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Enhancement of pentobarbital effect by continuous administration of morphine in the mouse
Pergamoa Prase
Life Sciences Vol . 19, pp .357-366, 1976 . Printed is the U .S .A .
ENHANCEMENT OF PENTOBARBITAL EFFECT BY CONTINUOUS ADMINISTRATION OF MORPHINE IN THE MOUSE I . K . Ho, I . Yamamoto, K . E . Becker, H . H . Loh and E . L . Way Departments of Pharniacology and Toxicology and Anesthesiology The University of Mississippi Medical Center Jackson, Mississippi 39216, U .S .A . Daiichi College of Pharmaceutical Sciences 93 Takamiya, Tamagawa-Cho, Fukuoka, Japan Department of Pharmacology University of California, San Francisco San Francisco, California 94143, U .S .A . (Received is final form Jtma 7, 1976) These studies demonstrated that continuous morphine treatment from implantation of a 75 mg morphine pellet for 3 days potentiated pentobarbital narcosis and enhanced pentobarbital hypothermia . In the morphine implant mice, sleeping time after two different doses of pentobarbital was greater than 2 .5 x the sleeping time in placebo pellet implant animals and also greater than sleeping time in animals treated acutely with morphine prior to pentobarbital . Moreover, in the morphine implant mice both the degree and duration of pentobarbital induced hypothermia were enhanced . The above findings were due to slower rate of metabolism of pentobarbital as evidenced by inhibition of hepatic N-demethylation, and higher levels of brain and rerun pentobarbital in the morphine implant mice compared to both placebo and acute morphine mice . Many drugs are known to affect the responses to barbiturates 1n various species (1-9) . In the rat, chronic administration of morphine depresses microsomal metabolism of many drugs (10-16), but only in sexually mature males (16 20) . Recently we demonstrated that morphine can inhibit microsomal N-danethylation of ethylmorphine in both male and female mice (21) . Since morphine and pentobarbital are often abused in combination, we have studied the effect of chronic morphine administration on pentobarbital-induced narcosis and hypothermia, and possible biochemical mechanisms underlying this effect . Experimental Procedures Animals . Male ICR mice (Simonsen Labs ., rendered two erant to and physically dependent plantation of a specially formulated morphine mice were implanted with a placebo pellet for
Gilroy, CA) weighing 27-30 g were on morphine by the subcutaneous impellet for 72 hours (22) . Control the same period of time .
Effects of chronic morphine administration on pentobarbital narcosis . 357
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The sleeping time of each group of mice was determined on the fourth day of pellet implantation . Sodium pentobarbital, both 60 and 75 mg/kg, i .p ., was given to 12 to 18 mice in each group. The duration of sleeping time was taken as the time from the loss of righting reflex to its return . The acute effect of morphine on pentobarbital sleeping time in the placebo implanted group was studied with a group of animals receiving morphine sulfate 10 mg/kg, s .c ., 30 min prior to the administration of Na-pentobarbital, 60 mg/kg, i .p . Effects of chronic morphine administration on pentobarbital-induced hypothermia . Pentobarbital induced hypôthérmia in morphine implant and placebo imp a~ nt mice was studied on the fourth day of pellet implantation . Rectal temperature was measured at different time intervals after the administration of Napentobarbital, 60 mg/kg, i .p ., in 10 mice in each group . The ambient temperature was 23 .2 t 0 .2oC . A Model 581C Digital Thermometer from United systems Corporation was used in this study. Effects of chronic administration of mor hive on liver wei ht liver rotein and he at c m crosoma meta sm o et mor ne . ice were mP ante w t e t er morphine or p acebo pe let for two ays as above . After the body weight of each animal was measured, it was sacrificed by decapitation . Livers were quickly removed and washed with ice-cold 1.15% KC1 . The microsomal fractions were prepared and the activity of hepatic drug metabolizing enzymes in microsomal fractions were determined by the N-demethylation method using ethylmorphine as substrate (23) . The microsomal protein was determined by the method of Lowry et al . (24) . Effects of morphine pellet implantation on the pentobarbital level in both serum and brain 1 . Pentobarbital - 14C Assa . In animals receiving either morphine or placebo péllet imp antat on, Na-pentobarbital, 60 mg/kg, containing pentobarbital-14C (50 uCi/kg) was injected intraperitoneally . The mice were decapitated at 5, 15, 30 and 60 min . Blood and brains were collected for assay of pentobarbital by the method of Kuntzman et al . (25) . Brains were homogenized in 2 ml of 2 M acetate buffer (pH 5 .0) . f~e~omogenate was further diluted with 1 .15% KC1 to provide a 10% homogenate . Pentobarbital was extracted with petroleum ether containing 1 .5% isoa~l alcohol . One-half ml of serum was diluted with 2 .0 ml of ~~etate buffer and 2 .5 ml of 1 .15% KC1 and extracted as above. PentobarbitalC assays were performed in a Beckman LS-150 liquid scintillation counter.
é6
2 . Pentobarbital as chromato ra assay. In animals receiving either morphine or p act pe e~ an~~a-pentobarbital, 60 mg/kg, was injected intraperitoneally . A third group of placebo animals was injected with morphine sulfate, 10 mg/kg, s .c ., 30 min prior to the administration of pentobarbital . Mice were decapitated at 30 and 60 minutes . Brains were removed and weighed, and blood from animals in each group was pooled and serum separated . The brains were homogenized in 3 ml of 0.1 N HC1 . Twenty ug of Na-secobarbital in 0.2 cc 2N HC1 was added to each sample of brain or serum prior to extraction with 5 ml of nanograde toluene . The barbiturates were then reextracted into trimethylpher~ylammonium hydroxide in methanol after the method of Kananen et al . (26) . Recovery by this method was 98% or better . A Tracor gas chromatograprl-with dual flame ionization detectors and a 1 .83 M 2 mm i .d . glass column containing 3% 5E-30 on chromasorb W, AW, HP, DMSC was used . The concentration of pentobarbital was determined by comparing the peak height ratio of pentobarbital/secobarbital with standard curves . Results Effects of chronic morphine administration on pentobarbital narcosis . The continuous administration of morphine by 3 days of pellet implantation potentiat-
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Pentobarbital and Continuous Morphine
35 9
ed the effects of pentobarbital on sleeping time . As shown in Fig . 1, mice receiving a morphine pellet implant for 72 hpurs exhibited a sleeping time after In placebo Na-pentobarbital several-fold longer than that of the control group . implant mice, the sleeping time after a single challenge dose of 60 mg/kg, i .p ., of Na-pentobarbital was 113 .4 t 23 .4 min . In morphine implant mice, the sleeping time after the same Na-pentobarbital challenge dose was increased 2 .5-fold to 288 .8 f 32 .3 min . In mice receiving a higher challenge dose of Na-pentobarbital 75 mg/kg, i .p ., the sleeping times were 185 .4 t 32 .2 min for placebo mice and 433 .7 t 47 min for morphine . 500
400
300
ioo
eo maiko
rs mai~
PeMobarbNal Soä~m,i .p . Figure 1 Effects of continuous morphine administration on pentobarbital sleeping time . The mice were implanted with either morphine (solid lines) or placebo pellet (broken lines) for 3 days . The sleeping time of a mouse after a single dose of Na-pentobarbital 60 or 75 mg/kg, i .p ., was taken as the time between the loss of righting reflex of the mouse and its return . The bracketed vertical lines represent S .E . The nunber of mice for each group was 12-14 . In order to eliminate the possibility that the enhancement of pentobarbital sleeping time by continuous morphine treatment might be due to depression produced from both drugs, a group of placebo-implanted mice was administered a dose of morphine sulfate, 10 mg/kg, s .c ., 30 min prior to a challenge dose of Na-pentobarbital, 60 mg/kg, i .p . The brain level of morphine 30 min after a single dose of
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morphine sulfate 10 mg/kg, s .c ., has béen shown in our laboratory to be comparable to that obtained 72 hours after morphine pellet implantation . In mice receiving an acute injection of morphine sulfate, the sleeping time after a single challenge dose of Na-pentobarbital was 192 .1 t 37 .0 min . This was significantly higher than the placebo group receiving only pentobarbital (P < 0 .05), and significantly lower than the sleeping time in mice receiving 3 days of morphine pellet implantation (P < 0 .05) . Effects of chroni~ c morp~hine administration on entobarbital-induced hypotherm~hine pe el t imp antat on en ance t t e egree an uration of pentobarbital-induced hypothermia . As shown in Fig . 2, pentobarbital, 60 mg/kg, i .p ., caused a sharp decrease (7 .2oC in 1 hour) in the rectal temperature of placebo control mice . The body t~nperature gradually returned to normal within 3 hours . In mice receiving a morphine pellet implant for 3 days, the rectal temperature was 37 .0 t 0 .3°C which was significantly lower than that of the control, 38 .4 t 0 .3oC (P < 0 .005) . When the morphine implant mice received the same challenge dose of pentobarbital, the same degree of hypothermia occurred at one hour (7 .2 °C) . However, the duration of hypothermia in the morphine pellet im-
Figure 2 Effects of continuous morphine administration on pentobarbital induced hypothermia . The mice were implanted with either morphine (solid lines) or placebo pellet (broken lines) for 3 days . The rectal temperature of each animal was taken at various time intervals after the administration of Na-pentobarbital 60 mg/kg, i .p . The bracketed vertical lines represent S .E . Ten mice were used in each group .
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planted group was much longer than the placebo control group . At 2, 3 and 4 hours after the administration of Na-pentobarbital, the rectal temperature of morphine implant animals was still significantly lower than that of control animals . The inhibition of hepatic microsomal metabolism of ethylmorphine after morp~hine e_ et m antat on . ~ e continuous a n stration o morphine decreased the weight o iver, re uced the microsomal protein content in the liver and significantly inhibited the hepatic microsomal metabolism of ethylmorphine in the mouse . As shown in Fig . 3, in mice receivin a morphine pellet implant for 3 days, the liver weight was decreased to 82% ~P < 0 .02) of that of the placebo implant group ; however, the body weights of both groups were not significantly different . Morphine pellet implantation also caused a 17% decrease in microsomal protein content of the liver . In morphine pellet implant mice the hepatic microsomal N-demethylation of ethylmorphlne was inhibited 36% (6 .43 t 0 .07 }uncles HCHO formed per gram liver per hour) co ared to the placebo group (8 .22 t 0 .61 umoles HCHO formed per gram liver per hour .
PiACEBO PELLET
. , : aoa .. v ~ oce
MOAPHIrE PELLET
30
Body ~d
Liver rYYiyM
N-D~Ilylolbn
üïaaanol praleYr
Figure 3 Effects of chronic morphine administration on body and liver weights, hepatic microsomal protein contents and N-demethylase activity . One group of mice were implanted with morphine pellets for 3 days . The control group received placebo pellet . Ethylmorphine was used as the substrate to determine N-demethylase activity . Microsomal roteins were determined according to the method of Lowry et a1 . (24~ . Six mice were used in each group .
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Effects of mor hine ellet im lantation on the entobarbital levels in brain and serum . ont nuous a n strat ono morp ine y pe et mp antat on ncrease~~ain and serum levels of pentobarbital after a single dose of this drug . As shown in~Fig . 4, after the administration of 60 mg/kg, i .p ., Na-pentobarbital, the brain levels of pentobarbital in the morphine pellet implanted group at 15, 30 and 60 min were 15, 42 and 56% higher in the brain and 50, 23 and 20% higher in the serum compared to placebo implant mice .
Mo% Mice.
60 P 50
~ ~ ~~ _ _ P~bersbio
40
30 60
40
30
I 5
I 15
I 30 Time (mlrutee)
i 60
Figure 4 Effects of continuous morphine administration and serum levels of pentobarbital . Three days after the implantation of either morphine or placebo pellet, the animals received sodium pentobarbital 60 mg/kg, containing C 14-pentobarbital with specific activity of 50 uCi/kg . Serum and brain levels of pentobarbital were determined according to the method of Kuntzman et al . (25) at different time inBracketed lines indicattérvals after the administration of thedrug . ed S .E . The number of animals per point was 5-6 . Shown in Table 1 are additional results obtained by gas chromatography . Serum pentobarbital levels at 30 and at 60 min were essentially the same in the placebo and placebo plus acute administration of morphine groups . Serum levels in the group administered morphine continuously were 26% higher at 30 min and 46% higher at 60 minutes compared to the placebo group . Brain levels of pentobarbital were similar at 30 min in the placebo and the placebo plus acute administration of morphine groups . At 60 min, brain pentobarbital levels in the
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Pentobarbital and Continuous Morphine
36 3
group administered morphine acutely were significantly higher than the placebo group (P < 0 .026) . However, in the group administered morphine continuously, brain pentobarbital levels were markedly higher at both 30 and 60 min when compared to the placebo group (P < .00001, P < .000001) and to the placebo plus acute administration of the morphine group (P < .0001, P < .004) . Thus, brain levels of pentobarbital in the chronically morphine-treated group remained at much higher levels for much longer periods of time . There was an increase in the ratio of brain/serum levels in the chronic morphine vs placebo groups . But little or no increase in the brain/serum level in the acute morphine group vs placebo group . Table 1 Brain and serum levels of pentobarbital G roup
Pooled 30 mi n
serum (ug/ml) Brain (yg/gm) * 6 0 min 30 min 60 min
Brain/plasma ratio 30 min 60 min
Placebo
22 .5
21 .5
29 .511 .1
28 .310 .8
1 .3
1 .3
Acute morphine
22 .6
24 .0
29 .411 .5
34 .412 .7
1 .3
1 .4
Morphine pellet
28 .4
31 .5
50 .110 .6
48 .212 .0
1 .8
1 .5
*Mean t standard error . Discussion The present studies demonstrate that continuous treatment with morphine increased pentobarbital-induced narcosis and hypothermia . These studies also showed that an increase in pentobarbital response was related to inhibition of N-de methylase activity in the liver which was used as an indirect measurement of hepatic drug metabolizing enzyme activity . The increased brain uptake of pentobarbital in morphine treated animals may also contribute to the enhancement of pentobarbital response . The continuous administration of morphine by three days of pellet implantation potentiated the effect of pentobarbital on sleeping time with two different dosages of sodium pentobarbital . This could possibly be 2o to existing depres sion from residual morphine . However, we have repeatedly shown in our laboratory that the brain levels of morphine after 72 hours of pellet implantation are comparable to the brain levels of morphine 30 min after a single morphine dose of 10 mg/kg subcutaneously . At these similar brain morphine levels, the enhancement of pentobarbital sleeping time from morphine pellet implantation was approximately 1 .5 x higher than that from acute morphine treatment . Therefore, the prolongation in pentobarbital sleeping time from continuous morphine administration was not solely due to depression from residual morphine . The hypothermia induced by pentobarbital administration also supported these findings . In morphine implant mice, the hypothermia induced by administration of Na-pentobarbital was greater and more prolonged compared to hypothermia on control mice . It has been reported that morphine exhibits a sex-dependent action on microsamal drug metabolism in the rat, but not the other species, by impairing androgen-induced stimulation of the hepatic drug metabolizing enzyme system (20) . Our recent studies have shown that continuous administration of morphine by pellet Implantation inhibits drug microsomal activity in both males and females (21) . The present study confirms our previous study (21) and shows that continuous administration of morphine decreases the hepatic N-demethylase activity,
364
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liver weight and microsomal protein . The present study demonstrates markedly increased brain and serum levels and brain/serum ratios of pentobarbital after continuous but not after single acute administration of morphine . These findings may result from the inhibition of he patic drug metabolizing enzyme activity . However, the possibility of an increase in pentobarbital uptake into the brain cannot be excluded since the brain pentobarbital levels in morphine-treated animals remain at much higher levels for a longer period of time and since the ratio of brain/serum pentobarbital is elevated in the morphine-treated mice . Finally, with the 14 C assay (Fig . 1), brain pentobarbital levels in the morphine implant group were constant at 30 and 60 min, while serum levels fell . This probably represents assay of b~~h pentobarbital and metabolites of pentobarbital in serum and brain since the C assay does not completely separate the two . The GC assay measures only intact pentobarbital . With this assay, brain and serum levels of ntobarbital at 30 vs 60 min were similar and serum levels lower than with the ~~C assay, thus supporting the argument that the 14 C assay is including metabolites of pentobarbital . The present studies may have clinical implication . Barbiturates, for exSince chronic morphine ample, are often abused in combination with narcotics . and heroin administration are effective microsomal enzyme inhibitors, if the sub ject took narcotics and barbiturates together, the toxic effects of both drugs would be additive and the duration of action of the barbiturates prolonged, thus leading to increased toxicity . Acknowledgments I . K. These studies were supported by NIDA Grants DA-01310 and DA-01403 . Ho is a recipient of a Faculty Development Award in Pharmacology from the Pharmaceutical Manufacturers' Association Foundation and H . H . Loh is a reci pient of a NIMH Research Scientist Development Award . References , W . C . Lubawy and H . B . Kostenbauder . 1 . S . A . Stavchans 1539 (1974 . 2 . I . H . Stevenson and M . J . Turnbull .
Life Sçi . _14, 1535-
Brit . J . Pharmacol . 50, 499-511 (1974) .
3 . R . E . Johnston, R . C . Schnell and T . S . Miya . 90-95 (1974) . 4 . G . Depasquale, P . Welaj and C . L . Rassaert . Pharniacol . 9, 253-263 (1974) .
Toxicol . Appl . Pharmacol . 30, Res . Comm . Chem . Patho l
5 . A . J . Siemens, H . Kalant, J . M . Khanna, J . Marshman and G . Ho . Pharmacol . 23, 477-488 (1974) . 6 . G . B . Chesher, D . M . Jackson and G . A . Starmer . 593-599 (1974) .
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7 . E . Buchar, K . Masek, I . Janku, J . Seifert and R . U . Ostrovskaya . Neurochem . 23, 447-449 (1974) . 8 . G . J . Patel, R . P . Schatz, S . M . Constantinides and H . Lal . Pharmacol . 24, 57-60 (1975) . 9.
I . K . Ho .
Proc . West . Pharmacol . Soc .
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18, 260-264 (1975) .
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Biochem .
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Pantqbarbital aad Coatinuovs Morphine
10 . J . Axel rod .
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11 . G . J . Mannering and A. E . Takemore . (1959) . 12 . J . Cochin and J . Axelrod. 13 . D. H. Clouet .
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144,
125,
187-190
105-110 (1959) .
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14 . D. H. Clouet and R. Ratner .
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15 . R. Kato and J . R. Gillette .
J . Pharmacol . Exp. Ther . 150, 285-291 (1965) .
16 . R. Kato and K. I . Onoda and A. Takenaka . (1971) . 17 . H . Remmer and B. Alsleben .
Biochem . Pharmacol . 20, 1093-1099
Klin . Woschr . 36, 332-333 (1958) .
18 . R . Kato, K. Onoda and M. Sasajima .
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20 . N . E . Sladek, M. D . Chaplin and G. J . Mannering . Disposition 2, 293-300 (1974) .
Drug Metabolism _and
21 .
I . K. Ho, I . Yamamoto, H . H . Loh and E . L . Way. 358 (1976) .
22 . E . L . Way,
H. H . Loh and F. H. Shen .
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23 . G . J . Mannerin . I n "Selected Pharmacological Testing Methods", ed . A . Berger, ~Marcél Dekker, New York), p . 51-119 (1968) . 24 . 0 . H . Lowry, N . J . Rosebrough, A . L. 193, 265-275 (1951) .
Farr and R. J . Randall .
25 . R . M. Kuntzman, M. Ikeda, M. Jacobson and A. H . Conney . Ther . 157, 220-226 (1967) . 26 . G . Kananen, R. Osiewicz and I . Sunshine . 287 (1972) .
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Life Sciences Vol . 19, pp .357-366, 1976 . Printed is the U .S .A .
ENHANCEMENT OF PENTOBARBITAL EFFECT BY CONTINUOUS ADMINISTRATION OF MORPHINE IN THE MOUSE I . K . Ho, I . Yamamoto, K . E . Becker, H . H . Loh and E . L . Way Departments of Pharniacology and Toxicology and Anesthesiology The University of Mississippi Medical Center Jackson, Mississippi 39216, U .S .A . Daiichi College of Pharmaceutical Sciences 93 Takamiya, Tamagawa-Cho, Fukuoka, Japan Department of Pharmacology University of California, San Francisco San Francisco, California 94143, U .S .A . (Received is final form Jtma 7, 1976) These studies demonstrated that continuous morphine treatment from implantation of a 75 mg morphine pellet for 3 days potentiated pentobarbital narcosis and enhanced pentobarbital hypothermia . In the morphine implant mice, sleeping time after two different doses of pentobarbital was greater than 2 .5 x the sleeping time in placebo pellet implant animals and also greater than sleeping time in animals treated acutely with morphine prior to pentobarbital . Moreover, in the morphine implant mice both the degree and duration of pentobarbital induced hypothermia were enhanced . The above findings were due to slower rate of metabolism of pentobarbital as evidenced by inhibition of hepatic N-demethylation, and higher levels of brain and rerun pentobarbital in the morphine implant mice compared to both placebo and acute morphine mice . Many drugs are known to affect the responses to barbiturates 1n various species (1-9) . In the rat, chronic administration of morphine depresses microsomal metabolism of many drugs (10-16), but only in sexually mature males (16 20) . Recently we demonstrated that morphine can inhibit microsomal N-danethylation of ethylmorphine in both male and female mice (21) . Since morphine and pentobarbital are often abused in combination, we have studied the effect of chronic morphine administration on pentobarbital-induced narcosis and hypothermia, and possible biochemical mechanisms underlying this effect . Experimental Procedures Animals . Male ICR mice (Simonsen Labs ., rendered two erant to and physically dependent plantation of a specially formulated morphine mice were implanted with a placebo pellet for
Gilroy, CA) weighing 27-30 g were on morphine by the subcutaneous impellet for 72 hours (22) . Control the same period of time .
Effects of chronic morphine administration on pentobarbital narcosis . 357
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The sleeping time of each group of mice was determined on the fourth day of pellet implantation . Sodium pentobarbital, both 60 and 75 mg/kg, i .p ., was given to 12 to 18 mice in each group. The duration of sleeping time was taken as the time from the loss of righting reflex to its return . The acute effect of morphine on pentobarbital sleeping time in the placebo implanted group was studied with a group of animals receiving morphine sulfate 10 mg/kg, s .c ., 30 min prior to the administration of Na-pentobarbital, 60 mg/kg, i .p . Effects of chronic morphine administration on pentobarbital-induced hypothermia . Pentobarbital induced hypôthérmia in morphine implant and placebo imp a~ nt mice was studied on the fourth day of pellet implantation . Rectal temperature was measured at different time intervals after the administration of Napentobarbital, 60 mg/kg, i .p ., in 10 mice in each group . The ambient temperature was 23 .2 t 0 .2oC . A Model 581C Digital Thermometer from United systems Corporation was used in this study. Effects of chronic administration of mor hive on liver wei ht liver rotein and he at c m crosoma meta sm o et mor ne . ice were mP ante w t e t er morphine or p acebo pe let for two ays as above . After the body weight of each animal was measured, it was sacrificed by decapitation . Livers were quickly removed and washed with ice-cold 1.15% KC1 . The microsomal fractions were prepared and the activity of hepatic drug metabolizing enzymes in microsomal fractions were determined by the N-demethylation method using ethylmorphine as substrate (23) . The microsomal protein was determined by the method of Lowry et al . (24) . Effects of morphine pellet implantation on the pentobarbital level in both serum and brain 1 . Pentobarbital - 14C Assa . In animals receiving either morphine or placebo péllet imp antat on, Na-pentobarbital, 60 mg/kg, containing pentobarbital-14C (50 uCi/kg) was injected intraperitoneally . The mice were decapitated at 5, 15, 30 and 60 min . Blood and brains were collected for assay of pentobarbital by the method of Kuntzman et al . (25) . Brains were homogenized in 2 ml of 2 M acetate buffer (pH 5 .0) . f~e~omogenate was further diluted with 1 .15% KC1 to provide a 10% homogenate . Pentobarbital was extracted with petroleum ether containing 1 .5% isoa~l alcohol . One-half ml of serum was diluted with 2 .0 ml of ~~etate buffer and 2 .5 ml of 1 .15% KC1 and extracted as above. PentobarbitalC assays were performed in a Beckman LS-150 liquid scintillation counter.
é6
2 . Pentobarbital as chromato ra assay. In animals receiving either morphine or p act pe e~ an~~a-pentobarbital, 60 mg/kg, was injected intraperitoneally . A third group of placebo animals was injected with morphine sulfate, 10 mg/kg, s .c ., 30 min prior to the administration of pentobarbital . Mice were decapitated at 30 and 60 minutes . Brains were removed and weighed, and blood from animals in each group was pooled and serum separated . The brains were homogenized in 3 ml of 0.1 N HC1 . Twenty ug of Na-secobarbital in 0.2 cc 2N HC1 was added to each sample of brain or serum prior to extraction with 5 ml of nanograde toluene . The barbiturates were then reextracted into trimethylpher~ylammonium hydroxide in methanol after the method of Kananen et al . (26) . Recovery by this method was 98% or better . A Tracor gas chromatograprl-with dual flame ionization detectors and a 1 .83 M 2 mm i .d . glass column containing 3% 5E-30 on chromasorb W, AW, HP, DMSC was used . The concentration of pentobarbital was determined by comparing the peak height ratio of pentobarbital/secobarbital with standard curves . Results Effects of chronic morphine administration on pentobarbital narcosis . The continuous administration of morphine by 3 days of pellet implantation potentiat-
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Pentobarbital and Continuous Morphine
35 9
ed the effects of pentobarbital on sleeping time . As shown in Fig . 1, mice receiving a morphine pellet implant for 72 hpurs exhibited a sleeping time after In placebo Na-pentobarbital several-fold longer than that of the control group . implant mice, the sleeping time after a single challenge dose of 60 mg/kg, i .p ., of Na-pentobarbital was 113 .4 t 23 .4 min . In morphine implant mice, the sleeping time after the same Na-pentobarbital challenge dose was increased 2 .5-fold to 288 .8 f 32 .3 min . In mice receiving a higher challenge dose of Na-pentobarbital 75 mg/kg, i .p ., the sleeping times were 185 .4 t 32 .2 min for placebo mice and 433 .7 t 47 min for morphine . 500
400
300
ioo
eo maiko
rs mai~
PeMobarbNal Soä~m,i .p . Figure 1 Effects of continuous morphine administration on pentobarbital sleeping time . The mice were implanted with either morphine (solid lines) or placebo pellet (broken lines) for 3 days . The sleeping time of a mouse after a single dose of Na-pentobarbital 60 or 75 mg/kg, i .p ., was taken as the time between the loss of righting reflex of the mouse and its return . The bracketed vertical lines represent S .E . The nunber of mice for each group was 12-14 . In order to eliminate the possibility that the enhancement of pentobarbital sleeping time by continuous morphine treatment might be due to depression produced from both drugs, a group of placebo-implanted mice was administered a dose of morphine sulfate, 10 mg/kg, s .c ., 30 min prior to a challenge dose of Na-pentobarbital, 60 mg/kg, i .p . The brain level of morphine 30 min after a single dose of
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morphine sulfate 10 mg/kg, s .c ., has béen shown in our laboratory to be comparable to that obtained 72 hours after morphine pellet implantation . In mice receiving an acute injection of morphine sulfate, the sleeping time after a single challenge dose of Na-pentobarbital was 192 .1 t 37 .0 min . This was significantly higher than the placebo group receiving only pentobarbital (P < 0 .05), and significantly lower than the sleeping time in mice receiving 3 days of morphine pellet implantation (P < 0 .05) . Effects of chroni~ c morp~hine administration on entobarbital-induced hypotherm~hine pe el t imp antat on en ance t t e egree an uration of pentobarbital-induced hypothermia . As shown in Fig . 2, pentobarbital, 60 mg/kg, i .p ., caused a sharp decrease (7 .2oC in 1 hour) in the rectal temperature of placebo control mice . The body t~nperature gradually returned to normal within 3 hours . In mice receiving a morphine pellet implant for 3 days, the rectal temperature was 37 .0 t 0 .3°C which was significantly lower than that of the control, 38 .4 t 0 .3oC (P < 0 .005) . When the morphine implant mice received the same challenge dose of pentobarbital, the same degree of hypothermia occurred at one hour (7 .2 °C) . However, the duration of hypothermia in the morphine pellet im-
Figure 2 Effects of continuous morphine administration on pentobarbital induced hypothermia . The mice were implanted with either morphine (solid lines) or placebo pellet (broken lines) for 3 days . The rectal temperature of each animal was taken at various time intervals after the administration of Na-pentobarbital 60 mg/kg, i .p . The bracketed vertical lines represent S .E . Ten mice were used in each group .
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Pentobarbital aad Coatinuous Morphine
361
planted group was much longer than the placebo control group . At 2, 3 and 4 hours after the administration of Na-pentobarbital, the rectal temperature of morphine implant animals was still significantly lower than that of control animals . The inhibition of hepatic microsomal metabolism of ethylmorphine after morp~hine e_ et m antat on . ~ e continuous a n stration o morphine decreased the weight o iver, re uced the microsomal protein content in the liver and significantly inhibited the hepatic microsomal metabolism of ethylmorphine in the mouse . As shown in Fig . 3, in mice receivin a morphine pellet implant for 3 days, the liver weight was decreased to 82% ~P < 0 .02) of that of the placebo implant group ; however, the body weights of both groups were not significantly different . Morphine pellet implantation also caused a 17% decrease in microsomal protein content of the liver . In morphine pellet implant mice the hepatic microsomal N-demethylation of ethylmorphlne was inhibited 36% (6 .43 t 0 .07 }uncles HCHO formed per gram liver per hour) co ared to the placebo group (8 .22 t 0 .61 umoles HCHO formed per gram liver per hour .
PiACEBO PELLET
. , : aoa .. v ~ oce
MOAPHIrE PELLET
30
Body ~d
Liver rYYiyM
N-D~Ilylolbn
üïaaanol praleYr
Figure 3 Effects of chronic morphine administration on body and liver weights, hepatic microsomal protein contents and N-demethylase activity . One group of mice were implanted with morphine pellets for 3 days . The control group received placebo pellet . Ethylmorphine was used as the substrate to determine N-demethylase activity . Microsomal roteins were determined according to the method of Lowry et a1 . (24~ . Six mice were used in each group .
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Pentobarbital and Continuous Morphine
Effects of mor hine ellet im lantation on the entobarbital levels in brain and serum . ont nuous a n strat ono morp ine y pe et mp antat on ncrease~~ain and serum levels of pentobarbital after a single dose of this drug . As shown in~Fig . 4, after the administration of 60 mg/kg, i .p ., Na-pentobarbital, the brain levels of pentobarbital in the morphine pellet implanted group at 15, 30 and 60 min were 15, 42 and 56% higher in the brain and 50, 23 and 20% higher in the serum compared to placebo implant mice .
Mo% Mice.
60 P 50
~ ~ ~~ _ _ P~bersbio
40
30 60
40
30
I 5
I 15
I 30 Time (mlrutee)
i 60
Figure 4 Effects of continuous morphine administration and serum levels of pentobarbital . Three days after the implantation of either morphine or placebo pellet, the animals received sodium pentobarbital 60 mg/kg, containing C 14-pentobarbital with specific activity of 50 uCi/kg . Serum and brain levels of pentobarbital were determined according to the method of Kuntzman et al . (25) at different time inBracketed lines indicattérvals after the administration of thedrug . ed S .E . The number of animals per point was 5-6 . Shown in Table 1 are additional results obtained by gas chromatography . Serum pentobarbital levels at 30 and at 60 min were essentially the same in the placebo and placebo plus acute administration of morphine groups . Serum levels in the group administered morphine continuously were 26% higher at 30 min and 46% higher at 60 minutes compared to the placebo group . Brain levels of pentobarbital were similar at 30 min in the placebo and the placebo plus acute administration of morphine groups . At 60 min, brain pentobarbital levels in the
Vol . 19, No . 3
Pentobarbital and Continuous Morphine
36 3
group administered morphine acutely were significantly higher than the placebo group (P < 0 .026) . However, in the group administered morphine continuously, brain pentobarbital levels were markedly higher at both 30 and 60 min when compared to the placebo group (P < .00001, P < .000001) and to the placebo plus acute administration of the morphine group (P < .0001, P < .004) . Thus, brain levels of pentobarbital in the chronically morphine-treated group remained at much higher levels for much longer periods of time . There was an increase in the ratio of brain/serum levels in the chronic morphine vs placebo groups . But little or no increase in the brain/serum level in the acute morphine group vs placebo group . Table 1 Brain and serum levels of pentobarbital G roup
Pooled 30 mi n
serum (ug/ml) Brain (yg/gm) * 6 0 min 30 min 60 min
Brain/plasma ratio 30 min 60 min
Placebo
22 .5
21 .5
29 .511 .1
28 .310 .8
1 .3
1 .3
Acute morphine
22 .6
24 .0
29 .411 .5
34 .412 .7
1 .3
1 .4
Morphine pellet
28 .4
31 .5
50 .110 .6
48 .212 .0
1 .8
1 .5
*Mean t standard error . Discussion The present studies demonstrate that continuous treatment with morphine increased pentobarbital-induced narcosis and hypothermia . These studies also showed that an increase in pentobarbital response was related to inhibition of N-de methylase activity in the liver which was used as an indirect measurement of hepatic drug metabolizing enzyme activity . The increased brain uptake of pentobarbital in morphine treated animals may also contribute to the enhancement of pentobarbital response . The continuous administration of morphine by three days of pellet implantation potentiated the effect of pentobarbital on sleeping time with two different dosages of sodium pentobarbital . This could possibly be 2o to existing depres sion from residual morphine . However, we have repeatedly shown in our laboratory that the brain levels of morphine after 72 hours of pellet implantation are comparable to the brain levels of morphine 30 min after a single morphine dose of 10 mg/kg subcutaneously . At these similar brain morphine levels, the enhancement of pentobarbital sleeping time from morphine pellet implantation was approximately 1 .5 x higher than that from acute morphine treatment . Therefore, the prolongation in pentobarbital sleeping time from continuous morphine administration was not solely due to depression from residual morphine . The hypothermia induced by pentobarbital administration also supported these findings . In morphine implant mice, the hypothermia induced by administration of Na-pentobarbital was greater and more prolonged compared to hypothermia on control mice . It has been reported that morphine exhibits a sex-dependent action on microsamal drug metabolism in the rat, but not the other species, by impairing androgen-induced stimulation of the hepatic drug metabolizing enzyme system (20) . Our recent studies have shown that continuous administration of morphine by pellet Implantation inhibits drug microsomal activity in both males and females (21) . The present study confirms our previous study (21) and shows that continuous administration of morphine decreases the hepatic N-demethylase activity,
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Pentobarbital and Continuous Morphiae
liver weight and microsomal protein . The present study demonstrates markedly increased brain and serum levels and brain/serum ratios of pentobarbital after continuous but not after single acute administration of morphine . These findings may result from the inhibition of he patic drug metabolizing enzyme activity . However, the possibility of an increase in pentobarbital uptake into the brain cannot be excluded since the brain pentobarbital levels in morphine-treated animals remain at much higher levels for a longer period of time and since the ratio of brain/serum pentobarbital is elevated in the morphine-treated mice . Finally, with the 14 C assay (Fig . 1), brain pentobarbital levels in the morphine implant group were constant at 30 and 60 min, while serum levels fell . This probably represents assay of b~~h pentobarbital and metabolites of pentobarbital in serum and brain since the C assay does not completely separate the two . The GC assay measures only intact pentobarbital . With this assay, brain and serum levels of ntobarbital at 30 vs 60 min were similar and serum levels lower than with the ~~C assay, thus supporting the argument that the 14 C assay is including metabolites of pentobarbital . The present studies may have clinical implication . Barbiturates, for exSince chronic morphine ample, are often abused in combination with narcotics . and heroin administration are effective microsomal enzyme inhibitors, if the sub ject took narcotics and barbiturates together, the toxic effects of both drugs would be additive and the duration of action of the barbiturates prolonged, thus leading to increased toxicity . Acknowledgments I . K. These studies were supported by NIDA Grants DA-01310 and DA-01403 . Ho is a recipient of a Faculty Development Award in Pharmacology from the Pharmaceutical Manufacturers' Association Foundation and H . H . Loh is a reci pient of a NIMH Research Scientist Development Award . References , W . C . Lubawy and H . B . Kostenbauder . 1 . S . A . Stavchans 1539 (1974 . 2 . I . H . Stevenson and M . J . Turnbull .
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