Miriam Krischke

Tyrosine phosphatase inhibition induces loss of blood–brain barrier integrity by matrix metalloproteinase-dependent and -independent pathways

Tight junctions between endothelial cells of brain capillaries form the structural basis of the blood-brain barrier (BBB), which controls the exchange of molecules between blood and CNS. Regulation of cellular barrier permeability is a vital and complex process involving intracellular signalling and rearrangement of tight junction proteins. We have analysed the impact of tyrosine phosphatase inhibition on tight junction proteins and endothelial barrier integrity in a primary cell culture model based on porcine brain capillary endothelial cells (PBCEC) that closely mimics the BBB in vitro. The tyrosine phosphatase inhibitor phenylarsine oxide (PAO) induced increased matrix metalloproteinase (MMP) activity, which was paralleled by severe disruption of cell-cell contacts and proteolysis of the tight junction protein occludin. ZO-1 and claudin-5 were not affected. Under these conditions, the transendothelial electrical resistance (TEER) was markedly reduced. PAO-induced occludin proteolysis could be prevented by different MMP inhibitors. Pervanadate (PV) reduced the TEER similar to PAO, but did not increase MMP activity. Cell-cell contacts of PV-treated cells appeared unaffected, and occludin proteolysis did not occur. Our results suggest that tyrosine phosphatase inhibition can influence barrier properties independent of, but also correlated to MMPs. Evidence is given for a role of MMPs in endothelial tight junction regulation at the BBB in particular and probably at tight junctions (TJs) in general.

Minimization of the preanalytical error in plasma samples for pharmacokinetic analyses and therapeutic drug monitoring--using doxorubicin as an example.

Background: There are many sources of variability in plasma samples drawn for pharmacokinetic analyses or therapeutic drug monitoring. In this article, methods are proposed on how to prevent sample dilution (Part I) and contamination effects (Part II) in plasma samples, using doxorubicin as an example. Methods: Experiments were performed in the laboratory setting to identify factors that could influence plasma samples in clinical practice. In part I, it was hypothesized that saline solution left in a catheter could lead to a dilution of samples drawn through this catheter. The impact of 2 different sampling techniques, the “discard method” and the “push–pull method”, was examined. In part II, an infusion system was filled with a 1 mg/mL solution of doxorubicin. After rinsing the system with increasing volumes of saline solution, the drug concentration of the fluid left in the system was analyzed. Furthermore, plasma samples were drawn through the drug administration catheter, and the contamination of these samples with doxorubicin left in the catheter was measured. Results: In part I, a discard volume of plasma equal to 4 dead volumes of the sampling line was necessary to avoid dilution of a sample taken from a port or double-lumen catheter filled with saline solution (“discard method”). Pulling up and down the same volume through the catheter 5 times (“push-pull method”) was proved to be an alternative with no need to discard blood. In part II, after rinsing the infusion system with a volume of saline solution corresponding to 4 dead volumes of the system and after discarding a volume of plasma corresponding to 4 sampling line volumes, the doxorubicin contamination in the samples was negligibly small. Conclusions: Under the described conditions, the push-pull method delivered the same results as the discard method to prevent sample dilution. To avoid contamination in plasma samples, development of standardized sampling procedures seems to be essential and feasible.

Minimization of the preanalytical error in pharmacokinetic analyses and therapeutic drug monitoring: focus on IV drug administration.

Background: The conduct of multicenter pharmacokinetic (PK) analyses for long-established drugs entails specific problems, because samples have to be obtained within daily clinical practice. Practices for intravenous (IV) drug administration vary between hospitals, including the use of different infusion devices, the use of infusion line systems with different line volumes, and different priming and rinsing procedures. Methods: Variables of IV drug administration that could influence concentration data obtained in PK analyses were evaluated. Kinetics of drug delivery during initiation and cessation of IV infusions were simulated in vitro for a drop-counter and a syringe-driven infusion system at different flow rates. Furthermore, the percentages of the target drug dosage remaining in the infusion line after different rinsing periods were investigated in vitro and in clinical practice. Results: Varying times required for the drug to migrate from the bag/syringe to the cannula and to reach a steady-state drug administration rate were observed. Time to steady state ranged from almost immediate to 48 minutes depending on the infusion system and flow rate. The longest times were seen for the drop-counter system at low flow rates and were associated with large drug concentration gradients in the infusion line, which makes it difficult to accurately determine start and end of the infusion. For most systems, when rinsing at the end of infusion was performed with once the volume of the infusion line, <5% of the total drug dosage was discarded. Larger variability was seen for slow infusion rates and small infusion volumes. Conclusions: The choice of the infusion apparatus, standardized infusion systems, and standardized operating procedures for drug administration are important when performing postmarketing PK analyses in multicentric studies.

Sources of preanalytical error in pharmacokinetic analyses – focus on intravenous drug administration and collection of blood samples

Introduction: Pharmacokinetic (PK) studies for long-established drugs are generally performed outside the well-standardized settings of pharmaceutical industry trials. Instead, such studies are usually performed within daily clinical practice of hospitals. Areas covered: This article describes aspects of intravenous (i.v.) drug administration and blood sampling that contribute to potential sources of preanalytical errors for PK investigations. Parameters that bias determination of start and end time of i.v. infusions, as well as consistent rate of drug delivery, are discussed. Causes for drug loss in the infusion device, including adsorption and insufficient flushing, are outlined. The advantages and disadvantages of different blood sampling techniques are reviewed, with an emphasis on pediatric studies. Expert opinion: For PK studies that are integrated into the general hospital routine, a variety of potential sources of error exist. Potential pitfalls depend on the specific drug and trial characteristics and they must be anticipated and discussed in advance. Working procedures need to be developed that address the anticipated problems and in detail describe procedures that need compliance between bed and bench.

Towards a Model-Based Dose Recommendation for Doxorubicin in Children

Following the publication of our paper regarding a population-based model of doxorubicin pharmacokinetics in children in Clinical Pharmacokinetics last year (Voller et al. 54:1139–1149, 2015), we have received many inquiries on the practical clinical consequences of this model; however, a population-based model is only one of the aspects to be taken into account when developing dosing algorithms. In addition, any new method of dose calculation would need clinical validation and, subsequently, a new clinical trial. However, such a trial, especially with regard to burden to the children involved, requires optimal preparation and the selection of the best hypotheses. The European Paediatric Oncology Off-Patent Medicines Consortium (EPOC), represented by the authors, would therefore like to initiate an interdisciplinary discussion on the clinical and pharmacological goals for dose calculation. This current opinion summarizes the existing knowledge on the pharmacokinetics and pharmacodynamics of doxorubicin. Our aim was to define the clinical needs as precisely as possible, with the intention of stimulating discussion between the clinical pediatric oncologist and the pediatric pharmacologist. By doing so, we hope to define surrogates for best practice of a common doxorubicin dose in children. The intent is for a trial to validate a rational dose calculation rule, leading to a regulatory process and subsequent labeling.

EUROPEAN PAEDIATRIC ONCOLOGY OFF-PATENT MEDICINES CONSORTIUM (EPOC): A PHARMACOKINETIC STUDY OF DOXORUBICIN IN CHILDREN WITH CANCER

Background Doxorubicin is a key component of a number of treatment regimens used in paediatric oncology. The pharmacology data on which current dosing regimens are based are very limited. Methods We conducted a multicentre, multinational pharmacokinetic study investigating age-dependency in the clearance of doxorubicin in children with solid tumours and leukaemia. Blood samples for measurement of doxorubicin and its metabolite doxorubicinol were collected after 2 administrations, with 5 samples collected in children 3 yrs. A population pharmacokinetic approach was used for analysis, including pharmacogenetic covariates. NT-proBNP and cardiac troponin T were measured to evaluate their role as early indicators of cardiotoxicity. Results 101 children could be recruited including 27 patients less than 3 years and among those 5 infants younger than 1 year. Overall, the patient acceptance of the trial was very good. Age dependence of doxorubicin clearance was demonstrated, with children less than 3 years having a lower clearance (21.1±5.8 l/h/m2) than older children (26.6±6.7 l/h/m2) (p=0.0004), after correcting for body weight. Pharmacogenetic variants, including those in transporters and drug metabolizing enzymes, had little influence on pharmacokinetic parameters. Natriuretic peptides plasma levels increased significantly shortly after doxorubicin administration, whereas cardiac troponin levels increased only with the administered cumulative anthracycline dose. Only limited correlation could be observed between their blood levels and doxorubicin pharmacokinetics. Conclusion The paediatric need concerning missing PK-data could be addressed with limited burden for the patients. Empirically used dose adaptations for infants were found to be justified based on our PK analyses.

Abstract 4625: Age dependence of doxorubicin pharmacokinetics in pediatric cancer patients; results of an FP7-funded clinical study

Doxorubicin is a key component of a number of treatment regimens used in paediatric oncology, despite the very limited pharmacology data on which current dosing regimens are based. We conducted a multicentre, multinational phase II pharmacokinetic study investigating age-dependency in the clearance of doxorubicin in children with solid tumours and leukaemia. The study was funded under the Framework Program 7 of the European Commission. 101 patients treated with doxorubicin according to a tumour-specific national or European therapeutic trial were recruited to the study, with a particular focus on children less than 3 years. Absolute doses of doxorubicin ranged from 2.4 to 57 mg, administered, with infusion durations ranging from 0.25 to 24 hours. Samples were collected after 2 administrations, with 5 samples collected in children Data were available from all but 5 patients, with samples lost due to withdrawn consent, analytical issues or sample stability related to sample transport. A three compartment model was sufficient to characterize the pharmacokinetics of doxorubicin, with a further compartment to describe those of the major metabolite doxorubicinol. All parameters of the population model were scaled to body surface area. Age dependence of doxorubicin clearance was demonstrated, with children less than 3 years having a lower clearance (21.1±5.8 l/h/m2) than older children (26.6±6.7 l/h/m2) (p=0.0004), even after correcting for body weight. No other patient-related covariate, including liver function, was found to influence the parameters of the model. While the model provided a good fit to the doxorubicinol data, no covariate was identified which influenced the parameters of the metabolite model. Pharmacogenetic variants, including those in transporters and drug metabolizing enzymes, had little influence on pharmacokinetic parameters. Likewise, pharmacokinetics had only limited impact on markers of cardiotoxicity such as troponins or on measured cardiac function. This study yields new pharmacokinetic data on doxorubicin pharmacokinetics in very young patients, suggesting that lower doses may be appropriate in children less than 3 years. The data presented her may be useful for refining dosage regimens in this patient group. Citation Format: Alan V. Boddy, Nicolas Andre, Gianni Bisogno, Joachim Boos, Maurizio D9Incalci, Nina Kontny, Miriam Krischke, Swantje Voeller, Georg Hempel. Age dependence of doxorubicin pharmacokinetics in pediatric cancer patients; results of an FP7-funded clinical study. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4625. doi:10.1158/1538-7445.AM2014-4625

Abstract 2219: The European Paediatric Oncology off-patent medicines Consortium (EPOC) pharmacokinetic/pharmacodynamic study of doxorubicin.

Background: Doxorubicin is a key component of a number of treatment regimens used in paediatric oncology despite the very limited pharmacology data on which current dosing regimens are based. In recognition of this, doxorubicin was included on the EMA priority list for paediatric off-patent medicines for which more information was required, particularly on pharmacokinetics. Methods: The EPOC consortium, with collaborators in the UK, France, Germany and Italy, performs paediatric oncology pharmacology clinical trials. This study is a multicentre, multinational phase II pharmacokinetic study investigating age-dependency in the clearance of doxorubicin in children with solid tumours and leukaemia. All patients are being treated according to a tumour-specific national or European therapeutic trial. The aim was to recruit 100 patients, with a particular focus on children less than 3 years. The plasma levels of markers such as troponin T, are being measured to evaluate their use as clinical markers for cardiotoxicity. Data is being analysed using a population pharmacokinetic approach and the impact of pharmacogenetics is being investigated. Results: The study has been open to recruitment in 20 clinical centres across the four participating countries. The target 100 patients have been recruited from all the contributing study protocols, with 26 patients less than 3 years. Of these 26 patients, 5 patients are less than 1 year. An interim pharmacokinetic analysis has been performed on the data from the first 31 patients. The results of the analysis are consistent with the primary aims of the overall study. Conclusion: The data generated during this study should provide comparative data on the age-dependent pharmacokinetics and pharmacogenetics of doxorubicin. The results will contribute to optimising the safe use of doxorubicin in children with cancer. Acknowledgements: Study funded under the seventh framework programme of the European Commission (FP7) Citation Format: Alan V. Boddy, Alison J. Steel, Joachim Boos, Miriam Krischke, Nicolas Andre, Gianni Bisogno, Georg Hempel, Gerlind Bode, Maurizio D9Incalci, Nina Kontny. The European Paediatric Oncology off-patent medicines Consortium (EPOC) pharmacokinetic/pharmacodynamic study of doxorubicin. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2219. doi:10.1158/1538-7445.AM2013-2219