- Email: [email protected]
Screening methods of drug dependence liability using small animals
TIPS - Octoher 1981
2,,-
down, e.g. by DMSO peel reatment, w,ually leads to incre ~sed toxicity. A skm'ing dolin of the Inelabolism. e.g. by toluene. decreases the toxic effects. TIle first product Of oxidation is an ui1stable, highly reaclive benzene +lxIxide
(Fig. I }. Snyder et
el. ~
suggesled thai this
melabolite is responsible for most of the
of inlcstlgalitm ha~c iildicated it dcrailgcd intenitlkm betlieei1 ~tem cell',, prilge.,lihlr cells and rcgulaling micro e m i r o n m e m l .
A n i n l p o r l a n l que~titln i+ the ix~,•ihlc carcinogcnieil) of hl.-nzene. Itere. an imD~rlanl difference hetmcen e x w r i m e n l a l anim;ils and man is the fact that. de,,pile titan) allempls, no maligllam:y hit+ k ' e n iilduced b.v ben/cue in i'il'o in ailimals. In contrail. the --pidcmiologi~il el ideilce mentioned earlier indicates th:il k ' n # t ' n c - e x ~ + d human indi+iduals shoe an i i l t ' r e a + d incidence llf leukemia. This might iildical¢ thai in human+, additional hl%.icil.% i+ alllicled on the hacmalOl~fietic • t i m call.
loxi¢ily in I,ivo, by onvalent binding to protein and iludeic acids. The enzyme sysieni in lhis palhway is ai3lhydrocartlonhydroxyla,~ (a mixed function oxida.~, A H H ) which is present in Ihe liver and I~mc-marrow. The inducibility of this enzyme in the mouse is under genetic onntrol by' the Ah locus, Ivhieh heals Ilit.o alleles (Ah ~ and Ah~). Ah" codes for high R e a d i . I list I ilaak. It. i . 4 IqSli| ( ?in. ?hwma#,d "~ /,-'1 +i',I'+ inducibility and Ah a fiw much lower 2 (;old,.lu~n. B I ) | 1 t l " T ) J I+,lu+d Inttr,,n inducibilityL The.~ alleles arc aim present I h'ahh .%~*ppL 2. Aq- l i IO on human eelk. This mechanism might 3 Vi,,Iman %. R.( It~77)J /-,~l/¢oLtt.nltn,,I Ih.ahh offer a basis for thc imt~wtailce of the gene.%uppL 2. ~3--hx 4 I-Icrnt~crgS.. Sa~ilakli..%1..AMman.K..rod -%,p. tic background. .%.(19h6) Rr. I. bid..l~,ed. 23.21i4-211~ Another pathway leading to a deranged 5 A I ~ l % . ,it,|. (19~7) ,li~,. I~mnhu/( Ontrtb ( hn ~, DNA metabolism was suggested by 1"~ 3 - 1 4 Freedma#. It was shown that benzene or h .%n_~dcr. R.. [_ec. U. %%_ K ~ ' ~ i , , . . I . J . a n d %%ilrnL.r. its metabnlites inhibit aminolevulinic acid synthetase, a rate-limitiilg enzyme in haem synthesis. Haemin binds to a dercpresmr molecule of globin and non-globin protei-~ syimhesis. A decrease in available harm couM then lead to a geilcral repression of protein synthesis at the level of translation. This in turn could lead to a deranged DNA repair mechanism. Cytogenetic effects As mentioned earlier, bone-marro~ and lymphocyte e h r o m o m m e analysis show a
wide variety of chromosome and chmmatid breaks and gaps. It was emphasized by Wolman ~ that most damage meres to be toxic rather than mutational. In view of the suspected carcinogeilicity of bei1zeilc, several mutation induction studies have been performed. No increased frequency of mutations was observed in the bacterial system by benzene or its melabolites. It is of interest that Morimoto and Wolff ha~e recently hmild that the induction of sister chromatid exchanges (SEE) in human cells was nol aft'ccted by Ix,-n/cilc or pllcnol. However hydroquinol and catechlll were very potenl inducers of s e E , indicating a major interference with DNA-synthesis during S-pha~e t°. Summarizing the experimental part, wc tint; that benzene induces haematopoietic hlilare at the level of the matutatiilg progenit,~r cell, beyoud the pluripotent slem cells. This is expressed by progressive.dysplaslic chan~es in I~mne-marrow and peripheral blood. In addition, several line,~
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Screening methods of drug dependence liability using small animals Ken-ichi Yamamoto and Kohji Yoshimura lJtvt+itm ,iJ .'%'ettrolihtirmzicotol~l..llht< .h~lgt Rt'~carcit l~th,,rat,,ri,'+ %+z.'.,tt, ,~.'+,++;d ( ,+ l td I ,,k., ,:.',;., ~., + +,,.~ .IS.i, Japan
Varkius animals, such a~ mice ral~. dogs and monkeys, have been used t++ pri, dict
dependence Iiabilil)on drugs. G e n e r a l k rhesus monke)-s are thought to Ix." the ino~t suhable animals for imorphin¢-t~ i ~ e~lx'n ments. I%lan~ ne~ ('NS-affe.-ting dru~, are coiltiuuousl) being synlhe.~ized :lild nccd to be checked lor dep2ndence liabilil) it+ confirm their salet). I iol~e~ er. continti¢, l ,,upp l y O f tJle~C m o n k e y s
dot."~ n o t ~CCi11 i ' , r o m -
ising and also such e%lt, riillcnt~ arc i c e e x t t ' n s i l e aild lal~,~r-con~ulning. ]+lh'r+'ll,lrc, a scrceiling nlelhod l o t drug dt i~.'ndeuce thai call clt~il) ) i d d llceurale rt,+,.lll+ needs to Ix: established using small aninlais such as mio: and rats instead tit monkey',. This paper pre~:nls o b ~ r x a l i o i ~ ol -~mle general screening i11elhod.' for
morphine-type ph)~ical dependence u~ing i11iCe a n d r i l l s a n d c o m p a r e s
them
~l i~11, ~ur
findings obtained receilll> ~iiill t i l x .
Te~Is using mitt+ The j u mp i n g method dc~clol~:d h~ X~ a~ t.t aL t lla', l~.'cn ~udck t,...'d ,l. u icll,,l~l¢ ifld{i'ahlr in ql.lai1111} iflg Ih¢ c%.lCllt .q ph}-,cal dcl~ndcncc in nncc Junq'.n~ ll:duccd l'~,, al'rupt or prc¢ipit,ltcd ~itlldua~ d m i11orphih¢ ix'llct-mq+kmtcd r:lcc i, regarded ,I, an ltldlca[Ol ol ~ l ; h d r ~ . d .ign,. i h c i n t l d d n ¢ c tit i u n l t . l i l g l,~llo'.~illg iibrtipl i~ ilhdrilx~dl i~ Ilmi-d¢l'~n,Kni. lhc ic~tklll.,c I~:llld llld%lnl,il l ~ , h ,dice fClllOl,iI o l l i l t morl~hlnc lalq¢l..~tl~lupl ilbMigi'ncc I~ qU,liltllied bx pliicInL' the alillmal~ tin ,I ¢irl'uldr lil,llhffm arid li%'oidnl ! the Ip~.'r¢t'llla~C ol anililal+ thai leap oil xlitliin !+4 niin. [ h i ' dcgrec ol ph,,~ieJ depeildeilcc can Ix" asw~.~d b.~ plotting th,: tx:rcx-ntage of re~lxm~c against time and determining the a,¢a undcr tl:c tar're. -[he ilre-l I~ I l t e L l ~ u r c d ~ ) etlltillg oUt the ciir~+ from the graph p a l t ' r and x~ciglling It. Irl
TIPS - October
2~bt DOSES OF MORPHINE
OF
1981
SPONTANEOUS MOTOR ACTIVITY
GROWTH CURVE
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ABRUPT WITHDRAWAL
l'ig. I Rel.uionUIIp b e t . een t zrcadicm rhythu o.[~t~,mtaneolt~ . I ~lor atlivit.v at d ('hat g¢,.~#1 hot#, .'eight U! morphble.depemlcnl rat.~
prccipilated withdrawal with the illorphine antagonist, naloxone, the degree (ff physical dependence on morphine was assessed without re~" valot the pellet by esiimating the median effective dose (EDse) of nalt~xone needed to cause jumping from the platform. Marshall et ul. a used thc' number ,)f jumps in addition to their incidence as an indicator of physical dependenct, in mice repeatedly treated with morphine• Kaneto et al. a reported that actual withdra~al caused various withdnlwal signs involving jumping, wet shakes and weight loss in mice injected with morphine at increasing d ~sesof I 0--I 0(1 mg kg-~ twice a day fl~r I 0 days. The degree of ~ilhdrawal signs was totalled for groups of five mice each for a 2{)-rain monitoring D;riod and expressed as a sum of scores. According to this estimation, the intensity of the abstinence state reached a plateau 5--6 h after the morphine administration. The same
xvithdra~al signs also appeared following morphine-dependent mice may be actual withdrawal after !-I 0 h of continu- regarded an behaviorally excitable ous intraver:ous injections, five subcutane- changes. ous injections every hour or a single injection of 100 mg kg -t morphine. These Tests using ra*.s phenomena were potentiated by naloxone. In rats repeatedly injected with mor0~1 ~he other hand, it takes a long time to phine, abrupt withdrawal produces make mice dependent with oral adminis- sedation-like hypoactivity and ptosis as tration, of morphine. Significantly lower x~ellas excited behavior such as aggression, latencie,, for myoclonic and clonic convul- vocalization, hyperactivity, and convulsive behavior induced by flurothyl in sions, or both changes in excited and sedamorphine-dependent mice were also asses- tive activities. Thus, the withdraw~d signs sod as hdieative of physical dependence. rel~)rted differ considerably among the which i~, characterized by increased CNS investigators. exeitabillity4. Hosoya e was the first to prove inducRecently, Wiley and Downs~ reported tively, based on experimental fact, that that naloxone-induced jumping after a weight loss l~di~wing the withdrawal of single ie/cction of opioid in mice might be morphine is a useful t(~l fi~r ~reening the of value in the rapid identification of drugs physical dependence liability in rats. Later, with a propensity to produce morphine- this was confirmed by many investigators like physical dependence. and applied ~'ltisfactorily to the screening In any case. withdrawal signs in of morphine-type dependence. However,
1 1 P 5 - e h ' t o h e r i +t81
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Ihis weight loss occurs even i0 v+ilhdra,~al
implant,ilion, and the longer the mter,+al bet~,~,¢eu ;,inlagt ~ni',,l i n i c t l . m ,llh:l the hnal mt~rphine. Ihe le',,, tlne incidence ,d the resp, m~_" ( ) n lhe other h a n d Y a n o c t a l " su[zgesli.'d lhal hislologlcallt, morphologyinduced by precipitalcd withdrav+al have caU c h a r g e s ol mil~:hondrla in a d r c m ~ : o f hcen quanlilatively cv:llualcd and :0sscssed tical cells are uv,:ful as intk'xe,. , I ph.~,.ical as a criterion of drug delm:ndence in rals d e p e n d e n c e liabilil.,, ol m-rphine, l h u s . subcutaneously implanted with morphine ',arious behavioral change,, hkr lumping t~eilcts as in miccL Ho~,evcr. many inves- Ilil~,+e b e e u considered sUilablc signs I,,, lhe tigators have claimed that Ihem: we! shakes e t alu;ntion o [ ph.~,,Ical d e p e n d e n c e t d I11,,r-
from barbilurates and IranqoilizeP, ~,nd. thereh)re, is not al~ays specilic to signs of morphine withdrawal. Also. changes m behavior such as wet shakes and jumping
or jumping responses are essentially dillcrenl from wilhdrawal signs; they conslitutc simply anolhcr kind of antagonistic activity, becau.~ they have been induced hy the injection of antagonist even immediately after morphine injection or during pellel
phine in rals. a h h o u g h r c l ~ r l s hat+: been lira!led to record~ ol Ih¢ t~ilhdra~,l ..ign~ ~ith considerable ~ar0ation Irom a u t h ~r I.. author.
We ",tudied .+~hal kind,+ of ,,ign,, I+~.',,id¢', u, eighl hv,,, +,~mld I~." u ~ l u l ;+1,,ol'qccli~c
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+'-~II mg k.d t. t+otk+n11~,dt dlt~.-~ ,it 1 4 ~ I m~ kg ' .rod d-pr, q~,~pi~.+m: .'i+-l + " ++1'.:"k++,' ' t~,¢*.' ;t d,t} her ~ - | 2 ~+c. k', R, ¢l,d ~¢m|~. ~;llllrc did first ~'iLJI~C o n ';dotup+ +t ill+dr;ire +d. bul d,.'cr+a%'d 2 1 ( . +'4 ( a + l d +t '+ ( I,,1
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d r a ~ a l and m a r k e d X+tlh K.+;~lhqph++n induced mllhdrax~al ol m o l p h m c .+led ¢,~Jcmc Ih¢~: l~:,~ull+..,u+~,:tc.q Ilhfl ¢h.+lt~¢in I~KI~, tcml~.-l+alure ,;lAd 1fl+lI|'+II++)flo! ,+ ¢lmdillimed l~..,,pln,~., a r c U'~.lU+ +is +nd+cators for ¢xalu+fllng dtu,J dcl~.'nd~ nov + Xcxt. x~- cxamm~+d x~hcthc; ,st not the natix¢ o r c a d m n rhxthm ,,1 rat,, ,:+-,,Meal h,+ Animcx and F i : ( i ~,,uk! I~- u~'tul a~ an ohwt-tlX¢ indii.:Jtor |,t,l dndtx",l', ol dru# dep~ndcno.'_ %lalc Xt. i,.Li; .+++~1- ~ ith implanicd ,.-hroni¢ clvctrodc, x~cr¢ -ub+
cuLam.'ouq,+ in]¢dL-d v+flh morphine at increasing do-e,, ot 5-511 mg k,.." ~ tx~u'c ,~ day h r ~ ~cck-. The p . ~ ' c - . *.: dcl~+n dencc dc....Iopmcm of mt,rphinc and the aPt~arancc ot ~'thdra~tal ,.n~n. ~tcnc .,,ntinuou,,I.x r¢+.'oldcd bt .-~nxrncx and E L ( i " ' Control rub tr+.'atcd with tch~clc .ho~ c~rcadian rhxthm **ith dccrca-cd attar It5 .rod at tendcnc~ to ",loop in the Eli( i during the daytime t~.'th sexeral t~.',lk~ ot m,+tor a,:tr'. ilX and more ,,+aking qa,_':'+ In the |:[+(' a: night. Single iniecthm t,7 morphine at ,:
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:DISSOCIATIONBE'twin+ EEG et~lO8£HCWIOR
Fig. 2. ,Sh,ep-+vakeJi~h~ess('ych'x and EE( ; h'rHs ol a morphme-dept'ndt'tzt rat fi,r 7 h turm¢ the d, O.
oi the drug. and lhc RI:M ..h..cpnl¢,la~. decrea..ed trom lhL' 3rd da} oI Inl¢Clh+'r (Fig. 2b+ The,< change, ,.con m the I I-Xi might i~ regarded a- brain electrical atticit} corre~,ponding tt, thc increa,,e m motor actixit.x. AI~+. the injection- ot morphine interestingly induced a temt~war,, aptx'arante of high ~oltagc FI-.G .I,+x~ N.r,t. accompanied bx I~'ha~ ioral alcrtne.- x, lth the cxc~ oleo'ned v, idc.. Thi+ phcnomc'~on
270
TIPS BEHAVIORAL ALERTNESS
oosr-s OF B E F O R E MORPHINE
o.~j~ ;0
HII~OC,;AMPUS
NEOCORTEX
MORPHINEsL 42mia
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October
108!
prehensively as~ssed by analysis of hod) temperature, spontaneous molar activity, and electrophysiology such as EEG and hody weight in addition to biochemical changes such as in brain neurotransmitters which arc not diseus~d here. We have al~) studied the physical dependence of other drugs besides morphine-type ones using the neurochemical method in. addition to the ahove method and also the psychological dependence of CNS-affecting drugs using intravenous self-adminislration in rats. Our results indicate that rats are as good as rhesus monkeys for use as subjects in evaluating drug dependence.
Readinglist ~oJ
~h~
~g. 3. I:'E(?palterns ~f a morphim,-dependent rae in behm,i¢~ratalerfnexs'.
appeared with repeated injections of morphine, especially markedly and continuously with injection of the drug again after abrupt withdrawal, and was clearly regarded as evidence of disg)ciation beIween EEG and behavior (Fig. 3). Abrupt withdrawal of morphine produced a consistent decrea~ in motor activii) in both daylime and night-fime, and the circadian rhythm seen before injection and during the course of repeated injections of morphine disappeared. This phenomenon appeared more marked during 1-2 days following withdrawal after 8 weeks of repealed injections ~han after 4 weeks. This rhythm with reduced movements in hath daytime and night-time .seems to be accompanied by behavioral changes such as sedation and hypoactivity following abrupt withdrawal~. But on the 3rd day alter the witadrawal, motor activity during the dayfim¢~ increased and the circadian rhythm with increased activity in bot h daytime and night-time was formed. Afterwards, the rhythm returned gradually to the control level (Fig. I). On the other hand. the EEG pattern such as of rest and light sleep waves predominated from about 17 h after the final morphine injection and the waking stage increased and slow-wave sleeping and REM stages decreased in
both daytime and night-time in comparigm
with tho~ before abrupt withdrawal (Figs. 2, 3). Rat brain was unexpectedly found to have high activity i~ spite of behavioral ~dation and sleep after abrupt withdrawal, As reported previously", rhesus monkeys treated with 3 mg kg -~ of morphine every 6 h showed sleeping-likebehavior and a waking pattern in the EEG fi>r 3-4 h after the final morphine injL,clion, but an increzse in slow-wave sleeping stage 6-10 h .post. injection followed by insomnia. In other words, this confirmed that the withdrawal in morphine-dependent rats caused the same EEG changes as in monkeys. Thus, a clear relationship was tbund between the circadian rhythm recorded by EEG and Animex, and analysis of the EEG supplied detailed infiwmation on brain activity that could not be grasped by only behavioral analysis. Sarah el aL ~ analysed the development of tolerance and dependence using unit activity, that is, a single injection of m a r phine to depress the sptmtaneous firing in cortex neurons of rats. The inhibitory effect was antagonized by naioxone. Repeated morphine application resulted in a reduction of the inhibitory effect and favored the excitatory effect. Drug dependence should be cam-
I Way. E L., Lob, H. H. and Shen, F.-H. (1969) J. PhaemacoL Exp. Thee. 167, I-8 2 Marshall. I. and W~:instock, M. (1971) Nature (London) 234, 223-234 3 Kaneto, H.. Koida. M., Nakanishi. H. and Sasano. H. ( I973)Jpn J. Phannacol. 23, 701-707 4 Grcer. C. A,, AIpern, H. P. and Collins, A. C. ( I ')76) Life Set. 18, 1375--I 382 5 Wiley, J. N. and Downs, D. A. (197t~) Life SoL 2Y,, 7~)7-801 Host~ya, E, (195~)) Pharnrac,logist I. 77 7 Bl{isig, J., Herz, A., Reinhold, K. and Zicglg,/.insb~rger, S. (1973) Psychopharmacol¢~gia 33. 19-38 8 Yano, I., Nisl~h~o. H.. Yamamt~zo, H. and. Murano. T. (1'473) Jpn J. I%annacol. 23, 201-215 9 Yoshimura, K. and Yamamllt~), K. (l~';'q)Folin Phnrma('ol. Jpn 75,805-828 I0 Y~himura. K and Yamamoto. K. (ITS80) Folia Pharmacr~l. JWI 76, 373--4 [ I II Yoshimura. K. and Yamamoto, K. (|977) Profeedings o f tlJe hrternational Medi(al Symposhcm on Alcolrol and Drug Depead~.nce. ICAA SvmI~Sinm in Japan, 25 12 Sarah. M., Zieglg~,lnsberger, W., l=nes. W. and Herz. A. (1974) Brain Res. 82,378-382
Dr Ken-ichi YamamottJ graduau,d from the Tohoku UniversiO, in 1952. He has worked mttre Shhmagi Research Laborntorh.s since 1952 to the pres. ent. He suMied botlr m the University c~fKyoto and tile University o f Tokyo from 1957to 1960 and at dw Uni. venity o f Michigan Medical Sch(ml from 1963 to I q65. He is currently lice Directtbr o f the Department o f Pharmacology at the ,~ihianogi Research Laharatories. His inleresLvinclade neuro-psycht~pharn~acology, particalarly E~..G and hehm.ioml analysis ¢~fCNS at'ling drngs. Dr Ktdrji Yoshinnmr graduated from tire master cmtr~'e in Veterinary Science o f Osaka Prefecture University b; 1968. He has" ~ r k e d m tire Shicmngi Resec{rclt Laboratories since 1968 and has also studied at the Medical School o f Gifu University since 1978. His majcw research interest it in tire mechanism t~f drag dependence development in animals.
2,,-
down, e.g. by DMSO peel reatment, w,ually leads to incre ~sed toxicity. A skm'ing dolin of the Inelabolism. e.g. by toluene. decreases the toxic effects. TIle first product Of oxidation is an ui1stable, highly reaclive benzene +lxIxide
(Fig. I }. Snyder et
el. ~
suggesled thai this
melabolite is responsible for most of the
of inlcstlgalitm ha~c iildicated it dcrailgcd intenitlkm betlieei1 ~tem cell',, prilge.,lihlr cells and rcgulaling micro e m i r o n m e m l .
A n i n l p o r l a n l que~titln i+ the ix~,•ihlc carcinogcnieil) of hl.-nzene. Itere. an imD~rlanl difference hetmcen e x w r i m e n l a l anim;ils and man is the fact that. de,,pile titan) allempls, no maligllam:y hit+ k ' e n iilduced b.v ben/cue in i'il'o in ailimals. In contrail. the --pidcmiologi~il el ideilce mentioned earlier indicates th:il k ' n # t ' n c - e x ~ + d human indi+iduals shoe an i i l t ' r e a + d incidence llf leukemia. This might iildical¢ thai in human+, additional hl%.icil.% i+ alllicled on the hacmalOl~fietic • t i m call.
loxi¢ily in I,ivo, by onvalent binding to protein and iludeic acids. The enzyme sysieni in lhis palhway is ai3lhydrocartlonhydroxyla,~ (a mixed function oxida.~, A H H ) which is present in Ihe liver and I~mc-marrow. The inducibility of this enzyme in the mouse is under genetic onntrol by' the Ah locus, Ivhieh heals Ilit.o alleles (Ah ~ and Ah~). Ah" codes for high R e a d i . I list I ilaak. It. i . 4 IqSli| ( ?in. ?hwma#,d "~ /,-'1 +i',I'+ inducibility and Ah a fiw much lower 2 (;old,.lu~n. B I ) | 1 t l " T ) J I+,lu+d Inttr,,n inducibilityL The.~ alleles arc aim present I h'ahh .%~*ppL 2. Aq- l i IO on human eelk. This mechanism might 3 Vi,,Iman %. R.( It~77)J /-,~l/¢oLtt.nltn,,I Ih.ahh offer a basis for thc imt~wtailce of the gene.%uppL 2. ~3--hx 4 I-Icrnt~crgS.. Sa~ilakli..%1..AMman.K..rod -%,p. tic background. .%.(19h6) Rr. I. bid..l~,ed. 23.21i4-211~ Another pathway leading to a deranged 5 A I ~ l % . ,it,|. (19~7) ,li~,. I~mnhu/( Ontrtb ( hn ~, DNA metabolism was suggested by 1"~ 3 - 1 4 Freedma#. It was shown that benzene or h .%n_~dcr. R.. [_ec. U. %%_ K ~ ' ~ i , , . . I . J . a n d %%ilrnL.r. its metabnlites inhibit aminolevulinic acid synthetase, a rate-limitiilg enzyme in haem synthesis. Haemin binds to a dercpresmr molecule of globin and non-globin protei-~ syimhesis. A decrease in available harm couM then lead to a geilcral repression of protein synthesis at the level of translation. This in turn could lead to a deranged DNA repair mechanism. Cytogenetic effects As mentioned earlier, bone-marro~ and lymphocyte e h r o m o m m e analysis show a
wide variety of chromosome and chmmatid breaks and gaps. It was emphasized by Wolman ~ that most damage meres to be toxic rather than mutational. In view of the suspected carcinogeilicity of bei1zeilc, several mutation induction studies have been performed. No increased frequency of mutations was observed in the bacterial system by benzene or its melabolites. It is of interest that Morimoto and Wolff ha~e recently hmild that the induction of sister chromatid exchanges (SEE) in human cells was nol aft'ccted by Ix,-n/cilc or pllcnol. However hydroquinol and catechlll were very potenl inducers of s e E , indicating a major interference with DNA-synthesis during S-pha~e t°. Summarizing the experimental part, wc tint; that benzene induces haematopoietic hlilare at the level of the matutatiilg progenit,~r cell, beyoud the pluripotent slem cells. This is expressed by progressive.dysplaslic chan~es in I~mne-marrow and peripheral blood. In addition, several line,~
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Screening methods of drug dependence liability using small animals Ken-ichi Yamamoto and Kohji Yoshimura lJtvt+itm ,iJ .'%'ettrolihtirmzicotol~l..llht< .h~lgt Rt'~carcit l~th,,rat,,ri,'+ %+z.'.,tt, ,~.'+,++;d ( ,+ l td I ,,k., ,:.',;., ~., + +,,.~ .IS.i, Japan
Varkius animals, such a~ mice ral~. dogs and monkeys, have been used t++ pri, dict
dependence Iiabilil)on drugs. G e n e r a l k rhesus monke)-s are thought to Ix." the ino~t suhable animals for imorphin¢-t~ i ~ e~lx'n ments. I%lan~ ne~ ('NS-affe.-ting dru~, are coiltiuuousl) being synlhe.~ized :lild nccd to be checked lor dep2ndence liabilil) it+ confirm their salet). I iol~e~ er. continti¢, l ,,upp l y O f tJle~C m o n k e y s
dot."~ n o t ~CCi11 i ' , r o m -
ising and also such e%lt, riillcnt~ arc i c e e x t t ' n s i l e aild lal~,~r-con~ulning. ]+lh'r+'ll,lrc, a scrceiling nlelhod l o t drug dt i~.'ndeuce thai call clt~il) ) i d d llceurale rt,+,.lll+ needs to Ix: established using small aninlais such as mio: and rats instead tit monkey',. This paper pre~:nls o b ~ r x a l i o i ~ ol -~mle general screening i11elhod.' for
morphine-type ph)~ical dependence u~ing i11iCe a n d r i l l s a n d c o m p a r e s
them
~l i~11, ~ur
findings obtained receilll> ~iiill t i l x .
Te~Is using mitt+ The j u mp i n g method dc~clol~:d h~ X~ a~ t.t aL t lla', l~.'cn ~udck t,...'d ,l. u icll,,l~l¢ ifld{i'ahlr in ql.lai1111} iflg Ih¢ c%.lCllt .q ph}-,cal dcl~ndcncc in nncc Junq'.n~ ll:duccd l'~,, al'rupt or prc¢ipit,ltcd ~itlldua~ d m i11orphih¢ ix'llct-mq+kmtcd r:lcc i, regarded ,I, an ltldlca[Ol ol ~ l ; h d r ~ . d .ign,. i h c i n t l d d n ¢ c tit i u n l t . l i l g l,~llo'.~illg iibrtipl i~ ilhdrilx~dl i~ Ilmi-d¢l'~n,Kni. lhc ic~tklll.,c I~:llld llld%lnl,il l ~ , h ,dice fClllOl,iI o l l i l t morl~hlnc lalq¢l..~tl~lupl ilbMigi'ncc I~ qU,liltllied bx pliicInL' the alillmal~ tin ,I ¢irl'uldr lil,llhffm arid li%'oidnl ! the Ip~.'r¢t'llla~C ol anililal+ thai leap oil xlitliin !+4 niin. [ h i ' dcgrec ol ph,,~ieJ depeildeilcc can Ix" asw~.~d b.~ plotting th,: tx:rcx-ntage of re~lxm~c against time and determining the a,¢a undcr tl:c tar're. -[he ilre-l I~ I l t e L l ~ u r c d ~ ) etlltillg oUt the ciir~+ from the graph p a l t ' r and x~ciglling It. Irl
TIPS - October
2~bt DOSES OF MORPHINE
OF
1981
SPONTANEOUS MOTOR ACTIVITY
GROWTH CURVE
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ABRUPT WITHDRAWAL
l'ig. I Rel.uionUIIp b e t . een t zrcadicm rhythu o.[~t~,mtaneolt~ . I ~lor atlivit.v at d ('hat g¢,.~#1 hot#, .'eight U! morphble.depemlcnl rat.~
prccipilated withdrawal with the illorphine antagonist, naloxone, the degree (ff physical dependence on morphine was assessed without re~" valot the pellet by esiimating the median effective dose (EDse) of nalt~xone needed to cause jumping from the platform. Marshall et ul. a used thc' number ,)f jumps in addition to their incidence as an indicator of physical dependenct, in mice repeatedly treated with morphine• Kaneto et al. a reported that actual withdra~al caused various withdnlwal signs involving jumping, wet shakes and weight loss in mice injected with morphine at increasing d ~sesof I 0--I 0(1 mg kg-~ twice a day fl~r I 0 days. The degree of ~ilhdrawal signs was totalled for groups of five mice each for a 2{)-rain monitoring D;riod and expressed as a sum of scores. According to this estimation, the intensity of the abstinence state reached a plateau 5--6 h after the morphine administration. The same
xvithdra~al signs also appeared following morphine-dependent mice may be actual withdrawal after !-I 0 h of continu- regarded an behaviorally excitable ous intraver:ous injections, five subcutane- changes. ous injections every hour or a single injection of 100 mg kg -t morphine. These Tests using ra*.s phenomena were potentiated by naloxone. In rats repeatedly injected with mor0~1 ~he other hand, it takes a long time to phine, abrupt withdrawal produces make mice dependent with oral adminis- sedation-like hypoactivity and ptosis as tration, of morphine. Significantly lower x~ellas excited behavior such as aggression, latencie,, for myoclonic and clonic convul- vocalization, hyperactivity, and convulsive behavior induced by flurothyl in sions, or both changes in excited and sedamorphine-dependent mice were also asses- tive activities. Thus, the withdraw~d signs sod as hdieative of physical dependence. rel~)rted differ considerably among the which i~, characterized by increased CNS investigators. exeitabillity4. Hosoya e was the first to prove inducRecently, Wiley and Downs~ reported tively, based on experimental fact, that that naloxone-induced jumping after a weight loss l~di~wing the withdrawal of single ie/cction of opioid in mice might be morphine is a useful t(~l fi~r ~reening the of value in the rapid identification of drugs physical dependence liability in rats. Later, with a propensity to produce morphine- this was confirmed by many investigators like physical dependence. and applied ~'ltisfactorily to the screening In any case. withdrawal signs in of morphine-type dependence. However,
1 1 P 5 - e h ' t o h e r i +t81
2+,"
Ihis weight loss occurs even i0 v+ilhdra,~al
implant,ilion, and the longer the mter,+al bet~,~,¢eu ;,inlagt ~ni',,l i n i c t l . m ,llh:l the hnal mt~rphine. Ihe le',,, tlne incidence ,d the resp, m~_" ( ) n lhe other h a n d Y a n o c t a l " su[zgesli.'d lhal hislologlcallt, morphologyinduced by precipitalcd withdrav+al have caU c h a r g e s ol mil~:hondrla in a d r c m ~ : o f hcen quanlilatively cv:llualcd and :0sscssed tical cells are uv,:ful as intk'xe,. , I ph.~,.ical as a criterion of drug delm:ndence in rals d e p e n d e n c e liabilil.,, ol m-rphine, l h u s . subcutaneously implanted with morphine ',arious behavioral change,, hkr lumping t~eilcts as in miccL Ho~,evcr. many inves- Ilil~,+e b e e u considered sUilablc signs I,,, lhe tigators have claimed that Ihem: we! shakes e t alu;ntion o [ ph.~,,Ical d e p e n d e n c e t d I11,,r-
from barbilurates and IranqoilizeP, ~,nd. thereh)re, is not al~ays specilic to signs of morphine withdrawal. Also. changes m behavior such as wet shakes and jumping
or jumping responses are essentially dillcrenl from wilhdrawal signs; they conslitutc simply anolhcr kind of antagonistic activity, becau.~ they have been induced hy the injection of antagonist even immediately after morphine injection or during pellel
phine in rals. a h h o u g h r c l ~ r l s hat+: been lira!led to record~ ol Ih¢ t~ilhdra~,l ..ign~ ~ith considerable ~ar0ation Irom a u t h ~r I.. author.
We ",tudied .+~hal kind,+ of ,,ign,, I+~.',,id¢', u, eighl hv,,, +,~mld I~." u ~ l u l ;+1,,ol'qccli~c
MORPHINE nosES
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+'-~II mg k.d t. t+otk+n11~,dt dlt~.-~ ,it 1 4 ~ I m~ kg ' .rod d-pr, q~,~pi~.+m: .'i+-l + " ++1'.:"k++,' ' t~,¢*.' ;t d,t} her ~ - | 2 ~+c. k', R, ¢l,d ~¢m|~. ~;llllrc did first ~'iLJI~C o n ';dotup+ +t ill+dr;ire +d. bul d,.'cr+a%'d 2 1 ( . +'4 ( a + l d +t '+ ( I,,1
m,,rphinL+. ¢{,dcmc +rod d-p.,p+xxphcm-+ IC~I~'¢IP.cIs, with I¢+alh,rpI}an-mduced +llthdram,d. h l h i h t l . m ,d Ih¢ +~+,,.L+n+c +~l lhc lcarncd i++,k• dtmbln++.: .,,ndfl+,,n+d It"+i'k;A'~" in lal~, +++;t~,m i l d + ith +d+rupl ++~lh-
d r a ~ a l and m a r k e d X+tlh K.+;~lhqph++n induced mllhdrax~al ol m o l p h m c .+led ¢,~Jcmc Ih¢~: l~:,~ull+..,u+~,:tc.q Ilhfl ¢h.+lt~¢in I~KI~, tcml~.-l+alure ,;lAd 1fl+lI|'+II++)flo! ,+ ¢lmdillimed l~..,,pln,~., a r c U'~.lU+ +is +nd+cators for ¢xalu+fllng dtu,J dcl~.'nd~ nov + Xcxt. x~- cxamm~+d x~hcthc; ,st not the natix¢ o r c a d m n rhxthm ,,1 rat,, ,:+-,,Meal h,+ Animcx and F i : ( i ~,,uk! I~- u~'tul a~ an ohwt-tlX¢ indii.:Jtor |,t,l dndtx",l', ol dru# dep~ndcno.'_ %lalc Xt. i,.Li; .+++~1- ~ ith implanicd ,.-hroni¢ clvctrodc, x~cr¢ -ub+
cuLam.'ouq,+ in]¢dL-d v+flh morphine at increasing do-e,, ot 5-511 mg k,.." ~ tx~u'c ,~ day h r ~ ~cck-. The p . ~ ' c - . *.: dcl~+n dencc dc....Iopmcm of mt,rphinc and the aPt~arancc ot ~'thdra~tal ,.n~n. ~tcnc .,,ntinuou,,I.x r¢+.'oldcd bt .-~nxrncx and E L ( i " ' Control rub tr+.'atcd with tch~clc .ho~ c~rcadian rhxthm **ith dccrca-cd attar It5 .rod at tendcnc~ to ",loop in the Eli( i during the daytime t~.'th sexeral t~.',lk~ ot m,+tor a,:tr'. ilX and more ,,+aking qa,_':'+ In the |:[+(' a: night. Single iniecthm t,7 morphine at ,:
"'""
do',,t,." +,)t .~ nlg k~ I g,ltC r l - - lo,+¢dath,n J n d h~poactkn+x x+hll¢ I t ~+dax rcp+atcJ in.lcctiom, of the drug produced h} l%'ra,.+[t~ 36"
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potentiated
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10 40 60 O0 100 (1)
~ZZ~ : ,o.u++ +,,.0-*,~
:DISSOCIATIONBE'twin+ EEG et~lO8£HCWIOR
Fig. 2. ,Sh,ep-+vakeJi~h~ess('ych'x and EE( ; h'rHs ol a morphme-dept'ndt'tzt rat fi,r 7 h turm¢ the d, O.
oi the drug. and lhc RI:M ..h..cpnl¢,la~. decrea..ed trom lhL' 3rd da} oI Inl¢Clh+'r (Fig. 2b+ The,< change, ,.con m the I I-Xi might i~ regarded a- brain electrical atticit} corre~,ponding tt, thc increa,,e m motor actixit.x. AI~+. the injection- ot morphine interestingly induced a temt~war,, aptx'arante of high ~oltagc FI-.G .I,+x~ N.r,t. accompanied bx I~'ha~ ioral alcrtne.- x, lth the cxc~ oleo'ned v, idc.. Thi+ phcnomc'~on
270
TIPS BEHAVIORAL ALERTNESS
oosr-s OF B E F O R E MORPHINE
o.~j~ ;0
HII~OC,;AMPUS
NEOCORTEX
MORPHINEsL 42mia
.
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-
October
108!
prehensively as~ssed by analysis of hod) temperature, spontaneous molar activity, and electrophysiology such as EEG and hody weight in addition to biochemical changes such as in brain neurotransmitters which arc not diseus~d here. We have al~) studied the physical dependence of other drugs besides morphine-type ones using the neurochemical method in. addition to the ahove method and also the psychological dependence of CNS-affecting drugs using intravenous self-adminislration in rats. Our results indicate that rats are as good as rhesus monkeys for use as subjects in evaluating drug dependence.
Readinglist ~oJ
~h~
~g. 3. I:'E(?palterns ~f a morphim,-dependent rae in behm,i¢~ratalerfnexs'.
appeared with repeated injections of morphine, especially markedly and continuously with injection of the drug again after abrupt withdrawal, and was clearly regarded as evidence of disg)ciation beIween EEG and behavior (Fig. 3). Abrupt withdrawal of morphine produced a consistent decrea~ in motor activii) in both daylime and night-fime, and the circadian rhythm seen before injection and during the course of repeated injections of morphine disappeared. This phenomenon appeared more marked during 1-2 days following withdrawal after 8 weeks of repealed injections ~han after 4 weeks. This rhythm with reduced movements in hath daytime and night-time .seems to be accompanied by behavioral changes such as sedation and hypoactivity following abrupt withdrawal~. But on the 3rd day alter the witadrawal, motor activity during the dayfim¢~ increased and the circadian rhythm with increased activity in bot h daytime and night-time was formed. Afterwards, the rhythm returned gradually to the control level (Fig. I). On the other hand. the EEG pattern such as of rest and light sleep waves predominated from about 17 h after the final morphine injection and the waking stage increased and slow-wave sleeping and REM stages decreased in
both daytime and night-time in comparigm
with tho~ before abrupt withdrawal (Figs. 2, 3). Rat brain was unexpectedly found to have high activity i~ spite of behavioral ~dation and sleep after abrupt withdrawal, As reported previously", rhesus monkeys treated with 3 mg kg -~ of morphine every 6 h showed sleeping-likebehavior and a waking pattern in the EEG fi>r 3-4 h after the final morphine injL,clion, but an increzse in slow-wave sleeping stage 6-10 h .post. injection followed by insomnia. In other words, this confirmed that the withdrawal in morphine-dependent rats caused the same EEG changes as in monkeys. Thus, a clear relationship was tbund between the circadian rhythm recorded by EEG and Animex, and analysis of the EEG supplied detailed infiwmation on brain activity that could not be grasped by only behavioral analysis. Sarah el aL ~ analysed the development of tolerance and dependence using unit activity, that is, a single injection of m a r phine to depress the sptmtaneous firing in cortex neurons of rats. The inhibitory effect was antagonized by naioxone. Repeated morphine application resulted in a reduction of the inhibitory effect and favored the excitatory effect. Drug dependence should be cam-
I Way. E L., Lob, H. H. and Shen, F.-H. (1969) J. PhaemacoL Exp. Thee. 167, I-8 2 Marshall. I. and W~:instock, M. (1971) Nature (London) 234, 223-234 3 Kaneto, H.. Koida. M., Nakanishi. H. and Sasano. H. ( I973)Jpn J. Phannacol. 23, 701-707 4 Grcer. C. A,, AIpern, H. P. and Collins, A. C. ( I ')76) Life Set. 18, 1375--I 382 5 Wiley, J. N. and Downs, D. A. (197t~) Life SoL 2Y,, 7~)7-801 Host~ya, E, (195~)) Pharnrac,logist I. 77 7 Bl{isig, J., Herz, A., Reinhold, K. and Zicglg,/.insb~rger, S. (1973) Psychopharmacol¢~gia 33. 19-38 8 Yano, I., Nisl~h~o. H.. Yamamt~zo, H. and. Murano. T. (1'473) Jpn J. I%annacol. 23, 201-215 9 Yoshimura, K. and Yamamllt~), K. (l~';'q)Folin Phnrma('ol. Jpn 75,805-828 I0 Y~himura. K and Yamamoto. K. (ITS80) Folia Pharmacr~l. JWI 76, 373--4 [ I II Yoshimura. K. and Yamamoto, K. (|977) Profeedings o f tlJe hrternational Medi(al Symposhcm on Alcolrol and Drug Depead~.nce. ICAA SvmI~Sinm in Japan, 25 12 Sarah. M., Zieglg~,lnsberger, W., l=nes. W. and Herz. A. (1974) Brain Res. 82,378-382
Dr Ken-ichi YamamottJ graduau,d from the Tohoku UniversiO, in 1952. He has worked mttre Shhmagi Research Laborntorh.s since 1952 to the pres. ent. He suMied botlr m the University c~fKyoto and tile University o f Tokyo from 1957to 1960 and at dw Uni. venity o f Michigan Medical Sch(ml from 1963 to I q65. He is currently lice Directtbr o f the Department o f Pharmacology at the ,~ihianogi Research Laharatories. His inleresLvinclade neuro-psycht~pharn~acology, particalarly E~..G and hehm.ioml analysis ¢~fCNS at'ling drngs. Dr Ktdrji Yoshinnmr graduated from tire master cmtr~'e in Veterinary Science o f Osaka Prefecture University b; 1968. He has" ~ r k e d m tire Shicmngi Resec{rclt Laboratories since 1968 and has also studied at the Medical School o f Gifu University since 1978. His majcw research interest it in tire mechanism t~f drag dependence development in animals.