND KOPIN, I.: Effect of probenicid on free 3-methoxy-4-hydroxyphenylethyl- ene glycol (MHPG) and its sulfate in human cerebro- spinal fluid. Biochemical Medicine it: 32-—40 , 1974. GRAHAME-SMITH, D.G. AND GREEN, A.R.: The role of brain dOpamine in the hyperactivity syndrome produced in rats after administration of L-tryptophan and a monoamine oxidase inhibitor. Proc. Brit. Pharm. Soc. 2nd-4th Jan., 442-443, 1974. GURIN, S. AND DELLUVA, A.M.: The biochemical synthesis of radioactive adrenaline from phenylalanine. J. Biol. Chem. 170: 545-550, 1947. HILL, R.T., KOOSIS, I., MINOR, M.W. AND SIGG, E.B.: Potentiation of methylphenidate by imipramine, ami- triptyline and their desmethyl analogues. Pharma- coloqist g: 75, 1961. HILL, R.T. AND TEDESCHI, D.H.: Animal testing and screen- ing procedures in evaluating psychotrOpic drugs, £2: "An Introduction of PsychOpharmacology", Rech, R.H. and Moore, K.E., Eds., Raven Press, New York, pp. 237- 288, 1971. HONIGFELD, G. AND HOWARD, A.: "Psychiatric drugs: A desk referencel' Academic Press, New York, 1973. HOOK J.B. AND MOORE, K.E.: The renal handling of a-methyl- tyrosine. J. Pharmacol. Exp. Ther. 168: 310-314, 1969. HOUSLAY, M.D., TIPTON, K.F. AND YOUDIM, M.B.H.: Multiple forms of monoamine oxidase: Fact and artifact. Life Sci. t2: 467-478, 1976. IVERSEN, S.D. AND IVERSEN, L.L.: "Behavioral Pharmacology", Oxford University Press, New York, 1975. 128 JAIN, M.: Monoamine oxidase: Examination of multiple forms. Life Sci. 39: 1925-1934, 1977. JOHNSTON, J.P.: Some observations upon a new inhibitor of monoamine oxidase in brain tissue. Biochem. Pharmacol. 11: 1285-1297, 1968. KADZIELAWA, K. AND WIDY-TYSZKIEWICZ, E.: Electroencephal- ographic analysis of the central effects of dihydroxy- phenylalanine. Electroenceph. Clin. NeurOphysiol. .gg: 259-265, 1970. KAROUM, D., GILLIN, J.C., WYATT, R.J. AND COSTA, E.: Mass-fragmentography of nanogram quantities of biogenic amine metabolites in human cerebrospinal and whole rat brain. Biomed. Mass Spectrom. 12: 183-189, 1975. KILTS, C.D., VRBANAC, J.J., RICKERT, D.E. AND RECH, R.H.: Mass fragmentographic determination of 3,4-dihydroxy- phenylethylamine and 4-hydroxy-3-methoxyphenyl- ethylamine in the caudate nucleus. J. Neurochem. g5: 465-467, 1977. KOSLOW, S.H., CATTABENI, F. AND COSTA, E.: Norepinephrine and dopamine: Assay by mass fragmentography in the picomole range, Science 176: 177-180, 1972. KULKARNI, S.K. AND DANDIYA, P.C.: Effects of antide- pressants on open-field behavior in rats. Psycho- pharmacol. 2;: 333-338, 1973. LAPIN, I.P.: Qualitiative and quantitative relationship between the effects of imipramine and chlorpromazine on amphetamine group toxicity. PsychOpharmacol.3; 413-416, 1962. LHUGUENOT, J.B. AND MAUME, B.F.: Improvements in quantita- tive gas phase analysis of catecholamines in the pico- mole range by electron-capture detection and mass fragmentography of their pentafluorobenzyliminetrimeth- y1311yl derivatives. J. Chromatogr. Sci. t2: 411-418, '1974. LINDSTROEM, B., SJOEQUIST, B. AND ANGGARD, B.: Preparation of deuterium labeled catecholamines, catecholamine precursors and metabolites for use as internal stan- dards in mass fragmentography and for turnover studies. J. Labeled Comp. 19: 187-193, 1974. LOOMER, H.P., SAUNDERS, J.C. AND KLINE, N.S.: Psychiat. Res. Rep. Amer. Psychiat. Ass. 129: 1952. 129 LOWRY, O.H., ROSENBROUGH, N.J., FARR, A.L. AND RANDALL, R.J.: Protein measurement with the Folin phenol reagent. MAITRE, L., DELINI-STULA, A. AND WALDMEIER, P.C.: Relation between the degree of monoamine oxidase inhibition and some psychoPharmacological responses to monoamine oxidase inhibitors in rats, £3: Ciba Found. Symp. on "Monoamine Oxidase and Its Inhibition", Elsevier- Excerpta Medica, Amsterdam, pp. 247-267, 1976. MCFADDEN, W.H.: "Techniques of Combined Gas Chromatography/ Mass Spectrometry: Applications in Organic Analysis", New York, Wiley and Sons, Inc., 1973. MCKEARNEY, J.W.: The ralative effects of g-amphetamine, imipramine and harmaline on tetrabenazine suppression of schedule-controlled behavior in the rat. J. Pharmacol. Exp. Ther. tég: 429-440, 1968. MEEK, J. AND WERDINIUS, B.: Hydroxytryptamine turnover decreased by the antidepressant drug chlorimipramine. J. Pharm. Pharmacol. gg: 141-143, 1970. MOORE, K.E.: Biochemical correlates of the behavioral effecgs of drugs, £3: "An Intruduction to PsychOpharm- acology", Rech, R.H. and Moore, K.E., Eds., Raven Press, New York, pp. 79-136, 1971. MOORE, K.E. AND RECH, R.H.: Antagonism by monoamine oxi- dase inhibitors of a-methyltyrosine-induced catechol- amine depletion and behavioral depression. J. Pharm- acol. Exp. Ther. ttg: 70-75, 1967. MOORE, K.E., WRIGHT, F.P. AND BERT, J.K.: Toxicoloqical studies with a-methyltyrosine, an inhibitor of tyrosine hydroxylase. J. Pharmacol. Exp. Ther. tit: 506-515, 1967. NARASIMHACHARI, N.: Selective gas chromatography-mass spec- trometric methods for the quantitation of normetane- phrine (NM), metanephrine (MN) and vanillylmandellic acid (VMA). J. Chromatogr. 29: 163-176, 1974. NEFF, N.H. AND GORIDIS, C.: Neuronal monoamine oxidase: Specific enzyme types and their rates of formation, 1&3 "Advances in Biochemical PsychOpharmacology", Volume 5, Costa, E. and Sandler, M., Eds., Raven Press, New York, pp. 307-323, 1972. NEFF, N.H. AND YANG, Y-Y.T.: Another look at the monoamine oxidases and the monoamine oxidase inhibitor drugs. Life Sci. t1: 2061-2074, 1974. 130 NEUBURG, J. AND THUT, P.D.: Comparison of the locomotor stimulant mechanisms of action of g-amphetamine and g-amphetamine plus L-DOPA: Possible involvement of serotonin. Biol. Psychiat. t: 139-150, 1974. NG, K.Y., CHASE, T.N., COLBURN, RJW. AND KOPIN, I.J.: L-DOPA-induced release of cerebral monoamines. Science 170: 76 77, 1970. OLDENDORF, W.H.: Blood-brain barrier permeability to drugs. Ann. Rev. Pharmacol. ti: 239-248, 1974. PAYSON, H.E.: Drug therapy of mental illness: Treatment of psychiatric depression, In: "An Introduction to PsychOpharmacology", RecHT R.H. and Moore, K.E., Eds., Raven Press, New York, pp. 321-339, 1971. PEARSON, J.D.M. AND SHARMAN, D.E.: A gas-liquid chromato- graphic method for the estimation of the acidic meta- bolites of dOpamine in cerebrospinal fluid and brain tissues. Proc. Br. Pharmacol. Soc., Southhampton, 28-29 March: p114, 1974. PIRCH, J.H., RECH, T.H. AND MOORE, K.E.: Depression and recovery of the electrocorticogram, behavior and brain amines in rats treated with reserpine. Neuropharmacol. g: 375-385, 1967. PLANZ, G., QUIRING, K. AND PALM, D.: Rates of recovery of irreversibly inhibited monoamine oxidases: A measure of enzyme protein turnover. Naunyn-Schmiede- berg's Arch. Pharmacol. 213: 27-42, 1972. RECH, R.H.: Antagonism of reserpine behavioral depression by g-amphetamine. J. Pharmacol. Exp. Ther. 146: 369-376, 1964. RECH, R.H.: Enhanced avoidance in poor performers as a model for evaluating antidepressant agents. Psycho- pharmacol. Bull. lg: 19, 1974. RECH, R.H.: Experimental aminal models in evaluating antidepressant drugs. Panel presentation, 8th Annual Winter Conference on Brain Research, Steamboat Springs, Colorado, 1975. RECH, R.H., BORYS, H.K. AND MOORE, K.E.: Alteration in behavior and brain catecholamine levels in rats treated with a-methyltyrosine. J. Pharmacol. Exp. Ther. ttg: 412-419, 1966. RECH, R.H., CARR. L.A. AND MOORE, K.E.: Behavioral effects of a-methyltyrosine after prior depletion of brain catecholamines. J. Pharmacol. Exp. Ther. lgg: 326- 335, 1968. 131 RECH, R.H. AND STOLK, J.M.: Amphetamine-drug interactions that relate brain catecholamines to behavior, ta: "Amphetamines and Related Compounds", Costa, E. and Garattini, S., Eds., Raven Press, New York, pp. 385- 413, 1970. RECH, R.H. AND THUT, P.D.: Mechanisms of L-DOPA effects on EEG and behavior, It: "Drugs and Central Synaptic Transmission", Bradley, P.B. and Dhawan, B.N., Eds. Mac Millan Press, London, pp. 175-191, 1976. SANDLER, M..AND YOUDIM, M.B.H.: Multiple forms of mono- amine oxidase: Functional significance. Pharmacol. Rev. 31: 331-348, 1972. SNADLER, M. AND YOUDIM, M.B.H.: Monoamine oxidases: The present status. Int. Pharmac0psychiat. g: 27- 34, 1974. SCHANBERG, S.M., SCHILDKRAUT, J.J. AND KOPIN, I.J.: Tge effects of psychoactive drugs on norepinephrine- H metabolism in brain. Biochem. Pharmacol. it: 393-399, 1967. SCHECKEL, C.L., BOFF, E. AND PAZERY, L.M.: Hyperactive states related to the matabolism of norepinephrine and similar biochemicals. Ann. N.Y. Acad. Sci. 159: 939-958, 1969. SCHEEL-KRUGER, J.: Behavioural and biochemical comparison of amphetamine derivatives, cocaine, benztrOpine and tricyclic antidepressants. Eur. J. Pharmacol. it: 63-73, 1972. SCHILDKRAUT,:LJ.: Affective Disorders. Ann. Rev. Med. gg: 333-343, 1974a. SCHILDKRAUT, J.J.: Norepinephrine metabolism in the patho- physiology and classification of depressive disorders. £23 "Psychopathology and PsychOpharmacology", Cole, J.O. and Friedman, A., Eds., Johns Hopkins Press, Baltimore, 1975. SCHILDKRAUT, J.J., SCHANBERG, S.M., BREESE, G.R. AND KOPIN, I.J.: Effects of psychoactive drugs on the metabolism of intracisternally administered serotonon in the rat brain. Biochem. Pharmacol. lg: 1971-1978, 1969. SCHILDKRAUT, J.J.: The chatecholamine hypothesis of af- fective disorders: A review of supporting evidence. Amer. J. Psychiat. 122: 509-522, 1965. 132 SCHILDKRAUT,J.J.: "NeurOpharmacology of the Affective Disorders", Little, Brown and Co., Boston, 1970. SCHILDKRAUT, J.J., WINOKUR, A. AND APPELGATE, C.: Nor- epinephrine turnover and metabolism in rat brain after long term administration of imipramine. Science tgt: 867-869, 1970. SCHUBERT, J.: Effect of chronic lithium treatment on monoamine metabolism in rat brain. Psychopharm. .gg: 301-311, 1973. SCHUBERT, J., NYBACK, H. AND SEDVALL, G.: Effect of anti- depressant drugs on accumulation and disappearance of monoamines formed t3 vivo from labeled precurssors in mouse brain. J. Pharm. Pharmacol. 33‘ 136-139, 1970. SCHUBERT, J. AND SEDVALL, G.: Effect of amphetamines on tryptOphan concentrations in mice and rats. J. Pharm. Pharmacol. g5: 53-62, 1972. SHASKIN, E. AND SNYDER, S.H.: Kinetics of serotonin accumu- lation into Slices from rat brain: Relationship of catecholamine uptake. J. Pharmacol. Exp. Ther. 175: 404-418, 1970. SHOPSIN, B., WILK, S., GERSHON, S., DAVIS, K. AND SUHL, M.: Cerebrospinal fluid MHPG: An assessment of norepi- nephrine metabolism in affective disorders. Arch. Gen. Psychiat. gt: 230-233, 1973. SIGG, E.B.: Pharmacological studies with Tofranil. Can. Psychiat. Assoc. J. 5: 75-85, 1959. SIGG, E.B., GYERMEK, L. AND HILL, R.T.: Antagonism of reserpine-induced depression by imipramine, related psychoactive drugs, and some autonomic agents. PsychOpharmacol. 1: 144-148, 1965. SJOQUIST, B. AND ANGGARD, E.: Determination of homovanillic acid in cerebrOSpinal fluid by gas chromatoqraphy with electron capture or mass spectrometric detection. Anal. Chem. 15: 2297-2301, 1972. SJOQUIST, B,. DAILEY, J., SEDVALL, G. AND ANGGARD, B.: Mass fragmentographic assay of homovanillic acid in brain tissue. J. Neurochem.gg: 729-733, 1973. SMITH, C.B.: Enhancement by reserpine and a-methyltyrosine of the effects of g-amphetamine upon the locomotor activity of mice. J. Pharmacol. Exp. Ther. 113‘ 343-350, 1963. 133 SMITH, C.B. AND DEWS, P.B.: Antagonism of locomotor suppressant effects of reserpine in mice and rats. Psychopharm. 3: 55-59, 1962. SMITH, T.E., WEISSBABACH, H. AND UDENFRIEND, 8.: Studies on the mechanism of action of monoamineoxidase: Metabolism on N,N-dimethyltryptamine and N,N-di- methyltryptamine-N-oxide. Biochem. J. 1: 137-143, 1962. SOKAL, R.R. AND ROHLF, F.J.: "Biometry", W.H. Freeman, Co., San Francisco, pp. 396-397, 1969. SPECTOR, S., GORDON, R., SJOERDSMA, A AND UDENFRIEND, 5.: End product inhibition of tyrosine hydroxylase as a possible mechanism for regulation of norepinephrine syn- thesis. Mol. Pharmacol. 3: 549-555, 1967. SPECTOR, S., HIRSCH, C.W. AND BRODIE, B.B.: .Association of behavioral effects of pargyline, a non-hydrazine MAO inhibitor with increase in brain norepinephrine. Int. J. Neuropharmacol. 3: 81-92, 1963. SPECTOR, S., SJOERDSMA, A. AND UDENFRIEND, S.: Blockage of endogenous norepinephrine synthesis by d-methyl- tyrosine, an inhibitor of tyrosine hydroxylase. J. Pharmacol. Exp. Ther. 86-95, 1965. STEIN, L. AND SEIFERT, J.: Possible mode of antidepressant action of imipramine. Science 134: 286-287, 1961. STOLK, J.M. AND RECH, RJH.: Enhanced stimulant effects of g-amphetamine in rats treated chronically with reser- pine. J. Pharmacol. Exp. Ther. 163: 75-83, 1968. SUCHOWSKY, G.K., PEGRASSI, L., BONSIGNORI, A., BERTAZZOLI, C. AND CHIELE, T.: Some pharmacological properties of 4-H-3-methylcarboxamide-one, 3-benzoxazine-2-one (F16654). Eur. J. Pharmacol. g: 327-332, 1969a. SUCHOWSKY, G.K., PEGRASSI, L., MOTETTI, A. AND BONSIGNORI, A.: The effect of 4-H-3-methylcarboxamide-one, 3-benzoxa- zine-2-one (FI6654) on monoamine oxidase and cerebral 5-HT. Arch. Int. Pharmacodyn. 182: 332-340, l969b. SUCHOWSKY, G.K. AND PEGRASSI, L.: The pharmacology of 4H-3-methylcarboxamide-3,4-benzoxazine-2-one. A compound acting on the central nervous system. Arzneimittel-Forschung Aulendorf Wurtt t2: 643-648, l969c. 134 SULSER, F., WATTS, J.S. AND BRODIE, B.B.: Blocking of reserpine action by imipramine, a drug devoid of sti- mulatory effects in normal animals. Fed. Proc. gg: 321, 1961. SULSER, F., WATTS, J.S. AND BRODIE, B.B.: On the mechanism of antidepressant action of imipramine-like drugs. Ann. N.Y. Acad. Sci. 2g: 279-281, 1962. SUMMERS, R.J.: The effect of monoamine oxidase inhibitors on the rectal temperature of the rat. J. Pharm. Pharmacol. g9: 335-343, 1974. TAYLOR, S.M. AND SNYDER, S.H.: Amphetamine: Differentiation by g and t isomers of behavior involving brain nore- pinephrine and dopamine. Science 168: 1487-1489, 1970. TIPTON, K.F.: Some properties of monoamine oxidase. Adv. Biochem. PsychOpharmacol.‘5: 11-24, 1972. THUT, P.D.: An analysis of the locomotor stimulation and the ECoG effects of L-DOPA. Ph.D. dissertation, Dartmouth College, Hanover, N.H., 1970. THUT, P.D. AND RECH, R.H.: Role of peripheral effects and central serotonin (5-HT) release in the locomotor activity (MA) due to L-DOPA. Fed. Proc. g9: 250, 1971. THUT, P.D. AND RECH, R.H.: Effects of L-DOPA on the EEG and brain amines of unrestrained rat. Exp. Neurol. gg: 13-29, 1972. UDENFRIEND, S., WEISSBACK, H. AND BOGDANSKI, D.E.: Effect of iproniazid on serotonin metabolism t3 vivg. J. Pharmacol. Exp. Ther. 120: 255-260, 1957. USDIN, E.: "NeuropsychOpharmacology of Monoamines and Their Regulatory Enzymes", Costa, E. and Sandler, M., Eds., Raven Press, New York, 1974. VALZELLIIL., CONSOLO, S. AND MORPURGO, C.: Influence of imipramine-like drugs on the metabolism of amphetamine, lg: "Antidepressant Drugs", Garattini, S. and Dukes, M.N.G., Eds., Excerpta Medica Foundation, Amsterdam, p. 61, 1967. VAN ROSSUM, J.M. AND HURKMANS, A.T.: Mechanism of action of psychomotor stimulant drugs: Significance of d0pamine in locomotor stimulation. Int. J. NeurOpharm. ;: 227- 239, 1964. VRBANAC, J.J., KILTS, C.D. AND RECH, R.H.: The anti-reser- pine activity of caroxazone: A new antidepressant drug. J. Pharmacol. Expt. Ther., submitted (1978a). 135 VRBANAC, J.J., KILTS, C.D., WEBER, A.H. AND RECH, R.H.: Prevention of a-methyltyrosine (dMT) behavioral de- pression by caroxazone. Pharmacol. Biochem. Behav., submitted (1978b). VRBANAC, J.J., KILTS, C.D., ALPER, R.H. AND RECH, R.H.: L-DOPA interaction with caroxazone; a new antidepressant drug. PsychOpharmacol., submitted (1978c). VRBANAC, J. J. AND TILSON, H. A.: Effects of d-amphetamine (_d—A) and 2, 5- -dimethoxy-4-methy1amphetamine (DOM) on the Ln vivo release of l4-C-norepinephrine (NE) into the lateral ventricle of rats. Fed. Proc. 23: 815, 1973. VRBANAC, J.J., TILSON, H.A., MOORE, K.E. AND RECH, R.H.: comparison of 2, 5- -dimethoxy-4-methylamphetamine (DOM) and d-amphetamine for in vivo efflux of catecholamines from_ rat brain. Pharmacol. Biochem. Behav. ;: 57- 64,1975. WALDMEIER, P.C., DELINI-STULA, A. AND MAITRE, L.: Pre- ferential deamination of dopamine by an A type mono- amine oxidase in rat brain. Naunyn-Schmiedeberg's Arch. Pharmacol. 223; 9-14, 1976. WALDMEIER, P. C. AND MAITRE, L.: Lack of significance of MAO B for Ln vivo deamination of dopamine. Naunyn- Schmiedeberg' s Arch. Pharmacol. 287: R2-R10, 1975. WALDMEIER, P.C. AND MAITRE. L.: Comparison of short and long-lasting effects of pargyline on cerebral dOpamine metabolism. Naunyn-Schmiedeberg's Arch. Pharmacol. 325: 133-140, 1976. WATSON, E. AND WILK, S.: Derivatization and gas chromato- graphic determination of some biologically important acids. Anal. Biochem. 59: 441-451, 1974. WIEGAND, R.G. AND PERRY, J.B.: Effect of L-DOPA and N- methyl-N-benzyl-2-pr0pyny1amine-HC1 on L-DOPA, dopamine, norepinephrine, epinephrine, and serotonin levels in mouse brain. Biochem. Pharmacol. 1: 181-186, 1961. WURTMAN, R.J. AND AXELROD, J.: A sensitive and Specific assay for the estimation of monoamine oxidase. Biochem. Pharmacol. lg: 1439-1440, 1964. YANG, H.-Y.T. AND NEFF, N.H.: B-Phenylethylamine: A Specific substrate for type B monoamine oxidase of brain. J. Pharmacol. Exp. Ther. 187: 365-371, 1973. 136 YANG, H.-Y.T. AND NEFF, N.H.: The monoamine oxidase of the brain: Selective inhibition with drugs and the consequences for the metabolism of the biogenic amines. J. Pharmacol. Exp. Ther. 182: 773-740, 1974. YOUDIM, M.B.H.: Heterogeneity of rat brain mitochondrial monoamine oxidase, in: “Advances in Biochemical Psycho- pharmacology", Volume 11, Costa, E., Gessa, O.L. and Sandler, M., Eds., Raven Press, New York, pp. 59-63, 1974. YOUDIM, M.B.H.: Multiple forms of monoamine oxidase and their prOperties. Adv. Biochem. Psychopharmacol. 5: 67-77, 1972a. YOUDIM, M.B.H.: Multiple forms of mitochondrial monoamine oxidase. Brit. Med. Bull. 22: 120-122, 1972b. YOUDIM, M.B.H., HOLZBAUER, M. AND WOODS, F.H.: Physico- chemical properties, development, and regulation of central and peripheral monoamine oxidase activity, in: "Neur0psych0pharmacology of Monoamines and Regulation Enzymes", Usdin, E., Ed., Raven Press, New York, pp. ll-28, 1974. YOUDIM, M.B.H. AND SANDLER, M.: Isoenzymes of soluble monoamine oxidase from human placental and rat liver mitochondria. Biochem. J. 105: 43F, 1967. ZELLER, E.A., BARSKY, J., FORTS, J.R. AND VAN ORDEN, L.S.: Influence of isonicotinic acid hydrazide (INH) and l-isonicotinyl—Z-iSOprOpyl hydrazide (IIH) on bacterial and mammalian enzymes. Experientia 8: 349-350, 1952. APPENDAGE TO THE BIBLIOGRAPHY: GENERAL REFERENCES GENERAL REFERENCES AARON, H.: "The Medical Letter on Drugs and Therapeutics", Drug and Therapeutic Information, Inc., New York, New York, biweekly publication. ACHESON, G.H.: "Second Symposia on catecholamines", Phar- macological Reviews, VOlume 18, Part I, pp 1-803, 1966. BOURNE, G.H.: "The Structure and Function of Nervous Tissue", Academic Press, Inc., New York, New York, 1972. BIANCI-IINE, J.R. AND SUNYAPRIDAKUL, L.: Interactions Bet- tween levodopa and other drugs: significance in the treatment of Parkinson's disease. Drugs 6: 364-388, 1973. DIKSTEIN, 8.: "Fundamentals of Cell Pharmacology", Charles C. Thomas, Pub., Springfield, 111., 1973. D'ARCY, F.P. AND GRIFFIN, J.P.: "Iatrogenic Diseases", Oxford University Press, London, 1972. FEATHERSTONE, B.M.: "A Guide to Molecular Pharmacology- Toxicology", Marcel Dekker, Inc., New York, N. Y. (Part I and Part II), 1973. GOULD, R.F.: "Advances in Chemistries Series—Molecular Mod- ification in Drug Design", American Chemical Society, Washington, D.C., 1964. GOLDSTEIN, A., ARONOW, L. AND KALMAN, S.M.: "Principles of Drug Action; The Basis of Pharmacology", Harper and Row, Pubs., New York, N.Y., 1968. GOODMAN, L.S. AND GILMAN, A.: "The Pharmacological Basis of Therapeutics", MacMillan Co., New York, N.Y., 1970 and 1975. HILLHARP, N.A., FUXE, K. AND DAHLSTROM, A: Demonstration and mapping of central neurons containing dopamine, noradrenaline, and 5-hydroxytryptamine and their re- actions to psychOpharmaca. Pharmac. Rev. 18: 727- 741, 1966. HORNYKIEWIEZ, O.: DOpamine (3-hydroxytyramine) and brain function. Pharmac. Rev. 18: 925-964, 1966. 137 138 LEVINE, R.R.: "Pharmacology: Drug Actions and Reactions", Little, Brown and Company, Boston, 1973. MALETZ, S.: "L-Dopa and Behavior", Raven Press, New York, N.Y., 1972. MELMON, K.L. AND MORELLI, H.E.: "Clinical Pharmacology", MacMillan Publishing Company, Inc., New York, N.Y., 1972. PORTER, R. AND O'CONNOR, M.: "Molecular Properties of Drug Receptors“, J. and A. Churchill, Ltd., London, 1970. SOURKES, T.L.: "Central actions of dOpa and dOpamine. Revue can. Biol., 3;: 153-168, 1972. SHUSTER, L.: "Readings in Pharmacology", Little, Brown and Company, Boston, 1062. SYMPOSIUM: "International Symposium on Amphetamines and related Compounds: Proceedings of the Mario Negri Institute for Pharmacological Research, Milan, Italy" (Editors: E. Costa and S. Garattini), Raven Press, New York, N.Y., 1970. : "Proceedings of the Third International Phar- macological Meeting",(Editors: J. Chymol and J.R. Boissier), Pergamon Press, Oxford, 1968. : "Biochemistry and Pharmacology of the Basal Ganglia;Proceedings on the Second Symposium on the Parkinson's Disease Information and Research Center" (Editors: E. Costa, L.J. Kote and M.D. Yahr) , Raven Press, New York, N.Y., 1966 : "Frontiers in Neruology and Neuroscience Res- earcn; First International Symposium of the Neuro- science Institute of the University of Toronto" (Edi- tors: P. Seeman and G.M. Browm) , Published by The Neuroscience Institute, University of Tbronto, Toronto, Canada, 1974. : "Monoamine Oxidase and Its Inhibition; CIBA Foundation Symposium", Elsevier-Exerpta Medica, Amster- dam: North Holland, 1976. : "NeuropsychOpharmacology of the Monoamines and their Regulatory Enzymes" (Editor: E. Usden), Raven Press, New York, N.Y., 1974. 139 : "Monoamine Oxidases-New Vistas" (Editors: E. Costa and M. Sandler), Raver Press, New York, N.Y., 1972. : "Proceedings of the Second International Phar- macology, vo1. 4, Drugs and Enzymes" (Editors: Brodie, 3.3. and Gillette, J.R.), Pergamon Press, Ltd., OX- ford, 1965 : "Absorption and Distributrition of Drugs" (Editor: Binns, T.B.), The Williams and Wilkins Company, Baltimore, 1964. : "Antidepressant Drugs" (Editors: 8. Garattini and M.N.G. Dukes), Excerpta Medica Foundation, Amsterdam: North Holland, 1967. : "Drugs and Central Synaptic Transmission" (Edi- tors: P.B. Bradley and B.N. Dhawan), MacMillan Press, London, 1976. : "Advances in Biochemical Psychopharmacology" (Editors: E. Costa and P. Greengard), Ravin Press, New York, N.Y., 1972. : "Advances in Biochemical Psychopharmacology" (Editors: E. Costa, O.L. Gessa and M. Sandler), Raven Press, New York, N.Y., 1974. SIGG, E.B.: "PsychOpharmacology: A Review of Progress", Government Printing Office, Washington, D.C., 1968. TALALAY, P.: "Drugs in Our Society", The Johns Hopkins Press, Baltimore, 1964. VARLEY, H. AND GOWENLOCK, A.H.: "The Clinical Chemistry of Monoamines", Elsevier Publishing Company, Amster- dam: North Holland, 1963. WAGNER, J.G.: "BiOpharnaceutics and Relevant Pharmaco- kinetics", Drug Inteligence Publications, Hamilton, 111., 1971.