Validation and clinical application of a multiplex high performance liquid chromatography - tandem mass spectrometry assay for the monitoring of plasma concentrations of 12 antibiotics in patients with severe bacterial infections.

OBJECTIVE: Unpredictable pharmacokinetics of antibiotics in patients with life-threatening bacterial infections is associated with drug under- or overdosing. Therapeutic drug monitoring (TDM) may guide dosing adjustment aimed at maximizing antibacterial efficacy and minimizing toxicity. Rapid and accurate analytical methods are key for real-time TDM. Our objective was to develop a robust high-performance liquid chromatography-tandem mass spectrometry method (HPLC-MS/MS) for multiplex quantification of plasma concentrations of 12 antibiotics: imipenem/cilastatin, meropenem, ertapenem, cefepime, ceftazidime, ceftriaxone, piperacillin/tazobactam, amoxicillin, flucloxacillin, rifampicin, daptomycin. METHODS: A single extraction procedure consisting in methanol plasma protein precipitation and H2O dilution was used for all analytes. After chromatographic separation on an Acquity UPLC HSS-T3 2.1 × 50 mm, 1.8 µm (Waters®) column, quantification was performed by electro-spray ionisation-triple quadrupole mass spectrometry with selected reaction monitoring detection. Antibiotics were divided in two pools of calibration according to the frequency of analyses requests in the hospital routine antibiotic TDM program. Stable isotopically-labelled analogues were used as internal standards. A single analytical run lasted less than 9 min. RESULTS: The method was validated based on FDA recommendations, including assessment of extraction yield (96-113.8%), matrix effects, and analytical recovery (86.3-99.6%). The method was sensitive (lower limits of quantification 0.02-0.5 µg/mL), accurate (intra/inter-assay bias -11.3 to +12.7%) and precise (intra/inter-assay CVs 2.1-11.5%) over the clinically relevant plasma concentration ranges (upper limits of quantification 20-160 µg/mL). The application of the TDM assay was illustrated with clinical cases that highlight the impact on patients' management of an analytical assay providing information with short turn-around time on antibiotic plasma concentration. CONCLUSION: This simple, robust high-throughput multiplex HPLC-MS/MS assay for simultaneous quantification of plasma concentrations of 12 daily used antibiotics is optimally suited for clinically efficient real-time TDM.

Clinical usefulness of therapeutic concentration monitoring for imatinib dosage individualization: results from a randomized controlled trial.

PURPOSE: This study assessed whether a cycle of "routine" therapeutic drug monitoring (TDM) for imatinib dosage individualization, targeting an imatinib trough plasma concentration (C min) of 1,000 ng/ml (tolerance: 750-1,500 ng/ml), could improve clinical outcomes in chronic myelogenous leukemia (CML) patients, compared with TDM use only in case of problems ("rescue" TDM). METHODS: Imatinib concentration monitoring evaluation was a multicenter randomized controlled trial including adult patients in chronic or accelerated phase CML receiving imatinib since less than 5 years. Patients were allocated 1:1 to "routine TDM" or "rescue TDM." The primary endpoint was a combined outcome (failure- and toxicity-free survival with continuation on imatinib) over 1-year follow-up, analyzed in intention-to-treat (ISRCTN31181395). RESULTS: Among 56 patients (55 evaluable), 14/27 (52 %) receiving "routine TDM" remained event-free versus 16/28 (57 %) "rescue TDM" controls (P = 0.69). In the "routine TDM" arm, dosage recommendations were correctly adopted in 14 patients (median C min: 895 ng/ml), who had fewer unfavorable events (28 %) than the 13 not receiving the advised dosage (77 %; P = 0.03; median C min: 648 ng/ml). CONCLUSIONS: This first target concentration intervention trial could not formally demonstrate a benefit of "routine TDM" because of small patient number and surprisingly limited prescriber's adherence to dosage recommendations. Favorable outcomes were, however, found in patients actually elected for target dosing. This study thus shows first prospective indication for TDM being a useful tool to guide drug dosage and shift decisions. The study design and analysis provide an interesting paradigm for future randomized TDM trials on targeted anticancer agents.

Population pharmacokinetic modelling and evaluation of different dosage regimens for darunavir and ritonavir in HIV-infected individuals.

OBJECTIVES: Darunavir is a protease inhibitor that is administered with low-dose ritonavir to enhance its bioavailability. It is prescribed at standard dosage regimens of 600/100 mg twice daily in treatment-experienced patients and 800/100 mg once daily in naive patients. A population pharmacokinetic approach was used to characterize the pharmacokinetics of both drugs and their interaction in a cohort of unselected patients and to compare darunavir exposure expected under alternative dosage regimens. METHODS: The study population included 105 HIV-infected individuals who provided darunavir and ritonavir plasma concentrations. Firstly, a population pharmacokinetic analysis for darunavir and ritonavir was conducted, with inclusion of patients' demographic, clinical and genetic characteristics as potential covariates (NONMEM(®)). Then, the interaction between darunavir and ritonavir was studied while incorporating levels of both drugs into different inhibitory models. Finally, model-based simulations were performed to compare trough concentrations (Cmin) between the recommended dosage regimen and alternative combinations of darunavir and ritonavir. RESULTS: A one-compartment model with first-order absorption adequately characterized darunavir and ritonavir pharmacokinetics. The between-subject variability in both compounds was important [coefficient of variation (CV%) 34% and 47% for darunavir and ritonavir clearance, respectively]. Lopinavir and ritonavir exposure (AUC) affected darunavir clearance, while body weight and darunavir AUC influenced ritonavir elimination. None of the tested genetic variants showed any influence on darunavir or ritonavir pharmacokinetics. The simulations predicted darunavir Cmin much higher than the IC50 thresholds for wild-type and protease inhibitor-resistant HIV-1 strains (55 and 550 ng/mL, respectively) under standard dosing in >98% of experienced and naive patients. Alternative regimens of darunavir/ritonavir 1200/100 or 1200/200 mg once daily also had predicted adequate Cmin (>550 ng/mL) in 84% and 93% of patients, respectively. Reduction of darunavir/ritonavir dosage to 600/50 mg twice daily led to a 23% reduction in average Cmin, still with only 3.8% of patients having concentrations below the IC50 for resistant strains. CONCLUSIONS: The important variability in darunavir and ritonavir pharmacokinetics is poorly explained by clinical covariates and genetic influences. In experienced patients, treatment simplification strategies guided by drug level measurements and adherence monitoring could be proposed.

Clinical usefulness of electronic drug-drug interaction checking in the care of cardiovascular surgery inpatients.

OBJECTIVES: Drug-related problems (DRPs) are events or circumstances involving drug therapy that actually or potentially interfere with desired health outcomes. This study tested the applicability of clinical decision support software in identifying and managing DRPs among cardiovascular surgery inpatients. METHODS: Two clinical pharmacologists attended ward rounds on a low-dependency cardiovascular surgery ward every 2 weeks over a 7-month period. Three hundred and three patients were assessed. On average, patients received 17 scheduled and 'as required' medicines. DRPs were identified 'manually' via assessment of electronic prescription charts and patient records and 'electronically' using clinical decision support software (Pharmavista). The numbers of alerts for optimizing medication safety generated by the two methods were compared. RESULTS: Manual checking identified 346 DRPs leading to 346 alerts in 201 patients (overall 1.1 alerts/patient). Relevant interactions accounted for 44% of DRPs detected by clinical pharmacologists. Clinical decision support software, which could only report interactions, however, generated 1,370 alerts (average 4.5 alerts/patient). Only 147 (11%) drug-drug interaction alerts were identical to those identified by manual checking; the remaining 89% were considered not clinically relevant. CONCLUSIONS: Compared to identification of DRPs by clinical pharmacologists, the clinical decision support software performed poorly due to over-alerting and inability to assess for problems not caused by drug-drug interactions.

Therapeutic Drug Monitoring of the new targeted anticancer agents imatinib, nilotinib, dasatinib, sunitinib, sorafenib and lapatinib by LC tandem mass spectrometry.

The treatment of some cancer patients has shifted from traditional, non-specific cytotoxic chemotherapy to chronic treatment with molecular targeted therapies. Imatinib mesylate, a selective inhibitor of tyrosine kinases (TKIs) is the most prominent example of this new era and has opened the way to the development of several additional TKIs, including sunitinib, nilotinib, dasatinib, sorafenib and lapatinib, in the treatment of various hematological malignancies and solid tumors. All these agents are characterized by an important inter-individual pharmacokinetic variability, are at risk for drug interactions, and are not devoid of toxicity. Additionally, they are administered for prolonged periods, anticipating the careful monitoring of their plasma exposure via Therapeutic Drug Monitoring (TDM) to be an important component of patients' follow-up. We have developed a liquid chromatography-tandem mass spectrometry method (LC-MS/MS) requiring 100 microL of plasma for the simultaneous determination of the six major TKIs currently in use. Plasma is purified by protein precipitation and the supernatant is diluted in ammonium formate 20 mM (pH 4.0) 1:2. Reverse-phase chromatographic separation of TKIs is obtained using a gradient elution of 20 mM ammonium formate pH 2.2 and acetonitrile containing 1% formic acid, followed by rinsing and re-equilibration to the initial solvent composition up to 20 min. Analyte quantification, using matrix-matched calibration samples, is performed by electro-spray ionization-triple quadrupole mass spectrometry by selected reaction monitoring detection using the positive mode. The method was validated according to FDA recommendations, including assessment of extraction yield, matrix effects variability (<9.6%), overall process efficiency (87.1-104.2%), as well as TKIs short- and long-term stability in plasma. The method is precise (inter-day CV%: 1.3-9.4%), accurate (-9.2 to +9.9%) and sensitive (lower limits of quantification comprised between 1 and 10 ng/mL). This is the first broad-range LC-MS/MS assay covering the major currently in-use TKIs. It is an improvement over previous methods in terms of convenience (a single extraction procedure for six major TKIs, reducing significantly the analytical time), sensitivity, selectivity and throughput. It may contribute to filling the current knowledge gaps in the pharmacokinetics/pharmacodynamics relationships of the latest TKIs developed after imatinib and better define their therapeutic ranges in different patient populations in order to evaluate whether a systematic TDM-guided dose adjustment of these anticancer drugs could contribute to minimize the risk of major adverse reactions and to increase the probability of efficient, long lasting, therapeutic response.

Relationship of imatinib-free plasma levels and target genotype with efficacy and tolerability.

Imatinib has revolutionised the treatment of chronic myeloid leukaemia (CML) and gastrointestinal stromal tumours (GIST). Using a nonlinear mixed effects population model, individual estimates of pharmacokinetic parameters were derived and used to estimate imatinib exposure (area under the curve, AUC) in 58 patients. Plasma-free concentration was deduced from a model incorporating plasma levels of alpha(1)-acid glycoprotein. Associations between AUC (or clearance) and response or incidence of side effects were explored by logistic regression analysis. Influence of KIT genotype was also assessed in GIST patients. Both total (in GIST) and free drug exposure (in CML and GIST) correlated with the occurrence and number of side effects (e.g. odds ratio 2.7+/-0.6 for a two-fold free AUC increase in GIST; P<0.001). Higher free AUC also predicted a higher probability of therapeutic response in GIST (odds ratio 2.6+/-1.1; P=0.026) when taking into account tumour KIT genotype (strongest association in patients harbouring exon 9 mutation or wild-type KIT, known to decrease tumour sensitivity towards imatinib). In CML, no straightforward concentration-response relationships were obtained. Our findings represent additional arguments to further evaluate the usefulness of individualizing imatinib prescription based on a therapeutic drug monitoring programme, possibly associated with target genotype profiling of patients.

Population pharmacokinetics of imatinib and the role of alpha-acid glycoprotein.

AIMS: The aims of this observational study were to assess the variability in imatinib pharmacokinetics and to explore the relationship between its disposition and various biological covariates, especially plasma alpha1-acid glycoprotein concentrations. METHODS: A population pharmacokinetic analysis was performed using NONMEM based on 321 plasma samples from 59 patients with either chronic myeloid leukaemia or gastrointestinal stromal tumours. The influence of covariates on oral clearance and volume of distribution was examined. Furthermore, the in vivo intracellular pharmacokinetics of imatinib was explored in five patients. RESULTS: A one-compartment model with first-order absorption appropriately described the data, giving a mean (+/-SEM) oral clearance of 14.3 l h-1 (+/-1.0) and a volume of distribution of 347 l (+/-62). Oral clearance was influenced by body weight, age, sex and disease diagnosis. A large proportion of the interindividual variability (36% of clearance and 63% of volume of distribution) remained unexplained by these demographic covariates. Plasma alpha1-acid glycoprotein concentrations had a marked influence on total imatinib concentrations. Moreover, we observed an intra/extracellular ratio of 8, suggesting substantial uptake of the drug into the target cells. CONCLUSION: Because of the high pharmacokinetic variability of imatinib and the reported relationships between its plasma concentration and efficacy and toxicity, the usefulness of therapeutic drug monitoring as an aid to optimizing therapy should be further investigated. Ideally, such an approach should take account of either circulating alpha1-acid glycoprotein concentrations or free imatinib concentrations.

Severe pustular eruption associated with imatinib and voriconazole in a patient with chronic myeloid leukemia.

Imatinib is a specific and potent inhibitor of the BCR-ABL tyrosine kinase. Several clinical trials have demonstrated the efficacy of imatinib in chronic myeloid leukemia. Adverse cutaneous reactions induced by imatinib are frequent and may be dose related. We report a case of an unusual pustular eruption in a patient with chronic myeloid leukemia, who received high doses imatinib for blast crisis and later voriconazole for invasive pulmonary aspergillosis. At the time of his skin eruption, elevated plasma levels of imatinib were recorded. Imatinib is primarily metabolized by the cytochrome CYP3A4. Voriconazole is a cytochrome CYP3A4 inhibitor and can lead to high plasma levels of imatinib. This case suggests that severe drug reactions to imatinib may be related not only to imatinib doses, but also to elevated plasma drug levels resulting from pharmacokinetic interactions. The monitoring of imatinib plasma levels may be of help for identifying patients at risk for severe toxicity.

Determination of imatinib (Gleevec) in human plasma by solid-phase extraction-liquid chromatography-ultraviolet absorbance detection.

A sensitive HPLC method has been developed for the assay of imatinib in human plasma, by off-line solid-phase extraction followed by HPLC coupled with UV-Diode Array Detection. Plasma (750 microl), with clozapine added as internal standard, is diluted 3 + 1 with water and subjected to a solid-phase extraction on a C18 cartridge. After matrix components elimination with 2000 microl of water (in two aliquots of 1000 microl), imatinib is eluted with 3 x 500 microl MeOH. The resulting eluate is evaporated under nitrogen at room temperature and is reconstituted in 180 microl 50% methanol. A 50 microl volume is injected onto a Nucleosil 100-5 microm C18 AB column. Imatinib is analyzed using a gradient elution program with solvent mixture constituted of methanol and water containing both 0.05% ammonium acetate. Imatinib is detected by UV at 261 nm. The calibration curves are linear between 0.1 and 10 microg/ml. The limit of quantification and detection are 0.05 and 0.01 microg/ml, respectively. The mean absolute recovery of imatinib is 96%. The method is precise with mean inter-day CVs within 1.1-2.4%, and accurate (range of inter-day deviations -0.6 to +0.7%). The method has been validated and is currently being applied in a clinical study assessing the imatinib plasma concentration variability in a population of chronic myeloid leukemia- and gastro-intestinal stromal tumor-patients.

Research and development to improve naval shipboard waste management using a compact closed-loop-controlled waste incinerator.

Research has been conducted into the application of forced acoustics for enhancing the performance of a pyrolyzed waste afterburner configured as a dump combustor. Subscale studies showed that acoustic forcing of an air jet entering a dump chamber could trigger the formation of coherent vortices generated by entrainment of ambient gases. Subsequent studies showed that combustible gases could be introduced into the coherent vortices, and with additional modulation this configuration would lead to an enhanced combustion rate with low emissions of pollutants. The acoustically forced burner concept was scaled up to practical levels and tested as an afterburner on a commercial waste incinerator operating in pyrolysis mode. Results show that the afterburner can promote both compactness, due to the rapid combustion rate, and low pollutant emissions resulting from enhanced mixing prior to combustion.