Neur05c1ence• 8106ehav10ra1Rev1ew5.V01. 15, pp. 135-139. Per9am0n Pre55 p1c. 1991. Pr1nted 1n the U.5.A.
0149-7634191 $3.00 + .00
7he R01e 0f Car60xy1e5tera5e 1n 5pec1e5 Var1at10n 0f 0x1me Pr0tect10n A9a1n5t 50man D 0 N A L D M. M A X W E L L A N D K A R E N M. 8 R E C H 7
U.5. Arm), Med1ca1 Re5earch 1n5t1tute 0 f Chem1ca1 Defen5e A6erdeen Pr0v1n9 6r0und, MD 21010-5425
MAXWELL, D. M. AND K. M. 8RECH7. 7he r01e 0f car60xy1e5tera5e 1n 5pec1e5 var1at10n 0f 0x1me pr0tect10n a9a1n5t50man. NEUR05C1 8108EHAV REV 15(1) 135-139, 1991.--0x1me pr0tect10n a9a1n5t 50man, a h19h1y t0x1c ant1ch011ne5tera5e a9ent, wa5 exam1ned 1n m1ce and 9u1nea p195.7he max1ma1 pr0tect10n pr0duced 6y the 0x1me5 PAM and H1-6 var1ed a5 much a5 6-f01d 6etween the5e 5pec1e5. 51nce end09en0u5 ear60xy1e5tera5e (CaE) 15 "kn0wn t0 6e an 1mp0rtant determ1nant 0f 5pec1e5 var1at10n 1n 50man t0x1c1ty, the pr0tect10n 0f PAM and H1-6 a9a1n5t 50man wa5 a150 mea5ured 1n an1ma15 wh05e end09en0u5 CaE wa5 1nh161ted w1th cre5y16en20d10xaph05ph0r1n 0x1de. 1n CaE-1nh161ted an1ma15 the 50man LDa0 va1ue5 were 51m11ar 1n unpr0tected m1ce and 9u1nea P195 (10.2 v5. 12.2 p.9/k9) and 0x1me-pr0tected m1ce and 9u1nea p195 (38.1 v5. 40.3 ~9/k9 f0r PAM; 159 v5. 151 1-L9/k9 f0r H1-6). 7he 1eve150f 0x1me pr0tect10n 065erved 1n CaE-1nh161ted an1ma15 a9reed w1th prev10u5 exper1ment5 1n 0ther 5pec1e5 that have n0 end09en0u5 p1a5ma CaE. 7he 4--5 t1me5 9reater 1n v1v0 pr0tect10n a9a1n5t 50man 0f 1-11-6v5. PAM 1n CaE-1nh161ted an1ma15 c0rre1ated w1th 1n v1tr0 exper1ment5 1n wh1ch 1-11-6pr0duced 3-5 t1me5 m0re 0x1me react1vat10n 0f 50man-1nh161ted AChE than PAM. 0x1me pr0tect10n
50man
Car60xy1e5tera5e
5pec1e5var1at10n
t10n 0f new therapeut1C 0X1me5 (3, 12, 17). 0ne 0f the m05t eXten51ve1y 5tUd1ed new 0X1me5 15 1-(((4(am1n0Car60ny1) pyr1d1n0)-meth0Xy) methy1-2-(hydr0Xy1m1n0) methy1) pyr1d1n1Um d1Ch10r1de (H1-6), Wh1Ch 15 c1ear1y 5Uper10r t0 PAM a5 an 1n v1tr0 reaCt1Vat0r 0f 50man-1nh161ted AChE (10). When te5ted 1n V1v0, H1-6 1n C0m61nat10n W1th atr0p1ne pr0tected a9a1n5t 1.9 LD50 0f 50man 1n m1Ce, 2.1 LD50 1n rat5, 3.5 LD50 1n 9U1nea p195, 9.0 LD50 1n d095, and 5 LD50 1n rhe5U5 m0nkey5 (3,17). 7h15 var1at10n 1n the pr0tect10n 0f H1-6 a9a1n5t 50man may re5U1t fr0m 5peC1e5 d1fference5 1n the pharmaC01091Ca1 re5p0n5e t0 e1ther H1-6 0r 50man. 5pec1e5 var1at10n 1n the pharmac0k1net1c5 a5 we11 a5 the pharmac0dynam1c5 0f H1-6 and 50man have 6een rep0rted. F0r examp1e, the 5erum e11m1nat10n ha1f-11fe f0r H1-6 var1ed 1n m1ce, rat5, and d095 (7, 20, 21, 25, 26), and the pharmac0k1net1c5 0f 50man var1ed 1n rat5, 9u1nea p195, and marm05et5 (1). W1th re9ard t0 5pec1e5 var1at10n 1n the phamaac0dynam1c5 0f H1-6 and 50man, the re5t0rat10n 6y H1-6 0f n0rma1 funct10n after 50man-1nduced neur0mu5cu1ar 610ckade var1ed am0n9 human5, d095, rat5, and 9u1nea p195 (27,32), and the LD50 f0r 50man var1ed 13-f01d am0n9 m1ce, rat5, 9u1nea p195, ra661t5, d095, and marm05et5 (3, 11, 22). 1n an attempt t0 under5tand the nature 0f 5pec1e5 var1at10n 1n 0x1me pr0tect10n, we eva1uated the de9ree 0f pr0tect10n that c0u1d 6e ach1eved a9a1n5t 50man w1th pharmac01091ca11y e4u1va1ent
7HE t0x1c1ty 0f 0r9an0ph05ph0ru5 a9ent5 15 6e11eved t0 6e due t0 the1r 1nh161t10n 0f acety1ch011ne5tera5e (ACHE), an en2yme that term1nate5 the act10n 0f the neur0tran5m1tter acety1ch011ne 6y cata1y21n9 1t5 hydr01y515. A1th0u9h 1nh161t10n 0f AChE pr0duce5 a var1ety 0f ch011ner91c effect5 6y 1ncrea51n9 acety1ch011ne at ch011ner91c 5ynap5e5, 1nh161t10n 0f AChE 1n 6ra1n and d1aphra9m 15 part1cu1ar1y 1mp0rtant 6ecau5e death fr0m 0r9an0ph05ph0ru5 a9ent5 15 u5ua11y due t0 re5p1rat0ry fa11ure (31). 51nce the d15c0very 0f pyr1d1ne-2-a1d0x1me methy1ch10r1de (PAM), 0x1me5 1n c0m61nat10n w1th atr0p1ne (A7R) have 6ec0me the 5tandard therapy a9a1n5t the t0x1c1ty 0f 0r9an0ph05ph0ru5 a9ent5 (12,31). React1vat10n 0f 0r9an0ph05ph0nate-1nh161ted AChE 6y 0x1me5 can 9enerate en0u9h act1ve ACHE, part1cu1ar1y 1n d1aphra9m, t0 re5t0re n0rma1 ch011ner91c neur0tran5m15510n (5, 6, 24, 30). A1th0u9h 0x1me5 have pr0duced pr0tect10n a9a1n5t 0r9an0ph05ph0ru5 a9ent5 1n many mamma11an 5pec1e5, c0n51dera61e 5pec1e5 var1at10n ha5 6een rep0rted f0r 0x1me pr0tect10n a9a1n5t p1nac01y1methy1 ph05ph0n0f1u0r1date (50man), 0ne 0f the m05t refract0ry 0r9an0ph05ph0ru5 a9ent5. F0r examp1e, PAM 1n c0m61nat10n w1th atr0p1ne pr0tected a9a1n5t 1.3 LD50 0f 50man 1n m1ce, 1.8 LD50 1n rat5, 2.1 LD50 1n 9u1nea p195, and 3.5 LD50 1n d095 (3). 7h15 5pec1e5 var1at10n 1n the a6111ty 0f 0x1me5 t0 pr0tect a9a1n5t 50man ha5 made 1t d1ff1cu1t t0 extrap01ate 0x1me pr0tect10n fr0m an1ma15 t0 human5, wh1ch ha5 h1ndered the ad0p-
J7he 0p1n10n5 0r a55ert10n5 c0nta1ned here1n are the pr1vate v1ew5 0f the auth0r5 and are n0t t0 6e c0n5trued a5 0ff1c1a1 0r a5 ref1ect1n9 the v1ew5 0f the Army 0r the Department 0f Defen5e. 1n c0nduct1n9 the re5earch de5cr16ed 1n th15 rep0rt, the 1nve5t19at0r5 adhered t0 the ••6u1de f0r the Care and U5e 0f La60rat0ry An1ma15,•• Nat10na1 1n5t1tute5 0f Hea1th pu611cat10n 85-23.
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136
MAXWELL AND 8RECH7
d05e5 0f PAM 0r H1-6 and e4u1t0x1c d05e5 0f 50man 1n m1ce and 9u1nea p195. 7he5e tw0 5pec1e5 exh161t the 9reate5t var1at10n 1n 0x1me pr0tect10n a9a1n5t 50man 0f the an1ma15 ava11a61e 1n 5uff1c1ent 4uant1ty f0r d05e-re5p0n5e eva1uat10n5. 7he u5e 0f pharmac01091ca1 and t0x1c01091ca1 e4u1va1ence wa5 5ucce55fu11n exp1a1n1n9 the 5pec1e5 var1at10n 1n car6amate pr0tect10n a9a1n5t 50man (23), and we fe1t that th15 appr0ach c0u1d 6e e4ua11y u5efu1 f0r under5tand1n9 5pec1e5 var1at10n 1n 0x1me pr0tect10n. 7he cr1ter10n f0r pharmac01091ca1 e4u1va1ence 6etween 5pec1e5 f0r PAM and H1-6 wa5 the ach1evement 0f max1ma1 pr0tect1ve effect a9a1n5t 50man 1n each 5pec1e5. E4u1t0x1c d05e5 0f 50man (1.e., e4ua1 LD50) were ach1eved 1n each 5pec1e5 6y pretreat1n9 an1ma15 w1th 2-(0-cre5y1) 4H-1 : 3 : 2 6en20d10xaph05ph0r1n-2-0x1de (C8DP). C8DP 15 an 1nh161t0r 0f car60xy1e5tera5e (CaE) (2), a 50man-det0x1fy1n9 en2yme that 15 an 1mp0rtant determ1nant 0f 5pec1e5 var1at10n 1n 50man t0x1c1ty (22). 7he purp05e 0f the pre5ent 5tudy wa5 1) t0 determ1ne the 1n v1v0 d05e-re5p0n5e 0f 0x1me therapy 1n c0m61nat10n w1th atr0p1ne a9a1n5t 50man 1n m1ce and 9u1nea p195, 2) t0 c0mpare the max1ma1 1n v1v0 pr0tect1ve effect 0f 0x1me/atr0p1ne therapy 1n c0ntr01 and CaE-1nh161ted an1ma15, and 3) t0 exam1ne the re1at10n5h1p 6etween 1n v1tr0 0x1me react1vat10n 0f 50man-1nh161ted AChE and 1n v1v0 0x1me pr0tect10n. ME7H0D
An1ma15 A t0ta1 0f 480 hea1thy ma1e C r 1 : C 0 8 5 (HA) 9u1nea p195
(Cav1a p0rce11u5) we19h1n9 330 t0 360 9, and 800 ma1e Cr1:C085 CD-1(1CR) m1ce (Mu5 mu5cu1u5) we19h1n9 22 t0 26 9 were 06ta1ned fr0m Char1e5 R1ver La60rat0r1e5 (W11m1n9t0n, MA). 7he5e an1ma15 were 4uarant1ned 0n arr1va1 and 5creened f0r ev1dence 0f d15ea5e. 7he 9u1nea p195 were 1nd1v1dua11yh0u5ed wh11e the m1ce were h0u5ed 1n 9r0up5 0f ten 1n p01ycar60nate 5h0e60x ca9e5 (La6 Pr0duct5, Mayw00d, NJ) 0n 8eta-Ch1p hardw00d ch1p c0ntact 6edd1n9 (N0rthea5tern Pr0duct5 C0rp., Warren56ur9, NY) w1th c0mp1ete ca9e chan9e tw0 t1me5 per week. An1ma15 were pr0v1ded w1th c0mmerc1a1 cert1f1ed 9u1nea p19 0r r0dent ch0w (21e91er 8r05., 6ardner5, PA), a5 appr0pr1ate, and tap water ad 116. An1ma1 r00m5 were ma1nta1ned at 20-22°C and re1at1ve hum1d1ty 0f 50•+ 10% w1th at 1ea5t 10 c0mp1ete a1r chan9e5 per h0ur 0f 100% c0nd1t10ned fre5h a1r. A11 an1ma15 were 0n a 12-h0ur 119ht/dark fu11 5pectrum 119ht1n9 cyc1e w1th n0 tw1119ht.
Mater1a15 50man wa5 06ta1ned fr0m the Chem1ca1 Re5earch, Deve10pment and En91neer1n9 Center (A6erdeen Pr0v1n9 6r0und, MD) and wa5 98.6% pure. 7he 0x1me5 H1-6 d1ch10r1de and PAM ch10r1de were 06ta1ned fr0m the Defence Re5earch E5ta6115hment (5uff1e1d, Canada) and Ayer5t La60rat0r1e5 (New Y0rk, NY), re5pect1ve1y. C8DP wa5 5ynthe512ed 6y 5tark5 A550c1ate5 (8uffa10, NY) and wa5 99.5% pure. Atr0p1ne 5u1fate, acety1th10ch011ne, and 7r1t0n X-100 were purcha5ed fr0m 519ma Chem1ca1 C0. (5t. L0u15, M0). 7he chem1ca1 pur1t1e5 0f C8DP and 50man were determ1ned 6y 31p-nuc1ear ma9net1c re50nance 5pectr05c0py. A11 0ther dru95 0r chem1ca15 were U5P 0r rea9ent 9rade, re5pect1ve1y.
An1ma1 Exper1ment5 PAM ch10r1de, H1-6 d1ch10r1de, and atr0p1ne 5u1fate were adm1n15tered 1M a5 501ut10n5 1n 150t0n1c 5a11ne. C8DP wa5 adm1n15tered 5C a5 a 501ut10n 1n pr0py1ene 91yc01 c0nta1n1n9 5% ethan01. 50man wa5 adm1n15tered 5C a5 a 501ut10n 1n 150t0n1c 5a11ne. 1nject10n v01ume5 were 1 m1/k9 1n m1ce and 9u1nea p195. An1ma15
rece1v1n9 mu1t1p1e 1nject10n5 were 1njected 1n a1ternate h1nd 11m65. LD50 va1ue5 were ca1cu1ated 6y pr061t ana1y515 (15) 0f death5 0ccurr1n9 w1th1n 24 h after adm1n15trat10n 0f 50man at f1ve d1fferent d05e5 w1th e1ther 51x 9u1nea p195 0r ten m1ce per d05e.
AChE React1vat10n 0x1me react1vat10n exper1ment5 were perf0rmed 0n 501u61112ed AChE preparat10n5 fr0m 60th 6ra1n and d1aphra9m 0f 9u1nea p195 and m1ce. M1ce and 9u1nea p195 were euthan12ed w1th car60n d10x1de. Wh01e 6ra1n and d1aphra9m were 4u1ck1y rem0ved and r1n5ed 5evera1 t1me5 w1th 1ce-c01d 0.15 M NaC1. 8ra1n t155ue wa5 h0m09en12ed 1n 9 v01ume5 0f 1ce-c01d 0.01 M ph05phate 6uffer, pH 7.4, c0nta1n1n9 0.15 M NaC1 and 1% 7r1t0n X-100 6y u51n9 a P0tter-E1vehjem t155ue h0m09en12er. D1aphra9m t155ue wa5 h0m09en12ed 1n 4 v01ume5 0f 1ce-c01d 0.01 M ph05phate 6uffer, pH 7.4, c0nta1n1n9 0.15 M NaC1 6y u51n9 a P01ytr0n h0m09en12er (8r1nkman 1n5trument5, We5t6ury, NY). After P01ytr0n h0m09en12at10n each d1aphra9m h0m09enate rece1ved an am0unt 0f 7r1t0n X-100 e4ua1 t0 1% 0f 1t5 v01ume and wa5 h0m09en12ed a9a1n u51n9 a P0tter-E1vehjem h0m09en12er. 8ra1n and d1aphra9m h0m09enate5 were centr1fu9ed at 30,000 × 9 f0r 30 m1n at 4-6°C, and the re5u1tant 5upematant5 c0nta1n1n9 501u61112ed AChE were 5t0red at - 20°C. 7he react1vat10n 0f 50man-1nh161ted AChE 6y 0x1me wa5 perf0rmed 6y the meth0d 0f De J0n9 et a1. (9). 501u61112ed AChE that had 6een d11uted 1n 3 v01ume5 0f 0.01 M ph05phate 6uffer, pH 7.4, c0nta1n1n9 0.15 M NaC1 wa5 1nh161ted w1th 10 nM 50man f0r 10 m1n at 37°C. Exce55 1nh161t0r wa5 rem0ved 6y three extract10n5 w1th an e4ua1 v01ume 0f hexane. 1nh161ted AChE wa5 react1vated f0r 30 m1n 6y add1t10n 0f 4 v01ume5 0f 0.05 M ph05phate 6uffer, pH 7.4, c0nta1n1n9 0.33 mM H1-6 0r PAM. AChE act1v1ty wa5 mea5ured 6y the meth0d 0f E11man (14) w1th acety1th10ch011ne a5 5u65trate.
Data Ana1y515 519n1f1cant d1fference5 6etween pa1r5 0f LD50 va1ue5 were 1dent1f1ed 6y the Newman-Keu15 te5t (18). En2yme act1v1t1e5 were c0mpared 6y 5tudent•5 t-te5t. D1fference5 were c0n51dered 519n1f1cant 1f p
0149-7634191 $3.00 + .00
7he R01e 0f Car60xy1e5tera5e 1n 5pec1e5 Var1at10n 0f 0x1me Pr0tect10n A9a1n5t 50man D 0 N A L D M. M A X W E L L A N D K A R E N M. 8 R E C H 7
U.5. Arm), Med1ca1 Re5earch 1n5t1tute 0 f Chem1ca1 Defen5e A6erdeen Pr0v1n9 6r0und, MD 21010-5425
MAXWELL, D. M. AND K. M. 8RECH7. 7he r01e 0f car60xy1e5tera5e 1n 5pec1e5 var1at10n 0f 0x1me pr0tect10n a9a1n5t50man. NEUR05C1 8108EHAV REV 15(1) 135-139, 1991.--0x1me pr0tect10n a9a1n5t 50man, a h19h1y t0x1c ant1ch011ne5tera5e a9ent, wa5 exam1ned 1n m1ce and 9u1nea p195.7he max1ma1 pr0tect10n pr0duced 6y the 0x1me5 PAM and H1-6 var1ed a5 much a5 6-f01d 6etween the5e 5pec1e5. 51nce end09en0u5 ear60xy1e5tera5e (CaE) 15 "kn0wn t0 6e an 1mp0rtant determ1nant 0f 5pec1e5 var1at10n 1n 50man t0x1c1ty, the pr0tect10n 0f PAM and H1-6 a9a1n5t 50man wa5 a150 mea5ured 1n an1ma15 wh05e end09en0u5 CaE wa5 1nh161ted w1th cre5y16en20d10xaph05ph0r1n 0x1de. 1n CaE-1nh161ted an1ma15 the 50man LDa0 va1ue5 were 51m11ar 1n unpr0tected m1ce and 9u1nea P195 (10.2 v5. 12.2 p.9/k9) and 0x1me-pr0tected m1ce and 9u1nea p195 (38.1 v5. 40.3 ~9/k9 f0r PAM; 159 v5. 151 1-L9/k9 f0r H1-6). 7he 1eve150f 0x1me pr0tect10n 065erved 1n CaE-1nh161ted an1ma15 a9reed w1th prev10u5 exper1ment5 1n 0ther 5pec1e5 that have n0 end09en0u5 p1a5ma CaE. 7he 4--5 t1me5 9reater 1n v1v0 pr0tect10n a9a1n5t 50man 0f 1-11-6v5. PAM 1n CaE-1nh161ted an1ma15 c0rre1ated w1th 1n v1tr0 exper1ment5 1n wh1ch 1-11-6pr0duced 3-5 t1me5 m0re 0x1me react1vat10n 0f 50man-1nh161ted AChE than PAM. 0x1me pr0tect10n
50man
Car60xy1e5tera5e
5pec1e5var1at10n
t10n 0f new therapeut1C 0X1me5 (3, 12, 17). 0ne 0f the m05t eXten51ve1y 5tUd1ed new 0X1me5 15 1-(((4(am1n0Car60ny1) pyr1d1n0)-meth0Xy) methy1-2-(hydr0Xy1m1n0) methy1) pyr1d1n1Um d1Ch10r1de (H1-6), Wh1Ch 15 c1ear1y 5Uper10r t0 PAM a5 an 1n v1tr0 reaCt1Vat0r 0f 50man-1nh161ted AChE (10). When te5ted 1n V1v0, H1-6 1n C0m61nat10n W1th atr0p1ne pr0tected a9a1n5t 1.9 LD50 0f 50man 1n m1Ce, 2.1 LD50 1n rat5, 3.5 LD50 1n 9U1nea p195, 9.0 LD50 1n d095, and 5 LD50 1n rhe5U5 m0nkey5 (3,17). 7h15 var1at10n 1n the pr0tect10n 0f H1-6 a9a1n5t 50man may re5U1t fr0m 5peC1e5 d1fference5 1n the pharmaC01091Ca1 re5p0n5e t0 e1ther H1-6 0r 50man. 5pec1e5 var1at10n 1n the pharmac0k1net1c5 a5 we11 a5 the pharmac0dynam1c5 0f H1-6 and 50man have 6een rep0rted. F0r examp1e, the 5erum e11m1nat10n ha1f-11fe f0r H1-6 var1ed 1n m1ce, rat5, and d095 (7, 20, 21, 25, 26), and the pharmac0k1net1c5 0f 50man var1ed 1n rat5, 9u1nea p195, and marm05et5 (1). W1th re9ard t0 5pec1e5 var1at10n 1n the phamaac0dynam1c5 0f H1-6 and 50man, the re5t0rat10n 6y H1-6 0f n0rma1 funct10n after 50man-1nduced neur0mu5cu1ar 610ckade var1ed am0n9 human5, d095, rat5, and 9u1nea p195 (27,32), and the LD50 f0r 50man var1ed 13-f01d am0n9 m1ce, rat5, 9u1nea p195, ra661t5, d095, and marm05et5 (3, 11, 22). 1n an attempt t0 under5tand the nature 0f 5pec1e5 var1at10n 1n 0x1me pr0tect10n, we eva1uated the de9ree 0f pr0tect10n that c0u1d 6e ach1eved a9a1n5t 50man w1th pharmac01091ca11y e4u1va1ent
7HE t0x1c1ty 0f 0r9an0ph05ph0ru5 a9ent5 15 6e11eved t0 6e due t0 the1r 1nh161t10n 0f acety1ch011ne5tera5e (ACHE), an en2yme that term1nate5 the act10n 0f the neur0tran5m1tter acety1ch011ne 6y cata1y21n9 1t5 hydr01y515. A1th0u9h 1nh161t10n 0f AChE pr0duce5 a var1ety 0f ch011ner91c effect5 6y 1ncrea51n9 acety1ch011ne at ch011ner91c 5ynap5e5, 1nh161t10n 0f AChE 1n 6ra1n and d1aphra9m 15 part1cu1ar1y 1mp0rtant 6ecau5e death fr0m 0r9an0ph05ph0ru5 a9ent5 15 u5ua11y due t0 re5p1rat0ry fa11ure (31). 51nce the d15c0very 0f pyr1d1ne-2-a1d0x1me methy1ch10r1de (PAM), 0x1me5 1n c0m61nat10n w1th atr0p1ne (A7R) have 6ec0me the 5tandard therapy a9a1n5t the t0x1c1ty 0f 0r9an0ph05ph0ru5 a9ent5 (12,31). React1vat10n 0f 0r9an0ph05ph0nate-1nh161ted AChE 6y 0x1me5 can 9enerate en0u9h act1ve ACHE, part1cu1ar1y 1n d1aphra9m, t0 re5t0re n0rma1 ch011ner91c neur0tran5m15510n (5, 6, 24, 30). A1th0u9h 0x1me5 have pr0duced pr0tect10n a9a1n5t 0r9an0ph05ph0ru5 a9ent5 1n many mamma11an 5pec1e5, c0n51dera61e 5pec1e5 var1at10n ha5 6een rep0rted f0r 0x1me pr0tect10n a9a1n5t p1nac01y1methy1 ph05ph0n0f1u0r1date (50man), 0ne 0f the m05t refract0ry 0r9an0ph05ph0ru5 a9ent5. F0r examp1e, PAM 1n c0m61nat10n w1th atr0p1ne pr0tected a9a1n5t 1.3 LD50 0f 50man 1n m1ce, 1.8 LD50 1n rat5, 2.1 LD50 1n 9u1nea p195, and 3.5 LD50 1n d095 (3). 7h15 5pec1e5 var1at10n 1n the a6111ty 0f 0x1me5 t0 pr0tect a9a1n5t 50man ha5 made 1t d1ff1cu1t t0 extrap01ate 0x1me pr0tect10n fr0m an1ma15 t0 human5, wh1ch ha5 h1ndered the ad0p-
J7he 0p1n10n5 0r a55ert10n5 c0nta1ned here1n are the pr1vate v1ew5 0f the auth0r5 and are n0t t0 6e c0n5trued a5 0ff1c1a1 0r a5 ref1ect1n9 the v1ew5 0f the Army 0r the Department 0f Defen5e. 1n c0nduct1n9 the re5earch de5cr16ed 1n th15 rep0rt, the 1nve5t19at0r5 adhered t0 the ••6u1de f0r the Care and U5e 0f La60rat0ry An1ma15,•• Nat10na1 1n5t1tute5 0f Hea1th pu611cat10n 85-23.
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MAXWELL AND 8RECH7
d05e5 0f PAM 0r H1-6 and e4u1t0x1c d05e5 0f 50man 1n m1ce and 9u1nea p195. 7he5e tw0 5pec1e5 exh161t the 9reate5t var1at10n 1n 0x1me pr0tect10n a9a1n5t 50man 0f the an1ma15 ava11a61e 1n 5uff1c1ent 4uant1ty f0r d05e-re5p0n5e eva1uat10n5. 7he u5e 0f pharmac01091ca1 and t0x1c01091ca1 e4u1va1ence wa5 5ucce55fu11n exp1a1n1n9 the 5pec1e5 var1at10n 1n car6amate pr0tect10n a9a1n5t 50man (23), and we fe1t that th15 appr0ach c0u1d 6e e4ua11y u5efu1 f0r under5tand1n9 5pec1e5 var1at10n 1n 0x1me pr0tect10n. 7he cr1ter10n f0r pharmac01091ca1 e4u1va1ence 6etween 5pec1e5 f0r PAM and H1-6 wa5 the ach1evement 0f max1ma1 pr0tect1ve effect a9a1n5t 50man 1n each 5pec1e5. E4u1t0x1c d05e5 0f 50man (1.e., e4ua1 LD50) were ach1eved 1n each 5pec1e5 6y pretreat1n9 an1ma15 w1th 2-(0-cre5y1) 4H-1 : 3 : 2 6en20d10xaph05ph0r1n-2-0x1de (C8DP). C8DP 15 an 1nh161t0r 0f car60xy1e5tera5e (CaE) (2), a 50man-det0x1fy1n9 en2yme that 15 an 1mp0rtant determ1nant 0f 5pec1e5 var1at10n 1n 50man t0x1c1ty (22). 7he purp05e 0f the pre5ent 5tudy wa5 1) t0 determ1ne the 1n v1v0 d05e-re5p0n5e 0f 0x1me therapy 1n c0m61nat10n w1th atr0p1ne a9a1n5t 50man 1n m1ce and 9u1nea p195, 2) t0 c0mpare the max1ma1 1n v1v0 pr0tect1ve effect 0f 0x1me/atr0p1ne therapy 1n c0ntr01 and CaE-1nh161ted an1ma15, and 3) t0 exam1ne the re1at10n5h1p 6etween 1n v1tr0 0x1me react1vat10n 0f 50man-1nh161ted AChE and 1n v1v0 0x1me pr0tect10n. ME7H0D
An1ma15 A t0ta1 0f 480 hea1thy ma1e C r 1 : C 0 8 5 (HA) 9u1nea p195
(Cav1a p0rce11u5) we19h1n9 330 t0 360 9, and 800 ma1e Cr1:C085 CD-1(1CR) m1ce (Mu5 mu5cu1u5) we19h1n9 22 t0 26 9 were 06ta1ned fr0m Char1e5 R1ver La60rat0r1e5 (W11m1n9t0n, MA). 7he5e an1ma15 were 4uarant1ned 0n arr1va1 and 5creened f0r ev1dence 0f d15ea5e. 7he 9u1nea p195 were 1nd1v1dua11yh0u5ed wh11e the m1ce were h0u5ed 1n 9r0up5 0f ten 1n p01ycar60nate 5h0e60x ca9e5 (La6 Pr0duct5, Mayw00d, NJ) 0n 8eta-Ch1p hardw00d ch1p c0ntact 6edd1n9 (N0rthea5tern Pr0duct5 C0rp., Warren56ur9, NY) w1th c0mp1ete ca9e chan9e tw0 t1me5 per week. An1ma15 were pr0v1ded w1th c0mmerc1a1 cert1f1ed 9u1nea p19 0r r0dent ch0w (21e91er 8r05., 6ardner5, PA), a5 appr0pr1ate, and tap water ad 116. An1ma1 r00m5 were ma1nta1ned at 20-22°C and re1at1ve hum1d1ty 0f 50•+ 10% w1th at 1ea5t 10 c0mp1ete a1r chan9e5 per h0ur 0f 100% c0nd1t10ned fre5h a1r. A11 an1ma15 were 0n a 12-h0ur 119ht/dark fu11 5pectrum 119ht1n9 cyc1e w1th n0 tw1119ht.
Mater1a15 50man wa5 06ta1ned fr0m the Chem1ca1 Re5earch, Deve10pment and En91neer1n9 Center (A6erdeen Pr0v1n9 6r0und, MD) and wa5 98.6% pure. 7he 0x1me5 H1-6 d1ch10r1de and PAM ch10r1de were 06ta1ned fr0m the Defence Re5earch E5ta6115hment (5uff1e1d, Canada) and Ayer5t La60rat0r1e5 (New Y0rk, NY), re5pect1ve1y. C8DP wa5 5ynthe512ed 6y 5tark5 A550c1ate5 (8uffa10, NY) and wa5 99.5% pure. Atr0p1ne 5u1fate, acety1th10ch011ne, and 7r1t0n X-100 were purcha5ed fr0m 519ma Chem1ca1 C0. (5t. L0u15, M0). 7he chem1ca1 pur1t1e5 0f C8DP and 50man were determ1ned 6y 31p-nuc1ear ma9net1c re50nance 5pectr05c0py. A11 0ther dru95 0r chem1ca15 were U5P 0r rea9ent 9rade, re5pect1ve1y.
An1ma1 Exper1ment5 PAM ch10r1de, H1-6 d1ch10r1de, and atr0p1ne 5u1fate were adm1n15tered 1M a5 501ut10n5 1n 150t0n1c 5a11ne. C8DP wa5 adm1n15tered 5C a5 a 501ut10n 1n pr0py1ene 91yc01 c0nta1n1n9 5% ethan01. 50man wa5 adm1n15tered 5C a5 a 501ut10n 1n 150t0n1c 5a11ne. 1nject10n v01ume5 were 1 m1/k9 1n m1ce and 9u1nea p195. An1ma15
rece1v1n9 mu1t1p1e 1nject10n5 were 1njected 1n a1ternate h1nd 11m65. LD50 va1ue5 were ca1cu1ated 6y pr061t ana1y515 (15) 0f death5 0ccurr1n9 w1th1n 24 h after adm1n15trat10n 0f 50man at f1ve d1fferent d05e5 w1th e1ther 51x 9u1nea p195 0r ten m1ce per d05e.
AChE React1vat10n 0x1me react1vat10n exper1ment5 were perf0rmed 0n 501u61112ed AChE preparat10n5 fr0m 60th 6ra1n and d1aphra9m 0f 9u1nea p195 and m1ce. M1ce and 9u1nea p195 were euthan12ed w1th car60n d10x1de. Wh01e 6ra1n and d1aphra9m were 4u1ck1y rem0ved and r1n5ed 5evera1 t1me5 w1th 1ce-c01d 0.15 M NaC1. 8ra1n t155ue wa5 h0m09en12ed 1n 9 v01ume5 0f 1ce-c01d 0.01 M ph05phate 6uffer, pH 7.4, c0nta1n1n9 0.15 M NaC1 and 1% 7r1t0n X-100 6y u51n9 a P0tter-E1vehjem t155ue h0m09en12er. D1aphra9m t155ue wa5 h0m09en12ed 1n 4 v01ume5 0f 1ce-c01d 0.01 M ph05phate 6uffer, pH 7.4, c0nta1n1n9 0.15 M NaC1 6y u51n9 a P01ytr0n h0m09en12er (8r1nkman 1n5trument5, We5t6ury, NY). After P01ytr0n h0m09en12at10n each d1aphra9m h0m09enate rece1ved an am0unt 0f 7r1t0n X-100 e4ua1 t0 1% 0f 1t5 v01ume and wa5 h0m09en12ed a9a1n u51n9 a P0tter-E1vehjem h0m09en12er. 8ra1n and d1aphra9m h0m09enate5 were centr1fu9ed at 30,000 × 9 f0r 30 m1n at 4-6°C, and the re5u1tant 5upematant5 c0nta1n1n9 501u61112ed AChE were 5t0red at - 20°C. 7he react1vat10n 0f 50man-1nh161ted AChE 6y 0x1me wa5 perf0rmed 6y the meth0d 0f De J0n9 et a1. (9). 501u61112ed AChE that had 6een d11uted 1n 3 v01ume5 0f 0.01 M ph05phate 6uffer, pH 7.4, c0nta1n1n9 0.15 M NaC1 wa5 1nh161ted w1th 10 nM 50man f0r 10 m1n at 37°C. Exce55 1nh161t0r wa5 rem0ved 6y three extract10n5 w1th an e4ua1 v01ume 0f hexane. 1nh161ted AChE wa5 react1vated f0r 30 m1n 6y add1t10n 0f 4 v01ume5 0f 0.05 M ph05phate 6uffer, pH 7.4, c0nta1n1n9 0.33 mM H1-6 0r PAM. AChE act1v1ty wa5 mea5ured 6y the meth0d 0f E11man (14) w1th acety1th10ch011ne a5 5u65trate.
Data Ana1y515 519n1f1cant d1fference5 6etween pa1r5 0f LD50 va1ue5 were 1dent1f1ed 6y the Newman-Keu15 te5t (18). En2yme act1v1t1e5 were c0mpared 6y 5tudent•5 t-te5t. D1fference5 were c0n51dered 519n1f1cant 1f p
