Neuropharmacology Vol. 30, No. 10, pp. 1059--1064, 1991
`Printed in Great Britain. All rights reserved
`
`0028-3908/91 $3.00 + 0.00
`Copyright© 1991 Pergamon Press pic
`
`ANTAGONISM OF MORPHINE-INDUCED RESPIRATORY
`DEPRESSION BY NOVEL ANTICHOLINESTERASE
`AGENTS
`
`ESTHER ELMALEM, 1 M. CHOREV2 and MARTA WEINSTOCK 1*
`1Departments of Pharmacology and 2Medicinal Chemistry, School of Pharmacy, Hebrew University,
`Ein Kerem, Jerusalem, Israel
`
`(Accepted 13 May 1991)
`
`Summary-This study compared the effects of 3 novel antiAChE agents (derivatives of dimethy(cid:173)
`laminoethyl-phenyl carbamate) with that of physostigmine on the respiratory depression induced by
`morphine in rabbits. Each drug, RA6, (I mg i.v., 2 mg s.c.) RA7 (I or 2 mg i.v.); RA 1s (0.25 or 0.5 mg i.v.),
`physostigmine (0.05 or 0.1 mg i.v.) or saline (I ml), was injected simultaneously with morphine (8 mg i.v.)
`to groups of 6-10 rabbits. Respiration rate, blood gases and pH were monitored for 3 hr. Plasma ChE
`was measured before and at 15 min intervals after injection. The 4 antiAChE's were given to 40 other
`rabbits, which were sacrificed at the time of maximal antagonism of the respiratory depressant effect of
`morphine, in order to measure the activity of AChE in the medulla, cortex and hippocampus.
`Physostigmine (0.1 mg) only antagonized the increase in paC02 induced by morphine at 15 and 30 min.
`The drugs RA 1s (0.5 mg), RA6 (2.5 mg) and RA7 (2 mg) almost completely prevented the respiratory
`depression, without obvious signs of peripheral cholinergic hyperactivity, for at least 3 hr. There was no
`relationship between the degree of antagonism of the effects of morphine with any drug and that of
`inhibition of ChE in plasma. In contrast, a highly significant correlation (P < 0.01) was found between
`the former and the amount of inhibition of AChE in the medulla.
`It is suggested that the novel carbamates may have potential therapeutic application in reducing the
`respiratory depression of opiates, without impairing analgesia.
`
`Key words-respiratory depression, cholinesterase inhibition in medulla, carbamates, rabbit.
`
`In previous studies in human subjects and experimen(cid:173)
`tal animals it was shown that physostigmine could
`reduce the respiratory depressant effect of morphine,
`without
`interfering with
`the analgesic effect
`(Snir-Mor, Weinstock, Bahar and Davidson, 1983;
`Weinstock, Erez and Roll, 1981a; Weinstock,
`Davidson, Rosin and Schnieden, 1982). However, as
`potential therapy for concomitant use in patients
`receiving opiates, physostigmine has a number of
`serious disadvantages. The most important of these is
`its relatively high toxicity, which results in the
`appearance of distressing side effects at therapeutic
`doses (Christie, Shering, Ferguson and Glenn, 1981).
`Its low chemical stability and short duration of action
`also necessitate frequent administration.
`In an
`attempt to overcome these drawbacks, a number of
`novel anticholinesterase agents were synthesized in
`this laboratory. These agents readily penetrate the
`central nervous system, have a greater chemical stab(cid:173)
`ility and longer duration of action than that of
`physostigmine and several of them also have signifi(cid:173)
`cantly higher therapeutic ratios (Weinstock, Razin,
`Chorev and Tashma, 1986).
`
`*Address correspondence to Professor Marta Weinstock,
`Department of Pharmacology, The Hebrew University
`Hadassah Medical School, Jerusalem 91010, Israel.
`
`The purpose of this study was twofold; to compare
`the abilities of three of these novel anticholinesterase
`agents with that of physostigmine to antagonize the
`respiratory depressant effect of morphine and to
`determine whether there is a correlation between the
`degree of such antagonism and the amount of inhi(cid:173)
`bition of acetyl-cholinesterase (AChE) in the medulla
`oblongata.
`
`METHODS
`
`Antagonism of the cardiovascular and respiratory de(cid:173)
`pressant effects of morphine by the anticholinesterase
`compounds
`Male and female rabbits, weighing 2.5-3 kg, were
`prepared with catheters in the central ear artery and
`marginal ear vein, as previously described (Weinstock
`et al., 198la). Rectal temperature was monitored on
`a telethermometer with the aid of a thermistor probe
`inserted into
`the rectum. Respiration rate was
`counted visually for periods of 30 sec. Blood gases
`and pH were measured on a blood gas analyzer
`(Instrumentation Laboratories) after correction for
`the appropriate body temperature from samples of
`blood taken from the ear artery. Blood pressure and
`heart rate were monitored on a Brush Gould
`recorder.
`
`1059
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`1060
`
`EsTHER ELMALEM et a/.
`
`Each of the following drugs, physostigmine, (0.05
`and 0.1 mg/kg); RA6 (0.5 and 1 mg/kg); RA 7 (1 and
`2 mg/kg) and RA 15 (0.25 and 0.5 mg/kg), was injected
`to
`intravenously (i.v.) with morphine (8 mg/kg)
`groups of 6-10 rabbits per drug. Nine other rabbits
`were given morphine alone with 0.1 ml/kg saline. An
`additional group of 6 rabbits received morphine
`(8 mg/kg) plus RA6 , (2.5 mg/kg) subcutaneously
`(s.c.). Blood samples were taken for blood gas analy(cid:173)
`sis, at least twice before administration of drug, 5, 15
`and 30 min after injection and thereafter at 30 min
`intervals, for 3 hr.
`
`Measurement of anticholinesterase activity in different
`areas of the brain of rabbits
`Rabbits were injected intravenously with either
`physostigmine or each of the above drugs, in the
`doses designated, or with RA6 , (s.c.). A minimum of
`4 animals was used for each treatment group. At
`stated times after the injection, the animals were
`sacrificed by air embolism. Eight additional rabbits
`were injected with saline or morphine (8 mg/kg) and
`sacrificed at the same times as the rabbits treated with
`the anticholinesterases, i.e. 4 after 60 min and two
`each after 15 and 30 min. The brain was removed and
`the frontal cortex, hippocampus and medulla were
`rapidly dissected out on ice, weighed individually and
`homogenized in phosphate buffer (0. 1 M) pH 8,
`containing 1% Triton. The mixture was centrifuged
`at 1000 g and the supernatant, which contained most
`of the solubilized enzyme, was used for the determi(cid:173)
`nation of the activity of AChE by the method of
`Ellman, Courtney, Andres and Featherstone (1961).
`The percentage inhibition of AChE by the drugs
`was computed by comparison with the pooled mean
`value for each of the appropriate saline-treated con(cid:173)
`trols.
`
`Estimation of plasma cholinesterase
`Blood (0.5 ml) was withdrawn into a heparinized
`syringe, during the control period and at 5, 15, 30, 60,
`90, 120, 150 and 180 min after injection of the AChE
`inhibitors. The blood was centrifuged at 4oC for
`5 min at 1000 g and the activity of AChE of the
`plasma was measured by the method of Ellman et at.
`(1961).
`
`Drugs
`The agents tested were RA6 (N -ethyl-3[1-( dimethyl(cid:173)
`amino)ethyl]phenyl carbamate) HCI. RA7 (N-ethyl,
`N -methyl-3[1-( dimethylamino )ethyl] phenyl carba(cid:173)
`mateHCI. RA 15(N -propyl-3( 1-dimethylamino )-ethyl](cid:173)
`phenyl carbamate HCI. Physostigmine salicylate
`(Sigma Ltd); Morphine HCl (Teva Pharmaceuticals,
`Israel). All drugs were made up freshly in sterile
`saline, which included an equal weight of sodium
`metabisulphite, to prevent oxidation. All doses are
`expressed as mg per kg of body weight of the
`appropriate salt.
`
`RESULTS
`Antagonism of the respiratory depressant effect of
`morphine by antiAChE
`Intravenous injection of morphine (8 mg) caused a
`significant fall in respiration rate of about 50% and
`a rise in paC02 of 54% within 15 min, which lasted
`for 2-3 hr. The pa02 was significantly reduced from
`114 + 6 to 85 + 5 at 15 and 30 min, while the pH fell
`from-7 .45 + o:007 to 7.27 ± 0.01 at 15--{)0 min. Mor(cid:173)
`phine also reduced the heart rate by 70-120 beats per
`min during the first hour and caused a small
`(5.2 ± 1.8 mmHg) but significant decrease in blood
`pressure during this period. Although control paC02
`values were very similar in all the treatment groups,
`the respiration rates varied from 67 ± 4 to 86 ± 6.
`These differences were normalized by representing the
`values after administration of drug as a percentage
`change from the resting rate for each animal.
`Physostigmine (0.05) had no significant influence
`on the respiratory depression induced by morphine at
`any time after injection, but caused mild salivation in
`all the rabbits. At a dose of 0.1 mg, physostigmine
`reduced the elevation in paC02 only at 15 and 30 min
`after injection and the fall in respiration rate at
`15 min (Fig. 1 ). Physostigmine potentiated the bray(cid:173)
`cardia induced by morphine at 15 and 30 min. These
`effects were accompanied by signs of peripheral
`cholinergic hyperactivity, including salivation, defae(cid:173)
`cation and slight muscular twitches.
`The drug RA15 (0.25 mg) significantly reduced the
`elevation in paC02 and the fall in respiratory rate
`after morphine, only at 15 min after injection (Fig. 2).
`At a dose of 0.5 mg, both the change in paC02 and
`in respiration rate, induced by morphine, were signifi(cid:173)
`cantly antagonised for 3 hr (Fig. 2) but the brady-
`
`---- ~11\E --+- ~11\E •···~·· ~11\E +
`t8mg)
`PHY$.0, 1 mQ
`PI-IVS.O.O&ng
`
`40
`
`25L-~~~--~----~~------~~
`LU 55
`f-
`<l a:
`e; 45
`!l!
`
`A···············b.···············A·····
`
`~ 25L_--~----~~----~~~~---:
`0
`2
`3
`
`TIME AFTER INJECTION (hours)
`
`Fig. I. The influence of physostigmine on the respiratory
`depressant effect of morph~ne. Physosti~ine. ~as _injected
`intravenously at the same ttme as morphme. Stgmficantly
`different from morphine alone, P < 0.05.
`
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`

`Opiate antagonism by anticholinesterases
`
`1061
`
`I to 3 hr after injection (Fig. 3). Side effects were only
`present in a mild degree.
`The drug RA7 had no effect against morphine, at
`a dose of 1 mg but completely prevented the respirat(cid:173)
`ory depression from 30 min to more than 3 hr when
`2 mg were given (Fig. 4). There were no signs of
`peripheral cholinergic activity at this dose, neither
`was the bradycardia potentiated. Moreover, the hy(cid:173)
`potensive effect of morphine was abolished and the
`reduction in pH markedly attenuated.
`
`Inhibition of plasma cholinesterase by the novel carbo(cid:173)
`mates and physostigmine
`The control value for plasma cholinesterase in
`these rabbits was 24 ±21-1M acetylcholine (ACh)
`hydrolysed/ml/hr. Physostigmine caused a maximum
`inhibition of 40% of this enzyme, 5 min after injec(cid:173)
`tion of a dose of 0.1 mg and this declined to 11% by
`2 hr (Table 1). In contrast, RA15 (0.5 mg) caused more
`than 70% inhibition of cholinesterase in plasma from
`5-90 min and more than 50% after 2 hr. Both RA6
`and RA7 also caused a maximum inhibition of more
`than 70% at 15 min, which declined slowly, like that
`of RA 15, over the next 3 hr (Table 1).
`
`The inhibition of AChE by the carbamates in the
`medulla of rabbits
`The antiAChE agents caused between 37 and 63%
`inhibition in cholinesterase, in the medulla oblongata,
`depending on the drug, the dose and the time of
`administration. In order to determine whether there
`was any correlation between the degree of inhibition
`of cholinesterase in the brainstem and the extent of
`
`......... ~ _._~·-··~·
`emo
`,.....,eo.2emo
`,.. ... ,~.omg
`
`40
`
`30
`
`25
`55
`
`45
`
`35
`
`25
`0
`
`·•·-·-·-·A
`*
`*
`
`,.•·-·-....... _ ·-......
`
`•
`*""'
`·--- .•. ,.,•'"4
`
`.f_,-f
`
`TIME AFTER INJECTION ChOU"s)
`
`2
`
`3
`
`Fig. 2. The influence of RA 15 on the respiratory depressant
`effect of morphine. The RA 15 was injected at the same time
`as morphine. *Significantly different from morphine alone,
`p <0.05.
`
`cardia was only increased at 15 min. Peripheral
`cholinergic activity was present to a lesser degree than
`with physostigmine. Only a very small antagonistic
`effect was shown by RA6 (1 mg) on the respiratory
`depressant effect of morphine. At a dose of 2 mg
`(i.v.), it caused marked cholinergic hyperactivity and
`therefore was not given together with morphine.
`However, when given subcutaneously, RA6 (2.5 mg)
`abolished the effect of morphine on respiration from
`
`- - - MOFIA-fi"E
`(Bmg)
`
`-·-· MOFPHI".e' +- ..... MC:IFR-IfrE: +
`FIA-e l1mol
`AAe 12.efTICI)
`
`40
`
`N
`
`.t· *
`-~--:.:.• .. ==-:-----.... ,.,------...-.------•-................. _______ ...,
`*
`""'•
`~30
`...
`.:'
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`· .......................... -·-*
`l
`*
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`II
`•I
`
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`t!! 55
`~
`9j45
`.........
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`*
`*
`....
`ii::
`035
`.................
`..... *
`*
`~
`~ 25 ~.-. ____ ..__ _______ .._~----~
`
`0
`
`2
`
`3
`
`__.._ MOFPI-!1""1:
`(Bmg)
`
`-·-· MOFFI-ilf\E ~- •·· MOAPf-lrtE +
`FIA-7 (1mg)
`AA-7 (2mg)
`
`40 , ......
`
`I ... _______ ..,.. ______ "'+-------~---
`'* u•.
`r '·*
`1
`-....
`-.,
`
`*
`.............. .: ............. .; ......... -~----·-·.:
`
`24~----~--~--~~~~--~--~
`60
`
`~ 50
`9i
`~ 40
`ai 30
`
`,......,,.
`__ .... ___
`.. ............... ---~---__..,~
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`..---·-·---..... ______ ...
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`0
`
`··-.•. _
`·---..... ,.-·'·~·
`~ 20~--------._--~----~------~
`3
`2
`0
`
`TINE AFTER INJECTION (hours)
`
`TIIVE AFTER IN..ECTJON (hours)
`
`Fig. 3. The influence of RA6 on the respiratory depressant
`effect of morphine. The RA6 (I mg) was injected intra(cid:173)
`venously and (2.5 mg) subcutaneously, at the same time as
`morphine. *Significantly different from morphine alone
`P < 0.05. The values of paC02 for morphine+ RA6
`(2.5 mg) were not significantly different from the pretreat-
`ment values from I to 3 hr.
`
`Fig. 4. The influence of RA7 on the respiratory depressant
`effect of morphine. The RA7 was injected intravenously at
`the same time as morphine. *Significantly different from
`morphine alone, P < 0.05. The values of the paC02 for
`morphine + RA7 (2 mg) was not significantly different from
`pretreatment values from 30 min to 3 hr.
`
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`

`1062
`
`EsTHER ELMALEM et a[.
`
`Drug
`Physo
`
`RA"
`
`RA7
`
`RAo
`
`Table I. Inhibition of plasma cholinesterase by physostigmine
`(Physo) and the novel carbamates
`Percentage
`inhibition
`(±SE)
`32.5 ± 1.6
`7.6 ± 1.8
`43.2 ± 3.6
`11.2 ± 3.5
`56.2 ± 1.2
`31.4 ± 1.1
`78.2 ± 0.9
`51.1 ± 2.2
`68.0 ± 0.8
`41.7 ± 1.2
`78.1 ± 0.6
`57.5 ± 0.8
`42.3 ± 1.1
`77.4 ± 0.6
`43.2 ± 1.2
`79.2 ± 1.0
`65.4 ± 1.1
`50.5 ± 0.9
`
`Dose
`(mg/kg)
`0.05
`0.05
`0.1
`0.1
`0.25
`0.25
`0.5
`0.5
`1.0
`1.0
`2.0
`2.0
`2.0
`1.0
`1.0
`2.5(s.c.)
`2.5
`2.5
`
`Time
`(min)
`5
`120
`5
`120
`5
`120
`5
`120
`15
`120
`15
`120
`180
`15
`120
`30
`120
`180
`
`the antagonism of the respiratory depressant effect of
`morphine, the increase in paC02 from a mean control
`value of 26.7-40.1 mmHg after morphine,
`i.e.
`13.4 mmHg, was designated as = 100%. Then the
`effect of each of the antiAChE agents at different
`times after injection was expressed in relation to the
`effect of morphine alone; e.g. the combination of
`morphine and RA7 (2 mg i.v.) at 30 min resulted in a
`paC02 of 28.4 mmHg, i.e. a 16% increase above the
`control value of 26.7 mmHg.
`inhibition
`Since
`the measurement of enzyme
`necessitated the sacrifice of each animal this was only
`measured at the time of peak antagonism of the effect
`of morphine and a whole time course was not per(cid:173)
`formed for each drug. The time after administration
`of drug that the medulla was removed and the activity
`of AChE was measured is shown in Figure 5. This
`shows a highly significant correlation (r = - 0.948;
`P < 0.001) between the percentage reduction in the
`maximum increase in paC02 with the antiAChe
`agents and morphine, and the percentage inhibition
`of AChE in the medulla of the brainstem. It also
`shows that this enzyme must be inhibited by at least
`
`40% before any significant reduction in the respirat(cid:173)
`ory depressant effect of morphine can be observed
`and that complete antagonism of the latter was
`achieved when the enzyme was inhibited by 60%.
`
`The inhibition of AChE by the carbamates in other
`areas of the brain
`The amount of AChE activity in three areas of
`brain, 30 min after an injection of saline or morphine
`(8 mg) is shown in Table 2. Morphine did not appear
`to cause any significant inhibition. The degree of
`enzyme inhibition by the carbamates in the frontal
`cortex, hippocampus and medulla of rabbits, sac(cid:173)
`rificed at the time of peak antagonism of morphine,
`is shown in Table 3. While no difference was seen in
`the 3 areas of brain with physostigmine, 15 min after
`injection, the inhibition became significantly greater
`in the hippocampus and medulla at 30 min and had
`almost completely worn off by 90 min. On the other
`hand, RA 15 showed a greater effect in the latter areas
`of the brain than in the cortex at 15 min, which
`became similar in them all at 30 min.
`At 60 min, RA6 (1 and 2.5 mg) caused similar
`degrees of inhibition in the cortex and hippocampus
`but the smaller dose had a much smaller effect in the
`medulla. The inhibition of enzyme, induced by RA7
`at 60 min, was dose-related and similar in all three
`regions.
`
`DISCUSSION
`
`Hypercapnia stimulates respiration by increasing
`the release of ACh from neurones in the medulla (Dev
`and Loeschcke, 1979; Metz, 1966). Acetylcholine,
`applied directly to this area (Miller, 1949) and cen(cid:173)
`trally acting cholinomimetics or anticholinesterases,
`administered parenterally, also stimulate respiration
`(Weinstock, 1981; Weinstock, Roll and Zilberman,
`1981 b). Conversely, narcotic analgesics are thought
`to depress respiration by inhibiting the release of
`ACh (Domino and Wilson, 1973), thereby reducing
`the sensitivity of the respiratory centre to C02
`(Florez, McCarthy and Borison, 1968). Since choli(cid:173)
`nomimetics and physostigmine have analgesic ac(cid:173)
`tivity, when given alone to experimental animals and
`man (Pleuvry and Tobias, 1971; Sitaram, Buchsbaum
`and Gillin, 1977) and, unlike naloxone, do not impair
`that of morphine, physostigmine was tested for its
`ability to reverse narcotic-induced respiratory de(cid:173)
`pression in post-operative patients (Weinstock et a/.,
`1982; Bourke, Rosenberg and Allen, 1984). A signifi(cid:173)
`cant but short-lived antagonism was observed in
`some but not in all subjects. Failure to obtain a
`consistent effect of adequate duration may have been
`
`Table 2. Activity of AChE in areas of the brain in rabbits injected
`with saline or morphine (JlM ACh hydrolysed/g/hr)
`Saline (8)
`Morphine (2)
`
`45 ± 2
`57± 4
`84±5
`
`40 ±4
`55± 5
`79±5
`
`• FIA7 (2mg)
`
`C\1
`
`100
`8 eo
`
`<(
`Q.
`
`~ 60
`~
`<( 40
`
`~
`
`'II.
`
`20
`
`0
`30
`
`D
`
`•
`
`..
`
`r • O.Q48
`
`p < 0.001
`
`+
`
`0
`
`A
`
`40
`
`50
`
`60
`
`70
`
`4
`
`0
`
`+
`
`D
`
`60min
`FIA6 (2.15mg)
`60min
`FIA 15 (0.5mg)
`ao min
`I>HYS (0. 1 mg)
`30 min
`I>HYS.(O. 1 mg)
`15 m•n
`
`..
`• FIA6 (1mg)
`
`60min
`!'VI. 115 (0.5mg)
`30 min
`
`• FV>.7 (1mg)
`
`60mln
`
`%
`
`INHIBITION OF ACI-E
`
`Fig. 5. Relationship between the percentage increase in
`paC02, induced by a combination of morphine and anti(cid:173)
`ChEs, to the percentage inhibition of AChE in the medulla,
`at the time of maximum antagonism of the respiratory
`depression r = 0.962, P < 0.001.
`
`Area
`Cortex
`Hippocampus
`Medulla
`
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`

`Opiate antagonism by anticholinesterases
`
`1063
`
`Table 3. Inhibition of AChE by physostigmine and novel carbamates in different
`areas of the brain
`
`Percentage inhibition (±SE)
`
`Drug dose
`
`(mg/kg)
`
`Time
`
`(min)
`
`Physo (0.1)
`
`RAIS (0.5)
`
`RA6 (1.0)
`
`(2.5) S.C.
`
`RA7 (1.0)
`
`(2.0)
`
`15
`
`30
`
`90
`
`15
`
`30
`
`60
`
`60
`
`60
`
`60
`
`Cortex
`
`Hippocampus
`
`Medulla
`
`37.0± 3.4
`
`44.1 ±2.1
`
`44.0 ± 1.5
`
`35.3 ± 2.9
`
`55.0 ± 1.9°
`
`54.1 ± 4.5•
`
`17.0± 1.8
`
`40.2 ± 0.9
`
`10.0 ± 5.3
`59.9 ± u•
`
`16.5±5.1
`
`53.2 ± 1.9°
`
`40.0 ± 3.3
`
`38.2 ± 3.8
`
`39.2 ± 3.7
`
`62.5 ± 0.9
`
`56.8 ± 3.4
`
`38.8 ± 2.4°
`
`56.3 ± 2.6
`
`61.1 ± 3.4
`
`55.4 ± 1.0
`
`37.9 ± 6.8
`
`48.5 ± 2.6
`
`33.2 ±4.0
`
`66.1±3.9
`
`71.9 ± 4.6
`
`63.5 ± 3.1
`
`due to the short half-life of the drug (Giacobini,
`Somani, Mcllhany, Downen and Hallak, 1987) and
`its narrow therapeutic window (Christie eta/., 1981).
`In the present study, physostigmine (O.l mg/kg),
`administered together with morphine to rabbits, sig(cid:173)
`nificantly decreased the rise in paC02 for 30 min but
`intensified the reduction in blood pH and bradycar(cid:173)
`dia. The acidosis may have been due to production of
`lactic acid in muscle in response to stimulation of
`nicotinic receptors by the increased levels of ACh
`(Weinstock eta/., l98lb). Muscle fasciculations, of a
`small intensity, were seen at this dose in some of the
`rabbits and these became pronounced at 0.2 mg/kg.
`Physostigmine inhibited cholinesterase in plasma by
`a maximum of 40%, 5 min after injection, which
`rapidly declined to about 10% within 2 hr. Peak
`inhibition of AChE (54%) did not occur in the
`medulla until 30 min.
`All three novel carbamates produced a long-lasting
`antagonism of the respiratory depressant effect of
`morphine, with only very mild signs of peripheral
`cholinergic hyperactivity. This was minimized with
`RA6, through ensuring a slower absorption, by ad(cid:173)
`ministering the drug subcutaneously. In contrast to
`physostigmine, RA 7 completely prevented the respir(cid:173)
`atory depression from 30 to 180 min and also reduced
`the acidosis, bradycardia and hypotensive effect of
`morphine. This suggests that the degree of inhibition
`of AChE in the heart and skeletal muscle was
`relatively less with this drug than with physostigmine
`and that the increase in central cholinergic activity
`and resultant sympathetic stimulation, overcame
`the cardiovascular depressant effects of morphine
`(Weinstock, Zavadil, Chieuh and Kopin, 1979). The
`data agree with previous findings with RA7 in the rat,
`in which it was demonstrated that AChE was inhib(cid:173)
`ited, to a significantly greater extent in the brain, than
`in the heart and skeletal muscle (Weinstock et a/.,
`1986).
`
`Unlike reports with physostigmine in other species
`(Giacobini eta/., 1987; Hallak and Giacobini, 1986),
`there was no direct relationship between the degree of
`inhibition of cholinesterase in plasma in the rabbit
`and the amount of antagonism of the respiratory
`depression or of the severity of peripheral cholinergic
`side effects. Both RA6 and RA7 (l mg) showed only
`minimal antagonism of morphine and no cholinergic
`symptoms, in spite of more than 60% inhibition of
`cholinesterase in plasma, which lasted for at least
`I hr. In contrast, physostigmine only reduced the
`activity of cholinesterase by a maximum of 40%, for
`a few minutes but produced peripheral cholinergic
`symptoms and antagonism of morphine.
`A comparison of the relative degrees of inhibition
`by the four drugs in these areas of brain also revealed
`significant differences between them. While physo(cid:173)
`stigmine and RA 15 were more active in the hippo(cid:173)
`campus and medulla, RA6 (I mg) caused a greater
`inhibition in the cortex and hippocampus. This
`suggested that the drugs may either be distributed to
`these areas at different rates, or that the enzymes
`within these regions may show unequal sensitivity to
`the inhibitors. There is some evidence in favour of the
`former suggestion from the studies of Hallak and
`Giacobini (1986) and Giacobini et a/. (1987), who
`showed that the concentration of physostigmine in
`the brain of the rat varied considerably in different
`areas and this produced differences in the degrees of
`inhibition of AChE and in the resultant levels of
`ACh. It was also found that RA7 showed a greater
`inhibition in vitro, in solubilized preparations of
`AChE, prepared from the cortex and hippocampus,
`than from the striatum (Weinstock, Kay, Razin and
`Enz, 1987). This indicates that there may also be
`differences in the sensitivity of the enzymes to the
`antiChE agents in various regions of the brain.
`It was
`therefore most relevant
`to determine
`whether there was a direct relationship between the
`
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`ESTHER ELMALEM et a/.
`
`magnitude of the desired pharmacological effect, i.e.
`antagonism of the respiratory effect of morphine and
`the degree of inhibition of AChE in the area of the
`respiratory centre. A highly significant correlation
`was found for all 4 drugs between the prevention of
`the rise in paC02 by morphine and inhibition of
`AChE in the medulla. Moreover, it was found that
`the enzyme had to be inhibited by more than 40% in
`order to achieve any significant antagonism of the
`effects of morphine and that complete suppression of
`the respiratory depression by the opiate could be
`obtained at 60% inhibition.
`The findings in this study show that the novel
`carbamates may prove to be effective in combating
`somnolence and respiratory depression in human
`subjects receiving opiates, without causing any im·
`pairment of the analgesic activity. In view of their
`lower toxicity and smaller potential than physostig(cid:173)
`mine for causing inhibition of AChE in the heart and
`skeletal muscle, their use for the above purpose may
`also be relatively free of peripheral side effects.
`
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