Regulatory Considerations Toward Orphan Drug Designation and Orphan Drug Exclusivity in the United States and European Union: Structural Similarity, Clinical Superiority/Significant Benefit, and Case Studies.

The U.S. Food and Drug Administration and European Commission have developed successful orphan drug legislation to promote the research, development, and marketing approval of drugs to treat rare diseases. Central to these regulations are the concepts of structural similarity and clinical superiority/significant benefit to achieve orphan drug exclusivity. However, differences in health authority expectations remain regarding the qualification for an orphan drug designation, defining structural similarity, and demonstrating clinical superiority/significant benefit. These differences can create sponsor company uncertainty regarding the approvability of products (e.g., blocking risk by an existing orphan product) and divergent orphan drug decisions among health authorities. A comprehensive assessment of current regulations, case studies in exclusivities, and recommendations for improvement are presented.

Driving gradual endogenous c-myc overexpression by flow-sorting: intracellular signaling and tumor cell phenotype correlate with oncogene expression.

Insulin-exposed rat mammary cancer cells were flow sorted based on a c-myc reporter plasmid encoding a destabilized green fluorescent protein. Sorted cells exhibited gradual increases in c-myc levels. Cells overexpressing c-myc by only 10% exhibited phenotypic changes attributable to c-myc overexpression, such as cell cycle disturbances, increased cell size, and overexpression of the S6 ribosomal protein. Cells overexpressing c-myc by 70% exhibited additional phenotypic changes typical of c-myc overexpression, such as increased histone H3 phosphorylation, and reduced adherence. Sorted cells also exhibited overexpression of the IGF-1R, and slightly elevated expression of the IR. Increased susceptibility to the mitogenic effect of insulin was seen in a small proportion of the sorted cells, and insulin was more effective in activating the p44/42 MAPK pathway, but not the PI3K pathway, in the sorted cells than in the nonsorted cell population. To our knowledge, this is the first in vitro system allowing functional coupling between mitogenic signaling by a well-defined growth factor and gradual overexpression of the normal, endogenous c-myc gene. Thus, our flow-sorting approach provides an alternative modeling of the receptor-mediated carcinogenic process, compared to the currently used approaches of recombinant constitutive or conditional overexpression of oncogenic transmembrane receptor tyrosine kinases or oncogenic transcription factors.

MCF-7 human mammary adenocarcinoma cells exhibit augmented responses to human insulin on a collagen IV surface.

Human mammary cell lines are extensively used for preclinical safety assessment of insulin analogs. However, it is essentially unknown how mitogenic responses can be optimized in mammary cell-based systems. We developed an insulin mitogenicity assay in MCF-7 human mammary adenocarcinoma cells, under low serum (0.1% FCS) and phenol red-free conditions, with 3H thymidine incorporation as endpoint. Based on EC50 values determined from 10-fold dilution series, beta-estradiol was the most potent mitogen, followed by human IGF-1, human AspB10 insulin and native human insulin. AspB10 insulin was significantly more mitogenic than native insulin, validating the ability of the assay to identify hypermitogenic human insulin analogs. With MCF-7 cells on a collagen IV surface, the ranking of mitogens was maintained, but fold mitogenic responses and dynamic range and steepness of dose-response curves were increased. Also, PI3K pathway activation by insulin was enhanced on a collagen IV surface. This study provided the first determination and ranking of the mitogenic potencies of standard reference compounds in an optimized MCF-7 assay. The optimized MCF-7 assay described here is of relevance for in vitro toxicological testing and carcinogenicity safety assessment of new insulin compounds.