ropyAD

STEREOSPECIFICITY OF ANTIDOTES AND THEIR MECHANISM OF

ACTION IN INTOXICATIONS WITH ORGANOPHOSPHORUS

ANTICHOLINESTERASES(V)NANNUAL REPORT

B. HOLMSTEDT, B. KARLEN, I. NORDGREN and L. PALMERN

FEBRUARY 1990

Supported by

U.S. ARMY MEDICAL RESEARCH AND DEVELOPMENT COMMAND

Furt Detrick, Frederick, Maryland 21701-5012

Grant No. DAMD17-87-G-7007

Department of Toxicology, Karolinska Institutet,

Box 60400, S-104 01 Stockholm, Sweden

Approved for public release; distribution unlimited

The findings in this report are not to be construed as an official

Department of the Army position unless so designated by other

authorized documents.

DTICAnV1 n iCTE L-

SECURITY CLASSIFICATION OF THIS PAGEForm Approved

REPORT DOCUMENTATION PAGE OMBNo.d07040188

Ia. REPORT SECURITY CLASSIFICATION lb RESTRICTIVE MARKINGS

Unclassified

2a. SECURITY CLASSIFICATION AUTHORITY 3 DISTRIBUTION IAVAiLABILITY OF REPORT

Approved for public release;b. OE.LASSIFICA~TON /DOWNGRADING SCHEDULE distribution unlimited

4. PERFORMING ORGANIZATION REPORT NUMBER(S) S MONITORING ORGANIZATION REPORT NUMBER(S)

6a. NAME OF PERFORMING ORGANIZATION |6b. OFICE SYMBOL 7a. NAME OF MONITORING ORGANIZATION

Department of Toxicology (If applicable)

Karolinska Ins titutet

6c. ADDRESS (City, State, and Z!P Code) 7b ADDRESS (City, State, and ZIP Code)

Box 60400S-104 01 STOCKHOLMSweden

Ba. NAME OF FUNDING/SPONSORING 8b. OFFICE SYMBOL 9 PROCUREMENT INSTRUMENT IDENTIFICATION NUMBERORGANIZATION U.S. Army Medical (Ift applicable)

esearch and Development CommandDAD7-87-G-7007

8c. ADDRESS (City. State, and ZIP Code) 10 SOURCE OF FUNDING NUMBERSFort Detrick, Frederick, PROGRAM PROJECT TASK IWORK UNIT

Maryland 21701-5012 ELEMENT NO NO 3Ml- NO ACCESSION NO62787A 62787A875 BA 375

11. TITLE (Include Security Classification)

Stereospecificity of antidotes and their mechanism of action in intoxications with

organophosphorus anticholinesterases12. PERSONAL AUTHOR(S)

Holmstedt, Bo; Karl~n, Bo; Nordgren, Ingrid; and Palmer, Lena

13a. TYPE OF REPORT 13b. TIME COVERED 14 DATE OF REPORT (Year, Month'Oay) 15 PAGE COUNT

Annual IFROM 2/1/89 TOI/31/90 1 1990 February 7 27

16. SUPPLEMENTARY NOTATION

17. COSATI CODES 18 SUBJECT TERMS (Continue on reverse if necessary and identify by block number)

FIELD GROUP SUB-GROUP Acetylcholine, Choline, Diazepam, Hyoscyamine, HI-6,

06 15 Cholinesterase inhibitors, RA 5

06 11 I19. ABSTRACT (Continue on reverse if necessary and identify by block number)

-Our program during the last year has mainly been concerned with studies on*- the effect of diazepam on l-hyoscvamine binding in the old stock of sick and healthy

mice and new stock of healthy mice,2. the usability of I-hyoscyamine binding for studies of specific receptor distribution in

brain.3. effect of different combinations of antagonists (I-hyoscyamine, diazepam, HI-6) on

acetylcholine turnover.

4. duration of the effect'of diazepam on acetylcholine turnover.

There is a significant difference between the old and new stock of mice as far as the Iinfluence of diazepam on l-hyoscyamine binding is concerned. In order that optimal

parameters can be determined, therefore, the model needs further development in the

new stock of mice.20 DISTRIBUTION/AVAILABILITY OF ABSTRACT 21 ABSTRACT SECURITY CLASSIFICATION

0 UNCLASSIFIEDAJNLIMITEO 6a SAME AS RPT 0 DTIC USERS22s. NAME ?F RESPONSIBLE INtIVIDUAL 22b TjlkPONE (Include Area Code) c 22c. OFFICE SYMBOL

Mary Erances ostan 1 0uA- 663-7325 @ I SGRD-RMI-S

0O Form 1473, JUN 86 previous editions are obsolete. SEC)RITY CLASSIFICATION OP THIS PAGE

19. ABSTRACT

In testing the ability of the model to detect muscarinic receptors of specific subtypes,we have studied the kinetics of displacing the ligand l-hyoscyamine. So far we have per-formed experiments on the interaction of the unspecific ligand scopolamine with thisbinding. The displacement of l-hyoscyamine is time-dependent and proceeds until abouthalf the l-hyoscyamine pool has been displaced.

l-Hyoscyamine treatment results in an increase in the synthesis rate of acetylcholine(ACh). This is probably an unfavourable feature of a protecting agent. Diazepam, onthe other hand, lowers the turnover rate of ACh. The combination of diazepam and1-hyoscyamine decreases the turnover of ACh to the same extent as diazepam alone does.When l-hyoscyamine and diazepam are combined with HI-6, the dominant result is anincrease of choline levels, with a slight decrease of ACh turnover.

The duration of the effect of diazepam has been followed for 4 h. After 20, 30 and 45min, levels of acetylcholine are significantly increased, while they are returning tonormal after 4 h. The fractional rate constant seems to be somewhat lowered at 4 h. Thedata indicate a duration of effect on acetylcholine dynamics of at least 4 h.

LTIC T. _Ura ru ced

3ust feat :

By-A t4r lbt.u/ . ...

. . . ~. . . . . . I

,J _"' " 2 , c

SUMMNARY

During the past year we have studied 1) I-hyoscyamine binding in an old

and a new stock of mice; 2) conditions for displacement of 1-hyoscyamine

in vivo by scopolamine and size of the muscarinic receptor pool after

chronic treatment with scopolamine; 3) effect of the antidotes I-hyoscy-

amine, diazepam and HI-6 on acetylcholine turnover separately and in

combination; and 4) duration of the effect of diazepam on acetylcholine

turnover.

1) In 1988 the stock of mice used in our studies contracted mouse hepa-

titis and our models had to be re-established in a new stock of animals.

l-Hyoscyarnine binding in vivo in mouse brain had been shown to be

decreased by about 40% with diazepam. We were unable to repeat these

results in the new stock of mice. In a preliminary study and in order to

establish new experimental conditions for this model, we repeated the

experimenits in "old" (now healthy) and "new" stocks of mice under the

same experimental conditions. The decrease in "old" healthy stock was

now 25% (significant) and in "new" stock 12% only (not significant).

2) Studies of 1-hyoscyamine binding as a model for assessment of re-

ceptor subpopulations (MI1 \1,, etc.) and receptor pool size after

chronic treatments with agonists and antagonists are in progress. The

feasability of the model for the study of subpopulations was assessed by

testing the ability of the known in vitro antagonist scopolarmine to dis-

place I-hyoscyamine. Doses of scopolamine (4-50 mg/kg) administered

before 1-hyoscyamine (2 mg/kg, i.v.) displace this ligand by about 50 .

When I-hyoscyamine is given before scopolamine, 1 h is needed to dis-

place I-hyoscyamine to the same extent.

In the experiment to measure receptor pool size, scopolamine (25 mg/kg,

i.v.) was injected into mice for 12 days. After an additional 2 days,

1-hyoscyamine (2 mg/kg, i.v.) was injecteu and then measured in brain

after 2 hours. Compared to controls no difference in receptor pool size

could be detected. The fact that no increased concentration was

2-

observed may have been due to residual sccpolamine still occupying

receptors after 2 days. This will be evaluated by simultaneous measure-

ment of the scopolamine concentration.

3) I-Hyoscyamine decreases acetylcholine, increases choline and increases

the rate of synthesis of acetyicholine. Diazepam increases choline and

decreases acetylcholine turnover. HI-6 increases choline. The combination

of the three agents increases choline and marginally decreases the turn-

over of acetylcholine. These experiments were conducted to provide

background data for the experiments in which the protecting effect of

these three antidotes against soman poisoning and their effect on acetyl-

choline dynamics will be studied.

4) The effect of diazepam (2 mg/kg, i.v.) on levels of acetylcholine and

choline lasts for about I h. The effect on acetylcholine turnover lasts

for about 4 h.

-3-

FOREWORD

In conducting the research described in this report, the investigators

adhered to the "Guide for the Care and Use of Laboratory Animals"

prepared by the Committee on Care and Use of Laboratory Animals of the

Institute of Laboratory Animal Resources, Nationl Research Council

(DHEW Publication No. (NIH) 86-23, Revised 1985).

Citations of commercial organizations and trade names in this report do

not constitute an official Department of the Army endorsement or approv-

al of the products or services of these organizations.

-5-

TABLE OF CONTENTS

page

SL;wii ry 1

Forewurc3

Introouction 7

Materials and Methods 9

Results and Discussion 12

Conclusions 17

Fig. 1. Effect of diazeparm on specific bindino of

l-hyoscyamine in brain of old and nev. mouse

stock .

Fig. 2. Influence of scopolamine on specific bindinrj

of l-hyoscyamine in mouse Drain 19

Table I. Effect of diazepam, l-hyoscyamine and

HI-6 on dynamics of acetylcholine in

mouse brain 20

Tdble 2. Duration of effect of Jiazepam on dynamics

of acetylcholine in mouse brain 21

-6-

References 23

Abbreviations 25

Distributioni List 27

-7-

INTRODUCTION

In accordance with our plans in connection with the grant we are eluci-

dating :he mechanism of the protecting effect of a combination of atro-

pine, diazepam and an oxime against organophosphorus poisoning. For

this purpose, we use different in vivo models in mice. Since intoxication

by organophosphorus cholinesterase inhibitors is mediated by effects on

the chulinergic nervous system, we study the effects of the protecting

agents on this system. For the study of the biochemical processes, we

use turnover of acetylcholine in mouse brain. We also study binding of

the receptor ligand l-hyoscyamine to muscarinic receptors in brain in-

volved in the dynamics of acetylcholine. These biochemical processes are

then correlated with the signs of intoxication observed in the animals

after the treatments.

Our research within the framework of this grant has so far described:

- development of a method to study binding in vivo to muscarinic

receptors by measuring I-hyoscyariine in brains of mice

- study of the protecting effect of diazepam on the intoxication syndrorne

induced in mice by cholinergically active substances

- correlation of the protecting effect of diazepam in vivo with its effect

on acetylcholine dynamics. This was studied by following choline and

acetylcholine levels and acetylcholine turnover in the mouse brain

using a CC-MS method developed in our laboratory

- correlation of the protective effect of diazepam with its effect on

I-hyoscyamine binding in vivo after cholinergic timulation

- development of a method to measure diazepam in brain and blood

- study of the impact of the formulation vehicle on the distribution of

diazepam to mouse brain after intravenous injection of diazeparn in

different formulations

During the last year, our work has mainly concenirated un studies on 1)

the effect of diazepam on l-hyoscyamine binding in "old" stock of sick

- a -

and "new" stock of healthy mice (These experiments have been made tu

determine suitable experinental conditions for the new stock.); 2) the

usability of I-hyoscyamine binding for the study of specific receptor

distribution in brain (muscarine M1, %1 2'M etc.) (For this purpose,

the ability of a second ligand to displace already-bound I-hyoscyamine

after injection of scopolamine has been studied.); 3) the effect of

different combinations of antagonists - i.e., diazupam, 1-hyoscyamine and

HI-6 - on acetylcholine turnover (These experiments represent part of

the study on the protective effect of the combination of all three anti-

dotes, which will be studied after cholinesterase inhibition.); and 4) the

duration of the effect of diazepam on acetylcholine turnover.

-9-

NIATERIALS AND METHODS

Animals

Male NNMRI mnice weighing 20-25 g were used.

Chemicals

Deuter ium- libelled ACh and choline (Ch) were synthesized according to

Karl&n et al. (1) . Diazepam was in the form of Diazemuls R( KabiVitrum

AB, Sweden). The reagients used for the analysis of ACh anid Ch were

prepared as described by Karl~n et al. (1 ) . I-Hyoscyamine was obtained

from Sigma Chemical Company, USA. The synthesis of deuterium-labelled

atropine is described in Palm~r Lt al. (2). Scopolamine was obtained froin

Aldrich Chernie, West-Germany. HI-6 ([[f (4-amniirio-carboniyl )pyridinio -

methioxyimethyl i-2-[ (hydroxyininjo)methy i-pyrioinriumn dichloride) was

gift from Astra %leditec, Swed-2n. All other chem,.icals wereof a ndlytic.al

arade.

Drugs anid their administration

Diazepami (as Diazemuls R) was given) in doses of 1 mg/kq, i.v. I-Iyoscy-

amine (base) was given i.v. in doses of 2 rriqkci. MI-6 (as di-CI mono-

hydrate) was administered i.v. in doses of 25 mg/kg i.v. Scopolamine

(base) was injected in doses of 14 or 50 mg/kg i.v. or 25 mg/kg i.[ The

drugs, except diazepam, were dissolved in saline and given in 3 volume

of 5 mI/kg.

Analysis of ACh anid Ch in mouse brain

ACh anid Chi were anialyzed Using gas chromatography-mass spectrometry

(G C-MIS) and deuterated internal standards, accoroing to lKarl~n et 1l.

(1) . The mice were killed by, focused miicrowave irradiation on the licau

with 2.5 k' v for 0.68 sec (Xletabostato, Geriling-dloore, Pailo Alto, CAl ii

order to inactivate enzymes rapidly anid to prevent postmortem chanycs

- 10 -

of A-Ch amc Ch. The brain was homogenized in 4~ ml 0.4 '0\ HCIO1 2 4

LH 1 ACi, (N- (2-acetoxyethiyl)I-N, N, N-tni 1- H iethylanimioniumn iodlide)

and.- [ 2H 9ICh N -( 2-hydr-oxyethyl )-N, N, N- triL H, methylamrnoniUrn ionlde )

.yere added as internal standards. The homogeniates were centrituged for

.,u ni at 100 ,000 x g. ACh anid Ch,. together with their deuterated

troieta5, were extracted into methylene chloride as ion pairs with) dipi-

cr-domjnio 12 ,4,6,2' ,41',61-hexantitrodiphenylaminec) (DPA) . anid wore then

Jernethy lated with sodium benzenethiolate to form the corresponldirra-

tertiary amnines, which were analyzed by CC-MS using a 25-r-, HP-5

capillary column.

Turnover of ACh in mouse brain

The ACh turnover (TR A ) in brain was studied by follo,\ nwc the in-

c:orporation of 116 jCh ( N-( 2-hy droxyoethl ,) N, N, N-tr-i[ H H l, ietlhy

jrizmoniurn. iodide) into ACh after an i . v . injection of 1 11 6 C1

(10 -,.nol i/kg) , inl a Vol urnoL of 5 nil / k) adinin istered during 1 sec. The

I-,rnover rote of ACh was calcukted from the specific aictivities Ot

IL V, 6 ACh LN-t2-aceloxyethyl)-Nl,N ,NI'--til Hf2 jnetI ylanrnonlUIum iodiciel2 2

(S ACh) anid [ H6I(S Ch)20 anid 50 soc after the [ H 6 Ch injection,

aiccording to Zilversinit (3) , as described by Karl6rr et al . (4) and

Nordlgren et al. (5).

A\nalysis of I-h-yoscyamine

The inice were killed by focused microwave irradiation, ais dlescrihedi

above. The brain tissue was hoimogenrizedi in 2 ml 0. 4 V, IlCIO4 contailled,-

1 13 ]jatropirie as internal standard. The homogenates were centrifucnod

fo r 20 mmi at 100,.000 x g . The concentraition of !-hyoscyanme ~determined accoruing to Palmer et al . (6) , with the followiry nodifi-

cations: to the supernatant 0.25 nil 5 M Na301 ind 6 nil dliethy I ethri

-Nere added. After agitation anid centrifugation, the ether layer %mrs

evaporated to dryness. Thirty microliters of N, Q-bistririethylsilyl Iceta-

;nide (B3SAJ reagent was auided to the re.Sidue, andi after reaction for

30 min I*, Go0 C, the excess of reaigent was evaporated. The residue was

dissolved in 25 -ili ethylene chloride and analyzed by 6C-MS. The gas-

chromatogratphic columrn used was a 1 2-rn SE-54 fused silica capillary

Cal LM1n. Commni temperature was 2 l0L)C.

RESULTS AND DISCUSSION

I-Hyoscyamine binding

As previously reported, our suppliers ot experimental animals had to

change the animal stock due to a hepatitis infection. This upset the

parameters of our study models. Ve hove also reported certain problems

In adjusting the experimental conditions in the receptor binding studies

with I-hyoscyamine. Vie have now been able to compare the two stocks of

,nice in this respect.

Mice were pretreated for 2 mi with 1 mg/kg of diazepam given as Diaze-

nuls R . I-Hyoscyamine, 2 mg/kg i.v., was then given arid the mice were

killed 2 h later by microwave irradiation. Results are compiled in Figure

1 arid comprise the effect of diazepam on I-hyoscyamine binding inl the

new stock of mice, the old stock of healthy mice generated at the same

time (a limited number of healthy progeny of the survivors were avail-

able), and data generated in 1986 in the old stock of sick ;nice. Com-

parisons have been made with animals treated with saline in all three

sets of animals. Results now generated in the old healthy stock (Fig. 1;

columns 1 and 2) are very similar to those generated in the same stock

in 1986 (columns 5 and 6). Thus, a decrease of I-hyoscyaminc bindirg

after diazepar is clearly demonstrated. When the experiment is performed

on the new stock of mice, a marginal, non-significant, decrease is

observed. Therefore, and in order to find optimal parameters, the model

will need further development in the new stock of mice.

In order to test the ability of the model to detect muscarinic receptors ol

specific subtypes, we performed experiments on the interaction of

scopolamine with I-hyoscyamine binding. Thus, after establishing stable

I-hyoscyamine binding in brain in vivo (2 mg/kg, i.v.; 2 h), it should

be possible to displace this binding with different subreceptor specific

ligands for mapping of regions in bruin rich in such subtypes (7). In

our first experiments, we have used the unspecific ligand scopolamine

13 -

for such studies in order to test the feasibility of the experimental

design. When scopolamine, 4 mg/kg i.v., was given 1 min before or 1

min after I-hyoscyamine and its concentration was analyzed 2 ih later,

about 30 ng/g were found (compared to about 60 ng/g in controls). If,

instead, scopolamine was given 1 h 50 min after I-hyoscyamine, no

displacement was obtained. The same held true if the scopolamine dose

was increased to 50 mg/kg. Therefore, the design of the experiment was

changed and displacing doses (50 mg/kg i.v.) of scopolamine were given

1 h 50 min, 1 h 30 in and I h after 1-hyoscyamine. The results of

these experiments are depicted in Figure 2. It can be seen that the

displacement is time-dependent and proceeds to about 30 ng/g when

treatment is allowed to last for 1 h (column 4). This is probably the

highest amount possible to displace since pretreatment with scopolamine

gave the same level. This type of experiment will be carried out using

receptor specific ligands.

These experiments confirm the concept that 1-hyoscyamine binding in

vivo represents specific binding to muscarinic receptors possible to

displace with other antimuscarinic receptor ligands. The experiments

were performed to enable study of, e.g., the size of this receptor pool

and its dependence on chronic stimulation or blockade. In order to study

the latter phenomenon, mice were treated with daily i.p. doses (25

mg/kg) of scopolamine for 12 days. They were then left untreated for 2

days, whereafter they were injected with I-hyoscyamine i.v. (2 mg/kg),

sacrificed 2 h later and analyzed for brain 1-hyoscyamine. Concentrations

in six animals (62, 51, 51, 56, 59, arid 53 ng/g; mean 55 ng/g) were

marginally reduced compared to concentrations in two control animals

(58, 60 ng/g). The control values are consistent with those previously

reported. The experimental design was very similar to one published, iw

which animals (rat) treated chronically in vivo with scopolamine were

analyzed for specific binding in vitro (8). The lack of demonstrable

increase in receptor pool size in our experiments may depend on residual

bound scopolamine masking the increase of the pool size. To investigate

- 14-

this possibility, scopolamine concentrations in brain will be measured

under the same conditions.

Antidotes and acetylcholine turnover

In accordance with our plans, we have studied the effect of combinations

of antidotes on central cholinergic mechanisms in mice. Vve have studied

l-hyoscyamine, diazepain and HI-6 separately and in different combina-

tions. Data on acetylcholine turnover after administration of saline,

diazepain (1 mg/kg, i.v.), 1-hyoscyamine (2 mg/kg, i.v.), or HI-6 (25

mg/ky, i.v.) are collected in Table 1.

As previously shown by us and others, l-hyoscyamine decreases the

endogenous level of ACh; presumably ACh output is increased (9,10).

However, when diazepam is added to the injection solution, the endo-

genous ACh levels are normalized. Diazepam separately does not affect,

or very slightly affects (increase), the ACh level. The endogenous

choline (Ch) levels are increased after administration of diazepam, I-

hyoscyamine, and a combination of the two.

The concentration of L 2H 6 ]-ACh is markedly decreased in animals admini-

stered diazepam and diazepam in combination with 1-hyoscyamine, while

I-hyoscyamine alone does not seem to have any effect. The specific

activity of L 2H 6 ]-ACh (SAch) is decreased to the same extent in animals

treated with diazepam and those treated with the combination diazepam/l-

hyoscyamine, while an increase is seen in animals administered I-hyoscy-

amine alone.

The increased Ka value seen in animals pretreated with 1-hyoscyamine

indicates an increased synthesis rate of ACh, which is consistent with

earlier findings in our laboratory and with literature data. If the in-

creased release of ACh is a sustained effect, the synthesis rate has to

be increased in order to balance the increased release anid maintain the

new steady state. The net TRAC h is, however, kept constant since after

-t5 -

I-hyoscyamine a smaller amount of ACh has to be renewed. Diazepan

lowers the turnover rate of ACh, as we have shown previously (11).

Interestingly, the combination of diazepam ano I-hyoscyamine decreascs

TRACh to the same extent as diazepam alone does, which is in great

contrast to the effect of I-hioscyamine alone. Thus, the effects of

diazepam dominate in this combination of doses. The increased turnover

of ACh after atropine is probably an unfavourable feature of a [)ro--

tecting agent. These results may therefore, at least partly explain the

advantage of combining atropine with diazepam in cases of nerve gas

poisoning.

HI-6 alone induces a marginally increasing effect on the acetylcholine

level, a statistically significant increasing effect on choline levels aild a

slight increasing effect on acetylcholine turnover.

\ihen the three treatments are combined in the san-e injection, the domi-

nant result is an increase of choline levels, with a slinht decrease of the

acetylcholine turn;over. In this last combination, the differing effect, of

diazepam and 1-hyoscyamiie on acetylcholine level seem to be balatncen.

The choline level is increased and the mole ratio of labelled choline is

reduced, reflecting the effects of diazepam, I-hyoscyamirie and 1-1-6.

Consequently, the acetylcholine turnover is somewhat decreased. The aim

of these experiments has been to collect background data on the protec-

tive effect of this three-drug combination on soman poisoning ad the

correlation of the effect with acetylcholine dynamics.

An important issue for the mechanism of protection with antidotes is the

duration of effect and how this correlates with the dynamics of acetyl-

choline. We have already found that I-hyoscyamine concentration persists

for a long time (up to at least 30 h). We know from our exper-imeiits that

the conceiitration in brain of diazepam is much more short-lived. When

high doses of diazepam (25-35 mg/kg) are given, the levels of diazepam

are down to a few .ig/g after 30 min. Thus, the half-life is very short,

which is in accordance with previous findings (12).

- 16 -

In the above experiments, diazepam was given 10 min before 2 H6 ]-Ch

and its effect on acetylcholine turnover evaluated 20 and 50 sec later.

Experiments have also been performed to study the duration of this

effect (Table 2). After 20, 30 and 45 min, levels of acetylcholine are

significantly increased, while they are returning to normal after 4 h.

The tendetcy for an increase seen at 10 min is, thus, fully developed at

20 min and persists for at least 45 min. The levels ot choline also stay

elevated for 45 rain, but have become normalized after 4 h. The frac-

tional rate constant is maximally decreased after 10 min and seems to be

somewhat lowered, still, at 4 h. The data tend to iodicate a duration of

effect on the acetylcholine dynamics (K a ) of at least 4 h.

17

CO NCLU S I U' S

Studies of specific binding of 1-hyoscyamine to muscarinic receptors in

the mouse brain have been made in stocks of old healthy and new

healthy mice. Displacement of I-hyoscyamine with diazepam seen in old"sick" stock was repeated in old healthy stock. Optimal experimental

conditions for new stock have yet to be found.

The study of muscarinic subreceptor pools in mouse brain has started. It

has been snown possible to displace about 50%5 of I-hyoscyatnine either by

pretroatmett with scopolamine or by displacement ("chase") of an estab-

lished coticentration by treatment with scopolamine for I h.

/he effect of I-hyoscyanine, diazepam and HI-6 on acetylcholine turnovcr

has been studied separately after each antidote and after combinations of

these bloc;kers. The effect of 1-hyoscyamine and diazepam given sepa-

rately has been reported earlier. HI-6 increases endogenous choline

levels and slightly increases the turnover of acetylcholine. The combina-

tion of the three andidotes increases choline and slightly decreases the

turnover of acetylcholine.

The effect of oiazepam oi acetylcholine turnover lasts for ,bout L h.

The effect on choline and acetylcholine levels lasts for about 1 1h.

18 -

FIGURE 1

Effect of diazepamn on specific binding of l-hyoscyamnine in brain of old

and new mouse stocks. The animals were pretreated with saline or

,Iiazepamn (I mg/kg, i.v.) 2 min before the injection of 1-hyoscvamine (2R

,ngikg, t.v.). Diazepain is given as Diazemuls

80-7 57 60 53(9) (9)

70 4 3

©60 4

0/)

2 50

0 40

1 2 3 4 5 6

1) Old mouse stock saline (healthy progeny)

2) Old mouse stock Diazemuls (healthy progeny)

3) New mouse stock saline

4) New mouse stock Diazemuls

5) 1986 values saline (old stock of sick mice)

6) 1986 values Diazemuls (old stock of sick mice)

Two-tailed Student's t-test of saline treatment meais in comparison to

diazepam treatment means.

*** 2p<0.001, ** 2 pK0.01, n.s. = not significant.

Figures are mean vulues, with number of animdls within parentheses.

The bars show the standard deviation.

19

FIGURE 2

Intluence of scopolamine on specific binuing of l-hyoscyamine in rouse

brain. Tihe animals were administered I-hyoscyarnine (2 my/kg, i V.) dci

iniected scopolamine (50 mg/kg, i.v.) 1 h 50 Mr), I h 30 min or 1 h

later.

80-1 (6) 59! (4)

C0 6

/C 60

E

2 40

20

1 ) Controls

2) Scopolamine h 50 tin after I-hyoscyamine

3) Scopolamine 1 h 30 min after I-hyoscyamine

3) Scopolamine 1 h after I-hyoscyasaine

Figures are mean values, with number of animals within paretheses.

The bars show the standard deviation.

-20-

CI0 0+ 0 0 0 0

v+, mnL Ca

0

0 0 .40 -0 ..>C 04) z00 00 1

E. y a: 0 00 0 0 0 0 '

03 U 10

0 < 00 0

, cc

CC-C C0 4

o . 0 . 4 0 0 0 0 0 0

o 33 ++ 3, 1 3 ++ 3+

EU - 2

~~' 2 ~~ 0 . . .. C 4 C1: 00 020 0 +0

u00 ' t uO. 01 .3 4

3+4 l -C "m 0 00 0 00

- 0 0'30 04 00

0 0 C

0 LU

U0 + 0o 04)

3+ + C S: 41 >4o .( +3 3 +1 + +1 1.1 3 w330 3C~ 0. (I C C 0clo0 0

0E CE00 o U E ~ -Z, - 30

C A 5, C

E< C c E.. *4 4 4 . 1

3+0

;. c; c; a N 611 1; 0+0I

10~~~~ 4. 0i 31 *+ ++3 41 + .1 341

*i~ ~~~ ~~ C. JC 0 34 03=+ E04 ., ~ 00 00 0- ~ 0.cE

2 o-'~ jL+-

-0 0 1 1 0.

0 '

C U C: C f . , ,0 < ~ C,.C .C

C U E CE 00 00 40 40 00 0o , C o

U E

-c 0

.~~ -0* - -t - 0 -ru > 0 .- C ,0 .- 0 0 00 0

0U - O - r. 0 0

0 u a). E 0 0 o o t o o o.

o 0 < C I . . . . 0 0 0

< ' E, c

-~E wu .~

0.S-< o m 0 0 0 0 0 0

c .Co E0IA L C .

<-E--0 00m C. 0 00 0

< c p >>10 CC 0 00 r 0

I- . . . . . . . . . . . . . . . . .

cm~ ~ ~ <I t

Co E E u . 4* 4 .

0 E

o C 0 o O

6 00 0 0o .C 1 4 c* 1 * 1 0 40E 0c Ct E t.60 0

Ec.2E

0~ c c SClC 0 cc 00 0 00 00 0 C a

U 0 EC - 1 ~C C -

<0 (A .- y 0f

- 21 -

D04 CC C C 00 0

0 E I--_

0 mC ME I

000

00000

04

0'c 00 CC1

C .0 V

0 cE%~

oo C 2 u. -- -r, C C 0 C C N

mC CL 04

41 .

E X,

u m

m 0--- r~ H

c 3 0 .- 0 o- 0

0. 00 C O - 0 C

z 0 u. . 44 04 0 04 C 040

0 <~ m E0 E CC CO - NC CC

C -

~-E X-

N C C 44 0CC tn

0.0 440 C30 M 0,. 00 0 N

~~~ 0" C

CO C! 400

C~~~~~ C- 40 0 ON 0 0

>4. 44 C 00 C 00 0 0CIO

CC'n Oc

ON- 0~ Nt 04 f cO020 o n~ N-t 440 CC .C 2D

02 -0

0zJo A0 7 007

0 E' 0L, C',r o

:3 0 3: i

I-i wnl ZtE .co a 0 < -a o 0

a -

.C

0 0 -0

c 0

-a H en0r- 0 r. 0 0 o I

u E 000

ON <0 00 00 E 00 0

a Y

* Z; Cz C

:3 c

C0

0~0. 0 T

0 0n tae 0 0 c- . 0 0

VE Ea~~~~~~c wa.O at 00 0 0 0

-c-. 2 c-c~i. 0 4-n C

Li1 1 ic-

2... tc

0 i

enO 0 00 0

torn enO o~ 0 00 00 0 io

.2 U Li

Eu <0 0u0 cCoo< Oo o oo n o V) n a

23 -

.K fFERENCES

kor; r,, B., Luodgren, G., Nordgren, I. and Holmstedt, B.: Ion

flair extraction imd gas phase analysis of acetylcholine and cholire.

In: Chuline and Acety choline. Handbook of Chemical Assay

,lethods. I. Halin (Ed.). Raven Press, New York, 1974i, pp.

163-179.

Pahm6r, L. , Edgar, J., Lundgren, G., Karl6n, B. and Hermansson,

J.: Atropine in mouse brain and plasma quantified by mass frag-

,-itography. Acta Pharmacol. Toxicul. 1981 , 49, 72-76.

3. Zilverswit, 19.B.: The design and inalysis of isotope experiments.

A.. leu. 1960, 29, 832-348.

rdarl6n, B., Lut.rgren, G., Lundin, J. ane Holmstedt, B.: On the

tor-i over of acetylcholine in mouse brain: Influence of oose size of

douteriuri labelled choiino given as precursor. Biochem. Pharmacol.10,82, 31(18), 20;67-2872.

S. Norderen, I., Lundgren, C., Puu , G. KarlV .. holh ,tedt,

3.: Distributio a nd elimination of "i, stereoisorers ot roan d

their effect on brain acetyichiline. Fund. Appl. Tuxicol. 1985, 5,

S252-S259.

6. Palm6r, L., Lundgren, C. anid Karl6n, B.: A method using 1-hyo-

scyamine for the study of muscarinic acetylcholine receptor binding

in vivo. Pharmacology and Toxicology, 19F7, 60, 5i-57.

-241 -

liammiar, k. , tM'onterini , FE. , DeConti , L., Giraldo, FE. , Schiavi,

6.B3. cino Ladinskv , H. :Distinct iuscarinic receptor subtypes in

the heart and in exocrine glands. In: Cellular arid Molecular Basis

uf Cholinergic Functions leci. Dowdall anid Hawthorne) Ellis Horwood

Ltd, Chichester, Englano, anid VCH, 4einheirn,, FRC, 1987, pp.

56-o3.

8\ar chii, MI. a nd Reuter , M. :Presynaptic noscarinic receptors:

Change oI Sensibi litv,, ClUnit long-term cirug treatnent. In: Dyna-

mics of C:-im ergic Function (0(d. 1, Hinin) Plenumn Press, Nev,

York, 1986, pp. 4~23-428.

9. Ci ar manr , N.J. and Pepeu , C. :Drug-indlucc changes in brain

acetyicholine. Brit. J . Pharnacol . 1562, 19, 216-234.

U. %,itchell. J. F.: The spontaneous anid evokecl releaise of acetylcholine

from the curebral cortex. J. Physiol. 1963, 165, 98-116.

11 . ILonugren, G., Nordgreii, 1. , lKanl6n, B. and Jacubsson G.

Effects, of diazepamn on bloou choline and ace',Y'lcholino turnover in

braiin of nice. Pharmacology and Toxicolugy,. 1967, CO, 96-99.

I.van der K lin. :Kinetic-; uf dlistribu tion and metaibolism, of din .epam

anid chiordiazepoxide in mice. Arch. I nt. Pharmnacody~n. 1969, 178,

193-215.

ABBRVIAT ION S

Ch, Choline

G C-.\'b, Gas chromatography - -nass spectrometry

UPA, 2,4,6.2',41,6-H exanitr-odiphien-yj loin

T ~'Acet ylcholinie turnover

sACh' Specific activity of [ 2H. jACh

s Ch' Specific activity of H 6 'Ch

BSA, N ,O0-E3i strivnethyl si lylacetainidle

F1 1-6, [ [ (4-ornino-carbony I) pyridino j methoxy ! mothyII - 2-

(hycdr,:xyim ino)rimethy I ,-pyrid in ium cichiloride

- 27 -

DISTRIB3UTION LIST

I cupy: Commander

US Army Mvedical Research and Developmient Conoj-ancil

ATTN-. SCRD-RMI-S

Fort Detrick, Frederick, NMD 21 702-5U1 2

2 copius: Administrator

Defense Technical Information Center

ATTN: DTIC-FDA

Cameron Station, Alexandria, Virginia 2,31-

1 copy: Commandant

Academy of Health Sciences, US Arm,,

ATTN-. AHS-CDM

Fort Sam Houston, Texas 78224

1 copy Dean, School of Medicine

Uniformed Services University at the Health Sciernces

4301 Jones Bridge Road

Bethesda, NID 20014

'4 copies: Commander

US Army Medical Research Institute of Chemica lDefense (USAMRICD)

ATTN: SGRD-UV-RC

Aberdeen Proving Ground, W) 21010