Abstract
Stereoselective disposition of sulbenicillin (SBPC) epimers in healthy human volunteers was studied in order to clarify the differences in pharmacokinetic behavior between the epimers. Stereospecific high-performance liquid chromatography was used for the determination of SBPC epimers. Plasma protein binding was measured in vitro with an ultrafiltration method. The binding was stereoselective, with the unbound fraction (fu) of the R-epimer being approximately 1.3-fold greater than that of the S-epimer. SBPC was administered intravenously to human volunteers, and concentrations of SBPC in plasma and urinary excretion rates were measured. Renal clearance (CLR) for the unbound drug (approximately 400 ml/min) was greater than the glomerular filtration rate (GFR) (approximately 109 ml/min) for both epimers, suggesting that both epimers are secreted at the renal tubules. Renal tubular secretion appeared to be greater for the S-epimer. When probenecid was coadministered, the CLR values of both epimers were significantly reduced and were approximately equal to the GFR values. CLR was greater for the S-epimer (37.5 and 49.8 ml/min for R-SBPC and S-SBPC, respectively), which was simply due to the greater fu of the S-epimer in plasma. In contrast, total body clearance was greater for the R-epimer (67.8 and 56.3 ml/min for R-SBPC and S-SBPC, respectively) because of the stereoselective degradation of the R-epimer in plasma. It was revealed that stereoselective degradation in the body had significant influence on the disposition of SBPC epimers.
It has been revealed that stereoisomers are recognized by the body as distinct chemical entities and that they exhibit different pharmacological activities, toxicities, and pharmacokinetics. For many β-lactam antibiotics, stereoisomers exist because of chirality in the side chains. Some of these epimeric β-lactams are used as mixtures of stereoisomers, although there are differences in pharmacokinetics between the epimers. For example, stereoselectivity has been reported in the plasma protein binding of moxalactam (22, 23), carbenicillin (12), and ceftibuten (20). Intestinal absorption of cephalexin (15, 21) and ceftibuten (17, 27) is also stereoselective. Moreover, it has been reported that carbenicillin (1, 8, 10), ticarcillin (10), cefsulodin (5), moxalactam (8, 9), and ceftibuten (20) may isomerize in the body as well as in aqueous solution. However, information on stereoselectivity in the pharmacokinetics and pharmacodynamics of epimeric β-lactams is very limited (14).
Sulbenicillin (SBPC) is a semisynthetic β-lactam antibiotic which has a broad antibacterial spectrum and is effective against both gram-positive and gram-negative bacteria. SBPC has been used clinically as a mixture of two epimers (R-SBPC and S-SBPC) because of the chirality of a (phenylsulfoacetyl)amino group attached to the 6 position of a penicillanic acid (Fig. 1). The R-epimer is approximately 40 times more potent than the S-epimer in antibiotic activity (18), and the ratio of R-epimer to S-epimer (R/S ratio) in the commercial preparation is approximately 3 (3, 18, 25). The half-life of SBPC in humans following intravenous administration is approximately 1 h, which was obtained by a microbiologic assay method (7). However, the pharmacokinetic characteristics of each epimer have not been clarified to date, since almost all of the previous studies employed a microbiologic assay method due to a lack of reliable and convenient stereospecific analytical methods (2, 7, 25).
FIG. 1.
Chemical structures of SBPC.
Stereospecific high-performance liquid chromatography (HPLC) methods have been developed in our laboratory for the analysis of some epimeric β-lactam antibiotics in biological fluids (11, 12, 14). Similar analytical methods were used in this study. Since many β-lactam antibiotics are secreted at the renal tubules, the effects of probenecid on the urinary excretion of SBPC epimers were also studied.
MATERIALS AND METHODS
Volunteer study protocol.
This study was approved by the Ethical Review Board of the School of Pharmaceutical Sciences, Kitasato University. Four male volunteers participated in the present study, and all of them gave written, informed consent. The volunteers were between 22 and 37 years old, with body weights of 63.0 ± 9.0 kg (mean ± standard deviation [SD]; n = 4). They had no evidence of disease, as determined by physical examination, urinalysis, and blood chemical tests. The following studies (control and probenecid studies) were conducted with a crossover study design and a 2-week washout period.
For the control study, 2 g of SBPC (R-SBPC/S-SBPC = 2.78) (Lilacillin; Takeda Pharmaceutical Industries Co., Osaka, Japan) was dissolved in 15 ml of saline and injected into a forearm vein over a 4-min injection time. Blood was collected in heparinized disposable syringes from the vein of the contralateral forearm before SBPC injection and at 0.25, 0.5, 0.75, 1, 1.5, 2.5, 3.5, and 5 h following injection of SBPC. Plasma was obtained immediately by centrifugation and stored at −80°C until it was analyzed. Urine was collected before SBPC injection as well as at the following time intervals after the injection: 0 to 0.5, 0.5 to 1, 1 to 2, 2 to 3, 3 to 4, 4 to 6, 6 to 12, and 12 to 24 h. Immediately after each collection, the urine volume was recorded and was diluted 50-fold with deionized water. The diluted urine samples were stored at −80°C until analyzed.
For the probenecid study, 1 g of probenecid (Probenemid tablets; Banyu Pharmaceutical Co., Tokyo, Japan) was administered orally at 12 and 1 h before SBPC injection. SBPC was administered in the same manner as in the control study. Blood was collected before SBPC injection and at 0.25, 0.5, 0.75, 1, 1.5, 2.5, 3.5, 5, and 9 h after injection. Urine was collected at the same time intervals as in the control study. Urine and plasma samples were stored in the same manner as in the control study.
Sample preparation for HPLC analysis.
Solid-phase extraction methods, which were modifications of previously reported methods, were used to prepare plasma and urine samples for HPLC analysis. A solid-phase extraction column (Bond Elut-SAX; Analytichem International, Harbor City, Calif.) was used for the plasma samples obtained in the control study and for the urine samples obtained both in the control and probenecid studies. Another type of solid-phase extraction column (Bond Elut-Certify II; Analytichem International) was used for the plasma samples obtained in the probenecid study, since the interfering substances could not be removed from the probenecid-treated plasma samples with the Bond Elut-SAX column.
A bond Elut-SAX column was preconditioned with 2 ml of methanol, followed by 2 ml of distilled water. A plasma sample was filtered through a Cosmonice filter (pore size, 0.45 μm) (type W; Nihon Millipore Kogyo, Tokyo, Japan), and an aliquot (0.5 ml) was mixed with 5 ml of 0.05 M CH3COONH4. One hundred microliters of carbenicillin aqueous solution (100 μg/ml) was added as an internal standard. The mixture was loaded onto a preconditioned SAX column and was drawn through the column under vacuum. The column was flushed with 3 ml of a mixture consisting of 0.5 M CH3COOH-CH3CN (1:1 [vol/vol]) and then with 2 ml of a mixture of 0.1 M CH3COONH4-CH3OH (1:1 [vol/vol]), and the flushing mixtures were discarded. This procedure was necessary to eliminate interfering substances on the HPLC chromatogram. The sample was then eluted with 0.5 ml of a mixture of 10% LiCl-CH3OH (3:2 [vol/vol]), and a 20-μl portion of the final eluent was injected into the HPLC. For the preparation of urine samples, 500 μl of a diluted urine sample was mixed with 5 ml of 0.05M CH3COONH4, and the sample was prepared for HPLC analysis by the same procedure as that described above for plasma samples.
A Bond Elut-Certify II column was preconditioned with 5 ml of a mixture consisting of 10% LiCl-CH3OH (3:2 [vol/vol]), followed by 2 ml of methanol, and then 2 ml of distilled water. An aliquot (0.5 ml) of the filtered plasma sample (as described above) was mixed with 2 ml of 0.05 M CH3COONH4. One hundred microliters of carbenicillin aqueous solution (100 μg/ml) was added as an internal standard. The mixture was loaded onto a preconditioned Certify II column and was drawn through the column under vacuum. The column was flushed with 3 ml of a mixture consisting of 0.5 M CH3COOH-CH3CN (1:1 [vol/vol]) and then with 2 ml of a mixture of 0.1 M CH3COONH4-CH3OH (1:1 [vol/vol]), and the flushing mixtures were discarded. The sample was then eluted with 1 ml of a mixture of 10% LiCl-CH3OH (3:2 [vol/vol]), and a 40-μl portion of the final eluent was injected into the HPLC.
HPLC conditions for SBPC determinations.
An HPLC was used to determine the concentrations of SBPC epimers. The HPLC system consisted of a dual piston pump (model LC10AD), a UV detector (model SPD-10A), and an integrator (model C-R4A), all from Shimadzu Co., Kyoto, Japan. A Cosmosil column (5C18-AR, 4.6 by 250 mm; Nacalai Tesque Co., Kyoto, Japan) was used as an analytical column. The mobile-phase compositions were 0.05 M phosphate buffer (pH 7.0)–CH3OH (8:1 [vol/vol]) for the plasma samples in the control study and for the urine samples both in the control and probenecid studies, and 0.05 M CH3COONH4-CH3OH (7:1 [vol/vol]) for the plasma samples in the probenecid study. The flow rate was 0.9 ml/min, and SBPC epimers were detected at 254 nm.
Plasma protein binding study.
For both the control and probenecid studies, plasma was obtained from each volunteer 15 min before the SBPC injection and was used for in vitro binding studies. Binding of SBPC in human plasma in vitro was measured by an ultrafiltration method. Amicon Centrifree was used as an ultrafiltration device with a type YMT membrane (Amicon Division, W. R. Grace & Co., Beverly, Mass.). One milliliter of the plasma sample (pH adjusted to 7.4 ± 0.1 with 1 N HCl) was mixed with 50 μl of various concentrations of SBPC (R/S = 2.78) aqueous solution, and an aliquot (0.1 ml) was prepared for HPLC, as previously described, to determine the total (bound plus unbound) concentration of each epimer. The remainder of the sample (ca. 0.95 ml) was centrifuged at 1,000 × g for 4 min at 37°C, and a 100-μl aliquot of the filtrate was prepared for HPLC analysis in a manner similar to that described above for the plasma samples in order to determine the unbound concentration of each epimer. A Bond Elut-Certify II or SAX column was used for samples with and without probenecid pretreatment, respectively. A customized Himac 15D centrifuge (Hitachi, Tokyo, Japan) was used to control the temperature during ultrafiltration.
Pharmacokinetic analysis.
Plasma protein binding data were analyzed according to a Langmuir equation with a single class of binding sites:
 |
1 |
where r is the number of bound drug per albumin molecule, n is the number of binding sites, K is the binding constant, and Cf is the unbound concentration of the drug. Binding parameters were obtained by a nonlinear least-squares method (MULTI) (26). Parameter values were calculated for each subject both in the control and probenecid studies.
The unbound (free) fraction (fu) of each epimer at a given concentration was calculated with the following equation:
 |
2 |
where n is the number of binding sites, K is the binding constant, Pt is the plasma albumin concentration (assumed to be 0.6 mM), and Ct is the total plasma concentration (bound plus unbound) of each epimer in plasma. Equation 2 was derived by solving the equation for fu, which was reported by Martin (16), as a function of Ct.
SBPC concentration in plasma versus time curves were analyzed by a nonlinear least-squares method (MULTI) (26) with a weight of 1/C2 for each datum point, where C is the concentration in plasma. Total body clearance (CL) was calculated as the dose divided by the area under the curve.
The amount of each epimer excreted in urine during each collection interval was determined as the concentration of each epimer in the urine multiplied by the urine volume. Renal clearance (CLR) was calculated as the renal excretion rate divided by the concentration in plasma at the midpoint of each urine collection interval. Moreover, renal clearance for unbound drug (CLR,u) was calculated as CLR/fu.
CLR can generally be expressed by the following equation:
 |
3 |
where CLR is the renal clearance, GFR is the glomerular filtration rate, fu is the unbound fraction in plasma, QR is the renal plasma flow rate, CLint,sec is the intrinsic clearance by renal tubular secretion, and Rabs is the extent of reabsorption (6, 19). However, reabsorption was negligible (Rabs = 0) for both epimers because the CLR,u values were almost equal to the GFR when renal secretion was blocked with probenecid. Therefore, intrinsic clearance by renal tubular secretion (CLint,sec) was calculated with equation 4, which is obtained by solving equation 3 for CLint,sec. The same equation has been used for the analysis of carbenicillin in humans (12):
 |
4 |
Stability of SBPC epimers in plasma.
An aqueous solution of SBPC was added to the plasma obtained from the control subjects, and the mixture was transferred to a glass tube and incubated at 37°C. The pH was controlled by filling the glass tube with O2-CO2(95:5). Concentrations of SBPC epimers were determined at appropriate times with HPLC as described above.
Determination of GFR.
Creatinine concentrations in plasma and urine were determined with a Wako Creatinine-Test kit (Wako Pure Chemicals, Osaka, Japan). The GFR was determined as the urinary excretion rate of creatinine divided by the creatinine concentration in plasma.
Statistical analysis.
Statistical analyses were conducted by using the Wilcoxon signed rank test and the Mann-Whitney U test for paired and unpaired comparisons, with a P value of less than 0.05 considered statistically significant.
RESULTS
According to the analytical method used in the present study, the detection limit of SBPC was approximately 0.5 μg/ml for each epimer. Calibration curves were linear over the range of 0 to 300 μg/ml, with the correlation coefficients being greater than 0.999. Recovery ranged from 97.2 to 99.4% for R-SBPC and 93.3 to 98.1% for S-SBPC. Interday variabilities were 3.6 to 9.0% and 3.3 to 5.8% for R-SBPC and S-SBPC, respectively, over the range of 10 to 200 μg/ml. Intraday variabilities were 2.6 to 7.3% and 2.4 to 5.1% for R-SBPC and S-SBPC, respectively, over the range of 50 to 200 μg/ml.
The fu measured in vitro was plotted against the concentration of each epimer (Fig. 2). The fu values were concentration dependent, and the data were analyzed according to a Langmuir equation with a single class of binding sites since the Scatchard plots for both epimers were linear. As shown in Fig. 2, there was a difference in the extent of binding between the two epimers; that is, fu was significantly greater for S-SBPC than for R-SBPC in the plasma of both control and probenecid-treated volunteers (P < 0.05 [paired]). The fu of each epimer in the plasma of the probenecid-treated subjects was slightly (less than 10%) greater than that in the plasma of the control subjects. The binding parameters were calculated according to equation 1, and the values are listed in Table 1. The parameter values obtained for each subject were used to calculate fu of SBPC at a given concentration with equation 2.
FIG. 2.
Unbound fractions of R-SBPC (○) and S-SBPC (•) in plasma of control (A) and probenecid-treated (B) subjects. Binding was measured at nine different concentrations for each subject.
TABLE 1.
Binding parameters of R-SBPC and S-SBPC in plasma of control and probenecid-treated subjects
| Study group | Epimer | na | 10−3K (M−1)b |
|---|---|---|---|
| Without probenecid | R-SBPC | 1.29 ± 0.11 | 3.89 ± 0.43 |
| S-SBPC | 0.41 ± 0.07 | 8.58 ± 0.27 | |
| With probenecid | R-SBPC | 1.11 ± 0.18 | 3.52 ± 0.53 |
| S-SBPC | 0.39 ± 0.09 | 5.09 ± 1.57 |
Number of binding sites. Values are means ± SDs for four subjects.
Binding constant.
R-SBPC and S-SBPC concentration profiles in plasma following SBPC injection with and without probenecid coadministration are shown in Fig. 3. In the control study, drug concentration profiles in plasma were analyzed with a one-compartment open model, which showed a better fit than that obtained with a two-compartment open model. In the probenecid study, drug concentration profiles in plasma were analyzed with a two-compartment open model. When probenecid was coadministered, a small distribution phase was observed and the two-compartment open model gave smaller values of AIC (Akaike’s information criterion) (26).
FIG. 3.
Concentrations of R-SBPC (○) and S-SBPC (•) in plasma following intravenous injection of 2 g of SBPC (R/S = 2.78) to healthy volunteers with (B) and without (A) probenecid coadministration. The means and SDs of four subjects are shown.
In the control study, R-SBPC concentrations in plasma were approximately threefold greater than S-SBPC concentrations at all time points. The threefold difference between R-SBPC and S-SBPC concentrations reflected the R-SBPC/S-SBPC ratio in the administered preparations. The volumes of distribution (V) for R-SBPC and S-SBPC were 10.3 ± 0.5 and 13.1 ± 0.6 liters, respectively (mean ± SD; n = 4), with the V of S-SBPC being slightly but significantly greater than that of R-SBPC (P < 0.05 [paired]). The difference in V between the epimers reflected that in the fu, as mentioned above. The CL of S-SBPC was significantly greater than that of R-SBPC (Table 2).
TABLE 2.
CL, CLR, and CLNR of R-SBPC and S-SBPC following intravenous injection of 2 g of SBPC to healthy volunteers with and without probenecid coadministrationa
| Study group | CL (ml/min/163 kg)
|
CLR (ml/min/163 kg)b
|
CLNR (ml/min/163 kg)c
|
|||
|---|---|---|---|---|---|---|
| R-SBPC | S-SBPC | R-SBPC | S-SBPC | R-SBPC | S-SBPC | |
| Without probenecid | 129 ± 11 | 157 ± 9d | 112 ± 11 | 159 ± 20 | 18 ± 7 | —e |
| With probenecid | 67.8 ± 7.0f | 56.3 ± 6.2f | 37.5 ± 7.6f | 49.8 ± 7.2f | 30.2 ± 8.3 | 6.5 ± 8.7 |
Each value is the mean ± SD for four subjects in each group.
Calculated as the mean of four urine collection intervals (0.5 to 1, 1 to 2, 2 to 3, and 3 to 4 h).
Calculated as CL minus CLR.
Significantly greater than R-SBPC (P < 0.01) according to a paired t test.
This value was not calculated, since the CLR value was slightly greater than the CL value.
Significantly different from the control (P < 0.01) according to a paired t test.
In the probenecid study, R-SBPC concentrations were approximately threefold greater than those of S-SBPC shortly after the injection. However, the concentration of R-SBPC in plasma became closer to that of S-SBPC, and the concentrations of R-SBPC and S-SBPC were almost equal at 9 h after the injection. This was reflected in the greater CL value of R-SBPC (Table 2). On the other hand, the CL values were significantly smaller than those in the control study, suggesting that elimination of both epimers from the body is inhibited by probenecid (Table 2).
Urinary excretion profiles of SBPC epimers are shown in Fig. 4A and B for the control and probenecid studies, respectively. In the control study, all of the administered S-epimer was recovered in the urine within 24 h, whereas the urinary recovery of the R-epimer was ca. 80% in 24 h. The results suggested that urinary excretion is the major elimination pathway for both epimers.
FIG. 4.
Cumulative excretion (percentage of dose) of R-SBPC (○) and S-SBPC (•) in urine following intravenous injection of 2 g of SBPC (R/S = 2.78) to healthy volunteers with (B) and without (A) probenecid coadministration. The means and SDs of four subjects are shown.
In the probenecid study, more than 90% of the S-epimer was excreted in the urine within 24 h, while only 60% of the R-epimer was excreted in the urine during the same time period. When probenecid was coadministered, urinary excretion rates of both epimers were smaller than those in the control study. On the other hand, there were significant differences (P < 0.05 [paired]) in the urinary recovery of SBPC epimers both in the control and probenecid studies, and the difference was greater in the probenecid study.
The R/S ratios in urine accumulated for 24 h were 2.30 ± 0.02 and 1.88 ± 0.11 (mean ± SD; n = 4) in the control and probenecid studies, respectively. These values were significantly smaller than the R/S ratio of 2.78 ± 0.05 (mean ± SD; n = 8) in the administered preparations (P < 0.05 [unpaired]). The differences in urinary excretion between the epimers suggested a stereoselective elimination from the body, which was more marked when probenecid was coadministered.
The CLR values are listed in Table 2. In the control study, the CLR of S-SBPC was greater than that of R-SBPC, which was consistent with the greater CL of S-SBPC. Also, the CLR was slightly but significantly greater for S-SBPC in the probenecid study. This observation, however, disagreed with the fact that the CL of S-SBPC was smaller than that of R-SBPC in the probenecid study. The greater CL of R-SBPC was due to the greater nonrenal clearance (Table 2; also see Discussion).
The CLR was further divided by the fu, which was calculated with equation 2, and the values obtained are shown as CLR,u in Fig. 5. The CLR,u values for both epimers were much greater than the GFR (109 ± 19 ml/min [mean ± SD; n = 4]), suggesting that both epimers are secreted at the renal tubules. When probenecid was coadministered, the CLR,u values of both epimers were almost equal to the GFR values. The results suggested that secretion of both epimers is almost completely blocked by probenecid and that reabsorption is negligible for both epimers.
FIG. 5.
CLR,u of R-SBPC (□) and S-SBPC (▪) following intravenous injection of 2 g of SBPC (R/S = 2.78) to healthy volunteers with (B) and without (A) probenecid coadministration. The means and SDs of four subjects are shown. Solid lines and shaded areas represent the means and SDs of the GFR values.
Intrinsic clearance for renal tubular secretion (CLint,sec) was calculated for each time interval according to equation 4. For the calculation of CLint,sec, the renal plasma flow (QR) was assumed to be 10 ml/min/kg of body weight (4). The CLint,sec values obtained were 378 ± 70 and 461 ± 66 ml/min (mean ± SD; n = 4) for R-SBPC and S-SBPC, respectively. The CLint,sec values of S-SBPC were significantly greater than those of R-SBPC (P < 0.05 [paired]). Although equation 4 is widely accepted (6, 19), it may be more accurate to use QR − GFR instead of QR to calculate CLint,sec, since the plasma flow rate at the proximal renal tubules is represented more accurately by QR − GFR. However, when the QR − GFR value was used for the calculation of CLint,sec, the obtained CLint,sec values were only 3 to 7% greater than the values mentioned above.
SBPC was incubated with human plasma at 37°C, and the concentrations of the epimers were measured at appropriate times. The results are shown in Fig. 6. R-SBPC disappeared at a much higher rate than S-SBPC, indicating stereoselective degradation of R-SBPC. The degradation of R-SBPC appeared to be a first-order process.
FIG. 6.
Disappearance of R-SBPC (○) and S-SBPC (•) in human plasma in vitro at 37°C.
DISCUSSION
Binding of SBPC epimers in human plasma was stereoselective, as has been reported for other epimeric β-lactam antibiotics such as moxalactam (22) and carbenicillin (12). However, the stereoselectivity in the binding of SBPC was opposite to those of moxalactam and carbenicillin; the fu of the R-epimer is greater for moxalactam and carbenicillin. Since the concentrations of R-SBPC are approximately threefold greater than those of S-SBPC in the present binding studies, there is a possibility that S-SBPC may be displaced by R-SBPC from the binding sites, which may result in the greater fu of S-SBPC. However, when the binding was measured with isolated SBPC epimers in our preliminary study, the fu values were similar to those observed in the present study (data not shown). The results suggested that the greater fu of S-SBPC is not because of the displacement by R-SBPC with threefold-greater concentrations and that the observed stereoselectivity may indeed reflect the intrinsic stereoselectivity in the binding of SBPC epimers.
The parameter values listed in Table 1 may be apparent values, because the binding of each epimer was measured in the presence of the other epimer. According to our study, serum albumin is the predominant binding component for the binding of SBPC in plasma (data not shown), and only a small portion (less than 20%) of the binding sites are occupied by SBPC epimers at therapeutic concentrations. Under these conditions, mutual displacement between the epimers is not significant (13). Indeed, the fu values were successfully calculated at therapeutic concentrations with the present parameter values.
The fu values in the probenecid study may be overestimated because the protein binding was measured with plasma samples shortly before SBPC administration. The probenecid concentration in plasma should decrease following SBPC administration, which may result in reduced effects on the binding of SBPC. However, considering the relatively long half-life of probenecid (6 to 12 h in humans), the influence of probenecid on the binding of SBPC may not change drastically during the course of the administration study.
On the other hand, potential misestimation of fu values may influence the calculation of CLint,sec but not the calculation of the CL and CLR values. The CL values were calculated from the plasma concentration profiles, and the CLR values were calculated from the plasma concentration and urinary excretion data. Therefore, the difference between the CL and CLR in the probenecid study truly resulted from the degradation of R-SBPC (see below), not from the misestimation of fu values.
Since the CLR,u values of both SBPC epimers were much greater than the GFR values in the control study (Fig. 5), it was confirmed that SBPC epimers are actively secreted in the renal tubules. This was further supported by the observations in the probenecid study, in which the CLR,u values were similar to the GFR values. The results suggest that the secretion of SBPC epimers by the organic anion transport system is almost completely blocked by probenecid and that reabsorption of the epimers is negligible. These observations are consistent with previous results on the disposition of carbenicillin, which is also a dianionic β-lactam. In humans, carbenicillin was secreted but not reabsorbed from the renal tubules, and the secretion was almost completely blocked by probenecid under the same conditions (12).
Recovery of S-SBPC in the 24-h urine appeared to be slightly greater than 100% in the control study (Fig. 4A), which may be partly due to the epimerization of R-SBPC in the body. In our preliminary study, approximately 2.5% of R-SBPC was converted to S-SBPC in 10 h in the presence of 4% human serum albumin (HSA) at 37°C (data not shown). Since R-SBPC concentrations in plasma were approximately threefold greater than those of S-SBPC following intravenous injection, the 2.5% conversion of R-SBPC corresponds to 7.5% of S-SBPC being generated in the body.
In the control study, the nonrenal clearance (CLNR) of S-SBPC [CLNR(S)] was negligible since the CL of S-SBPC was almost equal to the CLR of S-SBPC, whereas the CLNR of R-SBPC [CLNR(R)] was 18 ml/min (Table 2). In the probenecid study, the CLNR(S) was 6.5 ml/min. Although this value was not significantly different from zero (P > 0.05 [Student’s t test]), the increased CLNR(S) may be because of the greater contribution of other elimination pathways (e.g., bile excretion, etc.) in the disposition of SBPC, which resulted from the increase in fu. On the other hand, the CLNR(R) in the probenecid study was 30.2 ml/min (Table 2). Assuming that the contribution of other elimination pathways to CLNR(R) is also 6.5 ml/min, 23.7 ml/min of CLNR(R) is still unaccounted for in the probenecid study. This value is similar to the CLNR(R) in the control study (18 ml/min). Therefore, it is suggested that approximately 20 ml/min of stereoselective elimination clearance for R-SBPC exists both in the control and probenecid studies.
When SBPC was incubated in human plasma, it was observed that the R-epimer degraded stereoselectively (Fig. 6). The degradation was facilitated by HSA and was dependent on the HSA concentration (data not shown). The apparent first-order rate constant for the degradation of R-SBPC in plasma was 0.178 h−1, which was almost equal to that observed in 4% HSA solution (pH 7.4; 37°C). The extent of epimerization of R-SBPC was much lower than the extent of degradation; less than 3% of the disappeared R-epimer was converted to the S-epimer in the presence of 4% HSA in our preliminary study (data not shown).
In order to estimate the influence of the stereoselective degradation on the disposition of R-SBPC, the following assumptions were made: (i) SBPC epimers are distributed only in plasma and interstitial fluids, and (ii) the first-order degradation rate constant of R-SBPC is proportional to the HSA concentration. Since it has been reported that most β-lactams are distributed only in plasma and interstitial fluids (22), the first assumption may hold true for SBPC. The second assumption is also likely to hold true because the degradation of R-SBPC was dependent on HSA concentration in our preliminary study.
With these assumptions, the clearance for the degradation of R-SBPC [CLdeg(R)] was calculated by the following equation:
 |
5 |
where Kdeg(R) is the first-order degradation rate constant in 4% HSA solution, Vp is the plasma volume in the body, VIFi is the volume of the interstitial fluid of the ith tissue sample, and ARi is the reported ratio of the albumin concentration in the interstitial fluid to that in plasma in the ith tissue (19). The Vp value was calculated as 63 kg · (1/13) · 0.55 = 2.67 liters, where the ratio of the plasma volume to the whole blood volume was assumed to be 0.55. Clearance for the degradation of R-SBPC calculated for each tissue type is listed in Table 3, together with other physiological parameters. Calculation was conducted for a 63-kg human, since the average weight of the volunteers was 63 kg. The CLdeg(R) value calculated according to equation 5 was 23.0 ml/min (Table 3), which was similar to the stereoselective elimination clearance for R-SBPC (approximately 20 ml/min), as mentioned above. Therefore, it is very likely that at least approximately 20 ml/min of CLNR(R) results from the degradation of R-SBPC in the body.
TABLE 3.
Physiological parametersa and degradation clearance values of R-SBPC in various tissues of a 63-kg human
| Tissue type | Vp or VIF (ml) | AR | Kdeg(R) · VIF (or Vp) · AR (ml/min) |
|---|---|---|---|
| Blood pool | 2,670b | 1.0 | 7.9 |
| Lung | 108 | 0.5 | 0.2 |
| Heart | 36 | 0.5 | 0.1 |
| Muscle | 3,240 | 0.6 | 5.8 |
| Skin | 810 | 1.0 | 2.4 |
| Bone | 864 | 1.0 | 2.6 |
| Gut | 90 | 0.9 | 0.2 |
| Liver | 90 | 0.5 | 0.1 |
| Kidney | 41 | 0.5 | 0.1 |
| Carcass | 2,421 | 0.5 | 3.6 |
| Total [CLdeg(R)]c | 23.0 |
Physiological parameters were obtained from reference 18, with the VIF and Vp adjusted for a 63-kg human.
Vp. All other volumes are VIF.
CLdeg(R) calculated with equation 5 (see text).
Stereoselective degradation of R-SBPC may also account for the reduced urinary excretion of R-SBPC. The ratios of CLR(R)/[CLR(R) + CLdeg(R)] were 0.83 and 0.62 for the control and probenecid studies, respectively, similar to the observed urinary recoveries of approximately 0.87 and 0.66, respectively.
The present study revealed that the R-epimer of SBPC degrades stereoselectively in plasma, which plays a significant role in the stereoselective disposition of SBPC epimers. Since many chiral drugs are currently used as mixtures of stereoisomers with little information on stereoselective behavior in the body, it is important to clarify the differences in pharmacokinetics and pharmacodynamics between stereoisomers. The results obtained in the present study should provide valuable information for understanding the stereoselective dispositions of chiral drugs.
ACKNOWLEDGMENT
Part of this study was financially supported by the Japan Research Foundation for Clinical Pharmacology.
REFERENCES
- 1.Aso Y, Yoshioka S, Takeda Y. Epimerization and racemization of some chiral drugs in the presence of human serum albumin. Chem Pharm Bull. 1990;38:180–184. doi: 10.1248/cpb.38.180. [DOI] [PubMed] [Google Scholar]
- 2.Benoni G, Bertazonni M E, Cantelli F G. Comparative investigation of pharmacokinetic properties of sulbenicillin and carbenicillin in rat. Drugs Exp Clin Res. 1982;VIII(1):71–77. [Google Scholar]
- 3.Bird A E, Steele B R. Nuclear magnetic resonance and circular dichroism of penicillins derived from disubstituted acetic acids. J Chem Soc Perkin Trans I. 1982;1982:563–569. [Google Scholar]
- 4.Bischoff K B, Dedrick R L, Zaharko D S, Longstreth J A. Methotrexate pharmacokinetics. J Pharm Sci. 1971;60:1128–1133. doi: 10.1002/jps.2600600803. [DOI] [PubMed] [Google Scholar]
- 5.Fujita T, Koshiro A. Kinetics and mechanisms of the degradation and epimerization of sodium cefsulodin in aqueous solution. Chem Pharm Bull. 1984;32:3651–3661. doi: 10.1248/cpb.32.3651. [DOI] [PubMed] [Google Scholar]
- 6.Gibaldi M, Perrier D. Pharmacokinetics. 2nd ed. New York, N.Y: Marcel Dekker, Inc.; 1982. Clearance concepts; pp. 319–353. [Google Scholar]
- 7.Hansen I, Jacobson E, Weis J. Pharmacokinetics of sulbenicillin, a new broad spectrum semisynthetic penicillin. Clin Pharmacol Ther. 1975;17:339–347. doi: 10.1002/cpt1975173339. [DOI] [PubMed] [Google Scholar]
- 8.Hashimoto N, Tanaka H. Epimerization kinetics of moxalactam, its derivative, and carbenicillin in aqueous solution. J Pharm Sci. 1985;74:68–71. doi: 10.1002/jps.2600740118. [DOI] [PubMed] [Google Scholar]
- 9.Hashimoto N, Tasaki T, Tanaka H. Degradation and epimerization kinetics of moxalactam in aqueous solution. J Pharm Sci. 1984;73:369–373. doi: 10.1002/jps.2600730320. [DOI] [PubMed] [Google Scholar]
- 10.Hoogmartens J, Roets E, Janssen G, Vanderhaeghe H. Separation of the side-chain diastereomers of penicillins by high-performance liquid chromatography. J Chromatogr. 1982;244:299–309. [Google Scholar]
- 11.Ishida M, Tsuda Y, Onuki Y, Itoh T, Yamada H. High-performance liquid chromatographic determination of carbenicillin epimers in plasma and urine. J Chromatogr B. 1994;652:43–49. doi: 10.1016/0378-4347(93)e0378-4. [DOI] [PubMed] [Google Scholar]
- 12.Itoh T, Ishida M, Onuki Y, Tsuda Y, Shimada H, Yamada H. Stereoselective renal tubular secretion of carbenicillin. Antimicrob Agents Chemother. 1993;37:2327–2332. doi: 10.1128/aac.37.11.2327. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Itoh T, Nakashima K, Tsuda Y, Yamada H. Stereoselective binding of carbenicillin epimers to human serum albumin. Chirality. 1996;8:201–206. doi: 10.1002/(SICI)1520-636X(1996)8:2<201::AID-CHIR5>3.0.CO;2-L. [DOI] [PubMed] [Google Scholar]
- 14.Itoh T, Yamada H. Diastereomeric β-lactam antibiotics: analytical methods, isomerization and stereoselective pharmacokinetics. J Chromatogr A. 1994;694:195–208. doi: 10.1016/0021-9673(94)00932-y. [DOI] [PubMed] [Google Scholar]
- 15.Kramer W, Girbig F, Gutjahr U, Kowalewski S, Adam F, Schiebler W. Intestinal absorption of β-lactam antibiotics and oligopeptides. Eur J Biochem. 1992;204:923–930. doi: 10.1111/j.1432-1033.1992.tb16713.x. [DOI] [PubMed] [Google Scholar]
- 16.Martin B K. Potential effect of the plasma proteins on drug distribution. Nature. 1965;207:274–276. doi: 10.1038/207274a0. [DOI] [PubMed] [Google Scholar]
- 17.Muranushi N, Yoshikawa T, Yoshida M, Oguma T, Hirano K, Yamada H. Transport characteristics of ceftibuten, a new oral cephem, in rat intestinal brush-border membrane vesicles: relationship to oligopeptide and amino β-lactam transport. Pharm Res. 1989;6:308–312. doi: 10.1023/a:1015946407709. [DOI] [PubMed] [Google Scholar]
- 18.Nomura H, Kawamura K, Shinohara M, Masuda Y, Okada Y, Fujii S. Studies on diastereomers of sulbenicillin by nuclear magnetic resonance. J Takeda Res Lab. 1972;31(4):442–452. [Google Scholar]
- 19.Rowland M, Tozer T N. Clinical Pharmacokinetics—concepts and applications. Philadelphia, Pa: Lea & Febiger; 1980. Clearance and renal excretion; pp. 48–64. [Google Scholar]
- 20.Shimada J, Hori S, Oguma T, Yoshikawa T, Yamamoto S, Nishikawa T, Yamada H. Effects of protein binding on the isomerization of ceftibuten. J Pharm Sci. 1993;82:461–465. doi: 10.1002/jps.2600820506. [DOI] [PubMed] [Google Scholar]
- 21.Tamai I, Ling H, Timbul S, Nishikido J, Tsuji A. Stereospecific absorption and degradation of cephalexin. J Pharm Pharmacol. 1988;40:320–324. doi: 10.1111/j.2042-7158.1988.tb05259.x. [DOI] [PubMed] [Google Scholar]
- 22.Tsuji A, Yoshikawa T, Nishide K, Minami H, Kimura M, Nakashima E, Terasaki T, Miyamoto E, Nightingale C H, Yamana T. Physiologically based pharmacokinetic model for β-lactam antibiotics. I. Tissue distribution and elimination in rats. J Pharm Sci. 1983;72:1239–1252. doi: 10.1002/jps.2600721103. [DOI] [PubMed] [Google Scholar]
- 23.Yamada H, Ichihashi T, Hirano K, Kinoshita H. Plasma protein binding and urinary excretion of R- and S-epimers of an arylmalonylamino 1-oxacephem. I. In humans. J Pharm Sci. 1981;70:112–113. doi: 10.1002/jps.2600700130. [DOI] [PubMed] [Google Scholar]
- 24.Yamada H, Ichihashi T, Hirano K, Kinoshita H. Renal excretion of R- and S-epimers of moxalactam in dogs. J Pharm Sci. 1981;70:112. doi: 10.1002/jps.2600700130. [DOI] [PubMed] [Google Scholar]
- 25.Yamaoka K, Narita S, Nakagawa T, Uno T. High-performance liquid chromatographic analyses of sulbenicillin and carbenicillin in human urine. J Chromatogr. 1979;168:187–193. doi: 10.1016/s0021-9673(00)80708-6. [DOI] [PubMed] [Google Scholar]
- 26.Yamaoka K, Tanigawara T, Nakagawa T, Uno T. A pharmacokinetic analysis program (MULTI) for microcomputer. J Pharmacobio-Dyn. 1981;4:879–885. doi: 10.1248/bpb1978.4.879. [DOI] [PubMed] [Google Scholar]
- 27.Yoshikawa T, Muranushi N, Yoshida M, Oguma T, Hirano K, Yamada H. Transport characteristics of ceftibuten (7432-S), a new oral cephem, in rat intestinal brush-border membrane vesicles: proton-coupled and stereoselective transport of ceftibuten. Pharm Res. 1989;6:302–307. doi: 10.1023/a:1015994323639. [DOI] [PubMed] [Google Scholar]