Clofazimine as a substitute for rifampicin improves efficacy of Mycobacterium avium pulmonary disease treatment in the hollow-fiber model.

Mycobacterium avium complex pulmonary disease is treated with an azithromycin, ethambutol, and rifampicin regimen, with limited efficacy. The role of rifampicin is controversial due to inactivity, adverse effects, and drug interactions. Here, we evaluated the efficacy of clofazimine as a substitute for rifampicin in an intracellular hollow-fiber infection model. THP-1 cells, which are monocytes isolated from peripheral blood from an acute monocytic leukemia patient, were infected with M. avium ATCC 700898 and exposed to a regimen of azithromycin and ethambutol with either rifampicin or clofazimine. Intrapulmonary pharmacokinetic profiles of azithromycin, ethambutol, and rifampicin were simulated. For clofazimine, a steady-state average concentration was targeted. Drug concentrations and bacterial densities were monitored over 21 days. Exposures to azithromycin and ethambutol were 20%-40% lower than targeted but within clinically observed ranges. Clofazimine exposures were 1.7 times higher than targeted. Until day 7, both regimens were able to maintain stasis. Thereafter, regrowth was observed for the rifampicin-containing regimen, while the clofazimine-containing regimen yielded a 2 Log10 colony forming unit (CFU) per mL decrease in bacterial load. The clofazimine regimen also successfully suppressed the emergence of macrolide tolerance. In summary, substitution of rifampicin with clofazimine in the hollow-fiber model improved the antimycobacterial activity of the regimen. Clofazimine-containing regimens merit investigation in clinical trials.

Results of the first international quality control programme for oral targeted oncolytics.

AIMS: With the rising number of oral targeted oncolytics and growing awareness of the benefits of therapeutic drug monitoring (TDM) within the field of oncology, it is expected that the requests for quantifying concentrations of these drugs will increase. It is important to (cross-)validate available assays and ensure its quality, as results may lead to altered dosing recommendations. Therefore, we aimed to evaluate the performance of laboratories measuring concentrations of targeted oral oncolytics in a one-time international quality control (QC) programme. METHODS: Participating laboratories received a set of plasma samples containing low, medium and high concentrations of imatinib, sunitinib, desethylsunitinib, pazopanib, cabozantinib, olaparib, enzalutamide, desmethylenzalutamide and abiraterone, with the request to report their results back within five weeks after shipment. Accuracy was defined acceptable if measurements where within 85%-115% from the weighed-in reference concentrations. Besides descriptive statistics, an exploratory ANOVA was performed. RESULTS: Seventeen laboratories from six countries reported 243 results. Overall, 80.7% of all measurements were within the predefined range of acceptable accuracy. Laboratories performed best in quantifying imatinib and poorest in quantifying desethylsunitinib (median absolute inaccuracy respectively 4.0% (interquartile range (IQR) 1.8%-6.5%) and 15.5% (IQR 8.8%-34.9%)). The poorest performance of desethylsunitinib might be caused by using the stable-isotope-labelled sunitinib instead of desethylsunitinib as an internal standard, or due to the light-induced cis(Z)/trans(E) isomerization of (desethyl)sunitinib. Overall, drug substance and performing laboratory seemed to influence the absolute inaccuracy (F = 16.4; p < 0.001 and F = 35.5; p < 0.001, respectively). CONCLUSION: Considering this is the first evaluation of an international QC programme for oral targeted oncolytics, an impressive high percentage of measurements were within the predefined range of accuracy. Cross-validation of assays that are used for dose optimization of oncolytics will secure the performance and will protect patients from incorrect advices.

Measuring Tumour Imatinib Concentrations in Gastrointestinal Stromal Tumours: Relevant or Redundant?

Imatinib plasma trough concentrations are associated with efficacy for patients treated for advanced or metastatic KIT-positive gastrointestinal stromal tumours (GISTs). This relationship has not been studied for patients treated in the neoadjuvant setting, let alone its correlation with tumour drug concentrations. In this exploratory study we aimed to determine the correlation between plasma and tumour imatinib concentrations in the neoadjuvant setting, investigate tumour imatinib distribution patterns within GISTs, and analyse its correlation with pathological response. Imatinib concentrations were measured in both plasma and in three regions of the resected primary tumour: the core, middle part, and periphery. Twenty-four tumour samples derived from the primary tumours of eight patients were included in the analyses. Imatinib tumour concentrations were higher compared to plasma concentrations. No correlation was observed between plasma and tumour concentrations. Interpatient variability in tumour concentrations was high compared to interindividual variability in plasma concentrations. Although imatinib accumulates in tumour tissue, no distribution pattern of imatinib in tumour tissue could be identified. There was no correlation between imatinib concentrations in tumour tissue and pathological treatment response.

High-Dose Intermittent Treatment with the Multikinase Inhibitor Sunitinib Leads to High Intra-Tumor Drug Exposure in Patients with Advanced Solid Tumors.

Patients with advanced cancer refractory to standard treatment were treated with sunitinib at a dose of 300 mg once every week (Q1W) or 700 mg once every two weeks (Q2W). Tumor, skin and plasma concentrations were measured and immunohistochemical staining for tumor cell proliferation (TCP), microvessel density (MVD) and T-cell infiltration was performed on tumor biopsies before and after 17 days of treatment. Oral administration of 300 mg sunitinib Q1W or 700 mg Q2W resulted in 19-fold (range 5-35×) and 37-fold higher (range 10-88×) tumor drug concentrations compared to parallel maximum plasma drug concentrations, respectively. Patients with higher tumor sunitinib concentrations had favorable progression-free and overall survival than those with lower concentrations (p = 0.046 and 0.024, respectively). In addition, immunohistochemistry of tumor biopsies revealed an induction of T-cell infiltration upon treatment. These findings provide pharmacological and biological insights in the clinical benefit from high-dose intermittent sunitinib treatment. It emphasizes the potential benefit from reaching higher tumor drug concentrations and the value of measuring TKI tumor- over plasma-concentrations. The finding that reaching higher tumor drug concentrations provides most clinical benefit in patients with treatment refractory malignancies indicates that the inhibitory potency of sunitinib may be enforced by a high-dose intermittent treatment schedule. These results provide proof of concept for testing other clinically available multitargeted tyrosine kinase inhibitors in a high-dose intermittent treatment schedule.

Standard therapy of Mycobacterium avium complex pulmonary disease shows limited efficacy in an open source hollow fibre system that simulates human plasma and epithelial lining fluid pharmacokinetics.

OBJECTIVES: Mycobacterium avium complex (MAC) bacteria can cause chronic pulmonary disease (PD). Current treatment regimens of azithromycin, ethambutol and rifampicin have culture conversion rates of around 65%. Dynamic, preclinical models to assess the efficacy of treatment regimens are important to guide clinical trial development. The hollow fibre system (HFS) has been applied but reports lack experimental details. METHODS: We simulated the human pharmacokinetics of azithromycin, ethambutol and rifampicin both in plasma and epithelial lining fluid (ELF) in a HFS, exposing THP-1 cells infected with M. avium to the triple-drug regimen for 3 weeks. We accounted for drug-drug interactions and protein-binding and provide all laboratory protocols. We differentiated the effects on the intracellular and extracellular mycobacterial population. RESULTS: The antibiotic concentrations in the HFS accurately reflected the time to peak concentration (Tmax), the peak concentration (Cmax) and half-life of azithromycin, rifampicin and ethambutol in plasma and ELF reported in literature. We find that plasma drug concentrations fail to hold the MAC bacterial load static (ΔLog10 CFU/mLControl:Regimen = 0.66 ± 0.76 and 0.45 ± 0.28 at 3 and 21 days); ELF concentrations do hold the bacterial load static for 3 days and inhibit bacterial growth for the duration of the experiment (ΔLog10 CFU/mLControl:Regimen = 1.1 ± 0.1 and 1.64 ± 0.59 at 3 and 21 days). DISCUSSION: In our model, the current therapy against MAC is ineffective, even when accounting for antibiotic accumulation at the site of infection and intracellularly. New treatment regimens need to be developed and be compared with currently recommended regimens in dynamic models prior to clinical evaluation. With the publication of all protocols we aim to open this technology to new users.

Simple and Rapid Quantification of the Multi-Enzyme Targeting Antifolate Pemetrexed in Human Plasma.

BACKGROUND: Pemetrexed is an antifolate cytostatic drug that targets multiple enzymes involved in folate biosynthesis and is indicated for treatment of non-small-cell lung cancer and malignant pleural mesothelioma. As evidence for an exposure-response/toxicity relationship is accumulating, dose individualization using therapeutic drug monitoring may be a feasible strategy to optimize treatment. The purpose of this study was to develop a simple, sensitive, high-performance liquid chromatography method with UV detection for quantification of pemetrexed levels in human plasma. METHOD: The method involves a simple protein precipitation, followed by ultra-performance liquid chromatography with ultraviolet detection at a wavelength of 254 nm. Pemetrexed was separated using a mobile phase with a linear gradient and a run time of only 7 minutes. RESULTS: The assay has been validated over the concentration range 0.25-500 mg/L of pemetrexed. Accuracy for this assay ranged from -4.50% to 1.78%, and the within- and between-run coefficients of variation were <3.57%. Pemetrexed in plasma was proven to be stable for 8 months at -40°C. CONCLUSIONS: The bioanalytical method we developed proved to be simple, accurate, precise, and fast. This analytical method is successfully in use for therapeutic drug monitoring and will be used for pharmacokinetic studies.

Manual punch versus automated flow-through sample desorption for dried blood spot LC-MS/MS analysis of voriconazole.

Dried blood spot (DBS) sampling is a patient-friendly alternative for plasma sampling for the purpose of therapeutic drug monitoring (TDM). To speed up the analysis time, an automated flow-through desorption method of DBS samples may be beneficial. This article describes the cross-validation of a manual punch DBS method with an automated desorption (DBS autosampler, DBSA) method for the DBS analysis of the antifungal drug voriconazole, followed by cross-validation of both DBS methods with a plasma-based method, and an assessment of agreement between DBS/DBSA and regular plasma concentration measurements (gold standard) in samples from patients on voriconazole treatment. DBS and DBSA LC-MS/MS assays for voriconazole were validated according to the latest guidelines on bioanalytical method validation (FDA, EMA). Additional DBS-specific validation parameters included hematocrit effect and the influence of spot volume. Passing-Bablok regression and Bland-Altman plots were used to cross-validate the punch DBS, DBSA and plasma methods. The assessment of agreement between DBS/DBSA and plasma concentration measurements involved the performance of DBS/DBSA measurements to predict voriconazole plasma concentrations in patient samples. Both DBS methods complied with all validation parameters. Sample pre-processing time was reduced from 1.5 h to 3 min when using the DBSA. Cross-validation of both DBS methods showed a proportional bias and a correction factor was needed to interchange voriconazole concentrations of both DBS methods. Similarly, the punch DBS method required a factor to correct for proportional bias compared to the plasma method, but the DBSA and plasma assays showed no bias. Limits of agreement of the DBS/DBSA and plasma assays in Bland-Altman analysis were relatively wide, i.e. 0.75-1.28 for the DBS punch method versus plasma method and 0.57-1.38 for the DBSA versus plasma assay. Interpretation of DBS, DBSA and plasma samples in terms of concentrations in or outside of the voriconazole therapeutic range agreed in 82-86% of the cases. The variability in paired DBS/DBSA and plasma concentration measurements is considered high for TDM purposes and this limitation should be balanced against the advantages of DBS sampling of voriconazole and the speed of flow through desorption.

Determination of protein-unbound, active rifampicin in serum by ultrafiltration and Ultra Performance Liquid Chromatography with UV detection. A method suitable for standard and high doses of rifampicin.

Rifampicin is the most important antibiotic in use for the treatment of tuberculosis (TB). Preclinical and clinical data suggest that higher doses of rifampicin, resulting in disproportionally higher systemic exposures to the drug, are more effective. Serum concentrations of rifampicin are the intermediary link between the dose administered and eventual response and only protein-unbound (free) rifampicin is pharmacologically active. The objective of this work was to develop an ultra performance liquid chromatography assay for protein-unbound rifampicin in serum with ultrafiltration, carried out at a sample temperature of 37°C, suitable for measurement of concentrations achieved after currently used and higher doses of rifampicin. Human serum was equilibrated at 37°C and ultrafiltrated at the same temperature in a Centrifree YM-30 ultrafiltration device, followed by dilution of the ultrafiltrate with methanol and ascorbic acid. Unbound rifampicin was analyzed using ultra performance liquid chromatography with a BEH C18 column, isocratic elution and ultra-violet (UV) detection. The run time was 5min. The assay was linear over the concentration range of 0.065-26mg/L rifampicin in ultrafiltrate. Accuracies for measurement of rifampicin in ultrafiltrate were 97% and 102% at the higher and lower limits of quantitation. Accuracy of the ultrafiltration process cannot be established, as it is not possible to spike blank serum with known amounts of protein-unbound rifampicin. Within- and between-day precision of the method including ultrafiltration as well as after ultrafiltration were within prespecified limits (CV<10%). Dilution of the ultrafiltrate with methanol and ascorbic acid kept rifampicin in solution and prevented it from degradation. Rifampicin loss during the ultrafiltration process and variation in analytical results when using two different batches of ultrafiltration devices were both limited. Processed ultrafiltrate samples were stable for 3days in the autosampler. The developed method can be applied in pharmacokinetic research, studying exposure-response relationships for rifampicin when administered at higher than currently used doses.