Benedicte Kiehr

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, β‐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. Copyright

Assay for levormeloxifene, a selective estrogen receptor modulator, in human and monkey plasma employing high-performance liquid chromatography and solid-phase extraction.

Assays for levormeloxifene, a new selective estrogen receptor modulator, and its 7-desmethyl metabolite in human and cynomolgus monkey plasma are described. Plasma was extracted on mixed-mode bonded sorbent material (C8/SCX) and the extracts were analysed by high-performance liquid chromatography with fluorescence detection. Recoveries of levormeloxifene and the metabolite exceeded 70%. Within and total assay precision calculated as a coefficient of variation (C.V.) were <8% for both compounds at all concentration levels, except at the limit of quantitation (LOQ) where the C.V. was 15%. Within and total-assay accuracy calculated as a percentage of the nominal value were between 90 and 114% for both analytes. The LOQ was for levormeloxifene and 7-desmethyllevormeloxifene, respectively, 1.5 and 2.5 ng/ml (man) and 5.2 and 6.9 ng/ml (monkey). In the monkey plasma assay, human plasma could substitute monkey plasma as blank plasma.

Characterization of high molecular weight plasma protein complexes induced by clotting factor rFXIII‐treatment in the cynomolgus monkey

Summary.  Background: In cynomolgus monkeys, suprapharmacological doses of clotting recombinant factor XIII (rFXIII) cause a generalized coagulopathy, associated with formation of circulating high molecular weight protein complexes (HMEX). HMEX consist of plasma protein substrates cross‐linked by FXIII transglutaminase activity. Objective: To characterize HMEX, with a view to develop safety biomarker assays. Methods: Cynomolgus monkeys received single i.v. injections with vehicle or rFXIII at 1, 3 and 10 mg kg−1. Plasma HMEX were analyzed by sodium dodecylsulfate–polyacrylmide gel electrophoresis, silver staining, Western blotting and quantitative dissociation‐enhanced lanthanide fluoroimmunoassay. Plasma FXIII antigen was analyzed by quantitative ELISA. Human HMEX were made in vitro, by spiking plasma with thrombin‐activated rFXIII. Results: Maximal circulating HMEX levels were reached within 1 h of rFXIII treatment, and remained stable over 24 h. HMEX above 250 kDa contained fibrinogen α‐chains and fibronectin. Fibrinogen γ‐chain was detected only in HMEX below 250 kDa. The total plasma concentration of HMEX was in the low μg mL−1 range, distributed on less than 20 main species. Human and cynomolgus HMEX were similar. HMEX formation increased with rFXIII dose in a disproportionate manner, with 3‐fold and fortyfold increases in HMEX exposure associated with rFXIII dose increments from 1 to 3 and 3 to 10 mg kg−1, respectively. Conclusions: The disproportionate HMEX formation parallels the steep toxicity dose response previously reported for rFXIII in cynomolgus monkeys, supporting a mechanistical role for HMEX in the generalized coagulopathy seen in rFXIII toxicity. Our findings support that HMEX constitute candidate (potential) safety biomarkers in rFXIII treatment.

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.

Preclinical toxicity biomarkers for combination treatment with clotting factors rFXIII and rFVIIa

Abstract Combination treatment with the clotting factors recombinant activated factor VII (rFVIIa), serine protease, and recombinant factor XIII (rFXIII), protransglutaminase, is being explored for haemostatic therapy. We performed a single-dose toxicology study in the cynomolgus monkey, with four dose groups receiving 0.1 + 0.34 mg kg−1 (group 1), 0.33 + 1.12 mg kg−1 (group 2), 1.67 + 5.60 mg kg−1 (group 3) and 5.00 + 16.80 mg kg−1 (group 4) of a rFVIIa + rFXIII combination. In the three lower dose groups, no clinical, histopathological or blood chemistry changes were observed. In group 4, the animals died at 4 h post-dosing, with histopathology revealing a systemic coagulopathy resembling, but distinct from, disseminated intravascular coagulation. Due to the absence of toxicity warning signs, toxicity biomarkers were identified by a Western blot-based screening of approximately 20 plasma proteins known to be involved in the clotting cascade. Three of the examined proteins were specifically affected by rFVIIa + rFXIII treatment. Fibronectin and fibrinogen exhibited dose-dependent reductions from less than 10% reduction (group 2) to more than 90% reduction (group 4). These reductions were reversible, and specific. For vitronectin, a dose-dependent conversion to the 65-kDa form was found to occur in groups 3 and 4. Thus, fibrinogen, fibronectin and vitronectin represent the first biomarkers for clotting factor toxicity.

Pharmacokinetics of levormeloxifene in young versus elderly postmenopausal women

BACKGROUND Physiologic changes of aging may affect processes of drug absorption and distribution, in some cases necessitating age-dependent dose adjustment. OBJECTIVE The possibility of age dependence in the pharmacokinetic behavior and tolerability of levormeloxifene was investigated in a single-center, open-label study. METHODS The study comprised 2 groups of healthy postmenopausal women: group A included younger subjects (50-60 years) and group B included elderly subjects (> or = 66 years). All subjects received a single 40-mg tablet of levormeloxifene base. Blood samples were collected immediately before drug intake and at several points after administration, through day 34. Peak plasma concentration, time to maximum plasma concentration, area under the plasma concentration-time curves from zero to the last quantifiable value and to infinity, and terminal half-life were calculated for levormeloxifene and compared between age groups. RESULTS Of 29 subjects enrolled, 28 (15 group A, 13 group B) completed the study. The ages of the women in group A ranged from 50 to 58 years and in group B from 66 to 79 years. No serious adverse events were reported. Ten subjects experienced 17 adverse events, of which 2 (abdominal pain and vaginal hemorrhage) were judged to be possibly related to study drug. There was no noticeable difference between age groups in the frequency of adverse events or laboratory abnormalities. The plasma concentration-time curves of levormeloxifene were indistinguishable between age groups up to 48 hours after dosing. From 72 hours onward, the mean plasma concentration-time curve was approximately 20% higher and the area under the curve was approximately 19% greater in the older subjects compared with the younger subjects. However, no statistically significant differences were observed between groups in any of the pharmacokinetic parameters, except for the elimination rate constant. The difference in mean elimination half-life was 25 hours (group A, 126 hours; group B, 151 hours). CONCLUSION The findings suggest that it is not necessary to adjust the dose of levormeloxifene on the basis of age in postmenopausal women. However, these results need confirmation in a multiple-dose setting under steady-state conditions--that is, as the drug is intended to be used clinically.

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

SummaryWe 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.