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The intensive use of various antibiotics for clinical and agricultural purposes has resulted in their widespread use in wastewater treatment plants. However, little research has been conducted on the effects of antibiotics on nitrite accumulation, antibiotic degradation pathways, or the microbial community structure in nitrification systems. In this study, a laboratory-scale sequencing batch reactor was used to treat wastewater containing cefalexin (CFX) at different doses (5, 10, 15, and 20 mg/L). The results showed that the nitrification performance was gradually inhibited with increasing CFX concentration. Ammonia-oxidizing bacteria (AOB) are more tolerant to CFX than nitrite-oxidizing bacteria (NOB). Under 15 mg/L of CFX, NOB were completely suppressed, whereas AOB were partially inhibited, as evidenced by an ammonium removal efficiency of 60 % and a 90 % of nitrite accumulation ratio. The partial nitritation was achieved. CFX can be degraded into 2-hydroxy-3phenylpyrazine and cyclohexane through bacterial co-metabolism, and CFX degradation gradually diminishes with decreasing nitrification performance. The abundance of Nitrospira gradually decreased with increasing CFX concentration. Ferruginibacter, Hydrogenophaga, Thauera, and Pseudoxanthomonas were detected at relative abundances of 13.2 %, 0.4 %, 0.9 %, and 1.3 %, respectively, indicating their potential roles in antibiotic degradation. These findings provide insight into the interactions between antibiotics and microbial communities, which are beneficial for a better understanding of antibiotic degradation in nitrification systems., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Irinotecan causes severe gastrointestinal damage, which may affect the expression of intestinal transporters. However, neither the expression of peptide transporter 1 (Pept1) nor the pharmacokinetics of Pept1 substrate drugs has been investigated under irinotecan-induced gastrointestinal damage. Therefore, the present study quantitatively investigated the effects of irinotecan-induced gastrointestinal damage on the intestinal expression of Pept1 and absorption of cephalexin (CEX), a typical Pept1 substrate, in rats. Irinotecan was administered intravenously to rats for 4 days to induce gastrointestinal damage. The expression of Pept1 mRNA and the Pept1 protein in the upper, middle, and lower segments of the small intestine of irinotecan-treated rats was assessed by quantitative real-time polymerase chain reaction (PCR) and western blotting, respectively. The pharmacokinetic profile of CEX was examined after its oral or intravenous administration (10 mg/kg). In irinotecan-treated rats, ∼2-fold increases in Pept1 protein levels were observed in all three segments, whereas mRNA levels remained unchanged. The oral bioavailability of CEX significantly decreased to 76% of that in control rats. The decrease in passive diffusion caused by intestinal damage may have overcome the increase in Pept1-mediated uptake. In conclusion, irinotecan may decrease the intestinal absorption of Pept1 substrate drugs; however, it increased the expression of intestinal Pept1., (© 2023 The Authors. Biopharmaceutics & Drug Disposition published by John Wiley & Sons Ltd.)

Nitrifying system is an effective strategy to remove numerous antibiotics, however, the contribution of ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea (AOA) and heterotrophs for antibiotic removal are still unclear. In this study, the mechanism of β-lactam antibiotic (cefalexin, CFX) removal was studied in a nitrifying sludge system. Results showed that CFX was synergistically removed by AOB (Nitrosomonas, played a major role) and AOA (Candidatus_Nitrososphaera) through ammonia monooxygenase-mediated co-metabolism, and by heterotrophs (Pseudofulvimonas, Hydrogenophaga, RB41, Thauera, UTCFX1, Plasticicumulans, Phaeodactylibacter) through antibiotic resistance genes (ARGs)-encoded β-lactamases-mediated hydrolysis. Regardless of increased archaeal and heterotrophic CFX removal with the upregulation of amoA in AOA and ARGs, the system exhibited poorer CFX removal performance at 10 mg/L, mainly due to the inhibition of AOB. This study provides new reference for the important roles of heterotrophs and ARGs, opening the possibilities for the application of ARGs in antibiotic biodegradation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The structure of the skin only allows those hydrophobic elements to penetrate through the depth of the skin with low molecular weight (less than 500 Da) and low daily dose (less than 100 mg/day). Skin penetration of many drugs such as antibiotics at a high daily dose remains an unresolved challenge. In this study a transdermal patch using cephalexin as an antibiotic drug model was developed. Cephalexin was loaded into α-tocopherol succinate-based solid lipid nanoparticles (SLNs). Cephalexin-loaded SLNs with a drug/lipid ratio of 20%, diameter of 180 ± 7 nm, and drug loading 7.9% led to the greatest inhibition zone of Staphylococcus aureus and showed the highest skin permeation capabilities. Cephalexin-loaded SLNs were distributed into poly-iso-butylene adhesive solution and final patches prepared using solvent casting. The physico-chemical characteristics, in vitro drug release, antimicrobial efficacy, and skin cell proliferation properties of patches were evaluated. Results indicated that the optimal transdermal patch formulation containing 90% adhesive solution, 7% cephalexin, and 3% cephalexin-loaded SLNs (with antibiotic content approximately 28% less) inhibited growth of S.aureus better than the formulation containing 90% adhesive solution and 10% cephalexin. In vitro evaluation of the growth of human fibroblast skin cells in media with the optimal patch exhibited greater proliferation (about 25.5%) than those in media without the patch., (Copyright © 2022 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

The kinetics of cephalexin synthesis and hydrolysis of the activated acyl-donor precursor phenylglycine methyl ester (PGME) were characterized under a broad range of substrate concentrations. A previously developed model by Youshko-Svedas involving the formation of the acyl-enzyme complex followed by binding of the nucleophilic β-lactam donor does not fully estimate the maximum reaction yields for cephalexin synthesis at different concentrations using initial-rate data. 7-aminodesacetoxycephalosporanic acid (7-ADCA) was discovered to be a potent inhibitor of cephalexin hydrolysis, which may account for the deviation from model predictions. Three kinetic models were compared for cephalexin synthesis, with the model incorporating competitive inhibition due to 7-ADCA yielding the best fit. Additionally, the βF24A variant and Assemblase® did not exhibit significantly different kinetics for the synthesis of cephalexin compared to the wild-type, for the concentration range evaluated and for both initial-rate experiments and time-course synthesis experiments. Lastly, a continuous stirred-tank reactor for cephalexin synthesis was simulated using the model incorporating competitive inhibition by 7-ADCA, with clear tradeoffs observed between productivity, fractional yield, and PGME conversion., (© 2022 Wiley Periodicals LLC.)

Purpose: Serotonin (5-HT) is important for gastrointestinal functions, but its role in drug absorption remains to be clarified. Therefore, the pharmacokinetics and oral absorption of cephalexin (CEX) were examined under 5-HT-excessive condition to understand the role of 5-HT., Methods: 5-HT-excessive rats were prepared by multiple intraperitoneal dosing of 5-HT and clorgyline, an inhibitor for 5-HT metabolism, and utilized to examine the pharmacokinetics, absorption behavior and the intestinal permeability for CEX., Results: Higher levels of 5-HT in brain, plasma and small intestines were recognized in 5-HT-excessive rats, where the oral bioavailability of CEX was significantly enhanced. The intestinal mucosal transport via passive diffusion of CEX was significantly increased, while its transport via PEPT1 was markedly decreased specifically in the jejunal segment, which was supported by the decrease in PEPT1 expression on brush border membrane (BBM) of intestinal epithelial cells. Since no change in antipyrine permeability and significant increase in FITC dextran-4 permeability were observed in 5-HT-excessive rats, the enhanced permeability for CEX would be attributed to the opening of tight junction, which was supported by the significant decrease in transmucosal electrical resistance. In 5-HT-excessive rats, furthermore, total body clearance of CEX tended to be larger and the decrease in PEPT2 expression on BBM in kidneys was suggested to be one of the reasons for it., Conclusions: 5-HT-excessive condition enhanced the oral bioavailability of CEX in rats, which would be attributed to the enhanced permeability across the intestinal mucosa via passive diffusion through the paracellular route even though the transport via PEPT1 was decreased., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Peptide transporter 1 (PepT1), highly expressed on the apical membrane of enterocytes, is involved in energy balance and mediates intestinal absorption of peptidomimetic drugs. In this study, we investigated whether and how diabetes affected the function and expression of intestinal PepT1. Diabetes was induced in rats by combination of high-fat diet and low dose streptozocin injection. Pharmacokinetics study demonstrated that diabetes significantly decreased plasma exposures of cephalexin and acyclovir following oral administration of cephalexin and valacyclovir, respectively. Single-pass intestinal perfusion analysis showed that diabetes remarkably decreased cephalexin absorption, which was associated with decreased expression of intestinal PepT1 protein. We assessed the levels of bile acids in intestine of diabetic rats, and found that diabetic rats exhibited significantly higher levels of chenodeoxycholic acid (CDCA), cholic acid (CA) and glycocholic acid (GCA), and lower levels of lithocholic acid (LCA) and hyodeoxycholic acid (HDCA) than control rats; intestinal deoxycholic acid (DCA) levels were unaltered. In Caco-2 cells, the 6 bile acids remarkably decreased expression of PepT1 protein with CDCA causing the strongest inhibition, whereas TNF-α, LPS and insulin little affected expression of PepT1 protein; short-chain fatty acids induced rather than decreased expression of PepT1 protein. Farnesoid X receptor (FXR) inhibitor glycine-β-muricholic acid or FXR knockdown reversed the downregulation of PepT1 expression by CDCA and GW4064 (another FXR agonist). In diabetic rats, the expression of intestinal FXR protein was markedly increased. Oral administration of CDCA (90, 180 mg·kg -1 ·d -1 , for 3 weeks) dose-dependently decreased the expression and function of intestinal PepT1 in rats. In conclusion, diabetes impairs the expression and function of intestinal PepT1 partly via CDCA-mediated FXR activation.

The present study proposes the synthesis and characterization of graphene oxide (GO) and its application in the adsorption of the antibiotic cephalexin (CFX) in aqueous solution. The characterization of graphene oxide was obtained by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and zeta potential. The influence of pH on the batch adsorption process was investigated by analysing adsorption equilibrium isotherms and adsorption kinetics. The images obtained by SEM and TEM presented the typical morphology attributed to GO sheets. The kinetic adsorption tests showed that equilibrium was reached in 420 min, and an adsorption capacity of 164 mg g -1 was obtained. The models that best fit the experimental data were pseudo-second as well as the Langmuir isotherm. Therefore, GO was effective for removing the CFX antibiotic from aqueous solution by using a batch adsorption process.

The nitrifying systems have been reported to be able to biodegrade micropollutants, yet it is still unclear about the cometabolism of ammonia-oxidizing bacteria (AOB) towards micropollutants, in particular their enzyme and transcriptional responses under exposure of micropollutants. This study investigated cometabolic biodegradation of a selected antibiotic, cephalexin (CFX), by an enriched nitrifying culture through a series of batch experiments, together with the assessments of enzymatic activity, key gene expression, and biotoxicity of the degradation products. More than 99% CFX with an initial concentration of 50 μg/L could be removed with the presence of ammonium, while <44% of CFX removal was observed in the absence of ammonium, suggesting the cometabolic degradation of CFX by ammonia-oxidizing bacteria (AOB). After the addition of 50 μg/L CFX, the ammonia oxidizing rate (AOR) decreased from 36.6 to 11.0 mg N/(L·h·g VSS), followed by a slight recovery when CFX concentration decreased to below 8 μg/L. Ammonia monooxygenase (AMO) activity showed a similar trend with that of AOR. The quantitative reverse transcription PCR assay indicated that the expression level of amoA gene was significantly upregulated (up to 3-fold, p < 0.05) due to the addition of CFX, while decreased to the normal level once CFX was degraded, suggesting a mechanism of AOB to neutralize the toxicity of CFX by metabolizing ammonia more effectively. Meanwhile, the biotoxicity test showed the degradation products of CFX did not exhibit any antibacterial impacts in terms of cell viability, compared to the parent compounds. Our finding shed a light on AMO-mediated cometabolic biodegradation of antibiotics in nitrifying cultures., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Biodegradation of pharmaceuticals active compounds (PACs) in secondary effluents by using B. subtilis 2012WTNC as a function of β-lactamase was optimized using response surface methodology (RSM) designed by central composite design (CCD). Four factors including initial concentration of bacteria (1-6 log 10 CFU mL -1 ), incubation period (1-14 days), incubation temperature (20-40 °C) and initial concentration of PACs (1-5 mg L -1 ) were investigated. The optimal operating factors for biodegradation process determined using response surface methodology (RSM) was recorded with 5.57 log 10 CFU mL -1 of B. subtilis, for 10.38 days, at 36.62 °C and with 4.14 mg L -1 of (cephalexin/amoxicillin) with R 2 coefficient of 0.99. The biodegradation was 83.81 and 93.94% respectively. The relationship among the independent variables was significant (p < 0.05) with 95% of confidence level at the best operating parameters. The bioassay for PACs after the degradation process revealed that no residual antibiotic activity was detected of amoxicillin and cephalexin against E. coli and S. aureus after degradation using B. subtilis which reflects the higher potential of bacteria to biodegrade PACs in secondary effluents. B. subtilis has the potential for biodegradation of PACs in the secondary effluents., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Objectives: This study reports the draft genome sequence of Xenophilus sp. E41, a strain resistant to multiple antimicrobials isolated from an activated sludge reactor treating wastewater with a high cephalexin concentration., Methods: Genomic DNA of Xenophilus sp. E41 was extracted and sequenced using an Illumina NovaSeq 6000 system. The generated sequence reads were assembled using MEGAHIT in combination with SOAPdenovo. Mauve and CompareM were used to align the Xenophilus sp. E41 genome to other draft genomes of the genus Xenophilus in order to determine their evolutionary relationships. The draft genome was annotated using the Rapid Annotation using Subsystem Technology (RAST) server and the nr database, whilst antimicrobial resistance genes (ARGs) were identified using the SARG 2.0 database, RAST server and nr database., Results: Xenophilus sp. E41, with a genome length of 5919552bp, harbours seven types of ARGs involving resistance to β-lactams, tetracycline, aminoglycosides, sulfonamides, chloramphenicol, teicoplanin and bleomycin. No virulence factors or plasmids were identified., Conclusion: The genome sequence reported here will provide useful information for a better understanding of antimicrobial resistance profiles in this strain and the genus Xenophilus., (Copyright © 2018 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.)

Emergence of antibiotic resistance due to New Delhi metallo-β-lactamase (NDM-1) bacterial enzymes is of great concern due to their ability to hydrolyze a wide range of antibiotics. There are ongoing efforts to obtain the atomistic details of the hydrolysis mechanism in order to develop inhibitors for NDM-1. In particular, it remains elusive how drug molecules of different families of antibiotics are hydrolyzed by NDM-1 in an efficient manner. Here we report the detailed molecular mechanism of NDM-1 catalyzed hydrolysis of cephalexin, a cephalosporin family drug, and meropenem, a carbapenem family drug. This study employs molecular dynamics (MD) simulations using hybrid quantum mechanical/molecular mechanical (QM/MM) methods at the density functional theory (DFT) level, based on which reaction pathways and the associated free energies are obtained. We find that the mechanism and the free energy barrier for the ring-opening step are the same for both the drug molecules, while the subsequent protonation step differs. In particular, we observe that the mechanism of the protonation step depends on the R2 group of the drug molecule. Our simulations show that allylic carbon protonation occurs in the case of the cephalexin drug molecule where Lys211 is the proton donor, and the proton transfer occurs via a water chain formed (only) at the ring-opened intermediate structure. Based on the free energy profiles, the overall kinetics of drug hydrolysis is discussed. Finally, we show that the proposed mechanisms and free energy profiles could explain various experimental observations.

TEM-1 beta-lactamases can accurately catalyze the hydrolysis of the beta-lactam rings in beta-lactam antibiotics, which make beta-lactam antibiotics lose its activity, and the prerequisite for the hydrolysis procedure in the binding interaction of TEM-1 beta-lactamases with beta-lactam antibiotics is the beta-lactam rings in beta-lactam antibiotics. Therefore, the binding of TEM-1 beta-lactamase to three beta-lactam antibiotics including penicillin G, cefalexin as well as cefoxitin was explored here by frontal affinity chromatography in combination with fluorescence spectra, adsorption and thermodynamic data in the temperature range of 278-288K under simulated physiological conditions. The results showed that all the binding of TEM-1 beta-lactamase to the three antibiotics were spontaneously exothermic processes with the binding constants of 8.718×10 3 , 6.624×10 3 and 2.244×10 3 (mol/L), respectively at 288K. All the TEM-1 beta-lactamases were immobilized on the surface of the stationary phase in the mode of monolayer and there existed only one type of binding sites on them. Each TEM-1 beta-lactamase bound with only one beta-lactam antibiotic and hydrogen bond interaction and Van der Waals force were the main forces between them. This work provided an insight into the binding interactions between TEM-1 beta-lactamases and beta-lactam antibiotics, which may be beneficial for the designing and developing of new substrates resistant to TEM-1 beta-lactamases., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Serum albumins are the most abundant carrier proteins in blood plasma and participate in the binding and transportation of various exogenous and endogenous compounds in the body. This work was designed to investigate the recognition and binding of three typical β-lactam antibiotics including penicillin G (Pen G), penicillin V (Pen V) and cefalexin (Cef) with bovine serum albumin (BSA) by frontal affinity chromatography in combination with UV-vis absorption spectra, fluorescence emission spectra, binding site marker competitive experiment and molecular docking under simulated physiological conditions. The results showed that a BSA only bound with one antibiotic molecule in the binding process, and the binding constants for Pen G-BSA, Pen V-BSA and Cef-BSA complexes were 4.22×10(1), 4.86×10(2) and 3.32×10(3) (L/mol), respectively. All the three β-lactam antibiotics were mainly inserted into the subdomain IIA (binding site 1) of BSA by hydrogen bonds and Van der Waals forces. The binding capacity between the antibiotics and BSA was closely related to the functional groups and flexibility of side chains in antibiotics. This study provided an important insight into the molecular recognition and binding interaction of BSA with β-lactam antibiotics, which may be a useful guideline for the innovative clinical medications and new antibiotic designs with effective pharmacological properties., (Copyright © 2016 Elsevier B.V. All rights reserved.)

With few exceptions, all living organisms encode the same 20 canonical amino acids; however, it remains an open question whether organisms with additional amino acids beyond the common 20 might have an evolutionary advantage. Here, we begin to test that notion by making a large library of mutant enzymes in which 10 structurally distinct noncanonical amino acids were substituted at single sites randomly throughout TEM-1 β-lactamase. A screen for growth on the β-lactam antibiotic cephalexin afforded a unique p-acrylamido-phenylalanine (AcrF) mutation at Val-216 that leads to an increase in catalytic efficiency by increasing kcat, but not significantly affecting KM. To understand the structural basis for this enhanced activity, we solved the X-ray crystal structures of the ligand-free mutant enzyme and of the deacylation-defective wild-type and mutant cephalexin acyl-enzyme intermediates. These structures show that the Val-216-AcrF mutation leads to conformational changes in key active site residues-both in the free enzyme and upon formation of the acyl-enzyme intermediate-that lower the free energy of activation of the substrate transacylation reaction. The functional changes induced by this mutation could not be reproduced by substitution of any of the 20 canonical amino acids for Val-216, indicating that an expanded genetic code may offer novel solutions to proteins as they evolve new activities.

Pharmaceuticals have recently been regarded as contaminants of emerging concern. To date, there is limited knowledge about antibiotic-degrading microorganisms in conventional activated sludge treatment systems and their characteristics toward antibiotic degradation especially in the presence of a pharmaceutical mixture. As such, antibiotic-degrading microorganisms were investigated and isolated from the activated sludge, and their degradation capabilities were evaluated. Two strains of cefalexin-degrading bacteria CE21 and CE22 were isolated and identified as Pseudomonas sp. in the collected activated sludge. Strain CE22 was able to degrade over 90% of cefalexin, while CE21 was able to remove 46.7% of cefalexin after incubation for 24h. The removal efficiency of cefalexin by CE22, different from that of CE21, was not significantly affected by an increase in cefalexin concentration, even up to 10ppm, however the presence of 1ppm of other pharmaceuticals had a significant effect on the degradation of cefalexin by CE22, but no significant effect on CE21. The degradation product of cefalexin by the two strains was identified to be 2-hydroxy-3-phenyl pyrazine. Our results also indicated that CE21 and CE22 were able to degrade caffeine, salicylic acid and chloramphenicol. Moreover, CE21 was found to be capable of eliminating sulfamethoxazole and naproxen., (Copyright © 2014 Elsevier B.V. All rights reserved.)

We designed and prepared nanoreactors based on a poly(2-methyloxazoline)-block-poly(dimethylsiloxane)-block-poly(2-methyloxazoline (PMOXA-b-PDMS-b-PMOXA) amphiphilic triblock copolymer encapsulating the enzyme penicillin acylase for local and controlled production of antibiotics.

Staphylococcus aureus is a widespread Gram-positive opportunistic pathogen, and a methicillin-resistant form (MRSA) is particularly difficult to treat clinically. We have solved two crystal structures of penicillin-binding protein (PBP) 3 (PBP3) from MRSA, the apo form and a complex with the β-lactam antibiotic cefotaxime, and used electrospray mass spectrometry to measure its sensitivity to a variety of penicillin derivatives. PBP3 is a class B PBP, possessing an N-terminal non-penicillin-binding domain, sometimes called a dimerization domain, and a C-terminal transpeptidase domain. The model shows a different orientation of its two domains compared to earlier models of other class B PBPs and a novel, larger N-domain. Consistent with the nomenclature of "dimerization domain", the N-terminal region forms an apparently tight interaction with a neighboring molecule related by a 2-fold symmetry axis in the crystal structure. This dimer form is predicted to be highly stable in solution by the PISA server, but mass spectrometry and analytical ultracentrifugation provide unequivocal evidence that the protein is a monomer in solution., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The comparative degradation behavior of cefalexin (CLX) using advanced oxidation processes (AOPs) with the aim of improving CLX biodegradability was studied. Among the AOPs used, RuO(2)/Ti anodic oxidation (AO), AO in the presence of electro-generated H(2)O(2) (AO-H(2)O(2)), and the electro-Fenton (EF) process, the EF process was the most effective. In the EF process an activated carbon fiber (ACF) was used as a cathode. Different input variables, including catalyst concentration, pH, and current density were evaluated to find the optimum conditions for the EF. The most suitable operational conditions were as follows: a current density of 6.66 mA/cm(2), a pH of 3, and a concentration of 1 mM of Fe(2+) as the catalyst. Different CLX concentrations were analyzed as well of different reaction times to assess the degree of mineralization. The change in biodegradability was evaluated by the BOD(5)/COD and the BOD(14)/COD ratios. The EF did not effectively remove the COD, but removed enough to achieve suitable biodegradability for a further biological process., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The purpose of this study was to construct stably transfected HeLa cells with human peptide transporters (hPEPT1/hPEPT2) and to identify the function of the transfected cells using the substrate JBP485 (a dipeptide) and a typical substrate for PEPTs, glycylsarcosine (Gly-Sar). An efficient and rapid method was established for the preparation and transformation of competent cells of Escherichia coli. After extraction and purification, hPEPT1/hPEPT2-pcDNA3 was transfected into HeLa cells by the liposome transfection method, respectively. HeLa-hPEPT1/hPEPT2 cells were selected by measuring the protein expression and the uptake activities of JBP485 and Gly-Sar. A simple and rapid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of JBP485 and Gly-Sar in biological samples. The Michaelis-Menten constant (K(m)) values of Gly-Sar uptake by the hPEPT1 and hPEPT2-expressing transfectants were 1.03 mM and 0.0965 mM, respectively, and the K(m) values of JBP485 uptake were 1.33 mM for PEPT1 and 0.144 mM for PEPT2. The uptake of Gly-Sar was significantly inhibited by JBP485 with a K(i) value of 8.11 mM (for PEPT1) and 1.05 mM (for PEPT2). Maximal uptake of Gly-Sar were detected at pH 5.8 (for PEPT1) and pH 6.5 (for PEPT2), suggesting that both HeLa-hPEPT1 and HeLa-hPEPT2 were H(+) dependent transporters. Stably transfected HeLa-hPEPT1/HeLa-hPEPT2 cells were constructed successfully, and the functions of hPEPT1/hPEPT2 were identified using their substrates, JBP485 and Gly-Sar. The transfected cells with transporters were used to investigate drug-drug interactions (DDIs) between JBP485 and other substrates (cephalexin or lisinopril) of PEPT1 and PEPT2., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Cimetidine, an H₂ receptor antagonist, has been used to investigate the tubular secretion of organic cations in human kidney. We report a systematic comprehensive analysis of the inhibition potency of cimetidine for the influx and efflux transporters of organic cations [human organic cation transporter 1 (hOCT1) and hOCT2 and human multidrug and toxin extrusion 1 (hMATE1) and hMATE2-K, respectively]. Inhibition constants (K(i)) of cimetidine were determined by using five substrates [tetraethylammonium (TEA), metformin, 1-methyl-4-phenylpyridinium, 4-(4-(dimethylamino)styryl)-N-methylpyridinium, and m-iodobenzylguanidine]. They were 95 to 146 μM for hOCT2, providing at most 10% inhibition based on its clinically reported plasma unbound concentrations (3.6-7.8 μM). In contrast, cimetidine is a potent inhibitor of MATE1 and MATE2-K with K(i) values (μM) of 1.1 to 3.8 and 2.1 to 6.9, respectively. The same tendency was observed for mouse Oct1 (mOct1), mOct2, and mouse Mate1. Cimetidine showed a negligible effect on the uptake of metformin by mouse kidney slices at 20 μM. Cimetidine was administered to mice by a constant infusion to achieve a plasma unbound concentration of 21.6 μM to examine its effect on the renal disposition of Mate1 probes (metformin, TEA, and cephalexin) in vivo. The kidney- and liver-to-plasma ratios of metformin both were increased 2.4-fold by cimetidine, whereas the renal clearance was not changed. Cimetidine also increased the kidney-to-plasma ratio of TEA and cephalexin 8.0- and 3.3-fold compared with a control and decreased the renal clearance from 49 to 23 and 11 to 6.6 ml/min/kg, respectively. These results suggest that the inhibition of MATEs, but not OCT2, is a likely mechanism underlying the drug-drug interactions with cimetidine in renal elimination.

Metallo-β-lactamase from Bacillus anthracis (Bla2) catalyzes the hydrolysis of β-lactam antibiotics which are commonly prescribed to combat bacterial infections. Bla2 contributes to the antibiotic resistance of this bacterium. An understanding of it is necessary to design potential inhibitors that can be introduced with current antibiotics for effective eradication of anthrax infections. We have purified Bla2 using Ni(2+)-affinity chromatography with over 140-fold increase in activity with a yield of 3.5%. The final specific activity was 19,000 units/mg. Purified Bla2 displays different K ( m ), V ( max ), and (k ( cat ) /K (M)) with penicillin G and cephalexin as substrates and is also sensitive to pH, with maximum activity between pH 7.0-9.0. The IC(50) (50% inhibition concentration) value of EDTA against Bla2 is 630 nM, which can be understood by observing its three-dimensional interaction with the enzyme.

Objective: To determine the effect of protein binding on the pharmacokinetics and distribution from plasma to interstitial fluid (ISF) of cephalexin and cefpodoxime proxetil in dogs., Animals: 6 healthy dogs., Procedures: In a crossover study design, 25 mg of cephalexin/kg or 9.6 mg of cefpodoxime/kg was administered orally. Blood samples were collected before (time 0) and 0.33, 0.66, 1, 2, 3, 4, 6, 8, 10, 12, 16, and 24 hours after treatment. An ultrafiltration device was used in vivo to collect ISF at 0, 2, 4, 6, 8, 10, 12, 16, and 24 hours. Plasma and ISF concentrations were analyzed with high-pressure liquid chromatography. Plasma protein binding was measured by use of a microcentrifugation technique., Results: Mean plasma protein binding for cefpodoxime and cephalexin was 82.6% and 20.8%, respectively. Mean ± SD values for cephalexin in plasma were determined for peak plasma concentration (Cmax, 31.5±11.5 μg/mL), area under the time-concentration curve (AUC, 155.6±29.5 μg•h/mL), and terminal half-life (T½, 4.7±1.2 hours); corresponding values in ISF were 16.3±5.8 μg/mL, 878±21.0 μg•h/mL, and 3.2±0.6 hours, respectively. Mean±SD values for cefpodoxime in plasma were 33.0±6.9 μg/mL (Cmax), 282.8±44.0 μg•h/mL (AUC), and 5.7±0.9 hours (T1/2); corresponding values in ISF were 4.3±2.0 μg/mL, 575±174 μg•h/mL, and 10.4±3.3 hours, respectively., Conclusions and Clinical Relevance: Tissue concentration of protein-unbound cefpodoxime was similar to that of the protein-unbound plasma concentration. Cefpodoxime remained in tissues longer than did cephalexin.

There is a marked trend in pharmaceutical industry towards the replacement of classical organic methods by "green" alternatives that minimize or eliminate the generation of waste and avoid, where possible, the use of toxic and/or hazardous reagents and solvents. In this work the kinetically controlled synthesis of cephalexin by soluble and penicillin G acylase immobilized in sol-gel micro-particles with magnetic properties was performed in aqueous media with PGME and 7-ADCA as substrates, at different concentrations of substrate, temperature, pH, enzyme to substrate ratio and acyl donor to nucleophile ratio. Excess acyl donor had a strong effect on cephalexin productivity. A PGME/7-ADCA ratio of 3 was considered optimum. A maximum specific productivity of 5.9 mmol h(-1), gbiocatalyst(-1) at 160 mM 7-ADCA, 480 mM PGME and low enzyme to substrate ratio at 32.5 U mmol(-1) 7-ADCA was obtained with immobilized PGA in full aqueous medium, suggesting that diffusional limitations were minimized when compared with other commercial biocatalysts. A half-life of 133 h for the immobilized biocatalyst was estimated during cephalexin synthesis in the presence of 100 mM 7-ADCA and 300 mM PGME, in 50 mM Tris/HCl at pH 7.2 and 14°C. These results compare quite favorably with those previously reported for the kinetically controlled synthesis of cephalexin., (© 2010 Wiley Periodicals, Inc.)

TEM-1 beta-lactamase is the most common plasmid-encoded beta-lactamase in Gram-negative bacteria and is a model class A enzyme. The active site of class A beta-lactamases share several conserved residues including Ser(70), Glu(166), and Asn(170) that coordinate a hydrolytic water involved in deacylation. Unlike Ser(70) and Glu(166), the functional significance of residue Asn(170) is not well understood even though it forms hydrogen bonds with both Glu(166) and the hydrolytic water. The goal of this study was to examine the importance of Asn(170) for catalysis and substrate specificity of beta-lactam antibiotic hydrolysis. The codon for position 170 was randomized to create a library containing all 20 possible amino acids. The random library was introduced into Escherichia coli, and functional clones were selected on agar plates containing ampicillin. DNA sequencing of the functional clones revealed that only asparagine (wild type) and glycine at this position are consistent with wild-type function. The determination of kinetic parameters for several substrates revealed that the N170G mutant is very efficient at hydrolyzing substrates that contain a primary amine in the antibiotic R-group that would be close to the Asn(170) side chain in the acyl-intermediate. In addition, the x-ray structure of the N170G enzyme indicated that the position of an active site water important for deacylation is altered compared with the wild-type enzyme. Taken together, the results suggest the N170G TEM-1 enzyme hydrolyzes ampicillin efficiently because of substrate-assisted catalysis where the primary amine of the ampicillin R-group positions the hydrolytic water and allows for efficient deacylation.

The genus Chryseobacterium and other genera belonging to the family Flavobacteriaceae include organisms that can behave as human pathogens and are known to cause different kinds of infections. Several species of Flavobacteriaceae, including Chryseobacterium indologenes, are naturally resistant to beta-lactam antibiotics (including carbapenems), due to the production of a resident metallo-beta-lactamase. Although C. indologenes presently constitutes a limited clinical threat, the incidence of infections caused by this organism is increasing in some settings, where isolates that exhibit multidrug resistance phenotypes (including resistance to aminoglycosides and quinolones) have been detected. Here, we report the identification and characterization of a new IND-type variant from a C. indologenes isolate from Burkina Faso that is resistant to beta-lactams and aminoglycosides. The levels of sequence identity of the new variant to other IND-type metallo-beta-lactamases range between 72 and 90% (for IND-4 and IND-5, respectively). The purified enzyme exhibited N-terminal heterogeneity and a posttranslational modification consisting of the presence of a pyroglutamate residue at the N terminus. IND-6 shows a broad substrate profile, with overall higher turnover rates than IND-5 and higher activities than IND-2 and IND-5 against ceftazidime and cefepime.

The oligopeptide transporter PEPT1 (SLC15A1) is responsible for absorption of peptidic nutrients in the small intestine. Although the L-diastereomer of the beta-lactam antibiotic cephalexin (L-cephalexin) is likely to be transported by PEPT1, there has been no direct demonstration of PEPT1-mediated L-cephalexin transport. Indeed, after the incubation with L-cephalexin, the intact form of L-cephalexin has not been identified inside vesicles/proteoliposomes prepared from brush border membrane of intestinal epithelial cells or cultured cell lines exogenously transfected with PEPT1 gene. Thus, it appears that L-cephalexin is rapidly metabolized by PEPT1 or PEPT1-associated proteins. Here, we attempted to verify whether L-cephalexin is transported by PEPT1 and whether it is hydrolyzed by PEPT1 itself, by using budded baculovirus expressing PEPT1 protein. Marked uptake of L-cephalexin in PEPT1-expressing budded baculovirus, compared with wild-type virus, indicated that L-cephalexin is a substrate for PEPT1. The uptake was found to be pH sensitive, and was strongly inhibited by the D-diastereomer of cephalexin and glycylsarcosine, but not by glycine. Thus, L-cephalexin is transported by PEPT1 itself. Upon the transport of both L- and D-cephalexin by PEPT1, dose-dependent membrane depolarization was observed; the EC(50) values of 0.18 and 2.9 mM, respectively, indicate that the affinity of L-cephalexin for PEPT1-mediated transport is much higher than that of the D-diastereomer. On the other hand, the L-cephalexin metabolite 7-aminodesacetoxycephalosporanic acid was not detected in PEPT1-expressing or wild-type virus at either pH 6.0 or 7.4. We conclude that L-cephalexin is transported by PEPT1 with high affinity, but is not metabolized by PEPT1 itself.

The purpose of this project was to assess the validity of a novel Electroporation and transcutaneous sampling (ETS) technique for sampling cephalexin from the dermal extracellular fluid (ECF). This work also investigated the plausibility of using cephalexin levels in the dermal ECF as a surrogate for the drug levels in the synovial fluid. In vitro and in vivo studies were carried out using hairless rats to assess the workability of ETS. Cephalexin (20 mg/kg) was administered (i.v.) through tail vein and the time course of drug concentration in the plasma was determined. In the same rats, cephalexin concentration in the dermal ECF was determined by ETS and microdialysis techniques. In a separate set of rats, only intraarticular microdialysis was carried out to determine the time course of cephalexin concentration in synovial fluid. The drug concentration in the dermal ECF determined by ETS and microdialysis did not differ significantly from each other and so as were the pharmacokinetic parameters. The results provide validity to the ETS technique. Further, there was a good correlation ( approximately 0.9) between synovial fluid and dermal ECF levels of cephalexin indicating that dermal ECF levels could be used as a potential surrogate for cephalexin concentration in the synovial fluid., (Copyright 2009 Wiley-Liss, Inc.)

Cell-free extracts of Streptomyces sp. DRS I converted ampicillin to cephalexin, presumably due to the activity of the enzyme, expandase. The extract was fractionated and characterized by colorimetric and chromatographic measurements coupled with disc-agar diffusion bioassay against an ampicillin-resistant, cephalexin-sensitive E. coli strain. Though expandase could not be identified, the presence of a hitherto unreported expandase in Streptomyces sp. DRS I is suggested.

The present work focuses on the development and basic characterization of a new magnetic biocatalyst, namely penicillin G acylase (PGA), immobilized in sol-gel matrices with magnetic properties, ultimately aimed for application in cephalexin (CEX) synthesis. A mechanically stable carrier, based on porous xerogels silica matrixes starting from tetramethoxysilane (TMOS), was prepared leading to micro-carriers with medium sized particles of 30 microm, as determined by scanning electron microscopy. An immobilization yield of 95-100% and a recovered activity of 50-65% at 37 degrees C, as determined by penicillin G (PG) hydrolysis (pH STAT method), were observed. These results clearly exceed those reported in a previous work on PGA immobilization in sol-gel, where only 10% of activity was recovered. The values of activity were kept constant for 6 months. Immobilized PGA (682 U/g(dry weight)) retained high specific activity throughout ten consecutive runs for PG hydrolysis, suggesting adequate biocatalyst stability. The CEX synthesis was performed at 14 degrees C, using the free and immobilized PGA in aqueous medium. Phenylglycine methyl ester was used as acyl donor at 90 mM and 7-aminodeacetoxycephalosporanic acid was the limiting substrate at 30 mM. The CEX stoichiometric yield after 1-h reaction was close to 68% (23 mM CEX/h) and 65% (19 mM CEX/h), respectively.

Different types of calcium phosphate cements (CPCs) have been studied as potential matrices for incorporating different types of antibiotics. All of these matrices were morphologically microporous whereas macroporosity is essential for rapid cement resorption and bone replacement. In this study, liberation of cephalexin monohydrate (CMH) from a macroporous CPC was investigated over 0.5-300 h in simulated body fluid and some mathematical models were fitted to the release profiles. Macroporosity was introduced into the cement matrix by using sodium dodecyl sulfate molecules as air-entraining agents and the effect of both surfactant and CMH on basic properties of the CPC was studied. Incorporation of CMH into the CPC composition increased the setting time, decreased the crystallinity of the formed apatite phase, and improved the injectability of the paste. The use of both CMH and sodium dodecyl sulfate did not affect the rate of conversion of the reactants into apatite phase while soaking the cements in simulated body fluid. Results showed that the liberation rate of the drug from porous CPC was higher than that of the nonporous CPC but same release patterns were experienced in both types of cements, that is, like to nonporous CPC, a time-dependent controlled release of the incorporated drug was obtained from macroporous CPC. The Weibull model was the best fitting-equation for release profiles of all cements. The liberated CMH was as active as fresh cephalexin. It is concluded that this macroporous CPC can be successfully used as drug carrier with controlled release profile for the treatment of bone infections., ((c) 2008 Wiley Periodicals, Inc.)

The H+-coupled peptide cotransporter 1 (PepT1) mediates absorption of peptides and peptidomimetic drugs. Acute luminal leptin was reported to induce translocation of PepT1 to the enterocyte membrane in vitro and in vivo in the rat, resulting in enhanced peptide and peptidomimetic drug absorption. In this study, we analyzed chronic effects of leptin and leptin deficiency on PepT1 activity and expression in the small intestine. Wistar rats and ob/ob mice were used. Activity of PepT1 was determined by monitoring [3H]glycyl-sarcosine (Gly-Sar) transport using the jejunal loop method. The levels of PepT1 mRNA and protein were quantified by real-time quantitative reverse transcription-polymerase chain reaction and Western blot analysis, respectively. Induction of chronic hyperleptinemia in rats (1 microg/g/day for 7 days; subcutaneous continuous infusion), caused a significant 25% increase (P < 0.05 versus control) in Gly-Sar transport and uptake. This effect was associated with a significant 2-fold increase in the abundance of PepT1 protein and a 6-fold increase in the levels of PepT1 mRNA. In the leptin-deficient ob/ob mice, PepT1 activity and expression were significantly reduced, and replacement of leptin (10 microg/day for 7 days; subcutaneous continuous infusion) completely restored full PepT1 expression and activity. Moreover, we showed that a 7-day challenge of the Caco-2 cells with 0.2 nM leptin induced a significant increase in PepT1 activity and protein expression, arguing for a direct action. These data demonstrate, for the first time, an impaired activity/expression of PepT1 in leptin-deficient ob/ob mice that could be restored by leptin replacement. These findings may have relevance in modulation of dietary nitrogen supply and PepT1 substrate bioavailability in obesity.

The substrate specificities of human (h) multidrug and toxin extrusion (MATE) 1 and hMATE2-K were examined to find functional differences between these two transporters by the transfection of the cDNA of hMATE1 and hMATE2-K into HEK293 cells. Western blotting revealed specific signals for hMATE1 and hMATE2-K consistent with a size of 50 and 40kDa, respectively, in the transfectants as well as human renal brush-border membranes under reducing conditions. In the presence of oppositely directed H(+)-gradient, the transport activities of various compounds such as tetraethylammonium, 1-methyl-4-phenylpyridinium, cimetidine, metformin, creatinine, guanidine, procainamide, and topotecan were stimulated in hMATE1- and hMATE2-K-expressing cells. In addition to cationic compounds, anionic estrone sulfate, acyclovir, and ganciclovir were also recognized as substrates of these transporters. Kinetic analyses demonstrated the Michaelis-Menten constants for the hMATE1-mediated transport of tetraethylammonium, 1-methyl-4-phenylpyridinium, cimetidine, metformin, guanidine, procainamide, topotecan, estrone sulfate, acycrovir, and ganciclovir to be (in mM) 0.38, 0.10, 0.17, 0.78, 2.10, 1.23, 0.07, 0.47, 2.64, and 5.12, respectively. Those for hMATE2-K were 0.76, 0.11, 0.12, 1.98, 4.20, 1.58, 0.06, 0.85, 4.32, and 4.28, respectively. Although their affinity for hMATE1 and hMATE2-K was similar, the zwitterionic cephalexin and cephradine were revealed to be specific substrates of hMATE1, but not of hMATE2-K. Levofloxacin and ciprofloxacin were not transported, but were demonstrated to be potent inhibitors of these transporters. These results suggest that hMATE1 and hMATE2-K function together as a detoxication system, by mediating the tubular secretion of intracellular ionic compounds across the brush-border membranes of the kidney.

Gastrointestinal absorption of the beta-lactam antibiotic cephalexin (CEX) is highly stereoselective: l- and d-CEX are both taken up by intestinal epithelial cells through the brush-border membrane, most likely via oligopeptide transporter PEPT1, but l-CEX is not found in serum or urine after administration p.o. because of its rapid intestinal metabolism, whereas d-CEX is well absorbed in the unchanged form. We examined the contribution of PEPT1 to the stereoselective uptake and metabolism of CEX. We observed stereoselective metabolism of CEX after exogenous transfection of PEPT1 alone into mammalian cell lines: l-CEX, but not d-CEX, was metabolized to 7-aminodesacetoxycephalosporanic acid (7-ADCA) in HeLa and human embryonic kidney 293 cells stably and transiently expressing human PEPT1, respectively, whereas such metabolism was minor in cells expressing the vector alone. The formation rate of 7-ADCA depended on the amount of PEPT1 cDNA transfected. l-CEX metabolism was rapid because only 7-ADCA was found inside and outside the cells during incubation with l-CEX. The characteristics of PEPT1-mediated metabolism of l-CEX were similar, but not identical, to those of PEPT1-mediated transport. PEPT1-mediated metabolism was also observed in permeabilized cells expressing PEPT1, in which PEPT1-mediated intracellular substrate accumulation was negligible, suggesting that the increase in l-CEX metabolism by PEPT1 transfection cannot be fully explained by an increase in uptake and subsequent exposure to intracellular hydrolases. The present findings show that stereoselectivity in CEX absorption can be fully explained in terms of PEPT1, implying that the l-CEX hydrolase is PEPT1 itself or is induced by PEPT1.

Several genes for the enzyme penicillin G acylase, as isolated from four different micro-organisms (Alcaligenes facaelis, Escherichia coli, Kluyvera cryocrescens or Providencia rettgeri) were modified at their carboxy-termini to include His-tag fusions, then were expressed from the plasmid pET-24a(+) in E. coli JM109(DE3) cells. All fusion proteins were next purified to homogeneity in a single step by agar-based Co-IDA chromatography, and were then evaluated as catalysts for the synthesis of cephalexin by a kinetically controlled strategy. We find here that the penicillin G acylase enzyme from K. cryocrescens shows a higher intrinsic synthesis/hydrolysis ratio, when compared to three other enzymes from A. facaelis or P. rettgeri, or E. coli.

A complete, integrated process for the production of an innovative formulation of penicillin G acylase from Providencia rettgeri(rPAC(P.rett))of industrial applicability is reported. In order to improve the yield of rPAC, the clone LN5.5, carrying four copies of pac gene integrated into the genome of Pichia pastoris, was constructed. The proteinase activity of the recombinant strain was reduced by knockout of the PEP4 gene encoding for proteinase A, resulting in an increased rPAC(P.rett) activity of approximately 40% (3.8 U/mL vs. 2.7 U/mL produced by LN5.5 in flask). A high cell density fermentation process was established with a 5-day methanol induction phase and a final PAC activity of up to 27 U/mL. A single step rPAC(P.rett) purification was also developed with an enzyme activity yield of approximately 95%. The novel features of the rPAC(P.rett) expressed in P.pastoris were fully exploited and emphasized through the covalent immobilization of rPAC(P.rett). The enzyme was immobilized on a series of structurally correlated methacrylic polymers, specifically designed and produced for optimizing rPAC(P.rett) performances in both hydrolytic and synthetic processes. Polymers presenting aminic functionalities were the most efficient, leading to formulations with higher activity and stability (half time stability >3 years and specific activity ranging from 237 to 477 U/g (dry) based on benzylpenicillin hydrolysis). The efficiency of the immobilized rPAC(P.rett) was finally evaluated by studying the kinetically controlled synthesis of beta-lactam antibiotics (cephalexin) and estimating the synthesis/hydrolysis ratio (S/H), which is a crucial parameter for the feasibility of the process., ((c) 2005 Wiley Periodicals, Inc.)

We investigated the effects of progesterone and norethisterone on the apical-to-basolateral and basolateral-to-apical transports of cephalexin, a typical peptide transporter PEPT1 substrate, and the PEPT1 mRNA and protein expression levels, using the human intestinal cell line, Caco-2. Caco-2 cell monolayers (passages 50 to 60) were cultured on permeable membrane, plastic culture dish and culture tube. The Caco-2 cell monolayers were pretreated with progesterone and norethisterone (3, 10, 30 microM) for 24 h. After the pretreatment, the apical-to-basolateral and basolateral-to-apical transports of cephalexin were measured, and the densities of PEPT1 mRNA and protein expression levels were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, respectively. The apical-to-basolateral transport of cephalexin was significantly decreased by the progesterone and norethisterone (30 microM each) pretreatments. By contrast, the basolateral-to-apical transport of cephalexin was not altered by the same pretreatments. The densities of PEPT1 mRNA and protein expressions were significantly decreased by progesterone and norethisterone (each at 3 and 10 microM) pretreatments compared with those of the non-treated Caco-2 cells. The results suggest that the transcription of the PEPT1 gene is downregulated by the progesterone and norethisterone pretreatments. Further studies are needed to clarify whether the inhibition of the PEPT1 gene transcription by progesterone pretreatment proceeds via sigma1-receptor or progesterone receptor.

Cephalexin synthesizing enzyme (CSE) of Gluconobacter oxydans ATCC 9324 was purified up to about 940-fold at a yield of 12%. CSE biosynthesis in G. oxydans was found inducible in the presence of D-phenylglycine but not its substrate phenylglycine methyl ester. The purified enzyme was shown homogeneous on SDS-PAGE and exhibited a specific activity of 440 U per mg protein. The apparent molecular mass of the native enzyme was estimated to be 70 kDa over a Superdex 200 gel filtration column and 68 kDa on SDS-PAGE, indicating that the native enzyme is a monomer. Its isoelectric focusing point is 7.1, indicating a neutral character. The enzyme had maximal activity around pH 6.0 to 6.5, and this activity was thermally stable up to 40 degrees C. Synthesis of cephalexin from D-phenylglycine methyl ester and 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) by the purified CSE was demonstrated. Its L-enantiomer was not accepted by CSE. Apart from cephalexin, ampicillin was also synthesized by the purified CSE from its acyl precursors and 6-aminopenicillanic acid (6-APA). Substrate specificity studies indicated that the enzyme required a free alpha amino group and an activated carboxyl group as a methyl ester of D-form phenylglycine. Interestingly, the purified enzyme did not catalyze hydrolysis of its products, e.g., cephalexin, cephradine, and ampicillin, in contrast to enzymes from other strains of Pseudomonadaceae.

The rat proximal kidney tubule cell line SKPT-0193 cl.2 (SKPT) expresses the di-/tripeptide transporter PEPT2 (rPEPT2) and has been used to study PEPT2-mediated transport. Traditionally, SKPT cells have been cultured in growth media supplemented with epidermal growth factor (EGF), apotransferrin, dexamethasone, and insulin. It was recently demonstrated that omission of EGF from the culture media caused a drastic increase in the expression of rPEPT2. The hypothesis was therefore that the SKPT cell line might be able to differentiate and express rPEPT2 in the absence of the four agonists traditionally added. The aim of the study was thus to characterize Gly-Sar transport parameters in SKPT cells cultured in basic growth media (conventional media without added agonists). Morphology was studied using confocal laser scanning microscopy (CLSM) and immunohistochemistry. Monolayer integrity was evaluated using transepithelial electrical resistance (TEER) measurements and [(3)H]-mannitol permeabilities. Di-/tripeptide transporter activity was studied using [(14)C]-glycylsarcosine ([(14)C]-Gly-Sar). SKPT cells grown in basic media for 4 days formed confluent monolayers with a TEER of 5.03 +/- 0.33 kOmega.cm(2) (n = 5). Apical Gly-Sar uptake peaked after 3-6 days in culture. Uptake at day 4 was 5.89 +/- 0.30 pmol.cm(-2).min(-1) (n = 3). Di-/tripeptide uptake displayed an optimum at approximately pH 6. Affinity values for cephalexin, kyotorphin, and delta-aminolevulinic acid were comparable to those obtained in other PEPT2-expressing model systems. It can be concluded that SKPT cells grown in the absence of the agonists traditionally added to the culture media retain all necessary properties for PEPT2-mediated peptide uptake studies. Furthermore, the absence of the agonists might facilitate studies of hormonal regulation of PEPT2 expression and transport activity.

In the present study, detection, isolation, and characterization of beta-lactamases from Bacillus cereus PTCC 1015 were investigated. B. cereus was inoculated in nutrient broth containing ampicillin (50 mg x ml(-1)) for 24 h (35 degrees C, 200 rpm). Activity measurements were carried out against ampicillin (0.1 mg x ml(-1)) and cephalexin (0.08 mg x ml(-1)) by a spectrophotometric method at different conditions (pH 6-10, temperatures 25-45 degrees C). Maximum penicillinase and cephalosporinase activity was observed at pH 7. The optimized temperatures for penicillinase and cephalosporinase activity were 30 and 40 degrees C, respectively. At the above conditions, maximum enzymatic activity was calculated as 0.89 +/- 0.014 and 0.037 +/- 0.001 units against ampicillin and cephalexin.

We investigated whether cephalexin transport in Caco-2 cells is regulated by insulin. After the insulin pretreatment, cephalexin uptake, and transport as well as PEPT1 mRNA and protein expression in the cells were measured. Cephalexin uptake was significantly increased by the insulin pretreatment. Insulin significantly increased cephalexin saturable uptake, but had no significant effect on the non-saturable one. PEPT1 protein expression on the apical membrane, but not PEPT1 mRNA expression, was increased by the insulin pretreatment. The enhancement of cephalexin uptake by the insulin pretreatment was inhibited by genistein, a tyrosine kinase inhibitor, and colchicine, an agent that disrupts protein translocation. Apical-to-basolateral transport of cephalexin has increased by the insulin pretreatment at the apical side and long-term insulin pretreatment at the basolateral side. It is considered that insulin mainly binds to its receptor on the apical and basolateral membranes, thereby promoting PEPT1 translocation from the intracellular pool to the apical membrane surface; consequently, PEPT1 protein expression on the apical membrane is increased.

Modern biotechnology, in combination with chemistry and process technology, is crucial for the development of new clean and cost effective manufacturing concepts for fine-chemical, food specialty and pharmaceutical products. The impact of biocatalysis on the fine-chemicals industry is presented, where reduction of process development time, the number of reaction steps and the amount of waste generated per kg of end product are the main targets. Integration of biosynthesis and organic chemistry is seen as a key development. The advances in bioseparation technology need to keep pace with the rate of development of novel bio- or chemocatalytic process routes with revised demands on process technology. The need for novel integrated reactors is also presented. The necessary acceleration of process development and reduction of the time-to-market seem well possible, particularly by integrating high-speed experimental techniques and predictive modelling tools. This is crucial for the development of a more sustainable fine-chemicals industry. The evolution of novel 'green' production routes for semi-synthetic antibiotics (SSAs) that are replacing existing chemical processes serves as a recent and relevant case study of this ongoing integration of disciplines. We will also show some challenges in this specific field.

In this study the influence of diffusion limitation on enzymatic kinetically controlled cephalexin synthesis from phenylglycine amide and 7-aminodeacetoxycephalosporinic acid (7-ADCA) was investigated systematically. It was found that if diffusion limitation occurred, both the synthesis/hydrolysis ratio (S/H ratio) and the yield decreased, resulting in lower product and higher by-product concentrations. The effect of pH, enzyme loading, and temperature was investigated, their influence on the course of the reaction was evaluated, and eventually diffusion limitation was minimised. It was found that at pH >or=7 the effect of diffusion limitation was eminent; the difference in S/H ratio and yield between free and immobilised enzyme was considerable. At lower pH, the influence of diffusion limitation was minimal. At low temperature, high yields and S/H ratios were found for all enzymes tested because the hydrolysis reactions were suppressed and the synthesis reaction was hardly influenced by temperature. The enzyme loading influenced the S/H ratio and yield, as expected for diffusion-limited particles. For Assemblase 3750 (the number refers to the degree of enzyme loading), it was proven that both cephalexin synthesis and hydrolysis were diffusion limited. For Assemblase 7500, which carries double the enzyme load of Assemblase 3750, these reactions were also proven to be diffusion limited, together with the binding-step of the substrate phenylglycine amide to the enzyme. For an actual process, the effects of diffusion limitation should preferably be minimised. This can be achieved at low temperature, low pH, and high substrate concentrations. An optimum in S/H ratio and yield was found at pH 7.5 and low temperature, where a relatively low reaction pH can be combined with a relatively high solubility of 7-ADCA. When comparing the different enzymes at these conditions, the free enzyme gave slightly better results than both immobilised biocatalysts, but the effect of diffusion limitation was minimal., (Copyright 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 331-340, 2002.)

The proton-coupled oligopeptide transporter (PEPT1) has been shown to mediate mucosal cell transport of di- and tripeptide, and some peptidomimetic drugs. In this study, we determined the correlation between PEPT1 protein expression and the permeability of cephalexin, a substrate of PEPT1, in human PEPT1 (hPEPT1)-overexpressed Caco-2 cells (Caco-2/hPEPT1 cells) and rat jejunum. Caco-2/hPEPT1 cells with various levels of hPEPT1 expression were established by an adenoviral transfection system. The effective intestinal permeability (P(eff)) in rat jejunum was evaluated using a single pass in situ perfusion method. The level of PEPT1 in Caco-2/hPEPT1 cells and rat intestinal mucosal samples was quantitated by densitometry after immunoblotting and enhanced chemiluminescence detection. In Caco-2/hPEPT1 cells, an excellent correlation was observed between cephalexin uptake and hPEPT1 expression (R2 = 0.96, P < 0.005). This demonstrates that cephalexin uptake is directly proportional to hPEPT1 expression. In the rat perfusion study, the mean P(eff) +/- S.D. (n = 15) of cephalexin was 3.89 +/- 1.63 x 10(-5) cm/s. A very significant correlation between PEPT1 expression and cephalexin permeability with an R2 = 0.63 (P < 0.001) was observed. This indicates that the variation in PEPT1 expression is one of the major factors accounting for variable intestinal cephalexin absorption. To our knowledge, this is the most direct evidence that variation of PEPT1 expression is correlated with absorption permeability variation of peptide-like compounds in vitro and in vivo.

Dietary proteins are mostly absorbed as di- and tripeptides by the intestinal proton-dependent transporter PepT1. We have examined the effects of leptin on PepT1 function in rat jejunum and in monolayers of the human enterocyte-like 2 cell Caco-2. Leptin is produced by the stomach and secreted in the gut lumen. We show here that PepT1 and leptin receptors are expressed in Caco-2 and rat intestinal mucosal cells. Apical (but not basolateral) leptin increased Caco-2 cell transport of cephalexin (CFX) and glycylsarcosine (Gly-Sar), an effect that was associated with increased Gly-Sar uptake, increased membrane PepT1 protein, decreased intracellular PepT1 content, and no change in PepT1 mRNA levels. The maximal velocity (Vmax) for Gly-Sar transport was significantly increased by leptin, whereas the apparent Michaelis-Menten constant (Km) did not change. Furthermore, leptin-stimulated Gly-Sar transport was completely suppressed by colchicine, which disrupts cellular translocation of proteins to plasma membranes. Intrajejunal leptin also induced a rapid twofold increase in plasma CFX after jejunal perfusion with CFX in the rat, indicating enhanced intestinal absorption of CFX. These data revealed an unexpected action of gastric leptin in controlling ingestion of dietary proteins.

Background: Cephalexin and amoxicillin are semisynthetic beta-lactam antibiotics with a broad spectrum of antibacterial activity against gram-positive and gram-negative microorganisms. Both antibiotics are produced by a new 'green' process in which enzyme technology is used to combine the intermediate structure and the side chain in an aqueous medium to yield cephalexin or amoxicillin, thus avoiding the use of several chemical reagents and volatile organic solvents. As a result of the enzyme technology a new residual protein impurity has been identified. To check for the sensitizing capacity of the residual protein, a mouse IgE test was used to detect differences in the production of specific IgE by chemical or enzymatic preparations of the antibiotics., Methods: Balb/c female mice were immunized intraperitoneally with alum and conjugates of different amoxicillins or cephalexins with ovalbumin (OVA). After 16 days, the amoxicillin mice were injected with one half the original amount of antigen. After 19-23 days, the sera were tested for specific IgE by the passive cutaneous anaphylaxis assay in Sprague-Dawley rats. The greatest dilution of sera which resulted in a positive response was the titer of specific IgE., Results: No significant differences were found between the titers of specific IgE caused by the chemically and enzymatically produced beta-lactam antibiotics, indicating that the antibiotics are equal in allergenicity., Conclusions: The data show that a residual level of 35 ppm protein did not affect the allergenic potency of these beta-lactam antibiotics as determined by the mouse allergenicity model., (Copyright 2001 S. Karger AG, Basel)

Deacetoxycephalosporin C synthase from Streptomyces clavuligerus catalyses the conversion of the five-membered penicillin ring to the unsaturated six-membered cephem ring of deacetoxycephalosporin C. The effects on enzyme activity of the penicillin substrate sidechain and various cofactors were investigated using a continuous spectrophotometric assay. The conversion of penicillin G to phenylacetyl-7-aminodeacetoxycephalo sporanic acid (G-7-ADCA) was confirmed, and further details of the reaction were elucidated. The conversion of ampicillin to cephalexin was faster than that of acetyl-6-APA to acetyl-7-ADCA kcat = 0.120 +/- 0.001 s(-1) versus 0.035 +/- 0.001 s(-1), but they had similar Km values: 4.86 +/- 0.12 and 3.28 +/- 0.26 mM, respectively. Amoxycillin and penicillin V were also converted at low levels. Conversion was not detected for penicillanate, 6-aminopenicillanate, carbenicillin, temocillin, ticarcillin or benzylpenicilloic acid, suggesting that the enzyme has a relatively strict selectivity for the sidechain of the penicillin substrate.

Cephalexin is a constituent of the cephalosporin group used for the treatment of bronchitis and other heart diseases due to its enhanced oral activity. The effluent from these industries contains a disintegrated form of the drug contributing high chemical oxygen demand (COD), volatile solids and organic solvent. A laboratory-scale study was conducted to evaluate the efficiency of a fluidized bed reactor operated under anaerobic condition with bioaugmentation to treat the cephalexin containing pharmaceutical factory effluent. 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 COD of 12,000-15,000 mg/l, was diluted and studied in single and multiple inoculation experiments with hydraulic retention times of 3-12 h. The removal efficiency after inoculation from an anaerobic sequencing batch reactor was related to influent concentration, mass of inoculum and hydraulic retention time characterized by calculating the initial food to microorganism ratio. Continuous COD removal efficiency 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.

1. Methotrexate (MTX), an anticancer drug, has been shown to induce acute injury in the small intestine. The present study was designed to investigate the in vivo absorptive function of the small intestine injured by MTX using an amino-beta-lactam antibiotic cephalexin (CEX). Time-dependent changes in diamine oxidase (DAO) and alkaline phosphatase (ALP) activity in the small intestine and histopathological findings were also measured in rats treated with MTX (20 mg/kg). 2. Most severe mucosal damage was observed 2 days after MTX treatment and the area under the plasma concentration-time curve of CEX (AUC(CEX)) following oral administration of 20 mg/kg tended to decrease. Thereafter, the AUC(CEX) increased significantly and the histopathological changes diminished within 5 days. 3. Both villus height and mucosal weight followed the same pattern, decreasing in the first 2 or 3 days following treatment, increasing on the 5th day and returning to control levels by the 10th day. Methotrexate-induced changes in the mucosal wet weight/whole intestinal weight ratio were significantly correlated with those of AUC(CEX), but did not correlate with mucosal DAO and ALP activity. 4. These findings provide evidence that the change in the total amount of CEX is an index of the active transport function, probably by intestinal peptide transporter (PEPT1), and is well reflected by histopathological changes in the intestinal mucosa induced by MTX. In addition, there is a possibility that this method could be applied in the clinical setting for diagnosis of intestinal status and absorptive function.

A molecularly imprinted polymer was prepared using cefalexin as the template molecule and 2-(trifluoromethyl)acrylic acid as the functional monomer. The bulk polymer was ground, sieved and investigated in an equilibrium binding experiment to evaluate the binding characteristics of the cefalexin-imprinted polymer for a better understanding of the mechanisms of recognition in molecularly imprinted polymers. Scatchard analysis showed that two classes of binding sites were formed in the imprinted polymer under the concentration studied. The dissociation constants were estimated to be 0.14 mmol/L and 2.38 mmol/L. The polymer gave much higher binding capacity for cefalexin than the non-imprinted polymer with the same chemical composition. The selectivity was evaluated by distribution coefficients of cefalexin and other structurally similar compounds. The results showed that the imprinted polymer exhibited high affinity for cefalexin among the tested compounds.