Retrospective Analysis of Bioanalytical Method Validation Approaches in Biosimilar Biological Product Development.

Development and validation of a bioanalytical method for biosimilar biological product development (BPD) can be challenging. It requires the development of a bioanalytical method that reliably and accurately measures both proposed biosimilar and reference products in a biological matrix. This survey summarizes the current state of bioanalysis in BPD. Bioanalytical data from 28 biosimilar biologic license applications submitted to U.S. Food and Drug Administration (FDA) up to December 2018 were analyzed. The aim of the analysis was to provide (i) a summary of the bioanalytical landscape for BPD, (ii) a cumulative review of bioanalytical method validation approaches to aid in understanding how a specific method was selected, and (iii) a summary of data regarding bioanalytical bias differences between products. Results show diversity of the bioanalytical approaches used, as well as the observed differences in bioanalytical bias. Our findings highlight the need for understanding the critical aspects of BPD bioanalysis and clarifying BPD bioanalytical best practices, which could help ensure consistent method validation approaches in the BPD community.

Ceremonies of gratitude following the dissection course: A report on procedures in departments of anatomy in German speaking countries.

The departments of anatomy in Germany, Austria and the German-speaking part of Switzerland were sent comprehensive (18 items) questionnaires requesting details on memorial ceremonies held at the close of the dissection course in the medical curriculum, including objectives, organization, number of participants and the role of the medical students. The response rate was very high (95%). In more than 95% of instances a ceremony is held, initiated mainly after 1970. The titles of the ceremony range from commemoration ceremony (42%), service of mourning (19%) memorial service (19%) to ceremony of gratitude (7%). The number of participants exceeds 300 in 15% of these ceremonies. The invitation comes mostly from the student group organizing the ceremony (62%). The ceremony is offered mainly for the students of the course (23%), for student tutors (16%), relatives of the body donors (23%) and scientific staff (15%). The students actively participate with musical contributions (19%), gestures such as candles (17%) and flowers (12%), speeches (17%) and readings (12%). The relevance of the practical dissection course and body donation programs is also discussed. The results are compared to ceremonies in various countries with different religious backgrounds. This dissection course is unique among all courses in the medical curriculum as it obviously also has spiritual aspects.

Exposure-response relationship of T-DM1: insight into dose optimization for patients with HER2-positive metastatic breast cancer.

Exposure-response (E-R) analyses for ado-trastuzumab emtansine (T-DM1, Kadcyla) were performed using data from a randomized, active control (lapatinib plus capecitabine) trial in patients with human epidermal growth factor 2-positive metastatic breast cancer. Kaplan-Meier survival analyses stratified by T-DM1 trough concentration on day 21 of cycle 1 (Cmin,C1D21) were performed for overall survival (OS) and progression-free survival (PFS). E-R analyses indicated that after adjusting for baseline risk factors, higher T-DM1 exposure is associated with improved efficacy. T-DM1-treated patients with Cmin,C1D21 lower than the median value had values of OS and PFS comparable to those of the active control arm. The percentage of patients who received T-DM1 dose adjustments was similar across the exposure range and was lower than that of the active control arm. Our findings suggest that there may be an opportunity to optimize Kadcyla dose in the patient subgroup with low T-DM1 exposure for improved efficacy with acceptable tolerability.

Pharmacological interaction of the calcium channel blockers verapamil and flunarizine with the opioid system.

We evaluated the opioid antinociceptive mechanism of the calcium channel blockers verapamil and flunarizine in groups of mice with the hotplate test. Both produced a naloxone-sensitive dose-dependent analgesia. The antinociceptive effect of both was reversed by beta-FNA, (mu1 and mu2 antagonists), and both enhanced the antinociceptive activity of morphine, implying a role for mu receptors. Furthermore, since the analgesic effect of flunarizine, but not verapamil, was reversed by naloxonazine (mu1 antagonist), we suggest that the mu1 subtype is involved in flunarizine analgesia, but not in verapamil analgesia. Studies with the selective delta opioid agonist DPDPE and the selective antagonists naltrindole indicated that the antinociceptive activity of verapamil is also mediated by delta receptor agonistic activity (primarily following i.c.v. administration); flunarizine, by contrast, exhibited antagonistic activity at this receptor. Verapamil amplified the antinociceptive activity of kappa1 (U50,488H) and kappa3 (nalorphine) agonists, but its known analgesic activity was inhibited only partially by the kappa1 antagonist Nor-BNI, indicating partial involvement of kappa1 receptor. Flunarizine, however, demonstrated antagonistic activity at both kappa1 and kappa3 receptors, with more prominent inhibitory activity at the latter one. These findings suggest that verapamil and flunarizine elicit analgesia at both the spinal and supraspinal levels. Verapamil's analgesia was explained by agonistic activity at the mu, delta and may also be kappa3 receptor subtypes. Flunarizine exhibited a mixed agonistic-antagonistic opioid activity as shown by its agonistic activity at the mu1 receptor and antagonistic activity at delta, kappa1 and kappa3 receptor subtypes.

Calcineurin mediates human tumor necrosis factor alpha gene induction in stimulated T and B cells.

The tumor necrosis factor alpha (TNF-alpha) gene is rapidly transcribed in activated T cells via a calcium-dependent pathway that does not require de novo protein synthesis, but is completely blocked by the immunosuppressive drugs cyclosporin A (CsA) and FK506. Here we show that calcineurin phosphatase activity is both necessary and sufficient for TNF-alpha gene transcription in T cells, and identify the factor that binds to the kappa 3 element of the TNF-alpha gene promoter as the target for calcineurin action. The ability of analogues of CsA and FK506 to block calcineurin phosphatase activity correlates completely with their ability to inhibit induction of TNF-alpha mRNA, induction of a TNF-alpha promoter reporter plasmid in transiently transfected T cells, and induction of the kappa 3 binding factor in an electrophoretic mobility shift assay. Moreover, a cDNA encoding the constitutively active form of calcineurin is sufficient to activate the TNF-alpha promoter and the kappa 3 element. TNF-alpha gene transcription is also highly inducible, CsA-sensitive, and protein synthesis-independent in B cells stimulated through their surface immunoglobulin receptors. Using the panel of CsA and FK506 analogues, we show that calcineurin participates in the induction of TNF-alpha transcription in activated B cells. These results extend our previous demonstration that the kappa 3 binding factor is related to NFATp, the preexisting subunit of nuclear factor of activated T cells, and suggest that calcineurin-mediated modification of the kappa 3 binding factor in T cells is of key importance in the induction of TNF-alpha transcription.