Degradation of cefradine in alga-containing water environment: a mechanism and kinetic study.

Large quantities of antibiotics are manufactured, used, and eventually discharged into alga-containing water environment as prototypes, by-products, or transformation products. Different activities of Chlamydomonas reinhardtii toward cefradine (CFD) were studied, and the results indicated that CFD is resistant (removal rate of 5.45-14.72%) in simulated natural water environment. Cefradine was mainly removed by hydrolysis, adsorption, desorption, photodecarboxylation, and photoisomerization. The effects of C. reinhardtii density, initial solution pH, and different light sources on CFD removal efficiency were investigated. The optimum conditions occurred at a density of algae 10 × 104 cells/mL, a solution pH of 9.0, and the ultraviolet (UV) light. Additionally, the removal kinetics under 16 different conditions was explored. The results showed that the removal of CFD fits well with a pseudo-first-order kinetic, and the half-life times are from 0.8 to 261.6 days. This study summarizes the CFD removal mechanisms in alga-containing water environment, highlights the important role played by light irradiation in eliminating CFD, and obtains the important kinetic data on CFD removal.

Computational design of cephradine synthase in a new scaffold identified from structural databases.

Computational enzyme design exhibits excellent performance for identifying potential scaffolds from structural databases and creating new enzymatic catalysts from naught. Using the active site-matching algorithm ProdaMatch, we identified a new scaffold cocaine esterase from Rhodococcus sp. that showed modest activity (kcat/Km = 0.018 M-1 s-1) towards the hydrolysis of ß-lactam antibiotic cephradine. The identified cocaine esterase scaffold afforded low sequence identity (<30%) with the known ß-lactam synthases, such as penicillin G acylase or α-amino acid ester hydrolase, and was able to catalyze the condensation reaction between d-dihydrophenylglycine methyl ester and 7-aminodesacetoxycephalosporanic acid to produce cephradine via a kinetically controlled synthesis. By virtue of the computational enzyme design protocol, hundreds of sequences were predicted in the cocaine esterase scaffold to promote the catalytic activity towards the hydrolytic reaction of cephradine. Moreover, a single mutant (F261T) was experimentally confirmed to have improved the catalytic efficiency by ten times (kcat/Km = 0.193 M-1 s-1), indicating that the novel scaffold cocaine esterase may be potentially redesigned to become an industrially useful cephradine synthase.

Access the toxic effect of the antibiotic cefradine and its UV light degradation products on two freshwater algae.

Two common freshwater algae Microcystis aeruginosa and Scenedesmus obliquus were employed as test organism to evaluate the toxic effects of the widely used antibiotic, cefradine. In general, cefradine had significantly toxic effect on population growth and chlorophyll-a accumulation of two algae and the cyanophyceae was more sensitive than the chlorophyceae. In addition, cefradine UV light degraded products had adverse effect on M. aeruginosa's growth and chlorophyll-a accumulation. In comparison, even if S. obliquus had growth ability when exposed to cefradine UV light-degradation products, the algal photosynthesis function was also disrupted.

Biotic and abiotic degradation of four cephalosporin antibiotics in a lake surface water and sediment.

Cephalosporins are widely used veterinary and human antibiotics, but their environmental fate and impacts are still unclear. We studied degradation of four cephalosporins (cefradine, cefuroxime, ceftriaxone, and cefepime) from each generation in the surface water and sediment of Lake Xuanwu, China. The four cephalosporins degraded abiotically in the surface water in the dark with half-lives of 2.7-18.7d, which were almost the same as that in sterilized surface water. Under exposure to simulated sunlight, the half-lives of the cephalosporins decreased significantly to 2.2-5.0d, with the maximal decrease for ceftriaxone from 18.7d in the dark to 4.1d under the light exposure. Effects of dissolved organic matter (DOM) and nitrate on photodegradation of the cephalosporins were compound-specific. While DOM (5 mg L(-1)) stimulated the photodegradation of only cefradine (by 9%) and cefepime (by 34%), nitrate (10 microM) had effects only on cefepime (stimulation by 13%). Elimination rates of the cephalosporins in oxic sediment (half-lives of 0.8-3.1d) were higher than in anoxic sediment (half-lives of 1.1-4.1d), mainly attributed to biodegradation. The data indicate that abiotic hydrolysis (for cefradine, cefuroxime, and cefepime) and direct photolysis (for ceftriaxone) were the primary processes for elimination of the cephalosporins in the surface water of the lake, whereas biodegradation was responsible for the elimination of the cephalosporins in the sediment. Further studies are needed on chemical structure, toxicity, and persistence of transformation products of the cephalosporins in the environment.

Lack of interaction between the peptidomimetic substrates captopril and cephradine.

Intestinal peptide transporters, including hPEPT1, facilitate the absorption of cephalosporins and angiotensin-converting enzyme inhibitors, and have been investigated as a means to improve oral drug absorption. Renal peptide transporters including hPEPT2, may also facilitate renal reabsorption of such compounds. In vitro and animal studies suggest that co-administration of peptidomimetic compounds may alter oral pharmacokinetics, although this has not been well studied in humans. The purpose of this study was to determine whether co-administration of the hPEPT substrates captopril and cephradine alters the oral pharmacokinetics of either agent. Nine healthy male volunteers received a single oral 25-mg dose of captopril, a single oral 500-mg dose of cephradine, or concurrent ingestion of captopril and cephradine in a cross-over manner. Venous blood samples were taken and captopril and cephradine pharmacokinetics were determined using noncompartmental analyses. No significant differences were observed in captopril or cephradine pharmacokinetics when administered together as compared to each agent alone (a marginal decrease in C(max) was observed for both captopril and cephradine during co-administration [5-15%]; however, differences were not statistically significant). The results of our study suggest that hPEPT1 and hPEPT2 are unlikely to contribute to clinically important drug interactions in humans.

Do cell culture conditions influence the carrier-mediated transport of peptides in Caco-2 cell monolayers?

Despite the fact that different laboratories have reported large differences in permeability for actively transported substrates, Caco-2 cell monolayers are widely used as in vitro model to study small intestinal drug transport. Therefore, we investigated the effect of cell culture conditions, such as time in culture, membrane support, seeding density and supplements to the medium, on the morphology, the formation of tight junctions, as well as the expression of two peptide transporters (PepT1, HPT1) and the efflux pump, P-glycoprotein (Pgp), in Caco-2 cell monolayers. Tight junction formation was assessed by transepithelial electrical resistance measurements; multi-cell layer formation by confocal laser scanning microscopy, the expression of transporters by RT-PCR and the permeability of the PepT1 substrate, cephradine. Both morphology and the expression of carrier-mediated transporters, varied strongly as a function of culture conditions. An increase of differentiation, as documented by tight, homogeneous cell monolayer formation displaying a strong expression of all carrier-mediated transporters, was found up to 3 weeks post seeding. One week later, multi-layer structures were observed and the expression of Pgp decreased. Polyester and polyethylene terephthalate membrane supports decreased the paracellular transport rates substantially, while collagen-coating of PC inserts showed no influence on the morphology and even increased carrier-mediated transporter expression. An average seeding density of 6x10(4) cells/cm(2) seemed to be most favorable, since lower seeding densities led to thin monolayers with altered tight junctions and higher seeding densities to the formation of multilayers. In summary, the expression of carrier-mediated transporters was strongly affected by the culture conditions. The full differentiation was reached after 21 days on collagen-coated polycarbonate inserts at an initial seeding density of 6x10(4) cells/cm(2).

Treatment of anti-osmotic drug based pharmaceutical effluent in an upflow anaerobic fluidized bed system.

During the production of Cephradine (a main constituent of anti-osmotic drug) a large quantity of concentrated effluent was produced. The main polluting compounds in this effluent are osmotic drug, acetic acid and ammonia. The feasibility of using a fluidized bed reactor under anaerobic condition with bioaugmentation to treat anti-osmotic drug based pharmaceutical effluent was evaluated. The main objective of the study was to show that bioaugmentation could be used to promote biological treatment to applications where conventional operation might be difficult or unfavourable. The effluent with chemical oxygen demand (COD) of 14000-18000 mg/l was treated in a fluidized bed reactor with a hydraulic retention time of 3-12 h. The reactor was unable to maintain consistent removal in conventional mode of operation due to an inability to retain and grow biomass. The COD reduction (%) after inoculation from a sequencing batch reactor was related to influent concentration, mass of inoculum and hydraulic retention time characterized by calculating the initial food to microorganism ratio. The role of volatile fatty acid (VFA) as cosubstrate was assessed with respect to COD reduction (%). Continuous COD reduction (%) attained a maximum value of 88.5% using bioaugmentation through periodic addition of acclimated cells every 2 days with 30-73.2 g of cells from an off-line enricher reactor.

Application of fractal kinetics for carrier-mediated transport of drugs across intestinal epithelial membrane.

PURPOSE: Fractal kinetics was used for the analysis of the carrier-mediated transport for drugs across the intestinal epithelial cells. METHODS: The transport was examined under various agitation rates using a monolayer of Caco-2 cells and rabbit ileum sheets. RESULTS: The passive transport of antipyrine across Caco-2 cells was increased with the increasing rate of agitation and was supposed to be caused by a change in the thickness of the unstirred water layer. On the contrary, in the case of L-lactic acid transport, which follows a carrier-mediated transport mechanism, the more the agitation rate controlling the fractal dimension was increased, the more the permeability rate across the Caco-2 cells was decreased. Fractal kinetic analysis of L-lactic acid transport indicated that the permeability was caused by a single saturable process. Similar agitation effects with L-lactic acid transport were observed in the transport of phenylalanine and cephradine in Caco-2 cells. However, the permeability rates of benzoic acid and 3-O-methyl-D-glucose across Caco-2 cells and L-lactic acid transport across the rabbit ileum tissue indicated the maximum levels at a designated agitation rate. This phenomenon was likely to be caused by the agitation effects controlling not only the fractal environment but also the unstirred water layer. CONCLUSIONS: Carrier-mediated transports are well defined by fractal kinetics rather than classical kinetic analysis. Fractal kinetics are one of the important areas for understanding and confirming the properties of a carrier-mediated transport process.

Inhibitory effects of angiotensin-converting enzyme inhibitor on cefroxadine uptake by rabbit small intestinal brush border membrane vesicles.

Effects of angiotensin-converting enzyme (ACE) inhibitors, captopril, enalapril maleate and quinapril, on the uptake of aminocephalosporin antibiotic, cefroxadine, by rabbit small intestinal brush border membrane vesicles were examined. These ACE inhibitors significantly inhibited the uptake of cefroxadine, which is transported by H+/dipeptide transporter in the membrane, in the order of captopril < enalapril < quinapril in the presence of an inward H+ gradient. Inhibitory effect of quinapril was more marked than that of aminocephalosporin cephradine, while in the absence of an inward H+ gradient inhibition by these ACE inhibitors was much less. Dixon plot analysis showed that the inhibition by enalapril and quinapril in the presence of an inward H+ gradient occurred in a competitive manner and estimated inhibition constants of these two drugs to be 5.3 mM and 0.46 mM, respectively. These results suggested the strong affinity of these ACE inhibitors, especially quinapril, on the H+/dipeptide transporter.

Identification of the histidine residues involved in substrate recognition by a rat H+/peptide cotransporter, PEPT1.

The LLC-PK1 cells stably transfected with a rat PEPT1 cDNA transported ceftibuten (anion) and cephradine (zwitterion), both oral beta-lactam antibiotics, in a H+-gradient-dependent manner. Diethylpyrocarbonate, a histidine residue modifier, abolished ceftibuten uptake. This inhibition was prevented in the presence of glycylsarcosine or cephradine. When expressed in Xenopus oocytes, replacement of either histidine 57 or histidine 121 of the rat PEPT1 with glutamine by site-directed mutagenesis eliminated ceftibuten and [14C]glycylsarcosine transport activities. Immunostaining of oocyte sections indicated that insertion of the mutant transporters in the plasma membranes was not impaired. These findings suggest that both histidine 57 and histidine 121, which are conserved in the rat, rabbit and human PEPT1, are involved in substrate recognition of this molecule.

Expression and characterization of the ponA (ORF I) gene of Haemophilus influenzae: functional complementation in a heterologous system.

The coding sequence of the Haemophilus influenzae ORF I gene was amplified by PCR and cloned into different Escherichia coli expression vectors. The ORF I-encoded protein was approximately 90 kDa and bound 3H-benzyl-penicillin and 125I-cephradine. This high-molecular-weight penicillin-binding protein (PBP) was also shown to possess transglycosylase activity, indicating that the ORF I product is a bifunctional PBP. The ORF I protein was capable of maintaining the viability of E. coli delta ponA ponB::spcr cells in transcomplementation experiments, establishing the functional relevance of the significant amino acid homology seen between E. coli PBP 1A and 1B and the H. influenzae ORF I product. In addition, the physiological functioning of the H. influenzae ORF I (PBP 1A) product in a heterologous species established the ability of the enzyme not only to recognize the E. coli substrate but also to interact with heterologous cell division proteins. The affinity of the ORF I product for 3H-benzylpenicillin and 125I-cephradine, the MIC of beta-lactams for E. coli delta ponA ponB::spcr expressing the ORF I gene, and the amino acid alignment of the PBP 1 family of high-molecular-weight PBPs group the ORF I protein into the PBP 1A family of high-molecular-weight PBPs.

Transcellular transport of oral cephalosporins in human intestinal epithelial cells, Caco-2: interaction with dipeptide transport systems in apical and basolateral membranes.

The transport characteristics of p.o. cephalosporin antibiotics by monolayers of the human intestinal epithelial cell line Caco-2 were examined by measuring intracellular accumulation and transcellular transport. In the presence of an inward H+ gradient (at pH 6.0 of the apical medium), cephalosporins were accumulated by the monolayers in the following order: ceftibuten (anion) > cephradine (zwitterion) > cephalexin (zwitterion) > cefixime (anion). The accumulation rate of ceftibuten was more rapid than that of cephradine, whereas both appeared at the same rate in the basolateral compartment. The efflux of ceftibuten from the monolayers to the basolateral compartment was lower than the efflux of cephradine. The accumulation rate of ceftibuten from the basolateral side was markedly lower than that of cephradine. The accumulation of ceftibuten from both the apical and basolateral compartment was significantly inhibited by excess dipeptides. The kinetic parameters indicated that ceftibuten has higher affinity for the apical dipeptide transport system in the presence of a pH gradient, whereas it has much lower affinity for the basolateral dipeptide transport system at physiological pH of 7.4 than cephradine. These results suggest that both cephradine and ceftibuten are transported via the H+/dipeptide cotransport system localized in the apical membranes, and that the flux of these drugs across the basolateral membranes is also mediated by the dipeptide transport system in an H+ gradient-independent manner, exhibiting a rate-limiting step for their transcellular transport in Caco-2 monolayers.

Transport of bestatin in rat renal brush-border membrane vesicles.

Bestatin [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-L-leucine] is a dipeptide, comprising L-leucine and an unusual beta-amino acid. We studied its transport mechanism in rat renal brush-border membrane vesicles. Uptake of cephradine, an aminocephalosporin, by isolated brush-border membrane vesicles was trans-stimulated and cis-inhibited by bestatin, indicating that these drugs are transported via the same transport system(s). The uptake of bestatin was trans-stimulated by preloading the vesicles with glycylsarcosine, and was cis-inhibited by substrates for the H+/dipeptide cotransport system. Bestatin inhibited tetraethylammonium (an organic cation) uptake, and bestatin uptake was cis-inhibited by substrates for the H+/organic cation antiport system. In addition, bestatin uptake was stimulated by an outward H+ gradient (the driving force for the H+/organic cation antiport system). These findings suggest that bestatin, in spite of being a dipeptide, is transported via not only the H+/dipeptide cotransport system but also the H+/organic cation antiport system in rat renal brush-border membrane.

Copolymers of lysine and polyethylene glycol: a new family of functionalized drug carriers.

Poly(PEG-Lys), a new, water-soluble poly(ether urethane), derived from L-lysine and poly(ethylene glycol) was investigated as a precursor for the preparation of polymeric drug conjugates. To facilitate a wide variety of coupling chemistries, the pendent carboxyl groups of poly(PEG-Lys) were converted to other reactive functional groups (amino, hydroxyl, active ester, and aldehyde) in high yield. These reactive pendent chains were then used as anchors for the covalent attachment of penicillin V and cephradine, two clinically used antimicrobial agents. Coupling to the carrier was achieved in good yields and the chemical versatility of this system was demonstrated by the preparation of conjugates having antibiotic ligands linked via biostable or biodegradable linkages to the carrier, either directly or via a spacer. Conjugate 4, poly(PEG-Lys-penicillin V ester), was obtained by linking penicillin V to the polymer backbone via hydrolytically labile ester bonds. This conjugate exhibited activity similar to that of the parent drug against three clinically important strains of bacteria. Drug activity coincided with the release of the drug from the carrier. Hydrolytically stable cephradine-containing conjugates were prepared by three different coupling methods but showed no antibiotic activity. 14C-labeled poly(PEG-Lys) was injected into mice and its biodistribution was monitored for 48 h. The carrier showed no preferential uptake by liver, spleen, or kidney. No signs of acute toxicity were evident in mice or rats when poly(PEG-Lys) was administered iv and ip at doses up to 10 g/kg. These results indicate that poly(PEG-Lys) is a promising precursor for the preparation of soluble drug conjugates.

Pharmacokinetics of cefradine, sulfamethoxazole and trimethoprim and their metabolites in a patient with peritonitis undergoing continuous ambulatory peritoneal dialysis.

Cefradine and co-trimoxazole pharmacokinetics were studied in a patient with peritonitis that complicated continuous ambulatory peritoneal dialysis (CAPD). Concentrations in the plasma reached after oral administration of 500 mg cefradine four times daily and 400/80 mg co-trimoxazole four times daily were for cefradine 100 micrograms/ml, for trimethoprim 15 micrograms/ml, and for sulfamethoxazole 100 micrograms/ml, respectively. In the dialysate concentrations were reached of 35-70 micrograms/ml cefradine, 2-5 micrograms/ml trimethoprim and 8-17 micrograms/ml sulfamethoxazole. The values for sulfamethoxazole are regarded too low to be clinically effective. Half-lives, protein binding values and CAPD clearances are presented. Low CAPD clearances were obtained during the night and high values during the day. The dosage yielded too high plasma trimethoprim concentrations, while sulfamethoxazole dialysate concentrations were too low. It seems questionable therefore whether co-trimoxazole can be used orally for the treatment of CAPD peritonitis.

Comparison of the pharmacokinetics of cephradine and cefazolin in pregnant and non-pregnant women.

The pharmacokinetics of cephradine, a cephalosporin with a low degree of protein binding, was studied in 12 women after oral and intravenous administration of the drug during and after pregnancy. Six of the 12 women also received a cephalosporin with a high degree of protein binding, cefazolin, intravenously during and after pregnancy. For both drugs most pharmacokinetic parameters were altered in pregnancy. The area under the plasma concentration-time curve (AUC) following intravenous administration was smaller for both drugs during as compared to after pregnancy (mean change 39% for cephradine and 31% for cefazolin). Half-lives of both drugs were significantly shorter during compared with after pregnancy (mean change 26% for cephradine and 35% for cefazolin). Consequently, total body clearance was increased during pregnancy. A significant negative correlation between length of gestation and total clearance per kg bodyweight was seen for cephradine. The bioavailability of oral cephradine did not differ significantly during compared with after pregnancy. It is concluded that the dosage of both cefazolin and cephradine should be increased when treating infections in pregnant women in order to obtain the same antibacterial effect as when treating non-pregnant women.

Bone levels of cephradine and cefuroxime after intravenous administration in patients undergoing total hip replacement.

A randomised, comparative study is reported of single intravenous doses of cephradine 2 g or cefuroxime 1.5 g given as prophylactic cover for total hip replacements in 40 patients. The serum and bone levels of cephalosporin achieved were higher in the cephradine treated group in proportion to the higher dose employed. Both agents provided adequate bone levels on average, cephradine 25.34 mcg/g, cefuroxime 17.39 mcg/g, although bone penetration was more variable with cefuroxime.

H+ coupled uphill transport of aminocephalosporins via the dipeptide transport system in rabbit intestinal brush-border membranes.

The transport characteristics of aminocephalosporin antibiotics, possessing an alpha-amino group and a carboxyl group, in brush-border membranes isolated from rabbit small intestine have been studied by a rapid filtration technique. The uptake of cephradine by brush-border membrane vesicles was stimulated by the countertransport effect of dipeptides, which indicates the existence of a common carrier transport system. An inward H+ gradient ([pH]i = 7.5 to 8.4, [pH]o = 6.0) stimulated cephradine uptake against a concentration gradient (overshoot phenomenon), and this stimulation was reduced when the H+ gradient was subjected to rapid dissipation by the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone, a protonophore. A valinomycin-induced K+ diffusion potential (interior-negative) stimulated H+ gradient-dependent cephradine uptake without altering the equilibrium value. The uptake of other aminocephalosporins (cefadroxil, cefaclor, cephalexin) was also stimulated in the presence of an inward H+ gradient, while the uptake of cephalosporins without the alpha-amino group (cefazolin, cefotiam) was not changed in the presence or absence of the H+ gradient. These results suggest that the transport of aminocephalosporins can be driven actively by an inward H+ gradient via the dipeptide transport system in the intestinal brush-border membranes, and that the process results in the transfer of a positive charge.

Effect of inflammation on antibiotic penetration into the anterior segment of the rat eye.

Bacterial infections were established in the right cornea of rats. Animals infected with Staphylococcus aureus were given cephradine intravenously (IV) (40 mg/kg) or topically (50 mg/ml) to both eyes. Animals infected with Pseudomonas aeruginosa were given gentamicin sulfate IV (40 mg/kg) or topically (10 mg/ml). Antibiotic concentrations in cornea and aqueous humor were measured for 4 hrs following dosing using bioassay and radioimmunoassay. In general, infection significantly increased the concentrations obtained soon after dosing. Topically applied cephradine passed through infected eyes more quickly than through normal eyes. Of the pharmacokinetic parameters derived, the permeability of the corneal epithelium to gentamicin in the rat more closely agrees with reported human values than does the rabbit, while the coefficient of elimination from aqueous in the rat is considerably greater than that for either humans or rabbits. This suggests that there are both advantages and disadvantages in using the rat for therapeutic studies of ocular disease.

Comparative pharmacokinetics of cefamandole, cefuroxime and cephradine during total hip replacement.

Serum, bone and tissue levels of the three cephalosporins, cefamandole, cefuroxime and cephradine were determined in 60 patients given a single 1 g dose intravenously before total hip surgery and two similar doses at 8 h intervals. The levels achieved were comparable for all three antibiotics, hence the choice of prophylactic antibiotic for total hip replacement should be guided by the pathogens causing infection and cost of the antibiotic.