ANTICO‘NVULSANT, BEHAVIORAL, TOXICOLOGICAL ,
AND ‘ANTIEPILEPT'IC CHARACTERIZATION; ,
OFDIPHENYLSILANEDIOL ‘ *

Dissertation for the Degree Of Ph. D.
MICHIGAN STATE UNIVERSITY -
VERNE D. H‘ULCE ‘
1976 '

 

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This is to certify that the

thesis entitled

ANTICONVULSANT, BEHAVIORAL, TOXICOLOGICAL
AND ANTIEPILEPTIC CHARACTERIZATION
OF DIPHENYLSILANEDIOL

presented by

Verne D. Hulce

has been accepted towards fulfillment
of the requirements for

Ph.D. Pharmacology

degree in

 

 

MM we 2

Major professor

Date October 12, 1976

 

0-7639

 

 

 

ABSTRACT
ANTICONVULSANT, BEHAVIORAL, TOXICOLOGICAL

AND ANTIEPILEPTIC CHARACTERIZATION
OF DIPHENYLSILANEDIOL

By

Verne D. Hulce

The research discussed in this dissertation began as an
investigation of the general pharmacology of organosilicon compounds
that possessed some feature in their structures that made them struc—
turally different from carbon compounds. Diphenylsilanediol is a
geminal—diol. No analogue with carbon substituted for silicon exists
for this compound.

Emphasis was placed on diphenylsilanediol as it became apparent
that this compound was a broad spectrum anticonvulsant. Anticonvulsant
activity appears in its ability to antagonize convulsions due to general
electrical stimulation (maximal electroshock), general chemical stimu-
lation (pentylenetetrazol, strychnine, and picrotoxin), and both elec—
trical and chemical focal stimulation of the neocortex. These results
were obtained with the rat, gerbil, and mouse as subjects. Depending
on subject species, test procedure and route of administration, these
anticonvulsant effects appeared in the dose range of from 2 mg./kg. to
100 mg./kg. Studies on probable anticonvulsant mechanisms for this
compound showed that one of its actions is the inhibition of post—

tetanic potentiation.

Verne D. Hulce

Behavioral evaluation revealed a behavioral toxicity, in terms
of rotating rod performance depression, for rodents at doses ranging
from 100 mg./kg. to 300 mg./kg. as effective doses-~fiftieth percentile
(ED-50). Operant behavioral test models identified an anti-anxiety
effect beginning at 10 mg./kg. after intraperitoneal administration
in the rat.

There was no significant tolerance development to the anti-
convulsant effects after periods up to three months following chronic
oral administration of diphenylsilanediol. Drug withdrawal hyperexcit-
ability was observed only as a small change in seizure threshold after
dosing with diphenylsilanediol was stopped. This occurred only after
long term administration of a high (behaviorally toxic) dose.

Toxicity studies demonstrated a lethal dose (LD-SO) of
2,500 mg./kg. after single dose administration. With chronic adminis-
tration, a dose-dependent eosinophilia was detected. Other hematologic
effects included a dose-dependent increase in reactive lymphocytes. A
histopathologic observation of swollen liver cells was examined further
using liver function tests and electron microscopic procedures. The
liver was not functionally impaired and the cell changes could be
attributed to a proliferation of smooth endoplasmic reticulum. This
ultrastructural change is not considered to be a pathological change,
but only an adaptation due to an increase in metabolic activity of
these cells in processing a foreign substance for excretion.

Antiepileptic testing was done using the epileptic gerbil and

the epileptic dog as test subjects. Seizure control was observed for

Verne D. Hulce

both of these species after the administration of diphenylsilanediol.
The canine subjects were subjected to constant clinical laboratory mon-
itoring which verified the hematological changes seen in the toxicity
study done with mice. In addition, an increase in serum alkaline phos—
phatase levels was observed. There is no apparent explanation as to
its origin and it appears that it is not associated with any impairment
as shown by other routine determinants of liver function.

The compound diphenylsilanediol appears to be an effective
anticonvulsant having pharmacological properties that make it similar
to the range of anticonvulsant drugs currently in use but not entirely
like any one single drug. It inhibits post-tetanic potentiation as
does diphenylhydantoin, while being able to antagonize the effects of
chemical convulsants as does phenobarbital. The compound also pos-
sesses anti-anxiety activity similar to diazepam yet appears to

possess less motor depressant activity at anticonvulsant dose levels.

ANTICONVULSANT, BEHAVIORAL, TOXICOLOGICAL
AND ANTIEPILEPTIC CHARACTERIZATION

OF DIPHENYLSILANEDIOL

By

Verne D. Hulce

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Pharmacology

1976

ACKNOWLEDGMENTS

The author wishes to express his gratitude to the members
of his committee, and to certain other individuals, for their con—
tributions to this dissertation effort.

Thanks are due to Dr. D. Bennett, Dr. R. Rech, Dr. G. Gebber,
Dr. T. Brody, Dr. J. Cunningham, Dr. T. Pinnavia, Dr. A. Meininger,
and Mr. C. Kilts for their aid in this research.

Special thanks are due to my parents and to my wife and
children for their tolerance and sacrifices that made the pursuit

of this dissertation and the Ph.D. program possible.

ii

TABLE OF CONTENTS

Page
LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . v

LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . viii

PREFACE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I
INTRODUCTION . . . . . . . . . . . . .,. . . . . . . . . . . . . . 2
Organosilicon Chemistry . . . . . . . . . . . . . . . 2
Pharmacology of Organosilicon Compounds . . . . . . . . . . . 6
Diphenylsilanediol--Chemistry, Properties . . . . . . . . . 8
Plan for the Characterization of Diphenylsilanediol . . . . . 1n

ANTICONVULSANT CHARACTERIZATION . . . . . . . . . . . . . . . . . 20

General Remarks . . . . . . . . . . . . . . . . . . . . . . . 20
Maximal Electroshock Generalized Seizures . . . . . . . . . . 21
Methods . . . . . . . . . . . . . . . . . . . . . . . . . 22
Results . . . . . . . . . . . . . . . . . . . . . . . . . 24
Chemical Stimulation Models . . . . . . . . . . . . . . . . . 31
Methods . . . . . . . . . . . . . . . . . . . . . . . . . 31
Results . . . . . . . . . . . . . . . . . . . . . . . . . 3%
Focal Stimulation Models . . . . . . . . . . . . . . . . . . . 34
General Remarks . . . . . . . . . . . . . . . . . . . . 3”
Focal Electrical Stimulation of the Neocortex . . . . . . 36
Methods . . . . . . . . . . . . . . . . . . . . . . . 37

Results . . . . . . . . . . . . . . . . . . . . . . . 39
Post-Tetanic Potentiation Model . . . . . . . . . . . . . 39
Methods . . . . . . . . . . . . . . . . . . . . . . . Ml

Results . . . . . . . . . . . . . . . . . . . . . 43
Tolerance and Physical Dependence Studies . . . . . . . . . . 45
Methods . . . . . . . . . . . . . . . . . . . . . . . . . H6
Results . . . . . . . . . . . . . . . . . . . . . . . . 50
Discussion of Anticonvulsant Effects . . . . . . . . . . . . . 55

BEHAVIORAL CHARACTERIZATION . . . . . . . . . . . . . . . . . . . 60

General Remarks . . . . . . . . . . . . . . . . . . . . . . . 60
Rotating Rod Effects . . . . . . . . . . . . . . . . . . . . . 62
Methods . . . . . . . . . . . . . . . . . . . . . . . . . 63
Results . . . . . . . . . . . . . . . . . . . . . . . . . 64

iii

 

Page

Operant Behavioral Effects . . . . . . . . . . . . . . . . . . 64
Methods . . . . . . . . . . . . . . . . . . . . . . . . . 66
Results . . . . . . . . . . . . . . . . . . . . . . 67

Discussion of Behavioral Effects . . . . . . . . . . . . . . . 78

TOXICOLOGICAL STUDIES . . . . . . . . . . . . . . . . . . . . . . 87

General Remarks . . . . . . . . . . . . . . . . . . . . . . . 87
Acute Toxicity . . . . . . . . . . . . . . . . . . . . . . . . 87
Methods . . . . . . . . . . . . . . . . . . . . . . . . . 87
Results . . . . . . . . . . . . . . . . . . . . . . . . . 88
Chronic Toxicity . . . . . . . . . . . . . . . . . . . . . . . 91
Methods . . . . . . . . . . . . . . . . . . . . . . . . . 91
Results . . . . . . . . . . . . . . . . . . . . . . . . . 92
Gross and Microscopic . . . . . . . . . . . . . . . . 92

Hematological Observations . . . . . . . . . . . . . . 104
Supplementary Toxicity Studies . . . . . . . . . . . . . . . . 109
Electron Microscopic Examinations . . . . . . . . . . . . 109
Methods . . . . . . . . . . . . . . . . . . . . . . . 109

Results . . . . . . . . . . . . . . . . . . . . . 110
I2_Vivo Liver Function Tests . . . . . . . . . . . . . . . 110
Methods . . . . . . . . . . . . . . . . . . . . . . . 113

Results . . . . . . . . . . . . . . . . . 11a
Discussion of Toxicological Results . . . . . . . . . . . . . 119

ANTIEPILEPTIC STUDIES IN SEIZURE DISORDER ANIMALS . . . . . . . . 125

Introduction . . . . . . . . . . . . . . . . . . . . . . . 125
Epileptic Gerbil Model . . . . . . . . . . . . . . . . . . . . 128
Methods . . . . . . . . . . . . . . . . . . . . . . . . . 129
Results . . . . . . . . . . . . . . . . . . . . . . . . 131
Epileptic Dog Model . . . . . . . . . . . . . . . . . . . . . 131
Methods . . . . . . . . . . . . . . . . . . . . . . . . . 13”
Results . . . . . . . . . . . . . . . . . . . 141
Discussion of Antiepileptic Studies . . . . . . . . . . . . . 148

GENERAL DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . 150
Results of the Characterization Program . . . . . . . . . . . 150
Some Considerations of Diphenylsilanediol as an
Anticonvulsant Compound . . . . . . . . . . . . . . . . . . 15”

Some Considerations of Diphenylsilanediol as an
Antiepileptic Drug . . . . . . . . . . . . . . . . . . . . . 160
RECOMMENDATIONS . . . . . . . . . . . . . . . . . . . . . . . . . 165

BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . . . . . . . . 167

iv

Table

10.

11.

12.

13.

14.

15.

16.

17.

LIST OF TABLES

Page
Chemical Comparison of Silicon with Carbon . . . . . . . . 3
Desirable Properties for an Antiepileptic Drug . . . . . . _16
Plan for Characterization of Diphenylsilanediol (DPSD) . . 18
Anticonvulsant Screen Results for DPSD Against Seizures
in Mice (MES , MET) o o o o o o o o o o o o o o o o o o o o 26
Time Course for the Anticonvulsant Action of DPSD . . . . . 27
Route of Administration Effects on the Antagonism of
MES Seizures by DPSD in Mice . . . . . . . . . . . . . . . 29
Comparative Electroconvulsive Shock Seizure Antagonism . . 3O
Antagonism of Drug Induced Seizures in Rats by DPSD . . . . 35
Inhibition of Cortical Electrical After-Discharge by
DPSD (30 mg./kg. i.p.) in the Rat . . . . . . . . . . . . . #0
Inhibition of Post-Tetanic Potentiation by DPSD . . . . . . nu
Design for Study to Evaluate Potential for Tolerance
and Physical Dependence for DPSD . . . . . . . . . . . . . 48
Pentylenetetrazol "First Twitch" Threshold Changes
Associated with DPSD Administration for 1H Days . . . . . . 51
Pentylenetetrazol "First Twitch" Threshold Changes
Associated with DPSD Administration for 90 Days . . . . . . 53
Summary of Anticonvulsive Action of DPSD . . . . . . . . . 56
Comparison of the Anticonvulsive Action of
Anticonvulsant Compounds . . . . . . . . . . . . . . . . . 57
Rotating Rod Performance Decrement Due to DPSD . . . . . . 65
Two Hour FR-MO Response Totals After Dosing Rats
With DPSD O O O O O O O I C O O O O O O O O O 0 O O O O O O 68

Table

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

3M.

35.

Page
Two Hour FR—HO Response Totals After Dosing Rats
with Diphenylhydantoin (DPH) . . . . . . . . . . . . . . . 71
Two Hour FR-HO Response Totals After Dosing Rats
with Diazepam . . . . . . . . . . . . . . . . . . . . . . . 73
Two Hour Conditioned Emotional Suppression (CES)
Response Totals After DPSD Dosing . . . . . . . . . . . . . 76
Two Hour Conditioned Emotional Suppression (CES)
Response Totals After DPH Dosing . . . . . . . . . . . . . 79
Two Hour Conditioned Emotional Suppression (CES)
Response Totals After Diazepam Dosing . . . . . . . . . . . 81
Results and Comparisons of Behavioral Effects . . . . . . . 83
Lethal Effects of DPSD (i.p., Mice) . . . . . . . . . . . . 89
Lethal Effects of DPSD Administered Subacutely in Food . . 90
Design for Chronic Toxicity Study in Mice . . . . . . . . . 93
Organ and Body Weight Changes After Chronic Dosing
with DPSD for Three Months . . . . . . . . . . . . . . . . 99
Summary of Gross and Microscopic Observations on Organs
from Mice Dosed with DPSD (6.9 mg./gm. food) for
90 days I O O O O O O O O O I C C O O O O D O O O O O O O O 9 5
Peripheral Leukocyte Changes After Chronic Dosing
with DPSD for Three Months . . . . . . . . . . . . . . . . 105
Dose—Dependent Leukocyte Changes After Dosing with
DPSD for Three Months . . . . . . . . . . . . . . . . . . . 106
Plasma Level of BSP Calibration Data . . . . . . . . . . . 115
Plasma Levels of BSP in Control Mice-~Time Course . . . . . 116
Plasma Levels of BSP in Treated vs. Control Mice . . . . . 120
Acute Lethality Comparisons of DPSD with Other
Anticonvulsive Compounds . . . . .p. . . . . . . . . . . . 121
Specific Toxicity Responses to the Chronic
Administration of Anticonvulsant Compounds . . . . . . . . 123

Table

36.

37.

38.

39.

40.

41.

42.

43.

41+.

45.

Page

Animals, In Addition to Man, Displaying Idiopathic
Seizures . . . . . . . . . . . . . . . . . . . . . . . . 126

Seizure Characteristics for Gerbil Colony . . . . . . . . 130

Effects of Anticonvulsant Drugs on the Idiopathic
Seizure of Mongolian Gerbils . . . . . . . . . . . . . . 132

Case Summary on Epileptic Dog A.G. Treated with
DPSD . . . . . . . . . . . . . . . . . . . . . . . . . . 135

Case Summary on Epileptic Dog R.D. Treated with
DPSD O O O O O 0 O 0 O O O O O O O O I O O O O O O O 0 0 138

Case Summary on Epileptic Dog B.S. Treated with
DPSD . . . . . . . . . . . . . . . . . . . . . . . . . . 192

Summary of Treatment Results with Epileptic Dogs
Treated With DPSD O O O I O O O O O O O O O O O O O O O 0 1””

Summary of Anticonvulsant Characterization Program
for DPSD . . . . . . . . . . . . . . . . . . . . . . . . 151

Acute Dose Safety Comparisons of DPSD with Other
Anticonvulsant Compounds . . . . . . . . . . . . . . . . 162

Spectrum of Action Comparisons of DPSD with Other
Anticonvulsant Compounds . . . . . . . . . . . . . . . . 16H

vii

Figure

10.

ll.

l2.

13.

19.

LIST OF FIGURES

Page
Examples of Uniquely Stable Compounds of Silicon . . . . . 5
Synthesis of Diphenylsilanediol from Silicon . . . . . . . 10
60MHz. Nuclear Magnetic Resonance Spectrum of a
Solution of Diphenylsilanediol in Dimethylsulfoxide-D5 . . 11
Infrared Absorption Spectrum of Diphenylsilanediol--
Sample Suspended in a Hydrocarbon/Halocarbon Mull . . . . l2
Schematic of Shocker Used to Deliver Maximal
Electroshock for Convulsive Seizures . . . . . . . . . . . 23
Time Course for the Anticonvulsant Action of
Diphenylsilanediol . . . . . . . . . . . . . . . . . . . . 28
Device for the Application of Penicillin to the Rat
Neocortex . . . . . . . . . . . . . . . . . . . . . . . . 33
Method for Cortical After-Discharge Studies . . . . . . . 38
Diagrammatic Summary of the Post-Tetanic Potentiation
(PTP) Method and Data Collection . . . . . . . . . . . . . H2
Pentylenetetrazol (MET) Seizure Threshold During and
After DPSD Administration (0. 4 mg. /gm. food) for
1” Days 0 O O O 0 O 0 O 0 C O O O O O O I O O O O O O O 52
Pentylenetetrazol (MET) Seizure Threshold During
and After DPSD Administration (6. u mg. /gm. food)
for 90 Days . . . . . . . . . . . . . . . . . . . . . 54
Dose Response on Two Hour Operant FR-MO Performance
with Rats After Dosing with Diphenylsilanediol . . . . . . 69
Dose Response on Two Hour Operant FR—HO Performance
with Rats After Dosing with Diphenylhydantoin . . . . . . 72
Dose Response on Two Hour Operant FR—MO Performance
with Rats After Dosing with Diazepam . . . . . . . . . . . 7H

viii

Figure

15a.

15b.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

Mechner Diagram for the Combined FR—40 and CBS

Behavioral Paradigm Used as an Anxiety State Model .

Cumulative Record of a Rat Performing Under a
Combined FR—40 and CES Paradigm for Behavior .

Responding During Conditioned Emotional Suppression

(CES) Periods After Dosing with Diphenylsilanediol .

Responding During Conditioned Emotional Suppression
(CES) Periods After Dosing with Diphenylhydantoin

Responding During Conditioned Emotional Suppression
(CES) Periods After Dosing with Diazepam . . .

Section of Spleen from a Mouse Treated with DPSD
for 90 Days 0 O O C O O O O O O O O O O O O 0

Section of the Adrenal Gland from a Mouse Treated
With DPSD for 90 Days 0 O O O O O O 0 O O O O 0

Section from Kidney of a Mouse Treated with DPSD
for 90 Days . . . . . . . . . . .

Bone Marrow Smear from the Femur of a Mouse
Treated with DPSD for 90 Days . . . . .

Section from Cerebellum of a Mouse Treated with
DPSD for 90 Days (Original Magnification 180X) .

Section from the Cerebellum of a Mouse Treated
With DPSD for 90 Days (Original Magnification
lOOOX) o o o o o o o o o o o o o o o o o o 0

Section of Liver from a Mouse on the Control Diet
for 90 Days . . . . . . . . . . . . . .

Section of Liver from a Mouse Treated with DPSD
for 90 Days 0 O O O O I O O C O O O O O O O O O O

Eosinophil and Lymphocyte from Peripheral Blood
of Mouse Treated with DPSD for 90 Days . . .

Toxic Neutrophil (with Doehle Body) from Blood
of Mouse Treated with DPSD for 90 Days . . .

ix

Page

75

75

77

8O

82

97

97

98

99

100

101

102

103

107

107

 

Figure Page

29. Reactive Lymphocyte from Peripheral Blood of Mouse

Treated with DPSD for 90 Days . . . . . . . . . . . . . . 108
30. Electron Micrograph of Liver from Control Mouse . . . . . 111
31. Electron Micrograph of Liver from Mouse After

Treatment with DPSD for Three Months . . . . . . . . . . . 112
32. Calibration Curve for Plasma Levels of BSP

(phenoltetrabromophthalein sodium sulphonate) . . . . . . 117
33. Disappearance of BSP from Mouse Plasma: Normal

Control Group Subjects . . . . . . . . . . . . . . . . . . 118
34. Structural Forms of Phenyl-Containing Anticonvulsant

Drugs 0 O O O O O O O O O O O O O C I O O O O O O O O O O 15 S
35. Comparison of Alcoholic Forms of Selected Diphenyl—

Containing Anticonvulsant Compounds . . . . . . . . . . . 157

x

PREFACE

Diphenylsilanediol is an organosilicon compound. The current
biological investigation of this compound began as an attempt to
examine the central nervous system pharmacology of organosilicon
compounds selected on the basis of having a unique chemical structure
which could not be duplicated with a carbon substituted analogue. Two
other compounds, phenyl—(2,2',2"-Nitrilotriethoxy) silane and di-Ef
butylsilane, had received some preliminary attention in earlier studies
by the author of this manuscript. These compounds had local anesthetic
and possible anti-depressant properties respectively. Each might have
equally satisfied my interest in the pharmacology of organosilicon
compounds but diphenylsilanediol was selected because of its potential

usefulness as an anticonvulsant and antiepileptic compound.

INTRODUCTION

Organosilicon Chemistry

 

Silicon and its compounds are ubiquitious constituents of the
planet earth and the universe. Silicon composes 27.7% of the earth's
crust making it the second most abundant element, whereas oxygen is
first. Carbon, considered as the basis for life on this planet, is
fourteenth and comprises 0.03% of the earth's crust (Weast, 1971).
Like carbon, silicon is a group four element in the periodic table.
As a third period element it has some important properties which make
it different from carbon (Table 1).

The electronic ground state of silicon as 3323p2 resembles the
carbon 2822p2 configuration on initial examination, but the atomic
number of 14 instead of 6 for carbon provides the 3d atomic orbitals
in an energetic position of easy availability. These d—orbitals play
an important role in the chemistry of silicon. 0n the Pauling scale,
silicon has an electronegativity of 1.8 compared with 2.5 for carbon
and 2.1 for hydrogen. This makes silicon the more electropositive
element in a carbon-silicon bond. With silicon—hydrogen bonding, the
hydrogen appears in part as a hydride ion and reacts accordingly
(Bazant 3:.Eir’ 1965). When hydrocarbon compounds are compared with
analogous hydrogen-silicon compounds on reacting with oxygen, for

example, the silicon compound is found to be more reactive, which

Table 1

Chemical Comparison of Silicon With Carbon

Comparison
Aspect

Group

Period

Atomic Number
.Atomic weight
Natural Isotopes

Electronic
Configuration

Hybridization
States

Electronegativity
(Pauling Scale)

Bond Energies
(Kcal./Mol.)

Carbon

1v
2

6

12.01

12, 13, 14

lSZZSZsz

5P9 5P2: 5P3

2.5

(C-C) 82.5
(c—Si) 76

(C-0) 85.5
(C-H) 98.7

Silicon

IV

3

14

28.09

28, 29, 30

1522522p63323p2

3

Sp 9 513st 51-)st

1.8

(Si-Si) 53
(Si-C) 76
(Si-O) 108
(Si-H) 76

Sources: (Eaborn, 1966; Reberts and Caserio, 1964).

would be anticipated for metal hydrides (Roberts et_al,, 1965). A
third factor which serves to differentiate silicon from carbon in its
chemistry and compounds is physical size. Carbon has an atomic radius
of 0.77 A compared with 1.77 A for silicon. Though the more common
electronic hybridized state for both carbon and silicon is the tetra-
hedral spa, carbon can form double and triple bonds with carbon, oxygen,
nitrogen, and sulfur commonly through the overlap of sp2 or sp hybrid—
ized orbitals. For thermodynamic reasons, silicon is unable to assume
these hybridization states and consequently does not form the more
classical double bonds (Burger, 1973). Silicon is able to use its
d—orbitals for back—bonding which imparts some multiple bond character
to 81-0 and Si-F bonds (Eaborn, 1960). 'Silicon is also able to utilize
the trigonal—bipyramid, sp3d, hybridization state for five coordinate
compounds and the square—bipyramid spad2 for six coordinate bonding
(Burger, 1973).

The implications of this discussion are graphically illustrated
in Figure 1 where four compounds of silicon are listed which have mark-
edly increased stability compared to any carbon analogues. Hexamethyl-
disiloxane is a stable compound due to the high bond energy of the
silicon oxygen bonds. The carbon analogue, di—tfbutyl ether is subject
to rapid hydrolysis to tfbutanol. Diphenylsilanediol exists as a
geminal diol. The carbon analogue of this compound does not exist
and if attempted to be synthesized would rapidly be converted to a
diphenyl ketone with the loss of water. Compounds C and D in Figure 1
are five and six coordinate compounds of silicon, respectively. No

carbon analogues are known or theoretically stable.

2H3 1H3
CH3- $1 - o - $1 - CH3
I I
CH3 CH3

A. Hexamethyldisiloxane

B. Diphenylsilanediol

/ ‘ 7
Si.(- 0 - CHZ- CH2)3N

C. Pheny1-(a,2'2"-n1trilotriethoxy)-silane

o 2'
s1<o
° 3

D. Silicon tricatecholate

 

Figure 1. Examples of Uniquely Stable Compounds of Silicon

Pharmacology of Organosilicon Compounds

Though an extensive coverage of the world's literature on
organosilicon pharmacology will not be attempted here, a brief review
of recent developments in this field will hopefully provide a perspec-
tive for the examination of diphenylsilanediol pharmacology and
toxicology which makes up the majority of this dissertation.

Despite the abundance of silicon in the earth's crust, only
recent discoveries have implicated this element as being an essential
element important to life in organisms more advanced than the diatoms.
The verification of silicon as an essential element had eluded inves-
tigators due, principally, to its large abundance and the difficulty
in producing silicon-free environments for laboratory research. The
independent reporting by Carlisle (1970) and Schwarz and associates
(Milne £3 31., 1972) showed that silicon-deficient diets in chicks and
in the rat led to reduced growth rates and structural deformities.
More recent studies (Schwarz, 1973; Carlisle, 1974) suggested that
silicon participates in the formation of bone and connective tissue
through the crosslinking of polysaccharides by siloxane bridges.

Our discussion of the pharmacology will begin with an
examination of two organosilicon compounds, 1-phenylsilatrane and
2,6-Eisfdiphenylhexamethylcyclotetrasiloxane, which are beginning
to be characterized as having well—defined pharmacological activity.

Some recent reviews (Fessenden and Fessenden, 1967; Garson
and Kirchner, 1971; Voronkov gt_al,, 1971; Voronkov, 1975) attest to

a developing interest in the pharmacology of silicon compounds. The

Russian book, Kremnii i Zhizn, (Silicon and Life) by Voronkov, Zelchan,

 

and Lukevits (1971) includes more than 5,000 references in its bibliog—
raphy. Unfortunately, most of the data on organosilicon compounds are
descriptive toxicological responses.

The compound 1—phenylsilatrane (see Figure 1.C), a trivial name
of phenyl-(2,2',2"-nitrilotriethoxy) silane, was one of the earliest
(Baltkais 23 21,, 1964) organosilicon compounds reported to possess
significant acute toxicity. The LD-50 was 0.3 mg./kg. This property
was commercialized by MEET Chemicals, Inc., Rahway, New Jersey, as a
rodenticide. This was based on the extremely toxic nature of this
compound and on its susceptibility to hydrolysis in aqueous media
(Greaves gt_al,, 1974). An examination of this compound by Hulce
and Rech (1973; 1974) identified this compound to be a potent local
anesthetic. This activity appears to account for the acute toxicity
of l-phenylsilatrane.

Bennett, Gorzinski, and LeBeau (1972) and LeVier and Jankowiak
(1972) isolated and characterized the compound 2,6-gisrdiphenylhexa—
methylcyclotetrasiloxane as having anti-androgenic and estrogenic
activity. More recent investigations by LeVier and Boley (1975) have
attempted to localize this activity in the hypophysis and hypothalamus
through the control of dopamine release at the level of the hypothalamic-
median eminence pathways.

Some anticonvulsant organosilicon compounds have appeared in the
literature, but, though interesting, are silicon—substituted analogues

of known organic anticonvulsants. Belsky 33.2}: (1968) were able to

demonstrate anticonvulsant activity in sila derivatives of acetylurea
and barbiturate anticonvulsants. Fessenden and Coon (1965) showed the
same anticonvulsant and muscle relaxant properties for silameprobamate
as meprobamate. The sila derivative had a shorter duration of action

after i.p. administration and was not effective p.o.
Diphenylsilanediol-—Chemistry, Properties

Having current commercial use, diphenylsilanediol has also
played a role in the history of organosilicon pharmacology. Originally
synthesized in 1905 by Dilthey and Eduardoff (1904), the compound was
not well characterized until 1912 when Kipping (1912) purified it as
a crystalline solid from benzene solutions.

Today silicon compounds bearing methyl and phenyl groups com-
prise the majority of commercially available silicon compounds and still
dominate the organosilicon chemistry literature. Diphenylsilanediol is
a commonly produced intermediate in the silicones industry (Noll, 1968)
with the hydrolysis of diphenyldichlorosilane being the most common
synthesis. This latter compound is one of the products of phenyl-
containing silicon compound production by the the direct process
(Rochow, 1945) or Grignard (Kipping, 1904) synthetic routes. This
makes it an industrial compound of modest production volume.

Most dichlorosilanes and dihydroxysilanes (diols) are used in
the synthesis of silicones through polymerization (Eaborn, 1960; N011,
1968). Diphenylsilanediol is difficult to polymerize (Tyler, 1952)

due to the steric effects of the bulky phenyl groups, but does find

commercial use as a filler in making silicone elastomers where it
serves as a structure control additive (Peter §t_§l,, 1953), preventing
hardening in silicone rubber materials filled with a reinforcing silica.

Figure 2 summarizes a typical commercial synthesis of diphenyl-
silanediol from raw material and via the direct process (Rochow, 1945).
Currently all organosilicon compounds are prepared from elemental
silicon which is prepared by the reduction of sand by coke in an
electric furnace—-Reaction I. The direct process reactions (II in
Figure 2) yield a mixture of phenyl-containing organosilicon compounds.
Fractional distillation yields diphenyldichlorosilane which can be
reacted with methanol, Reaction III, to yield diphenyldimethoxysilane.
Hydrolysis of this product yields diphenylsilanediol which may be
further purified by recrystallization (Noll, 1968).

Chemically, diphenylsilanediol is a white crystalline solid
having an empirical formula of C12H1202Si. Its melting point is not
well-defined as the compound has a tendency to change its hydration
state and to polymerize at higher temperatures (Eaborn, 1960). Purity
can be characterized by reference to standardized infrared absorption
spectra (Anderson, 1974) or nuclear magnetic absorption spectra
(Williams, 1974). Figure 3 shows the nuclear magnetic resonance
spectrum for a pure sample of diphenylsilanediol. Figure 4 shows
the infrared absorption spectra for the same sample which was obtained
from D. R. Bennett at Dow Corning Corporation, Midland, Michigan and

used as a reference standard in testing.

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The presence of both phenyl and hydroxy groups in
diphenylsilanediol enables this compound to be soluble in both
polar and non-polar solvents. Though poorly soluble in water, this
compound is readily soluble in alcohols, hydrocarbons, ethers, etc.

In both acid and alkali solutions, there is a tendency for this
compound to undergo polymerization by silanol condensation.

Note that diphenylsilanediol is structurally a geminal diol.
Carbon compounds with geminal diol structures tend to be unstable
unless stabilized by strong electron withdrawing groups (i.e., as
in chloral hydrate). The carbon compound tends to form a ketone, but,
due to the inability of silicon to form stable double bonds, it is
unable to make this rearrangement.

Three samples of diphenylsilanediol were obtained for use in
the investigations discussed here. The reference sample, obtained from
D. R. Bennett of the Dow Corning Corporation, Midland, Michigan, and
identified as E-580—63-1, was labeled as being 99% pure. The material
used for the majority of the tests discussed below was obtained from
Aldrich Chemical Company, Milwaukee, Wisconsin, and identified as lot
number 102717. A third sample, obtained as General Electric PC Powder,
proved to be impure and was not used except occasionally in the
epileptic dog study when the Aldrich material was not available.

The infrared absorption spectra of these three materials were
similar except that the General Electric material showed some additional
absorption bands. Nuclear magnetic resonance spectra for these mate-

rials was examined closely to estimate purity. In Figure 3 note that

14

the hydrogen absorption bands are labeled A, B, C, D, and E. The lines
above the absorption spectra are the integrations for the absorption
curve areas and are directly proportional to the number of hydrogen
protons undergoing absorption state changes. The bottom line in this
figure, labeled 8 to 0, is an index of the chemical shift of the
absorbing proton nuclei with peak B being the standard (tetramethyl
silane—TMS). Peak complex A is due to the phenyl groups on diphenyl-
silanediol; peak B, the silanol hydrogens; peak C, the water in the
samples; and peak D attributed to the solvent which was deuterated
DMSO (dimethylsulfoxide—DS). Comparing the silanol hydrogen chemical
shifts, the standard was 6.88 ppm; the Aldrich sample was 6.87; and
the General Electric material was 6.80. The phenyl to silanol ratios
(theoretically 5:1) were 5.1:1 for the standard; 5.2:1 for the Aldrich
sample and 6.1:1 for the General Electric sample. The higher phenyl
concentrations are probably attributable to the existence of polymeric
forms of diphenylsilanediol being present.

Plan for the Characterization
of Diphenylsilanediol

 

 

With the demonstration by Putnam and Merritt (1937) that
diphenylhydantoin elevated the electroconvulsive shock threshold in
the cat and their later (Merritt and Putnam, 1938) proof of its effec—
tiveness in epileptic patients, the possibility of identifying new
drugs in the laboratory for the treatment of epilepsy seemed apparent.

In a review paper eight years later (Merritt and Putnam, 1945), these

15

authors presented a list of no less than 1,000 compounds that they
had screened in the cat yielding 75 that showed exceptional seizure
protection. The majority of these compounds contained phenyl groups,
or were otherwise aromatic, which supported their 1943 postulate that
phenyl-containing compounds had the best potential as anticonvulsant
agents. Considering structure-activity relationships for clinically
useful antiepileptic drugs, Toman and Goodman (1948) concluded that
phenyl substitution was essential for drugs used to treat grand mal
or psychomotor epilepsy but was also either non—essential or detrimental
to drugs used in the treatment of petit mal seizure types. This posi—
tion has been supported through the years in the structure activity
reviews by Close and Spielman (1961), Spinks and Waring (1963),
Delgado and Isaacson (1970), and Mercier (1973).

The properties of a compound which could disqualify it as a
potential antiepileptic, even though it may appear to be an effective
anticonvulsant, are its acute and chronic toxicity, its behavioral
effects, and its duration of action.

Toman (1970) writing in Goodman and Gilman's textbook states,
"The ideal antiepileptic drug should be capable of complete suppression
of seizures at a dosage level that does not cause sedation or other
undesired central toxicity. Since it must be used continuously for
months or years, it should be well tolerated by the oral route,
inexpensive, long acting, and incapable of inducing tolerance or
withdrawal signs. It should be devoid of systemic toxicity. . . ."

From this definition one can extract 10 properties of an

antiepileptic drug as shown in Table 2.

 

16

Table 2

Desirable Properties for an Antiepileptic Drug

8.
9.

10.

Adapted From Toman (1970)
(Reference in List of References)

The Ideal Antiepileptic Drug:
suppresses all seizure types.
is effective orally.
is long acting.
shows no tolerance to its action.
shows no withdrawal effects.
has no sedation nor CNS toxic action.
shows no systemic toxicity
shows no idiosyncratic toxicity.
has a wide nargin of safety.

is inexpensive to buy.

17

The procedures and programs outlined below, to evaluate the
anticonvulsant action of diphenylsilanediol, are guided by Toman's
definition of an ideal antiepileptic drug. This, of course, is
evaluated in the light of data on currently available antiepileptic
compounds. Suppression of seizures is to be evaluated with a variety
of acute and chronic seizure models. Both dose-response and response—
time data after intraperitoneal and per 03 doses will facilitate com-
parisons between drugsand with behavioral and toxicological parameters.
Sedation and central toxicity will be evaluated by behavioral methods
which provide sensitive and standardized measurements. Oral dosing
will also be evaluated chronically to observe accumulation, tolerance,
and physical dependence possibilities. Determination of organ and
system toxicity was also planned as part of this research and will
be incorporated with the chronic-dosing studies. It was anticipated
that, at the completion of these studies, the anticonvulsant and sup-
porting properties of diphenylsilanediol should be sufficiently well
defined to permit a statement that this compound is similar to
existing anticonvulsants or has its own unique spectrum of action.

Table 3 lists the major phases of this research and the
objectives to be accomplished in each phase. Each of these five
phases also represent the chronology for a systematic characterization
of diphenylsilanediol. The primary concern is for the anticonvulsive
action which, upon being sufficiently interesting, justifies the

pursuit of the remaining phases.

18

Table 3

Plan. for the Characterization of Diphenylsilanediol (DPSD)

II.

III.

IV.

Anticonvuls ant Testing

Objectives: anticonvulsive action spectrum,
comparisons with known compounds.

Behavioral Characterization

Objectives: neurotoxicity screen, behavioral
aspects of general pharmacology.

Toxicological Studies:

Objectives: acute lethality dose range, organ
system chronic toxicity.

Antiepi leptic Studies:

Objectives: establishnent of efficacy results
in clinical disease state.

General Pharmacology:

Objectives: pharmacokinetics, tolerance phenomena,
organ system pharmacology.

19

Published toxicity data in the Soviet literature (Kelman 33_
21., 1968) showed diphenylsilanediol to be relatively non—toxic when
administered to rats and mice. The LD—SO was 2,200 mg./kg. During
discussions with the then manager of biomedical research at Dow Corning
Corporation, Dr. Donald R. Bennett, it was learned that primary screens
conducted at the Dow Chemical Company, Zionsville, Indiana, and Gruppo
Lepetit, Milano, Italy, suggested that this compound might show anti-
convulsant activity. The potential for a new anticonvulsant agent
having a novel chemical structure and a large therapeutic index
coupled with the possibility of a potentially new mechanism of
action created the interest leading to these investigations

summarized herein.

ANTICONVULSANT CHARACTERIZATION

General Remarks

 

There are perhaps as many, if not more, techniques to evaluate
anticonvulsant drugs as there are anticonvulsant drugs. One of the
problems with this state of affairs is that few compounds have been
evaluated by all procedures and few procedures have evaluated all
compounds. Recent summary articles (Naquet and Lanior, 1973; Swinyard,
1973; Woodbury, 1972) attest to this difficulty. The procedures
explained below which were adopted for use in characterizing the
anticonvulsant properties of diphenylsilanediol (DPSD) have the
advantages of either having been used widely, and thus permit compar-
isons of data from other compounds, or show some unique activity for
a particular drug, thus justifying their use. Most of the procedures
involve acute observations even though epilepsy itself is a chronic
phenomenon. Acute studies are designed to cover a range of convulsant
types. These are categorized as whole brain or focal mechanisms and
involve both electrical and chemical stimulation. There are very few
chronic models in use, and most of these involve focal lesions. One
chronic model to be discussed later is the epileptic dog. Also part
of this effort is the evaluation of the effects of chronic oral dosing
on the anticonvulsant action observed for DPSD. The purpose of chronic

administration is to evaluate accumulation of effect, tolerance, and

20

21

whether physical dependence develops. Details of rationale and

methodologies are given below.

Maximal Electroshock Generalized Seizures

 

Earlier studies had revealed that DPSD was effective in
antagonizing maximal electroshock induced seizures in the mouse
(Bennett, 1972, personal communication). The ED-50 was reported
to be 16.5 mg./kg. after i.p. administration in methyl cellulose.

As a preliminary investigation, the re-determination of this ED—50
value was undertaken for further comparisons of data. Using maximal
electroshock antagonism as a dependent variable, an estimate of some
temporal parameters of the action of DPSD could also be determined.
Time of peak effect, half—time and duration of action were explored
for estimating doses and times for future experiments.

The mouse and rat are standardized subjects for anticonvulsant
testing of drugs (Swinyard, Brown, and Goodman, 1952) and, except for
the oral administration of diphenylhydantoin (Swinyard, and Toman,
1950), have similar dose-response patterns. With the introduction
of the gerbil as an epilepsy model (Loskota, Lomax, and Rich, 1974),
and with the establishment of a colony of these rodents in our lab-
oratory, it was felt important to have comparable dose—response data
for this species.

For direct comparison, the antagonism of maximal pentylene—
tetrazol seizures was also determined. The objectives of these early

studies were to (1) compare the maximal seizure antagonism of DPSD

 

22

against maximal electroshock and maximal pentylenetetrazol seizures;
(2) evaluate the time course for the anticonvulsant action of a single
«dose of DPSD; (3) establish the influence of route of administration on
time anticonvulsant action of DPSD; and (4) examine the possibility of

saignificant species variation in the anticonvulsant action of DPSD.

Methods

Subjects were rats, mice (Spartan Laboratory Animals, Haslett,
blichigan), and gerbils (Meriones unguiculatus). The gerbils were
cflatained from a number of sources on the Michigan State University
caunpus and maintained as a breeding colony in the Life Sciences
Btuilding. The mice, rats, and gerbils were adult females with weights
Of: 20-30 grams, 200-250 grams, and 70-90 grams, respectively. Groups
0f: six rodents were administered either 10, 30, 100, or 300 mg./kg. of
DPS”) in 0.5% methyl cellulose solution in distilled water. Control
Stflbjects received the methyl cellulose solution only. Electroshock
Wei; delivered with a Woodbury—Davenport Shocker, Figure 5 (Woodbury
anti Davenport, 1952) through corneal electrodes according to the
mettuod of Toman, Swinyard, and Goodman (1946). With the administration
0f END Hz. A.C. interrupted shocks of 0.2 second duration and 50 mA.,
allu'ntreated mice demonstrated tonic extension of the hind limbs.

Electroconvulsive shock was administered for 0.2 seconds with
a Curment level of 50 mA. for mice, 100 mA. for gerbils, and 150 mA.
for rats. Groups of six subjects were scored as number protected per

Ilumber treated. Seizure protection was scored as the inhibition of

23

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Electroshock for Convulsive Seizures.

24

tonic extension (Swinyard, Brown, and Goodman, 1952) and, for time
effects, groups of six mice were tested at repeated intervals. One
group was tested at O, 30, and 300 minutes, another at 7.5, 60, and
480 minutes, and a third at 15, 120, and 600 minutes after dosing.
Groups of six mice were also run in triplicate for statistical reasons.
Response time experiments were done with a single dose of 50 mg./kg.

The comparison with maximal pentylenetetrazol seizures used
the same end point of the antagonism of tonic hind limb extension but
here the seizures were caused by the administration of 95 mg./kg.
pentylenetetrazol via the subcutaneous route in mice. This convulsant
was administered 30 minutes after the DPSD administration and was
expected to produce seizures in 15 minutes. Animals not displaying
tonic hind limb extension by 30 minutes after pentylenetetrazol
administration were scored as protected.

Routes of administration were i.p. injection, oral gavage with
the methyl cellulose vehicle, and administration with a powdered food
in the diet. After i.p. administration animals were tested between
30 minutes and one hour post dose; after oral gavage, the time was
from 45 minutes to 105 minutes post dose; and the subjects with DPSD
in their food were tested after they had been on the food and drug

mixture for 24 hours.

Results
A direct comparison of the dose response antagonism by DPSD

Of tonic hind limb extension due to maximal electroshock or to

25

pentylenetetrazol in mice is shown in Table 4. The maximal
electroshock ED-SO value is 20 mg./kg. i.p. while the pentylenetetrazol
seizure antagonism ED—50 is 48 mg./kg. The time course data are
‘reported in Table 5 and plotted in Figure 6. The Table 5 entries are
the individual responses of treatment groups at the times indicated.
The mean values for the three determinations at each time period are
recorded at the lower portion of the table. From an examination of
Figure 6, the peak time for anticonvulsant action is at 30 minutes post
dose and there is some activity up to 600 minutes, or ten hours, post
dose. An apparent half-time for the elimination phase of this action
is on the order of four hours.

Table 6 summarizes the effects of differing routes of
administration of the DPSD antagonism of maximal electroshock tonic
hind limb extension. The i.p. route ED-SO is 20 mg./kg., the oral
gavage ED-SO is 2.9 mg./kg., and the oral ED-SO for drug mixed with
food is 0.41 mg./gm. food or estimated as 68.3 mg. (DPSD)/kg. (body
weight)/day (24 hr.).

Table 7 gives comparative results for the rat, mouse, and
gerbil. Each of these values are similar in that each of the 95%
confidence intervals have extensive overlap. The calculated ED—SO
values are 20 mg./kg. in the mouse, 42 mg./kg. in the gerbil, and

30 mg./kg. in the rat.

26

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31

Chemical Stimulation Models

 

According to Swinyard (1973) and Woodbury (1972), no single
anticonvulsant test procedure is adequate to identify the spectrum of
a potential antiepileptic drug by itself. Only through an examination
of anticonvulsant action against a variety of seizure mechanisms can
the spectrum of action for an anticonvulsant drug be determined.

Diphenylhydantoin is very poor at antagonizing chemical con-
vulsions (Everett and Richards, 1944), contrasted with phenobarbital,
which is very effective. The anticonvulsant action of DPSD against
pentylenetetrazol, strychnine, and picrotoxin was thus determined.

Topical penicillin G to the brain neo—cortex of experimental
animals produces a temporary lesion that discharges to produce con-
vulsive seizures which occur repeatedly over a 2” hour period and
then cease (Prince, 1972). Diphenylhydantoin (Lousi e£_al:, 1968)
and diazepam (Sharer and Kutt, 1971) effectively antagonize these
focally—induced clonic seizures. Phenobarbital (Bdmonds et_al,, 197%)
apparently is ineffective. The effectiveness of DPSD against this

seizure was also evaluated.

Methods

Subjects were female Sprague—Dawley rats (Spartan Laboratory
Animals, Haslett, Michigan) weighing between 200 and 260 grams.
Pentylenetetrazol, picrotoxin, and strychnine were obtained from
Aldrich Chemical Company. Animals were treated with i.p. doses of

DPSD suspended in 0.5% methyl cellulose one-half hour before injection

32

with one of the convulsant drugs via the subcutaneous (s.c.) route
at an ED—99 dose. Pentylenetetrazol testing was done according to
the method of Swinyard (1949), picrotoxin by the method of Hahn and
Oberdorf (1962), and strychnine by the method of Everett and Richards
(iguu). The end points for scoring groups of six rats were no clonic
seizures for the pentylenetetrazol tests during a 30-minute period post
dose. This was also the criterion for scoring the strychnine test
animals. With picrotoxin, the end point was protection from the
seizures produced by this compound. ED—SO values and their 95%
confidence intervals were determined using Finney's method (1952)
for probit analysis.

Penicillin G, sodium salt, was applied to the cerebral cortex
in the parietal lobe using an implanted cannula depicted in Figure 7.
The inner cannula was packed with crystals of penicillin and inserted
into the outer cannula for focal cortical stimulation with this topical
convulsant drug. Only four rats were implanted with these cannulae
which were fabricated from stainless steel hypodermic needles. Upon
the insertion of the inner cannula continuing penicillin, each animal
would begin having episodic seizures within 15 minutes. These seizures
would decrease in frequency over a lQ—hour period. For the purpose of
anticonvulsant testing, DPSD was adminiStered 30 minutes before the
inner cannula was inserted. Animals not having seizures within 30
minutes after the cannula was inserted were scored as being protected
by the anticonvulsant medication. Each rat received the dose vehicle

(0.5% methyl cellulose solution), 30 mg./kg. DPSD and 100 mg./kg. DPSD

 

 

33

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separated by a two-day inter-trial period. The order of dosing was

permuted for the purposes of experimental design.

Results

Table 8 summarizes the data from the chemical—convulsant tests.
Part A of this table shows that DPSD was able to antagonize seizures
produced by pentylenetetrazol, strychnine, and picrotoxin. The ED-SO
values for pentylenetetrazol and strychnine antagonism were very sim-
ilar, being 58 mg./kg. and 60 mg./kg., respectively. The picrotoxin
activity antagonism was produced at a higher ED-SO dose level of 150
mg./kg. An examination of Part B of Table 8 provides evidence that
DPSD offers some protection from focal stimulation with penicillin.
Both the number of seizures and the number of animals having seizures
show a dose dependent reduction in penicillin convulsions due to DPSD.
An ED-SO was not calculated as only two doses of DPSD were used. A
reasonable estimate is that the ED-SO value lies between 30 and 100
mg./kg. With its ability to antagonize chemically—induced seizures,

DPSD is unlike diphenylhydantoin and shows a broader spectrum of action.

Focal Electrical Stimulation Models

General Remarks

 

With the seizure models discussed above, the dependent variable
was, in each case, a gross observation of the animal's behavior. With
the focal electrical stimulation models discussed below, the attempt was
not to produce gross seizure and convulsions, but to observe electrical

phenomena in the nervous system in response to electrical stimulation.

     

35

Table 8
Antagonism Of Drug-Induced Seizures In Rats By DPSD
A. Protection From Systemic Convulsants

Dose of DPSDQ (mg./kg.):

Convulsant 10 30 100 300 80-50 95% C.I.
(Dose, Route)

Pentylenetetrazol 0/6* 2/6 4/6 6/6 58 mg./kg. (13-103)
(80 mg./kg., ioPO)

Strychinine 1/6 3/6 3/6 5/6 60 mg./kg. (7-113)
(1.5 mg./kg., s.c.)

Picrotoxin 0/6 1/6 3/6 4/6 150 mg./kg. (34—266)

(5 mg./kg. , s.c.)
Notes: *Table entries are protected/tested rats.

{DPSD given i.p. in 0.5% methyl cellulose.

B. Protection From Topical Penicillin To The Neocortex

Dose of DPSD} (mg./kg.)

Rat Seizures 0 30 100 (Sequence)@
Bu in 30 min.: 5 o o (o, 30, 100)
R in 30 min.: 13 l 1 (100, 30. 0)
G in 30 min.: 6 5 O (30, 0, 100)
El in 30 min.: 4 4 0 (0. 100, 30)
Total seizures 28 10 1

Quantel Score 0/4* 1/4 3/4

Notes: #DPSD given i.p. in 0.5% methyl cellulose, 30 min. before penicillin.
@0rder in which doses were administered separated by 2 days.

*Protected from seizures/treated.

 

 

36

These methods permitted observations on the effect of an anticonvulsant
compound on the initiation and spread of an electrical disturbance to
the nervous system.

Of the many procedures available (Purpura et_§13, 1972; Naquet
and Lanoir, 1973), focal electrical stimulation of the neocortex
(Penfield and Jasper, 1954) and post-tetanic potentiation (Esplin,
1957) of the superior sympathetic ganglion were selected.

Focal Electrical Stimulation of the
Neocortex

 

Electrical stimulation of discrete sites in, and on, the brains
of laboratory animals and humans (Penfield and Jasper, 1954) can evoke
a sustained electrical discharge from cortical neurons that outlasts
the stimulus duration by a wide margin and is not accompanied by gross
motor seizures in the subject. These discharges can only be evoked by
trains of high frequency stimulation above threshold values. Wilder,
King, and Schmidt (1969) have shown that normal seizure propagation
from an epileptic focal discharge has similar properties. This is
strongly suggestive of the post—tetanic potentiation that Esplin (1957)
demonstrated to be highly sensitive to the action of diphenylhydantoin.
Most of these studies have been done in the cat (Schallek and Kuehn,
1963), the monkey (Delgado g£_§13, 1956), or the human (Penfield and
Jasper, 1954) to demonstrate drug effects. After—discharge is inhib—
ited best by diphenylhydantoin, next by phenobarbital, and somewhat
equivocally by diazepam. The rat has recently become a useful subject
for post stimulus after-discharge phenomena (Marsan, 1972) and was

selected for use in this study with DPSD.

 

 

 

37

Methods. Adult female Sprague-Dawley rats (Spartan Laboratory
Animals) weighing 200-225 grams were implanted with epidural silver—
silver chloride electrodes, according to the method of Pirch and Rech
(1968), at least two weeks before testing. Additional electrodes made
from a short section of stainless steel wire were placed 2 mm. anterior
to the left-hand side recording electrode for stimulation of the cortex,
and a stainless steel screw placed into the left side nasal bone to
serve as a ground reference. Recording electrodes were located over
parietal cortex using stereotaxic coordinates. Recording of the
spontaneous electrocorticogram (ECoG) and evoked after—discharges
were accomplished with a Grass Model 7 polygraph through a 7P5 wide
band preamplifier. Biphasic stimulation was delivered at 60 Hz. with
a Grass S-8 stimulator. Voltage and train duration were adjusted to
evoke an after-discharge for a particular animal. Once these parameters
were obtained, they reliably produced an after-discharge in a subject.
Duration of the after—discharge was determined to the nearest 10 sec.
by an examination of the ECoG record. Drugs were administered after
a baseline response was obtained. Animals were retested 24 hours after
the dose of DPSD was administered. DPSD was administered via the i.p.
route in a 0.5% methyl cellulose solution 30 minutes before an after—
discharge was evoked. Figure 8 shows the recording and stimulation

techniques used in this study.

38

 

 

 

 

 

Electrical
Stimulator:
Grass 888
Polygraph
Amplifier: '1 ECoG.
Grass 7 ~ Record.
implanted
cortical ----,
electrodes
0 Sp rague-Dawl ey
4’4 Rat
l.)
I»,

Figure 8. Method for Cortical After-Discharge Studies.

 

 

 

39

Results. Table 9 summarizes the results from the
after—discharge experiments with three rats which received 30 mg./kg.
of DPSD. There was a significant decrease in the duration of the after—
discharge. Note that this drug was very dramatic in abolishing the
after—discharge response. One subject also received a stimulus of
twice the duration and twice the voltage, but this only served to
evoke a tonic—clinic seizure in the animal and not a seizure—free,
sustained discharge. These results strongly suggested that a more

classical post—tetanic potentiation experiment be done.

Post—Tetanic Potentiation Effects

 

It was the purpose of the post—tetanic potentiation experiments
using the cat sympathetic superior cervical ganglion preparation to
test the hypothesis that DPSD, like diphenylhydantoin, acts to inhibit
post-tetanic potentiation mechanisms.

With the demonstration by Esplin (1957) that post—tetanic
potentiation in the spinal cord of the cat was markedly attenuated by
anticonvulsant doses of diphenylhydantoin, an explanation for the action
of this drug was developed. Toman (1970) notes that patients with
epilepsy caused by a focal discharge still have the pre-seizure aura,
but are without the seizure when treated with diphenylhydantoin. This,
he surmised, is evidence that post—tetanic potentiation inhibition is
an important clinical mechanism for diphenylhydantoin. Later studies
showed that trimethadione (Esplin and Curto, 1957) and phenobarbital

(Esplin, 1972) do not inhibit post—tetanic potentiation. The ability

no

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of diphenylhydantoin to inhibit post-tetanic potentiation is seen
at the neuromuscular junction (Raines and Standaert, 1969) and the
various sympathetic ganglia (Esplin and Zablocka, 1969; Esplin, 1972)

in addition to the spinal cord.

Methods. Because Esplin and Zablocka (1969) had shown that
diphenylhydantoin blocks post-tetanic potentiation in cat sympathetic
ganglia, and because of the relative simplicity of this procedure (over
a spinal root preparation), it was employed using the technique of
Gebber and Volle (1966); see also Volle (1962). Cats were surgically
prepared under diallylbarbiturate anesthesia with the superior cervical
ganglion isolated with its afferent sympathetic trunk and efferent
sympathetic nerves intact. Stimulating electrodes were placed on the
ascending sympathetic trunk and recording electrodes on one of the
:sympathetic branches (e.g., the external carotid nerve). The evoked
nuanosynaptic potential was displayed on an oscilloscope and recorded
cum a Grass polygraph for analysis.

Figure 9 depicts the recording and stimulation procedures

Lused to elicit post-tetanic potentiation in the cat superior cervical
ganglion. An idealized output record is also shown describing the peak
Eunplitude and tetanization duration measures discussed below. The peak
Efluplitude of the tetanization record is identified and measured in
Inillivolts. With a prior determination of baseline amplitude for
reference, the time for the return of the potentiated response to
baseline is also noted. These three parameters of baseline amplitude,

Peak potentiated amplitude, and the time for the potentiation to

42

METHOD FOR PTP \X/ITH CAT GANGLIA
PREP. IN SITU .

$ su P. CERVICAL
, 3 ®

_SYMPATHETIC TRUNK) LO.)
CAROTI D NERVE

RECORD ON POLYGRAPH

 

 

 

 

 

11 MV.
RESP IHH ”HHHHHHHHHHHHHHHHHHHHHHHHHI
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ST'M' SOHZ. O.5HZ. ..__. 10$EC.‘

Figure 9. Diagrammatic Summary of the Post Tetanic Potentiation
(PTP) Method and Data Collection.

 

 

43

return to baseline amplitude are recorded for each subject both
before and after drug administration. Each datum point consists

of the mean of three successive determinations. Administered drug
was suspended in a 0.5% methylcellulose vehicle at doses of 0.0 mg./kg.,
25 mg./kg., and 50 mg./kg. of DPSD via the i.p. route. Three animals
were used as control subjects, three as low-dose subjects, and three
as high-dose subjects. The initial dose selected was 50 mg./kg. of
DPSD as this represented a high anticonvulsant dose in mice and rats.
A total of sixteen cats were prepared surgically for post-tetanic
potentiation experiments. Only those animals showing consistent,
repeatable, evoked—responses between 500 and 1,500 microvolts and
tetanization durations close to one minute were included in this

study.

Results. Table 10 is a compilation of the post—tetanic
potentiation results with nine cats both before and after dosing
with vehicle alone, 25 mg./kg. of DPSD, and 50 mg./kg. 0f DPSD. At
the bottom of this table the means and standard error values for each
dose level are presented. In order to facilitate examination of this
data, the differences in before-dosing and after—dosing amplitudes of
the maximum post—tetanic potentiation amplitudes in millivolts are
examined separately. The mean values for these parameters show a sig—
nificant difference between the 25 mg./kg. and the 50 mg./kg. values
when compared with the control (vehicle) values. The overall F sta—

tistic for a completely randomized analysis of variance design was not

 

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significant at the p<:.05 level; but an application of Dunnett's
modified t-test showed both values to be significantly different from
the control value at the above alpha level. Notice, however, that the
two amplitude values are not different from each other. In addition,
there is a dose-related decrease in post-tetanic potentiation duration
due to DPSD. Here all values were found to be significantly different
using a combination of F-test and Dunnett's t—test procedures.

Thus, DPSD inhibits post-tetanic potentiation as part of its

action.
Tolerance and Physical Dependence Studies

The wide range of anticonvulsant activity observed for DPSD,
as discussed above, suggests a therapeutic potential for this compound
as an anticonvulsant or antiepileptic agent. The latter therapeutic
indication demands long term administration. Under these conditions,
the possibility of tolerance development to the DPSD anticonvulsant
action needs to be examined. In addition, the potential for physical
dependence and a resulting withdrawal syndrome upon cessation of the
DPSD administration also needs to be seriously considered.

In this section an attempt was made to design a study which
would give some indication of the potential for both tolerance and
physical dependence associated with DPSD administration. McQuarrie
and Fingl (1958) were able to demonstrate a lowering of seizure
threshold to low-frequency electroshock seizures and to pentylene—

tetrazol administered intravenously after withdrawal from alcohol

46

administration for as little as one day. Using these techniques,

or modifications of them, some degree of physical dependence, as
evidenced by lowered seizure threshold, has been demonstrated for

a number of anticonvulsant drugs (Dzhagatspanyan and Klygul, 1971;
Rumke, 1967; Swinyard g£_al,, 1957). These articles also show an
associated tolerance development evidenced in a decreasing anticon-
vulsant action with repeated dosing of mice with meprobamate (Swinyard
§E_§l,, 1957), phenobarbital, primidone, trimethadione, diazepam,
chlordiazepoxide, and diphenylhydantoin (Dzhagatspanyan and Klygul,
1971; Rumke, 1967). There are, of course, variations in the degrees
of both tolerance development and physical dependence with each of the
individual anticonvulsant drugs. (A discussion of these variations is
considered below in the light of results from the DPSD tolerance and

dependence studies.)

Methods

The procedures used here were modeled after a study by
Dzhagatspanyan and Klygul (1971) in which tolerance and physical
dependence were shown for diazepam and chlordiazepoxide in comparison
with phenobarbital. The seizure threshold method used there, and below,
is the timed intravenous infusion of pentylenetetrazol technique of
Orloff e: 313 (1989). Threshold dose of pentylenetetrazol to produce
convulsive seizures is determined before treatment with an anticonvul—
sant test drug, at various times during anticonvulsant drug treatment,
and for a number of days after the anticonvulsant drug treatment has

been stopped. Three types of information are gained from this type of

 

 

Ln

experimental design. An elevation of the seizure—threshold dose of
pentylenetetrazol, when control periods are compared with the treatment
periods, indicates an anticonvulsant action against pentylenetetrazol
for the test compound. The second type of information is tolerance
development. During the daily dosing period, a drop in seizure thresh—
old dose for pentylenetetrazol during the dosing period is evidence
that tolerance to the drug's anticonvulsant action has occurred. An
increasing threshold during this same time period is indicative of
either accumulation of the drug in the organism or sensitization
("negative tolerance") to the action of the drug. The third type of
information focuses on the change in threshold after the compound has
been withdrawn. A decrease in threshold dose for pentylenetetrazol
would be indicative of physical dependence if the threshold level
dropped below the control values established before dosing with the
anticonvulsant drug. By following the time course of this decrease

in seizure threshold, the onset and duration of a physically dependent
state can be characterized. Table 11 summarizes these observation
criteria for tolerance and physical dependence.

Subjects were female Swiss Webster mice (Spartan strain),
housed in groups of six animals, each weighing from 25—30 grams, and
fed ad libitum on ground rat food (Purina Rat Chow, Ralston Purina Co.,
St. Louis, Missouri) and water. The rat food also served as a dosing
vehicle for the DPSD. Two doses of DPSD were administered to the mice.
The lower dose was the ED—SO anticonvulsant dose that antagonized max—

imal electroshock induced tonic hind—limb extension (see Table 6 above)

 

 

 

 

l+8

Table 11
Design For Study To Evaluate Potential For
Tolerance And Physical Dependence For
DPSD

Method: -- From McQuarrie and Fingl (1958) and Dzhagatspanyan and Klygul
(19 71). '

 

Tolerance: - DecreaSe in elevated pentylenetetrazol threshold values
from the increase established after acute test drug with.
repeated administration of the test drug.

PAhysical Dependence: - Decrease in pentylenetetrazol threshold values
below pre-dosing values after the test drug is
abruptly discontinued.

 

Pentylenetetrazol Threshold: - First twitch value (Orloff etial. 1949).
Subjects: - Mice (Swiss Webster, Spartan Strain): females; 25-30 gm.
Test Drug: ~ Diphenylsilanediol (DPSD) .

Route of Administration: - Dispersed in food.

 

Dose of DPSD: - 0.4 mg./gm. food; (MES ED—SO - 0.41 'mg./gm. food)

 

6.4 mg./gm. food; (RotaRod ED-SO = 6.62 mg./gm. food)
Duration of Dosing: - 14 days and 90 days.
Test Times: - Before-Dosing period: 1 day before

During-Dosing period: days 2, 5, 14, 30, 90
After-Dosing period: days 1, Z, 3.

 

 

1+9

when administered in the food for 24 hours. The higher dose was the
rotating rod depression ED-SO value (see Table 16 below). Food was
prepared once a week by grinding the DPSD into the powdered food on
a milligram of DPSD per gram of food basis. The lower dose was 0.4 mg.
DPSD/gm. food and the higher dose was 6.4 mg. DPSD/gm. food. The food
was placed into porcelain feeding dishes which were filled daily with
the ground food and placed in the cages with groups of six mice.
Eighty—four mice were used in this experiment with forty-two in

the low dose group and the other forty—two in the high dose group.

Six animals maintained in one cage were tested together for
seizure threshold during intravenous infusion of pentylenetetrazol
(Sigma Chemical Co., St. Louis, Missouri). The pentylenetetrazol
was dissolved in 0.9% saline solution and administered via the lateral
tail vein through a 27 gauge hypodermic needle connected to a syringe
driven by a syringe pump (Harvard Apparatus Co., Inc., Millis, Massa-
chusetts). The 1% solution of pentylenetetrazol was administered at
a rate of 19.4 m1./hr. At the start of the infusion, a stopwatch was
started and the time to the first twitch (Orloff 23.3l3’ 1949) was
recorded. The dose of pentylenetetrazol to produce this event was
calculated from infusion time, infusion rate, and body weight for
individual animals.

The low dose groups were tested on the day that DPSD was to
be added to their food, at the end of the first full day of dosing,
day 2, after seven days of dosing, after fourteen days of dosing, and

one, two, and three days after dosing with DPSD was discontinued. At

 

 

50

any particular test time, the individual mice from a cage of six

were tested using the timed intravenous infusion of pentylenetetrazol
technique. The mice were restrained in a clear plastic holder (No. 2,
rat holder, Narco Biosystems, Inc., Houston, Texas) during these
observations which permitted limited movements of the mice and clear
observation of the seizure activity.

The high dose groups were tested on the day that DPSD was added
to their food; at the end of their first full day of dosing, on the
fifth day, fourteenth day, thirtieth day, and on the ninetieth day of
dosing; and again on the first (9lst.) and third (93rd.) days after
dosing with DPSD had been discontinued. As above, one cage of six
mice was tested at any one time period and the time (i.e., dose) for
the first twitch after pentylenetetrazol administration was observed
for each animal.

Table 11 summarizes the methods design as discussed above in

this section.

Results

Data on the pentylenetetrazol threshold changes associated
with administration and withdrawal of 0.H mg./gm. food are listed in
Table 12 and plotted in Figure 10. Though there was an elevation in
pentylenetetrazol threshold associated with DPSD administration, there
is no evidence of tolerance or physical dependence. The more severe
dosing conditions of 6.4 mg./gm. food for 90 days produced the results
listed in Table 13 and plotted in Figure 11. The threshold for

pentylenetetrazol threshold seizures was elevated to 126 mg./kg. i.v.

 

 

 

 

Table 12

Pentylenetetrazol "First Twitch” Threshold
Changes Associated With DPSD Administration For 14 Days
(Dosing started at 0.4 mg./gm. food on day one and stopped on day 14.)

Test Number Infusion Total Dose Standard

D_ay_ 135—ted? Time# _MEE £731: Error
1 6 21.7 1.17 30 4.6
2 5 53.6 2.88 103 11.6
7 4 51.2 2.76 95 18.3
14 S 58.3 3.28 106 17.3
15 6 26.7 1.46 49 4.2
16 S 25.4 1.37 44 2.9

Notes: @Number of mice in group on test day.

#Time to first twitch observation in seconds with i.v.
infusion of 1% pentylenetetrazol solution to the
lateral tail vein.

*Calculated total dose of pentylenetetrazol GWET) in Hg.

+Calculated mg./kg. dose of MET admisistered i.v.

 

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53

Table 13
Pentylenetetrazol "First Witch" Threshold
Changes Associated With DPSD Administration

For 90 Days
(Dosing started at 6.4 mg._/gm. food on day one and stopped on day 90.)

Test Number Infusion Total Dose Standard

 

2% w Time# MET$ w Error

21 5 21.1 1.13 40 5.1
5 4 64.7 3.48 134 10.8
14 4 70.4 3.79 126 17.5
30 5 69.7 3.75 121 14.7
90 4 70.1 3.77 125 16.2
91 5 16.2 0.87 29 4.9
93 5 15.4 0.82 27 6.0

Notes: @Number of mice in group on test day.
#Time in seconds to first twitch observation with i.v.
infusion of 1% pentylenetetrazol (MET) solution at
0.3233 m1./min. to the lateral tail vein.
$Ca1cu1ated total dose of pentylenetetrazol (MET) in mg.

+Ca1cu1ated mg./1<g. dose of MET administered i.v.

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55

and can be compared with the elevation to 103 mg./kg. i.v. with the
lower dose of DPSD as it appears in Table 12. In both cases the control
(before test drug) values were approximately 40 mg./kg. Again there is
little evidence of tolerance development on this dosage regimen. The
decrease in seizure threshold to 29 mg./kg. and 27 mg./kg. at one and
three days after the DPSD is withdrawn is evidence for some physical
dependence activity. There were, however, signs of spontaneous

increases in excitation level or seizures.
Discussion of Anticonvulsive Effects

As a result of these anticonvulsive activity studies, one can
<:onclude that DPSD has a broad spectrum of anticonvulsant action. This
(zompound is able to protect rodents from the tonic seizures produced
1337 electroshock and pentylenetetrazol and the clonic seizures from

peantylenetetrazol, strychnine, picrotoxin, and penicillin. It is also
alxle to elevate the threshold for seizures produced by pentylenetetrazol
arnfl for focal electrical stimulation of the brain. In addition to
Stuppressing neocortical after-discharges to electrical stimulation, it
ixfliibits post-tetanic potentiation in sympathetic ganglia. Furthermore,
DPSD is able to suppress seizure phenomena with chronic dosing showing
little evidence suggestive of rapid tolerance development and no major
withdrawal effects. These results and associated doses are summarized
in Table 14. A comparison of these results for DPSD with the published
results for some clinically useful anticonfulsive agents is summarized

in Tablfa 15 and discussed below.

 

 

56

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Comparisons are made with three phenyl—containing anticonvulsive

drugs--diphenylhydantoin, phenobarbital, and diazepam. An examination

of the activity profiles for these four compounds, as shown in Table 15,
reveals a strong similarity between DPSD and phenobarbital except for
the post-tetanic potentiation effects and the apparent minimal with—
drawal effects of DPSD.

Since Esplin (1957) demonstrated the inhibition of post—tetanic
potentiation by diphenylhydantoin, many other compounds have been tested

for their effects on this model (Esplin, 1972; Suria and Costa, 1974;

Polc 3:.El39 1974). In addition to diphenylhydantoin, only diazepam

showed some degree of post—tetanic potentiation inhibition. One could

:speculate about what similarities these three compounds DPSD, diphenyl—
Irydantoin, and diazepam might have in common in terms of molecular size,
Eilectronic properties, etc., but a simple observation is that they are
eaich diphenyl-containing compounds (see GENERAL DISCUSSION, p. 154, for
Incnre elaborations). Increased seizure susceptibility is a common
ptnanomenon associated with the withdrawal of anticonvulsant medication
(Rumke, 1967; Swinyard 33 31:, 1957; Dzhagatspanyan and Klygul, 1971).
TTue small effect seen with DPSD is encouraging as it implies the pos-
sibility of minimizing or eliminating this potential hazard of anti—
convulsant medication.
The inhibition of after-discharge phenomena in the brain is a
common property of many anticonvulsants (Shallek and Kuehn, 1963;
DPSD as with diazepam,

Racine SEE 3&3, 1974; Wise and Chineraman, 1974).

the balubiturates, trimethadione, and diphenylhydantoin, also has this

 

 

 

59

property. Unlike diphenylhydantoin, DPSD also elevates this seizure
threshold.

The inhibition of the clonic portion of seizures produced by
chemical convulsants (Stone, 1972) has been used to predict muscle
relaxant (Roszkowski, 1964), antianxiety (Hanson and Stone, 1964)
and petit mal antiepileptic activity (Swinyard et_alf, 1974). This
activity for DPSD gives support for a wide spectrum of anticonvulsant
action.

Elevation of pentylenetetrazol seizure threshold (Jenny and
Pfeiffer, 1956) is another action that differentiates anticonvulsant

drugs. DPSD, unlike diphenylhydantoin, elevates this threshold.

 

BEHAVIORAL CHARACTERIZATION

General Remarks

Another important area of consideration for anticonvulsant
drugs is their effect on behavior. This evaluation was conducted for
two reasons. One is to determine any behavioral toxicity that may be
associated with the use of an anticonvulsant. The second is to iden-
tify any behavioral activity that could be therapeutically beneficial.

Diazepam, originally developed as a minor tranquilizer and
hypnotic, has found recent use as an anticonvulsant drug (Brown and
Penry, 1973), both in the treatment of status epilepticus and as an
adjunct to antiepileptic therapy. On the other hand, diphenylhydantoin
originally introduced as an anticonvulsant drug (Merritt and Putnam,
1938), has found current use as an anti-anxiety drug to treat psycho—
neurotic symptoms (Uhlenhuth and Stephens, 1972). The majority of
anticonvulsant drug evaluations usually rely only on the inclined
screen or rota—rod performance as a measure of behavioral effects
(Swinyard, 1949; Toman, 1964; Hudson and Wolpert, 1970). As early as
1952, Swinyard, Brown, and Goodman had recommended that other behavioral
measures be used in testing anticonvulsant drugs as the current tech—
niques were sometimes inadequate. Laboratory and clinical assessments

of the behavioral effects of diphenylhydantoin have been few and far

60

 

 

 

 

 

61

between. Most of these have shown mild tranquilizer or
sedative—hypnotic effects for this drug. Early operant studies

with the rat (Gordon, 1968; Doty and Dalman, 1969) were aimed at
demonstrating that diphenylhydantoin could improve learning in animals
impaired by age or other disability. On performance measures, this drug
has been found to decrease fighting in mice (Tedeschi e£_al., 1959)
decrease the toxicity of amphetamine with aggregated mice (Pink and
Swinyard, 1962), reduce motor activity levels (Millichap and Boldrey,
1967), and to depress shuttlebox performance (Izquierdo and Nasello,
1973). Human studies have centered around the cerebellar ataxia pro—
duced by this drug (Stephens et_al., 1974). Weinreich and Clark (1970)
and Domino and Olds (1972) have also demonstrated that diphenylhydantoin
inhibits responding under intracranial self—stimulation when tested on
rats having electrodes placed in the medial forebrain bundle.

There are elaborate and lengthy schemes for the identification
of psychotropic drug action (Nodine and Siegler, 1964; Siegler and
Moyer, 1967) but selected procedures should be adequate to evaluate
anticonvulsant drugs. Primidone and trimethadione (Woodbury, 1972),
as well as the minor tranquilizer anticonvulsants, including methaqua—
lone (Swift and Becker, 1959), have general depressant and anti—anxiety
behavioral activity. With diphenylhydantoin there appears to be general
depression at high doses while lower doses produce ataxia and cerebellar
dysfunction syndromes (Kutt and McDowell, 1968). Compounds can be
evaluated for neuromuscular relaxation and general depressant prop—

erties using rota—rod techniques (Moore and Rech, 1967).

 

62

Operant conditioning techniques, such as conditioned suppression
procedures, introduced by Estes and Skinner (1941), provide convenient
methods for detecting anti—anxiety properties of drugs that do not
produce profound stimulation or depression in the disinhibiting dose

range.

Rotating Rod Effects

 

The disruption of rotating rod performance in the mouse and
rat is a widely used measure of neurotoxicity with anticonvulsant and
sedative drugs (Raines et_al:, 1973; Tislow, 1968). Gross, Tripod,
and Meier (1955) suggested that this measure is the best predictor of
the dose level at which humans appear drowsy and ataxic. The three
species used in the characterization studies of DPSD are the mouse,
gerbil, and rat. As a measure of the neurotoxic effects of this drug
and, also, for a simple behavioral comparison in these three species,
the dose dependent effects of DPSD on rota-rod performance was eval—
uated. Two routes of administration were used. The intraperitoneal
route is consistent with the majority of the anticonvulsant procedures
discussed above. An oral route, involving administering the drug in
the diet as a powdered suspension, provides a comparison for the
anticonvulsant (MES) data after oral administration above, and an

oral dosage level comparison for a toxicity study in mice (see below).

 

 

 

 

63

Methods

Subjects were 20—30 gram mice, 200—250 gram rats, and 70-90
gram gerbils. Mice (Swiss—Webster) and rats (Sprague—Dawley) were
obtained from Spartan Laboratory Animals (Haslett, Michigan). Gerbils,

Meriones unquiculatus, (random bred) were from the Michigan State

 

University colony located in the Life Sciences Building. Rota—rod

was run according to the method of Moore and Rech (1967) on a 4 inch
diameter, sand—paper covered rod that was rotated at 8 revolutions per
minute. Animals were trained to a criterion of three minutes on the
rod without falling off for each of three consecutive trial periods
before drug testing. Groups of six animals were dosed with i.p. DPSD,
suspended in 0.5% methyl cellulose, one hour before rotating rod test—
ing. For the special tests after powdered food administration in mice,
the food-DPSD mixture was administered for 24 hours before rotating rod
testing (see anticonvulsant section for the food composition and
preparation procedures).

Subjects were scored with a one or zero if they completed or
failed to complete three minutes on the rota-rod. A particular dose
score would then be the number of subjects falling per the number
tested in a group. These quantal data were evaluated using Finney's
probit analysis method (Finney, 1952), thus giving ED—50 values

together with their confidence intervals.

 

 

64

Results

Table 16 shows the dose response data for rota-rod depression
with these rodents. ED—SO values and their 95% confidence intervals
were calculated using Finney's method. Section A of this table (Table
16A) shows the rotating rod response values for the rat, gerbil, and
mouse after i.p. administration of DPSD. The gerbil was the most
sensitive to the depressant effects of DPSD, i.e., ED—SO of 80 mg./kg.
vs. 170 and 200 for the rat and mouse, respectively. The gerbil also
had the most difficulty in learning this task. A likely explanation
is that the gerbil, having large flat hind feet, is ill—prepared to
climb on moving surfaces unlike the rat and mouse. The mouse and rat
data show essentially identical dose—response values.

An examination of Table 16B shows 6.62 mg. DPSD/gm. of food
was the calculated value for the rotating rod ED—SO utilizing this oral
route of administration. One can estimate the equivalent total daily
dose for this food level by using a daily food consumption value of
16.7% of body weight for the mouse (Bell, 1962) to calculate a daily

drug dosage of 1,105 mg./kg./day for DPSD.
Operant Behavioral Effects

As a further examination of the behavioral toxicity of DPSD
the performance effect of this compound on an ongoing operant behavior
was evaluated. Performance baselines under fixed ratio (FR) operants
are relatively resistant to depression (Thompson and Schuster, 1968)

until highly sedative doses of depressant drugs are reached.

 

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66

Concomitant with this performance, a superimposed measure of
experimental anxiety is highly sensitive to anti—anxiety drug effects
(Estes and Skinner, 1941; Geller and Seifter, 1962; Blum, 1970).
Anti-anxiety agents such as diazepam produce a release of conditioned
emotional suppression (CES) at doses that are widely separated from
those that produce a depression of response rate on a fixed ratio
performance baseline. The barbiturates, on the other hand, produce

both effects in similar dose ranges.

Methods

Adult female Sprague-Dawley rats were obtained from Spartan
Laboratory Animals (Haslett, Michigan) and trained to bar press for
feed using standard procedures (Thompson and Schuster, 1968). They
were stabilized at a FR—4O schedule, one food pellet for 40 bar presses,
and then trained on an Estes and Skinner (1941) type of CES paradigm.
On an average of once every two minutes, a tone was sounded in the
animal chamber for 15 seconds; this was followed by a brief, 0.5 sec.
shock of 0.6 mA. delivered to the feet of the rats through a shock
scrambler. After training, subjects made very few responses while the
tone was on. Animals were tested for two hours per day in a continuous
run with these two paradigms, FR—4O and CES, superimposed. Electro—
mechanical counters kept track of the total number of responses made
by the animal during the two—hour background period and the total
number of responses made while the tone was on. A depression of FR
responding was determined as a statistically significant change from

baseline after a given dose. CES effects were measured as significant

 

 

67

increases in the number of bar presses made during the "tone—on"
Eperiod in drug-treated subjects compared with non—treated levels.
IDoses of DPSD were administered via the i.p. route at 15 minutes
loefore testing. The drugs were suspended in a 0.5% methyl cellulose
esolution. Diazepam and diphenylhydantoin were also suspended in a
rnethyl cellulose solution and administered via the i.p. route. Two
flour response data were evaluated for significance using analysis of
\rariance techniques (Guenther, 1964) and followed, if ANOVA was sig—
riificant, by an examination of the individual treatment means using
I)uncan's Multiple Range Test (Brunning and Kintz, 1968). There were
1:hree rats in each group.

Figure 15a depicts a Mechner diagram (Mechner, 1959) which
lirriquely and clearly describes both the FR-40 and CES behavioral
puaxnadigms. Figure 15b shows the response pattern of a typical control
r231: responding under this combined schedule. CES responses are those
URacie during the ”tone—on" period. The tone—on periods are shown as
dcnvrnvard excursions of the bottom tracing shown in this figure. The
cunnilative record is standard with upward excursion indicating responses

and.<iiagonal excursions indicating reinforcements.

32%

Table 17 gives the two hour response totals for the FR—4O
reSPOnding part of the paradigm for DPSD. The actual two hour counts
are eXpressed together with the means of these values calculated as a
percerltage of the control group totals. These normalized values are

plottead.in Figure 12 to show the dose response trend. In a similar

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70

manner the FR-4O results for diphenylhydantoin are shown in Table 18
and plotted in Figure 13. Also, the diazepam results appear in
Table 19 and Figure 14.

For the DPSD data an F ratio of 15.8 was calculated, using
a completely randomized design which was significant at the p < 0.05
level. Duncan's test revealed that only the high dose, 300 mg./kg. ,
was significantly different. There appears to be a small stimulation
in responding at the 10 mg./kg. dose level but it is not statistically
significant. The values for O, 10, 30, and 100 mg./kg. are statis-
tically identical and represent no detectable behavioral impairment
over this dose range. Discussion of the behavioral results for
diphenylhydantoin and diazepam will be deferred to the next section,
"Discussion of Behavioral Effects."

Table 20 summarizes the conditioned emotional suppression
(CES) responses during a two-hour period after DPSD administration.
The number of responses during the tone-on periods for the individual
animals are presented, as well as these values normalized to the control
group responding. An increase in CES responding is interpreted as an
anti—anxiety effect according to the Estes-Skinner models (Estes and
Skinner, 1941). The normalized means and standard error values for
CBS responses are plotted in Figure 16. Analysis of variance on these
data produced an P value of 22.3 which was significant at the p < 0.05
level, Subsequent testing with Dunnett's t showed that all CES
responses at greater than 10 mg./kg. of DPSD were significantly

diff:eli‘ent from the control values with an alpha level of p < 0.05.

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Operant
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100
Responses

16 Minutes

 

 

Figure 15b. Cumulative Record of a Rat Performing Under a
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Thus at doses as low as 30 this compound is a very active anti-anxiety

agent.

Conditioned emotional suppression (CES) results for diphenyl-
hydantoin and diazepam are shown in Tables 21 and 22 and plotted in
Figures 17 and 18, respectively. The discussion and comparison of

these data are, also, deferred to "Discussion of Behavioral Effects"

section below.

Discussion of Behavioral Effects

 

With the introduction of the rotating rod apparatus (Dunham
and Miya, 1957), a simple and easily standardized device was made
available to quantitate observations concerning the neurotoxicity of
anticonvulsant and other central nervous system depressant drugs and
compounds. The rota-rod data for DPSD produced an BD-SO value of 200
mg./kg. with the mouse and 170 mg./kg. with the rat. Literature values
for phenobarbital and diphenylhydantoin (Raines gt 2&3, 1973) in the
.mouse are similar in magnitude. For phenobarbital an ED-SO of 126
Ing./kg. is given and for diphenylhydantoin the value is 133 mg./kg.
Table 23, part A, summarizes these results with their 95% confidence
intervals. Mice data for diazepam yield an anticonvulsant BD-SO of
3.1+ mg./kg. and a rotating rod depression BD—SO of 3.9 mg./kg., giving
a therapeutic index of only 1.1 for this compound. The sedative-ataxic
effects of diazepam are definitely overlapping with its anticonvulsive
effects. This is observed in clinical practice too (Hollister, 1973)

where diazepam finds use in treating status epilepticus but only at

 

 

79

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Table 23

Results and Comparisons of Behavioral Effects,

A. Rotating rod data from mice after i.p. administration.

Compound Rotatinngod ED-SO and 95% C.I.

DPSD 200 mg./kg.
Phenobarbital 126 mg./kg.
DPH 133 mg./kg.
Diazepam 3.9 mg./kg.

B. Operant performance on

(72-328)
(78-201.6)
(81.5—216.8)
(2.3-5.8)

FR-4O with rats.

 

Compound Dose for Performance
Increase Decrease

DPSD lO mg.7k . 300 mg.7kg.

DPH not seen 40 mg./kg.

Diazepam 0.3 mg./kg.

5.0 mg./kg.

(2. Conditioned emotional suppression (CES) results

 

Compound

Initial Dose
DPSD 10 mg./kg.
DPH none

Diazepam 0.3 mg./kg.

Antianxietpresponses

Peak Dose 'Peak Amplitude

 

100 mg./kg. 2156% of control

none none
2.5 mg./kg. 877% of control

8M

doses that produce drowsiness and ataxia in human patients. DPSD
appears to have the neurotoxic dose range well-isolated from the
anticonvulsive range, making its therapeutic index more akin to
diphenylhydantoin than to diazepam or phenobarbital (i.e., a
therapeutic index of about 10).

For discussion of the behavioral characterization results
tising operant techniques it will be necessary to more closely examine
tlie figures in the results portion of this section above. The
comparisons to be made are summarized in Table 23, part B.

Baseline performance on FR—uO reinforcement paradigms is
pneesented as a dose—response curve for DPSD in Figure 12, diphenyl—
hydantoin in Figure 13, and diazepam in Figure 14. The overall shapes
of: these three curves are similar. The diazepam and DPSD curves each
stuaw an initial response increase before the decrease. The doses at
which these increases and decreases occur are summarized in Table 23,
pard: B. DPSD showed an increase in responding at 10 mg./kg., while a
decrease became statistically significant (p‘<0.05 level) at 300 mg./kg.
Corrmasponding data for the diphenylhydantoin showed no increase and a
decrtuase at 40 mg./kg. For diazepam the increase occurred at 0.3
mgo/kég. and the decrease at 5.0 mg./kg. The overall similarity of
DPSD 'to diazepam is apparent from these figures. Of more interest
is thee conditioned emotional suppression data.

Results on the conditioned emotional suppression studies for
DPSD: <iiphenylhydantoin, and diazepam are shown in Figures 16, 17, and

18’ reHSpectively. Numerical comparisons are listed in Figure 23, part

 

 

85

C. An initial comparison of the shapes of these three figures reveals
the similarities between the diazepam and DPSD dose-response data and
the dissimilarity in these two with respect to the diphenylhydantoin
data. The diphenylhydantoin data, Table 21 and Figure 17, show no
conditioned emotional suppression rate increase, thus indicating no
.anti-anxiety properties for this compound.

Both DPSD and diazepam serve to facilitate the performance
ch rats under the conditions of experimental anxiety, making their
<alassification as anti—anxiety compounds clear.

The magnitude of this effect can be seen through a comparison
lxatween the initial (threshold) increase of Operant responding and the
rmaximal (peak) observed increase in operant responding. These data,
Sinmmarized in Table 23, have been extracted from Tables 20 and 22. For
diiazepam the first increase is observed at 0.3 mg./kg., reaching a peak
at 22.5 mg./kg. with an increase to 877% above the control level. For
DPSD, the first increase is at 10 mg./kg., the peak occurring at 100
mg‘./ kg. , having increased to 2,156% above the control level. For both
Of these compounds, the portion of the dose—response curves up to the
Peak::responding on the conditioned emotional suppression paradigm shows
no overlap with the baseline FR—LLO depressant effects shown in Figures
12 and IN.

In conclusion, the behavioral evaluation of DPSD reveals no
Signiiiicant neurotoxicity or behavioral impairment in the antiConvulsant
dose twange of 10 to 100 mg./kg. on intraperitoneal administration.

There .is an additional pharmacological activity that occurs in the

 

 

86

anticonvulsant dose range. This anti-anxiety effect is not to be

considered as a neurotoxic or behavioral impairment. Both the rotating
rod apparatus and the operant FR—HO response rate measures show varying
levels of impairment at doses above 100 mg./kg. These results tend to
indicate that DPSD has behavioral properties that make it appear more
like diazepam than other anticonvulsant drug types. An anticonvulsant
acrtion is obtained in DPSD with an improved therapeutic index and an
associated anti—anxiety response. These statements are, of course,
based on comparisons with the few anticonvulsant compounds for which
"there are behavioral data (Millenson and Leslie, 197a; Raines E£.E£"
Swinyard 2:.Ei" 1952; Zbinden and Randall, 1967). Further examination

of these comparisons will be made in the general discussion section at

the end of this thesis.

TOXICOLOGICAL STUDIES

General Remarks

 

The assessment of potential toxicity for a chemical compound
having pharmacological activity provides both an indication of the
safety, or potential risk factors, associated with its use and perhaps
some insight into the mechanism of action of the compound as a thera-
peutic agent. There are standardized procedures for the assessment of
toxicity in the initial stages (Abrams 33.3Er’ 196%; Nelson, 1965).

An attempt to identify these basic data is reported in this section.
Only simple observations on acute toxicity and the observations on a

chronic (90 day) feeding study are reported below.

Acute Toxicity

 

The primary objective here is to assess the LD-50, or lethal
dose, fiftieth percentile, and to make some observations on the rela-

tive toxicity of DPSD when it is compared with other anticonvulsants.

Methods

Groups of six Swiss Webster mice (Spartan Laboratory Animals,
Haslett, Michigan) were treated via i.p. injection with 0.5% methyl
cellulose suspensions of DPSD. Animals were treated with freshly

Prepared solutions (injection within 30 minutes after preparation).

87

 

88

With each dose and each group of six mice the number surviving
after eight hours was noted. A particular dose-treatment group was
scored as the number dead per number injected. As a variation on this
procedure, groups of six mice received DPSD in their diet for a six day
period to assess a basic lethality on very short term administration.
Here the compound was mixed with ground rat food (Rat Chow, Ralston
Purina Co., St. Louis, Missouri) and a small amount of chocolate flavor-
ing added (Carnation Hot Cocoa Mix, Carnation Food Co., Kansas City,
Missouri) to facilitate its consumption by the mice (pilot observations
showed that the mice would not eat the higher dose mixtures unless a

flavoring agent was added).

Results

Table 29 shows the results from an acute i.p. administration
of DPSD to mice. The LD-SO was calculated to be 2,599 mg./kg. using
the method of Finney (1952). The slope of this curve is evidenced in
the values for the LD-lO at 1,068 mg./kg. and the LD-90 at 6,319 mg./kg.
which suggest a wide range of values for the dose of DPSD which produce
lethal effects. Animals that died did so without any signs of a stimu-
lation phase; only general depression of central nervous system activity
was observed.

Table 25 shows values of the food administration levels of DPSD
and the observed deaths over six day period with mice on these diets.
The far right column lists the last day in the six day observation
Period in which a mouse was found dead. A calculated LD-SO of

32.4 mg. DPSD/gm. of food was found using Finney's probit analysis

 

 

89

Table 24
Lethal Effects Of DPSD (i.p., Mice)
Dose (mg./kg. i.p.! Number Dosed. Number Dead# Percent Deadé

vehicle* Alone 6 0 0.0
500 6 0 0.0

1,000 6 1 16.7
1,500 6 1 16.7
3,000 6 3 50.0
5,0001 6 5 83.3
10,000 6 6 100.0

Notes: *Methyl cellulose 0.5% in. saline.
#Dead animals counted 8 hours post dose.
@Calculated.1ethal dose values (mg./kg.):
LD value Lower 95% CI Upper 95% CI

 

LD-lO = 1068 351 1657 A
LD-SO = 2599 1681 4167
LD-90 = 6319 3992 21139

 

 

90

Table 25
Lethal Effects Of DPSD Administered Subacutely In Food

Food Concentration@ Daily Number Number Day+

 

 

 

Dose# 'Dosed Dead*

6.4 1067 6 0 -
12.8 2133 6 0 -
25.6 4267 6 2 5
51.2 8533 6 5 3

102.4 17067 6 6 2

Notes: @Food formulation: x.x mg. DPSD/gm. food
50 mg. Carnation Hot
Cocoa Mix
To make 1 gm. Purina Rat
Chow.

#Calculated dose (mg./kg.) based on 166 mg.
food/gm. body weight daily food consumption.

* Total number dead animals during 5 day dosing
period.
Calculated LD-SO: 32.4 mg./gm. food

+Day post-dose that last animal died.

 

91

techniques. This corresponds to an ingested dose of 5,410 mg./kg./day
based on an estimated food consumption of 16.7% of body weight (Bell,

1962).

Chronic Toxicity Studies

 

As potential drugs to control epileptic seizures are generally
administered on a chronic basis, the importance of evaluating the
toxicity associated with longer term administration of a drug is
apparent. The toxicological responses for diphenylhydantoin, primidone,
phenobarbital, and diazepam (Woodbury g£_al,, 1972) are well known.
Standardization procedures (Hayes, 1972) recommend that a chronic dosing
schedule continue for at least 10% of the animal's lifetime. Drug
administration for a three month period in mice meet these criteria

and objectives.

Methods

Subjects were Swiss Webster mice from Spartan Laboratory
Animals, Haslett, Michigan. DPSD was administered as a mixture with
pulverized rat chow (Ralston Purina Co., St. Louis, Missouri) at 1.6
and 6.4 mg./gm. of food. These doses were selected to represent an
acute ED—90 for anticonvulsant action (the 1.6 mg./gm. food), and an
ED-SO for rota-rod depression (the 6.4 mg./gm. food). This food was
available on an ad_libitum basis and was replenished once a day.
During the dosing period the subjects were periodically examined for
signs of gross toxicity in terms of appearance, mobility, and food

annd water consumption. During the last day of the drug administration

 

 

92

the subjects were weighed, euthanized by cervical dislocation,
peripheral blood smears obtained after cutting off the tip of the
tail, autopsied according to standardized techniques, and histological
specimens prepared. At autopsy gross observations were made and organ
weights determined. Organ weights on brain, liver, kidney, spleen,
and adrenals were determined after blotting the tissue on a piece of
filter paper to remove excess moisture. Organ samples were taken from
the absorptive organs (stomach, small intestine, large intestine), the
metabolic organs (liver, kidney, lung), and other organs including
blood, bone marrow, spleen, lymph nodes, adrenal glands, gonads, cere—
brum, and cerebellum. These were fixed in formalin except for the
brain tissues which were fixed in buffered glutaraldehyde. Staining
was with hematoxolin and eosin according to standard histological
procedures (Humason, 1966). Blood and bone marrow smears were stained
with Wright's stain. Table 26 summarizes the experimental design for

this 90 day toxicity study.

Results

Gross and microscopic. The high dose group receiving 6.4

 

mg./kg. food was examined thoroughly in comparison with the control
group not receiving DPSD in their diets. Table 27 lists the organ
weights of selected organs for both treated and control groups. Only
the adrenal and spleen weights were statistically different between
these two groups. The smaller size of these organs was not a very
great difference but was observable. As listed in Table 28 these

<iifferences in weight did not reflect any histological differences.

93

Table 26
Design For Chronic Toxicity Study In Mice
Compound: Diphenylsilanediol (DPSD)

Subject: Adult Mice, 20-30, gram body weights, 12 per group (6 male,
‘ 6 female), 3 groups (36 mice).

Doses and Dosing Schedule:

Compound in food (ad lib.); food replenished daily; food
batches prepared weeEIy

'Ihree Doses: Control Diet (no compound)
MES ED-90 Diet (1.6 mg._/gm. food)
Rota—Rod ED-SO Diet (6.4 mg._/ gm. food)
Duration of Dosing: Three months (90 days)
Planned Observations:
During Dosing -
Body weight, general health, deaths
At End of Dosing Period -

Whole body and specific organ weights gross and microscopic
examination of:

a. Absorptive organs: stomach, small intestine,
large intestine.

b. Metabolic organs: liver, kidney, lung.

c. HematOpoietic organs: blood, bone marrow, spleen,
lymph nodes.

d. Endocrine and reproductive organs: adrenal, gonads.
e. Brain: cerebrum, cerebellum

f. Electron microscopic examination of selected organs.

94

18016 27

Organ And Body Weight Changes After Chronic Dosing

Treatment
Control
Die t

Mean Values
Std. Error
Treatment
treated

diet

6.4 mg./gm.
foodfi

Mean Values

Std. Error

Notes: # - Average drug consumption 1067 mg./kg./day.
* - Sigiificant difference from control (p < 0.05 level).

With DPSD For Three Months

Body and Organ Weights in Grams

 

Subject Body Liver Kidney Adrenal Spleen
00-1 35 1.810 0.435 0.020 0.220
00-2 59 1.572 0.500 0.057 0.135
00-3 34 1.545. 0.448 0.021 0.215
00-4 33 1.504 0.459 0.020 0.125
00-5 31 1.920 0.376 0.018 0.241
CC-6 52 1.466 0.460 0.028 0.137

34 1.619 0.446 0.022 0.179

1.1 .079 .016 .003 .021

'04-7 31' 1.684 0.435 0.014 0.116
1H-8 33 1.887 0.449 0.019 0.132
1H-9 30 1.482 0.408 0.016 0.081
TH-O 30 1.578 0.426 0.010 0.170

’TH-l 31 1.493 0.443 0.018 0.077
anz 32 1.541 0.411 0.023 ' 0.112

31* 1.611 0.429 0.017* 0.115*
1.2 .063 .007 .002 .014

 

 

Brain

0.479
0.519
0.436
0.472
0.461
0.472

.013
0.470
0.492
0.415
0.486
0.483
0.396
0.457

.017

95

Table 28

Summary Of Gross And Microscopic Observations On

Organs From Mice Dosed With DPSD (6.4 mg./gm. food)

Organ or Tissue

Absorptive Organs:
Stomach
Duodenum
Jejunum
Ileum
Cecum
Colon

Metabolic Organs:
Liver

Kidney
Lung

HematOpoietic:
Blood

Bone Marrow
Spleen
‘Lymph Nodes

Endocrine and Reproductive:
Slightly Smaller

Adrenal
Gonads Male
Gonads Female

Other Tissues:
Visceral Fat

Skeletal Muscle

Cardiac Muscle
Brain

For 90 Days

Gross

Normal
Normal
Normal
Normal
Normal
Normal

Microscopic

Normal
Normal
Normal

Enlarged Lymphatics

Normal
Normal

Gall Bladder Light: Congestion

Large
Normal
Normal
Normal

Normal

Slightly Smaller

Normal

Normal
Normal

Normal
Normal
Normal
Normal

E.M.: Smooth ER. mild

proliferation
mild Congestion
Normal

EosinOphilia
Reactive Lymphs.
Normal Ratios
Normal

Normal

Normal
Normal
Normal

Normal
Normal
Normal
Normal

 

96

Figure 19 shows the spleen from a representative animal in the high
dose DPSD group. There is no apparent histological change to account
for the smaller mass. Figure 20 in a similar manner shows the adrenal
gland for this treated animal. The kidney weights were normal, Table
27, and there was no major gross or microscopic (Figure 21) abnormal—
ities observed except for a mild congestion. Bone marrow smears
appeared normal with a balance of red and white cells in blast,
immature, and mature stages as shown in Figure 22. The brains were
examined closely with particular attention to the cerebellum area.
Del Cerro et_213 (1967) demonstrated that diphenylhydantoin had a
disorganizing effect on the cerebellum caused by a depletion of the
cerebellar Purkinje cells. Figure 23 shows a low magnification section
of the cerebellum. The boxed-in region is enlarged in Figure 24 showing
the Purkinje cells clearly. There is no apparent abnormality in either
of these slides.

The only readily apparent abnormality observed on gross
examination of the mouse was the enlarged gall bladders seen in 30%
of the animals treated at the high dose. The enlargements appeared
to be due to increased bile storage. Two of these gall bladders
appeared hemorrhagic. The livers in these animals all appeared to
be congested mildly and to have some cloudy appearance in their
cellular structures.

Figure 25 shows a liver section representative of the tissues
observed in the control group. By comparison, the treated group slides,

Figure 26, showed enlarged sinusoids and swollen cloudy cells.

 

 

 

Figure 19. Section or Spleen from a Mouse Treated with DPSD
for 90 Days. (Original Magnification 200 X).

 

Figure 20. Section of the Adrenal Gland from a Mouse Treated
with DPSD for 90 Days. (Original Magnification ho X).

98

 

Figure 21. Section from Kidney of a Mouse Treated with DPSD
for 90 Days. (Original Magnification 200 X).

99

 

Figure 22. Bone Marrow Smear from the Femur of a Mouse
Treated with DPSD for 90 Days.
(Original Magnification 1000 X).

 

 

Figure 23. Section from Cerebellum of a Mouse Treated with
DPSD for 90 Days. (Original Magnification 160 X).

lOl

 

Figure 214 Section from Cerebellum of a Mouse Treated with
DPSD for 90 Days.(0riginal Magnification 1000 X).

 

102

 

Figure 25. Section of Liver from a Mouse on the Control Diet
for 90 Days.(0riginal Magnification 200 X).

 

Figure 26. Section of Liver from a Mouse Treated with DPSD
for 90 Days.(0rigina1 Magnification 200 X).

 

Hematological observations. The other major histological

 

abnormalities observed, listed in Table 28, were in the blood. Table 29
lists the white blood cell differential counts for the control, low dose
DPSD, and high dose DPSD treated mice. The reactive (appearing immuno—
logically activated) lymphocytes are listed as a percentage of the
lymphocytes for comparison purposes. These differential counts are
summarized in Table 30. Representative blood cells from the high dose
treated animals are shown in Figures 27, 28, and 29.

Two parameters increased in the treated groups. There is an
increase in the percentage of reactive lymphocytes. These differences
are statistically significant also (ANOVA and Dunnett's t—test). They
both appear to be dose dependent in that the eosinophil count increases
from 3.6% in the control group, to 4.8% in the low dose treated group,
and 8.5% in the high dose treated group. Similarly, the reactive
lymphocyte levels increase from 0.5% to 2.8% to 3.7%.

In the high dose treated group, some of the neutrophils
contained cytoplasmic inclusion bodies (Doehle Body) as shown in
Figure 28 and none were seen in the control group. A typical reactive
lymphocyte is shown in Figure 29. This can be compared with a more

normal appearing lymphocyte in Figure 27.

105

Table 29
Peripheral Leukocyte Changes After Chronic Dosing
With DPS) For Three Months

Leukocyte Differential Counts (Percent):

 

 

Treatment
"18o DPSD/ gm. Subject , 7 .
Food 1.92113; m M392; ESE}; E93; React. Lymph,
0 1 (20-1 70 2 20 3 1
0 cc-z 77 4 17 2 0
0 CC-3 83 3 10 4 0
0 CC~4 77 4 16 3 1
0 CC-S 75 10 13 2 1
0 CC-6 61 8 2 8 3 0
1.6 TL—l 75 1 17 7 5
1.6 TL-Z 79 2 16 2 2
1.6 TL-3 82 0 16 3 4
1.6 TL-4 75 0 21 4 1
1.6 TL-S 39 0 6 5 1
1.6 TL-6 31 0 11 3 4
6. 4 TH- 7 69 3 18 10 7
6. 4 "IN-8 30 3 7 10 1
6 . 4 111-9 79 2 12 7 3
6. 4 TH-O 7s 5 15 5 4
6 . 4 TH-l 78 2 14 6 3

6.4 111-2 61 6 19 13 4

106

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Figure 27. Eosinophil and Lymphocyte from Peripheral Blood
of Mouse Treated with DPSD for 90 Days.
(Original Magnification 1000 X).

 

Figure 28. Toxic Neutrophil (with Doehle Body) from Blood
of Mouse Treated with DPSD for 90 Days.
(Original Magnification 1000 X).

108

 

Figure 29. Reactive Lymphocyte from Peripheral Blood of
Mouse Treated with DPSD for 90 Days.
(Original Magnification 1000 X).

109

Supplementary Toxicity Studies

 

Electron Microscopic Examination

 

Observed changes in the livers of mice treated with DPSD for
three months were not well-defined after light microscopy examination
of liver tissue taken from these same mice at autopsy. Swollen cloudy
cells can be ultrastructurally characterized further as having enlarged
mitochondria, being vacuolated, having a disorganized rough endoplasmic
reticulum, possessing fat droplets, having a proliferated smooth endo-
plasmic reticulum, or some combination of these primary changes (Sand—
writter and Wartman, 1973). The clarification of these potentially

toxic changes is attempted below.

Methods. Liver specimens were obtained from animals treated as
discussed above and outlined in Table 26. Tissues were prepared using
standard electron microscopic preparation techniques. Immediately after
removal, small specimens were minced in a buffered 6% glutaraldehyde
solution for primary fixation, washed with the phosphate buffer, post—
fixed with a 1% osmium tetroxide solution, and dehydrated with increas-
ing concentrations of ethyl alcohol. After dehydration, tissues were
embedded in an epoxy resin (Epon 812) after clearing the pr0pylene
oxide mixed with increasing concentrations of the epoxy resin. After
trimming the epoxy resin-embedded tissues, they were sectioned with a
Porter—Blum MT-2 ultramicrotome using freshly prepared glass knives.
These sections were transferred to carbon-collodion coated copper

electron microscopy grids, stained with lead citrate and uranyl acetate,

110

and examined using a Hitachi HS-7S electron microscope. Three liver
specimens from each of six treated and six control mice were examined.
Only the high dose (6.4 mg./gm. food) treated animals were examined in
the treated group. Electron micrographs were taken of representative
liver areas in both the treated and control groups for further

comparisons.

Results. A careful examination of both treated and control
liver sections under the electron microscope revealed that there was
only one major, consistent, and recurring difference between tissues
in these two groups. The smooth end0plasmic reticulum was found to be
more abundant in the cells of liver obtained from treated animals.
Figures 30 and 31 compare electron micrographs of the control and
treated liver sections. In Figure 31, note the abundance of smooth
endoplasmic reticulum. Even close to the nucleus, where smooth E.R.
rarely occurs normally, the smooth endoplasmic reticulum is clearly

seen .

In Vivo Liver Function Tests

 

With the observation, discussed in the previous section,
that chronic administration of high doses of DPSD tends to produce
liver congestion and maybe some gall bladder enlargement, an exami—
nation of liver function was considered appropriate. Of the available
test methods and procedures (Hoe, 1968) commonly used, the bromsul—
phalein (BSP) excretion method was chosen as it is both relatively

easy to perform and has been shown to correlate with histopathological

 

lll

 

Figure 30. Electron Micrograph of Liver from Control Mouse -
Control Diet for three Months. (13,800 X).

ll2

 

Figure 31 . Electron Micrograph of Liver from Mouse after
Treatment with DPSD for three Months. (13,800 X).

ll3

examination of the liver (Kutob and Plaa, 1962). The procedure

involves the administration of this dye to a test animal and determining
the time course of its elimination. As this compound is almost totally
excreted via the liver into the bile (Hoe, 1968), its elimination at a

control rate indicates some competency of the hepatobiliary system.

Methods. The treated subjects were six mice which had received
6.4 mg. DPSD/gm. food as their diet. The fifteen control mice had been
on the same diet without the DPSD. The dye, phenoltetrabromophthalein
(Bromsulphalein BSP, Hynson, Westcott and Dunning, Inc., Baltimore,
Maryland), is supplied as a sodium sulphonate salt and was dissolved
in physiological saline for intravenous administration (via the lateral
tail vein in the mouse). A standard dose of 100 mg./kg. BSP was admin-
istered i.v. and blood samples drawn from groups of mice by cardiac
puncture with the animals lightly anesthetized with diethyl ether.
Blood samples were collected into heparinized test tubes and then spun
on a laboratory centrifuge (10 min. at 2,000 rpm) to provide plasma
samples. Analysis of BSP content was done by measuring the 580 nm.
absorptance of alkalinized plasma samples on a Beckman DB-GT Grating
Spectrophotometer in quartz cuvettes. A calibration curve was prepared
based on 0.1 ml. of plasma alkalinized with 0.5 ml. of 0.1N NaOH and
then diluted to 2 ml. with physiological saline. A plasma sample from
an untreated mouse was used as a blank in the spectrophotometer and
received the same regents. First, a calibration curve was constructed
that permitted the calculation of BSP levels in mg./100 plasma. Next

the control mice were treated with BSP and, using groups of three mice

114

each, an elimination curve for BSP from plasma was plotted. From this
curve a decision was made to test the plasma content of BSP in the DPSD
treated mice after 30 minutes post-administration of the BSP. These
results would then be compared with the control group data at 30 min-

utes using a t-statistic at an alpha level of 0.05 probability.

Results. Table 31 shows the absorptance data for the
calibration samples of BSP in plasma. These data are plotted in
Figure 32. A regression equation was calculated as BSP (mg./lOO ml.) =
32.45 O.D. - 1.249 to be used in calculating BSP levels for other
samples.

In Table 32, Part B, the optical density (O.D.) readings for
the plasma samples for the 15 control mice are presented. Blood was
drawn from three mice at each time period of 5, 10, 15, 20, and 30
minutes post dose. The actual concentration of BSP was calculated
via the equation given above. The calculated values, means, and stan-
dard errors are shown in Table 32, Part B; these are also tabulated in
Figure 33. In this figure note that the BSP concentration is expressed
on a logarithmic scale against a linear time scale. The logarithmic
data points fit a straight line which becomes reasonable to fit to a
linear regression model. The logarithmic numbers are found on Table 2
together with a linear regression equation calculated from these data.
This equation is log (BSP) = -0.0465 (Time) + 1.7734.

Using the above time course as a guide, six mice that had
received 6.4 mg. DPSD/gm. food (or 1,267 mg./gm. food) were injected

with BSP and their blood examined for BSP levels as a function of time.

115

Table 31

Plasma level. of BSP Calibraticn@ Data

BSP" mg./100 ml. Plasma Absorptance# (O.D.)
10 0.341
20 0.663
30 0.966
40 1.270
50 1.570
60 1.893

Notes: *BSP = phenoltetrabromphthalein sodium.
sulphonate.

#Plasma samples are alkalinized (0.1 ml.
plasma, 0.5 m1. of 0.1N NaOH, diluted
to 2 ml. with physiological saline) and
the absorptance at 580 nm. measured on
a Beckman DB-GT Grating Spectrophoto-
meter.

@Calculated calibration:

BSP (mg./100 ml.) = 32.45 x 0.0. = 1.249.

116
Table 32

Plasma Levels of BSP in Control Mice - Time Course

A. Concentration Calculated from O.D. Values

Time After BSP" (Minutes)

5 10 15 20 30
Data Values 43.3 19.1 10.3 1.8 4.0
Values 47.0 27.6 12.4 17.0 2.4
mg. BSP per 100 ml./p1asma 33.3 16.6 10.4 4.5 2.7
Mean 41.2 21.1 11.0 7.8 0.5
S.E. of Mean 4.1 3.3 0.7 4.7 0.5

B. Optical Density Measurements (O.D.) at 580 nm.

Time After BSP* (Minutes)

5 10 15 20 30
Individual 1.37 0.63 0.35 0.09 0.16
0.0. 1.43 0.89 0.42 0.56 0.11
Values 1.06 0.55 0.36 0.13 0.12

C. Calculated Log (BSP) Values for Time Calculations

Time After BSP" (Minutes)

5 10 15 20 30

1.637 1.281 1.013 0.255 0.602
1.762 1.441 1.093 1.230 0.380
1.522 1.220 1.017 0.653 0.431

These numbers provide the equation

l.og(BSP) = --0.0465 X (Time) + 1.773

Note: *100 mg./kg. BSP given i.v. at time zero.

 

 

117

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Figure 33. Disappearance of BSP from Mouse Plasma: Normal
Control Group Subjects.

119

Only one time spot was identified in the testing of these animals.
Thirty minutes (30 min.) was chosen as a good time to determine the

BSP levels. These data are shown in Table 33 together with the control
group values. The means are compared with a Student's t-test and found
to be statistically identical. The mean for the treated animals was 3.1
whereas the control group was 3.0. A calculated t-value of p = 0.054
was much less than the theoretical value of 2.365, acceptable for
significant difference. The raw absorptance (O.D., optical density)
data for these samples are shown on this table as well. From these
results it is apparent that the liver is not functionally impaired,

at least as determined by this procedure.

Discussion of Toxicological Results

 

Acute lethality data for DPSD provided a calculated LD-SO
(Lethal Dose-~50th percentile) of 2,599 mg./kg. when administered i.p.
to mice. The reported LD-50 from the Russian literature (Kelman et_al:,
1968) was 2,150 mg./kg. which is within the 95% confidence interval for
this value (see Table 24) obtained here.

Acute lethality (LD-SO) comparisons of DPSD with other anti—
convulsant compounds appears in Table 34. Of these four compounds
(diphenylsilanediol, phenobarbital, diphenylhydantoin, and diazepam),
DPSD is definitely the least toxic in having the greatest LD-50 on
single—dose administration. A further comparison of these four com-
pounds on anticonvulsant and neurotoxic parameters will be covered

below in the general discussion section.

 

120

Table 33

Plasma levels of BSP in Treated vs. Control Mice

Control Mice# Treated Mice#
(Untreated diet for 90 days) (6.4 mg. DPSD/gm. Food for 90 days)
0. D. Calculated O. D. Calculated
_____ mg./100 ml. __ Egg/100 ml.
0.16 4.0 0.054 0.63
0.11 2.4 0.12 2.7
0.12 2.7 0.16 4.0
0.82 1.4
1.44 6.7
0.14 3.3
Mean: 3.0* 3.1"
S.E.: . 0.49 0.87

”6Dose Retained: S . 1

Note

U1
N

: #Bloodplasma samples at 30 min. post dose.

*No significant difference (1'. a 0.054).

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122

Chronic toxicity comparisons between compounds are less easily
made but a simple summary appears in Table 85. First, however, a brief
summary of the observed toxicological responses to DPSD is required.

A re-examination of Table 28 provides a summary of the observed
gross and microscopic differences between the treated and control
groups that might be attributed to DPSD administration. Gross obser-
vations in treated animals compared with controls showed the body,
adrenal, and spleen weights were slightly lower. Some large and
hemorrhagic gall bladders were seen. On a microscopic level there
were enlarged lymphatics in the small intestine, liver congestion with
smooth endoplasmic reticulum proliferation, some mild kidney congestion,
and hematological changes. The blood changes included an increase in
the number of circulating eosinophils and reactive lymphocytes. The
liver congestion and the gall bladder enlargements were examined further
but there was no significant functional change utilizing BSP in a liver
function test. The hematological changes could be serious toxicological
effects and are discussed in some detail below.

Though eosinopenia is a common manifestation of drug toxicity,
the occurrence of eosinophilia is not (Zbinden, 1963; Finch, 1972).

The literature on eosinophilia shows that this state is a common
response to most parasitic and allergic conditions (Donohugh, 1966).
The physiological role of this blood cell is not well understood. It
displays chemotaxis to histamine and has antihistaminic properties in
its ability to render histamine inactive (Archer, 1963). These cells

display only a small fraction of their total numbers in the circulating

blood. The ratio of blood to tissue eosinophils has been estimated at

 

 

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123

Table 35

Specific Toxicological Responses to the Chronic Administration

Compound

Diphenylsilanediol

Phenobarbital

Diphenylhydantoin

Diazepam

of Anticonvulsant Compounds.

Observation

eosinophilia
reactive lymphocytes
swollen liver cells

gall bladder enlargements

megaloblastic anemia
liver cell changes

allergy and skin rash

megaloblastic anemia
encephalopathies
connective tissue
disorders

no specific animal
organ toxicities

rare idiosyncratic
effects in man

Reference

this investigation

Mannheimer et al., 1952
Herdson et al., 196M

McGeachy et al., 1953

Mannheimer et al., 1952

del Cerro et al., 1967

Hauck et al., 1972

Randall et al., 1960

Wilcox, 1962; Gaul, 1961

124

1:200 (Rytomaa, 1960). From these basic data one can speculate on a
variety of mechanisms for the observed eosinophilia associated with
DPSD administration. These include such possibilities as DPSD being ‘3
chemotactic for the eosinophils and drawing them from the tissue stores
into the blood or selectively displacing these cells from the intestine
or other tissues where they appear in large numbers (Vilpo 2:.Eixa 1970).
As the activation of complement components 5, 6, and 7 (Ward, 1969) is
also chemotactic to eosinophils, these reactions might be mediated by
an antigen-antibody reaction involving the DPSD. The possibility of
some immunological—type mechanism is supported by the observed increase
in reactive lymphocytes associated with DPSD administration. The pri—
mary role of lymphocytes is in humoral antibody formation and cellular
immunity (Schalm 33.2133 1975). The cytological changes seen in these
cells have been associated with an increase in the production of immuno—
globulins (Henry et a1}, 1972) which is considered the functional role
of this increase in reactive lymphocyte numbers.

The other observed organ and tissue changes may be significant
but appear to offer no serious threat when compared with the observed
toxicities of known anticonvulsant compounds (see Table 35). Additional
discussion of these comparisons appears in the GENERAL DISCUSSION sec—
tion below. The observations summarized in this table were selected to
represent what Zbinden (1963) calls "specific toxicity." These effects
appear to be a direct result of a compound's activity and_notlan
indirect effect through, for example, a reduction in food or water

consumption or behavioral activity level.

ANTIEPILEPTIC STUDIES IN SEIZURE DISORDER ANIMALS

Introduction

 

The human epilepsies have been the source of much research
and speculation from early times to the present (O'Leary and Goldring,
1976). In this section, the evaluation of DPSD as an antiepileptic
agent is attempted, not in man, but in animals that exhibit spontaneous
epileptiform-type seizures.

It is relatively easy to produce seizures in animals by
chemical or electrical means. The section on anticonvulsant charac-
terization above discusses some of these methods. In these situations
one is forced to justify the experimental results by saying that the
induced seizure has certain prOperties that make it appear to mimic
an epileptic seizure state.

The existence of seizure phenomena in animals that is of a
spontaneous nature, or triggered by phenomena that are relatively
innocuous to non-epileptic animals, provides another opportunity
to evaluate chemicals and procedures as antiepileptic agents.

A search of the scientific literature revealed only a few
animals that have seizure states of an epileptic nature and that bear
resemblance to the human maladies in being idiopathic and reasonably
well-documented. Table 36 summarizes the seizure phenomena observed

in several animal species. Three of these occur in rodents (the rat,

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127

rnouse, and gerbil). The rodent seizures are perhaps the best
<1ocumented. The baboon and dog, as advanced mammals, have been
rnoderately well investigated. The newest model to be examined is

'the chicken (Johnson 33.31:, 1974), which is of course a non-mammalian
species .

Audiogenic seizures in the mouse (Hall, 1997) and the rat
(Maier and Glaser, 1940) have been used for the purposes of anticon-
‘Vulsant drug characterization (Collins, 1972). The necessity for high
intensity sound stimulation to evoke the seizures make them unlike
seizures in man (Brazier, 1973). The patterning of the seizure (run—
:ning followed by tonic—clonic seizures and then a major tonus followed
13y death) further make this animal model unlike the human disease.

The gerbil seizures are a more recent observation (Kaplan and
Ediezejeski, 1972) and have received little attention in anticonvulsant
cirug evaluation (Loskota 23.21:, 1979). The seizure in these animals
<Dccurs spontaneously but can be triggered by stress and photic stimu—
Jiation making these seizures similar to those in man (Harris and
Dfiawdsley, 1974). The epileptic gerbil was one of the models chosen
txo be used for further anticonvulsant and antiepileptic character—
ization of DPSD .

The photomyoclonic baboon has received much attention as a
Tmodel for human epilepsy due principally to the fact that this seizure
(Killam 33 al., 1966) can be evoked by intermittent light stimulation.
Though human seizures can often be evoked by photic stimulation

(Brazier, 1973), myoclonic seizures are a rare phenomenon in human

epilepsies (Schmidt and Wilder, 1968).

 

 

128

Canine epileptic animals are a common neurological observation
to the veterinary practitioner (Hoerlein, 1965) and are managed by the
sanmaanticonvulsant medications used for humans (Siegmund gt_al,, 1967).
'Fhe dog shows a wide range of seizure types (Cunningham, 1971) and
aappears only rarely to require some external stimulation to evoke a
seizure. This, then, was an additional model chosen for use in the

evaluation of DPSD.

 

Epileptic Gerbil Model

The mongolian gerbil, Meriones unguiculatus, as found in

 

laboratories in the United States, displays convulsive seizure
phenomena in 2% to 20% of the adult population (Kaplan and Miezejeski,
1972). The seizure displayed is idiopathic and of a grand mal (major
motor) type displaying generalized symmetrical electroencephalographic
signs (Loskota g£_al,, 1972). A feature that makes these seizures both
interesting and convenient for experimental study is that they can be
evoked in susceptible animals by stressing them. Given the highly
territorial nature of these rodents, an adequate stress is placement
into a new environment (Arrington 35.31:, 1973; Loskota et_al,, 197M).
Typical animals can be induced to have seizures every two to four days.
Loskota and Lomax (1979) have shown that these seizures are controlled
by treatment with antiepileptic drugs. As an epilepsy model, this

seizure type was used to examine the effects of DPSD.

129

Methods

Gerbils were obtained from various laboratories on the M.S.U.
(uampus, principally physiology and zoology, and screened over a four-
vneek period for seizure susceptibility. Testing was done by placing
'the gerbil in a large rat cage lined with paper towels. Seizures were
«elicited in susceptible animals within 30 minutes. All animals were
tested daily. The animals showing seizures were marked for identi-
fication and their seizures recorded.

Table 37 lists the seizure histories for gerbils selected as
having seizure susceptibility. Gerbils were selected for this exper-
iment as those with short seizure latencies and seizure rates of at
least one every three days. Twenty-four animals were randomly assigned
to one of four treatment groups of four animals each. Group I received
2 ml./kg. body weight of 0.5% methyl cellulose solution; Group II,

50 mg./kg. of a methyl cellulose suspension of diphenylhydantoin;
Group III, 20 mg./kg. of phenobarbital in methyl cellulose; and
Groups IV, V, and VI received DPSD at 20, 50, and 100 mg./kg. as
methyl cellulose suspensions.

Each animal received daily injections of the drug and was
observed for seizures for two hours (one hour before and one hour
after dosing). The last hour was accompanied by the animal being
placed in a rat box as noted above. The seizures of each animal

over a four-day period were noted and recorded.

130

Table 37
Seizure Characteristics for Gerbils in Colony
Summary from Four week Observation Period

 

 

 

 

Longest
Total Number Number of Shortest Time ' Time Between
of Seizures Gerbils* Between Seizures Seizures
0 36 28 days 28 days
1 10 28 days 28 days
2 16 1 day 22 days
3 9 2 days 15 days
4 9 1 day 17 days
5 6 1 day 12 days
6 7 1 day 8 days
7 8 1 day 6 days
8 6 1 day 6 days
9 9 1 day 5 days
10 5 1 day 4 days
11 1 2 days 5 days
12 2 1 day 5 days
13 l 2 days 5 days
14 3 1 day 4 days
15 3 1 day 3 days
16 l 1 day 4 days
17 5 1 day 3 days
18 6 1 day 3 days
19 8 1 day 3 days
20 2 1 day 3 days

Notes: *Number of animals having the seizure totals
listed in Column 1 (Total "epileptic"
gerbils b 117).

131

Results

Table 38 summarizes the incidence of seizure for the epileptic
gerbils in each of the treatment groups. At the doses tested, all
«compounds reduced seizure frequency over the four-day test period.
Each animal had had a seizure on day zero. And each would have been
expected to have another seizure before day four. The protection by
diphenylhydantoin and phenobarbital corresponds with the data obtained
by Loskota and Lomax (1974) on their inbred strain of epileptic gerbils
(WJL/UC). The DPSD data suggests and antiepileptic activity for this
compound. As apparent ED-SO was calculated to be 32 mg./kg. from these

data using Finney's probit method (Finney, 1952).

Epileptic Dog Model

 

The domestic dog, Canis familiaris, displays a wide variety

 

of neurological disorders (Hoerlein, 1965) many of which have direct
correspondence to human heurological disorders (Foster, 1973). Some

of the more common canine neurological diseases include seizure dis-
orders, intervertebral disc abnormalities, brain tumors, meningitis,

and hydrocephalus. Observations from the Veterinary Medical Teaching
Hospital of the University of California, Davis (Holliday 3: a1}, 1970),
revealed that epilepsy-like disorders in dogs comprise about 1% of the
total canine diseases diagnosed at that clinic. Furthermore, convulsive
seizure is the most commonly seen neurologic symptom for the dog

requiring veterinary medical treatment (Cunningham, 1971a).

 

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133

Dogs, like man, show symptomatic as well as idiopathic seizures
(Cunningham, 1971b; Hoerlein, 1965). The canine idiopathic seizure
shows genetically determined trends in offspring seizure frequency
(Cunningham, 1971a), as is known for the human disorder (Schmidt and
'Wilder, 1968). Dogs generally have one of three types of seizure
displays: generalized, partial, or psychomotor (Cunningham, 1971a;
Siegmun E£.§lr’ 1967). The most common generalize seizure is a major
motor (grand mal) type which has motor components, shows loss of con-
sciousness, autonomic signs, and complex behavior signs developing
immediately before or after a generalized seizure (Cunningham, 1971a).
Partial seizures tend to be asymmetric in appearance; lifting one paw,
facial twitching, etc., and frequently display a localizing sign sug-
gesting an intracranial lesion. Psychomotor seizures have infrequently
been carefully diagnosed (Hoerlein, 1965; Siegmund e: 313’ 1967) but
can be suspected in dogs displaying episodes of sudden and inappropriate
fright, disorientation, with barking and stereotyped motor patterns.

The diagnostic criterion for idiopathic generalized seizures
is usually one of exclusion of probable causes that would identify the
seizure as being symptomatic. In the methods section below, the role
of most of the diagnostic tests in the selection of dogs to receive
DPSD was to eliminate animals showing symptomatic seizures. This was
done principally to eliminate dogs from the study that might have
disease states showing poor prognosis (i.e., brain tumor) and to avoid

confounding the results.

 

 

134

Methods

The three dogs used in this study were clinical research
patients of the neurology teaching unit of the Michigan State University
Small Animal Veterinary Medicine Clinic. Each animal was recommended
for therapy after having failed to be controlled by conventional anti—
convulsant medication regimens (Hoerlein, 1965; Schmidt and Wilder,
1968). It was decided to begin treatment of each dog with DPSD doses
of 20 mg./kg. two times per day orally as powdered DPSD—filled gelatin
capsules. DPSD was used as supplied by Aldrich Chemical Company (Lot
102717) and pulverized in a mortar and pestle before being packed into
empty gelatin capsules (Eli Lilly and Company, Indianapolis, Indiana).
The dose and dosage sequence was estimated from the mouse data obtained
against maximal electroshock seizures (see Maximal Electroshock General-
ized Seizures, page 21, above). The plan for treatment was to increase
the dose if an animal had a seizure close in time after an individual
dose was given and to shorten the dosing period if an animal had a
seizure in the middle of a dosing period (12 hours between doses). The
initial dose of DPSD was to be added to the current medications, which
were then sequentially withdrawn by dividing the dose by two each day
and starting with the compound having the least potential for physical
dependence liability (see Tolerance and Physical Dependence Studies,
page 45, above) and progressing to that compound with the most potential
for physical dependence. Thus, as an example, with patient A.G., Table
39, on diphenylhydantoin, primidone, and phenobarbital, the diphenyl—

hydantoin would be withdrawn first, followed by the primidone, and

 

 

135

Table 39

Case Summary on Epileptic Dog A.G. Treated with DPSD

General and Diagnostic Summary:

Breed: German Shepherd, Age: 1.5 yrs., Sex: Male,
Wt.: 25 kg.
Primary Diagnosis: Idiopathic Epilepsy-six month
duration.
Control Period Medication: 500 mg. Diphenylhydantoin
tid.
250 mg. Primidone tid.
32.4 mg. Phenobarbital tid.
Control Period Observations: 6 seizures in 1 month
(l/wk.)
Control Period Laboratory Results:
EEG: Occasional spikes, bilaterally symmetrical.
Spinal Tap: 135 mm. CSF pressure, 6 mg. % protein.

Chemistries: BUN = 13 mg. %, SGPT =. 20 IU.,
Fast. Glu. = 94 mg. %, BSP é 1%
retention.

Hematology: WBC = 7100, Diff.: Neut. 67 (Seg. 60),
Lymph. 22, Mono. 7, Eos. 4, Baso. 0.
Hb. 15.5 gm. %, TP. 6.7 gm. %, PCV. 54%

Treatment Summaries on DPSD:
Week 1

Dose: 500 mg. bid. (20mg./kg.)
Seizure: none
Clinical Laboratory Results:
Chemistries: BUN= 18mg. %, SGPT = 62 IU.
Hematology: WBC = 15800, Diff.: Neut. 68
( Seg.67), Lymph. 24, Mono. 5,
Eos.3, Baso. 0. Hb. 15 gm. %.
TP. 6.7 gm %, PCV. 46 %.

Week 2

Dose: 500 mg. bid. ( 20mg./kg.)
Seizures: none
Clinical Laboratory Results:
Chemistries: BUN= 12mg. %, SGPT== 51 IU
Hematology: WBC = 9700, Diff.: Neut. 66 (Seg.66),
Lymph. 20, Mono. 9, Eos. 5, Baso. 0.
Hb. 15.5 gm. %, TP. 6.7 gm. %,
PCV. 45%

 

136

Table 39 (Continued)

Case Summary on Epileptic Dog A.G. Treated with DPSD

week 3

Dose: 250 mg. bid. (10 mg./kg.)

Seizures: none

Clinical Laboratory Results:

Chemistries:
Hematology:

Necropsy:

BUN =11 mg. %, SGPT = 34 IU.

WBC = 7400, Diff. : Neut. 67 (Seg. 67)
Lymph. 17, Mono. 10, Eos. 6, Baso. 9.
Hb. 17 gm. %, TP. 6.9 gm. %, PCV. 54%
Patient euthanized, no gross nor micro-
scopic evidence of drug toxicity.

137

finally the phenobarbital. If seizures were exhibited during this
withdrawal period, the plan was to stop the next scheduled reduction
and to increase the dose of DPSD before starting up the withdrawal
sequence again. The criterion for DPSD efficacy was to be a comparison
of seizure frequency before DPSD treatment with the seizure frequency
while the patient was on DPSD treatment. A reduction of seizure
incidence associated with the DPSD therapy would be indicative of
antiepileptic efficacy.

Patients were selected having a diagnosis of idiopathic
epilepsy based on history, physical and neurological examination,
clinical laboratory data, and baseline electroencephalogram results
(Hoerlein, 1965; Cunningham, 1971a). Patient A.G. was a male, German
Shepherd, age 1.5 years, with a seizure history of six months duration.
This animal was donated to the clinic to be euthanized if not needed
and was the first dog to be treated with DPSD. The diagnostic and
history data are summarized for this animal in Table 39. Physical
and neurological examination showed no focal deficit evidence nor
symptoms of other disease states which might display seizures as
part of a complex syndrome. This patient was started on 20 mg./kg.
DPSD twice a day with capsules given at 8:00 a.m. and 8:00 p.m.

Patient R.D. was a male, Great Dane, age 2.5 years, with a
seizure history of one year duration. This animal was being considered
for euthanasia by its owners when introduced into this test program.
Table 40 summarizes the diagnostic and history data for this animal.

Therapy was begun with 10 mg./kg. of DPSD twice a day at 8:00 a.m. and

 

 

138

Table 40
Case Summary on Epileptic Dog R.D. Treated with DPSD
General and Diagnostic Summary:

Breed: Great Dane, Age: 2.5 years, Sex: Male, Wt.: 60 kg
Primary Diagnosis: Idiopathic Epilepsy- 1 year duration.
Control Period Medication: 500 mg. Diphenylhydantion qid
750 mg. Primidone qid
10 mg. Diazepam qid
Control Period Observations: 15 seizures in 6 months
( 1/2 weeks)
Control Period Laboratory Results:

EEG: Occasional spikes, multiple spikes, symmetrical
Spinal Tap: 160 mm. CSF pressure, 5 mg. % Protein
ChemistrieszBUN 11 mg. %, SGPT 4 IU.,
Fast Glu. 107 mg. %, Alk. Phos. 100 IU..
BSP. 0% retention
Hematology: WBC 5400, Diff.: Neut. 76 (Seg. 70),
Lymph. 9, Mono. ll, Eos. 4, Baso. 0.
Hb. 17.5 gm. %, TP. 6.0 gm. %, PCV. 54%

Treatment Summaries on DSPD:

Two week Period 1

Dose: 600 mg. bid ( 10 mg. /kg.) + 750 mg. Primidone
bid . '
Seizures: one episode with status
Clinical Laboratory Results:
Chemistries: BUN 15 mg. %, SGPT 8 IU,
Alk. Phos. 86 IU., BSP 0% retention.
Hematology: WBC 8300, Diff.: Neut. 76 (Seg. 67),
Lymph.1l, Mono. 3, Eos. 10, Baso. 0.
Hb. 16.0 gm. %, TP 7.0, PCV 48%.

Two Week Period 2

Dose: 1200 mg. bid ( 20 mg./kg.) + 750 mg. Primidone
bid
Seizures: none
Clinical Laboratory Results:
EEG: Occasional spikes, spike wave complexes,
symmetrical.
Chemistries: BUN 11 mg. %, SGPT 8 IU.
Hematology: WBC 8000, Diff.: Neut. 75 ( seg. 65),
Lymph. 13, Mono. 0. Eos. 12, Baso.0
Hb. 16.3 gm. %, TP 5.9 gm. %,
PCVs 45%, RBC 4.6 M.

139

Table 40 (Continued)
Case Summary on Epileptic Dog R.D. Treated with DPSD

Two Week Period 3

 

Dose: 1200 mg. bid ( 20 mg./kg.) + 750 mg. Primidone
bid.
Seizures: One episode, 21 days since last.
Laboratory Results:
Chemistries: BUN 13 mg. %, SGPT 19 IU.
Alk. Phos. 66 IU, BSP. 0% retention.
Hematology: WBC 7000, Diff. : Neut. 66 ( Seg. 55),
Lymph.20, Mono.0, Eos. 14, Baso O.
Hb. 16.4 gm. %, TP 6.4 gm. %,
PCV. 48%, RBC 6.56M.

Two week Period 4

 

Dose: 2400 mg. bid ( 40mg./kg.) + 750 mg. Primidone
- bid
Seizures: none
Clinical Laboratory Results:
Chemistries: BUN 16 mg. %, SGPT 26 IU,
BSP 4 % retention
Hematology: WBC 10,500 Diff.: Neut. 62 (Seg. 61),
Lymph. l4, Mono.4, Eos. 20, Baso. 0.,
Hb.14.3 gm. %, TP 6.1 gm. %, PCV. 43%,
RBC 5.74M.

Two Week Period 5

 

Dose 6000 mg. bid ( 100 mg./kg.) + mg. Primidone bid
Seizures: One episode, 16 days since last.
Clinical Laboratory Results:
Chemistries: BUN 14 mg. %, SGPT na., BSP 3% retention
Hematology: WBC 6000, Diff. : Neut. 59 (Seg. 58),
Lymph. 18, Mono. 5, Eos. 17, Baso. l,
Hb. 15.0 gm. %, TP 4.6 gm. %, PCV 45%,
RBC 6.54M.

Two Week Period 6

 

Dose: 6000 mg. bid ( 100 mg./kg.) + 750 mg. Primidone
bid
Seizures: none
Clinical Laboratory Results:
Chemistries: BUN 24 mg. %, SGPT 10 IU.,
Alk. Phos. 970 IU.
Hematology: WBC 9400, Diff.: Neut. 64 ( Seg. 62),
Lymph 15, gmMono S, Eos. l6, Baso ).
H 17.2 m. %, TP 6. 2 gm. %, PCV 51%,
RBC 7. 08M.

 

 

140

Table 40 ( Continued)
Case Summary on Epileptic Dog R.D. Treated with DPSD

Two Week Period 7

 

Dose: 6000 mg. bid ( 100 mg./kg.) + 750 mg. Primidone
bid.
Seizure: none ~
Clinical Laboratory Results:
Chemistries: BUN na., SGPT 34 IU., Alk Phos. 1840 IU.,
BSP 0% retention.
Hematology: WBC 11400, Diff : Neut. 73 (Seg. 69),
Lymph.1l, Mono.2, Eos. 14, Baso. 0,
RBC 6.44M, Hb 16.7 gm. %, TP 6.0 gm. %,
PCV 50%.

Two Week Period 8

 

Dose: 4200 mg. bid ( 70 mg./kg.) + 750 mg. Primidone
bid
Seizures: One episode, 56 days since last.
Clinical Laboratory Results:
Chemistries: BUN 12 mg. %, SGPT 8 IU., Alk. Phos.
679 IU.
Hematology: WBC 11400, Diff.: Neut. 80 (Seg. 73),
Lymph. 10, Mono.2. Eos. 8, Baso. 0.
Hb. 17.1 gm. %, TP 5.5 gm %,
PCV 50 %, RBC 5.65M.
NecrOpsy: Patient euthanized, no gross or micro-
scopic evidence of drug toxicity. Brain
showed signs of anoxic damage.

141

8:00 p.m. The lower dose was based on the results seen with patient
A.C. (see Results and Discussion sections below).

Patient 8.8. was a female, Laborador Retriever, age 4.5 years,
with a seizure history of eight months duration. The diagnostic and
history data for this animal are summarized in Table 41. Note also
that this animal has a second diagnosis of hepatitis possibly of viral
origin. The decision to include this animal in the DPSD study was
prompted, in part, by the hepatitis diagnosis. An assumption was made,
that if DPSD showed any liver toxicity directly, it would be likely to
show up in an animal with an already impaired liver (Becker, 1974).
This patient was donated to the clinic for experimental purposes and
maintained as a research animal. Therapy was begun with 20 mg./kg.

DPSD twice a day at 8:00 a.m. and 8:00 p.m.

Results

Table 42 summarizes the treatment results for these three
canine patients. In each case the addition of diphenylsilanediol to
the therapy for these animals either eliminated seizures completely or
reduced the seizure frequency. The reduction in seizure frequency for
these three patients as a group was both statistically and clinically
significant. Each of these animals will be discussed with regard to
seizure data, observations relating to potential toxicity, and
incidental findings that could become important or could be of

little consequence.

142

Table 41
Case Summary on Epileptic Dog B.S. Treated with DPSD

General and Diagnostic Summary:
Breed: Labrador Retriever, Age: 4.5 years, Sex: Female
Wt.: 30 kg.
Primary Diagnosis: Idiopathic Epilepsy— 8 mOnth
duration.
Control Period Medication: 600 mg. Diphenylhydantion
tid
32.4 mg. Phenobarbital tid
5 mg. Diazepam tid
Control Period Observations: 11 seizures in 4 months.
(1 seizure / two weeks).
Control Period Laboratory Results:
EEG: Occasional Spikes, Symmetrical
Spinal Tap: 135 mm CSF pressure, 25 mg. % protein.
Chemistries: BUN 11 mg. %, SGPT 17 IU., Fast Glu.
96 mg. %, BSP 0% retention, NH3 54 gm.
%.
Hematology: WBC 7800, Diff.: Neut. 72 ( Seg. 68),
Lymph.20, Mono.l, Eos. 7, Baso.0.
Hb. 17.3 gm. %, TP 6.1 gm. %, PCV 48%.
Secondary Diagnosis: Hepatitis, Chronic, relapsing.
Laboratory Results on Secondary Diagnosis:
Chemistries: SGPT 148 IU., Alk. Phos. 158 IU., BSP
19% retention, T Bili. 0.4 mg. %,
NH3 117 g. %.
Hematology: WBC 20,000, Diff.: Neut. 84 ( seg. 71)
Lymph. 10, Mono 6, Eos. 0, Baso. 0.

Treatment Summaries on DPSD:

Month 1

Dose: 600 mg. bid ( 20 mg./kg.) + 32.4 mg. phenobarb.
ital
Seizure: None
Clinical Laboratory Results:
Chemistries: SGPT 66 IU., Alk. Phos. 949 IU.,
BSP. 6% retention.
Hematology: WBC 7600, Diff.: Neut. 77 ( seg. 74),
Lymph 20, Mono 1, Eos. 2, Baso. 0.
Hb. 17.1 gm. %, TP 7.0 gm. %, PCV 50%

 

 

143

Table 41 ( Continued)

Month 2

Dose: 600 mg. bid ( 20 mg./kg.) + 32.4 mg. phenobarb-
ital
Seizures: None
Clinical Laboratory Results: '
Chemistries: SGPT 140 IU. Alk. Phos. 1790 IU., BSP
0% retention. '
Hematology: WBC 9400, Diff.: Neut 75 ( Seg.73),
Lymph. l9, Mono.4, Eos. 2, Baso. 0.
Hb. 16.3 gm. %, TP 6.8 gm. %, PCV 46%.

Month 3

Dose: 600 mg. bid ( 20 mg./kg.) + 32.4 mg. phenobarb-
ital
Seizures: None
Clinical Laboratory Results:
Chemistries: SGPT 700 IU., Alk. Phos. 3070 IU., BSP
22.5% retention.
Hematology: WBC 8300, Diff.: Neut. 71 ( Seg. 66),
Lymph. 23, Mono. 3, Eos. 3, Baso.0.
Hb. 16.4 gm. %, TP 5.76 gm. %, PCV 47%

Month 4

Dose: 600 mg. bid ( 20 mg./kg.) + 32.4 mg. phenobarb-
ital
Seizures: None .
Clinical Laboratory Results:
Chemistries: SGPT 400 IU., Alk. Phos. 2620, BSP 17%
Hematology: WBC 6000, Diff.: Neut. 66 ( seg. 66),
Lymph. 31, Mono. 0, Eos. 3,.Baso. 0.
Hb. 16.1 gm. %, TP 5.8 gm. %, PCV 48%

Month 5

Dose: 600 mg. bid ( 20 mg./kg.) + 32.4 mg. phenobarb—
ital
Seizures: None
Clinical Laboratory Results:
Chemistries: SGPT 90 IU., Alk. Phos. 420 IU., BSP
17% retention.
Hematology: WBC 6000, Diff. : Neut. 73 ( Seg. 70),
Lymph.22, Mono. 2, Eos. 3, Baso. 0.
Hb. 15.9 gm. %, TP 6.3 gm. %, PCV 45%

 

 

 

141+

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145

Table 42 and Table 39 show the treatment results with DPSD
given to canine patient A.G. While being treated with 500 mg. of
diphenylhydantoin, 250 mg. of primidone, and 32.4 mg. of phenobarbital
three times per day, this animal had at least one seizure per week.
During the first week on DPSD, this animal had no seizures and the
other three drugs were completely withdrawn during the same period.
The second week showed no seizures either, so that the dose was reduced
to 10 mg./kg. for the third week. Still no seizures occurred. From
the previous history and the controlled observation period, this dog
was expected to have had at least three seizure episodes during the
same three week period. At the end of the third week the DPSD was
withdrawn and the animal had a seizure 26 hours after the last dose
and then began having multiple daily seizure episodes. The dog A.G.
was euthanized with an intravenous dose of pentobarbital and examined
at necropsy using both gross and microscopic techniques. The histo-
pathology exam revealed no abnormalities. Routine clinical laboratory
results obtained during the control and DPSD observation periods
revealed no large significant changes. Table 42 has a note indicating
a slight eosinophilia associated with the DPSD dosing period. The
slight rise in eosinophil (eos.) white blood cell count is noted in
the light of the marked eosinophilia noted in patient R.D. below.

Tables 40 and 42 summarize the results obtained with patient
R.D. This patient was being treated with 500 mg. of diphenylhydantoin,
750 mg. of primidone, and 10 mg. of diazepam four times per day. Even

though this patient was drowsy and mildly ataxic, he still managed to

146

have one seizure episode, usually status epilepticus, every two weeks.
By the end of the first two weeks of dosing with DPSD at 10 mg./kg.,
the diphenylhydantoin and the diazepam had been completely removed and
the primidone had been reduced to 750 mg. twice a day, but the dog had
a seizure episode. For the second two—week period the dose of DPSD was
increased to 20 mg./kg. and no seizures were seen. During the third
two-week period there was another seizure episode so the dose of DPSD
was increased to 40 mg./kg. At the beginning of two-week period five
the dog had another seizure episode, so the dose was increased to 100
mg./kg. of DPSD twice a day. On this dose there were no seizures during
the next two (6 and 7) two-week periods. This prompted a reduction of
the DPSD dose to 70 mg./kg. during two-week period eight. The dose
reduction was followed shortly by another episode of seizures which
occurred 35 days after the last seizure episode. The animal was
transferred to another experimental program and, as he was having
repeated episodes of status epilepticus during attempts to withdraw
him from the DPSD, was euthanized with an intravenous dose of pento—
barbital three days later. At necropsy most tissues and organs
appeared normal but the brain showed areas of damage in the cerebral
hemispheres that appeared to be similar to those seen after anoxic
episodes (Sandritter and Wartman, 1973). These areas of encephalo—
malacia were confined to cerebral grey matter and at the junctions of
white and grey matter. The clinical laboratory data associated with
this therapeutic program identified two areas for concern. The serum

enzyme alkaline phosphatase was found to be markedly elevated during

147

DPSD therapy. The blood—formed element, eosinophil, was found to
have increased in number. The resulting eosinophilia showed a maximum
of 20%, compared with 4% for the control period, during two-week period
number four. The serum alkaline phosphatase level increased from a
baseline level during the control period of 100 IU. to 1,840 IU. during
two—week period number seven. The implications of these two elevated
values is not clear but some hypothetical considerations are covered
in the discussion below.

Table 41 and 42 summarize the treatment results for patient
B.S. This dog is still under treatment and has not experienced a
seizure since beginning therapy with DPSD five months ago. Before
therapy with DPSD was begun this patient was being treated with 600
mg. diphenylhydantoin, 32.4 mg. phenobarbital, and 5 mg. diazepam
three times per day. On this treatment regimen the animal was having
a seizure episode every two weeks. ‘As with the previous patient, this
dog showed a marked elevation in serum alkaline phosphatase levels
during DPSD treatment. The maximum value was recorded during the
third month of treatment at a level of 3,070 IU. compared with 158 IU.
recorded earlier and which was elevated due to the associated hepa-
titis. There was no evidence of an eosinophilia seen here. All other
drugs except the phenobarbital had been withdrawn by the end of the
second week of DPSD therapy. The phenobarbital was left at 32.4 mg.
twice a day to provide enzymatic stimulation of the liver and some

mild sedation of the animal.

148

Discussion of the Antiepileptic Studies

 

It is apparent from these investigations with the epileptic
gerbil and dog that DPSD also controls seizures in epileptic animals.
The results with the dog, even though the number of animals is small,
provides some evidence that this compound may be albe to antagonize
convulsions which are not well—controlled by the conventional anti—
convulsant drugs. Some speculation on this possibility appears below
(GENERAL DISCUSSION).

The dog also showed some signs of eosinophilia, with a large
increase in one animal (R.D.), a small increase in another (A.G.), and
no increase in the third (B.S.). This serves as additional support for
the observed increase in these cells in the circulation in the mouse.
Refer to the Toxicological Studies section (pages 87ff.) of this report
for a discussion of the potential implications of this eosinophilia.
Two dogs showed a very large increase in serum alkaline phosphatase
levels (B.S. and R.D.). The third dog (A.G.) did not have this param—
eter determined as it was the first subject to be studied. With the
introduction of B.S. and R.D. to the study, an attempt was made to
monitor more clinical parameters due to the planned longer period of
administration.

This huge increase in alkaline phosphatase deserves some
discussion as to possible implications of its significance. Alkaline
phosphatase enzyme activity in the blood serum has three major sources:

bone, liver, and intestine (Fishman, 1974). In females there is also

149

an important contribution from the placenta. Normally the elevation
of alkaline phosphatase levels is interpreted as indicating liver
damage (Hoe, 1969). For the dogs discussed here, other parameters

of liver function (BSP excretion, SGPT levels, serum bilirubin levels,
and plasma protein concentration) appear normal. This tends to direct
attention to other sources for this enzyme elevation. Despite the
extreme values for alkaline phosphatase observed, there was no evidence
that any of the dogs experienced any debilitation that might be
expected to generate this enzyme from bone to intestinal damage.

Until a detailed examination of the possible isoenzymes for the origin
of this enzyme elevation is accomplished, its source will remain
unknown. It may evern be that the enzyme concentration has not been

increased but that its activity is somehow potentiated by DPSD.

 

GENERAL DISCUSSION

In this section, an attempt will be made to consider the
characterization program for DPSD. First, an overall summary of the
effects of DPSD on the test methods conducted in this investigation
will be presented to provide a convenient reference for comparison.
Secondly, discussions of this compound as an anticonvulsant will center
around speculations about mechanism of action and structure-activity
relations compared to conventional anticonvulsant compounds. Finally,

DPSD as a potential antiepileptic drug will be addressed.
Results of the Characterization Program

Table 43 summarizes each of the test procedures on which DPSD
was evaluated. Table 48 has five major divisions: Anticonvulsant
Testing, Behavioral Characterization, Toxicological Studies, Anti-
epileptic Studies, and General Pharmacology. The general pharmacology
of DPSD was not systematically pursued in these studies and only rep—
resents findings incidental.tx>-the other four major investigational
areas. Table 43 lists the test nmmhod used, the species of the animal
used, What measurement was made, and the dose level at which the
observations were made. A single dose indicates an LID—.50 value
calculated from the data. A range of values indicates those doses

WhiCh were actually tested. IDoses that were administered in the food

150

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152

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154

are shown together with a calculated estimate of the total daily

dose received by an animal or group of animals.

Some Considerations of DPSD as
an Anticonvulsant Compound

 

 

With the demonstration by Putnam and Merritt (1937) that
diphenylhydantoin elevated the electroconvulsive shock threshold to
produce tonic seizures in cats, the potential for developing new anti~
convulsive drugs in the laboratory became apparent. Supported by their
clinical data, Merritt and Putnam (1938) demonstrated the effectiveness
of diphenylhydantoin in man. In a review paper eight years later
(Merritt and Putnam, 1945), the screening of over 1,000 compounds
resulted in approximately 75 that showed good seizure protection. The
majority of these compounds contained phenyl groups or were otherwise
aromatic. This tended to support an earlier (1937) postulate that
phenyl-containing compounds appeared to have the best potential as
anticonvulsant drugs. Toman and Goodman (1948), after a consideration
of structure-activity relationships for clinically effective anticon-
vulsant drugs, concluded that phenyl—substitution was essential for
drugs used to treat grand mal seizures or psychomotor displays, but
was either non-essential or detrimental to drugs used in the treatment
of petit mal seizure types. This position has tended to be supported
throughout many reviews of anticonvulsant structure-activity data
(Close and Spielman, 1961; Spinks and Waring, 1963; Delgado and
Isaacson, 1970; Mercier, 1973). Some of these phenyl-containing

compounds are shown in Figure 34 together with DPSD for comparison

155

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156

purposes. If one attempts to make direct structural comparisons
between DPSD and these other compounds, the phenyl groups become
obvious. On the other hand, what might be said about the remaining
structures? Most of the organic anticonvulsants have the common

feature of 0 f{
H l
O C-C-N-C

and if one considers keto-enol tautomerism, an alternate version of

this common feature becomes

O-H
I
O c-c=N-c.

With the success of diphenylhydantoin therapy, there has been some
renewed interest in other diphenyl-containing compounds. Diphenyl—
barbituric acid was originally introduced as a sedative-hypnotic and
dismissed because of its low potency (McElvain, 1935). A current
examination of this compound as an anticonvulsant (Raines, 1973;'
Raines et_§l,, 1973) shows it to be potentially a broad spectrum
anticonvulsant compound having a better safety margin, in terms of
therapeutic indices, than phenobarbital. Many years ago Berger (1951)
showed that the propanediol, 2,2-diphenyl—l,3npropanediol, antagonized
electroshock seizures in mice. This compound was ignored due to its
short duration of action. Attempts to prolong the action of the
propanediols ultimately produced the carbamate esters of these
compounds. Of these, meprobamate became the major one of interest
(Berger, 1952). Figure 35 shows a collection of anticonvulsant com—

pounds compared with DPSD. The classical keto forms are redrawn as

157

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158

enol forms showing a direct comparison with DPSD. The larger size
of the silicon atom appears to place the phenyl and alcohol groups
in a position making it similar to each of these compounds.

As is currently true for nearly all central nervous system
acting drugs, the mechanism of action for any anticonvulsant drug has
not been well defined. Most attempts to deal with mechanisms for these
compounds have to contend with a wealth of conflicting electrophysiolog—
ical and biochemical studies. Woodbury (1969) suggested that there may
be three general types of anticonvulsant effects. These include (1)
effects of non-neural systems to prevent changes which may precipitate
or facilitate seizure activity, (2) effects confined to the patholog-
ically altered neurons of the seizure focus to prevent their seizure
discharge, and (3) effects on normal neurons to prevent their excessive
discharge by the seizure focus. Anticonvulsant drugs probably have
their most significant action attributable to the latter general
mechanism. There are probably some mechanistic subcategories based
on how the various anticonvulsant drugs accomplish this suppression
of excessive discharge in normal neurons by so-called "epileptic
neurons." Such subcategories can includea general depression of
neural activity through neurotransmitter or electrogenic means or
a facilitation of physiologically active inhibitory mechanisms.
Circumstantial evidence obtained from the results of various anti—
convulsant test methods permits inferences about possible mechanisms

of action for each drug tested.

159

Investigations into the mechanisms of action of these drugs
is coupled with investigation into the mechanisms of the seizures
themselves. Phenobarbital has been investigated most extensively
though diphenylhydantoin appears to have been best defined in terms
of its action. Investigations on diazepam have tended to center around
its behavioral effects and not its anticonvulsant properties.

Early studies on phenobarbital implicated it as an inhibitor
of brain respiration (Jowett and Quastel, 1937). Brody and Bain (1951)
were able to show that the barbiturates uncouple oxidative phosphoryla—
tion lending support to the effects of these compounds on brain metab-
olism. Nachmanson and Wilson (1951) suggested that the barbiturates
inhibit acetylcholine synthesis and produce its seizure-inhibiting
effects through this mechanism. The net effect of the barbiturates
in general, and phenobarbital in particular, is the stabilization of
the neuron in reference to the generation of transmitted impulses
(Seeman, 1972). This property is evidenced in the effectiveness of
phenobarbital in antagonizing seizures generated by a variety of means
(see discussion in anticonvulsant section above). The observation that
levels of brain serotonin in animals are increased by 50% or more after
treatment with phenobarbital (Bonnycastle et_al:, 1957) adds another
inference that altered transmitter functions are involved in the mech—
anism of action of phenobarbital. In support of this idea, the pre-
treatment of animals with iproniazid has been found to increase the
anticonvulsant activity of phenobarbital (Yen 23.513’ 1960).

Unlike phenobarbital, diphenylhydantoin does not have a wide

spectrum of activity in anticonvulsant test methods. Esplin (1957)

160

was able to demonstrate that diphenylhydantoin inhibited post-tetanic
potentiation in the spinal cord of cats.

More refined mechanistic studies for these actions of
diphenylhydantoin (Woodbury, 1969) have shown a decrease in the
concentration of intracellular sodium and a lowered ratio of intra-
cellular to extracellular sodium in brain neurons even after maximal
electroshock seizures.

Diazepam shows the same wide spectrum of anticonvulsant
activity as does phenobarbital but electrophysiological studies show
a number of regional differences for these two compounds. Though both
inhibit after—discharges in the neocortex (Schallek 33.21:, 1969), the
benzodiazepines appear to actually lower the threshold for after-
discharges evoked from the amygdala. Effects on the electrocorticogram
are also different for these two compounds. Phenobarbital tends to
produce a synchronization effect or lowering of the observed fre—
quencies, while diazepam produces an "arousal" (desynchronization)
effect with a shift to higher frequencies.

In terms of its anticonvulsant action, DPSD appears to share
similarities with each of these three compounds.

Some Considerations of DPSD as
an Antiepileptic Drug

Looking once again at Toman's definition of an ideal anti-
epileptic drug (Toman, 1970) as summarized by Table 2, we can re-
examine DPSD in the light of these criteria: (1) suppresses all

seizure types, (2) is effective orally, (3) is long acting, (4) shows

161

no tolerance to its action, (5) shows no withdrawal effects, (6) has
no sedation or CNS toxic action, (7) shows no systemic toxicity,

(8) shows no idiosyncratic toxicity, (9) has a wide margin of safety,
and (10) is inexpensive to buy.

Looking at these in reverse order, consider first the cost
of DPSD. We can estimate from the dog data a therapeutic dose of
20 mg./kg./twice a day or 6.8 grams per day for an average man (weight
of 70 kg.). Based on the Aldrich Chemical Company price of $10.00 per
100 grams, this would cost the patient 68 cents per day or about $248
per year.

Table 44 illustrates margin of safety considerations where
therapeutic indices are compared for DPSD, diazepam, phenobarbital,
and diphenylhydantoin. Three indices are calculated based on the
maximal electroshock antagonism ED—SO, the rotating rod depression
TD—SO and the lethal dose LD-SO. When the neurotoxic dose is compared
with the anticonvulsant dose, both DPSD and diphenylhydantoin appear
similar and are safer than either diazepam or phenobarbital. Comparing
the neurotoxic dose with the lethal dose (LD-SO/TD-SO), one sees an
improved safety margin of DPSD over diphenylhydantoin. The seemingly
large safety margin for diazepam is counter balanced by the poor
separation of anticonvulsant from neurotoxic doses for this compound.

Idiosyncratic toxicity probably cannot be evaluated except in
an extensive clinical trial.

Systemic toxicity for DPSD is not well—defined at this point
but the observations made with the rat and the dog appear to require

some cautious additional study.

162

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163

The sedation and CNS toxicity issues were already covered
in brief discussion of margin of safety aspects. It appears that
DPSD is at least as free of neurotoxicity as diphenylhydantoin which
appears to be the standard anticonvulsant in this regard.

The lack of withdrawal effects of any significant magnitude
with DPSD is perhaps one of the more interesting observations made on
this compound. .

Tolerance development appears to be mild and, over the longest
time tested (90 days), appears to be insignificant.

An estimated elimination half-time of four hours is not really
impressive, but due to the large therapeutic indices for this compound
it is possible to elevate the dose to obtain whatever dosing regimen is
required. Experience with the dog suggests that a twice a day dosing
regimen is adequate. This animal normally requires the administration
of diphenylhydantoin three or four times per day for effective treatment
due to its rapid excretion in this animal.

Both the mouse and dog data attest to DPSD being effective
orally, and well tolerated by this route.

The therapeutic indication for DPSD at this point is for the
major motor seizures, i.e., grand mal and psychomotor types. The
possibility for adjunctive use in minor seizures (petit mal or absence
types) should be considered based on the wide spectrum of action for
this compound.

An overall comparison of DPSD with diphenylhydantoin,

phenobarbital and diazepam is given in Table 45.

 

 

 

 

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RECOMMENDATIONS

In this section an attempt will be made to discuss casual
observations about the activity of DPSD that are not well substantiated.
In addition, some suggestions will be made as to where future investi-
gations on this organosilicon compound should be directed.

The most pressing problems are an explanation of the observed
toxicological responses to DPSD. The hematological changes of reactive
lymphocytes and eosinophilia suggest some immunological effects for this
compound. A systematic examination of DPSD on immune mechanisms should
be undertaken. Note that silicosis can be considered an immune complex
disease and DPSD has both the silicon and the silanols common to silica.
An exploration of the increases in alkaline phosphatase is also required.
An initial approach could be the identification of the isoenzyme frac—
tion which is selectively increased for a hint as to which tissue is
involved here.

There is an additional observation concerning the toxicity of
DPSD that deserves mention, though it is not well documented. This
compound may have some centrally—acting emetic properties. Three dogs
dosed orally with this compound exhibited emesis an hour or so after
initial dosing. An additional dog that was dosed with DPSD intrave-
nously vomited shortly after the dose was administered. If this is an
important property it appears to tolerate out relatively rapidly.

However, emesis may become a problem with human administration.

165

166

The structure~activity comparisons of DPSD with other
anticonvulsant drugs suggests that a direct comparison of this compound
with diphenylbarbituric acid and with 2,2-diphenyl—l,3-propanediol is
in order.

Based on the strong anti-anxiety effect observed for DPSD, the
behavioral properties of this compound could become more therapeutically
useful than its broad anticonvulsant activity spectrum of action. This
pharmacological activity has been much examined for the benzodiazepines
(Garattini and Shore, 1967; Zbinden and Randall, 1967) for which the
role of endogenous inhibitory mechanisms seems to be of paramount
importance. It may be that the anticonvulsant as well as the anti-
anxiety effects of DPSD could ultimately be attributable to the effects
of this compound on normal inhibitory mechanisms.

Based on the general observation that most anticonvulsant
compounds are also antiarrhythmic agents (Morgan and Mathison, 1976),
it would be beneficial to examine the antiarrhythmic activity for DPSD.
Of course, the general cardiovascular pharmacology for this compound
needs examination in depth to further characterize this compound.

Further characterization of other areas of pharmacology is
definitely required. Pharmacokinetics and metabolism studies are
needed to unravel some of the implications of the tendency of DPSD
to polymerize. There is always the possibility that the polymeric
forms of this compound are responsible for its spectrum of action or

account for aspects of its toxicity.

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