Nonpeptidic Z360-Analogs Tagged with Trivalent Radiometals as Anti-CCK2R Cancer Theranostic Agents: A Preclinical Study

(1) Background: Theranostic approaches in the management of cholecystokinin subtype 2 receptor (CCK2R)-positive tumors include radiolabeled gastrin and CCK motifs. Moving toward antagonist-based CCK2R-radioligands instead, we herein present three analogs of the nonpeptidic CCK2R-antagonist Z360, GAS1/2/3. Each was conjugated to a different chelator (DOTA, NODAGA or DOTAGA) for labeling with medically relevant trivalent radiometals (e.g., Ga-68, In-111, Lu-177) for potential use as anti-CCK2R cancer agents; (2) Methods: The in vitro properties of the thee analogs were compared in stably transfected HEK293-CCK2R cells. Biodistribution profiles were compared in SCID mice bearing twin HEK293-CCK2R and wtHEK293 tumors; (3) Results: The GAS1/2/3 analogs displayed high CCK2R-affinity (lower nM-range). The radioligands were fairly stable in vivo and selectively targeted the HEK293-CCK2R, but not the CCK2R-negative wtHEK293 tumors in mice. Their overall pharmacokinetic profile was found strongly dependent on the radiometal-chelate. Results could be visualized by SPECT/CT for the [111 In]In-analogs; (4) Conclusions: The present study highlighted the high impact of the radiometal-chelate on the end-pharmacokinetics of a new series of Z360-based radioligands, revealing candidates with promising properties for clinical translation. It also provided the impetus for the development of a new class of nonpeptidic radioligands for CCK2R-targeted theranostics of human cancer

PI3 kinases p110α and PI3K-C2β negatively regulate cAMP via PDE3/8 to control insulin secretion in mouse and human islets

Objectives: Phosphatidylinositol-3-OH kinase (PI3K) signalling in the endocrine pancreas contributes to glycaemic control. However, the mechanism by which PI3K modulates insulin secretion from the pancreatic beta cell is poorly understood. Thus, our objective was two-fold; to determine the signalling pathway by which acute PI3K inhibition enhances glucose-stimulated insulin secretion (GSIS) and to examine the role of this pathway in islets from type-2 diabetic (T2D) donors. Methods: Isolated islets from mice and non-diabetic or T2D human donors, or INS 832/13 cells, were treated with inhibitors of PI3K and/or phosphodiesterases (PDEs). The expression of PI3K-C2β was knocked down using siRNA. We measured insulin release, single-cell exocytosis, intracellular Ca2+ responses ([Ca2+]i) and Ca2+ channel currents, intracellular cAMP concentrations ([cAMP]i), and activation of cAMP-dependent protein kinase A (PKA) and protein kinase B (PKB/AKT). Results: The non-specific PI3K inhibitor wortmannin amplifies GSIS, raises [cAMP]i and activates PKA, but is without effect in T2D islets. Direct inhibition of specific PDE isoforms demonstrates a role for PDE3 (in humans and mice) and PDE8 (in mice) downstream of PI3K, and restores glucose-responsiveness of T2D islets. We implicate a role for the Class II PI3K catalytic isoform PI3K-C2β in this effect by limiting beta cell exocytosis. Conclusions: PI3K limits GSIS via PDE3 in human islets. While inhibition of p110α or PIK-C2β signalling per se, may promote nutrient-stimulated insulin release, we now suggest that this signalling pathway is perturbed in islets from T2D donors. Author Video: Author Video Watch what authors say about their articles Keywords: PI3K, cAMP, PDE, Beta cells, Insulin secretion, T2

Enhanced Peptide Stability Against Protease Digestion Induced by Intrinsic Factor Binding of a Vitamin B₁₂ Conjugate of Exendin‑4

Peptide digestion from proteases is a significant limitation in peptide therapeutic development. It has been hypothesized that the dietary pathway of vitamin B₁₂ (B₁₂) may be exploited in this area, but an open question is whether B₁₂-peptide conjugates bound to the B₁₂ gastric uptake protein intrinsic factor (IF) can provide any stability against proteases. Herein, we describe a new conjugate of B₁₂ with the incretin peptide exendin 4 that demonstrates picomolar agonism of the glugacon-like peptide-1 receptor (GLP1-R). Stability studies reveal that Ex-4 is digested by pancreatic proteases trypsin and chymotrypsin and by the kidney endopeptidase meprin β. Prebinding the B₁₂ conjugate to IF, however, resulted in up to a 4-fold greater activity of the B₁₂-Ex-4 conjugate relative to Ex-4, when the IF-B₁₂-Ex-4 complex was exposed to 22 μg/mL of trypsin, 2.3-fold greater activity when exposed to 1.25 μg/mL of chymotrypsin, and there was no decrease in function at up to 5 μg/mL of meprin β