BackgroundA validation is crucial to ensure the quality of an analytical method and its results. However, the validation is only a first step, further quality assessment has to be utilised to ensure high quality research. Specifications for the validation process, but also for the assessment of data, acquired in a study setting, are given by the EMA and FDA to ensure highest quality of the data.1 2 MethodsA multi-level analytical quality system was established. Data of the calibration standards (CSs), quality control samples (QCs), and incurred sample reanalysis (ISR) were evaluated according to the specifications given by the EMA and FDA guidelines.[1,2] For a run to be considered valid ≥6 levels or 75% of the CSs and 67% of the QCs (≥50% per level) had to vary ≤±20% (LLOQ ≤±25%) from their nominal concentration.[1,2] For the ISR analysis at least 67% of the ISR samples have to lay in ±30% to the nominal concentration of the mean of the original and reanalysed value.[1]ResultsSeventy analytical runs were conducted, applying the quality measures, 79% runs were classified as valid and were used to determine unknown samples in a paediatric study. The high quality of the acquired data is reflected in the high conformity of the CSs and QCs to the EMA and FDA guidelines, 99% of the CSs and 95% of the QCs were accepted. Further underlining the high quality of the acquired data, 85% of the IRS have also been accepted. The assay was successfully used over a time period of 29 months.ConclusionThe results of the quality assessment confirmed the robustness of the aldosterone assay throughout the whole study duration. Thus, the samples measured by this assay are reliable and facilitate the high quality research in the paediatric population.ReferencesGuideline on bioanalytical method validation. European Medicines Agency, London, UK (2011).Guidance for Industry: Bioanalytical Method Validation. US Department of Health and Human Services, US FDA Rockville, MD, USA (2018).Disclosure(s)Nina Makowski, Ilja Burdman, Mohsin Ali, Bartel A, Bjoern B. Burckhardt declare that there is no conflict of interest. The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007–2013) under grant agreement n°602295 (LENA).
BackgroundThe European multicentre paediatric trials for the drug development programme of LENA (FP7 Grant agreement No. 602295); ‘Labeling of Enalapril from Neonates up to Adolescents’ require the determination of laboratory safety parameters. It was anticipated that the laboratory normal reference values and age range classifications vary depending on the clinical site. Thus, the objective was a seamless and clear depiction of the laboratory parameters to allow an adequate subsequent analysis of data.MethodsFourteen haematological and biochemical safety parameters plus the biomarker N-terminal pro-brain natriuretic peptide were considered. The laboratory normal reference values received from eight clinical sites were screened on data gaps, uncertainties, misclassifications and overlap of age range classifications. These aspects were revised. If further data were necessary for clarification the responsible person of the respective laboratory was contacted by email or telephone.ResultsData gaps and uncertainties of the laboratory normal reference values such as missing data for one sex, missing data for an age range classification, missing data for a parameter or overlap of age range classification were identified. All issues were solved by communication with the sites. Each laboratory parameter was categorized in between 1 and 23 age range classifications between an age from birth to 4744 days depending on the classification of the clinical site. Furthermore, up to 4 various units were recorded per laboratory parameter and subsequently harmonised into one unit.ConclusionThe developed seamless depiction of the laboratory parameters will allow the assessment and classification of the paediatric trial data and are essential for the adequate subsequent analysis.Disclosure(s)Samieh Farahani, Elisabeth Feles, Bjoern B. Burckhardt, Stephanie Laeer declare that there is no conflict of interest. The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007–2013) under grand agreement n°602295 (LENA)
BackgroundIn cell line experiments, the selective NK-1-receptor antagonist Aprepitant was able to inhibit the cardiotoxic adverse effects of Doxorubicin,1 a common cytostatic used in paediatric cancer therapies. Cytostatic therapy is one of the principal reasons for toxic cardiomyopathy in children, resulting in dilated cardiomyopathy and consequently leading to heart failure. However, Aprepitant is currently licenced for adults and children and is indicated amongst others in the antiemetic supportive therapy of Doxorubicin regimes. To address the hypothesis of any indication of Aprepitant in preventing cardiotoxic adverse effects of Doxorubicin, systematic literature research is needed.MethodsSystematic literature research was examined using PubMed in January 2019. Selected inclusion criteria were: ‘Substance P’ or ‘Aprepitant’ and ‘Doxorubicin’/’cardiac inflammation’/’cardiomyopathy’ or ‘Aprepitant’ and ‘paediatrics’/’neonates’/’children’. The use of Aprepitant in adults and children as an antiemetic agent and the involvement of Substance P in (neurogenic) inflammation, cardiac infarction or diabetes led to the exclusion of publications.ResultsThe PubMed search resulted in 220 identified publications whereby 33 were relevant concerning the potential use of Aprepitant in the prevention of cardiotoxic adverse effects of Doxorubicin. It emphasises the potential use of Aprepitant in the prevention of toxic cardiomyopathy by antagonizing the inflammatory effects of the endogenous NK-1-agonist Substance P regarding cell and animal models.1 2 Based on these models, Substance P is associated with adverse cardiac remodelling and cardiac inflammation. However, in children, Aprepitant was only used as an antiemetic agent and no off-label indication was described.ConclusionSince toxic cardiomyopathy is a severe adverse effect in the Doxorubicin therapy in children, the evaluation of the role of Substance P is a promising and worthy approach to condense the knowledge about a potential use of Aprepitant in preventing paediatric toxic cardiomyopathy.ReferencesRobinson P, Kasembeli M, Bharadwaj U, et al. Substance P receptor signaling mediates doxorubicin-induced cardiomyocyte apoptosis and triple-negative breast cancer chemoresistance. Biomed Res Int2016; 2016. doi:10.1155/2016/1959270Levick SP, Soto-Pantoja DR, Bi J, et al. Doxorubicin-induced myocardial fibrosis involves the neurokinin-1 receptor and direct effects on cardiac fibroblasts. Heart Lung Circ. Published Online First: September 2018. doi:10.1016/j.hlc.2018.08.003Disclosure(s)Martin Feickert and Bjoern B. Burckhardt declare that there is no conflict of interest. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
BackgroundPhysiological and pathophysiological circumstances of the paediatric renin-angiotensin-aldosterone-system (RAAS) are still inadequately understood. Due to the limited paediatric data available, the LENA (Labeling of Enalapril from Neonates up to Adolescents) project aimed to comprehensively investigate the drug enalapril and its effect on humoral parameters of the RAAS. Examination of four humoral parameters, including plasma renin activity (PRA), was conducted regarding cardiac diseased paediatric population receiving enalapril, of which 60% were below 1 year of age. To fully address the agreed Paediatric Investigation Plan (EMEA-001706-PIP) of the LENA project, reliable small-volume assays for pharmacodynamics determination were mandatory to ensure the reliable data sets.Materials and methodsA commercial PRA enzyme linked immunosorbent assay (ELISA) was tailored for paediatric application and validated according to European Medicine Agency (EMA) and U.S. Food and Drug Administration (FDA) bioanalytical guidelines.1 2 In this context, accuracy, precision, total error, linearity, parallelism, matrix effects and stability were investigated.ResultsThe adopted bioanalytical PRA-assay was successfully validated. Between-run precision (CV) and accuracy (relative error) ranged from 1.6% to 19.6% and -13.0% to +11.2% respectively. Samples of five different human sources showed no substantial matrix effect and facilitated the assay’s application to heterogeneous populations. The obtained precision of parallelism of five dilution steps ranged from 7.7% to 8.3% allowing to dilute high samples within the calibration range. Stability measurements proved four freeze and thaw cycles plus short-term and long-term (37 weeks) stability. Overall, all results were complied with guideline requirements.ConclusionThe FDA/EMA-compliant PRA assay is able to accurately and precisely quantify PRA values in 50 µL plasma and is applicable for GCLP-compliant clinical studies enabling sophisticated investigations in children within the LENA project.ReferencesCommittee for Medicinal Products for Human Use (CHMP): EMEA/CHMP/EWP/192217/2009 Rev. 1 Corr. 2** Guideline on bioanalytical method validation, June, 2015U.S. Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER) Center for Veterinary Medicine (CVM): Bioanalytical Method Validation Guidance for Industry, May 2018Disclosure(s)Fabian K. Suessenbach, Martin Feickert, Jutta Tins and Bjoern B. Burckhardt declare that there is no conflict of interest. The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007–2013) under grant agreement n°602295 (LENA).
BackgroundSince sample volume is limited in children, innovative bioanalytical methods and enrichment procedures are highly required. The analysis of endogenous substances by liquid chromatography coupled to mass spectrometry is a highly specific method because of its selectivity and accuracy. However reliable detection of endogenous substances can only be achieved by a hybrid assay approach combining immunocapture and mass spectrometry. Key element of the immunocapture procedure is the selection of the appropriate antibody for capturing the desired antigen. This study is meant to identify the most suitable antibody that facilitates the development of an hybrid assay approach concerning reliable detection of endogenous prorenin in paediatric samples.MethodsDynabeads magnetic beads were coupled to three different antibodies from three different vendors (GeneTex, Molecular Innovations, R&D systems). 500 µL human plasma which was spiked with 20 ng recombinant human prorenin (Cayman chemicals). The immunocapture step was followed by protease digestion and a custom-made µelution solid-phase extraction protocol. The digest was analyzed by Shimadzu Nexera LC-system coupled with Sciex TripleTOF 6600 mass spectrometer.ResultsThe analysis of the captured prorenin was performed by the surrogate peptide approach. In this case the surrogate peptide was identified as unique. The comparison of the three available antibodies showed that one antibody did not ensure reliable binding properties in human matrix. Among the two remaining antibodies only one showed sufficient binding capacities to be applied in small sample volumes commonly available in paediatric samples. Using this hybrid approach enabled the enrichment of the required volume by factor of 20.ConclusionThis study identified the most suitable antibody for the immunocapture procedure of the prorenin hybrid approach. This is now followed by further mass spectrometric method development and validation prior to its application in paediatric samples.Disclosure(s)Declaration of interest: none Ilja Burdman and Bjoern B. Burckhardt declare that there is no conflict of interest. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors
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