A Whole-Body Physiologically Based Pharmacokinetic Model for Colistin and Colistin Methanesulfonate in Rat.

Colistin is a polymyxin antibiotic used to treat patients infected with multidrug-resistant Gram-negative bacteria (MDR-GNB). The objective of this work was to develop a whole-body physiologically based pharmacokinetic (WB-PBPK) model to predict tissue distribution of colistin in rat. The distribution of a drug in a tissue is commonly characterized by its tissue-to-plasma partition coefficient, Kp . Colistin and its prodrug, colistin methanesulfonate (CMS) Kp priors, were measured experimentally from rat tissue homogenates or predicted in silico. The PK parameters of both compounds were estimated fitting in vivo their plasma concentration-time profiles from six rats receiving an i.v. bolus of CMS. The variability in the data was quantified by applying a nonlinear mixed effect (NLME) modelling approach. A WB-PBPK model was developed assuming a well-stirred and perfusion-limited distribution in tissue compartments. Prior information on tissue distribution of colistin and CMS was investigated following three scenarios: Kp was estimated using in silico Kp priors (I) or Kp was estimated using experimental Kp priors (II) or Kp was fixed to the experimental values (III). The WB-PBPK model best described colistin and CMS plasma concentration-time profiles in scenario II. Colistin-predicted concentrations in kidneys in scenario II were higher than in other tissues, which was consistent with its large experimental Kp prior. This might be explained by a high affinity of colistin for renal parenchyma and active reabsorption into the proximal tubular cells. In contrast, renal accumulation of colistin was not predicted in scenario I. Colistin and CMS clearance estimates were in agreement with published values. The developed model suggests using experimental priors over in silico Kp priors for kidneys to provide a better prediction of colistin renal distribution. Such models might serve in drug development for interspecies scaling and investigate the impact of disease state on colistin disposition.

New aerosol formulation to control ciprofloxacin pulmonary concentration.

Ciprofloxacin (CIP) apparent permeability across a pulmonary epithelium model can be controlled by the affinity of its complex with a metal cation. The higher the complex affinity, the larger is the reduction in CIP apparent permeability. The aim of this study was to evaluate if the control of the CIP apparent permeability observed in vitro could be transposed in vivo to control the CIP lung-to-blood absorption rate and CIP concentrations in the lung epithelial lining fluid (ELF) after intratracheal (IT) administration. Two types of innovative inhalable microparticles loaded with the low-affinity CIP-calcium complex (CIP-Ca) or with the high-affinity CIP-copper complex (CIP-Cu) were formulated and characterized. Then, ELF and plasma pharmacokinetics of CIP were studied in rats after IT administration of these two types of microparticles and of a CIP solution (2.5mg/kg). The presence of Cu2+ had little effect on the microparticle properties and the dry powder had aerodynamic properties which allowed it to reach the lungs. CIP concentrations in ELF were much higher after CIP-Cu microparticles IT administration compared to the other two formulations, with mean AUCELF to AUCu,plasma ratios equal to 1069, 203 and 9.8 after CIP-Cu microparticles, CIP-Ca microparticles and CIP solution pulmonary administration, respectively. No significant modification of lung toxicity markers was found (lactate dehydrogenase and total protein). CIP complexation with Cu2+ seems to be an interesting approach to obtain high CIP concentrations in the ELF of lungs after dry powder IT administration.

Pharmacokinetics of nebulized colistin methanesulfonate in critically ill patients.

Objectives: Optimal dosing for nebulized colistin methanesulfonate (CMS), the prodrug of colistin, is unknown. We describe the pulmonary and systemic pharmacokinetics of CMS and colistin following nebulization of 0.5 million IU (MIU) of CMS in ventilated patients. Methods: Twelve critically ill patients received 0.5 MIU of CMS administered every 8 h as 30 min nebulizations. Blood samples were collected immediately before and until 8 h after first nebulization; mini-bronchoalveolar lavage (mini-BAL) was performed at 1 and 5 h or 3 and 8 h (six patients each) post-dose. Pharmacokinetic analysis was performed for CMS and colistin plasma concentrations using a non-compartmental method. ClinicalTrials.gov: NCT01060891. Results: After nebulization, CMS concentrations in epithelial lining fluid (ELF) were much higher (100- to 1000-fold) than those in plasma. Concentrations of colistin in ELF should be considered with caution because when <6 mg/L in BAL, colistin bound to mini-BAL devices. Nevertheless, CMS and colistin concentrations in ELF were much lower than expected from previous results with a 2 MIU dose. From CMS plasma pharmacokinetics it was shown that CMS systemic bioavailability was only slightly decreased for the 0.5 MIU dose compared with 2 MIU. Conclusions: This study shows that CMS concentrations were much higher (100- to 1000-fold) in ELF than in plasma after a 0.5 MIU aerosol of CMS, but much lower (10-fold) than expected from previous results with a 2 MIU dose. Therefore, until new pharmacokinetic and pharmacodynamic assessments of the treatment of ventilator-associated pneumonia with nebulized CMS are performed, the 2 MIU dose should be preferred to the 0.5 MIU dose.

Pulmonary pharmacokinetics of levofloxacin in rats after aerosolization of immediate-release chitosan or sustained-release PLGA microspheres.

A comparative pharmacokinetic study was conducted in rats after intratracheal aerosolization of levofloxacin, as a solution, as immediate-release chitosan microspheres or as sustained-release PLGA microspheres. A pharmacokinetic model was constructed to model levofloxacin concentrations both in plasma and in the lung epithelial lining fluid (ELF). The plasma and ELF experimental concentration profiles versus time were similar for the intravenous and intratracheal levofloxacin solutions and for the intratracheal levofloxacin-loaded chitosan microsphere dry powder, indicating that levofloxacin diffused almost instantaneously through the broncho-alveolar barrier and that the chitosan microspheres released levofloxacin very rapidly, as anticipated from in vitro release studies. The bioavailability for the intratracheal levofloxacin solution and intratracheal chitosan microspheres was estimated to be 98% and 71%, respectively, both with a direct release into the ELF compartment. The ELF-to-unbound plasma AUC ratios were slightly above 2 and may result from an efflux transport. For the intratracheal PLGA microspheres, a high ELF-to-unbound plasma AUC concentration ratio (311) was observed and high levofloxacin concentrations were maintained in ELF for at least 72h in consistency with the in vitro release studies. The bioavailability was 92%, with 19% of the dose released immediately (burst release) into the ELF and 73% released slowly into the ELF from a depot compartment, i.e. the PLGA microspheres, according to a Weibull model. These results highlight the benefit of using sustained-release microspheres administered as aerosols to provide and to maintain high pulmonary concentrations of an antibiotic characterized with a high permeability profile through the broncho-alveolar barrier. The sustained-release microsphere dry powder aerosol may therefore provide advantages over solutions or pure drug dry powders for inhalation in terms of treatment efficiency, ease of use and frequency of administration.

Biopharmaceutical Characterization of Nebulized Antimicrobial Agents in Rats. 4. Aztreonam.

The aim of this study was to determine aztreonam (ATM) membrane permeability using Calu-3 cells and its plasma and pulmonary epithelial lining fluid (ELF) pharmacokinetics in rats after intratracheal nebulization and intravenous administration (15 mg · kg(-1)). ATM exhibits low Calu-3 permeability (0.07 ± 0.02 × 10(-6) cm · s(-1)), and a high area under the ELF/unbound plasma concentration time curve between 0 and infinity (AUCELF/AUCu,plasma) ratio of 1,069 was observed after nebulization in rats. These results confirm that ATM is a low-permeability molecule and a good candidate for nebulization.

Biopharmaceutical Characterization of Nebulized Antimicrobial Agents in Rats: 3. Tobramycin.

The aim of this study was to determine the biopharmaceutical characteristics of tobramycin (TOB) after nebulization in rats. TOB was administered by intravenous (i.v.) bolus or intratracheal nebulization (3 mg · kg(-1)), and concentrations were determined in plasma and epithelial lining fluid (ELF) by liquid chromatography-tandem mass spectrometry. The ratio of the TOB concentration in ELF to the plasma area under the curve (AUC) was more than 200 times as high after NEB as after i.v. bolus administration, indicating that TOB nebulization offers a biopharmaceutical advantage over i.v. administration.

Pharmacokinetics of Colistin Methansulphonate (CMS) and Colistin after CMS Nebulisation in Baboon Monkeys.

PURPOSE: The objective of this study was to compare two different nebulizers: Eflow rapid® and Pari LC star® by scintigraphy and PK modeling to simulate epithelial lining fluid concentrations from measured plasma concentrations, after nebulization of CMS in baboons. METHODS: Three baboons received CMS by IV infusion and by 2 types of aerosols generators and colistin by subcutaneous infusion. Gamma imaging was performed after nebulisation to determine colistin distribution in lungs. Blood samples were collected during 9 h and colistin and CMS plasma concentrations were measured by LC-MS/MS. A population pharmacokinetic analysis was conducted and simulations were performed to predict lung concentrations after nebulization. RESULTS: Higher aerosol distribution into lungs was observed by scintigraphy, when CMS was nebulized with Pari LC® star than with Eflow Rapid® nebulizer. This observation was confirmed by the fraction of CMS deposited into the lung (respectively 3.5% versus 1.3%).CMS and colistin simulated concentrations in epithelial lining fluid were higher after using the Pari LC star® than the Eflow rapid® system. CONCLUSIONS: A limited fraction of CMS reaches lungs after nebulization, but higher colistin plasma concentrations were measured and higher intrapulmonary colistin concentrations were simulated with the Pari LC Star® than with the Eflow Rapid® system.

Feasibility of microdialysis for determination of protein binding and target site pharmacokinetics of colistin in vivo.

Tissue pharmacokinetics and plasma protein binding of colistin have not been described in humans in vivo. Colistin concentrations in plasma, muscle, and subcutis of healthy volunteers were measured by microdialysis after a single dose of 2.5 million IU of colistin methanesulfonate. In vitro microdialysis experiments and an in vivo pilot study were performed prior to the in vivo main study. Concentration-time profiles of total colistin in plasma were comparable with previously described values. The unbound fraction of colistin in plasma (f(u)) ranged from 2.8% to 14.1%. Low plasma f(u) correlated with low unbound colistin concentrations in muscle and subcutis. In vitro, mean relative recovery of microdialysis probes was higher in the reverse dialysis setting compared to the forward dialysis mode (71 ± 9% vs. 45 ± 14%, respectively); mean overall recovery in the main study in vivo was 49 ± 5%. Present data suggest that colistin is extensively protein bound in plasma and poorly distributed into soft tissue. However, differences in relative recovery between forward and reverse dialysis in vitro indicate that results might have been influenced by adhesion of colistin to microdialysis equipment. Microdialysis should be considered as a semiquantitative method for the estimation of unbound colistin levels in soft tissue.

Comparison of intrapulmonary and systemic pharmacokinetics of colistin methanesulfonate (CMS) and colistin after aerosol delivery and intravenous administration of CMS in critically ill patients.

Colistin is an old antibiotic that has recently gained a considerable renewal of interest for the treatment of pulmonary infections due to multidrug-resistant Gram-negative bacteria. Nebulization seems to be a promising form of administration, but colistin is administered as an inactive prodrug, colistin methanesulfonate (CMS); however, differences between the intrapulmonary concentrations of the active moiety as a function of the route of administration in critically ill patients have not been precisely documented. In this study, CMS and colistin concentrations were measured on two separate occasions within the plasma and epithelial lining fluid (ELF) of critically ill patients (n = 12) who had received 2 million international units (MIU) of CMS by aerosol delivery and then intravenous administration. The pharmacokinetic analysis was conducted using a population approach and completed by pharmacokinetic-pharmacodynamic (PK-PD) modeling and simulations. The ELF colistin concentrations varied considerably (9.53 to 1,137 mg/liter), but they were much higher than those in plasma (0.15 to 0.73 mg/liter) after aerosol delivery but not after intravenous administration of CMS. Following CMS aerosol delivery, typically, 9% of the CMS dose reached the ELF, and only 1.4% was presystemically converted into colistin. PK-PD analysis concluded that there was much higher antimicrobial efficacy after CMS aerosol delivery than after intravenous administration. These new data seem to support the use of aerosol delivery of CMS for the treatment of pulmonary infections in critical care patients.

Biopharmaceutical characterization of nebulized antimicrobial agents in rats: 1. Ciprofloxacin, moxifloxacin, and grepafloxacin.

The aim of this study was to evaluate the biopharmaceutical characteristics of three fluoroquinolones (FQs), ciprofloxacin (CIP), moxifloxacin (MXF), and grepafloxacin (GRX), after delivery via a nebulized aerosol to rats. Bronchoalveolar lavages (BAL) were conducted 0.5, 2, 4, and 6 h after FQ intravenous administration and nebulized aerosol delivery to estimate epithelial lining fluid (ELF) drug concentrations. Plasma drug concentrations were also measured, and profiles of drug concentrations versus time after intravenous administration and nebulized aerosol delivery were virtually superimposable, attesting for rapid and complete systemic absorption of FQs. ELF drug concentrations were systematically higher than corresponding plasma drug concentrations, whatever the route of administration, and average ELF-to-unbound plasma drug concentration ratios post-distribution equilibrium did not change significantly between the ways of administration and were equal: 4.0 ± 5.3 for CIP, 12.6 ± 7.3 for MXF, and 19.1 ± 10.5 for GRX (means ± standard deviations). The impact of macrophage lysis on estimated ELF drug concentrations was significant for GRX but reduced for MXF and CIP; therefore, simultaneous pharmacokinetic modeling of plasma and ELF drug concentrations was only performed for the latter two drugs. The model was characterized by a fixed volume of ELF (VELF), passive diffusion clearance (QELF), and active efflux clearance (CLout) between plasma and ELF, indicating active efflux transport systems. In conclusion, this study demonstrates that ELF drug concentrations of these three FQs are several times higher than plasma drug concentrations, probably due to the presence of efflux transporters at the pulmonary barrier level, but no biopharmaceutical advantage of FQ nebulization was observed compared with intravenous administration.

Biopharmaceutical characterization of nebulized antimicrobial agents in rats: 2. Colistin.

The purpose of this study was to investigate the pharmacokinetic properties of colistin following intrapulmonary administration of colistin sulfate in rats. Colistin was infused or delivered in nebulized form at a dose of 0.35 mg/kg of body weight in rats, and plasma drug concentrations were measured for 4 h after administration. Bronchoalveolar lavages (BAL) were also conducted at 0.5, 2, and 4 h after intravenous (i.v.) administration and administration via nebulized drug to estimate epithelial lining fluid (ELF) drug concentrations. Unbound colistin plasma concentrations at distribution equilibrium (2 h postdosing) were almost identical after i.v. infusion and nebulized drug inhalation. ELF drug concentrations were undetectable in BAL samples after i.v. administration, but they were about 1,800 times higher than unbound plasma drug levels at 2 h and 4 h after administration of the nebulized drug. Simultaneous pharmacokinetic modeling of plasma and ELF drug concentrations was performed with a model characterized by a fixed physiological volume of ELF (VELF), a passive diffusion clearance (QELF) between plasma and ELF, and a nonlinear influx transfer from ELF to the central compartment, which was assessed by reducing the nebulized dose of colistin by 10-fold (0.035 mg kg(-1)). The km was estimated to be 133 µg ml(-1), and the Vmax, in-to-Km ratio was equal to 2.5 × 10(-3) liter h(-1) kg(-1), which was 37 times higher than the QELF (6.7 × 10(-5) liter h(-1) kg(-1)). This study showed that with the higher ELF drug concentrations after administration via nebulized aerosol than after intravenous administration, for antibiotics with low permeability such as colistin, nebulization offers a real potential over intravenous administration for the treatment of pulmonary infections.

Passive and active strategies for transdermal delivery using co-encapsulating nanostructured lipid carriers: in vitro vs. in vivo studies.

This work aimed at designing a formulation based on nanostructured lipid carriers (NLC) for transdermal co-administration of olanzapine and simvastatin, using passive and active strategies in a combined in vitro/in vivo development approach. NLC were prepared by two distinct methods, namely solvent emulsification-evaporation (SE/E) and high pressure homogenization (HPH). HPH was selected on the basis of a better performance in terms of drug loading and in vitro permeation rate. Several mathematical models were used to elucidate the release mechanisms from lipid nanoparticles. In vitro release kinetics was shown to be driven by diffusion, but other mechanisms were also present, and supported the feasibility of using NLC for sustained drug delivery. The in vitro skin studies showed that the chemical penetration enhancers, limonene and ethanol, added to the NLC formulations, promoted a synergistic permeation enhancement of both drugs, with olanzapine exhibiting a higher permeation than simvastatin. Transdermal administration to rats resulted in steady-state levels reached at around 10h and maintained for 48h, again with olanzapine exhibiting a better permeation rate. The pharmacokinetic parameters indicated that the NLC dispersion displayed a better in vivo performance than the gel, which was consistent with the in vitro results. These differences were, however, negligible in the flux values, supporting the use of gel as a final, more convenient, formulation. The in vivo experiments in rats correlated well with in vitro findings and revealed that the combined use of ethanol and limonene, incorporated in the NLC formulation, provided the main driving force for drug permeation. The Dermaroller® pretreatment did not significantly enhance drug permeation, supporting the use of passive methods as suitable for a transdermal delivery system. Furthermore, this work may provide a promising proof-of-concept for further clinical application in the treatment of schizophrenia and associated disorders, combined with dyslipidemia.

Convulsions and apnoea in a patient infected with New Delhi metallo-ß-lactamase-1 Escherichia coli treated with colistin.

There has been a resurgence of interest in the use of colistin for the treatment of multidrug-resistant Gram-negative bacterial infections. A more favorable infection outcome is observed when colistin is used in combination with carbapenems. We present a patient with severe New Delhi metallo-ß-lactamase-1 Escherichia coli infection who developed convulsions rapidly followed by acute respiratory muscle weakness and apnoea during treatment with colistin and meropenem. Chromatographic assay showed a "trough" colistin level that was approximately fourfold higher than previously reported maximum steady-state colistin plasma levels in critically ill patients. The patient's renal clearance never necessitated dose adjustments, suggesting that the observed high plasma colistin level might be due to impaired non renal elimination. Although meropenem itself has very low neurotoxic potential, its concomitant use with colistin may have elicited colistin neurotoxicity.

Aerosol therapy with colistin methanesulfonate: a biopharmaceutical issue illustrated in rats.

The aim of this study was to evaluate the biopharmaceutical behavior of colistin methanesulfonate (CMS) with special focus on colistin presystemic formation after CMS nebulization in rats. CMS was administered (15 mg x kg(-1) of body weight) either intravenously for systemic pharmacokinetic studies (n = 6) or as an intratracheal nebulization for systemic pharmacokinetic studies (n = 5) or for CMS and colistin concentration measurements in epithelial lining fluid (ELF) at 30, 120, and 240 min after nebulization (n = 14). CMS and colistin concentrations were determined by a new liquid chromatography (LC)-tandem mass spectrometry (MS/MS) assay. Pharmacokinetic parameters were estimated by noncompartmental analysis. CMS and colistin pharmacokinetic data were consistent with previously published values when comparisons were possible. The fraction of the CMS dose converted systematically into colistin after intravenous CMS administration was estimated to be 12.5% on average. After CMS nebulization it was estimated that about two-thirds of the dose was directly absorbed within the systemic circulation, whereas one-third was first converted into active colistin, which was eventually absorbed. As a consequence, the colistin area under curve (AUC) reflecting systemic availability was about 4-fold greater after CMS intratracheal nebulization (607 +/- 240 microg x min x ml(-1)) than after CMS intravenous administration (160 +/- 20 microg x min x ml(-1)). CMS concentrations in ELF at 30 min and 120 min postnebulization were very high (in the order of several mg/ml) due to the limited volume of ELF but were considerably reduced at 240 min. Although lower (15% +/- 5% at 120 min) in relative terms, colistin concentrations in ELF could be high enough for being active against microorganisms following CMS nebulization.

Dose-ranging pharmacokinetics of colistin methanesulphonate (CMS) and colistin in rats following single intravenous CMS doses.

OBJECTIVES: The aim of this study was to evaluate the effect of colistin methanesulphonate (CMS) dose on CMS and colistin pharmacokinetics in rats. METHODS: Three rats per group received an intravenous bolus of CMS at a dose of 5, 15, 30, 60 or 120 mg/kg. Arterial blood samples were drawn at 0, 5, 15, 30, 60, 90, 120, 150 and 180 min. CMS and colistin plasma concentrations were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The pharmacokinetic parameters of CMS and colistin were calculated by non-compartmental analysis. RESULTS: Linear relationships were observed between CMS and colistin AUCs to infinity and CMS doses, as well as between CMS and colistin C(max) and CMS doses. CONCLUSIONS: CMS and colistin pharmacokinetics were linear for a range of colistin concentrations covering the range of values encountered and recommended in patients even during treatment with higher doses.

Assay of colistin and colistin methanesulfonate in plasma and urine by liquid chromatography-tandem mass spectrometry.

A rapid high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed for the routine quantification of colistins A and B and their prodrugs, colistin methanesulfonate (CMS) A and CMS B, respectively, in human plasma and urine by using polymyxin B1 as the internal standard (IS). CMS concentrations were determined indirectly by subtracting the colistin concentrations determined in biological samples from the whole colistin concentrations determined after sample treatment with sulfuric acid in order to hydrolyze CMS into colistin. After extraction on a solid-phase extraction column, the colistins were separated on an XBrigde C(18) column with isocratic elution (run time, 3.8 min). The mobile phase was 0.1% (vol/vol) formic acid in acetonitrile-0.1% (vol/vol) formic acid in water (20:80, vol/vol), run at a 0.2-ml/min flow rate. Ions were detected in the turbo-ion-spray-positive and multiple-reaction-monitoring modes. The ions monitored (precursor [M + 2H](2+) to product ions) were m/z 585.5/101.2 for colistin A, m/z 578.5/101.2 for colistin B, and m/z 602.5/241.2 for IS. Prevalidation studies demonstrated the stability of CMS in biological samples and extracts, a key point for the reliable quantification of colistin and CMS. The assay was accurate and reproducible for the quantification of colistins A and B and CMSs A and B in plasma samples over concentration ranges appropriate for pharmacokinetic studies: 0.024 to 6.144, 0.015 to 3.856, 0.029 to 7.492, and 0.010 to 2.508 microg/ml, respectively. In urine samples, the assay was validated over the same concentration ranges for colistins and over concentration ranges of 0.058 to 7.492 microg/ml and 0.020 to 2.508 microg/ml for CMSs A and B, respectively.