Comparison of the ARK Immunoassay With High-Performance Liquid Chromatography With Ultraviolet Detection for Therapeutic Drug Monitoring of Linezolid.

BACKGROUND: An enzymatic immunoassay is under development by ARK Diagnostics, Inc for the quantification of plasma concentrations of linezolid (LZD). In this study, the authors aimed to assess the performance of this immunoassay using a validated high-performance liquid chromatography (HPLC) ultraviolet method as reference. METHODS: Within- and between-day in vitro inaccuracy and imprecision of the ARK LZD assay were firstly tested using spiked quality controls (QC) provided by the kit manufacturer. Subsequently, the performance of the immunoassay was verified in vivo by analyzing 170 trough LZD plasma samples from patients on antibiotic therapy. RESULTS: Imprecision of the spiked QCs resulted in every instance less than 7.0% and the inaccuracy ranged from -1.5% to 6.6%. The linear correlation between the 2 methods was documented by the Pearson analysis of plasma samples from patients on LZD therapy (coefficient = 0.9619). By Bland-Altman comparison, 8.2% of the patient samples resulted out of the limits ranging from -27.0% to +33.5%, with most of them having LZD concentrations exceeding 10 mg/L. CONCLUSIONS: Acceptable analytical performance of the ARK LZD immunoassay has been demonstrated both with spiked QC and patients' samples, making it a viable alternative to HPLC for the therapeutic drug monitoring of LZD in clinical practice in laboratory hospitals that do not have HPLC equipment.

Therapeutic drug monitoring of second-generation antipsychotics in pediatric patients: an observational study in real-life settings.

PURPOSE: Available guidelines on therapeutic drug monitoring of second-generation antipsychotics were designed for adults; therefore, they cannot be transferred as such in pediatric patients, who may have different drug absorption, distribution, metabolism, and elimination. Moreover, available tools that guide dosing in neuropsychiatric pediatric patients are scant, leading to the possibility of reduced efficacy and/or increased risks of toxicity. Here we describe the results of observational therapeutic drug monitoring conducted in three pediatric neuropsychiatry units across Italy in 2012-2014, with the following aims: (1) to describe the distribution of plasma concentrations of second-generation antipsychotics in our pediatric patients and (2) to identify clinical covariates associated with plasma drug levels. METHODS: Five hundred fifty-six plasma trough concentrations of the second-generation antipsychotics risperidone (plus 9-hydroxy-risperidone), aripiprazole, olanzapine, and quetiapine were measured from 172 pediatric outpatients overall. The distribution of drug concentrations was described and correlated with drug doses and clinical variables. RESULTS: Risperidone plasma levels were lower than in adults (median 13.6 ng/ml), with a high inter-patient (78.9%) but lower intra-patient (34.2%) variability. In multiple regression analyses, risperidone plasma levels depended only on drug dose (p < 0.001). Aripiprazole plasma levels were similar to those described in adults (median 165.8 ng/ml) and were widely distributed, with an inter-patient variability of 81.1%, while the intra-patient variability was much lower (29.3%). Multiple regression analyses indicated that aripiprazole plasma levels were influenced by the daily doses (p < 0.001) and by the number of concomitant drugs (p < 0.01). CONCLUSION: Our study described the distribution of plasma levels of SGAs in a real-life setting involving pediatric patients, significantly increasing the amount of available data for this fragile population. If confirmed in larger dataset, these data may contribute to the definition of optimal therapeutic window for risperidone and aripiprazole plasma levels in pediatric patients.

Comparison of the QMS Analyzer With HPLC-UV for the Quantification of Lamotrigine Concentrations in Human Plasma Samples.

BACKGROUND: Recently, a turbidimetric immunoassay method has been developed for use in the form of a QMS lamotrigine (LTG) commercial immunoassay. This study was designed to evaluate the performance of this immunoassay using a validated high-performance liquid chromatography-ultraviolet (HPLC-UV) method as the reference. METHODS: The performance of QMS was initially tested using drug-free plasma spiked with different amounts of LTG and, subsequently, by analyzing 61 trough plasma samples from epileptic patients given the drug as part of their maintenance antiepileptic therapies. RESULTS: The correlation between LTG concentrations measured by QMS and HPLC was good, with a Pearson coefficient of 0.968 (P < 0.0001). The Bland-Altman approach showed that LTG concentrations measured with QMS exceeded HPLC on an average by 15.6% (limits of agreement, -18% to +63%), with a concentration-dependent performance (mean percent bias, 49.5 ± 8.2% and 0.6 ± 12.7% for concentrations less than 2 mg/L and greater than 14.9 mg/L, respectively). CONCLUSIONS: The QMS provided acceptable analytical performance across a wide concentration range for routine LTG measurements, being at least comparable with the other commercial immunoassays. It could be, therefore, considered as a viable alternative to HPLC methods for routine LTG monitoring in the clinical practice, although its suitability for accurate analysis of samples with low concentration is limited.

Comparison of the pharmacokinetics of raltegravir given at 2 doses of 400 mg by swallowing versus one dose of 800 mg by chewing in healthy volunteers: a randomized, open-label, 2-period, single-dose, crossover phase 1 study.

BACKGROUND: The pharmacokinetics of raltegravir (RAL) in HIV patients is characterized by high interindividual and intraindividual variability. We documented previously that HIV patients taking RAL at 400 mg bid by chewing the tablets had significantly higher drug absorption and reduced pharmacokinetic variability than patients taking the drug by swallowing the tablets. This study extends our previous findings. METHODS: An open-label, 2-period crossover study compared the pharmacokinetics of 2 doses of RAL given at 400 mg every 12 hours (that mimics a bid administration) by swallowing with 1 dose of 800 mg (that mimics a qd administration) by chewing the tablets in 12 healthy volunteers. RAL plasma concentrations were measured by a chromatographic method coupled with mass spectrometry. RESULTS: Subjects taking RAL by chewing had significantly higher drug exposure (RAL area under the curve[AUC](0-24): 40722 ± 14843 versus 21753 ± 12229 ng · h/mL, P < 0.0001) and reduced pharmacokinetic variability compared with those taking the drug by swallowing the whole tablet, with no difference in the minimum RAL concentrations (RAL C(min): 36 ± 23 versus 43 ± 23 ng/mL, P = 0.298). Subjects taking RAL by chewing the tablets had significantly higher drug absorption and reduced pharmacokinetic variability compared with those taking the drug by swallowing. No differences were observed in the minimum RAL concentrations. CONCLUSIONS: RAL at 800 mg once daily by chewing the tablets may represent a novel therapeutic option for the treatment of HIV being associated with higher drug absorption, reduced pharmacokinetic variability, and potentially better compliance compared with patients swallowing the 400-mg bid intact tablets.

Lights and shadows of the actual European guidelines on bioanalytical method validation: the case of raltegravir.

BACKGROUND: Recently, the European Medicines Agency (EMA) has released new guidelines on the validation of bioanalytical methods. In this work, we compared the analytical performance of 2 high-performance liquid chromatography with tandem mass spectrometry methods designed for the quantification of the antiretroviral drug raltegravir (RAL) that fully accomplish the criteria according to the new EMA guidelines. METHODS: The first method was developed with the goal of separating RAL from its main metabolite, whereas in the second method, we deliberately did not discriminate the parent drug from its metabolite. After validation, both methods were used for the quantification of plasma samples from HIV-infected patients on RAL-based maintenance antiretroviral therapy. RESULTS: Incurred reanalysis of samples obtained from patients receiving RAL as therapy evidenced optimal performance for both methods. Similarly, the comparison of both methods performed by the Deming test showed that they correlate significantly with each other (Pearson coefficient of regression 0.97, P < 0.0001) with no significant deviation from linearity according to the Cusum test. The Bland-Altman test, however, showed a mean difference between the 2 methods of 54.1% (limits of agreements of ±1.96 SD ranged from -163.3% to +271.5%). Such differences were significantly affected by interindividual variations in the production of RAL metabolites. CONCLUSIONS: We conclude that the recently released EMA guidelines on bioanalytical method validation present some limitations related to the process of method development. To have confidence in the drug-concentration measurements, laboratories must demonstrate their performance through the participation in international proficiency testing schemes that must include patient samples.

Comparison of the in vivo pharmacokinetics and in vitro dissolution of raltegravir in HIV patients receiving the drug by swallowing or by chewing.

The pharmacokinetics of raltegravir (RAL) in HIV patients is characterized by high interpatient/intrapatient variability. We investigated the potential contribution of the drug pharmaceutical formulation to RAL pharmacokinetics. We first compared in vivo the pharmacokinetics of RAL for 67 patients to whom the drug was administered by swallowing the intact tablet with those obtained from 13 HIV-infected patients who chewed the RAL tablet due to swallowing difficulties. Subsequently, we evaluated in vitro the dissolution of RAL tablets under different conditions. In the in vivo study, we found that patients given RAL by chewing the tablets presented pharmacokinetic profiles characterized by significantly higher RAL absorption than did patients receiving the drug by swallowing. The in vitro studies showed that when the whole tablets were exposed to an acidic medium, the release of RAL was very low, whereas when the tablets were crushed, the profiles presented significantly higher concentrations of RAL. Crushed tablets tested in water or in a pH 6.8 buffer exhibited prompt and complete dissolution of RAL. HIV-infected patients receiving RAL by chewing the tablet showed higher drug absorption and reduced pharmacokinetic variability compared with patients swallowing the intact tablet. This is related to problems in tablet disintegration and to erratic drug absorption. The amelioration of the RAL pharmaceutical formulation could improve drug pharmacokinetics.

Inter- and intra-patient variability of raltegravir pharmacokinetics in HIV-1-infected subjects.

OBJECTIVES: Limited studies in healthy volunteers and in HIV-1-infected patients have shown that raltegravir pharmacokinetics are characterized by high inter-patient variability. Only scanty data are, however, available on intra-patient raltegravir variability. The present study was designed to evaluate in parallel the inter- and intra-patient variability of raltegravir pharmacokinetics in HIV-1-infected patients during routine therapeutic drug monitoring (TDM). METHODS: Fifteen HIV-infected patients treated with highly active antiretroviral therapy containing 400 mg of raltegravir twice daily were included in the study. Pharmacokinetic evaluations were performed during two consecutive visits. Only patients given raltegravir for at least 1 month and with no changes in antiretroviral and concomitant therapy between the two pharmacokinetic evaluations were considered. Raltegravir plasma concentrations were determined by a validated HPLC method. Blood samples were collected at 0, 1, 2, 3 and 4 h after the morning drug dose. Raltegravir AUC(0-12) was estimated using a recently developed algorithm. RESULTS: The pharmacokinetic evaluation was repeated after an average of 52 ± 68 days. Raltegravir AUC(0-12) values ranged from 1495 to 49 051 ng ·â€Šh/mL. The main finding was that intra-patient variability was a large component of the overall variability in raltegravir pharmacokinetics. In some instances the difference between raltegravir AUC(0-4) and AUC(0-12) measured in the same patient during two consecutive evaluations exceeded 110% and 75%, respectively. CONCLUSIONS: The pharmacokinetics of raltegravir in HIV-1-infected subjects are characterized not only by inter-patient variability but also by high intra-patient variability. This condition limits the application of TDM for raltegravir, and might potentially affect patient outcome.

Co-administration of raltegravir reduces daily darunavir exposure in HIV-1 infected patients.

The potential drug-to-drug interaction between darunavir and raltegravir in the setting of HIV infection is a highly debated issue still unresolved. In the present study we have evaluated the pharmacokinetics of darunavir and ritonavir in 53 HIV-1 infected patients with or without concomitant raltegravir administration. The assessment of trough plasma drug concentrations was carried out in all subjects and the potential influence of raltegravir on darunavir and ritonavir disposition, assessed by specific pharmacokinetic evaluations in a subgroup of 25 patients. No significant differences on darunavir and ritonavir plasma trough levels were observed between patients receiving or not raltegravir. Co-administration of raltegravir was, however, associated with a 40% reduction in darunavir C(max) and estimated AUC(0-24), as well a 60% increase in the estimated darunavir clearance compared with values measured in patients not given raltegravir. Notably, this interaction was independent of the dosage of darunavir and not due to effects of raltegravir on the pharmacokinetics of ritonavir. These results should be taken into account when darunavir-based regimens are implemented in the setting of HIV, especially considering that this drug is usually administered at fixed daily dose and no therapeutic drug monitoring is performed in most centres.

Different effects of PCB101, PCB118, PCB138 and PCB153 alone or mixed in MCF-7 breast cancer cells.

Polychlorinated biphenyls (PCBs) are ubiquitous, persistent environmental contaminants that can be a potential health hazard. In the present study we analyzed the potential estrogenic effect in MCF-7 cells of four biologically relevant PCB congeners, alone or in mixtures, present in dairy products, vegetable oil and fish: PCB101, PCB118, PCB138 and PCB153. The mixture of four PCB was tested at seven different concentrations. We investigated the ability of these PCBs, alone or mixed, to induce cell proliferation, and the level of estrogen-regulated protein pS2, in human MCF-7 breast cancer cells. PCB153 (35 microM) stimulated cell proliferation from 48 h up to day 6, PCB118 (40 microM) only at 48 h, but PCB101 (45 microM) and PCB138 (15 microM) applied to the cells for 6 days had no effect. In contrast, the various concentrations of mixtures significantly reduced cell proliferation at different times. No change in pS2 levels was seen after treatment with the PCBs alone or mixed. In exploring the mechanism of these events, we found that PCB153 induced mitogen-activated protein kinase (MAPK) ERK1/2 at 4, 8 and 12 h, while the antiproliferative effect seemed to be related to an apoptotic action beginning at 12 h and ending at 48 h. These findings indicate that these PCBs alone or mixed have no estrogenic effect in MCF-7 cells, although PCB153 induce an ERK1/2-mediated mitogenic effect. On the contrary the mixture of PCBs induces an antiproliferative effect, ascribable to an apoptotic action.

Is it time to revise antiretrovirals dosing? a pharmacokinetic viewpoint.

We document our experience with therapeutic drug monitoring (TDM) of antiretroviral agents (1807 determinations) carried out as day-by-day clinical practice for the optimization of drug dosing in HIV-infected patients. A significant proportion of patients had lopinavir, atazanavir and nevirapine trough concentrations exceeding the upper therapeutic threshold. Further studies are needed to identify good candidates/drugs for TDM, eventually allowing the selection of patients who may benefit from TDM-driven adjustments in antiretrovirals dosage.

Combined isosorbide dinitrate and ibuprofen as a novel therapy for muscular dystrophies: evidence from Phase I studies in healthy volunteers.

We designed two Phase I studies that assessed healthy volunteers in order to evaluate the safety and to optimize the dosing of the combination of the drugs isosorbide dinitrate, a nitric oxide donor, and ibuprofen, a nonsteroidal antiinflammatory drug. We designed these studies with the aim of designing a Phase II trial to evaluate the drugs' efficacy in patients affected by Duchenne muscular dystrophy. For the first trial, ISOFEN1, a single-dose, randomized-sequence, open-label, active control, three-treatment cross-over study, was aimed at comparing the pharmacokinetics of ibuprofen 200 mg and isosorbide dinitrate 20 mg when given alone and concomitantly. The pharmacokinetics of ibuprofen given alone versus ibuprofen given concomitantly with isosorbide dinitrate were similar, as documented by the lack of statistically significant differences in the main drug's pharmacokinetic parameters (time to maximal concentration [Tmax], maximal concentration [Cmax], area under the curve [AUC]0-t, and AUC0-∞). Similarly, we found that the coadministration of ibuprofen did not significantly affect the pharmacokinetics of isosorbide dinitrate. No issues of safety were detected. The second trial, ISOFEN2, was a single-site, dose titration study that was designed to select the maximum tolerated dose for isosorbide dinitrate when coadministered with ibuprofen. Eighteen out of the 19 enrolled subjects tolerated the treatment well, and they completed the study at the highest dose of isosorbide dinitrate applied (80 mg/day). One subject voluntarily decided to reduce the dose of isosorbide dinitrate from 80 mg to 60 mg. The treatment-related adverse events recorded during the study were, for the large majority, episodes of headache that remitted spontaneously in 0.5-1 hour - a known side effect of isosorbide dinitrate. These studies demonstrate that the combination of isosorbide dinitrate and ibuprofen does not lead to pharmacokinetic interactions between the two drugs; they also demonstrate that the combination of isosorbide dinitrate and ibuprofen has optimal tolerability and safety profiles that are similar to those previously reported for isosorbide dinitrate and ibuprofen given alone.

Limited sampling strategies for the estimation of atazanavir daily exposure in HIV-infected patients.

Stepwise multiple regression analyses were applied to 44 atazanavir pharmacokinetic profiles from 44 HIV-1 infected patients concomitantly treated with raltegravir with the goal of identifying limited sampling strategies for the prediction of drug AUC(0-12) . Atazanavir trough-based equations failed to reliably predict daily drug exposure in patients with low drug bioavailability. Conversely, different algorithms based on few samples and associated with good correlation, acceptable bias and imprecision with the measured atazanavir AUC(0-12) were identified. These models could be used to predict atazanavir exposure for clinic or research purposes.

Linezolid plasma concentrations and occurrence of drug-related haematological toxicity in patients with gram-positive infections.

Retrospective studies have documented a significant association between linezolid (LNZ) plasma concentrations and drug-related haematological toxicity. However, the safe upper threshold level for LNZ plasma trough concentrations (Cmin values) has not been defined with certainty. A prospective observational study was performed aimed at comparing LNZ Cmin values in patients developing drug-related side effects with those measured in patients not experiencing LNZ toxicity. LNZ Cmin values were measured from the first week after starting therapy and were repeated periodically up to the end of treatment. Fifty patients, for a total of 210 LNZ Cmin evaluations, were considered. All patients (n=9) who developed drug-related haematological toxicity also had significantly higher plasma LNZ Cmin values during the first week of therapy (9.0±6.4 mg/L vs. 4.9±3.7 mg/L; P<0.01) and thereafter (9.3±5.4 mg/L vs. 4.4±3.4 mg/L; P<0.01). The significant association between LNZ plasma concentrations and haematological toxicity was also confirmed by multivariate logistic regression analysis including age, serum creatinine and concomitant medications as independent variables. A causal relationship between LNZ concentrations and the risk of developing drug-related haematological toxicity was observed. Accordingly, application of therapeutic drug monitoring may improve the safety outcome of patients receiving LNZ therapy.

Low body weight in females is a risk factor for increased tenofovir exposure and drug-related adverse events.

Treatment with tenofovir sometimes leads to non-reversible kidney and/or bone diseases. Factors associated with these drug-related adverse events are poorly characterized. Our objective was to investigate such factors in patients treated long term with daily tenofovir. One-hundred Caucasian HIV-positive patients with basal creatinine clearance >80 mL/min treated with tenofovir for at least 6 months and with at least one assessment of tenofovir plasma trough concentrations were considered. Tenofovir-associated adverse events were defined as the appearance of pathological proteinuria, worsening of renal function or bone demineralization. By multivariate regression analysis, we found that serum creatinine (p = 0.003) and body weight (p = 0.002) were the factors independently associated with plasma tenofovir concentrations. In particular, women with body weight<50 kg had significantly higher plasma tenofovir concentrations than those weighting >50 Kg (160±93 vs.71±52 ng/mL, p<0.001). High tenofovir plasma trough concentrations and the age of the patients were independently associated with the development of drug-related kidney and bone toxicity. In this retrospective study we have shown that HIV-infected women with low body weight are at risk to be exposed to high tenofovir plasma trough concentrations, ultimately resulting in a significant hazard to develop long-term tenofovir complications.

Limited sampling strategies for the estimation of raltegravir daily exposure in HIV-infected patients.

Stepwise multiple regression analyses were applied to 50 raltegravir pharmacokinetic profiles from 50 HIV patients with the goal to identify limited sampling strategies for the prediction of drug area under the time-concentration curve (AUC(0-12)). Raltegravir single sampling point-based equations failed to reliably predict daily drug exposure. Conversely, different algorithms based on few samples and associated with good correlation, acceptable bias, and imprecision with the measured raltegravir AUC(0-12) were identified. These models could used to predict raltegravir exposure for clinic or research purposes.

Genotoxic effects of polychlorinated biphenyls (PCB 153, 138, 101, 118) in a fish cell line (RTG-2).

Polychlorinated biphenyls (PCBs) are persistent pollutants in aquatic environments, often causing the decline or disappearance of wild populations. The primary aim of this study was to investigate the genotoxic effects of some PCBs (PCB153 (2,2',4,4',5,5'-hexachlorobiphenyl) and 138 (2,2',3,4,4',5'-hexachloro-biphenyl), both non-dioxin-like compounds, and the pentachlorobiphenyls PCB118 (2,3',4,4',5-) and 101 (2,2',4',5,5'-), the former an ortho-substituted, low-affinity dioxin-like compound and the latter a non-coplanar congener classified as non-dioxin-like) in fish cells (RTG-2). These congeners are mostly present in surface waters and in edible aquatic organisms and the loss of DNA integrity in vitro serves as a sensitive biomarker of cytogenetic alterations and is considered as an initial step for the identification of genotoxic effects. The alkaline comet assay and the micronucleus test show clear genotoxic damage after short and longer exposure (2 and 24h) to maximum soluble, non-cytotoxic doses, evident sooner with PCBs 101 and 118. Oxidative stress situations involving ROS release, reduction in total GSH, lipid peroxidation and alteration to superoxide dismutase, seen after exposure with all the congeners, though with different kinetics, seem the most likely explanation for the genotoxic damage. This appears to be confirmed by the modified comet assay (pH 10) for detection of oxidized bases using endonuclease III. The increased generation of intracellular ROS might explain the apoptosis seen after treatment with the single PCBs and evaluated on the basis of the rise in 3-7 caspase activity. Therefore both the non-coplanar, non-dioxin-like PCBs (153, 138, 101) and the low-affinity dioxin-like compound PCB118 cause evident genotoxic damage, probably as a consequence of oxidative stress.