Cefadroxil Transport Research Articles

Oral Availability of Cefadroxil Depends on ABCC3 and ABCC4

Some cephalosporins, such as cefadroxil, are orally available. H(+)-coupled peptide transporter 1 mediates the transport of cephalosporins across the apical membrane of enterocytes. It is not known which mechanism(s) is responsible for the subsequent transport of cephalosporins across the basolateral membrane toward the circulation. We have tested whether ATP-binding cassette (ABC) transporters ABCC3 and/or ABCC4 are involved in the latter process. Transport experiments with plasma membrane vesicles expressing these transporters were used to determine whether ABCC3 and ABCC4 can transport cephalosporins in vitro. The involvement of Abcc3 and Abcc4 in the transport of cefadroxil from enterocytes was subsequently studied using intestinal explants from wild-type, Abcc3(-/-), Abcc4(-/-), and Abcc3(-/-)/Abcc4(-/-) mice in an Ussing chamber setup. Finally, appearance of cefadroxil in portal blood was investigated in vivo after intrajejunal administration of cefadroxil in wild-type, Abcc3(-/-), Abcc4(-/-), and Abcc3(-/-)/Abcc4(-/-) mice. ABCC3- and ABCC4-mediated transport of estradiol-17β-glucuronide was dose-dependently inhibited by cephalosporins in vesicular transport experiments. Furthermore, transport of cefadroxil by ABCC3 and ABCC4 was saturable with K(m) values of 2.5 ± 0.7 and 0.25 ± 0.07 mM, respectively. Transport of cefadroxil from the apical to the basolateral side of jejunal tissue explants was unchanged in Abcc3(-/-) but significantly reduced (approximately 2-fold) in Abcc4(-/-) and Abcc3(-/-)/Abcc4(-/-) when compared with wild-type tissue. Upon instillation of cefadroxil in the jejunum, portal and peripheral blood concentrations were similar in Abcc3(-/-) and Abcc4(-/-) but approximately 2-fold reduced in Abcc3(-/-)/Abcc4(-/-) compared with wild-type mice. Our data demonstrate that intestinal absorption of cefadroxil depends partly on ABCC3 and ABCC4.

Effect of Different Metal Ions on the Biological Properties of Cefadroxil.

The effect of different metal ions on the intestinal transport and the antibacterial activity of cefadroxil [(6R,7R)-7-{[(2R)-2-amino-2-(4-hydroxyphenyl)acetyl]amino}-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid] was investigated. The [14C]Gly-Sar uptake via PEPT1 was inhibited by Zn2+ and Cu2+ treatment in a concentration-dependent manner (Ki values 107 ± 23 and 19 ± 5 µM, respectively). Kinetic analysis showed that the Kt of Gly-Sar uptake was increased 2-fold in the presence of zinc sulphate (150 µM) whereas the Vmax value were not affected suggesting that zinc ions inhibited Gly-Sar uptake by PEPT1 in a competitively manner. Ni2+ exhibited moderate inhibitory effect, whereas Co2+, Mg2+, Al3+ ions showed no inhibitory effect on Gly-Sar uptake via PEPT1. Subsequently, we examined the effect of Zn2+ and Al3+ ions on the transepithelial transport of cefadroxil across Caco-2 cells cultured on permeable supports. The results showed that zinc ions inhibited the transepithelial flux of cefadroxil at Caco-2 cell monolayers while Al3+ ions had no effect. The interaction of cephalosporins with the metal ions could suggest negative effects of some metal ions on the clinical aspects of small intestinal peptide and drug transport. Finally, the effect of Zn2+, Cu2+ and Al3+ ions on the antibacterial activity of cefadroxil was tested. It was found that there is no significant difference between the activity of cefadroxil and the cefadroxil metal ion complexes studied against the investigated sensitive bacterial species.

Molecular interactions between dipeptides, drugs and the human intestinal H+ -oligopeptide cotransporter hPEPT1.

The human intestinal proton-coupled oligopeptide transporter hPEPT1 has been implicated in the absorption of pharmacologically active compounds. We have investigated the interactions between a comprehensive selection of drugs, and wild-type and variant hPEPT1s expressed in Xenopus oocytes, using radiotracer uptake and electrophysiological methods. The beta-lactam antibiotics ampicillin, amoxicillin, cephalexin and cefadroxil, the antineoplastics delta-aminolevulinic acid (delta-ALA) and bestatin, and the neuropeptide N-acetyl-Asp-Glu (NAAG), were transported, as judged by their ability to evoke inward currents. When the drugs were added in the presence of the typical substrate glycylsarcosine (Gly-Sar), the inward currents were equal or less than that induced by Gly-Sar alone. This suggests that the drugs are transported at a lower turnover rate than Gly-Sar, but may also point towards complex interactions between dipeptides, drugs and the transporter. Gly-Sar and the drugs also modified the kinetics of hPEPT1 presteady-state charge movement, by causing a reduction in maximum charge (Qmax) and a shift of the midpoint voltage (V0.5) to more negative potentials. Our results indicate that the substrate selectivity of hPEPT1 is: Gly-Sar > NAAG, delta-ALA, bestatin > cefadroxil, cephalexin > ampicillin, amoxicillin. Based on steady-state and presteady-state analysis of Gly-Sar and cefadroxil transport, we proposed an extension of the 6-state kinetic model for hPEPT1 function that globally accounts for the observed presteady-state and steady-state kinetics of neutral dipeptide and drug transport. Our model suggests that, under saturating conditions, the rate-limiting step of the hPEPT1 transport cycle is the reorientation of the empty carrier within the membrane. Variations in rates of drug cotransport are predicted to be due to differences in affinity and turnover rate. Oral availability of drugs may be reduced in the presence of physiological concentrations of dietary dipeptides in the gut, suggesting that oral delivery drugs should be taken on an empty stomach. The common hPEPT1 single-nucleotide polymorphisms Ser117Asn and Gly419Ala retained the essential kinetic and drug recognition characteristics of the wild type, suggesting that neither variant is likely to have a major impact on oral absorption of drugs.

PEPT2 (Slc15a2)-Mediated Unidirectional Transport of Cefadroxil from Cerebrospinal Fluid into Choroid Plexus

Cefadroxil is a cephalosporin antibiotic used in the treatment of infection. However, cerebrospinal fluid (CSF) concentrations of cefadroxil and other aminocephalosporins are not adequate for the treatment of bacterial meningitis. To evaluate the relevance of PEPT2 in affecting the exposure of aminocephalosporins in brain, we investigated the transport properties of cefadroxil at the blood-CSF interface using primary-cultured epithelial cells and isolated whole tissues of choroid plexus. Our results indicated that cefadroxil was preferentially taken up from the apical as opposed to basal side of the monolayer (5-fold), and its apical uptake was stimulated by an inwardly directed proton gradient. The concentration-dependent apical uptake of cefadroxil was characterized by a high-affinity/low-capacity transport system (Km = 39.0 +/- 22.7 microM; Vmax = 22.9 +/- 6.6 pmol/mg/min) and a nonsaturable component (Kd = 0.15 +/- 0.01 microl/mg/min); in contrast, only a nonsaturable component was found for the basal uptake of cefadroxil (Kd = 0.14 +/- 0.01 microl/mg/min). The apical-to-basal transepithelial transport of 2 microM cefadroxil was greater than its basal-to-apical transport, but no differences were observed in directionality when 5 mM concentrations of cefadroxil were studied. Moreover, the cellular efflux of cefadroxil was not saturable in either direction (i.e., to apical or basal side). Finally, no differences were observed in the choroid plexus tissue efflux of 2 microM cefadroxil from wild-type and PEPT2 null mice. These findings demonstrate that PEPT2 has an important role in limiting the exposure of cefadroxil in CSF. Located at the apical membrane of choroid plexus epithelium, PEPT2 acts in a unidirectional (as opposed to bidirectional) manner in transporting cefadroxil from CSF into the cell.

Active Drug Delivery by Heterologous Expression of Membrane Transporters

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Active drug delivery by heterologous expression of membrane transporters

The oligopeptide transporter PEPT1 is predominantly expressed in the brush-border membranes of small intestinal epithelial cells, where it plays pivotal roles in the efficient absorption of di-/tripeptides. PEPT1 has enormous potential as an oral drug delivery target, because it also mediates the intestinal absorption of peptide-mimetic and nonpeptide substrates. We demonstrated that the peptide derivation of amino acid-related drugs is applicable to improve their intestinal absorption. We have found that oligopeptide transport activity is also expressed in cancer cell lines. The tissue distribution of bestatin was significantly increased in solid tumors that overexpress PEPT1 in nude mice. Orally administered bestatin strongly suppressed tumor growth. These results provided the first demonstration of the tumor-selective delivery of a drug by specific transport activity. The absolute degree of PEPT1 mRNA expression in the small intestine was determined using real-time PCR in rats. Starvation of the animals increased the mRNA expression level profoundly in the upper small intestine. The longitudinal expression pattern was well correlated with the intestinal transport of cefadroxil in rats. We constructed a recombinant adenovirus vector encoding PEPT1 cDNA. Heterologous expression of PEPT1 in the liver was successfully achieved by simple intvavenous. Administration of the vector, resulting in increased liver distribution of [3H] carnosine. In situ perfusion of the brain with the vector doubled the brain distribution of cefadroxil. Heterologous expression of the drug transporter in vivo could be a useful approach for drug delivery.

Expression cloning and functional characterization of the kidney cortex high-affinity proton-coupled peptide transporter.

The presence of a proton-coupled electrogenic high-affinity peptide transporter in the apical membrane of tubular cells has been demonstrated by microperfusion studies and by use of brush border membrane vesicles. The transporter mediates tubular uptake of filtered di- and tripeptides and aminocephalosporin antibiotics. We have used expression cloning in Xenopus laevis oocytes for identification and characterization of the renal high-affinity peptide transporter. Injection of poly(A)+ RNA isolated from rabbit kidney cortex into oocytes resulted in expression of a pH-dependent transport activity for the aminocephalosporin antibiotic cefadroxil. After size fractionation of poly(A)+ RNA the transport activity was identified in the 3.0- to 5.0-kb fractions, which were used for construction of a cDNA library. The library was screened for expression of cefadroxil transport after injection of complementary RNA synthesized in vitro from different pools of clones. A single clone (rPepT2) was isolated that stimulated cefadroxil uptake into oocytes approximately 70-fold at a pH of 6.0. Kinetic analysis of cefadroxil uptake expressed by the transporter's complementary RNA showed a single saturable high-affinity transport system shared by dipeptides, tripeptides, and selected amino-beta-lactam antibiotics. Electrophysiological studies established that the transport activity is electrogenic and affected by membrane potential. Sequencing of the cDNA predicts a protein of 729 amino acids with 12 membrane-spanning domains. Although there is a significant amino acid sequence identity (47%) to the recently cloned peptide transporters from rabbit and human small intestine, the renal transporter shows distinct structural and functional differences.

Expression cloning of a cDNA from rabbit small intestine related to proton-coupled transport of peptides, ßlactam antibiotics and ACE-inhibitors

Injection of poly(A)+ RNA from rabbit small intestine into Xenopus laevis oocytes resulted in expression of pH dependent transport of the aminocephalosporin cefadroxil. A cDNA library constructed from a 2.2 to 5 kb fraction was screened for expression of cefadroxil transport after injection of the corresponding cRNA synthetized in vitro from different pools of clones. The single clone identified stimulated uptake of cefadroxil into oocytes about 50-fold at pH 6.5. Kinetic analysis of expressed transport activity revealed a saturable transport system shared by amino beta-lactam antibiotics, dipeptides and selected angiotensin converting enzyme inhibitors. Evidence for rheogenic cefadroxil/H(+)-cotransport was obtained by a) The demonstration that cefadroxil influx increased the inward current in oocytes clamped at a holding potential of -60 mV in sodium-free medium and b) A decrease of intracellular pH in oocytes caused by cefadroxil uptake. Current-voltage relationships in the presence of glycylsarcosine or cefadroxil showed that transport activity is dependent on the membrane potential. Sequencing of the cDNA revealed its identity with the recently cloned peptide transporter from rabbit small intestine designated PepT1.

Transport of cefadroxil, an aminocephalosporin antibiotic, across the small intestinal brush border membrane

The transport characteristics of cefadroxil, an aminocephalosporin antibiotic, across the brush border membrane of rat small intestine were investigated by a rapid filtration technique. The uptake of cefadroxil was not affected by Na + gradient, suggesting the absence of a cotransport system between cefadroxil and Na + in the brush border membrane. The uptake was slightly inhibited by HgCl 2 pretreatment and stimulated by the countertransport effect, where cyclacillin played a role as an elicitor. These results suggest the existence of a carrier-mediated transport system for cefadroxil in the brush border membrane, which is shared with cyclacillin. Papain treatment increased the specific transport activities for the antibiotic. This may be the first step of purification of the cefadroxil transport carrier.

Characterization of aminocephalosporin transport across rat small intestine.

The absorption mechanism of aminocephalosporins was investigated in rat small intestine. Cephalosporins chosen in this study were cephalexin, cephradine, SCE-100, cefroxadin, cefatrizine, and cefadroxil. Absorption experiments were carried out by both in situ and in vitro techniques. In situ recirculation experiments through the whole small intestine demonstrated that the absorption rates of the antibiotics were saturable (except cephalexin) and inhibited by the short-term pretreatment of the intestinal mucosa with a low concentration of HgCl2, suggesting the participation of proteins and/or sulfhydryl groups within the brush border membrane. Among the tested antibiotics, cefadroxil showed the highest absorption, , in spite of its lowest lipophilicity, and its absorption was inhibited by the simultaneous perfusion of other aminocephalosporins and aminopenicillins. So, the carrier system for cefadroxil seems to be common, at least in part, to amino-β-lactam antibiotics. Also, comparison of the apparent directional permeabilities of the jejunum and ileum by using the everted sacs and noneverted ones in vitro indicated that this drug penetrates more rapidly from mucosa to serosa (M-to-S) than from serosa to mucosa (S-to-M) in both segments. The (M-to-S)/(S-to-M) ratios at the concentration of 0.1 mM were 2.26 and 4.82 in the jejunum and ileum, respectively. The M-to-S flux of the drug was saturable, Na+-dependent, and inhibited by 2, 4-dinitrophenol Thus, it is suggested that energy is required for M-to-S cefadroxil transport. The location of SH-groups of the brush border membrane essential for cefadroxil transport was discussed.