79 390 PREVENTION AM TURETMENT OF VESICATION fIU POISONIN ICAUSED BY fKSENICfLS(U) ARIZONA UNIV TUCSON DEPT OFCELLULI AM DEVELOPMENTAL BIOLOGY H V RPOSHIN

UNCLSSIFIED 15 SEP 62 DHDI7-66-C-0052 FIG 6/15 .

EEEEEEEEEEEEEEeEEEEEEEeEEEllllllllll

IIIIIIIIII

IIN 1111 L1.0 2.08

i1

of Veuicandtzmamea oan Paimcning Cauus by Arasmca's

C0 Covr 9 m~t~h pecics f= eb. 1, 1981 -

Sftb 15, 1982

II~ ~vw4~by

U .S. A~y mo~a Famearch and Deval.1 C~m

Frt De±ckd

Ccta No. DMM17-80-C-0052

Chiversity of Ad Z

TLbmm, AzrJza or

85721 ~ ~

A~,edMAR 3 1987Dm i szbjm±t Sa

mi eriImt±i MU4vitd

Ohe XU*tt fOr thiS d=cintxumt be xatfm toth

C~mne, U.S. Aty MedcalPlusinrc and Dwevs1mt Cen

Attm: SGF-SI1fRt D~.ck, Frukrick, Mary~am 2.1701

Tef=i~±zs = this rpt are o to be c~tued as anoffical-jq% of the Aimy posiio imlsa so desi gm-mi

by othr aizho.zed doums

DISCLAIMER NOTICE

THIS DOCUMENT IS BEST QUALITYPRACTICABLE. THE COPY FURNISHEDTO DTIC CONTAINED A SIGNIFICANTNUMBER OF PAGES WHICH DO NOTREPRODUCE LEGIBLY.

V

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R~TEPORT DOCUMENTATION PAGE 13ZFRE COPTCTORMNAOI U140ER 2.GVT ACESIOCO. J ZPIENT'S CATALOGG NUMSER

(4. TITLE (and SUMUO) 5. TYPE OF REPORT & P9RICO CoveREOAnnual Summary Report

PREVENTION AND TREA~r 'ENT OF VESICATION Feb. 1, 1981 - Oct. 31,1H 1AND POISONING CAUSED BY ARSENICALS 4. PEARuN ORo. REPORT ?iUMsalt

7. AUTHORWa 4. -CONTRACT OR GRANT NMS&R(O)

R. Vasken Aposhian, Ph.D. DAMD17-80-C-0052

9. PERPRMMNCGAGANIZATION NAM19 AND ACCRE5S 10. PROGRAM EL.&MENT. PROJECCT. TASK

University of Arizona, Dept. of Cellularand Developmental Biology, Tucson,AZ 85721

11. CONTROLLING OPPiCK NAME AND ADDRESS IS. REPORT CATE -U.S. Army MedicalI Research and Development~ . 1982Comman-d, Fort Detrick, Frederick, HD 13. NUM89R Of PAGES

21701 _ _ _ _ _ _ _ _ _ _ _ _ _ _

1-. MONITORING AGENCY NIM & ACOOREII dtiU.*E 1 C..gmjjW OtW..) IL. SECURITY CLASS. (.1 (hie mpen)

Unclassified

I". 09CLASSIF1CATION/OOWNGRAOING5CHC OULE

16. OIS1RIUTfON STATEMENT (6tga hI pm

)Approved for public release; distribution unlimited

17. OhSIRIGUTION STATUMCU? f.1 the s.ief a =soft 20,11 EI0114W m A810800

IS6 SUPPLEMENTARY NOT193

19. KEY WGOS (Canmis ev towwadd .It ifnag...-, Md Iia.Utp &P block Mower)

Dimercaptopropanesulfonic acidDimercap to succinic acidLevisiteArsenic

20.. A011RACr (bwinwd ogwwm ~d N~ noe a" Idnafir Or 610k n~bin)

PAM I AM-LISI= AC=VIT AM S7ATY CF MEC-I4RA"rU=

Cudecposu~inie ar-id (EPS) and the sodim salt of 2,3-A!-imrcap-l-pmpaumaJfcnic acid (DM) are analogous in chimca. struc--re to

(EBritish Azti-Lawisite). Diverrapro. was au~g the fiJst~.'~~ma~ca~yusful1 meutal chelating ageunts and was develcped =r.gially as an

anti-lwisite agenit. Either CM~A or MTS protect rabbits fram the lethal.

DO s'.vco" 14731 EDofl@W or? iO £35 6s is ssag

SCI~gmi CL.AWP1FCATIO' OP THIS P49g (IW ikm Denii. fae*

-2

(No. 20 con't.)

-~ v sstatic action Of didloro(2-CcoroinYl) arsine (29. 7=ros/kg, aiso ]wn aslewisite. 12* analogs are acti1ve In this resPect when given either sc: or po.Theu stability of each of the three dlimrcMaptcl CiPouds in distilled HO, PH 7.0at 24, hag been, exmie for seven days. rU4SA retained 82% of itS marcaptogr=p6, but no ttAale AmmrcaptO gv3s remined in the aeS or BAL solutions.At PH 5.0, however, there was no striking dif!ference in the stability of th.etbhlee dinarcapto mowx (78-87%) over a seven day period. DMA and cm~WWu=t furti iesgaI as water soluble tal1 binding agents in both inVivo and in vitro exe :31ts

PAM 11 - Cptica1 Isomrs of 2, 3-imrcap:tn-j-propnsloaeAntidotal. Activity,= inVitro and in vivo,

against Sodium Arsenite.

MIS (2, 3-dimarcaprw- 1-propane suLfonte, Na salt) is an =portant watersoluble analog of diu -rcaprol. All investigations of this antidote for heavymtal uinWoacain have dealt only With the racemc mixture. In the presentrepoirt, the optical iscm@rs of CWS have been separated and the arsenic-antidoteactivity of the bev-rctatory (-) is e, the detrorotatory (+) isatur and theracaic mbeture of CWS havie been investigated in vivo and in vitro. =-a

MndVida optical isomrs; and the rac zix ef alPS are' ezectiveequally, in vitro, in orvetr the inhibition by sodium arsenite of theaactivity of u s kidney pyrmva d"=-rogq as comzplex: (PDH) . In additicn,when PEE is inhibited, in vitro, by sodium arsenite, any of the three a-eSprg; xations will reverse the .niito e.ull well. The in. vitro evicenceOsxgests that tw of WtAS are required to prevent t-M =a=s of onemolemle of sodiu arsmniite. Neither the tflOs nor the ED50s of each of thethree f~of W~4S differ significanitiy when measured i.p. in mice. ZnIaddition, therm is no string differenca betwm the effectiveness of the Jlevo-or dexto-rotoy W4S When given orally to mi.ce calge wihsodi=arsenita. 'fIhs, the use of the iivdal oca..isnr of OMS does not

aerto have any advantage over the rawc rrdxue as an arsenic antidoteurxer thes coditions.

PACIT - Theu PI is Iing this space to Point out that the in vi= assayminitioned in PAT I is now the first screwn used In this oraczy fordiJscoverzi Ag coous with arsenic Or Ileisite antidote activity. It is fast,inexensive, very acurate and uses fewer animals than the In VIVO S~een. =h.ein vivo screen i o u eodsrm

I i IDistrib~tion Statement A is correct for this

4 report.-- Per Mrs. Varginia Miller, AMD/SGRD-4RiI--S

"'C --po"

.COPI.

-3-

3IUI!Vl CL.ASS1PICATION OF T14IS 0AGEC(Wh. DaW ta ie.)

Prevention and Tfreatment of Vesiationard Poisoning Caused by ~ Arwia

Aximal Summary RportCovers 9 month period fr Feb. 1, 1981-

Oct. 31, 1981

September 15, 1982

-apotd by

U.S. Aity Medical. Research and DvczntCarnmand

Fort Detrick

Frederick, Maryland 21701

Conract No. DAMD17-80-C-0052

0 University of Arizona

Tucon, Arizona

85721

DCID Distribution StatwmitApproved for Public Releas

Distribution Unlimited

A-

Sept.er 15, 1982

Other requests for this docmenmust be referred to the

Candier, U.S. Amy MicalResearch and Deveopmnt C uand

Fort Detrick, Frederick, MaryLand 21701

The f indings in ttis report are not to be cmnsAed as anoffi ci a I Deparen -,ALOf tta Army posi.tion unless so designated

V

by other aut±~rized doc~ents.

-4-

DCD D Sta A. S*N ~ -.

to find ways to prevent vesication and poisoning caused by arsenicals~~nfl liite.

Sm 4e t s Prtection of rabbits against the lethaleof lewisie; of mice against lethal effects of sodium.arsenite: and prev and reversal of PM Jiibiticn as in vit screen.

Smmry of results: PART I ANTI-LEWISIE A=vf= AND S'T IITY OFbMDMCMM aCACID AND 2,3-DInqCAPT-I-PRCPANES52MNIC A=I

-" acid (MMA) and the sodimn salt of 2,3-dimr-- pt-ropanw-1' o4,i- acid (EPS) are analogous in cmical stuctre todT1r )I (BAL, British Anti-Laisite). DimIr;rol was amumg the firsttIa .nLca.y useful metal chelatiM agents and was developed originally as ananmti-lewisite agent. Either CMSA or U4PS prtects rabits from the lethalsystemc action of dicdloro(2-clorovinyl)arsine (29.7umols/kg, also knw aslwisite. ~ analogs are active in this respect when given either sc or no.Th stability of each of the t1zee Air c t- compounds in distil.led E1o, pH 7.0at 240, has b1n exmined for seven days. MS retained 82% of itS .wercancg7MUPS, but no titratable mercapto gxoIus rmained in the DMS or BAL solutions.At pH 5.0, however, there was no stik diffaence in the stability of thethree Aimm a com ds (78-87%) over a seven day period. -A SA and MPSmuzat futher investigation as water soluble mal binding agents in oth in

vi.va and in vitro rimmem"

PAR I CpticaJ Isomers of 2,3-Dinrcapt-l-propanesuflfnate:Antidotal ,tivity, in vito and in vivo, against Sodium Arsente.

-O1MPS (2,3-dim r to-l-prpan sulfanate, Na salt) is an imrant watersoluble anal of imarcaprol. All investigations of this antidote for heavymetal inmccatioa have dmlt only with the racemc mibxre. In the presentreport, the optical is of EMS have een separated and the arsenic antidoteactivity of the lava-rtatory (-) isr, the dext= .rotatory (+) iser and theracmc extre of CM have bee investigated in viv and in vitro Meindividual optical ismrs and the raceme mix or U---e2ectiveequally, in vitr, in the inhibition by sodium arsenite of theactivity o7 nme kidndy pyravate d cgenase complex (PE). In additicn,when PEH is inhihited, in vitro, by sodium arsenite, any of the three Z2Spreparations will reverse th71-nhibition equally will. Te in vitro enridencsuggests that tw EM r of ICMP are requaired to prevent thW e ~!ts o f cneamolecule of sodiun arsenite. Neither the LD50s nor the M50s of each of thethree fm= of MMPS differ sic ly e asured i.p. in .ice.adition, there is no stricng difference betehen the effectiveness of -e -evo-or d@c o-Atdvy_ MS wIwn given orally to c challenge with sodi .narsenis. Ths, ' use of the individual optical isomers of CM does "cta to have J6y advantage over the raciic mixure as an arsenic antcote,.uner these ~to s

0PATII - The PI is using this space to point o=t that them -,I vitro assaymwsnend in PART I is now the first s used in this M -abrtcr, :ord erung compud with arsenic or l2 ite antidote activit. : .s fast,

" 5 --

izureiv, very accuate and fewer animl than the in vivo screen. Thein vivo scz-en is now otr ssm.d

C==1nosign: It wculd ajppear that , S and o4ther mcapto cmpwidswm=ant lrd= sbtdil and evmnUl&Uy cical t for

a t- aimby-arsenic, sei.~ against ].mwisite ga,,.kThemea beu used in ltn therapy in the Soviet Urnicn and Cn.a. -;* tinvestigatcr and Wst Gegm investigat=s have reccamended that it repace BALfor treatment of heavy metal poiscnig

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0 , Q ", .• -.° .-, "D "J ."..°- " ' '. % " ' o, % '."'m i'% ," %b'%.",'%,'.". ... *"'

In the research described in this report, the inestigator (s)adbered to the "Olide for the Care and Use of Laboratory Arjimals," prepared bythe Committme on Care and Use of ba Anxail of the Instiute of

Anilml 3eorces, Nationaa Research Council (D Publication No.(NI) 78-23, RPrised 1978).

Citatio of cca Ji organizations and trade nams in this report do notsti .e an offi c ial of the Aty endorsemnt oapproval of the

products or services of teeoanizains

-7-

*©. -::r~~ *K>

TABLE CF C q

Suary 5

Forwrd 75V

7V

Table of Cntits 8

Part 3:- Anti-lewisite ACti.vity and Stability 9of W)-iuraosucianic acid and2, 3-dimarcpto--pupanesulfcnic acid

Materials & mthods for Part 1 10

Results for Part I 12

Table I 13

Table I 14

Figure 1 16

Di scssim for Part I 17

eferences for Part I 19

Part - Optica I of 2,3-Dimrcapto-l- 22pr z2SLlfoflte: Antidotal. Activity, in vitro and S

in vivo, against Sod3 Amieite. S

Matrials & .thods fomr Part 11 24

Results for Part IT 27

Figure 1 28

Figure 2 29

Figure 3 31

Table 1 32

Table II 33

Table I= 34

Discussion for Part =I 24

SReferecs for Part I 37

Part 1= - Ansmrs to possible questi.ons as to scope 4iand.. ,ress of contact.

-8-

* ~~ - .- & .~s d r ~ . . . . . * *- **P .

PAEW I ANTI-LE-SIE ACrT AND STABILITY CF MESO-DD IM C SUC=C %

A= AND 2,3-DIMERCAPO-I- RCPANEZ C A= %

Meso-di-captosucm c acid (M4SA) and the sodiumsalt of 2,3- d-mercapto-1-propanesulfanic acid (DOPS) areanalogotm in cwmical strucure to dinrcaprol (BAL,British Anti-Lrwisite). Dimarcaprol was among the first

ra1tically useful nital. chelating agents and wasdevelped oiginal as an anti-lewisite agent. EitheromSA or 4M protects rabbits from the lethal systmicaction of dichloro(2-chiorovinyl)arsine (29.7umols/kg,also known as lewisite. The analogs are active in this %respect wien given either sc or po. The stability ofeach of the three dimrap =mpou s in distilled OfpH 7.0 at 240, has been eiamired for see days. Sptie82% of it brt group, b o t±iratable

merapto groups r-ined in the MS or BAL solutios.At pH 5.0, haever, there was no stiking difference inthe stability of the three Aims ccpounds (78-87%)over a seven day period. CMSA and CPS warrent .r-Pnerinvestigation as water solubl mtal binding agents inboth in vivo and in vitro Zeiwms.

British Anti-Lwisite (BAL, dimercprol) was developed in the 1940's as

an antice to dichloro- (2-chlorovirryl) -arsine, commnly called 1ewisite

(1,2). Th lethal action of lewisite is believed to be '.e result of ,.s

oining with n or more suLfhydryl grous and t.s inactivat r essen-za.

sulfydryl-coitaining enzyms (3). It is the arsenic in the iewlslte

molae1 that reacts with sulfhydryl moieties.

At the time of its in-zaction into clinica2. midic .ne, BAL -.as

cnsidered by many to be the long sought, universal antidote for heavy reta.

poisoning. In subseqmut years, however, less tcic and =e soe=ic meta.

binding ag--ts have been soght and investigated. Som have ,mt the

-9-

and standards necessary for clinical use. Others have not. For example, BAL

glucside was introduced (4) as a result of a search for water soluble and

less tmcsc analogs of BAL. Although it was found to be less txic than BAL

for iv use, (probably because of its low lipoid solubility), it did not

beauD established as a clinical agent because it is unstable chnica.y.

Other cC -I nids, which are less analogous in chmical strucue, iave

replaced BAL for sam of its more specific therapeutic uses. For example,

D-A--ipe 1=min is used to mobilize and increase the excreticn of coper ,

pat.ents with Wilson' s Disease (5). Its N-acetyl derivative is effective as

a mercury antidote (6,7). BAL has remained, hwever, the drug of choice in

the U.S. for the tatmt of arsenic poisoning.

-~Z'sod~;raptosucciniec acidl (CMA) (8) and the sodium sal1t of

2,3-d±mmrm %to ]- acid (IMPS) (9) are prmsing replacements

f,= BAL. The oceponds are very siuul ar in cheical stucte to BAL and

are so-tu s refezed to as water soluble and/or orally-effective analogs of

BAL. To our kXmwledr,h the anti-lewisite activity of these two

imortant chcal analogs has not been deterined. either are any

pub]ished data available ce =ing the stabiity of aqueous solutzcns of

these dierrpto acupolms. Evidence for the anti-lewisite ac=vatvl and

stability of MTS and 24SA are presented in this paper.

Materials and thods

Male New Zealand white stock rabbits weighing 2.3-3.3 kq _re zur-c-ased

fr= Dutchland aboratories Inc., Denver, ?A and Davidscn M i .,

Zav r, ,U and caged ndividualy. Food (PL- Rabbina Cat w arand 5322)

and water were available ad libitm except in the case of t-ose a-.~als who

reived therapy orally. Aminals receiving therapy po _r-e f 6r n

hrs prior to the first administat-in to I hr after -he last adn=m-Lticn

10

on day one. Cn days t and three, animals were fasted fr 1 r prior to

the aming a-ini -at=in to 1 hr after the evening administration,

approxately 7 hours.

When dithiol therapy was given sc, the animais were anesthetized fif-teen

mintes before lewisite administration by administering im 0.50 ml of

a t ic solution per kg. The animals were anesthetized to reduce =7e pa.in

expected to be caused by lewisite. Subsequently, it was observed t'.at

neither pain nor discmfort was apparent. Ms, anesthesia was .ot used in

the experinunts when dithiols were given po. The anesthe-ic sclutacn was.

prepared by mixing 5 parts Ketamine HC1 (100 mg/mL) and I part of )Cylazine

(100 mg/ml).

A 5 ml GiLson Pipetman was used to give the dtiols; by mouth. The

rabbit was placed in a short restaining box. The box was placed on its end

so that the rabbit was in a vertical position with its head at t.7'a t=p.

Pipetun was filled with the desired vo iume of the drug soluticn. The

plastic tip was gently inserted betwen the lips at one coner of the -.cuth

and the liquid delivered slowly into the back of the -abbit's mouth. -h2s

method did not appear to cause any trauma or injur. :t was easier arn

faster to perfor than the use of polyethylene stmach abes.

NaLI4PS was a gift of Heyl and Co., Berlin. Since each lcIec:e -.as a

molecuil of E20 associated with it, a molecultar weight of 228.2 -was ;sedI

calcula11 ons. CMSA was a gift of Joimson and Jchson, Ski2,zi, N.J. 2ct

cmds ware ph-a,'uceica2. grade purity. LPS and :MSA were tt-.rat. d " -_

iodine in order to measure -parity and ,rmerapto content. By -- s -_:er'_n,

C) each preparation was judged to be greater than 99% pure. -ie r .cunds wrer.

given by mouth were dissived in water. Zn order to d.issol-ve mSA, t.-e

aqueus was adjusted to =H 5.5 with NaCH. When '.-ern -c, --e

-1]. -

solutions were preared the sa way except that the ccoLmds were disolIved

in 0.9% NAI-5% NAHM 3. Unless otherwise stated, the cenrations of CS

or MA were such that the rabit received 1. 0 ml of solution per kg of body

weight, per adrdnistraticn. Dimrcaprol Injection, USP (BAL in Oil Ampules)

was a gift of Hynson, Wsctt & Dmining, Baltimre, M. .

Lisite was 97-99.6% pure as judged by NMR-spectroscopy as weJ.I as !: .v.

iodine titratin. Analysis by the former method also indicated that t e

forms of laeisite that ware present ware trans (97.7%), cis (1.7%) and di.rs

(0.5%). taisite is a hazardous material with which to wrk since i.t :s a .

potent vesicant. All handling of lewisite was done in an exemly -wel

vented chemcal exhaust hood. Safety glasses and thick neopren gloves wre

h stability of IMPS, WA or BAL was e od using iodwetric

titration. To 2.50 ml of a 0.10 M edir-c;-o solution, 10 drops of starch

indicatr soli=ion wre added. The solution was titrated using 0.025 N

iodine solution until the blue color appeared and persisted for at least 10

sec.

Results S.

Anti-ewisie At ivityt

The data of Table I early shw that both SA and a2%S :-ave

anti-lewisite activity when given subcutaneously. As IZle as

20uos/bI/kq adarnistered sc, acording to the stated rwgiran, pr=-tec-s

against the lethal actions Of ].eWivlte. M~s, DMA and UVS are analLocs

tO BAL ot Only in ch w.cal struc ure but also with respect , ant.- ew-.is e

activity. In addition, MI and MPS have anti-lewisite act:v .ty w-wen g.ven

orally (Table 2). ,'7 0-z

- 12 - '5

TAME I

IIAu Antli-lisite Ativity of Mso-Dimarcapsucinic Acid and2,3,-Dimarpto-i-Propanesulfnate when given sc to rabbits

Group wmols Ikgj survivel t a survival

1 1/182 LE + 75.0 IMSAc 12/12 IJ003 LEW + 37.5 UFSA 6/6 1004 LE + 20.0 S 6/6 1005 LEW + 10.0 MA 1/6 1 7

- - - -- - - - - - - - - - -

6 LEW + 75.0 M9Sc 10/12 83 p.

7 LEW + 37.5 MS 5/6 83

8 L 75.0 BALC 8/12 679 LEW + 37.5 BAL 3/6 50

0 t of this the data reresent the

cobne emlsof a mbr of separate ~ce~uns ThisWBA * to save s . The roan for the 6zber of animalin scm gxzap differing from the m~er in other grous ofthe am tabl is that very often the combined data are theresult of ft= 2-3 sepaate xts. Otherwise, the-Iriits ,e-e perforand i r idmictl c± oi.=s. The

suxviva.1 -erd 1 11n thi.s table is that for 7 days after

b Lewisite (29.7 =Is/kg) ws given sc at tim zero.

c All agents given sc except BAL, whii ws g-ivn im.Di 1 r1;- ccepods given at +1 nn, +90 min, +180 min,+360 min after lwisite and at 8 a.m. and 4 p.m. on day 2and 3. dmzihistaion of these ammuts of dinucaptoc=ound at the ms cited above did not cause anyfataI ities in cot ol anim.Ls that did not receve lewsi e(data no shown).d Pair-wise c=arison: p < 0.0001 for I vs 2; p-0.C0I

for 1 vs 3 and 1 vs 4; p < 0.001 for . vs 6 and I vs ; ? -0.001 for I vs 8 and 0.01 for I vs 9.

- 13 -

S..

0

Me o-Oizmrcaptosucinic Aci.d or 2, 3-Dimrcpto--oaeufatis effective, wtm given by muth., in pro ting rabbits

again the lethal. effects of Lwisite

Grp u:ls/kg survive/start % survival

1 L a+ 0/12 0b2 LEW + 400 SA 5/6 833 LE + 200 W 4/6 674 LEW + 400 MPs 6/6 1005 LEf + 200 MTS- 4/6 67

6 LEW 4 - 1/6 177 LEW + MSA c 4/6 678 LEW + C 1/6 17

a Lewisits (29.7 uols/kg) was given sc at tim zero.b Diuurceo cwpnds given po at -45, -2, +90 and +300 nn.

ter isita and 8 a.m. and 4 p.m. on cday 2 and 3. No

fatalit.It o==ed in control anim l that received themse amontof * rmz11- cwund, po, (b= no awis!-t) at the times citedabve. Survival was foliw and recorded for 7 days afterlisita dnmistratin.

DimmrcaPt comPunds given po as follmws: 400umcls ofdr o ound /kg at 5 rmin before lewisite, and 200=unls/kgat each of the following time after lowisite: I hr., 2.5 1hrs.and 5 hrs. on the first day plus 8 a.m. and 4 p.m. on day 2 and3.d "For pair-wise ris : p - 0.001 for 1 vs 2; p -0.01 for I

vs 3; p < 0.001 for 1 vs 4 and p - 0.01 for 1 vs 5

'diti , al s=udies have dmtns aed that a single po adinis"atz~cn zf

DEA (400 uncles/k) 15 in prior to lisvi.te was ineffective since cni,y 1

6 ani ma1-s survived for 7 days. In the exrents of Table 1 and 2, 'cst z:

the rabbits that received lewisite and no t therapy died wit= "

- 14 -

........_| 3

hrs. If amimal -c died after receiving lewisite plus d ;nrcapto therap)y, they

usuall~y died bewen the first and fifth day of theexeiu.

Stability, StMdies

MA stabilities of ESA, IMPS and BAL in 0.10M solutions at pH 5.0 and

7.0 were eamned (Fig 1). The mercapto grots of these capounds, .n

aqueous solutims at pH 5.0, are stable (Fig 1). Even after 7 days at roca

tmera , fr=n 78 to 87% of the marcapto, groups rmain titratable. At =H

7.0, h1mver, the greater stability of 2!SA is evdet with 82% of t"e

nurcapo grous remaining after 7 days.

15,.

;.:

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0 ,_ , ' .,.. . " - ,. ,j ,,. . . . . -. . . . , . , . ' . ' - " .., " ._ ", ._" .. ' % ... .' ,. ' .

00

500

4I Mu u

-Day

~. 0FIG.1I

Stblt ofS 1MA MadBLatp . r70

adutdt4p . 701an to~ a ia .--tano

0.0olm.1 Souin wer prpae using

Sytahlityn ofd ucer~ teIs andtn none at tpH 5.0a or= 7.0.

a soutin, f 5 NaM3 wheinso eth fozen for 4 paysard, enad ..r

eac da fodastetaie p 825. o 7.0 anoainal cpt grops of sm:

solutca wre h~eld at indicated tium4sdand, th e tainedp76 atn 9 f t

mrcapS o A grus, ed.Other sAis ta rotu sof ON) indicte tha= t'IAr1

stable (92-95% of original) when either frozen, or frozen and tha~wd each day

16 -I

for 7 days, after three days at r!! tPeratore no titatable rrercapto

groups Wee evident.

Ectfiff~Ve clinical eaeiec with MMS and MTS as anti~dotes and

prophylactics for mercury, lead, arsenic and other heavy m~etals has been

reported in Soviet and maini~d Chinese literature (10,11,12,13). In the

Soviet Union, CM4 has beow for ma.ny years an official drug ca I Ied Unithil.

Pecartly, there has 1ea a great deal of interest in both of these water

soluble chmical1 analogs of dimurcaprol in the United States and abroad

(14,15,16,17,18). This has resulted in the confi~nation and extension

(19,20,21,22) of earlier reports dealing with both the basic and clinica.

O investigations of MME and MR4S.

Altouh d~Ai -I is a nm relatively easily identified in the field,

of teaatics, the cmound is kwwn most norly in other areas as

British Anti-LImasite. It seemeid reasonable to expect that a tru.e analog Ie

aqcnist mdit also have Ati-Lewisite Activity.

Th present eprntsclearly show that either MS or MKPS wi.

Protect. rabbits against. the lethal systmic effects of bcnus.*

wiinistered lewisite (Table I and 2). Therefore, 1a1SA and aIPS can 6e

considered to be njot only analogous in chemical strct~re but &--sc -r

anti-lewisite. activity. In addition, CMSA and Ot*1S are effective inen g;:-Pn

by mouth; a route not. re-m al Id for BAL amuinstrati on.

The dose schedule for administering OMSA and t1'IPS was ba sed =n a t~irs-e

day ruginxrc~ur in the L.iterature for the use of these metal

agents. Subsequent studies (Aposhian, =.p~blished) have demonstated t.at as

C)little as on dose of 40 umls/kg of eithier drug given i.m -ne rniiite a&ftwr

Lwi'site wi.,. result in the survival of 4 out of 6 rabbi.ts. :n addit~cn --men

-17 -

MA therapy is delayed until 90 min after lewisite, 6 of 6 rabbits,

survied. T purpose of these studies was to deteine whether CSA or WS

have anti-lawisite activity. No att.zpt has beu made to quantitate their

relative efficacy anst lewisite.

Not cnly are these analogs crystalline and readily water soluble, thy

are less tmic than BAL. e results of a rm er of differet irestgatins

in rots have led to the ccnclusion that the acute tdccity of ZA is less

than that of MOS which is uuch less than that of BAL (19,23t24,25).

M stability st~dies (Fig 1) were initiated for tw reasons. Many

investigators believe that OMP and MS are unstable beca.se of t.-r

d r to 3CMre. Since soluions of thse cazrounds were being used

th x. t the day, for cple see Table 1 and 2, it has been csi red

necessary by a moer of investigators (17,20) to preare solutions

inudately before uAe. T stability of solitions of these diuur

c=;aznds Is suwhat suxpri.si~ng since lmercpt cxzpounds are usua.y thoughto be redily coddized.

In a ditio to amny older rerts in the Soanet and Cunese Litara4re

(10,12,26) dealing wi3th MIPS and ZMA in limyn therapy, such use -as been

stx.igtieed by roemyt papers= ti~n data fx= clinical r.sgat-;ons.

For eu ple, ICM has bew used recntly in the treatnunt, of a 46 yr. z..d

mnn who ingested 2000 mg of arsenic in a suicide attet (27). T:eaent .

300 rq of C every 6 hrs po for 3 days caused an ncmase .e in-r ',

wetJon of arsenic and evenual recovery. aA =neased the ex=_.= =

lad in the urine of selter workers and was effec-ve .n _esatug -,- sr:.-ns

and symptcm of lead poisonng (8). :t ranp=.d -was ",.

tolerated and no signs of c:i.ty w r evident. -e use-.nss of :,' S ad,'

other metal binding agents in the trea nt of 'arc=uz m ca.an result.,"

- 18 -

f te Za mercury disaster has been oc-muned recently (18). MS, as

DVVAL, is xww an aped drug in West Gemany for the treaimt of mrcuxy

poison. woe - iter soluble analogs of BAL, analogou in activity as

well as ich a2 structure, active when given by mmuth and of low tcici,.y,

warrant citimmd investigation as possible replacements for BAL.

i FOR PART I

1. R.A. PE , L.A. STIKJ, and R.H.S. MOOS , Nature 156 616-619

(1945). 2. L.L. W and C. STOCK, Scienc 102 601-606 (1945).

3. L.A. SCN, and S.H.s. TOWSM, Physiol. ev. 29 168-192 (1949). V

4. J.P. DA14ELI, M. OANLI, J.B. FRASER, P.D. _MU , L.N. COWE, and G.

SW, Biocd. J. 41 325-333 (1947).

5. J.M. WATSH, Amer. J. Md. 21 487-495 (1956).

6. H.V. APOMIN and LM. APOSMAN, J. Piarmacl. Exp. Tar. 126 131-135

(159).

7. R.A.P. K, D.C. PO6WE, J.D. BLUZZCK, and G. BOYLE, N. Engl. J.Ma., 285 10-16 (1971). ;"

8. E. F=M4I, J.R. DaSILVA, and A.V. NIM-24S, Amr. J. a-op. ftd. W.q3 .5

714-727 (1954).

9. V.mE. PE a , Ukr. Khim. Zh. 22 603-607 (1956). "'

10. s.I. AMMM, In Tioloy soedn Ta v meditsine (N. N. LUianisk;:, 7-...

Petrnkin, P. V. RIdionov and A. J. Cerkes, eds.) pp. 161-168. Grs.

-Md.

Izd. crain, SSR, Kiev (1959).

11. L.K. K=IWA, Farmakol. Taksikol. 21 53-59 (1958).

12. W. S-EaD , T. KtM -SMC, and W. (X '<M, C2=i. Md. . 34 437-439

(1965)..

- 1.9 - -

13. I.Y. OIMIS3NIMM, Gig. Tr. Prof . Zabol 9 38-43 (1965).

14. L FRE , and C. CMI, J. Pbam 9t,- ,l. 27 624-626 (1975).

15. G.C. BATrLS.T E, R.A. MILLER, and M. RMBIN, In Clinical Cisty and

T¢emiIac1 Tcixi ogy of Mtals (S. S. Brown, ed.) 221-224. Elsevier .ress

(1977).

16. W. HAUE, and N. WG, In 7th Int-maticnal Congress of P.hanacology.

Paris (J. R. Bosir et al., eds.). Pergamm, El1ford, N.Y (1978).

17. C.H. MUM, and H.V. AP N, Biochem. Biaphys. Res. Cmn. 94 501-507

(1980).

18. T.W. CLARKSON, L. MAGOS, C. CCX, M.R. GREENWODOD, L. AQ%=4-ZAKI, %LA."

M=k3- , and S.F. Al-CUZSI, J. Phazma . Expt. Therap. 248 74-83

(1981).

19. H.V. AP N, C.H. LNX.=, and T.E. MD, Tcxicol. Appl. P ,am.cci. 61

385-392 (1981).

20. M.M. JCNES, M.A. WMSE, A.D. %MVE, C.M. DAVIS, and W.K. VAU , l:es.

Cemm. C . Patol. Pha=g,. 27 363-372 (1980).

21. J.H. GRAZIAW, D. MOM, and E. ,EE:,, J. Pha ,l. Expt. Thrap.

207 1051-1055 (1978).

22. A. .hYLOR, LL. LALLCNE, J. Nucl. Med. 21 1190-1193 (1980).

23. F. MAOS-BE, B. GMPA , and E.H. M , Arxned.-Forsch. 30, 9

1291-1294 (1980).

24. L. SZCZ, W. MV=, U. HELL, and N. WEGE, (Drug Research, Zn ?ress).

25. P. ZVI IS, and R.I. E , Taoicol. AFpI. Pha.=amc. 36 397-399 (--976).

26. D.M. ZISLIN, I.E. O 1 , G.N. SAZ.v nUZCA, and A.S. VXRCOU'SCVA,

O) Gig. ~. Prof, Zabol 12 17-21 (1968).

27. K. LENZ, K. HIEW, W. Da2V, A. ErM, A. GAS=, G. - .

PC1 , and M. W Arch. Tc l. 47 241-243 (1981).

- 20 -

i_, ,. ,- ., .,= ., L...-k -.- ,.- . , .-. , , - .......- . ... - . '. V -.. .'. . . " .•.- .. . - -. " ..

028. E. ,5 ,R ,N4 J.H. zmAN, D. m, D. AOI, and B. KARIL, Lancet

If, 1234-1235 (1978).

.

"5'

*5-

°J

SI.

,

..

- ,, , .j %.. % .. _ - .'a' j . .%,. , j., . .% % - / ,.% . . • , - - % .° ,' ," .'-'- "%'- , %" - ',,",-.• .I;.

0PA I Cptical Isazmrs of 2, 3-Dimarcapto-1-propanesulfcnate:

Anti A ctvity, in vitro and Ln vivo,

against: Sodi= Arsenite.

SM9

DMS (2,3-dimircapto-1-propane sulfonate, Na salt) is an iuortant water

soluble analog of dizercaprol. All investigaticms of this antidote for heavy

matal n tcicn have dealt only with the raceac mixtre. In t present

report, the optuical iscmrs of DMPS have been separated and the

arsenic-antidote activity of the lev -rotatary (-) Lsar, te

dextro-rotatory (+) iszar and the racemic mixre of OMPS have been

investigated in vivo and in vitro. The individual optical isarers and the

r . mixure of EMS are effective equally, in vitro, in Preventna e

inhibitin by sodimu arsenite of the activity of mase kidney pynavate

deyAjoe~nafs cmpleax (PDH). In addition, when PDH is inhibited, in v.tro,

by sodium arsenite, any of the three CM p aations will reverse "t-e

inhibition equally well. The in vitro evidence suggests that t; -miecules

of OS are required to prevent the effects of one molecule of sodium

arsenite. Neither the ML50s nor the M50s of each of the three forms of :S

differ sinificantly when measured i.p. in mice. In additicn, there -s no

striing difference beten the effectiveness of the levo- or dext--o ator

CMS when given orally to mice challenged with sodium arsenite. -s, z-e

use of the individual optic.al isacmrs of WZ4PS does rot appear to rave any

advantage over the ra m ic re as an arsenic ant.dote under .ese

o , iti2s.

- 22 -

.. -. *.*~~*~'~ :~ .

CMPS (2, ,3-durcp 1-propanesulfonate, Na salt) is a water soluble

analog of dimarprl (BAL) (Petrumiin, 1956). Nt only is it analogous in

chemcal stuctue but it is also analogous in its activity as an antidote

for lewisite, 2-chloroethenyl-arsonous dichloride (Aposhian et al., 1982).

OMPS is an official drug of the Soviet Union (Klimova, 1958) wiaere it is used

clinically and is kino as Unithiol. It is used in West Ge~nany as an

antidote ffor mercury intoxication in humans and it is receiving inc-reasq

attntion in the USA, England and Western Europe. A recent revie of Its

pbalOg-- a, . and tberaps iti prop erties has apeared (Aposban, 1981). :t6-

is effective p.o., s.c. and i.m. as an antidote against heavy ,etai.

i ntt ation, in against arsenic, (xanazski et al., 1957; Tadlo6k

and Aposbian, 1980), mrczry, (Kostygov, 1958; Gabard, 1976; Clarkson et al.,

1981), lead (Anatovskaya, 1962) and gold (Gabard, 1980). Since the n=cer 2

carbon atrm of MPS s asyj~ir ic, tw op tCal isamers or enan rs of MS

exist. Hetofore, published repots dealing with UAPS have dealt only U

the phancological prpe rties of its racenic mixture or its stc- --raL.

isomr, isoMPS (Mizyukova and Lokantsev, 1960). Te biological propert.es

of the optical is rs of MPS have not been studied.

The optical Isomers of other metal binding or cheiating agents ofz.

differ in their properties and usefulness (Aposhian, 1961). D-ieni -e,

for exMPle, is less t~Cxc than l-penicLLLWnine because - '-as Zess

ant3i-vitanin B6 activity. us, I-penicillaine is a potent .inbzr of

cYstein desul Iydrase while d-penicilLanine is -ct (AposLian, -96).)

difference in tocity s one of the reascns that d-cezuc"-.a e, and -.cz

its l-iscmr or racemc mixre, has been the drug of =ce fr -i-e

teatmot of hepatolenticul-r d raion, Wilson's 01se se.

-. 23 .0 ' . "' ,'. ' " . " . ' -. " . ' ,' , " .. " . , ",''" 4. " .,.' . " .. 'r . ''. '' ," . ,' . ', .- •

Te present paper rqrts the separation and saiu of the antidotal

prertieas of the individa optical is rs of MS. Their activity, in

vitro, in prevmting and reversing the sodium arsenite inhibitin of the

pruvate e muLtienzyme complex (PEH) has been denstrated.

Secondly, the LD50, the D50 and the t uti index of each CMPS fomn have

been temt~ned when given i.p., and found to be essential.ly the same.

Finally, the isoars do not appear to differ in their antidotal activity w.en,."

given P.O.

Animals. CD-1 imle albino mic- ere purhased from C=ares River

Breedin Labs (Wilmingtn, MA). y were intained as described previcusly

(Mloc- and A oshian, 1980). At the time of their use for these experients

they weighed 26-31 g. The amt of sodium arsenite injected was equal lo

the a=roximte l100. M- caaceration of the %AsO 2 solution was sucn

that a 25g mnse received 0.050 ml s.c. The dimercapto unds -were

dissIved in 0.9% saline imdiately before use and the solutions ,are

adjusted t pH 5.5. The cocentration of the dinurcapto solutzons was uch

that a 25g mouse received 0.20 ml by the intaperitoneaJ or 0.20 mI -y z-,e

oral routs. For oral amni.station, the animls were fasted for 16 .- s and

curved 18 gauge oral feeding needles, purchased from Ppper & Sns, N.ew .,de

Park, N.Y., were used.

-UMeicals. 1MPS contains 1 ,molecule of H20 of r-e-ysta. _:.zatn. --e

enantiars and the racemic mixture (D L h r -traed "--h .:-..e .

order to measure mercapto cotent and puriy te2~ reaat~zns -ere

judged to be greater than 99% pure by ti.s =i-ter;..

-7..

Thiamne pyropksphata, D+ , CcA and sodium pyruvate were purchased

f=au Sigm Chmical Co. (St. Louis, MO). [l- 14C]-odim pyruvate was

purchased from New England Nuclear, Boston, MA.

of optical isomers of Oms

T reaction of dl-MPS with the opticaly active base L(+) arginine leads to

the diastexecmeric salts of D4PS. They can be separated by f--mc-ticnal,

cryst ization in methanol. The pure optical isaners of OMPS can ze

isolated by teating each of the diastereoisareric arginine salts w'ith lead

acetate folowe by de~psiticn of the resulting lead salts with Tld.--gen

sulfide. The separation of the isavars of EMPS was also successful aszig

brucine as the optically active base. In this way it was possible -

substantiate the physical poperties of = and IV.

Pb-2,3-9)-d rpel-sulfanate (Petnmkin, 1956), 49.6 q, was

s; q edwith sti xmq in 400 ml methanol. The suspm.ion was sazrarnd

with hykdLzgm sulfide. The lead sulfide was rmmved by filtration and wased

twice with 100 ml methanol. To the cbined filtrates was added (-) -

arginine, 9.5g, until pH 4.5 was reached. The addition of hydrgen sulfide

and the filtration must be carried out quickly. The filtered soluticr -as

then evarated and the residue recrystallized frz 250 ml methanol to gie

9.2 g of (H) 2,3--dimercato-Propane sulfonic acid, (+) arginine salt (prouc--

1) m.p.: 172 - 1730C; [C]20 : + 7.5 ± 1.0 (water, C = Ig/00ml). aodamer-a c

titratimn of mercapto groups ndimcated that the pur.fied -atariaiL =ns.s-e

of equal parts of arginine and (+) DMPS.

The mther liquor, f== which I was ciotalned, was evaporated =dar

reduced pressure. The residue was reystallized _= 50 ml met-z-anc -

obtain 8.4g of (-) 2,3-d±mrcapto-Propane sulfanic acid, (-) argini.e sa.-

(proct ) m.p.: 200-2020 C; [ 20 0 t I (water,

Z5 - "

ToAvPI titration of SH-groups shwed that th c mpositicn of the

diaster-zaric salt consisted of equal parts of arginine and -) DMPS.

A solution of (+) S- (+) arginine salt, 9.2g in 200 ml of a-0, was

acidified with acetic acid to pH 3. The solution was stirred at 600C. An

aqueous solution taini. 14.2g of lead acetate was heated at 600 C and

added to the reaction mix .trTe yellw

Pb-2,3- " ra,-opeslzat precipitate was filtered, washed

thoroXhly with acidified 0, 12 0 and methanol. The lead salt was suscended

in 250 ml methanol and de~osed with hydrogen sulfide. The lead sulfide

was filtered, washed twice with methanol and discarded. NaH 3 was added to

the cined filrates to give a pH of 4.5. The solution was then evaporated

OIder reuced pressure and the residue was reystallized frm 90% ethanol to

give 4.5g of +) EMPS, sodim- salt ( 1. [a]20: +2.5±1 ° (water, C=

Ig/1OOml) .Contm±: 98% SH - i Stric titration.

Using the sam procedre that was used to separate =II, 8 g of (-) 2MS-

(+) arginine salt was preared and hydrolyzed to give (-) CMPS, sodiium salt

20(IV). Yield: 3.7g; [a] 2 0 - 2.5 ± 1* (water, C - Ig/lOOml).Content: 98% M -

Midonuic titration

ioration of naie kzcekw extact for PDH complex activitv. Eight .euse

kidneys (about 4 grars) ware sliced and washed with deicnized water. -".

tissue slice were stored in a refrigerator for 2 days (Linn et al., .972).

The tissue slices and 4 ml of 0.25 M sucrose were hc=genized at 4°C "-n a 3.

Braun Melsungen ha,:genizer using 20 stokes at 650 E04. '.e homogenate was

cyifuged for 20 min in the SS-34 rotor of SorvalL. FC2-B cen -..±.ce at

3000x; and 40C. The supexatant was collected and stored at -70°C '..'

needed.

Z6,"

Assy of Pyxvt Deh:droetme gzplex Th te =.xture for assaylng

PM ccpla was prepar d by adding 0.08 ml of Tzis-H=. buffer, (pH 8.1),14C '

which cmitained the cofactors, to a test tabe tiIx -yrvt n h

sqernata tr of the mouse kidney extract. The final mt cnta:ned 0.' :4

is-H. (PH 8.1), 1 mt4 l-, 0.5M aC ,12 0.5 at EDM, 0.2 m- thia:Le..

pyrphospat:, 2.5 mK WD 2 M cysteine-Ha, 0.13 mM coenz,,n A, and 2 -ft

(1-C1 4 l- sodium pyruwate (0.5 mCi/mle) (Kresze and Stebe , :979) .n a

total vome of 0.24 ml. After inoatiLi at 300C for 10 min, 0.24 :MI of 20%

14TCA was added. Th 140 2 prdue was m e to measure PMH =cmPIA

activity. h 14CO2as trap by the mthod of Palma.tier et al., ('970)

and coumtd in a Packard TriCar scintillation counter.

In vivo stxzies - .nats of the LD50 and ED50 of the mriou fo=Ls of

a4ps we perfoed and anal.yz statistically by the m. m.ethds as

outL.nd in te biological stmdie and statistical analysis sectons of - :

pper by Aposhian et al., (1981).

Prevention and Reverml of the Inhibition of OH The imhihit=cn of --e ?ZH

enzyme _x. ix activity by sodian arsu.ete is -- ztatd Jx Fig. I.

dtannmn e er t-se canditios, wheuher the dIf.ert f=ms of OKIS =an

v the inhibitory activity, sodium arsenite (0.25 -tM) and .rza:..-us

amnts of M were i .ncubated with te PDH =zplex. X,.:-n was ,-

deta ned as a measure of H activity. The ammunt of 2MPS needed for C%

pr of the inhibitory act.lviy of 0.25 mM sodium arsentie was .. z

be about 0.50 iM (Fig. 2). The amotat needed for 130% prtec.cn .s

essnxiaUy the sime regardless of whth er ttw (-) -iscar, ---e - - er :r

the racin xtze is used. The of dl-, -) -, or -- 2'S .

needed for 50% prev otic2, a tr quantitavve value, was f-rnd z e .24 7M4

-27-,".., . .".-. i.-'. ','.. ..- ., :' .. ' ............................................- ".".....".."'.'.'" "...

100 ''1 1 1

80-

ele

o 40

20

0 0-2 0.~4 0.6 0.8 1.0

NaAsO2, M

Fig. 1. nb'lim± (Of m~Se kjiey pyruvic acid de±±ydragenase =r.pexactivitY bY godiuin arseite. Sodium arsezuite, PDH ca1pex (2.3 mg protear : frrmm kira ex~act) and other =Lqtit-en of t.h-e PCH lr~ex revac-.Icn,O txtur wre incubated as dsczted in Msthc=ds.

-28-

y~ w -w.-w-., w.a w ...- f- .WW - ~ - nl f ' .* - .a~u.-- -S.~-~100 -rrr r ,.~s lu r ns - -

10

0 0.2 0.4 .8 08 1.

DMP% m

Fi .2. d -C r t eat~ms 4il8peen0actiity f =ue k r~y DH c plexby odi= arseite

(02 m, mcpm(23m rten- osf c rc.a0mz

- 29

(Fig. 2). It appears that a CPS:As ratio of 2:1 is necessary to prevent

mletely the inhibition of the activity of PDH omplex imder these

coniticns.

h relative activity of each foam of CM in r the inhib-itn

by sodiian arsenite of the PDH complex activity was deteined next. '.e

sodium arsenite and PDE complex were incubated first for 5 rins at 30°C; then

different aounts of EMPS were added (Fig. 3) and the incuba cnued

for 5 rans. T reversal activity of the different forms of CS are equaCL,

(Fig. 3). To obtain 50% reversal of the inhibitory properties of 0.20 7

NaO 2 , 0.26 to 0.28 mM IMPS is required; 100% raversal requi.red

a~r~c±Lteily 0.40 nt4 CM4. Since the 50% value Mmi.tiplied by tw is more

O accra t, it appears that a CMS:As ratio of 2.7 or realisticaly

between 2.5 and 3.0 is necessary for 100% reversal of the inhibition by

sodin arsenite of the activity of the PH complex. The three fo=Ls of aPS

e to hae equal reversal activity.

Protetion, in vivo, against the Lethal Action of Sodium Arsenite. Mie

ther-tic i ndex fr the different fr of PS in mice was d extine. d by

dividing the LD50 of the di-e rcaTo pomu by its ED50. The latter ralue

is definad as the ammt of rp powd (rmrl/kg) proec tng 30% of

the animals against the lethal effects of 0.15 nmol NaAso2ikg. 7x -1i-s

laboratory, this dose of NaAsO2 killed 100% of the animals. Mhe i.p. -Z-90

for the three CS forms are given in Table I and the =50 and t!eArmeutc

index values in Table 2.

Zn the above ieriamts, OPS was g.ven i.p. Mhe antidota ac -.v i- Of

(-N the three CPS fomu was dete=Jmed when given p.o. also (Table 3). No

strik=ir difference n the antidotal activity of ( ) or (-)- fomn was ft-n.d. %

- 30

SI.,

w0 --W ------ wv

100-

80-

h60

~40

*(+)O0MPSH (m-OMPS

~di-OMPS

0 0.2 0.4 0.6

DMPS, mMFig- 3. X-eral by d1.-Cmw or wmsuat±mmrs of t±,Ie Inh.l.i of t..ec~lplex by sodii. arsmte. Fm complex rmeacticn m==xe (0.24 .ctaning 2.58 mg of muse k±iey extrcot prate=2. and 60 :=1es -Zso.arsuiita wer. incubated at 300C for 5 min. L4-An 0.06 Ml Of Sit.fC cItair n 0. 1 14 Tris-IH. buff or (pH 8. 1) , Z -,4 pvruvate, of ac-.ors anuma ad and the =3cbat=~ cont~ued for 5 .- r= m=.s --. -ea a ssMw~pd with 0.30 ml of 20% T.. 'fla SPCIfiC aC-.vlt Of -,7. PMH ==Iax

wca s 2.47 nacwls Co2I/m.n/mc pxotein.

TABLE 1. LD5O of the optical isomers and racemic mixture ofDMPS given i.p.

95% No. of DoseLD5O confidence mice range

(mmol/kg) interval used (mmol/kg)

(-)-DMPS 5.88 4.676-7.214 85 4.0- 3.3

(+)-DMPS 6.02 5.121-6.490 76 4.0-i0.

di-OMPS 6.53 5.494-7.706 88 4.0-0.,

3Za

-,

©

Il

- 32 - a

TABLE 2. ED50 and therapeutic index of the optical isomersand racemic mixture of DMPS for protecting miceagainst the lethal action of 0.15 mmol sodiumarsenite/kg.

(-)-DMPS (+)-DMPS dl-DMPS

ED50 (mmol/kg)a 0.045 0.051 0.055 "

Confidence interval (0.006- (0.0084- (0.0261-0.0824) 0.0877) 0.0820)

Therapeutic index 131 118 119

a The dose range of DMPS to obtain the ED50 value was 0.012

to 0.120 mmol/kg and the number of animals used was 140for each DMPS form. DMPS given i.p.; sodium arsenitegiven s.c.

%%

f..

- 33 -

TABLE 3. DMPS enantiomers are active p.o. in protecting ;3

mice against sodium arsenite lethality.

I 7-day survivala

no. surv./no. start survived

(-) -DMPS 11/16 69

(+)-DMPS 9/16 56

dI-DMPS 14/16 88

(H2 0) 0/16 0

a DMPS (0.12 nuol/kg) or H 0 was given p.o. 15 min prior to

NaAsO (0.15 muol/kg) s.3. Control animals receivingDMPS J0.12 mol/kg) p.o. had 100% survival.

344

0 4-

- 3 ¢, -

J4

' " . ,w , flV %,v-' f W. a.r rlc.- ~ r ~ S -r',--r ,5w ~ W rrrw ,r ~r r

In the ClAuAical1 wark on,.- aru lc wisite and dim.rcaprol. by Peters and

o-Ia (Stockmi and flmsm, 1949), pyravate cxcidase was believed to be

the most sensitive enzyme. ore recent studie have show that pyruvate

= edase is not a sigle enzym. It is wr~n now as the PDH rutienzyme

cmlex. T hemdative d cylaticm of pyruvate catalyzed by t * pyr-v-ats

dekiydrog.eaase comlex can be smrized as follows:

Pyrmte + ND+ + CcAH CCASSc NDH H + CO2

Th mJ~ii--ezym cclex cotains marry copies of each of othe tre

enzym CCOt; pyravate dehydrogenase, (Haa et al., 1976),

dihydrolippyl transactylase, (Emnaa et al., 1975), and dinyoipcvl

1hydrogwa (Sakurai, et. al., 1970). In a ition, pyruvate dehyd

0 k~ jnae andi pyrivate deyzgenase pkxosphatase (Bater et al., 1978; Lizm et

al., 1972) are preset in the ccmzlex.

T pregmut farijeJmMi.s using the PM naluatienzy. wplex ac--,-, 'iv

dm~istaltes that I PS can prevent and reverse the sodium arsenite inhibit-="

of this enzyme activity. All three foms of MPS are equa.Uy active -n -nis

respect. It sxgests that 2 molecules of OS are required to prevent =a

inhibitoy activity of 1 moa of arsmite while saruwhat -mre Is

necessary to obtain reversal (Figs. 2 and 3). It does -ot appear that e

simple classical picture of chelate or ring fomatin with the -esu-=

thcarsenit type of stuce is app.icable, in t Ls case. MA .q -e.eate

would be expected to have a 1:. ratio of chelator to As. We are -- _. c

isolate the MS-arsmric stuctures in order to detesne th.Ar a . .-e

three f 1 of CS have si rdIAr LDS0 and ED50 values -n .mice. --!-e

C turaintic index of each form as a sodi.ur arsmiite antidote is -.nden:dw.-

of Mar is ric stcte (Table I and 2).

-35-

7U V WE W -- *.WW- - Trwr '.1' -W, - -. VWW'r. .j %- W V I.W '-~ VV "* *

It has been reported that rMWPS is not ompletely absorbed from the GI

tract (Gabard, 1978; Wiedemann et al., 1982). The extent of absorpticn has

bw reported to be anywbere betwen 30-60% depending on the species used.

Te activity of the M enn in protecting against arsenite let-a.--=z

was sudied p.o. to detenine wbether the 30-60% absorption of DL--'-PS . u~g

be due to one of the isanmrs in the racm.c mixtre not being absorbed -

the GI tract. U this were so, that isamr when given p.o. wuld r= pr--tect

the animil against sodilan arsenite. This does rmt appear to ze =..e =as

(Table 3). There does not appear to be any stiking differenc ber _n -- ,

individuaal t of CM as far as their activity p.o. under -- se

C~deitons.

Very little is known ab=a the bitransf ~ation of UAPS or its scamrs.

Preliminazy s~ldies have indicated a possible onersion to the disulfide and

t... ,1l4,ii in biological s yS (I nSki4 and Tsboda, 1960). ,.reth-

each of the optical isawas of MPS would be aoidized enzymaticai..lv at tne

saw rate is not kmwn and must wait for more sop icated .eat).s Cfz

separation and analysis that are not available at present. These t.--s are,

at present being develoed in this labratory. ;=n such ethds are _m.,

and perfected, additional tdie of the optical s rs of this -r-a

water soluble metal binding agent that is effec=.i.ve orally shcud ce -f

interest.

- 36 -

REFERENCES FOR PART II

ANATOVSKAYA, V.S.: The use of unithiol in the treatment of chronic

lead intoxication. Gig. Tr. Prof. Zabol. 29: 50-56, 1962.

APOSHIAN, H.V. : Biochemical and Pharmacological Properties of the

Metal-binding Agent Penicillamine (National Academy of Sciences

Symposium on Metal Binding), Fed. Proc. 20: 195, 1961.

APOSHIAN, H.V. : DMSA and OMPS - Water soluble antidotes for neaw

metal poisoning. Ann. Rev. Pharmacol. and Toxicol. 1983, in press.

APOSHIAN, H.V., TADLOCK, C.H., and MOON, T.E.: Protection of -nice

against the lethal effects of sodium arsenite - a quantitative

comparison of a number of chelating agents. Toxicol. Appl. Pharm.acol.

C 61: 385-392, 1981.

APOSHIAN, H.V., MERSHON, M.M., BRINKLEY, F.B., HSU, C.A., and HACKLZZY,

B.E.: Anti-lewisite activity and stability of meso-dimercaptosucciniC

acid and 2,3-dimercapto-l-propanesulfonic acid. Life Sciences, 1982,

in press.

BAXTER, M.A. and COORE, H.G.: The Mode of Regulation of ?yr=:vate

Dehydrogenase of Lactating Rat Mammary Gland. Biochem. J. -74:

553-561, 1978.

CLARKSON,T.W., MAGOS, L., GREENWOOD, M.R., AMIN-ZAKX, L. MA - , A.A.

and AL-DAMLUJI, S.F.: Tests of efficacy of antidotes for remcva "-

methylmercury in human poisoning during the Iraq out-reak.

Pharmacol. Exp. Ther. 218: 74-83, 1981.

GABARD, B.: Treatment of methylmercury poisoning I.n te rat-.:-n

C sodium 2 ,3-dimercaptopropane-l-sulfonate: :nfluence of dose and mcde

of administration. Toxicol. Appl. Pharmacol. 38: 415-424, 1976.

- 37 -

"..I ' " ' " .

8%

GABARD, B.: Removal of internally deposited gold by 2,3

dimercapto-propane sodium sulfonate, (Dimaval). Br. J. Pharmacol. 69:

607-610, 1980.

HAMADA, M., OTSUKA, K-I.,TANAKA, N., OGASAHARA, K., KOIK., K.,

HIRAOKA, T., and KOIKE, M.:Purification, Properties, and Subunit:

Composition of Pig Heart Lipoate AcetyItransferase. J. Biochem. 79:

187-197, 1975.

HAMADA, M. , HIRAOKA, T., KOIKE, K., OGASAHARA, K. , KANZAK, T. and

KOIKE, M. : Properties and subunit structure of pig heart py.ravate

dehydrogenase. J. Biochem. 79: 1273-1285, (1976).

KLIMOVA, L.K.: Pharmacology of a new unithiol antidote. Farnako. -

Toksikol. (Moscow) 21: 53-59, 1958./-.-KOSTYGOV, N.M.: The antidotal action of mercaptosuccinic acid and

Unithiol against mercury. Farmakol. Toksikol. (Moscow) 21: 64-69,

1958.

KRESZE, G.B., and STEVER L.: Inactivation and disassembly cf -.e l

pyruvate dehydrogenase multienzyme complex from bovine kidney by

limited proteolysis with an enzyme from rat liver. Eur. J. Biocnen. l

95: 567-578, 1979.

LIN, T.C., PELT.Y, J.W., PETTIT, F.H. HUCHO, F., RNDAL., : .D., and

REED, L.J.: )Keto Acid Dehydrogenase Complexes. W. Puri::a:icn and

Properties of the Component Enzymes of the Pyruvate Dehvdrcqenase

Complexes from Bovine Kidney and Heart. Arch. Biochem. 3.cphys. i43:

327-342, 1972.

LUGANSKII, N.I., LOBODA, Y.I.: Transformation if ' ni-.niol .n :.ne :cdv.

C Farmakol. Toksikol. (Moscow) 23:349-55, 1960.

- 38 -

0LUGANSKII, N.I., MIZYUKOVA, I.G. LOKANTSEV, D.S.: The mechanism of

antidotal activity of unithiol in poisoning with arsenic compounds.

Tiolovye Soedinen V. Med. Ukrain. Nauch-Issledovatel. Sanit-Khim Inst.

Trudy Nauch Knof. Kiev, pp. 115-130, 1957.

MIZUKOVA, I.G., LOKANTSEV, D.S.: A comparative essay of toxic and

antidotal activity of some mercaptoalkanesul fonate der vat-ves.

Farmakol. Toksikol. (Moscow) 23: 355-361, 1960.

PALMATIER, R.D. , MCCROSKEY, R.P. , and ABBOTT, M.T.: The enzvma-c

conversion of uracil 5- carboxylic acid to uracil and carbon dizx-ie.

J. Biol. Chem. 245: 6706, 1970.

PETRUNKIN, V.E.: Synthesis and properties of dimercapto derivatives :

alkylsulfonic acids. 1: Synthesis of sccd.m

O 2,3-dimercaptopropylsulfonate (unithiol) and scdi'.m

2-mercaptoethylsulfonate. Ukr. Khim. Zh. 22: 603-607, 1956.

SAKURAI, Y., FUKUYOSHI, Y., RAMADA, M., HAYAKAWA, T., and KOIZ,

M. :Mammalian a-Keto Dehydrogenase Complexes. J. Biol. Chem. 243,

4453-4462, 1970.

STOCKEN, L.A., and THOMPSON, R.H.S.: React-.ons of 3rl-_.sn

Anti-Lewisite with arsenic and other metals in living systems.

Physiol. Rev. 29, 168-192, 1949.

TADLOCK, C.H., and APOSHIAN, H.V.: Protection of mice aga ns- -he

lethal effects of sodium arsenite by 2,3 dimercapto-1-cr:pane-suL7z-n-

acid and dimercaptosuccinic acid. Biochem. 3iophys. Res. 2omrn. "4:

501-507, 1980.

- 39 -

-- - , ' '..-- r .,-..'.' .',. " ,-,--' .- ,....'.' .- '. . . .. -. . . . " .. . . .

WIZEMANN, P., FICHTL, B., and Szinicz, L.: Pharmacokinetics of14 C-DMPS (sodium-,3 314 C-2, 3 dimercaptopropane--sulfonate) in beagle

dogs. Biopharm. Drug Dispos. 1982, in press.

40.

PART III -

Answers to possible questions as to scope and progress of contract

Questions to be answered about scope and what was and what wasnot done during the contract from February 1, 1980 to October 31,1981.

Why didn't we test more compounds? One page 21 of contractproposal dated November 26, 1979, we stated that we would test 23compounds for protective activity against the lethal activity ofsodium arsenite. We actually tested only compounds numbered 1, 2, 6,7 and 10 of that list. Therefore 5 compounds on the list were tested.We also tested 9 other compounds not on the list. Seven of these arepublished in Table 3 of our paper that appeared in Toxicology andApplied Pharmacology, 61, 385-392 (1981) and in our first annua-"report.

Therefore we tested 14 compounds. Why didn't we test -he othercompounds on the list? For two reasons. First of all, the protectiveactivity of DMPS and DMSA far surpassed our expectation. Because DMPSand DMSA were so successful, it was decided by conversation with Armypeople that we should push ahead and do LD50, ED50 and therapeuticindex studies and compare the effectiveness of DMPS and DMSA. Thesestudies were not even alluded to in the original proposal. They are ',costly both in number of animals, personnel and computer time andevaluation. Once the effectiveness of OMPS and DMSA were found, these2 compounds became the primary interest of the PI and his lab. It wasdeemed very important that their anti-Lewisite activity be determined.The compounds, DMPS and DMSA, were studied by the PI at Edgewood andwere found to have anti-Lewisite activity.

Why were the other compounds of Table 1 not tested? There aremany reasons: 1. We became more sophisticated and realized thatmonothiols would not work. 2. We tried polythiols that we did not ,even know existed at the time of our original proposal. '-

In summary, looking at page 37 of our original contract proposal",two phases of research were proposed under methods Phase I and Phase11. Under Phase I we assayed 5 of 23 compounds and 9 others that werenot listed. We did not study mixed ligand chelates because -eeffectiveness of mixed ligand chelates in treatment of heav-. -e-a ..poisoning was retracted by Shubert.

Phase 11, Part i -- DMPS and DMSA were shown in preliminary experi.nen-sat Edgewood to protect pigs against Lewisite burns. Experiments weretried with mice and Lewisite at Edgewood but they were not success-'_because of factors out of the control of the PI.

Phase 11, Part ii and Part iii were not performed because we ran :.;t I37 money.

It should be kept in mind that the PI showed that DMPS and :MSA 4

have anti-Lewisite activity. There were no such reports :: -z-activity in the world literature.

-41-.- -~, * **.

";W: .4"'.''--,",' -''" " . - ."""", % .- "*. * " .-" *.- .,** * .- .- w ". " . . ", "." " .' . *. " -' . 5 . r .-",-".."- , ", "- -. -', ," - ,.

The PI is told that $109,000 was originally awarded for thascontract and the contract from February 1, 1980 to October 31, 1981 .spent a total of $213,000. The difference between what was originallyawarded and the total spent was primarily for the purchase ofequipment. The purchase of new equipment was justified in letters to .the manager of Area V research and/or the COTAR for this contract.These letters as justification for equipment needs are in the Army'sfiles.

It also should be kept in mind that the work from February ,1980 to October 31, 1981 in Aposhian's lab was carried out by the ?:,Mrs. Boxhorn and 2 honor students. The two honor students worked a -minimum of 20 hours a week without salary and were indispensable tc ,the work covered by the original contract.

In addition one member of a site team has criticized us for us:nc %mice from Temco Lab and mice from Charles River instead of stick-4.g t= .one source. We started with Temco mice since the company is locatednear us in Houston, Texas but we soon found them to be very unrei abieas far as ability to deliver. We continued to use them even though i.was inconvenient until a logical point came where future exper,*e.ien-swould not depend on having the same strain of mice. We then switchedto Charles River mice. There has been absolutely no indication thatthe switch changed any results in either quality or specificity cfdata.

'

42

%%

Distribution List

4 copies: CommanderUS Army Medical Research and Development CommandATTN: SGRD-RMSFort Detrick, Frederick, MD 21701

5 copies: CommanderUS Army Medical Research and Development CcrmandATTN: SGRD-RMSFort Detrick, Frederick, MD 21701

12 copies: AdministratorDefense Technical Information CenterATTN: DTIC-DDACameron StationAlexandria, VA 22314

I copy: CommanderAcademy of Health Sciences, US ArmyATTN: AHS-CDMFort Sam Houston, TX 78234

I copy: Dean, School of MedicineUniformed Services University

of the Health Sciences4301 Jones Bridge RoadBethesda, MD 20014

3,

9.

IE.