Clinical Validation of Therapeutic Drug Monitoring of Imipenem in Spent Effluent in Critically Ill Patients Receiving Continuous Renal Replacement Therapy: A Pilot Study

Objectives The primary objective of this pilot study was to investigate whether the therapeutic drug monitoring of imipenem could be performed with spent effluent instead of blood sampling collected from critically ill patients under continuous renal replacement therapy. Methods A prospective open-label study was conducted in a real clinical setting. Both blood and effluent samples were collected pairwise before imipenem administration and 0.5, 1, 1.5, 2, 3, 4, 6, and 8 h after imipenem administration. Plasma and effluent imipenem concentrations were determined by reversed-phase high-performance liquid chromatography with ultraviolet detection. Pharmacokinetic and pharmacodynamic parameters of blood and effluent samples were calculated. Results Eighty-three paired plasma and effluent samples were obtained from 10 patients. The Pearson correlation coefficient of the imipenem concentrations in plasma and effluent was 0.950 (P<0.0001). The average plasma-to-effluent imipenem concentration ratio was 1.044 (95% confidence interval, 0.975 to 1.114) with Bland-Altman analysis. No statistically significant difference was found in the pharmacokinetic and pharmacodynamic parameters tested in paired plasma and effluent samples with Wilcoxon test. Conclusion Spent effluent of continuous renal replacement therapy could be used for therapeutic drug monitoring of imipenem instead of blood sampling in critically ill patients.


Introduction
were met: 1) adult; 2) admission to ICU; 3) treatment with imipenem and CRRT simultaneously. Patients who were judged inappropriate to get frequent blood samples by the attending physician were excluded from this study. The study protocol was approved by the Research Ethics Committee of Beijing Friendship Hospital. All of the eligible patients or their legal guardians were informed the essentials of this study, and written informed consent was obtained before enrolling each patient into this study.

Medications
Patients enrolled into this study received imipenem-cilastatin (500mg/500mg, Merck Sharp & Dohme Corp., USA) as part of their medical care. Dosage regimen was determined by the attending physicians according to clinical indication and institutional dosing guidelines. Dosages represented by the quantity of imipenem of 500 mg every 6 h, 500 mg every 8 h and 1 g every 8 h were commonly prescribed. Both of the doses of 500mg and 1 g were suspended and transferred to 100 ml of an appropriate infusion solution, and administered by intravenous infusion pump over 1 h. Detailed dosage regimen and specific administration time were recorded for each enrolled patient.

CRRT Procedures
All patients were treated with CRRT using the Gambro PrismaFlex system with Gambro Prismaflex M100 AN-69 hemofilter sets. Vascular access was obtained by introduction of an ARROW Two-Lumen Hemodialysis Catheterization Set (12Fr, 20cm, Arrow International Inc., USA) into the jugular vein or an ARROW Large-Bore Multi-lumen Central Venous Catheterization Set (12Fr, 16cm, Arrow International Inc., USA) into the femoral vein. The type and dosing of CRRT were managed by the attending physicians. The parameters such as blood flow rate, dialysate rate and ultrafiltration rate were set and adjusted by therapeutic needs. Replacement fluids were delivered by a combination of pre and post filter. The extracorporeal circuit was anticoagulated with sodiumcitrate. The uptime of the hemofilter sets was generally 24-48 h. The types and parameters of CRRT were recorded for each patient in detail. Urine output was obtained from the nursing records.

Sample Collection and Storage
In order to obtain plasma imipenem concentrations at or near steady state, sampling was performed at least 24 h after initiation of the CRRT and imipenem therapy [35]. Blood and effluent samples were collected pairwise before and 0.5, 1, 1.5, 2, 3, 4, 6, and 8 h (for the dose of 1 g every 8 h) after imipenem administration. Blood samples (0.6 mL) were drawn from the red access port of the central line for CRRT, and collected into tubes containing heparin as anticoagulant. Effluent samples (approximately 1.2 mL) were collected from the CRRT apparatus directly into polypropylene tubes. All of the samples were placed in ice box immediately, and processed within 2 h. Because imipenem is rapidly hydrolyzed in plasma through a pH-dependent reaction [36], morpholinopropanesulfonic acid (MOPS, ultra pure grade; Amresco, USA) buffer (0.126 M, pH 6.8) was served as stabilizing solution. Blood samples were centrifuged (3,000×g, 10 min) and aliquots of plasma were mixed 1:1 with stabilizing solution [37]. Effluent samples were directly mixed 1:1 with stabilizing solution. Stabilized plasma and effluent samples were stored at -70°C until analysis. If CRRT was interrupted, sampling was halted, and resampling was allowed after CRRT was reestablished. Complete profiles of medical history, physical examination and laboratory tests were obtained and reviewed prior to collection of samples.

Sample Analysis and Validation
Analysis of imipenem concentrations in plasma and effluent were based on previously validated high performance liquid chromatography-ultraviolet detection (HPLC-UV) methods [37][38][39] with a few modifications. Chromatography was performed on a ZORBAX SB-C8 column (5μm, 4.6 x 250 mm; Agilent, USA), maintained at 30°C. The ultraviolet detector was set at 298 nm. A gradient elution of ammonium acetate buffer (0.5 M, pH 6.8) and acetonitrile was used as the mobile phase with a flow rate of 1 mL/min. Imipenem monohydrate (I1K226, 0.932 mg/mg; USP) was used for the preparation of standard solutions, and ceftazidime was used as the internal standard.
Two hundred microliters of stabilized plasma and effluent samples were mixed with 20 μL of 500 μg/mL ceftazidime, respectively. The effluent samples were injected directly into the HPLC system. The plasma samples were deproteinized using Amicon Ultra-0.5 centrifugal filters (3 kDa, Millipore, USA), and the filtrates were injected into the HPLC system.
The lower limit of quantification (LOQ) was 0.3 μg/mL for both of the plasma and effluent samples. The linearity of the standard curve was assessed with 1/x 2 weighting over a concentration range of 0.3 to 200.0 μg/mL. Accuracy and precision were evaluated with quality control samples at concentrations of 0.5, 2.0, 30.0, and 75.0 μg/mL in triplicate. Stability was assessed by storing stabilized quality control samples (2.0, 30.0, and 75.0 μg/mL) at −70°C and 20°C for 30 days and 6 h, respectively. The stability of patients' samples stored in ice box (2-8°C) for 2 h was also evaluated.

Pharmacokinetic and Pharmacodynamic Analysis
Pharmacokinetic analysis was performed by Drug and Statistics (DAS, version 2.0, Mathematical Pharmacology Professional Committee of China, Shanghai, China) using non-compartment model [40]. As a β-lactam, the pharmacodynamic predictor of clinical efficacy and risk of developing microbial resistance to imipenem is commonly indicated by the percentage of free drug concentrations remain above the minimum inhibitory concentration (MIC) of the pathogen (f T > MIC), and a target of at least 40% was recommended [7,[41][42][43]. For critically ill patients, the pharmacodynamic predictor of the β-lactams was not clearly defined. It was indicated that f T > 4-5 × MIC could maximize the likelihood of clinical cure in patients with severe infections [19,20], and the target of at least of 60% is suggested for bolus infusion [44]. Therefore, both of f T>MIC and f T > 4 × MIC were calculated according to the methods of Fish, et al [35]. For the patient without an identified pathogen or without the MIC for imipenem, the pharmacodynamic predictor was not calculated.

Statistical Analysis
In the process of analytical method validation, bias was defined as the difference between the analytical concentrations and the nominal concentrations, expressed as a percentage. The effluent analysis was clinically validated by comparing the imipenem concentrations in paired plasma and effluent samples from patients by Passing-Bablok regression and Bland-Altman analysis using MedCalc, version 11.4.2 (MedCalc Software, Ostend, Belgium). Furthermore, the Pearson correlation was calculated to determine the correlation between the concentrations in plasma and effluent samples. The Wilcoxon test for paired samples was applied to the comparisons of pharmacokinetic and pharmacodynamic parameters. A P value of <0.05 was considered statistically significant.

Subject Characteristics
A total of 10 patients were enrolled in this study and completed the scheduled sampling. The median age and weight of the patients were 64 years (range, 32 to 87 years) and 70 kg (range, 45-90 kg), and 8 patients (80.0%) were male. The characteristics of the patients are summarized in Table 1. Eight of the patients were diagnosed with severe sepsis, and the other two patients were diagnosed with serious abdominal infection and severe acute pancreatitis, respectively. Eight of the patients were diagnosed with acute kidney injury. Half of the patients were with hypoalbuminaemia. The isolated pathogens were Enterobacter aerogenes in 2 patients, Acinetobacter baumannii in 2 patients (20.0%), Escherichia coli in 2 patients (20.0%), Klebsiella pneumoniae in 1 patient (10.0%), Pseudomonas aeruginosa in 1 patient (10.0%), and not specified in 3 patients (30.0%). The dosages of imipenem were 500 mg every 6 h in 7 patients (70.0%), 1 g every 8 h in 2 patients (20.0%) and 0.5 g every 8 h in 1 patient (10.0%).
Urine output and details of CRRT of the patients were illustrated in Table 2. Eight patients received CRRT because of AKI, two for severe sepsis. Nine patients were treated with CVVHDF, and one with CVVH.

Validation of Analytical Methods
The plasma and effluent analytical method showed a good linearity over the imipenem concentration range. Correlation coefficients were within the range of 0.9990 to 1.0000 for the entire process of analysis. The mean measured concentrations were between 93.96% and 105.25% of the nominal concentration for plasma and between 97.24% and 106.48% for effluent. The within-day variations were between 1.04% and 3.03% for plasma and between 0.31% and 1.73% for effluent, and the day-to-day variations were between 0.63% and 5.11% for plasma and between 0.63% and 1.13% for effluent. Both stabilized plasma and effluent samples were stable at −70°C and 20°C for 30 days and 6 h, respectively. The patients' samples were stable in ice box for 2 h. All variations were well within the desired limits of 15%.

Clinical Validation
Eighty-three paired plasma and effluent samples from 10 patients were included in the clinical validation of the effluent analysis. The Pearson correlation showed a correlation coefficient of 0.950 (P<0.0001) for the imipenem concentrations in plasma and effluent. Passing-Bablok regression between the plasma and effluent imipenem concentrations showed a proportional bias of 0.997 (95% confidence interval [CI], 0.935 to 1.078) and a constant bias of -0.039 (95% CI, -0.514 to 0.247) (Fig 1). A linear relationship between plasma and effluent imipenem concentrations was indicated by the Cusum linearity test (P>0.10). Bland-Altman assessment showed a good agreement between analyses of imipenem concentrations in plasma and effluent, with 4.8% (4/83) of observations for imipenem falling outside 95% limits of agreement (Fig 2). The observed bias for the mean plasma-to-effluent imipenem concentration ratio versus the mean concentration in plasma and effluent was 1.044 (95% CI, 0.975 to 1.114, Fig 2).

Pharmacokinetic and Pharmacodynamic Evaluation
Plasma and effluent concentration-versus-time profiles for imipenem during CRRT for each patient are shown in Fig 3. Pharmacokinetic parameters of imipenem in plasma and effluent are displayed in Table 3. The Wilcoxon test for paired samples showed no statistically significant difference between medians of all pharmacokinetic parameters in plasma and effluent samples. Pharmacodynamic parameters of imipenem for the patients are displayed in Table 4. No statistically significant difference was found between medians of f T>MIC or f T>4 × MIC in plasma and effluent samples, respectively. Both of f T>MIC in plasma and effluent samples showed that the pathogens with a MIC of 1 μg/mL would be adequately treated with the doses of 0.5 g every 6 h and 1 g every 8 h. However, when f T>4 × MIC was used, only 2 patients (50.0%) with the doses of 0.5 g every 6 h and the patient with the dose of 1 g every 8 h were adequately treated. It was also found that these were the only three patients survived, the other two patients predicted undertreated by f T>4 × MIC both passed away. For the pathogens with a MIC of 16 μg/mL, neither of the two patients (with the doses of 0.5 g every 6 h and 1 g every 8 h, respectively) was adequately treated based on the values of both f T>MIC and f T>4 × MIC, and both passed away.

Discussion
To our knowledge, this is the first study to investigate the possibility of using spent effluent of CRRT to perform TDM of imipenem for critically ill patients instead of blood samples. We found the imipenem concentration in effluent samples was in good agreement with the plasma imipenem concentration. The mean ratio of effluent and plasma imipenem concentrations was approximately 1. It has been shown that the drug concentration in ultrafiltrate or dialysate divided by that in plasma was mainly influenced by plasma protein binding of the drugs [24,45,46]. Therefore, this result may be explained by the low plasma protein binding of imipenem, which is showed as 9% in healthy volunteers [47]. Hypoproteinemia is very common in critically ill patients, and even lower protein binding of imipenem may be expected [24,48]. Previous study illustrates a strong correlation between plasma free and dialysate piperacillin concentrations in patients receiving CVVHD [34]. Protein binding of piperacillin is about 20-30% in healthy volunteers and patient populations [49]. Taking these into account, it may be  possible to use the effluent of CRRT to perform TDM of other drugs with low or moderate plasma protein binding, especially for critically ill patients.
Moreover, though the mean ratio of effluent and plasma imipenem concentrations was approximately 1, the limits of agreement of the ratio were between 0.417 and 1.671, which had a discrepancy of approximately 3-fold, and a larger bias was seen in the plasma-to-effluent concentration ratio in the low-concentration area. The bias may be mainly explained by the factors Analysis of Imipenem Concentration in CRRT Effluent for Critically Ill Patients of potential adsorption by filter membrane and the disparity of filter duration for each patient [45,50,51]. It is indicated that a proteinaceous secondary membrane is formed when blood exposes to the filter, possibly as soon as the first few minutes of CRRT [52]. And the formation of the proteinaceous membrane over the filter may hinder convective solute removal and reduce transmembrane clearance as the proteinaceous membrane thickens [51]. The time limits for the filters are typically set at 24-48 h. However, we may extend the duration of filters up to 96 h in the spirit of cost containment for some patients, unless of clotting. Limited importance is attached to the issue of filter performance over time, because of the difficulty to assess. The ratio of effluent and plasma urea nitrogen is commonly recommended as a surrogate marker of filter performance [53]. However, effluent creatinine/serum creatinine is suggested to be a better marker than the ratio of urea nitrogen in a recent study, because of a better sensitivity for filter performance [51]. It is a pity that we did not evaluate the performance of the filter in this study. This will be included in our further studies to verify that effluent is well equilibrated with plasma.
In this study, we found no statistically significant difference between medians of all pharmacokinetic and pharmacodynamic parameters in plasma and effluent samples, which further validated the rationality of using effluent samples to perform imipenem analysis instead of blood samples. However, it should be noted that pharmacodynamic parameters was significantly underestimated by effluent sampling (approximately three times) in one patient (subject 9). The reason for this is not clear, and further investigation is needed. Besides that, we found f T>4×MIC may be a better indicator of clinical efficacy for imipenem in critically ill patients. For the pathogens with MIC = 16μg/mL, the doses of 0.5 g every 6 h and 1 g every 8 h could not achieve the therapeutic target and may cause therapeutic failure. Because of the limited samples in this study, these findings need to be justified by further investigations.
There are a few limitations to be taken in consideration in this study. Limited number of patients and only the CRRT types of CVVHDF and CVVH were included. Furthermore, though we tried to limit the influential factors as minimal as possible, the factors, such as the patient's condition and the duration of the hemofilter were not well controlled, this may cause the bias.
In conclusion, a method for analyzing imipenem in spent effluent in critically ill patients with CRRT is developed and clinically validated in this pilot study conducted in a real-world clinical setting. It suggests that reliable measurement of drug concentrations in spent effluent of CRRT may facilitate therapeutic drug monitoring of imipenem in this population. Further investigation is needed to justify the clinical value of our results.