Polymyxin B2

Polymyxin B, a last line antibiotic for extensively drug resistant gram-negative bacteria, therapeutic drug monitoring (TDM) is recommended to minimize its nephrotoxicity and improve efficacy. In the present study, we developed a novel quantification method of polymyxin B in dried blood spots (DBS) using liquid chromatography coupled with mass spectrometry (LC-MS/MS), which was performed on a Shimadzu Prominence HPLC system coupled with a 4500 triple quadrupole mass spectrometer. An aliquot of 50 μL whole blood sample was spotted on Whatman 903® paper cards. Each DBS sample was cut off into a 6 mm diameter disc and extracted by acetonitrile in water (30% in volume, containing 6% formic acid, v/v). Both intra and inter-batch accuracy was in the range of 92.6%-111.0% for polymyxin B1 and 91.5%-111.5% for polymyxin B2. The precision was in the range of 5.2%-12.2% for polymyxin B1 and 4.9%-13.1% for polymyxin B2. The matrix effects for polymyxin B1 and polymyxin B2 at low, medium and high concentrations were ranged from 102.2%-107.9% and 99.8%-106.2%, respectively. The extraction recoveries were >85.4%. Stability results showed that DBS cards can be transported at room temperature within 2 days and was stable in sealed plastic bags for 38 days at -70 °C. Bland-Altman analysis demonstrated that concentrations of polymyxin B measured in DBS and plasma methods were in moderate agreement with 95.1% samples within the 95% confidence interval of limits of agreement. The DBS method was successfully applied in clinic for TDM of polymyxin B, which can be an alternative approach in clinic.

Background: Polymyxin B is used as the last treatment resort for multidrug-resistant gram-negative bacterial infections. This study aimed to develop and validate a simple and robust liquid chromatography with tandem mass spectrometry analytical method for therapeutic drug monitoring of plasma and cerebrospinal fluid (CSF) polymyxin B1 and B2. Methods: Plasma and CSF polymyxin B1 and B2 were chromatographically separated on a Thermo Hypersil GOLD aQ C18 column and detected using electrospray ionization mode coupled with multiple reaction monitoring. Blood and CSF samples for pharmacokinetic analysis were collected from 15 polymyxin B-treated patients. Results: The calibration curve showed acceptable linearity over 0.2-10 mcg/mL for polymyxin B1 and 0.05-2.5 mcg/mL for B2 in the plasma and CSF, respectively. After validation, according to the Food and Drug Administration (FDA) method validation guideline, this method was applied for polymyxin B1 and B2 quantification in over 100 samples in a clinical study. Conclusions: A simple and robust method to measure polymyxin B1 and B2 in human CSF was first exploited and validated with good sensitivity and specificity, and successfully applied in polymyxin B pharmacokinetic analysis and therapeutic monitoring in Chinese patients.

Novel antimicrobials or new treatment strategies are urgently needed to treat Pseudomonas aeruginosa (P. aeruginosa) related infections and especially to address the problem of antibiotic resistance. We propose a novel strategy that combines the human antimicrobial peptide (AMP) LL37 with different antibiotics to find synergistic AMP-antibiotic combinations against P. aeruginosa strains in vitro. Our results showed that LL37 exhibited synergistic inhibitory and bactericidal effects against P. aeruginosa strains PAO1 and PA103 when combined with the antibiotics vancomycin, azithromycin, polymyxin B, and colistin. In addition, LL37 caused strong outer membrane permeabilization, as demonstrated through measurement of an increased uptake of the fluorescent probe N-phenyl-1-naphthylamine. The membrane permeabilization effects appear to explain why it was easier to rescue the effectiveness of the antibiotic toward the bacteria because the outer membrane of P. aeruginosa exhibits barrier function for antibiotics. Furthermore, the change in the zeta potential was measured for P. aeruginosa strains with the addition of LL37. Zeta potentials for P. aeruginosa strains PAO1 and PA103 were -40.9 and -10.9 mV, respectively. With the addition of LL37, negative zeta potentials were gradually neutralized. We found that positively charged LL37 can interact with and neutralize the negatively charged bacterial outer membrane through electrostatic interactions, and the process of neutralization is believed to have contributed to the increase in outer membrane permeability. Finally, to further illustrate the relationship between outer membrane permeabilization and the uptake of antibiotics, we used LL37 to make the outer membrane of P. aeruginosa strains more permeable, and minimum inhibitory concentrations (MICs) for several antibiotics (colistin, gentamicin, polymyxin B, vancomycin, and azithromycin) were measured. The MICs decreased were twofold to fourfold, in general. For example, the MICs of azithromycin and vancomycin decreased more than fourfold when against P. aeruginosa strain PAO1, which were the greatest decrease of any of the antibiotics tested in this experiment. As for PA103, the MIC of polymyxin B2 decreased fourfold, which was the strongest decrease seen for any of the antibiotics tested in this experiment. The increased uptake of antibiotics not only demonstrates the barrier role of the outer membrane but also validates the mechanism of synergistic effects that we have proposed. These results indicate the great potential of an LL37-antibiotic combination strategy and provide possible explanations for the mechanisms behind this synergy.