Introduction

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Conjugation with glucuronic acid to yield 1-O-acyl--D-glucopyranuronates (1-O-acyl glucuronides) is a major metabolic route for many carboxylate drugs including nonsteroidal anti-inflammatory and hypolipidemic drugs (Dutton, 1980). It is well known that 1-O-acyl glucuronides are labile and reactive. These compounds undergo both hydrolysis and internal acyl migration. In the acyl migration, the aglycone is transferred to the C-2, C-3, or C-4 position of the glucuronic acid ring (Spahn-Langguth and Benet, 1992) (fig. 1). In general, acyl migration has been observed to predominate over hydrolysis under physiological conditions (Akira et al., 1997a; Blanckaert et al., 1978; Bradow et al., 1989; Hansen-Moller et al., 1988; Iwakawa et al., 1988; Iwaki et al., 1995; Nicholls et al., 1996; Sidelmann et al., 1996a). Furthermore, the 1-O-acyl glucuronides of many carboxylate drugs have been shown to react with proteins to form covalent adducts (Dickinson and King, 1991; Dubois et al., 1993; Kretz-Rommel and Boelsterli, 1994; Presle et al., 1996; Volland et al., 1991). Covalent binding seems to occur mainly via the formation of a Schiff's base linkage between the free aldehyde of the open-chain acyl-migrated glucuronide and a nucleophilic amine group on the protein, in the drug acyl glucuronides that readily undergo acyl migration (Dickinson and King, 1991; Ding et al., 1995; Kretz-Rommel and Boelsterli, 1994; Smith et al., 1990). The isomeric glucuronides differ from one another in reactivity for protein adduct formation (Dickinson and King, 1991). Currently, there is speculation that protein adducts are at least partially responsible for immunological side effects of carboxylate drugs (Spahn-Langguth and Benet, 1992; Worral and Dickinson, 1995; Zia-Amirhosseini et al., 1995).

View larger version (8K): [in this window] [in a new window]   Fig. 1.   Reaction scheme for the acyl migration of 1-O-acyl glucuronides.

1-O-Acyl glucuronides are usually excreted in the bile and then hydrolyzed by -D-glucuronidase in the small intestinal tract, where the unconjugated drugs are reabsorbed into the systemic circulation (enterohepatic circulation). In addition, 1-O-acyl glucuronides are hydrolyzed by tissue and serum esterases. The aglycones formed by these enzymes are then again available for other biotransformations as well as glucuronide conjugation. The isomeric glucuronides formed by acyl migration have often been described as being resistant to -D-glucuronidase (Blanckaert et al., 1978). Dickinson et al. (1986) have suggested that the isomeric glucuronides of valproic acid are resistant to serum esterase. Therefore, the amount of drug regenerated by these enzymes can be decreased if the drug 1-O-acyl glucuronides can undergo rapid acyl migration. Thus, acyl migration is intimately related to drug disposition.

The lability of 1-O-acyl glucuronides themselves has been successfully investigated by reversed phase high performance liquid chromatography (HPLC)¹ using acidic mobile phase (Spahn-Langguth and Benet, 1992), where the 1-O-acyl glucuronides are relatively stable and the 1-O-acyl glucuronides and the aglycones are easily distinguished from the isomeric glucuronides. However, details of the formation and degradation of each isomer following the degradation of 1-O-acyl glucuronides have been ambiguous because HPLC separation of the various isomers to one another is much more difficult. Moreover, the isomers can ring-open and mutarotate giving - and -anomers, which further complicates the HPLC separation. Thus, a more direct and specific method is required to assess the overall reactivities of 1-O-acyl glucuronides, including the formation and degradation of the isomers.

Nuclear magnetic resonance (NMR) spectroscopy is suitable for the determination of such unstable compounds because it allows reaction mixtures to be analyzed without extraction and chromatographic separations. NMR spectroscopy also allows a "real-time" analysis of the reactions that proceed in the NMR tube under defined physicochemical conditions. Bradow et al. (1989) have examined the reactivity of 1-O-acyl glucuronide by ¹H NMR spectroscopy. However, spectral resolution was not sufficient because of the spectral complexity owing to the formation of many isomers, the small chemical shift range, and the disturbance by residual water. Although ¹⁹F NMR spectroscopy has been effectively used to monitor the acyl migration of 1-O-acyl glucuronides of 2-, 3-, and 4-(trifluoromethyl)benzoic acids (Nicholls et al., 1996), it is not a general method as it can not be applied to non-fluorinated compounds. ¹³C NMR has a chemical shift range that is large and comparable with that of ¹⁹F NMR, but in addition the range of drugs that can be studied is virtually unlimited. A major disadvantage is that the sensitivity of ¹³C NMR is poor because of the low natural abundance (1%) of ¹³C and the low gyromagnetic ratio. However, ¹³C NMR can be effectively used together with ¹³C labeling (approximately 100% enrichment) (London, 1988). We have demonstrated that the NMR approach with ¹³C labeling is useful in pharmacokinetic research in terms of sensitivity and specificity (Akira and Shinohara, 1996; Akira et al., 1993; Baba et al., 1995). The reactivity of benzoyl glucuronide has been recently elucidated by this ¹³C NMR approach (Akira et al., 1997a).

Ketoprofen (KP, see fig. 2) is one of the chiral 2-arylpropionic acids (profens), an important group of nonsteroidal anti-inflammatory drugs (NSAIDs), which is clinically used as the racemate and eliminated predominantly as diastereomeric acyl glucuronides in humans (Foster et al., 1988a). Upton et al. (1980) were the first to describe the susceptibility of (RS)-KP acyl glucuronides to chemical hydrolysis. Subsequently, the hydrolysis half-lives of (R)- and (S)-KP acyl glucuronides were reported to be greater than 24 hr under physiological conditions (Hayball et al., 1992). However, susceptibility to acyl migration was not investigated in these studies because the glucuronides were not directly analyzed using HPLC. Thus, in the present study, we have investigated in detail the stereoselective reactivity of (R)- and (S)-KP acyl glucuronides under physiological conditions using the ¹³C labeling in the ester carbonyl group of the glucuronides and NMR.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Structures of 13C-labeled KP and KP glucuronide. The asterisk indicates an assymetric center.

	Materials and Methods

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Chemicals. (R)- and (S)-KP acyl glucuronides ¹³C-labeled in the ester carbonyl carbon [1-O-((R)-2-(3-benzoylphenyl)-[1-¹³C]propyl)--D-glucuronic acid and 1-O-((S)-2-(3-benzoylphenyl)-[1-¹³C]propyl)--D-glucuronic acid] were prepared according to the previously reported method (Akira et al., 1997b). N²H₃ ²H₂O solution (16 M, 99.0 atom % ²H) was purchased from Aldrich (Milwaukee, WI). Other reagents including ²H₂O (>99.80 atom % ²H) and 40% NaO²H ²H₂O solution (99.0 atom % ²H) were purchased from Kanto Chemical (Tokyo, Japan).

NMR Spectroscopic Monitoring of Degradation. (R)-[¹³C]KP acyl glucuronide (2 mg), (S)-[¹³C]KP acyl glucuronide (2 mg), or a mixture of both labeled diastereomers (each 1 mg) was dissolved in 475 µl of 0.1 M phosphate buffer (pH 7.4). After adding of 25 µl of ²H₂O to provide a ²H signal for field frequency lock, each solution was transferred to a 5-mm NMR tube with a coaxial capillary tube (1.7-mm o.d., 1.0-mm i.d.) containing 1,4-dioxane as the reference for chemical shifts and quantitation. After the pH of the solution was measured (7.20 ± 0.05), the NMR tube was immediately inserted in the NMR probe set at 310 K, and the reaction was monitored by ¹³C NMR spectroscopy

Quantitation Method. ¹³C Resonance heights of [¹³C]KP acyl glucuronide (ester carbonyl), its isomers (ester carbonyl), and aglycone (carboxyl) in the reaction mixture were measured, and the height ratios of these signals relative to that of dioxane (internal standard) were calculated. These ratios were assumed to reflect the relative proportions of the various compounds contained in the reaction mixture (see text). The ratios were converted to micromoles, assuming that the sum of the ratios at each time point corresponds to the amount of [¹³C]KP acyl glucuronide initially dissolved (4.6 µmol).

Identification of the Isomeric Glucuronides by Two-Dimensional NMR Spectroscopy. (R)- or (S)-[¹³C]KP acyl glucuronide (3 mg) dissolved in 500 µl of ²H₂O was transferred into a 5-mm NMR tube. To the solution was added 5 µl of 3.2 M N²H₃ ²H₂O solution to decompose the [¹³C]KP acyl glucuronide to its isomers and aglycone. The reaction was followed by ¹³C NMR spectroscopy. When the relative amounts of the isomers had stabilized, the sample was freeze-dried to remove the alkali and stored at 20°C until analyzed. The residue was reconstituted in 450 µl of ²H₂O, and then the resultant solution was transferred to a 5-mm NMR tube after filtration, followed by two-dimensional NMR spectroscopy (HMBC, COSY, TOCSY) using a Bruker AM500 spectrometer, operated at 500 MHz. The internal H²HO signal was used as a lock reference, and shift assignments were made relative to it (¹H 4.78). The composition of the reaction mixture was almost constant during the time-consuming two-dimensional NMR measurements.

Kinetic Analyses. The differential equations fitted to the model (see fig. 8) describing the degradation kinetics of KP glucuronides were constructed, assuming that the acyl migration, hydrolysis, and anomerization follow first-order kinetics (see Appendix). The equations were solved by a kinetic simulation program, which uses the Runge-Kutta method as an algorithm and the steepest descent method for the optimization.

	Results

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The lability of KP glucuronides in phosphate buffer (pH 7.4) at 37°C was directly examined in the NMR tube. With time, the intensity of ¹³C signals due to the 1-O-acyl glucuronides decreased with concurrent and sequential appearance of several signals at other chemical shifts as shown in figs. 3 and 4. The signal at 185.8, which was well separated from other resonances, was assigned to the aglycone formed by hydrolysis by comparison of the chemical shift with that of the authentic [¹³C]KP. Other signals were obviously because of the isomeric glucuronides (2-O-acyl, 3-O-acyl, and 4-O-acyl), as they completely disappeared and the signal due to the aglycone increased by addition of alkaline to the sample (not shown). The signals of the positional isomers were tentatively assigned based on the order in which they were formed in the incubation mixture, assuming that the 2-O-acyl isomer is necessarily formed before the 3-O-acyl isomer etc. This successive acyl migration between the neighboring hydroxyl groups has been demonstrated in numerous drugs (Blanckaert et al., 1978; Bradow et al., 1989; Hansen-Moller et al., 1988; Nicholls et al., 1996; Sidelmann et al., 1996b). Splitting of the signals for the isomeric glucuronides, except for (R)-4-O-acyl isomer, was observed because of formation of both - and -anomers (approximately 1:1 proportion) by mutarotation after acyl migration (Sidelmann et al., 1996a, 1996c). The (R)-4-O-acyl isomer appeared as a single peak in all the experiments.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   13C NMR spectra as a function of time, showing degradation of (R)-[13C]KP glucuronide in phosphate buffer (pH 7.4) at 37°C. All spectra were plotted out at the fixed resonance height of the internal standard. The spectra correspond to the following times: A, 15-25 min; B, 60-70 min; C, 135-145 min; D, 270-280 min; E, 450-460 min.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   13C NMR spectra as a function of time, showing degradation of (S)-[13C]KP glucuronide in phosphate buffer (pH 7.4) at 37°C. All spectra were plotted out at the fixed resonance height of the internal standard. The spectra correspond to the following times: A, 15-25 min; B, 120-130 min; C, 240-250 min; D, 450-460 min; E, 860-870 min.

The assignments of the acyl ¹³C signals due to each isomeric glucuronide was confirmed by measurements of the two-dimensional NMR spectra of the reaction mixture. The reaction mixture containing all the isomeric glucuronides was obtained by addition of N²H₃ to the solution of [¹³C]KP glucuronide followed by freeze-drying and reconstitution in ²H₂O. First, the acyl ¹³C signals were assigned to the signals owing to the protons on the acylated carbons based on the HMBC spectrum (fig. 5). Although the signals owing to the protons on the acylated carbons of (R)- and (S)-2-O-acyl isomers were obscured because of the H²HO resonance, the chemical shifts were determined based on the cross peaks. Subsequently, the ¹H-¹H COSY and TOCSY spectra of the reaction mixture were measured. The signals owing to protons around the glucuronide ring for each isomeric glucuronide were assigned based on chemical shifts, spin-spin coupling constants, and integrals (Kaspersen and van Boeckel, 1987) and on the connectivity information from the COSY and TOCSY experiments (table 1). The protons on the acylated carbons were thus assigned to the individual isomeric glucuronides. From these experimental results, the acyl ¹³C signals were assigned to the individual isomeric glucuronides. Consequently, the initial tentative identification based on the order of isomer appearance proved to be correct. Therefore, successive acyl migration from the 1 position to the 4 position for (R)- and (S)-KP glucuronides can be considered to be established. Also, the splitting of the ¹³C signals was confirmed as a result of anomerization. The ¹³C signals for the (R)-4-O-acyl and (R)-4-O-acyl isomers were found to be spectrally coincident.

View larger version (46K): [in this window] [in a new window]   Fig. 5.   HMBC spectra of the equilibrium mixtures of the isomeric glucuronides formed from (R)-[13C]KP glucuronide (A) and (S)-[13C]KP glucuronide (B).

The above experimental results demonstrated that almost all the isomeric glucuronides including - and -anomers can be discriminated by the combined use of ¹³C NMR and ¹³C labeling of the ester carbonyl carbon. Fig. 6 shows ¹³C NMR spectra of a mixture of (R)- and (S)-KP glucuronides dissolved in phosphate buffer (pH 7.4). The signals owing to KP glucuronides and their isomers formed by acyl migration were mostly discriminated from one another, although 3- and 3-O-acyl isomers were not discriminated between their R- and S-antipodes. These results show that the reactivity of diastereomeric glucuronides can be compared under identical physicochemical conditions using ¹³C labeling and NMR. It would be impossible to obtain such a high specificity of detection by HPLC.

View larger version (22K): [in this window] [in a new window]   Fig. 6.   13C NMR spectra of an equimolar mixture of (R)- and (S)-[13C]KP glucuronides in phosphate buffer (pH 7.4) at 37°C. The spectra correspond to the following times: upper trace, 20-40 min; lower trace, 420-440 min. The signal due to KP appeared at 185.8 as shown in figs. 3 and 4.

The amounts of KP glucuronide, its isomers, and aglycone present in the NMR tube were calculated using the resonance-height ratios of these nuclei vs. the internal standard (dioxane ¹³C 70). Because the monitored nuclei were all quaternary carbons, NMR characteristics (nuclear Overhauser enhancements and spin-lattice relaxation times) and sensitivity were considered to be very similar. In support of this assumption, the sum of the ratios at each time point was almost constant (coefficient of variation 4%). Therefore, the ¹³C NMR sensitivity of KP glucuronide, its isomers, and aglycone can be regarded as virtually the same and the ratios directly compared to one another. The ratios were converted to micromoles as described in Methods. The time-course of the acyl migration and hydrolysis of each KP glucuronide is shown in fig. 7. (R)- and (S)-KP glucuronides showed pseudo first-order degradation kinetics and apparently disappeared at 4.0 and 5.6 hr after dissolution, respectively. These results indicate that acyl migration from the 1 position to the 2 position of both glucuronides is irreversible, which is consistent with the notion that acyl migration of the 2-O-acyl isomer to the 1-O-acyl isomer is thermodynamically unfavorable. In contrast, the acyl migration reactions between 2-O-acyl and 3-O-acyl isomers, and 3-O-acyl and 4-O-acyl isomers are probably reversible. These observations are consistent with those of other workers (Blanckaert et al., 1978; Bradow et al., 1989; Nicholls et al., 1996) except for the study of diflunisal glucuronide isomers assayed with HPLC (Hansen-Moller et al., 1988). Acyl migration of KP glucuronide to the 2-O-acyl isomer was found to be a major pathway of transformation for the degradation, whereas the competing reaction of hydrolysis to KP was only minor under the conditions examined.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   An example of profiles for acyl migration, hydrolysis, and mutarotation reactions of (R)-[13C]KP glucuronide (A) and (S)-[13C]KP glucuronide (B) in phosphate buffer (pH 7.4) at 37°C. As the individual NMR spectra were accumulated over 10 min, the midpoint was used as the time data point. A: , 1; , 2; , 2; , 3; , 3; , 4 and 4; ×, KP. B: , 1; , 2; , 2; , 3; , 3; , 4; , 4; ×, KP. The simulated curves obtained from the calculated reaction rate constants are represented by the dotted lines.

The disappearance of (R)-KP glucuronide (t_1/2 = 0.66 ± 0.092 hr) was found to be much faster than that of (S)-KP glucuronide (t_1/2 = 1.26 ± 0.074 hr). To evaluate the stereoselective reactivity of (R)- and (S)-KP glucuronides in detail, the individual rates of acyl migration, hydrolysis, and anomerization were calculated based on the kinetic model shown in fig. 8 using a kinetic simulation program. The model assumes that the acyl migration reactions are reversible except for the initial acyl migration (12) and occur between the neighboring glucuronic acid hydroxyl groups via ortho-acid ester intermediates. In the case of (R)-KP glucuronide, the 4-O-acyl and 4-O-acyl isomers were not discriminated because of spectral coincidence. Sidelmann et al. (1996b) have constructed a kinetic model describing the degradation kinetics of 1-O-acyl glucuronide of a model drug assuming that no hydrolysis of the isomeric glucuronides occurs. However, in our case, the concentrations of aglycone significantly increased after KP glucuronide had disappeared, implying that the isomeric glucuronides were susceptible to hydrolysis (Volland et al., 1991). Thus, the above kinetic model includes the hydrolysis pathways of isomeric glucuronides. Differential equations fitted to the model were constructed and solved as described in Methods. The calculated rate constants for acyl migration, hydrolysis, and anomerization reactions are presented in table 2. The simulated curves obtained from those calculated rate constants are shown as dotted lines in fig. 7.

View larger version (21K): [in this window] [in a new window]   Fig. 8.   Kinetic model for the acyl migration reactions of KP glucuronides. In the case of (R)-KP glucuronide, the 4-O-acyl and 4-O-acyl isomers are not discriminated because of spectral coincidence, and thus the model is simplified.

The rate constant for acyl migration of 12 was much larger than that for hydrolysis of 1KP in all KP glucuronides. The rate constant of acyl migration 12 of (R)-KP glucuronide was two times larger than that of (S)-KP glucuronide, whereas the hydrolysis 1KP was not stereoselective. Thus, stereoselectivity in the disappearance of KP glucuronide is because of 12 acyl migration. Acyl migration of all the isomeric glucuronides showed relatively similar rate constants, which were smaller than those for the initial 12 acyl migration. The rate constants for hydrolysis were one order of magnitude smaller than those for acyl migration, although 1KP hydrolysis was significantly faster than the hydrolysis of other isomeric glucuronides. Anomerization of all the isomeric glucuronides showed similar rate constants for reaction in either direction, or , and were much larger than those for acyl migration and hydrolysis. These results are consistent with the appearance of twin resonances due to - and -anomers on the spectra shown in figs. 3 and 4 and indicated that only 12 acyl migration has significant stereoselectivity.

	Discussion

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The resolution of ¹³C-labeled compounds by NMR spectroscopy largely depends on the labeled position. In our previous paper (Akira and Shinohara, 1996), the lability of KP glucuronides was investigated using a diastereomeric mixture of [methyl-¹³C]KP glucuronides and ¹³C NMR spectroscopy with the aid of methyl--cyclodextrin as a shift reagent. The results of this study suggested that the glucuronides were susceptible to acyl migration as well as hydrolysis under physiological conditions, although NMR spectral resolution was poor. Recently, the individual glucuronides of (R)- and (S)-[carboxyl-¹³C]KP were prepared in our laboratory (Akira et al., 1997b) to improve spectral resolution between the glucuronide, aglycone, and isomeric glucuronides. In this paper, we have investigated the difference in reactivity between the diastereomers and the reaction kinetics of acyl migration and hydrolysis using these labeled compounds and ¹³C NMR spectroscopy.

Acyl glucuronides can be considered as reactive metabolites as they can irreversibly bind to endogenous proteins. Stereoselective covalent binding with plasma protein has been established in vitro for KP glucuronides (Presle et al., 1996), although covalent binding of KP glucuronides to proteins in vivo has not been reported. In contrast, drug-protein adducts have been observed in vivo with fenoprofen (Volland et al., 1991). Our results have shown that (R)-KP glucuronide is more susceptible to the covalent binding to proteins via acyl migration than its corresponding antipode. In healthy humans, KP is extensively metabolized to diastereomeric KP glucuronides and readily excreted in the urine (ca. 70% of dose in 24 hr). The diastereomeric ratio (S/R) of urinary KP glucuronides is ca. 1.2 (Foster et al., 1988a), which has been suggested to be because of a limited bioinversion of the R-enantiomer to the S-enantiomer (Jamali et al., 1990). Glucuronidation has been found to be stereoselective for several profens (Hamdoune et al., 1995; Mouelhi et al., 1987; Spahn, 1988). However, the formation ratio of the (R)- and (S)-KP glucuronides was almost 1 in human liver microsomes (Chakir et al., 1994). Whereas only negligible concentrations of KP glucuronides were present in the plasma of healthy humans after oral administration of racemic KP (Foster et al., 1988b), significant concentrations of KP glucuronides were detected in the plasma of patients with impaired renal function (Foster et al., 1988b; Grubb et al., 1996). In addition, the area under the plasma concentration vs. time curve for (S)-KP glucuronide was much higher than that for (R)-KP glucuronide in the patients. The reason for the stereoselectivity in these patients may be that (R)-KP glucuronide is more labile than (S)-KP glucuronide, which has been described in this paper. Dubois-Presle et al. (1995) have reported the presence of a stereoselective esterase activity toward the (R)-KP glucuronide in human serum albumin. Stereoselective hydrolysis could provide an alternative explanation for the lower plasma concentration of (R)-KP glucuronide.

The lability of acyl glucuronides of profens including fenoprofen (Volland et al., 1991), naproxen (Iwaki et al., 1995), benoxaprofen (Bradow et al., 1989; Spahn et al., 1989), carprofen (Iwakawa et al., 1988), and flunoxaprofen (Spahn, 1988) has been investigated under physiological conditions (pH 7.4, 37°C) by other workers using HPLC. In all the profen glucuronides, including the KP glucuronides investigated here, the degradation rate constant of the (R)-conjugate was 1.5 to 2 times larger than that of its (S)-antipode. The glucuronides of carprofen, benoxaprofen, naproxen, and KP underwent predominant acyl migration but only minor hydrolysis similar to most other drug acyl glucuronides (Akira et al., 1997a; Blanckaert et al., 1978; Bradow et al., 1989; Hansen-Moller et al., 1988; Iwakawa et al., 1988; Iwaki et al., 1995; Nicholls et al., 1996; Sidelmann et al., 1996a). (R)-Fenoprofen glucuronide undergoes both reactions equally, whereas (S)-fenoprofen glucuronide is predominantly subject to hydrolysis. Therefore, (R)-profen glucuronide seems to be much more susceptible to acyl migration than the corresponding (S)-profen glucuronide.

The present paper has elucidated the stereoselective reactivity of diastereomeric KP glucuronides. Using ¹³C labeling of the ester carbonyl carbon and NMR, the R-conjugate was found to be more susceptible to 12 acyl migration than its S-antipode under physiological conditions. This ¹³C NMR approach is a highly appropriate method to follow the overall reactions of acyl glucuronides because of the high specificity of detection and the lack of a requirement for pretreatment. Although the technique requires the synthesis of ¹³C-labeled drugs, labeling the carboxyl carbon of carboxylate drugs, such as acidic NSAIDs, is relatively easy (Akira et al., 1997b), and the labeled precursors for such compounds are inexpensive and available. The direct approach of using ¹³C labeling and NMR as presented in this paper could also provide insight into the reactivities of other labile drug acyl glucuronides and their isomeric glucuronides.