Impact of Changing Regulations and the Dynamic Nature of European Risk Management Plans for Human Medicines on the Lifecycle of Safety Concerns.

INTRODUCTION: The European Risk Management Plan (EU-RMP) is a proactive planning tool for identification, characterisation and management of important risks and missing information throughout the lifecycle of a medicinal product. Over the past 15 years the EU-RMP has been a part of the pharmacovigilance practice in Europe, but there are no published studies assessing impact of the growing experience and evolving regulatory framework on the content and focus of the EU-RMP. OBJECTIVES: The objectives were to study the real-world impact of evolving pharmacovigilance guidelines on the proactive lifecycle management of important risks and missing information through EU-RMPs, and to further explore the impact of different resources on the management of the benefit-risk profile. METHODS: A retrospective study based on the review of 64 EU-RMPs dated between 01 January 2006 and 01 October 2020 for seven human medicinal products for which Boehringer Ingelheim holds the Marketing Authorisation in the European Union. Data on the timing and rational behind changes (i.e., inclusion, reclassification, removal) to the safety concerns (Important Identified Risks, Important Potential Risks, Missing Information) and associated additional Pharmacovigilance activities and/or Risk Minimisation measures were collected and assessed. RESULTS: The analysed EU-RMPs included a total of 197 safety concerns, 129 of which were removed and 19 were reclassified during the observation period. The implementation of the Guidelines on Good Pharmacovigilance Practices Module V in 2012 and Revision 2 in 2017 resulted in a noticeable decrease in the number of safety concerns. Clinical trial, non-clinical and routine post-marketing data were common sources that influenced the safety concern dynamics, and results from dedicated post-authorisation studies lead to the removal of 21 important risks and missing information. Many safety concerns were related to pharmacological class effect (n = 55) and target population characteristics (n = 37). CONCLUSIONS: This study demonstrated that the growing knowledge regarding benefit-risk of approved products and the introduction of new or revised regulatory guidelines influenced the EU-RMP lifecycle of safety concerns, and moreover, the results emphasise that exchange of knowledge about the pharmacological class and target population between stakeholders are important for keeping an up-to-date understanding of a medicinal product's safety profile. The aim of improving the efficiency of risk management has leveraged the accumulation of knowledge leading to revision of regulatory guidelines and increasingly, proactive Risk Management Plans focused on safety concerns that are important for patients and public health.

different Roles for the axin interactions with the SAMP versus the second twenty amino acid repeat of adenomatous polyposis coli.

Wnt signalling is prevented by the proteosomal degradation of ß-catenin, which occurs in a destruction complex containing adenomatous polyposis coli (APC), APC-like (APCL), Axin and Axin2. Truncating mutations of the APC gene result in the constitutive stabilisation of ß-catenin and the initiation of colon cancer, although tumour cells tolerate the expression of wild-type APCL. Using the colocalisation of overexpressed Axin, APC and APCL constructs as a readout of interaction, we found that Axin interacted with the second twenty amino acid repeat (20R2) of APC and APCL. This interaction involved a domain adjacent to the C-terminal DIX domain of Axin. We identified serine residues within the 20R2 of APCL that were involved in Axin colocalisation, the phosphorylation of truncated APCL and the down-regulation of ß-catenin. Our results indicated that Axin, but not Axin2, displaced APC, but not APCL, from the cytoskeleton and stimulated its incorporation into bright cytoplasmic dots that others have recognised as ß-catenin destruction complexes. The SAMP repeats in APC interact with the N-terminal RGS domain of Axin. Our data showed that a short domain containing the first SAMP repeat in truncated APC was required to stimulate Axin oligomerisation. This was independent of Axin colocalisation with 20R2. Our data also suggested that the RGS domain exerted an internal inhibitory constraint on Axin oligomerisation. Considering our data and those from others, we discuss a working model whereby ß-catenin phosphorylation involves Axin and the 20R2 of APC or APCL and further processing of phospho-ß-catenin occurs upon the oligomerisation of Axin that is induced by binding the SAMP repeats in APC.

Cytokeratin 8-MHC class I interactions: a potential novel immune escape phenotype by a lymph node metastatic carcinoma cell line.

Defective human leukocyte antigen (HLA) class I expression in malignant cells facilitates their escape from destruction by CD8(+) cytotoxic T lymphocytes. In this study, a post-translational mechanism of HLA class I abnormality that does not involve defects in the HLA subunits and antigen processing machinery components was identified and characterized. The marked HLA class I downregulation phenotype of a metastatic carcinoma cell line can be readily reversed by trypsin, suggesting a masking effect by serine protease-sensitive HLA class I-interacting factors. Co-immunoprecipitation, combined with LC-tandem mass spectrometry and immunoblotting identified these factors as cytokeratin (CK) 8 and its heterodimeric partners CK18 and CK19. Ectopic CK8/18 or CK8/19 expression in HEK293 cells resulted in surface CK8 expression with an HLA class I downregulation phenotype, while redirecting CK8/18 and CK8/19 to the endoplasmic reticulum (ER) had no such effect. This observation and the failure to constrain CK8/18 and CK8/19 membrane trafficking by an ER-Golgi transport inhibitor suggested an ER-independent route for CK8 access to HLA class I molecules. Monoclonal antibody mapping revealed a potential CK8 blockade of HLA class I-CD8 and -TCR contacts. These findings, along with the emerging role of cell surface CK8 in cancer metastasis, may imply a dual strategy for tumor cell survival in the host.

Functional comparison of human adenomatous polyposis coli (APC) and APC-like in targeting beta-catenin for degradation.

Truncating mutations affect the adenomatous polyposis coli (APC) gene in most cases of colon cancer, resulting in the stabilization of ß-catenin and uncontrolled cell proliferation. We show here that colon cancer cell lines express also the paralog APC-like (APCL or APC2). RNA interference revealed that it controls the level and/or the activity of ß-catenin, but it is less efficient and binds less well to ß-catenin than APC, thereby providing one explanation as to why the gene is not mutated in colon cancer. A further comparison indicates that APCL down-regulates the ß-catenin level despite the lack of the 15R region known to be important in APC. To understand this discrepancy, we performed immunoprecipitation experiments that revealed that phosphorylated ß-catenin displays a preference for binding to the 15 amino acid repeats (15R) rather than the first 20 amino acid repeat of APC. This suggests that the 15R region constitutes a gate connecting the steps of ß-catenin phosphorylation and subsequent ubiquitination/degradation. Using RNA interference and domain swapping experiments, we show that APCL benefits from the 15R of truncated APC to target ß-catenin for degradation, in a process likely involving heterodimerization of the two partners. Our data suggest that the functional complementation of APCL by APC constitutes a substantial facet of tumour development, because the truncating mutations of APC in colorectal tumours from familial adenomatous polyposis (FAP) patients are almost always selected for the retention of at least one 15R.