INTRODUCTION

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Cephalosporins usually exhibit poor bioavailabilities when they are given orally. Higher values are obtained only with cephalosporins that are taken up by carrier systems or when the polarity of the carboxylic acid group in the 4 position is reduced by esterification (10). Esterification of the carboxylic acid group produces derivatives which can be absorbed by passive diffusion. Therapeutically useful compounds can, however, be obtained only if the absorbed prodrug ester is readily converted back to the active drug. The success of the prodrug ester approach depends vitally on the solubility and lipophilicity of the prodrug ester as well as its stability to chemical and enzymatic ester cleavage.

Cephalosporin prodrug esters exhibit oral bioavailabilities of approximately 50%. Premature hydrolysis in the intestine before absorption has been discussed as a possible reason for their incomplete bioavailability (4, 6, 8). Hydrolysis can proceed either by enzyme-catalyzed direct hydrolysis to the parent cephalosporin (6) or via a reversible base-catalyzed isomerization yielding the 2-cephalosporin ester which is rapidly cleaved to give the biologically inactive 2-cephalosporin (7). Both pathways will remove some of the cephalosporin available for absorption. The result is incomplete bioavailability of the oral prodrug ester. However, direct hydrolysis to the nonabsorbable but biologically active cephalosporin in the gut lumen could affect the gut flora, causing undesirable intestinal side effects (3).

To date, no studies have assessed the relative importance of the two pathways. Acquiring a better understanding of these pathways would be beneficial in designing cephalosporin prodrug esters with improved bioavailabilities and intestinal tolerability characteristics. Therefore, we studied the degradation of three clinically relevant cephalosporin prodrug esters (cefetamet pivoxil, cefuroxime axetil, and cefpodoxime proxetil) in human intestinal juice and phosphate buffer (a well-understood model for chemical hydrolysis [5, 7-9]).

	MATERIALS AND METHODS

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Chemicals and reference compounds. All chemicals and solvents were of analytical grade. They were all purchased from Carlo Erba, Nanterre, France.

View larger version (17K): [in this window] [in a new window]   FIG. 1.   Chemical structures of cephalosporin prodrug esters.

Instrumentation. Nuclear magnetic resonance (¹H NMR) spectra were recorded on a Bruker AM 400 spectrometer (400 MHz) in D₂O. The high-performance liquid chromatography (HPLC) system consisted of a Shimadzu LC-6A pump and a Shimadzu SIL-6A autoinjector (Shimadzu Corporation, Kyoto, Japan), a Beckman DAD 168 detector, and a UV spectrophotometer (DU-64; Beckman).

Intestinal juice collection. Four male subjects (average age, 28 years) were recruited from the medical personnel of the University of Kentucky. They were healthy as evidenced by a complete medical history. Informed consent was obtained from all study participants. After a 12-h fasting period each subject received with the help of a radiologist a 14 French Bilboa-Dotter tube (Cook Inc., Indianapolis, Ind.). The proximal holes of the tube were taped shut through the nose. The tube was guided into the duodenum by standard fluoroscopy. After the tube had reached the duodenum, gallbladder contraction was stimulated by a 10-min intravenous infusion of 20 ng of cholecystokinin octapeptide (Sincalide Kinevac; Squibb, Princeton, N.J.) per kg of body weight. During this period and the following 15 min, all duodenal secretions were collected. A volume of 5 to 10 ml of intestinal juice (pH, approximately 7.0, as determined with pH paper) was collected from each patient. Immediately after sample collection, the tube was removed and a standard breakfast was provided to the volunteers.

Analytical scale incubation with phosphate buffer (pH 7.4). The following were added sequentially to a test tube: 1.5 ml of deionized water, 300 µl of 0.6 M phosphate buffer (pH 7.4), and 20 µl of an acetonitrile solution of either cefetamet pivoxil, cefuroxime axetil, or cefpodoxime proxetil at a concentration of 3 mg/ml. The mixture was stirred well and was incubated for 24 h in a temperature-controlled water bath at 37°C. At 0, 10, and 30 min as well as at 1, 2, 4, 6, 10, and 24 h, samples of 150 µl were withdrawn from the incubation solution, mixed with 150 µl of 0.5 M perchloric acid, and centrifuged at 3,000 × g for 10 min. A 50-µl sample of the supernatant was then injected onto an analytical HPLC column for analytical separation (see below). All analytical incubations were performed in duplicate.

Analytical scale incubation with intestinal juice (pH 7.4). The following were added sequentially to a test tube: 300 µl of freshly thawed intestinal juice, 60 µl of 0.6 M phosphate buffer (pH 7.4; the pH of the mixture of intestinal juice and phosphate buffer was 7.4), and 20 µl of an acetonitrile solution of either cefetamet pivoxil, cefuroxime axetil, or cefpodoxime proxetil at a concentration of 1 mg/ml. The following procedures were performed exactly as described above for the phosphate buffer study.

Preparative scale incubation with intestinal juice (pH 7.4). The following were added sequentially to a test tube: 600 µl of freshly thawed intestinal juice, 120 µl of 0.6 M phosphate buffer (pH 7.4), and 50 µl of an acetonitrile solution of prodrug ester at a concentration of 20 mg/ml. The mixture was stirred well and was incubated for 15 h in a temperature controlled water bath at 37°C. The incubation was stopped by the addition of 80 µl of 5 M perchloric acid. After centrifugation at 3,000 × g for 15 min, the supernatant was evaporated with nitrogen at 40°C to a volume of approximately 100 µl. The material from two incubations in 150 µl of fluid was subjected to HPLC for preparative separation of the metabolites (see below).

Analytical separation after incubation with phosphate buffer or intestinal juice. The chromatographic conditions for the analytical separation of the metabolites of cefetamet pivoxil and cefuroxime axetil were as follows: analytical column, C₁₈ Nucleosil (5 µm, 150 by 4.6 mm; Interchim, Montluçon, France) maintained at room temperature; mobile phase I, 0.002 M perchloric acid; mobile phase II, acetonitrile; flow rate, 1 ml/min; and detector wavelength, 265 nm. The eluent for the separation consisted of 84% mobile phase I (16% mobile phase II) for the first 6.5 min, and then it decreased from 84 to 62% mobile phase I at between 6.5 and 9 min and continued with this composition until 28.5 min. The column was then washed with 84% mobile phase I up to 40 min.

Preparative separation after incubation with intestinal juice. The chromatographic conditions for the preparative separation were the same as those described above in the analytical section except that trifluoroacetic acid was used in place of perchloric acid in mobile phase I. The two major polar peaks that formed during the incubation (for cefetamet pivoxil, ~6.1 and 7 min; for cefuroxime axetil, ~11 and 12.2 min; for cefpodoxime proxetil, ~7.3 and 7.7 min) were collected, evaporated under nitrogen at 40°C to ~150 ml, and further purified by HPLC on the same C₁₈ Nucleosil (5 µm, 150 by 4.6 mm) column by using an isocratic mobile phase separation (flow rate, 1 ml/min with a mobile phase of 88% water and 12% acetonitrile for cefuroxime axetil and cefpodoxime proxetil). For cefetamet pivoxil, a mixture of 90% water and 10% acetonitrile was used. The fraction containing major acidic metabolites was collected, concentrated with a stream of nitrogen to ~100 µl, and stored at 18°C. Immediately before initiating ¹H NMR analysis in D₂O, each fraction was evaporated to dryness by freeze-drying.

Quantification of the incubation results. At specific time points during the in vitro incubation with phosphate buffer and intestinal juice, samples were withdrawn and separated on an analytical HPLC column (for a description of the procedure, see above). For the quantification of the metabolites, the relative percentage of the areas under the main peaks at 265 nm was measured. Quantification of the prodrug esters and their metabolites neglects the small differences in their molar absorption at 265 nm (for cefetamet,  [265 nm] = 0.99 × 10⁺⁴ (for the 2 isomer and 1.19 × 10⁺⁴ for the 3 isomer).

Kinetic rate constants and t_1/2s. The kinetic degradation rate constants (K_DEG) of the different prodrug esters were determined as 1 times the slope. The slope was determined by linear regression of the linear portion of the ln (prodrug ester concentration)-versus-time curves. The half-life t_1/2 degradation values were determined by ln 2 divided by K_DEG.

	RESULTS

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The time courses and the patterns of the products of degradation differed significantly between phosphate buffer and intestinal juice at pH values of 7.4. Similar differences were observed with all three prodrug esters. The time course of disappearance of the prodrug esters and the appearance of the 3 and 2 acids for the hydrolysis in phosphate buffer is shown in Fig. 3a, 4a, and 5a, and those for the hydrolysis in intestinal juice are shown in Fig. 3b, 4b, and 5b. All degradation studies were performed over a 24-h period. The pathways of degradation of prodrug esters following enzymatic and weak aqueous base hydrolysis are shown in Fig. 2.

View larger version (10K): [in this window] [in a new window]   FIG. 2.   Degradation of prodrug esters following enzymatic and base catalyzed hydrolysis.

View larger version (12K): [in this window] [in a new window]   FIG. 3.   Incubation of cefetamet pivoxil in phosphate buffer (a) and intestinal juice (b) at pH 7.4. Shown are the relative percentages of the areas of the 3 ester (cefetamet pivoxil), the 3 acid (cefetamet), and 2 acid (inactive isomer of cefetamet) at 265 nm.

View larger version (15K): [in this window] [in a new window]   FIG. 4.   Incubation of cefuroxime axetil in phosphate buffer (a) and intestinal juice (b) at pH 7.4. Shown are the relative percentages of the areas of the two 3 esters (diastereoisomers of cefuroxime axetil), the 3 acid (cefuroxime), and the 2 acid (inactive isomer of cefuroxime) at 265 nm.

View larger version (15K): [in this window] [in a new window]   FIG. 5.   Incubation of cefpodoxime proxetil in phosphate buffer (a) and intestinal juice (b) at pH 7.4. Shown are the relative percentages of the areas of the two 3 esters (diastereoisomers of cefpodoxime proxetil), the 3 acid (cefpodoxime), and the 2 acid (inactive isomer of cefpodoxime) at 265 nm.

Cefetamet pivoxil. Degradation of cefetamet pivoxil progressed much faster in intestinal juice than in phosphate buffer. Cefetamet pivoxil degraded in intestinal juice with a K_DEG of 8.86 × 10¹ h¹, while in phosphate buffer the K_DEG was only 1.62 × 10¹ h¹. The corresponding t_1/2s were 0.78 h in intestinal juice and 4.3 h in phosphate buffer.

Cefuroxime axetil. In line with the other two prodrug esters the diastereoisomers of cefuroxime axetil degraded faster in intestinal juice than in phosphate buffer. Hence, there were significant differences in K_DEG and t_1/2 between the two incubation media (Table 1). Both diastereoisomers degraded in phosphate buffer with the same K_DEG of approximately 4.2 × 10¹ h¹ (t_1/2 = 1.6 h). However, in intestinal juice, one diastereoisomer declined with a K_DEG of 7.45 × 10¹ h¹ (t_1/2 = 0.93 h) and the other declined with a K_DEG of 18.8 × 10¹ h¹ (t_1/2 = 0.37 h).

Cefpodoxime proxetil. Like the other two prodrug esters, the two diastereoisomers of cefpodoxime proxetil degraded considerably faster in intestinal juice than in phosphate buffer (Table 1). In phosphate buffer, both diastereoisomers degraded with approximately the same K_DEGs (2.74 × 10¹ h¹ [t_1/2 = 2.5 h] and 3.13 × 10¹ h¹ [t_1/2 = 2.2 h]). In intestinal juice, however, one diastereoisomer degraded much faster than the other one (K_DEG = 38.7 × 10¹ h¹ [t_1/2 = 0.18 h] versus K_DEG = 7.10 × 10¹ h¹ [t_1/2 = 0.98 h]).

	DISCUSSION

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Hydrolysis of cephalosporin esters is a well-studied process. The C-4 ester group of 3 esters is rather resistant to hydrolysis, whereas the C-4 ester group of the 2 isomers is easily hydrolyzed. It is known that at a pH above 6, cleavage of the hydrolytically stable 3 prodrug ester occurs via isomerization to the hydrolytically unstable 2 ester. Isomerization is followed by rapid hydrolysis to the 2-cephalosporin (5, 7, 9). It is generally assumed that this degradation pathway is active in the intestine (2, 4). Whether or not active 3-cephalosporin will reach the blood following oral administration of the prodrug is believed to depend on the relative rate of the 3 to 2 isomerization versus the rate of cleavage of the C-4 ester group (9). Thus, the bioavailability of cephalosporin prodrug esters is seen simply as a function of the kinetics of the 3 to 2 isomerization whereby a high isomerization rate means low bioavailability. This model has actually been used for optimization of oral cephalosporin prodrug esters (4, 5, 8, 9).

Enzymatic hydrolysis of prodrug esters was not considered a critical factor influencing the bioavailability of the prodrug esters, although it is responsible for the release of active 3-cephalosporin in intestinal tissue once the prodrug esters are absorbed. Indeed, the hydrolytic activity of homogenates of intestinal tissue was high enough for prodrug esters to be completely hydrolyzed within minutes (4). In contrast, the content of the intestinal lumen of mice was shown to contain only about 1% of the hydrolytic activity of the intestinal tissue. This ester-cleaving activity was considered the source of fecal excretion of 3 acids, but it was not considered a critical factor for the bioavailability of prodrug esters (4).

Our studies have shown that the current theory has grossly overestimated the importance of the isomerization mechanism in the absorption process of oral cephalosporin prodrug esters. With all the compounds studied (cefetamet pivoxil, cefuroxime axetil, and cefpodoxime proxetil), rapid hydrolytic cleavage to the biologically active 3-cephalosporin was observed in intestinal juice without the concomitant formation of significant amounts of the 2 isomers. Spontaneous base-catalyzed isomerization to the 2 ester was significantly slower than hydrolytic cleavage to the 3 acid. Thus, the isomerization process is not efficient enough to compete with enzymatic ester cleavage. Therefore, the incomplete bioavailability of the prodrug esters is simply a consequence of the efficient enzymatic hydrolysis and not the result of the 3 to 2 isomerization and spontaneous hydrolysis of 2 esters.

A further relevant observation is the high stereoselectivity of the enzymatic cleavage reaction. With cefuroxime axetil and cefpodoxime proxetil, both of which are diastereoisomer mixtures, one of the diastereoisomers was hydrolyzed faster than the other one by factors of 2.5 and 5.5, respectively (Table 1). The presence of a cefuroxime axetil esterase in the intestinal tissue of dogs, rats, and humans has previously been demonstrated to be stereoselective. It was considered to contribute to the incomplete bioavailability of cefuroxime axetil (1, 6).

In view of the great difference in the hydrolytic rates of the diastereoisomers seen in our study with cefuroxime axetil and cefpodoxime proxetil, it can be concluded that the observed stereoselectivity of the intestinal enzyme activity is of general importance for the prodrug ester concept. By using the most stable diastereoisomer of the prodrug ester instead of a mixture, higher bioavailabilities can be achieved for these compounds. Furthermore, elimination of the more rapidly hydrolyzed isomer should reduce the amount of biologically active cephalosporin formed in the gut, leading to improved intestinal tolerability.