- Email: [email protected]
Modulation of metabolic effects of morphine-6-glucuronide by morphine-3-glucuronide
Brain ResearchBulletin,Vol. 38, No. 4, pp. 325-329, 1995 Copyright~' 1995ElsevierScienceInc. Printedin the USA.All rightsreserved 0361-9230/95 $9.50 + .00
Pergamon 0361-9230(95)00104-2
Modulation of Metabolic Effects of Morphine-6Glucuronide by Morphine-3-Glucuronide YOJIRO HASHIGUCHI,*I- PATRICIA E. MOLINA*-~1 AND NAJI N. ABUMRAD*I-
*Department of Surgery, SUNY at Stony Brook, Stony Brook, NY 11794-8191, USA ?~Departmentof Research, VAMC, Northport, Northport, NY 11768, USA [Received 6 March 1995; Accepted 19 May 1995] and kidney [23]. In humans it is estimated that 55% of morphine is transformed to M3G, whereas a lower (15%) amount is metabolized to morphine-6-glucuronide(M6G) [3]. Both M3G and M6G have been isolated from plasma and CSF after oral, intramuscular, or spinal administration of morphine [7,16,2], suggesting either crossover of the blood-brain barrier or CNS conversion of morphine to its glucuronides in the brain. These metabolites have been also shown to accumulate in plasma in higher levels than those of the parent compound after chronic oral dosing [20] as well as in individuals with impaired renal function [12]. M3G does not bind to opioid receptors [4] and is devoid of analgesic activity [21 ]. Nevertheless, it causes behavioral excitation, hyperalgesia, and respiratory stimulation by nonopioid mechanisms [9,24]. Studies have recently focused on morphine glucuronides because of the increased degree and duration of the effects of morphine with elevated levels of glucuronides reported in patients with renal dysfunction [ 13]. Recent reports in the literature have suggested that M3G has an antagonizing effect on the antinociceptive and respiratory depressant effect of morphine and M6G [22,6,5]. In contrast, M6G binds to receptors with affinities similar to those of morphine [4,14], and its intracerebroventricular (ICV) administration elicits a 13- to 200-fold greater analgesic effect than morphine [1,15]. In addition, recent studies f~'omour laboratory have demonstrated potent hyperglycemic effects of M6G, suggesting important modulatory roles of glucuronidation on morphine's effects on glucose metabolism [1 1]. The purpose of this study was to determine if M3G at a dose that does not alter glucose homeostasis would antagonize or attenuate the metabolic effects of M6G.
ABSTRACT: Modification of pharmacological effects of morphine by its glucuronides has been recently reported. Morphine6-glucuronide (M6G) is a more potent opioid agonist than morphine, whereas morphine-3-glucuronide (M3G) has no opioid effects and has been suggested to be an antagonist of morphine's antinociceptive and respiratory depressive effects. This study addressed the metabolic effects of direct central nervous system administration of M3G and its interaction with the hyperglycemic effects of M6G. Hormonal and whole body glucose metabolic effects of M3G, M6G, and M3G + M6G ICV administration were studied in conscious unrestrained chronically catheterized rats. Whole body glucose kinetics were assessed with a primed constant intravenous infusion of 3[~H]glucose in rats injected intracerebroventricularly (ICV) with H2O (5 pl), M3G (1 pg), M6G (1 pg), or M3G (1/~g) + M6G (1 pg). A significant rise in plasma glucose level was observed after ICV injection of M6G (28%), and M3G + M6G (41%), but not after M3G as compared to time-matched H20 control. Early increases in the rate of glucose appearance (Ra) and whole body glucose utilization (Rd) were observed (58% and 48%, respectively) 30 min after M3G + M6G administration, whereas the increases after M6G injection were progressive and reached values 47% higher than basal 180 min after injection. M3G administration enhanced the M6G induced increase in plasma glucose level (+21%), Ra (+29%), Rd (+26%), and plasma lactate level (+21%). Though no significant hormonal change was observed in H20, M3G, and M6G injected animals, the combination of M3G + M6G resulted in a significant increase in circulating catacholamine levels with no aRerations in plasma corticosterone, insulin, and glucagon. These results indicate that M3G at a dose that does not produce hyperglycemia enhanced the hyperglycemic effects of M6G through accelerated release of catecholamines. These findings suggest that the glucuronidated metabolites of morphine influence each other's biological effects.
MATERIALS AND METHODS KEY WORDS: Morphine, Morphine-3-glucuronide, Morphine-6glucuronide, Glucose, Catecholamines, Hormones, Rats, ICV.
Animal Preparation Male Sprague-Dawley rats (275-350 g, Charles River, Wilmington, MA) were housed in a controlled environment, exposed to a 12:12 h light-dark cycle, and fed standard rat diet (Purina Rat Chow, Ralston Purina, St. Louis, MO) for 1 week before being used in an experiment. Five days before the experiment, animals were anesthetized with an intramuscular injection of ketamine and xylazine (90 rag/ kg and 9 mg/kg, respectively) and positioned in a stereotaxic
INTRODUCTION Morphine-3-glucuronide (M3G) is a major product of morphine glucuronidation in all species. The glucuronidation of morphine occurs not only in the liver but may also take place in the brain
Requests for reprints should be addressed to Patricia E. Molina, Department of Surgery, SUNY at Stony Brook, Stony Brook, NY 117948191, USA. 325
326
apparatus (David Kopf Instruments, Tujunga CA). The overlying skin and connective tissue were cleared from the skull. A hole was drilled in the skull and a 22-gauge stainless steel guide cannula (Plastic Products, Richmond VA) was inserted unilaterally into the lateral ventricle of the brain. The stereotaxic coordinates used for cannula placement were 0.3 mm posterior to bregma, 1.3 mm lateral from midline, and 4.25 mm below the surface of the skull. The cannula was secured using dental cement (Lang's Dental Manufacturing, Chicago IL) and anchored to the skull with one stainless steel screw. A wire dummy cannula was used to seal the guide cannula until the time of the experiment. The animals were provided with food and water ad lib and allowed to recover in individual cages. The day after surgery, cannula placement was assessed by determining whether angiotensin |I (100 ng/rat, Sigma, St. Louis, MO) stimulated dipsogenesis. Proper positioning of the cannula was verified at the end of the experiment by ICV injection of trypan blue. Animals continued their recovery for 3 more days before catheters were implanted in the right jugular vein and left carotid artery. On the day before the experiment, animals were anesthetized with an intramuscular (0.25 ml) injection of ketamine (9 mg/ 100 g) and xylazine (0.9 mg/100 g). Using aseptic surgical procedures, a catheter (PE-50) was implanted in the left carotid artery, and advanced to the arch of the aorta, and another catheter placed in the right jugular vein. After surgery, the animals were returned to individual cages, fasted overnight, and provided water ad lib. Animals were fasted to minimize gut absorption as contributor to glucose appearance. Experiments were started between 0600 and 0800 h the following day, with rats conscious and unrestrained throughout the duration of the protocol.
Glucose Kinetics On the day of the experiment a primed constant intravenous (IV) infusion of [3-3H]glucose (37 Ci/mmol; HPLC purified, #NET-100C, Dupont-NEN, Boston, MA.) was initiated via the venous catheter, and continued throughout the experimental procedure (1 ml/h). Isotopic equilibrium was achieved within 90 min after starting the [3H]glucose infusion; thereafter blood samples were taken at 100 and 120 min for the determination of basal glucose metabolism and after drug treatment throughout the remainder of the experimental period.
HASHIGUCHI, MOLINA AND ABUMRAD
10-
§
§
#*
#*
+
i- t f # -
90 o
(.9 m
#.
+
8+
E
.. 7
E fl)
--
+
÷
--
6-
n
!
0
i 30
I 60
1 90
I 120
I 150
I 180
Time (min)
§ 55--
+
3+,..+ T
50m t~ t/)
O o
r-
E
#
#*
45-
d~
#
40--
0
__3 E (.9
35-30-25--
: 0
[ 30
I 60
I 90
I 120
150
l 180
Time (min) FIG. 1. Plasmaglucose(mM) (top) and glucoserate of appearance(rmol/ kg/min) (bottom) of H20 (©), M3G (O), M6G (G), and M3G + M6G (A) injected animals as a function of time after ICV administration.Values are means _+ SEM, (N = 6-7 per group). + p < 0.05 compared to preinjection values. *p < 0.05 compared to time-matchedH20 control. #p < 0.05 compared to time-matched M3G ICV injection. §p < 0.05 compared to time-matchedM6G 1CV injection.
Experimental Protocol After the 120 sample, one of the following combinations of drugs was injected ICV: M3G (1 #g), M6G (1 #g), M3G + M6G (1 #g each), and H20 alone (5/zl). Four groups of rats (n = 6 7 per each group) were used in this study . Additional arterial blood samples (0.35 ml) were taken at 15, 30, 60, 90, 120, and 180 min after the ICV injection for analysis of plasma lactate and glucose concentrations, and glucose specific activity. The blood withdrawn was replaced with an equal volume of sterile saline. Because of blood volume limitations, arterial blood samples (1.5 ml) were obtained only at 180 rain after drug administration for determinations of plasma insulin, glucagon, corticosterone, and catecholamines. At the end of the experiment, muscle (gastrocnemius) and a portion of liver were freeze clamped, immediately immersed in liquid nitrogen, and subsequently frozen at -80°C until samples were analyzed for glycogen content.
Analytical Procedures Neutralized supernatants of deproteinized plasma were used to determine glucose and lactate concentrations, and to estimate
glucose specific activity. An aliquot of the supernatant was used to determine [~H]glucose radioactivity (Wallac LSC 1409, Gaithersburg, MD) after tritiated water was removed by evaporation (RC 10-10, Jouan, Labrepco Inc., Southampton, PA). Liver glycogen content was determined by measuring the glucose residues after treatment of tissue with amyloglucosidase and expressed as milligrams of glucose per gram wet weight of tissue [19]. Blood samples for determination of plasma hormone levels were collected in chilled syringes containing aprotinin (Sigma, St. Louis, MO, 500 KIU/ml), and the plasma stored at -90°C until assayed. Plasma immunoreactive insulin was measured by radioimmunoassay using a double antibody method (ICN Biomedical, Costa Mesa, CA). Immunoreactive glucagon was assayed using the ICN double antibody method, using porcine standards and tzsIlabelled glucagon. Plasma corticosterone was assayed using a double antibody kit method from ICN. Catecholamines were analized by HPLC. Briefly; 3.4-dihidroxybenzylamine (DHBA) was added to plasma as an internal standard, and catecholamines absorbed onto alumina at pH = 8.5 and eluted with perchloric acid (PCA; 0.1 M). The processed samples were quantified by
M O D U L A T I O N OF M E T A B O L I C EFFECTS
327
55 -~ 50-o'E"
45+
40(3 v
353025 J
I 0
[ 30
1 60
I 90
I 120
I 150
[ 180
T i m e (min) 400
-
§
(p < 0.05). A significant 28% rise in glucose level was observed 90 min after M6G injection (p < 0.0t vs. basal and p < 0.05 vs. time-matched H20 and M3G), which lasted until the end of the experiment. A greater 41% increase in plasma glucose level was evident after M3G + M6G injection from 6 ___ 0.3 mM during basal to 9 -+ 0.6, mM peaking at 60 min after ICV drug administration (p < 0.01 vs. basal and p < 0.05 vs. time-matched other three groups). Thereafter, plasma glucose concentrations of the M3G + M6G group gradually decreased and were not different from those of M6G at the end of the experiment. ICV administration of water or M3G did not result in significant alterations in glucose rate of appearance (Ra), disappearance (Rd), or metabolic clearance rate (MCR). ICV injection of M6G produced a progressive increase in glucose Ra from basal values of 32 _+ 2 mol/kg • min to 46 _+ 3 mol/kg • min at 180 rain (p < 0.05 vs. basal and time-matched H20) (Fig. 1; bottom). M3G + M6G produced an early, yet transient (30 min) 58% increase in glucose Ra (p < 0.05 vs. basal and time-matched values of other groups). Thereafter, glucose Ra values were still
Liver -J
o
~E E ~ if)
2OO
•. .
to fit.
600
-
500
-
400
-
30O
-
200
-
¢/)
100
-
c03 ° ~
0
•
100-
0
i 30
I t I 60 90 120 T i m e (min)
I 150
I 180
FIG. 2. Glucose rate of disappearance (rmol/kg/min) (top) and plasma lactate (rmol/l) (bottom) of H20 (©), M3G (O), M6G (A), and M3G + M6G (&) injected animals as a function of time after ICV administration. Values are means _+ SEM, (N = 6-7 per group). +p < 0.05 compared to basal preinjection value. *p < 0.05 compared to time-matched H20 control• #p < 0.05 compared to time-matched M3G ICV injection. §p < 0.05 compared to time-matched M6G ICV injection• HPLC using a chromatographic analyzer with catecholarnine column and an electrochemical detector (Bioanalytic Systems, West Lafayette, IN). Using this system, the detection limit for epinephrine, norepinephrine, and DHBA was 20 pg.
The rate of glucose appearance (Ra) and disappearance (Rd) were calculated using the nonsteady state equations of Steele. The glucose metabolic clearance rate (MCR) was calculated by dividing glucose Rd by the prevailing plasma glucose concentration. Statistical differences were analyzed by A N O V A followed by N e w m a n - K e u l s test to determine treatment effect. Statistical significance was set at p < 0.05. RESULTS
Glucose Metabolic Response ICV Water injection resulted in a small 14% increase in glucose concentrations toward the end of the experiment (p < 0.05) (Fig. 1; top). M3G injected animals showed a similar 16% increase in plasma glucose concentrations from an average of 6 _+ 0.2 mM during basal to 7 -+- 0.1 mM at the end of the experiment
#
UO
1
I
(D
Muscle 03
300
-
0
250
-
200
-
150
-
100
-
E
Calculations
T
T
500
f,
H20
M3G
M6G
M3G+M6G
FIG. 3. Glycogen content (#mol/g wet weight tissue) of liver (top) and muscle (bottom) glycogen content of rats injected with H2O (open bar), M3G (hatched), M6G (cross-hatched), and M3G + M6G (solid bar). Values are means _+ SEM, (N = 6-7 per group). *p < 0.05 compared to time-matched H20 lCV injection, #p < 0.01 compared to time-matched H20 ICV injection.
328
HASHIGUCHI, MOLINA AND ABUMRAD
higher than time-matched H20 injected, but not statistically different from those of M6G injected rats. Changes in glucose Rd were similar to those of glucose Ra. There was a significant 48% increase in glucose Rd 30 min after ICV M3G + M6G injection. However, the peak of glucose Rd in M3G + M6G rats was smaller than that of Ra, and there was no statistical difference from Rd in M6G group (Fig. 2; top). No significant alteration was detected in MCR in any of the experimental groups. Plasma lactate levels were not altered after ICV H20 injection (Fig. 2; bottom). ICV M3G resulted in a moderate elevation in lactate levels from 147 ___22 tool/1 at basal to 198 +_ 22 mol/l at 30 min (p < 0.05). ICV M6G injection resulted in a significant increase in plasma lactate level from 148 _+ 20 mol/l during basal to 239 _+ 35 mol/1 60 min after administration. M3G + M6G resulted in a more marked rise in plasma lactate levels from 165 _+ 9 mol/l to 324 _+ 64 mol/1 30 min after injection (p < 0.05 vs. basal and time-matched values of other groups). Thereafter, plasma lactate levels decreased and were not different from those of M6G.
Liver and Muscle Glycogen There was no significant difference in liver glycogen content in M3G and H20 injected rats (Fig. 3; left). M6G and M3G + M6G 1CV injected rats had significantly lower liver glycogen content and averaged 278 _+ 72/zmol/g wet weight tissue in M6G (17 < 0.05) and 244 +_ 27/zmol/g wet weight tissue in M3G + M6G treated animals (p < 0.01). No significant difference was observed in muscle glycogen content (Fig. 3; right).
Hormone Concentrations Plasma insulin, glucagon, and corticosterone levels were not altered after injection of M3G, M6G, and M3G + M6G, and thus, were not different from H20 injected rats (Table 1). No significant alterations in circulating catecholamine levels were observed after ICV injection of M3G or M6G alone. However, M3G + M6G injection produced a 5-fold elevation in norepinephrine (NE) and a 6-fold rise in epinephrine (EP) levels compared to time matched H20 infused rats (p < 0.05 vs. other groups). DISCUSSION The hyperglycemic effects of morphine are predominantly dependent on stimulation of opiate-receptors, in a dose-dependent manner, and are mediated through large increases in epinephrine and small increases in norepinephrine [8,10,17]. Previous studies from our laboratory have demonstrated that the hyperglycemic effects of M6G are at least 80 times more potent than morphine
[11]. In this study the dose of M6G injected as a bolus was a more modest amount than that used previously, yet it was sufficient to produce a significant hyperglycemic response. The dose of M3G used in these studies was determined in preliminary studies in which a dose-response relationship was established by assessing glucose levels and behavioral responses to ICV M3G injection at 1, 2.5, 5, and 10/zg. Hyperglycemia within 15 rain after ICV M3G injection were observed with doses in the range of 2.5 /zg to 10/zg. Remarkable excitatory effects such as salivation, aggression, and general hyperresponsiveness to stimuli were also observed with doses equal to and higher than 2.5 /zg. ICV M3G at 10 /zg resulted in seizures in all rats in addition to general excitation. Thus 1/zg of M3G, a dose without remarkable hyperglycemic or excitatory effects was chosen to investigate its interaction with the metabolic effects of M6G. In this study a slight increase in glucose levels and lactate levels were observed after M3G injection, though the time course and magnitude of elevation was similar to H20 injected rats. These, however, were not accompanied by any detectable changes in glucose kinetics. Although these changes were also not accompanied by any alterations in the hormonal milieu at the time points examined, there remains, however, a slight possibility that elevations in circulating epinephrine and norepinephrine could have occurred which were not detected in our studies. The hyperglycemic effects of M6G resulted from progressive increases in hepatic glucose output (glucose Ra), suggesting accelerated hepatic gluconeogenesis and glycogenolysis. The contribution of hepatic glycogenolysis to this response was supported by the decreased glycogen content in the liver at the end of the experiment. Although the elevations in plasma catecholamines and corticosterone after M6G injection were modest, it is possible that these hormonal actions contributed to the hepatic glycogenolytic and gluconeogenic response. Interestingly, although M3G did not result in significant metabolic effects after its administration, it appears to have enhanced the metabolic and hormonal effects of M6G, which presumably are mediated through the/z-receptor. The glucose kinetics demonstrate that the enhanced glucose metabolism in the M3G + M6G treated rats was the result of enhanced hepatic glucose output within 60 min after 1CV injection. Thereafter, there was no apparent difference in glucose Ra between M6G and M3G + M6G groups. These findings suggest that an earlier and larger amount of release of catecholamine induced by the combined injection of M3G and M6G stimulated hepatic glycogenolysis. The significant contribution of glycogenolysis to accelerated hepatic glucose production is supported by the marked decrease in hepatic glycogen content in M3G + M6G rats at the end of the
TABLE 1 HORMONAL ALTERATIONS ICV GLUCURONIDE-INDUCED HORMONAL ALTERATIONS |t20
Insulin (/zU/ml) Glucagon (pg/ml) Corticosterone (ng/ml) Epinephrine (pg/ml) Norepinephrine (pg/ml)
22 136 216 353 369
+_ 2 ± 13 _+ 61 ± 85 ± 66
M3G
M6G
M3G + M6G
23 +_+ 2 143 ± 7 446 + 44 489 _+ 50 598 ± 82
22 ± 1 153 +_ 15 389 +_ 80 1096 ± 336 820 _+ 108
25 ± 3 168 _+ 20 422 +_ 76 2053 ± 543*¢$ 1798 ± 578"t$
Hormonal alterations in response to H20, morphine 3-glucuronide, morphine 6-glucuronide, and morphine 3- and morphine 6-glucuronide administration, presented as mean ± SEM 3 h after ICV administration, n = 6-7 per group. * p < 0.05 compared to water-infused rats, t P < 0.05 compared to morphine-3-glucuronide-infused rats, p < 0.05 compared to morphine-6-glucuronide-infused rats.
M O D U L A T I O N OF METABOLIC EFFECTS
experiment. Although liver glycogen content in M6G + M3G was identical to that in M6G, one can speculate that the hyperlactacidemia in M3G + M6G treated rats favored a shift from glycogenolysis to gluconeogenesis as the main source of hepatic glucose production and thus resulted in a similar content of liver glycogen to M6G injected rats [18]. In contrast to our observations of enhanced metabolic effects, M3G has been reported to antagonize M6G and morphine's antinociceptive and ventilatory depressing effects. The mechanism of these antagonistic effects of M3G is still unclear. However, it has been hypothesized that central excitatory effects of M3G antagonize the sedative effects of morphine and M6G. Our paradoxical results suggest that M3G does not antagonize the binding of M6G to #-receptor, but that it modulates the opioid effects by alternate central nervous system pathways. Thus, it is possible that antinociception or respiratory depression is antagonized through neural excitation, in addition to simultaneous sympathetic nervous system stimulation. In conclusion, combined injection of M3G + M6G resulted in enhanced hyperglycemia, hepatic glucose production, peripheral glucose utilization, and lactacidemia. Our findings clearly demonstrate that M3G does not inhibit the hyperglycemic effects of M6G, but rather potentiates its effects, most probably through enhanced release of catecholamines. Thus, together with M6G, M3G may play a role in modifying not only the analgesic but also the metabolic effects of morphine. ACKNOWLEDGEMENTS
329
7. 8.
9. 10. II.
12. 13. 14. 15.
16.
The authors would like to thank Rebecka Naukam, Dawn Sasvary, Barbara Tyndall, Mohamed Ajmal, and Yuqun Hong for their excellent technical assistance.
17.
REFERENCES
18.
I. Abbot, F. V.; Palmour, R. M. Morphine-6-glucuronide: Analgesic effects and receptor binding profile in rats. Life Sci. 43:1685-1695; 1988. 2. Bigler, D.; Christensen, C. B.; Eriksen, J.; Jensen, H. H. Morphine, morphine-6-glucuronide concentrations in plasma and cerebrospinal fluid during long-term high-dose intrathecal morphine administration. Pain 41:15-18; 1990. 3. Boerner, U.; Abbott, S.; Roe, R. L. The metabolism of morphine and heroin in man. Drug Metab. Rev. 4:39-73; 1975. 4. Christiansen, C. B.; Jorgensen, L. N. Morphine-6-glucuronide has high affinity for the opioid receptor. Pharmacol. Toxicol. 60:75-76; 1987. 5. Gong, Q. L.; Hedner, J.; Bj6rkman, R.; Hedner, T. Morphine-3glucuronide may functionally antagonize morphine-6-glucuronide induced antinociception and ventilatory depression in the rat. Pain 48:249-255; 1992. 6. Gong, Q. L.; Hedner, T.; Hedner, J.; BjOrkman, R.; Nordberg, G. Antinociceptive and ventilatory effects of the morphine metabolites:
19. 20.
21. 22. 23. 24.
Morphine-6-glucuronide and morphine-3-glucuronide. Eur. J. Pharmacol. 193:47-56; 1991. Hand, C. W.; Blunnie, W. P.; Claffey, L. P.; McShang, A. J.; McQuay, H. J.: Moore, R. A. Potential analgesic contribution from morphine-6-glucuronide in CSF. Lancet ii:1207-1208; 1987. Johansen, O.; Tonnesen, T.; Jensen, T.; Jorde, R.; Burhol, P. G.; Reikeras, O. Increments in glucose, glucagon and insulin after morphine in rats and naloxone blocking of this effect. Life Sci. 51 : 12371242; 1992. Labella, F. S.; Pinsky, C.; Havlicek, V. Morphine derivatives with diminished opiate receptor potency show enhanced central excitatory activity. Brain Res. 174:263-271; 1979. Molina, P. E.; Abumrad, N. N. Metabolic effects of opiates and opioid peptides. Adv. Neuroimmunol. 4:105-116; 1994. Molina, P. E.; Hashiguchi, Y,; Ajmal, M.; Mazza, M.; Abumrad, N. N. Differential hemodynamic, metabolic and hormonal effects of morphine and morphine-6-glucuronide. Brain Res. 664:126-132; 1994. Osborne, R.; Joel, S.; Grebenik, K.; Trew, D.; Maurice, S. The pharmacokinetics of morphine and morphine glucuronides in kidney failure. Clin. Pharmacol. Ther. 54:158-167; 1993. Osborne, R. S.; Joel, S. P.; Slevin, M. L. Morphine intoxication in renal failure: The role of morphine-6-glucuronide. Br. Med. J. 292:1548-1549; 1986. Patemak, G. W.; Bodnar, R. J.; Clark, J. A.; Inturrisi, C. E. Morphine-6-glucuronide: A potent mu antagonist. Life Sci. 41:28452849; 1987. Paul, D.; Standifer, K. M.; Inturrisi, C. E.; Paternak, W. Pharmacological characterization of morphine-6/3-glucuronide, a very potent morphine metabolite. J. Pharmacol. Exp. Ther. 251:477-483; 1989. Poulain, P.; Moran Ribon, A.; Hanks, G. W.; Hoskin, P. J.; Agerne, G. W.; Chapman, D. J. CSF concentration of morphine-6-glucuronide after oral administration of morphine. Pain 41 : 115-116; 1990. Radosevich, P. M.; Williams, P. E.; Lacy, D. B.; McRae, J. R.; Steiner, K. E.; Cherrington, A. D.; Lacy, W. W.; Abumrad, N. N. Effects of morphine on glucose homeostasis in the conscious dog. J. Clin. Invest. 74:1473-1480; 1984. Radziuk, J.; Pye, S.; Zfiang, Z. Substrates and the regulation of hepatic glycogen metabolism. Adv. Exp. Med. Biol. 334:235-252; 1993. Roe, J. H.; Dailey, R. E. Determination of glycogen with the anthrone reagent. Anal. Biocfiem. 15:245-250; 1966. Sawe, J. Morphine and its 3- and 6-glucuronides in plasma and urine during chronic oral administration in cancer patients. In: Foley, K. M.; Inturrisi, C. E., eds. Advances in pain research and therapy, vol.8. New York: Raven Press; 1986:45-54. Shimomura, K.; Kamata, O.; Ueki, S.; Oguri, K.; Yoshimura, H.; Tsukamoto, H. Analgesic effect of morphine glucuronides. Tohoku J. Exp. Med. 105:45-52; 1971. Smith, M. T.; Watt, J. A.; Cramond, T. Morphine-3-glucuronide: A potent antagonist of morphine analgesia. Life Sci. 47:579-585; 1990. Wahlstri~m, A.; Winblad, B.; Bixo, M.; Rane, A. Human brain metabolism of morphine and naloxone. Pain 35:121 - 127; 1988. Yaksh, T. L.; Harty, L. G.; Onofrio, B. M. High doses of spinal morphine produce a nonopiate receptor-mediated hyperesthesia: Clinical and theoretic implications. Anesthesiology 64:590-597; 1986.
Pergamon 0361-9230(95)00104-2
Modulation of Metabolic Effects of Morphine-6Glucuronide by Morphine-3-Glucuronide YOJIRO HASHIGUCHI,*I- PATRICIA E. MOLINA*-~1 AND NAJI N. ABUMRAD*I-
*Department of Surgery, SUNY at Stony Brook, Stony Brook, NY 11794-8191, USA ?~Departmentof Research, VAMC, Northport, Northport, NY 11768, USA [Received 6 March 1995; Accepted 19 May 1995] and kidney [23]. In humans it is estimated that 55% of morphine is transformed to M3G, whereas a lower (15%) amount is metabolized to morphine-6-glucuronide(M6G) [3]. Both M3G and M6G have been isolated from plasma and CSF after oral, intramuscular, or spinal administration of morphine [7,16,2], suggesting either crossover of the blood-brain barrier or CNS conversion of morphine to its glucuronides in the brain. These metabolites have been also shown to accumulate in plasma in higher levels than those of the parent compound after chronic oral dosing [20] as well as in individuals with impaired renal function [12]. M3G does not bind to opioid receptors [4] and is devoid of analgesic activity [21 ]. Nevertheless, it causes behavioral excitation, hyperalgesia, and respiratory stimulation by nonopioid mechanisms [9,24]. Studies have recently focused on morphine glucuronides because of the increased degree and duration of the effects of morphine with elevated levels of glucuronides reported in patients with renal dysfunction [ 13]. Recent reports in the literature have suggested that M3G has an antagonizing effect on the antinociceptive and respiratory depressant effect of morphine and M6G [22,6,5]. In contrast, M6G binds to receptors with affinities similar to those of morphine [4,14], and its intracerebroventricular (ICV) administration elicits a 13- to 200-fold greater analgesic effect than morphine [1,15]. In addition, recent studies f~'omour laboratory have demonstrated potent hyperglycemic effects of M6G, suggesting important modulatory roles of glucuronidation on morphine's effects on glucose metabolism [1 1]. The purpose of this study was to determine if M3G at a dose that does not alter glucose homeostasis would antagonize or attenuate the metabolic effects of M6G.
ABSTRACT: Modification of pharmacological effects of morphine by its glucuronides has been recently reported. Morphine6-glucuronide (M6G) is a more potent opioid agonist than morphine, whereas morphine-3-glucuronide (M3G) has no opioid effects and has been suggested to be an antagonist of morphine's antinociceptive and respiratory depressive effects. This study addressed the metabolic effects of direct central nervous system administration of M3G and its interaction with the hyperglycemic effects of M6G. Hormonal and whole body glucose metabolic effects of M3G, M6G, and M3G + M6G ICV administration were studied in conscious unrestrained chronically catheterized rats. Whole body glucose kinetics were assessed with a primed constant intravenous infusion of 3[~H]glucose in rats injected intracerebroventricularly (ICV) with H2O (5 pl), M3G (1 pg), M6G (1 pg), or M3G (1/~g) + M6G (1 pg). A significant rise in plasma glucose level was observed after ICV injection of M6G (28%), and M3G + M6G (41%), but not after M3G as compared to time-matched H20 control. Early increases in the rate of glucose appearance (Ra) and whole body glucose utilization (Rd) were observed (58% and 48%, respectively) 30 min after M3G + M6G administration, whereas the increases after M6G injection were progressive and reached values 47% higher than basal 180 min after injection. M3G administration enhanced the M6G induced increase in plasma glucose level (+21%), Ra (+29%), Rd (+26%), and plasma lactate level (+21%). Though no significant hormonal change was observed in H20, M3G, and M6G injected animals, the combination of M3G + M6G resulted in a significant increase in circulating catacholamine levels with no aRerations in plasma corticosterone, insulin, and glucagon. These results indicate that M3G at a dose that does not produce hyperglycemia enhanced the hyperglycemic effects of M6G through accelerated release of catecholamines. These findings suggest that the glucuronidated metabolites of morphine influence each other's biological effects.
MATERIALS AND METHODS KEY WORDS: Morphine, Morphine-3-glucuronide, Morphine-6glucuronide, Glucose, Catecholamines, Hormones, Rats, ICV.
Animal Preparation Male Sprague-Dawley rats (275-350 g, Charles River, Wilmington, MA) were housed in a controlled environment, exposed to a 12:12 h light-dark cycle, and fed standard rat diet (Purina Rat Chow, Ralston Purina, St. Louis, MO) for 1 week before being used in an experiment. Five days before the experiment, animals were anesthetized with an intramuscular injection of ketamine and xylazine (90 rag/ kg and 9 mg/kg, respectively) and positioned in a stereotaxic
INTRODUCTION Morphine-3-glucuronide (M3G) is a major product of morphine glucuronidation in all species. The glucuronidation of morphine occurs not only in the liver but may also take place in the brain
Requests for reprints should be addressed to Patricia E. Molina, Department of Surgery, SUNY at Stony Brook, Stony Brook, NY 117948191, USA. 325
326
apparatus (David Kopf Instruments, Tujunga CA). The overlying skin and connective tissue were cleared from the skull. A hole was drilled in the skull and a 22-gauge stainless steel guide cannula (Plastic Products, Richmond VA) was inserted unilaterally into the lateral ventricle of the brain. The stereotaxic coordinates used for cannula placement were 0.3 mm posterior to bregma, 1.3 mm lateral from midline, and 4.25 mm below the surface of the skull. The cannula was secured using dental cement (Lang's Dental Manufacturing, Chicago IL) and anchored to the skull with one stainless steel screw. A wire dummy cannula was used to seal the guide cannula until the time of the experiment. The animals were provided with food and water ad lib and allowed to recover in individual cages. The day after surgery, cannula placement was assessed by determining whether angiotensin |I (100 ng/rat, Sigma, St. Louis, MO) stimulated dipsogenesis. Proper positioning of the cannula was verified at the end of the experiment by ICV injection of trypan blue. Animals continued their recovery for 3 more days before catheters were implanted in the right jugular vein and left carotid artery. On the day before the experiment, animals were anesthetized with an intramuscular (0.25 ml) injection of ketamine (9 mg/ 100 g) and xylazine (0.9 mg/100 g). Using aseptic surgical procedures, a catheter (PE-50) was implanted in the left carotid artery, and advanced to the arch of the aorta, and another catheter placed in the right jugular vein. After surgery, the animals were returned to individual cages, fasted overnight, and provided water ad lib. Animals were fasted to minimize gut absorption as contributor to glucose appearance. Experiments were started between 0600 and 0800 h the following day, with rats conscious and unrestrained throughout the duration of the protocol.
Glucose Kinetics On the day of the experiment a primed constant intravenous (IV) infusion of [3-3H]glucose (37 Ci/mmol; HPLC purified, #NET-100C, Dupont-NEN, Boston, MA.) was initiated via the venous catheter, and continued throughout the experimental procedure (1 ml/h). Isotopic equilibrium was achieved within 90 min after starting the [3H]glucose infusion; thereafter blood samples were taken at 100 and 120 min for the determination of basal glucose metabolism and after drug treatment throughout the remainder of the experimental period.
HASHIGUCHI, MOLINA AND ABUMRAD
10-
§
§
#*
#*
+
i- t f # -
90 o
(.9 m
#.
+
8+
E
.. 7
E fl)
--
+
÷
--
6-
n
!
0
i 30
I 60
1 90
I 120
I 150
I 180
Time (min)
§ 55--
+
3+,..+ T
50m t~ t/)
O o
r-
E
#
#*
45-
d~
#
40--
0
__3 E (.9
35-30-25--
: 0
[ 30
I 60
I 90
I 120
150
l 180
Time (min) FIG. 1. Plasmaglucose(mM) (top) and glucoserate of appearance(rmol/ kg/min) (bottom) of H20 (©), M3G (O), M6G (G), and M3G + M6G (A) injected animals as a function of time after ICV administration.Values are means _+ SEM, (N = 6-7 per group). + p < 0.05 compared to preinjection values. *p < 0.05 compared to time-matchedH20 control. #p < 0.05 compared to time-matched M3G ICV injection. §p < 0.05 compared to time-matchedM6G 1CV injection.
Experimental Protocol After the 120 sample, one of the following combinations of drugs was injected ICV: M3G (1 #g), M6G (1 #g), M3G + M6G (1 #g each), and H20 alone (5/zl). Four groups of rats (n = 6 7 per each group) were used in this study . Additional arterial blood samples (0.35 ml) were taken at 15, 30, 60, 90, 120, and 180 min after the ICV injection for analysis of plasma lactate and glucose concentrations, and glucose specific activity. The blood withdrawn was replaced with an equal volume of sterile saline. Because of blood volume limitations, arterial blood samples (1.5 ml) were obtained only at 180 rain after drug administration for determinations of plasma insulin, glucagon, corticosterone, and catecholamines. At the end of the experiment, muscle (gastrocnemius) and a portion of liver were freeze clamped, immediately immersed in liquid nitrogen, and subsequently frozen at -80°C until samples were analyzed for glycogen content.
Analytical Procedures Neutralized supernatants of deproteinized plasma were used to determine glucose and lactate concentrations, and to estimate
glucose specific activity. An aliquot of the supernatant was used to determine [~H]glucose radioactivity (Wallac LSC 1409, Gaithersburg, MD) after tritiated water was removed by evaporation (RC 10-10, Jouan, Labrepco Inc., Southampton, PA). Liver glycogen content was determined by measuring the glucose residues after treatment of tissue with amyloglucosidase and expressed as milligrams of glucose per gram wet weight of tissue [19]. Blood samples for determination of plasma hormone levels were collected in chilled syringes containing aprotinin (Sigma, St. Louis, MO, 500 KIU/ml), and the plasma stored at -90°C until assayed. Plasma immunoreactive insulin was measured by radioimmunoassay using a double antibody method (ICN Biomedical, Costa Mesa, CA). Immunoreactive glucagon was assayed using the ICN double antibody method, using porcine standards and tzsIlabelled glucagon. Plasma corticosterone was assayed using a double antibody kit method from ICN. Catecholamines were analized by HPLC. Briefly; 3.4-dihidroxybenzylamine (DHBA) was added to plasma as an internal standard, and catecholamines absorbed onto alumina at pH = 8.5 and eluted with perchloric acid (PCA; 0.1 M). The processed samples were quantified by
M O D U L A T I O N OF M E T A B O L I C EFFECTS
327
55 -~ 50-o'E"
45+
40(3 v
353025 J
I 0
[ 30
1 60
I 90
I 120
I 150
[ 180
T i m e (min) 400
-
§
(p < 0.05). A significant 28% rise in glucose level was observed 90 min after M6G injection (p < 0.0t vs. basal and p < 0.05 vs. time-matched H20 and M3G), which lasted until the end of the experiment. A greater 41% increase in plasma glucose level was evident after M3G + M6G injection from 6 ___ 0.3 mM during basal to 9 -+ 0.6, mM peaking at 60 min after ICV drug administration (p < 0.01 vs. basal and p < 0.05 vs. time-matched other three groups). Thereafter, plasma glucose concentrations of the M3G + M6G group gradually decreased and were not different from those of M6G at the end of the experiment. ICV administration of water or M3G did not result in significant alterations in glucose rate of appearance (Ra), disappearance (Rd), or metabolic clearance rate (MCR). ICV injection of M6G produced a progressive increase in glucose Ra from basal values of 32 _+ 2 mol/kg • min to 46 _+ 3 mol/kg • min at 180 rain (p < 0.05 vs. basal and time-matched H20) (Fig. 1; bottom). M3G + M6G produced an early, yet transient (30 min) 58% increase in glucose Ra (p < 0.05 vs. basal and time-matched values of other groups). Thereafter, glucose Ra values were still
Liver -J
o
~E E ~ if)
2OO
•. .
to fit.
600
-
500
-
400
-
30O
-
200
-
¢/)
100
-
c03 ° ~
0
•
100-
0
i 30
I t I 60 90 120 T i m e (min)
I 150
I 180
FIG. 2. Glucose rate of disappearance (rmol/kg/min) (top) and plasma lactate (rmol/l) (bottom) of H20 (©), M3G (O), M6G (A), and M3G + M6G (&) injected animals as a function of time after ICV administration. Values are means _+ SEM, (N = 6-7 per group). +p < 0.05 compared to basal preinjection value. *p < 0.05 compared to time-matched H20 control• #p < 0.05 compared to time-matched M3G ICV injection. §p < 0.05 compared to time-matched M6G ICV injection• HPLC using a chromatographic analyzer with catecholarnine column and an electrochemical detector (Bioanalytic Systems, West Lafayette, IN). Using this system, the detection limit for epinephrine, norepinephrine, and DHBA was 20 pg.
The rate of glucose appearance (Ra) and disappearance (Rd) were calculated using the nonsteady state equations of Steele. The glucose metabolic clearance rate (MCR) was calculated by dividing glucose Rd by the prevailing plasma glucose concentration. Statistical differences were analyzed by A N O V A followed by N e w m a n - K e u l s test to determine treatment effect. Statistical significance was set at p < 0.05. RESULTS
Glucose Metabolic Response ICV Water injection resulted in a small 14% increase in glucose concentrations toward the end of the experiment (p < 0.05) (Fig. 1; top). M3G injected animals showed a similar 16% increase in plasma glucose concentrations from an average of 6 _+ 0.2 mM during basal to 7 -+- 0.1 mM at the end of the experiment
#
UO
1
I
(D
Muscle 03
300
-
0
250
-
200
-
150
-
100
-
E
Calculations
T
T
500
f,
H20
M3G
M6G
M3G+M6G
FIG. 3. Glycogen content (#mol/g wet weight tissue) of liver (top) and muscle (bottom) glycogen content of rats injected with H2O (open bar), M3G (hatched), M6G (cross-hatched), and M3G + M6G (solid bar). Values are means _+ SEM, (N = 6-7 per group). *p < 0.05 compared to time-matched H20 lCV injection, #p < 0.01 compared to time-matched H20 ICV injection.
328
HASHIGUCHI, MOLINA AND ABUMRAD
higher than time-matched H20 injected, but not statistically different from those of M6G injected rats. Changes in glucose Rd were similar to those of glucose Ra. There was a significant 48% increase in glucose Rd 30 min after ICV M3G + M6G injection. However, the peak of glucose Rd in M3G + M6G rats was smaller than that of Ra, and there was no statistical difference from Rd in M6G group (Fig. 2; top). No significant alteration was detected in MCR in any of the experimental groups. Plasma lactate levels were not altered after ICV H20 injection (Fig. 2; bottom). ICV M3G resulted in a moderate elevation in lactate levels from 147 ___22 tool/1 at basal to 198 +_ 22 mol/l at 30 min (p < 0.05). ICV M6G injection resulted in a significant increase in plasma lactate level from 148 _+ 20 mol/l during basal to 239 _+ 35 mol/1 60 min after administration. M3G + M6G resulted in a more marked rise in plasma lactate levels from 165 _+ 9 mol/l to 324 _+ 64 mol/1 30 min after injection (p < 0.05 vs. basal and time-matched values of other groups). Thereafter, plasma lactate levels decreased and were not different from those of M6G.
Liver and Muscle Glycogen There was no significant difference in liver glycogen content in M3G and H20 injected rats (Fig. 3; left). M6G and M3G + M6G 1CV injected rats had significantly lower liver glycogen content and averaged 278 _+ 72/zmol/g wet weight tissue in M6G (17 < 0.05) and 244 +_ 27/zmol/g wet weight tissue in M3G + M6G treated animals (p < 0.01). No significant difference was observed in muscle glycogen content (Fig. 3; right).
Hormone Concentrations Plasma insulin, glucagon, and corticosterone levels were not altered after injection of M3G, M6G, and M3G + M6G, and thus, were not different from H20 injected rats (Table 1). No significant alterations in circulating catecholamine levels were observed after ICV injection of M3G or M6G alone. However, M3G + M6G injection produced a 5-fold elevation in norepinephrine (NE) and a 6-fold rise in epinephrine (EP) levels compared to time matched H20 infused rats (p < 0.05 vs. other groups). DISCUSSION The hyperglycemic effects of morphine are predominantly dependent on stimulation of opiate-receptors, in a dose-dependent manner, and are mediated through large increases in epinephrine and small increases in norepinephrine [8,10,17]. Previous studies from our laboratory have demonstrated that the hyperglycemic effects of M6G are at least 80 times more potent than morphine
[11]. In this study the dose of M6G injected as a bolus was a more modest amount than that used previously, yet it was sufficient to produce a significant hyperglycemic response. The dose of M3G used in these studies was determined in preliminary studies in which a dose-response relationship was established by assessing glucose levels and behavioral responses to ICV M3G injection at 1, 2.5, 5, and 10/zg. Hyperglycemia within 15 rain after ICV M3G injection were observed with doses in the range of 2.5 /zg to 10/zg. Remarkable excitatory effects such as salivation, aggression, and general hyperresponsiveness to stimuli were also observed with doses equal to and higher than 2.5 /zg. ICV M3G at 10 /zg resulted in seizures in all rats in addition to general excitation. Thus 1/zg of M3G, a dose without remarkable hyperglycemic or excitatory effects was chosen to investigate its interaction with the metabolic effects of M6G. In this study a slight increase in glucose levels and lactate levels were observed after M3G injection, though the time course and magnitude of elevation was similar to H20 injected rats. These, however, were not accompanied by any detectable changes in glucose kinetics. Although these changes were also not accompanied by any alterations in the hormonal milieu at the time points examined, there remains, however, a slight possibility that elevations in circulating epinephrine and norepinephrine could have occurred which were not detected in our studies. The hyperglycemic effects of M6G resulted from progressive increases in hepatic glucose output (glucose Ra), suggesting accelerated hepatic gluconeogenesis and glycogenolysis. The contribution of hepatic glycogenolysis to this response was supported by the decreased glycogen content in the liver at the end of the experiment. Although the elevations in plasma catecholamines and corticosterone after M6G injection were modest, it is possible that these hormonal actions contributed to the hepatic glycogenolytic and gluconeogenic response. Interestingly, although M3G did not result in significant metabolic effects after its administration, it appears to have enhanced the metabolic and hormonal effects of M6G, which presumably are mediated through the/z-receptor. The glucose kinetics demonstrate that the enhanced glucose metabolism in the M3G + M6G treated rats was the result of enhanced hepatic glucose output within 60 min after 1CV injection. Thereafter, there was no apparent difference in glucose Ra between M6G and M3G + M6G groups. These findings suggest that an earlier and larger amount of release of catecholamine induced by the combined injection of M3G and M6G stimulated hepatic glycogenolysis. The significant contribution of glycogenolysis to accelerated hepatic glucose production is supported by the marked decrease in hepatic glycogen content in M3G + M6G rats at the end of the
TABLE 1 HORMONAL ALTERATIONS ICV GLUCURONIDE-INDUCED HORMONAL ALTERATIONS |t20
Insulin (/zU/ml) Glucagon (pg/ml) Corticosterone (ng/ml) Epinephrine (pg/ml) Norepinephrine (pg/ml)
22 136 216 353 369
+_ 2 ± 13 _+ 61 ± 85 ± 66
M3G
M6G
M3G + M6G
23 +_+ 2 143 ± 7 446 + 44 489 _+ 50 598 ± 82
22 ± 1 153 +_ 15 389 +_ 80 1096 ± 336 820 _+ 108
25 ± 3 168 _+ 20 422 +_ 76 2053 ± 543*¢$ 1798 ± 578"t$
Hormonal alterations in response to H20, morphine 3-glucuronide, morphine 6-glucuronide, and morphine 3- and morphine 6-glucuronide administration, presented as mean ± SEM 3 h after ICV administration, n = 6-7 per group. * p < 0.05 compared to water-infused rats, t P < 0.05 compared to morphine-3-glucuronide-infused rats, p < 0.05 compared to morphine-6-glucuronide-infused rats.
M O D U L A T I O N OF METABOLIC EFFECTS
experiment. Although liver glycogen content in M6G + M3G was identical to that in M6G, one can speculate that the hyperlactacidemia in M3G + M6G treated rats favored a shift from glycogenolysis to gluconeogenesis as the main source of hepatic glucose production and thus resulted in a similar content of liver glycogen to M6G injected rats [18]. In contrast to our observations of enhanced metabolic effects, M3G has been reported to antagonize M6G and morphine's antinociceptive and ventilatory depressing effects. The mechanism of these antagonistic effects of M3G is still unclear. However, it has been hypothesized that central excitatory effects of M3G antagonize the sedative effects of morphine and M6G. Our paradoxical results suggest that M3G does not antagonize the binding of M6G to #-receptor, but that it modulates the opioid effects by alternate central nervous system pathways. Thus, it is possible that antinociception or respiratory depression is antagonized through neural excitation, in addition to simultaneous sympathetic nervous system stimulation. In conclusion, combined injection of M3G + M6G resulted in enhanced hyperglycemia, hepatic glucose production, peripheral glucose utilization, and lactacidemia. Our findings clearly demonstrate that M3G does not inhibit the hyperglycemic effects of M6G, but rather potentiates its effects, most probably through enhanced release of catecholamines. Thus, together with M6G, M3G may play a role in modifying not only the analgesic but also the metabolic effects of morphine. ACKNOWLEDGEMENTS
329
7. 8.
9. 10. II.
12. 13. 14. 15.
16.
The authors would like to thank Rebecka Naukam, Dawn Sasvary, Barbara Tyndall, Mohamed Ajmal, and Yuqun Hong for their excellent technical assistance.
17.
REFERENCES
18.
I. Abbot, F. V.; Palmour, R. M. Morphine-6-glucuronide: Analgesic effects and receptor binding profile in rats. Life Sci. 43:1685-1695; 1988. 2. Bigler, D.; Christensen, C. B.; Eriksen, J.; Jensen, H. H. Morphine, morphine-6-glucuronide concentrations in plasma and cerebrospinal fluid during long-term high-dose intrathecal morphine administration. Pain 41:15-18; 1990. 3. Boerner, U.; Abbott, S.; Roe, R. L. The metabolism of morphine and heroin in man. Drug Metab. Rev. 4:39-73; 1975. 4. Christiansen, C. B.; Jorgensen, L. N. Morphine-6-glucuronide has high affinity for the opioid receptor. Pharmacol. Toxicol. 60:75-76; 1987. 5. Gong, Q. L.; Hedner, J.; Bj6rkman, R.; Hedner, T. Morphine-3glucuronide may functionally antagonize morphine-6-glucuronide induced antinociception and ventilatory depression in the rat. Pain 48:249-255; 1992. 6. Gong, Q. L.; Hedner, T.; Hedner, J.; BjOrkman, R.; Nordberg, G. Antinociceptive and ventilatory effects of the morphine metabolites:
19. 20.
21. 22. 23. 24.
Morphine-6-glucuronide and morphine-3-glucuronide. Eur. J. Pharmacol. 193:47-56; 1991. Hand, C. W.; Blunnie, W. P.; Claffey, L. P.; McShang, A. J.; McQuay, H. J.: Moore, R. A. Potential analgesic contribution from morphine-6-glucuronide in CSF. Lancet ii:1207-1208; 1987. Johansen, O.; Tonnesen, T.; Jensen, T.; Jorde, R.; Burhol, P. G.; Reikeras, O. Increments in glucose, glucagon and insulin after morphine in rats and naloxone blocking of this effect. Life Sci. 51 : 12371242; 1992. Labella, F. S.; Pinsky, C.; Havlicek, V. Morphine derivatives with diminished opiate receptor potency show enhanced central excitatory activity. Brain Res. 174:263-271; 1979. Molina, P. E.; Abumrad, N. N. Metabolic effects of opiates and opioid peptides. Adv. Neuroimmunol. 4:105-116; 1994. Molina, P. E.; Hashiguchi, Y,; Ajmal, M.; Mazza, M.; Abumrad, N. N. Differential hemodynamic, metabolic and hormonal effects of morphine and morphine-6-glucuronide. Brain Res. 664:126-132; 1994. Osborne, R.; Joel, S.; Grebenik, K.; Trew, D.; Maurice, S. The pharmacokinetics of morphine and morphine glucuronides in kidney failure. Clin. Pharmacol. Ther. 54:158-167; 1993. Osborne, R. S.; Joel, S. P.; Slevin, M. L. Morphine intoxication in renal failure: The role of morphine-6-glucuronide. Br. Med. J. 292:1548-1549; 1986. Patemak, G. W.; Bodnar, R. J.; Clark, J. A.; Inturrisi, C. E. Morphine-6-glucuronide: A potent mu antagonist. Life Sci. 41:28452849; 1987. Paul, D.; Standifer, K. M.; Inturrisi, C. E.; Paternak, W. Pharmacological characterization of morphine-6/3-glucuronide, a very potent morphine metabolite. J. Pharmacol. Exp. Ther. 251:477-483; 1989. Poulain, P.; Moran Ribon, A.; Hanks, G. W.; Hoskin, P. J.; Agerne, G. W.; Chapman, D. J. CSF concentration of morphine-6-glucuronide after oral administration of morphine. Pain 41 : 115-116; 1990. Radosevich, P. M.; Williams, P. E.; Lacy, D. B.; McRae, J. R.; Steiner, K. E.; Cherrington, A. D.; Lacy, W. W.; Abumrad, N. N. Effects of morphine on glucose homeostasis in the conscious dog. J. Clin. Invest. 74:1473-1480; 1984. Radziuk, J.; Pye, S.; Zfiang, Z. Substrates and the regulation of hepatic glycogen metabolism. Adv. Exp. Med. Biol. 334:235-252; 1993. Roe, J. H.; Dailey, R. E. Determination of glycogen with the anthrone reagent. Anal. Biocfiem. 15:245-250; 1966. Sawe, J. Morphine and its 3- and 6-glucuronides in plasma and urine during chronic oral administration in cancer patients. In: Foley, K. M.; Inturrisi, C. E., eds. Advances in pain research and therapy, vol.8. New York: Raven Press; 1986:45-54. Shimomura, K.; Kamata, O.; Ueki, S.; Oguri, K.; Yoshimura, H.; Tsukamoto, H. Analgesic effect of morphine glucuronides. Tohoku J. Exp. Med. 105:45-52; 1971. Smith, M. T.; Watt, J. A.; Cramond, T. Morphine-3-glucuronide: A potent antagonist of morphine analgesia. Life Sci. 47:579-585; 1990. Wahlstri~m, A.; Winblad, B.; Bixo, M.; Rane, A. Human brain metabolism of morphine and naloxone. Pain 35:121 - 127; 1988. Yaksh, T. L.; Harty, L. G.; Onofrio, B. M. High doses of spinal morphine produce a nonopiate receptor-mediated hyperesthesia: Clinical and theoretic implications. Anesthesiology 64:590-597; 1986.