Pharmacokinetics of colistin after nebulization or intravenous administration of colistin methanesulphonate (Colimycin®) to cystic fibrosis patients.

OBJECTIVES: Colistin, administered as the prodrug colistin methanesulphonate (CMS), is an antibiotic frequently administered as aerosol in cystic fibrosis (CF) patient. Our aim was to assess the plasma PK of colistin in CF patients treated with CMS administered intravenously or as aerosol and to compare these results with those previously reported in healthy volunteers. METHODS: Six CF patients were included, CMS and colistin concentrations were measured in plasma, urine and sputum. Either after single intravenous administration of 2 Million International Unit (MIU) or after repeated nebulizations of 2 MIU of CMS. PK of CMS and colistin were assessed by a mixed effect modeling approach. RESULTS: Renal clearance of CMS was lower in CF patients compared to that previously reported in healthy volunteers (64.3 mL/min (RSE = 15%) vs. 103 mL/min (RSE = 8%)). However, apparent clearance of colistin was higher in CF patients compared to healthy volunteers (124 mL/min (RSE = 13%) vs. 48.7 mL/min (RSE = 15%)), resulting in reduced systemic exposure to colistin (dose normalized AUC (2 MIU) of 7.4 h.mg/L/MIU vs. 11.2 h.mg/L/MIU). After repeated nebulizations, colistin concentrations were very low in plasma (<0.21 mg/L). CONCLUSIONS: Although our study suggests a lower median dose normalized colistin plasma concentrations in CF patients compared with healthy controls, this difference was not significant and a larger study is needed to substantiate this.

Breaking the Vicious Cycle of Antibiotic Killing and Regrowth of Biofilm-Residing Pseudomonas aeruginosa.

Biofilm-residing bacteria embedded in an extracellular matrix are protected from diverse physicochemical insults. In addition to the general recalcitrance of biofilm bacteria, high bacterial loads in biofilm-associated infections significantly diminish the efficacy of antimicrobials due to a low per-cell antibiotic concentration. Accordingly, present antimicrobial treatment protocols that have been established to serve the eradication of acute infections fail to clear biofilm-associated chronic infections. In the present study, we applied automated confocal microscopy on Pseudomonas aeruginosa to monitor dynamic killing of biofilm-grown bacteria by tobramycin and colistin in real time. We revealed that the time required for surviving bacteria to repopulate the biofilm could be taken as a measure for effectiveness of the antimicrobial treatment. It depends on the (i) nature and concentration of the antibiotic, (ii) duration of antibiotic treatment, (iii) application as monotherapy or combination therapy, and (iv) interval of drug administration. The vicious cycle of killing and repopulation of biofilm bacteria could also be broken in an in vivo model system by applying successive antibiotic dosages at intervals that do not allow full reconstitution of the biofilm communities. Treatment regimens that consider the important aspects of antimicrobial killing kinetics bear the potential to improve control of biofilm regrowth. This is an important and underestimated factor that is bound to ensure sustainable treatment success of chronic infections.

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.

Characterization of urinary metabolites as biomarkers of colistin-induced nephrotoxicity in rats by a liquid chromatography/mass spectrometry-based metabolomics approach.

Colistin is a polypeptide antibiotic that effectively treats infections caused by multidrug-resistant Gram-negative bacteria, but its clinical use is limited due to nephrotoxicity. The purpose of the present study was to identify biomarkers of colistin-induced nephrotoxicity and to further characterize the mechanisms underlying this process by analyzing urinary metabolites using untargeted metabolomic approach. Rats receiving intraperitoneal administration of colistin sodium methanesulfonate (CMS) (25 or 50mg/kg) exhibited histopathological changes in the kidney and increased blood urea nitrogen levels. Additionally, the levels of phenylalanine, tryptophan, and tyrosine in the urine of the CMS-treated group were significantly higher than those of the control group, suggesting that colistin caused proximal tubular damage. Urinary acetylcarnitine and butyrylcarnitine levels also increased after CMS treatment, but the levels of purine metabolites and metabolites related to the tricarboxylic acid cycle were reduced. The most significant increase in the CMS-treated groups was observed in creatine levels. CMS-induced selective nephrotoxicity may be attributed to relatively high tissue concentrations of colistin in the kidney. Taken together, our results indicate that high levels of colistin in the kidney caused perturbations in the tricarboxylic acid cycle, amino acid metabolism, creatine metabolism, and purine metabolism and ultimately led to kidney injury.

Ocular penetration of intravenously administered colistin in rabbit uveitis model.

PURPOSE: The purpose of this study was to evaluate the ocular distribution of intravenously administered colistin in a rabbit uveitis model. METHODS: Colistin, a polypeptide antibiotic against the multidrug-resistant (MDR) Gram-negative organisms, was given intravenously to rabbits at 5 mg/kg of body weight starting 24 h after induction of uveitis by intravitreal endotoxin injection. Colistin concentrations were determined by high-performance liquid chromatography-mass spectrometry assay in the aqueous humor, vitreous humor, and plasma 0.5, 3, 6, and 24 h after administration of a single dose. RESULTS: The maximum colistin concentrations (mean±standard deviation) were found 0.5 h after the end of the intravenous administration and were 9.48±2.0 µg/mL in plasma and 0.62±0.07 µg/mL in the aqueous humor of the inflamed eye. After 24 h, no drug was detectable in the aqueous of the inflamed eyes. Colistin was undetectable in the aqueous of contralateral normal eyes at all time points. Drug concentrations in all the vitreous samples from both inflamed and normal eyes were undetectable, except at the 3-h inflamed eye group, and a colistin concentration of 0.02±0.01 µg/mL was found. Plasma levels of colistin fell to 0.93±0.07 and 0.24±0.08 µg/mL, after 3 and 6 h, respectively, and were not detectable 24 h after the given dose. CONCLUSIONS: In our model, colistin did not reach therapeutically relevant levels in the aqueous and in the vitreous humor of rabbit eyes. The findings suggest a limited role for intravenously administered colistin in the treatment of Gram-negative bacterial endophthalmitis.

Determination of colistin A and colistin B in human plasma by UPLC-ESI high resolution tandem MS: application to a pharmacokinetic study.

The resistance of gram-negative bacteria to most available antibiotics and the lack of new antimicrobial agents have prompted the re-emergence of colistin (CS) as potent treatment against most gram-negative microorganisms. Optimal dosing with CS suffers from poor pharmacokinetic characterization mainly due to the analytical challenge of assaying CS in biological fluids and the limited information on quantitative analysis of CS in plasma using high resolution mass spectrometry (MS). Hence, a rapid, simple and accurate analytical method based on ultra performance liquid chromatography (UPLC) combined with electrospray ionization (ESI) tandem mass spectrometry (MS/MS) on a hybrid quadrupole time of flight (QTOF) instrument has been developed and fully validated for the quantification of CS in human plasma. After the pretreatment of plasma samples by solid phase extraction (SPE) and the addition of the internal standard (reserpine, RSP) the analytes were chromatographed on an Acquity BEH C8 column (100 mm × 2.1 mm, 1.7 µm) using gradient elution with 0.5% aqueous acetic acid (AcOH) and acetonitrile with 0.5% AcOH (with CSA and CSB eluting at 1.39 and 1.31 min, respectively). Accurate mass measurement correction was performed on line using the leukine-enkephaline standard. The method presented good fit (regression coefficient≥0.998) over the quantitation range of 0.2-300 and 0.03-4.5 µg mL(-1) with the lower limit of quantitation (LLOQ) being 0.02 and 0.03 µg mL(-1) for CSA and CSB in human plasma, respectively. The intra- and inter-day precision, measured as %relative standard deviation, was better than 10%, whereas the accuracy expressed as %relative error was also better than 10%. The short term, freeze-thaw (three cycles) and in process stability showed non-significant degradation of CS under these conditions. The validation results showed that the developed method demonstrated adequate selectivity and sensitivity. The method has been successfully applied to plasma samples from patients suffering from cystic fibrosis and treated with CS, and the pharmacokinetic profile has been calculated.

Dry powder inhalation of colistin sulphomethate in healthy volunteers: A pilot study.

BACKGROUND: Pulmonary administration of the antimicrobial drugs colistin sulphomethate and tobramycin has been shown to be effective in slowing down pulmonary deterioration in cystic fibrosis (CF) patients. Both drugs are administered by liquid nebulisation, a technique known to have disadvantages. Dry powder inhalation may be an attractive alternative. We investigated inhalation of colistin sulphomethate dry powder using a newly developed Twincer device in healthy volunteers. METHODS: Eight healthy volunteers inhaled a single dose of 25mg colistin sulphomethate dry powder each, using the Twincer inhaler. The median diameter (X(50)) of the dry powder was 1.6 microm (X(10)=0.7 microm, X(90)=3.1 microm), measured by laser diffraction technique. Pulmonary function tests were performed before, 5 and 30 min after inhalation. Serum samples were drawn at t=15 min, 45 min, 1.5h, 2.5h, 3.5h, 5.5h, 7.5h and 24h after inhalation. RESULTS: The colistin sulphomethate dry powder inhaler was well tolerated: no clinically relevant effect on FEV(1) was observed nor did the volunteers experience adverse effects. CONCLUSION: Dry powder inhalation of colistin sulphomethate using the Twincer inhaler is well tolerated by healthy volunteers. A pilot study in cystic fibrosis patients is therefore considered safe in developing a dry powder inhalation of colistin for everyday CF treatment.

Development of a novel assay method for colistin sulphomethate.

Increased use of colistin therapy for infections caused by Pseudomonas aeruginosa has indicated a need for a more robust microbiological assay technique. This report describes a quick and simple microbiological assay for quantifying levels of colistin sulphomethate in serum and urine samples from cystic fibrosis patients. The technique uses no specialised or costly equipment and is suitable for use in all routine diagnostic microbiology laboratories.

The pharmacokinetics of colistin in patients with cystic fibrosis.

The safety and pharmacokinetics of colistin were determined after first dose (n = 30) and again under steady-state conditions (n = 27) in 31 patients with cystic fibrosis receiving the drug as a component of their treatment for an acute pulmonary exacerbation of their disease. Patients ranged in age from 14 to 53 years and received colistin for 6 to 35 days. Each patient was started on colistin 5 to 7 mg/kg/day administered intravenously in three equally divided doses. Elimination half-life (t1/2), mean residence time (MRT), steady-state volume of distribution (Vdss), total body clearance (Cl), and renal clearance (Clr) after first-dose administration averaged 3.4 hours, 4.4 hours, 0.09 l/kg, and 0.35 and 0.24 ml/min/kg, respectively. No differences in colistin disposition characteristics between first-dose and steady-state evaluations were observed. Sputum sampling was incomplete and confounded by previous aerosol administration but revealed colistin concentrations that markedly exceeded observed plasma concentrations. Twenty-one patients experienced one or more side effects attributed to colistin administration. The most common reactions involved reversible neurologic manifestations, including oral and perioral paresthesias (n = 16), headache (n = 5), and lower limb weakness (n = 5). All of these apparent colistin-induced neurologic adverse effects, though bothersome, were benign and reversible. Intermittent proteinuria was observed on urinalysis in 14 patients, and 1 patient developed reversible, colistin-induced nephrotoxicity. No relationship between the occurrence of any colistin-associated adverse effect and plasma colistin concentration or colistin pharmacokinetic parameter estimate was observed. These data provide no basis for routine monitoring of colistin plasma concentrations to guide dosing for patient safety and suggest slow upward dose titration to minimize the incidence and severity of associated side effects.

Safety and tolerability of bolus intravenous colistin in acute respiratory exacerbations in adults with cystic fibrosis.

OBJECTIVE: To assess the safety and tolerability of bolus intravenous doses of colistin during acute respiratory exacerbations in adults with cystic fibrosis and chronic Pseudomonas aeruginosa infection. METHODS: Twelve patients with acute exacerbations of cystic fibrosis were enrolled in a Phase I open-label study. On day 1, patients received three doses of colistin 2 mega-units (160 mg), reconstituted in 50 mL of NaCl 0.9%, by infusion over 30 minutes three times daily. On days 2, 3, and 4, the same dose of colistin was administered by bolus injection three times a day over five minutes after reconstitution in 20, 15, and 10 mL of NaCl 0.9%, respectively. The injection was given by a nurse or physician using a hand-held syringe. If the latter dose was tolerated, it was continued for the remaining eight days of the study. If any dose was not tolerated, treatment reverted to the previously tolerated concentration, which was continued throughout the remainder of the study. RESULTS: No serious adverse events occurred during the course of the trial. Patients without total indwelling venous access systems experienced mild to moderate injection pain. There were no clinically significant changes in renal function. CONCLUSIONS: This study indicates that the administration of bolus intravenous colistin as 2 mega-units (160 mg) in 10 mL of NaCl 0.9% three times a day is safe. It is well-tolerated by patients with total indwelling venous access systems.

High-performance liquid chromatographic method for the determination of colistin in serum.

Inhalation therapy of colistin is widespread in patients with cystic fibrosis. To date, no pharmacokinetic data of colistin after inhalation are available. To optimize the inhalation therapy, pharmacokinetic data of colistin are necessary. In this study, the authors describe a chromatographic analysis for measurement of colistin concentrations in serum. After protein precipitation, the colistin sample is treated with orthophthalaldehyde for derivatization. The sum of the peak areas of the two main components of colistin (polymyxin E1 and E2) were used for quantitation. The performance of the analytical method was assessed by determining the lower limit of quantitation, the selectivity of the method, the intra-assay variation, the reproducibility, the interassay variation, and the accuracy. The lower limit of quantitation was 28 microg/L. Ceftazidime, aztreonam, piperacilline, or tobramycin showed no interference with the colistin assay. In a pilot study, the authors found a trough value of approximately 10 microg/L and peak values of approximately 100 microg/L after inhalation of 160 mg colistin in serum samples of a representative patient. These values show that the method can be used to design further experiments. The applicability of the method was also tested on urine and sputum samples. Colistin was detectable but further validation experiments are required to confirm the usefulness of the method in these biologic matrices. To the authors' knowledge this is the first study in which serum concentrations are described after inhalation of colistin in patients with cystic fibrosis.