Pressure-Driven Insulin Release Overcomes Limitations of Diffusion for Encapsulated Islet Cell Therapy.

Cadaveric islet and stem cell-derived transplantation show great promise as therapeutic approaches for type 1 diabetes. To address the immunocompatibility challenge, numerous cellular macroencapsulation techniques, which rely upon diffusion to transport insulin across the immunoprotective barrier, have been proposed. Although several of these devices were advanced to human clinical trials, they uniformly failed to achieve physiologic glucose control or insulin independence. Indeed, based upon mathematical modeling and empiric evidence, diffusion-based encapsulation devices are fundamentally incompatible with homeostatic on-demand insulin delivery and physiologic glucose regulation. To realize the potential of achieving insulin independence through macroencapsulated cell-based therapy, we propose the necessity of a second driving force. Herein, we provide both theoretical proof and experimental demonstration that modest (11-kPa) micropump-applied pressure considerably enhances insulin flux across immunoisolation membranes by nearly three orders of magnitude, enabling precise delivery of both bolus and basal insulin. Furthermore, pressure-driven insulin efflux from encapsulated mouse and human islets is fast and repeatable. As such, we urge caution against further advancement of diffusion-based immune-isolating macroencapsulation devices that do not incorporate a secondary driving force for precise temporal regulation of peptide delivery. One Sentence Summary: Diffusion-based insulin delivery from macroencapsulated islet cells is incompatible with physiologic glucose control, a constraint addressed through pressure-based insulin delivery.

Novel Pathogenic De Novo INS p.T97P Variant Presenting With Severe Neonatal DKA.

Pathogenic INS gene mutations are causative for mutant INS-gene-induced diabetes of youth (MIDY). We characterize a novel de novo heterozygous INS gene mutation (c.289A>C, p.T97P) that presented in an autoantibody-negative 5-month-old male infant with severe diabetic ketoacidosis. In silico pathogenicity prediction tools provided contradictory interpretations, while structural modeling indicated a deleterious effect on proinsulin folding. Transfection of wildtype and INS p.T97P expression and luciferase reporter constructs demonstrated elevated intracellular mutant proinsulin levels and dramatically impaired proinsulin/insulin and luciferase secretion. Notably, proteasome inhibition partially and selectively rescued INS p.T97P-derived luciferase secretion. Additionally, expression of INS p.T97P caused increased intracellular proinsulin aggregate formation and XBP-1s protein levels, consistent with induction of endoplasmic reticulum stress. We conclude that INS p.T97P is a newly identified pathogenic A-chain variant that is causative for MIDY via disruption of proinsulin folding and processing with induction of the endoplasmic reticulum stress response.

Generation of highly potent DYRK1A-dependent inducers of human ß-Cell replication via Multi-Dimensional compound optimization.

Small molecule stimulation of ß-cell regeneration has emerged as a promising therapeutic strategy for diabetes. Although chemical inhibition of dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) is sufficient to enhance ß-cell replication, current lead compounds have inadequate cellular potency for in vivo application. Herein, we report the clinical stage anti-cancer kinase inhibitor OTS167 as a structurally novel, remarkably potent DYRK1A inhibitor and inducer of human ß-cell replication. Unfortunately, OTS167's target promiscuity and cytotoxicity curtails utility. To tailor kinase selectivity towards DYRK1A and reduce cytotoxicity we designed a library of fifty-one OTS167 derivatives based upon a modeled structure of the DYRK1A-OTS167 complex. Indeed, derivative characterization yielded several leads with exceptional DYRK1A inhibition and human ß-cell replication promoting potencies but substantially reduced cytotoxicity. These compounds are the most potent human ß-cell replication-promoting compounds yet described and exemplify the potential to purposefully leverage off-target activities of advanced stage compounds for a desired application.

Zinc-Chelating Small Molecules Preferentially Accumulate and Function within Pancreatic ß Cells.

Diabetes is a hyperglycemic condition characterized by pancreatic ß-cell dysfunction and depletion. Whereas methods for monitoring ß-cell function in vivo exist, methods to deliver therapeutics to ß cells are lacking. We leveraged the rare ability of ß cells to concentrate zinc to preferentially trap zinc-binding molecules within ß cells, resulting in ß-cell-targeted compound delivery. We determined that zinc-rich ß cells and islets preferentially accumulated TSQ (6-methoxy-8-p-toluenesulfonamido-quinoline) in a zinc-dependent manner compared with exocrine pancreas. Next, we asked whether appending a zinc-chelating moiety onto a ß-cell replication-inducing compound was sufficient to confer preferential ß-cell accumulation and activity. Indeed, the hybrid compound preferentially accumulated within rodent and human islets in a zinc-dependent manner and increased the selectivity of replication-promoting activity toward ß cells. These data resolve the fundamental question of whether intracellular accumulation of zinc-chelating compounds is influenced by zinc content. Furthermore, application of this principle yielded a proof-of-concept method for ß-cell-targeted drug delivery and bioactivity.

CC-401 Promotes ß-Cell Replication via Pleiotropic Consequences of DYRK1A/B Inhibition.

Pharmacologic expansion of endogenous ß cells is a promising therapeutic strategy for diabetes. To elucidate the molecular pathways that control ß-cell growth we screened ∼2400 bioactive compounds for rat ß-cell replication-modulating activity. Numerous hit compounds impaired or promoted rat ß-cell replication, including CC-401, an advanced clinical candidate previously characterized as a c-Jun N-terminal kinase inhibitor. Surprisingly, CC-401 induced rodent (in vitro and in vivo) and human (in vitro) ß-cell replication via dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) 1A and 1B inhibition. In contrast to rat ß cells, which were broadly growth responsive to compound treatment, human ß-cell replication was only consistently induced by DYRK1A/B inhibitors. This effect was enhanced by simultaneous glycogen synthase kinase-3ß (GSK-3ß) or activin A receptor type II-like kinase/transforming growth factor-ß (ALK5/TGF-ß) inhibition. Prior work emphasized DYRK1A/B inhibition-dependent activation of nuclear factor of activated T cells (NFAT) as the primary mechanism of human ß-cell-replication induction. However, inhibition of NFAT activity had limited effect on CC-401-induced ß-cell replication. Consequently, we investigated additional effects of CC-401-dependent DYRK1A/B inhibition. Indeed, CC-401 inhibited DYRK1A-dependent phosphorylation/stabilization of the ß-cell-replication inhibitor p27Kip1. Additionally, CC-401 increased expression of numerous replication-promoting genes normally suppressed by the dimerization partner, RB-like, E2F and multivulval class B (DREAM) complex, which depends upon DYRK1A/B activity for integrity, including MYBL2 and FOXM1. In summary, we present a compendium of compounds as a valuable resource for manipulating the signaling pathways that control ß-cell replication and leverage a DYRK1A/B inhibitor (CC-401) to expand our understanding of the molecular pathways that control ß-cell growth.