Structure-activity studies on polymyxin derivatives carrying three positive charges only reveal a new class of compounds with strong antibacterial activity.

Recent years have brought in an increased interest to develop improved polymyxins. The currently used polymyxins, i.e. polymyxin B and colistin (polymyxin E) are pentacationic lipopeptides that possess a cyclic heptapeptide part with three positive charges, a linear "panhandle" part with two positive charges, and a fatty acyl tail. Unfortunately, their clinical use is shadowed by their notable nephrotoxicity. We have previously developed a polymyxin derivative NAB739 which lacks the positive charges in the linear part. This derivative is better tolerated than polymyxin B in cynomolgus monkeys and is, in contrast to polymyxin B, excreted into urine in monkeys and rats. Here we have conducted further structure-activity relationship (SAR) studies on 17 derivatives with three positive charges only. We discovered a remarkably antibacterial class, as exemplified by NAB815, that carries two positive charges only in the cyclic part.

Determination of Colistin and Colistimethate Levels in Human Plasma and Urine by High-Performance Liquid Chromatography-Tandem Mass Spectrometry.

BACKGROUND: Colistin is a polypeptide antibiotic from the polymyxin E group used for the treatment of infections caused by multidrug-resistant gram-negative bacteria. The main constituents, accounting for approximately 85% of this mixture, are colistin A (polymyxin E1) and colistin B (polymyxin E2). The aim of this study was to develop and validate new and fast methods of quantification of colistin A and B and its precursors [colistin methanesulfonate sodium (CMS) A and B] by ultraperformance liquid chromatography-tandem mass spectrometry in plasma and urine with short pretreatment and run times. METHODS: Chromatography was performed on an Acquity UPLC-MS/MS system (WATERS) with a WATERS Acquity UPLC C18 column (4.6 × 150 mm, 3.5 µm particle size). The pretreatment of samples consists of precipitation and extraction into microcolumns plate and HLB 96-well plate 30 µm-30 mg (OASIS) with a Positive Pressure-96 (WATERS). RESULTS: Quantification was performed using a multiple reaction monitoring of the following transitions: m/z 390.9 → 385.1 for colistin A, m/z 386.2 → 101.0 for colistin B, and m/z 602.4 → 241.1 for polymyxin B1 sulfate. In plasma and urine, calibration curves were linear from 30 to 6000 ng/mL for colistin A and from 15 to 3000 ng/mL for colistin B. With an acceptable accuracy and precision, the lower limit of quantification were set at 24.0 ng/mL and 12.0 ng/mL for colistin A and B in plasma, and at 18.0 ng/mL and 9.0 ng/mL for colistin A and B in urine. CONCLUSIONS: These LC-MS/MS methods of quantification for colistin A and B and its precursors (CMS A and B) in plasma and urine are fast, simple, specific, sensitive, accurate, precise, and reliable. Furthermore, they are linear and repeatable. These procedures were successfully applied to a pharmacokinetic study of a critically ill patient suffering from ventilator-associated pneumonia, who was treated with nebulized CMS.

Simultaneous quantitation of polymyxin B1, polymyxin B2 and polymyxin B1-1 in human plasma and treated human urine using solid phase extraction and liquid chromatography-tandem mass spectrometry.

Two liquid chromatographic-tandem mass spectrometric (LC-MS/MS) methods have been developed and validated for the quantitative determination of polymyxin B1, polymyxin B2 and polymyxin B1-1 concentrations in human plasma and treated urine. During method development, technical challenges such as the separation of structural isomers polymyxin B1and polymyxin B1-1 and nonspecific binding in urine samples were encountered and overcome. Two automated solid phase extraction methods were used to extract plasma samples (100µL) and urine samples (200µL) and the resulting extracts were analyzed using reversed phase LC-MS/MS with an electrospray (ESI) interface and selected reaction monitoring (SRM) in the positive ionization mode. Both methods were validated over a calibration curve range of 5.00-2000ng/mL with a linear regression and 1/x(2) weighting. The between-run relative standard deviation (%RSD) ranged from 4.5 to 9.5% for the plasma assay and from 1.1 to 7.1% for the urine assay. For the plasma assay, the between-run accuracy ranged from 100.5 to 115.2% of nominal at all QC concentrations including the LLOQ. For the urine assay, the between-run accuracy ranged from 92.0 to 106% of nominal at all QC concentrations including the LLOQ. The extraction recoveries for all polymyxins in both assays were between 54.0 and 64.2%. Long term matrix storage stability for all polymyxins was established at both -20°C and -70°C for up to 85 days in human plasma and for up to 55 days in treated human urine. Both assays were used for the measurement of polymyxin B1, polymyxin B2 and polymyxin B1-1 concentrations in human plasma and treated urine for the determination of bioequivalence and toxicokinetic parameters in clinical studies.