Comprehensive structure-activity-relationship of azaindoles as highly potent FLT3 inhibitors

Acute myeloid leukemia (AML) is characterized by fast progression and low survival rates, in which Fms-like tyrosine kinase 3 (FLT3) receptor mutations have been identified as a driver mutation in cancer progression in a subgroup of AML patients. Clinical trials have shown emergence of drug resistant mutants, emphasizing the ongoing need for new chemical matter to enable the treatment of this disease. Here, we present the discovery and topological structure-activity relationship (SAR) study of analogs of isoquinolinesulfonamide H-89, a well-known PKA inhibitor, as FLT3 inhibitors. Surprisingly, we found that the SAR was not consistent with the observed binding mode of H-89 in PKA. Matched molecular pair analysis resulted in the identification of highly active sub-nanomolar azaindoles as novel FLT3-inhibitors. Structure based modelling using the FLT3 crystal structure suggested an alternative, flipped binding orientation of the new inhibitors.     

Breast MRI in nonpalpable breast lesions: a randomized trial with diagnostic and therapeutic outcome – MONET – study

Background

In orthodontic treatment, anchorage control is a fundamental aspect. Usually conventional mechanism for orthodontic anchorage control can be either extraoral or intraoral that is headgear or intermaxillary elastics. Their use are combined with various side effects such as tipping of occlusal plane or undesirable movements of teeth. Especially in cases, where key-teeth are missing, conventional anchorage defined as tooth-borne anchorage will meet limitations. Therefore, the use of endosseous implants for anchorage purposes are increasingly used to achieve positional stability and maximum anchorage.

Methods/Design

The intended study is designed as a prospective, multicenter randomized controlled trial (RCT), comparing and contrasting the effect of early loading of palatal implant therapy versus implant loading after 12 weeks post implantation using the new ortho-implant type II anchor system device (Orthosystem Straumann, Basel, Switzerland). 124 participants, mainly adult males or females, whose diagnoses require temporary stationary implant-based anchorage treatment will be randomized 1:1 to one of two treatment groups: group 1 will receive a loading of implant standard therapy after a healing period of 12 week (gold standard), whereas group 2 will receive an early loading of orthodontic implants within 1 week after implant insertion. Participants will be at least followed for 12 months after implant placement. The primary endpoint is to investigate the behavior of early loaded palatal implants in order to find out if shorter healing periods might be justified to accelerate active orthodontic treatment. Secondary outcomes will focus e.g. on achievement of orthodontic treatment goals and quantity of direct implant-bone interface of removed bone specimens. As tertiary objective, a histologic and microtomography evaluation of all retrieved implants will be performed to obtain data on the performance of the SLA surface in human bone evaluation of all retrieved implants. Additionally, resonance frequency analysis (RFA, Osstell? mentor) will be used at different times for clinically monitoring the implant stability and for histological comparison in order to measure the reliability of the resonance frequency measuring device.

Trial registration

Current Controlled Trials ISRCTN97142521.     

Abscisic acid controls embryo growth potential and endosperm cap weakening during coffee (<Emphasis Type="Italic">Coffea arabica</Emphasis> cv. Rubi) seed germination

The mechanism and regulation of coffee seed germination were studied in Coffea arabica L. cv. Rubi. The coffee embryo grew inside the endosperm prior to radicle protrusion and abscisic acid (ABA) inhibited the increase in its pressure potential. There were two steps of endosperm cap weakening. An increase in cellulase activity coincided with the first step and an increase in endo--mannanase (EBM) activity with the second step. ABA inhibited the second step of endosperm cap weakening, presumably by inhibiting the activities of at least two EBM isoforms and/or, indirectly, by inhibiting the pressure force of the radicle. The increase in the activities of EBM and cellulase coincided with the decrease in the force required to puncture the endosperm and with the appearance of porosity in the cell walls as observed by low-temperature scanning electronic microscopy. Tissue printing showed that EBM activity was spatially regulated in the endosperm. Activity was initiated in the endosperm cap whereas later during germination it could also be detected in the remainder of the endosperm. Tissue printing revealed that ABA inhibited most of the EBM activity in the endosperm cap, but not in the remainder of the endosperm. ABA did not inhibit cellulase activity. There was a transient rise in ABA content in the embryo during imbibition, which was likely to be responsible for slow germination, suggesting that endogenous ABA also may control embryo growth potential and the second step of endosperm cap weakening during coffee seed germination.     

The long-term results of ruby laser depilation in a consecutive series of 346 patients

The goal of this study was to prospectively assess the long-term results of ruby laser depilation in 346 consecutive patients who underwent hair removal at 402 anatomical sites. The patients were treated using a ruby laser, with mean power ranging from 8.6 J to 15.7 J according to skin type. Results were assessed using two outcome measures-the percentage reduction in hair density and the hair-free interval. The median reduction in hair density was 55 percent (range, 0 to 100 percent) at a median time of 1 year after the last treatment session. The median hair-free interval was 8 weeks. Patients underwent a median number of four treatment sessions. Forty-three of the 346 patients were treated at more than one anatomical site. Of the sites treated, 75 percent reduction in hair density was achieved in 22 percent, 90 percent reduction was achieved in 2.2 percent, and complete depilation was achieved in only 0.7 percent. Darker colored hair was more effectively treated. Treatment efficacy was not affected by anatomical site, with the exception of the faces of male patients, which were found to be particularly resistant to treatment. There was a significant correlation between the number of treatments given and the outcome. The overall complication rate was 9.0 percent (36 of 402 sites) with respect to pigmentary changes and blistering, but varied according to Fitzpatrick skin type. The complication rate was highest in skin types V and VI (24.7 percent), with no complications in skin type I. Although a greater than 50 percent reduction in hair density was achieved in half of the 346 patients treated, complete depilation was achieved in only an extremely limited number of patients.     

Diffusional properties of methanogenic granular sludge: 1H NMR characterization

The diffusive properties of anaerobic methanogenic and sulfidogenic aggregates present in wastewater treatment bioreactors were studied using diffusion analysis by relaxation time-separated pulsed-field gradient nuclear magnetic resonance (NMR) spectroscopy and NMR imaging. NMR spectroscopy measurements were performed at 22 degrees C with 10 ml of granular sludge at a magnetic field strength of 0.5 T (20 MHz resonance frequency for protons). Self-diffusion coefficients of H(2)O in the investigated series of mesophilic aggregates were found to be 51 to 78% lower than the self-diffusion coefficient of free water. Interestingly, self-diffusion coefficients of H(2)O were independent of the aggregate size for the size fractions investigated. Diffusional transport occurred faster in aggregates growing under nutrient-rich conditions (e.g., the bottom of a reactor) or at high (55 degrees C) temperatures than in aggregates cultivated in nutrient-poor conditions or at low (10 degrees C) temperatures. Exposure of aggregates to 2.5% glutaraldehyde or heat (70 or 90 degrees C for 30 min) modified the diffusional transport up to 20%. In contrast, deactivation of aggregates by HgCl(2) did not affect the H(2)O self-diffusion coefficient in aggregates. Analysis of NMR images of a single aggregate shows that methanogenic aggregates possess a spin-spin relaxation time and self-diffusion coefficient distribution, which are due to both physical (porosity) and chemical (metal sulfide precipitates) factors.     

Expression of somatostatin,cortistatin, and somatostatin receptors in human monocytes,macrophages, and dendritic cells

Increasing evidence suggests that neuropeptides play a role in the regulatory mechanisms between the neuroendocrine and immune systems. A differential expression of the five known somatostatin (SS) receptors (sst1-5) has been demonstrated in human immune cells and tissues. However, little is known concerning regulation and expression of sst1-5 and the peptide SS. Therefore, we investigated the expression and the time-dependent regulation of sst1-5, SS, and cortistatin (CST), a novel SS-like peptide, in human monocytes (MO), monocyte-derived macrophages (MP), and dendritic cells (DC) in the basal and lipopolysaccharide (LPS)-activated state. MO, MP, and DC selectively expressed sst2 mRNA. SS mRNA was not detectable, whereas all samples expressed CST mRNA. Expression levels of sst2 and CST mRNA showed marked differences and were in the rank order of MP>DC>MO. LPS stimulation did not induce expression of SS or sst1,3,4,5. However, sst2 mRNA expression was upregulated significantly by stimulation with LPS. CST mRNA was upregulated as well. During differentiation of MO in MP or DC, time-dependent, significantly increasing sst2 and CST mRNA levels were found. By confocal microscopy, the presence of sst2 receptors was demonstrated on MP, but not on DC. This study demonstrates for the first time a selective and inducible expression of the recently discovered CST, as well as sst2, in human monocyte-derived cells, suggesting a role for a CST-sst2 system rather than a SS-sst2 system in these immune cell types.     

In vitro characterisation of a monovalent and bivalent form of a fully human anti Ep-CAM phage antibody

 Antibodies to tumour-associated antigens are increasingly being used as targeting vehicles for the visualisation and for therapy of human solid tumours. The epithelial cell adhesion molecule (Ep-CAM) is an antigen that is overexpressed on a variety of human solid tumours and constitutes an attractive target for immunotargeting. We set out to obtain fully human antibodies to this antigen by selecting from a large antibody repertoire displayed on bacteriophages. Two single-chain variable antibody fragments (scFv) were identified that specifically bound recombinant antigen in vitro. One of the selected antibodies (VEL-1) cross-reacted with extracellular matrix components in immunohistochemistry of colon carcinoma, whereas the other scFv (VEL-2) specifically recognised colon cancer cells. The latter antibody was further characterised with respect to epitope specificity and kinetics of antigen-binding. It showed no competition with the well-characterised anti Ep-CAM MOC-31 monoclonal antibody and had an off-rate of 5 × 10⁻² s⁻¹. To obtain an antibody format more suitable for in vivo tumour targeting and to increase the apparent affinity through avidity, the genes of scFv VEL-2 were re-formatted by fusion to a human (γ1) hinge region and CH3 domain. This “minibody” was expressed in Escherichia coli, specifically bound the Ep-CAM antigen and showed a 20-fold reduced off-rate in surface plasmon resonance analysis. These results show that phage antibody selection, combined with antibody engineering, may result in fully human antibody molecules with promising characteristics for in vivo use in tumour targeting. Received: 13 July 2000 / Accepted: 12 October 2000