EJP 57771
Antiepileptic effects of CN 6024 a novel thyrotropin-releasing hormone analog, on absence-like and tonic seizures of spontaneously epileptic rats Xie
Renming, Kumatoshi Ishihara, Masashi Sasa, Hisamitsu Ujihara, Toshihiko Momiyama, Yasuhiko Fujita, Naoyuki Todo, Tadao Serikawa “, Junzo Y:lmada ” and Shuji Takaori
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Received IO June IYYZ. revised MS rrceivsd 75 August 1901. accepted1 Seplemher lYY7
The cffccts of CNK-602A IN~[~6-mcthyl-S-oxo-3-thiomorpholinyl~ carhonyll-L-histidyl-L-prolinamido). a novel thyrotropin-rclcasing hormone rclatcd analog. wcrc invcstigatcd on abscncc-like scizurc and sonic convulsion in the spontanconsly epileptic rat ISER), which is :I gcncticaliy dcfincd double-mutant. When CNK-6O2A of 0.2-l mg/kg was given intravenously to the animal, thcrc wcrc no changes in the background EEG cxccpt for an incrcasc in low-voltage fast waves concomitant with behavioral alcrtncss. Howcvcr. CNK-602A supprcsscd ahscncc-like scizurc and tonic convulsion in a dose-dcpcndcnt manner for oc’cr I IL Thcsc anticpilcptic cffccts of CNK-6OiA on hoth scizurcs wcrc antagonized by prctrcatmcnt with halopcridol (I mg/kg. i.p.). It was found, using iI brain in viva microdialysis method. that CNK-WA at a dose of I mg/kg, which inhihits the scizurcs. incrcnscd the rclcasc ofdopaminc in the caudatc nucleus. Thcsc results suggest that CNK-602A inhibits the seizures of SER in a similar manner to thyrotropin-rclcasing hormone (TRH), prohahly hy increasing the rclcnsc of dopaminc in the central nclvous system. In addition. ~hc anticpilcptic cffccts of CNK402A wcrc more potent and I;l:itcd iongcr than those r:l’ TRI 1.
Sponlancously
cpilcptic rat (SER); Thyrotropin-rclcasing hormone a~nalogs:
1. Introduction
Thyrotropin-releasing hormone (TRH) and its rcarc known to be widely distrihutcd in the central nervous system (Winokur and Utigcr, 1974; ccptors
Manaker et al., 10X5; Mantyh and Hunt, 1c)HSI . TRH is also known to modulate the rcleasc of ncurotransmitters such as dopamine and noradrenaline (Horita ct al., 1086; Lamour ct al., 1085; Takatani et al., 1087: Keller et al.. 1074). In addition, TRH rcportcdly has anticpilcptic activity in cxperimcntal animals, such as the kindling model of epilepsy C&to et sl., 1084: Sakai ct al., 1991), as well as in human epilepsy, such as the Lennox-Gastaut syndrome (Ucda ct al., IOX3) and scvcrc childhood epilepsy !Matsumoto et al., 1087). Prcviously, we also found that TRH supprcsscd abscncclike scizurcs and tonic convulsions in the spontu-
C’orrcspondcncc to: M. SilSiIa Dep;u?tncnt Al’ I’ll;lrln;lrology. r(jshitn;l University School ol’ Mcdicinc, Iliro41in~ 7.34. J;I~xII~.
I li-
C’NK-WA;
Anticpilcptic
cl’f’cct; Dnpaminc
ncously cpilcptic rat (SER) (Ujihara ct al.. 1991). This animal is a gcnctically defined double mutant (zi/zi, Im/tm) and is useful for the evaluation of anticpilcptic action of the drugs, since cthosuximide and trimcthadion suppress absence-like scizurc and phcnytoin inhibits tonic convulsion:;, while valproatc and phcnobarbital suppress both types of scizurc in this animal (Scrikawa and Y:;mada. 19x6; Sass cl al., 1988). However, tbc effects of TRH arc weak and short, compared with those of conventional anticpilcptics. prob;lbly due to the fast metabolism ot this drug. TRH itnalogs. QN-1417 and YM-14073. wcrc reported lo h::vc a more potent and longer action on scizurcs in the kindled animal than did TRH (S:tto ct al., 1084: Yatsugi and Yalllilnlol0, 1091). Thcrcforc. wc cxamincc! tiic cftccts of a novel TRH analog, CNK-hO2A (N-[(O-methyl-Soxo-.3-thiomorpliolinyl)carbonyl]-L-histidyl-L-pr~~linilmidc), on cpilcptic seizures in SER. Furthcrmorc, using an in viva Inicrodialyhis nettled, WC attcmptcd to dctcmminc whcthcr or not the cffu% of CNK-hO2A arc due to an increase in the rcleiisc 01 dopitt?litlc.
2. Mlterinfs
and nlrtheds
2. i. :flr.‘rt!rrh SEK
wcrc hrcd in our instilutc
iIs :q3orU
prcvi-
oualy (Scrikawa and Yi~m;d;~. 19Hh). ‘fhc anirnds kcpr individually
in shot-box
wcrc’
type cages in the animal
7’ WC and 5O--h5% humidity. Commcrquartcrs al __-_ cial fooJ pcflcts (F-_.7 f%naflashi f“arm. Japiln) rlnJ wrrtcr wcrc y&n id fibitum. ‘l’hc room hild 12-h lightdark. with lights on from X:(HI to 2O:OOh.
Under urcthanc (I.5 g/kg $1 ancsrhcsia. the SEK wHS fixed in the stcrcotaxic i\pparatus. and it microdialysis pmhc with a dialyzing mcmhranc 2.0 mm in length
and 0.S mm in diamctcr (CMA/fO.
Carncgic Mcdicin.
Swcdon) was then insertcd stcnanaxicafly &tc
into \hc au-
nucleus (P. 0.3: I,. 3.0; H. 5.0 from cortical SUT-
filcc) (Paxinos and Watson. IWO). Kingcr solution was continuously pcrfuscd ill a flow t?llC of 2 pf/min. using im injection pump (CMA/ J-en m;blc and twclvc fcmnlc SF.K wcrc USC~ for the EEC, cupcrimcnl. Under sadium pcntihiirl~ital iIllCS(llcSiia [_30
mg/kg
i.p_1.
il
sifvcr
hall-tippcd
and an cn;m~cl-~OillCJ SlilinlCsS-SbXl &rrrrlically
into
impfilntCd
clcctrodc
clcctrodc
rllc ICfr flIMltill cortcs
wcrc’ illld
tor iI stabilization callcctcd
Io[), Carncgic Mcdicin).
Af-
period of over I h. the didysatc
wils
cvcry 20 min using il microsamplcr
200. Carncgic Mcdicin). for
I h and thcrcaftcr the drug was injcctcd into the f3ICll dialysis Silmpk WZi automatically
Cillldill vein.
from hrcpma).
injcctcd into HPLC.
using iIn injcclion
rcspcctivdy. according to the iIlliIs c)f Paxinos id Watson ( IOXh). An indiffcrcnt &Xtrodlc WJS placed on
UAS. Japan). Afkr
separation
cholamincs
ihc frt)nl;ll
tcclor
I&t
hippttciimpus
(P.
1.5:
1,. 2.41: kj.
2.5
cranium.
Scvcn days ;rftcr implantation of Ihc clcclrodcs. each animal was placed ill iI sk)und-altcnuittcd BOX (40 x 40 x 40 cm) with ;I small window for olwx-ving t!lc 5sh;lvior of tfic animal ;tnd recording the EEG
(CMA/
B~~slrl rclctlsc wiis dctcrmincd
pump (PM-ho,
of the sample
catc-
wcrc dctoctcd with an elcctrochcmical
(LC-46,
RAS.
Jitpan).
dopaminc and its mcldditcs
and
the
contents
dcof
wcrc rhcn rccordcd in
thu chr~m~a~ogriun. 7%~’ dopaminc peak was t;ofcd 3.X min ihfkr injection.
with a pen-writing pr~lygraph (Nihtm Kohdcn. KMfQOII). Frcqucncy ilnillysis of’ EEG wits done using ;I trend
monitor
(Nihon
Kohdcn.
OEE-7102).
‘fhc
‘f’hc numb
of scizurcs
and the duration
of catch
recording prtlccdurc for fhc EEG itnLl the method of ;ipplic;ltion of !hc drugs wcrc similar to Ihosc dcscrihcd
scizurc wcrc nntcd for 5 min. and the Iotal duration of
previously (Ujiharn ct al.. IV11 1. Hricfly. ‘f’hc frcqucncy of the occurrcncc of ahscncc-like sburcs and tonic convulsions. as well ils the duration of cilch scizurc. wcrc mcasurcd on lhc EEG. sine the chibngcs in l+G
WiIS then Ci~lCUl~tCd cvcry 5 min before and after injection of the drug. ‘I‘hc mc;Ln total duration of
during
the
abscntdikc
scizurc
and tonic convulsion
alwitys cr)rrclihtcd with the rcspcclivc hchavitrrnl changes. as dcscrihcd previously (SilsiI Ct al.. I98H). When
;I S-7
Hz
spike-wavefikc
the sciznrcs
(summation
of duration
of each scizurc)
sci7urrs during il S-min grcriod WilS CiliCUliltCd hy IIVCTilgillg h! V;dll~s ohtilind CVCly 5 min (1S- IO, 10-S ilnd S-0
min hcforc drug administration).
CllilIlgCS ilftcr
drug idministr;dion
The pcrccnt
wcrc tIlliS d~lihN!d
from the mean value.
complch in corlicitl
and hipptwitmpid EEG fiislcd for (:vcr I S, this wits regarded as an ahscncc-liks scizurc. I’hc compfcxcs with ;I short lime interval (Icss than I s) bctwccn two spike-wavefikc compfcxcs WC’T(: rcgardcd its iI sin& scizurc. ‘I’0 iriilucc an arousal state and ;I consistsnl
CNK-602A
ionic scizurc. cacli animal rcccivcd a mild tiu3ilc slimu-
CO
CONI+
lus on the hack cvcry 5 min. One dose of CNK-MEA was injcclcd (i.v.1 inlo the tail vein of each zlnirnal. alone or in combnation with halopcridol (i.p.1. l’hc second &)SC trf CNK-OOZA or Ihc comhinittian wits given to rhc animal at Icast I week after the final in,iicction. CNK-ho3A or hillopcridol WBF injcctcd after
30 min
reading of it control EEG following I-h hahiiuabon of the imimi lo the box. When CNK4I12A
wils lipplied in combination with hEA wiIS administcrcd 3O min
h&~pcridol. CNKirflcr itljcclion of
TRH
0
C3NHa H
IS7
The pcrccnt change in dopaminc rclcasc induced by CNK-MEA
or TRH
was obtained by dividing the value
after drug treatment
by the basal rclcasc for the I-h
period before drug injection.
CNK-601A (1 mg/kg) was dctcrmincd by Student’s t-tat. Antagonism by haloperidol of the effects 01 CNK-MEA statistical
was dotermincd significance
with Student’s
t-test. ‘l‘hc
of increases in the rclcasc of
dopaminc was compared with the value bcforc drug administration using ;1 paired t-test. The statistical significance of the diffcrcncc between the means of the total duration saline-treated wits
dctcrmincd
(ANOC’A! significance
for the physiological
group and the CNK-607A-trcatcd by
one-way
followed by Student’s of the time
analysis
of
t-test. The
group variance
statistical
course of the inhibition
A Conrrof
CNK-602A
lmglkg i.v. ldmin
-0-1
6 Control Cl
t
s
CNK-602A
lmg/kg i.v. 15min
by
CNK-hO2A
(Nippon
Shinyaku.
Kyoto.
Japan; CG-
370.7, Gruncnthar. GmbH, Aachcn, F.R.G.. fig. I) was dissolved in saline and injected into the caudal vein iit doses of 0.04, 0.2 and 1 mg/kg with ;1 volume of 0.1
or gr(~oming. No ~ilt~r~lti(~ns of b~ckgr~)un~ EEG obscrvcd with 1 mg/kg of the drug. Tahlc 1 shows the offccts of CNK-602A i.v.1 on abscncc-like scizurcs
male itnd female SEK.
and fcmafc SEfi
Inhibitory
-fXH
like seizures
f(JgiciIf saline was itIS~x!cI> Md.
vicwb ol 0.
I IO.
ItIc
all;l~og(I)N_lJ17) ,111 hI* 31.
K. Ifl;lllil!il. IOHZ.(‘linkil
Cl’lCCIhOl 7 RI
1
I.~mIox syndrome. .I. J:IP. ~Pilcp~Y SOL