Introduction

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-1-O-Acyl glucuronides are common phase II metabolites of drugs with a carboxylic acid functionality (Spahn-Langguth and Benet, 1992), including the widely used non steroidal anti-inflammatory drug S-naproxen (S)-6-methoxy--methyl-naphthalene acetic acid (Upton et al., 1980; Wilson and Ismail, 1986; Vree et al., 1992).

-1-O-Acyl glucuronides have been shown to be reactive metabolites that will hydrolyze, acyl migrate, and bind covalently to proteins causing potential toxicity (Spahn-Langguth and Benet, 1992; Boelsterli et al., 1995). This toxicity may be caused by formation of immuno-reactive glucuronide protein adducts (Worrall and Dickinson, 1995), but toxicity mechanisms involving modification of active sites of enzymes (Terrier et al., 1999) and interaction with structural proteins (Bailey et al., 1998) has also been suggested. No common toxicity mechanism for -1-O-acyl glucuronides has been established, as the -1-O-acyl glucuronides of different drugs bind covalently to different proteins (Bailey and Dickinson, 1996) and on different sites on specific proteins (Qiu et al., 1998). The covalent binding to proteins proceeds predominantly via the acyl-migrated isomers (Dickinson and King, 1991; Ding et al., 1995; Liu et al., 1998; Qiu et al., 1998).

The stability and protein reactivity of S-naproxen--1-O-acyl glucuronide (S-nap-g²; Fig. 1) have been studied by several groups (Vree et al., 1992; Bischer et al., 1995; Iwaki et al., 1999). The generalized scheme of acyl migration is shown in Fig. 1, where the initial acyl-migration step from the -1-O-acyl glucuronide to the -2-O-acyl isomer (which can then mutarotate to the -2-O-acyl isomer) is considered to be irreversible, whereas the rearrangement between the /-2-, /-3-, and /-4-O-acyl isomers is reversible (Spahn-Langguth and Benet, 1992).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   The degradation rearrangement scheme for S-naproxen -1-O-acyl glucuronide. The minor -1-O-acyl isomer (see Results) is included. For clarity the /-2-, /-3-, and /-4-O-acyl isomer /-anomerization reactions have been omitted. The hydrolysis end product of all the rearrangement isomers is S-naproxen aglycone.

Most investigators have mainly been concerned with the total degradation rate of the -1-O-acyl glucuronide (Dickinson et al., 1994; Bischer et al., 1995; Castillo and Smith, 1995). The interest in the degradation of the -1-O-acyl glucuronide was focused by a study in which the in vitro degradation rate gave a linear correlation with in vitro covalent protein binding for the nine different -1-O-acyl glucuronides studied (Benet et al., 1993). The -1-O-acyl glucuronides of S- and R-naproxen also fit this linear correlation (Bischer et al., 1995). However, as the covalent protein binding predominantly proceeds via the acyl-migrated isomers, the degradation rate of the -1-O-acyl glucuronide per se may not be the most relevant bioactivity parameter. If the degradation is mainly due to hydrolysis, only small amounts of acyl-migrated isomers will be formed, and thus covalent binding to proteins will only be a minor reaction. It is consequently desirable to elucidate the complex kinetics of the acyl-migration rearrangement scheme (Sidelmann et al., 1996; Akira et al., 1998). A distinction between hydrolysis (k_1-D) and acyl migration (k_1-2) at the very least is necessary to evaluate the relative formation of acyl-migrated isomers compared with hydrolysis.

HPLC-¹H NMR has been used to identify the individual isomers in mixtures of acyl-migrated 2-, 3-, and 4-fluorobenzoic acid glucuronides (Sidelmann et al., 1995a,b) and R- and S-phenylpropionic acid glucuronides (Akira et al., 2000). This procedure eliminates the need for preparative HPLC of the unstable isomers and subsequent off-line NMR spectroscopy to assign the chromatographic peaks in mixtures of acyl-migrated glucuronide isomers (Hansen-Møller et al., 1988; Bradow et al., 1989).

The interconversion kinetics of 2-fluorobenzoic acid glucuronide isomers was followed by HPLC-¹H NMR in dynamic stopped-flow mode (Sidelmann et al., 1996). The chromatographic run was stopped with the peak(s) of interest in the NMR flow probe, and the degradation was followed over time. With this approach physical collection and purification of the individual isomers as done for the /-2-O-acyl isomer of S-nap-g (Iwaki et al., 1999) and the /-2-, /-3-, and /-4-O-acyl isomers of diflunisal--1-O-acyl glucuronide (Dickinson and King, 1991) is not necessary.

The reaction medium in dynamic HPLC-¹H NMR studies is the HPLC mobile phase containing D₂O to minimize solvent suppression artifacts in the ¹H NMR spectrum and acetonitrile for the chromatographic separation. These modifications compared with the normal in vitro conditions (pH 7.40 with no D₂O or acetonitrile) must be considered when comparing kinetics obtained by other methods.

Recently the acyl-migrated isomers of S-nap-g were assigned in two chromatographic systems using stopped-flow HPLC-¹H NMR in which the -1-O-acyl isomer was determined (Mortensen et al., 2001). In the present paper HPLC based on UV-detection (HPLC-UV) and the previously determined elution order is applied to elucidate the interconversion kinetics between the acyl-migration isomers of S-nap-g, and the results are compared with dynamic stopped-flow HPLC-¹H NMR methods. Also, the influence of D₂O and increasing concentrations of acetonitrile is studied to clarify the effect on the kinetics.

	Materials and Methods

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All chemicals used were of analytical reagent grade or higher from commercial suppliers. Water was purified using a Milli-Q Plus water purification apparatus (Millipore A/S, Glostrup, Denmark). Deuterium oxide for HPLC-¹H NMR was from Goss Scientific Instruments Ltd. (Essex, England). Acetonitrile for the HPLC-¹H NMR experiments was NMR CHROMASOLV grade from Riedel de Haën (Sigma-Aldrich UK, Dorset, England). Biosynthetic S-nap-g was isolated from human urine using solid phase extraction and preparative HPLC as previously described (Mortensen et al., 2001).

Reaction Media. The degradation kinetics of S-nap-g at 37°C was monitored by HPLC-UV in the following reaction media: 25 mM potassium phosphate buffer pH 7.40 (1); 25 mM potassium phosphate buffer in D₂O pD 7.40 (2); and 25 mM potassium phosphate buffer in D₂O pD 7.40/acetonitrile 80:20 v/v (3). Reaction medium 1 was prepared by adjusting a 25 mM KH₂PO₄ solution with 1 M KOH to pH 7.40. Reaction medium 2 was prepared by adjusting a 25 mM KH₂PO₄ solution in D₂O with 1 M KOH in D₂O to a pH meter reading of 7.40, using normal water-based calibration standards with pH 7.00 and 10.00 to calibrate the pH meter. Reaction medium 3 identical to the HPLC-¹H NMR mobile phase was prepared by mixing reaction medium 2 with acetonitrile in the ratio 80:20 v/v. The effect of acetonitrile was studied in mixtures of reaction medium 1 and acetonitrile in the following ratios: 90:10 v/v, 80:20 v/v (identical to the mobile phase used for the degradation studies monitored by HPLC-UV), 70:30 v/v, 60:40 v/v, and 50:50 v/v.

Kinetic Experiments by HPLC-UV. A HP 1100 series chromatographic system with HP ChemStation software was used (Agilent Technologies Denmark A/S, Birkerød, Denmark). This system was isocratic with a single-wavelength UV/visible-detector. The column was a Hibar LiChrospher 100 RP-C₁₈ column with a 5-µm particle size and 250 mm × 4-mm i.d. (Merck, Darmstadt, Germany) operated at ambient temperature. The flow rate was 1 ml/min, and the detection wavelength was 272 nm. The mobile phase consisted of reaction medium 1 mixed with acetonitrile in a ratio of 80:20 v/v.

Directly-Coupled 600-MHz HPLC-¹H NMR Spectroscopy. The LiChrospher 100 RP-C₁₈ column was used, with the following hardware: a Bruker LC-22 pump (Bruker, Rheinstetten, Germany), a Bruker photodiode array detector (J & M Analytische Mess- und Regeltechnik GmbH, Aalen, Germany), a Bruker column oven-operated at 25°C, and a Bruker BPSU-36 flow control unit. The outlet of the detector was connected to the HPLC-¹H NMR flow probe via an inert polyether-ether ketone capillary (3.1 m × 0.25-mm i.d.). HPLC-NMR-mass spectroscopy software (Hystar v1.1, Bruker) controlled the flow dynamics of the system and stored the chromatographic data.

NMR Spectroscopy. The ¹H NMR spectra were acquired using a Bruker AVANCE600 spectrometer operating at 600.13-MHz ¹H frequency equipped with a ¹H-¹³C inverse detection Z-gradient HPLC flow probe containing a 4-mm i.d., 120-µl cell. ¹H NMR spectra of the individual acyl-migrated glucuronide isomers were obtained in stopped-flow mode at 600.13 MHz and 37°C probe temperature. Dual solvent suppression of the acetonitrile and residual HDO signals was achieved using the standard one-dimensional nuclear Overhauser effect spectroscopy presaturation pulse sequence (Bruker) with relaxation and mixing delays of 2.0 and 0.1 s, respectively. Next, 1024 free induction decays were collected into 64 K computer data points with a spectral width of 20 ppm, corresponding to an acquisition time of 2.73 s. Each dynamic stopped-flow HPLC-¹H NMR degradation experiment ran for 11 to 16 h with a time resolution of 90 min. Before Fourier transformation, an exponential apodization function was applied to the free induction decays, corresponding to a line broadening of 2 Hz. Chemical shifts were referenced to the acetonitrile signal at  2.0 and thus may differ slightly from signals referenced to HDO at  4.7.

Isomer Concentration Determination by NMR. Selected diagnostic NMR peaks from the individual glucuronide isomers were integrated relative to those of the aromatic region of the spectra. The aromatic region NMR peak integral was set to six hydrogens, and the amounts of the individual isomers were determined from the relative areas of their selected diagnostic peaks. The selected NMR signals with assignments are given in Table 1. As the 2-, 3-, and 4-O-acyl isomers all exist as /-anomers in an equilibrium on the NMR time scale (Sidelmann et al., 1996), diagnostic signals for the individual /-anomers of each positional O-acyl isomer were integrated and then added to get the total concentration of each O-acyl isomer. For S-naproxen aglycone and the -1-O-acyl isomer a well resolved doublet from the S-naproxen methyl group at  1.5 was chosen as the diagnostic signal. Because a methyl group gives a three-proton signal, those integrals were divided by 3 to give the correct relative amount. The multiplet at  5.11 used for the -3-O-acyl isomer quantitation is a superposition of the signals from the 1' and 3' protons, and the integrated signal is thus divided by 2 to give the correct relative amount. All other characteristic signals chosen corresponded to individual protons and thus needed no correction. Before kinetic analysis the responses were corrected to give the percentage of each compound.

View this table: [in this window] [in a new window]   TABLE 1 Characteristic 1H NMR signals used for the quantitation of isomers in the dynamic 1H NMR degradation experiments Chemical shifts are referenced to acetonitrile at  2.0. Glucuronide ring protons are numbered according to Fig. 1. The signal at  5.11 for the -3-O-acyl isomer is a superposition of the H1' and H3' protons; thus, the integrated signals are divided by 2 prior to kinetic analysis to get the signal corresponding to one proton. The CH3 group used for quantitation of the -1-O-acyl isomer and S-naproxen aglycone is the -methyl group in the acetic acid moiety of S-naproxen. This signal is divided by 3 prior to kinetic analysis to get the signal corresponding to one proton. For the -1-O-acyl glucuronide and the /-2-, /-3-, and /-4-O-acyl isomers this methyl group appears as an unresolved band in the region  1.51 to 1.53 and cannot be used for quantitation.

Data Fitting. Kinetic rate constants for the acyl-migration reactions were fitted for the individual experiments using the program Gepasi 3.21 (Mendes, 1993, 1997; Mendes and Kell, 1998) using the kinetic scheme depicted in Fig. 1 with first order mass-action reaction kinetics. The hydrolysis rate constants for the acyl-migrated isomers were fitted as one parameter k_X-D (k_X-D = k_1-D = k_2-D = k_3-D = k_4-D). It is not possible to distinguish between those hydrolysis rate constants when the concentrations of the isomers are in the same order of magnitude because the product for all the reactions is the aglycone S-naproxen.

	Results

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The kinetic profiles of the degradation of S-nap-g in reaction media 1, 2, and 3 determined by HPLC-UV are given in Fig. 2. Points are experimental data, and lines are the simulated curves using the fitted kinetic rate constants given in Table 2. The effect of substituting the aqueous phosphate buffer (reaction medium 1, Fig. 2A) with phosphate buffer made up in D₂O (reaction medium 2, Fig. 2B) was an overall decrease in reactivity, both for acyl migration (k_1-2) as the maximum concentration of the /-2-O-acyl isomer is reached later and for hydrolysis (k_1-D) as the formation rate of S-naproxen was decreased. Adding acetonitrile to the phosphate buffer made up in D₂O in 80:20 (v/v) buffer/acetonitrile (reaction medium 3, Fig. 2C) further slowed down the rearrangement kinetics.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   D2O and acetonitrile effect on the degradation of S-naproxen--1-O-acyl glucuronide. The degradation was monitored by HPLC-UV. The time scale is linear, and the normalized response scale is logarithmic. Data points are HPLC-UV experimental data, and lines are the kinetic simulations using the fitted rate constants (Table 2). A, 25 mM potassium phosphate pH 7.40 (reaction medium 1); B, 25 mM potassium phosphate in D2O pD 7.40 (reaction medium 2); C, 25 mM potassium phosphate in D2O pD 7.40/MeCN 80:20 v/v (reaction medium 3). , -1-O-acyl glucuronide; , -1-O-acyl isomer; +, 2-O-acyl isomers; ×, 3-O-acyl isomers; , 4-O-acyl isomers; , S-naproxen.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   1H NMR-monitored degradation of S-naproxen--1-O-acyl glucuronide and its acyl-migrated 2-, 3-, and 4-O-acyl isomers. The degradation of S-naproxen--1-O-acyl glucuronide was monitored by dynamic 1H NMR, and the degradation of the acyl-migration isomers was monitored by dynamic stopped-flow HPLC 1H NMR. The time scale is linear, and the normalized response scale is logarithmic. Data points are determined by NMR, and lines are predicted by kinetic simulation with the rate constants determined from the degradation experiments monitored by HPLC-UV (Table 2). Integration of the NMR signals corresponding to less than 5% of one proton was not very reproducible due to the limited signal/noise ratio, and thus the data points in the lower parts of the figures are not determined with a great accuracy. A, S-naproxen--1-O-acyl glucuronide in 25 mM potassium phosphate in D2O pD 7.40 (reaction medium 2). B, /-2-O-acyl isomers in 25 mM potassium phosphate in D2O pD 7.40/MeCN 80:20 v/v (reaction medium 3). C, /-3-O-acyl isomers in 25 mM potassium phosphate in D2O pD 7.40/MeCN 80:20 v/v (reaction medium 3). D, /-4-O-acyl isomers in 25 mM potassium phosphate in D2O pD 7.40/MeCN 80:20 v/v (reaction medium 3). , -1-O-acyl glucuronide; , -1-O-acyl isomer; +, 2-O-acyl isomers; ×, 3-O-acyl isomers; , 4-O-acyl isomers; , S-naproxen.

View this table: [in this window] [in a new window]   TABLE 2 First order kinetic rate constants fitted for the degradation experiments in reaction media 1, 2, and 3 monitored by HPLC-UV Rate constants are shown in the reaction scheme depicted in Fig. 1. X-D is the hydrolysis of the acyl-migrated isomers to S-naproxen aglycone (D = drug), which is fitted as one parameter (kX-D = k1-D = k2-D = k3-D = k4-D). The pseudoequilibrium concentration ratios between the -1-, /-2-, /-3-, and /-4-O-acyl isomers are calculated by the ratio of the reversible acyl-migration first order rate constants in the equilibrium. Ratio am/h is the ratio between acyl migration (k1-2) and hydrolysis (k1-D) for S-nap-g. s2 is the sum of squares from the kinetic fitting to the analytical data for each reaction medium.

The kinetic rate constants for the individual experiments as determined by data fitting with the kinetic simulation program Gepasi 3.21 are shown in Table 2 for the acyl-migration rearrangement scheme in Fig. 1. The observed changes in reactivity shown in Fig. 2 are reflected in the fitted kinetic rate constants. The ratio am/h is the ratio between acyl migration and hydrolysis of S-nap-g. The pseudoequilibrium concentration ratios between the -1-, /-2-, /-3-, and /-4-O-acyl isomers are calculated by the ratio of the reversible acyl-migration first order rate constants in the equilibrium.

The degradation kinetics of S-nap-g in reaction medium 2 determined by dynamic ¹H NMR is shown in Fig. 3A. The degradation kinetic profiles of the individual acyl-migrated isomers in the HPLC-¹H NMR mobile phase, reaction medium 3, determined by dynamic stopped-flow NMR are shown in Fig. 3, B (/-2-O-acyl isomer), C (/-3-O-acyl isomer), and D (/-4-O-acyl isomer). In Fig. 3, A, B, C, and D individual data points are experimental data from the dynamic NMR experiments while the lines are the predicted values using the kinetics derived from the degradation experiments as monitored by HPLC-UV (Fig. 2, B and C).

The degradation of S-nap-g in reaction medium 1 mixed with increasing ratios of acetonitrile was also monitored by HPLC-UV. The fitted first order kinetic rate constants for hydrolysis (k_1-D) and acyl migration (k_1-2) of the -1-O-acyl glucuronide are given in Table 3. As the ratio of phosphate buffer/acetonitrile in reaction medium 1 was varied from 100:0 to 50:50 (v/v) the magnitude of the rate constants for hydrolysis and acyl migration decreased by equal amounts, and thus no effect on the ratio between acyl migration and hydrolysis of S-nap-g (ratio am/h in Table 3) was observed. All nine first order kinetic rate constants in the overall migration scheme (Fig. 1) were decreased by acetonitrile as well (data not shown).

View this table: [in this window] [in a new window]   TABLE 3 First order kinetic rate constants fitted for the degradation experiments in reaction medium 1 with acetonitrile added as monitored by HPLC-UV The dependence of the first order degradation rate constants k1-D (hydrolysis) and k1-2 (acyl migration) of S-naproxen--1-O-acyl glucuronide in mixtures of 25 mM potassium phosphate buffer, pH 7.40, and increasing amounts of acetonitrile. Ratio am/h is the ratio between acyl migration (k1-2) and hydrolysis (k1-D) for S-nap-g. All nine first order rate constants in the equilibrium (data not shown) are slowed down by increasing amounts of acetonitrile.

	Discussion

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The Effects of D₂O and Acetonitrile on the Degradation Kinetics of S-nap-g. The possible kinetic effect of acetonitrile and D₂O in the HPLC-¹H NMR mobile phase should be considered when performing dynamic stopped-flow HPLC-¹H NMR kinetic analysis of the degradation of -1-O-acyl glucuronides (Sidelmann et al., 1996). Because the addition of acetonitrile as well as the substitution of H₂O with D₂O is a necessary prerequisite for the stopped-flow HPLC-¹H NMR experiment, the study of those modifications is not possible by HPLC-¹H NMR. Thus, in the present degradation study HPLC-UV experiments were used to monitor the effects of those modifications of the reaction medium on the degradation kinetics of S-nap-g. Table 2 shows that all rate constants are slowed down by D₂O as well as 20% (v/v) acetonitrile. Table 3 shows the effect of increasing the acetonitrile content in reaction medium 1. Acetonitrile is seen to decrease the hydrolysis as well as the acyl-migration reaction rates. In addition, the ratio between the major acyl-migration reaction and the minor hydrolysis reaction is unchanged on addition of 0 to 50% acetonitrile (v/v). Thus, acetonitrile slows down the reaction rates but does not affect the ratio between the two parallel reactions.

Comparison of HPLC-UV and Dynamic NMR Methods in the Study of Acyl-Migration Kinetics. In the present study of reversible kinetics the initial concentrations of the involved compounds in the NMR-probe are not important because the rate constants in the overall kinetic scheme are the same irrespective of which compound is the starting material. Thus, it is possible by kinetic analysis to derive the rate constants for the overall scheme by incubating the biosynthetic -1-O-acyl glucuronide; therefore, dynamic stopped-flow HPLC-¹H NMR experiments of the individual isomers are not necessary since no new kinetic information is obtained with these experiments.

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1-D

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1-D

Kinetic Results Regarding the Formation of the -1-O-Acyl Isomer. The -1-O-acyl isomer was found to be the least stable species in the overall reaction scheme, as the acyl-migration rate from the -1-O-acyl isomer to the /-2-O-acyl isomer (k_1-2) was the highest in the overall equilibrium irrespective of the reaction medium (Table 2). The rapid equilibrium between the -1-O-acyl isomer and the /-2-O-acyl isomer can also be observed in Fig. 2A, in which the concentration of the -1-O-acyl isomer closely follows the /-2-O-acyl isomer concentration, whereas the concentration of the /-3-O-acyl isomer lags behind.

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1-2