Enzymes as pretreatment drugs for organophosphate toxicity

Neur05c1ence• 8106ehav10ra1Rev1ew5.V01. 15. pp. 123-128. Per9am0n Pre55p1c. 1991. Pr1nted1n the U.5.A.

"0149"~/631t/91:$~3.00 + .00

En2yme5 a5 Pretreatment Dru95 f0r 0r9an0ph05phate 70x1c1ty 8 H U P E N D R A P. D 0 C 7 0 R , * L1LY R A V E H , ~ A L A N D. W 0 L F E , * D 0 N A L D M . MAXWELL•1" A N D Y A C 0 V A5HAN1~:

*D1v1510n 0f 810chem15try, Wa1ter Reed Arm)~ 1n5t1tUte 0f Re5earCh, Wa5h1n9t0n, DC 20307-5100 ~Un1ted 5tate5 Arm), Med1Ca1 Re5earCh h25t1tUte 0f Chem1Ca1 Defen5e A6erdeen Pr0v1n9 6r0und, MD 21010-5425 and +,-15rae11n5t1tute 0f 8101091Ca1 Re5earch, P.0. 80a 19, Ne55-210na, 15rae1

D0C70R, 8. P., L. RAVEH, A. D. W0LFE, D. M. MAXWELL AND Y. A5HAN1. En2yme5a5 pretream2entdru95f0r 0r9an0ph05phate t0x1c10,. NEUR05C1 8108EHAV REV 15(1) 123-128, 1991.--We have 5ucce55fu11y dem0n5trated that ex09en0u51y adm1n15tered acety1- 0r 6utyry1ch011ne5tera5e(ACHE, 8ChE re5pect1ve1y) w1115e4ue5ter 0r9an0ph05phate5 (0P5) 6ef0re they reach the1r phy5101091ca1tar9et5. 1n add1t10n, a th1rd en2yme, end09en0u5 car60xy1e5tera5e 15 kn0wn t0 6e capa61e 0f 5caven91n9 0P5. 1n the5e 5tud1e5, we have adm1n15tered AChE and 8ChE t0 three d1fferent 5pec1e5 0f an1ma15 (m1ce, marm05et5 and m0nkey5) wh1ch were cha11en9ed w1th three d1fferent 0P5 (VX, MEPQ and 50man). Re5u1t5 06ta1ned fr0m the5e 5y5temat1c 5tud1e5 dem0n5trate that: (a) a 4uant1tat1ve 11nearc0rre1at10n ex15t5 6etween 6100d AChE 1eve15and the pr0tect10n aff0rded 6y ex09en0u51y adm1n15teredChE5 1n an1ma15cha11en9ed w1th 0P, (6) appr0x1mate1y 0ne m01e 0f e1ther AChE 0r 8ChE 5e4ue5ter5 0ne m01e 0f 0P, (c) 5uch pr0phy1act1c mea5ure5 are 5uff1c1ent t0 pr0tect an1ma15 a9a1n5t 0P5 w1th0ut the adm1n15trat10n0f any 5upp0rt1ve dru95.7hu5 the 0P d05e, the 6100d-1eve1 0f e5tera5e, the rat10 0f the c1rcu1at1n9en2yme t0 0P cha11en9e, and the rate 0f react10n 6etween them determ1ne the 0vera11 eff1cacy 0f an en2yme a5 a pretreatment dru9. 7he 610chem1ca1 mechan15m under1y1n9 the 5e4ue5trat10n 0f var10u5 0P5 6y the u5e 0f ex09en0u51y adm1n15tered 5caven91n9 e5tera5e5 15 the 5ame 1n a11 5pec1e5 0f an1ma15 5tud1ed. 7heref0re, the extrap01at10n 0f the re5u1t5 06ta1ned 6y the u5e 0f ChE pr0phy1ax15 1n an1ma15 t0 human5 5h0u1d 6e m0re re11a61e and effect1ve than extrap01at1n9 the re5u1t5 fr0m current1y u5ed mu1t1dru9 ant1d0ta1 m0da11t1e5. Acety1ch011ne5tera5e 8utyry1ch011ne5tera5e

0r9an0ph05phate t0x1c1ty Car60xy1e5tera5e VX

En2yme5 a5 pretreatment dru95 MEPQ

50man

5caven9er

E5tera5e

the t0x1C a9ent. 5ec0nd, 1t 5h0u1d have a 10n9 ha1f-11fe 1n v1v0. 7h1rd, 1t 5h0u1d 6e read11y ava11a61e 1n 5uff1C1ent 4uant1t1e5. F0urth, 1t 5h0u1d n0t 6e 1mmun0react1ve. F1na11y, the 5t01ch10metry 6etween 5caven9er en2yme and t0x1c a9ent5 5h0u1d appr0ach 1:1 f0r 13-e5tera5e5. Much Pr09re55 ha5 6een made 1n the pa5t f1ve year5, 6y 5evera1 9r0up5 0f 1nve5t19at0r5, 1n exp10r1n9 the p0tent1a1 u5e 0f an en2yme t0 c0unteract the t0x1c1ty 0f 0P5. Am0n9 the en2yme5 wh1ch h01d pr0m15e a5 5caven9er5 0f h19h1y t0x1c 0 P nerve a9ent5, 519n1f1cant advance5 have 6een made u51n9 ChE5. 51nce the 610chem1ca1 mechan15m under1y1n9 the pr0phy1ax15 6y ex09en0u5 e5tera5e5 5uch a5 ChE5 15 e5ta6115hed and te5ted 1n 5evera1 5pec1e5, th15 c0ncept 5h0u1d ena61e a re11a61e extrap01at10n 0f re5u1t5 fr0m an1ma1 exper1ment5 t0 human app11cat10n. 1n th15 rep0rt, we rev1ew the re5u1t5 0f 1nve5t19at10n5 1n wh1ch acety1ch011ne5tera5e (ACHE) and 6utyry1ch011ne5tera5e (8ChE) were 5ucce55fu11y u5ed f0r the 5e4ue5trat10n 0f h19h1Y t0x1c 0 P ant1-ChE5, w1th0ut the 0ccurrence 0f 51de effect5 0r perf0rmance decrement5.

PRE5EN7 treatment f0r p0150n1n9 6y 0r9an0ph05phate ant1ch011ne5tera5e c0mp0und5 c0n515t5 0f a c0m61nat10n 0f dru95 5uch a5 car6amate5, ant1mu5car1n1c5, react1vat0r5 and ant1c0nvu15ant5 1n pre- and p05texp05ure m0da11t1e5 (9, 12, 18). M05t 0f the5e re91men5 are effect1ve a9a1n5t many ant1ch011ne5tera5e5. H0wever, p05texp05ure 5ympt0m5, 5uch a5 trem0r5, c0nvu1510n5, apnea, fa5c1cu1at10n5 and 9enera1 1ncapac1tat10n, are c0mm0n1y 065erved and re5u1t 1n perf0rmance decrement5 1n human 5u6ject5. 0 n e effect1ve appr0ach t0 a11ev1ate the5e 51de effect5 15 the u5e 0f an en2yme a5 a pretreatment dru9 (2, 3, 5, 7, 8, 10, 16, 17), rather than the c1a551ca1 mu1t1p1e dru9 re91men5. F0r examp1e, 0ne can env1510n the u5e 0f the cata1yt1c act1v1ty 0f en2yme5 5uch a5 ph05ph0ry1ph05phata5e5 [e.9., DFPa5e, 50mana5e, etc. (10)] 0r e5tera5e5 (2, 3, 7, 8, 16, 17) t0 rap1d1y hydr01y2e ant1ch011ne5tera5e5 t0 n0nt0x1c pr0duct5. 7hu5, ch011ne5tera5e5 (ChE5) and car60xy1e5tera5e (CaE) may 6e u5ed a5 5caven9er5 t0 rem0ve 0r9an0ph05phate5 6ef0re they reach the1r phy5101091ca1 tar9et5 (2, 3, 5, 7, 8, 14, 16, 17). Ant160d1e5 t0 0r9an0ph05phate5 1n the1r tran51t10n 5tate5 may a150 6e de519ned and 9enerated t0 cata1y2e the hydr01y515 0f the5e c0mp0und5. 7here are certa1n re4u1rement5 f0r an en2yme t0 6e an effect1ve 5caven9er f0r 0r9an0ph05phate t0x1c1ty 1n v1v0. F1r5t, 1t 5h0u1d have a re1at1ve1y h19h rate 0f react1v1ty w1th 0r9an0ph05phate5; the h19her the rate, the fa5ter the en2yme w111 neutra112e

DEM0N57RA710N 0F U5E 0F E57ERA5E A5 5CAVEN6ER 0F 0R6AN0PH05PHA7E5 7he f1r5t 5ucce55fu1 dem0n5trat10n 0f the u5e 0f e5tera5e a5 a 5caven9er 0f 0 P 1n m1ce wa5 5h0wn 6y W01fe et a1. (17) wh0

123

124

D 0 C 7 0 R E7 AL.

7A8LE 1

7A8LE 2

PR07EC710N 0F M1cE A6A1N57 vX 8Y PRE7REA7MEN7 w17H F85-AChE

EFFEC7 0F C8DP 0N 50MAN 70X1C17Y* C0ntr01

VX D05e LD5051

5urv1v0r5 (+ ACHE)

1.00

5/5 5/5 4/5 1/5 0/5

2.00 3.00 4.24 5.99

M1ce were adm1n15tered 1P 10.89 nm01 F85-AChE appr0x1mate1y 20 h 6ef0re cha11en9e w1th VX (1ML 1 LD50=56.3 nm01 VX/k9:1 LD50 w1th F85-AChE = 201.3 nm01 VX/k9. Pr0tect1verat10= 3.6. Repr0duced w1th perm15510n fr0m Fundam. App1. 70x1c01. (17).

u5ed 1ar9e 4uant1t1e5 0f AChE pur1f1ed fr0m feta1 60v1ne 5erum (6.15) a5 a 5caven9er f0r VX (7aMe 1) and t0 50me de9ree a9a1n5t 50man. 7he5e re5u1t5 5u65tant1ated the hyp0the515 that AChE c0u1d 5erve a5 a pretreatment dru9/pr0phy1act1c a9ent t0 prevent 0 P p0150n1n9.7hey were a61e t0 pr0tect m1ce a9a1n5t appr0x1mate1y 3.5 LD50 0f VX w1th n0 apparent 519n5 0f c1a551ca1 0 P t0x1c1ty. 7w0 0ther 065ervat10n5 fr0m the5e 5tud1e5 were 0f 1mp0rtance: 1) 1n v1v0 1nh161t10n 0f ex09en0u51y adm1n15tered AChE 1n 6100d wa5 pr0p0rt10na1 t0 the am0unt 0f 0 P adm1n15tered a5 cha11en9e, a re5u1t c0n515tent w1th 1n v1tr0 exper1ment5, and 2) the am0unt 0f AChE re4u1red t0 pr0tect m1ce a9a1n5t VX wa5 c0n51dera61y 1e55 than t0 pr0tect a9a1n5t 50man [the LD505 (1M) 0f VX and 50man 1n m1ce are 56 and 577 nm01/k9 re5pect1ve1y]. Human 5erum 8ChE ha5 a150 6een dem0n5trated t0 c0nfer pr0tect10n a9a1n5t 50man p0150n1n9 1n m1ce wh1ch were pretreated w1th ex09en0u5 e5tera5e5 (2,3). Car60xy1e5tera5e 15 an examp1e 0f an end09en0u5 e5tera5e capa61e 0f 5e4ue5ter1n9 0P5 1n c1rcu1at10n. F0r examp1e, the t0x1c1ty 0f 50man appear5 t0 vary am0n9 d1fferent 5pec1e5 0f an1ma15 (14) and th15 var1at10n 15 attr16uted t0 the pre5ence 0f d1fferent am0unt5 0f car60xy1e5tera5e 1n the1r p1a5ma. 7he am0unt 0f 50man re4u1red t0 cau5e 1etha11ty para11e1ed the p1a5ma CaE 1eve15 1n the5e an1ma15.7he ev1dence f0r the 5e4ue5trat10n 0f 50man 6y CaE wa5 pr0v1ded 6y 5tud1e5 (4,5) 1n wh1ch the CaE 1nh161t0r, 2-(0-cre5y1)-4H- 1:3:2-6en20d10xaph05ph0r1n-2-0x1de (C8DP) (4) wa5 adm1n15tered t0 an1ma15 1n 0rder t0 1nh161t 5erum CaE pr10r t0 the adm1n15trat10n 0f 50man (14). 7he5e 5tud1e5 5h0wed that 5uch pretreatment 1ncrea5ed the t0x1c1ty 0f 50man (10wered the LD50) 1n an1ma15 1n pr0p0rt10n t0 the am0unt 0f CaE 1n the1r p1a5ma. 7a61e 2 5ummar12e5 the5e re5u1t5.1n add1t10n, m1ce were pretreated w1th 60th C8DP (2 m9/k9) and F85-AChE (257 nm01/ k9), and cha11en9ed w1th 50man. 7a61e 3 5h0w5 that the adm1n15trat10n 0f C8DP, a10ne, t0 m1ce reduced the LD50 0f 50man fr0m 620 nm01/k9 t0 53 nm01/k9. When m1ce were pretreated w1th F85-AChE, 1n add1t10n t0 C8DP, the LD50 0f 50man 1ncrea5ed t0 120 nm01/k9, thu5 y1e1d1n9 a pr0tect1ve rat10 0f 2.26. 1N v1v0 57A81L17Y 0F cH0L1NE57ERA5E5 1n 0rder f0r an en2yme t0 6e an effect1ve pretreatment dru9 f0r pr0tect10n a9a1n5t t0x1c c0mp0und5, 5uch a5 pe5t1c1de5 0r chem1ca1 warfare a9ent5, 1t 5h0u1d have a re1at1ve1y 10n9 ha1f-11fe 1n v1v0. 1n add1t10n, 1t5 therma1 5ta6111ty 0n pr010n9ed 5t0ra9e and 1t5 r0ute 0f adm1n15trat10n are 0f pr1me 1mp0rtance t0 1t5 u5efu1ne55 a5 an ant1d0te. 1t 5h0u1d 6e phy5101091ca11y and 1mmun01091ca11y c0mpat161e, and 6e ava11a61e 1n 1ar9e 4uant1t1e5.0ur 5e1ect10n

LD50 (p.9/k9)

5pec1e5 M0u5e Rat 6u1nea p19 Ra661t

125.0 116.0 32.3 22.8

2 m9/k9 C8DP

16 m9/k9 C8DP

LD50 % Decrea5e LD50 % Decrea5e (V.9/k9) w1thC8DP (p.9/k9) w1thC8DP 12.2 15.6 12.3 11.8

90 87 62 48

7.5 13.3 9.3 8.4

94 89 71 70

*C8DP wa5 adm1n15tered5C 1 h 6ef0re 50man (1M1. Repr0duced w1th perm15510n fr0m 70x1c01. Lett. (14). 0f the appr0pr1ate f0rm5 0f ch011ne5tera5e5, F85-AChE and human 5erum 8ChE t0 u5e a5 pretreatment te5t dru95 wa5 6a5ed 0n the fact that 60th the5e en2yme5 are 9106u1ar 1n f0rm, ea511y pur1f1ed fr0m 5erum, and are re1at1ve1y 5ta61e en2yme5 (6,15). 7he 1n v1v0 ha1f-11fe 0f F85-AChE 1n m1ce wa5 determ1ned 6y 1V 1nject10n and mea5urement 0f the 6100d en2yme 1eve1 0ver a per10d 0f 72 h0ur5 (16). 7he 5ame am0unt 0f en2yme wa5 a150 adm1n15tered 1P t0 c0mpare the r0ute5 0f adm1n15trat10n. 7he re5u1t5 5h0wn 1n F19. 1 1nd1cate that 6100d AChE 15 ma1nta1ned at a c0n5tant 1eve1 dur1n9 a per10d 0f 2-5 h, re9ard1e55 0f r0ute 0f adm1n15trat10n (7here 15 a fa9 0f a60ut 1.5 h after 1P 1nject10n 6ef0re the 6100d AChE 1eve1 peak5). A11 1n v1v0 0 P t1trat10n5 were c0nducted dur1n9 the per10d when the ex09en0u51y adm1n15tered AChE 1eve15 reached the1r p1ateau, there6y av01d1n9 the err0r5 1ntr0duced 6y var1at10n 1n 1eve15 due t0 c1earance 0f en2yme. C0n51dera61e en2yme act1v1ty wa5 065erved even after 72 h. 7he pr0f11e f0r c1earance 0f en2yme wa5 a60ut the 5ame u51n9 e1ther r0ute 0f adm1n15trat10n. 51m11ar re5u1t5 were 06ta1ned u51n9 human 5erum 8ChE 1n m1ce (2) and F85-AChE 1n marm05et5 and rhe5u5 m0nkey5 (7,8). 80th the5e en2yme5 have re1at1ve1y 10n9 ha1f f1ve5 and appear phy5101091ca11y c0mpat161e, pr06a61y 6ecau5e they 60th are 5erum pr0te1n5. 7he re1at1ve1y 510w c1earance 0f ChE5 fr0m 6100d 15 0f 9reater 519n1f1cance when 1t 15 c0mpared t0 the c1earance rate5 0f 0ther c1a551ca1 pr0phy1act1c and treatment dru95 5uch a5 car6amate5 and 0x1me5. F1na11y, 1t 5h0u1d 6e n0ted that ra151n9 the 6100d 1eve1 0f th15 en2yme m0re than a th0u5and-f01d d0e5 n0t appear t0 have any adver5e effect 0n a11 5pec1e5 0f an1ma15 te5ted. 1N v17R0 AND 1N v1v0 5701cH10ME7RY 0F 0P: 5cAvEN6ER 1N7ERAc710N Up0n 5u65tant1at10n 0f the hyp0the515 that ex09en0u51y ad7A8LE 3 EFFEC7 0F PRE7REA7MEN70F M1cE W17H c8DP AND F85-AchE 0N 50MAN 70x1c17Y 50man 70x1c1ty LD50 Pretreatment

1x9/k9

C0nf1dence L1m1t5

nm01/k9

N0ne +C8DP +C8DP + F85-AChE

113.0 9.6 21.8

( 9 4 . 6 - 135.2) ( 6 . 0 - 12.6) (17.9 - 25.0)

620 53 120

F85-AChE (257 nm01/k9) wa5 adm1n15tered 1P t0 9r0up 0f 5 m1ce 19 h0ur5 pr10r t0 adm1n15trat10n 0f C8DP (2 m9/k9). 7he an1ma15 were cha11en9ed 1M w1th 50man 0ne h0ur after C8DP adm1n15trat10n. C0nf1dence 11m1t5at p<0.01. 7he Pr0tect1ve Rat10:120/53 = 2.26.

EN2YME5 A5 PRE7REA7MEN7 DRU65 F 0 R 0 P 70X1C17Y

125

~.~ 900

~:)

• 100 ~ . ] ~ 75

~ AChE= 3.55 X 10-7 M 1MEPQ) J•• AChE= 3.39 X 10-7 M (PH05PH0L1NE1 L~.AChE= 3.11X 10-7M (PARA0X0N1

•r

700

--

v 0

~ 1

1 2

1 3

1 4

, 5

1 11 m ,, ~ ,1.-..1:-6 24•• 48 • 72

0

71ME ( h0ur5)

2

3

AN71--ChE X 10 -7 M

F16. 1. Avera9e wh01e 6100d-AChE 1eve15f0110w1n91V 0r 1P adm1n15trat10n 0f F85-AChE. Key: (0) m1ce adm1n15tered920 un1t5/m0u5e6y 1V r0ute (n=2), and ([7) m1ce adm1n15tered 760 un1t5/m0u5e 6y 1P r0ute (n = 3). Var1at10n5am0n91nd1v1dua1an1ma15adm1n15teredthe 5ameam0unt 0fAChEwa5<15%~8100dV01UmeWa5a55Umedt06e7"5%0f60dy we19ht. Repr0duced w1th perm15510nfr0m 810chem. Pharmac01. (16).

1

a 1r10 "-- [~ 1

75

4,17 nm01eAChE/m0me

1• 4.11 nm01eAChE/ma2e

1 ~

104~78--AC-~-hE/~1~1---1~28

m1n15tered AChE can effect1ve1y 5e4ue5ter 1n v1v0 p0tent 0P5 6ef0re they reach the1r phy5101091ca1 tar9et5, we c0nducted 5y5temat1c 5tud1e5 t0 e5ta6115h a 6a51c, mean1n9fu1 and 4uant1tat1ve c0rre1at10n 6etween the 6100d 1eve15 0f ex09en0u51y adm1n15tered en2yme and the de9ree 0f pr0tect10n a9a1n5t p0150n1n9 6y 0 P (16). We 5e1ected an 0P, 7-(methy1eth0xyph05ph1ny10xy)-1-methy14u1n011n1um10d1de (MEPQ), wh05e d15tr16ut10n 15 re5tr1cted t0 extrace11u1ar 5pace (1,13). A150, the 512e 0f the ChE5 m01ecu1e5 re5tr1ct5 1t t0 extrace11u1ar 5pace. 8a1anced 5tud1e5 c0nducted a5 part 0f the5e 1nve5t19at10n5 have c0nf1rmed th15 5upp051t10n 6y 5h0w1n9 that a11 0f the 1V-1njected AChE can 6e acc0unted f0r 1n the 6100d. MEPQ 1nh161t5 F85-AChE at an extreme1y h19h rate, w1th a 1:1 5t01ch10metry. Rap1d and c0mp1ete 1nh161t10n 0f the en2yme 0ccur5 at re1at1ve1y 10w c0ncentrat10n5 0f MEPQ and AChE (16). F19ure 2 5h0w5 the re5u1t5 0f 1n v1tr0 (2A) and 1n v1v0 (28) t1trat10n5 0f F85-AChE w1th ant1-ChE MEPQ (16). 5t01ch10metr1c am0unt5 0f MEPQ react w1th F85-AChE, thu5 60th enant10mer5 0f MEPQ d15p1ay a 51m11arrate 0f 1nh161t10nt0ward5 F85-AChE. W1th re5pect t0 1n v1v0 t1trat10n, a 11near re1at10n5h1p 6etween the re51dua1 6100d AChE act1v1ty and the d05e 0f MEPQ wa5 065erved. 7h15 11near c0rre1at10n wa5 further 5u65tant1ated 6y determ1n1n9 the pr0tect1ve rat10 c0nferred 6y AChE a9a1n5t MEPQ p0150n1n9.7a61e 4 5ummar12e5 the re5u1t5 0f the determ1nat10n 0f 1V LD50 0f MEPQ 1n F85-AChE-treated m1ce c0mpared t0 the t0x1c1ty 0f MEPQ 1n unpr0tected m1ce. 7he ca1cu1ated pr0tect1ve rat10 wa5 f0und t0 6e 1n rea50na61e a9reement w1th the 065erved pr0tect1ve rat10. (LD50 1n pr0tected an1ma15 d1v1ded 6y the LD50 1n unpr0tected an1ma15.) 1t appear5 that c0mp1ete 1nh161t10n0f the cata1yt1c act1v1ty 0f the ex09en0u5 AChE re4u1re5 appr0x1mate1y 2.5 nm01 0f add1t10na1 MEPQ per m0u5e 0ver the ca1cu1ated 1eve1 a55um1n9 a 1:1 5t01ch10metry. 1t 15 5urpr151n9 t0 n0te that LD50 0f MEPQ 1n untreated m1ce (wt. = 30 9) 15 a150 appr0x1mate1y 2.3 nm01/m0u5e. A5 ment10ned ear11er, MEPQ 15 a per1phera11y act1ve and hydr0ph111c 0P, wherea5 0P5 5uch a5 50man and 5ar1n are 11p0ph111c and cr055 the 6100d-6ra1n 6arr1er. 8ecau5e 0f 1t5 11p0ph111c1ty, the pharmac0k1net1c5, d15tr16ut10n and c1earance 0f 50man w0u1d 6e expected t0 6e d1fferent than VX and MEPQ wh1ch are char9ed m01ecu1e5. 70 dem0n5trate the un1ver5a1 eff1c1ency 0f ch011ne5tera5e a5 an 1n v1v0 5caven9er 0f a11 type5 0f ha2ard0u5 0P5, we

50

~

25 ~-

0

~

2

4

0

t

8

MEPQ, nrn01e/M0U5E F16. 2 (A) 1n v1tr0 t1trat10n0f F85-AChE w1th ant1ch011ne5tera5edru95. 70 the 5ame am0unt 0f AChE (137 un1t5/m11n 2 mM ph05phate, pH 8.0, 25°), 1ncrea51n9am0unt5 0f ant1ChE dru9 were added and 1ncu6atedunt11 c0mp1et10n 0f the 1nh161t10nreact10n (MEPQ, 30 m1n; ph05ph011ne, 2 h; para0x0n, 20 h). 7he 1n5etf19ure5de5cr16e the m01arc0ncentrat10n0f the three 1nh161t0r5re4u1red f0r 100% 1nh161t10n0f en2yme act1v1ty. Repr0duced w1th perm15510nfr0m 810chem. Pharmac01. (16). (8) 1n v1v0 t1trat10n 0f 6100d AChE 1n m1ce pretreated 1V w1th F85-AChE. C0n5ecut1ve 1nject10n5were adm1n15teredat 15 m1n t1me 1nterva15, f0110wed6y a55ay f0r 6100d AChE re51dua1 act1v1ty. Repre5entat1ve re5u1t5 5h0wn are f0r three 1nd1v1dua1m1ce w1th 1n1t1a16100d AChE 1eve15a5 dep1cted 1n the 1n5et. 71trat10n5 were 1n1t1ated 2-3 h after the adm1n15trat10n0f F85ACHE, 1n acc0rdance w1th the t1me pr0f11e50f 6100d AChE 1eve155h0wn 1n F19. 1. Repr0duced w1th perm15510nfr0m 810chem. Pharmac01. (16).

c0mpared the extent t0 wh1ch pretreatment w1th F85-AChE and human 5erum 8ChE c0u1d c0nfer pr0tect10n a9a1n5t 50man and MEPQ (2,3). 7he 1n v1tr0 t1trat10n 0f 8ChE w1th e1ther 50man 0r MEPQ 5h0wed d1fferent t1trat10n curve5 (F19.3A). 7he m01ar c0ncentrat10n 0f 50man re4u1red t0 cau5e 100% 1nh161t10n 0f 8ChE 0r F85-AChE wa5 appr0x1mate1y 1.5- and 2.0-f01d h19her re5pect1ve1y, than the am0unt re4u1red f0r t0ta1 1nh161t10n0f the 5ame en2yme 6y MEPQ (F19. 3A,8). 7h15 065ervat10n 15 c0n515tent w1th the rep0rt (11) that the rat10 0f the rate 0f 1nh161t10n0f h0r5e 5erum 8ChE 6y f0ur enant10mer5 0f 50man 15 1:0.075:0.075: 0.003, wherea5 the rat10 f0r 1nh161t10n 0f AChE 15 1:0.23: <0.0001:0.0001. 80th enant10mer5 0f MEPQ appear t0 1nh161t

126

D 0 C 7 0 R E 7 AL.

7he 1n v1v0 t1trat10n 0f human 5erum 8 C h E and F 8 5 - A C h E w1th 50man c105e1y re5em61e5 the t1trat10n 0f the5e en2yme5 w1th MEPQ. 1n each ca5e the t0ta1 am0unt 0f 0 P 5e4ue5tered 1n v1v0 6y ex09en0u51y adm1n15tered ChE wa5 the 5t01ch10metr1c am0unt neutra112ed acc0rd1n9 t0 1n v1tr0 t1trat10n5 p1u5 the t0x1c1ty 0f 0 P 1n untreated m1ce. 7 h e 5hape 0f the 1n v1v0 t1trat10n curve, 1n th15 part1cu1ar ca5e, 5u99e5t5 that a 5119ht exce55 (0ver the ca1cu1ated va1ue) 0f 50man, w111 6e re4u1red t0 c0mp1ete1y neutra112e c1rcu1at1n9 F85-AChE. 7he5e re5u1t5 1nd1cate that c0mp1ete 1nh161t10n 0f ex09en0u51y adm1n15tered e5tera5e5 re4u1re5 an add1t10na1 am0unt 0f e1ther 50man 0r MEPQ 0ver the 5t01ch10metr1c am0unt5 pred1cted fr0m the c0m61ned re5u1t5 06ta1ned fr0m the 1n v1tr0 t1trat10n5 and the 1n v1v0 neutra112at10n 0f 0P5 1n na1ve m1ce (1.e., 50man t0x1c1ty 1n untreated m1ce). 7h15 15 m0re ev1dent f0r the 1n v1v0 5e4ue5trat10n 0f MEPQ and 50man 6y human 5erum 8ChE. Un11ke 50man, the am0unt 0f MEPQ neutra112ed 6y human 5erum 8 C h E c0u1d 6e acc0unted f0r 0n the 6a515 0f 1n v1tr0 t1trat10n pr0f11e5 p1u5 1t5 t0x1c1ty 1n na1ve m1ce. 51nce the MEPQ d15tr16ut10n 15 11m1ted t0 per1phera1 t155ue5 (1,13), 1t may 6e ar9ued that 1t 15 m0re ava11a61e t0 the c1rcu1at1n9 e5tera5e5, 1n c0mpar150n w1th the 11p1d-501u61e 50man. A150, 0ne w0u1d ant1c1pate that the 1nh161t10n 0f ex09ene0u51y adm1n15tered 8 C h E 0r AChE 1n m1ce 6y repeated adm1n15trat10n 0f 0P5 w111 eventua11y decrea5e the apparent rate 0f the 5e4ue5trat10n. H0wever, 1n the ca5e 0f the 11p1d 501u61e 50man, 5uch a decrea5e may a110w m0re 0 P t0 e5cape the 5caven9er and reach 0ther 51te5 wh1ch are capa61e 0f react1n9 w1th

7A8LE 4 PR07EC710N-RA710 C0NFERRED 8Y AChE A6A1N57 MEPQ P0150N1N6* ACHE•[" Un1t5/M0u5e Unpr0tected m1ce 474 - 7 (1.2) 808 - 23 (2.0) 1387 - 19 (3.5) 2246 - 46 (5.6) 2606 - 48 (6.5)

Pr0tect1ve Rat10•

LD~0:~ (1V) 1.~9/k9

065erved

Ca1cu1ated•

30 (28-33)# 46 (38-56) 60 (53-69) 80 (66-96) 121 (111-132) 124 (108-141)

1.0 1.5 2.0 2.7 4.0 4.1

-1.5 1.9 2.5 3.5 3.9

*MEPQ wa5 adm1n15tered (1V) 15 m1n f0110w1n91V 1nject10n0f ACHE. /•Avera9e - 5EM 6100d-AChE act1v1typr10r t0 MEPQ 1nject10n;N = 12. 7he f19ure5 1n parenthe5e5 are nm01/m0u5e (a55um1n9 1 nm01=400 un1t5). :[:95% c0nf1dence 11m1t5. ~LD50 1n treated m1ce d1v1ded 6y LD50 1n untreated m1ce. •Ca1cu1ated 0n the a55umpt10n 0f 1: 1 5t01ch10metry 0f MEPQ 5e4ue5ter1n9 6y AChE and an avera9e LD50 0f 2.27 nm01 MEPQ per m0u5e. #MEPQ t0x1c1ty 1n C8DP-treated m1ce: LD~0=9.3 (8.2-10.4) 1J.9/k9 (determ1ned 90-120 m1n f0110w1n9 an 1M 1nject10n 0f 5 m9/k9 C8DP). Repr0duced w1th perm15510n fr0m 810chem. Pharmac01. (16).

human 5erum 8 C h E at a 51m11ar rate, 51nce a 5t01ch10metr1c am0unt 0f racem1c MEPQ react5 w1th 8ChE. 7he 1n v1v0 t1trat10n 0f human 5erum 8 C h E w1th MEPQ (F19. 3C) wa5 51m11ar t0 the 1n v1v0 t1trat10n 0f F 8 5 - A C h E w1th MEPQ (5ee F19. 28). A near1y 11near re1at10n5h1p 6etween re51dua1 human 5erum 8 C h E act1v1ty and MEPQ d05e wa5 065erved. A9a1n, 1n 0rder t0 c0mp1ete1y 1nh161t the ex09en0u51y adm1n15tered 8 C h E an add1t10na1 2 nm01e MEPQ/m0u5e wa5 re4u1red. 7h15 065ervat10n 15 c0n515tent w1th the 1V LD50 0f MEPQ 1n untreated m1ce (2.2 nm01/m0u5e).

8uChE

=~

50man.

When a h19h d05e 0f e1ther 50man 0r MEPQ wa5 adm1n15tered 1n an 1V 601u5 1nject10n t0 m1ce pretreated w1th human 5erum 8 C h E 0r w1th F85-AChE, pr0tect10n aff0rded 6y the ex09en0u5 e5tera5e wa5 5119ht1y 1e55 effect1ve, 1n c0mpar150n w1th the 5ame am0unt 0f 0P5 adm1n15tered 1n 3-5 c0n5ecut1ve d05e5 (2,3). 7h15 065ervat10n 5u99e5t5 that pr0tect10n 0f an1ma15 a9a1n5t 0 P p0150n1n9 w1th 5caven9er pr0phy1ax15 depend5, am0n9 0ther fact0r5, 0n 60th the pharmac0k1net1c pr0pert1e5 0f the 0 P cha11en9e and the rate 0f 1t5 1n v1v0 5e4ue5trat10n 6y the c1rcu1at1n9 e5tera5e.

AChE

100-

~00

75

75

75

50

50

50

25 •

25

25

38

3C

100

8uChE*

3

6

9

Anf1-ChE X 10 "8 M

12

10 15 Ant1-ChE X 10 "7 M

=1

110 115 2•0 Ant1•ChE, nm01e/m0U5e

215

F16. 3 (A, 8). 1n v1tr0 t1trat10n 0f 8ChE and F85-.~,ChE 6y MEPQ and 50man. Human 5erum 8ChE (4.5 U/m1) and F85-AChE (280 U/m1), 1n 2 mM ph05phate 6uffer, pH 8.0, were 1ncu6ated w1th 1ncrea51n9 am0unt5 0f MEPQ and 50man. Repr0duced w1th perm15510n fr0m 810chem. Pharmac01. (3). (C) 1n v1v0 (N=3) t1trat10n5 0f F85-AChE and human 5emm 8ChE 6y 50man and MEPQ. m: F85-AChE (6.2 nm01e1m0u5e) t1trated w1th 50man. ~: Human 5erum 8ChE (5.16 nm01e5/m0u5e) t1trated w1th MEPQ. Q: Human 5erum 8ChE (10.2 nm01e/m0u5e) t1trated w1th 50man. Cumu1at1ve d05e 0f 0P wh1ch reduced ChE act1v1ty t0 the 1nd1cated f1na1 1eve15exceeded the am0unt 0f ChE adm1n15tered, 5u99e5t1n9 the 1nv01vement 0f end09en0u5 e5tera5e. Repr0duced fr0m 810chem. Pharmac01. (3).

EN2YME5 A5 PRE7REA7MEN7 D R U 6 5 F 0 R 0 P 70X1C17Y

120. A

/ e

4A

80.

M0u5e

51-.-

/

120- 48

M0u=e /e R0t

Rat

80-

8E 0 0

40, /• • 0

0

6u1nea p19

6u1ne0 p19

Ra661t 1

2

1

1

4 6 P10=m0C0E(uM)

8•

00.0 C01F •

01.5 1•.0 tC1rC(UM m 1 n )

F16. 4 (A) Effect 0f p1a5ma CaE 0n 50man LD50 5C 1n d1fferent 5pec1e5. (8) Effect 0f the pr0duct 0f p1a5ma CaE and c1rcu1at10n t1me (CaEXtc1rc) 0n 5pec1e5 var1at10n 1n 50man LD50 5C. C1rcu1at10n t1me5 1n 5pec1e5 (m0u5e, 0.14 m1n; rat, 0.24 m1n, 9u1nea p19, 0.27 m1n and ra661t, 0.43 m1n1.

7heref0re, f0r d1fferent 0P5, a var1at10n 1n the en2yme: 0 P rat10 1n c1rcu1at10n may 6e nece55ary t0 c0nfer the 5ame pr0tect1ve rat10 1n 0P-cha11en9ed an1ma15. De5p1te a p0tent1a1 reduct10n 1n the re1at1ve aff1n1ty 0f 8ChE f0r the f0ur enant10mer5 0f 50man 1n m1ce, 1 m01e 0f human 5erum 8ChE 15 capa61e 0f neutra1121n9 appr0x1mate1y 0ne m01e 0f a racem1c m1xture 0f 50man, 1n a manner ana1090u5 t0 F85-AChE. 7heref0re, ex09en0u5 adm1n15trat10n 0f e1ther 8ChE 0r AChE can 6e an effect1ve pr0phy1act1c mea5ure, 5uff1c1ent t0 pr0tect m1ce fr0m mu1t1p1e LD505 0f the h19h1y p0tent ant1-ChE, 50man, w1th0ut the adm1n15trat10n 0f 5upp0rt1ve dru95. EFFEC7 0 F C1RCULA710N 71ME 0 F E57ERA5E 0 N 175 5CAVEN6ER EFF1C1ENCY

When re1at10n5h1p5 6etween p1a5ma CaE and 50man LD50 va1ue5 were eva1uated 1n d1fferent 5pec1e5, a d1rect pr0p0rt10na11ty wa5 n0t 065erved [F19. 4A; (14)]. P1a5ma CaE 5caven9er5 were n0t a5 effect1ve 1n 5ma11 an1ma15 (e.9., m1ce) a5 1n 1ar9er an1ma15 (e.9., ra661t5). 51nce the react10n 6etween p1a5ma 5caven9er and 50man 15 dependent 0n 60th p1a5ma CaE 1eve1 and the t1me t0 react, the r01e 0f c1rcu1at10n t1me 0n 5caven9er eff1c1ency wa5 c0n51dered. 1f the p1a5ma CaE 1eve1 wa5 mu1t1p11ed 6y the c1rcu1at10n t1me, wh1ch 15 the t1me ava11a61e f0r the p1a5ma CaE t0 react w1th 50man 6ef0re 1t 9et5 t0 the t0x1c 51te ACHE, the 50man LD50 wa5 d1rect1y pr0p0rt10na1 t0 the pr0duct 0f p1a5ma CaE and c1rcu1at10n t1me (F19.48). 7he5e re5u1t5 dem0n5trate the fact the c1rcu1at10n t1me f0r ChE5 p1ay5 a p1v0ta1 r01e 1n determ1n1n9 the eff1cacy 0f var10u5 5caven9er5 f0r 0r9an0ph05phate5. 5EQUE57RA710N 0 F 0 R 6 A N 0 P H 0 5 P H A 7 E 5 8Y F85-ACHE 1N N0NHUMAN PR1MA7E5

5ucce55fu1 dem0n5trat10n 0f a5ympt0mat1c pr0tect10n 0f m1ce a9a1n5t three d1fferent 0P5 6y pretreatment w1th tw0 ChE5 5u99e5t5 a p0tent1a1 ut111ty f0r 5uch pr0phy1ax15 1n h19her mamma15. 7heref0re an eva1uat10n 0f 5e4ue5trat10n 0f 0P5 6y ChE 1n a n0nhuman pr1mate 6ecame de51ra61e. We c0nducted pre11m1nary 1nve5t19at10n5 (7), u51n9 a 11m1ted num6er 0f marm05et5 (Ca/11thr1x jacchu5), pur1f1ed F85-AChE a5 a pretreatment dru9, and MEPQ a5 an 0 P 51nce 1t5 d15tr16ut10n 15 11m1ted t0 per1phera1 5pace5. Adm1n15trat10n (1V) 0f appr0x1mate1y 5000 un1t5 0f pur1f1ed F85AChE t0 a marm05et and m0n1t0r1n9 0f 6100d AChE 1eve1 0ver a 23-h per10d 5h0wed e55ent1a11y the 5ame pr0f11e a5 de5cr16ed 1n

127

F19. 1 f0r m1ce. A c0n5tant 1eve1 0f en2yme wa5 ma1nta1ned dur1n9 1-4 h p05t1nject10n 1n marm05et5. 1n an0ther 5et 0f exper1ment5 appr0x1mate1y 25-30 nm01e5 0f AChE act1ve 51te5 wa5 adm1n15tered t0 each 0f tw0 an1ma15.7he an1ma15 were cha11en9ed w1th tw0 1V 601u5 d05e5 0f MEPQ at a60ut ha1f h0ur 1nterva15. 7he re5u1t5 (n0t 5h0wn) 1nd1cate that the ex09en0u51y adm1n15tered F85-AChE 15 effect1ve1y a61e t0 5e4ue5ter MEPQ 1n marm05et5. A 11near c0rre1at10n ex15t5 6etween the re51dua1 6100d AChE 1eve1 and the am0unt 0f MEPQ adm1n15tered, 1.e., 0ne m01e 0f F85-AChE act1ve 51te 15 capa61e 0f 5e4ue5ter1n9 appr0x1mate1y 0ne m01e 0f MEPQ. A5 065erved 1n m1ce, an add1t10na1 am0unt 0f MEPQ w111 6e needed t0 c0mp1ete1y 1nh161t ex09en0u51y adm1n15tered F85-AChE. 7h15 re4u1rement 0f the add1t10na1 am0unt 0f MEPQ may 6e a5cr16ed t0 1t5 det0x1f1cat10n 1n na1ve an1ma1 p1a5ma. Hav1n9 06ta1ned the5e data fr0m pre11m1nary 1nve5t19at10n5 w1th marm05et5, we then carr1ed 0ut 5tud1e5 1n rhe5u5 m0nkey5, u51n9 F85-AChE a5 a pretreatment dru9 and 50man a5 the cha11en91n9 0 P (8). E55ent1a11y the 5ame pr0t0c01 wa5 u5ed w1th m0nkey5 and marm05et5, except that the m0nkey5 were tra1ned t0 perf0rm 5er1a1 pr06e rec09n1t10n (5PR) te5t5 t0 mea5ure the1r perf0rmance. 7he m0nkey5 were re4u1red t0 perf0rm the5e te5t5 (a) w1th0ut any treatment, (6) after 1V adm1n15trat10n 0f F85-AChE a10ne, and (c) after 1V adm1n15trat10n 0f F85-AChE and cha11en9e w1th a t0ta1 am0unt 0f 50man e4u1va1ent t0 an LD100 a5 the 5um 0f tw0 d05e5 [(LD50 0f 50man (1V)=6.5 p.9/k9]. A5 expected, pre11m1nary re5u1t5 a9a1n 5h0wed that 1) a 11near c0rre1at10n ex15t5 6etween re51dua1 6100d AChE 1eve1 and the 50man cha11en9e d05e, 1.e., 0ne m01e 0f F85-AChE act1ve 51te 15 capa61e 0f 5e4ue5ter1n9 0ne m01e 0f 50man 1n m0nkey5, 2) the m0nkey5 5urv1ved the LD100 cha11en9e 0f 50man and 5h0wed n0 5ympt0m5 0f 0 P t0x1c1ty de5p1te the a65ence 0f 5upp0rt1ve dru95, and 3) the 5PR 5h0wed the m0nkey5 d1d n0t exper1ence any perf0rmance decrement5 a5 a re5u1t 0f the adm1n15trat10n 0f en2yme 0r the 5u65e4uent cha11en9e w1th an LD100 d05e 0f 50man.

AN1MAL 7 0 MAN EX7RAP0LA710N

Extrap01at10n fr0m an1ma1 t0 man 0f the eff1cacy 0f the m1xture 0f ant1d0ta1 dru95 a9a1n5t 0 P p0150n1n9 mu5t 6e v1ewed w1th extreme caut10n, 60th 6ecau5e 0f the t0x1c01091ca1 d1fference5 f0110w1n9 0 P p0150n1n9 am0n9 the exper1menta1 an1ma15 and 6ecau5e 0f 5pec1e5 d1fference5 1n the a650rpt10n, d15tr16ut10n, and meta60115m 0f ant1d0te5 u5ed. Re5u1t5 de5cr16ed here 5u99e5t that the en2yme 5caven9er pr0phy1ax15 appr0ach 15 n0t 0n1y an eff1c1ent 51n91e dru9 ant1d0te 6ut a150 a p0tent1a1 pr0phy1act1c mea5ure f0r human app11cat10n. 0 f part1cu1ar 1mp0rtance, 1n th15 re5pect, 15 the a6111ty t0 pred1ct re5u1t5 1n human5 fr0m an1ma1 exper1ment5. 1n c0ntra5t t0 the mu1t1dru9 treatment appr0ach, extrap01at10n 0f re5uh5 fr0m an1ma15 t0 human5 appear5 t0 6e 5afer and m0re re11a61e f0r the 5caven9er, 51nce the 0 P 15 c0unteracted 1n c1rcu1at10n 6ef0re 1t can reach the tar9et 0r9an. Man1fe5tat10n 0f t0x1c 5ympt0m5 can 6e prevented and at the 5ame t1me the pr061em 0f 5pec1e5-dependent re5p0n5e t0 the p0150n 1t5e1f 15 a150 e11m1nated. Furtherm0re, c0m61nat10n therapy c0n515t1n9 0f var10u5 dru95, wh1ch are kn0wn t0 have 5pec1e5 dependent eff1cacy, 15 n0t needed. 7he 5ame 610chem1ca1 pr1nc1p1e under11e5 the eff1cacy 0f the 5caven9er c0ncept 1rre5pect1ve 0f the 5pec1e5 ut1112ed. 1n v1v0 5t01ch10metry and c0rre1at10n 6etween p1a5ma ChE 1eve15 and pr0tect10n dem0n5trate that ex09en0u5 e5tera5e 5h0u1d pr0v1de 51m11ar pr0tect10n 1n a11 an1ma15. 7hu5, the5e 065ervat10n5, a10n9 w1th the 1nf1uence 0f c1rcu1at10n t1me 1n d1fferent 5pec1e5 0n 5caven9er effect1vene55, 5h0u1d a110w 5afe extrap01at10n fr0m an1ma15 t0 human5.

128

D0C70R

E 7 AL.

REFERENCE5 1. A5han1, Y.; Levy, D.; Heyman, E.; 6runfe1d, Y.; Kadar, 7.; 5p1e9e15te1n, M. A 4uartemary ant1ch011ne5tera5e pr06e f0r determ1n1n9 the 1nte9r1ty 0f the 6100d-6ra1n 6arr1er. L1fe 5c1. 36:493-497; 1985. 2. A5han1, Y.; 5hap1ra, 5.; D0ct0r, 8. P.; Levy, D.; Raveh, L. 1n v1v0 5t01ch10metry 0f ch011ne5tera5e pr0phy1ax15 a9a1n5t 0r9an0ph05phate p0150n1n9. Pr0c. 3rd 1nt. 5ymp. A9a1n5t Chem1ca1 Warfare A9ent5. Umea, 5weden; 3:227-234; 1989. 3. A5han1, Y.; 5hap1ra, 5.; Levy, D.; W01fe, A. D.; D0ct0r, 8. P.; Raveh, L. 8utyry1ch011ne5tera5e and acety1ch011ne5tera5e pr0phy1ax15 a9a1n5t 50man p0150n1n9 1n m1ce. 810chem. Pharmac01., 1n pre55; 1991. 4. 805k0v1c, 8. 7he 1nf1uence 2-(0-cre5y1)-4 H-1:3:2:-6en20d10xaph05ph0r1n-2-0x1de (C8DP) 0n 0r9an0ph05phate p0150n1n9 and 1t5 therapy. Arch. 70x1c01. 42:207-216; 1979. 5. C1ement, J. 6. 1mp0rtance 0f a11e5tera5e a5 a det0x1f1cat10n mechan15m f0r 50man (p1nac01y1 methy1ph05ph0n0-f1u0r1date) 1n m1ce. 810chem. Pharmac01. 33:3807-3811; 1984. 6. De La H02, D.; D0ct0r, 8. P.; Ra15t0n, J. 5.; Ru5h, R. 5.; W01fe, A. D. A 51mp11f1edpr0cedure f0r the pur1f1cat10n 0f 1ar9e 4uant1t1e5 0f mamma11an acety1ch011ne5tera5e. L1fe 5c1. 39:195-199; 1986. 7. D0ct0r, 8. P.; A5han1, Y.; Raveh, L.; W01fe, A. D.; Maxwe11, D. M. 5e4ue5trat10n 0f 0r9an0ph05phate 6y feta1 60v1ne 5erum acety1ch011ne5terae5 1n marm05et5. 1n preparat10n; 1991. 8. D0ct0r, 8. P.; F1n9er, A.; Maxwe11, D. M.; McMa5ter, 5.; 501ana, R. L.; W01fe, A. D.; W00dard, C. Pr0tect10n 0f rhe5u5 m0nkey5 fr0m 50man t0x1c1ty 6y pretreatment w1th feta1 60v1ne 5erum acety1ch011ne5tera5e. 1n preparat10n; 1991.

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