Verapamil and low-dose anti-mouse thymocyte globulin combination therapy stably reverses recent-onset type 1 diabetes in NOD mice by acting on the beta cell and immune axes.

AIMS/HYPOTHESIS Verapamil, a calcium channel blocker, and low doses of anti-thymocyte globulin (ATG) have individually shown efficacy in preserving beta cell function in people with recent-onset symptomatic type 1 diabetes (stage 3). We hypothesised that combining interventions with complementary modes of action and different targets would increase their efficacy in arresting beta cell demise and promoting disease recovery. METHODS Continuous administration of verapamil via drinking water, combined with a short course of low-dose rabbit-anti-mouse ATG (mATG), was studied in female recent-onset diabetic NOD mice for its potential to induce disease remission and mechanism of action. RESULTS Verapamil stably reversed diabetes in 3 out of 15 mice (20%) by day 56 after therapy start. Low-dose mATG reversed diabetes in 7 out of 18 mice (39%) by day 7 after therapy start, yet the effect waned to 3 out of 18 mice (17%) by day 56. The combination of verapamil with mATG induced durable diabetes reversal in 9 out of 20 mice (45%) by day 56, which was associated with preserved beta cell function, higher pancreatic insulin content and increased total beta cell volume with decreased severe insulitis. mATG, both alone and in combination, induced a temporary depletion of lymphocytes in peripheral blood on day 3 after therapy start, which largely recovered by day 14, when naive cells had shifted to a memory phenotype in both CD4+ and CD8+ T cells. Only in combination-treated mice was a higher CD4+ regulatory T cell to CD8+ effector memory T cell ratio observed in the pancreatic draining lymph nodes. The expression of the glucose-induced gene encoding thioredoxin-interacting protein (Txnip), a key regulator of beta cell apoptosis and dysfunction, was reduced in pancreatic beta cells in reversed mice, irrespective of whether they received verapamil or not. CONCLUSIONS/INTERPRETATION The combination of verapamil and low-dose mATG outperformed monotherapy in reversing recent-onset type 1 diabetes in NOD mice. This approach targets both the beta cell and immune axes, suggesting a promising strategy for disease reversal in human type 1 diabetes.