Cefazolin Binding Research Articles

Interspecies variability in protein binding of antibiotics basis for translational PK/PD studies-a case study using cefazolin.

For antimicrobial agents in particular, plasma protein binding (PPB) plays a pivotal role in deciphering key properties of drug candidates. Animal models are generally used in the preclinical development of new drugs to predict their effects in humans using translational pharmacokinetics/pharmacodynamics (PK/PD). Thus, we compared the protein binding (PB) of cefazolin as well as bacterial growth under various conditions in vitro. The PB extent of cefazolin was studied in human, bovine, and rat plasmas at different antibiotic concentrations in buffer and media containing 20-70% plasma or pure plasma using ultrafiltration (UF) and equilibrium dialysis (ED). Moreover, bacterial growth and time-kill assays were performed in Mueller Hinton Broth (MHB) containing various plasma percentages. The pattern for cefazolin binding to plasma proteins was found to be similar for both UF and ED. There was a significant decrease in cefazolin binding to bovine plasma compared to human plasma, whereas the pattern in rat plasma was more consistent with that in human plasma. Our growth curve analysis revealed considerable growth inhibition of Escherichia coli at 70% bovine or rat plasma compared with 70% human plasma or pure MHB. As expected, our experiments with cefazolin at low concentrations showed that E. coli grew slightly better in 20% human and rat plasma compared to MHB, most probably due to cefazolin binding to proteins in the plasma. Based on the example of cefazolin, our study highlights the interspecies differences of PB with potential impact on PK/PD. These findings should be considered before preclinical PK/PD data can be extrapolated to human patients.

Adequate cefazolin therapy for critically ill patients: can we predict active concentrations from given protein-binding data?

Abstract Objectives Therapeutic drug monitoring of β-lactam antibiotics has become an important tool for treatment of severe infections, especially for critically ill patients who often exhibit altered PK/PD. Therapeutic targets are based on MIC, which refers to the active concentration of the drug. Cefazolin, a β-lactam agent used for treating of MSSA bacteraemia, has a protein binding of approximately 80 %. Therefore, a reliable determination of the active, non-protein-bound concentration is required to ensure optimal therapeutic outcome. Methods From seven critically ill patients who received an initial dose of 2 g cefazolin, followed by a continuous 24 h infusion, a total of 24 serum samples were obtained. The non-protein-bound concentration was directly measured after ultrafiltration and compared to prediction based total concentrations and protein binding values from the literature. For the analysis, a rapid and reliable LC-MS3 based assay was established, offering maximum sensitivity and specificity. Results The measured non-protein-bound concentration varied over a wide range (7.6–118 mg/L), with 22 out of 24 samples exhibiting cefazolin levels above the therapeutic target values (8–16 mg/L). Additionally, the observed protein binding ranged from 29 to 78 % (median 66.8 %), which was significantly lower than that reported in the literature. When comparing the measurements to the predictive performance of total concentrations and protein binding values, poor results were obtained. Conclusions The results show a high variability in plasma protein binding of cefazolin in critically ill patients. Therefore, the “one-dose-fits-all” principle can no longer be considered up to date. For personalised cefazolin therapy based on therapeutic drug monitoring (TDM) it is recommended to determine the active, non-protein-bound drug concentration, as calculations from the total fraction yield poor results.

Post-operative agranulocytosis caused by intravenous cefazolin: A case report with a discussion of the pathogenesis

A case of post-operative agranulocytosis which occurred in a 66-year-old woman following surgery for endometrial carcinoma is reported. The agranulocytosis had a rapid onset, being detected on the first post-operative day. The causative agent, cefazolin was given to the patient intraoperatively. The agranulocytosis persisted until the 22nd postoperative day. A bone marrow biopsy performed on post-operative day four showed a left-shifted myeloid maturation pattern but not a maturation arrest. The pathogenesis of drug-induced neutropenia/agranulocytosis is discussed. It is postulated that reversible binding of cefazolin to albumin accounts for the prolonged duration of agranulocytosis.

The anti-inflammatory potential of cefazolin as common gamma chain cytokine inhibitor

A continuing quest for specific inhibitors of proinflammatory cytokines brings promise for effective therapies designed for inflammatory and autoimmune disorders. Cefazolin, a safe, first-generation cephalosporin antibiotic, has been recently shown to specifically interact with interleukin 15 (IL-15) receptor subunit α (IL-15Rα) and to inhibit IL-15-dependent TNF-α and IL-17 synthesis. The aim of this study was to elucidate cefazolin activity against IL-2, IL-4, IL-15 and IL-21, i.e. four cytokines sharing the common cytokine receptor γ chain (γc). In silico, molecular docking unveiled two potential cefazolin binding sites within the IL-2/IL-15Rβ subunit and two within the γc subunit. In vitro, cefazolin decreased proliferation of PBMC (peripheral blood mononuclear cells) following IL-2, IL-4 and IL-15 stimulation, reduced production of IFN-γ, IL-17 and TNF-α in IL-2- and IL-15-treated PBMC and in IL-15 stimulated natural killer (NK) cells, attenuated IL-4-dependent expression of CD11c in monocyte-derived dendritic cells and suppressed phosphorylation of JAK3 in response to IL-2 and IL-15 in PBMC, to IL-4 in TF-1 (erythroleukemic cell line) and to IL-21 in NK-92 (NK cell line). The results of the study suggest that cefazolin may exert inhibitory activity against all of the γc receptor-dependent cytokines, i.e. IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21.

Effects of cardiopulmonary bypass on the disposition of cefazolin in patients undergoing cardiothoracic surgery.

The aim of the study was to evaluate the disposition of plasma unbound cefazolin in patients undergoing cardiothoracic surgery with cardiopulmonary bypass (CPB). Adult patients undergoing cardiothoracic surgery with CPB were enrolled in the study. Cefazolin sodium was given intravenously before skin incision (1 g) and at the beginning of CPB (2 g). Thereafter, an additional dose (1 g) was given every 4 hours. Seven to ten blood samples were collected before and during surgery. Plasma total and unbound (ultrafiltrated) cefazolin concentrations were analyzed using an HPLC‐UV method. Plasma protein binding was analyzed with the Langmuir model. Twenty‐seven patients (aged 70 ± 12 years, body weight 62 ± 12 kg, mean ± SD) with GFR >30 mL min−1 completed the study. There was a significant (P < 0.001) increase in median plasma unbound fraction of cefazolin from 21% before skin incision to 45% during CPB (P < 0.001), which was accompanied by a significant (P < 0.001) reduction in median plasma albumin concentration from 36 to 27 g L−1. Plasma concentrations of unbound cefazolin exceeded the assumed target thresholds of 2 μg mL−1 in all samples and of 8 μg mL−1 in all but one of 199 samples. The increased plasma unbound fraction of cefazolin would be attributable to dilutional reduction of serum albumin at the beginning of CPB and to saturable plasma protein binding of cefazolin. These data reveal CPB may alter the plasma protein binding and possibly distribution of cefazolin. Further studies are warranted to reappraise the protocol of antimicrobial prophylaxis with cefazolin in patients undergoing surgery with CPB.

Population Pharmacokinetic Study of Cefazolin Used Prophylactically in Canine Surgery for Susceptibility Testing Breakpoint Determination.

This study aimed to determine the population pharmacokinetic (Pop PK) parameters of cefazolin administered prophylactically at 25 mg/kg intravenously (IV) 30 min before surgery in a canine population of 78 dogs and assess whether covariates, such as sex, age, body weight (BW), breed, health status, creatinine level, and surgery time, have an influence on cefazolin disposition. The ultimate goal was to compute PK/PD cut off values and subsequently establish a specific clinical breakpoint (CBP) for the development of an antimicrobial susceptibility test (AST) of cefazolin in dogs according to the VetCAST approach. Two to 11 blood samples were collected from each dog from 5 to 480 min after cefazolin administration. A two-compartment model was selected, and parameterization was in terms of serum clearance (CL), intercompartmental CL(s) (Q) and volume(s) of distribution (V). The percentage of cefazolin binding to serum protein was 36.2 ± 5.3%. Population primary parameter estimates V1, V2, CL, and Q were (typical value ± SE) 0.116 ± 0.013 L/kg, 0.177 ± 0.011 L/kg, 0.0037 ± 0.0002 L/kg/min, and 0.0103 ± 0.0013 L/kg/min, respectively. Cefazolin presented rapid distribution and elimination half-lives (mean ± SE) 4.17 ± 0.77 min and 57.93 ± 3.11 min, respectively. The overall between-subject variability (BSV) for estimated primary parameters ranged from 36 to 42%, and none of the seven explored covariates were able to reduce this variability by an amplitude clinically relevant. By Monte Carlo simulation, the probability of a PK/PD target attainment (here to achieve a free serum concentration exceeding the MIC for 50% of the dosing interval in 90% of dogs) was computed with a dosage of 25 mg/kg administered IV every 6 h for 4 administrations in 24 h. The computed PK/PD cut off value was 2 mg/L. In conclusion, cefazolin administered prophylactically in surgical dogs at 25 mg/kg IV every 6 h was deemed effective against pathogens with a MIC value ≤ 2 mg/L and from a PK/PD perspective, can be recommended in a wide range of canine patient populations with no necessary dose adjustment for special dog subpopulations.

Pharmacokinetics and protein binding of cefazolin in morbidly obese patients

The aim of this study was to assess the pharmacokinetics and protein binding of cefazolin in morbidly obese patients undergoing bariatric surgery, to study the influence of bodyweight measures and age on pharmacokinetic parameters and to evaluate unbound cefazolin concentrations over time in this population. Twenty morbidly obese patients (bodyweight 112-260 kg, body mass index 38-79 kg m(-2)) were studied following the administration of cefazolin 2 g at induction of anaesthesia. Blood samples were collected up to 4 h post-dosing to determine total and unbound plasma cefazolin concentrations. Non-compartmental pharmacokinetic data analysis was performed. Cefazolin clearance was 4.2 ± 1.0 L h(-1) (mean ± standard deviation) and showed a negative correlation with age (p = 0.003) but not with bodyweight measures (p > 0.05). Volume of distribution was 13.0 ± 3.1 L and correlated positively with bodyweight measures (p ≤ 0.001). Saturable protein binding was observed with a median protein binding of 79% (interquartile range 74-82), which proved similar to reported protein binding in non-obese patients. In all patients, unbound cefazolin concentrations remained above 1 mg L(-1) (minimal inhibitory concentration for 90% (MIC(90)) of methicillin-sensitive isolates of Staphylococcus aureus in Europe) until 4 h post-dosing. Younger age--and not bodyweight--was significantly associated with higher cefazolin clearance. However, as in all patients with bodyweights up to 260 kg, unbound plasma cefazolin concentrations remained above 1 mg L(-1) until 4 h after the intravenous administration of a 2-g dose. As such, re-dosing within 4 h or dosing with another antibiotic class should only be considered in the case of a higher MIC(90) of the local isolates.

Protein binding of cefazolin is saturable in vivo both between and within patients

The aims of the study were a) to determine if there is evidence of saturable protein binding of cefazolin in plasma across the range of concentrations achieved clinically (between patient variability) and b) to investigate whether saturable protein binding is also evident from trough and peak concentrations in the same patient (within patient variability). Unbound and total plasma concentrations were measured in patients who were treated with cefazolin intravenously by continuous infusion or intermittent injection. In study (i) single random samples were taken from one series of patients. In study (ii) paired samples (troughs and peaks) were taken from a second series of patients. Thirty-one patients were included in study (i). Linear regression analysis of the percentage unbound vs. unbound plasma concentrations revealed a slope significantly different from zero, suggesting saturable protein binding. Mean values for percentage unbound ranged from 9% at low concentrations (8.5 mg l(-1)) to 51% at high concentrations (140 mg l(-1)). Twelve patients were investigated in study (ii). Values for protein binding ranged from 85% at low concentrations (2.7 mg l(-1)) to 52% at high concentrations (200.3 mg l(-1)). The percentage unbound was significantly higher (P < 0.0001) at high (peak) concentrations than at lower (trough) concentrations, confirming saturable protein binding. The protein binding of cefazolin is saturable in vivo in humans, both between and within patients.

Penetration of antibiotics into the surgical wound in a canine model.

The dose and timing of antimicrobial agents given for surgical wound prophylaxis should be based on the concentration-time profile of the drug in tissue at the site of contamination. However, concentrations of antimicrobial agents in surgical wounds are difficult to determine accurately. Since a surgical wound is a unique extravascular compartment with increased vascular permeability and a high surface area/volume ratio, antibiotic concentrations in sera and surgical wounds should be similar. To test this hypothesis, the pharmacokinetics of single intravenous doses of cefazolin (40 mg/kg) and gentamicin (4 mg/kg) in sera and surgical wounds in a clinically relevant surgical model using dogs were compared. Drug concentrations were determined in interstitial fluid in muscle biopsies taken randomly from wound surfaces and serial wound fluid samples collected after the incisions were closed. Protein binding of cefazolin and gentamicin in sera and wound fluids was low (less than or equal to 29 +/- 9%) in this canine model. Cefazolin and gentamicin equilibrated rapidly (less than or equal to 30 min) between serum and the surgical wound, and concentrations in the two sites declined in parallel. Values for the area under the concentration-time curve, mean residence time, and terminal half-life in serum and the surgical site for each drug were similar. Cefazolin concentrations in serum underestimated the time during which concentrations in surgical wounds exceeded the susceptibility breakpoint MIC for important pathogens by an average of 58 min (range, 26 to 109 min; P = 0.036); for gentamicin, the underestimation averaged 30 min (range, 10 to 60 min; P = 0.036). These data support the concept that the concentration-time profiles of antimicrobial agents in serum may prove valuable clinically as guides to determining the and timing of antibiotic administration necessary for effective antimicrobial prophylaxis in surgery. Further studies are needed to determine the surgical wound pharmacokinetics of highly protein-bound antibodies.

Cefazolin serum protein binding and its inhibition by bilirubin, fatty acids and other drugs

This paper describes the protein binding of cefazolin to human serum and to human serum albumin (HSA) using equilibrium dialysis. The drug is exclusively bound to HSA with a moderate affinity, K a = 16600 ± 1600 M −1, and one saturable binding site, n = 0.73 ± 0.02. Moreover cefazolin shows a dose-dependent binding leading a possible increase of the free fraction (when its total concentration increases). This antibiotic is displaced by free fatty acids (FFA) and bilirubin. Cefazolin binding to human serum and human serum albumin (HSA) was studied in presence of acidic drugs. At low concentrations clofibric acid and phenylbutazone both exhibiting high affinity for HSA displace strongly cefazolin. Valproic and salicylic acids, sulfamethoxazole, cefoperazone which have approximately the same affinity as cefazolin, must be used at higher concentrations to displace this antibiotic. A particular phenomenon was observed with cefazolin on HSA when associated with furosemide. A low concentration (5–25 μM) of this drug induces a positive cooperativity of binding between cefazolin and HSA. But at a molar ratio of furosemide to albumin greater than one, such cooperative interaction disappears and a competitive inhibition of cefazolin binding occurs. For all drugs studied, a competitive inhibition was found except for tryptophan. Finally, it is concluded that cefazolin shares the warfarin binding site on HSA.

Age-related change of cefazolin binding to rat serum proteins and its relation to the molar ratio of free fatty acid to serum albumin.

The binding of cefazolin to rat sera has been studied as a function of age. A significant difference was observed in the cefazolin binding to serum protein among 1-, 2-, 3-, 5-, 7-, 50- and 100-week-old rats. There was a good correlation between the dissociation constants of cefazolin binding and the molar ratio of free fatty acid to albumin concentration in sera. This suggests that both changes of concentration of albumin and free fatty acid, which could be a major endogenous inhibitor of cefazolin binding, play an important role in the age-related changes of the serum protein binding. Removal of free fatty acid in 1- and 2-week-old rat sera showed marked increases of the cefazolin binding. On the contrary, addition of oleic acid to 7-week-old rat serum produced significant reduction of cefazolin binding to rat serum protein. Accordingly, free fatty acid could effectively inhibit the cefazolin binding in the physiological concentration range with increasing age, and the age-related changes of cefazolin binding to rat serum protein appear to be due to the fluctuation of the molar ratios of free fatty acid to albumin concentration in sera.

Pharmacokinetics and protein binding of cefazolin and cephalothin in patients with cirrhosis.

The effects of liver disease on the pharmacokinetics and protein binding of cefazolin and cephalothin were studied in patients with cirrhosis, chronic active hepatitis or normal liver function. The T1/2 and mean residence time of cefazolin were significantly shorter in cirrhosis. Cephalothin clearance was decreased by cirrhosis. Plasma protein binding of cefazolin, but not cephalothin was significantly reduced in cirrhosis. It is suggested that no dose reduction is necessary for either drug in severe hepatic impairment.

The interaction of cephalosporin antibiotics with renal cortex of rats: Accumulation to cortical slices and binding to purified plasma membranes

The interaction of cephalosporin antibiotics with renal cortex of rats has been examined in vitro by means of cortical slice uptake and binding assay to plasma membranes and other subcellular fractions, including the comparative study with p-aminohippurate. The uptakes of cephalexin and cephaloridine by cortical slices were concentrative, and were strongly inhibited in the presence of 2, 4-dinitrophenol, ouabain, nitrogen gas and probenecid. In the case of cefazolin, the degree of concentrative uptake and influence of inhibitors were low. Cephalexin, cephaloridine and p-aminohippurate specifically bound to the basolateral membranes, compared to brush border membranes and other subcellular fractions. Cefazolin binding to basolateral membranes was relatively small. These results suggest that the specificity of cephalexin and cephaloridine bindings to the basolateral membranes could be related to the interaction with the organic acid transport system at the antiluminal side. Thus, an examination of cephalosporin interaction with plasma membranes from renal cortex could offer an appropriate in vitro model system to study the renal transport of these antibiotics.

Pharmacokinetics and Comparative Pharmacology of Cefoxitin and Cephalosporins

Features of the distribution, metabolism, elimination, and pharmacokinetics of the cephalosporins and cefoxitin must be considered when concentrations of these drugs in biological fluids are interpreted. The extensive (approximately 86%) binding of cefazolin to plasma protein may account for the smaller volume of distribution and slower rate of renal clearance than are observed for cefoxitin, which is less extensively (73%) bound to protein. Results of microbiological assays of drug in urine may be influenced by the extent of metabolism of the drugs, which is 33% for cephalothin but less than 2% for cefoxitin. Elimination of cephalosporins and cefoxitin occurs by both glomerular filtration and tubular secretion and can be inhibited by the concurrent administration of probenecid. The pharmacokinetics of cefoxitin may be described by a linear, two-compartment, open model that has been used to predict levels of drug achieved in serum and urine after various dose regimens, including administration by intravenous bolus or infusion. The bioavailability of intramuscularly administered cefoxitin is equivalent to that of intravenously administered cefoxitin and is 90% complete within 3-4 hr after the dose is given.

Concentrations of cefazolin in human skeletal muscle.

Tissue concentrations of cefazolin were measured in 18 patients undergoing urological operations. Specimens of skeletal muscle and blood were taken simultaneously at predetermined intervals. The concentrations in serum and homogenized skeletal muscle were determined by means of the agar well diffusion method. A comparison of standard curves obtained with phosphate buffer solution and the supernatant of muscle homogenate revealed no substantial binding of cefazolin to the supernatant of muscle homogenate. Because of methodological difficulties in determining the different compartments of a tissue specimen concentrations in tissue were not corrected. Within two hours after a short infusion (25 min.) of 2 g of Cefazolin in muscle tissue a peak concentration of 20 mug/g was reached. A tissue level above 10 mug/g maintained for three hours. This concentration is high enough to inhibit nearly all strains of E. coli, Klebsiella, Salmonella, Shigella and a large part of Proteus mirabilis, whereas nearly all gram-positive bacteria are inhibited at a lower concentration.

Protein Binding and Concentrations of Cephaloridine and Cefazolin in Serum and Interstitial Fluid of Dogs

Percentages of free and protein-bound cefazolin and cephaloridine in serum and interstitial fluid of dogs were determined by ultrafiltration and microbiologic assay. The percentages of cephaloridine and cefazolin bound to protein in serum were 10% and 80%, respectively. In interstitial fluid accumulating within tissue-embedded polypropylene capsules, 29% of cefazolin was bound to protein, and cephaloridine was unbound. Both antibiotics rapidly penetrated the interstitial fluid and attained measurable levels 5 min after intravenous administration. Levels of total cefazolin in the interstitial fluid were generally higher than those of cephaloridine; however, concentrations of free cephaloridine in the fluid exceeded the levels of free cefazolin after the first 15 min. Binding of anitbiotics by serum proteins does not restrict such agents to the intravascular space, since a highly protein-bound compound has been shown to penetrate interstitial fluid as readily as one that is minimally bound. It should be noted, however, that this penetration may be due primarily to the slow rate of binding of cefazolin to serum proteins.