A Study of Regulatory Challenges of Pediatric Oncology Phase I/II Trial Submissions and Guidance on Protocol Development.

The purpose of this study was to identify key deficiencies in pediatric oncology early phase clinical trial protocols in Germany and to provide guidance for efficient trial protocol development. A systematic review of the response letters of German competent authorities (CAs) and Ethics Committees to phase I/II pediatric oncology trial submissions in the period from 2014 to 2019 was performed. Documents were requested from all five Society for Paediatric Oncology and Haematology in Germany (GPOH) phase I/II trial networks plus all nine German Innovative Therapies for Children with Consortium Cancer (ITCC) centers. A blinded dataset containing aggregated data from 33 studies was analyzed for validation. All deficiencies were reviewed, listed, and weighted using a structured matrix according to frequency, category, significance, and feasibility. In total, documents of 17 trials from 6 different sites were collected. Two hundred fifty deficiencies identified by the CAs were identified and categorized into eight categories. "Toxicity and safety" was the most prominent category (27.6%), followed by "Manufacturing and Import" (18%). The majority of deficiencies were categorized as minor and potential measures as easy to address, but an important group of major and difficult to implement deficiencies was also identified. The blinded validation dataset confirmed these findings. The majority of the EC deficiencies could be resolved by changing the wording in the patient-facing documents. In conclusion, this study was able to detect a pattern of key deficiencies. Most of the shortcomings can be anticipated by minor changes in the protocol and increased awareness can prevent time-consuming revisions, withdrawals, or even rejections. A corresponding guideline describing key regulatory aspects is provided.

Nerve growth factor regulates the expression of the cholinergic locus and the high-affinity choline transporter via the Akt/PKB signaling pathway.

Nerve growth factor (NGF) is a trophic and survival factor for cholinergic neurons, and it induces the expression of several genes that are essential for synthesis and storage of acetylcholine (ACh), specifically choline acetyltransferase, vesicular ACh transporter (VAChT), and choline transporter. We have found previously that the phosphatidylinositol 3'-kinase pathway, but not the MEK/MAPK pathway, is the mediator of NGF-induced cholinergic differentiation. Here we demonstrate, in the rat pheochromocytoma cell line PC12 and in primary mouse neuronal cultures, that NGF-evoked up-regulation of these three cholinergic-specific genes is mediated by the anti-apoptotic signaling molecule Akt/protein kinase B. Inhibition of Akt activation by the pharmacological inhibitor 1L-6-hydroxymethyl-chiro-inositol 2(R)-2-O-methyl-3-O-octadecylcarbonate (HIMO), or by a peptide fragment derived from the proto-oncogene TLC1, eliminated NGF-stimulated increases in cholinergic gene expression, as demonstrated by RT-PCR and reporter gene assays. Moreover, treatment with HIMO reversed NGF-evoked increases in choline acetyltransferase activity and ACh production. In co-transfection assays with the reporter construct, a dominant-negative Akt plasmid and Akt1-specific small interfering RNA also attenuated NGF-induced cholinergic promoter activity. Our data indicate that, in addition to its well-described role in promoting neuronal survival, Akt can also mediate signals necessary for neurochemical differentiation.

Differential regulation of the high affinity choline transporter and the cholinergic locus by cAMP signaling pathways.

Synthesis, storage and release of acetylcholine (ACh) require the expression of several specialized enzymes, including choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT) and the high-affinity choline transporter (CHT). Extracellular factors that regulate CHT expression and their signaling pathways remain poorly characterized. Using the NSC-19 cholinergic cell line, derived from embryonic spinal cord, we compared the effects of the second messenger cAMP on the expression of CHT and the cholinergic locus containing the ChAT and VAChT genes. Treatment of NSC-19 cells with dbcAMP and forskolin, thus increasing intracellular cAMP levels, significantly reduced CHT mRNA expression, while it upregulated ChAT/VAChT mRNA levels and ChAT activity. The cAMP-induced CHT downregulation was independent of PKA activity, as shown in treatments with the PKA inhibitor H-89. The alternative Epac-Rap pathway, when stimulated by a specific Epac activator, led to significant downregulation of CHT and ChAT, and, to a lesser extent, VAChT. In contrast, the PKA activator 6-BNZ-cAMP stimulated the expression of all three genes, but with varying concentration-dependence profiles. Our results indicate that elevations of intraneuronal cAMP concentration have differential effects on the cholinergic phenotype, depending on the involvement of different downstream effectors. Interestingly, although CHT is expressed predominantly in cholinergic cells, its regulation appears to be distinct from that of the cholinergic locus.

RNA aptamers directed against release factor 1 from Thermus thermophilus.

An in vitro selection/amplification (SELEX) was used to generate RNA aptamers that specifically bind Thermus thermophilus release factor 1 (RF1). From 31 isolated clones, two groups of aptamers with invariable sequences 5'-ACCU-3' and 5'-GAAAGC-3' were isolated. Chemical and enzymatic probing of the structure indicate that in both groups the invariable sequences are located in single-stranded regions of hairpin structures. Complex formations between RF1 and aptamers of both groups were identified by electrophoretic shift assay and chemical footprinting. Deletion of the invariable sequences did not effect the secondary structure of the aptamers but abolished their binding to RF1. RNA motifs matching the invariable sequences of the aptamers are present as consensus sequences in the peptidyl transferase center of 23S rRNAs. T. thermophilus RF1 recognizes UAG stop codons in an Escherichia coli in vitro translation system. Aptamers from both groups inhibited this RF1 activity.