Morphine-3-glucuronide - a potent antagonist of morphine analgesia

Life Sciences, Vol. 47, pp. 579-585 Printed in the U.S.A.

Pergamon Press

MORPHINE-3-GLUCURONIDE - A POTENT ANTAGONIST OF MORPHINE ANALGESIA *Maree T. Smith, Julie A. Watt & Tess Cramond *Department of Pharmacy and Division of Anaesthetics, University of Queensland, St. Lucia, Queensland, Australia, 4072. (Received in final form June ii, 1990)

Summarv In this study, morphine-3-glucuronide (M3G), the major plasma and urinary metabolite of morphine, was shown to be a potent antagonist of morphine analgesia when administered to rats by the intra-cerebroventricular (i.c.v.) route. The antagonism of morphine analgesia was observed irrespective of whether i.c.v. M3G (2.5 or 3.0/~g) was administered 15 mins prior to or 15 mins after i.c.v, morphine (20/~g). When M3G (10mg) was administered intraperitoneaUy (i.p.) to rats 30-40 mins prior to morphine (1.5mg i.p.), the analgesic response was significantly reduced compared to administration of morphine (1.5mg i.p.) alone. It was further demonstrated that i.c.v. M3G (2.0/~g) antagonized the analgesic effects of subsequently administered i.c.v, morphine-6-glucuronide (0.25ug). Morphine is a potent opioid analgesic which has been used traditionally for the treatment of acute pain and for the long-term treatment of severe intractable pain associated with malignant disease. Morphine is metabolized predominantly to two metabolites, morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) (1,2). M3G is the major plasma and urinary metabolite of morphine which has previously been reported to be devoid of analgesic activity (3,4) and which has been shown to produce hyperaesthesia/hyperalgesia in rats, when administered by the intrathecal (5,6,7) and i.c.v, routes (8). In contrast, M6G, has been reported to be 20-40 times more potent an analgesic than morphine itself, when administered to rats by the i.c.v, route (3,4). Given that the areas under the plasma M3G concentration-time curves in humans after chronic high oral doses of morphine, exceed those of morphine and M6G by factors of 20 and 1-2 respectively (9), we hypothesized that "M3G may antagonize the analgesic effects of morphine and M6G and that cumulation of M3G in blood and CSF with time relative to morphine and M6G, may make a significant contribution to the development of tolerance to morphine analgesia". Thus, the purpose of this study was to examine the effect of M3G administration on morphine and M6G analgesia in the rat. 0024-3205/90 $ 3 . 0 0 + .00 Copyright

(c)

1990 P e r g a m o n P r e s s

plc

580

M3G as Antagonist

of Morphine Analgesia

Vol. 47, No. 6, 1990

Methods The animal experiments described in this study were approved by The University of Queensland Animal Ethics Review Committee. Male Sprague-Dawley-derived rats (250g) were purchased from The University of Queensland Medical School Animal House. M3G was purchased from Sigma Chemical Co. (Dorset, U.K.) and M6G was purchased from Salford Ultrafine Chemicals (Salford, U.K.). Morphine HC1 was purchased from the Royal Brisbane Hospital Pharmacy (Brisbane, Australia). Sur~cal Procedure: Before surgery, rats were anaesthetized with a mixture of xylazine (14mg/kg) and ketamine HC1 (90mg/kg), administered i.p. in two equal portions, 5 mins apart. This combination dose provided deep and stable anaesthesia for the duration of the surgical procedure. Guide cannulae were cut from 21 gauge stainless steel disposable needles (Terumo) to a length of 8mm, and filed to a 40°bevel. The guide cannulae were inserted stereotaxically to a depth of 1.0 mm above the left lateral ventricle and secured in position with dental cement in a manner similar to that described by Brady & Holtzman (10). A guide cannula plug, which extended 0.5mm beyond the tip of the implanted cannula, was kept in place except during drug injections. Following surgery, rats received Bicillin all-purpose injection (150,000 U intramuscularly) and were allowed to recover for one week prior to testing. Drug injections were made using an SGE 5gl syringe with the attached needle extending lmm beyond the tip of the guide cannula. I.C.V. Dosing and I.P. Dosin~ All drugs used were dissolved in isotonic sterile saline. For i.c.v, dosing, rats were administered either isotonic sterile saline (2.0/~1), Morphine HCI (20~g), M3G (2.5 or 3.0/~g), or M6G (0.25~g). Injections administered by the i.c.v, route were infused at the rate of 0.5 /~1 per 15 seconds and did not exceed 2.5/~1 in volume. Morphine HC1 (1.5-2.0mg) and M3G (10mg) were also administered by the i.p. route using volumes between 0.15ml and 2.0ml. Tail Flick Latencies All rats were housed in a restraining cage for the 3h duration of the experiment and analgesia was assessed by a tail-flick method similar to that described by Brady & Holtzman (10). Radiant heat was focussed on the dorsum of the rat's tall, the intensity of which gave predrug reaction times of approximately 2.0-2.5 secs in untreated control animals. A maximum tail flick latency of 6 secs was used to minimize tissue damage to the rat's tail. Tall flick latencies were determined prior to (an average of 3 readings taken 5 mins apart) and at the following times subsequent to the i.c.v, or i.p. injection of morphine/M6G: 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 2.0, 2.5, 3.0 h. For i.c.v, administration of morphine, all rats received at least one dose of morphine alone and/or one dose of M3G 15 mins prior to or 15 mins after morphine. Similarly, for the i.c.v. M6G studies, rats received 0.25/~g M6G and/or 2.0/~g M3G followed 15 mins later by 0.25gg M6G. For the i.p. studies, rats were administered either morphine (1.5 or 2.0mg) and/or M3G (10rag) 30-40 mins prior to morphine. All studies were performed at least 5-7 days apart, in random order.

Vol. 47, No. 6, 1990

M3G as Antagonist of Morphine Analgesia

581

D e ~ e e of Analgesia Develoned The degree of analgesia developed was expressed as a percentage of Maximum Possible Effect (%MPE) according to the formula (10): % MPE = post-drug latency - pre-drug latency

x 100

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

maximum latency - pre-drug latency

(6.00 secs)

1

The degree of analgesia developed (expressed as a percentage of maximum) was plotted against time for each dosing experiment and the Area Under the Degree of Analgesia versus Time Curve (AUC) was calculated using trapezoidal integration. The AUC's thus calculated for morphine administration alone were then compared with the AUC's calculated for M3G plus morphine administration, using the appropriate statistical test. Results Our results show that M3G is a potent antagonist of morphine analgesia when administered to rats by the i.c.v, route, irrespective of whether M3G (2.5#g or 3.0#g) is administered 15 mins prior to (Figure 1) or 15 mins after (Figure 2) i.c.v, morphine (20#g). This has been clearly demonstrated by the significant reduction in the AUC's for i.c.v. M3G plus morphine compared to i.c.v, morphine alone (Duncan's M-range test, p < 0.05). When 3.0~g M3G was administered i.c.v. 15 mins prior to i.c.v, morphine (20ug), the degree of analgesia developed (%MPE) was less than after a 2.5#g M3G dose. However, this decrease was not statistically significant (Duncan's M-range test, p > 0.05). When the i.c.v. M3G dose was increased to 3.5 and 4.0#g, it was not possible to obtain reliable tail flick reaction times, as the adverse effects produced by M3G (severe whole body jerks, Straub tail and seizures) interfered with the proper functioning of the test. M3G administered alone by the i.c.v, route, produced a range of apparently dose-dependent pharmacological effects in the rats. In ascending order of severity, the pharmacological effects observed in our rats were: hypersensitivity to normal stimuli such as sound and touch (allodynia), hyperalgesia, excessive grooming behaviour, tremor, wet dog shakes, Straub tail, seizures and death. An apparently dose-dependent reduction in the severity of these effects was observed after the subsequent administration of i.c.v. morphine. For i.c.v. M3G doses up to 10#g, the subsequent dose of i.c.v, morphine required to reverse the adverse effects of M3G was approximately 7 times the administered M3G molar dose. Our observations are consistent with previous reports that M3G administered intrathecally or i.c.v, to rats, causes allodynia/hyperalgesia in rats (5,6,7,8). The tail flick reaction times of control rats which received 2.0/~1 of i.c.v, isotonic saline, did not change significantly (paired Wilcoxon's test, p > 0.05) from the pre-injection reaction times (Figure 3), indicating that the experimental setup did not affect the reaction times for the 3h duration of the experiment.

582

M3G as Antagonist

of Morphine Analgesia

Vol. 47, No. 6, 1990

100 80

.

20gg M

,,=, e

?,

60

2.Spg M3G + 20pgM

E

20

3.0pg M3G ! 2 0 ~ g M ~ , ~

,

0.5

1.0

1.5

2.0

2.5

g.O

-20

Time After Morphine Administration -40

(h)

FIG. 1 The Mean (-+ s.e.m.) Degree of Analgesia versus time curves in rats after single i.c.v, administration of: ( H ) 20~g Morphine HCI (n = 12) (o-c) 2.5/~g M3G followed 15 mins later by 20/~g i.c.v. Morphine HC1 (n = 6) and (o-c~) 3.0~g M3G foLlowed 15 mins later by 20~g i.c.v. Morphine HC1 (n=6). 100 20pg M

80

60

40 20 0 0.5 -20 I -40

1.0

1.5

2.0

2.5

3.0

Time After Morphine Administration (h)

FIG. 2 The Mean (-+s.e.m.) Degree of Analgesia versus time curves in rats after single i.c.v, administration of (: -) 20#g Morphine HCI alone (n = 12) and 20#g Morphine HC1 followed 15 rains later by (o--o) 2.5#g M3G and (o--o) 3.0#g M3G.

Vol. 47, No. 6, 1990

M3G as Antagonist of Morphine Analgesia

583

Furthermore, when i.c.v. M3G (2.0/~g) was administered to rats 15 mins prior to i.c.v. M6G (0.25~g), the degree of analgesia was reduced significantly (unpaired Wilcoxon's test, n= 13, p<0.05) compared to i.c.v, administration of the same dose of M6G alone (Figure 4). It was also noted that at this dose of i.c.v. M6G, the rats showed symptoms of mild respiratory depression at approximately 1-1.5h after dosing. When the i.c.v, dose of M6G was increased to 0.5/~g, the rats exhibited maximum analgesia by 15 mins and profound respiratory depression by 1-1.5h post-dosing. The results obtained after i.p. administration of M3G (10mg) 30-40 mins prior to i.p. administration of morphine (1.5-2.0mg) show a significant reduction in analgesia in rats (paired Wilcoxon's test, n = 8, p < 0.05) when compared with the same dose of i.p. morphine administered alone (Figure 5). M3G (10rag i.p.) was administered to rats because this dose produced observable pharmacological effects (e.g. excessive grooming behaviour and mild tremor of the face) by 30-40 mins post-dosing. 100 -

,~

80

@ W

• 0 a.

E

60 ~

40

X

X

2o Normal Saline

T'"y" 0.5

t 1.0

1.5

2.0

2.5

-20 Time After Saline Administration -40

--

(h)

FIG. 3 The Mean (-* s.e.m.) Degree of Analgesia versus time curve in rats after i.c.v, administration of single 2.0/~1 doses of sterile isotonic saline (n = 5).

Morphine is a potent opioid analgesic, administered widely for the treatment of acute pain and for the longterm treatment of severe pain in patients with malignant disease. Morphine undergoes extensive first-pass metabolism forming two major metabolites, viz. morphine-3-glucuronide (the predominant metabolite) and morphine-6-glucuronide (the lesser metabolite). Morphine itself has been the focus of much scientific endeavour over the past several decades and more recently the reported analgesic potency of M6G (3,4) has focussed the attention of researchers on this metabolite. However, relatively little attention has been paid to M3G, due to it being devoid of analgesic potency (3,4).

584

M3G as Antagonist

of Morphine Analgesia

100 _

Vol. 47, No. 6, 1990

0.25~gM 6 G

40 ,

o

uJ _e £ w o.

60

S

_s

m¢ @

20

0 0.5 "20 t

1.0

1,5

2.0

2.5

3.0

Time After M6G Administration

-40

(h)

FIG. 4 The Mean (~s.e.m.) Degree of Analgesia versus time curves in rats after single i.e.v, injections of (m---m)0.25/~g M6G (n = 7) and (o---o) 2.0pg M3G followed 15 mins later by 0.25/~g M6G (n=6).

100 ~ ~ q ~ g

M

80

J/ llmM

&u • £

o Q.

60 w

E _s

40

:E

20

I

0.5

1.0

1.5

-20 Time After -41

-

Morphine

2.0

~.5 b

3.0

Administration

(h)

FIG. 5 A representative Degree of Analgesia versus time curve obtained in one rat after single i.p. injections (one week apart) of: (~-m) 1.5mg Morphine HCI and (o-o) 10rag M3G followed 35 minutes later by 1.5mg Morphine HC1.

Vol. 47, No. 6, 1990

M3G as Antagonist

of Morphine Analgesia

585

Our/n v/voobservations clearly show that i.c.v. M3G is a potent antagonist of the analgesic effects of i.c.v, morphine/M6G in the rat. However, it has been reported that M3G has very poor affinity for mu-, delta- and kappa-opioid receptors in /n v/tro binding studies (4,11). On this basis, it would appear unlikely that M3G antagonizes morphine/M6G analgesia by competing for mu-receptors. Rather, it is possible that morphine/M6G and M3G exert their pharmacological actions via different receptor mechanisms, with M3G mediating hyperalgesia/hyperaesthesia and morphine/M6G (in the doses administered) mediating analgesia. Although the i.p. studies showed a reduced analgesia when M3G was administered with morphine, the interpretation of these results may be complicated by systemic metabolism of these compounds occurring simultaneously with their entry into the CSF and their subsequent pharmacological effects. Together with Woolf, Yaksh and Labella (5,6,7,8) we have observed that M3G can produce effects in rats normally associated with morphine withdrawal symptoms. These results and observations raise the question of '~what is the contribution made by M3G to morphine tolerance and to the adverse effects of morphine observed in clinical practice?" We propose that tolerance to morphine analgesia at the functional level may develop as M3G accumulates with time, relative to morphine and M6G. The delayed (relative to the onset of analgesia) respiratory depression observed in rats administered i.c.v, doses (0.25-0.5/~g) of M6G also requires further investigation, particularly with regard to the implications of this finding for patients to whom morphine is administered. References 1.

2. 3. 4. .

6. 7. 8. 9. 10. 11.

K. IWANATO, C.KLAASSEN, J. Pharmacol. Exp. Ther. 200 236-244 (1977). K.W. BOCK, G. BRUNNER, M. HOENSCH, E. HUBER, D. JOSTING, Eur. J. Clin. Pharmacol. 14 367-373 (1978). H. YOSHIMURA, S. IDA, K. OGURI, J. TSUKAMOTO, Biochem. Pharmacol. 22 1423-1430 (1973). G.W. PASTERNAK, R.J. BODNAR, J.A. CLARK, C.E. INTURRISI, Life Sci. 41 2845-2849 (1987). C.J. WOOLF, Brain Research 209 491-495 (1981) T.L. YAKSH, G.J. HARTY, B.M. ONOFRIO, Anesthiol. 64 590-597 (1986). T.L. YAKSH, G.J. HARTY, J. Pharmacol. Exp. Ther. 244 501-507 (1988). F.S. LABELLA, C. PINSKY, V. HAVLICEK, Brain Research 174 263-271 (1979). J. SAWE, J.O. SVENSSON, A. RANE, Br. J. Clin. Pharmac. 16 85-93 (1983). L.S. BRADY, S.G. HOLTZMANN, J. Pharmacol. Exp. Ther. 222 190-197 (1982). K. OGURI, I. YAMADA-MORI, J. SHIGEZANE, T., HIRANO, H. YOSHIMURA, Life Sci 41 1457-1464 (1987).