Biotransformation of Morphinone and Its Glutathione Adduct Derived from Morphine by Anaerobic Gut Microbes in Guinea Pigs

Abstract

Morphinone (MO) and its glutathione adduct (MO-GSH) are excreted into bile of guinea pigs after subcutaneous administration of morphine (M). In the present study, we examined metabolites of M in guinea pig feces. Surprisingly, minimal amounts of MO and MO-GSH were excreted into the feces, whereas dihydromorphine (DHM) and dihydromorphinone (DHMO), which are not found in bile of guinea pigs administered M, were detected in the feces. Incubation of MO and MO-GSH with the contents of the large intestine under anaerobic conditions resulted in their conversion into DHMO. These results suggest that MO-GSH undergoes C–S cleavage by gut microbes to form MO, which is anaerobically reduced to DHMO excreted into feces.

INTRODUCTION

Morphine (M) is narcotic analgesic whose metabolism has been extensively studied to understand its pharmacological actions and side-effects. Morphine-3-glucuronide (M-3-G) is a major metabolite of M,¹⁾ while dihydromorphine (DHM) and hydromorphone, also known as dihydromorphinone (DHMO), are urinary metabolites.^2,3) Our group previously purified a nicotinamide adenine dinucleotide (phosphate) [NAD(P)]-dependent enzyme from guinea pig cytosol that catalyzes the conversion of M to morphinone (MO).⁴⁾ We subsequently developed a method for simultaneous quantitation of M and its metabolites using HPLC and found that MO and its glutathione adduct (MO-GSH), were excreted in the bile of guinea pigs given M (25 mg/kg), whereas the biliary excretions of DHM and DHMO were minimal.⁵⁾ Since biliary metabolites are excreted into feces through the intestinal tract, the present study examined the fecal excretion of biliary metabolites of M and the biotransformation of MO and MO-GSH by gut microbes in guinea pigs.

MATERIALS AND METHODS

Chemicals

M was obtained as a hydrochloride salt from Takeda Chemical Industries, Ltd. (Osaka, Japan). MO and MO-GSH were synthesized using the methods of Rapoport et al.⁶⁾ and Ishida et al.,⁷⁾ respectively. DHM and DHMO were synthesized using the methods of Rapoport et al.⁸⁾

Determination of M from Its Related Metabolites Using HPLC

M, MO, MO-GSH, DHM, and DHMO were separated using HPLC, following the method of Kumagai et al.⁵⁾

Purification and Identification of Fecal Metabolites of M

This study was approved by the Center for Experimental Animals at Fukuoka University and carried out in accordance with the guidelines of the center. Ten male guinea pigs (Hartley strain, 400–500 g) were subcutaneously administered 25 mg/kg M every 2 d (10 doses in total), with feces being collected 24 h after each injection. The feces were homogenized in five volumes of 1N HCl, then an equal volume of methanol was added and the samples stored at −80 °C until use. The homogenates were centrifuged at 5000 × g and the supernatant was concentrated using a rotary evaporator. After the resulting aqueous layer was neutralized using NaOH and 0.2 M sodium phosphate buffer (pH 7.4), 10 mL volumes were poured onto a Sep-Pak C18 cartridge (Waters Corporation, U.S.A.), then the cartridge was washed with 10 mL of water to remove polar components of the feces and metabolites were eluted with 2 mL methanol. The eluates were combined and evaporated to dryness under N₂ gas. Fecal metabolites in the residue were isolated according to procedures as described in legend of Fig. 1A. Two highly purified metabolites, designated M-1 and M-2, were analyzed using 1) a Silica Gel 60 F₂₅₄ TLC plate (0.25 × 100 × 50 mm, Merck, Germany) with chloroform/methanol (9 : 2 v/v) as a solvent and Dragendorff’s reagent or 2,4-dinitrophenylhydrazine used for detection, and 2) FAB-MS, as reported previously.⁷⁾

Fig. 1. Identification of Fecal Metabolites of Morphine (M)

A. Purification procedure for metabolites M-1 and M-2 in feces of guinea pigs administered M. Solvent a, 10 mM sodium phosphate buffer (pH 6.8)/acetonitrile (3 : 2 v/v); solvent b, 10 mM sodium phosphate buffer (pH 6.8)/acetonitrile (5 : 2 v/v); solvent c, 10 mM sodium phosphate buffer (pH 6.5)/acetonitrile (1 : 2 v/v). B. Chromatograms of extracts of feces from guinea pigs administered M. Two different HPLC systems were used. Left panel: YMC-Pack AL-312 ODS column (5 µm particles; 150 × 6 mm I.D.; solvent, 10 mM sodium phosphate buffer (pH 6.8)/acetonitrile (3 : 2 v/v). Peaks 1, 2, and 3 are assigned to M, dihydromorphine (DHM) and dihydromorphinone (DHMO), respectively. Right panel: Waters Nova-Pak C18 column (5 µm particles; 100 × 8 mm I.D.; solvent, 10 mM sodium phosphate buffer (pH 3.15) containing 1 mM sodium lauryl sulfate/acetonitrile (3 : 1 v/v). Peaks 1 is assigned to M and/or DHM, and peak 2 is assigned to DHMO. C. Thin-layer chromatograms of M (1), DHM (2), DHMO (3), M-1 (4) and M-2 (5). Left panel, sprayed with Dragendorff’s reagent; right panel, sprayed with 2,4-dinitrophenylhydrazine. Rf: retention factor. The second lane from the right is a co-chromatogram of synthetic DHMO and the metabolite assigned to DHMO. D. Fast atom bombardment mass spectra of DHMO (upper panel) and M-1 (bottom panel). E. Fast atom bombardment mass spectra of DHMO (upper panel) and M-2 (bottom panel).

Incubation of M and Its Related Metabolites with Gut Microbes

Contents of large intestines harvested from three guinea pigs were immediately collected and homogenized in four volumes of 0.1 M phosphate buffer (pH 7.4), then stored under N₂ gas on ice before use. The incubation mixture (10 mL) consisted of M or its related metabolites, the contents of the large intestine (equivalent to 0.2 g wet weight) and 50 mM sodium phosphate buffer (pH 7.4). N₂ gas was pumped into one side of an Erlenmeyer flask, and gas was aspirated from another side to maintain anaerobic conditions. The reaction was carried out at 37 °C for 60 min and then terminated by exposing the mixture to air.

Purification and Identification of Metabolites of MO Produced by Contents of the Large Intestine

A reaction mixture (300 mL) consisting of 0.2 mM MO, contents of the large intestine (equivalent to 20 g wet weight) and 50 mM sodium phosphate buffer (pH 7.4) was incubated at 37 °C for 4 h under anaerobic conditions. The reaction was terminated by exposure to air, then membrane-filtered. Metabolites in the filtrate were purified using the same procedure applied to fecal metabolites of M, as described above.

RESULTS

We previously established a method for simultaneous separation of free and bound metabolites of M using two different HPLC systems.⁵⁾ Using such systems with chemical standards, we found that M, DHM and DHMO were excreted into feces of guinea pigs administered M, whereas MO and MO-GSH, which are excreted into the bile,⁵⁾ were not detected (Fig. 1B). Following the procedure in Fig. 1A, we were able to purify M-1 and M-2 as fecal metabolites of M. While both M-1 and M-2 reacted positively to Dragendorff’s reagent, only M-1 reacted negatively to 2,4-dinitrophenylhydrazine on the TLC plate (Fig. 1C). In addition, the retention times of M-1 and M-2 were identical to those of authentic DHM and DHMO, and co-chromatography of all compounds supported the assignment of M-1 as DHM and M-2 as DHMO (Fig. 1C, second lane from right). FAB-MS spectra of M-1 and M-2 exhibited [M + H]⁺ peaks at m/z values of 288 and 286, respectively, consistent with the respective values for synthetic DHM and DHMO (Figs. 1D, E). Furthermore, high-resolution FAB-MS analysis indicated that the molecular weights of synthetic DHM (288.1599 for C₁₇H₂₂N₁O₃) and DHMO (286.1463 for C₁₇H₂₀N₁O₃) were almost identical to those of M-1 (288.1593 for C₁₇H₂₂N₁O₃) and M-2 (286.1459 for C₁₇H₂₀N₁O₃), respectively.

MO-GSH (a major metabolite of M) and MO are known to be excreted into bile in guinea pigs.⁵⁾ We therefore postulated that MO-GSH and MO are biotransformed into unknown metabolites in the intestinal tract prior to excretion into feces. To address such an issue, chemical standards were incubated with the contents of small and large intestines under aerobic or anaerobic conditions and analyzed using HPLC. Under aerobic conditions, minimal metabolite formation from M, MO, MO-GSH, M, DHM or DHMO was measured (data not shown). However, anaerobic incubation of the contents of the large intestine (but not small intestine) with MO-GSH resulted in formation of MO and DHMO as determined by HPLC (Fig. 2A), and incubation with MO resulted in DHMO formation (data not shown). Incubation of either MO-GSH or MO with the boiled contents of the large intestine did not produce DHMO, suggesting anaerobic bacteria participate in metabolite formation. Following the purification procedure for fecal metabolites of M (Fig. 1A), we confirmed using TLC (Fig. 2B) and FAB-MS (Fig. 2C) that the C7–C8 double bond of MO could undergo reduction in the large intestine to yield DHMO under anaerobic conditions. High-resolution FAB-MS analysis further supported the assignment of the metabolite to DHMO (observed molecular weights: authentic DHMO, 286.1463 for C₁₇H₂₀N₁O₃; metabolite, 286.1471 for C₁₇H₂₀N₁O₃).

Fig. 2. Identification of Products of Morphinone Reaction with the Contents of the Large Intestine of Guinea Pigs

A. High-performance liquid chromatogram of the products of reaction of the MO-GSH adduct with the contents of guinea pig large intestines under anaerobic conditions. Peaks 1 and 2 are assigned to MO and DHMO, respectively. Column, YMC-Pack AL-312 ODS (5 µm particles; 150 × 6 mm I.D.); solvent, 10 mM sodium phosphate buffer (pH 6.8)/acetonitrile (3 : 2 v/v); flow rate, 2 mL/min. B. Thin-layer chromatograms of MO (1), synthetic DHMO (2) and the metabolite assigned to DHMO (3). Left panel, sprayed with Dragendorff’s reagent; right panel, sprayed with 2,4-dinitrophenylhydrazine. C. Fast atom bombardment mass spectra of synthetic DHMO (upper panel) and the metabolite assigned to DHMO (bottom panel). D. Metabolism of M in guinea pig liver and large intestine. AKR, aldo-keto reductase; GSH, glutathione; GST, GSH S-transferase.

DISCUSSION

We previously found that aldo-keto reductase isozymes catalyze oxidation of M to an electrophilic metabolite MO⁴⁾ that covalently binds to the low-molecular-weight nucleophile glutathione with and without glutathione S-transferases to form MO-GSH.⁵⁾ It was also shown that MO and MO-GSH are excreted into the bile of guinea pigs administered M (25 mg/kg) and that the bile concentrations of M-3-G and MO-GSH reach approximately 7–10% of the administered dose, suggesting that MO-GSH is a major metabolite of M in guinea pigs.⁵⁾ The present findings indicate that further biotransformation of excreted MO to DHMO is mediated by gut microbes in the large intestine. Although DHMO is a urinary metabolite of M,³⁾ this is first study to show that DHMO is formed via the reduction of the C7–C8 double bond of MO. Notably, the gut microbes in the contents of the large intestine were capable of cleaving the C–S bond of MO-GSH to convert it back to MO, which is further metabolized to DHMO under anaerobic conditions (Fig. 2D). Several studies have characterized C–S bond-cleaving enzymes in intestinal microorganisms^9–11); these enzymes catalyze β-elimination reactions of a xenobiotic–cysteine adduct, leading to formation of a xenobiotic–SH adduct and alanine. In addition, Tomizawa et al.⁹⁾ reported that the C–S lyase purified from Fusobacterium varium has a high substrate specificity for S-(p-bromophenyl)-L-cysteine and S-phenyl-L-cysteine, whereas it has low substrate specificity for S-methyl-L-cysteine and L-cysteine. This suggests that other enzymes may be involved in the biotransformation of MO-GSH to MO.

MO is electrophilic metabolite with α,β-unsaturated carbonyl group and found to covalently bind to cellular proteins including opioid receptors^11,12) and to cause cytotoxicity.¹³⁾ In contrast, DHMO without α,β-unsaturated carbonyl group appears to be inactive metabolite. Consistent with this notion, MO covalently modified Keap1 and activated transcription factor nuclear factor-E2-related factor 2 (Nrf2), whereas DHMO had little effect on the activation of Keap1/Nrf2 pathway in HepG2 cells (Matsuo et al., unpublished observation). Thus, the present study suggests that MO is detoxified through conversion of DHMO by anaerobic gut microbes.

Acknowledgments

I thank Reiko Hirose, University of Tsukuba for editing of the figures of this manuscript. This work was supported by Grants-in-Aid (#18H05293 to Y.K.) for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Conflict of Interest

The author declares no conflict of interest.

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