Introduction

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NM441 [(±)-6-fluoro-1-methyl-7-[4-(5-methyl-2-oxo-1,3-dioxolen-4yl)methyl-1-piperazinyl]-4-oxo-4H-[1,3]thiazeto[3,2-a]quinoline-3carboxylic acid], a prodrug of the new antibacterial agent NM394 [(±)-6-fluoro-1-methyl-4-oxo-7-(1-piperazinyl)-4H-[1,3]thiazeto[3,2-a] quinoline-3-carboxylic acid], has a dioxolenylmethyl group (fig. 1). After oral administration to rats and humans, NM441 is well absorbed and hydrolyzed to NM394, mainly in the serum and liver (Okuyama et al., 1997; Kondo et al., 1986). The enzyme involved in the hydrolysis of NM441, however, has yet to be determined.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Chemical structure of NM441 and its postulated enzymatic conversion to NM394.

Esterases have major roles in the hydrolysis of a number of prodrugs in humans and experimental animals (Satoh, 1987; Obermeier et al., 1996; Tang and Kalow, 1995; Senter et al., 1996; Krasny et al., 1995). They are classified in three groups (A-, B-, and C-esterases) on the basis of their reactivity with organophosphorus compounds such as paraoxon [O,O-diethyl-O-(p-nitrophenyl)phosphate] and DFP¹ (Aldrich, 1953). B-esterases, including acetylcholinesterase (EC 3.1.1.7) and nonspecific carboxylesterase (EC 3.1.1.1), are inhibited by organophosphates, whereas A-esterases, including arylesterase (EC 3.1.1.2), rapidly hydrolyze organophosphates. C-Esterases such as acetylesterase (EC 3.1.1.6) do not interact with organophosphates. Interindividual variation in the activity of the esterases is an important factor that influences both the pharmacological and toxicological effects of prodrugs in humans (Williams, 1985). Large interindividual variations in esterase activity have been reported for carbonic anhydrase (EC 4.2.1.1) (Verpoorte et al., 1967), butylcholinesterase (EC 3.1.1.8) (McGuire et al., 1989), carboxylesterase (EC 3.1.1.1) (Hosokawa et al., 1995), paraoxonase/arylesterase (EC 3.1.8.1) (Playfer et al., 1976), and S-formylglutathione hydrolase (EC 3.1.2.12) (Eiberg and Mohr, 1986).

In a previous study, NM441-hydrolase activity in rat and human sera was inhibited by PCMB and EDTA (fig. 2). This suggests that A-esterases have a major role in the hydrolysis of NM441. Although there have been numerous studies on the enzymatic characterization of paraoxonase (an A-esterase), most have focused on its role in the detoxification of organophosphates such as paraoxon and chlorpyrifosoxon [O,O-diethyl-O-(3,5,6-trichloro-2-pyridyl)phosphate]. There have been no reports on the contribution of A-esterases to the activation of prodrugs. The purpose of this study was to purify and characterize the enzyme (NM441-hydrolase) responsible for the conversion of NM441 to NM394 in rats. We also examined interindividual variations in NM441-hydrolase activity in the sera of 67 healthy volunteers, because some A-esterases show genetic polymorphism in humans (Playfer et al., 1976).

View larger version (30K): [in this window] [in a new window]   Fig. 2.   Effects of DFP, PCMB, and EDTA on NM441-hydrolase activities in rat serum, rat liver microsomes, and human serum. Bars, mean ± SD (N = 3).

	Materials and Methods

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Chemicals. NM441 and NM394 were synthesized in our laboratories as reported previously (Okuyama et al., 1997). DFP, PCMB, EDTA, and Achromobacter protease I were purchased from Wako Pure Chemicals (Osaka, Japan). Emulgen 911, KBr, and paraoxon were products of Kao Corp. (Tokyo, Japan), Nacalai Tesque (Kyoto, Japan), and Sigma Chemical Co. (St. Louis, MO), respectively. The other reagents were all of analytical grade.

Animals. Male Sprague-Dawley rats, weighing 180-200 g, were obtained from Japan SLC Inc. (Hamamatsu, Japan). They were acclimatized to the laboratory conditions for >1 week before use.

Serum Samples. Under light ether anesthesia, blood samples were withdrawn from the abdominal aortas of the rats. Serum was separated from whole blood by centrifugation and was stored at 4°C until used.

Preparation of Liver Microsomes. Rats were killed by decapitation, after which their livers were removed and rinsed in 1.15% KCl. Liver microsomes, prepared according to the method of Hosokawa et al. (1990), were stored in a freezer at 80°C until used.

Purification of the NM441-Hydrolase from Rat Serum. A HDL fraction prepared by sequential flotation ultracentrifugation (Havel et al., 1955) was dialyzed against 20 mM Tris-HCl buffer (pH 7.4) containing 1 mM CaCl₂ (buffer A). The dialyzed sample was applied to a heparin-Sepharose column (Pharmacia Biotech, Uppsala, Sweden) equilibrated with buffer A. When the column was washed with buffer A, the NM441-hydrolase/HDL fraction passed through it. The fraction was suspended in buffer A containing 0.2% Emulgen 911 and 40% glycerol (buffer B). After centrifugation, the supernatant was subjected to HPLC using an anion exchange column (Mono Q HR 5/5; Pharmacia Biotech). HPLC was performed at a flow rate of 0.5 ml/min, with a linear salt gradient composed of buffer B and buffer B containing 0.5 M NaCl (buffer C). Protein was detected by monitoring the absorbance at 254 nm. The fraction containing NM441-hydrolase was dialyzed against buffer A and concentrated by centrifugation (Centricon-30 membrane; Amicon, Beverly, MA).

Measurement of NM441-Hydrolase Activity. The reaction mixture consisted of the enzyme solution, NM441 (50 nmol), and 0.1 M Tris-HCl buffer (pH 7.4) containing 1 mM CaCl₂, in a final volume of 0.5 ml. After incubation at 37°C for 5 min, the reaction was stopped by the addition of 0.5 ml of acetonitrile. After centrifugation, the concentration of NM394 in the supernatant was measured by HPLC using a C₁₈ reverse-phase column (Capcell pack SG120; Shiseido, Tokyo, Japan). The mobile phase consisted of acetonitrile/methanol/0.05 M phosphate buffer adjusted to pH 2.0 (10:20:65). The flow rate was 1 ml/min, and the effluent was monitored at 270 nm.

Inhibition Studies. Fifty microliters of microsomal suspension (20 mg protein/ml) or plasma (10 µl) were incubated for 5 min at 37°C with 0.1 mM DFP, 0.1 mM PCMB, or 2 mM EDTA before the addition of 100 µl of aqueous NM441 solution (0.5 mM). Control samples without the inhibitors were also incubated.

Analysis of Amino-Terminal and Internal Amino Acid Sequences. To determine its amino-terminal amino acid sequences, NM441-hydrolase was electrophoresed on 10% acrylamide gels and then transferred electrophoretically to a polyvinylidene difluoride membrane. The enzyme was then stained with Coomassie Brilliant Blue, after which its sequences were analyzed with a protein sequencer (PSQ-1; Shimadzu, Kyoto, Japan). To determine the internal amino acid sequences, the hydrolase was digested with Achromobacter protease I. Of the digests obtained, two lysyl peptides (peptides a and b) were purified by HPLC with a C₁₈ reverse-phase column (µ-Bondasphere; Waters, Milford, MA) (fig. 3) and analyzed in a protein sequencer (model 477A; Applied Biosystems, Foster, CA).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   HPLC profile of the lysyl peptides from NM441-hydrolase. Peptides produced by cleavage of pyridylethylated NM441-hydrolase (50 µg) with Achromobacter protease I were purified by reverse-phase HPLC with a linear gradient of buffer D (0.05% trifluoroacetic acid) to 60% buffer E (0.05% trifluoroacetic acid in acetonitrile).

Paraoxonase Activity and Phenotyping. Serum samples were obtained from 67 nonfasting healthy volunteers. Paraoxonase activity was measured by the method of Gan et al. (1991). All incubations were at 21°C. Each serum sample was phenotyped as type A (low activity), type AB (moderate activity), or type B (high activity) according to the relative ratio of the hydrolysis activity of paraoxon in the presence of 1 M NaCl to that of phenyl acetate (Eckerson et al., 1983).

Other Methods. Protein concentrations were measured by the method of Lowry et al. (1951). Spectrophotometric measurements were made with an Hitachi U-3300 spectrometer. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed by the method of Laemmli (1970). Proteins were stained with Coomassie Brilliant Blue.

	Results

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Inhibition Studies. To identify the esterases involved in the hydrolysis of NM441 to NM394, we studied the inhibitory effects of DFP, PCMB, and EDTA on the reactions catalyzed by rat serum, rat liver microsomes, and human serum. DFP inhibits the activities of cholinesterase and carboxylesterase (B-esterases), and PCMB inhibits the activity of A-esterases with an -SH group (cysteine) at the active center (Aldrich, 1953). EDTA also inhibits the activity of A-esterases (Playfer et al., 1976).

Purification of the NM441-Hydrolase from Rat Serum. In our preliminary experiment with rat serum, NM441-hydrolase and lipoprotein were eluted from a Sephacryl S-300 column (Pharmacia Biotech) in the void fraction. This suggests that the enzyme is associated with lipoprotein. In fact, most of the NM441-hydrolase activity (90%) in the serum was located in the HDL fraction prepared by ultracentrifugation (table 1). Emulgen 911, which is generally used to purify cytochrome P450 isozymes, solubilized NM441-hydrolase from HDL without loss of its activity (table 2).

View larger version (68K): [in this window] [in a new window]   Fig. 4.   Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the NM441-hydrolase purified from rat serum. Lane 1, molecular mass markers; lane 2, purified NM441-hydrolase (2 µg). The molecular mass markers were -galactosidase (116 kDa), bovine serum albumin (66 kDa), aldolase (42 kDa), and carbonic anhydrase (30 kDa).

Amino-Terminal and Internal Amino Acid Sequences. As shown in fig. 5, the amino-terminal and internal amino acid sequences of rat serum NM441-hydrolase are almost identical to those of mouse serum paraoxonase (Sorenson et al., 1995) and are homologous to those of human and rabbit serum paraoxonases (Furlong et al., 1991; Hassett et al., 1991).

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Comparison of the amino-terminal and internal amino acid sequences of rat NM441-hydrolase with those of mouse, rabbit, and human serum paraoxonases. Dashes, amino acid residues of paraoxonases identical to those of NM441-hydrolase. The deduced amino acid sequence data for mouse paraoxonase are from the report by Sorenson et al. (1995), and those for the human and rabbit paraoxonases are from the work of Furlong et al. (1991) and Hassett et al. (1991).

Characteristics of the NM441-Hydrolase. The apparent molecular mass of the NM441-hydrolase, estimated as 46 kDa (fig. 4), is close to that of human paraoxonase (44.7 and 47.9 kDa) (Hassett et al., 1991). The optimum pH for NM441-hydrolase activity was 8.6 (fig. 6). Similar values have been reported for sheep and human serum paraoxonases (Smolen et al., 1990). Enzyme activity was inhibited by EDTA, and NM441-hydrolase required calcium ions (data not shown). The characteristics of NM441-hydrolase and various mammalian serum paraoxonases are given in table 3. These characteristics of NM441-hydrolase are similar to those of the known paraoxonases (Hassett et al., 1991; Smolen et al., 1990; Kuo and La Du, 1995).

View larger version (11K): [in this window] [in a new window]   Fig. 6.   Effects of pH on the activity of purified NM441-hydrolase. NM441-hydrolase activity was measured in 0.1 M phosphate buffer (pH 5.9 or 6.8), 0.1 M Tris-HCl (pH 7.3, 8.0, 8.5, or 9.0), or 0.1 M glycine-NaOH buffer (pH 9.7).

Paraoxonase Phenotypes. Human paraoxonase has a genetically determined polymorphism with low-activity (type A), moderateactivity (type AB), and high-activity (type B) phenotypes (Smolen et al., 1990). These phenotypes were determined by the method of Eckerson et al. (1983). The numbers of individuals with type A, AB, and B phenotypes among the 67 healthy volunteers were 2 (3%), 42 (63%), and 23 (34%), respectively (fig. 7).

View larger version (34K): [in this window] [in a new window]   Fig. 7.   Histogram of the paraoxonase phenotyping ratios in sera from human subjects. Serum samples from 67 healthy volunteers were phenotyped by the paraoxonase activity in the presence of 1 mM NaCl/arylesterase activity ratios (phenotyping ratios). For types A, AB, and B, the phenotype ranges are 0.9-2.5, 2.6-7.5, and 7.6-12.0, respectively.

NM441-Hydrolase and Paraoxonase Activities. A positive correlation (r = 0.653, p < 0.005) was found between the NM441-hydrolase and paraoxonase activities in this population (fig. 8). Interindividual variation in paraoxonase activity was 9-fold (29.8-264.6 nmol/ml/min), whereas that in NM441-hydrolase activity was only 2-fold (6.69-14.55 nmol/ml/min).

View larger version (13K): [in this window] [in a new window]   Fig. 8.   Correlation between NM441-hydrolase and paraoxonase activities in sera from 67 healthy volunteers. The assay conditions are described in Materials and Methods.

	Discussion

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Many ester prodrugs have been developed, and the enzymes that catalyze the hydrolytic activation of these prodrugs have been purified and characterized (Tang and Kalow, 1995; Senter et al., 1996; Krasny et al., 1995). We purified, from rat serum, and characterized the enzyme (NM441-hydrolase) responsible for the hydrolytic activation of NM441. The amino-terminal amino acid sequence and characteristics of this NM441-hydrolase are similar to those of the known serum paraoxonases (Sorenson et al., 1995; Furlong et al., 1991; Hassett et al., 1991; Smolen et al., 1990; Kuo and La Du, 1995). We conclude that the NM441-hydrolase from rat serum is rat serum paraoxonase.

In human serum, NM441-hydrolase and paraoxonase activities were present in the lipoprotein fraction (78%). Some hydrolytic enzymes [lecithin cholesterol acyltransferase (EC 2.3.1.43) (Lui and Subbaiah, 1986), lipoprotein lipase (EC 3.1.1.34) (Komaromy and Schotz, 1987), and platelet-activating factor acetylhydrolase (EC 3.1.1.48) (Karabina et al., 1994)] are found in the serum lipoprotein fraction. These enzyme activities are inhibited by organophosphates such as DFP. In contrast, serum NM441-hydrolase activity was not sensitive to DFP (fig. 2), indicating that these hydrolytic enzymes do not contribute to the hydrolysis of this enzyme. Serum albumin has weak esterase activity (Augustinsson, 1959). Human serum albumin also had NM441-hydrolase activity, but the contribution of albumin to the total NM441-hydrolase activity in serum was <5% (data not shown). Thus, human paraoxonase is mainly responsible for the hydrolysis of NM441.

Because human paraoxonase shows polymorphism, NM441-hydrolase activities may exhibit marked interindividual differences and this variation may influence both the pharmacological and toxicological effects of NM394. We therefore examined whether paraoxonase polymorphism causes marked interindividual variation in NM441-hydrolase activities. In the phenotyping study of sera from 67 healthy volunteers, the frequency of type A (low-activity group) was the lowest among the three phenotypes (fig. 7). This is consistent with the finding (Geldmacher-von Mallinckrodt et al., 1983) that only 10% of the Japanese population has low activity. In our study, we found a positive correlation between the NM441-hydrolase and paraoxonase activities in this population (r = 0.653, p < 0.005) (fig. 8), which supports the contention that human paraoxonase contributes to the hydrolysis of NM441. EDTA and PCMB did not completely inhibit NM441-hydrolase activity in human serum (fig. 2), suggesting that other esterases contribute, in part, to the conversion of NM441 to NM394. This may be one of the reasons why the correlation with paraoxonase activity in human serum was not so strong (r = 0.653). The interindividual variation in NM441-hydrolase activities was smaller than that for paraoxonase activities (fig. 8). Eckerson et al. (1983) reported that the polymorphism of human paraoxonase is substrate dependent; the enzyme activity has bimodal distribution when paraoxon is the substrate but has unimodal distribution when phenyl acetate and chlorpyrifosoxon are the substrates (Furlong et al., 1989). Our findings suggest that NM441, like phenyl acetate, is a nonpolymorphic substrate, as reported by Eckerson et al. (1983). Interindividual differences in NM441-hydrolase activities seem to depend on the protein concentration, rather than genetic polymorphism. We conclude that paraoxonase polymorphism does not affect NM441-hydrolase activity.

In conclusion, we showed that paraoxonases have a major role in the hydrolytic activation of NM441 in rats and humans. The genetic polymorphism of human serum paraoxonase does not cause the marked interindividual variations in NM441-hydrolase activities.