Computational design of new enzymes for hydrolysis and synthesis of third-generation cephalosporin antibiotics.

Engineering active sites in inert scaffolds to catalyze chemical transformations with unnatural substrates is still a great challenge for enzyme catalysis. In this research, a p-nitrobenzyl esterase from Bacillus subtilis was identified from the structural database, and a double mutant E115A/E188A was designed to afford catalytic activities toward the hydrolysis of ceftizoxime. A quadruple mutant E115A/E188A/L362S/I270A with enhanced catalytic efficiency was created to catalyze the condensation reaction of ethyl-2-methoxy-amino-2-(2-aminothiazole-4-yl) acetate with 7-amino-3-nor-cephalosporanic acid to produce ceftizoxime in a fully aqueous medium. The catalytic efficiencies of the computationally designed mutants E115A/E188A/L362S/I270A and E115A/Y118 K/E188 V/I270A/L362S can be taken as starting points to further improve their properties towards the practical application in designing more ecology-friendly production of third-generation cephalosporins.

Extrapolation of Drug Clearance in Children ≤ 2 Years of Age from Empirical Models Using Data from Children (> 2 Years) and Adults.

BACKGROUND: The application of modeling and simulation approaches in clinical pharmacology studies has gained momentum over the last 20 years. OBJECTIVES: The objective of this study was to develop six empirical models from clearance data obtained from children aged > 2 years and adults to evaluate the suitability of the models to predict drug clearance in children aged ≤ 2 years (preterm, term, and infants). METHODS: Ten drugs were included in this study and administered intravenously: alfentanil, amikacin, busulfan, cefetamet, meperidine, oxycodone, propofol, sufentanil, theophylline, and tobramycin. These drugs were selected according to the availability of individual subjects' weight, age, and clearance data (concentration-time data for these drugs were not available to the author). The chosen drugs are eliminated by extensive metabolism by either the renal route or both the renal and hepatic routes. The six empirical models were (1) age and body weight-dependent sigmoidal maximum possible effect (Emax) maturation model, (2) body weight-dependent sigmoidal Emax model, (3) uridine 5'-diphospho [body weight-dependent allometric exponent model (BDE)], (4) age-dependent allometric exponent model (ADE), (5) a semi-physiological model, and (6) an allometric model developed from children aged > 2 years to adults. The model-predicted clearance values were compared with observed clearance values in an individual child. In this analysis, a prediction error of ≤ 50% for mean or individual clearance values was considered acceptable. RESULTS: Across all age groups and the ten drugs, data for 282 children were compared between observed and model-predicted clearance values. The validation data consisted of 33 observations (sum of different age groups for ten drugs). Only three of the six models (body weight-dependent sigmoidal Emax model, ADE, and semi-physiological model) provided reasonably accurate predictions of clearance (> 80% observation with ≤ 50% prediction error) in children aged ≤ 2 years. In most instances, individual predicted clearance values were erratic (as indicated by % error) and were not in agreement with the observed clearance values. CONCLUSIONS: The study indicated that simple empirical models can provide more accurate results than complex empirical models.

A metallo-ß-lactamase is responsible for the degradation of ceftiofur by the bovine intestinal bacterium Bacillus cereus P41.

Ceftiofur is a highly effective veterinary cephalosporin, yet it is rapidly degraded by bacteria in the gut. The goal of this work was to directly determine the mechanism of ceftiofur degradation by the bovine intestinal isolate Bacillus cereus P41. B. cereus P41 was isolated from the feces of a cow that had not been treated with cephalosporins, and was found to rapidly degrade ceftiofur in culture. Analysis of spent culture media by HPLC/UV and HPLC/MS revealed one major metabolite of ceftiofur, with a negative ion m/z of 127. Comparison of ceftiofur, ceftriaxone, and cefpodoxime degradation suggested that the major stable ceftiofur metabolite was the thiofuroic acid group eliminated from the C-3 position of the drug after hydrolysis by ß-lactamase. Genomic DNA from B. cereus P41 was cloned into Escherichia coli, and the transformants were screened for growth in the presence of ceftiofur. DNA sequencing of the plasmid pHSG299-BC-3 insert revealed the presence of a gene encoding a metallo-ß-lactamase. Incubation of ceftiofur with either the E. coli transformant or a commercial B. cereus metallo-ß-lactamase showed degradation of the drug and formation of the same major metabolite produced by B. cereus P41. These data demonstrate that a metallo-ß-lactamase plays a major role in the degradation of ceftiofur by the bovine intestinal bacterium B. cereus P41.

Natural gum as mucoadhesive controlled release carriers: evaluation of cefpodoxime proxetil by D-optimal design technique.

The present study deals with the development of mucoadhesive controlled release tablets of Cefpodoxime Proxetil to increase the gastric residence time and thus prolong drug release, reduce dosing frequency and improve oral bioavailability. Tablets were prepared using sodium alginate and karaya gum, a natural polymer, with a synthetic polymer hydroxypropylmethylcellulose (K100LV) and Karaya gum with HPMC K100LV in various ratios to optimize the drug release profile using D-Optimal technique. Pre- and post-compression parameters of tablets prepared with various formulations (S1-S9, C1-C9) were evaluated. The FTIR and DSC studies revealed that no physiochemical interaction between excipients and drug. The formulation S7 showed prolonged drug release, and the mechanism of drug release from the optimized formulation was confirmed using the Korsmeyer-Peppas model to be non-Fickian release transport and n value was found 0.605 indicating both diffusion and erosion mechanism from these natural gums. The optimized formulation showed mucoadhesive strength >35 g. An in vivo study was performed on rabbits using an X-ray imaging technique. The radiological evidence suggests that the tablets adheres (more than 10 hours) to a rabbit's stomach. No significant changes were found in the physical appearance, drug content, mucoadhesive study and in vitro dissolution pattern after storage at 40 °C/75% relative humidity for 3 months.

Highly sensitive and selective high-performance liquid chromatography method for bioequivalence study of cefpodoxime proxetil in rabbit plasma via fluorescence labeling of its active metabolite.

Cefpodoxime proxetil (CFP), a broad-spectrum third-generation cephalosporin, has been used most widely in the treatment of respiratory and urinary tract infections. For bioequivalence study of CFP in rabbit plasma, it was necessary to develop a highly sensitive and selective high-performance liquid chromatographic (HPLC) method with fluorescence (FL) detection. The pre-column labeling of cefpodoxime acid (CFA) (active metabolite) with an efficient benzofurazan type fluorogenic reagent, 4-N,N-dimethyl aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (DBD-F) was carried out in the present study in 100mM borate buffer (pH=8.5) at 50°C for 15min. The obtained fluorescent products were separated on C18 column with an isocratic elution of the mobile phase, which consists of 10mM phosphate buffer (pH=3.5)/CH3CN (70:30, v/v). The fluorescent product (DBD-CFA) was detected fluorimetrically at 556nm with an excitation wavelength of 430nm. Cefotaxime sodium was used as internal standard. The method was validated according to the requirements of US-FDA guidelines. The correlation coefficient of 0.999 was obtained in the concentration ranges of 10-1000ngmL(-1). The limits of detection and quantification (S/N=3) were 3 and 10ngmL(-1), respectively. Plasma CFA levels were successfully determined in rabbit with satisfactory precision and accuracy. The proposed HPLC-FL method was successfully applied to study bioequivalence in rabbits for two formulations of different brands contained CFP (prodrug) in a randomized, two-way, single-dose, crossover study and all pharmacokinetic parameters for the two formulations were assessed.

Gastro-retentive dosage form for improving bioavailability of Cefpodoxime proxetil in rats.

Cefpodoxime proxetil (CP) is a prodrug with poor oral bioavailability because of its metabolism to Cefpodoxime acid (CA) in luminal contents and intestinal epithelial cells. In the present investigation, regional variability in different segments of the gastrointestinal tract vis-à-vis solubility and metabolism were investigated, and the results indicated potential for a gastro retentive (GR) dosage form. Suitability of a GR dosage from for CP and finally in vivo efficacy were investigated. Thereafter, an effervescent floating GR dosage form was developed for CP and evaluated in rats. The GR dosage form improved the oral bioavailability of CP significantly by about 75%, hence providing a proof-of-concept. The Tmax value increased to 1.43+/-0.24 h from 0.91+/-0.23 h of pure drug, while Cmax values of 4735+/-802 ng/ml and 3094+/-567 ng/ml were obtained for the GR dosage form and pure drug respectively.

Cefoselis, a beta-lactam antibiotic, easily penetrates the blood-brain barrier and causes seizure independently by glutamate release.

Cefoselis is a widely used beta-lactam antibiotic, but occasionally induces seizures and convulsion in elder and renal failure patients. However, beta-lactams are known not to pass through the blood-brain barrier (BBB). In this study, we examined the BBB penetration of cefoselis in normal and renal failure rats by means of brain microdialysis. Cefoselis was dose-dependently appeared in brain extracellular fluid in proportion to its blood level. The elimination constant from brain extracellular fluid (apparent) was slightly lower than that from blood. These results indicated that cefoselis might penetrate the BBB or be discharged by a certain transport system. In contrast to the result of cefoselis, cefazolin, a leading drug of cephalosporins, could not be detected in the brain extracellular fluid after an intravenous injection. In renal dysfunction rats, the elimination half-lives of cefoselis from both blood and brain were extensively prolonged. This would be one of responsible factors inducing seizures seen in patients. However, the additional factor, such as decrease in brain function related to aging, would be involved in seizures in patient received cefoselis, because an extremely high dose was required to induce seizures even in renal failure rats. A local administration of cefoselis into the hippocampus through the microdialysis probe caused a striking elevation of extracellular glutamate, with a minimum increase in gamma-aminobutyric acid (GABA). However, a systematic cefoselis administration via the tail vein did not elevate extracellular glutamate and GABA concentrations in the hippocampus of renal failure rats that exhibited marked seizures. These results suggested that not the stimulation of glutamate release, but the blockade of GABA receptors might be responsible for the seizure induced by cefoselis.

Occurrence and detection of extended-spectrum beta-lactamases in members of the family Enterobacteriaceae at a veterans medical center: seek and you may find.

A total of 907 consecutive isolates of members of the family Enterobacteriaceae recovered during a 20-week period were tested for production of extended-spectrum beta-lactamases (ESBLs) by the double-disk (DD) potentiation method. Of 84 DD-positive isolates, 83 (9.2%) produced ESBLs based on isoelectric focusing. SHV-derived ESBLs and several TEM-derived ESBLs were present in nine species, including the first isolate of Citrobacter koserii and Morganella morganii known to harbor an SHV-derived ESBL. Results of testing 58 nonrepeat isolates for ESBL production by several recommended methods were as follows (percent detected in parentheses): DD method with aztreonam (95), ceftazidime (79), ceftriaxone (88), or cefpodoxime (90); broth microdilution method with ceftazidime (86) or cefotaxime (91) alone or in combination with clavulanate; and the standard disk diffusion method with new breakpoints and standard concentrations of aztreonam (78), ceftazidime (79), ceftriaxone (83), or cefpodoxime (98) or a novel concentration (5 microg) of ceftazidime (88). In three instances during an extended part of the study, an ESBL-producing isolate and a non-ESBL-producing isolate of the same species were recovered from a single blood culture bottle. These data indicate that ESBLs occur in several species of Enterobacteriaceae and at a relatively high incidence at our institution and that the standard disk diffusion method with cefpodoxime and the DD method with several beta-lactams are practical and cost-effective methods for detecting ESBL-producing isolates of Enterobacteriaceae.

In vitro activity of FK037, a new parenteral cephalosporin, against anaerobic bacteria.

The activity of FK037, a new parenteral cephalosporin, was compared with those of cefpirome, ceftazidime, and flomoxef against 322 recent clinical isolates of anaerobic bacteria. A fastidious facultative anaerobe, Gardnerella vaginalis, was also studied. FK037 inhibited 90% of isolates of Peptostreptococcus anaerobius, Peptostreptococcus asaccharolyticus, Clostridium perfringens, Mobiluncus spp., G. vaginalis, and Porphyromonas gingivalis at < or = 0.78 micrograms/ml. The MICs of FK037 for 50 and 90% of Bacteroides fragilis isolates were 25 and > 200 micrograms/ml, respectively; the activity of FK037 was comparable to those of cefpirome and ceftazidime but lower than that of flomoxef. The activity of FK037 against Fusobacterium nucleatum, Fusobacterium varium, and Bilophila wadsworthia decreased when inoculum size was increased from 10(6) to 10(8) CFU/ml. Little influence of inoculum size on the activity of FK037 was observed for other isolates tested. Medium pH affected the activity of FK037 against F. varium (MICs at pHs 5 and 7, 3.13 and 100 micrograms/ml, respectively) and Bacteroides gracilis (MICs at pHs 5 and 7, 12.5 and 1.56 micrograms/ml, respectively) but not against other organisms tested. FK037 was less resistant than flomoxef to hydrolysis by beta-lactamase group 2e derived from B. fragilis GAI 0558 and GAI 10150.

Differential protein binding of ceftizoxime in cord versus maternal serum.

Ceftizoxime concentrations are higher in cord blood and amniotic fluid than in maternal blood. More avid binding to fetal serum proteins is a suggested mechanism. We measured ceftizoxime protein binding in fetal and maternal blood and documented significantly less protein binding to fetal proteins (21.9% vs 57.8%).

Structure-binding relationship and binding sites of cephalosporins in human serum albumin.

The binding of some cephalosporins to human serum albumin (HSA) was studied by an ultrafiltration technique. Changes in C-3 side chain resulted in marked changes in the binding to HSA, but changes in C-7 side chain did not. Cephalosporins were classified into three groups by C-3 side chain: (i) Cationic side chain with low affinity for HSA; (ii) anionic side chain with high affinity for HSA; (iii) non ionized side chain, in which binding to HSA was dependent on lipophilicity. These findings suggest that electrostatic and hydrophobic forces play a role in the binding affinity of cephalosporins for HSA. The binding of cephalosporins with high HSA affinity was displaced significantly by warfarin but not by phenylbutazone, L-tryptophan, or diazepam. The interaction of the cephalosporins with high affinity for HSA with chemically modified HSA was investigated to clarify the amino acid residues of HSA involved in the cephalosporin binding sites. The binding of the cephalosporins decreased remarkably with the modification of the tyrosine residues. These results suggest that the binding site of cephalosporins is located in the vicinity of warfarin binding site rather than benzodiazepine binding site and that tyrosine residues are involved in the cephalosporin binding site.

In vitro activity, beta-lactamase stability and PBP affinity of RU 51,746-2, the active metabolite of the new orally absorbed cephalosporin ester, RU 51807.

The activity of RU 51,746-2 was determined against strains of Escherichia coli producing different types of beta-lactamases or showing alterations of permeability. The production of TEM 1, OXA 2, CARB 3 and PSE 1 beta-lactamases had no influence on susceptibility to the antibiotic, whereas the synthesis of TEM 2, SHV 1 and OXA 1 beta-lactamases increased minimum inhibitory concentrations (MIC) by 2-4 times. Highly resistant to the antibiotic was a strain producing CEP 1 beta-lactamase. E. coli mutans deficient in Omp F but not in Omp C had a decreased susceptibility to RU 51,746-2. RU 51,746-2 showed a good affinity for high molecular weight penicillin binding proteins (PBPs) of E. coli. PBP 3 was found to be the target for growth inhibition, whereas the additional saturation of PBP 1 and 2 was required for obtaining the best bactericidal activity.

Concentrations of cefpodoxime in plasma and pleural fluid after a single oral dose of cefpodoxime proxetil.

Eighteen patients of either sex with pleural effusions underwent aspiration 3, 6 or 12 h after receiving a single oral dose of cefpodoxime proxetil equivalent to 200 mg cefpodoxime. The mean concentrations of cefpodoxime in pleural fluid were, respectively, 0.62, 1.84 and 0.78 mg/l for these three time intervals, the corresponding ratios between pleural fluid and plasma concentrations being 0.24, 0.67 and 1.07. The findings indicate that there is good penetration of cefpodoxime into pleural fluid. Concentrations between 3 and 12 h after dosing were equal to or above the MIC90 for most of the organisms commonly found in lower respiratory tract infections.

Concentrations of cefpodoxime in plasma and tonsillar tissue after a single oral dose of cefpodoxime proxetil.

Seventeen patients undergoing tonsillectomy received cefpodoxime proxetil orally in a dose equivalent to 100 mg cefpodoxime 4, 7 or 12 h before operation. Plasma and tonsillar tissue concentrations of cefpodoxime were assayed by a microbiological method. Tonsillar tissue concentrations after 4 and 7 h were 0.24 and 0.09 mg/kg respectively--being 23% of the plasma concentration. The tonsillar tissue concentration after 12 h was less than 0.06 mg/kg. As the MIC for Streptococcus pyogenes is less than 0.06 mg/l, cefpodoxime proxetil may be of value in acute tonsillitis.

In vitro antibacterial properties of cefetamet and in vivo activity of its orally absorbable ester derivative, cefetamet pivoxil.

The in vitro activity of cefetamet, the microbiologically active metabolite of the orally administered prodrug cefetamet pivoxil, was compared with that of cephalexin, cefaclor, cefuroxime and amoxicillin. Cefetamet was highly active against Enterobacteriaceae, Neisseria spp., Vibrio spp., Haemophilus influenzae and streptococci other than enterococci. Cefetamet inhibited cefaclor-resistant species such as Proteus vulgaris, Providencia stuartii, Providencia rettgeri and Serratia marcescens. Staphylococci, Pseudomonas aeruginosa and cephalosporinase-overproducing strains of Enterobacter cloacae were resistant to cefetamet. The superior activity of cefetamet compared with older oral beta-lactam antibiotics against a large number of gram-negative pathogens correlated with enhanced stability towards beta-lactamases. In accordance with the in vitro findings, cefetamet pivoxil showed good activity in experimental infections in the mouse and rat, suggesting satisfactory bioavailability in these animals after oral administration.

Comparison of the in vitro and in vivo antibacterial activities of cefepime (BMY-28142) with ceftazidime, cefuzonam, cefotaxime and cefmenoxime.

Cefepime (BMY-28142), a new semisynthetic cephalosporin, was evaluated for in vitro and in vivo antibacterial activities in comparison with ceftazidime, cefuzonam, cefotaxime and cefmenoxime. Cefepime showed a well-balanced, broad spectrum of activity against a number of clinical isolates collected in Japan. The activity of cefepime against Gram-positive bacteria was several times greater than that of ceftazidime, nearly comparable to cefotaxime and cefmenoxime, and slightly weaker than cefuzonam. Against Enterobacteriaceae, cefepime showed superior activity to the reference cephalosporins against Proteus inconstans, Providencia rettgeri, Morganella morganii, Citrobacter freundii and Enterobacter cloacae. The activity of cefepime against Pseudomonas aeruginosa was nearly comparable to that of ceftazidime. Cefotaxime, cefuzonam and cefmenoxime were substantially less active against P. aeruginosa. Cefepime was more stable than cefuzonam, cefotaxime and cefmenoxime to various types of beta-lactamases from Gram-negative bacteria. The high in vitro activity of cefepime was reflected in its in vivo efficacy against experimental infections in normal and immuno-suppressed mice. Cefepime was the most effective among the cephalosporins tested against four Gram-negative bacterial infections.