Comparative in vitro activity of faropenem against staphylococci

The anti-staphylococcal activity of faropenem, a novel beta-lactam, was examined and compared with that of amoxicillin, cefuroxime, clindamycin and vancomycin using the agar dilution method. A total of 234 staphylococci, including a large number of clonally different methicillin-resistant strains and a representative number of Staphylococcus aureus small colony variants, were tested. While the activity of faropenem was independent of the staphylococcal phenotype, the novel penem was up to eight times more active against methicillin-susceptible strains compared with the other agents tested. In addition, faropenem was active against many methicillin-resistant strains of S. aureus and coagulase-negative staphylococci.     

Effect of protein binding on the pharmacological activity of highly bound antibiotics

During antibiotic drug development, media are frequently spiked with either serum/plasma or protein supplements to evaluate the effect of protein binding. Usually, previously reported serum or plasma protein binding values are applied in the analysis. The aim of this study was to evaluate this approach by experimentally measuring free, unbound concentrations for antibiotics with reportedly high protein binding and their corresponding antimicrobial activities in media containing commonly used protein supplements. Free, unbound ceftriaxone and ertapenem concentrations were determined in bacterial growth medium with and without bovine/human serum albumin, as well as adult bovine serum and human plasma using in vitro microdialysis. The corresponding antimicrobial activity was determined in MIC and time-kill curve experiments using Escherichia coli ATCC 25922 and Streptococcus pneumoniae ATCC 6303 as test strains. A semimechanistic maximum effect model was simultaneously fitted to the data and respective EC₅₀ (concentration at half-maximum effect) values compared. Protein binding differed significantly for ceftriaxone (P < 0.05) between human plasma (76.8 ± 11.0%) and commercially available bovine (20.2 ± 8.3%) or human serum albumin (56.9 ± 16.6%). Similar results were obtained for ertapenem (human plasma, 73.8 ± 11.6%; bovine serum albumin, 12.4 ± 4.8%; human serum albumin, 17.8 ± 11.5%). The MICs and EC₅₀s of both strains were significantly increased (P < 0.05) for ceftriaxone when comparing human and bovine serum albumin, whereas the EC₅₀s were not significantly different for ertapenem. Free, unbound antibiotic concentrations differed substantially between plasma and protein supplements and correlated well with antimicrobial efficacy. Therefore, free, active concentrations should be measured in the test system instead of correcting for literature protein binding values.     

Significance of "extravascular" protein binding for antimicrobial pharmacodynamics in an in vitro capillary model of infection

The effect of protein binding in an "extravascular" space on antimicrobial pharmacodynamics was studied in an in vitro capillary model of infection. Simulated 500-mg oral doses of dicloxacillin (approximately 96% bound) or cephalexin (less than 5% bound) were administered every 6 h for four doses. A 10-fold-higher dose of dicloxacillin was also studied to determine the effect of drug concentration on the reduction of bacterial killing in the presence of protein. Staphylococcus aureus ATCC 25923 was inoculated into peripheral chambers filled with either Mueller-Hinton broth or Mueller-Hinton broth plus 25% human serum. Serial samples for bacterial counts were collected over 24 h. The presence of serum in the chambers significantly reduced bacterial killing by dicloxacillin but not by cephalexin during the first 6 h (two-way analysis of variance, F = 6.04, P less than 0.05) but not at 24 h. Reduction of dicloxacillin activity in serum-containing chambers persisted with the higher dose. These data suggest that despite attaining higher total drug concentrations in protein-containing extravascular spaces with highly bound drugs, protein binding reduces bactericidal activity during the early stages of treatment in this model.     

Effect of protein binding of daptomycin on MIC and antibacterial activity.

A higher rate of clinical failures in patients treated with daptomycin (2 mg/kg of body weight, given once daily) compared with rates in patients treated with conventional regimens caused early termination of this comparative clinical trial. One explanation for these failures could be that daptomycin is highly protein bound and that the concentration of the unbound active drug is too low for antibacterial activity. To assess this explanation, we studied the binding of daptomycin to proteins by using an ultrafiltration method. pH (7.0 to 7.4), temperature (25 or 37 degrees C), or daily freezing and thawing over 2 months had no effect on binding of daptomycin to proteins. We found that daptomycin was bound to albumin (90%) at 4 g/100 ml. Binding of daptomycin was not concentration dependent (2.5 to 80 micrograms/ml). In human serum samples spiked with daptomycin, average binding was 94% +/- 2.4%. In 6 subjects given an intravenous infusion of daptomycin (3 mg/kg), average binding was 90% +/- 2.1%. Susceptibility studies showed that a concentration in serum 20 times the unbound concentration was needed to equal the MIC of the total drug. These results indicate that daptomycin is highly bound (90 to 94%) to albumin and that clinical failure to daptomycin can in part be explained by the low concentration of the unbound drug.     

Effect of protein binding on simulated intravascular and extravascular kinetics of cefotaxime in an in vitro model

The simulated intravascular and extravascular kinetics of cefotaxime were studied in an in vitro model to evaluate the effect of antibiotic protein binding in the "intravascular" and "extravascular" space. Intravascular fluid consisted of either phosphate-buffered saline, which has no cefotaxime binding, or 3% bovine albumin, which has 63% cefotaxime binding. Extravascular spaces were filled with phosphate-buffered saline, 1.5% bovine albumin (46.6% cefotaxime binding), or 3% bovine albumin. Cefotaxime (80 mg per dose) was infused every 3 h for eight doses, and intravascular and extravascular drug concentrations were measured after doses one and eight. The corresponding intravascular and extravascular spaces were at (phosphate-buffered saline) or approaching (3% bovine albumin) equilibrium by dose eight. There were marked differences in drug concentrations achieved in the various extravascular spaces, but all could be explained on the basis of differing amounts of albumin present and the resultant differences in cefotaxime binding.     

Influence of protein binding under controlled conditions on the bactericidal activity of daptomycin in an in vitro pharmacodynamic model

OBJECTIVE: Daptomycin exhibits bactericidal activity against clinically significant Gram-positive bacteria despite being highly bound to human proteins. Evaluations characterizing the effect of protein on daptomycin pharmacodynamics are warranted. METHODS: We utilized an in vitro pharmacodynamic model to simulate daptomycin regimens of 6 mg/kg/day under controlled conditions of pH, calcium and/or protein. Free concentrations were simulated in broth, whereas total concentrations were simulated in broth supplemented with human albumin. Bacterial density was profiled over 48 h for two methicillin-resistant Staphylococcus aureus (MRSA) and two vancomycin-resistant Enterococcus faecium (VREF) clinical isolates. RESULTS: Daptomycin exhibited bactericidal activity against both MRSA isolates, with time to 99.9% killing occurring at 0.5 h and 8 h in broth and in albumin-supplemented broth, respectively. Initial kill was observed against both VREF isolates followed by regrowth. There was no statistical difference (P>0.05) in extent of bacterial kill at 24 or 48 h between the different media. CONCLUSIONS: Although delayed, the extent of kill for daptomycin was unaltered against all isolates in albumin-supplemented broth. Further antimicrobial studies that incorporate protein are warranted to assess the influence of protein in the pharmacodynamic evaluation of antimicrobials.     

Effect of altered plasma protein binding on pharmacokinetics and pharmacodynamics of propranolol in rats after surgery: role of alpha-1-acid glycoprotein

The pharmacokinetics and pharmacodynamics of propranolol in rats 2 days after laparotomy were compared to control animals. The apparent volumes of distribution and the systemic clearance of propranolol were decreased to about 20 to 40 and 70% of control values, respectively. The area under the blood concentration-time curve (AUC) of propranolol after p.o. administration showed a marked elevation after surgery and its availability increased about 2-fold at doses of 5.0 and 12.5 mg/kg. These changes were associated with a decreased plasma unbound fraction of propranolol after surgery. Immunological determination of alpha-1-acid glycoprotein (AGP) revealed a marked increase after laparotomy and a linear relationship was found between the plasma AGP concentration and the binding capacity of high-affinity binding site for propranolol in plasma (r = 0.961, P less than .001). AUC of p.o. administered propranolol was also correlated with plasma AGP concentration. The beta-blocking activity of propranolol assessed by the reduction in the isoproterenol-induced tachycardia was decreased in rats after laparotomy when it was evaluated in terms of the total plasma concentration of propranolol. In contrast, its activity evaluated by the unbound plasma concentration showed no difference between control and laparotomized rats, suggesting the dependence of the pharmacological activity of propranolol on its unbound level in plasma. Thus, laparotomy-induced changes in both pharmacokinetics and pharmacodynamics could be considered largely due to an increase in its binding to the increased plasma level of AGP.     

Effect of heparin on digitoxin protein binding

Reduction of digitoxin binding to plasma proteins after heparin has been reported. Our aim was to determine whether this reduction is an in vivo effect or occurs only after blood collection as a result of heparin-induced lipolysis that increases levels of nonesterified fatty acids in vitro. The effect of heparin on digitoxin protein binding was studied in 10 patients undergoing hemodialysis receiving digitoxin maintenance therapy. Digitoxin free fraction increased after heparin, from 2.5% +/- 0.7% to 4.4% +/- 1.1%, but after inhibition of in vitro lipolysis with diethyl p-nitrophenyl phosphate (2mM), a potent lipase inhibitor, there was no increase in the free fraction (2.3% +/- 0.4% before heparin and 2.4% +/- 0.5% after heparin). Digitoxin salivary levels were also unchanged (0.41 +/- 0.08 ng/ml before heparin and 0.41 +/- 0.08 ng/ml after heparin [n = 8]). These data indicate that the binding of digitoxin to plasma proteins in vivo is not altered by heparin. The reduced binding reported elsewhere was a result of heparin-induced in vitro lipolysis.     

头孢曲松-舒巴坦复方制剂体外药动学/药效学研究

目的 通过体外药动学/药效学(PK/PD)研究,确定头孢曲松-舒巴坦(4:1)对产超广谱β内酰胺酶(ESBL)肠杆菌科细菌作用特点,评价其组方的合理性.方法 建立体外PK/PD模型,模拟人体单剂静脉注射头孢曲松、头孢曲松-舒巴坦(4:1)(同步与非同步消除)、头孢哌酮、头孢哌酮-舒巴坦(1:1)药动学过程,比较药物对肺...     

Effect of temperature on protein binding of cortisol

OBJECTIVES: Biological effects of cortisol are substantially determined by protein binding of the hormone. The aim of our study was to characterize temperature effects on cortisol protein binding by use of an equilibrium dialysis method. DESIGN AND METHODS: Serum samples obtained from ten healthy volunteers were submitted to equilibrium dialysis. Each sample from the individuals was incubated for 16 h at 37 degrees C, 38 degrees C, 39 degrees C, 40 degrees C and 41 degrees C, respectively. In the dialysate samples obtained, cortisol concentrations were measured by immunoassay. RESULTS: For samples incubated at 37 degrees C, a mean dialysate cortisol concentration of 0.41 microg/dL (SD=0.14) was found. Gradual increase of dialysate cortisol concentration was observed with increasing incubation temperatures. For samples incubated at 41 degrees C, a mean dialysate cortisol of 0.75 microg/dL (SD=0.24) was found. Thus, the mean percentage of free-to-total cortisol increased by about 80% from 3.7% (SD=1.1) at 37 degrees C to 6.7% (SD=1.8) at 41 degrees C. CONCLUSIONS: The results of our in vitro experiments suggest that during fever the free-to-total ratio of cortisol is increased substantially compared to normal conditions, and that administration of antipyretic drugs might potentially be associated with substantial changes in the bioavailability of cortisol.     

In vitro activity and pharmacodynamics of azithromycin and clarithromycin against Streptococcus pneumoniae based on serum and intrapulmonary pharmacokinetics

BACKGROUND: Multidrug-resistant strains of Streptococcus pneumoniae are increasingly common worldwide, but the clinical significance of their resistance to the macrolide antibiotics is controversial. Applying pharmacokinetic and pharmacodynamic principles can assist in the selection of appropriate antimicrobial therapy. OBJECTIVES: The purpose of this study was to determine the in vitro activity of penicillin, azithromycin, clarithromycin, and clindamycin against clinical isolates of S. pneumoniae and to evaluate the pharmacodynamics of azithromycin and clarithromycin based on serum and epithelial lining fluid (ELF) concentrations. METHODS: The minimum inhibitory concentrations (MICs) of penicillin, azithromycin, clarithromycin, and clindamycin were determined for 307 isolates of S. pneumoniae using broth microdilution. Using serum and ELF concentrations after standard dosing, we calculated the proportion of isolates against which it would be possible to obtain a ratio of azithromycin area under the curve to MIC > or =25 and clarithromycin concentrations that exceeded the MIC for > or =40% of the dosing interval. RESULTS: Overall, 19.5%, 25.4%, 25.1%, and 7.2% of the 307 pneumococcal isolates were resistant to penicillin, azithromycin, clarithromycin, and clindamycin, respectively. However, 71.7% of penicillin-resistant strains were also resistant to azithromycin and clarithromycin. Based on serum concentrations, clarithromycin achieved its pharmacodynamic target in 76.9% of isolates, compared with 59.9% for azithromycin. Based on ELF concentrations, clarithromycin achieved its pharmacodynamic target in 93.5% of isolates, compared with 74.6% for azithromycin. Based on ELF concentrations, clarithromycin achieved its pharmacodynamic target in 86.7% of penicillin-resistant isolates, compared with 28.3% for azithromycin. CONCLUSIONS: On the basis of serum and ELF concentrations, clarithromycin achieved pharmacodynamic targets against a greater proportion of S. pneumoniae isolates than did azithromycin. Clinical studies are needed to determine the efficacy of these agents against pneumococci that demonstrate in vitro resistance using current susceptibility breakpoints.     

In vitro activities of faropenem against 579 strains of anaerobic bacteria

The activity of faropenem, a new oral penem, was tested against 579 strains of anaerobic bacteria by using the NCCLS-approved reference method. Drugs tested included amoxicillin-clavulanate, cefoxitin, clindamycin, faropenem, imipenem, and metronidazole. Of the 176 strains of Bacteroides fragilis group isolates tested, two isolates had faropenem MICs of 64 micro g/ml and imipenem MICs of >32 micro g/ml. Faropenem had an MIC of 16 micro g/ml for an additional isolate of B. fragilis; this strain was sensitive to imipenem (MIC of 1 micro g/ml). Both faropenem and imipenem had MICs of < or=4 micro g/ml for all isolates of Bacteroides capillosus (10 isolates), Bacteroides splanchnicus (13 isolates), Bacteroides ureolyticus (11 isolates), Bilophila wadsworthia (11 isolates), Porphyromonas species (42 isolates), Prevotella species (78 isolates), Campylobacter species (25 isolates), Sutterella wadsworthensis (11 isolates), Fusobacterium nucleatum (19 isolates), Fusobacterium mortiferum/varium (20 isolates), and other Fusobacterium species (9 isolates). Faropenem and imipenem had MICs of 16 to 32 micro g/ml for two strains of Clostridium difficile; the MICs for all other strains of Clostridium tested (69 isolates) were < or =4 micro g/ml. Faropenem had MICs of 8 and 16 micro g/ml, respectively, for two strains of Peptostreptococcus anaerobius (MICs of imipenem were 2 micro g/ml). MICs were < or =4 micro g/ml for all other strains of gram-positive anaerobic cocci (53 isolates) and non-spore-forming gram-positive rods (28 isolates). Other results were as expected and reported in previous studies. No metronidazole resistance was seen in gram-negative anaerobes other than S. wadsworthensis (18% resistant); 63% of gram-positive non-spore-forming rods were resistant. Some degree of clindamycin resistance was seen in most of the groups tested.     

Effect of blood sampling schedules on protein drug binding in the rat

The effect of repetitive blood sampling schedules on the plasma protein binding of a highly protein-bound drug, dicumarol, was investigated in the rat. Blood samples were withdrawn at pre-determined intervals and hematocrit, total plasma protein, plasma albumin and free fatty acids were measured. Plasma protein binding of [14C]dicumarol was assessed by equilibrium dialysis. Withdrawal of 1 ml of blood every hour for 12 hr produced a significant decrease at 2 hr in hematocrit (42%), total plasma protein (14.5%), plasma albumin (31.4%) and an increase in free fatty acids (238%) compared to the control (O time) levels. The free fraction of dicumarol in the plasma increased 1350%, from 0.38 to 5.13%. Sampling schedules involving blood withdrawal of 0.5 mg/2 hr and 1 ml/2 hr producing less dramatic changes, but in all cases the free fraction of dicumarol was elevated at the 12-hr time period. An inverse relationship was found between plasma albumin concentrations and dicumarol-free fraction. The blood sampling schedule was found to alter the pharmacological response (prothrombin time) and pharmacokinetics of dicumarol after an 8 mg/kg i.v. dose. These results illustrate the influence multiple blood sampling can exert on pharmacodynamic parameters in studies involving small laboratory animals and highly protein-bound drugs.     

Effect of fluconazole on the pharmacokinetics and pharmacodynamics of nateglinide

OBJECTIVE: Our aim was to investigate the possible effects of fluconazole on the pharmacokinetics and pharmacodynamics of nateglinide, a new short-acting meglitinide analog antidiabetic drug. METHODS: In a randomized, double-blind, crossover study with 2 phases, 10 healthy volunteers took 200 mg fluconazole (400 mg on day 1) or placebo once daily for 4 days. On day 4, they ingested a single 30-mg dose of nateglinide. Plasma nateglinide and blood glucose concentrations were measured for up to 7 hours. RESULTS: Fluconazole raised the total area under the plasma concentration-time curve from time 0 to infinity of nateglinide by 48% (range, 20%-73%; P <.00001) and prolonged its half-life from 1.6 to 1.9 hours (P <.05), but the peak plasma nateglinide concentration remained unchanged. The peak plasma concentration of the M7 metabolite of nateglinide was reduced by 34% by fluconazole (P <.001), and its half-life was prolonged from 2.2 to 3.5 hours (P <.05). No significant differences were seen in the blood glucose response to nateglinide between the phases. CONCLUSIONS: Fluconazole raised the plasma concentrations and reduced the systemic elimination of nateglinide probably by inhibiting its cytochrome P4502C9-mediated biotransformation. Concomitant use of fluconazole with nateglinide may prolong its blood glucose-lowering effect.