A method for rapid screening of interactions of pharmacologically active compounds with albumin.

We determine the association constants for ligand-protein complex formation using the flow injection method. We carry out the measurements at high flow rates (F=1 mL min(-1)) of a carrier phase. Therefore, determination of the association constant takes only a few minutes. Injection of 1 nM of the ligand (10 µL of 1 µM concentration of the ligand solution) is sufficient for a single measurement. This method is tested and verified for a number of complexes of selected drugs (cefaclor, etodolac, sulindac) with albumin (BSA). We obtain K=4.45×10(3) M(-1) for cefaclor, K=1.00×10(5) M(-1) for etodolac and K=1.03×10(5) M(-1) for sulindac in agreement with the literature data. We also determine the association constants of 20 newly synthesized 3ß- and 3α-aminotropane derivatives with potential antipsychotic activity--ligands of 5-HT1A, 5-HT2A and D2 receptors with the albumin. Results of the studies reported here indicate that potential antipsychotic drugs bind weakly to the transporter protein (BSA) with K≈10(2)-10(3) M(-1). Our method allows measuring K in a wide range of values (10(2)-10(9) M(-1)). This range depends only on the solubility of the ligand and sensitivity of the detector.

Two plate-based colorimetric assays for screening α-amino acid ester hydrolase with high synthesis/hydrolysis ratio.

α-Amino acid ester hydrolases (AEHs) are enzymes of interest to the semi-synthesis of ß-lactam antibiotics with α-amino, such as cephalexin and cefaclor. An undesired side reaction, the hydrolysis of α-amino acid ester, had hindered applications in antibiotics synthesis. Although the enzymes' S/H ratio can be increased by protein engineering, such approaches require a suitable screening assay. Such a screening assay has not yet been described for AEHs. In this paper, we report a 96-well plate format screening procedure for AEHs based on two spectrophotometric assays. To reduce the hydrolysis reaction while maintaining synthesis activity, and to evaluate the effectiveness of the screening strategy, we introduced random mutations in part of the aeh gene from Xanthomonas rubrillineans by error-prone PCR. By a parallel plate-based screening strategy, three mutants with improved S/H ratio, R87L, T132N and N219I, were obtained.

Interactions of amoxicillin and cefaclor with human renal organic anion and peptide transporters.

Amoxicillin and cefaclor are two of the widely used beta-lactam antibiotics in the treatment of urinary tract infections. Both drugs are eliminated mainly by the kidney and rely on renal excretion to exert their antibacterial activities in the urinary tract. Previous studies have suggested the involvement of organic anion and oligopeptide transporters in membrane transport of beta-lactams. The objective of the current study was to examine the kinetics of amoxicillin and cefaclor interactions with human renal transporters human organic anion transporter 1 (hOAT1), human peptide transporter 1 (hPepT1), and human peptide transporter 2 (hPepT2) in detail, both as substrates and as inhibitors. Using fluorescence protein tagging and cell sorting, we established Madin-Darby canine kidney cell lines stably expressing highly functional hOAT1, hPepT1, and hPepT2. Amoxicillin and cefaclor inhibited hOAT1-mediated [(3)H]para-aminohippuric acid uptake (K(i) = 11.0 and 1.15 mM, respectively). However, our uptake study revealed that neither drug was transported by hOAT1. Amoxicillin and cefaclor competitively inhibited hPepT2-mediated [(3)H]glycylsarcosine uptake (K(i) = 733 and 65 muM, respectively), whereas much lower affinity for hPepT1 was observed with both antibiotics. Direct uptake studies demonstrated that amoxicillin and cefaclor were transported by hPepT1 and hPepT2. Kinetic analysis showed that hPepT2-mediated uptake of both drugs was saturable with K(m) of 1.04 mM for amoxicillin and 70.2 muM for cefaclor. hPepT2, and to a lesser extent hPepT1, may play an important role in apical transport of amoxicillin and cefaclor in the renal tubule. hOAT1, in contrast, is not involved in basolateral uptake of these antibiotics.

Novel mechanism of hydrolysis of therapeutic beta-lactams by Stenotrophomonas maltophilia L1 metallo-beta-lactamase.

Stopped-flow tryptophan fluorescence under single turnover and pseudo-first-order conditions has been used to investigate the kinetic mechanism of beta-lactam hydrolysis by the Stenotrophomonas maltophilia L1 metallo-beta-lactamase. For the cephalosporin substrates nitrocefin and cefaclor and the carbapenem meropenem, a substantial quench of fluorescence is observed on association of substrate with enzyme. We have assigned this to a rearrangement event subsequent to formation of an initial collision complex. For the colorimetric compound nitrocefin, decay of this dark inter- mediate represents the overall rate-determining step for the reaction and is equivalent to decay of a previously observed state in which the beta-lactam amide bond has already been cleaved. For both cefaclor and meropenem, the rate-determining step for hydrolysis is loss of a second, less quenched state, in which, however, the beta-lactam amide bond remains intact. We suggest, therefore, that the mechanism of hydrolysis of nitrocefin by binuclear metallo-beta-lactamases may be atypical and that cleavage of the beta-lactam amide bond is the rate-determining step for breakdown of the majority of beta-lactam substrates by the L1 enzyme.

Indirect polarographic and cathodic stripping voltammetric determination of cefaclor as an alkaline degradation product.

Cefaclor is not reducible at a mercury electrode, but it can be determined polarographically and by cathodic stripping voltammetry as its initial alkaline degradation product which is obtained in high yield by hydrolysis of cefaclor in Britton-Robinson (B-R) buffer pH 10 at 50 degrees C for 30 min (reduction peak at pH 10, -0.70 V). Differential pulse polarographic calibration graphs are linear up to at least 1 x 10(-4) mol/l(-1). Recoveries of 93% of the cefaclor (n = 3) were obtained from urine spiked with 38.6 microg/ml(-1) using this polarographic method with 1 ml urine made up to 10 ml with pH 10 buffer. Using cathodic stripping voltammetry and accumulating at a hanging mercury drop electrode at - 0.2 V for 30 s, linear calibration graphs were obtained from 0.35 to 40 microg/ml(-1) cefaclor in B-R buffer pH 10. A relative standard deviation of 4.2% (eta = 5) was obtained, and the limit of detection was calculated to be 2.9 ng/ml(-1). Direct determination of cefaclor in human urine (1 ml of urine was made up to 10 ml with pH 10 buffer) spiked to 0.39 microg/ml(-1) was made (recovery 98.6%).

Pharmacokinetic profile of cefaclor.

Cefaclor is a well-absorbed oral cephalosporin antibiotic. Peak concentrations in serum are attained within 30-60 minutes. Food intake reduces the rate, but not the extent of absorption. Cefaclor is not metabolized to a significant degree, but it degrades chemically in the body with an approximate half-life of 2 hours. Most of the drug is excreted unchanged in the urine, the serum half-life after oral administration is 0.5-0.7 hours. Due to the chemical degradation, cefaclor does not accumulate to the same degree as other cephalosporins in case of renal impairment.

Cefaclor uptake by the proton-dependent dipeptide transport carrier of human intestinal Caco-2 cells and comparison to cephalexin uptake.

The human Caco-2 cell line spontaneously differentiates in culture to epithelial cells possessing intestinal enterocytic-like properties. These cells possess a proton-dependent dipeptide transport carrier that mediates the uptake of the cephalosporin antibiotic cephalexin (Dantzig, A.H. and Bergin, L. (1990) Biochim. Biophys. Acta 1027, 211-217). In the present study, the uptake of cefaclor was examined and found to be sodium-independent, proton-dependent, and energy-dependent. The initial rate of D-[3-phenyl-3H]cefaclor uptake was measured over a wide concentration range; uptake was mediated by a single saturable transport carrier with a Km of 7.6 mM and a Vmax of 7.6 nmol/min per mg protein and by a non-saturable component. Uptake was inhibited by dipeptides but not amino acids. The carrier showed a preference for the L-isomer. The effect of the presence of a 5-fold excess of other beta-lactam antibiotics was examined on the initial rates of 1 mM cefaclor and 1 mM cephalexin uptake. Uptake rates were inhibited by the orally absorbed antibiotics, cefadroxil, cefaclor, loracarbef, and cephradine and less so by the parenteral agents tested. The initial uptake rates of both D-[9-14C]cephalexin and D-[3-phenyl-3H]cefaclor were competitively inhibited by cephalexin, cefaclor, and loracarbef with Ki values of 9.2-13.2, 10.7-6.2, and 7.7-6.4 mM, respectively. Taken together, these data suggest that a single proton-dependent dipeptide transport carrier mediates the uptake of these orally absorbed antibiotics into Caco-2 cells, and provide further support for the use of Caco-2 cells as a cellular model for the study of the intestinal proton-dependent dipeptide transporter.

Molecular basis of the efficacy of cefaclor against Haemophilus influenzae.

Cefaclor sustained its inhibitory activity against a beta-lactamase-producing strain of Haemophilus influenzae. Although a relatively high permeability coefficient was calculated for ampicillin compared with that calculated for cefaclor, the resulting periplasmic concentration of cefaclor was 5.7 times that of ampicillin. The efficacy of cefaclor may be due to its higher beta-lactamase resistance, which allows it to achieve a greater periplasmic concentration and adequate binding to crucial penicillin-binding proteins.

Characterization of penicillin-binding protein 2 of Staphylococcus aureus: deacylation reaction and identification of two penicillin-binding peptides.

Penicillin-binding protein (PBP) 2 is the major PBP of five that have been identified in susceptible strains of Staphylococcus aureus. Beta-lactam antibiotic binding to PBP 2 is important for the antibacterial effect. Antibiotic binding to PBP 2 in strain 209P was examined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis in competition assays using [3H]penicillin as the radiolabel. Clavulanic acid, which is specifically bound by PBP 2, and cefaclor, which is specific for PBP 3, were studied. Cefaclor, which alone appeared not to bind PBP 2, in combination inhibited PBP 2 binding of clavulanic acid. By varying the temperature during radiolabeling with [3H]penicillin in cefaclor competition assays and in direct radiolabeling assays with [3H]cefaclor, it was shown that cefaclor was bound by PBP 2 with high affinity (50% inhibitory concentration, less than or equal to 0.1 microgram/ml) and that the apparent low-affinity binding (50% inhibitory concentration, greater than 10 micrograms/ml) in competition assays performed at 37 degrees C was due to rapid deacylation. Two penicillin-binding peptides of PBP 2 also were identified in fluorographs of PBPs separated by nonequilibrium pH gradient gel and two-dimensional electrophoresis. Rapid deacylation for some antibiotics and the presence of two penicillin-binding peptides are two properties of PBP 2 that should be considered when correlating results of binding assays with effects of beta-lactam antibiotics on S. aureus.

Pharmacokinetic analysis of the effects of different foods on absorption of cefaclor.

Cefaclor is an oral cephalosporin antibiotic which has a broad antibacterial spectrum. The purpose of this study was to investigate the effect of food on the absorption of cefaclor and to analyze kinetically the absorption process of this drug. Cefaclor was given to eight volunteers at five test times: after overnight fasting, after two rice meals (350 and 700 cal [1 cal = 4.184 J]), and after two bread meals (500 and 1,000 cal). Urinary recoveries of cefaclor and concentrations of the drug in plasma were determined for each administration. Areas under the concentration-time curves and urinary recoveries were not affected by food intake, but the maximum concentration of drug in serum was reduced and the time to maximum concentration of drug in serum was prolonged depending on the type and the quantity of the meal. The larger the quantity of the meal, the more the maximum concentration of drug in serum and the time to maximum concentration of drug in serum were affected. The rice meals affected the absorption process of cefaclor more than the bread meals. The concentrations of cefaclor in plasma following administration after overnight fasting were well fitted to a conventional one-compartment model with a first-order absorption process, but those after the other administrations were not fitted to the model. For the pharmacokinetic analysis of those data, it was necessary to introduce a transfer process from administration site to absorption site to the conventional model. The concentrations in plasma after rice and bread meals were best fitted to the model with a zero-order transfer process than to that with a first-order process. The velocity of the transfer process depended on the type and volume of the meal.

High-performance liquid chromatographic determination of loracarbef, a potential metabolite, cefaclor and cephalexin in human plasma, serum and urine.

A high-performance liquid chromatographic (HPLC) method is reported for the determination of a new carbacephem antibiotic, loracarbef, a hydroxylated analogue, and two cephalosporins, cefaclor and cephalexin, in plasma, serum, and urine. The antibiotics are extracted from plasma by means of C18 solid-phase cartridges. Urine samples are diluted with water and directly injected on the HPLC system. The HPLC system utilizes a Supelcosil LC-18-DB (250 mm x 4.6 mm I.D.) reversed-phase column and ultraviolet detection at 265 nm. The limit of quantitation is 0.5 micrograms/ml for each compound. Excellent correlation of plasma concentrations is shown between results determined by HPLC and those obtained by microbiological agar-well diffusion assays. Stability studies of loracarbef in human plasma show the antibiotic to be stable for at least 24 h at room temperature and for at least twelve months at -20 degrees C.

[Comparative study of the penetration of penicillin V, amoxicillin, cefaclor and josamycin in the tonsils]. / Etude comparée de la pénétration de la pénicilline V, de l'amoxicilline, du céfaclor et de la josamycine dans les amygdales.

Fourty patients undergoing tonsillectomy for recurrent tonsillitis were administered penicillin V, amoxicillin, cefaclor or josamycin. Antibiotic concentrations in serum and tonsillar tissues were determined by microbiological assay. Cefaclor demonstrated a superior diffusion than penicillin V and amoxicillin, but nevertheless inferior to that of josamycin.

In vitro activity and beta-lactamase stability of LY163892.

Susceptibility testing of clinical isolates of several gram-negative and gram-positive species showed LY163892 to be more active than cefaclor and cephalexin. OXA-2, TEM-1, TEM-2, PSE-1, CEP-1, CARB-3 and SHV-1 beta-lactamases showed similar activity against LY163892 and cefaclor, whereas OXA-1 hydrolyzed the latter more rapidly. Organisms producing these beta-lactamases, but not TEM-2 and CEP-1, appeared to be more susceptible to LY163892 than cephalexin, although cephalexin proved to be more resistant to beta-lactamase activity. Strains producing TEM-2 and CEP-1 were resistant to LY163892, cefaclor and cephalexin.

Beta-lactamase hydrolysis and inhibition studies of the new 1-carbacephem LY163892.

A novel 1-carbacephem, LY163892, was determined to be more stable to plasmid-mediated beta-lactamases than cefaclor. Chromosomal-mediated Type Ia and IVc enzymes destroyed LY163892 at rates ranging from 16 to 93% that of nitrocefin. LY163892 showed minimal ability to inhibit beta-lactamases other than Type Ia (P99).

[Clinical studies on te concentration of cefaclor in sera and lung tissues of patients with respiratory diseases].

A 500 mg dose of cefaclor (CCL) was administered orally before surgery to each of patients with respiratory diseases and in fasting. Average concentrations of CCL in sera were 4.04 micrograms/ml at 1.5 hours, 3.03 micrograms/ml at 2 hours, 1.68 microgram/ml at 3 hours and 0.45 microgram/ml at 5 hours after administration. Average concentrations in lung tissues during operation were 0.120 microgram/g at 3 hours, 0.272 microgram/g at 4 hours and 0.297 microgram/g at 5 hours after administration. Ratios of concentrations of CCL in lung tissues to that in sera were from 7.1 to 66.0 percent. The CCL was considered to be a useful antibiotic for the treatment of patients with respiratory diseases.

Pharmacokinetic and therapeutic trial of sultamicillin in acute sinusitis.

Sultamicillin, an antibiotic combining ampicillin and the beta-lactamase inhibitor sulbactam, was administered to 13 patients diagnosed as having acute sinusitis. Specimens from sinus were obtained for all 13 patients by transantral puncture. Pharmacokinetics, bacteriology, and therapeutic efficacy were assessed. Eighty-five percent (11 of 13) were cured; two treatment failures were subsequently shown to have chronic (rather than acute) sinusitis during surgical exploration. Diarrhea was frequently encountered, and Clostridium difficile-associated enteritis was documented for one patient. Beta-lactamase-producing organisms were not encountered in this study; however, this study provides impetus for further controlled clinical trials.

[Fundamental and clinical studies of S 6472 (sustained release preparation of cefaclor) in the pediatric field].

Fundamental and clinical studies on S 6472 were carried out and following results were obtained. Serum concentrations after single oral administration showed 2 peaks at 1 or 2 hours and 5 or 6 hours in the cases with normal meal. Namely this drug has much more maintenance of serum concentration than normal cefaclor. In maintenance of serum concentrations after the administration, there were no obviously difference between normal and heavy meal. S 6472 was administered twice a day to 7 patients with various infections (bronchopneumonia 2 cases, acute bronchitis 1 case, purulent tonsillitis 4 cases) and clinical responses were all effective results. Pathogenic bacteria of S. aureus, S. pneumoniae, S. pyogenes and H. influenzae were completely eliminated in all cases. No significant side effects were observed. On the above results, this administration method of S 6472 twice a day was considered to be good response against mild or moderate bacterial infections in children.

In vitro activity of Ro 15-8074, a new oral cephalosporin, against Neisseria gonorrhoeae.

Ro 15-8074, a new cephalosporin the pivaloyloxymethylester of which (Ro 15-8075) is orally absorbable, showed greater in vitro activity than cefaclor against 48 Neisseria gonorrhoeae strains, including 25 penicillinase-producing strains. Unlike cefaclor, Ro 15-8074 was unaffected by increase in inoculum size, and it exhibited a remarkable stability against gonococcal beta-lactamase hydrolysis.

[Cefaclor dosage change with renal dysfunction].

Serum and/or urine levels of cefaclor (CCL) were studied in 4 patients during the therapy with CCL. In patients with severely impaired renal function, moderately higher serum and urine levels of CCL persisted, serum half-lives of CCL were moderately prolonged and urinary excretion of CCL slightly decreased. Although dosage modification of CCL is necessary in patients with renal dysfunction, multiple doses of 250 mg every 8 hours may be safe and effective even in patients with impaired renal function.

[Prolonged action preparation of cefaclor].

This study was conducted to develop a prolonged action preparation of cefaclor (CCL) which can offer, with the twice-a-day administration, as much effectiveness as its conventional preparation (Kefral capsule) with the 3 times-a-day administration. Absorption site of CCL in gastrointestinal tract, preparation form (enteric coated granules) which slowly release CCL, dissolution property of the form, and mixed ratio of the form and rapid release form (nonenteric coated granules) were studied and complex granules consisting of 40% of nonenteric coated granules and 60% of enteric coated granules which dissolve at pH 6 were chosen as a prolonged action preparation of CCL. Bactericidal activity of the prolonged action preparation (S6472) was confirmed to be the same as that of the conventional preparation by comparative viable cell count study in which concentrations of CCL simulated to plasma concentrations following the administration of S6472 at the dosage of 375 mg b.i.d. and the conventional preparation at the dosage of 250 mg t.i.d. were used. From the above, S6472 is considered to be a prolonged action preparation of CCL which serve our purpose. Since S6472 can be given with the twice-a-day administration, its daytime administration is not necessary. Therefore, S6472 is considered to be much useful preparation for the patients.