Comparison of morphine-6-glucuronide and morphine on respiratory depressant and antinociceptive responses in wild type and µ-opioid receptor deficient mice

R. Romberg1, E. Sarton1, L. Teppema1, H. W. D. Matthes2, B. L. Kieffer2 and A. Dahan*,1

1 Department of Anesthesiology, Leiden University Medical Center, 2300 RC Leiden, The Netherlands. 2 Institut de Génétique et de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique/Institut National de la Santé et de la Recherche Médicinale/Université Louis Pasteur, BP 163 67404 Illkirch, France

*Corresponding author. E-mail: a.dahan@lumc.nl

Accepted for publication: June 18, 2003


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Background. Morphine-6-glucuronide (M6G) is a metabolite of morphine with potent analgesic properties. The influence of M6G on respiratory and antinociceptive responses was investigated in mice lacking the µ-opioid receptor (MOR) and compared with morphine.

Methods. Experiments were performed in mice lacking exon 2 of the MOR (n=18) and their wild type (WT) littermates (n=20). The influence of M6G and morphine on respiration was measured using whole body plethysmography during three elevations of inspired carbon dioxide. Antinociception was assessed using tail flick and hotplate tests.

Results. In WT but not null mutant mice, a dose-dependent depression of the slope of the ventilatory carbon dioxide response was observed after M6G and morphine. Similarly, both opioids were devoid of antinociceptive effects in null mutant mice, but showed potent dose-dependent analgesia in WT animals. Potency differences between M6G and morphine in WT mice were of the same order of magnitude for analgesia and respiration.

Conclusions. The data indicate that the desired (antinociceptive) and undesired (respiratory depression) effects of M6G and morphine are linked to the same gene product; that is the MOR. Other opioid- and non-opioid-receptor systems may play a minor role in the actions of M6Gs and morphine. The clinical implications of our findings are that any agent acting at the MOR will invariably cause (potent) analgesia in combination with (variable) respiratory depression.

Br J Anaesth 2003; 91: 862–70

Keywords: analgesics opioid; measurement techniques, plethysmography; model, knockout mouse; ventilation; ventilation, control of breathing


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In humans the main metabolic pathway for morphine is glucuronidation in the liver.1 About 55% of morphine is metabolized to morphine-3-glucuronide (M3G), without significant activity in humans.2 Five to fifteen per cent is metabolized to morphine-6-glucuronide (M6G), a potent opioid acting predominantly at µ-opioid receptors (MOR) but also displaying actively at {delta}-opioid receptors (DOR).1 3 Animal studies indicate that activation of µ- or {delta} receptor systems is associated with respiratory depression.4 5 Although the contribution of DOR in morphine respiratory depression is of minor importance,5 other opioids may produce additional respiratory depression via the MOR system.4 Relative to morphine, M6G has similar affinity for the MOR and an increased affinity for the DOR.1 6

We recently tested the influence of morphine in MOR deficient mice and their wild type (WT) littermates and observed that morphine had no analgesic or respiratory depressant effect in the null mutant mice, while responses in the WT animals were ‘typical’ opioid responses, that is dose-dependent antinociceptive and respiratory depression.5 7 8 These findings indicate that the MOR gene is the molecular site of morphine analgesia and respiratory responses and that morphine produces its analgesic and respiratory effects via a common effector pathway. There are various reasons why it is of interest to examine the molecular mechanisms of M6G-induced respiratory depression. First, M6G plays an important role in analgesia and toxicity after repetitive and/or sustained morphine administration in cancer patients and in patients with renal failure,911 and secondly, and perhaps most important, M6G is currently undergoing phase III clinical trials and will be marketed in the near future as a postoperative analgesic. The involvement of the MOR vs the DOR system in particular but also vs non-opioid systems (for example, opioid-induced inhibition of cholinergic pathways)12 in M6G-induced respiratory depression is of importance in this respect.

In the current study, we compared the respiratory and antinociceptive effects of M6G with morphine in MOR deficient mice with a null mutation of exon 2 of the MOR gene.7 The effect of both drugs on respiration was tested by measuring resting ventilation and the ventilatory response to inspired carbon dioxide under unrestrained conditions using whole body plethysmography.5


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Subjects
Mice with a disruption of exon 2 of the MOR gene and their WT littermates were used.7 WT and MOR gene deficient mice (MOR–/–) were F1 from matings of Sv/C57BL/6 WT or MOR–/– breeding pairs, respectively (breeding of WT and knockout mice was not different from heterozygous matings). Experiments were performed in mice of either sex (weight 20–40 g) at ages 3–6 months. The total number of animals used in this study was 38 (20 WT and 18 mutants). In each experiment an equal number of male and female animals were tested. All experiments were performed with approval of the local Animal Ethics Committee. Animal care was in accordance with institutional guidelines.

Measurement of respiration
Respiration was measured by using whole body plethysmography with a continuous flow of dry gas through a reference and a measurement chamber (volume=600 ml).5 The flow and composition of the gas was set by three mass flow controllers (Bronkhorst High-Tec, Veenendaal, The Netherlands).13 The chambers were kept at room temperature (24–26°C). Following placements in the measurement chamber and a habituation period, data acquisition started. In each animal the effects of saline, M6G, and morphine were assessed on ventilation at four concentrations of inhaled carbon dioxide on a background of mild hyperoxia (oxygen 25% and carbon dioxide 0, 3, 5, and 8%). The inhalation of each gas mixture lasted at least 7 min. When on-line analysis revealed that steady-state ventilation had not been reached the duration of hypercapnia was extended. Tidal volume (VT), ventilatory frequency (RR), and minute ventilation (V, where V=VTxRR) were calculated per breath. The data were averaged over 50 breaths and stored on disc for further analysis. The slope of the hypercapnic ventilatory response (HCVR) was then determined from the slope of the relationship between V and the four levels of inspired carbon dioxide using least-squares regression analysis.5 13

Respiratory studies started 20 min after administration of each drug dose. During the study, the investigators were blinded to the animal genotype. Each mouse was tested on two occasions (morphine—M6G), at least 4 weeks apart.

In order to obtain an indication of the respiratory potency of morphine and M6G (ED50) in WT animals, we fitted the dose–effect data (slope of the HCVR) to the following simple function:14

S(d)=S0x[1 – 0.5x(d/ED50){gamma}]

where S(d) is the slope of the HCVR (units: ml min–1 per %CO2) at dose d (units: mg kg–1), S0 the slope after saline, ED50 the dose causing 50% depression of S and {gamma} a steepness parameter.

Measurement of antinociception
For the tail-immersion test, the tails of the mice were immersed (±2 cm) in water of 54°C and the latency time to a rapid tail flick was recorded. The cut-off time for this test was 15 s to prevent tissue damage. For the hotplate test, mice were placed on a rectangular metal plate heated to 52°C. The antinociceptive response was the latency time to hind paw licking, jumping, or vocalization, with a cut-off of 30 s to prevent tissue damage. After a positive response the mouse was immediately removed from the metal surface. If cut-off values were reached the mouse was not tested further. The tail-immersion test preceded the hotplate test with 30-s between tests. This procedure is identical to that of South and Smith.15

To obtain an indication of the antinociceptive potency of morphine and M6G in WT animals, we fitted the dose–effect data (latencies) to the following function:16

L(d)=L0x[1+(d/ED200){gamma}]

where L(d) is the latency (units: s) after dose d (units: mg kg–1), L0 the latency after saline, {gamma} a steepness parameter and ED200 the potency parameter or the dose causing a doubling of latency relative to baseline. Only doses causing latencies below the cut-off and the lowest dose causing the cut-off latency were used in the estimation of potency.

To test the effect of opioids against saline and between genotypes, the data were converted to percentage of maximum possible effect (MPE) using the following equation: %MPE=(post-drug latency – latency after saline)/(cut-off latency – latency after saline).

This approach has the advantage that a correction is performed for individual differences in baseline latencies. Note that 50% of MPE does not necessarily equal ED200.

Nociceptive studies started 20 min after administration of each drug dose. During the study, the investigators were blinded to the animals genotype. Each mouse was tested on two occasions (morphine—M6G), at least 4 weeks apart. The mice participating in these studies were not included in the respiratory studies.

Drugs
Morphine was produced locally by the hospital pharmacy; M6G was obtained from CeNeS Ltd (Cambridge, UK). Both opioids were dissolved in normal saline (NaCl 0.9%). The M6G solution contained no morphine or M3G as tested by the local toxicology laboratory. All drugs were administered intraperitoneally (i.p.) at a volume of 1 ml.

Study design
Mice participating in the respiratory studies (n=8 MOR–/– and 10 WT) were not included in the antinociceptive studies (n=10 MOR–/– and 10 WT) and vice versa. Initially, the effect of normal saline was assessed followed by a cumulative dose–response assessment (with 40 min between dosing). The doses of M6G tested were 10, 20, and 30 mg kg–1 in respiratory studies; and 1, 5, 10, 20, and 40 mg kg–1 in antinociceptive studies. The doses of morphine tested were 6, 20, and 100 mg kg–1 in respiratory studies and 10, 20, 40, 80, and 100 mg kg–1 in antinociceptive studies. Part of the morphine respiratory depression data has been published previously.5

Statistical analysis
Potency
Morphine and M6G ED50 and ED200 values were compared by paired-t-tests.

Treatment and genotype effect
A one-way analysis of variance (ANOVA) was performed on the slope of the HCVR, tidal volume at 0 inspired carbon dioxide, ventilatory frequency at 0 inspired carbon dioxide, minute ventilation at 0 inspired carbon dioxide, and antinociceptive effect (% MPE) in the tail flick and hotplate tests to test for treatment effects in animals with the same genotype. Post hoc analysis was by least significant differences test. Differences between genotype were tested with a two-way ANOVA (with factors treatment, genotype and treatmentxgenotype). A significant genotype effect was assumed when the interactive term was significant. Post hoc analysis was by least significant differences test. In the statistical analysis MPE values were set at 100% at doses not tested because of the attainment of cut-off at lower doses.

P-values <0.05 were considered significant. Values reported are mean (SD) unless otherwise stated.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Respiration
Examples of the effect of M6G on the slope (S) of the HCVR in a WT and a MOR–/– animal are shown in Figure 1, demonstrating dose-dependent decrease in S in WT animal only (Fig. 1B, the model fit is the line through the data points). No systematic M6G effect was observed in mice lacking µ-opioid the receptor gene (Fig. 1C and D). Table 1 shows mean effects of M6G and morphine in the WT and null mutant mice on tidal volume, ventilatory frequency, minute ventilation, and the slope of the HCVR. The MOR–/– mice did not show any response to morphine or M6G. In contrast, both morphine and M6G caused dose-dependent respiratory depression in WT animals, which was, for both agents, a result of reduction of ventilatory frequency and slope of the HCVR (Fig. 2). Tidal volume remained unaffected by both drugs, even at the highest doses tested (Table 1). As depicted in Figure 3B, M6G was more potent than morphine in depressing the slope of the HCVR by a factor of 3.7 in WT animals (ED50 M6G 20.5 (8.6) mg kg–1 vs ED50 morphine 76.0 (20.4) mg kg–1, P<0.01). There were no differences in respiratory effect between morphine and M6G in the MOR knockout mice (Fig. 3B).



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Fig 1 Typical effects of M6G on the ventilatory response to inspired carbon dioxide in a mouse with an intact MOR system (A and B) and a MOR gene deficient mouse (C and D). (A and C) HCVR at 0, 10, 20, and 30 mg kg–1 M6G in a WT animal (A) and a MOR–/– mouse (C). Lines are linear regression analyses. Note the dose-dependent reduction in ventilatory sensitivity to carbon dioxide in the WT mouse only. (B and D) M6G dose against slope of the HCVR. A function of the form S(d)=S0x[1 – 0.5x(d/ED50){gamma}], where S is the slope of the HCVR, S0 the slope after saline, d the drug dose, ED50 the dose causing 50% depression of S and {gamma} a steepness parameter, is fitted through the WT mouse data. In WT mice ED50 was 19.8 mg kg–1 (B); in the knockout mouse ED50 could not be determined (D).

 

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Table 1 Effects of M6G and morphine on breathing at 0% inspired carbon dioxide and slope of the HCVR (S) in WT and MOR deficient mice (MOR–/–). Values are mean (SD); treatment effect: *P<0.01 vs saline (0 mg kg–1) of the identical genotype, one-way ANOVA and least significant differences test. **Genotypextreatment effect, two-way ANOVA; NS, not significant; S slope of the HCVR; VT, tidal volume; RR, ventilatory frequency; V, minute ventilation
 


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Fig 2 Effects of M6G on the slope of the HCVR in mice lacking the MOR (MOR–/–, open circle) and mice with intact MOR (WT, filled circle). *P<0.001 vs saline (one-way ANOVA); **P<0.001 vs mice with intact receptors (two-way ANOVA). Values are mean (SEM).

 


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Fig 3 Typical effects of M6G and morphine on the slope of the HCVR in mice with intact MOR (left) and mice lacking the MOR gene and gene product (right). Note the 3-fold greater M6G sensitivity in depressing the HCVR relative to morphine in MOR intact mice, while no significant responses were seen with morphine and M6G in mice lacking the MOR. Values are percentage of control response (mean (SEM)).

 
Antinociception
Examples of the effect of M6G and morphine on the response latencies in hotplate and tail flick tests in WT and MOR knockout animals are shown in Figure 4. This shows a dose-dependent increase in latencies in WT animals only (the model fits are the continuous lines through the data points). The estimated potency values (ED200) in the WT genotype were 12.0 (4.5) mg kg–1 (morphine) and 2.3 (1.7) mg kg–1 (M6G) in the tail flick test (M6G vs morphine P<0.01), and 11.5 (5.1) mg kg–1 (morphine) and 3.6 (2.1) mg kg–1 in the hotplate test (M6G, P<0.01). In both nociceptive assays, neither morphine nor M6G caused an antiocieceptive response in mutant mice (Figs 4 and 5). However, M6G but not morphine, caused a small hyperalgesic effect in the hotplate test (Figs 4A and 5B). This effect was significant relative to saline and was dose-independent (Fig. 5B; P<0.01 vs saline). In contrast to the mutant mice, all WT animals displayed dose-dependent antinociception from both opioids in both tests (Fig. 5; MOR–/– vs WT: P<0.0001).



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Fig 4 Examples of the effect of M6G (A and B) and morphine (C and D) on antinociceptive responses in mice with intact MOR (WT) and mice lacking µ-opioid gene receptors (MOR–/–). A function of the form L(d)=L0x[1+(d/ED200){gamma}] was fitted to the WT animals data (thick continuous lines), where L(d) is the latency after dose d, L0 the latency after saline, {gamma} a steepness parameter and ED200 the potency parameter or the dose causing a doubling of latency relative to baseline. (A) M6G in the hotplate test. The ED200 of the WT animal was 2.6 mg kg–1. Note the hyperalgesic responses in the MOR–/– mouse. (B) M6G in the tail-immersion test. The ED200 of the WT animal was 4.5 mg kg–1. The MOR–/– mouse displayed no systematic response to M6G. (C) Morphine in the hotplate test. The ED200 of the WT animal was 32.3 mg kg–1. The MOR–/– mouse displayed no response to morphine. (D) Morphine in the tail-immersion test. The ED200 was 12.5 mg kg–1. The MOR–/– mouse displayed no response to morphine. In WT animals, opioid doses above the cut-off values (e.g. M6G doses of 20 and 40 mg kg–1 in example A and morphine doses of 80 and 100 mg kg–1 in example D) were not tested and their latency values consequently not taken into account in the estimation of potency.

 


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Fig 5 Antinociceptive responses to M6G (A and B) and morphine (C and D) in WT mice and MOR deficient mice. (A) M6G in the tail immersion test. MOR–/–, all doses not significant vs saline (0 mg kg–1); WT 1, 5, 10, 20, and 40 mg kg–1: P<0.0001 vs saline; MOR–/– vs WT: P<0.0001 (two-way ANOVA). (B) M6G in the hotplate test. MOR–/–, 1, 5, 10, 20, and 40 mg kg–1; P<0.001 vs saline; WT 1, 5, 10, 20, and 40 mg kg–1; P<0.0001 vs saline; MOR–/– vs WT: P<0.0001 (two-way ANOVA). (C) Morphine in the tail immersion test. MOR–/–, all doses not significant vs saline (0 mg kg–1); WT, 10, 20, 40, 80, and 100 mg kg–1: P<0.0001 vs saline; MOR–/– vs WT: P<0.0001 (two-way ANOVA). (D) Morphine in the hotplate test. MOR–/–, all doses not significant vs saline (0 mg kg–1); WT, 20, 40, 80, and 100 mg kg–1: P<0.0001 vs saline; MOR–/– vs WT: P<0.0001 (two-way ANOVA). Values are mean (SEM). MPE is maximum possible effect.

 

    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Data from studies in knockout mice and quantitative trait locus analysis in recombinant inbred mice indicate that the MOR gene is associated with morphine-induced analgesia.5 7 8 17 While it is known that opioids exert their respiratory effect at central sites (i.e. within the central nervous system),18 19 few studies have addressed the molecular sites of action of the respiratory actions of morphine,5 8 and none have addressed the respiratory effects of the morphine metabolite, M6G.

The present study shows that mice with a disruption of exon 2 of the MOR gene and consequently deficient in the MOR gene product did not display any respiratory effects in response to M6G. In contrast, M6G given to WT mice caused dose-dependent respiratory depression. In these respects, M6G behaved similarly to morphine although the potency of M6G was 3–4 times greater in mice with intact MOR. These findings indicate that the MOR gene product is the molecular site of action of the respiratory effect of M6G. In addition and in contrast to WT mice, MOR knockout animals did not display M6G and morphine-induced antinociception; hence, our data indicate that opioid analgesia and respiratory depression are mediated by a common MOR system.

Our data implicate the MOR gene as mediator of the respiratory effects of M6G. This is surprising taking into account M6G’s affinity for the DOR and the finding that DOR activation leads to respiratory depression.1 4 20 It may be argued that the loss of MORs throughout development caused some alterations and/or (functional) compensations in the DOR system. There are some indications that DOR-related effects are critically dependent on normally functioning MOR.4 The consequence of the loss of MOR activity may then be the absence or reduction of DOR effect in response to M6G administration. For example, mice that lack the MOR gene show less antinociception from {delta}-agonists despite a normal expression of DORs in the brain.8 21 However, there are various indications that DOR-related respiratory responses are intact in the exon 2 MOR gene knockout mice. We showed previously that naloxone increases ventilatory frequency and the slope of the HCVR equally in MOR deficient and MOR intact mice.5 We relate this effect to disinhibition of normal DOR activity within the ventilatory control system. Another study in the same mouse strain showed unaltered respiratory responses to putative {delta}2-receptor agonists and an augmented response of {delta}2-receptor agonists in respiratory neurons using brain slice preparations.20 These studies indicate that the lack of M6G respiratory effects in our null mutant mice was exclusively related to the absence of the MOR gene and not to abnormal function of the DOR system.

A potential drawback of our study is the fact that we had no information on the end-tidal or arterial gas concentrations in the animals. We performed experiments against a mild hyperoxic background to prevent hypoxaemia related to atelectasis or hypoventilation. Because of the lack of knowledge on carbon dioxide concentrations at the site of the chemoreceptors the observed reduction of the slope of the HCVR may not only be determined by depression of respiratory neurons but also by an effect of opioids on metabolism. However, as the direct effect of opioids on respiratory neurons is much greater than their metabolic effects on respiration,22 we believe that the lack of blood gas values in our study did not affect any of our conclusions.

Our observation that M6G is 3–4 times more potent in depressing ventilation in WT mice is in agreement with previous animal studies showing M6G over morphine potency ratios varying from 5:1 to 10:1 after i.v., s.c., or intracerebroventricular administration.18 2325 We believe, however, that as a result of the i.p. administration of opioids we may have underestimated the effect of morphine. Following i.p. injection most drugs are transported to the liver via the portal vein. Subsequently, part of morphine but not M6G is metabolized (note that mice, in contrast to humans, do not metabolize morphine to M6G). Moreover, as we did not measure blood opioid concentrations, it is best to view ventilatory frequency ratios in conjuction with analgesic ratios. We observed M6G/morphine analgesic ratios varying from 3:1 (hotplate test) to 5:1 (tail flick test). These values clearly overlap with the M6G over morphine respiratory potency ratios. The data indicate that at equipotent analgesia, morphine and M6G produce equipotent respiratory depression in our mice-strain. Our findings corroborate a recent human study showing M6G/morphine potency ratios for respiratory and analgesic effects of 1:10 (analgesia) to 1:20 (respiration).26 However, our findings are at variance with older human studies claiming absence of any significant respiratory effects of M6G compared with morphine or even some respiratory stimulatory effects of M6G.27 28 We believe that this is a result of the relatively low doses of M6G tested in these human studies (all M6G data would be on the flat part of the dose–response relationship) and a lack of comparison of M6G respiratory effect to its analgesic effects.

Recent studies suggest the existence of distinct MOR involved in morphine and M6G analgesia.29 30 For example, in rats treatment with antisense probes targeting exon 1 of the MOR significantly reduced morphine analgesia but failed to block M6G analgesia, while probes targeting exons 2 or 3 decreased M6G but not morphine analgesia.30 Similarly, specific probes targeting different G-protein subunits indicate a distinct effect on morphine and M6G analgesia.31 Our current study does not permit any conclusions regarding the involvement of MOR subsystems, receptor splice variants or specific G-proteins in morphine vs M6G respiratory effects. Further respiratory studies using antisense techniques are needed to clarify this issue.

In WT animals, M6G caused a small but systematic hyperalgesic response in the hotplate test. This effect was non-opioid-related as it was not reversed by naloxone (data not shown). Our observation is in agreement with an earlier finding by Kitanaka and colleagues32 showing dose-independent M6G and heroin hyperalgesic responses in the hotplate but not tail flick tests in a different strain of MOR knockout mice (exon 1 MOR knockout based on C57/129Sv genetic background). We observed no hyperalgesic responses with morphine. This suggests that the results are agent specific and that possibly the combination of a specific agent and methodological issues may have produced the observed phenotype. However, the hyperalgesic effects were small and dose-independent which assumes no direct relationship between the M6G dose and effect or a ceiling effect in the methodology. Further studies are needed to resolve this issue and to determine whether M6G has an important hyperalgesic component, which offsets part of its analgesic effect. If so, this could be one of the causes of the relatively low potency of M6G in human studies.11

In conclusion, our data obtained in exon 2 MOR knockout mice indicate that M6G and antinociception (desired) respiratory depression (undesired) are linked to the same gene product, the MOR. Other opioid- and non-opioid-receptor systems, such as the DOR system, may play a minor role in the respiratory effects of M6G and morphine. The clinical implications of our findings are that any agent acting at the MOR will invariably cause (potent) analgesia in combination with (variable) respiratory depression. M6G does not differ in this respect from morphine.


    Acknowledgement
 
The authors thank CeNeS Ltd, Cambridge for their generous donation of morphine-6-glucuronide.


    References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
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