Drug and Chemical Toxicology Downloaded from informahealthcare.com by McMaster University on 01/12/15 For personal use only.
DRUG AND CHEMICAL TOXICOLOGY, 1 4 ( 3 ) , 2 6 5 - 2 8 1 (1991)
PHYSOSTIGMINE (ALONE AND TOGETHER WITH ADJUNCT) PRETREATMENT AGAINST SOMAN, SARIN , TABUN AND vx INTOXICATION^ Larrel W. Harris, Brian G. Talbot, Willard J. Lennox, Dana R. Anderson and Richard P. Solana' US Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD 21010-5425
ABSTRACT A pretreatment for organophosphorus (OP) anticholinesterase (e.g., soman) intoxication should prevent lethality and convulsions (CNV) at 2 LD,,s and be behavioral-decrement-free when given alone. Behavioral-deficit-freepretreatment regimens '(PRGs) for guinea pigs consisted of Physostigmine (0.15 mg/kg, im) and adjunct. Adjuncts (mg/kg, im] tested were akineton [0.25], aprophen [ 8 ] , trihexyphenidyl [2], atropine [16], azaprophen [5], benactyzine [1.25], cogentin [4], dextromethorphan [7.5], ethopropazine [12], kemadrin [l], memantine (51, promethazine [ 5 ] , scopolamine [ 0 . 0 8 ] and vontrol [2]. PRGs were given 30 min before soman (60 ug/kg, sc; 2 LD,,s) or other OP agents. Animals were then observed and graded f o r signs of intoxication, including CNV at 7 time points and at 24 hr. Physostigmine alone reduced the incidence of CNV and lethality induced by 2 LD5,s of soman by 42 and 6 0 % , respectively. All of the PRGs tested abolished lethality and 12 shortened recovery time to 2 hr or less. Also, PRGs including azaprophen or atropine prevented CNV. When selected PRGs were tested against intoxication by sarin, tabun or VX, the efficacy was generally superior to that for soman. The data show that several PRGs are effective against soman intoxication in guinea pigs. 'In conducting the research described in this report, the investigators adhered to the "Guide for the Care and Use of Laboratory Animals," NIH Publication No. 85-23, revised 1985. 2The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Army or the Department of Defense. Part of this work was presented in October 1 9 8 8 at the ASPET meeting, Montreal, Canada.
265 Copyright 0 1991 by Marcel Dekker, Inc.
2 66
HARRIS ET AL.
INTRODUCTION
The
enzyme
acetylcholinesterase
(AChE)
is
the
primary
Drug and Chemical Toxicology Downloaded from informahealthcare.com by McMaster University on 01/12/15 For personal use only.
toxicological target for carbamates and the organophosphorus (OP) anticholinesterase agents, such as sarin, soman, tabun and VX. Exposure of animals to these OPs causes a buildup of acetylcholine (ACh) in the brain'!'.
The signs accompanying OP intoxication--
lacrimation, salivation, gastrointestinal disturbance, muscular twitching and weakness, prostration, convulsions and death through anoxia--maybe explained as resulting from overstimulation of the cholinergic system.
Successful treatment of OP intoxication
involves the use o f atropine plus an oxime (e.g. , 2-PAM). However, in the case of soman, conventional atropine plus 2-PAM therapy is only slightly effective3, and the lack of efficacy of this therapy is, in part, thought to be due to rapid aging o f the phosphonylated AChE and poor reactivation of the unaged enzyme4. Because carbamates and O P s occupy the esteratic site on the AChE, carbamates have been used successfully as pretreatment against
OPs.
The
carbamylated
phosphonylation of the AChE by OPs.
state
of
AChE
prevents
Spontaneous reactivation of
the carbamylated fraction of enzyme is then thought to provide a sufficient amount of AChE activity for normal function at some time after the OP has been bound and/or hydrolyzed.
In 1946, Koster5
was the first to use the carbamate physostigmine (Phy) to protect against intoxication by DFP. Since then, Phy in conjunction with atropine has been successfully used to antagonize soman intoxication3r6'*.
In 1978, Harris et a1'.
reported the
267
PHYSOSTIGMINE PRETREATMENT effectiveness of
a
pretreatment regimen containing
Phy
in
antagonizing soman-induced elevations-ofbrain ACh. In subsequent
Drug and Chemical Toxicology Downloaded from informahealthcare.com by McMaster University on 01/12/15 For personal use only.
studies it was shown that animals protected with a pretreatment regimen including Phy, but not pyridostigmine (a quaternary carbamate that does not readily penetrate the CNS'), overcame OP-induced physical debilitationlOvll.
rapidly
The positive
findings with Phy up to the current work suggested that a comprehensive study be conducted to identify adjuncts with adequate anticholinergic properties to antagonize potential side effects of Phy,
so
that the combined pretreatment regimen would be behavioral-
deficit-free (BDF)
in
unexposed
subjects.
Moreover,
such
pretreatment should prevent lethality and convulsions, and rapidly overcome the physical incapacitation resulting from a moderate challenge
(2 LD,,s)
of
OP
agent.
Herein, we
report
the
effectiveness o f novel pretreatment regimens on soman-,sarin-,VXand
tabun-induced
convulsive/subconvulsive
lethality, activity
convulsions
(apparently
and
incapacitating,
nonlethal intoxication). MATERIALS AND METHODS Male and female guinea pigs ( 3 5 0 - 4 0 0 g) were obtained from Charles River Laboratories and were used in all efficacy studies. Animals were provided
food and water ad lib, except during
experimentation (handling and drug administration). Physostigmine salicylate and all adjuncts were obtained from the Walter Reed Army Institute of Research. Physostigmine and adjuncts
2 68
HARRIS ET AL.
were each prepared in twice-distilled water just before use.
The
dosage o f Phy expected to provide 7 0 % inhibition of whole blood
Drug and Chemical Toxicology Downloaded from informahealthcare.com by McMaster University on 01/12/15 For personal use only.
AChE (I7o) at 30 min after intramuscu.lar (im) injection3 was used
in the present experiments.
A rat shuttle box test, modified for
guinea pigs,” was used to find the maximum BDF dose (in a series at 0.3 log intervals) of each adjunct to use in combination with the I,, of Phy. assigned
to
In efficacy studies, guinea pigs were randomly
individual
cages.
The
pretreatment
regimens,
consisting of Phy and the predetermined BDF dosage of adjunct, were concurrently administered im, in separate hind legs, 30 min before subcutaneous (sc) challenge with OP agent.
The im route for drug
administration was chosen because of the extensive data available for
Phy
and
atropine
sulfate
by
that
route.
Drugs
were
administered in a volume equivalent to 0.5 ml/kg of body weight and OP agent in a volume equivalent to 1.0 ml/kg. Each guinea pig was monitored by two observers. For the first
15 rnin after agent challenge, animals were continuously observed for convulsions, and then they were graded for selected, visible signs of intoxication at 0.25, 0.5, 1 , 1.5, 2 , 3 and 24 hr. A l s o , 24-hr mortality was recorded; if death occurred during the first three hours, the death time was noted to the nearest observation time.
In the same manner, a recovery time was recorded for each
survivor. Signs of intoxication were graded from 0 to 5 (0 = none of the selected signs present, 1
=
hyperactivity, 2
=
chewing and/or
269
PHYSOSTIGMINE PRETREATMENT salivation, 3
=
tremors and/or fasciculations, 4
and/or subconvulsive movements and 5
=
=
prostration
convulsions).
For each
Drug and Chemical Toxicology Downloaded from informahealthcare.com by McMaster University on 01/12/15 For personal use only.
observer, the scores for a given animal were summed across the observation times to obtain a severity score; the two such scores were averaged to obtain a final value for each survivor in a treatment group. For summary and analysis of results for each treatment group, mortality was expressed as a lethal fraction, or frequency symbol in the attached tables).
(#
In the initial tally of the
incidence o f convulsions, if one of the two observers indicated the presence of convulsions in a given animal, convulsions were considered present.
The incidence of convulsions was expressed
and analyzed as the fraction of survivors that convulsed; for tabular presentation, the incidence was converted to percent (of survivors) that convulsed
(%
C; see tables).
Each pair of death
times or recovery times for a given animal were averaged when the two values were each in the first three hours; otherwise, the shorter death time or the longer recovery time was taken as final. Simple median values (Md) and the observed extreme values were used to summarize group data for death time, severity score and recovery time. Differences between observed proportions (frequencies) were tested via 2 x 2 contingency table analysis.
sizes through N
=
For total sample
4 0 , differences were assessed using accurate
probability estimates for the Hypergeometric Distribution, as
2 70
HARRIS ET A L .
provided in tabular form by Natrella g
d 1 3 .
The Chi-square
approximation, with correction for continuity, was used for total
Drug and Chemical Toxicology Downloaded from informahealthcare.com by McMaster University on 01/12/15 For personal use only.
sample sizes greater than forty13. A l l comparisons of median values were done by estimating 90% confidence limits for each popi.lation median using the method as given by Tate and Clelland14; the upper or lower limit, as appropriate, was then utilized in a one-sided significance test.
Differences associated with probabilities of
0.05 and less were considered significant. RESULTS The data in Table 1 show the effects o f Phy alone and together with various adjuncts on mortality, percent o f
survivors that
convulsed, severity score and recovery time. Physostigmine alone protected approximately 60% of subjects from lethality and 42% from convulsions resulting from a 2 LD,, exposure to soman, sc. (In order for the pretreatment regimen to be significantly different (p < 0.05) when compared to Phy alone, no deaths or convulsions must occur, recovery times must be < 2-hr and the severity score must be < 14.) The data in the table also show that, when each of the adjuncts (mg/kg, im), atropine ( 1 6 ) , aprophen ( 8 ) , azaprophen ( 5 ) , benactyzine ( 1 . 2 5 ) ,
dextromethorphan ( 7 . 5 ) , ethopropazine
( 1 2 ) , or scopolamine ( 0 . 0 8 ) was paired with Phy in a pretreatment
regimen, complete protection against soman-induced lethality was obtained.
Also, median (Md) severity scores for the signs o f
intoxication were < 7, and Md recovery times were < 1.6 hours. Furthermore, a regimen including atropine or azaprophen prevented
271
PHYSOSTIGMINE PRETREATMENT TABLE 1
Drug and Chemical Toxicology Downloaded from informahealthcare.com by McMaster University on 01/12/15 For personal use only.
Effects of Physostigmine/Adjunct Pretreatment on Soman-Induced Lethality and Nonlethal Intoxication Adi unct1 (im route) (dosage)
#
C/Sub-C Effects' Recovery Time Mortalitv Time to % C Severity (hr) Death (hr) Score3 ( 0 - 4 0 ) Md(Extremes) Md(Extremes) Md(Extremes)
0/10
0
Azaprophen4 0/10 (5 m g / W
0
~copolamine~ O/IO ( 0 . 0 8 mg/kg)
Atropine4 (16 m g / W
0.0 (0.0; 8.5)
0.32 (0.25;2.0)
6.75 (0.0;18.0)
1.5 (0.5;3.0)
10
5.25 (0.O;ll.O)
1.0 (0.75;1.5)
Aprophen ( 8 mg/kg)
0/10
20
3.75 (1.0;12.5)
1.25 (0.5;1.5)
ETPZ5 (12 m g / W
0/10
20
6.0 (2.0;11.5)
1.0 (0.75;1.5)
6.0 (0.0;24.0)
(0.25;>24)
4.75 (0.0;11.5)
(0.5;1.75)
DXMP5 (7.5 m g / W
20
0/10
Benactyzine 0/10 (1.25 m g / W
30
None6
40/100
1.0 58 (0.25;24)
Adjunct and physostigmine (0.146 mg/kg, im) were given in opposite hind legs 30 min prior to soman (60 ug/kg; 2 x LD50, s c ) . C/Sub-C Effects = Convulsive/Subconvulsive Effects. Severity score for each 24-hr survivor: Mean (for the pair of observers monitoring the animal) of the sum of points for signs over 8 observation times after soman- 0.25,0.5,1, 1.5, 2 , 3 and 24 hr + 5 points if convulsions occurred (zero, otherwise) before 0.25 hr. Convulsions = 5 pts; prostration = 4 pts; tremors/or fasciculations = 3 pts; compulsive chewing/or salivation = 2 pts and increased locomotion = 1 pt. Maximum Severity Score = 40, or 8 observations times x 5 pts/observation time. p4