Comparative polymyxin B pharmacokinetics in critically ill patients with renal insufficiency and in continuous veno-venous hemodialysis.

PURPOSE: The aim of this study was to assess polymyxin B pharmacokinetics (PK) in patients with varying degrees of renal dysfunction and in patients who require continuous veno-venous hemodialysis (CVVHD). METHODS: The study enrolled 37 patients with sepsis, including 13 patients with glomerular filtration rate (GFR) below 80 mL/min and 11 patients on CVVHD. Each patient received a loading dose of polymyxin B (200-300 mg) and at least 3 subsequent doses of 100-150 mg every 12 h. For every patient, 6-8 blood samples were collected between doses. Polymyxin B (PMB) serum concentration was determined using enzyme-linked immunosorbent assay. RESULTS: In sepsis, patients with preserved renal function mean area under the curve over 24 h (AUC0-24 h) value reached 67.8 ± 9.8 mg*h/L, while in patients with GFR below 80 mL/min, mean AUC0-24 h was 87 ± 5.8 mg*h/L. PMB PK in patients with renal insufficiency was characterized by significantly lower clearance (CL) compared to the normal renal function group (2.1 ± 0.1 L/h vs 3.9 ± 0.4 L/h respectively). In patients on CVVHD, mean AUC0-24 h was 110.4 ± 10.3 mg*h/L, while CL reached 2 ± 0.23 L/h. The median recovery rate from dialysate constituted 22%. Simulation of different dosage regimens that indicate a fixed maintenance dose of 100 mg q12h with a loading dose of 200 mg is optimal for patients on CVVHD, and no dosage increase is required. CONCLUSION: This study demonstrates decreased clearance of PMB in patients with renal insufficiency, which puts them at risk of toxicity. Therefore, patients with extremes of renal function might benefit from therapeutic drug monitoring. For patients with anuria, who require CVVHD, we suggest a fixed dose of 100 mg q12h.

Comparative polymyxin B pharmacokinetics in patients receiving extracorporeal membrane oxygenation.

OBJECTIVES: To describe polymyxin B pharmacokinetics in patients receiving veno-venous extracorporeal membrane oxygenation (ECMO) in comparison with critically ill patients without ECMO support and to explore potential covariates that could affect the pharmacokinetics in this group of patients. PATIENTS AND METHODS: In 13 critically ill patients on ECMO and in 21 critically ill patients without ECMO support, 6-8 blood samples were collected during 12 h intervals after reaching steady state. Polymyxin B concentration in serum was determined using a previously developed ELISA. Protein binding was assessed by rapid equilibrium dialysis. RESULTS: In 13 critically ill patients on ECMO who received polymyxin B, the median area under the concentration-time curve over 12 h (AUC0-12h) was 48.38 mg/h/L for the total drug and 14.08 mg/h/L for the free drug. The unbound fraction was 0.35. Total body clearance was 1.16 L/h. In non-ECMO patients, the median AUC0-12h was 34.7 mg/h/L and the median CL was 1.76 L/h. The volume of distribution was significantly lower in ECMO patients (19.7 versus 30.4 L, respectively). We found a moderate negative correlation between the ECMO blood flow rate and AUC0-12h, a strong negative correlation between SOFA score and polymyxin B clearance and a moderate correlation between polymyxin B clearance and renal function in ECMO patients. CONCLUSIONS: Currently recommended polymyxin B dosage regimens are sufficient for patients receiving ECMO and no dosage increase is required. In our study, polymyxin B exposure was higher in ECMO patients compared with the control group.

Production of antibody and development of enzyme-linked immunosorbent assay for therapeutic drug monitoring of eravacycline.

Eravacycline (ERC) was approved for clinical use in 2018. It is more potent than other tetracyclines and can overcome resistance, making it an attractive option for combating multidrug-resistant bacterial infections. Intensive pharmacokinetic (PK) studies are currently being conducted to ensure the effectiveness and safety of ERC in various groups of patients, including those undergoing extracorporeal therapies. This study is the first attempt to develop a simple, efficient, and high-throughput immunoassay for quantifying ERC in human or animal serum. BSA-ERC conjugate as immunogen elicited antibody production in rabbits. Monitoring of the immune response and comparison of homologous and heterologous coating antigens allowed selection of immunoreagents and development of an assay that was selective for ERC possessing sensitivity (IC50), dynamic range (IC20-IC80) and detection limit equal to 3.3 ng/mL, 0.27-54 ng/mL and 0.09 ng/mL, respectively. The developed ELISA showed acceptable recovery of ERC (85-105 %) from rabbit and human serum in the clinically relevant concentration range of 0.1-3.0 mg/L. The method was used to quantify serum ERC concentration in the pilot PK study in Soviet chinchilla rabbits. The results were confirmed by HPLC-MS/MS.

Tigecycline Immunodetection Using Developed Group-Specific and Selective Antibodies for Drug Monitoring Purposes.

Tigecycline (TGC), a third-generation tetracycline, is characterized by a more potent and broad antibacterial activity, and the ability to overcome different mechanisms of tetracycline resistance. TGC has proven to be of value in treatment of multidrug-resistant infections, but therapy can be complicated by multiple dangerous side effects, including direct drug toxicity. Given that, a TGC immunodetection method has been developed for therapeutic drug monitoring to improve the safety and efficacy of therapy. The developed indirect competitive ELISA utilized TGC selective antibodies and group-specific antibodies interacting with selected coating TGC conjugates. Both assay systems showed high sensitivity (IC50) of 0.23 and 1.59 ng/mL, and LOD of 0.02 and 0.05 ng/mL, respectively. Satisfactory TGC recovery from the spiked blood serum of healthy volunteers was obtained in both assays and laid in the range of 81-102%. TGC concentrations measured in sera from COVID-19 patients with secondary bacterial infections were mutually confirmed by ELISA based on the other antibody-antigen interaction and showed good agreement (R2 = 0.966). A TGC pharmacokinetic (PK) study conducted in three critically ill patients proved the suitability of the test to analyze the therapeutic concentrations of TGC. Significant inter-individual PK variability revealed in this limited group supports therapeutic monitoring of TGC in individual patients and application of the test for population pharmacokinetic modelling.

Unbound serum polymyxin B in patients with sepsis: Detection approaches and limited sampling strategy for clinical practice and research.

Polymyxins are increasingly used to treat multidrug resistant bacteria in critically ill patients, however these drugs have a narrow therapeutic window. Highly variable antibiotic pharmacokinetics in critically ill patients puts them at high risks of toxicity and underdosing, therefore robust tools suitable for therapeutic drug monitoring and pharmacokinetic studies are in great demand. It is now recognized that only antibiotics not bound to serum proteins possess antimicrobial effects and free drug concentration is the most appropriate pharmacokinetic target. However, data on unbound polymyxin B (PMB) determination is very limited, especially in different cohorts of critically ill patients. Performance of rapid equilibrium dialysis and ultrafiltration in unbound PMB measurement, as well as different approaches to predict free PMB exposure based on a single measurement were explored in present study. In comparison to ultrafiltration rapid equilibrium dialysis demonstrated a lower degree of non-specific binding, which makes the latter a more reliable tool. Overall, unbound drug determination is a costly and labor-intensive procedure, hence different limited sampling strategies were proposed for further clinical application. Although pooled sample analysis is commonly used for this purpose, the results showed inconsistency between observed and predicted values. An alternative approach using 1-3 sampling points and Bayesian estimation is proposed.

Longitudinal multiparametric characterization of platelet dysfunction in COVID-19: Effects of disease severity, anticoagulation therapy and inflammatory status.

INTRODUCTION: Defects of platelet functional responses in COVID-19 were reported, but their origin and pathophysiological significance are unclear. The objective of this study was to characterize the thrombocytopathy in COVID-19. MATERIALS AND METHODS: Analysis of platelet functional responses to activation by flow cytometry and aggregometry in 46 patients with confirmed COVID-19 of different severity (non-ICU, ICU, and ECMO) over the course of hospitalization alongside with plasma coagulation, inflammatory markers (CRP, fibrinogen, NETosis assays in smears) was performed. RESULTS AND CONCLUSIONS: All patients had increased baseline percentage of procoagulant platelets (healthy: 0.9 ± 0.5%; COVID-19: 1.7 ± 0.6%). Patients had decreased agonist-induced platelet GPIb shedding (1.8 ± 0.7 vs 1.25 ± 0.4), P-Selectin exposure (1.51 ± 0.21 vs 1.1 ± 0.3) and aggregation. The values of these parameters among the non-ICU and ICU cohorts differed modestly, while the ECMO cohort differed significantly. Only ECMO patients had pronounced thrombocytopenia. While inflammatory markers improved over time, the observed platelet functional responses changed only moderately. SARS-CoV-2 RNA was found in 8% of blood samples and it did not correlate with platelet counts or responses. All patients had increased NETosis that moderately correlated with platelet dysfunction. High cumulative dosages of LMWH (average > 12,000 IU/day over 5 days) resulted in an improvement in platelet parameters. The observed pattern of platelet refractoriness was reproduced by in vitro pre-treatment of washed platelets with subnanomolar thrombin or perfusion of blood through a collagen-covered flow chamber. We conclude that platelet dysfunction in COVID-19 is consistent with the intravascular-coagulation-induced refractoriness rather than with an inflammation-induced mechanism or a direct activation by the virus.

Development of ELISA formats for polymyxin B monitoring in serum of critically ill patients.

Treating infections in critically ill patients often requires the use of last-line antibacterial drugs such as polymyxins with a narrow therapeutic window and high toxicity. In critically ill patients, the drug pharmacokinetics changes significantly, and as a result, the antibiotic concentrations in blood and infection foci become suboptimal, which leads to therapeutic failures or toxic manifestations. For timely dosage adjustments, a competitive ELISA-based method using antibodies to polymyxin В (PMB) was developed. Among the several considered assays, a direct antibody-coated format was selected for its short duration (1.5 h) and the best agreement with the LC-MS/MS data (R2 = 98 %). The assay dynamic measurement range (IC20-IC80) could be substantially shifted by changing the ratio of immunoreagents. To conveniently measure the therapeutic range of PMB concentrations, it was adjusted to 5.0-192 ng/mL, allowing the samples to be analyzed after a simple 100-fold dilution with the assay buffer. The ELISA sensitivity expressed in half-inhibition concentration (IC50) and the limit of detection were 30.6 and 1.8 ng/mL, respectively. The assay cross-reactivity towards the related analogue colistin (COL) was 95 %, and this compound could also be adequately quantified by the same assay. The PMB and COL recovery from the spiked serum samples was similar and constituted 98-109 %. The trial drug monitoring was carried out in 3 patients with Gram-negative sepsis, and the established pharmacokinetic profiles of PMB revealed the necessity for individual dosage adjustment.