Morphine hydrochloride was purchased from the Takeda Chemical Industries (Osaka, Japan). M3G was a generous gift from Prof. Hideyuki Yamada, Kyushu University (Fukuoka, Japan). Diclofenac, naloxone, uridine 5’-diphosphoglucronic acid (UDPGA), and alamethicin were purchased from Sigma-Aldrich (St. Louis, MO).

Male Fisher rats (7 - 10-week-old; 170 - 200 g) were purchased from Sankyo Laboratory Animal Co., Ltd. (Hamamatsu, Japan). Rats were housed under a 12-h light/dark cycle with free access to food and water. All animal procedures were carried out in accordance with the standards set forth in the Guidelines for the Care and Use of Laboratory Animals at the Takara-machi campus of Kanazawa University, and the experimental protocol was approved by the Institutional Animal Care and Use Committee of Kanazawa University, Japan.

Morphine glucuronosyltransferase activity was determined as described by Hara et al. [5] . A typical incubation mixture (0.2 mL total volume) contained 50 mM Tris-HCl buffer (pH 7.4), 5 mM MgCl₂, 5 mM UDPGA, 25 µg/mL alamethicin, 0.25 mg/mL microsomal protein, and 25 - 200 µM morphine. The reaction was initiated by addition of UDPGA. After incubation at 37˚C for 30 min, the reaction was terminated by the addition of 0.1-mL ice-cold perchloric acid. After removal of protein by centrifugation at 10,000 g for 5 min, at 4˚C, a 100-mL portion of the supernatant was subjected to HPLC (HPLC method is described below). All data were analyzed using the mean of triplicate determinations. Dixon plots were used for determining the type of inhibition. Kinetic parameters were determined by a nonlinear regression analysis using SigmaPlot 13 (Hulinks, Tokyo).

Change in intrinsic clearance (CLint) is expressed using the following equation [7] : CLint (+inhibitor)/CLint (−inhibitor) = 1/(1 + I/Ki), where I is the concentration of the inhibitor and Ki is the inhibition constant. Because data on liver concentrations and protein binding of diclofenac in tissues are not available, maximum plasma concentrations were used.

Blood samples were taken before drug administration as well as at 0.25, 0.5, 0.45, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, 12, 24, 35, and 36 h after subcutaneous (s.c.) administration of morphine and intraperitoneal injection (i.p.) of either saline (vehicle) or diclofenac from the jugular vein under light ether anesthesia. Blood samples were centrifuged after 30 min and serum was stored at −20˚C until analysis. Bile samples were collected via bile duct cannulation at 0 - 1.5, 1.5 - 3, 3 - 4.5, and 4.5 - 6 h after dose administration. Bile pools for each time interval were created by proportional mixing of samples from each rat. Urine was collected before drug administration and at 0 - 3, 3 - 6, and 6 - 12 h after drug administration. The volume was measured and an aliquot of each collection was stored at −20˚C until analysis.

Morphine and M3G concentrations were extracted by solid-phase extraction and measured by HPLC as described by Hasegawa et al. [8] . Solid-phase extraction was performed using Inertsep C18-C FF cartridges (GL Science Inc., Tokyo, Japan.). 200 μl Samples were added to 20 μl naloxone (internal standard) and loaded onto the cartridges. The plasma samples were loaded undiluted; urine samples were loaded after dilution in saline. The eluent was evaporated at 50˚C under reduced pressure. The residue was dissolved in 300 μl mobile phase, and an aliquot was injected onto the HPLC system. The reverse-phase column (TSKgel ODS-80TM., Tokyo, Japan) was maintained at 50˚C. The mobile phase was [0.1 M phosphate buffer (pH 2.5), acetonitrile, and methanol (72:24:2 v/v)] delivered at a constant flow of 1 ml/min for a total run time of 70 min. Morphine was detected using an electrochemical detector (Coulochem II; Esa Inc., Chelmsford, MA, USA). M3G was quantitated by a sensitive and specific HPLC method using fluorescence detection (excitation: 210 nm; emission: 350 nm).

Pharmacokinetic parameters were estimated using model-independent moment analysis as described by Yamaoka et al. [9] . Data were analyzed using Student’s t-test to compare the unpaired mean values of two datasets. The number of determinations is noted in each table and figure. p value <0.05 was considered to be statistically significant.

Analgesic effects of morphine with and without simultaneous administration of diclofenac were assessed using the Von Frey test as described by Shinoda et al. [10] . Briefly, the mechanical sensitivity of the plantar surface of the hind paw was assessed using Von Frey hairs [11] . To assess changes in mechanical nociceptive thresholds, rats were placed in cages with a mesh floor covered with transparent plastic boxes and were allowed to acclimate to their surroundings for a minimum of 15 min before testing in a temperature-controlled room (24˚C). The von Frey hairs were pressed against the plantar surface of the hind paw and withdrawal response frequency was measured from 5 trials. For each trial, the filament was applied at 1 min intervals. Paw-withdrawal threshold was defined as the minimum pressure needed to evoke a response in at least 60% of the trials. Subsequently, the changes from baseline were determined and for each study day the area under the pain threshold change versus time curve was calculated.

The anti-nociceptive effect was expressed as a percentage of the maximum possible effect (MPE): %MPE = (post-drug threshold − pre-drug threshold)/(maximum threshold − pre-drug threshold) × 100, where pre-drug threshold is the mean of the paw-withdrawal threshold in the saline group, post-drug threshold is the paw-withdrawal threshold of each animal treated with drug, and maximum threshold is the mean of the paw-withdrawal threshold in the sham-saline group. The area under the pain threshold versus time curves were presented as mean ± SD and were compared using Wilcoxon matched pairs test. p value <0.05 was considered significant.

Inhibitory effects of drugs on morphine glucuronosyltransferase activities in rat liver microsomes are shown in Figure 1. Our results indicated that morphine glucuronosyltransferase activities were strongly inhibited by diclofenac. The IC₅₀ value of diclofenac was 19.9 ± 3.80 μM, and diclofenac exhibited competitive inhibition for morphine glucuronosyltransferase activities.

We examined the possibility of drug-drug interactions via a metabolic process between morphine and diclofenac. The 1 + I/Ki values calculated for diclofenac were 2.65 for morphine glucuronosyltransferase activities, indicating that the change of plasma

concentration would be because of the inhibition of morphine glucuronidation by diclofenac.

Plasma concentrations of morphine after s.c. administration in rats pretreated with vehicle or diclofenac are shown in Figure 2. The plasma concentration of morphine in vehicle-pretreated rats reached a maximum (3.90 µg/ml) of 15 min after administration. (Figure 2(a)) In diclofenac-pretreated rats, the plasma concentration was 7.2 µg/ml at 15 min. The plasma concentration was markedly higher in diclofenac-pretreated rats than in vehicle-treated controls for all time points. AUC was also larger (2.14 fold) in diclofenac-pretreated rats than that in vehicle-pretreated rats. Diclofenac treatment significantly decreased the CL_total (by 54.6%) and volume of distribution of morphine (by 51.9%) (Table 1). The plasma concentration time profiles of M3G are shown in Figure 2(b). Concentration of M3G in the plasma in diclofenac-pretreated rats was increased by 1.38 fold at 30 min and 3.34 fold at 90 min compared to that in vehicle-pretreated rats (Figure 2).

The urinary and biliary excretion ratios of morphine and M3G are shown in Figure 3. The biliary excretion ratios of morphine and M3G and the urinary excretion of M3G were similar in the two groups. However, urinary excretion of morphine was significantly lower in diclofenac-pretreated rats [53.7% (0 - 3 h) of vehicle] (p < 0.05).

The anti-nociceptive effect of morphine was determined by the Von Frey test in vehicle- and diclofenac-pretreated rats as shown in Figure 4. The anti-nociceptive effect of morphine was 18.0% - 68.8% of the MPE in vehicle-pretreated rats. Administration of diclofenac increased the anti-nociceptive effect of morphine to 40.9% - 98.4 % MPE. The area under the anti-nociceptive effect time curve was 1.57-fold greater in diclofenac-pretreated rats than that in vehicle-pretreated rats (16800 ± 790 vs. 9700 ± 855 %MPE・min; mean ± SE; n = 6).