![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Division of Pharmaceutical Sciences, Graduate School of Natural Science and Technology, Kanazawa University, Kanazawa, Japan
(Received April 9, 2006; accepted November 27, 2006)
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
-amino-ß-lactam antibiotic, exists as two diastereoisomers, L-CEX and D-CEX (Fig. 1), but as with all the other commercially available ß-lactam antibiotics, only the D-isomer is used as an ethical drug. L-CEX is not used as a medicine because it is rapidly metabolized after administration p.o. (Tamai et al., 1988
), and it lacks antibacterial activity (Snyder et al., 1997). D-CEX is a highly hydrophilic compound, and it mainly exists in an ionized form in the vicinity of the brush-border membrane of intestinal epithelial cells, where the microclimate pH is 6.5 to 7.5 in the rat jejunum (Iwatsubo et al., 1986) and the pKa values of D-CEX are 2.56 and 6.88 (Yamana and Tsuji 1976). Nevertheless, permeability of D-CEX through the small intestine is good, and the oral bioavailability is more than 90% (Hardman and Limbird, 2001). Many studies have been performed to understand the mechanism(s) of efficient intestinal absorption, and it has been suggested that the transport of D-CEX through the intestinal membrane is governed by a carrier-mediated mechanism (Tsuji et al., 1981a,b; Nakashima et al., 1984; Dantzig and Bergin, 1990; Kramer et al., 1990), which also recognizes dipeptides and tripeptides.
|
). It was initially suggested that L-CEX is not transported by the oligopeptide transporter because L-CEX was not detected in rabbit intestinal brush-border membrane vesicles or proteoliposomes prepared from rabbit intestinal brush-border membrane vesicles (Kramer et al., 1992). However, it was shown that L-CEX binds to the peptide transporter with high affinity and L-CEX is transported by assessing substrate-induced intracellular acidification caused by H+ cotransported with the substrate in Caco-2 cells (Wenzel et al., 1995). Regarding affinity of CEX to the H+-coupled peptide transporter, L-CEX competitively inhibited D-CEX uptake more strongly than did D-CEX itself in a rat intestinal everted sac and human intestinal model cell line Caco-2 cells (Tamai et al., 1988; Wenzel et al., 1995; Snyder et al., 1997). Thus, although L-CEX seems to be transported via the same carrier system as D-CEX and the affinity of the transporter for L-CEX is higher than that for D-CEX, L-CEX is not detected inside the cells because it is metabolized very rapidly.
The oligopeptide transporter PEPT1 (SLC15A1) (Fei et al., 1994; Liang et al., 1995; Saito et al., 1995), which belongs to the proton/oligopeptide cotransporter family, has been shown to contribute to the uptake from the apical side of CEX and other ß-lactam antibiotics with peptide-type structures (Ganapathy et al., 1995; Tsuji and Tamai 1996; Tamai et al., 1997) in the small intestine. PEPT1 is expressed on the apical membranes of epithelial cells in the small intestine (Sai et al., 1996), and its expression level correlates with absorptive transport of ß-lactam antibiotics, as well as a dipeptide model compound, glycylsarcosine (Gly-Sar), in rats (Shiraga et al., 1999; Naruhashi et al., 2002). PEPT1 also mediates uptake of various other drugs, including certain angiotensin-converting enzyme inhibitors, an anticancer agent (ubenimex), and an antiviral agent (valacyclovir), as well as dipeptides and tripeptides derived from nutrients (Balimane et al., 1998; Rubio-Aliaga and Daniel, 2002; Tsuji, 2002; Daniel, 2004; Terada and Inui, 2004).
Although the pharmacokinetic properties of CEX have been well investigated, the molecular mechanism(s) responsible for the rapid metabolism of L-CEX has not yet been clarified. We hypothesized that PEPT1 would play a major role in the molecular mechanism of the stereoselective metabolism, as well as the membrane transport of CEX. Therefore, we examined the contribution of PEPT1 to the stereoselective uptake and metabolism of CEX using in vitro PEPT1-expressing cell lines.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
). The protein assay kit was purchased from Bio-Rad Laboratories, Inc. (Hercules, CA).
Cell Culture. HeLa/PEPT1 and mock cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Cansera International, Toronto, ON, Canada), 2 mM L-glutamine, and 1 mg/ml geneticin (G418), as described previously (Nakanishi et al., 2000). For the uptake and metabolism assay, each cell line was seeded on six-well plates (Nunc, Naperville, IL) and cultured for 3 days. HEK293 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1 mg/ml sodium pyruvate, 100 µg/ml streptomycin, and 100 unit/ml benzylpenicillin.
In Vitro Gene Transfer to HEK293 Cells. HEK293 cells were plated onto six-well plates for 24 h before cDNA transfection. Transfection was performed after the cells had reached 40 to 60% confluence by means of the calcium phosphate precipitation method (Watanabe et al., 2005). The uptake and metabolism assays were performed 40 to 48 h after transfection.
Uptake and Metabolism Experiments. The experiments that were conducted with HeLa cells were performed at 37°C in Hanks' balanced salt solution (HBSS) [136.7 mM NaCl, 5.36 mM KCl, 25 mM D-glucose, 0.952 mM CaCl2, 0.441 mM KH2PO4, 0.812 mM MgSO4, 0.385 mM Na2HPO4, and 10 mM 2-(N-morpholino)ethanesulfonic acid (MES) or HEPES] adjusted to pH 5.5 to 8.0. The osmolality of the HBSS was 305 ± 5 mOsm/kg. The experiments that were conducted with HEK293 cells were performed in MES buffer (125 mM NaCl, 4.8 mM KCl, 5.6 mM D-glucose, 1.2 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, and 25 mM MES) adjusted to pH 6.0. The osmolality of the MES buffer was 290 ± 5 mOsm/kg. Cultured cells were washed and preincubated in the buffer without test compounds for 20 min at 37°C and pH 7.4. The reaction then was initiated by replacing the medium with fresh buffer (2 ml) containing L-or D-CEX (28.8 µM). After incubation for desired times at 37°C, the experiment was terminated by removing the medium, followed by washing three times with ice-cold buffer.
To prepare digitonin-permeabilized cells, HeLa cells were treated in HBSS (pH 6.0) containing 125 µg/ml digitonin for 10 min at room temperature. HBSS was replaced with hypotonic buffer (10 mM MES, 25 mM D-glucose, adjusted to pH 6.0) for hypotonic treatment of the cells. Cellular protein was determined using a protein assay kit with bovine serum albumin as a standard.
Metabolism Experiments in Cellular Homogenates. Cells were collected with a cell scraper (Asahi Techno Glass Corporation Co. Ltd., Chiba, Japan) and suspended in 10 mM MES/NaOH, pH 6.0, or 10 mM Tris/HCl, pH 7.4. Cell suspensions were then homogenized on ice with 100 strokes of a 1-ml tapered tissue grinder (Potter-Elvehjem Teflon homogenizer, 358133, Wheaton Instruments, Millville, NJ) and centrifuged at 1500 rpm and 4°C for 5 min (Hitachi Himac CF15R, Tokyo, Japan). One hundred eighty microliters of supernatant was used for the assay. The reaction was initiated by adding the medium (20 µl) containing L-CEX (final concentration, 288 µM). After incubation for 60 min at 25°C, the experiment was terminated by adding 800 µl of ice-cold methanol.
High-Performance Liquid Chromatography Analysis. The medium sample was first centrifuged at 15,000 rpm and 4°C for 15 min. The cellular samples were collected by adding 500 µl of a mixture of mobile phase/methanol (1:4) and using a cell scraper, then sonicated for 30 min, and gently shaken at 4°C for 1 h for deproteinization with a petite rotor (model 2210, Wakenyaku Co. Ltd., Kyoto, Japan), followed by centrifugation at 15,000 rpm and 4°C for 15 min. Quantities of L-CEX, D-CEX, and 7-ADCA were determined by high-performance liquid chromatography (HPLC) as described previously (Tamai et al., 1988). The HPLC system consisted of a constant-flow pump (JASCO PU-2080 Plus), a UV detector (JASCO UV-2075 Plus), an automatic sample injector (JASCO AS-2057 Plus) (JASCO International Co., Ltd., Tokyo, Japan), and an integrator (Chromatopac C-R7A, Shimadzu Corporation, Kyoto, Japan). The UV detector was set at 260 nm. Reversed-phase columns, J'sphere ODS-M80 (4.6 x 150 mm; YMC Co., Ltd., Kyoto Japan) for L-CEX and 7-ADCA, and COSMOSIL 5C18-MS-II (4.6 x 150 mm; Nacalai Tesque, Inc.) for D-CEX were maintained at 35°C in a column oven (JASCO CO-2065 Plus; JASCO International Co., Ltd.). The mobile phase was 10 mM sodium phosphate buffer (pH 3.0) containing 10 mM ammonium acetate and 10 mM pentanesulphonic acid/methanol (10:90, v/v) for L-CEX, 10 mM ammonium acetate/methanol (22:78, v/v) for D-CEX, and 10 mM ammonium acetate and 10 mM tetra-n-butylammonium bromide/methanol (8:92, v/v) for 7-ADCA. The flow rate was 1.0 ml/min.
Measurement of Lactate Dehydrogenase Activity. Lactate dehydrogenase (LDH) activity in the medium was determined using an LDH-cytotoxicity test (Wako Pure Chemical Industries, Ltd., Osaka, Japan) according to the manufacturer's protocol. Cells treated with phosphate-buffered saline(–) containing 0.2% Tween or phosphate-buffered saline(–) alone were used as the positive and negative controls, respectively. The results were expressed as LDH-cytotoxicity ratio [(S – N)/(P – N) x 100 (%)] and corrected LDH-cytotoxicity ratio (100 – LDH-cytotoxicity ratio), where S, N, and P represent LDH activity observed under the tested condition and those of negative and positive controls, respectively.
Data Analysis. Metabolism of L-CEX was represented as generation of 7-ADCA during incubation with L-CEX. All the data were expressed as mean ± S.E.M. Statistical analysis was performed with Student's t test. The criterion of significance was taken to be p < 0.05.
|
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
). The HeLa/PEPT1 cells exhibited a marked increase in formation of 7-ADCA compared with mock cells (Fig. 2A). Disappearance of L-CEX from the medium of HeLa/PEPT1 cells was faster than from that of mock cells (Fig. 2B). Thus, exogenous transfection of PEPT1 gene resulted in an increase of L-CEX metabolism. Accumulation of 7-ADCA in HeLa/PEPT1 cells was significantly higher than in mock cells, whereas L-CEX was under the quantification limit (0.1 nmol/mg protein/180 min) in both cell lines (Fig. 2C). On the other hand, D-CEX was minimally hydrolyzed to 7-ADCA in both HeLa/PEPT1 and mock cells (Fig. 2, D and E). D-CEX was taken up as the unchanged form by HeLa/PEPT1 cells, whereas such uptake was extremely small in mock cells (Fig. 2F). To check the stability in the incubation medium, L- and D-CEX were incubated at pH 6.0 and 7.4 in the assay buffer without cells at 37°C. The decrease in L- and D-CEX concentration in the buffer after 180 min was less than 0.4% of initial dose, suggesting that both compounds are stable in the buffer.
To confirm such an increase in L-CEX metabolism by PEPT1 transfection, we next performed similar experiments in different host cells, HEK293 cells, which were transiently transfected with PEPT1 (HEK293/PEPT1). This is because HeLa/PEPT1 was a stable clone, and L-CEX metabolism observed in the HeLa/PEPT1 might represent any experimental artifact that may happen by selecting such specific clone of HeLa cells. For example, stable expression of PEPT1 may up-regulate endogenous peptidase, resulting in significant L-CEX metabolism. In addition, L-CEX metabolism in HeLa/PEPT1 may be HeLa cell-specific phenomenon. Consequently, to exclude these possibilities, we also performed metabolism studies using another cell line HEK293, which was transiently transfected with PEPT1. Very much higher metabolism of L-CEX was observed in HEK293/PEPT1 cells than in mock cells (Fig. 3, A and C). A similar increase in L-CEX metabolism was also observed after transient transfection of PEPT2, another proton/oligopeptide cotransporter family member (Fig. 3, A and C). Disappearance of L-CEX was much faster in HEK293/PEPT1 and HEK293/PEPT2 cells than in mock cells (Fig. 3B). Accumulation of L-CEX was not observed in HEK293/PEPT1 or HEK293/PEPT2 cells (Fig. 3C).
|
7 and 18 nmol/mg protein, respectively) were much lower than those in the medium (64 and 87 nmol/mg protein, respectively) (Figs. 2 and 3), appearance of 7-ADCA was measured only in the medium in the following studies to analyze metabolism of L-CEX. The L-CEX metabolic activity increased almost linearly in proportion to the amount of cDNA encoding PEPT1 or PEPT2 up to 2 µg/well (Fig. 4, A and B).
|
|
|
L-CEX Metabolism Does Not Solely Depend on Uptake Activity of PEPT1 in PEPT1-Expressing Cells after Membrane Permeabilization. To examine whether the increase in L-CEX metabolism following PEPT1 transfection can be simply explained by the increase in PEPT1-mediated uptake and consequent exposure of L-CEX to intracellular hydrolases, the plasma membranes of HeLa/PEPT1 cells were first perforated by exposure to hypotonic solution or digitonin-containing solution, and both metabolism and uptake activity were determined (Table 2). As an index of increased membrane permeability, the corrected LDH-cytotoxicity ratio was determined and was found to be 7 to 26% after the treatment with hypotonic or digitonin solution, indicating that the plasma membranes had been successfully permeabilized (Table 2). The concentration of digitonin was set at 125 µg/ml because in our preliminary analysis digitonin at this concentration exhibited the lowest value of the corrected LDH-cytotoxicity ratio (data not shown). In intact cells, exposure to L-CEX inside the cells could be much higher in PEPT1-transfected cells compared with that in mock cells because of PEPT1-mediated transport activity. On the other hand, it was considered that such permeabilization of the cells may increase the exposure to L-CEX in intracellular space of both cells, resulting in almost comparable exposure to L-CEX inside the cells. This was confirmed by the fact that PEPT1-mediated uptake of D-CEX was almost completely reduced in hypotonic (10% of control) and digitonin-treated (8% of control) conditions (Table 2). Even in such conditions, L-CEX metabolism was higher in HeLa/PEPT1 than that in mock cells, as indicated by PEPT1-mediated L-CEX metabolism, which was maintained at 30 to 50% of control level both in hypotonic and digitonin-treated conditions (Table 2). This suggests that L-CEX metabolism enhanced in HeLa/PEPT1 cells (Fig. 2) does not solely result from the increase in exposure to L-CEX inside the cells by PEPT1-mediated transport in HeLa/PEPT1 cells.
|
L-CEX Metabolism in Homogenates of PEPT1-Expressing Cell Lines. Metabolism of L-CEX was also examined in cellular homogenates prepared from HeLa/PEPT1 and mock cells. The generation of 7-ADCA at pH 6.0 in HeLa/PEPT1 and mock cells was 415 ± 20 and 135 ± 11 nmol/mg protein/60 min, respectively (mean ± S.E.M., n = 3–6), whereas that at pH 7.4 in HeLa/PEPT1 and mock cells was 305 ± 5 and 245 ± 3 nmol/mg protein/h, respectively (mean ± S.E.M., n = 3–6). PEPT1-mediated L-CEX metabolism, assessed by subtracting the formation of 7-ADCA in mock cells from that in HeLa/PEPT1 cells, was 281 ± 21 and 60.3 ± 5.1 nmol/mg protein/h at pH 6.0 and 7.4, respectively (mean ± S.E.M., n = 3–6). Thus, L-CEX metabolism observed in cellular homogenates was also more obvious at pH 6.0 than that at pH 7.4, as is the case of that found in intact cell lines (Fig. 5).
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
; Kramer et al., 1992). On the other hand, Wenzel et al. (1995) showed that L-CEX is a good substrate of oligopeptide transporter. Thus, it is likely that transport and metabolism of L-CEX occur almost simultaneously in vivo. Therefore, we hypothesized that oligopeptide transporter plays a fundamental role not only in transport of L-CEX but also in its metabolism. Because PEPT1 is thought to be a major oligopeptide transporter in small intestine, we examined whether PEPT1 could account for the stereoselective uptake and metabolism of CEX in the present study.
L-CEX hydrolysis was indeed dramatically stimulated by exogenous transfection of PEPT1 (Figs. 2 and 3). This phenomenon is independent of the type of host cell line or the transfection method because such enhancement of L-CEX metabolism by PEPT1 was observed in both HeLa and HEK293 cells (Figs. 2 and 3) and after both stable and transient transfection of the PEPT1 gene (Figs. 2 and 3). Activity of L-CEX metabolism depended on the amount of PEPT1 cDNA transfected (Fig. 4A). On the other hand, D-CEX was not metabolized even after transfection of PEPT1, although its uptake into the cells was enhanced by expression of PEPT1 (Fig. 2, D and F). These results suggest that PEPT1 alone is responsible for the stereoselective uptake and metabolism of CEX. It is noteworthy that the PEPT1-mediated stereoselective disposition of CEX observed in in vitro is quite similar to the stereoselective disposition observed in the small intestine after administration p.o. (Tamai et al., 1988), implying a predominant role of this transporter not only in transport but also in metabolism of L-CEX.
To characterize such PEPT1-mediated metabolism and uptake of CEX, we determined the pH dependence and substrate specificity of the two processes (Fig. 5; Table 2). Considering that PEPT1 exhibits proton-coupled transport of substrates, it is reasonable that uptake of CEX was greater at lower pH values (Fig. 5B). The optimal pH for L-CEX metabolism, on the other hand, was 6.0 to 6.5 (Fig. 5A), which is slightly higher than that for D-CEX uptake (Fig. 5B). As for the substrate specificity of the L-CEX metabolic activity, substrates of PEPT1 inhibited both L-CEX metabolism and D-CEX uptake (Table 1), whereas several other compounds, including L-Ala- L-Ala- L-Ala, enalapril, ubenimex, and EDTA, inhibited L-CEX metabolism much more strongly than D-CEX uptake (Table 1). Inhibition of D-CEX uptake by EDTA (Table 1) may imply possible involvement of metal ion(s) in PEPT1-mediated transport, and further studies are required for analyzing detailed mechanism(s). Thus, PEPT1-mediated metabolism and uptake have similar, although not identical, pH dependence and substrate recognition specificity.
The metabolism of L-CEX observed after PEPT1 transfection in in vitro transfectant systems (Figs. 2, 3, 4) can simply be explained if we hypothesize that the PEPT1-mediated uptake process of L-CEX is the rate-limiting step in its overall hydrolysis. It is possible that transfection of PEPT1 gene increases the membrane permeation of L-CEX, resulting in a higher exposure of L-CEX to intracellular peptidases. To examine whether uptake is the rate-limiting step, PEPT1-mediated metabolism of L-CEX was also examined after plasma membranes had been perforated using hypotonic or digitonin-containing solution (Table 2). Whereas PEPT1-mediated D-CEX uptake was greatly reduced under such highly permeabilized conditions, the decrease in L-CEX metabolism was less marked (Table 1), indicating that PEPT1-mediated metabolism cannot be fully explained by the hypothesis that the membrane permeation process is rate-limiting. This is consistent with our present finding that PEPT1-mediated metabolism also occurs in homogenates prepared from HeLa/PEPT1 cells (see under Results). If we assume that exogenous transfection of PEPT1 gene only increases PEPT1 protein, these results suggest that PEPT1 itself is directly involved in the hydrolysis of L-CEX. Up to now, this protein has been recognized only as a transporter for various substrates, including dipeptides and tripeptides and ß-lactams, and no information has been available on its potential for hydrolytic activity. Alternatively, it may be possible that exogenous transfection of PEPT1 gene increases the expression of some endogenous hydrolytic enzyme(s) and/or activates it. Because mammalian cell lines stably transfected with PEPT1 gene also express various types of undetermined endogenous proteins, it is difficult to finally conclude that PEPT1 itself is a metabolizing enzyme. A purified in vitro system may be useful to clarify whether PEPT1 itself exhibits hydrolytic activity for L-CEX. Several studies have aimed at construction of such in vitro system in which only PEPT1 protein purified from animal small intestine is present. Kramer et al. (1992) performed purification and reconstitution of the 127-kDa binding protein for ß-lactam antibiotics and oligopeptides expressed in brush-border membrane of rabbit small intestinal enterocytes. This liposome contained the binding protein, which exhibited stereospecific transport activity, but did not ensure a purity of the transporter, i.e., contamination of other proteins might be present. Iseki et al. (1998) also reported purification of oligopeptide transporter(s) from rat small intestinal brush border, although identification of this protein has not yet been fully characterized. Therefore, for the final demonstration of the hypothesis that PEPT1 may act as a metabolic enzyme, we need a novel experimental system that exclusively expresses PEPT1 protein.
Our results indicate a fundamental role of PEPT1 not only in transport but also in metabolism of oligopeptides. In humans, more than 80% of orally ingested protein was digested to small peptides (Adibi and Mercer, 1973), and the absorption rates of peptides mediated by the oligopeptide transport systems, including PEPT1, are greater than those of free amino acids (Adibi, 1971). Therefore, it seems reasonable that the uptake and subsequent hydrolysis processes would be tightly coupled, leading to efficient absorption of the nutrients.
7-ADCA is generated as a result of hydrolytic cleavage of the peptide bond of L-CEX (Tamai et al., 1988). Chemical degradation of the peptide bond of D-CEX was stimulated at higher pH, whereas the bond is quite stable at lower pH (Yamana and Tsuji, 1976). Considering that L-CEX is a diastereomer of D-CEX, it could also be stable under lower pH conditions. Because 7-ADCA generation was marked at pH 6 to 6.5 (Fig. 5A), hydrolysis of L-CEX is thought to be more likely to be mediated by metabolic enzyme(s) than by chemical degradation.
In the present study, exogenous transfection of PEPT2, which has 50% amino acid homology with PEPT1 (Meredith and Boyd, 2000; Daniel and Kottra, 2004; Smith et al., 2004) also increases the metabolism of L-CEX (Fig. 4B). PEPT2 is expressed at apical membranes of renal proximal tubules and epithelial cells of choroid plexus and is thought to play a role in (re)absorption of peptides and other substrates (Rubio-Aliaga et al., 2003; Shen et al., 2003). Considering the similar physiological roles of the two homologous transporters, it is reasonable that both proteins could be involved in the hydrolysis of L-CEX. Human peptide transporter 1 (HPT1, also named CDH17) is another peptide transporter that accepts cephalexin, ubenimex (Dantzig et al., 1994), and valacyclovir (Landowski et al., 2003) as substrates. In preliminary studies, we transfected human HPT1 gene (2 µg/well) into HEK293 cells but found no increase in 7-ADCA formation (10.9 ± 0.3 and 9.6 ± 0.8 pmol/min/mg protein in HPT1- and mock-transfected cells, respectively), suggesting that HPT1 is unlikely to be involved in CEX metabolism.
In the present study, the profiles of 7-ADCA formation in HeLa/PEPT1 cells (Fig. 2A) may exhibit an unusual one, the slope being faster after a certain lag time (30 min). The reason could be that metabolism of L-CEX may occur after it is taken up by the cells. In this case, such a lag time would be necessary for L-CEX molecules to permeate the plasma membranes and subsequently be exposed to the catalytic site of metabolizing enzymes inside the cells. A similar profile of the 7-ADCA formation was also observed in HEK293 cells expressing PEPT1 (Fig. 3A). If there is a lag time, after which 7-ADCA formation becomes much faster, and this is caused by the uptake and/or exposure processes of the parent compound in the cells, it would be difficult to perform Michaelis-Menten–type analysis of the 7-ADCA formation. Therefore, the experimental systems other than the intact cell lines are necessary for detailed analysis of the 7-ADCA formation.
In conclusion, PEPT1-mediated L-CEX metabolism has similar, although not identical, characteristics to those of PEPT1-mediated L-CEX transport in terms of pH dependence and substrate specificity, and can take place even when the PEPT1-mediated transport process is hindered, implying an intrinsic involvement of PEPT1 itself and/or PEPT1-associated peptidases in L-CEX metabolism in the small intestine.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
ABBREVIATIONS: CEX, cephalexin; L-CEX, L-stereoisomer of cephalexin, (6R,7R)-7-[[(2S)-2-amino-2-phenyl-acetyl]amino]-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid; D-CEX, D-stereoisomer of cephalexin, (6R,7R)-7-[[(2R)-2-amino-2-phenyl-acetyl]amino]-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid; 7-ADCA, 7-aminodesacetoxycephalosporanic acid, 7-amino-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid; PEPT1, H+/oligopeptide transporter 1; HEK, human embryonic kidney; HBSS, Hanks' balanced salt solution; MES, 2-(N-morpholino)ethanesulfonic acid; HPLC, high-performance liquid chromatography; LDH, lactate dehydrogenase; HPT1, human peptide transporter 1.
Address correspondence to: Akira Tsuji, Division of Pharmaceutical Sciences, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan. E-mail: tsuji{at}kenroku.kanazawa-u.ac.jp
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
, below the quantification limit.
