Binding of amino beta-lactam antibiotics to soluble protein from rat intestinal mucosa--I. Purification of drug-binding protein

Several amino beta-lactam antibiotics, including ampicillin, amoxicillin, cyclacillin, cephalexin, cephradine and cefadroxil, were found to bind in vitro to specific components in 105,000 g supernatant of homogenate obtained from rat intestinal mucosa. The major binding component (fraction b) was purified by chromatography on DEAE-cellulofine and by gel filtration on Sephadex G-50. The molecular weight of fraction b was determined by SDS polyacrylamide gel electrophoresis (15,000 Da). The binding behaviour of these amino beta-lactam antibiotics to fraction b were estimated by equilibrium dialysis. There were significant high affinities of all tested amino beta-lactam antibiotics which were well absorbed from intestine, but there was not a good correlation between binding and absorption of these drugs. It was also found that poorly absorbed cephalosporins which lack aminobenzyl group in their structure, cefazolin and cephaloridine, did not bind to fraction b.     

Uptake of amino beta-lactam antibiotics into rat intestinal brush border membrane vesicles

Uptake of amino beta-lactam antibiotics into rat intestinal brush border membrane vesicles has been examined for characterization of the transport of the antibiotics through the gut wall of the rat. The uptake of cephradine, cephalexin and ampicillin into membrane vesicles was similar, and there were no significant changes in the uptake in the presence of a NaCl or a KCl gradient. These results suggested that the carrier-mediated transport systems relating to amino acids and glucose were not concerned with the intestinal absorption mechanism of amino beta-lactam antibiotics.     

Binding of 125I-labeled beta-lactam antibiotics to the penicillin binding proteins of Escherichia coli

125I-Labeled derivatives of the beta-lactam antibiotics cephalexin, cephradine, cefaclor and 6-alpha-aminopenicillanic acid have been obtained by reacting these compounds with (125I)-Bolton-Hunter reagent. The following target proteins were found in Escherichia coli: (1) The derivatives of cephalexin, cefaclor and cephradine preferentially interact with the high molecular weight penicillin binding proteins ( PBP1a and PBP1b ); (2) The 125I- derivative of 6-alpha-aminopenicillanic acid is preferentially bound by the low molecular weight penicillin binding proteins 4 and 5/6. The iodinated derivatives showed a very high affinity of binding to their target proteins with apparent half-saturating concentrations in the nano -molar range.     

Effect of chlorpromazine on the permeability of beta-lactam antibiotics across rat intestinal brush border membrane vesicles

The effect of chlorpromazine on the membrane permeability of beta-lactam antibiotics (benzylpenicillin, ampicillin, cephradine and cephalexin) and actively transported substances (glycylglycine and D-glucose) has been studied using rat intestinal brush border membrane vesicles. Except for cephalexin, the initial uptakes at 25 degrees C of these antibiotics were significantly enhanced in the presence of chlorpromazine. In contrast, the transport of glycylglycine and D-glucose was significantly inhibited. These results suggest that the two groups, drugs and actively transported substances, have a different permeation process. The effect of chlorpromazine concentration on membrane lipid fluidity, as assessed by the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-anilino-8-naphthalene sulphonate (ANS), was also examined. The fluorescence polarization of ANS decreased with increasing concentration of chlorpromazine, while that of DPH increased suggesting an increase of membrane surface fluidity might affect the permeation of beta-lactam antibiotics and actively transported substances in a different manner.     

Comparison of transport characteristics of amino beta-lactam antibiotics and dipeptides across rat intestinal brush border membrane

The transport characteristics of amino beta-lactam antibiotics, ampicillin and cephradine, have been examined and compared with that of glycylglycine using brush border membrane vesicles isolated from rat small intestine. The initial rate of glycylglycine uptake was markedly stimulated in the presence of an inward H+ gradient compared with the uptake rates in the absence of an H+ gradient. With the same H+ gradient the stimulation of cephradine uptake was lower and ampicillin uptake was not altered. Cephradine uptake, however, was greater than that of glycylglycine in both vesicular conditions ((pH)i greater than (pH)o and (pH)i = (pH)o). Inhibitory effects of dipeptides, ampicillin and cephradine on the initial uptake of glycylglycine were also examined. Glycylglycine uptake was significantly decreased in the presence of L-phenylalanylglycine or carnosine. Ampicillin and cephradine did not alter the uptake of glycylglycine. These results suggest that the contribution of the inward H+ gradient to the permeation of ampicillin, cephradine and glycylglycine across the rat small intestinal brush border membranes is different for each of the substances examined.     

Interaction of 31 beta-lactam antibiotics with the H+/peptide symporter PEPT2: analysis of affinity constants and comparison with PEPT1.

The activity of the renal peptide transporters PEPT2 and PEPT1 determines-among other factors such as metabolic stability in liver and plasma-the circulatory half-life of penicillins and cephalosporins during therapy. This study was initiated to examine systematically the interaction of beta-lactam antibiotics with PEPT2. Interaction of 31 cephalosporins and penicillins with the carrier protein was characterized by measuring their ability to inhibit the uptake of [(14)C]Gly-Sar into renal SKPT cells. Cefadroxil, cefaclor, cyclacillin, cephradine, cephalexin and moxalactam were recognized by PEPT2 with very high affinity comparable to that of natural dipeptides (K(i)=3-100microM). Ceftibuten, dicloxacillin, amoxicillin, metampicillin, cloxacillin, ampicillin, cefixime, cefamandole, oxacillin and cefmetazole interacted with PEPT2 with medium affinity (K(i)=0.1-5mM). For the other beta-lactam antibiotics studied interaction was very low or not measurable (K(i)>5mM). The affinity constants of beta-lactam antibiotics at rPEPT2 and hPEPT1 are significantly correlated, but the rank orders are not identical. Decisive differences between PEPT1 and PEPT2 recognition of the N-terminal part of the compounds became evident. Moreover, this large data set of affinity constants of beta-lactam antibiotics will be useful for structure-transport (binding) analyses of PEPT2.     

Evidence for the existence of a common transport system of beta-lactam antibiotics in isolated rat hepatocytes

The inhibition effect of several beta-lactam antibiotics on the uptake of [14C]benzylpenicillin (PCG) into isolated rat hepatocytes was studied. Monobasic beta-lactam antibiotics such as apalcillin, cloxacillin, nafcillin, piperacillin, cefmetazole, cefoperazone, cefpiramide and cephalothin significantly inhibited the uptake of PCG, while amphoteric beta-lactam antibiotics such as amoxicillin, ciclacillin, cephradine, cephalexin and cephaloridine had a slight inhibitory effect on the uptake of PCG. Five monobasic compounds of these antibiotics used (apalcillin, nafcillin, piperacillin, cefmetazole and cefoperazone) which have a tendency to be excreted into bile to a large extent, inhibited the initial uptake rate of PCG in a fully competitive fashion according to the Lineweaver-Burk plots and the corresponding modified Inui-Christensen plots. Thus, it was concluded that almost all beta-lactam antibiotics have a common carrier system responsible for their uptake into isolated rat hepatocytes, but it is still uncertain whether or not amphoteric beta-lactam antibiotics have another specific transport system.     

Contribution of passive transport mechanisms to the intestinal absorption of beta-lactam antibiotics

The transport characteristics of aminopenicillins (ampicillin and amoxicillin), aminocephalosporins (cephalexin, cephradine and cefadroxil) and cefazolin have been compared with those of an actively transported substance (D-glucose) and a passively transported substance (L-glucose). Although the initial uptake of the aminocephalosporins was stimulated in the presence of an inward H+ gradient, there was no overshoot in the uptake of any of the drugs tested, even in the presence of an H+ gradient. Also, the time course and the degree of uptake of these drugs were similar to those of L-glucose, especially in the absence of an H+ gradient. These results suggest that the beta-lactam antibiotics tested, like L-glucose, pass through the rat intestinal brush border membrane mainly by passive diffusion. However, the differences in absorption between these drugs, like the differences in their disappearance from a proximal loop of rat intestine, cannot be explained by a simple permeation process alone.     

Stereoselective uptake of beta-lactam antibiotics by the intestinal peptide transporter.

1. The stereoselective transport of beta-lactam antibiotics has been investigated in the human intestinal epithelial cell line, Caco-2, by use of D- and L-enantiomers of cephalexin and loracarbef as substrates. 2. The L-isomers of cephalexin, loracarbef and dipeptides displayed a higher affinity for the oligopeptide/H(+)-symporter in Caco-2 cells than the D-isomers. This was demonstrated by inhibition of the influx of the beta-lactam, [3H]-cefadroxil. 3. By measurement of the substrate-induced intracellular acidification in Caco-2 cells loaded with the pH-sensitive fluorescent dye BCECF (2',7'-bis(2-carboxyethyl)-5-(6)-carboxy-fluorescein), it was demonstrated for the first time that L-isomers of beta-lactams not only bind to the peptide transporter with high affinity but are indeed transported. 4. Efficient proton-coupled transport of L-beta-lactam antibiotics was also shown to occur in Xenopus laevis oocytes expressing the cloned peptide transporter PepT1 from rabbit small intestine. 5. Both cell systems therefore express a stereoselective transport pathway for beta-lactam antibiotics with very similar characteristics and may prove useful for screening rapidly the oral availability of peptide-derived drugs.     

Transport studies of beta-lactam antibiotics and their degradation products across electrified water/oil interface

An electrochemical method for quantifying beta-lactam antibiotics (cephalexin and ampicillin) and their hydrolysis products is described. Cyclic voltammetry at the water/nitrobenzene interface in a four-electrode system was used. The zwitterionic compounds were ionized to the necessary electrochemical form by pH adjustment. The pH change, however, resulted also in hydrolysis of the antibiotics. Hydrolysis products were characterized across UV-vis spectrum. The various hydrolysis products as well as the ionized antibiotics were studied in voltammetric transfer from water to nitrobenzene using the method of the interface between two immiscible electrolyte solutions (ITIES). It was concluded that this electrochemical method is suitable for the quantification of beta-lactam antibiotics and their hydrolysis products.     

Direct photoaffinity labelling of binding proteins for beta-lactam antibiotics in rabbit intestinal brush border membranes with [3H]benzylpenicillin

Brush border membrane vesicles from rabbit small intestine were used to study the intestinal uptake system for beta-lactam antibiotics. Benzylpenicillin inhibited the H+-dependent uptake of alpha-aminocephalosporins in a concentration-dependent manner suggesting a common transport system for alpha-aminocephalosporins and benzylpenicillin. Benzylpenicillin is therefore a suitable probe to characterize this transport system. Irradiation of [3H]benzylpenicillin using light sources having their maximum of radiation at 300 or 254 nm resulted in a covalent incorporation of radioactivity into penicillin binding proteins as was shown with serum albumin. Hence [3H]benzylpenicillin can be used for direct photoaffinity labeling of penicillin binding proteins in different cells and tissues. In brush border membrane vesicles from rabbit small intestine predominantly a membrane polypeptide with an apparent molecular weight of 127,000 was labeled by [3H]benzylpenicillin. Competition labeling experiments demonstrated that beta-lactam antibiotics--penicillins and cephalosporins--specifically interact with this protein, whereas amino acids, sugars or bile acids had no effect on the labeling pattern. Compounds which decreased the labeling of the 127,000 molecular weight membrane polypeptide also inhibited the H+-dependent uptake of the alpha-aminocephalosporin cephalexin into intestinal brush border membrane vesicles. These results suggest that a polypeptide of molecular weight 127,000 in the brush border membrane from rabbit small intestine is a constituent of a common transport system responsible for the uptake of orally effective beta-lactam antibiotics and dipeptides. beta-Lactam antibiotics which are not absorbed from the small intestine also bind from the luminal site to this transport system, but are not transported across the brush border membrane.     

Kinetics and mechanism of in vitro uptake of amino-beta-lactam antibiotics by rat small intestine and relation to the intact-peptide transport system

By utilizing the everted jejunum of rats, the initial uptake rates of several antibiotics were measured over a wide range of concentrations. The uptakes followed mixed-type kinetics involving saturable and non-saturable processes in parallel. The pertinent kinetic parameters for the uptake of each antibiotic were determined. The effect of cephalexin on the uptake of cyclacillin obeyed competitive inhibition kinetics, and the inhibition constant Ki was found to be equal to the Michaelis constant Kt for the uptake of cephalexin itself. In a similar way, the uptake of cephalexin was inhibited by cyclacillin. Uptakes of both cyclacillin and cephalexin were reduced significantly by several metabolic inhibitors. From the effect of temperature on the uptakes of cyclacillin and cephalexin, activation energies of 24.8 and 23.1 kcal/mole were obtained respectively. These results indicate the involvement of an active transport mechanism for cyclacillin and cephalexin. It was found that several dipeptides markedly inhibited the uptakes of cyclacillin and cefadroxil. Furthermore, the uptake of glycylglycine, a typical dipeptide, was inhibited by cyclacillin, cefadroxil, cephalexin, and cephradine. The kinetics of mutual inhibition of the uptakes of cyclacillin and glycylglycine were consistent with competitive-type inhibition. This is the first report which establishes, from a kinetic point of view, the involvement of a common transport system in the in vitro uptakes of the dipeptides and the antibiotics.     

The bacterial outer-membrane permeability of beta-lactam antibiotics.

Two penicillins and 5 cephalosporins were evaluated for their ability to pass through the outer-membranes of Proteus morganii, Citrobacter freundii and Escherichia coli. Cefazolin, ceftezole and cephaloridine showed high permeability through the outer-membranes of these Gram-negative bacteria. Benzylpenicillin and cephalothin, on the contrary, showed low permeability. The outer-membrane permeability of ampicillin and cephalexin varied from species to species. C. freundii was found to have the highest barrier against both the penicillins and the cephalosporins, and E. coli appeared to have a low barrier against the cephalosporins. The hydrophobic character of the beta-lactam antibiotics, which was estimated by a reversed-phase thin-layer chromatography was closely related to the outer-membrane permeability. In general, the more hydrophilic antibiotic showed the higher outer-membrane permeability. However, cephaloridine, the most lipophilic compound among the antibiotics tested, showed good permeability.     

Antibacterial activity of beta-lactam antibiotics against extended-spectrum beta-lactamase producing bacteria

MICs of various beta-lactam antibiotics by themselves and in combination with beta-lactamase inhibitor (clavulanic acid) against extended spectrum beta-lactamase (ESBL) producing strains of Escherichia coli and Klebsiella pneumoniae which were isolated from clinical materials were investigated. Furthermore, based on the results obtained, a procedure to detect ESBL producing strains was proposed. The MICs of beta-lactam antibiotics against beta-lactamase producing strains were investigated. At first, beta-lactamase was investigated by the drug sensitivity pattern (MIC) to beta-lactam antibiotics and by the substrate profiles of beta-lactamase extracted from the transconjugant of E. coli K-12 strains. After that, we classified the beta-lactamase producing gene by PCR method. Furthermore, a proposal was made for an antibiotic to be used in the confirmation of mixed type beta-lactamase. The data obtained by the above investigations were compiled and used to determine the limit concentration of each beta-lactam against beta-lactamase producing strains including ESBL. By using beta-lactam antibiotics at the following concentrations, it is considered possible to classify beta-lactamase; ampicillin (64 micrograms/ml), ampicillin/clavulanic acid (32/5 micrograms/ml), piperacillin (64 micrograms/ml), cefotaxime (1 microgram/ml), cefpodoxime (2 micrograms/ml), ceftazidime (1 microgram/ml), cefmetazole (4 micrograms/ml), cefminox (2 micrograms/ml), cefepime (0.5 microgram/ml), aztreonam (1 microgram/ml) and imipeneme (1 microgram/ml). This method may be used as a reference in investigating the prevalence of beta-lactam resistant isolates by ESBL producing E. coli and K. pneumoniae.     

Comparative stability of cephalosporins in aqueous solution: kinetics and mechanisms of degradation.

The acidic, neutral, and alkaline degradations of six therapeutically useful cephalosporins (cephalothin, cephaloridine, cephaloglycin, cephalexin, cephradine, and cefazolin), 7-amino-cephalosporanic acid, 7-aminodeacetoxycephalosporanic acid, and some 7-substituted derivatives were followed by high-pressure liquid chromatographic, UV spectrometric, iodometric, and hydroxamic acid assays. The pH-rate profiles were determined at 35 degrees and mu = 0.5. The acidic degradation pathway for the 3-acetoxymethyl and 3-pyridinylmethyl derivatives was the specific hydrogen-ion-catalyzed hydrolysis of the beta-lactam bonds. The beta-lactam hydrolyses of these antibiotics exhibited half-lives of about 25 hr at pH 1.0 and 35 degrees. The acetyl functions of 3-acetoxymethylcephalosporins were hydrolyzed eight times faster than their beta-lactam moieties to yield the corresponding deacetyl intermediates, which were rapidly converted to the lactones. Deacetoxycephalosporins were fairly acid stable; e.g., cephalexin and cephradine were about 25 times more stable than cephalothin, cephaloridine, and cephaloglycin and about 180 times more stable than ampicillin at pH 1.0. In the neutral degradation of 3-acetoxymethyl compounds, the competitive reactions of the direct water attack and intramolecular catalysis by the side-chain amido upon the beta-lactams were proposed. The pH-rate profiles near pH 8 for cephaloglycin, cephalexin, and cephradine could be explained by the intramolecular-nucleophilic attack of the side-chain alpha-amino group upon the beta-lactam carbonyls to produce diketopiperazine-type compounds. The reactivity of the cephalosporins in the hydroxideion-catalyzed degradation was influenced significantly by the C-3 methylene substituents.     

Biophotometric investigations on the influence of cephradine and related cephalosporins on the growth of beta-lactamase-producing staphylococi.

Biophotometric growth studies were conducted with clinical isolates of beta-lactamase-producing staphylococi in the presence and absence of cephradine, cephalexin, cefazolin and cephacetrile. Of the four antibiotics, cephradine was the most effective in prolonging the suppression of bacterial growth resulting from antibiotic action while cefazolin was the least effective. Cephalexin and cephacetrile were intermediate in effect. The superiority of cephradine over cephalexin was shown to be related to the more rapid destruction of cephalexin by beta-lactamase. On comparing the results of these studies with clinical experience it was concluded that biophotometric studies have clinical relevance and consequently should be used for evaluating the efficacy of beta-lactam antibiotics against pathogenic staphylococi.     

Phospholipid bilayer permeability of beta-lactam antibiotics

Liposomes containing penicillinase or cephalosporinase were prepared from the phospholipids of Escherichia coli. After free beta-lactamase was inactivated by clavulanic acid or penicillanic acid sulfone followed by separation of inactivated enzyme and inhibitor from liposomes by gel filtration, the permeability of these liposomes to ampicillin, cefazolin and cephaloridine was estimated by measuring the hydrolysis of these antibiotics by the entrapped enzymes. The permeability parameter C (minute-1 microM lipid-1) of ampicillin, cefazolin and cephaloridine was calculated to be 2.35 X 10(-4), 0.33 X 10(-4) and 0.52 X 10(-4), respectively. The lipid bilayer permeability of these antibiotics was also measured by using the liposomes containing these antibiotics. About half of the initially entrapped ampicillin was released from the liposomes within 80 minutes, while no significant release of cefazolin and cephaloridine could be detected during the same period. These results clearly indicates that the lipid bilayer membrane is more permeable to ampicillin than cefazolin and cephaloridine, and they are consistent with the observations of Sawai et al., who showed that ampicillin was a more effective antibacterial drug than cefazolin and cephaloridine against the porin-deficient mutants.     

Studies on sub-MIC activities of beta-lactam antibiotics and aminoglycoside antibiotics against clinically isolated strain

Sub-MIC range of 8 kinds of beta-lactam antibiotics and 3 kinds of aminoglycoside antibiotics against strain of Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa isolated from clinical source were determined by nephlometic method, and following results were obtained. When 10 strains of S. aureus tested to ampicillin (ABPC), hetacillin (IPABPC), mecillinam (MPC), cephalexin (CEX), cefotaxime (CTX), latamoxef (LMOX), cefatrizine (CFT), cephapirin (CEPR), gentamicin (GM), dibekacin (DKB) and amikacin (AMK), ratio of MIC to MAC were 36.8, 53.6, 156.8, 29.6, 61.6, 34.4, 50.0, 111.2, 9.2, 20.0 and 13.6, respectively. When 10 strains of K. pneumoniae tested to MPC, CEX, CTX, LMOX, CFT, CEPR, GM, DKB and AMK, ratio of MIC to MAC were 409.6, 10.4, 34.4, 123.2, 39.2, 167.2, 5.2, 5.6 and 13.2, respectively. When 10 strains of P. aeruginosa tested against CTX, LMOX, GM, DKB and AMK, ratio of MIC to MAC were 16.8, 38.4, 6.8, 3.2 and 10.4, respectively.     

In vitro permeation of beta-lactam antibiotics across rat jejunum and its correlation with oral bioavailability in humans

AIMS: To investigate the correlation between in vitro permeation of 11 beta-lactam antibiotics across rat jejunum and their oral bioavailability in humans. METHODS: The absorptive and secretory permeation across rat jejunum was evaluated and apparent permeability coefficients (P(app)) were determined. RESULTS: A steep, sigmoid-type curve was obtained for the relationship between P(app) in the absorptive permeation and human oral bioavailability. When the ratios of P(app) in the absorptive direction to P(app) in the secretory direction were plotted against human oral bioavailability, a much improved correlation was obtained (r = 0.98, P < 0.001). The addition of glycylglycine to both mucosal and serosal media modified the permeation of ceftibuten and cephalexin from the absorptive to the secretory direction. CONCLUSIONS: For 11 beta-lactam antibiotics rat intestinal permeation correlated well with human oral bioavailability, especially when corrected for secretory transport.     

Transport characteristics of cephalosporin antibiotics across intestinal brush-border membrane in man, rat and rabbit.

The uptake of orally active cephalosporins, ceftibuten and cephradine, by intestinal brush-border membrane vesicles isolated from man, rat and rabbit was studied. In the presence of an inward H+ gradient, ceftibuten but not cephradine was taken up into intestinal brush-border membrane vesicles of man and rat against the concentration gradient (overshoot phenomenon). In rabbit jejunal brush-border membrane vesicles, the uptake of both cephalosporins in the presence of an inward H+ gradient exhibited the overshoot phenomenon. In human and rat vesicles, the initial uptake of ceftibuten was strongly inhibited by compound V, an analogue of ceftibuten, but the uptake of cephradine was not affected by any of the cephalosporins tested, whereas in the rabbit brush-border membrane vesicles, initial uptake of both ceftibuten and cephradine were markedly inhibited by all cephalosporins and dipeptides used. These results suggest that the transport characteristics of human and rat intestinal brush-border membrane for cephalosporins are comparable, and that rabbit is an inadequate animal for investigating the transport characteristics of beta-lactam antibiotics.