European Journal of Pain (1997) 1: 161-164

Short Communication

Morphine-3-glucuronide prevents tolerance to morphine-6-glucuronide in mice

C. C. Fauraarb, M. J. Olasoa and J. F. Horgaapb

aDepartamento de Farmacologia y Terapbutica and blnstituto de Neurociencias, Universidad Miguel Hernandez, Alicante, Spain

Daily subcutaneous (SC.) pretreatment with morphine-3-glucuronide (6 mg/kg) was found to reduce morphine- 6-glucuronide (4mg/kg s.c.)-induced antinociception, with no decrease in the effect over 5 days. Morphine-6- glucuronide administration (4 mg/kg s.c.) on Day 6, without morphine-3-glucorinide pretreatment, results in a significant increase in antinociception (from 24% on Day 5 to 70%; ~~0.05). Morphine-3-glucuronide may prevent tolerance to morphine-6-glucuronide by reducing morphine-6-glucuronide-induced antinociception.

INTRODUCTION

Morphine-6-glucuronide (M6G), the most im- portant active metabolite of morphine, binds to the p-opioid receptor, has a more potent anti- nociceptive effect than morphine and tolerance to this effect has also been proved (Faura et al., 1996). The major morphine metabolite, mor- phine-3-glucuronide (M3G), shows no affinity for the p-opioid receptor and has no analgesic activity; however, there are conflicting reports of its effects on morphine and M6G pharmacology (Smith et al., 1990; Gong et al., 1992; Ekblom et al., 1993; Faura et al., 1996).

Previously, the authors have shown that the acute antinociceptive effect of systemically ad- ministered M6G (4 mg/kg) was antagonised by M3G (6mg/kg) without affecting the anti- nociception induced by equipotent doses of mor- phine (lOmg/kg, Faura et al., 1996). Based on the above data, the aim of this study was to evalute if the reduction of the antinociceptive effect of M6G by M3G is followed by a decrease in the degree of tolerance to M6G.

Paper received 5 March 1997 and accepted in revised form 17 June 1997. Correspondence to: C. C. Faura, Departamento de Farmacologia TerapCutica, Universidad Miguel Hernandez, Campus de San Juan, 03080 Alicante, Spain.

1090-3801/97/020161+04 $12.00/O

MATERIALS AND METHODS

Male Swiss-Webster mice (2540 g) were housed in groups of 10 with food and water ad libitum and maintained on a 12/12 h light/dark cycle, with stable humidity and temperature (2%22°C). The ethical standards for the investigation of experimental pain in animals were followed.

All compounds for subcutaneous (s.c.) in- jections (0.25-0.40 ml) were dissolved in normal saline. Morphine hydrochloride was obtained from Alkaliber Lab., Spain, M6G from Ultrafine Chemicals, UK, and M3G from Sigma, USA.

The hot plate test was used to determine the degree of antinociception. The test was performed daily before the injection of the drug (the baseline latency was taken as the mean of two meas- urements, made 30 min apart), and 30 min after administration of morphine or 60 min after M6G (Faura et al., 1996). The mean value + SD of the daily baseline latencies was 10.4 f 1.1 s and the coefficient of variation was 10.6%. Animals with a baseline latency I 6 or 2 14 s were ex- cluded.

Different experiments were performed to study the development of tolerance to 4 mg/kg of M6G (equipotent dose to 10 mg/kg of morphine, Faura et al., 1996) alone and when co-administered with 6 mg/kg of M3G (Table 1). Five groups of mice

0 1997 European Federation of Chapters of the International Association for the Study of Pain

TABL

E 1.

Exp

erim

enta

l de

sign

and

antin

ocice

ptive

ef

fect

in

the

stud

ied

grou

ps

Day

1 Da

y 2

Day

3 Da

y 4

Day

5 Da

y 6

Day

7

Grou

ps

Abso

lute

%

Abso

lute

%

Abso

lute

%

Abso

lute

%

Abso

lute

%

Abso

lute

‘XC

Abso

lute

%

1 71

10

70

5110

50

51

10

50

4110

40

41

10

40

319"

33

.3

7/9a

77

.7

2 01

10

0 01

10

0 O/

IO

0 01

9"

0 O/

l 0

0 81

10

80

M 8

/11

72.7

3

5121

23

.8

5121

23

.8

6120

" 30

51

21

23.8

51

21

23.8

M

6G7I

lO

70

4 81

10

80

8110

80

61

10

60

5110

50

41

9"

44.4

31

8"

37.5

61

8"

75

5 01

11

0 l/I

O"

10

019"

0

o/loa

0

0/10

" 0

719"

77

.7

M6G

, m

orph

ine-6

-gluc

uron

ide;

M,

mor

phine

; Ab

solu

te,

num

ber

of

mice

wi

th

antin

ocice

ptive

ef

fect

/tota

l nu

mbe

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mice

st

udie

d.

%,

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ge

of a

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ice

exclu

ded

beca

use

base

line

late

ncie

s 16

or

2

14.

MORPHINE METABOLITES AND TOLERANCE 163

were treated daily (s.c.) over a 5-day period, as follows:- Group 1: 4 mg/kg of M6G (n = 10); Group 2: saline (n = 10); Group 3: M3G plus 4 mg/kg of M6G (n =21); Group 4: M3G plus 10 mg/kg of M6G (n = 10); and Group 5: M3G plus saline (n = 11). In Groups 3-5, M3G was administered on a daily basis 8 h before M6G or saline over the 5-day period. On Days 6 and 7, no M3G was administered. On Day 6, in order to assess the degree of cross-tolerance to morphine, 10 mg/kg of morphine (equipotent dose to 4 mg/ kg of M6G) was administered to Groups 1, 2, 4 and 5; animals from Group 3 received either 10 mg/kg of morphine (n = 11) or 4 mg/kg of M6G (n = 10). On Day 7, the morphine dose was doubled (20 mg/kg) in those groups (1 and 4) in which the previous morphine dose (10 mg/kg on Day 6) had not produced a significant degree of antinociception (<40%).

Antinociception was quantified as a two-fold or greater increase in the baseline latency for each individual animal (Faura et al., 1996); The results are expressed as percentage of anti- nociception [(number of mice with anti- nociceptive effect/total number of mice) x 1001. For statistical comparison, the Chi-square test was used in a Statview SE software.

RESULTS

Animals treated with either 4mg/kg of M6G alone (Group 1) or 10 mg/kg of M6G with M3G pretreatment (Group 4), showed a similar anti- nociceptive effect which decreased progressively over the 5 days of treatment (from 70 and 80% on Day 1 to 40 and 44% on Day 5, respectively). When morphine was administered instead of M6G on Day 6 to animals from Groups 1 and 4, the antinociceptive effect was similar to that observed on the previous day (33 and 37.5%, respectively). Doubling the dose of morphine on Day 7 increased the antinociceptive effect (Groups 1 and 4: 78 and 75%, respectively) when compared to Day 6 (Table 1).

In animals treated with 4 mg/kg of M6G with M3G pretreatment (Group 3), the low anti- nociceptive effect produced by M6G did not change over the 5 days of treatment; it ranged

from 24 to 30% antinociception. The ad- ministration on Day 6 of M6G (4 mg/kg) without M3G pretreatment produced an antinociceptive effect (70%) significantly higher 0, I 0.05) when compared with animals chronically treated with 4 mg/kg of M6G (33%, Group l), and similar to that observed in saline-treated animals (Groups 5 and 2: 78 and SO%, respectively). The ad- ministration of 10 mg/kg of morphine on Day 6 produced an antinociceptive effect (73%) similar to that observed on Day 6 with 4 mg/kg of M6G (Table 1).

M3G per se did not modify the hot plate latency time.

DISCUSSION

This study has found that the metabolite M3G administered at doses which significantly reduced M6G-induced antinociception, also prevents de- velopment of tolerance to M6G.

As reported previously (Faura et al., 1996), the effect of 4mg/kg of M6G was reduced by one-third in the presence of M3G, and its full effect was recovered by increasing the M6G dose to 10 mg/kg. In the present study, animals treated with 4mg/kg of M6G alone and with 10 mg/ kg of M6G with M3G pretreatment showed a similarity in acute and chronic antinociceptive effect, and followed a similar development of tolerance to M6G.

Although the antinociceptive effect of 4mg/ kg of M6G administered after M3G is low, it remained relatively constant during the 5-day period, and on Day 5 its effect was similar to that achieved with 4mg/kg of M6G alone or lOmg/kg of M6G with M3G pretreatment (Groups 1 and 4). The administration on Day 6 of morphine (10 mg/) or M6G (4 mg/kg) without M3G pretreatment to the animals from Group 3 produces the full antinociceptive effect, as would be expected from an initial dose of the opioid (e.g. Group 1 on Day 1). The difference in the antinociceptive effect between Days 5 and 6, in animals receiving 5 days M6G (4mg/kg) with M3G (6 mg/kg) pretreatment, followed by either morphine lOmg/kg or M6G 4mg/kg (Group 3), shows the absence of tolerance both to morphine and to M6G. The mechanism for

European Journal of Pain (1997). 1

C. C. FAURA ET AL.

to morphine appear, but if antinociception is

the effects of M3G are not clear, and a phar- macokinetic interaction may not be excluded

reduced (i.e. by the administration of M3G), the

since these glucuronides seem to be handled differently in the body (Van Crugten et al., 1991). According to previous reports (Fernandes et al., 1977), and independently of the mechanism be- hind M3G, it can be speculated that the intensity of drug exposure determines the degree of the effect, and plays an important role in the degree of tolerance achieved. The present results suggest that the effect of M3G on the tolerance to M6G is mainly dependent on its ability to decrease the antinociceptive effect, otherwise M3G would inhibit tolerance to M6G independently of the administered dose, and supports the idea that tolerance development might be dependent upon the initial level of antinociception for a given dose; if the antinociceptive effect of M6G is high, tolerance to M6G and cross-tolerance of M6G

was supported by a project grant (SAF93-0260) from CICYT, Spain.

REFERENCES

Ekblom M, Gardmatk M, Hammarlund-Udenaes M. Phar- macokinetics and pharmacodynamics of morphine-3-glu- curonide in rats and its influence on the antinociceptive effect of morphine. Biopharm Drug Dispos 1993; 14: l-l 1.

Faura CC, Olaso MJ, Garcia Cabanes C, Horga JF. Lack of morphine-6-glucuronide antinociception after morphine treatment. Is morphine-3-glucuronide involved? Pain 1996; 65: 25530.

Fernandes M, Kluwe S, Coper H. Quantitative assessment

curonide-a potent antagonist of morphine analgesia.

of tolerance to and dependence on morphine in mice. Naunyn Schmiedebergs Arch Pharmacoll917; 297: 53-60.

Gong QL, Hender J, Bjijrkman R, Hender T. Morphine-3-

Life Sci 1990; 47: 579-585. Van Crugten JT, Sallustio BC, Nation RL, Somogyi AA.

glucuronide may functionally antagonize morphine-6- glucuronide induced antinociception and ventilatory de- pression in the rat. Pain 1992; 48: 249-255. -’ Smith MT, Watt JA, Cramond T. Morphine-3-glu-

tolerance is prevented.

ACKNOWLEDGEMENTS

Renal tubular transport of morphine, morphine-6-glu- curonide, and morphine-3-glucuronide in the isolated perfursed rat kidney. Drug Metabol Dispos 1991; 19: 1087-1092.

The authors wish to thank Mr. S. Ingham for his critical review of this manuscript. The study

European Journal of Pain (1997), 1