The Ras Activator RasGRP3 Mediates Diabetes-Induced Embryonic Defects and Affects Endothelial Cell Migration

Fetuses that develop in diabetic mothers have a higher incidence of birth defects that include cardiovascular defects, but the signaling pathways that mediate these developmental effects are poorly understood. It is reasonable to hypothesize that diabetic maternal effects are mediated by one or more pathways activated downstream of aberrant glucose metabolism, since poorly controlled maternal glucose levels correlate with the frequency and severity of the defects

Correlation of Eemian sections in Lithuania and Belarus based on palaeomagnetic, radioisotope and palaeobotanic data

Eemian (Murava, Merkinė deposits at five exposed sections (Zaslavl, Zhukevichi, Ponemun, Snaigupėlė, and Netiesos) located in Lithuania and Belarus are described. Preliminary palaeomagnetic results show a record of the Brunhes epoch normal magnetic field and a short-term reversal – the Blake Event – is recognized in three of the five sections. The Blake Event recorded in the Netiesos section is characterized by a pattern consisting of three short reversed polarity intervals separated by two short normal polarity intervals. The directional changes of declination, inclination, and MS (magnetic susceptibility) are clear. ESR dating (112.5 ±10.8 and 112.1 ±25.9) and 230Th/U dates obtained from this section (108.8 ±12.0/9.9 ka for the L/L technique and 100.2 ±10.3/8.6 ka for the TSD technique) suggest that (Blake and post-Blake) palaeomagnetic excursions are present in this section. Palaeobotanical analysis and isotope dating of the Netiesos section suggest that the Blake Event occurred during the climatic optimum

Approaches to Improve the Pharmacokinetics of Radiolabeled Glucagon-Like Peptide-1 Receptor Ligands Using Antagonistic Tracers.

UNLABELLED The glucagon-like peptide-1 (GLP-1) receptors are important biomarkers for imaging pancreatic β-cell mass and detection of benign insulinomas. Using GLP-1 receptor antagonists, we aimed to eliminate the insulin-related side effects reported for all GLP-1 receptor agonists. Additionally, using a nonresidualizing tracer, (125)I-Bolton-Hunter-Exendin(9-39)NH2 ((125)I-BH-Ex(9-39)NH2), we aimed to reduce the high kidney uptake, enabling a better detection of insulinomas in the tail and head of the pancreas. METHODS The affinity and biodistribution of Ex(9-39)NH2-based antagonists, modified with DOTA or NODAGA chelators at positions Lys(27) and Lys(40) and labeled with (68)Ga and (125)I-BH-Ex(9-39)NH2, were compared with the reference GLP-1 receptor agonist [Nle(14),Lys(40)(Ahx-DOTA-(68)Ga)NH2]Ex-4. The inhibitory concentration of 50% (IC50) values were determined using autoradiography on human tissues with (125)I-GLP-1(7-36)NH2 as a radioligand. Pharmacokinetics and PET imaging were studied in nude mice bearing rat Ins-1E tumors. RESULTS Conjugation of DOTA and NODAGA chelators at positions Lys(27) and Lys(40) of Ex(9-39)NH2 resulted in a distinct loss of affinity toward GLP-1 receptor in vitro. Among the studied antagonists, [Lys(40)(NODAGA-(nat)Ga)NH2]Ex(9-39) showed the lowest IC50 value (46.7 ± 16.3 nM). The reference agonist [Nle(14),Lys(40)(Ahx-DOTA)NH2]Ex-4 demonstrated the highest affinity (IC50 = 0.9 ± 0.3 nM). Biodistribution of [Nle(14),Lys(40)(Ahx-DOTA-(68)Ga)NH2]Ex-4 at 1 h after injection demonstrated 40.2 ± 8.2 percentage injected activity per gram (%IA/g) uptake in Ins-1E tumor, 12.5 ± 2.2 %IA/g in the pancreas, and 235.8 ± 17.0 %IA/g in the kidney, with tumor-to-blood and tumor-to-kidney ratios of 100.52 and 0.17, respectively. Biodistribution of [Lys(40)(NODAGA-(68)Ga)NH2]Ex(9-39) showed only 2.2 ± 0.2 %IA/g uptake in Ins-1E tumor, 1.0 ± 0.1 %IA/g in the pancreas, and 78.4 ± 8.5 %IA/g in the kidney at 1 h after injection, with tumor-to-blood and tumor-to-kidney ratios of 7.33 and 0.03, respectively. In contrast, (125)I-BH-Ex(9-39)NH2 showed tumor uptake (42.5 ± 8.1 %IA/g) comparable to the agonist and 28.8 ± 5.1 %IA/g in the pancreas at 1 h after injection. As we hypothesized, the kidney uptake of (125)I-BH-Ex(9-39)NH2 was low, only 12.1 ± 1.4 %IA/g at 1 h after injection. The tumor-to-kidney ratio of (125)I-BH-Ex(9-39)NH2 was improved 20-fold. CONCLUSION Our results suggest that iodinated Ex(9-39)NH2 may be a promising tracer for imaging GLP-1 receptor expression in vivo. Because of the 20-fold improved tumor-to-kidney ratio (125)I-BH-Ex(9-39)NH2 may offer higher sensitivity in the detection of insulinomas and imaging of β-cell mass in diabetic patients. Further studies with (124)I-BH-Ex(9-39)NH2 are warranted

Radioiodinated Exendin-4 Is Superior to the Radiometal-Labelled Glucagon-Like Peptide-1 Receptor Probes Overcoming Their High Kidney Uptake - Fig 2

<p><b>Internalization (A) and binding (B) curves for [Nle</b>^<b>14</b><b>,</b>^<b>125</b><b>I-Tyr</b>^<b>40</b><b>-NH</b>_<b>2</b><b>]Ex-4 agonist and [Nle</b>^<b>14</b><b>,</b>^<b>125</b><b>I-Tyr</b>^<b>40</b><b>-NH</b>_<b>2</b><b>]Ex(9–39) antagonist in Ins-1E cells.</b> The plots show the percentage of specifically internalized and specifically bound peptide versus time. Values are mean±standard deviation of triplicate measurements.</p

Radioiodinated Exendin-4 Is Superior to the Radiometal-Labelled Glucagon-Like Peptide-1 Receptor Probes Overcoming Their High Kidney Uptake - Fig 3

<p><b>Radio-HPLC showing the radiochemical purity of formulated [Nle</b>^<b>14</b><b>,</b>^<b>124</b><b>I-Tyr</b>^<b>40</b><b>-NH</b>_<b>2</b><b>]Ex-4 without (A) and with co-injection of cold reference [Nle</b>^<b>14</b><b>,</b>^<b>127</b><b>I-Tyr</b>^<b>40</b><b>-NH</b>_<b>2</b><b>]Ex-4 (B)</b>. UV-and radio-detectors were in series, resulting in a lag time of about 15 sec for the radiotrace. Numbers in the chromatograms refer to the peak retention time in minutes.</p

[Nle¹⁴,¹²⁴I-Tyr⁴⁰-NH₂]Ex-4 tumor time-activity curves.

<p>The curves are derived from the PET image analysis and show the tumor uptake of [Nle¹⁴,¹²⁴I-Tyr⁴⁰-NH₂]Ex-4 versus time.</p

Biodistribution of [Nle¹⁴,¹²⁵I-Tyr⁴⁰-NH₂]Ex(9–39) in mice bearing Ins-1E xenografts.

<p>Biodistribution of [Nle¹⁴,¹²⁵I-Tyr⁴⁰-NH₂]Ex(9–39) in mice bearing Ins-1E xenografts.</p

<i>In vitro</i> saturation binding data.

<p>The graphs show the percentage of specifically bound [Nle¹⁴,¹²⁵I-Tyr⁴⁰-NH₂]Ex-4 (A) or [Nle¹⁴,¹²⁵I-Tyr⁴⁰-NH₂]Ex(9–39) (B) per 1×10⁶ cells after incubation of Ins-1E cells with increasing amounts of the peptide. Values are mean±standard error of triplicate measurements.</p

Mass dependence of [Nle¹⁴,¹²⁵I-Tyr⁴⁰-NH₂]Ex-4 biodistribution in nude mice bearing Ins-1E xenografts 1 h p.i.

<p>Mass dependence of [Nle¹⁴,¹²⁵I-Tyr⁴⁰-NH₂]Ex-4 biodistribution in nude mice bearing Ins-1E xenografts 1 h p.i.</p

The tetraamine chelator outperforms HYNIC in a new technetium-99m-labelled somatostatin receptor 2 antagonist

Abstract Background Somatostatin receptor targeting radiopeptides are successfully being used to image, stage, and monitor patients with neuroendocrine tumours. They are exclusively agonists that internalise upon binding to the relevant receptor. According to recent reports, antagonists may be preferable to agonists. To date, 99mTc-labelled somatostatin receptor antagonists have attracted little attention. Here, we report on a new somatostatin receptor subtype 2 (sst2) antagonist, SS-01 (p-Cl-Phe-cyclo(D-Cys-Tyr-D-Trp-Lys-Thr-Cys)D-Tyr-NH2), with the aim of developing 99mTc-labelled ligands for SPECT/CT imaging. SS-01 was prepared using Fmoc solid-phase synthesis and subsequently coupled to the chelators 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 6-carboxy-1,4,8,11-tetraazaundecane (N4), and 6-hydrazinonicotinic acid (HYNIC) to form the corresponding peptide-chelator conjugates SS-03, SS-04, and SS-05, respectively. SS-04 and SS-05 were radiolabelled with 99mTc and SS-03 with 177Lu. Binding affinity and antagonistic properties were determined using autoradiography and immunofluorescence microscopy. Biodistribution and small animal SPECT/CT studies were performed on mice bearing HEK293-rsst2 xenografts. Results The conjugates showed low nanomolar sst2 affinity and antagonistic properties. 177Lu-DOTA-SS-01 (177Lu-SS-03) and 99mTc-N4-SS-01 (99mTc-SS-04) demonstrated high cell binding and low internalisation, whereas 99mTc-HYNIC/edda-SS-01 (99mTc-SS-05) showed practically no cellular uptake in vitro. The 99mTc-SS-04 demonstrated impressive tumour uptake at early time points, with 47% injected activity per gram tumour (%IA/g) at 1 h post-injection. The tumour uptake persisted after 4 h and was 32.5 %IA/g at 24 h. The uptake in all other organs decreased much more rapidly leading to high tumour-to-normal organ ratios, which was reflected in high-contrast SPECT/CT images. Conclusions These data indicate a very promising 99mTc-labelled sst2-targeting antagonist. The results demonstrate high sensitivity of the 99mTc-labelling strategy, which was shown to strongly influence the receptor affinity, contrary to corresponding agonists. 99mTc-SS-04 exhibits excellent pharmacokinetics and imaging properties and appears to be a suitable candidate for SPECT/CT clinical translation