全 文 ：Chin J New Drug s Clin Rem , 2000 Nov , 19(6):433-437.
Antinociceptive effect of physostigmine and interaction of intravenous physostig-
mine with intrathecal morphine in rats
CHEN Lian-Hua1 , 　Alexander NEM IROVSK Y2 , 　GONG Qin-Yan3　(1 .Department of Anesthesiology ,
The Eye Ear Nose and Throat Hospital , Shanghai Medical University , SHANGHAI 200031 , China;2 .
Department of Anesthesiology , Los Angeles Medical Center of the Universi ty of South Cali fornia , CA90033 ,
USA ;3 .Department of Pharmacology , Shanghai Medical Universi ty , SHANGHAI 200032 , China)
[ Received date] 　2000-01-07
[ Accepted date] 　2000-07-25
[ Biography] 　CHEN Lian-Hua(1964-), female , the Zhejiang
pro vince people , associated professor , master , be engaged in
clinical anaesthesia and scientific research.
[ Correspondence author] 　CHEN Lian-Hua.Phn:86-21-
64377134 , ex t 511
[ KEY WORDS] 　phy sost igmine;morphine;pain
measurement;drug synergism
[ ABSTRACT] 　AIM:To investigate the antinoci-
cept ive ef fect of anticholinesterase agent physostig-
mine at different postoperative t ime , and the interac-
tion of int ravenous phy sostigmine wi th intrathecal
morphine in rats.　METHODS:Six groups of rats
w ere operated on fo r int rathecal and intravenous
catheterization.Nociceptive responses of hind paw s of
each animal w ere measured w ith “ plantar st imula-
tion” test 1-3 h and 3 d postoperatively.Animals re-
ceived int ravenous (iv)physostigmine , intrathecal
(ith) morphine , or combination of both.The
antinociceptive ef fect of each g roup w as converted to
the percent maximum possible effect (% MPE).　
RESU LTS:Administ ration of physostigmine 1-3 h
af ter operation resulted in dramatic increase in %
MPE.The ef fects of combinations of iv physostig-
mine and i th mo rphine w ere more pronounced at early
postoperative time.The potency of low dose combi-
nat ion w as significantly greater than that of double
doses of both drug s.The %MPE of the observed ef-
fects of all combinations w as signif icantly higher than
that of the expected addi tive effects.　CONCLU-
SION :The antinociception of physostigmine occurs
at early postoperative time.The interaction of iv
phy sostigmine wi th ith morphine indicates their syn-
ergistic effect.
[ Ucnumber] 　R 971.91;R 965
[ Document code] 　A
[ Article ID] 　1007-7669(2000)06-0433-05
　　Opioids activate opioid receptors and cause anal-
gesia.The effect of opioid recepto r activ ation may ul-
timately be mediated by some intermediate neuro-
t ransmit ters.It has been proved that one of the im-
portant inhibitory neurot ransmi tters in response to
nox ious stimulation is norepinephrine(NE).In sheep
and humans , cerebrospinal fluid (CSF) NE and
acetylcholine (Ach)concentrat ions increased after a-
cute painful stimulation[ 1] and int ravenous opioid ad-
minist ration[ 2] .In human volunteers , int rathecal
cholinesterase inhibitor neostigmine caused the in-
crease in CSF Ach concentrat ion , resulted in analgesia
and enhanced the effect of opioids injected systemical-
ly .It has been hypo thesized that either pain stimula-
tion or opioid administration may induce the release of
NE which in turn activates the cholinergic neuron in
spinal dorsal horn to release Ach and cause analgesi-
a
[ 4] .Therefore , the current study was designed to
investigate the ant inociceptive effect of ant i-
cholinesterase agent phy sostigmine at different post-
operative time in rats , and to evaluate the interact ion
of int ravenous (iv)physostigmine w ith int rathecal
(ith)morphine.
·433·中国新药与临床杂志(Chin J New Drug s Clin Rem), 2000 年 11 月 ,第 19 卷　第 6 期
MATERIALSAND METHODS
Animal preparation 　Male , 300-350 g Sprague-
Daw ley rats w ere used.Animals w ere housed individ-
ually in a temperature controlled room w ith a 12 h
light-dark cycle , and fed with both food and w ater
available ad l ibi tum .Experiments were performed
during the light cycle.Under halothane anesthesia , a
chronic catheterization of the lumbar subarachnoid
space w as performed for the ith injection.Briefly , a
PE 10 catheter was inserted through the atlanto-oc-
cipi tal membrane and introduced to a length of 10 cm
caudalw ard into the lumbar subarachnoid space.The
catheter w as then fixed to the back neck of the ani-
mal.A concurrent catheterization of the femoral vein
w as performed for intravenous injection.The animals
w hich exhibited no signs of neurological def icit af ter
recovery f rom the anesthesia underw ent the experi-
ment procedure.
Nociceptive threshold test　Nociceptive responses of
hind paws of each animal were measured by means of
“plantar stimulation” test using a device designed and
made by Yaksh s laborato ry[ 5](University of Califor-
nia , San Diego , USA).The rats were placed in a
clear plastic cage on an elevated floor of clear glass.
To reduce the variabili ty , the under-f loor temperature
w as maintained at 30℃.A radiant heat source f rom a
50 W , 8 V lamp was contained in a movable holder
placed beneath the glass f loor.The radiant heat diam-
eter w as 4 mm and bulb intensity w as controlled at
5.25 A .To initiate a test , the under-floor heat
source w as positioned to focus at the plantar surface of
one hind paw which w as completely in contact with
the g lass.The light was then started meanwhile the
timing circuit w as init iated automat ically .The noci-
cept ive response w as determined by the interval f rom
the application of the light beam to the hind paw
w ithdrawal.A cutof f t ime which w as three t imes of
the baseline nociceptive response latency was set in
o rder to avoid tissue burn injury.
Test Protocol 　Each rat w as used in one dosage
regime only.Initially , baseline nociceptive response
latency w as determined by the average of three mea-
surements.Subsequently , drug administ ration was
performed in a blind fashion.After drug injection ,
nocicept ive response latency of the hind paw was
measured at 5 min interv als until the baseline re-
sponse w as regained.The peak time of antinocicep-
tive ef fect and the durat ion of the ef fect w ere deter-
mined.Dif ferent g roups of rats w ere scheduled to re-
ceive iv physostigmine 50 or 100 μg·kg-1 , ith mor-
phine 1 or 2 μg , o r the combinations of iv phy sostig-
mine(50 or 100μg·kg-1)with concurrent ith mor-
phine 1μg , respectively.All drugs were dissolved in
Ringer s solution and administered in an inject ion
volume of 1 mL·kg-1 for iv and 10 μL for ith.Fol-
low ing each ith injection , the catheter w as f lushed
w ith 10 μL Ringer s solution to ensure the drug de-
livery into ith space.The tests w ere performed at 1-3
h af ter the i th and iv catheterization w hen the rats
completely recovered f rom anesthesia.To compare
the antinociceptive effect of each drug in the animals
w ith or wi thout postoperative stress , the tests for the
same drug doses were repeated three days later in the
same rats.
At the end of the experiments , all rats received
an i th injection of 10 μL of 2% lidocaine.Data f rom
rats w ho did not develop motor paralysis w ithin 2 min
were excluded.
Statistical analysis　The response latency w as con-
verted to the percent maximum possible ef fect (%
MPE)which was calculated by the follow ing fo rmu-
la:
% MPE=(postdrug response latency-baseline response latency)(cutoff time-baseline response latency)
Where postdrug response latency=the longest response la-
tency observed af ter drug administration , baseline response la-
tency=the average of three measures of the response latency be-
fore drug administration , and cutof f time=15 s.
Comparisons of the drug effects betw een differ-
ent postoperative time w ere perfo rmed.The observed
ef fects of the combinations were compared wi th the
expected addi tive effects calculated f rom the individu-
·434· 中国新药与临床杂志(Chin J New Drugs Clin Rem), 2000 年 11 月 , 第 19 卷　第 6 期
al drug s.All data w ere expressed as mean±standard
deviation.Statist ical analysis w as carried out with
student-t-test .A P value<0.05 was considered sig-
nif icant.
RESULTS
The nociceptive response latency of the six rats
w as tested preoperatively , 1-3 h and 3 d postoperativ-
ely.The results showed no significant differences ,
which meant the baseline nociceptive response w as
not noticeably affected by the operat ion.
Morphine ith significantly increased the %MPE ,
and no difference was found between the effects dur-
ing two postoperative periods.Physostigmine iv 50 o r
100μg·kg-1 injected 3 d af ter the operation did not
show obvious changes of the %MPE.But physostig-
mine iv 50 o r 100 μg·kg-1 administered 1-3 h af ter
the operation dramatically increased the %MPE.
Combined administ ration of iv physostigmine and ith
morphine resulted in marked increase in %MPE
which was more potent in higher phy sostigmine
dosage.The effects of the combinations were more
pronounced at early postoperat ive t ime.Table 1
shows the %MPE of each drug administered at dif-
ferent postoperative time.
Table 1　%MPE of each drug and combination administered
at 1-3 h and 3 d postoperatively
d rug dosage rat s 1-3 h 3 d
Mor 1μg 8 12±7a 17±7
Mor 2μg 7 29±8a 20±12
Phys 50μg·kg -1 8 19±7a 12±2
Phys 100μg·kg -1 6 35±10c 14±12
Phys 50+Mor.1 7 62±14c 33±8
Phys 100+Mor.1 8 90±9b 68±16
　　a P >0.05 , bP <0.05 , cP <0.01 vs the same dose injected 3 d
postoperatively.
Mor means i th morphine;Phys means iv physostigmine;Phys 50
+Mor 1 means iv physost igmine 50μg·kg -1+ith morphine 1μg;Phys
100+M or 1 means iv physostigmine 100μg·kg -1+ith morphine 1μg.
　　According to Berenbaum s[ 6] defini tion , non-ad-
di tive interaction of drug s is present when the ef fect
of a combination of drug s differs f rom that expected
from their individual dose response curves.If both
drug s act in an additive manner , then one of the
drug s can be replaced by the second in amount equief-
fective to the amount of the first drug .Therefore ,
the expected ef fect of the combinat ion lies between
the effects of double doses of bo th drugs and never ex-
ceeds bo th of them.If at any dose level response to
the combination of drugs is significantly greater than
the response to the double doses of bo th drug s , then
the interaction of the drugs is synergistic.Thus , in
this study , the observed effect of the combination of
small doses of mo rphine and physostigmine has been
compared to the ef fect of double doses of both drug s.
Figure 1 show s the time-response of the ith morphine
2μg , iv phy sost igmine 100 μg·kg-1 , and i th mor-
phine 1 μg +iv phy sostigmine 50 μg·kg-1.The ef-
fect of the low dose combination was obviously greater
than that of the double doses of both drugs.Figure 2
Fig 1　Time-response of the effect of intrathecal morphine 2
μg , intravenous physostigmine 100 μg·kg-1 , and morphine 1
μg+physostigmine 50μg·kg-1 on paw withdrawal latencies.
　　All points represent the mean response latencies of 6-8 an-
imals , and erro r bars denote SD.
Fig 2　Comparison of the observed combined effect and the
expected additive effect
　　The observ ed effect of each combination was significantly
greater than the expected additive effect (P<0.01).All bars
represent the mean % MPE of 6-8 animals , and error bars de-
no te SD.
·435·中国新药与临床杂志(Chin J New Drug s Clin Rem), 2000 年 11 月 ,第 19 卷　第 6 期
shows the comparison of the observed mixed effects of
the tw o doses of combinations with the expected addi-
tive effects calculated from the ef fect of morphine and
phy sostigmine injected separately.The potency of the
observed effects of the combinations was signif icantly
g reater than that of the expected additive effects.
DISCUSSION
Observations indicate that a descending inhibito-
ry pathway exists f rom the mid-brain and medulla to
the spinal cord dorsal horn in modulating analgesia.
Chief neurot ransmit ters involved are NE and Ach.
Plenty of evidence has been found.Ith administ ration
of the antag onist of acetylcholine muscarinic recepto r
at ropine pow erfully reduced the analgesia induced by
sy stemic morphine , but this effect failed to appear in
spinal co rd transected rats[ 7] .In sheep and humans ,
iv injection of morphine caused increase in concentra-
tion of NE and Ach in the CSF from lumbar spinal
space[ 2] .The exact relationship betw een NE and Ach
in analgesic mechanism is not clear yet .It has been
hypo thesized that any nox ious stimulation or opioid
administ rat ion may activate the pathway f rom
supraspinal central sites to spinal cord and cause the
release of NE.Then the released NE may mediate
cholinergic neurons to produce Ach , which diminishes
substance P released from primary af ferent neurons
and reduces response of dorsal ho rn neurons to nox-
ious stimulation[ 1 ,4] .This hypothesis becomes the
theoretical backg round fo r the application of anti-
cholinesterase agents in pain management .In the cur-
rent study , it is proved that physostigmine enhanced
the antinociception of morphine , which is coincident
w ith the previous reports
[ 3] .But the antinociception
of physostigmine alone appeared only at early postop-
erat ive t ime.Furthermo re , the combined effect of iv
phy sostigmine and ith morphine w as more pro-
nounced at early postoperative period.Since the
antinociceptive effect of morphine ith showed no sig-
nif icant changes during the two postoperative periods ,
the dif ferences of the antinocicept ion of phy sostigmine
and the combinat ion should no t be explained by the
tolerance of the drugs.The reason for this phe-
nomenon is probably due to the activation of endoge-
nous Ach by the postoperative st ress stimulation.
Physostigmine , which can pass the blood-brain barri-
er , may produce more effect on enhancement of the
decending inhibito ry analgesic modulat ion by further
increasing the Ach concentration.
Both opioids and anticholinesterase agents have
some dose-dependent side effects.The most danger-
ous side effect of opioids is respi ratory depression.
Physostigmine can cause sedation , nausea , leg w eak-
ness and even muscle t remble.If the combination of
the tw o drugs produces a synergistic ef fect , the desir-
able dosage of both drugs can be decreased , thus the
incidence of undesi rable effects can be reduced.Ac-
cording to pharmacological theory , if the combined
ef fect of the two drugs is obviously greater than the
expected addit ive ef fect of them at any dose level ,
then the interaction of the drugs is synergism[ 6] .
Therefore , the results of the present study suggest
that there is a synergistic interaction betw een iv
physostigmine and ith morphine.In addition , i t is re-
ported that anticholinesterase agents can antagonize
the respiratory depression induced by mo rphine[ 8] .I t
can be expected that this combination may lead to
some advancement in pain management .
Conclusions:anticholinesterase agent phy sostig-
mine produces antinociception under the condit ion
that exists postoperative st ress there.The combina-
tion of iv phy sostigmine and ith morphine results in
synergistic interaction.
[ REFERENCES]
[ 1] Eisenach JC , Detweiler DJ , Tong C , Angelo RD , Hood DD.Cere-
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[ 2] Bouaziz H , Tong C , Yoon Y , Hood DD , Eisenach JC.Int ravenous
opioids stimulate norepinephrine and acetylcholine release in spinal
cord dorsal horn [ J] .Anesthesiology , 1996 , 84:143-154.
[ 3] Hood DD , Mallak KA , James RL , Tutt le R , Eisenach JC.En-
hancement of analgesia f rom systemic opioid in humans by spinal
·436· 中国新药与临床杂志(Chin J New Drugs Clin Rem), 2000 年 11 月 , 第 19 卷　第 6 期
cholinesterase inhibition [ J] .J pharmacol Exp Ther , 1997 , 282:
86-92.
[ 4] Detw eiler DJ , Eisenach JC , Tong C , Jackson C.A cholinergic in-
teraction in alpha2 adrenocepter-mediated ant inociception in sheep
[ J] .J pharmacol Exp Ther , 1993 , 265:536-542.
[ 5 ] Naguib M , Yaksh TL.Antinociceptive ef fects of spinal
cholinesterase inhibi tion and isobolographic analysis of the interac-
tion w ith μ and α2 receptor systems [ J] .Anesthesiology , 1994 ,
80:1338-1348.
[ 6] Berenbaum MC.What is synergy ? [ J] .Pharmacol Rev , 1989 ,
41:93-141.
[ 7] Chiang CY , Zhuo M.Evidence for the involvem ent of a descending
cholinergic pathw ay in systemic morphine analgesia[ J] .Brain Res ,
1989 , 478:293-300.
[ 8] Elmalem E, C horev M , Weinstock M .Antagonism of morphine-in-
duced respi ratory depression by novel an ticholinesterase agents [ J] .
Neuropharmacology , 1991 , 30:1059-1064.
中国新药与临床杂志(Chin J New Drugs Clin Rem),
2000 年 11 月 , 19(6):433-437.
毒扁豆碱对大鼠的镇痛效应及静脉注
射毒扁豆碱与蛛网膜下腔注射吗啡的
相互作用
陈莲华1 ,Alexander NEM IROVSK Y2 ,贡沁燕3 　(1.
上海医科大学 眼耳鼻喉科医院 麻醉科 , 中国 上海 　
200031;2.美国南加州大学 洛杉机医疗中心 麻醉科 , 美国
洛杉机　90033;3.上海医科大学基础医学院 药理教研室 ,
中国 上海　200032)
[关键词] 　毒扁豆碱;吗啡;疼痛测定;药物协同作
用
[摘要] 　目的:观察在术后不同阶段静脉注射胆碱
酯酶抑制剂毒扁豆碱对鼠痛阈的影响 ,以及静注毒
扁豆碱和蛛网膜下腔注射吗啡的药物相互作用 。方
法:6组 SD大鼠 ,置入蛛网膜下腔和股静脉导管。
采用辐射热刺激诱发的鼠腿撤退试验测痛阈 ,测试
分别于术后 1 ～ 3 h和术后3 d进行。动物分别接受
静注毒扁豆碱 、蛛网膜下腔注射吗啡及两者联合给
药 。比较术后 1 ～ 3 h 和 3 d 各种药物对痛阈的影
响 、联合给药与单独给药对痛阈的影响 。药物的镇
痛效应以最大可能效应的百分比(%MPE)表示。
结果:毒扁豆碱术后 1 ～ 3 h 静注导致%MPE 明显
提高;毒扁豆碱与吗啡的联合给药效应在术后早期
更明显;小剂量联合给药的镇痛作用明显大于双倍
剂量单独给药的镇痛作用;联合给药的测得效应值
明显大于估计叠加效应值。结论:胆碱酯酶抑制剂
毒扁豆碱在术后早期产生镇痛作用;静注毒扁豆碱
与蛛网膜下腔注射吗啡的联合给药呈协同效应 。
[中图分类号] 　R971.91;R965
[文献标识码] 　A
[文章编号] 　1007-7669(2000)06-0433-05
第 7届全国生化药理会议召开　第7 届全国生化药理学术会议于 2000 年 9 月23 日-27 日在苏州召开 ,由苏州大学中药研究
所承办 ,由顾振伦 、朱秀媛 、丛铮 、池志强 、孙曼霁等教授主持。参加大会的有来自北京 、上海 、广东 、台北等各省市的同行 120
余人 ,孙曼霁院士 、台北中医药研究所陈介甫所长等 12位专家为大会作了特邀报告。同时大会进行了近 2 d(天)的分组讨论
和专题报告 ,分别就药物的生化作用机理 、药物代谢及药物代谢酶 、生物技术和分子生物学研究等多项专题展开讨论 ,并对生
化药理更广阔更纵深的发展作了展望。会议对青年优秀论文进行了评奖 、颁奖。
平喘新药多索茶碱片问世　浙江昂利康制药厂开发研制成功支气管扩张剂多索茶碱(商品名达复啉), 日前已批量生产上市
供应。多索茶碱系甲基黄嘌呤类衍生物 ,是一种支气管扩张剂 ,通过抑制平滑肌内的磷酸二酯酶 , 松弛平滑肌支气管 , 达到解
痉平喘功效。临床表明 ,本品临床治疗总有效率 、改善肺功能 、哮喘 、慢支哮喘 、过敏哮喘和感染性哮喘均优于茶碱控释片 , 且
不良反应明显低于茶碱控释片。临床用于支气管哮喘 、喘息性慢性支气管炎以及支气管痉挛引起呼吸障碍。
·437·中国新药与临床杂志(Chin J New Drug s Clin Rem), 2000 年 11 月 ,第 19 卷　第 6 期