Optimal dosing of cefotaxime and desacetylcefotaxime for critically ill paediatric patients. Can we use microsampling?

OBJECTIVES: To describe the population pharmacokinetics of cefotaxime and desacetylcefotaxime in critically ill paediatric patients and provide dosing recommendations. We also sought to evaluate the use of capillary microsampling to facilitate data-rich blood sampling. METHODS: Patients were recruited into a pharmacokinetic study, with cefotaxime and desacetylcefotaxime concentrations from plasma samples collected at 0, 0.5, 2, 4 and 6 h used to develop a population pharmacokinetic model using Pmetrics. Monte Carlo dosing simulations were tested using a range of estimated glomerular filtration rates (60, 100, 170 and 200 mL/min/1.73 m2) and body weights (4, 10, 15, 20 and 40 kg) to achieve pharmacokinetic/pharmacodynamic (PK/PD) targets, including 100% ƒT>MIC with an MIC breakpoint of 1 mg/L. RESULTS: Thirty-six patients (0.2-12 years) provided 160 conventional samples for inclusion in the model. The pharmacokinetics of cefotaxime and desacetylcefotaxime were best described using one-compartmental model with first-order elimination. The clearance and volume of distribution for cefotaxime were 12.8 L/h and 39.4 L, respectively. The clearance for desacetylcefotaxime was 10.5 L/h. Standard dosing of 50 mg/kg q6h was only able to achieve the PK/PD target of 100% ƒT>MIC in patients >10 kg and with impaired renal function or patients of 40 kg with normal renal function. CONCLUSIONS: Dosing recommendations support the use of extended or continuous infusion to achieve cefotaxime exposure suitable for bacterial killing in critically ill paediatric patients, including those with severe or deep-seated infection. An external validation of capillary microsampling demonstrated skin-prick sampling can facilitate data-rich pharmacokinetic studies.

Microsampling to support pharmacokinetic clinical studies in pediatrics.

BACKGROUND: Conventional sampling for pharmacokinetic clinical studies requires removal of large blood volumes from patients. This can result in a physiological/emotional burden for children. Microsampling to support pharmacokinetic clinical studies in pediatrics may reduce this burden. METHODS: Parents/guardians and bedside nurses completed a questionnaire describing their perception of the use of microsampling compared to conventional sampling to collect blood samples, based on their child's participation or their own role within a paired-sample pharmacokinetic clinical study. Responses were based on a seven-point Likert scale and were analyzed using frequency distributions. RESULTS: Fifty-one parents/guardians and seven bedside nurses completed a questionnaire. Parents/guardians (96%) and bedside nurses (100%) indicated that microsampling was highly acceptable and recommended as a method for collecting blood samples for pediatric patients. Responding to a question about the child indicating pain during the blood sampling procedure, 61% of parent/guardians reported no pain in their children, 14% remained neutral, and 26% reported that their child indicated pain; 71% of the bedside nurses slightly agreed that the children indicated pain. CONCLUSIONS: This study strongly suggests that parents/guardians and bedside nurses prefer microsampling to conventional sampling to conduct pediatric pharmacokinetic clinical studies. Employing microsampling may support increased participation by children in these studies. IMPACT: Pharmacokinetic clinical studies require the withdrawal of blood samples at multiple times during a dosing interval. This can result in a physiological or emotional burden, particularly for neonates or pediatric patients. Microsampling offers an important opportunity for pharmacokinetic clinical studies in vulnerable patient populations, where smaller sample volumes can be collected. However, microsampling is not commonly used in clinical studies. Understanding the perceptions of parents/guardians and bedside nurses about microsampling may ascertain if this technique offers an improvement to conventional blood sample collection to perform pharmacokinetic clinical studies for pediatric patients.

Development and validation of a UHPLC-MS/MS method to measure cefotaxime and metabolite desacetylcefotaxime in blood plasma: a pilot study suitable for capillary microsampling in critically ill children.

Critical illness has been shown to affect the pharmacokinetics of antibiotics, which can lead to ineffective antibiotic exposure and the potential emergence of resistant bacteria. The lack of studies describing antibiotic pharmacokinetics in critically ill children has led to significant off-label dosing. This is, in part, due to the ethical and physiological challenges of removing frequent, large-volume samples from children. Capillary microsampling facilitates the collection of small volumes of blood samples to conduct clinical pharmacokinetic studies. A sensitive, rapid, and accurate ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) bioanalytical method to measure cefotaxime and desacetylcefotaxime in 2.8 µL of plasma was developed and validated. Plasma samples were treated with acetonitrile and analytes were separated using a Kinetex C8 (100 × 2.1 mm) column. The chromatographic separation was established using a gradient method, with the mobile phases consisting of acetonitrile and ammonium acetate. An electrospray ionization source interface operated in a positive mode for the multiple reaction monitoring MS/MS analysis of cefotaxime, desacetylcefotaxime, and deuterated cefotaxime (internal standard). The bioanalytical method using microsample volumes met requirements for method validation for both analytes. Cefotaxime had precision within ± 7.3% and accuracy within ± 5% (concentration range of 0.5 to 500 mg/L). Desacetylcefotaxime had precision within ± 9.5% and accuracy within ± 3.5% (concentration range of 0.2 to 10 mg/L). The bioanalytical method was applied for the quantification of cefotaxime and its metabolite to 20 capillary microsamples collected at five time points in one dosing interval from five critically ill children.

Pharmacodynamics of once- versus twice-daily dosing of nebulized amikacin in an in vitro Hollow-Fiber Infection Model against 3 clinical isolates of Pseudomonas aeruginosa.

This study aims to compare the bacterial killing of once- versus twice-daily nebulized amikacin against Pseudomonas aeruginosa and to determine the optimal duration of therapy. Three clinical P. aeruginosa isolates (amikacin MICs 2, 8, and 64 mg/L) were exposed to simulated epithelial lining fluid exposures of nebulized amikacin with dosing regimens of 400 mg and 800 mg once- or twice-daily up to 7-days using the in vitro hollow-fiber infection model. Quantitative cultures were performed. Simulated amikacin dosing regimens of 400 mg twice-daily and 800 mg once-daily achieved ≥2-log reduction in the bacterial burden within the first 24-hours of therapy for all isolates tested. No dosing regimen suppressed the emergence of amikacin resistance. No difference in bacterial killing or regrowth was observed between 3- and 7-days of amikacin. Amikacin doses of 800 mg once-daily for up to 3-days may be considered for future clinical trials.

Pharmacodynamic Evaluation of Plasma and Epithelial Lining Fluid Exposures of Amikacin against Pseudomonas aeruginosa in a Dynamic In Vitro Hollow-Fiber Infection Model.

Given that aminoglycosides, such as amikacin, may be used for multidrug-resistant Pseudomonas aeruginosa infections, optimization of therapy is paramount for improved treatment outcomes. This study aims to investigate the pharmacodynamics of different simulated intravenous amikacin doses on susceptible P. aeruginosa to inform ventilator-associated pneumonia (VAP) and sepsis treatment choices. A hollow-fiber infection model with two P. aeruginosa isolates (MICs of 2 and 8 mg/liter) with an initial inoculum of ∼108 CFU/ml was used to test different amikacin dosing regimens. Three regimens (15, 25, and 50 mg/kg) were tested to simulate a blood exposure, while a 30 mg/kg regimen simulated the epithelial lining fluid (ELF) for potential respiratory tract infection. Data were described using a semimechanistic pharmacokinetic/pharmacodynamic (PK/PD) model. Whole-genome sequencing was used to identify mutations associated with resistance emergence. While bacterial density was reduced by >6 logs within the first 12 h in simulated blood exposures following this initial bacterial kill, there was amplification of a resistant subpopulation with ribosomal mutations that were likely mediating amikacin resistance. No appreciable bacterial killing occurred with subsequent doses. There was less (<5 log) bacterial killing in the simulated ELF exposure for either isolate tested. Simulation studies suggested that a dose of 30 and 50 mg/kg may provide maximal bacterial killing for bloodstream and VAP infections, respectively. Our results suggest that amikacin efficacy may be improved with the use of high-dose therapy to rapidly eliminate susceptible bacteria. Subsequent doses may have reduced efficacy given the rapid amplification of less-susceptible bacterial subpopulations with amikacin monotherapy.

Analysis of capillary microsamples obtained from a skin-prick to measure vancomycin concentrations as a valid alternative to conventional sampling: A bridging study.

A bridging study is presented to investigate the applicability of measuring vancomycin concentrations obtained by finger-prick. A total of 25 paired plasma samples, collected from finger prick as capillary microsampling and arterial plasma samples collected from an indwelling cannula as conventional sampling, were obtained from critically ill patients receiving vancomycin. The maximum concentration (Cmax) and the minimum concentration (Cmin) measured were 66.2 mg/L and 29.7 mg/L for capillary microsampling and 78.9 mg/L, 25.6 mg/L for conventional sampling, respectively. The area under the concentration-time curve from 0 to 6 h (AUC0-6h) ranged between 94.8 and 269 mg/L.h for capillary microsampling and from 106 and 303 mg/L.h for conventional sampling. The comparative study conducted was assessed using three different statistical approaches: Bland-Altman and Passing-Bablok regression analyses and the USFDA criterion for the incurred sample reanalysis. The results of this analysis revealed no significant bias and a strong correlation between both sampling methods, with 95% of the calculated concentrations from the paired plasma samples laying within 20% of difference of the mean. This bridging study verifies that capillary microsampling may serve as an alternative to conventional sampling techniques to support clinical applications for measuring vancomycin concentrations in plasma.

In-vitro adsorption and sieving coefficient of ticarcillin-clavulanate during continuous haemofiltration.

There are very limited data on ticarcillin-clavulanate elimination by haemofiltration. We measured in vitro ticarcillin-clavulanate adsorption to polyacrylonitrile (PAN) filters and the sieving coefficient using a well-described bench model of haemofiltration. The dose of ticarcillin-clavulanate was 60/2 mg or 180/3 mg, and 0 or 12 g albumin was added to the 1 L of circulating blood-crystalloid mixture to produce four different experimental conditions. The experiment was repeated four times under each condition. Median (interquartile range [IQR] ) ticarcillin adsorption varied from 28 (27-30) mg to 85 (78-90) mg. Adsorption was increased when the dose of ticarcillin was higher (P<0.001), but was not affected by the addition of albumin. Median (IQR) adsorption of clavulanate ranged from 0.67 (0.55-0.75) mg to 1.8 (0.33-3.5) mg and was neither dose dependent (P = 0.505) nor significantly affected by the addition of albumin. Median (IQR) ticarcillin sieving coefficient ranged from 0.73 (0.67-0.75) to 0.99 (0.97-1.03). It was significantly higher with a higher dose of ticarcillin (P = 0.021) and without addition of albumin (P = 0.015). Median (IQR) clavulanate sieving coefficient ranged from 1.03 (1.00-2.24) to 2.0 (1.98-2.47). Clavulanate sieving coefficient was not significantly affected by dose or the addition of albumin. These data indicate that significant adsorption of both ticarcillin and clavulanate occurs in vitro; however, this requires confirmation by clinical pharmacokinetic studies. The sieving coefficient data may help guide appropriate dosing of critically ill patients receiving haemofiltration until more extensive clinical pharmacokinetic data are available.

Clinical application of microsampling versus conventional sampling techniques in the quantitative bioanalysis of antibiotics: a systematic review.

Conventional sampling techniques for clinical pharmacokinetic studies often require the removal of large blood volumes from patients. This can result in a physiological or emotional burden, particularly for neonates or pediatric patients. Antibiotic pharmacokinetic studies are typically performed on healthy adults or general ward patients. These may not account for alterations to a patient's pathophysiology and can lead to suboptimal treatment. Microsampling offers an important opportunity for clinical pharmacokinetic studies in vulnerable patient populations, where smaller sample volumes can be collected. This systematic review provides a description of currently available microsampling techniques and an overview of studies reporting the quantitation and validation of antibiotics using microsampling. A comparison of microsampling to conventional sampling in clinical studies is included.

A UHPLC-MS/MS method for the simultaneous determination of piperacillin and tazobactam in plasma (total and unbound), urine and renal replacement therapy effluent.

Piperacillin-tazobactam is a beta-lactam/beta-lactamase combination antibiotic used in patients with moderate to severe infection. Dosing of piperacillin-tazobactam requires an understanding of this patient group to maximise the effectiveness of this antibiotic and limit a further emergence of resistant pathogens. This is the first method that measures piperacillin and tazobactam simultaneously, across this range of clinically-relevant biological matrices. The calibration line was linear across the concentration range of 0.5-500µg/mL for piperacillin and 0.625-62.5µg/mL for tazobactam. All validation testing for matrix effects, precision and accuracy, specificity and stability were within 15%. A calibration equivalence study was performed to investigate the suitability of applying calibration curves prepared in an alternative matrix, with a mean bias of -10.8% identified for the application of a calibration line prepared for tazobactam in plasma only. Bias for all other calibration lines prepared in alternate matrices was within the 5% acceptance criteria. The method was successfully applied to a pharmacokinetic study of a critically ill patient receiving renal replacement therapy, with the results included.

An LC-MS/MS method to determine vancomycin in plasma (total and unbound), urine and renal replacement therapy effluent.

AIM: Critical illness and medical interventions, such as renal replacement therapy, can cause changes to vancomycin pharmacokinetics and lead to suboptimal dosing. To comprehensively characterize vancomycin pharmacokinetic a method must measure vancomycin in a range of clinical matrices. RESULTS: A LC-MS/MS method was developed using hydrophilic interaction liquid chromatography and microsample volumes, where possible. For all matrices, the linear concentration range was 1-100 µg/ml, interassay accuracy and precision was within 15%, and recovery above 80%. No matrix effects were observed. Calibration equivalence may be applied for some matrix combinations. CONCLUSION: A method for the analysis of vancomycin in plasma (total, unbound), urine and renal replacement therapy effluent, suitable for use in any patient pharmacokinetic study, has been developed and validated.

A validated UHPLC-MS/MS method for the measurement of riluzole in plasma and myocardial tissue samples.

Through blocking the cardiac persistent sodium current, riluzole has the potential to prevent myocardial damage post cardiac bypass surgery. A sensitive UHPLC-MS/MS method was developed and validated for quantitation of riluzole and 5-methoxypsoralen in human plasma and myocardial tissue homogenate using a liquid-liquid extraction with dichloromethane. The chromatographic separation was achieved using Shimadzu Shim-pack XR-ODS III, 2.0 × 50 mm, 1.6 µm column with a gradient mobile phase comprising methanol and ammonium acetate buffer pH 3.6 in purified water. The analyte and internal standard were separated within 3.5 min. Riluzole quantitation was achieved using the mass transitions of 235-138 for riluzole and 217-156 for 5-methoxypsoralen. The method was linear for riluzole plasma concentrations from 0.2 to 500 ng/mL and myocardial tissue homogenate concentrations from 0.2 to 100 ng/mL. The method developed was successfully applied to a clinical study for patients receiving riluzole while undergoing cardiac bypass surgery.

Effect of time on recovery of plasma microsamples for the quantitative determination of vancomycin.

The reliability of extraction recovery of an analyte in bioanalysis is fundamentally important for downstream analytical testing. For dried format microsamples, if the recovery changes with time the concentration in clinical samples, derived from calibration standards and alongside quality control samples prepared following different drying protocols, may not reflect the true result. The purpose of this paper was therefore to evaluate changes to extraction recovery across time for one analyte, the glycopeptide antibiotic vancomycin, in plasma using two dried microsampling formats, dried plasma spots and volumetric absorptive microsampling.

Determination of Cefalothin and Cefazolin in Human Plasma, Urine and Peritoneal Dialysate by UHPLC-MS/MS: application to a pilot pharmacokinetic study in humans.

An ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the analysis of cefazolin and cefalothin in human plasma (total and unbound), urine and peritoneal dialysate has been developed and validated. Total plasma concentrations are measured following protein precipitation and are suitable for the concentration range of 1-500 µg/mL. Unbound concentrations are measured from ultra-filtered plasma acquired using Centrifree(®) devices and are suitable for the concentration range of 0.1-500 µg/mL for cefazolin and 1-500 µg/mL for cefalothin. The urine method is suitable for a concentration range of 0.1-20 mg/mL for cefazolin and 0.2-20 mg/mL for cefalothin. Peritoneal dialysate concentrations are measured using direct injection, and are suitable for the concentration range of 0.2-100 µg/mL for both cefazolin and cefalothin. The cefazolin and cefalothin plasma (total and unbound), urine and peritoneal dialysate results are reported for recovery, inter-assay precision and accuracy, and the lower limit of quantification, linearity, stability and matrix effects, with all results meeting acceptance criteria. The method was used successfully in a pilot pharmacokinetic study with patients with peritoneal dialysis-associated peritonitis, receiving either intraperitoneal cefazolin or cefalothin. Copyright © 2015 John Wiley & Sons, Ltd.