Integrative bioinformatics and proteomics-based discovery of an eEF2K inhibitor (cefatrizine) with ER stress modulation in breast cancer cells.

Eukaryotic elongation factor-2 kinase (eEF2K), a unique calcium/calmodulin-dependent protein kinase, is well known to regulate apoptosis, autophagy and ER stress in many types of human cancers. Therefore, eEF2K would be regarded as a promising therapeutic target; however, the eEF2K-regulated mechanism and its targeted inhibitor still remain to be discovered in cancer. Herein, we constructed a protein-protein interaction (PPI) network of eEF2K and achieved an eEF2K-regulated ER stress subnetwork by bioinformatics prediction. Then, we found that the differential protein expressions involved in ER stress in the context of si-eEF2K-treated MCF-7 and MDA-MB-436 cells by iTRAQ-based analyses, respectively. Integrated into these aforementioned results, we constructed a core eEF2K-regulated ER stress subnetwork in breast cancer cells. Subsequently, we screened a series of candidate compounds targeting eEF2K and discovered a novel eEF2K inhibitor (cefatrizine) with an anti-proliferative activity toward breast cancer cells. Moreover, we found that cefatrizine induced ER stress in both MCF-7 and MDA-MB-436 cells. Interestingly, we demonstrated that the mechanism of cefatrizine-induced ER stress was in good agreement with our bioinformatics and proteomics-based results. In conclusion, these results demonstrate that a novel eEF2K inhibitor (cefatrizine) induces ER stress in breast cancer cells by integrating bioinformatics prediction, proteomics analyses and experimental validation, which would provide a clue for exploring more mechanisms of eEF2K and its targeted inhibitors in cancer therapy.

In vitro activity of cefadroxil, cephalexin, cefatrizine and cefpirome in presence of essential and trace elements.

Evidences supporting the introduction of metallic elements in several biological processes are rapidly accumulating. Likewise, many drugs possess modified toxicological and pharmacological properties when in the form of metal complexes. In order to ascertain the role of various essential and trace element complexation on the antibacterial activity of various cephalosporins, the synergistic or antagonistic behavior of cefadroxil, cephalexin, cefatrizine and cefpirome in presence of essential and trace elements has been studied and compared with the parent drug. The essential and trace elements comprised of magnesium, calcium, chromium, manganese, ferric, cobalt, nickel, copper, zinc and cadmium in the form of their chloride. These studies were carried out by observing the minimum inhibitory concentration (MIC) using agar dilution method and compared with the MIC'S of the standard cephalosporins against various species of Gram (+) and Gram (-) microorganisms such as Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus faecalis, Escherichia coli, Proteus vulgaris, Pseudomonas aeruginosa, Salmonella typhi and Shigella dysenteriae. Different dilutions of cephalosporins and salts of essential and trace elements were used in these studies. The ratio of the drug and metal salts was 1:1 and the reactions were carried out at two different temperatures as 37 degrees C and 60 degrees C in order to study the complex formation. The aim of our study was on one hand to evaluate the changes in microbiological activity of the standard cephalosporins after in vitro metal interactions to study the synergetic or antagonistic behavior of the later through the difference in MICs values of these cephalosporins and on the other hand to access the bioassay directed extent of drug metal complexations. Our investigation reveal that interaction of above cephalosporins with essential and trace elements cause antagonistic effect in many cases which was shown by decrease in antimicrobial activity of cephalosporins and MIC values were increased.

[Infective pathogens and drug resistance in burned patients].

OBJECTIVE: To find out the changes in the infective pathogens and their drug resistance in burned patients. METHODS: The patients were divided into two groups. The first group was from July 1991 to June 1996, and the second group was from July 1996 to June 2001. The bacteria of burned body surface and blood were cultured, and the bacteria and their drug sensitivity were analyzed. RESULTS: Gram-negative bacteria were the major bacteria in burn infection, among which Pseudomonas aeruginosa ranked the top. Staphylococcus aureous ranked the first among the Gram-positive bacteria, and the isolation rate of methicillin resistant Staphylococcus aureus increased; The isolation rate of Enterobacter cloacae (10.4%), Escherichia coli (8.3%), Klebsiella pneumoniae (7.3%), and fungus (4.2%) all rose. The antibiotic resistant strains of Pseudomonas aeruginosa and Staphylococcus aureous increased. CONCLUSION: The changes in pathogens of burn infection and bacterial drug resistance are related to the wide use of broad spectrum antibiotics such as cefazidime and imepenem, suggesting that dynamic observation of changes in pathogenic strains and sensitivity of bacteria to antibiotics are useful for clinical prevention and cure of burn infection.

In-vitro activity of 21 beta-lactam antibiotics against penicillin-susceptible and penicillin-resistant Streptococcus pneumoniae.

MICs of 21 beta-lactams were determined by agar dilution against 283 penicillin-susceptible (pen-S), 122 intermediate (pen-I) and 23 fully penicillin-resistant (pen-R) pneumococci. MICs of all beta-lactams increased with increasing MICs of penicillin. Clometocillin was the most active penicillin against pen-I or pen-R pneumococci. All oral cephalosporins except cefuroxime and cefpodoxime were less active than penicillin and none was satisfactory against pen-I or pen-R pneumococci. The parenteral third- and fourth-generation cephalosporins (except ceftazidime) were similar in activity to penicillin against pen-S isolates. Cefpirome showed the lowest mean MICs against pen-I and pen-R strains.

Saturable rate of cefatrizine absorption after oral administration to humans.

This study examined the absorption kinetics of cefatrizine, an amino-beta-lactam antibiotic, after oral administration of a single 500-mg dose to 12 healthy volunteers. Plasma concentrations were determined by high performance liquid chromatography. The plots of the percentage of drug unabsorbed and the apparent rate of cefatrizine absorption as a function of time showed, first, a delay and, then, an almost constant rate of absorption with a tendency to move toward first-order kinetics at the end of the process. Three compartmental models incorporating a lag time and first-order elimination kinetics, but differing in their input rate, were used for analysis of the time course of cefatrizine plasma concentrations. The model with first-order absorption kinetics was clearly inadequate. The results were improved with the model for which the rate of absorption is constant, but a model incorporating saturable absorption kinetics of the Michaelis-Menten type improved the fit further. This last model was statistically superior to the constant-rate input model in 6 out of 12 subjects, according to the likelihood-ratio method. Because of the innovative feature of the model incorporating the Michaelis-Menten equation, simulations of the effect of altering the model parameters and the dose administered on the concentration-time profile, were performed. Different hypotheses which might explain why cefatrizine absorption kinetics fits the Michaelis-Menten equation were examined. The observation of saturable absorption kinetics is consistent with a carrier-mediated transport previously reported to occur in the gastrointestinal tract of rats.

Effect of cefatrizine and cephaloglycine on L-leucine absorption in rat jejunum.

1. The oral cephalosporins: cefatrizine and cephaloglycine inhibit the L-leucine absorption in vivo on rat jejunum. 2. This inhibition is dose and time dependent and the effect is irreversible. 3. These antibiotics have a systemic effect on L-leucine absorption. 4. The inhibition of these antibiotics affect only leucine transport, without affecting the diffusion. 5. Cefatrizine and cephaloglycine inhibit the basolateral (Na(+)-K+) ATPase activity in rat jejunum.

Clinical pharmacokinetics of five oral cephalosporins in calves.

The minimal inhibitory concentrations (MIC) of cephalexin, cephradine, cefaclor, cefatrizine and cefadroxil for Salmonella species, Escherichia coli and Pasteurella multocida isolated previously from young calves were determined. The MIC90 values for cephalexin, cephradine and cefadroxil ranged between 3.12 micrograms ml-1 and 12.5 micrograms ml-1, whereas those of cefatrizine and cefaclor were 3.12 micrograms ml-1 and 0.78 microgram ml-1, respectively. Each drug was administered intravenously and orally to groups of pre-ruminating calves and orally to early ruminating calves. Although the pharmacokinetic characteristics of the drugs after intravenous injection were similar to other beta-lactam antibiotics, significant differences between the cephalosporins examined were found in respect of certain kinetic parameters. The drugs showed rapid absorption into the systemic circulation after oral administration to pre-ruminating calves but the elimination half-life values (t1/2 beta) varied between three hours (cefaclor and cefadroxil) and nine hours (cefatrizine). The bioavailability of the drugs was about 35 per cent of the administered dose. Co-administration of probenecid with each antibiotic caused a twofold or greater increase in peak serum drug concentrations (Cmax) but the effect on t1/2 beta was variable. Cephalexin, cephradine and cefaclor given to the ruminating calves resulted in very low serum or plasma concentrations and their use should be restricted to younger calves. Cefadroxil was found to give the highest serum concentrations in this age group but had significantly lower bioavailability when compared with the unweaned calves. Provisional oral dosage regimens were computed for each cephalosporin on the basis of the MIC data and the kinetic parameters derived from intravenous and oral drug administration.

[Determination of the bactericidal potency of cefatrizine and other oral antibiotics against Haemophilus influenzae, Klebsiella pneumoniae and Streptococcus pyogenes]. / Valutazione del potere di batteriocidia della cefatrizina e altri antibiotici orali nei confronti di Haemophilus influenzae, Klebsiella pneumoniae e Streptococcus pyogenes.

The in vitro determination of the T log and the minimal bactericidal time of cefatrizine was evaluated against H. influenzae, Str. pyogenes A isolated from clinical specimens. and K. pneumonia ATCC 10031 and compared with that of amoxycillin, cefaclor, cefroxadine and miocamycin against the same bacterial strains. Cefatrizine demonstrate the shorter TMB against all bacterial strains and antibiotic tested with MBCs.

Cefatrizine: a clinical overview.

Cefatrizine, a new oral cephalosporin, proved effective in the treatment of a wide range of bacterial infections in both adult and paediatric patients. Adverse reactions mild and mainly limited to gastrointestinal disturbances.

Susceptibility of clinical isolates of gram-positive and gram-negative organisms to cefatrizine.

The in vitro activity of cefatrizine was evaluated against 294 Gram-positive and 270 Gram-negative bacteria isolated from clinical specimens. Cefatrizine had excellent activity against Gram-positive cocci, inhibiting all except enterococci at minimal inhibitory concentrations below 1 mcg/ml. Moreover, cefatrizine was an effective antibacterial agent for most major Gram-negative species. Cefatrizine activity was tested against 34 strains of H. influenzae and 37 strains of K. pneumoniae and compared with that of other orally administered cephalosporins. Cefatrizine MIC50 values were much lower than those recorded for cephalexin and cefadroxil.

Comparison of in vitro antibacterial activity of four oral cephems: cephalexin, cefaclor, cefatrizine and cefadroxil.

The in vitro antibacterial activity of four oral cephems: cephalexin, cefaclor, cefatrizine and cefadroxil against clinical isolates of staphylococci, Enterobacteriaceae and Pseudomonas aeruginosa was compared by agar dilution method. All drugs had comparable antistaphylococcal activity. Cefatrizine and cefaclor were more active than cephalexin and cefadroxil against Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis. All four drugs were relatively inactive against isolates of Enterobacter species and indole-positive Proteus. However, cefatrizine demonstrated the greatest activity of the four oral cephems against these organisms. None of these oral cephems was active against Serratia marcescens or Pseudomonas aeruginosa.

[A study on the disc sensitivity test for cefatrizine].

Susceptibilities of 227 strains of 34 bacterial species to cefatrizine (CFT) were determined by the 2-fold agar dilution method in parallel with the diameter of inhibition zones by the single-disc method, under the experimental condition established by Kanazawa. The experiments demonstrated significant correlation between MIC by the dilution method and diameter of inhibition zone in each of conventional assay of the over-night (about 16 hours) incubation, delayed assay (about 24 hours incubation), and rapid assay (after 3-4 or 5-6 hours incubation), thus confirming applicability of the single-disc assay for CFT. Analysis of the data obtained by using CFT disc containing 30 micrograms revealed the primary regression equation to be: D (diameter, mm) = 25.6--9.6 log MIC (micrograms/ml) in conventional assay, D = 33.2--13.2 log MIC (micrograms/ml) in delayed assay, D = 15.8--4.7 log MIC (micrograms/ml) in 3-4 hours rapid assay and D = 20.2--7.0 log MIC (micrograms/ml) in 5-6 hours rapid assay, respectively. The range of variations in MICs estimated from the diameter of inhibition zone by the disc test was then calculated in comparison with that in MIC determined by the 2-fold agar dilution assays, as reference for the experimental errors which may be involved in the estimation of MIC of CFT by the single-disc assay.

[Experimental and clinical studies of cefatrizine in oral infections (author's transl)].

Investigation was made on the therapeutic effect of a new antibiotic, cefatrizine(CFT), in oral infections according to the following procedure. Results are summarized as follows. Determination was made on the MIC of CFT against 8 clinical isolates of S. epidermidis, 5 of S. aureus and 7 of E. coli in comparison with those of CEX and PBPC. MICs of CFT against S aures were demonstrated 3.13 to 6.25 mcg/ml which were superior to those of CEX and ABPC. CFT was administered orally in a dose of 500 mg at an hour and a half before the extraction of the impacted wisdom tooth in the mandibula. During the operation, gingival and blood specimens were collected and each CFT level was determined. The mean CFT level in the gingiva reached to 0.95 mcg/ml and that in the blood to 5.77 mcg/ml. According to these experimental results, CFT was administered to patients with moderate oral infection at a dose of 500 mg and clinical assessment was made according to the criteria established by Japanese Society of Oral Surgeons. As the results, the effectiveness rate of CFT was 85%. No serious side effect was observed. From the results of the present study, CFT may be effective for moderate oral surgery infections.

The cephalosporins: activity and clinical use.

The cephalosporin antibiotics have been employed with increasing frequency since their introduction into clinical practice in the early 1960s. With the exception of cephaloridine, cephalosporin compounds are not associated with the production of significant untoward effects. The availability of newer cephalosporins, both oral and parenteral, with enhanced antibacterial activity, has expanded the clinical indications for administration of these antibiotics.

Cefatrizine activity compared with that of other cephalosporins.

Cefatrizine, a new orally administered cephalosporin, was tested against 400 clinical isolates. Cefatrizine had excellent activity against gram-positive cocci, inhibiting all except enterococci at minimal inhibitory concentrations below 1 mug/ml. Cefatrizine inhibited the majority of Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, and Salmonella at concentrations below 12.5 mug/ml. Although cefatrizine was not hydrolyzed by many beta-lactamases, it did not inhibit a number of strains of Enterobacter, Serratia, or indole-positive Proteus. Cefatrizine was more active than cephalothin or cephalexin against E. coli, Klebsiella, Enterobacter, Citrobacter, Salmonella, and Shigella. Its overall activity was less than that of cefoxitin against strains resistant to cephalothin, but its activity against cephalothin-susceptible strains was equivalent to that of cefamandole.

In vitro activities of five oral cephalosporins against aerobic pathogenic bacteria.

Cefaclor (Lilly 99638) and cefatrizine (BL-S640, SK&F 70771) are orally absorbed, broad-spectrum semisynthetic cephalosporins. They were compared in vitro with cephalexin, cephaloglycin, and cepharadine against a variety of aerobic pathogenic bacteria by an agar dilution procedure. Cefaclor and cefatrizine were found to be similar or superior to cephalexin, cephaloglycin, and cephradine in terms of activity against gram-positive cocci other than enterococci. Only cefatrizine demonstrated any potentially useful activity against some susceptible isolates of enterococci. Cefaclor and cefatrizine also were highly active, equally or more so than the other oral cephalosporins, against several gram-negative species including Escherichia coli, Enterobacter aerogenes, and Klebsiella pneumoniae. None of the cephalosporins were particularly active against Enterobacter cloacae. Both cefaclor and cefatrizine were active against Proteus mirabilis; cefatrizine was uniquely active against indolepositive Proteus species.

In vitro activity of five oral cephalosporins against anaerobic pathogenic bacteria.

The in vitro inhibitory activities of cefaclor and and cefatrizine, two new orally absorbed cephalosporin antibiotics, against 44 isolates of anaerobic pathogenic bacteria were measured using the agar dilution procedure of the World Health Organization-International Collaborative Study. Tests also were performed with cephalexin, cephaloglycin, and cephadrine, as well as with the parenteral cephamycin antibiotic cefoxitin. Cefoxitin was the most active antibiotic and inhibited the majority of isolates at a concentration of less than or equal to 4 microgram/ml. None of the oral cephalosporins was clearly superior against all of the anaerobic isolates; only cephadrine and cefatrizine appeared to have any potential clinical value.