O States
EPA-600/1-80-028
May 1980
                 »( j'' ' '
Toxicity, Interactions,
and Metabolism of
Formamidine Pesticides
in Mammals

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                               DISCLAIMER

     This report has been  reviewed by  the  Health  Effects  Research
Laboratory, U.S. Environmental Protection  Agency,  and  approved  for
publication.  Approval does  not  signify  that  the  contents necessarily
reflect the views  and policies of the  U.S.  Environmental  Protection
Agency, nor does mention of  trade names  or commercial  products
constitute endorsement or  recommendation for  use.
                                     ii

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                                    FOREWARD
     The many benefits of our modern,  developing,  industrial society are
accompanied by certain hazards.   Careful assessment of the relative risk
of existing and new man-made environmental hazards is necessary for the
establishment of sound regulatory policy.  These regulations serve to
enhance the quality of our environment in order to promote the public
health and welfare and the productive  capacity of our nation's population.

     The Health Effects Research Laboratory,  Research Triangle Park,
conducts a coordinated environmental health research program in toxicology,
epidemiology, and clinical studies using human volunteer subjects.  These
studies address problems in air  pollution, non-ionizing radiation, environ-
mental carcinogenesis and the toxicology of pesticides as well as other
chemical pollutants.  The Laboratory participates in the development and
revision of air quality criteria documents on pollutants for which national
ambient air quality standards exist or are proposed, provides the data for
registration of new pesticides or proposed suspension of those already in
use, conducts research on hazardous and toxic materials, and is primarily
responsible for providing the health basis for non-ionizing radiation
standards.  Direct support to the regulatory function of the Agency is
provided in the form of expert testimony and preparation of affidavits as
well as expert advice to the Administrator to assure the adequacy of health
care and surveillance of persons having suffered imminent and substantial
endangerment of their health.

     This report describes investigations of the mechanism(s) of acute
toxicity of formamidine pesticides in  mammals.  Formamidine pesticides are
a relatively new group of insecticide-acaricides which are particularly
useful for the control of Lepidoptera, Hemiptera,  phytophagous mites,
and cattle ticks.  Their widespread use for control of cotton insects and
cattle ticks make the investigation of their toxicity extremely important.
                                             F. Gordon Hueter
                                             Director
                                             Health Effects Research Laboratory
                                      iii

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                                ABSTRACT

     The overall goal of this  research  project  was  to  investigate  the
mechanism(s) of acute toxicity of  formaroidine  pesticides  in  mammals
using chlordimeform  (^'-(M-chloro-o-tolyD-^NUdimethylformamidine)  and
its metabolites as the  primary model  compound!?.  The role of
blotransformations,  particularly jN-demethylation  reactions,  in
generating potentially  toxic metabolites  was  also studied.
     By comparing  the  effects  of  hepatic microsomal  mixed  function
oxidase inducers and  inhibitors administered  Jin vivo on  the toxicity,
metabolism, and distribution of metabolites in mouse tissues,  it  was
concluded that although  NUdemethylation products are innately  more  toxic
than chlordimeform, they~are also less stable, and the best correlation
of toxicity was obtained  with  the total level of formamidines  in  the
brain, rather than with  the  level of any individual  metabolite.
     In a  series  of  studies  with dogs,  rabbits,  and cats,  the cause of
death was  found to be  cardiovascular collapse accompanied  by respiratory
arrest.  Cardiovascular  collapse resulted primarily from a peripheral
local anesthetic-like  effect of chlordimeform.   Monoamine  oxidase
inhibition was not a major  factor in lethality.   Respiratory arrest was
central in origin.   Several  other central effects of the formamidines
were described, some of  which may be local anesthetic actions, and a
behavioral profile for chlordimeform poisoning in the rat  was developed.
The effectiveness of various drug treatments as  potential  therapeutic
aids for formamidine intoxication were  studied.   Finally,  the
formamidines  were found  to  possess a number of aspirin-like actions
which resulted from  an ability to inhibit prostaglandln synthesis.
     This report  was  submitted  in  fulfillment of Research Grant No.
R-803965 by  Purdue  Research  Foundation under the sponsorship of the  U.S.
Environmental  Protection  Agency.   This report covers the period July
1st, 1975 to December  31st,  1978,  and the work was completed as of July
1st, 1979.
                                     iv

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                                CONTENTS
Foreword .  .	    ill
Abstract	     iv
List of Figures  .		     vl
List of Tables	   vlti
Abbreviations, ... 	      x
Acknowledgments	     xl

     1.  General Introduction  	    1
     2.  Conclusions	    4
     3.  Section 1 - Evaluation of Monoamine
           Oxldase as a Target for Formamidlne Pesticides.  .....    6
     4.  Section 2 - Actions of Chlordimeform on the
           Cardiovascular System ........ 	   20
     5.  Section 3 - The Central Actions of Chlordimeform.  .....   29
     6.  Section 1 - Local Anesthetic Properties of Chlordimeform
           and Potential Means of Alleviating Toxicity  	   38
     7-  Section 5 - Behavioral Effects of Chlordimeform  	   48
     8.  Section 6 - Inhibition of Prostaglandin Synthesis
           by Formamidines	   57
     9.  Section 7 - Blotransformation of Chlordimeform and  its
           Relation to Toxicity	   67

     References	93

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                                     FIGURES
Number
  1    Recovery of mouse  brain  mitochondrial  MAO from inhibition
       by CDM after  successive  washes  to  remove the inhibitor.  . .  .

  2    Time course of  inhibition  of MAO from  mouse intestine
       by chlordimeform,  N-demethyl chlordimeform, and
       j|I-formyl-4-chloro-o-toluidine ................      12

  3    Inhibition of MAO  from mouse intestine by CDM assessed
       with three MAO  substrates  ..................      13

  M    Inhibition of MAO  from mouse intestine by NFT assessed
       with three MAO  substrates  ..................      14
  5    Typical  records  showing the effects of CDM and lidocaine
       on heart  rate, cardiac  contractility,  and blood pressure
       in the pentobarbital-anesthetized dog  ............     21

  6    Initial  (1°)  and  secondary (11°)  effects of CDM and
       lidocaine on  blood  pressure and cardiac contractility ....     23

  7    The effects of CDM  and  lidocaine  on heart rate and cardiac
       contractility in  the  isolated rabbit heart preparation  ...     24

  8    The effects of intraarterial CDM  and lidocaine on the hind
       limb perfusion pressure in the pentobarbital-anesthetized
       dog .............................     25

  9    Typical  record showing  the effects of CDM on blood pressure
       and spike discharge frequency recorded preganglionically
       from the  superior cervical nerve  of the pentobarbital-
       anesthetized  cat  .......................    26

  10   Pressor  responses in  the carotid  artery after intraventric-
       ular injections  of  lidocaine or CDM in a rat anesthetized
       with urethane .........................   31

  11   Suppression of amygdala EEC upon  raphe stimulation and
       antagonism of this  suppresison by intraventricular lidocaine
       or CDM   ............................   32

  12   Duration  of suppression of amygdala EEC activity and
       antagonism of this  raphe-mediated EEC  suppression by
       lidocaine and CDM .......................   32
                                      vi

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13   Amygdala EEC spikes and blood pressure oscillation  recorded
     during overt CDM seizures 	   33

14   Antagonism by diazepam of the EEC and blood pressure effects
     of intraventricular lidocaine and CDM	   33

15   Respiratory and cardiovascular parameters of a urethane-
     anesthetized rat infused with a lethal iv dose of lidocaine  .  .   34

16   Same as Fig. 15 except drug infused was chlordimeform	   35

17   Chemical structure of chlordimeform and related local
     anesthetics	   38

18   Effect of chlordimeform, N-demethylchlordimeform, procaine,
     holocaine, and lidocaine on the action potential in frog
     sciatic nerve 	   40

19   Effect of chlordimeform at pHs 6.0 and 7.0 in the sheathed
     and at pH 7.0 in the desheathed nerve on action potential
     amplitude at 2 min intervals following drug application  ....   41

20   Dose response effect of PCA, CDM, and quipazine in  eliciting
     serotonergic syndrome and additional symptoms 	   50

21   Effect of cinanserin pretreatment on the serotonergic  syndrome
     and additional symptoms elicited by PCA, CDM, and quipazine  .  .   51

22   CDM-induced depression of the electrically stimulated  twitch
     height in guinea pig ileum preparation  	   59

23   Effects of naloxone, tolazoline and phentolamine on twitch
     depression by 3 x 10   M CDM.  Effects of PGE. on twitch
     depression induced by 10~  M indomethacin, CDM, or
     lidocaine	60

24   Effects of drug free wash on twitch depression induced by
     3 x 10"b M indomethacin or by 10   M CDM	60

25   Antagonism of yeast-induced fever in rats by acetylsalicylic
     acid, indomethacin, and two formamidine pesticides,
     chlordimeform and amitraz	61

26   Antagonism of the carrageenin-induced edema of rat  paw by
     the formamidines amitraz and chlordimeform	63

27   Metabolic map showing fate of chlordimeform in mammals	69

28   Rates of hydrolysis of chlordimeform and its N-demethylated
     metabolites at pH 7.4 and 37°	77
                                  vii

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                                     TABLES
Number
                                                                      Page
  1    Acute Toxicity  and MAO  Inhibition  In Vitro by
       Chlordimeform and  its Major  Metabolites  	      9

  2    Reversibility of Inhibition  of Mitochondrial MAO
       From Mouse Brain by  Chlordimeform  and its Metabolites
       on Washing	    11
                              111
  3    Recovery of  Injected [  C] Tryptamine After Pretreatment
       of Mice With MAO Inhibitors	    15

  4    The Effect of Time After  Dosing on the Ability of
       Chlordimeform to Increase the Recovery of Injected
       [  C] tryptamine From Mouse  Tissues	    16

  5    Time Course  of  the Primary and Secondary Blood Pressure
       and Cardiac  Contractility Responses in Chlordimeform-
       Treated Dogs	    22

  6    Effects of Anticonvulsants on CDM  Convulsions and
       Lethality in Mice	    42

  7    Effect of Other Pharmacological Agents on CDM Convulsions
       and Lethality in Mice	    44

  8    Toxicity in  Mice of  CDM Combined with Agents Possessing
       Local Anesthetic Activity  	    45

  9    Effect of Cinanserin on Serotonergic Syndrome Produced
       by PCA, CDM  and Quipazine	    52

  10   Effect of Cinaserin  on  Additional  Symptoms Produced by
       PCA, CDM and Quipazine	    53

  11   Effect of 5-HT  on  Serotonergic Syndrome and on Additional
       Behaviors in the Rat	    54

  12   Inhibition of PGE  Synthesis by Bovine Seminal Vesicle
       Microsomes With Two  Formamidines and Two Common
       Non-steroidal Anti-Inflammatory Agents 	    62

  13   Behavioral and  Pharmacological Profile of CDM in the Rat .  .    65
                                  viii

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14   The Effect of Several Pretreatments Affecting  Microsotnal
     Oxidations on the Toxicity of Formamidines to  Mice	      76

15   Rate Constants and Half Lives for the Hydrolysis of
     Chlordimeform and its N-demethylated Metabolites 	      78

16   Relative Activities of Mouse Tissue Microsomes  and  Other
     Cellular Subfractions in Degrading Chlordimeform 	      79
                                       14
17   Metabolites Formed in Reaction of   C-Chlordimeform With
     Mouse Liver Microsomes 	      80

18   ^n Vitro Metabolism of Chlordimeform and its N-demethylated
     Metabolites by Mouse Liver Microsomes   	      81

19   Effect of Several Pretreatments Affecting Microsomal
     Oxidations on the In Vitro Metabolism of Chlordimeform
     by Mouse Liver Microsomes  	      82

20   Total Radioactivity in Tissues After an Oral Dose
     of Chlordimeform	      83

21   Effect of Several Pretreatments Affecting Microsomal
     Oxidations on the ^n Vivo Metabolism and Distribution
     of Chlordimeform in Mice	      85

22   Effect of Several Pretreatments Affecting Microsomal
     Oxidations in the Mouse on the III Vivo Metabolism
     of Chlordimeform	      86

23   Comparison of the Effect of Pretreatments on the Toxicity
     of CDM to Mice and on the Levels of Selected Formamidines
     in their Tissue	      87
                                   ix

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                              LIST  OF  ABBREVIATIONS
ABBREVIATIONS

ACh
CAA
CDM
CDU
CNS
CT
DCDM
DCDU
DDCDM
DDCDU
DFP
EGG
EDTA
EEC
G.C.
GSH
5-HT
ia
ip
iv
ivc
LSD
MAO
MFO
NADPH

NE
NFA
NFT
PCA
PG
PGE
PGE
   1
sc
SD
SE(M)
TCA
TLC
acetylcholine
5-chloroanthranilic acid
chlordimeform
1 , 1-dimethyl-3-C*-chloro--o-tolyl)urea
central nervous system
4-chloro-o-toluidine
N-demethyl chlordimeform
1 -methyl-3- ( U-chloro-o-tolyl) urea
N^.II-didemethyl chlordimeform
M-chloro-o-tolylurea
diisopropylphosphorofluoridate
electrocardiograph
ethylenediamine tetr acetate
electroencephalograph
gas chromatography
glutathione
5-hydroxytryptamine (serotonin)
intraarterially
intraperitoneally
intravenously
intraventricularly
D-lysergic acid diethylamide
monoamine oxidase
mixed function oxygenase
reduced nicotinamide-adenine
dinucleotide phosphate
norepinephrine (noradrenaline)
N-formyl-5-chloroanthranilic acid
N-formyl-4-chloro-o-toluidine
parachloroamphetamine
prostaglandin
prostaglandin E
prostaglandin
subcutaneously
standard deviation
standard error (of mean)
trichloroacetic acid
thin-layer chromatography


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                              ACKNOWLEDGMENTS

     The collaboration, advice, and technical assistance of  the  following
people is gratefully recognized:  Dr. D. E. Blake, Dr. C.  Chinn,  Mr.  M.  P.
Holsapple, Mr. M. T. Lowy, Ms. V. Noland and Dr. W. R. Pfister and  Mr.  J.
Rolley of the Department of Pharmacology and Toxicology, Purdue  University;
Dr. D. E. Nichols of the Department of Medicinal Chemistry,  Purdue
University; and Mrs. K. Dersch, Dr. G. Ghali, Mr. J. Leister, Dr. A.  E.
Lund and Dr. D. L. Shankland of the Department of Entomology, Purdue
University.

     The following material is reprinted with the permission of  the
copyright holders, to whom we express our appreciation.  Some text
material, Figs. 1 through 4 and Tables 1 through 4 in Section 1  are
reproduced from Chemico-Biological Interactions, vol. 24,  pages  35-49,
copyright 1979 by Elsevier/North-Holland Scientific Publishers Ltd.,
Shannon, Ireland.  Some text material, Figs. 5 through 9 and Table  5  in
Section 2 are reproduced from Toxicology and Applied Pharmacology,  vol.  44,
pages 357-365, copyright 1978 by Academic Press, Inc., New York.  Some  text
material and Figs. 10 through 14 in Section 3 are reproduced from Neuro-
pharmacology, vol. 16, pages 867-871, copyright 1977 by Pergamon  Press,
Inc., New York.  Some text material, Figs. 17 through 19 and Tables 6
through 8 in Section 4 are reproduced from Pesticide Biochemistry and
Physiology, vol. 9, pages 148-156, copyright 1978 by Academic Press,  Inc»,
New York.  Some text material, Figs. 20 and 21, and Tables 9 through  11  in
Section 5 are reproduced from Communications in Psychopharmacology, vol. 2,
pages 287-295, copyright 1978 by Pergamon Press, Inc., New York.  Some  text
material, Fig. 25 and Table 12 in Section 6 are reproduced from  Life
Sciences, vol. 23, pages 2509-2515, copyright 1978 by Pergamon Press, Inc.,
New York.  Some text material, and Figs. 22 through 24 in  Section 6 are
reproduced from Prostaglandins and Medicine, vol. 2, pages 215-222,
copyright 1979 by Churchill Livingstone, Edinburgh, Scotland.
                                    xi

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                           GENERAL INTRODUCTION
     The formamidines are a relatively new group of insecticide-
acaricides which are particularly useful for the control  of Lepidoptera,
Hemiptera, phytophagous mites,  and cattle ticks (Hollingworth,  1976).   In
the U.S. two compounds are of primary interest, chlordimeform (I;
£«_(4_chloro-£-tolyl)-N^N-dimethylformamidine)) and amitraz (II;
l,5-di-(2,4-dimethylphenyl)-3-methyl-1,3,5-triazapenta-1,4-diene).
          N=C-N<
                     CH
Chlordimeform
(Fondol : Nor-Am/Schering)
(Galecron :  Ciba-Geigy)
                                             Amitraz
                                             (BTS-27419: Boots)
                                             (U-36059-.Upjohn)
     During this grant period both compounds were reported to be
carcinogenic in mice at high doses (Johnson, 1977).  This is probably due
to the metabolic release of carcinogenic anilines rather than because of
any innate carcinogenicity of the parent formamidines (Ghali, 1980).
Registration for amitraz has thus been limited to control of psylla on
pears (Johnson, 1979), although it is used widely outside the U.S., e.g.
for cattle tick control.  By voluntary agreement, chlordimeform (CDM) is
confined to use for control of cotton insects, in which situation it
remains an important insecticide.  It too has significant uses outside
the U.S.  A number of other compounds related to these formamidines are
currently undergoing field testing.

     The formamidines are structurally unlike other pesticides and their
mechanism(s) of toxic action in vertebrates and invertebrates appear to
be novel (Matsumura and Beeman, 1976; Lund et al., 1979b).  At the start
of this project it was known only that the symptoms of poisoning by

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lethal doses of  CDM  in  rodents  were  excitatory and  included
hyperexcitation, tremors,  and convulsions  preceding death (Beeman  and
Matsumura, 1973).  The  symptoms were described as  sympathomimetic  in
nature (Aziz and Knowles,  1973).   No cholinomimetic responses have been
reported.  The most  likely site of action  of formamidines was thought to
be monoamine oxidase (MAO).   Despite evidence that  MAO was inhibited in
v-'tro (Aziz and  Knowles,  1973;  Beeman and  Matsumura, 1973),  we felt that
tne rapid, excitatory poisoning symptoms were not  congruent  with the MAO
theory, nor were the symptoms predominantly sympathomimetic, though this
component was present.   In particular it seemed important to determine
the degree, duration, and  reversibility of MAO inhibition in vivo.  Not
only is this crucial in order to interpret the role of MAO in poisoning,
but also to evaluate the likelihood  of dangerous interactions with
dietary or medicinal amines (Sjoqvist, 1965) or of cumulative inhibition
of MAO on repeated exposure to  formamidines.

     A number of other  biochemical actions of formamidines have been
described, such  as mitochondrial uncoupling (Abo-Khatwa and  Hollingworth,
1973) and inhibition of RNA synthesis (Murakami and Fukami,  1974), but
these are not plausible as the  cause of the symptoms observed (Lund jit
al.. 1979b).  At the start of the project  the actual cause of death from
formamidines was unknown although Beeman and Matsumura (1974) noted
briefly that CDM caused a  depression of blood pressure in rabbits.

     Before and  during  the work presented  here a number of publications
appeared describing  the metabolism and fate of CDM in mammals (see
Section 7 for references).  A major  metabolic route for CDM in all
species studied  is N-demethylation to yield N-demethyl chlordimeform
(DCDM) (Knowles  and  Sen Gupta,  1970; Sen Gupta and Knowles,  1970;
Morikawa j5t £l.,  1975).  Subsequently a second N-demethylation to give
N^N-didemethyl chlordimeform (DDCDM) was reported  (Benezet and Knowles,
1976b).  Our preliminary results indicated that DCDM was more toxic than
CDM to mice (Hollingworth, 1976) and more  rapid in its actions.  Also,
piperonyl butoxide,  an  insecticide synergist which inhibits many
j^-demethylations, strongly antagonized some of the toxic effects of CDM,
but not of DCDM, in  cattle ticks, suggesting that  conversion of CDM to
DCDM is crucial  for  such toxicity (Knowles and Roulston, 1972).  If this
were true in mammals also, there are obvious implications regarding the
appropriate compounds to use in mode of action studies, and for
unexpected toxic interactions arising after exposure to mixed function
oxidase (MFO) inducers  or  inhibitors.

     The goals of this  project  were  therefore:

     (a).  To define the symptoms and mechanism(s)  of toxicity of CDM in
           mammals.

     (b).  To specifically examine the role of MAO in these processes and
           to evaluate  the possibility of  interactions or cumulative
           effects based  on MAO inhibition.

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(c).  To develop information leading to potential means of  therapy
      for formamidine overexposure.

(d).  To study other pharmacological and behavioral actions of
      formamidines which might have toxicological implications,
      indicate potential sites of action, or have utility as  an
      index of exposure to these pesticides.

(e).  To investigate the metabolism of CDM and the accumulation of
      CDM and its metabolites jin vitro and ^.n vivo.  Specific
      objectives in this respect were to determine whether
      N-demethylations are important activation reactions for
      formamidines, and to assess the possibility of significant
      interactions with MFO inducers and inhibitors.

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                                CONCLUSIONS
1.   Chlordimeform is a moderately  potent,  reversible  inhibitor  of
monoamine oxidase.  Inhibition  of monoamine oxidase  in vivo  is ephemeral
and never reaches high levels even  at  near  lethal  doses.   Several other
lines of evidence also indicate that monoamine  oxidase inhibition is  not
a major mechanism underlying acute  toxicity,  and  the possibility of
dangerous interactions resulting from  concurrent  exposure  to f ormamidines
and natural or medicinal  biogenic amines  is slight.  No evidence of such
interactions was seen in  cardiovascular  studies performed  with dogs.
Inhibition of monoamine oxidase is  unlikely to  be  cumulative with
continued daily exposure  to formamidines  because  of  their  rapid
metabolism and the reversibility of inhibition.

2.   Animals poisoned with chlordimeform  suffer concurrent cardiovascular
collapse and respiratory  arrest. When given intravenously,  chlordimeform
has a biphasic action on  blood  pressure  i.e. an intial depression
followed (in survivors) by an elevation.   Both  actions are shared with
lidocaine and are attributable  to a local anesthetic-like  effect of
chlordimeform.  The depressor effect results primarily from  vasodilation
and reduced cardiac contractility while  the pressor  response results
centrally from enhanced sympathetic outflow.   Respiratory  arrest is a
secondary effect arising  centrally, probably through depression  of  the
respiratory center following convulsions.

3.   The effects of chlordimeform and  lidocaine are  also similar when
administered intraventricularly in  rats  and when  applied to  the  isolated
frog sciatic nerve.   Intraventricular  chlordimeform  induces  EEC  seizure
discharges in the amygdala and  blocks  amygdalar inhibition by the raphe.
Evidence for actions  of formamidines on  the nervous  system other than as
local anesthetics exists  e.g. biogenic amine accumulation  through partial
inhibition of monoamine oxidase, a  direct excitatory effect  on some types
of axons, and a partial   -adrenergic agonist effect  of l£-demethylated
formamidines.  However, on the  basis of present evidence the local
anesthetic-like actions appear  to explain well  most  of the symptoms and
signs observed, including the primary  cardiovascular depression,
secondary increase in sympathetic outflow,  and  the convulsive and
respiratory effects of chlordimeform.   Like other  local anesthetics,
chlordimeform can induce  "pharmacological kindling"  i.e.  precipitation of
convulsions and other behavioral effects  after  repeated subliminal  doses.

4.   Therapeutic approaches to  local anesthetic overdose are limited.
Diazepam was clearly helpful in alleviating the central symptoms of

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chlordimeform poisoning in rats and dogs, but only at  low doses  of
diazepara.  A combination of therapeutic steps is likely to  be  necessary
and probably should include:

     (a).  Artificial resuscitation to combat respiratory depression.

     (b).  Low doses of diazepam.

     (c).  Vasoconstrictors and cardiac stimulants (e.g. epinephrine  and
           calcium gluconate) for the cardiovascular depression.

5.   In developing the "behavioral profile" for chlordimeform  to  serve as
an aid in detecting and diagnosing overexposure, it was found  to  induce
responses typical of the so-called "serotonergic behavioral syndrome".
Most of these and the other behavioral effects of chlordimeform  are not
induced by known local anesthetics.  However, further  study casts doubt
on the existence of this 'serotonergic syndrome1 as presently  defined  and
on any general role for serotonin in the behavioral effects of
chlordimeform.  The origins of these behavioral responses remains unclear
in most cases although some possibilities other than local anesthetic
action or presented in Conclusion 3.

6.   Because of their ability to inhibit prostaglandin synthesis, the
formamidines were found to have antipyretic, analgetic, and
anti-inflammatory effects which are comparable to those of aspirin.
However, they lack the potent gastric ulcerogenicity typical of
aspirin-like agents.

7.   The two successive metabolic N-demethylations of  chlordimeform yield
compounds of increased acute toxicity.  These N-demethylation  products
are produced rapidly by mouse liver microsomes ^n vitro and iin vivo.
Metabolic reactions which result in the alteration of  the formamidine
nucleus are detoxications, and, in part, appear to be  catalyzed  by
cytochrome P448 (3-methylcholanthrene inducible).  Studies with  several
mixed function oxidase inhibitors and inducers indicate that
N-demethylation in vivo tends to be a toxicologically  'neutral1  process.
Although the N-demethylation products are more toxic,  they  are also less
stable.  After dosing mice with chlordimeform, the best correlation of
toxicity is found with the total level of all formamidines  in  the brain
rather than with the level of any individual metabolite.

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                                SECTION  ONE

                     EVALUATION OF  MONOAMINE  OXIDASE

                 AS A  TARGET  FOR FORMAMIDINE  PESTICIDES

INTRODUCTION

     In considering the  possible biochemical  site of action of the
formamidines, much attention  has been directed  to the actions of
formamidines on monoamine-mediated  neurotransmission in both mammals and
arthropods  (Matsumura  and  Beeman, 1976; Knowles and Aziz,  1974).  In
particular  it has been reported that  these compounds inhibit monoamine
oxidase (MAO) activity in  rat liver (Beeman and Matsumura, 1973; Aziz
and Knowles, 1973) and in  rat brain (Benezet  .and Knowles,  1976a).  The
symptoms of acute poisoning  in the  rat  have been described as 'similar
to those elicited by sympathomimetic  agents including known MAO
inhibitors' (Aziz and  Knowles, 1973)  and as sympathomimetic in nature
and lacking normal cholinomimetic features (Beeman and Matsumura, 1973).
Beeman and  Matsumura (1973)  also reported that  treatment of rats with
chlordimeform (CDM) caused an elevation in the  serotonin and
norepinephrine levels  of the  brain  and  also antagonized certain symptoms
of reserpine intoxication  which are generally believed to  arise by
release of  biogenic amines from their presynaptic stores.   These
observations are stimulating  since  no other pesticides have been shown
to exert their primary effect through aminergic mechanisms.  Neumann and
Voss (1977), recently  have cast doubt on the  role of MAO inhibition in
the acute toxicity of  CDM  and some  related toluidine derivatives on the
basis of a  lack of correlation of anti-MAO potency and lethality to rats
among these compounds.  Additionally, Robinson  and Smith (1977),
selectively depleted rat brain serotonin (5-HT) and norepinephrine (NE),
or treated  rats with the a-adrenergic agonist,  phenylephrine.  None of
these pretreatments altered  the acute toxicity of CDM, and the authors
therefore concluded that MAO  was probably not involved in  its acute
toxicity.   Previously  they had shown  that pretreatments with reserpine,
to deplete  monoamines, or  with a-adrenergic and serotonergic blockers
also did not alter the LD^_ of CDM  (Robinson  ^t _al., 1975).  Meanwhile
the view that MAO inhibition  in particular, and interference with
aminergic transmission in  general,  play a central role in  generating the
poisoning syndrome has been  reiterated  (Matsumura and Beeman, 1976;
Knowles and Aziz, 1974;  Knowles, 1976). Until now there has been no
consideration of the potency  of CDM and its metabolites as MAO
inhibitors  in other vertebrates than  the rat, and no reports of their

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effectiveness as MAO inhibitors in vivo.   In the  investigation  presented
here such studies were made and the results related  to  the  hypothesis
that MAO inhibition is an important feature of the toxicology of the
formamidines in vertebrates.

MATERIALS AND METHODS

Chemicals

     The radiolabelled MAO substrates were tryptamine bisuccinate (48.5
and 53 Ci/mol), 5-hydroxytryptamine (48.5  and 51  Ci/mol), and
3-phenylethylamine (9.9 Ci/mol) from New England  Nuclear, and tyramine
hydrochloride (55 Ci/mol) from Amersham/Searle.   All were labelled with
  C in the side chain.  MAO inhibitors, tranylcypromine hydrochloride
and harmaline hydrochloride, were obtained from Sigma Chemical  Co.
Pheniprazine hydrochloride was the kind gift of Dr.  C.  Chinn.  The
formamidines JN'-(4-chloro-£-tolyl)-N^N-dimethylformamidine  (CDM),
N'-(4-chloro-o-tolyl)-Nrmethylformamidine  (DCDM), and
IJ-(4-chloro-o-tolyl)fonnamidine (DDCDM), and their metabolites
N-formyl-4-chloro-o-toluidine (NFT) and 4-chloro-o-toluidine  (CT) were
synthesized and purified as described in Section  7.  The identities and
purity of these synthesized products were  assessed by IR and  NMR
spectroscopy and by TLC on silica gel developed in benzene/diethylamine
(19:1).   Small amounts of NFT were sometimes present in the  parent
formamidines, particularly after storage.  The amount of NFT  did not
exceed 2-3% and, when present, did not significantly affect the results
obtained.  Particular care was taken before the MAO  assays  to reduce the
levels of NFT present in the formamidines.

Acute lethality

     Male Swiss white mice (20-25 g) were  obtained from Bellaire Acres,
Danville, IN.  The mice were starved for 4 hr before administration of
the toxicants by gavage in a corn oil vehicle at  0.01 ml/g  body wt.  At
least 5 doses with at least 10 mice/dose were utilized  and  the  tests
were replicated for each compound.  Mortality was determined  after 48 hr
and the LD   was determined by the method  of Litchfield and Wilcoxon
(1949).   5

Monoamine oxidase inhibition in vitro

     MAO activity was assayed by the general radiometric method of
Wurtman and Axelrod (1963).  Mouse tissues were dissected,  rinsed clean
in saline, weighed after drying, and homogenized  at  20  mg tissue/ml in
0.25 M sucrose containing 1 mM EDTA in a teflon-glass system.  A
mitochondrial fraction was isolated by differential  centrifugation,
initially at 600 g for 15 rain and with further centrifugation of the
supernatant at 12,000 g for 15 min.  The resulting pellet was washed by
resuspension in the same solution and recentrifugation. The washed
mitochondria were then resuspended in the  original volume of  Tris buffer
(50 mM, pH 7.5) containing 1 mM EDTA.

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     For the MAO assay, mitochondria  (0.3 ml)  were incubated with
inhibitor (^Q  1 in  ethanol)  for  30 min at 25° in a stoppered centrifuge
tube.  The   C-labelled amine substrate (tryptamine, tyramine,
e-phenylethylamine,  or 5-HT at a  final concentration of 10-30 PM) was
added in 10-20 yi  water and the MAO reaction allowed to proceed for
exactly 2 min before addition of  0.2  ml 2N HC1.  Toluene (5 ml) was
added, the tube shaken, and 3 ml  of the toluene layer was taken for
scintillation counting of  the amount  of deaminated product present.
Controls were run  in which only 10 Ul ethanol  was added to the enzyme.
Blank values obtained with 0.3 ml boiled enzyme were used to correct
these data.  In every MAO  assay enzymatic rates were determined in
triplicate and averaged.   Under these conditions the assay was linear
over several min with tissue concentrations up to 50 mg/ml.  I5Q values
were calculated graphically from  plots of percent inhibition or MAO
against log of inhibitor  concentration.  The I _ values reported are the
means of at least  three  such determinations.

     The reversibility of  the inhibition of MAO was examined by repeated
washing of the mitochondria to remove the inhibitor.   Mitochondria from
mouse brain were  incubated with 5 X 10   M COM.for 30 min.  One portion
was removed to assay for  MAO activity using [   C] tyramine, while the
rest was centrifuged, and  the mitochondrial pellet resuspended to its
original volume in inhibitor-free buffer.  After removing a further
portion for enzyme assay  the centrifugation and resuspension in fresh
buffer was repeated  several times more with samples removed for assay
each time.  An inhibitor-free MAO sample was treated similarly as a
control.  Protein  was determined  in each sample by a modified Lowry
method  (Schacterle and  Pollack, 1973) and the activities of MAO were
compared in the inhibited  and control samples after correction for any
loss of protein during  the washing procedures.

     In order to  study  the relation of MAO inhibition to time of
exposure to the inhibitor, intestinal_or brain mitochondria were
incubated with CDM or DCDM at 5 X 10"5M, or NFT at 5 X 10  M at 25 .
Portions were removed at  intervals from 20 sec-3 hr after addition of
the  inhibitor and  immediately assayed for MAO using tryptamine.
Activity was compared to  a control sample incubated without inhibitor.

MAO  inhibition in  vivo

     The effect of inhibitors on the  activity of MAO in mouse liver and
small intestine in vivo  was assayed indirectly from -the recovery of
injected C  C]-tryptamine.  The method was based on that of Wang Lu and
Domino  (1976) for  J<,N-dimethyltryptamine.  Potential MAO inhibitors were
given orally as their hydrochlorides  in water at sublethal doses except
for NFT which was  administered in corn oil.  Two hr later, labelled
tryptamine at 0.05 uCi/g  body wt. was injected intraperitoneally.  After
a further 5 min the mice  were killed  and the liver and intestine were
rapidly removed,  rinsed  with saline,  and homogenized in 4 ml cold IN
HC1.  The homogenate was  centrifuged  at 9000 g for 15 min, and the
pellet was washed  twice  by resuspension in 1.5 ml portions of 0.1N HC1
and centrifugation.   The  supernatants were combined and extracted with

                                       8

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10 ml ethyl acetate/toluene (1/1) and the aqueous phase was made
alkaline by mixing with 2 ml 10 N NaOH.  The residual tryptamine  was
then extracted with three 10 ml portions of the ethyl acetate/toluene.
The organic phase was pooled and 5 ml samples taken for scintillation
counting.  Chromatography of such extracts from liver on  silica gel
plates developed with n-butanol/acetic acid/water (25/4/10) showed that
the major peak of radioactivity cochromatographed with authentic
tryptamine.  When labelled tryptamine was added to the tissue during the
initial homogenization, recovery of the added tryptamine  ranged from
86-91% with a mean of 89%.  The results were not corrected for recovery
losses.  Preliminary experiments showed that the tissue levels of
tryptamine peaked at about 5 min after injection in untreated mice.
Further initial experiments were conducted as described above but with
various intervals between the administration of CDM.HC1 and tryptamine
from 15 min-24 hr to follow the change in MAO activity with time  after
exposure to the inhibitor.

RESULTS

Acute lethality of COM and its metabolites to mice

     The LD   values for CDM, its two N-demethylation products  (DCDM  and
DDCDM), and two hydrolysis products (NFT and CT) are presented  in Table
1.  It is notable that although CDM itself is only of medium potency
(LD,_0 = 267 rag/kg) successive N-demethylations yielded increasingly more
toxic metabolites with an LD Q for DDCDM of only 78 mg/kg.  With  CDM,

TABLE 1: ACUTE TOXICITY AND MAO INHIBITION IN VITRO BY CHLORDIMEFORM  AND
ITS MAJOR METABOLITES
                                                         MAOa,I5()(yM)
                                   LD__,mg/kg    Liver   Intestine   Brain
                                   (mouse,oral)
Chlordimeform (CDM) 267
N-Demethyl chlordimeform (DCDM) 163
N.N-Didemethyl chlordimeform (DDCDM 78.
N-Formyl-4-chloro-o-toluidine (NFT) 750
4-Chloro-o-toluidine (CT) >1000
47
21
133
4.5
88
121
61
73
2.8
185
122
69
104
3.2
253
 substrate:tryptamine bisuccinate.
 solvent:  propylene glycol.
DCDM, and DDCDM, all of which contain  the  formamidine nucleus, symptoms
developed in less than 1 hr after  dosage and  were excitatory in nature.

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Hyperexcitability, marked  tremors  in  the  head  and  limbs,  gasping,  and
rapid death following one  or more  convulsive  episodes were typical of
all three compounds.  The  other  two metabolites (NFT and  CT)  were  less
acutely toxic, slower-acting,  and  depressant  in their effect.  A gradual
loss of responsiveness  to  external stimuli  and hypothermia were noted in
each case.

Innibition of MAO  in vitro

     The anti-MAO  potency  of  COM and  these  metabolites was compared in
mitochondria from  three tissues, the  liver, brain, and small  intestine.  The
I   values are presented in Table  1.   Although the tissues differed somewhat
in their relative  sensitivities  to these  agents, the differences were not
extreme and no regular  pattern of differential sensitivity was apparent.  Most
I50 values were  in the  range  of  20-200 yM.   Successive N-demethylations in
tnis case did not  greatly  increase biological potency since although DCDM was
about twice as effective as a  MAO inhibitor compared to CDM,  DDCDM was similar
to CDM in its overall  effectiveness.   However, one metabolite, NFT, was
notably better than  any other  compound as a MAO inhibitor with all three
tissues, having  I,-0  values in  the 3-5 UM  range.  In contrast, the parent
toluidine, CT, was somewhat less effective  than CDM.

Reversibility of MAO inhibition

     The recovery of mouse brain MAO from inhibition by CDM on repeated
washing is shown in  Fig. 1.   The initial  level of inhibition  was 72%, this was
reduced to 28% after one wash, 1% after the second, and activity returned to
the control level  with  subsequent washes.  This behavior was  repeated with
DCDM and the more  potent inhibitor, NFT,  as shown in Table 2.  With these
compounds the mitochondria were  washed twice after exposure to the inhibitor
for 30 min at 25°.  In  each case inhibition was reversed essentially to
control levels.   The high  variability in  the data for the washed mitochondria
arises because of the  substantial increase  in MAO activity which occurred on
washing the mitochondria in both the treatments and controls.

The time course  of inhibition of MAO

     When incubated  with MAO  from brain or  intestine for periods from
20 sec-3 hr, all three  compounds tested (CDM, DCDM, and NFT)  gave essentially
instantaneous inhibition.   With  NFT,  inhibition after 20 sec  was the same as
after 3 hr and at  all  intermediate times.  However, the two formamidines
showed a more complex  behavior.   A large part of the final level of inhibition
achieved at 3 hr was obtained 'instantaneously1, but in each case a slow
progressive component  also was observed so  that the degree of inhibition rose
steadily about 7%/hr over  the 3  hr observation period.  This  behavior is
illustrated for  the  intestinal mitochondria in Fig. 2.
                                     10

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          1OO
                             NO.   WASHES
Figure 1.  Recovery of mouse brain mitochondria! MAO from inhibition by CDM
(50 UM) after successive washes to remove the inhibitor.

TABLE 2.  REVERSIBILITY OF INHIBITION OF MITOCHONDRIAL MAO FROM
MOUSE BRAIN BY CHLORDIMEFORM AND ITS METABOLITES ON WASHING
                                                  Mean % Inhibition (+SD)'
                                                 Unwashed
                    Washed
Chlordimeform (50
]}-Demethyl chlordimeform (50yM)
N-Formyl-4-chloro-o-toluidine (5yM)
68.5+4.9
86.1+0.7
84.1+2.1
 2.4+ 2.7
 8.9+14.2
-1.4+ 5.6
 Mean of three replicates.  Tyramine hydrochloride as substrate.
                                     11

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  Classification  of  CDM  and  its  metabolites  as MAO  inhibitors
        In  order  to  determine  whether  these  compounds are Type A or Type B
   inhibitors,  the  inhibition  of  MAO from  mouse  intestine was assayed over a wide
   1OO   '
   80

I
a  60
   40
   20
                                          __— DCDM
                                  	— 	   *
 >„---*
                                                                           CDM
                 3O
60
90
120
150
180
                                MINUTES
   Figure  2.   Time  course of inhibition  of  MAO  from mouse intestine by
   chlordimeform (CDM,  50yM), N-demethyl chlordimeform  (DCDM, 50yM), and
   N-formyl-4-chloro-o-toluidine (NFT,  5yM).

   range of inhibitor  concentrations  with three substrates, 5-HT (Type A),
   tryptamine  (Mixed A and B),  and  g-phenylethylamine (Type B).  The results are
   shown in Figs. 3 and 4, for  CDM  and  NFT  respectively, derived from three
   independent experiments in each  case.  DCDM behaved similarly to CDM.  For each
   of  the  inhibitors,  the MAO reaction  with 5-HT is most sensitive to inhibition
   and 3-pnenylethylamine is the substrate  least effective for inhibition, with
   tryptamine  intermediate.   The distinction between these three substances is
   most evident with NFT.  The  I-Q  values obtained for  the three substrates,
   5-HT, tryptamine, and 3-phenyIethylamine were 34,100, and  140  M respectively
   for CDM, 12, 33, and 39 yM for DCDM,  and 1.2,  3.0, and 12 yM for NFT.  In
   limited  studies  with tyramine, another mixed A and B type  substrate, the
   results  were very similar to those obtained  with tryptamine.

   Inhibition  of MAO in vivo

       The assumption  that  the inhibition  of MAO would allow more injected
   tryptamine  to survive intact in  the tissues  is confirmed by the results in
   Table 3.  Although  this method proved  effective for  assessing MAO activity in
   vivo in  liver and small intestine, the amount  of labelled  tryptamine present
                                       12

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in the brain was too small to allow accurate determinations for this tissue.
Tranylcypromine and pheniprazine are well-established and potent irreversible
MAO inhibitors, and with these compounds 4-6 times as much tryptamine was
recovered in the liver and intestine as in the untreated controls.  Since
tranylcypromine is an irreversible inhibitor, MAO inhibition was also assayed
by homogenization of tissues from treated mice (15 mg^kg) followed by our
normal MAO assays with isolated mitochondria using [  C] tryptamine as
substrate.  Under these conditions tranylcypromine caused 80% inhibition of
liver MAO and 92% inhibition of intestinal MAO in vivo 2 hr after dosage.

1OO
  80
  60
 4O
  20
     1O
       -6
1O'
10
                                                       .-4
1O"
                              INHIBITOR  CONC. (M)
Figure 3.  Inhibition of MAO from mouse intestine by COM assessed with three
MAO substrates:  5-Hydroxytryptamine, 0	• ; Tryptamine, X	x;
3-phenylethylamine, O	O •

     Harmaline, a reasonably potent reversible inhibitor of MAO, did not
protect tryptamine in the liver but did increase the amount of tryptamine
surviving in the intestine.  All of the standard MAO inhibitors were given at
doses which caused no mortality and little if any external sign of toxicity.

     CDM at 100 mg/kg also caused no strong poisoning symptoms and gave no
significant increase in tryptamine recoveries. At 200 mg/kg poisoning symptoms
were clear and an occasional death occurred.  In this case a significant
increase in tryptamine levels were seen, but this was much less than the
increase caused by tranylcypromine and pheniprazine at their symptomless
                                     13

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100
                            INHIBITOR  CONG. (M)

Figure 4.  Inhibition of MAO from mouse intestine by NFT assessed with three
MAO substrates:  5-Hydroxytryptamine, •	*; Tryptamine.B	•;
3-Phenylethylamine x—	-/.•


doses.  NFT as a more potent MAO inhibitor than CDM caused a greater
accumulation of tryptamine at  100 mg/kg than did CDM despite difficulties with
getting the dose of  NFT into solution  in corn oil at this concentration.

     The justification for the choice  of 2 hr as the time delay after CDM
dosage before administration of the tryptamine is illustrated by the data in
Table 4.  In this case varying times were left between the doses of
chlordimeform (150 mg/kg) and  tryptamine in order to evaluate the dynamics of
MAO inhibition in vivo.  The increase  in tryptamine levels was not great at
this dose but appeared to peak around  the 2-4 hr period after dosage and to be
declining by 6-12 hr with a return to  normal levels within 24 hr.
                                    14

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                                 1U
TABLE 3.  RECOVERY OF  INJECTED  [I4C]  TRYPTAMINE AFTER PRETREATMENT OF MICE
WITH MAO INHIBITORS
                                  (pmol  tryptamine  recovered/g tissue) —SD (n)
                                                                              a
                                   Dose           Liver              Intestine
                                   (mg/kg)
Tranylcypromine
Pheniprazine
Chlordimeform
Chlordimeform
N-Formyl-4-chloro-c>-tol uid ine
Harmaline
Control
15
20
200
100
100
50
	
562 + 183(8)°
536 + 90(8)
237 + 59(10)
118 +37(7) „
198 + 36(9)
83 + 24(9)
86 + 27(10)
632 + 126(8)C
482 + 84(8) h
233 + 47(10)°
152 + 50(7)
218 + 84(9)
359 + 155(9)
131 + 39(10)
 S.D.(n) = standard deviation (number of observations).
 Significantly different from control at P =  0.05.
 Significantly different from control at P =  0.01.

DISCUSSION

     The data presented in the initial section dealing with  MAO inhibition j.n
vitro establishes that with mitochondrial MAO from  several mouse tissues, CDM
is a readily reversible inhibitor of medium potency,  ^n  vivo it is rapidly
metabolized by successive N-demethylations to yield DCDM  and then DDCDM.
These formamidines are susceptible to hydrolysis to NFT and  then CT (see
Section 7).  These metabolites are in general rather  similar to CDM in potency
against MAO and, in the case of DCDM and NFT  at least, are also reversible
inhibitors.  The only exception to these generalizations  was the 10 to 40-fold
greater potency of NFT compared to CDM as a MAO inhibitor jln vitro. Thus the
metabolism of CDM to NFT is potentially an activation reaction as far as MAO
inhibition is concerned.  The I^Q values here are generally  similar to those
obtained by other investigators working with  MAO from rat liver (Beeman and
Matsumura, 1973; Aziz and Knowles, 1973) and  rat brain (Benezet and Knowles,
1976).  Benezet and Knowles (1976) in their more detailed study of the
mechanism of inhibition of MAO by formamidines also noted the greater potency
of NFT against rat brain, although in their earlier study with rat liver MAO,
NFT was not found to be more effective than CDM.  In  our  work with the mouse,
NFT was clearly more potent than CDM against  MAO from all tissues examined
including liver.  Otherwise MAO from the rat  and the  mouse responds to CDM and
its metabolites similarly.
                                      15

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     From the studies reported  here  it  is  evident  that  NFT  inhibits  MAO with
no progressive component when enzyme and  inhibitor are  preincubated  in  the
absence of substrate.  However,  superimposed  on  the 'instantaneous phase' with
CDM and DCDM was a slow progressive  increase  in  the degree  of inhibition
observed.  This effect was  also  noted by  Benezet and Knowles  (1976a)  for CDM
and DCDM with their rat brain MAO.   They  suggested that this  might be due to
tho slow generation of NFT  by hydrolysis  of the  formamidines.  Since NFT is a
more potent inhibitor, the  level of  inhibition would thus slowly rise.   This
is a plausible explanation  and  in accord  with the observed  rates of  hydrolysis
of these formamidines  (Section  7).   Our observation that NFT  itself  lacks a
progressive component of  inhibition  is compatible with  this explanation.

TABLE 4.  THE EFFECT OF TIME AFTER  DOSING ON  THE ABILITY OF CHLORDIMEFORM TO
INCREASE THE RECOVERY OF  INJECTED [C]TRYPTAMINE FROM  MOUSE  TISSUES
                                          Tryptamine recovered

                                        (pmol/g tissue)  + SD (n)
Hours after
CDM dose3
0
0.25
0.50
1.0
2.0
4.0
6.0
12.0
24.0
Liver
65 + 6(8)
95 + 34(6)
81 + 24(6)
85 + 16(5)°
125 + 32(5)°
106 + 28(5)°,
104 + 28(41°
92 + 8(4)a
64 + 6(5)
Intestine
105 + 20(8)
121 + 42(5)
97 + 21(6)
139 + 57(6)
136 + 16(5)°
163 •»• 66(6)c
142 + 39(5)°
90 + 15(8)
108 + 11(5)
.Dose was  150  mg/kg,  oral.
 SD  (n)  =  standard  deviation (number of observations)
°Significantly different from control at P = 0.05.
 Significantly different from control at P = 0.01.
     It  is  known  that mitochondrial MAO exists in several forms which may be
distinguished  by  their differential responses to inhibitors, thermal
inactivation,  and differing substrate specificities (Squires, 1968 and 1972;
Neff and Yang,  1974).  Particular attention has been focused on the division
of MAO enzymes  into two classes, type A (with 5-HT and NE as preferred
substrates)  and type B (with 3-phenylethylamine and benzylamine as preferred
substrates).   Several substrates are oxidized with similar efficiency by both
types of MAO e.g. tryptamine, tyramine, dopamine, and kynuramine.  Similarly
some inhibitors such as tranylcypromine and pheniprazine are not particularly
selective between the two forms of MAO while others are quite specific for one
                                      16

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of the types.  Thus harmaline and clorgyline are considerably more  potent  as
inhibitors of type A MAO while deprenyl is a relatively specific  inhibitor of
the B type.  Different tissues vary widely in their relative contents  of types
A and B MAO and such variations are not constant among different  species.   The
data in Figs. 3 and 4 show clearly that CDM, DCDM, and particularly NFT  are
more potent inhibitors when 5-HT is the substrate than when 3-phenylethylamine
is the substrate.  Thus they appear to be selective inhibitors of type A MAO,
although the degree of selectivity is quite low.  As would be expected with
tryptamine or tyramine as substrate, the potency is intermediate  since these
compounds act as efficient substrates for both types of MAO.  However, if  the
situation were as simple as the assumptions above indicate, one would  predict
that a plateau in the inhibition curve should be observed using tryptamine,
since the activity of the type A MAO against this substrate should  be
inhibited before that of the type B also present.  Such plateaus  are commonly
seen with more highly selective inhibitors such as deprenyl, clorgyline, and
harmine (Squires, 1972; Neff and Yang, 1974; Yang and Neff, 1974).   However
from these results it may be concluded that, like other selective inhibitors
of type A MAO such as clorgyline (Yang and Neff, 1974), CDM and its
metabolites jji vivo should cause the preferential accumulation of the
substrates for this form of MAO i.e. 5-HT, NE and dopamine if any high degree
of inhibition were induced.  Beeman and Matsumura (1973)t report  a  22%
increase in NE and a 70% increase in 5-HT in the brains of rats 1 hr after an
intraperitoneal dose of 200 mg/kg of CDM (LD Q).  The results discussed  above
were obtained with mouse intestinal mitochondria.  Recently Neumann and  Voss
(1977) obtained K. values for CDM against rat liver MAO using the same
substrates as in our study plus kynuramine.  No trends in the K.  values  with
substrate were found which would allow classification on the type A/type B
system.  However, using rat brain MAO, Benezet and Knowles (1976) obtained
results rather similar to ours with the same substrates.

     The data presented in this section offer several lines of evidence
regarding the probable contribution of MAO inhibition to the acute  lethality
of CDM.  We believe that this evidence strongly disfavors the idea  that  MAO is
the primary target.  Despite the fact that CDM and its metabolites  show  some
degree of potency as MAO inhibitors ^n vitro, there is no correlation  between
the ability to inhibit MAO and either the lethality or symptoms of  poisoning.
Sequential J4-demethylations decrease the LD_Q values in the formamidines (CDM,
DCDM, DDCDM), but there is no corresponding trend in the I   values for  MAO
from any of the tissues examined.  On the other hand NFT is easily  the best
MAO inhibitor, but is not particularly toxic.  Even the poorly toxic
toluidine, CT, is an inhibitor with activity in about the same range as  the
formamidines.  This suggests that MAO inhibition is more dependent  on  the
presence of the substituted toluidine moiety than on the presence of an  intact
formamidine nucleus.  A similar conclusion was reached by Neumann and  Voss
(1977) with a series of substituted chlorotoluidines including CDM  and NFT.
Furthermore, although CT is comparable to the formamidines in  its potency
against MAO, and NFT is more effective, their speed of action  and symptoms are
completely different from those of the formamidines.  Rapid mortality
accompanied by marked excitation was seen only for compounds with an intact
formamidine nucleus.  Since MAO is not an enzyme which is immediately
essential to the integrity of nervous functions and for survival  of the


                                     17

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organism, as compared  for  example  to  acetylcholinesterase,  the rapid  onset of
symptoms and strong excitatory  effects  of CDM  and  the  related  formamidines is
not in keeping with MAO  as the  site of  action.  Many potent MAO inhibitors
such as tranylcypromine  and  the hydrazines exert their acute toxic actions
through mechanisms other than MAO  inhibition  (Pletscher et  al., 1966) and CDM
probably resembles them  in this respect.

     The results obtained  here  with MAO inhibition in vivo  are entirely
consistent with this conclusion.   Since CDM is a readily reversible inhibitor
of MAO, estimation of  the  degree of inhibition _in vivo presents severe
problems because iin vitro  assays of MAO which  involve tissue homogenization
and dilution before assay  will  partially reverse the inhibition.  Thus an
indirect but potentially more realistic method of assessing the status of MAO
JJ1 vivo was adopted which  measured the  ability of injected  [  C]tryptamine to
survive in the tissues.  Judging by the observed actions of the known MAO
inhibitors, tranylcypromine, pheniprazine, and harmaline, this method is an
appropriate one for assessing the  status of MAO in. vivo*  The first two
compounds are  potent, irreversible MAO inhibitors which are not selective
between type A and type  B  MAO (Neff et^l., 1974).  In both liver and
intestine they cause  a substantial increase in the amount of tryptamine
recovered.  Harmaline  is a reversible inhibitor which is strongly selective
for type A MAO  (Neff  et_ al., 197M).   It was found to preserve tryptamine in
the intestine but  not  in the liver.   This is reasonable since while the MAO of
mouse  intestine is about 70% of the A type, this enzyme class is virtually
absent in mouse liver  (Squires, 1968  and 1972).  Thus, in the liver the
tryptamine is destroyed  by type B MAO which is insensitive to harmaline.

     Assayed in this  way,  CDM causes  only a moderate degree of MAO inhibition
in_ vivo even at near-lethal doses.  This is a degree of inhibition which other
established MAO inhibitors, such as  tranylcypromine and pheniprazine, easily
exceed without any apparent signs  of  poisoning.  It seems quite unlikely that
the level of MAO  inhibition after  an  oral dose of CDM as high as 200 mg/kg
approaches the 85% or  more necessary  to cause significantly damaging
accumulation of biogenic amines in the brain and other organs  (Pletscher gt
al., 1966.  Tranylcypromine at  15  mg/kg does cause this degree of inhibition,
but also leads to  a  far  greater accumulation of tryptamine than does CDM at
200 mg/kg.

     From the I_n  data of  Table 1, one would not expect CDM to be a more
effective inhibitor of brain MAO in vivo compared to liver or  intestinal MAO.
However, this could  not  be assessed  by the tryptamine recovery method and
further data are  needed  regarding the status of the MAO in the brain during
CDM poisoning.

     The data of  Table M reveal that  the MAO inhibition which does result from
dosage with CDM is not long-lasting,  being essentially reversed within  12-2*1
hr after exposure.  This is entirely what would be expected from a rapidly
metabolized reversible inhibitor such as CDM.   There thus seems to be little
risk of the cumulative effects  on  MAO and possible interactions with natural
or medicinal biogenic  amines which are seen with many of the irreversible
inhibitors (Sjoqvist,  1965). even  with repeated exposure to rather large
amounts of CDM.

                                      18

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     Although our work does not exclude the possibility that  under  some
circumstances, in some species, the MAO-inhibitory actions of COM may  present
some hazard or contribute to the symptoms observed, it does indicate that  this
risk is probably not great and that the biochemical lesion underlying  the
acute lethality of the fortnamidines to mammals must be sought elsewhere.   It
has been suggested that other effects on the regulation of biogenic amines may
be important in the lethality of COM (Matsumura and Beeman, 1976; Knowles  and
Aziz, 1974; Knowles, 1976).  Although this cannot be ruled out,  there  is
little direct evidence to support this view, and other workers have shown  that
blockade of serotonergic and a-adrenergic receptors, and prior depletion of
tissue stores of these amines did not reduce the lethality of CDM to rats
(Robinson and Smith, 1977; Robinson ^t jl., 1975).  A more plausible
hypothesis on the basis of current information relates the lethal actions  of
CDM and related formamidines to their local anesthetic-like actions (See
Section 4) with a consequent depressor effect on the cardiovascular system
(See Section 2) and marked central stimulation with enhanced  sympathetic
outflow which may account for the sympathomimetic symptomatology reported  by
other investigators (See Sections 3 and 4).  A local anesthetic-like action
could also account for the depression in motor end-plate sensitivity and
inhibition of the depolarization-contracture process observed with  the frog
neuromuscular junction (Wang^t^., 1975; Watanabe jjt al., 1976).  In fact
Watanabe et aL., (1975) briefly noted some similarities between  CDM and the
local anesthetic, procaine, in their actions on the acetylcholine-stimulated
contraction of the frog rectus abdominis muscle.  However the formamidines are
compounds with actions on multiple biochemical systems, and it is clearly
premature to ascribe all observed effects of these compounds  to  any single
type of biochemical action.
                                      19

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                                   SECTION TWO

             ACTIONS OF  CHLORDIMEFORM ON  THE CARDIOVASCULAR SYSTEM

INTRODUCTION

     As a first  step in  attempting to define the cause of death and toxic
mechanism with chlordimeform  we  started with the observation of Beeman and
Matsumura (1974) that  200  mg/kg   of COM,  ip, decreased the mean arterial bood
pressure in the  pentobarbital-anesthetized rabbit.   The purpose of this
investigation was therefore to  analyze further the  effects of CDM on the
cardiovascular system.   Parallel studies  in these laboratories (Sections 3 and
4) showed that CDM  shared  many  of the actions of local anesthetic agents,
hence the cardiovascular actions of CDM were compared to those of lidocaine to
determine if these  agents  had common sites and modes of action.

MATERIALS AND METHODS

Blood pressure,  heart  rate, and  cardiac contractility

     Fourteen dogs  of  both sexes, 7-12 kg each, were anesthetized with 30-35
mg/kg of pentobarbital  sodium iv.  The left femoral arterial bood pressure was
monitored via a  Statham  pressure transducer with a  Grass polygraph.  Drugs
were administered via  a  cannula  in the cephalic vein.  Lead II of the ECG and
beat-to-beat heart  rate  were  recorded on  an E & M Physiograph.  Artificial
respiration was  provided to the  dogs using a Harvard Apparatus respirator, and
right ventricular contractile force was monitored by means of a Walton-Brodie
strain gauge sewn onto  the right ventricle.

Blood pressure,  heart  rate, and  peripheral resistance

     Fifteen dogs of both  sexes, 7-12 kg each, were anesthetized with 30-35
mg/kg of pentobarbital  sodium iv.  The right carotid artery and the left
external jugular vein  of each animal were cannulated and arterial and venous
pressures were recorded.  Lead  II of the ECG was monitored along with the
heart rate on a  cardiotach.   The dogs were given 600-700 units/kg of heparin.
The right femoral artery was  cannulated both toward and away from the heart.
Blood was pumped from  the  femoral artery by a Harvard Apparatus
constant-perfusion  pump  and back into the leg via the femoral artery.
Perfusion pressure  was  monitored with a Statham pressure transducer distal to
the pump and was displayed on the polygraph.  The flow rate through the pump
was adjusted (usually  27 ml/min) so that  the perfusion pressure approximated
the carotid pressure.   The volume of the  perfusion  apparatus was 34 ml.  Drugs
were administered via  a  cannula  in the cephalic vein or intraarterially (1 ml
volume) into the perfusion apparatus distal to the  pump.
                                     20

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  Contractility of  isolated  rabbit  hearts

       Eighteen conventional  Lagendorf rabbit heart preparations were perfused
  with 38°, oxygenated  Locke-Ringer solution.  Contractility was displayed on a
  Physiograph using  a Type B,  E  & M myograph, and heart rate was monitored on
  another channel via a  cardiotach.  Drugs (1 ml volume) were injected into the
  perfusate as it entered the  aorta.

  Sympathetic outflow in the  cat

       Six cats, 2.6-3.4 kg,  were anesthetized with 30 mg/kg of pentobarbital
  sodium.  Both cervical sympathetic  nerves were exposed and placed on bipolar
  electrodes for recording,  and  the cervical region was filled with warm mineral
  oil.  Spike activity  was amplified  by a Tektronix 122 preamplifier and spike
  discharge frequency was recorded  on the polygraph.  Drugs were administered
  via a cannula in  the  cephalic  vein.

  RESULTS

  Blood pressure

       Chlordimeform or  lidocaine at  doses of 3-30 mg/kg iv. in dogs, caused
  dose-dependent decreases in  mean  arterial blood pressure within 1 min.  A
  secondary increase was observed after COM injection which returned to predrug
  values in less than 1  hr (Fig. 5, Table 5).  Hyperventilation, tremors,
  particularly in the forelimbs, and  occasionally clonic convulsions were
  associated with the transition from depressor to pressor responses.  Animals
  under light anesthesia, as  determined by the presence of active toe pinch and
  corneal reflexes,  exhibited  tremors and large secondary pressor responses
  after 10 or 30 mg/kg  of COM iv, while animals under deep anesthesia showed
  neither tremors nor pressor  responses,  indicating that these were of central
HI Rate 1<
Beals/min ft
Blood
Pressure
mm Hg
          f 30 mg/kg
          COM, i.v.
30 sec
TlOmg/kg
I Lido iv
  Figure 5.  Typical records  showing  the  effects of (a) CDM and (b> lidocaine  on
  heart rate, cardiac  contractility (in  arbitrary units), and blood pressure  in
  the pentobarbital-anesthetized  dog.

  origin.  Secondary pressor  responses were smaller and less frequently  observed
  following lidocaine.   Similarly,  somatic manifestations of CNS stimulation  by
                                       21

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    TABLE  5.   TIME COURSE OF THE PRIMARY (1°) AND SECONDARY  (11°)  BLOOD  PRESSURE  AND CARDIAC CONTRACTILITY


    RESPONSES IN CHLORDIMEFORM-TREATED DOGS
                                                                        Time  (min)  to:
t-0
NJ

Dose
( mg/kg iv)
3
10
30

n
9
9
6

1°
Depressor
response
0.7 + 0.2a
0.8 + 0.1
0.8 + 0.1
1°
Decreased
contractility
response
0.9 + 0.2
0.7 + 0.1
0.7 + 0.1

11°
Pressor
response
5.6 - 1.3
10.5 + 1.6
14.6 + 0.5
11°
Increased
contractility
response
3.8 + 1.3
9.5 + 1.6
14.2 + 0.5

Recovery
to control
12.8 + 0.7
29.5 + 4.0
53.3 + 4.3
      Mean + SE.

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   lidocaine were less intense and less frequently observed  than after CDM.

        Dose-response data were collected on animals  that  were  "light" by the
   above criteria.  Level of anesthesia appeared to have no  effect on the blood
   pressure depression caused by CDM or lidocaine.  Blood  pressure responses to
   CDM in four unanesthetized but restrained rabbits  were  qualitatively identical
   and quantitatively similar to those shown in Fig.  5.

        Lethal doses of lidocaine (>30 mg/kg) or CDM  (>50  mg/kg) caused an
   irreversible decrease in blood pressure.  Although respiratory arrest occurred
   almost simultaneously, cardiovascular collapse was assumed to be the primary
   cause of death since artificial respiration did not protect  the dogs against
   death.  Lead II of the ECG showed that neither CDM nor  lidocaine caused
   cardiac arhythmias at the doses used in these experiments.

   Heart Rate and Cardiac Contractility

        Figure 5 and Table 5 show that CDM caused a depression  of cardiac
   contractility concurrent with the decrease in mean blood  pressure, as well as
   a secondary increase concurrent with the increase  in blood pressure.  The
   dose-response curves for these events are shown in Fig. 6.   The inotropic
   effects did not appear to result from changes in diastolic filling of the
   heart since vena cava pressure recordings at the level  of the right atrium
   (six experiments) showed no decrease or increase in venous pressure associated
   with the decrease or increase in contractility, respectively.  Therefore, CDM
   must have some effect either on the autonomic nervous system or on the
   myocardium itself.
£  -
o
a
   20
   -40
   -60
               3         10
               mg/kg, i.v.
                                  80
  60 «-
                                             I
-20
             o
             a.
             tn
             &
              :-40
                                  20
                                    W
                                              o
                                             u
             °M-60
30
            3       10
             mg/kg. i.v.
                                              80
  60 s

     o
     a
     VI
     41
     at
  40 ~
     u
     O
                                   O

                                20°H
30
   Figure 6.  Initial I°(»	•), and secondary,  11°  (I
   CDM and lidocaine on blood pressure and cardiac contractility.
   expressed as a percentage of predrug control  (mean +_ SE,  number of
   observations).
                                    ),  effects of
                                    Results are
                                       23

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     Bilateral vagotomy (five experiments) or 1 mg/kg of atropine (three
experiments) had  no  effect on the cardiac depression caused by 10 or 30 mg/kg
of COM.  The same CDM doses had no effect on the positive chronotropic and
inotropic effects of the ganglionic stimulant DMPP (1 yg/kg, two experiments)
or the 3-adrenergic  agonist isoproterenol (1 yg/kg, three experiments).
Furthermore, CDM  caused a decrease in contractility very similar to that of
lidocaine in the  isolated rabbit heart (Fig. 7).  Therefore, CDM depressed
cardiac muscle independently of the autonomic nervous system in a way similar
to that of  lidocaine.

The secondary  increase in cardiac contractility was observed following CDM in
the dog but not  in the isolated rabbit heart preparation and therefore it is
not a direct cardiac effect.  The observations in the dog that propranolol (1
mg/kg, three experiments), hexamethonium  (10 mg/kg, three experiments), and
diazepam  (1 mg/kg, five experiments) all  blocked the increase in contractility
and blood pressure caused by 10 mg/kg of  CDM further suggests that the
positive  inotropic effect of CDM results  from sympathetic activity of central
origin.
                           M ml.ol
                           I I mM COM
             30 sec.
                       lOOr
                        10
10-*         IO'J
Molar Concentration
Figure 7.   The  effects of CDM (I
     I) and  lidocaine  (<
on heart
rate and  cardiac contractility (in arbitrary units)  in the  isolated rabbit
heart preparation (7b).   Doses are expressed as the  concentration of  1 ml
injected  into  the perfusate as it enters the aorta.  Results are expressed  as
a percentage of predrug  control (mean + SE, number of observations).  A
typical record is shown  in la.
                                     24

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Peripheral Resistance

     Chlordimeform or lidocaine  injected  into  the  femoral  artery caused a
decrease in perfusion pressure in the  perfused  hind  limb of the dog (Fig. 8a).
Figure 8b shows that CDM was approximately  10  times  more potent in this action
than lidocaine.  Propranolol at  2 mg ia  (two experiments)  or 5 mg of atropine
ia (two experiments) blocked the decrease in perfusion  pressure caused by 2 yg
of acetylcholine (ACh) ia, respectively, but neither blocking drug attenuated
the response to 10 mg of CDM ia.  The  increase  in  perfusion pressure caused by
2 yg of norepinephrine ia was not altered by 10 mg of CDM  ia (three
experiments).  Tripelennamine at 2 mg  ia blocked the decrease in resistance
caused by 5 yg of histamine ia,  but not  that caused  by  CDM (two experiments).
                           a
                       70
                       90
                     J
                       10
                         0.1
                              SOllM
                                      I mg COM
                                        10
                                                 30 sec
                                                    CDM
                                        1.0
                                      mg-i.a.
                                                      10
                                                      0  and lidocaine
Figure 8.  The effects of intraarterial  CDM  (•	
(•	•) on the hind limb perfusion  pressure  in the pentobarbital-
anesthetized dog (8b).  Results are  expressed as  a percentage of the predrug
control (mean +_ SE, number of observations).  A typical record is shown in 8a,

Finally, denervation of the perfused  limb by the  ligation of the femoral and
sciatic nerves had no effect on the  intraarterial  CDM response.  These
findings suggest that the decrease in perfusion pressure caused by
intraarterial CDM is caused by nonspecific relaxation of vascular smooth
muscle and is not the result of adrenergic blockage or of cholinergic,
3-adrenergic, or histaminergic stimulation.
                                     25

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     When  CDM or  lidocaine was injected iv at 10-30 mg/kg, hind limb perfusion
pressure decreased  simultaneously with the systemic blood pressure.  The
secondary  rise in systemic pressure caused by CDM was accompanied by a rise in
perfusion  pressure.   This rise was abolished by ligation of the femoral and
sciatic nerves (six  experiments), by 10 mg/kg of hexamethonium iv (one
experiment),  and  by  5 mg of phentolamine ia (one experiment).  Therefore, it
appears that  CDM  given iv in addition to its cardiac effects, caused a
decrease in  peripheral resistance that contributed to the depression of
orterial blood pressure, and a secondary increase in resistance apparently
mediated by  the sympathetic nervous system which probably contributes to the
secondary  pressor response.

Central Sympathetic Involvement

      CDM administered at doses of 1-30 mg/kg iv to cats produced an initial
decrease in  blood pressure followed by a secondary increase as in dogs.
Recordings of spike  discharge frequency from the cervical sympathetic nerves
in  six cats  showed  that 10 mg/kg of CDM caused a M6 +_ 28% increase in mean
discharge  rate over  the predrug control level (seven observations).  This
increase in  rate  was concurrent with the secondary blood pressure response,
thereby providing further evidence that the secondary pressor response results
from  a central sympathetic discharge (Fig. 9).  Surprisingly, the initial
depressor  response  was accompanied by a decrease in cervical sympathetic
discharge  rate, suggesting that some central nervous system action of CDM
might  contribute  to the initial blood pressure depression.  Another
observation  lending support to this idea is that 10 mg/kg of CDM iv blocked
the pressor  response caused by bilateral carotid occlusion (five dogs).
Blood
Pressure
(mm Hg) 100

       50
             t
10 mg/kg
CDM. i.v.
2 min.
Figure  9.   Typical record showing the effects of CDM on blood pressure and
spike discharge frequency (in impulses per second) recorded preganglionically
from the superior cervical nerve of the pentobarbital-anesthetlzed cat.
                                      26

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DISCUSSION

     The experiments reported here show that cardiac depression  and
vasodilation resulting in hypotension can account for death  in animals  exposed
to COM.  Respiration is depressed by COM but artificial respiration  does  not
alleviate or delay toxicity.

     Both the cardiac and vascular depression involve direct  effects of CDM on
cardiac and vascular smooth muscle since neither denervation  of  these
structures nor autonomic pharmacological manipulations reduced the depressor
responses.  Cardiovascular effects of toxic doses of local anesthetics  have
been well studied (for a review see Covino and Vassalo, 1976) and have  been
found to be identical to those described here for CDM.  Although qualitatively
identical effects are observed in this investigation with CDM and lidocaine,
interesting quantitative differences are present.  In concurrent studies
(Section 4) we found that lidocaine is 10 times more potent  than CDM in
blocking the action potential of the frog sciatic nerve.  Figures 6  and 7 show
that CDM and lidocaine depress the dog heart, isolated rabbit heart, and  blood
pressure with similar potency while Fig. 8 shows that CDM is  10  times more
potent than lidocaine in dilating peripheral blood vessels.   The lack of any
quantitative correlation between intrinsic anesthetic potency, as defined
using nerve conduction blockade, and potency of effects on other excitable
tissues is well documented for local anesthetics (Covino and  Vassalo, 1976).
It is therefore not particularly surprising that no such correlation is
observed between CDM and lidocaine.

     The increased contractility and vasoconstriction, which  were responsible
for the secondary pressor response, were blocked by sympatholytic agents and
were absent in the isolated rabbit heart and denervated perfused hind limb
preparations.  Other observations also indicate that the secondary pressor
response results from massive sympathetic discharge, which in turn is an.
autonomic component of CDM-induced seizures.  Indeed, the blood  pressure
oscillations and discharge patterns of the superior cervical  sympathetic nerve
were similar to those observed during picrotoxin and strychnine  seizures
(Polosa jet al., 1969).  Further, the CDM-induced secondary pressor response
was observed under conditions which favored the appearance of tremors and
clonic limb movements.  Both pressor and seizure responses were  observed
following the injection of 3 to 4 mg of CDM into the lateral  ventricles of
rats (Section 3).  Finally, diazepam, which antagonizes local anesthetic
seizures (de Jong, 1972), blocked both responses of intraventricular CDM
(Section 3) as well as the secondary pressor response of intravenous CDM.
Even the mydriasis induced by CDM in mice could be explained  better  by central
sympathetic discharge than by monoamine oxidase inhibition,  as  suggested by
Beeman and Matsumura (1973)f since the present studies  indicated that CDM had
little or no direct sympathomimetic or parasympatholytic activity.
Furthermore, CDM did not potentiate the action of the biogenic  amines assayed
on the cardiovascular system, an action which would be  expected  if  CDM
significantly inhibited MAO.
                                     27

-------
     Centrally mediated pressor  responses  were  also  observed  following
intraventricularly  administered  lidocaine (Section  3).   The  smaller and less
frequently observed secondary  pressor  response  to  lidocaine may be related  to
the weaker convulsive  action of  lidocaine  as  compared  to CDM  (Prince and
Wagman, 1966; Pfister, W.  F.,  Noland,  V.,  and Yim, G.  K. W.,  unpublished
observations).  The stronger convulsive  action  of  CDM  may also be due in part
to direct neuronal excitation  by CDM,  which has been observed in cockroach
nerve cord preparations (Beeman  and  Matsumura,  1973; Yamamoto and Fukami,
1976; Lund^t^l., 1979a).

     Clearly these studies  indicate  that the  cardiovascular toxicity of CDM is
similar to that of local  anesthetics.   This implies  that diazepam,
vasoconstrictors, cardiac  stumulants,  and  artifical  respiration should be
useful in treating cases  of CDM  poisoning.  Observations in these laboratories
with the dog suggest  that  this regimen is  effective  in reversing the
hypotension caused by doses of CDM that otherwise  would be marginally lethal,
but  is ineffective in antagonizing the severe cardiovascular  depression caused
by larger doses.  Additional  studies on this  topic are presented in detail  in
Section 4.
                                      28

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                                SECTION THREE

                    THE CENTRAL ACTIONS OF CHLORDIMEFORM

INTRODUCTION

     In rats and mice, chlordimeform produces hyperactivity, respiratory
depression and convulsions.  The mechanisms behind these central  nervous
system effects have not been elucidated.  Since the chemical structure  of
CDM is similar to that of the local anaesthetic phenacaine, and  since CDM
exhibits a local anesthetic effect on the isolated frog sciatic  nerve
(Section U) and cardiovasular system of several species (Section  2). the
possibility was entertained that it may have actions on the central  nervous
system similar to that of local anaesthetics.

     Lidocaine has been shown to produce an action on the amygdala which
initiates seizures in the cat (Wagman _et al., 1967).  In dogs, lidocaine
increases arterial blood pressure by a central mechanism (Kao  and Jalar,
1959).  This Section describes some central actions of chlordimeform in the
urethane-anaesthetized rat and compares them to those of lidocaine.  The
arterial blood pressure and electrical activity in the amygdala  were
examined after intraventricular injections of chlordimeform or lidocaine.
Since the raphe nucleus and serotonin exert inhibitory effects on seizures
and on arterial blood pressure (Boggan and Seiden, 1973; Baum  and
Shropshire, 1975), the effects of raphe stimulation were also  examined.

     Local anesthetics and CDM (Wang and Narahashi, 1975) depress
neuromuscular transmission although relatively high concentrations of  CDM
are needed (10  -10~^M).  Thus, studies were also initiated to identify
whether the respiratory depression caused by CDM was of central  or
peripheral origin.

MATERIALS AND METHODS

Central responses to intraventricular drugs

     Male Sprague-Dawley rats weighing 260-350g were used in all
experiments.  Rats were anaesthetized with pentobarbital sodium, 35  mg/kg,
given intraperitoneally and fixed in a Kopf stereotaxic apparatus.   Bipolar
stainless-steel electrodes having diameters of 0.010 in. (303  series.
Plastic Products Co.) were implanted in the dorsal raphe nucleus and left
basal lateral part of the amygdaloid nucleus according to the  stereotaxic
atlas of Pelligrino and Cushman (1967).  A right lateral ventricular cannula
                                   29

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was also implanted  for drug  injections.   The  lateral  ventricular  cannula was
cut from a 23-gauge  stainless-steel  needle  and  fitted with an indwelling
stylet.  The electrodes  and  cannula  were  fixed  to the calvarium by pouring
cranioplastic cement  around  them and over two stainless-steel screws which
had been inserted  into the skull.  After  a  minimum of 7 days recovery
period, the animals were tested.
     Rats prepared  as described  above were  anaesthetized with urethane,  1.25
rf/kg, given intraperitoneally.   The  external  jugular  vein was cannulated
with polyethylene  tubing for intravenous  injections,  and the carotid artery
cannulated for recording of  blood  pressure, using a Statham P 23 pressure
transducer.  Electrical  activity from the basolateral nucleus of the
amygdala was monitored on a  Grass  polygraph.   Using a Grass S-88 stimulator,
the raphe nucleus  was stimulated,  usually with a 10 sec train of square  wave
pulses  (0.1-1.5  V,  0.5 msec  duration, 10-50 Hz).  Intraventricular drug
injections were  made  from a  needle fitted to  polyethylene tubing connected
to a microsyringe.   Injection volumes were 10-20 ul.   Body temperature was
maintained at 37-38° with a  thermostatically controlled heating pad.

     Drugs used  in these experiments were chlordimeform, lidocaine HC1
(Astra  Pharmaceuticals,  Inc.),  and diazepam (commercially available Valium,
Roche,  Inc.)

Respiratory arrest

     Rats were lightly  anesthetized  with urethane (1.2 g/kg, ip), and the
test chemicals were infused  over a period of 20-30 min via the femoral vein
until  respiratory  arrest occurred.  During the progression of symptoms,  a
Grass  polygraph  was used to  monitor  the following:  Femoral arterial blood
pressure  (via a  Statham  P23A transducer); ECG via transthoracic electrodes;
diaphragmatic contractions (via a Grass FT03 force displacement transducer);
and end tidal pCO,, levels (via a catheter in a tracheal tube and Bee km an LB
medical gas analyzer).   The  phrenic  nerve was dissected free in the cervical
region  and an oil  pool  formed.   Phrenic nerve discharges were detected via a
pair of platinum electrodes, amplified (filter settings: 0.1-10 KHz),
displayed on a Tektronix storage oscilloscope, and fed to a Grass AM7 audio
monitor.  The output was also fed to a Grass P7 Integrator and the
integrated phrenic burst pattern was recorded on the polygraph.  Displays of
the phrenic discharges  on the storage oscilloscope were also photographed
using  a Tektronix  C5 oscilloscope camera.

     When respiratory arrest occurred the status of the phrenic nerve-
diaphragm system was assessed by delivering single and 2-4 sec long trains
of square wave pulses (0.1 msec duration,  10 Hz, 0.2-5V) to the phrenic
nerve  via the pair of platinum electrodes, in order to monitor neuromuscular
function.

RESULTS

Blood  pressure responses to  intraventricular injection of lidocaine or
chlordimeform
                                     30

-------
     In a total of 21 rats, the intraventricular  injection  of  300-1000 yg
lidocaine produced variable responses ranging from pure pressor  or  depressor
responses to biphasic ones.  Of 21 rats, 7 exhibited pressor,  10 depressor
and 4 biphasic responses.  Because of the variable responses,  it was not
feasible to quantify the data.  However, in any particular  animal,  the
effect produced was dose related.  A dose-dependent pressor response is
illustrated in Figure 10.  In 17 rats, the effects of  COM were also
variable:  9 being pressor, 3 depressor and 5 biphasic.  As with lidocaine,
for a particular response, the effect was dose-dependent.   Figure 10
illustrates the dose-dependent pressor response to COM.
       BP 200'
     mm Hg 100 •
                      LI DC
                      O.lSmt
        BP 200
      mm Hg  100"
                                                     1MIN
                 COM
                 0.3 mg
CDM
0.6 mf
Figure 10.  Pressor responses in the carotid artery  after  intraventricular
injections of lidocaine or CDM in a rat anaesthetized with urethane.

Effects of lidocaine and CDM on changes in amygdaloid electrical activity
induced by raphe simulation.

     When the dorsal raphe was stimulated, the amplitude of the spontaneous
electrical activity of the amygdala was suppressed.  This  suppression was
antagonized by lidocaine or CDM, and the  degree of antagonism was
dose-dependent.  These effects are illustrated in Figure  11.   The duration
of the EEC suppression was dependent on the frequency of raphe stimulation
and was antagonized by lidocaine or CDM given intraventricularly.  Such an
effect is illustrated in Figure 12.
                                     31

-------
         CONTROL
                                                  CONTROL
                I    t
                                  \      \
         AFTER LIOOCAINE O.J mg
                                                  AFTER  COM l.o mg
'     I
                                                      II
                                        100 HV
                                    S SEC
                                                  AFTER CDM Z.O mg

                                           VV^VVAfvVV*"-M/Wr^^

                                                      t      t

Figure  11.   Suppression of amygdala EEC upon raphe stimulation  and
antagonism  of this suppression  by  intraventricular lidocaine  or CDM.
Between  arrows:  10 sec of dorsal  raphe stimulation (1.5V. 0.5  msec.  20 Hz).
             120


             100


              80


              60


              40


              20
                      120


                      IOO


                      80


                      60


                      40


                      20
                                                10   20
                                    30
                                   Hz'
                                                            40
                                                                50
Figure  12.   Duration of suppression of amygdala EEC activity  and  antagonism
of this  raphe-mediated EEC suppression by lidocaine and CDM.   Panel A:
duration of suppression at varying frequencies of raphe stimulation, control
(•	•)  and after 0.5 mg  intraventricular lidocaine (O	o).  Panel
B; control  (•	•) and after  1.0 mg intraventricular  CDM  (o	o).
Vertical bars = SEM; n = 5.
                                      32

-------
  AMY
  IIO
                   SEIZUIfS
    A.-^V^VvY*^
  JOB-


  10O-


   0-
  Figure  13.   Amygdala EEC spikes and blood  pressure oscillation recorded
  during  overt CDM seizures.  Dose:  3  successive  1  mg intraventricular  doses
  Between arrows, the dorsal raphe was  stimulated  electrically at  1.5V,  0.5
  msec duration at 60 Hz.
  AMY
  EEO

Control
                                                                                 »°"*
MtoJL

 If 200
   ,00 •
LIDO
           MINUTES
 t
COM
  I  -t-«
  I    I
OIAZIMM  UDO
 LO«t/M  o.(«a
                                                        fe
                                                 MINUTES
                                                                  I
                                                                 CDM
                                                                 1-0 ma
  Figure  14.   Antagonism by diazepam of  the  EEC and blood pressure effects of
  intraventricular lidocaine and CDM.  Panels  A and B:  before; Panels  C and
  D:  after  intravenous diazepam, 1.0 mg/kg.

  Seizure activity induced by CDM and blockage by raphe stimulation

        The  successive injections of 3 to M intraventricular 1 mg doses  of CDM
  resulted  in clonic limb movements.  This seizure activity was accompanied by
  oscillations in the arterial blood pressure, and the amygdala EEC  was
  characterized by slow spikes (Fig. 13).  spindle bursts (Fig. 14A)  or
  high-amplitude rhythmic discharges (Fig. 14B).  During seizure activity,
                                       33

-------
   high  frequency electrical stimulation of  the  dorsal  raphe  nucleus inhibited
   seizure  activity along with its  EEC  and autonomic  concomitants.   This
   inhibition  persisted during the  entire  period  of  electrical  stimulation.
   Upon  cessation of raphe stimulation,  spiking  of the  EEC and  oscillation of
   the  blood  pressure reappeared.   Seizures  did  not  return in the 5  rats
   observed.   These effects are  illustrated  in Fig.  13.

   Effects  of  diazepam

         Intravenous diazepam,  1.0 mg/kg, blocked  the  effects  of intra-
   ventricular lidocaine or CDM  on  the  electrographic activity  of the amygdala
   of the 5 rats.  This dose of  diazepam also  antagonized  the centrally
   mediated blood pressure responses  produced  by lidocaine or CDM.  These
   results  are illustrated in  Figure  14.

   Mechanism of respiratory arrest

         The decreasing order of  lethality  and  dose resulting  in respiratory
    arrest were:  cocaine  (35.3 +. 11 mg/kg),  lidocaine (35.4 + 9.6 mg/kg), CDM
    (62.3 _+ 6.0 mg/kg), and morphine (93.5  +_  11.0 mg/kg).   In  lidocaine-treated
    rats, the amplitude and rate  of  diaphragmatic movements and  of phrenic nerve
    bursts gradually decreased  until respiratory  arrest  (Figure  15).
LIDOCAINE

  B
                                          ( 5 mg/ml - 0 I ml/mln )

                                                      C
        CONTROL
PHRENIC 50_
 RATE
counts/sec
      0
DIAPHRAMg
  FORCE
orbit units
  B P
 mm Kg
   25 MV
             MIN
        5 SEC
    Figure 15.  Respiratory  and  cardiovascular parameters of a urethane-
    anesthetized rat  infused  with  a  lethal  iv dose of lidocaine.  Measurements
    include:   End-tidal pCO   levels;  integrated discharge rate recorded from cut
    central end of phrenic nerve;  diaphragmatic contractions; carotid blood
    pressure; oscilloscope tracings  of phrenic bursts and diaphragmatic
    contractions.

-------
     In contrast, as  shown  in  Figure  16,  respiratory rate was initially
increased by cocaine  and COM.   Diaphragmatic  contractile force and the
amplitude of phrenic  nerve  bursts  remained  near control values, but
disappeared abruptly  upon respiratory  arrest.   The profile of morphine on
the phrenic nerve activity  was  unique  in  that  continous inter-burst
discharge preceded the abrupt  decrease in  phrenic nerve amplitude and
respiratory arrest.   Naloxone  reversed both actions of morphine but did  not
reverse the respiratory depression  induced  by  the other agents.  Following
respiratory arrest produced  by  all  of  these agents, the diaphragm and
gastrocnemius muscle  still  contracted  following electrical stimulation of
the phrenic and sciatic nerves,  respectively.   In contrast, central output
in the phrenic nerve  declined  and  disappeared  at the time of respiratory
arrest.  These results indicate  that  respiratory arrest induced by these
agents is central in  origin, and not a result  of neuromuscular blockade.
                               CHLORDIMEFORM  ( 10 mg/ml - 0 I ml/mm)

            ABC

      CONTROL                    L025                 LD50

                                i 19 mg/Kg               • 23 mg/Kg
 P   X
  C°2  0.
 L09S

 • 36 mg/Kg

LlL <,:..,,, L
PHRENIC
 RATE 50
counts/see
      OL
      3r-
DIAPHRAUt
 FORCE
orbit units '
      0.
    200p
 B P
 mmHgioo)- I
25 MV
          t INFUSION
           START
                                            ^|^^^L|,
           INSPIRATION
           I  MIN
      YYTmrmrv
                             YVYYYYVTVYYV
     5 SEC
Figure 16.  Same as  Fig.  15  except  drug infused was chlordimeform.

DISCUSSION

     As previously found  in  the  frog  sciatic nerve (Section U),  the  isolated
rabbit heart, and dog  blood  pressure  preparations (Section 2),  the  central
actions of chlordimeform  that  were  examined in the present study were
qualitatively indistinguishable  from  those produced by the local anaesthetic
lidocaine.  Thus, when  injected  intraventricularly in the anaesthetized rat,
both agents produced similar responses on blood pressure and  electrical
activity of the amygdala.   The antagonism by intravenous diazepam of the
effects of CDM and lidocaine were anticipated in view of diazepam's well
established effectiveness  in supressing local anaesthetic seizures  (de Jong,
1970).
                                      35

-------
     The studies of Wagman ^t _al.  (1967),  and  Eidelberg  et _al_.  (1963)  have
implicated the amygdala as the  probable  focus  of  seizure activity  induced by
the local anaesthetics, lidocaine  and  cocaine.  The  present  study  also
points to the importance of  the amygdala in  the action of lidocaine  as well
as chlordimeform.  The generally held  scheme for  explaining  local
anaesthetic seizures is that local  anaesthetics   selectively block tonic
inhibitory mechanisms, thereby  releasing excitatory  mechanisms  (de Jong,
19/0).  The observed block by lidocaine  or chlordimeform of  the suppression,
induced by raphe stimulation, of the electrical activity in  the amygdala
raises the possibility that  local  anaesthetics may produce seizures  by
blockade of raphe  inhibitory input  to  the  amygdala.

     Blockade by lidocaine and  CDM of  raphe  inhibition of the amygdala is an
attractive possibility in view  of  previous findings  of antagonism  of
5-hydroxytryptamine  (5-HT) inhibition  of cortical neurones by cocaine
(Phillis, 1970) and  antagonism  of  5-HT-mediated rhythmic activity  of
cockroach malphighian tubules  (Hollingworth, unpublished observations).
Spontaneous firing of amygdala  neurones  is reduced  following systemic
5-hydroxytryptophan  (Eidelberg  ^t  jl., 1967),  iontophoretic  5-HT or  dorsal
raphe stimulation  (Wang and  Aghajanian,  1977). Moreover, electrical
stimulation of the midbrain  raphe  inhibits seizures  induced  by  electrical
stimulation of the amygdala  or  by  pentylenetetrazole (Kovacs and Zoll,
1974), and 5-hydroxytryptophan  raises  the threshold  for  audiogenic seizures
and restores the reserpine-induced hypersensitivity  to noise in mice (Boggan
and Seiden, 1973).   Although reduction of brain 5-HT levels  facilitates,  and
elevation of brain 5-HT levels  antagonizes,  pentylenetretrazole or
electrically induced seizures  (Killian and Frey,  1973).  brain 5-HT levels
are elevated in CDM-treated  rats  (Beeman and Matsumura,  1973).   The  elevated
5-HT levels have been attributed  to MAO inhibition by CDM, but  more  recent
work suggests that the elevation  is slight compared  to standard MAO
inhibitors (Benezet ^t al.,  1978).   Since the 5-HT antagonist,  LSD,  also
causes an increase in [^f]5-HT  levels  following  [^H]tryptophan  injection
(Diaz and Huttunen,  1971). it  is  apparent that the  effectiveness of  CDM and
lidocaine in antagonizing 5-HT  and raphe inhibition  of amygdala neurones
must be directly examined  in order to  better assess  the  role of the  raphe
system in seizures induced by  CDM  and  lidocaine.

     The blood pressure oscillations occurring during seizures  induced by
CDM are similar to those  induced  by convulsants  such as  picrotoxin and
strychnine in the  cat and  described in detail by  Polosa  ^t  al.  (1969)  and
Polosa e£ al. (1972).  This  oscillatory behavior  has been observed to  be
associateid~with synchronized periodic  activity of sympathetic preganglionic
neurones and appears to be generated in  the  central  nervous  system.   Similar
discharges have been recorded  from peripheral sympathetic efferent nerves
during the pressor response  following  intravenous CDM (Section  2).  The
pressor and convulsant effects  of  intraventricularly applied lidocaine and
CDM might involve  similar mechanisms,  since  both  agents  also blocked raphe
inhibition of pressor responses evoked by stimulation of the amygdala.  If
the raphe system exerts a tonic inhibitory influence on  amygdala and other
nuclei responsible for vasomotor  tone, then  blockade of  such inhibitory
input could result in pressor responses  such as those seen after
                                     36

-------
intraventricular lidocaine and chlordimeform.  Since  serotonergic  neurones
also exert an inhibitory influence upon spinal sympathetic  neurones
(Neurmayr _et _al., 1974), blockade of this descending  inhibitory pathway may
also contribute to the increase in blood pressure observed  after CDM and
lidocaine.

     In regard to the mechanism underlying respiratory  arrest  induced  by
CDM, the results presented here clearly support the CNS rather  than a
blockade of neuromuscular junctions as the site of action.   In  CDM-poisoned
rats, neuromuscular function appears to be relatively unimpaired,  while CNS
output via the phrenic nerve is diminished and disappears,  indicating a
probable block of the respiratory center in the brain stem.
                                     37

-------
                                  SECTION  FOUR

                 LOCAL  ANESTHETIC PROPERTIES OF CHLORDIMEFORM

                 AND  POTENTIAL  MEANS OF ALLEVIATING TOXICITY
INTRODUCTION
      In the preceding  two  Sections,  it was shown that COM in both its
peripheral and central  actions  is qualitatively indistinguishable from the
local anesthetic,  lidocaine.   In addition, CDM, when injected into the
lateral ventricle, caused a delayed respiratory arrest that was consistent
with slow passage  of  CDM via  the third ventricle and subsequent depression of
the brain stem respiratory  "center" by a local anesthetic-like action of CDM.
CDM and local anesthetics have  common toxic symptoms consisting of
hyperexcitability, tremors, convulsions, and respiratory arrest (Beeman and
Matsumura, 1974; deJong,  1970).  Furthermore, the chemical structure of CDM
is similar to that of the local anesthetics, phenacaine and guanicaine (Fig.
17).

     The first objective of this study was to determine if CDM did have a
local anesthetic action on  the  frog sciatic nerve preparation.  Since
diazepam is an effective antagonist of convulsions and lethality induced by
local anesthetic agents (deJong, 1970; Richards et al., 1968), and was found
to reverse the hypotensive  effects of CDM in dogs (Section 2), a second
objective of this  study was to  examine diazepam and other agents for their
effects on CDM-induced  convulsions and lethality.
                                           3 Q
                                       DJ\   II     /C7H5
                                         }-N-C-CH,-N(C
                                                    2*5
                         CH
  v_n.

N=C-N/
                  PHENACAINE
                                      C,H50-
                                              -N=C( NH
                                         GUANICAINE
Figure 17-  Chemical  structure of chlordimeform and related local
anesthetics.
                                     38

-------
MATERIALS AND METHODS

Frog sciatic nerve preparation

     The hydrochloride salts of COM, DCDM, and of the local anesthetic  agents
phenacaine (Holocaine, Winthrop-Stearns, Inc., New York), lidocaine
(Xylocaine, Astra Pharmaceutical Products, Inc., Worcester, Massachusetts),
and procaine (Mallinckrodt, Inc., St. Louis, Missouri) were dissolved  in  frog
Ringer's solution (Wesseling et al., 1971) and the pH was adjusted to  7.0 _+
0.1 with 0.1 M NaOH.  Sheathed and desheathed sciatic nerves dissected  from
frogs (Rana pipiens) measuring 3 to 3.5 in. in length were placed  into
Harvard nerve chambers with pairs of platinum wire stimulating  and recording
electrodes spaced at 1 cm intervals.  Compound action potentials were  evoked
using supramaximal stimulation (0.6-4.0 V, 0.15 msec, 1 Hz) delivered  from a
Grass Model SD-5 stimulator.  The action potential amplitude was monitored on
a Tektronix 565 oscilloscope.  Conduction rate was estimated as the  ratio of
the change in latency to the distance between two sets of recording
electrodes.  During twin pulse stimulation, the interval between pulses that
resulted in 50% depression of the response to the second pulse  was used as a
measure of the relative refactory period.  Drug solutions were  applied  to a  5
mm segment of the nerve between the stimulating and recording electrodes.
Each nerve was used only once and six to eight nerves were run  per drug
concentration.  Three or four concentrations of each agent enabled the
calculation of the ED__ and standard error values by reverse regression
analysis (Aldrete and Daniel, 1972).

CDM-drug interactions in vivo

     White mice (25-30 g; Cox-Swiss, Laboratory Supply Co., Indianapolis,
Indiana) were used in the studies of the convulsant and lethal  actions  of
COM.  The mice were housed in groups under a 10 hr light-dark cycle  for at
least 1 week prior to testing.  They were pretreated with the potential
antagonists usually 30 min prior to being injected with approximate  LDQ(.  dose
of COM (100 mg/kg, ip).  When assessing agents for possible enhancement of
COM toxicity, the pretreated mice were challenged with the approximate LD
dose of CDM (80 mg/kg COM, ip).  Comparisons were made using Fisher's  metnod
of calculating exact probabilities (Schwartz, 1974).

     Diazepam (Valium:  Hoffman-LaRoche Inc.) and the other pretreatments
were injected intraperitoneally in a volume of 10 ml/kg.  Saline was the
solvent for diphenylhydantoin sodium, librium HC1 (Hoffman-LaRoche Inc.),
pentobarbital sodium (Nembutal, Abbott Laboratories), atropine  sulfate
(Nutritional Biochemicals Corp.), imipramine HC1 (Geigy Pharmaceuticals),
pyribenzamine HC1 (Ciba Pharmaceutical Co.), phenoxybenzamine HC1  (Smith,
Kline & French), chlorphenoxamine HC1 ( Pitman-Moore Co.), neostigmine
methylsulfate (Hoffman-LaRoche, Inc.), physostigmine (Eserine Salicylate,
Mallinckrodt Chemical Works), cycloheximide (Calbiochem), and cinanserin (SQ
10,643:  Squibb and Co.). Trimethadione (Tridione:  Abbott Laboratories).
mephenesin (K & K Laboratories, Inc.), and chlorpromazine HC1 (Thorazine:
Smith, Kline & French) were dissolved in  10? ethanol, 20% propylene  glycol,
70% saline mixture.
                                    39

-------
RESULTS

Effects of  CDM  and  DCDM on the frog sciatic nerve preparation

      Figure  18 shows the effect of CDM, DCDM, phenacaine, procaine, and
lidocaine on  the amplitude of the compound action potential of the frog
sciatic nerve.   The slopes of the dose-response relationship for all
co .pounds were  similar:  no significant departure from parallelism was
observed between the regression lines for holocaine, procaine, DCDM, or
lidocaine compared  to that of CDM.   The order of increasing potency for the
compounds studied was found to be DCDM < CDM < procaine < phenacaine <
lidocaine.  The ED   's + SE were 35+6.2 mM (DCDM); 15.0 + 3.7 mM (CDM); 8.7
+_ 2.5 mM (procaine?; 4.0 +_ 1.5 mM (phenacaine), and 1.9 +_ 0.3 mM (lidocaine).
Hence,  CDM  and  its  demethylated metabolite, DCDM, were 0.6 and 0.3 times as
potent  as procaine  respectively.  The effects of CDM and DCDM on all other
parameters  studied  were similar to those observed with procaine, phenacaine,
and  lidocaine.   After 15 min of exposure to either CDM or procaine, threshold
stimulus voltages and relative refractory periods were increased by
approximately 30 and 20%, respectively.  Conduction velocity was decreased by
15 and  20?  after 15 min of exposure to CDM (30 mM, pH 6.0) and procaine (10
mM,  pH  7.0).
   IOO
O  80
O  60

b
UJ
UJ

-------
     Repetitive or spontaneous firing was never  observed.   After complete
blockage of the nerve by CDM, partial recovery was  obtained  following
repeated washing of the nerve.  When the degree  of  ionization  of CDM was
increased by lowering the pH from 7.0 to 6.0, the nerve-blocking
effectiveness of 30 mM CDM was reduced by more than 5058  (Fig.  19).   With
desheathed nerves, 1 mM CDM (pH 7.0) caused 20%  depression  (Fig. 19).  Note
that with intact nerves (Fig. 18) approximately  5 mM  CDM was required to
cause 20% depression.  Thus CDM was about five times  more active on the
desheathed nerve.
              Sheathed Nerve    COM
                          pH 6.0,90 mM
                          pHTO.30 mM
              Desheathed Nerve
                O	O     pH70 , ImM
                          6      8      10     12     14     16

                        TIME IN  CHLORDIMEFORM (CDM)MINUTES
IB
20
Figure  19.  Effect  of  chlordimeform  (30  mM)  at pHs 6.0 and 7.0 in the
sheathed and  at pH  7.0 (1  mM)  in  the desheathed nerve on action potential
amplitude at  2 min  intervals  following drug  application.  Abscissa:  time
(min) following application of chlordimeform to the nerve.  Ordinate:  action
potential amplitude (percentage of control).  Each point represents the mean
+ SE  (30 mM,  pH 6.0; n =  7; 30 mM, pH 7.0; n = 6;  1 mM, pH 7.0; n = 6).

Diazepam and  other  agents on  CDM  convulsions and lethality

      The approximate LD   dose of CDM, 100 mg/kg,  ip, resulted in periodic
convulsive  jerks  (mean latency:  4 min—see  Table 6).  The frequency,
severity, and duration of the convulsive episodes increased until a
continuous  seizure  bout resulted  in  repiratory arrest and death (mean
latency:  6-12 min  for groups of  10  mice).   The LD,-0 (and 95? confidence
interval) for CDM was  85.5 (87.9-83-3) mg/kg).  The threshold dose for
inducing convulsions was  well below  50 mg/kg, a dose at which no acute
lethality was evident.

                                       41

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TABLE 6.  EFFECTS OF ANTICONVULSANTS  ON  COM  CONVULSIONS  AND  LETHALITY IN  MICE
     Pretreatments'
Dose No. convulsed/
Cr n pound (mg/kg) No. injected
Saline controls
Saline controls
Saline controls
Saline controls.
Vehicle control
Diphenylhydantoin
Trimethadione
Mephenesin
Mephenesin
Diazepam
Diazepam
Diazepam
Librium
Librium
Pentobarbital
Pentobarbital
	
	
	
	
	
40
400
100
200
15
15c
30°
15
40
15n
25°
10/10
10/10
10/10
10/10
10/10
10/10
10/10
8/10
10/10
10/10
10/10
10/10
10/10
10/10
10/10
4/10*
Mean
latency No. deaths/
(min) No. injected
3.6
4.3
3.7
2.3
3.0
2.2
2.8
	
4.6
3.8
4.7
5.1
3.2
4.3
___
10.0*
10/10
9/10
10/10
10/10
10/10
10/10
10/10
7/10
10/10
1/10*
3/10*
7/10
5/10**
10/10
9/10
6/10**
Mean
latency
(min)
6.0
12.7
8.0
6.2
14.6
6.2
10.2
8.7
8.7
15.0*
20.0*
25.4*
10.4
24.2*
	
11.6
 Mice were  pretreated  30  min prior to 100 mg/kg COM,  ip (LE>   dose).
Exceptions:  mephenesin  (15  min),  diphenylhydantoin (120 min?, trimethadione
(60 min).

  iO%  ethanol,  20% propylene ^lycol,  70%  saline.

°Resulted  in ataxia  and  decreased  motor activity.

* P <_ 0.001

** P <  0.01
                                      42

-------
      Pretreatment with the anticonvulsants diphenylhydantoin, trimethadione,
or mephenesin did not result in prevention of either the convulsions or
lethality that was observed following the LDq(. dose of COM (Table 6).  At  the
15 mg/kg dose, diazepam pretreatment reduced the severity and duration but
did not appreciably reduce the incidence nor prolong the onset time of the
CDM convulsions.  However, lethality was reduced about 20%, and the onset
time to death was lengthened to about 20 min.  The 30 mg/kg dose of diazepam
was also ineffective in preventing CDM convulsions, and it was less effective
than the 15 mg/kg dose in preventing CDM lethality.  Mice receiving this
higher dose of diazepam exhibited motor impairment, ataxia, decreased motor
activity, decreased responsiveness to sensory stimulation, and, in some
cases, loss of righting reflex.  Similar results were obtained with librium:
the 15 mg/kg dose attenuated CDM lethality, whereas the 40 mg/kg dose
prolonged the latency to death, but was ineffective in preventing CDM
lethality.  Pentobarbital reduced the incidence of convulsions and death,  but
only at the 25 mg/kg dose which was accompanied by marked sedation and
depression.

     The effects of other agents that were tested as possible antagonists  of
CDM are summarized in Table 7.  Chlorpromazine (20 mg/kg) increased the
latency to convulsions and the latency to death, but provided little
protection against CDM lethality.  Agents that did not reduce either the
severity of CDM convulsions or the incidence of CDM convulsions and lethality
included haloperidol, chlorphenoxamine, cycloheximide, neostigmine, and
physostigmine.  Physostigmine increased the latency to death.  Cinanserin
increased the onset time and decreased the severity and incidence of
convulsions as well as increased the latency to death and decreased the
incidence of lethality in mice treated with CDM.  Animals treated with the 50
mg/kg cinanserin were ataxic and exhibited ptosis and decreased muscle tone.
During preliminary cardiovascular studies with CDM, it appeared that atropine
and pyribenzamine intensified the hypotensive action of CDM.  Hence, possible
enhancement of CDM lethality by atropine and other agents that possess "local
anesthetic" activity was tested by pretreating mice with these agents, and
then challenging them with 80 mg/kg CDM (approximate LD_ dose).  This  dose of
CDM caused convulsions in  100? of the saline-pretreated mice  (mean latency:
5.2 ± 0.2 min - see Table 8).  As expected, no pretreatment reduced the
incidence of convulsions.  The onset time for CDM convulsions was reduced  in
the animals treated with imipramine, phenoxybenzamine, and atropine but  not
with the mice dosed with pyribenzamine.  CDM lethality was enhanced following
pretreatment with pyribenzamine, imipramine, and phenoxybenzamine, but  not
after atropine or lidocaine pretreatment.

DISCUSSION

     The acute toxic symptoms of CDM are similar to those observed  following
local anesthetic overdose, i.e., clonic convulsions, respiratory depression,
and cardiovascular collapse followed by death (dejong,  1970).   The
effectiveness of diazepam  and pentobarbitol in reducing lethality by  toxic
doses of CDM also parallels the effectiveness of these  agents in decreasing
lethality from local anesthetic agents such as lidocaine  (Richards  et  al.,
1968; Wesseling et al.t  1971; Aldrete et al., 1972).  The  ineffectiveness of
                                      43

-------
TABLE 7.  EFFECT OF OTHER  PHARMACOLOGICAL AGENTS ON COM CONVULSIONS AND
LETHALITY IN MICE
Pretreatments


Compound
Saline controls
Saline controls
Saline controls
Chlorpromazine
Chlorpromazine
Haloperidol
Haloperidol
Chlorphenoxamine
Cyloheximide
Neostigmine
Physostigmine
Physostigmine
Cinanserin
Cinanserin

Dose No.
(mg/kg) No.

	
	
5
20
1
5
5
0.7
0.1
0.25
0.50
25 K
50b

convulsed/
injected
10/10
10/10
10/10
9/9
5/10
9/10
10/10
10/10
8/10
10/10
10/10
8/8
8/10
6/10»*
Mean
latency
(min)
2.8
2.3
3.6
3.5
6.0*
3.1
3.6
2.4
3.8
2.0
2.9
3.6
6.0*
7.2*

No. deaths/
No. injected
10/10
9/10
10/10
9/9
7/10
9/10
10/10
9/10
8/10
10/10
10/10
6/8
9/10
6/10**
Mean
latency
(min)
6.4
6.2
6.0
	
15.3*
9.8
8.1
9.5
9.5
5.9
18.1*
14.0*
9.0*
13.8*
 Mice  were  pretreated 30 min prior to 120 mg/kg COM, ip (LD   dose)
Exceptions:  cycloheximide (2 hr), Chlorpromazine (3 hr).

 Resulted  in ataxia and decreased motor activity.

*  P  <_  0.001

** P < 0.01
                                      44

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     TABLE  8.   TOXICITY IN MICE OF COM COMBINED WITH AGENTS POSSESSING  LOCAL  ANESTHETIC ACTIVITY
•o
Ln
—
Pretreatment
Saline
Pyribenzamine
Pyribenzamine
Atropine
A tr opine
Imipramine
Imipramine
Phenox yben zam ine
Phenox yben zam ine
Lidocaine
Lidocaine
Dose
(mg/kg)

10
30
1
10
10
30
1
10
10
20
Latency to convulse
(min)
5.2+0.2
4.4+0.3
4.2+0.6
3.6+0.2*
3.0+0.2*
4.5+0.2
3.2+0.5*
3.1+0.3*
3.6+0.4**
5.2+0.4
4.0+0.2
No. deaths/
No. injected
7/70
5/10*
8/10*
2/20
3/20
4/10**
4/10**
4/10**
4/10**
4/30
3/20
     aMice  were  pretreated  15 min prior to 80 mg/kg COM ip (LDe

     30 mg/kg  (30 min).

      * P _< 0.001


      ** P  < 0.01
dose) except lidocaine (3 min);.pyribenzamine

-------
diphenylhydantoin,  trimethadione,  and mephenesin in reducing COM lethality
was anticipated  in  view of the previously reported inability of these agents
in antagonizing  local  anesthetic toxicity (deJong, 1970).  The observed
enhancement  of CDM  toxicity by the antihistaminic, pyribenzamine, the
a-adrenergic blocker,  phenoxybenzamine,  and the tricyclic antidepressant,
imipramine,  could  have been due to the local anesthetic actions of this
diverse  group of pharmacologic agents (Crescitelli and Geissman, 1951;
Guerrero and Molgo, 197*0.  However, CDM toxicity was not enhanced by
atropine,  which  also has comparable local anesthetic activity (Sheu et al.,
1969), or  by lidocaine.

      In  the  frog sciatic nerve preparation, CDM and its metabolite DCDM
produced effects similar to those of the local anesthetic agents studied:
amplitudes and  conduction velocities of the compound action potential were
depressed, and  threshold voltages and refractory periods were increased.
Reversibility of the nerve block,  increased effectiveness in desheathed
preparations, and  decreased potency at pHs favoring ionization of the
compounds (pKa  of  CDM is near 7.0) are additional characteristic actions
shared by CDM and  the local anesthetics (deJong, 1970; Ritchie and Greengard,
1966).

      In  our  comparisons of the actions of CDM and local anesthetics in vivo
(Sections 2 and  3), we found that both CDM and lidocaine caused similar
biphasic cardiovascular responses when injected intravenously in dogs, and
similar  amygdala EEC spike discharges, overt seizures, and blood pressure
responses when  injected into the lateral ventricle of rats.  Further, like
lidocaine and cocaine (Post est^l., 1975), CDM induced "pharmacological
kindling"  in rats  i.e. a type of reverse tolerance in which repeated daily
subconvulsant doses eventually precipitated generalized convulsions (Yim, et
al..  1977).   These results indicate that some of the pharmacological and
toxic actions of CDM are due to actions that are shared by local anesthetic
agents.

      Unlike  the  situation with local anesthetic seizures, which are readily
antagonized  by  diazepam (deJong, 1970), CDM seizures could not be prevented
even  at  doses higher than that which reduced CDM lethality.  This was
especially surprising since we found that diazepam did antagonize seizures
and  amygdala EEC spike discharges that resulted from intraventricular CDM
(Section 3).  We also observed that diazepam blocked the secondary pressor
response of CDM, an autonomic consequence of CDM-induced seizures (Section
2).   The latter  studies were carried out in anesthetized preparations and
perhaps  an additive anticonvulsant effect of the anesthetic? might explain
the  lack of protection observed in this study with unanesthetlzed mice.  The
general  CNS  depressant actions of pentobarbital and cinanserin might likewise
explain  their effectiveness against the CDM seizures and lethality.

      The resistance of CDM convulsions to diazepam might also reflect
manifestations  of  direct excitatory actions of CDM that are not shared by
local anesthetics.   Indeed, Beeman and Matsumura (1974), reported repetitive
firing from  the  cockroach ventral nerve cord treated with CDM.  Moreover,
repetitive firing  in cockroach giant axons (Lund _et al., 1979a) and increased
                                    46

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firing of caudate neurons (Pfister, W. and Yim, G. K. W.,  unpublished)  have
been noted following the application of CDM but not lidocaine.   The  greater
intensity of the CDM convulsions, as compared to lidocaine  seizures,  is also
consistent with this possibility.  These indications of  a  direct excitatory
action of CDM on neuronal membranes suggest that CDM may have  effects on
sodium and potassium conductances beyond the depression  characteristic  of
local anesthetics such as lidocaine and procaine.  The acute symptoms of CDM
toxicity are qualitatively similar to those produced by  DDT, which also
elicits repetitive firing in the cockroach giant axon (Narahashi and
Yamasaki, 1960).  Although cycloheximide is effective in preventing  acute
lethality and decreasing the severity of convulsions induced by DDT  (Hrdina
and Singhal, 1972). cycloheximide failed to protect the  mice from convulsions
and lethality induced by CDM.

     An understanding of the mechanisms of CDM-induced lethality must precede
the analysis of the diazepam-CDM interactions observed in  this  study.  In the
lightly anesthetized dog intravenous CDM can result in simultaneous
respiratory arrest and cardiovascular collapse, and the  irreversible
hypotension is mediated primarily by direct depressant effects  of CDM on the
heart and blood vessels (Section 2).  It seems unlikely  that diazepam would
prevent these direct cardiovascular depressant actions.  The report  by Wang
£t al.., (1975) of CDM block of acetylcholine depolarization at  the frog
neuromuscular junction raises the question of whether CDM-induced respiratory
arrest involves peripheral blockade of neuromuscular transmission.   The
observed inability of neostigmine and physostigmine to reduce  CDM lethality
does not support the possibility of a peripheral site of action, and  is in
keeping  with the conclusion already reached in Section  3.  It  is more  likely
that CDM-treated mice succumbed from postconvulsive depression  of the
respiratory "centers."  Diazepam at 15 mg/kg appeared to have  sufficiently
attenuated the severity of the CDM convulsions to  prevent  the  secondary
postical respiratory arrest and consequent death.  The ineffectiveness of 30
mg/kg of diazepam in reducing CDM lethality could  possibly be  due to additive
central respiratory depressant actions of CDM and  the higher dose of
diazepam. Block by diazepam of the secondary pressor response  of CDM could
also unmask an irreversible hypotensive action by  CDM.   The interactions of
diazepam with CDM and local anesthetic on central  respiratory  mechanisms
deserve further study.  An unequivocal interpretation of these results is not
presently possible.
                                     47

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                                 SECTION  FIVE

                      BEHAVIORAL  EFFECTS  OF  CHLORDIMEFORM

INTRODUCTION

     Recently, Jacobs described  a  behavioral  model  in  the  rat  consisting  of
a spectrum of symptoms  termed  the  "serotonergic  syndrome"  which  is  purported
specifically to reflect "increased central  serotonergic  transmission".
Regimens that induced the  syndrome include  release  of  serotonin  (5-HT)  by
parachloroamphetamine (PCA), or  direct  stimulation  of  postsynaptic  5-HT
receptors by L-tryptophan  plus monoamine oxidase (MAO)  inhibition (Jacobs,
1976).

     Interestingly,  sub-toxic  doses of  CDM  elicit some of  the  component
symptoms characteristic of the 5-HT syndrome  in  rats.   The symptoms of  CDM
behavioral toxicity  have previously been likened to serotonin  poisoning in
the presence of a  MAO inhibitor  (Beeman  and Matusumura,  1973).   In  addition,
CDM possesses moderate  MAO inhibitory potency (Section 1)  and  brain levels
of both 5-HT and norepinephrine  are elevated  in  rats receiving toxic doses
of CDM (Beeman and Matsumura,  1973), although recent work  suggests  that the
elevations are low compared to those induced  by  other  MAO  inhibitors (Beeman
et al.t 1978).Since  CDM inhibits MAO and elevates brain  5-HT levels, while
PCA (Jacobs, 1976) indirectly, and quipazine  (Hong^t^., 1969)  directly,
stimulate postsynaptic  5-HT receptors,  each should  be  capable  of eliciting
the 5-HT syndrome.

     The first aim of this study was to  determine if the serotonergic
syndrome could be  produced by  CDM, quipazine  and by intraventricular 5-HT.
The second objective was to determine if the  serotonergic  syndrome  produced
by these agents could be blocked by 5-HT antagonists (cinanserin and
methysergide).  An additional  objective  was to develop a "behavioral
profile" for the effects of lethal and  sublethal doses of  CDM  in the rat.

MATERIALS AND METHODS

     Adult, male Sprague-Dawley  derived  albino rats (350-450 g)  (Laboratory
Supply Co., Indianapolis,  IN)  were housed in  groups (8-10/cage)  except
animals with lateral  ventricular cannulas which  were housed individually in
air-conditioned rooms (24  ) in a quiet  environment, with lighting between
07:00 and 18:00 hours.   Animals  were supplied with  food  (Wayne Lab  Blox,
Allied Mills, Inc.,  Chicago, ID and water  ad libitum  for  approximately one
week prior to use.
                                      48

-------
     For the intraventricular injection of 5-HT, stainless steel cannulas
were implanted 0.5mm above the lateral ventricles.  The stereotaxic
coordinates were:  L = 1.5 mm; AP = 1.0 mm: H = 3.0 below dura.  5-HT,
dissolved in saline, was j.njected in a volume of 10 pi over a period of  30
sec through a stainless steel needle which protruded 1 mm below the tip  of
the cannula.  Doses are expressed as the total salt.

     The presence (or absence) of the following six components of the
"Serotonergic syndrome" was assessed:  rhythmic reciprocal forepaw treading;
slow lateral head weaving; hindlimb abduction (splayed out posture);
rigidity (manifested by increased resistance of the hindlimbs to passive
extension and flexion); Straub tail, and resting tremor (especially of the
head and forelimbs).  Positive control regimens for inducing the 5-HT
syndrome included PCA (5 mg/kg), and L-tryptophan (200 rag/kg, ip) 1 hr
following pargyline (50 mg/kg, ip).  Animals were injected with appropriate
drugs and observed at 30, 60, and 90 min, except animals injected with 5-HT
which were observed at 4 x 15 min intervals following injection.  The
animals were also observed for the presence of slow periodic backing
movements; periodic circling; hyperactivity (periodic movement about the
cages); hyperreactivity (startle reaction and/or vocalization accompanying
handling); hyperreflexia (periodic clonic spasms) and salivation.

     All drugs were prepared immediately prior to use and administered
intraperitoneally to rats in volumes of 1 ml/kg of body weight.
Chlordimeform HC1 (CDM); parachloroamphetamine HC1 (PCA) (Regis Chemical
Co., Chicago, IL); quipazine, 2-(l-piperazinyl)quinoline maleate (Miles
Laboratories, Elkhart, IN); cinanserin HC1 (E. R. Squibb and Sons, New
Brunswick, NJ); and pargyline (Eutonyl , Abbott Laboratories, North Chicago,
IL) were dissolved in physiological saline.  L-Tryptophan (Calbiochem, Los
Angeles, CA) and methysergide (Sandoz Inc., Hanover, NJ) were made up in a
1.5% suspension of methocellulose (USP) and acidified with a few drops of
0.1 N HC1.  5-Hydroxytryptamine creatine sulfate complex (5-HT, Sigma
Chemical Co., St. Louis, MO) was dissolved in physiological saline and
administered intraventricularly (ivc).

     Where applicable, data were analyzed for significance between treatment
groups by using Fisher's method for calculating exact probabilities (Fisher,
1970), calculating X  for 2x2 contingency tables, or using Student's £
test.  Three doses of each agent enabled the calculation, by reverse
regression analysis (Brownlee, 1965), of threshold doses and standard errors
for eliciting 4 out of 6 serotonergic or additional symptoms.

RESULTS

     Rats treated with both PCA and CDM exhibited the serotonergic syndrome
in a dose-related manner (Fig. 20).  The calculated threshold PCA and CDM
doses (+ SE) for eliciting 4 of the 6 symptoms were 4.7 + 0.8 and 94.8  + 6.7
mg/kg, respectively.  In addition, the slopes of the dose response lines of
PCA and CDM were not significantly different  (F    = 0.72; p >  0.5).   Since
pargyline plus L-tryptophan elicited behaviors indistinguishable  from  those
of PCA, all further comparisons were made between non-lethal sub-threshold
doses of PCA (5 mg/kg). CDM (80 mg/kg), and quipazine (20 mg/kg).

                                     49

-------
       m
       x
       X
       UJ
       en
       co  3
       UJ
       QD
SEROTONERGIC      ADDITONAL

          COM      O—--O
          PCA
          QUIPAZINE0	
                                                      I I
                             6  8  K)     20   40 60
                                  DOSE  mg/Kg
                       100   200
 Figure 20.   Dose response effect of PCA,  COM,  and quipazine in eliciting
 serotonergic syndrome (closed symbols) and additional symptoms (open
 symbols).  Each point represents the mean  + SE  number of symptoms observed  (n
 =  6-12/dose).

      The mean  number of symptoms exhibited per rat and the frequencies  with
 which each  of  the 6 different component symptoms of the 5-HT syndrome were
 exhibited  by the groups of rats treated with 5 mg/kg PCA,  80 mg/kg COM  and
 20  mg/kg quipazine are given in the first three columns of Table 9.  Thus
 the eleven  animals treated with 5 mg/kg of PCA exhibited an average  of  3.7 +
 0.5 of the  6 possible "5-HT-like" responses.   CDM at 80 mg/kg also        ~
 effectively elicited the 5-HT syndrome, as the 12 animals  averaged 4.3  + 0.3
 responses.   The  onset of PCA and CDM-induced behavioral alteration was  rapid
 (5-10  min),  and  of long duration (up to M hours at the highest doses
 tested).  Forepaw treading,  head weaving  and tremor were the most  frequently
 observed symptoms,  whereas Straub tail responses were infrequently observed.

     Since  the quipazine response changed from an excitatory to  a  depressant
 phase  after  about  30 min,  the quipazine-treated  rats were  observed several
 times during the  first  30  min.   In  contrast to PCA and CHM,  quipazine did
not effectively elicit  the serotonergic syndrome in rats;  no dose  response
relationship was  evident (Fig.  20).   The  mean  number of symptoms exhibited
by the 12 rats that  received  20  mg/kg quipazine  was only 0.8 + 0.3 of the 6
                                     50

-------
 possible  (Table 9).  The three responses seen  most  often with PCA and CDM
 (forepaw  treading, head weaving, and tremor) were absent in the  12
 quipazine-dosed rats.  Forepaw treading and  tremor  could be elicited only
 with  doses of quipazine producing acute lethality (HO  and 80 mg/kg).  Even
 with  the  40 and 80 mg/kg dose (Fig.  20), the overall incidence of the 6
 serotonergic symptoms was far below  the 4 of 6 criteria of Jacobs (1976).

      Cinanserin (20 mg/kg) effectively antagonized  the 5-HT syndrome induced
 by PCA in rats (Fig. 21). with the total incidence  of  the components of the
 syndrome  halved (3.7 + 0.5 vs 2.0 +  0.6, t  = 2.19, P < 0.05).  The dose of
 cinanserin employed (20 mg/kg) produced no overt behavioral symptoms of its
 own,  except a decrease in muscle tone.   Cinanserin  also completely prevented
 quipazine from inducing any signs of the 5-HT  syndrome (Table 9 and Fig.
 21).  In  contrast, the incidence of  the components  of  the 5-HT syndrome
 elicited by CDM was unchanged by pretreatment  with  cinanserin (20 mg/kg)
 (4.3  + 0.5 vs 3.8 + 0.4,  t   = 1.05,  p  < 0.05)-  Higher doses of cinanserin
 (40-60 mg/kg) could not be tested for their effectiveness in antagonizing
 the CDM-induced 5-HT symptoms, since  these higher doses of cinanserin alone
 induced hindlimb abduction as well as marked signs  of depression (decreased
 motor activity, diminished responsiveness to external  stimuli and
 respiratory depression).
  Q  6
  Ui
  £5
  o
  12
  CO   I
                             PRETREATMENTS
                    SALINE          B CINANSERIN  20 mg/Kg
SEROTONERGIC  SYNDROME
ADDITIONAL   BEHAVIORS
             PCA    CDM    QUIP      PCA    CDM    QUIP
Figure 21.   Effect of  cinanserin  (20 mg/kg) pretreatment (30 min)  on the
serotonergic syndrome  and  additional symptoms elicited by PCA (5 mg/kg). CDM
(80 mg/kg),  and  quipazine  (10 mg/kg).  Values = X +_ SE; (n = 6-12).
* p < 0.05,  ** p < 0.01.
                                    51

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    TABLE 9.   EFFECT  OF  CINANSERIN ON SEROTONERGIC SYNDROME PRODUCED BY  PCA,  CDM  AND  QUIPAZINI
S3

Treatments
Dose


PCA

Controls

After
Cinanserin
(20 mg/kg)
CDM Quip-
PCA
CDM
azine
mg/kg
//Symptoms X
Exhibited/Rat +SE
Symptoms
Forepaw Treading
Head Weaving
Hindi imb Abduction
Rigidity
Tremor
Straub Tail
Totals
Percent Incidence
5
3.7
+0.5

7/11
10/11
6/11
6/11
10/11
2/11
41/66
62$
80
4.3 0
+0.3 ±0

12/12
12/12
8/12
6/12
12/12
1/12
51/72
71*
20
.8
.3

0/12
0/12
2/12
6/12
0/12
2/12
10/72
14*
5
2.0*
+0.6

5/11
5/11*
1/11*
2/11
7/11
1/11
22/66
33*
80
3.8
+0.4

8/8
8/8
4/8
3/8
8/8
1/8
32/48
67*
Quip-
azine
20
0.0*
+0.0

0/8
0/8
0/8
0/8*
0/8
0/8
0/48
0*
After
Methysergide
(10 mg/kg)
PCA

5
3.0
+0.6

0/5*
1/5*
4/5
5/5
4/5
1/5
15/30
50*
CDM

80
3.6*
+0.2

0/10*
2/10*
10/10
10/10
4/10*
10/10
36/60
60*
       *  P  <  0.05 when compared to corresponding controls (Fisher, 1970).

-------
     TABLE  10.   EFFECT OF CINANSERIN ON ADDITIONAL SYMPTOMS PRODUCED BY PCA, COM AND QUIPAZINE.
to
Treatments
Dose
mg/kg
No. Symptoms X
Exhibited/ Rat +SE
Symptoms
Backing
Circling
Hyperactivity
Hyperreac tiv ity
Hyperreflexia
Salivation
Totals
Percent Incidence
Controls
PCA
5
4.3
+0.4
4/11
6/11
11/11
9/11
7/11
11/11
48/66
72%
CDM
80
3.8
+0.3
4/8
6/8
8/8
8/8
3/8
0/8
29/48
60}
Quip-
azine
20
2.0
+0.4
0/8
8/8
8/8
1/8
2/8
1/8
20/48
422
After
Cinanserin
(20 mg/kg)
PCA
5
0.7**
+0.3
0/11
1/11*
1/11*
2/11*
0/11*
2/11*
6/66
9%
CDM Quip-
azine
80 20
2.3*
+0.4
2/8
1/8*
7/8
7/8
2/8
0/8
19/48
40*
0.3**
+0.3
0/8
1/8
1/8*
0/8
0/8
0/8
2/48
4*
After
Methysergide
(10 mg/kg)
PCA
5
2.8*
+0.4
0/5
3/5
5/5
2/5
0/5*
4/5
14/30
47?
CDM
80
2.7
+0.2
2/10
0/10*
6/10
10/10
9/10
0/10
27/60
45*
      *  p  <  0.05  when compared to corresponding controls (Fisher, 1970).
    ** p < 0.01 when  compared to corresponding controls.

-------
     Methysergide (10 mg/kg) pretreatment  (30 min)  significantly  reduced  the
incidence of the components of  the  serotonergic  syndrome  in  CDM (80  mg/kg)
treated rats (4.3 ± 0.3 vs 3.6 ±  0.2,  t    =  2.31.  P <  0.05)  but not
following treatment with PCA (Table 9).  Methysergide  alone  did not  elicit
any overt behavioral effects.

In addition to the 5-HT syndrome, PCA, CDM,  and  quipazine elicited other
Affects which are categorized in  Table 10.   Only PCA and  CDM exhibited  these
effects in a dose related manner  (Fig. 20).  The calculated  doses (X +  SE)
for eliciting 4 of these 6 additional  effects were 4.7 +  1.0 and  91.5 + 6.9
mg/kg.  The fact that these doses are  not  significantly different from  doses
that elicit the serotonergic syndrome  raises the possibility that these
symptoms have common underlying mechanisms.

     As illustrated in Figure 21  and detailed in Table 10, cinanserin
pretreatment also reduced the overall  incidence  of the 6  additional
behaviors elicited by PCA (4.3  vs 0.7, t    = 10.2, p < 0.01);  by  CDM (3.8 vs
2.3, t ^ = 3.23, p < 0.01); and by  quipazine (2.0 vs 0.3, t.,  = 3.5, p  <
0.01).  CDM-induced hyperactivity was  noticeably resistant to  antagonism  by
cinanserin.  In contrast, hyperactivity and  circling induced by PCA  or
quipazine were almost completely  prevented by cinanserin. Methysergide (10
mg/kg) pretreatment did attenuate the  total  incidence  of  the additional
symptoms following PCA  (4.3 ± 0.4 vs 2.8 + 0.4,  t   =  2.48,  p < 0.05) but
not following CDM.                                1^

     When administered  into the lateral ventricles, 5-HT  depressed the
spontaneous activity of the rat but did not elicit the serotonergic  syndrome
(Table 11).  In particular, Straub  tail, tremor  and rigidity were not  .
observed.  Of the additional symptoms, circling  and head  twitches (pinnial
reflex) were present.

     TABLE 11.  EFFECT OF 5-HT  ON SEROTONERGIC  SYNDROME AND  ON ADDITIONAL
     BEHAVIORS IN THE RAT3

     Mean (+ SE) number of symptoms and percent  incidence
Dose ( g)

0
50
100
200
400
Serotonergic
Syndrome
0.3 + 0.2 ( 450
0.8 + 0.5 (13%)
2.0 + 0.4** (25*)
1.5 + 0.5* (25%)
1.5 -i- 0.3***(37%)
Additional
Symptoms
1.0 + 0.4 (17%)
0.5 + 0.3 (18%)
0.8 + 0.4 (17%)
0.3 + 0.3 (4%)
1.0 + 0.4 (17%)
     aSee Methods for classification  of Serotonergic Syndrome and
     Additional Symptoms.   Significant  differences from controls:
     * p < 0.05; ** p < 0.01;  ***  p < 0.001.
                                      54

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DISCUSSION

     The demonstrated ability of COM to induce the "5-HT syndrome" and  the
cinanserin block of the PCA-induced "5-HT syndrome" seem compatible with
Jacobs' view that "the 5-HT syndrome" is a model of increased 5-HT
transmission, since CDM is an MAO inhibitor and elevates brain 5-HT levels
(Beeman and Matsumura, 1973; Benezet jt jal., 1978) while cinanserin is  a
5-HT antagonist (Salas jit al., 1966).

     Clearly in our study central injections of 5-HT did not produce
hyperactivity but rather depressed motor activity, and this is consistent with
previous reports that both intraventricular (Green et jal., 1976a) and systemic
(Jacobs and Eubanks,  1974) administration of 5-HT depresses motor activity  and
produces sedation.

     Even though 5 yl injections into the lateral ventricle rapidly reach the
fourth ventricle, brain stem nuclei distant to the ventricular surfaces and
the spinal area would be inaccessible (Myers and Yaksh, 1968).  Hence,  the
ineffectiveness of intraventricular 5-HT in inducing the complete serotonergic
syndrome could well be due to 5-HT not reaching all the appropriate sites of
action.  In the case  of quipazine, however, no explanation is apparent  for  the
inability of this direct 5-HT agonist to induce the 5-HT syndrome.  Although
Green ^t al. (1976b)  reported that quipazine (25 and 50 mg/kg) exhibited a
motor syndrome similar to that induced by L-tryptophan following
tranylcypromine, they did not provide data detailing the frequencies with
which the component symptoms were observed.

     The ineffectiveness of the two 5-HT antagonists in blocking the 5-HT
syndrome induced by CDM likewise cannot be easily explained.  Since PCA was
about 20 times as potent as CDM in its ability to induce the syndrome,  it  is
unlikely that these results are due to greater affinity of CDM (as compared to
PCA) for the 5-HT receptors involved.  CPP, 6-chloro-2-(piperazinyl)-2-pyra-
zine, is a new agent  which also induces the 5-HT syndrome (Clineschmidt ^t
al., 1977).  In this  case also the 5-HT syndrome was not antagonized by
cinanserin.  One other confounding finding was that the higher doses of
cinanserin alone elicited hindlimb abduction, one of the components of  the
"5-HT syndrome"!  Clineschmidt et al. suggested that the 5-HT motor syndrome
could be due to central tryptaminergic activation to explain the
ineffectiveness of the specific 5-HT antagonist cinanserin.  However, this
possibility was not supported by the effective antagonism by cinanserin of  the
PCA-induced motor syndrome observed in this study.  Thus, these latter
findings are difficult to explain by the simple concept that the serotonergic
syndrome specifically reflects increased serotonergic transmission.

     The behavorial symptoms other than the components of the 5-HT  syndrome
(Table 11) deserve further comment.  Although the stereotypic behavior
observed with PCA, CDM and quipazine is usually associated with dopaminergic
agonists, this stereotypic activity was readily antagonized by cinanserin.
Quipazine-induced stereotypy had previously been  shown to be abolished  by
antiserotonergic agents as well as by dopamine receptor antagonists,  leading
Grabowska ^t a±. (197*0 to suggest that serotonergic as well as dopaminergic


                                      55

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pathways are essential  for  the  full  expression of the stereotypy.   Our
preliminary observation  of  a  block of CDM-induced stereotypy by both
cinanserin and by haloperidol is  consistent with Grabowska's suggestion.

     In view of  these contradictions and uncertainties,  the concept of a
spf.oific 5-HT syndrome  is questionable and needs further validation.  CDM
certainly has a  number  of behavioral effects in rats at  sublethal  doses, as
described above  and  summarized  in Table 13, but whether  they are wholly due to
serotonergic actions is dubious in view of the ineffectiveness of 5-HT
blockers in preventing  their  expression in most instances.  In those cases
where methysergide was  effective  i.e., forepaw treading, head weaving, and
tremor, even if  5-HT is involved, it remains to be shown whether this response
is due to an increase in brain 5-HT  through CDM's MAO inhibitory action or
whether other direct or indirect  serotonimimetic actions are occurring.
                                       56

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                                  SECTION SIX

             INHIBITION OF PROSTAGLANDIN SYNTHESIS BY FORMAMIDINES

INTRODUCTION

     As presented in the previous Sections, CDM possesses a number of  actions
(i.e. blockade of nerve conduction, inhibition of MAO, psychomotor
stimulation, and induction of stereotypic behavior) which are known to be
shared by cocaine.  This encouraged us to make a further comparison between
these compounds in regard to their relative antinociceptive (analgesic)
activity in rats.  CDM (40 mg/kg, ip) and cocaine (20 mg/kg, ip) both
increased tail flick latencies and raised the threshold for vocalization
induced by electrical stimulation of the tail.  The narcotic antagonist,
nalorphine (5 mg/kg, so), did not block CDM- or cocaine-induced
antinociception.  Lidocaine produced analgesic responses only at doses which
resulted in loss of the righting reflex (i.e. 60-80 mg/kg).  Because of this
non-narcotic analgesic effect of CDM, which is probably not related to its
local anesthetic actions, further studies of the mechanism of analgesia were
conducted to detect any aspirin-like analgesic properties of CDM using the
isolated guinea pig ileum.  Aspirin, a prostaglandin synthesis inhibitor,
cause depression of the electrically-induced ileum twitch which is reversed  by
prostaglandin E.. (PGE.) but not by the morphine antagonist, naloxone.   PGE..  is
believed to stimulate the release of the actual transmitter, acetylcholine,  in
this preparation (Ehrenpreis et _al.f 1976).  When CDM was found to block  the
ileum twitch, further studies were performed to define other aspirin-like
actions of CDM (antipyretic, anti-inflammatory) and its activity as a  PG
synthesis inhibitor.

MATERIALS AND METHODS

     Male Sprague-Dawley rats (300-400 g) were obtained from Laboratory Supply
Co., Indianapolis, Indiana.  Chlordimeform and amitraz were kindly provided  by
Ciba-Geigy Corporation and the Upjohn Co. respectively.  They were
recrystallized before use to a final purity of over 98J.

     The drugs included:  acetycholine iodide  (Calbiochem); clonidine  HC1
(Catapres, Boehringer Ingelheim, Ltd. Dist.);  indomethacin  (Indocin, Sigma
Chemical Co.); morphine sulfate  (Mallenkrodt); naloxone HC1  (Narcan, Endo
Laboratories); 1-norepinephrine bitartrate (Sigma Chemical  Co.); phentolamine
mesylate, USP (Regitine, Ciba Pharmaceutical Co.); tolazoline HC1  (Priscoline,
Ciba Pharmaceutical Co.); and prostaglandin E*  (PGE., Sigma Chemical Co.).
Indomethacin and PGE. were dissolved in 95% ethanol and stored  in  the  freezer
until needed.  All other compounds were prepared fresh in distilled water.
                                      57

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Guinea pig ileuro preparation

     Strips of longitudinal muscle  were  obtained  from the guinea pig ileum as
described by Rang  (1964).  The  strips  were equilibrated for one hour in 37°
Krebs solution aerated  with 95/S 0-5%  CO , and  were "field" stimulated as
described by Paton  (1957).  A Grass S88  stimulator was used to deliver square
wave pulses (60 V,  2-4  msec, 0.1  Hz)  and the submaximal (approximately 80%
maximum) contractions were recorded on a Grass  Model 5 polygraph via Grass
FT-03 transducers.   Postsynaptic  effects were assessed using cumulative ACh
concentration responses according to the method of Ariens ^t al^., (1964).

Antipyretic and anti-inflammatory assays

     Fever was induced  in 24-hour fasted rats by the subcutaneous injection of
a 15% suspension of dried brewer's  yeast (10 mg/kg).  Fifteen hours later the
test drugs, suspended  in 1.5J methylcellulose,  were injected intraperitoneally
and body temperature was monitored   rectally over a further 7 hours.  Edema
was induced by injection of  0.05  ml of 1% suspension of carrageenin
(Viscareen, Marine  Colloids)  into the plantar tissue of the left hind paw
(Winter et^a^.,  1962).   The  formamidines were administered as in the
antipyretic assay,  30 min before the carrageenin.  The resultant edema was
expressed  as the percent difference in paw volume, measured by mercury
displacement, compared  to the  volume of the untreated right hind paw.

Assay of prostaglandin  synthesis

     PG synthesis  was  assayed  using bovine seminal vesicle microsomes (Flower
et  aL.,  1973).   Lyophilized  microsomes (1.5 mg) were incubated with
^i-arachidonic  acid (5  Ci  per  mmole) at pH 8.2 in the presence of glutathione
(5  mM)  and  epinephrine (5 mM)  for 5 min.  The reaction (0.51 ml) was
terminated  with  0.25 ml 1 N  HC1, and extracted with 2 ml ethyl acetate.
Authentic  PGE? was added to  a  1 ml portion of the extract and the ethyl
acetate was removed.  The  residue was taken up in 0.1 ml ethanol and
chromatographed  on silica  gel  plates in ethyl acetate:acetone:acetic  acid
(90:10:1).  The  PGE_ spot was  visualized by exposure to  iodine vapor, scraped
off, and counted  in a  scintillation counter.  Control reactions with  a boiled
enzyme  sample were run  similarly, and the PGE2 counts from the other
incubations were  corrected  accordingly.  Corrected rates of synthesis of PGEp
(arachidonate at  0.4 yM final  concentration) in the absence of inhibitors
ranged  from 15-20  pmoles per 5  min per mg protein.  At least five
concentrations  of  each inhibitor were used, without preincubation with the
enzyme  before addition of 0.4 or 50 yM arachidonic acid.  From two  to five
replicates  of each assay were run.  I^Q (50% inhibitory) concentrations were
determined  graphically from  plots of % inhibition of PGE^ synthesis against
log of  inhibitor  concentration.

RESULTS

     COM produced  a dose-related inhibition of the twitch responses of the
guinea-pig  longitudinal muscle  preparation induced by electrical stimulation
(Figures 22 and  23).  The  ED50  as  determined by  reverse  regression  analysis


                                       58

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was 2.9  x  10   J* (7  trials,  Figure 22A), with a 95% confidence  interval of
2.4-3.6x  10   M.   At this concentration, ACh-induced contractions  were
reduced  by approximately 15%.

     As  illustrated  in  Figure  22B, ACh-induced contractions were  reduced by
approximately  30% in the presence of 10"  M CDM (8 trials), a concentration
that depressed  twitch responses by approximately 20* (Figure 22A).  ACh
contractions were enhanced by  the presence of 10J7 M PGE1  (8 trials.  Figure
22B).  ACh responses, in the presence of both 10   M CDM and 10   M PGE., were
not appreciably different from control ACh responses (8 trials, Figure  22B).
                                           B
                                                                       -i
   .--'*
                                                               I CONTROL (N-8)

                                                                 COM ICT4*) (N«8)

                                                                 PGE, ICT*M (N-8)

                                                                 CDM IOT*M +
                                                                 PGE, ICT*M (N-8)
3xKT«     lO*     3»0r»
       CHLORDIMEFORM (M)
                                             —I	
                                              3xl(T»
           icrT
ACETYUCHOUNE (M)
Figure 22. A:  CDM-induced  depression of the electrically stimulated  twitch
height.  Ordinate:  mean +  SE  (n =  7) percent depression from control height
spaced on a probit  scale.   Abscissa:   log cumulative concentration  of CDM.   B:
ACh responses in the  presence  of CDM  and PGE^  Ordinate:  mean + SE  (n = 8)
percent of maximum  control  ACh contraction.  Abscissa:  cumulative  ACh
concentration.
  _„ Twitch depression  induced  by CDM was not antagonized by naloxone  (3 x
10"' M to 10"  M,  3  trials,  Figure.,23A).   In contrast, twitch depression
produced by morphine  (ED  =  3 x  10"'  M),  was readily reversed by naloxone (3 x
10"  M, 2 trials). This concentration of naloxone did not affect  control
twitch height  (2 trials).   The  depression induced by 3 x 10   M CDM  (45.6
4.7% of control) was  reversed by  3  x 10 " M tolazolipe (117 +^ 5.3% of  control,
n = 5, Figure 23B), but not  by  phentolamine (3 x 10   M, 3 trials. Figure
23C).  Twitch depression  produced by norepinephrine or by clonidine  was
readily reversed^by either tolazoline (2 trials) or by phentolamine  (3
trials).  At 10~  M (3 trials), phentolamine alone had only a slight
depressant effect on  the  twitch response.  In contrast, tolazoline alone (3 x
10~  M) increased the twitch response to 143 +_ 4.5% of control  (n =  6).
                                       59

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          COM 3XJO-* M
                         NALLINE 3*IO'TM
   B
         COM
                          2.5»IO'*M

                          TOLAZOLINE
                        l.8xl
-------
     PGE.
(10   M) readily reversed  twitch depression induced by
*__ / f\ M	i_i_  r»j	 r\*\r\ \   	11	 4 n""  U *T\M 10*7 *7 -L  O
                                                                   10~4 M
indomethacin  (2  trials,  Figure 23D), and by  10"* M  COM (27.7 ± 2.3* of control
with COM:   86.9+3.6% of control with COM plus PGEJt  n = 9, Figure 23E).   The
depression  by  3  x 10~5 M CDM (49.M +. 1.8* of control)  was readily reversed  by
PGE  (10~°  M,  102.4 ± 2.1% of control, n = 6).  Washing the strip with fresh
drug-free Kreb's buffer readily reversed twitch depression induced by CDM,  but
not that induced by the irreversible PG synthesis  inhibitor, indomethacin
(Figure 24).   Depression of the twitch by lidocaine (50% at 10   M) was  only
partially and  erratically reversed by PGE., (10~  M, 5  trials, Figure 23F).

     The antipyretic activities of the formaraidines, CDM and amitraz, were
assessed against yeast-induced fever in rats and compared to the effects of
two known antipyretic inhibitors of PG synthesis,  indomethacin and aspirin.
Examination of the resulting dose responses  presented  in Fig. 25 yields  the
following rank-order of the compounds according to their antipyretic activity:
indomethacin  > chlordimeform > amitraz > aspirin.   The reduction by
chlordimeform of the rectal temperatures to  37° in the yeast-treated rats is
consistent  with the hypothermia effects of chlordimeform which we previously
observed in normal rats (Pfister and Yim,  1977).
      40
       39
     K
     o
     00
       36


       40




      ,39
      I


      i 38
     O
     CD
                      0 SALINE
                      • ASA 20
                      o 40mg/Kg
                      A 160
                      A 320
                 i—3-
                      D SALINE
                      • CDM Sing/Kg
                      O 10
                      A 20
                      A 40
-i	6-
                                                     INDOMETHACIN
                                                             B
                                            M=^=*~4
a SALINE
• INDO 0.23mg/Kg
o o.S
A I.O
A £0
a SALINE
• AMITRAZ 20mg/Kg
O 40
A 80
 ^T   2    3    4
  TIME FOLLOWING INJECTION  (HR)
                                 ir\
                     2345
                TIME FOLLOWING INJECTION (HR)
                         i-
 Figure 25.  Antagonism of yeast-induced fever  in  rats by acetylsalicylic
 acid (aspirin),  indomethacin, and two formamidine pesticides, chlordimeform
 and amitraz.  Normal  body temperature at_time  of  administration of yeast
 (-15 hr) is shown  in  A and C.  Values = X +  ;  n = 6.
                                        61

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              TABLE 12.  INHIBITION OF PGE  SYNTHESIS BY BOVINE
               SEMINAL VESICLE MICROSOMES WITH TWO FORMAMIDINES
            AND TWO COMMON NON-STEROIDAL ANTI-INFLAMMATORY AGENTS
                     Inhibitor           I   (yM + SD) at

                                         arachidonic acid

                                         concentration of:


                                0.4yM               50 yM
            Chlordimeform      34+19              145 +_ 36
            Amitraz           880 +  170             >1500
            Indomethacin      0.4+0.08            1.0+0.2
            Aspirin              790                  3900
     To assay the inhibition of PG  synthetase we used a  standard method
based on the conversion of  H-arachidonic  acid  to  PGEp by bovine seminal
vesicle microsomes.  The values in  Table  12  reveal  that  chlordimeform  is  an
inhibitor of PG synthesis of some potency  while amitraz  is  less active,
approximating aspirin  in its activity  under  these  conditions.  Figure  26
demonstrates that chlordimeform and  amitraz  are anti-inflammatory  agents.
The control curves show the time course of the  edema produced by the
injection of carrageenin into  the plantar  tissue of the  hind paw.   Paw
swelling was reduced by approximately  5Q%  in the chlordimeform-treated rats
(40 mg/kg). and by 25% in those treated with amitraz (80 rag/kg).   Aspirin  at
140 mg/kg reduced paw  swelling by 60-65%.  The  ability of aspirin  and
indomethacin to reduce the edema induced by  carrageenin  and other  irritants
is well documented (Winter, 1965).

DISCUSSION

     CDM produced a dose-related depression  of  the  electrically-stimulated
twitch responses of the guinea-pig  longitudinal muscle preparation.  In this
study, the concentration of CDM which  depressed the twitches by 50?
(3 x 10   M) and 70% (10~  M)  reduced  ACh-induced  contractions by  15%  and
30% respectively.  In  comparison, twitch depression by morphine (Paton,
1957), by NE (Paton and Vizi,  1969), by clonidine  (Kroneberg and Oberdorf,
1974), and by indomethacin (Ehrenpreis eit  _al.,  1974) primarily involve
decreased ACh release, with minimal  or no  postsynaptic depression  of ACh
responses.
                                       62

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                                                                 CONTROL
                                                                 ASA I60mg/Kg
o-o CONTROL
*-• AMITRAZ 80mg/Kg
                                                         o—o CONTROL
                                                         •-•COM 40mg/Kg
o—o CONTROL
•-• COM 40mg/Kg
      012345678     26
       TIME AFTER  CARRAGEENIN INJECTION (hr)
                     012345678
                      TIME AFTER CARRA6EENAN INJECTION (hr)
Figure 26.  Antagonism  of  the  carrageenin-induced edema of rat paw by  the
formamidines amitraz  and chlordimeform and aspirin (ASA). ^Compounds were
administered 30 minutes before the  carrageenin.  Values = X _+ SE; n =  6.

     The CDM-induced  twitch  depression was not like that produced by
narcotic-type analgetics,  because  it  was not antagonized by naloxone,  which
readily reversed the  twitch  depression by morphine (Paton, 1957).  In  the
rat tail flick preparation,  we had  already found that CDM-induced
antinociception is  likewise  not antagonized by naloxone.

     The CDM-induced  twitch  depression was also not like the depression
produced by appetite  stimulants,  clonidine, or another alpha-adrenergic
agonist, norepinephrine, since phentolamine, which is an alpha antagonist,
reversed the depression induced by either clonidine or norepinephrine, but did
not antagonize the  CDM-induced effect.  The ability of tolazoline, another
alpha-adrenergic antagonist, to readily reverse the depression of  the  twitch
height by CDM may possibly involve the previously reported anticholinesterase
action of tolazoline  (Dzoljic, 1967).   It is also unlikely that the  local
anesthetic action of  CDM is  primarily responsible for the depression  of the
                                        63

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ileal twitch responses  since  PGE  was  much more  effective in  antagonizing
CDM-induced than lidocaine-induced  depression.

     Thus, the present  studies  indicate  that  the twitch depression produced by
CDM was most similar  to that  produced by the  nonsteroidal anti-inflammatory
agent, indomethacin;  both  drugs were  consistently reversed by exogenously
add ,d PGE  .  Indomethacin  is  a  well-known irreversible inhibitor of
r.rostaglandin synthesis (Gryglewski,  1974).   The results above show that
chlordimeform is also an effective  inhibitor  of prostaglandin synthesis.  The
ICQ values reported here for  aspirin  and indomethacin with the bovine seminal
vesicle synthetase  fall within  the  range of  existing values  in the literature
(Gryglewski, 1974).   The mechanism  and kinetics of inhibition by the
formamidines remain to  be  established, but preliminary results indicate that
chlordimeform is a  reversible,  non-progressive  inhibitor.  In each case in
Table 12  the inhibitory potency decreased at  the higher substrate
concentration.  Normal  concentrations of arachidonate at the active site of
the synthetase jlri vivo  are hard to  estimate  since it is believed that the
fatty acid precursors for  PG  are  stored  as phospholipids and are released as
needed by the action  of phospholipases.   Tissue levels of the free acid are
very low  (Hinman,  1972).  It  is significant  that the potencies of the four
compounds assayed as  synthetase inhibitors are  directly related to their
antipyretic activities  as  shown in  Fig.  25.

     The  washing studies with the guinea pig ileum uncovered an important
difference between  the  actions  of CDM and indomethacin.  The persistence,
after washing with  fresh Kreb's buffer,  of the  twitch depressant action of
indomethacin, has been  attributed by Ehrenpreis et al., (1976), to
irreversible inhibition of PG synthesis (Ku  and Wasvary, 1975).  The
reversibility of the  twitch depression induced  by CDM is in keeping with the
conclusion that it  is a reversible  PG synthesis inhibitor.  Thus, the guinea
pig ileal preparation may  be  a  convenient screen to distinguish reversible
from irreversible inhibitors  of PG  synthesis.

     We have shown  that CDM has several aspirin-like actions (antinociception,
anti-inflammation,  antipyretic, and block of guinea pig ileum twitch).  It is
likely that aspirin achieves  most or all of these actions by inhibition of
prostaglandin synthesis (Vane,  1971;  Flower,  1974; Robinson and Vane, 1974),
an  inhibition which we  have found also to be shared by the formamidines.
Since aspirin and indomethacin  are  progressive, irreversible inhibitors of PG
synthetase (Gryglewski, 1974),  and  chlordimeform appears to be reversible and
non-progressive, a  direct  comparison of !,.« values can be misleading.
However,  we feel that inhibition  of PG synthesis can provide a reasonable
explanation of the  several aspirin-like pharmacological actions of the
formamidines.

     Our  present results are  of interest from a number of aspects:  (i) PG
synthetase inhibitors with the  general structural characteristics of the
formamidines have not been previously reported.  Most known inhibitors are
acids rather than bases (Robinson and Vane,  1974).  Further, the ability of
pesticides to interfere with  prostaglandin-dependent processes has not
apparently been the subject of  previous study,   (ii) Early death from single
                                      64

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high doses of the formamidines has been  attributed  to  direct depression
myocardial and vascular muscle and accompanying  respiratory depression, which
are local anesthetic-like actions.  It is  intriguing to  note in this regard
the recent report that PG can antagonize the  block  of  nerve conduction induced
with local anesthetics, and that the PG  synthetase  inhibitor indomethacin also
caused a conduction block which could be reversed by the addition of PGE..
(Horrobin et al_., 1977).  Thus there may be a connection between the PG
synthetase inhibiting and local anesthetic actions  of  the formamidines.  (iii)
Finally, the gastric ulcers, intestinal  hemmorhaging,  and death in 2-5 days
observed in animals treated with indomethacin and other  non-steroidal


 TABLE  13.   BEHAVIORAL AND PHARMACOLOGICAL PROFILE OF COM  IN THE  RAT


                                                   Dose     (mg/kg,  ip)
         Effects           1-20    21-40    41-60    61-80    81-100     200
 HYPERPHAGIA                +++        +
 HYPERGLYCEMIA                +       -M-      +-M-
 ANTIPYRESIS                  +       -M-
 ANALGESIA                    +       -M-
 ANTIINFLAMMATORY             +       -H-      -M-+
 ANOREXIA                                       +
 STRAUB TAIL                  -
 HYPERTONICITY                         +       ++
 HIND LIMB ABDUCTION                   +       -H-
 HYPERREACTIVITY              +        +       -n-      +++        +++     +-M-
 INCREASED MOTOR ACTIVITY     +        +       +++++        +++
 CIRCLING                     -        -        +       ++        +-M-
 BACKING
 TREMOR
 HEAD WEAVING
 SPASTICITY-HYPERREFLEXIA
 CONVULSIONS
 RESPIRATORY ARREST           -                                           -M-+
 CARDIAC ARREST
 ELEVATED BRAIN 5-HT
 ELEVATED BRAIN NE
 DEATH
 Effects are rated subjectively according to intensity:  +  =  present (low
 intensity); ++ = moderate intensity; +++ = marked intensity;  - = not present;
 empty space = not known.  The effects produced by CDM  are  arranged in
 decending rank order of appearance over the dose range indicated.
                                      65

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anti-inflammatory agents has been  attributed  to  the  inhibition of PG synthesis
(Robert, 1976).  This raises the question  of  whether the formamidines also may
initiate these toxic consequences  of  the  inhibition  of PG synthesis.

     Preliminary studies have  shown that  COM  in  fact is much less potent than
aspirin in inducing  gastric ulcers.   This  may be attributable to the
reversibility of the inhibition of PG synthesis  by CDM (Yanagi and Komatsu,
1/T6) and the fact that it  is  a base  rather than an  acid like aspirin (Brune
e;t ^L., 1976).  Thus, CDM  and  related formamidines comprise a new and
interesting class of nonsteroidal  anti-inflammatory  agents which lack some of
the undesirable side-effects of current compounds.

     A  summary of the behavioral  and  pharmacological actions of CDM in the rat
as observed in the studies reported in Sections  2 through 6, and in other
studies in our laboratories, is provided  in Table 13.
                                       66

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                                SECTION SEVEN

       BIOTRANSFORMATION OF CHLORDIMEFORH AND ITS RELATION TO TOXICITY

INTRODUCTION

Clearance and metabolism of chlordimeform in mammals.

     The metabolism of COM and some of its intermediate metabolites has  been
investigated in dogs and goats (Sen Gupta and Knowles, 1970), rats (Knowles
and Sen Gupta, 1970; Ahmad and Knowles, 1971a, 1971b; Morikawa et _al.,  1975;
Benezet and Knowles, 1976b) and mice (Knowles and Benezet, 1977).  In each
case, when CDM labelled with   C in the ring methyl was orally administered,
it was readily metabolized and rapidly eliminated in the first 24 hours,
primarily in the urine.  About 85% of the   C-CDM equivalents were eliminated
in the rat urine (Knowles and Sen Gupta, 1970), while 70-80% was eliminated
in the dog urine in the first 24 hours.  Slightly less than 5% of the
administered dose was accounted for in the bile of dogs with biliary cannulas
in 72 hours (Sen Gupta and Knowles, 1970).  The clearance of CDM was also
rapid in goats.  Excretion was faster in males, since 80% of the administered
dose was eliminated in the urine of male goats compared to only 65%
eliminated in the urine of lactating female goats in the same experimental
period of 24 hours.  The elimination of chlordimeform in feces after an  oral
dose is considered a minor route, since it accounted for only 7.556, 0.6% and
1.8% of the administered dose in 72 hours in rats, dogs and goats
respectively.

     The pathway of excretion seems to be dependant on the^route of
administration since, after ip administration to mice of   C-CDM, 39% of the
administered radiocarbon was eliminated in the urine and 45% in the  feces  in
24 hours (Knowles and Benezet, 1977), compared to about 81% recovered in the
urine of mice in the same experimental period when   C-CDM was given orally
(Ghali and Hollingworth, unpublished).

     The elimination of two chlordimeform metabolites after oral
administration was also rapid, but the relative^importance of the routes of
excretion were changed compared to CDM.  When   C-labeled DCDM or CT were
orally administered to rats, 35% and 71% of the radiocarbon from the two
compounds were eliminated in the urine and 64% and 24% were eliminated  in  the
feces respectively in 24 hours (Knowles and Sen Gupta, 1970; Ahmad and
Knowles, 1971 a).

     In all of these studies, the majority of the radioactive materials in
urine were unidentified water soluble materials, most likely glucuronide and
sulfate conjugates, which yielded to arylsulfatase/g-glucuronidase cleavage.


                                      67

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No individual conjugates  have  been  identified,  but  some of the  radiocarbon-
containing aglycones were identified  after  deconjugation as COM,  DCDM,  NFT,
and CT, in addition to  other unidentified materials (Sen Gupta  and  Knowles,
1970).  The organosoluble metabolites in  the  urine  account only for 10-25J  of
the total radiocarbon.  This relatively wide  range  of variation in  the  amount
of organosoluble metabolites indicates a  difference in the rates of
degradation of  COM by different  mammalian species.   The major  organosoluble
ratabolites recovered were NFT,  CT  and N^fortnyl-5-chloroanthranilic acid
(NFA).  In addition to  these major  metabolites, others such as  DCDM,  DDCDM,
and 5-chloroanthranilic acid (CAA)  were also  present in all mammalian species
studied.  Recently, Knowles and  Benezet (1977)  added three urea derivatives
to this list of metabolites, i.e.  1, l-dimethyl-S-CJ-chloro-o^tolyDurea
(CDU),  1-methyl-3-(M-chloro-£-tolyl)urea  (DCDU) and U-chloro-c>-tolylurea
(DDCDU).

      Iri vitro studies using various hepatic cellular subfractions have
indicated that  the maximum activity in degrading CDM in rats was associated
with  the microsomal subfraction  (Ahmad and  Knowles, 1971a; Morikawa et  al.,
1975).  The metabolites detected in vitro were  well correlated  qualitatively
with  those found  in vivo.  With  the elucidation of  the molecular structure  of
these metabolites, the  sites of  enzymatic attack on the formamidine molecule
can be  tentatively established and  a metabolic  pathway (Fig. 27) has been
postulated (Ahmad and Knowles, 1971a; Morikawa  jit al^., 1975; Knowles and
Benezet,  1977).

      The  initial  metabolism of CDM  is thought to be catalyzed  exclusively by
mixed function  oxidases (MFC).  One major pathway involves successive
N-demethylations  to yield DCDM and  DDCDM.   The  initial Nkdemethylation
reaction  is NADPH and 0  dependent, inhibited by SKF 525-A, and therefore is
probably  catalyzed by microsomal MFC (Ahmad and Kowles, 1971a).  However, it
is not  known whether the  second  Nj-demethylation reaction is catalyzed by  the
same  enzymes.   Oxidative  iN-demethylation  usually proceeds through an
N-hydroxymethyl intermediate  (McMahon, 1966).  This intermediate, however,
was not isolated  with CDM in vitro  (Ahmad and Knowles, 1971a;  Morikawa  eit
.al..  1975).

      Both of the  J^-demethylated  metabolites (DCDM and DDCDM),  as well as  CDM
itself, can be  cleaved  to NFT  in a  reaction which overall appears to be a
hydrolysis.  The  mechanism of  this  cleavage is  an unresolved issue.  Ahmed
and Knowles (1971 a) suggested  a  nonenzymatic  hydrolytic mechanism for
formamidine cleavage.   Morikawa  jet  al. (1975),  although not entirely
excluding this  possibility, provided evidence that  this reaction is probably
mediated by an  oxidative  mechanism.  This conclusion was based  on the fact
that  production of the  .N-formyl  derivative  in hepatic microsomal incubation
was significantly dependent on the  concentration of NADPH, and  was inhibited
by SKF  525-A, a known inhibitor  of  microsomal drug  oxidation.   However, since
the N-formyl derivative can be produced non-enzymatically even  more readily
from  the two JY-demethylated derivatives than  from CDM itself,  and since the
_N-demethylation reaction  is also impaired by microsomal inhibition, then  the
decrease in the rate of production  of the J^-formyl  derivative might also  be
an indirect result of the decrease  in the rate  of NU-demethylation.
                                      68

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      cm  J>-N=C-N
           CH3
    -a
                     X
                     X
                   COM
                 V
            CH
                     N.
                   DC DM
N=C-N
            CH3

                   DDCDM
                   NFA
                                 Cl
Cl
                            V 9   /CH3
                            N-C-N,
                                CDU
                            ? 9   XCH3
                            N-C-NX
                                    H
                                OCOU
                                             9
                                    H
                                                 x.
                         CH3
                                DDCDU
                                        CH3
                                              CT
                                            2

                                        COOH
                                CAA
Figure 27. Metabolic map showing fate of chlordimeform in mammals.
                             69

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     NFT can be deformylated  to  CT.  This  reaction  is  inhibited  by  the
organophosphate DFP, but not  by  SKF 525-A,  and  is probably  carried  out by  the
soluble liver formamidase which  normally catalyses  the deformylation  of
N^formylkynurenine, and is  inhibited by DFP (Mehler and Knox,  1950; Shinohara
and Ishiguro, 1970; Ahmad and Knowles,  1971b).   This enzyme was  also  capable
of deformylating the corresponding anthranilic  acid (NFA) to CAA, a common
metabolite from CDM.

     CDU, one of the three  urea  metabolites recently reported  by Knowles  and
Benezet (1977), may be  formed by hydroxylation  of the  central  (amidine)
carbon of CDM followed  by an  enol-keto  rearrangement.   The  other two  urea
metabolites could  be formed by a similar mechanism  from their  respective
formamidines or by sequential oxidative NN-demethylation of  CDU.   Oxidative
Nj-dealkylation is  known to  be an important pathway  in  the mammalian
metabolism of substituted phenylurea herbicides (Geissbuhler «it  al^.,  1975).
CT could also be produced by  a direct cleavage  of the  ureas.  Direct  cleavage
of substituted phenylureas  in mammals to yield  substituted  anilines has been
postulated but apparently is  not a major pathway (Geissbuhler €it a±., 1975).
Therefore, it seems likely  that  most of the 4-chloro-£-toluidine is derived
by deformylation of NFT.  In  addition to the production of  CT and NFT, which
with their conjugates,  are  major terminal  metabolites, an additional  reaction
has been observed.  This involves oxidation of  the  ring methyl moiety
yielding various anthranilic  acids  (Knowles and Benezet, 1977).   It is not
known at what stage of  metabolism the ring-methyl group is  oxidized,  but
ring-methyl oxidation products  (alcohol, aldehyde,  or  acid) with an intact
formamidine group  have  not  been  reported.

     CDM is not a  substrate for  mammalian  hepatic glutathione transferases
(Ghali, G. and Hollingworth,  R.  M.,  unpublished).   No  in vitro studies of the
conjugation of CDM or its metabolites by glucuronyl transferases,
sulfotransferases, or other conjugating enzymes have been reported, although
from the results described  on the aglycones present after urinary
deconjugation, direct conjugation is a  possible reaction with formamidines
(Sen Gupta and Knowles,  1970).

MATERIALS AND METHODS

Synthesis of chlordimeform  and  its  major metabolites

     Chlordimeform (CDM) and  its ^-demethylated metabolites, DCDM and DDCDM,
were synthesized by coupling  4-chloro-o-toluidine with N^-dimethylformamide,
N-methylformamide, or formamide  in  the  presence of  phosphorous oxychloride
(Hollingworth, 1976).

     In a typical  synthesis of  chlordimeform, (J.JY-dimethylformamide (19  g,
0.260 moles) in 25 ml of dry  benzene was added  to phosphorous oxychloride
(12 g, 0.078 moles) in  20 ml  of  dry benzene in  a 250 ml three-neck  flask
attached to a water condenser.   The mixture was stirred at  room  temperature
for 30 min, then 4-chloro-o-toluidine  (7.4 g, 0.052 moles)  in 30 ml of dry
benzene was added  at room temperature with stirring for a  further 3 hr.
                                     70

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     In the case of DCDM and DDCDM, the order of addition of reactants  was
changed in order to avoid the accumulation of a sticky intermediate  complex
i.e., the formamide and the toluidine were added simultaneously  to the
phosphorous oxychloride in the flask.  Furthermore, the formamide was
dissolved in acetonitrile and the stirring continued for only one hr at room
temperature.

     In the case of CDM and DCDM, the precipitate from this reaction (crude
hydrochloride salt) was collected by filtration and treated with 250 ml of
ice-cold 1 N sodium hydroxide under cooling and continuous stirring  for about
10 min.  The oily layer was then extracted with 100 ml of benzene.   The
benzene layer was washed with water and dried over anhydrousj.sodium  sulfate.
The compound was either redistilled (CDM; bQ _ 103-108°, nD    1.5918),  or
recrystallized (CDM, DCDM, and DDCDM) several times until purity was evident
by thin layer chromatography (Whatman K5F silica gel, developed  with
benzene:diethylamine, 95:5) and melting point (Fisher-Johns hot  plate).  CDM
and DCDM were recrystallized from benzene while DDCDM was recrystallized from
ether by the gradual addition of hexane.

     In the case of DDCDM, which is more easily hydrolyzed under basic
conditions, the precipitate was taken into a separatory funnel which
contained prechilled ether and an equal volume of ice-cold 0.5 N sodium
hydroxide, and shaken continuously for 2-3 min.  The ether phase was then
separated, washed several times with water, and dried over anhydrous sodium
sulfate.  The compound was recrystallized several times until  purity was
evident as before.  All three of these formamidines as the free  base gave
colorless cystals with melting points of 30-31°, 91-92°. and 88°
respectively.  The hydrochloride salts of these formamidines were made  by
passing HC1 gas into their ethereal solutions.  The salts were recrystallized
from ethanol-acetone (1:1) giving water soluble white crystals with  melting
points of 221-223°, 192-194° and 220-221° for CDM, DCDM and DDCDM
respectively.

     The formamidine metabolite, Nkformyl-4-chloro-<>-toluidine  (NFT),  was
synthesized by refluxing 4-chloro-o-toluidine with an excess of  formic  acid
in benzene with concomitant removal of the water produced  (Fieser  and  Jones,
1955).  After removing the solvent, the product was recrystallized  from 1:1
ethanol-acetone mixture producing colorless crystals with a sharp  melting
point at 120°C.

     The urea metabolite, 4-chloro-o-tolylurea (DDCDU). was synthesized by
the reaction of equimolar potassium cyanate and 4-chloro-o-toluidine in
acetonitrile (Geissbuhler jt _al. 1975) at room temperature.  The urea
produced in this reaction precipitated as a highly insoluble product in good
yield.  The product was purified by washing the precipitate several  times
with acetonitrile and recrystallization from dimethylsulfoxide.   The material
so obtained was a white powder  (m. 204 ).

     1-Methyl-3-(4-chloro-o-tolyl)urea (DCDU) was  synthesized  by the reaction
of equimolar amounts of 4-cihloro-o-toluidine and methyl  isocyanate in
benzene.  The isocyanate was added to the toluidine with  stirring  at 40°.
                                     71

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The precipitate was filtered  off  and  washed  with  water  and  then  with
acetonitrile and recrystallized  from  dimethylsulfoxide  (m.  213°).

     The structures of these  compounds  were  confirmed  by elemental  analysis,
and infrared, nuclear magnetic  resonance,  and   g.c.- mass spectroscopy.
4-Chloro-£-toluidine was  purified  from  the commercial  material  (Aldrich
ChCiflical Co.) by vacuum distillation.   A  further  metabolite,
j-chloroanthranilic acid,  was obtained  from  Ciba-Geigy Agrochemicals,
Greensboro, NC.

                                 14
     Chlordimeform  labeled with    C in  the ring methyl  group  with  specific
activity of 20 UCi  per mg (4  mCi  per  mmole)  and a radiochemical  purity of
96-99/1  (major impurity^T, formed on storage)  was obtained from Ciba-Geigy
Agrochemicals.  Other^ C-labeled metabolites  were biosynthesized  by
incubating 20 yCi of   C-chlordimeform  for 20  min with the  microsomal
fraction of mouse liver and NADPH (1.7  mM) in  Tris buffer (pH 7.6)  at  37°,
and subsequent extraction of  the  metabolites with chloroform, and
purification by preparative two-dimensional  TLC (Knowles and  Benezet,  1977).
Compounds were eluted  from the  TLC plates with acetone.

Effect  of mixed function  oxidase  inducers and  inhibitors on the  acute
toxicity of CDM and its N-demethylated  metabolites

     The mice were  pretreated with either of two  hepatic mixed function
oxidase (MFO) inhibitors  or with  one  of three  MFC inducers.  The inhibitors
SKF 525-A  (in 0.9%  NaCl)  and  piperonyl  butoxide (in corn oil) were
administered ip at  the rate of  50 and 400 mg/kg respectively two hours prior
to subsequent treatment with  graded oral  doses of CDM,  DCDM,  or  DDCDM  as
described before.   In  the case  of the inducers, phenobarbital (in  saline
solution) was administered ip at  four successive  daily doses of  50 mg/kg
prior to assay on day  five.   Aroclor 1254 (in corn oil), a polychlorinated
biphenyl mixture, was  given once  at 500 mg/kg  ip  five  days  prior to
treatment, and 3-methylcholanthrene  (in corn oil) was  administered ip  in two
successive daily doses of 40  mg/kg with the  second dose two days prior to the
toxicity tests.  Control  animals were pretreated  with  the vehicle  alone.
LD,. 's  were determined as described in  Section 1.

Subcellular fractionations

     Animals were killed  by a blow to the head.  The  desired tissues  such as
livers, brains, and lungs were  rapidly  removed and washed with cold 0.25 M
sucrose solution.   The tissues  were then  weighed, minced, and homogenized in
fresh prechilled sucrose  at the  rate  of 5 ml/g tissue  using a Potter-Elvehjem
type glass homogenizer fitted with a  teflon  pestle.  The system  was kept ice
cold throughout.  The microsomal  and  mitochondrial fractions were  isolated by
differential centrifugation,  initially  600 x g for 10  min,  then  the
supernatant was spun down at  12,000 x g for  another 10 min  to isolate  the
mitochondrial fraction, and then  by further  centrifugation  of the  supernatant
at 105,000 x g for  one hr using  a Beckman L2-65 ultracentrifuge  to separate
theomicrosomal fraction.   The resulting microsomal pellet was kept frozen at
-20  for a maximum  of  four days  before  determination  of the MFO  activities.
                                      72

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     The supernatant from the 105,000 g spin was used as the  source  of
soluble enzymes.  It and the mitochondrial fraction was used  within  two  hours
of preparation. Hepatic microsomes were also prepared from mice  pretreated
with MFO inducers or inhibitors as already described.

Metabolism of chlordimeform in vitro

     Microsomal pellets (or mitochondrial fractions) of hepatic,  brain,  orQ
lung tissues were resuspended in ice-cold 0.05 M Tris buffer  (pH  7.6 at  37 )
to a final concentration corresponding to 5 g of liver and 20 g  of brain or
lung tissues per 100 ml.  Soluble fraction of liver was diluted  5 times  with
buffer.  Rates of metabolism were determinedj.at.j37  in an incubating shaker
in open 50 x 5 mm tubes containing 4.2 x 10~ M   C-labeled substrate (CDM,
DCDM, or DDCDM), 10 yl 10"^M NADPH in the Tris buffer, and 50 yl  of
microsomal (or raitochondrial) suspension or liver soluble fraction.   The
final incubation volume was 60 yl.  The substrate was added as a  solution in
hexane and the hexane was carefully evaporated before addition of the other
components.  Five yl samples were taken from the incubating mixture  at
several different times over 10 min after the addition of enzyme,  spotted on
silica gel TLC plates (Whatman K5F, 250 y) along with the chromatographic
standards in some runs, and developed in a two dimensional system modified
from Knowles and Benezet (1977).  The first system consisted  of
benzene:acetonerdiethylamine (95:5:5). while the second system consisted of
benzene:dioxane:acetic acid (90:10:1).  Individual metabolite zones  were
located by autoradiography on X-ray film and scraped for scintillation
counting in a toluene base scintillation cocktail containing  0.4J
2,5-diphenyloxazole and 4$ Cab-0-Sil powder.  The nature of the  metabolites
was confirmed by cochromatography in the system above and by  mass
spectrometry in some cases.

     To study the effect of the absence of molecular oxygen on the microsomal
metabolism of CDM, a modified Warburg vessel with two gas valves was used.
The enzyme and NADPH were placed in one compartment while the substrate  was
placed in the other.  A source of nitrogen was connected to the  gas  inlet
while both valves were open.  The nitrogen was allowed to pass for 10 min
before the valves were closed.  This method gave equivalent results  to a
second one in which the flask was alternately evacuated and then flushed with
N_.  However, the latter method resulted in foaming and possible enzyme
denaturation.  The vessel was warmed to 37° and the enzyme  and NADPH were
admitted to the substrate compartment and mixed well before incubation for 10
min in a shaking water bath at 37°.  The reaction was stopped by dropping the
vessel in a cooling mixture (-20°).

     The effect of carbon monoxide on the microsomal metabolism  of CDM was
also studied by bubbling CO gas for 5 to 10 min at a rate which  did  not cause
foaming into a mixture of the enzyme and NADPH before adding  them to the
substrate in the usual incubation tubes.

Metabolism of chlordimeform in vivo

                                                             14
     Male Swiss white mice (23-25 g) were orally dosed with    C-chlordimeform
after pretreatment with piperonyl butoxide, phenobarbital,

                                      73

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3-methylcholanthrene, or  vehicle  as  previously described.   Four  mice were
used for each treatment and  each  mouse  was given  10 yCi  of the toxicant (1:1
mixture of labelled  and unabelled CDM)  in  0.25 ml corn oil.   This dose of COM
amounted to 40 mg/kg.  After 40 min,  the  animals  were  sacrificed.  Blood was
collected in heparinized  vials.   Livers and brains were  rapidly removed,
placed in preweighed  scintillation vials,  and  immediately  frozen in an
acetone/dry ice bath  and  stored at -20° for analysis.  Total tissue
 •adioactivity was  assayed using a 50 mg sample of well-macerated tissue in a
scintillation vial.   The  tissue was  digested overnight with 0.5 ml of
Soluene-100 (Packard  Instrument Co.,  Downers Grove, ID  at room temperature,
bleached with 0.5  ml  30%  hydrogen peroxide, and then  treated with 0.2 ml
glacial acetic acid  to overcome chemiluminescence.  Scintillation cocktail
(10 ml, Aquascint  1,  ICN) was added  to  the vials which were counted in a
scintillation spectrometer (Packard  Tri-Carb 3310).  Efficiency of counting
was determined with   C-toluene  internal  standard.

     To study the  nature  and magnitude  of the individual metabolites, about
200-400 mg of each tissue was weighed and homogenized  in 2 ml 2% HC1, 0.5 ml
10% trichloroacetic  acid  (TCA) was added,  and the mixture  was centrifuged for
30 min at 3,000 rpm.  The supernatant was transferred  to another tube and the
pellet re-extracted  with  another  2 ml of 2% HC1 followed by TCA and
centrifugation as  before.  The two supernatants were  combined, treated with
another 0.5 ml of  10% TCA and spun down for a further  30 min.  The clear
acidic extract was then transferred  to  a larger tube,  the  pH was carefully
adjusted to 9.0 using 1 N NaOH with  rapid mixing, and  the  solution was
extracted  three times with prechilled  ether-  The combined ether
extractswere dried over anhydrous sodium sulfate, and  evaporated to near
dryness using a gentle stream of  nitrogen  under cooling conditions to
minimize evaporative losses of volatile components.  The residual tissue
material from the  acidic  extractions was further extracted with two portions
of ether to remove any residual NFT.  These two ether  extracts were combined,
dried over anhydrous sodium sulfate, and evaporated to near dryness as
before.  The ethereal extracts were  analyzed by two-dimensional
TLC/scintillation  counting as already described.  The  nature of the
metabolites was determined initially by cochromatography on TLC.

Rates of hydrolysis  of chlordimeform and its N-demethylated metabolites

     The rates of  hydrolysis of CDM, DCDM, and DDCDM were  investigated at pH
7.4 and pH 8.7.  A 10 mM  stock solution of each compound was made in
methanol. One ml of  each  of these solutions was placed in  a 12 ml graduated
tube with glass stopper and made  to  10  ml using 0.01  M Tris buffer which was
prewarmed to 37°.  A 5 yl sample  (zero  time) was taken right after mixing and
before placing the tube in a shaking water bath at 37°.   Samples (5 ul) were
taken at 5 min intervals  over the first 30 min then less frequently for 3 hr
and every 24 hr until the reaction was  completed.

     The amount of the hydrolysis product, N_-fortnyl-4-chloro-o-toluidine
(NFT), was monitored  by injecting the samples immediately into a Water
Associates Liquid  Chromatograph equipped  with a 30 cm  x  4  mm i.d. Micron
Bondapak C-18 column, UV  absorbance  detector (Model 440) of a fixed
wavelength (245 nm),  and  a Water  Associates Pump Model M-6000A.  An

                                      74

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attenuation unit full scale of 0.05 to 0.1, a flow rate of 2 ml per  minute,
and a pressure of 2000 psi were used.  The mobile phase was a mixture  of
acetonitrile:water:methanol (45:45:10).  All solvents were "L.C.  Distilled  in
Glass" grade.  Retention time for the hydrolysis product under these
conditions was 3.5 min.  Retention times for the parent formamidines were
much longer (> 20 min).  The experiment was duplicated for each compound at
each pH and the results averaged.  The pH of the reactions was monitored and
was steady throughout the experiment.  In order to ensure that the reaction
had reached completion, the tubes were boiled for 30 min at the end  of the
reaction and the amount of the hydrolysis product was checked before and
after boiling.  However, no change in the amount of the hydrolysis product
was observed.

     The NFT peak heights were measured at each time interval and a  first
order plot relating time to log[(ha - h )/(ha - h )] was made, where ha is
the final peak height of NFT at ta and 80 is the peak height at t ,  and h   is
the peak height at a given time, t.  The pseudo first order rate  constant  (k)
was calculated from the equation, k = slope x 2.303, and the half life (
of each compound was calculated from the equation, t1/2 = 0.693/k.

                                   RESULTS

Toxicity and symptoms in mice after oral exposure to CDH and its  metabolites

     Data regarding the acute toxicity of COM and its major metabolites have
already been shown in Table 1.  Additional studies with two of the more minor
urea metabolites (DCDU and DDCDU) indicated LD,.. values over 500  mg/kg and  an
absence of the rapid excitatory symptoms typical of the three formamidines.

     The symptoms elicited by lethal doses of CDM and the two jf-demethylated
metabolities, although not identical, are very similar.  These compounds  are
rapidly acting and symptoms are manifested as restlessness, hyperreflexia,
tremors, particularly of the head and forelimbs, developing to one or  more
episodes of clonic convulsions.  Death occurs generally within the first  hour
during one of these convulsive episodes and is marked by labored  breathing
and gasping.  The tremors are not seen in the case of animals poisoned with
the NUdemethylated derivatives and the convulsive stage is significantly
longer.  The CDM-treated animals usually show signs of locomotor  difficulty
partly due to frequent hyperextension of the hind legs a later stages  of
poisoning, but this was not seen with DCDM and DDCDM.

     Since N_-demethylations appear to be the only metabolic reactions  leading
to more acutely toxic products, and these are most likely catalyzed  by
microsomal mixed function oxidases (MFO), the effect of ip pretreatment with
inhibitors or inducers of hepatic MFO systems on the toxicity of  CDM was
studied.  The results are shown in Table 14.  Surprisingly, the MFO
inhibitors (SKF 525-A and piperonyl butoxide) had no effect on the  toxicity
of CDM.  Neither did the MFO inducer, phenobarbital.  The other two  inducers,
3-methylcholanthrene and Aroclor 125M had the even more paradoxical  effect of
decreasing the toxicity of CDM about 2-fold.  The same trends were  also seen
in the effect of some of these pretreatments on the toxicity of DCDM and
DDCDM.  3-Methylcholanthrene had a clear protective effect, approximately

                                      75

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doubling the LD_Q for both DCDM  and  DDCDM.  Aroclor  1254  was  tested  only with
DCDM but had a comparable protective action.   Piperonyl  butoxide  pretreatment
again did not have any effect  on  the toxicity  of DCDM  or DDCDM.

TABLE 14.  THE EFFECT OF SEVERAL  PRETREATMENTS AFFECTING MICROSOMAL OXIDATIONS
ON THE TOXICITY OF FORMAMIDINES  TO MICE


                                            LD5  (±95?  Conf.  Intervals), mg/kg
Pretreatment                Formamidine          Control            Pretreated
Piperonyl butoxide
SKF 525-A
Phenobarbital
Aroclor 1254
3-Methylcholanthrene
Piperonyl butoxide
Aroclor 1254
3-Methylcholanthrene
Piperonyl butoxide
3-Methylcholanthrene
COM
COM
COM
COM
COM
DCDM
DCDM
DCDM
DDCDM
DDCDM
233(183-295)
290(223-377)
250(225-279)
295(268-324)
250(223-280)
185(162-209)
167(145-199)
167(145-199)
76(61-95)
100(81-123)
260(206-327)
280(215-364)
266(229-309)
510(425-612)
410(360-462)
190(168-214)
350(273-447)
310(258-371)
84(67-105)
170(132-210)
Non-enzymatic  hydrolysis of chlordimeform and its N—demethylated metabolites.

      Since  it  has  been  suggested that much of the NFT produced from these
formamidires arises by  non-enzymatic hydrolysis, and because this hydrolysis
is  a  clear  detoxication reaction (although potentially leading to
mutagenic/carcinogenic  anilines), we measured the rates of hydrolysis of CDM,
DCDM,  and DDCDM.   As shown in Fig. 28 plots of log C(ha - h )/(ha - h )]
against  time were  linear, indicating a pseudo first order rate of hydrolysis
for each compound.   First order kinetics were observed over at least 60% of
the total reaction.  The rate constants (k) and half lives (t^) calculated
from  these  plots at 37° and either pH 7.4 or 8.7 are given in Table 15.

      Under  'physiological conditions' i.e. pH 7.4 and 37°, the formamidines
are relatively stable with half lives ranging from 161 min for DDCDM to 538
min for  CDM.   In each case, raising the pH to 8.7 approximately triples the
rate  of  hydrolysis.  The hydrolysis product in each case, NFT, could also be
hydrolyzed  further  to CT.  However, under the reaction conditions and times
employed here,  NFT was  stable i.e. no hydrolysis was detected in 12 hr at pH
8.7.

Metabolism  of  formamidines by mouse enzymes in vitro

      In  order  to determine which tissue(s) and subcellular fraction(s) were
most  active in  degrading CDM, a preliminary study was conducted with mouse
liver, brain,  kidney, heart,  skeletal muscle, and lung homogenates fortified

                                      76

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    0.10
                                                 	.__CDM



                                                 	DCDM
o>
o
 .  0.03.,
0.02.
    0.01 ..
    0.00
             10  20  30  40  50  60  70  80  90  100  110  120 130


                                  Time ( min )
Figure 28.  Rates of  hydrolysis of chlordimeform and its N-demethylated

metabolites at pH 7.4 and 37 .
                                    77

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    TABLE 15.  RATE CONSTANTS AND  HALF  LIVES  FOR THE HYDROLYSIS OF CHLORDIMEFORM AND ITS _N-DEMETHYLATED
    METABOLITES.
                                           CDM                          DCDM                       DDCDM
                                 pH  7.4         pH 8.7         pH 7.4        pH 8.7         pH 7.4       pH 8.7
Rate constant (k, min~~ )
Half life (t1/2, min)
1.29 x 10~3
538
3.10 x 10~3
. 224
2.78 x 10~3
250
7.80 x 10"3
88.8
4.33 x 10~3
161
1.25 x 10~2
55.2
00

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with NADPH (1 mM) and GSH (5 mM). These, and all other data for metabolism .in
vitro were corrected for the slight spontaneous breakdown of formamidines  to
NFT, as measured by blanks run concurrently with the appropriate enzyme
preparation denatured by boiling.  Significant metabolism (loss of COM greater
than 5% in 20 rain) was seen only with the liver homogenate.  Further  studies
with subfractions from these tissues were conducted and the results are  shown
in Table 16.  Of the liver subfractions, CDM-metabolizing activity was
confined almost exclusively to the microsomal fraction.  It is significant
that no detectable metabolism of CDM was seen with brain microsomes.  The  lung
and liver microsomal fraction was virtually inactive in degrading CDM even
when prepared from mice pretreated with the MFO inducers, phenobarbital  and
3-methylcholanthrene.  Since the liver microsomal system is so predominant in
the biotransformation of CDM, further metabolic studies in vitro were confined
to this fraction.

TABLE 16.  RELATIVE ACTIVITIES OF MOUSE TISSUE MICROSOMES AND OTHER CELLULAR
SUBFRACTIONS IN DEGRADING CHLORDIMEFORM.
                                                                              g
Cellular subfraction                       % of added CDM recovered unchanged

Liver
     Microsomes                                         62.9
     Mitochondria                                       98.2
     Soluble                                            98.7

Brain
     Microsomes                                         99.1

Lung
     Microsomes                                         98.2

Control
     Buffer                                             99.4

 Means of two independent experiments with two replicates each.  Incubation
time:  10 min.

     The nature of the microsomal enzymes attacking CDM was investigated,  with
the results given in Table 17.  In the absence of NADPH, the rate of  CDM
degradation was reduced virtually to zero.  Replacement of 0_ by Np also
greatly decreased the rate of metabolism (80% inhibition).  Even in the
presence of atmospheric 0_ pretreatment of the microsomal suspension  with CO
gas inhibited the degradation of CDM by about 75%.

Comparative metabolism of formamidines by mouse liver microsomes

     The rates and pathways of microsomal metabolism of CDM, DCDM,  and  DDCDM
were compared at several times over 20 min.  The data presented  in  Table 18
for 10 min incubations are typical.  In addition to the parent compound  (CDM).
five other types of metabolites were assayed; DCDM, DDCDM,  NFT,  CT  and  a
'Polar1 fraction.  This last fraction stayed on or near the origin  in the
                                      79

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                                              1U
TABLE 17.  METABOLITES FORMED  IN  REACTION  OF   C-CHLORDIMEFORM  WITH  MOUSE
LIVER MICROSOMES.

      Reaction Conditions                  %  of Substrate recovered asa;
Enzyme       NADPH         Gas         CDM          DC DM        DDCDM         NFT
+ Air
+ - Air
+ + N
+ CO
62.3
98.4
91.5
89.3
26.8
0.0
3.2
7.6
1.1
0.0
0.1
0.2
2.4
0.4
0.2
0.6
 Means of two  independent  experiments  with two replicates each.   Incubation
Ortime:  10 min.

 2-dimensional TLC  system  employed.   The nature of the constituents was not
determined, but  rechromatography with  a more polar developing system showed at
least 7  components.  The nature of the major metabolities (CDM,  DCDM, DDCDM and
NFT) was confirmed  by GC-MS Analysis.   Rather small amounts of the urea
derivatives  (CDU,  DCDU,  and DDCDU) were detected (less than 5%).  Since these
compounds are  neither highly toxic,  nor major metabolites they were not
quantified  further.

   Liver microsomes degraded the three formamidines in the order
DDCDM>DCDM>CDM.   DDCDM was very readily metabolized since only 1556 of the added
DDCDM was recovered intact after 10 min, and 60$ was hydrolyzed to NFT, whereas
63? of the CDM added was  recovered unchanged.  Only small amounts of DDCDM were
recovered starting  from CDM and DCDM,  although this may reflect the instability
of DDCDM as much as a slow rate of synthesis.  When CDM was the substrate, the
major metabolite (27?) was DCDM with relatively small amounts of the other
metabolites.   Thus  the first N[-demethylation (CDM to DCDM) occurs readily.  The
rate of  the  second  N-demethylation (DCDM to DDCDM) is hard to evaluate because
of rapid further degradation of DDCDM, but it is toxicologically significant
that DDCDM does  not accumulate to any high level.


Effect of manipulations of MFO activity on the in vitro metabolism of
chlordimeform.

     Intraperitoneal pretreatments of mice with inducers and inhibitors of MFO
considerably affected the  rate and type of microsomal metabolism of CDM iji
vitro as shown in Table 19.  Despite their lack of effect on toxicity, the MFO
inhibitors,  SKF  525-A and  piperonyl butoxide, decreased both the rate of
destruction of CDM and the rate of accumulation of the apparently more toxic
metabolites  DCDM and DDCDM.  Phenobarbital, as expected, showed the reverse
effect,  causing  a much more rapid loss of CDM and a considerably greater
accumulation of  the two N-demethylation products.

     3-Methylcholanthrene  and Aroclor 1254 were even more effective inducers
as judged by the overall  loss of CDM.   However, in contrast to the other
compounds, these two inducers also considerably increased the rate of
production of  NFT as well  as the rate of jY-demethylation.  Compared to the
controls, more of both DCDM and DDCDM accumulated after  3-methylcholanthrene

                                       80

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     TABLE 18.  IN VITRO METABOLISM  OF  CHLORDIMEFORM  AND  ITS J4-DEMETHYLATED METABOLITES BY MOUSE LIVER
     MICROSOMES.
                                            Percentage  of substrate recovered as :
Substrate
CDM
DCDM
DDCDM
CDM DCDM DDCDM
62.72 + 1.58 26.88 +1.10 1.02 + 0.30
	 49.92 + 2.20 1.83 + 1.50
	 	 15.17 + 0.15
NFT
4.22 + 0.70
15.26 -i- 1.36
60.04 + 1.40
CT
1.10 + 0.30
7.43 + 0.40
9.23 + 0.60
Polar
2.33 + 0.70
12.90 + 1.35
10.66 + 0.90
00
      Mean (+SD) of two independent experiments with  two  replicates  each.   Incubation  time:   10 min.

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     TABLE  19.  EFFECT OF  SEVERAL  PRETREATMENTS AFFECTING MICROSOMAL OXIDATIONS ON THE _IN VIThO METABOLISM OF
     CHLORDIMEFORM BY MOUSE  LIVER  MICROSOMES.
                                                          Metabolites, percent as :
00
Pretreatment
Control
Phenobarbital
3-Methylcholanthrene
Aroclor 1254
Piperonyl butoxide
SKF 525-A

62.72
40.04
32.78
19.99
75.03
70.81
CDM
+ 1.53
+ 1.06
+ 2.18
+ 0.46
+ 6.20
+ 2.05
DCDM
26.88
44.86
37.37
54.26
16.86
19.18
+ 1.10
+ 0.12
+ 4.00
+ 0.50
•i- 4.71
-i- 0.16
DDCDM
1.02 + 0.30
4.09 + 0.37
1.40 + 0.17
7.69 + 0.30
0.61 + 0.14
0.67 + 0.03
NFT
4.22 -»-
5.85 +
16.18 +
8.54 +
3.93 +
3.76 +
0.70
0.07
1.33
0.38
0.40
0.01
CT
1.10 +
1.06 +
1.56 +
1.62 +
0.93 +
1.60 +
0.29
0.33
0.04
0.18
0.28
0.01
2.33
3.18
9.59
5.35
1.50
1.54
Polar
+ 0.69
+ 0.33
+ 0.33
+ 0.21
+ 0.57
+ 1.10
     a
      Means  of  two  independent  experiment with two replicates in each.  Control is the average of ten
      independent  experiments with two replicates in each.  Incubation time:  10 min.

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and Aroclor pretreatments.  However, 3-methylcholanthrene caused only  a  small
rise in the level of DDCDM compared to phenobarbital and Aroclor 1254.
3-Methylcholanthrene was also the most effective compound stimulating  the
pathways to NFT and polar metabolites.

Effects of manipulations of MFO activity on in vivo metabolism of
chlordimeform.

     The effect of pretreatment with the inducers phenobarbital and
3-methylcholanthrene and the inhibitor piperonyl butoxide on the levels  of  CDM
metabolites in the liver, blood and brain was determined 40 min after  dosage
with CDM.  The 40 min interval was chosen since this was the typical time for
the development of severe toxicity symptoms and death at lethal doses  of CDM.
Recoveries of added CDM, DCDM, DDCDM, and NFT by this method of extraction
ranged from 80-8556.  The data in Tables 21 and 22 are corrected for this level
of recovery.  The recovery of CT was not checked and no recovery correction of
the data for this compound was made.

     The percentage of the dose present in the tissues after each treatment is
shown in Table 20.  For the purposes of calculation the total blood volume  of
the mice was taken as 6.0% of their body weight.  Liver weight averaged  1.44 g
and brain weight 0.42 g.  Relatively large amounts of radioactivity were
present in the liver with much less in the blood and brain.

TABLE 20.  TOTAL RADIOACTIVITY IN TISSUES AFTER AN ORAL DOSE OF CHLORDIMEFORM.
                                                                         Q
                               Total radioactivity as percent of the dose
Pretreatment                 Liver             Brain              Blood
Control
Phenobarbital
3-Methylcholanthrene
Piperonyl butoxide
11.15 + 0.26
12.53 + 0.55
11.16 + 0.52
6.36 + 0.34
0.717 + 0.071
0.694 + 0.051
0.693 + 0.037
0.352 + 0.051
1.23 + 0.072
1.33 + 0.081
1.37 + 0.110
0.65 + 0.052
aMean ^ SD  (n = 3).  Time after dosage, 40 min.

     Pretreatment with phenobarbital or 3-methylcholanthrene  had  no effect on
the levels  of radioactivity in the tissues.  However, piperonyl butoxide
unexpectedly reduced the amount of radiolabel by about  50%  in all the tissues.
This effect proved to be reproducible in a replicate run.   The levels of the
individual metabolites in these tissues are presented in terms of the relative
percentage  they represent of the total radioactivity recovered (Table 21) and
in terms of their absolute levels  (Table 22).

     The data for the liver in Table 21 show that  pretreatment with
phenobarbital or 3-methylcholanthrene hardly affects the percentage of the
radioactivity present as the parent compound, CDM,  which averages about 10$.
However, the MFO inhibitor, piperonyl butoxide  radically changes  this picture,
reducing metabolism so that 56$ of the radioactivity is present  as CDM.
Piperonyl butoxide also increases the proportion of DCDM present  in the liver
                                      83

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but the other treatments  have little  effect in this regard.   However, the
proportion of DDCDM was much  reduced  by piperonyl butoxide,  and much increased
by the inducer  pretreatments.  NFT and  polar metabolites were greatly reduced
by piperonyl butoxide.  The most  notable change resulting from inducer
pretreatment was  the  140% increase in NFT after 3-methylcholanthrene which was
not seen with the other MFO  inducer,  phenobarbital.


     A rather similar  picture is  presented in the data for the blood and, to a
lesser extent,  for brain  also.   The few notable differences  are the low
percentage of DCDM in  the blood and brain after 3-methylcholanthrene
treatment, and  the high percentage of NFT in the brain compared to the other
tissues.  In fact, 45% of the radioactivity in the brain is  NFT in the
3-methylcholanthrene-treated  animals  compared to 18% in the  controls.  Clearly
3-methylcholanthrene  increases NFT levels in the brain at the expense of DCDM.
The same conclusion  holds, to a lesser  extent, in the blood  also.  As in the
liver, so in the  blood and brain, piperonyl butoxide greatly increases the
proportion of label  present  as COM, and, to a lesser extend, DCDM, while
radically decreasing  the  other metabolites.  Polar metabolites are less
important in the  brain,  perhaps because of the blood-brain barrier.

     Since the  total  levels  of radioactivity in these tissues were virtually
identical in the  control, phenobarbital, and 3-methylcholanthrene
pretreatments (Table  20), the above conclusions also apply when the data are
presented as the  percent  of  the total dose recovered in each form in Table 22.
However, because  piperonyl butoxide pretreatment reduced the total
radioactivity in  all  the  tissues, the data presented in Table 22 are
significantly different  for  this pretreatment.  It is these  data which should
be related to the observed toxicity in Table 1.  Although COM levels are still
quite  elevated  after  piperonyl butoxide, the DCDM concentration is lower than
the control  in  the blood  and  brain, and the amount of DDCDM in all tissues is
extremely low.  This  is  true  for the  other metabolites (NFT, CT and polar
fraction) also.

     On  the  basis of  the  data in Tables 1 and 22, an attempt was made to
correlate the effects of  the  several  pretreatments on the toxicity of COM with
the levels of formamidines singly or  in combination in liver, blood, or brain.
In this  comparison,  the  levels of COM,  DCDM, and DDCDM singly or in the
combinations DCDM plus DDCDM, and COM plus DCDM plus DDCDM (total
formamidines) were examined.   The only plausible correlations obtained are
shown  in Table  23.  The  effects on the toxicity of CDM are presented as a
ratio  of LD   (control)/LD  (pretreatment).  A ratio of greater than 1.0
indicates tnat  the pretreatment increased the toxicity of CDM while a value of
less than 1.0 reveals a decrease in toxicity.  Thus a direct correlation of
this toxicity ratio with  tissue levels of any 'active' compound(s) would be
expected.
                                      84

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TABLE 21 .  EFFECT OF SEVERAL PRETREATMENTS AFFECTING MICROSOMAL  OXIDATIONS  ON  THE
DISTRIBUTION OF CHLORDIMEFORM IN MICE.
                                                                                          VIVO METABOLISM AND
                                       —Metabolites as percent of total radioactivity in the liver
    Pretreatments
                           COM
DC DM
DDCDM
NFT
CT
Polar
00
Control
Phenobarbital
3-Methylcholanthrene
Piperonyl butoxide

Pretreatments
Control
Phenobarbital
3-Methylcholanthrene
Piperonyl butoxide

Pretreatments
Control
Phenobarbital
3-Methylcholanthrene
Piperonyl butoxide
10.11+2.79
9.39+1.03
10.79+0.50
56.47+1.47

CDM
7.27+1.03
10.06+0.91
10.25+2.79
61.59+1.98

CDM
6.78+2.03
4.89+0.26
6.92+1.46
33.05+2.70
14.30+1.86
17.88+0.62
14.85+4.11
26.11+1.55
Metabolites as
DCDM
11.34+2.90
8.16+1.69
4.93+0.13
13.15+0.88
Metabolites as
DCDM
27.99+4.65
30.18+0.37
8.84+1.03
32.80+1.95
6.92+0.99
15.66+1.34
13.47+0.47
3.88+0.25
percent of total
DDCDM
4.58+0.91
5.71+0.79
12.36+3.30
1.01+0.07
percent of total
DDCDM
4.37+1.17
5.28+0.44
5. 36+0. 34
1.16+0.11
7.70+1.20
8.58+1.44
18.72+1.58
3.49+1.33
radioactivity
NFT
11.13+0.39
10.82+3.16
19.78+0.47
3.74+1.20
radioactivity
NFT
18.23+0.89
30.33+1.76
45.06+6.32
4.27+1.20
3.26+0.19 30.67+3.97
6.32+0.87 22.17+1.89
2.89+0.51 26.72+2.24
3.09+0.84 4.52+1.34
in the blood3
CT
3.73+0.88
9.92+1.32
3.07+0.81
2.54+0.37
in the brain
CT
6.74+1.19
3.05+1.10
1.72+0.64
3.90+0.79

Polar
36.11+4.50
21.84+2.38
27.27+3.55
4.04+0.35

Polar
13.86+4.20"
12.06+2.91
15.78+5.26
4.62+1.29
    aMean  (+  SD).   Three  animals,  duplicate assays on each extract.

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TABLE 22.  EFFECT OF SEVERAL  PRETREATMENTS  AFFECTING MICROSOMAL OXIDATIONS IN THE MOUSE ON THE IN VIVO
METABOLISM OF CHLORDIMEFORM.
Pretreatments
Control
Phenobarbital
3-Methylcholanthrene
Piperonyl butoxide
Pretreatements
Control
Phenobarbital
3-Methylcholanthrene
Piperonyl butoxide
Pretreatments
Control
Phenobarbital
3-Methylcholanthrene
Piperonyl butoxide

CDM
1.12+0.28
1.16+0.14
1.20+0.11
3.59+0.11

CDM
0.089+0.015
0.134+0.019
0.141+0.045
0.398+0.022

CDM
0.049+0.
0.034+0.
0.048+0.
0.118+0.
Metabolites found
DCDM
1.59+0.17
2.21+0.11
1.64+0.04
1.66+0.17
Metabolites found
DCDM
0.137+0.030
0.109+0.027
0.066+0.006
0.086+0.011
Metabolites found
DCDM
016 0.202+0.046
002 0.210+0.016
009 0.062+0.003
022 0.116+0.024
in the liver
DDCDM
0.77+0.10
1.93+0.83
1.50+0.04
0.25+0.22
in the blood
DDCDM
0.056+0.008
0.075+0.012
0.167+0.032
0.006+0.001
in the brain
DDCDM
0.031+0.011
0.037+0.005
0.037+0.003
0.004+0.001
as percent of
NFT
0.86+0.12
1.06+0.19
1.86+0.02
0.23+0.10
as percent of
NFT
0.136+0.010
0.140+0.034
0.269+0.026
0.024+0.008
as percent of
NFT
0.130+0.016
0.210+0.004
0.314+0.060
0.015+0.006
total dose
CT
0.36+0.02
0.78+0.11
0.32+0.07
0.20+0.05
total dose
CT
0.046+0.012
0.132+0.020
0.045+0.009
0.016+0.003
total dose
CT
0.048+0.008
0.021+0.009
0.012+0.004
0.014+0.005

Polar
3.42+0.52
2.74+0.24
2.99+0.04
0.29+0.10

Polar
0.410+0.036
0.228+0.220
0.406+0.037
0.025+0.003

Polar
0.098+0.028
0.086+0.019
0.108+0.030
0.016+0.004
 Mean (+_SD).  Three  animals, duplicate  assays  on  each extract.

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TABLE 23.  COMPARISON OF THE EFFECT OF PRETREATMENTS ON THE  TOXICITY OF CDM TO
MICE AND ON THE LEVELS OF SELECTED FORMAMIDINES  IN THEIR TISSUE.

                                                   % of dose as  :
                                          DCDM in:
Pretreatraent
Toxicity Ratio"
Brain
Blood
Total Formamidines
      in Brain
Control
Phenobarbital
3-Methylcholanthrene
Piperonyl butoxide
1.00
0.94
0.61
0.89
0.202
0.210
0.062
0.116
0.137
0.109
0.066
0.086
0.282
0.281
0.147
0.238
Calculated as LD,-0(control )/LD   (pretreatment).  Data  from  Table  1.

     Table 23 shows that only three correlations relate toxicity to metabolite
levels in all the treatments with any degree of fidelity.  The  levels of DCDM
in brain and blood (which might reasonably be expected  to be correlated  with
each other) show the same general trend as the toxicity ratios  but are not
well correlated quantitatively with toxicity.  However, by combining  the
levels of all formamidines present in the brain, a correlation  is achieved
which matches the pattern of toxicity changes quite well quantitatively.

                                  DISCUSSION

     Toxicity tests with CDM and  its metabolites establish that only  those
compounds which retain the formamidine nucleus (CDM, DCDM, DDCDM) have a high
acute toxicity and induce strong  excitatory symptoms.   Only  after the initial
excitatory phase (and most mortality) is complete do the mice show the
prolonged depressed behavior typical of the non-formamidine  metabolites.
Because of the clear increase in  toxicity and more rapid toxic  action in the
order CDM
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3-methylcholanthrene actually decreased  the  toxicity  of  COM  to  about  half  its
normal value.

     Despite the unexpected  results  obtained  in  the toxicity studies,  the  MFC
inducers and inhibitors do have  their  predicted  effects  on hepatic microsomal
metabolism when administered in  vivo.  The data  in Table 19  show that
i> nibitor pretreatments of the mice  reduce the amount of COM destroyed by  the
isolated microsomes.   Even in the  control microsomes  the degradation  of  CDM
was quite rapid (39% in 10 min), but this was greatly increased in microsomes
from inducer-pretreated mice, with 60%,  67%,  and 80%  of  the  CDM destroyed
after the phenobarbital,  3-methylcholanthrene, and Aroclor treatments
respectively.  The  two MFO inhibitors  had similar effects on the pattern of
metabolites  produced from CDM.   All  metabolites  were  reduced,  although the
effect was rather small with NFT compared to the decrease in DCDM, DDCDM,  and
polar metabolites.  The three inducers,  however, produced rather different
changes in the spectrum of metabolites.   Phenobarbital and Aroclor
considerably increased the amounts of  DCDM and DDCDM, with less effect on  the
amount of NFT.  3-Methylcholanthrene on  the  other hand increased the  level of
NFT by 260%  but increased DCDM  only  by 35% and there  was np  increase  at  all
in the level of DDCDM.

     Two further conclusions can be  drawn from these  results.   First,
phenobarbital  in known to increase the activity  of those MFO reactions
catalyzed by cytochrome P-450 while  3-methylcholanthrene induces
predominantly  the related cytochrome P-448 (Conney,  1967; Alvares et  al.,
1973).  Aroclor 1254,  a mixture  of polychlorinated biphenyls,  induces both
types of cytochromes (Alvares et ^.,  1973).  All three  inducers increase  the
rate of N[-demethylation of CDM,  though the P-450 inducers are more effective,
but the hydrolysis  of  the formamidines to NFT appears to be  rather
selectively  increased  by  the P-448 inducers, particularly
3-methylcholanthrene.  The same  relationship is  true  for the 'polar1
fraction.  Thus much of the  cleavage of  the  formamidines to  NFT is probably
catalyzed by cytochrome P-448.

     It has  been suggested  (Ahmad  and  Knowles,  1971a), that  the hydrolytic
production of  NFT is largely non-enzymatic,  although  Morikawa est jil.  (1975)
concluded that the  MFO system also might be  involved. As pointed out
earlier, this  does  not prove that  the  MFO system produces NFT directly.
However, the data in Table  19 cannot be  interpreted  except in terms of a
direct MFO-catalyzed hydrolysis  of one or more of the formamidines.   The
rates of spontaneous hydrolysis  of CDM,  DCDM, and DDCDM  are  shown in  Table
15.  Although hydrolysis  to  NFT does occur,  it is relatively slow e.g. at  pH
7.4 and 37°, as used in the  MFO  studies  above, in the 10 min incubation
period employed, the percentage  hydrolysis of CDM, DCDM, and DDCDM to NFT
would be 1.3, 2.7,  and 4.2 respectively. This compares  to the  presence  of
16.2% of the added  CDM as NFT in the MFO assay after  the 3-methylcholanthrene
pretreatment, and this value has already been corrected  for  spontaneous
production of NFT (boiled enzyme blank). The mechanism  of this formamidine
cleavage reaction is unknown.  One possibility is that the primary MFO attack
in this case is by  hydroxylation of  the  amidine  carbon which may lead  either
to formamidine cleavage and  release  of NFT or, by tautomerization, to  the
corresponding urea  derivatives.

                                      88

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     The second conclusion in comparing Tables  1 and  19  is  that  it  is
difficult to relate these i£ vitro metabolic results  to  the effects of  the
same pretreatments on toxicity.  The two pretreatments which decrease the
toxicity of COM i.e. Aroclor and 3-methylcholanthrene do not reduce the level
of any metabolite, and Aroclor greatly increases the  level  of  the more  toxic
metabolites, DCDM and DDCDM.  These pretreatments do  reduce the  amount  of CDM
remaining, but the LD  's of the other four treatment were  not greatly
different, yet the amount of CDM unmetabolized  ranges from  40J to 75%.

     A partial answer to this lack of evident correlation of toxicity and
metabolism probably lies in Table 18 where the metabolism of CDM, DCDM, and
DDCDM is compared in hepatic microsomes from untreated mice.   Clearly the
JT-demethylated products are more rapidly degraded than CDM.  DDCDM, the most
toxic of these formamidines is also the least stable  with only 15%  of the
parent compound recoverable after 10 min and 60% conversion to NFT. Toxicity
and metabolic stability are therefore negatively correlated.   Thus  the  more
potent metabolites  (DCDM, DDCDM) are also the ones likely to have the
shortest survival times in the body.

     Because of the confusing metabolism-toxicity relations so far  developed,
a study was performed to analyze the levels of  each of the  critical
metabolites in liver, blood, and brain at a time chosen  to  coincide with the
peak of symptoms in mice given lethal doses of  CDM.   Because of  time and
radioisotope limitations this study was confined to phenobarbital,
3-methylcholanthrene, and piperonyl butoxide as the pretreatments.   The data
for recovery of total radioactivity in the three tissues again gave an
unexpected, but important, result.  Phenobarbital and 3rmethylcholanthrene
pretreatments have no significant effect on the total    C present in any
tissue compared to the control.  Piperonyl butoxide reduces this to half the
control value in each case.  Thus the piperonyl butoxide must  reduce uptake
of the oral dose of CDM.  Why this decreased uptake should  occur is not
obvious, especially since the piperonyl butoxide was  given  ip  and the CDM
orally.  One possibility is that the uptake of  CDM from  the gastrointestinal
tract is speeded by rapid metabolic destruction of CDM in the  liver. When
this metabolism is decreased by piperonyl butoxide, the  rate of  uptake  is
slowed.  This interesting effect deserves to be studied  further.

     Examination of the distribution of CDM and its metabolites  in  the  three
tissues when expressed as the % of the total metabolites recovered  (Table 21)
reveals a picture qualitatively similar to that seen  in  Table  19 with
metabolism by the hepatic microsomes in vitro,  but there are some notable
quantitative differences.  With the control and inducer  treatments  in  vivo
relatively little unchanged CDM was present in  the liver (about  10J of  the
quantitative differences.  With the control and inducer  treatments  jji  vivo
relatively little unchanged CDM was present in  the liver (about  10% of  the
recovered radioactivity) indicating a very rapid biodegradation  for this
compound.  DDCDM was found to be a much more prominent metabolite  in vivo
than JUi vitro.

     Further, major differences in the tissue levels  of  CDM between the
inducer pretreatments and control are not evident in  most cases jin  vivo.  A
further difference between the in vivo and J.n vitro results is the  high

                                      89

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effectiveness of piperonyl  butoxide  in  preventing  the  degradation  of COM
(e.g. in the brain; 56%   as COM  versus  10%  as  COM  in the  control).   In
particular the proportion of metabolites  present  as  DDCDM was greatly reduced
with this pretreatment.   Thus  it is  again evident  that in vitro metabolic
studies have only  a limited ability  to  predict rates of metabolism j.n vivo
although pathways  of metabolism  can  often be deduced in vitro.

     The crucial comparison in relating the toxicity of COM to its metabolism
lies with the data in  Table 22,  and  the further correlations in Table 23.
The concentration  of neither CDM nor DDCDM  in  any tissue  correlates well with
toxicity after the different pretreatments  e.g. if CDM itself were the  major
toxicant, the data in  Table 22 suggest  that the piperonyl butoxide
pretreatment should clearly increase the  toxicity of CDM  since the levels of
this compound are  elevated  2-3 fold  in  all' tissues compared to the control.
On the other hand, if  DDCDM were the major  toxicant, piperonyl butoxide
should greatly decrease  the toxicity of CDM because the tissue levels of
DDCDM are considerably lowered.   In  fact, neither of these results is seen;
piperonyl butoxide has only a  minor  effect  on  the toxicity of CDM.  Similar
comparisons with the other  pretreatments  lead  to the same general  conclusion.
Of the individual  formamidines,  the  trend in DCDM levels  can be related to
the toxicity quite closely  (Table 23) and a reasonable quantitative
correlation is obtained  between  the  toxicity ratio and DCDM in brain
(correlation coefficient,  r =  0.897) and  blood (r = 0.884) for the four
treatments.  However,  in most  cases  all three of the formamidines  are present
in the tissues at  roughly comparable concentrations.   The most reasonable
assumption, based  on our knowledge of their pharmacological actions, is that
all three have some innate  toxicity  and the overall effect on the  animal is
the sum of the effects of the  three  compounds acting in concert rather  than
the action of only one compound  such as DCDM.   This hypothesis receives
support from the data  in Table 23 where an  excellent quantitative  correlation
is obtained between the  toxicity ratio  after the various  pretreatments  and
the total level  of formamidines  in the  brain.   The correlation coefficient
for this relationship  is 0.985.   Further  support for this view comes from
preliminary experiments  where  we have observed that the toxicities of CDM,
DCDM and DDCDM are additive in mice  - each  can replace an equivalent toxic
fraction of another without a  change in lethality or symptoms.

     In this relationship,  the major factor underlying the lowered toxicity
of CDM after 3-methycholanthrene pretreatment is the strongly increased
activity in cleaving the toxic formamidines to the poorly toxic metabolite,
NFT, probably through  the induction  of cytochrome P-448.   The same reason may
underly the lower  toxicity  after Aroclor  treatment which  also induces P-448
and increases NFT  production.   Piperonyl  butoxide, as  one might expect, tends
to preserve the  formamidines  from further degradation  and should,  therefore,
increase the toxicity  of CDM by  our  hypothesis.  However, this effect is
balanced by the  reduced  rate of  penetration of CDM after  piperonyl butoxide
pretreatment so  that,  overall, the tissue levels of total formamidines  differ
little from those  of the control, and toxicity of CDM  is  not changed greatly.
Phenobarbital pretreatment  does  not  alter the  pattern  of  metabolites found in
the brain very significantly,  and thus  has  no  effect on the toxicity of CDM.
Our results offer  no support for the idea that ^-demethylation is  an
                                      90

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obligatory activation reaction for CDM, or that the most toxic compound,
DDCDM, is the sole or even major toxic species iin vivo.

     These conclusions must be tentative since only the determination of  a
time-concentration profile for each of the major metabolites in each tissue
containing important sites of action would be definitive in determining the
role of the individual formamidines in such a complex toxicological
relationship.

     The reason why DCDM and DDCDM are more toxic than CDM when given orally
(Table 1) is unclear.  Comparisons of the relative potencies of CDM and DCDM
on most biochemical and physiological systems which plausibly might be
involved in their toxicity show no striking differences between the
compounds. DDCDM, as a relatively newly discovered toxic metabolite, has,
unfortunately, been less widely studied.  As shown in Section  1, DCDM is  only
slightly better than CDM as an inhibitor of MAO.  In Section 4, DCDM was
found to be somewhat less effective than CDM as a local anesthetic, at least
on the frog sciatic nerve.  Although only preliminary experiments were
performed to assess the effects of DCDM on the cardiovascular system of the
dog, using the methods described for CDM in Section 2, DCDM was
quantitatively and qualitatively rather similar to CDM although some
differences in action were noted.  Further, the two compounds acted very
rapidly in this system (Table 5), suggesting that J^-demethylation of CDM  (or
DCDM) is not obligatory for the cardiovascular effects which arise through
peripheral actions.  For this reason we did not include further work with
DCDM in our cardiovascular studies.

     The only major difference in pharmacological potency between CDM and its
N^demethylation products so far known is in their a-adrenergic and octopa-
minergic actions.  DCDM has a much stronger effect than CDM as a partial
agonist at a-adrenergic sites (rabbit ear artery) as shown by Robinson and
Bittle (1979).  The same distinction is also found in their action as
octopaminergic agonists in invertebrates, a type of system which is analogous
to the a-adrenergic system of vertebrates.  In this case DDCDM is also
active, but somewhat less so than DCDM, while CDM itself is relatively
ineffective (Hollingworth and Murdock,  1980).

     However, although it is attractive to try to relate the higher toxicity
of DCDM and DDCDM to their greater potency in stimulating a-adrenergic neuro-
transmission, there are problems with this hypothesis.  a-Adrenergic agonists
(e.g. NE itself) are central depressants not central stimulants, as seen  with
formamidines at lethal levels (Feldberg, 1963).  Further, we found that phen-
tolamine, an a-adrenergic blocker, did  not prevent the peripheral cardiovas-
cular depression caused by CDM (Section 2).  Robinson et jd.  (1975) reported
that phentolamine given ip to rats did  not reduce the lethality of CDM,  and
concluded that "it seems improbable that death following chlordimeform
poisoning in rats results from stimulation of a-adrenergic  .  .  .receptors."
This view was strengthened by the subsequent observation  (Robinson and  Smith,
1977) that phenylephrine, itself an o-agonist, did not increase the toxicity
of CDM as might be expected if CDM, or  a metabolite, had important actions  at
the same site.
                                       91

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     The exact a-adrenergic  effects  of  DCDM  (and  probably DDCDM)  are
difficult to predict since partial agonists  are,  by  definition, partial
antagonists also and thus may  stimulate a-adrenergic transmission at  one  dose
and partially block it at a  higher dose.   This  action of  the  formamidines
deserves further study, particularly with  regard  to  the central effects of
CDM, but as pointed out above,  the evidence  presently available does  not
strongly support this type of  action as a  major factor in the acute toxicity
cf CDM, whereas local anesthetic-like effects can explain most of the
symptoms observed.  The greater toxicity of  DCDM  and DDCDM may therefore  lie
as much in the area of pharmacokinetics (more rapid  accumulation  at the
site(s) of action) as in  the area  of pharmacodynamics (greater potency at, or
a different, site of action).

     Overall, the toxicological actions of CDM, and  probably  related
formamidines, is rather complex, with several active compounds present
simultaneously, each of which may  have  several  significant actions on the
nervous system.  Much is  now known concerning the neurotoxicology of
formamidines and its relationship  to their toxicity, but  numerous questions
remain to be answered before a satisfactory  and complete  picture  of their
pharmacological and toxicological  effects  can emerge.
                                      92

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     demethylchlordimeform: A metabolite of chlordimeform.  Pestic. Biochem.
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                                                     14
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                                      1Q1

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                                    TECHNICAL REPORT DATA
                             (Please read Instructions on the reverse before completing)
i. REPORT NO.
  EPA-6QQ/i-an~n?ft
                                                             3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
  Toxicity,  Interactions, and Metabolism of
  Formamidine  Pesticides in Mammals
             6. REPORT DATE
                Mav 198Q
             8. PERFORMING ORGANIZATION COOK
7. AUTHCRIS)
  R. M. Hollingworth and G.K.W. Yim
                                                             8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS

  Purdue University
  Department  of Entomology
  West LaFayette,  Indiana  47907
              10. PROGRAM ELEMENT NO.
                1EA615
              11. CONTRACT/GRANT NO.
                                                                Grant No. R-803965
12. SPONSORING AGENCY NAME AND ADDRESS
  US Environmental Protection Agency
  Health Effects  Research Laboratory
  Research Triangle Park, NC  27711
              13. TYPE OF REPORT AND PERIOD COVERED
              14. SPONSORING AGENCY CODE
                                                                EPA 600/11
15. SUPPLEMENTARY NOTES
 16. ABSTRACT
      The overall  goal  of this research project was  to investigate the mechanism(s) of
 acute toxicity  of formamidine pesticides in mammals using chlordimeform (N/-(4-chloro-o-
 tolylJ-N^-dimethylformamidine) and its metabolites as the primary model compounds.  The
 role of biotransformations, particularly N-demethylation reactions,, in generating poten-
 tially toxic metabolites was also studied.                          !
      By comparing the  effects of hepatic microsomal mixed function oxidase inducers and
 inhibitors  administered in vivo on the toxicity, metabolism,  and distribution of metabo-
 lites in mouse  tissues, it was concluded that although N-demethylation products are
 innately more toxic than chlordimeform, they are also less stable, and the best correla-
 tion of toxicity  was obtained with the total level  of formamidines in the brain, rather
 than with the level of any individual metabolite.
      In a series  of studies with dogs, rabbits, and cats, the cause of death was found to
 be cardiovascular collapse accompanied by respiratory arrest.  Cardiovascular collapse
 resulted primarily from a peripheral local anesthetic-like effect of chlordimeform.
 Monoamine oxidase inhibition was not a major factor in lethality.  Respiratory arrest
 was central in  origin.  Several other central effects of the  formamidines were described,
 some of which may be local anesthetic actions, and  a behavioral  profile for chlordimeform
 poisoning in the  rat was developed.  The effectiveness of various drug treatments as
 potential therapeutic  aids for formamidine intoxication were  studied. Formamidines also
 nave asoirin-like actions due^tD_flJuLnahi 1 itv t.n inhibit. nrnst.aolaruHn synthesis	
17.
                                 KEY WORDS AND DOCUMENT ANALYSIS
                   DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS  C. COSATI Field/Group
      Formamidine pesticides
      Toxicity in mammals
      Administered In Vivo
                              06,F,T,
18. DISTRIBUTION STATEMENT
      RELEASE TO PUBLIC
19. SECURITY CLASS (THIS Report)
  UNCLASSIFIED
21. NO. Or PAUC9
   114
20. SECURITY CLASS (This page)

  UNCLASSIFIED
                                                                            22. PRICE
£PA Form 2220-1 'Rev. 4-77)   PREVIOUS EDITION IS OBSOLETE
                                               102

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