In vivo evaluation of a novel, orally bioavailable, small molecule growth hormone receptor antagonist.

IGF-I is regarded as the most sensitive marker of growth hormone (GH) secretion in both GH deficient individuals and in individuals with excessive GH production. Studies on the effect of inhibitors of GH action in normal experimental animals are difficult to evaluate due to the complex relationship and feed back mechanisms of the GH/IGF-I system and the hypothalamo-pituitary axis. To circumvent the GH/IGF-I feedback mechanisms, we have used hypophysectomized (HX) rats treated with GH to assess the potential of a new low molecular weight compound, BVT-A ((N-[5-(aminosulfonyl)-2-methylphenyl]-5-bromo-2-furamide), to act as a GH receptor antagonist in vivo. GH treatment of HX rats induced serum IGF-I, body weight and hepatic mRNA levels of IGF-I, IGFBP-3, ALS and the IGF-I and GH receptors. Co-treatment with BVT-A suppressed all the GH-induced effects. We conclude that the GH substituted HX rat is a useful model for studies on GH receptor antagonists, and for the first time, a small molecule GH receptor antagonist with in vivo activity has been revealed. This opens up for development of new drugs for diseases in which lowering of GH receptor activity would be beneficial.

Identification of AKN-032, a novel 2-aminopyrazine tyrosine kinase inhibitor, with significant preclinical activity in acute myeloid leukemia.

Aberrant signal transduction by mutant or overexpressed protein kinases has emerged as a promising target for treatment of acute myeloid leukemia (AML). We here present a novel low molecular weight kinase inhibitor, AKN-032, targeting the FMS-like tyrosine kinase 3 (FLT3) and discovered in a new type of screening funnel combining the target therapy approach with sequential cellular screens. AKN-032 was identified among 150 selected hits from three different high throughput kinase screens. Further characterization showed inhibitory activity on FLT3 enzyme with an IC(50) of 70 nM. Western blot analysis revealed reduced autophosphorylation of the FLT3-receptor in AML cell line MV4-11 cells after exposure to AKN-032. Flow cytometry disclosed cytotoxic activity against MV4-11, but not against non-malignant 3T3-L1 fibroblast cells. Using a fluorometric microculture cytotoxicity assay, AKN-032 was tested against 15 cell lines and displayed a potent cytotoxic activity in AML cell lines MV4-11 (IC(50)=0.4 µM) and Kasumi-1 (IC(50)=2.3 µM). AKN-032 was also highly cytotoxic in tumor cells from AML patients in vitro. Furthermore, AKN-032 demonstrated significant antileukemic effect in a relatively resistant in vivo hollow fiber mouse model. No major toxicity was observed in the animals. In conclusion, AKN-032 is a promising new kinase inhibitor with significant in vivo and in vitro activity in AML. Results from the hollow fiber mouse assay suggest a favorable toxicity profile. Future studies will focus on pharmacokinetic properties, toxicity as well as further clarifying the mechanisms of action of AKN-032 in AML.

Growth hormone replacement in hypophysectomized rats affects spatial performance and hippocampal levels of NMDA receptor subunit and PSD-95 gene transcript levels.

Clinical studies have demonstrated that growth hormone (GH) promotes learning and memory processes in GH-deficient (GHD) patients. In animal studies, GH also influences the N-methyl-D-aspartate (NMDA) receptor system in the hippocampus, an essential component of long-term potentiation (LTP), which is highly involved in memory acquisition. This study was designed to examine the beneficial effects of recombinant human GH (rhGH) on cognitive function in male rats with multiple hormone deficiencies resulting from hypophysectomy (Hx). The performance of an rhGH-treated group and an untreated control group was appraised in the Morris water maze (MWM). The rhGH-treated group performed significantly better in the spatial memory task than the control animals on the second and third trial days. Further training eliminated this difference between the groups. Hippocampal mRNA expression of the NMDA subunits NR1, NR2A and NR2B, insulin-like growth factor type 1 receptor (IGF-1R), and postsynaptic density protein-95 (PSD-95) was then measured in the animals by Northern blot analysis. The results suggest that there may be a relationship between the NMDA receptor subunit mRNA expression levels and learning ability, and that learning is improved by rhGH in Hx rats. Furthermore, a link between MWM performance and PSD-95 was also suggested by this study.

Reliability of the western ligand blot method for the simultaneous relative estimations of serum insulin-like growth factor binding proteins (IGFBPs) / Confiabilidad del método de \"western ligand blot\" para la determinación simultánea de las proteínas de unión de los factores de crecimiento similares a la insulina (IGFBPs) en suero

It is well established that nutrition is an important regulator of both serum insulin-like growth factor-I (IGF) and its binding proteins (IGFBPs). The Western ligand blot method (WLB) for simultaneous determinations of IGFBPs in serum or plasma sambples was evaluated and validated with emplasis on its reproducible capabilities. After electrophoretic separation and transfer, the membranes were incubated with a mixture of recombinant labeled human (GF-I/IGF-II(rhIGF-II) and band intensities measured by autoradiography. The typical electrophoretic profile for pig serum, as determined with molecular weight markers, showed four mainbands of approximately 42-39,32,30-28 and 24 kDa which seemed to correspond to IGFBP-3, IGFBP-2, IGFBP-1 and IGFBP-4 respectively. Likewise, a trinlet of approximately 42-39 kDa (IGFBP-3), a broad area called OGFBP-30 region (most probably IGFBP-1, -2 and -3 variants) and a third band of 24 kDa IGFBP-4) were seen in rat samples. Determination of IGFBP/2 and 1 in rat serum samples, as two separate bands on 12 por ciento gels was difficult due to their close electrophoretic migration and possibly to the reported lower levels of IGFBP-2 in adult rat serum. Dilutions tested on 0.2 µm nitrocellulose membranes with samples volumes between 0.25 to 1.5 µl(1:10-1:60 dilutions), showed IGFBPs curves with good linearity which suggest first, that there exist a quantitative relation betweem the amount of each protein and densitometric response and second, that the transfer of the proteins was linear across the range of 0.25 to 1.5 µl(1:10-1:60 dilutions). Moreover, the results also suggest that losses were aqually spread and that the proteins retained their binding pro[erties after the process. Reproducibility showed intra-assay coefficients of variation (CVs) of 15 per cent or lower using either a transfer device without cooling system or a combination of a transferdevice with cooling system and manually defined band boundaries. In summary, it was shown that the optimized experimental conditions here described for the WLB method, allow realiable simultaneous measurements of the main pig and rat serum IGFBPs and therefore, could be utilised to detect changes in the profile after dietary manipulations