Comparison of morphine-6-glucuronide and morphine on respiratory depressant and antinociceptive responses in wild type and μ-opioid receptor deficient mice  R Romberg, E Sarton, L Teppema, H.W.D. Matthes, B.L. Kieffer, A Dahan  British Journal of Anaesthesia  Volume 91, Issue 6, Pages 862-870 (December 2003) DOI: 10.1093/bja/aeg279 Copyright © 2003 British Journal of Anaesthesia Terms and Conditions

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)=S0×[1 – 0.5×(d/ED50)γ], 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 γ 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). British Journal of Anaesthesia 2003 91, 862-870DOI: (10.1093/bja/aeg279) Copyright © 2003 British Journal of Anaesthesia Terms and Conditions

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). British Journal of Anaesthesia 2003 91, 862-870DOI: (10.1093/bja/aeg279) Copyright © 2003 British Journal of Anaesthesia Terms and Conditions

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)). British Journal of Anaesthesia 2003 91, 862-870DOI: (10.1093/bja/aeg279) Copyright © 2003 British Journal of Anaesthesia Terms and Conditions

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)=L0×[1+(d/ED200)γ] was fitted to the WT animals data (thick continuous lines), where L(d) is the latency after dose d, L0 the latency after saline, γ 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. British Journal of Anaesthesia 2003 91, 862-870DOI: (10.1093/bja/aeg279) Copyright © 2003 British Journal of Anaesthesia Terms and Conditions

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. British Journal of Anaesthesia 2003 91, 862-870DOI: (10.1093/bja/aeg279) Copyright © 2003 British Journal of Anaesthesia Terms and Conditions