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.

Non-linear mixed effects modeling of sparse concentration data from rats: application to a glycogen phosphorylase inhibitor.

We investigated the use of non-linear mixed effects modeling in two preclinical studies of the glycogen phosphorylase inhibitor 1,4-dideoxy-1,4-imino-D-arabinitol (DAB). In a 28-day repeated-dose toxicity study rats were dosed once daily p.o. with 0, 20, 45, 100, or 470 mg/kg of DAB in aqueous solutions by oral gavage. Three blood samples were obtained from each animal using a staggered sampling scheme. During the cause of model development, data were included from a safety pharmacological cardiovascular study, in which rats were dosed once orally with 0, 4, 40, or 400 mg/kg of DAB thereby enabling an extension of the dose range of the model. DAB was assayed in plasma using a validated LC/MS/MS method. Non-linear mixed effects modeling was performed using the software NONMEM. The covariate analysis comprised dose, sex and time. Exposure results (Cmax, AUC) obtained by mixed effects modeling were compared to results from noncompartmental analysis using naïve pooling of data. The final model was a one-compartment model with first order absorption and a saturation-like dose dependent increase of the (oral) clearance (CL/f) and volume of distribution (V/f). Furthermore, V/f increased (by 55%) from Day 1 to Day 28. The dose dependencies of CL/f and V/f were most likely due to dose dependent decreases of the fraction systemically absorbed (f). The mechanism behind the dose dependencies may be saturation of a (putative) carrier mediated transport or modulation of tight junctions causing a reduced paracellular transport across the intestinal epithelium. Exposure results obtained from the model compared well with results obtained using noncompartmental analysis. An analysis of the data requirements for non-linear mixed effects modeling showed that at least three concentration values per animal were required for model development. We conclude that non-linear mixed effects modeling is feasible even with dose dependent pharmacokinetics in preclinical studies, such as 28-day toxicity studies in rodents. Supplementing data from additional preclinical studies may be required in order to extend the dose range. Non-linear mixed effects models may prove to be valuable tools in early PK and PK-PD modeling during drug development.

Levormeloxifene: safety, pharmacodynamics and pharmacokinetics in healthy postmenopausal women following single and multiple doses of a new selective oestrogen receptor modulator.

AIMS: The safety, pharmacodynamics and pharmacokinetics of levormeloxifene, a selective oestrogen receptor modulator (SERM), were investigated in postmenopausal women following single doses and multiple dosing once daily up to 56 days. METHODS: The two randomized, double-blind, placebo controlled studies of six single ascending doses and at four multiple dose levels, respectively, included a total of 104 healthy postmenopausal women. Safety assessments comprised vital signs, ECG, haematology, clinical chemistry and reporting of adverse events. The pharmacodynamic properties were investigated after multiple dosing by assessment of the short-term effects on bone and lipid metabolism and on the hypothalamic-pituitary axis. Blood samples for pharmacokinetic analysis were collected at intervals until 648 h (27 days) after single and multiple dosing. RESULTS: Levormeloxifene was tolerated well after single doses in the range of 2.5--320 mg and multiple once daily dosing in the range of 20--160 mg. Adverse events reported were generally mild or moderate. The most frequent adverse events after multiple dosing were headache, abdominal pain and leukorrhea with the highest frequency reported after the highest daily dose of 160 mg levormeloxifene. Five weeks of treatment with 20--160 mg levormeloxifene and 8 weeks of treatment with 40 or 80 mg levormeloxifene reduced the biochemical marker of bone turnover, the collagen I C-terminal telopeptide (CrossLaps) by 44.4% [95% CI: 11.3, 65.1] and 35.5% [95% CI: 14.0, 51.6], respectively, without any dose-dependent decrease in the studied dose range. The total cholesterol and LDL-cholesterol concentrations were significantly reduced by 19--25% and 28--35%, respectively, when compared with placebo. HDL-cholesterol and triglyceride concentrations were not affected. An oestrogen-like effect on the hypothalamic-pituitary axis was observed with approximately 50% reductions of FSH and LH after 8 weeks of treatment. No clinically significant changes of other safety variables were observed. The pharmacokinetic analysis demonstrated a rapid absorption (mean tmax: 2--3 h), a slow elimination (mean t1/2: 4.8--8.4 days) and dose linearity of Cmax and AUC for doses up to 160 mg. As expected for a drug with slow elimination given frequently, the relative fluctuation around the steady state plasma concentration was small and the drug accumulation considerable (RA: 3--5). CONCLUSIONS: Short-term administration of levormeloxifene in postmenopausal women was well-tolerated at doses that elicited a favourable pharmacodynamic response suggesting oestrogen-like bone preserving and antiatherogenic effects. Little variation of peak-trough plasma concentrations was observed during daily administration due to a plasma half-life of approximately 1 week.