Palmitate is a potent inducer of endoplasmic reticulum (ER) stress in beta-cells. In type 2 diabetes, glucose amplifies fatty-acid toxicity for pancreatic beta-cells, leading to beta-cell dysfunction and death. Why glucose exacerbates beta-cell lipotoxicity is largely unknown. Glucose stimulates mTORC1, an important nutrient sensor involved in the regulation of cellular stress. Our study tested the hypothesis that glucose augments lipotoxicity by stimulating mTORC1 leading to increased beta-cell ER stress.

Vascular endothelial cell (VEC) senescence is considered an early event in the development of atherosclerotic lesions. Stressful stimuli, in particular oxidative stress, have been linked to premature senescence in the vasculature. Foam cells are a major source of reactive oxygen species and may play a role in the induction of VEC senescence; hence, we investigated their involvement in the induction of VEC senescence in a co-culture transwell system. Primary bovine aortic endothelial cells, exposed to the secretome of THP-1 monocyte-derived foam cells, were analysed for the induction of senescence. Senescence associated β-galactosidase activity and the expression of p16 and p21 were increased, whereas phosphorylated retinoblastoma protein was reduced. This senescent phenotype was mediated by 4-hydroxnonenal (4-HNE), a lipid peroxidation product secreted from foam cells; scavenging of 4-HNE in the co-culture medium blunted this effect. Furthermore, both foam cells and 4-HNE increased the expression of the pro-oxidant thioredoxin-interacting protein (TXNIP). Molecular manipulation of TXNIP expression confirmed its involvement in foam cell-induced senescence. Previous studies showed that peroxisome proliferator-activated receptor (PPAR)δ was activated by 4-hydroalkenals, such as 4-HNE. Pharmacological interventions supported the involvement of the 4-HNE-PPARδ axis in the induction of TXNIP and VEC senescence. The association of TXNIP with VEC senescence was further supported by immunofluorescent staining of human carotid plaques in which the expression of both TXNIP and p21 was augmented in endothelial cells. Collectively, these findings suggest that foam cell-released 4-HNE activates PPARδ in VEC, leading to increased TXNIP expression and consequently to senescence.

Accumulation of misfolded proinsulin in the β-cell leads to dysfunction induced by endoplasmic reticulum (ER) stress, with diabetes as a consequence. Autophagy helps cellular adaptation to stress via clearance of misfolded proteins and damaged organelles. We studied the effects of proinsulin misfolding on autophagy and the impact of stimulating autophagy on diabetes progression in Akita mice, which carry a mutation in proinsulin, leading to its severe misfolding. Treatment of female diabetic Akita mice with rapamycin improved diabetes, increased pancreatic insulin content, and prevented β-cell apoptosis. In vitro, autophagic flux was increased in Akita β-cells. Treatment with rapamycin further stimulated autophagy, evidenced by increased autophagosome formation and enhancement of autophagosome-lysosome fusion. This was associated with attenuation of cellular stress and apoptosis. The mammalian target of rapamycin (mTOR) kinase inhibitor Torin1 mimicked the rapamycin effects on autophagy and stress, indicating that the beneficial effects of rapamycin are indeed mediated via inhibition of mTOR. Finally, inhibition of autophagy exacerbated stress and abolished the anti-ER stress effects of rapamycin. In conclusion, rapamycin reduces ER stress induced by accumulation of misfolded proinsulin, thereby improving diabetes and preventing β-cell apoptosis. The beneficial effects of rapamycin in this context strictly depend on autophagy; therefore, stimulating autophagy may become a therapeutic approach for diabetes.

Pridopidine is an oral drug in clinical development for treatment of patients with Huntington's disease. This study examined the interactions of pridopidine with in vitro cytochrome P450 activity and characterized the effects of pridopidine on CYP2D6 activity in healthy volunteers using metoprolol as a probe substrate. The effect of food on pridopidine exposure was assessed.

SD-1077, a selectively deuterated precursor of dopamine (DA) structurally related to L-3,4-dihydroxyphenylalanine (L-DOPA), is under development for treatment of motor symptoms of Parkinson's disease. Preclinical models have shown slower metabolism of central deuterated DA. The present study investigated the peripheral pharmacokinetics (PK), metabolism and safety of SD-1077.

TV-46000 is a long-acting subcutaneous antipsychotic that uses a novel copolymer drug delivery technology in combination with a well-characterized molecule, risperidone, that is in clinical development as a treatment for schizophrenia. A population pharmacokinetic (PPK) modeling and simulation approach was implemented to identify TV-46000 doses and dosing schedules for clinical development that would provide the best balance between clinical efficacy and safety. The PPK model was created by applying pharmacokinetic data from a phase 1 study of 97 patients with a diagnosis of schizophrenia or schizoaffective disorder who received either single or repeated doses of TV-46000. The PPK model was used to characterize the complex release profile of the total active moiety (TAM; the sum of the risperidone and 9-OH risperidone concentrations) concentration following subcutaneous injections of TV-46000. The PK profile was best described by a double Weibull function of the in vivo release rate and by a 2-compartment disposition and elimination model. Simulations were performed to determine TV-46000 doses and dosing schedules that maintained a median profile of TAM concentrations similar to published TAM exposure following oral risperidone doses that have been correlated to a 40% to 80% dopamine-D2 receptor occupancy therapeutic window. The simulations showed that therapeutic dose ranges for TV-46000 are 50 to 125 mg for once-monthly and 100 to 250 mg for the once every 2 months regimens. This PPK model provided a basis for prediction of patient-specific exposure and dopamine-D2 receptor occupancy estimates to support further clinical development and dose selection for the phase 3 studies.

The dynamic regulation of autophagy in β-cells by cycles of fasting-feeding and its effects on insulin secretion are unknown. In β-cells, mechanistic target of rapamycin complex 1 (mTORC1) is inhibited while fasting and is rapidly stimulated during refeeding by a single amino acid, leucine, and glucose. Stimulation of mTORC1 by nutrients inhibited the autophagy initiator ULK1 and the transcription factor TFEB, thereby preventing autophagy when β-cells were continuously exposed to nutrients. Inhibition of mTORC1 by Raptor knockout mimicked the effects of fasting and stimulated autophagy while inhibiting insulin secretion, whereas moderate inhibition of autophagy under these conditions rescued insulin secretion. These results show that mTORC1 regulates insulin secretion through modulation of autophagy under different nutritional situations. In the fasting state, autophagy is regulated in an mTORC1-dependent manner, and its stimulation is required to keep insulin levels low, thereby preventing hypoglycemia. Reciprocally, stimulation of mTORC1 by elevated leucine and glucose, which is common in obesity, may promote hyperinsulinemia by inhibiting autophagy.

Introduction: Understanding drug exposure at disease target sites is pivotal to profiling new drug candidates in terms of tolerability and efficacy. Such quantification is particularly tedious for anti-tuberculosis (TB) compounds as the heterogeneous pulmonary microenvironment due to the infection may alter lung permeability and affect drug disposition. Murine models have been a longstanding support in TB research so far and are here used as human surrogates to unveil the distribution of several anti-TB compounds at the site-of-action via a novel and centralized PBPK design framework. Methods: As an intermediate approach between data-driven pharmacokinetic (PK) models and whole-body physiologically based (PB) PK models, we propose a parsimonious framework for PK investigation (minimal PBPK approach) that retains key physiological processes involved in TB disease, while reducing computational costs and prior knowledge requirements. By lumping together pulmonary TB-unessential organs, our minimal PBPK model counts 9 equations compared to the 36 of published full models, accelerating the simulation more than 3-folds in Matlab 2022b. Results: The model has been successfully tested and validated against 11 anti-TB compounds-rifampicin, rifapentine, pyrazinamide, ethambutol, isoniazid, moxifloxacin, delamanid, pretomanid, bedaquiline, OPC-167832, GSK2556286 - showing robust predictability power in recapitulating PK dynamics in mice. Structural inspections on the proposed design have ensured global identifiability and listed free fraction in plasma and blood-to-plasma ratio as top sensitive parameters for PK metrics. The platform-oriented implementation allows fast comparison of the compounds in terms of exposure and target attainment. Discrepancies in plasma and lung levels for the latest BPaMZ and HPMZ regimens have been analyzed in terms of their impact on preclinical experiment design and on PK/PD indices. Conclusion: The framework we developed requires limited drug- and species-specific information to reconstruct accurate PK dynamics, delivering a unified viewpoint on anti-TB drug distribution at the site-of-action and a flexible fit-for-purpose tool to accelerate model-informed drug design pipelines and facilitate translation into the clinic.

We have recently generated lipophilic D-xylose derivatives that increase the rate of glucose uptake in cultured skeletal muscle cells in an AMP-activated protein kinase (AMPK)-dependent manner. The derivative 2,4:3,5-dibenzylidene-D-xylose-diethyl dithioacetal (EH-36) stimulated the rate of glucose transport by increasing the abundance of glucose transporter-4 in the plasma membrane of cultured myotubes. The present study aimed at investigating potential antihyperglycaemic effects of EH-36 in animal models of diabetes. Two animal models were treated subcutaneously with EH-36: streptozotocin-induced diabetes in C57BL/6 mice (a model of insulin-deficient type 1 diabetes), and spontaneously diabetic KKAy mice (Kuo Kondo rats carrying the A(y) yellow obese gene; insulin-resistant type 2 diabetes). The in vivo biodistribution of glucose in control and treated mice was followed with the glucose analogue 2-deoxy-2-[(18) F]-D-glucose; the rate of glucose uptake in excised soleus muscles was measured with [(3) H]-2-deoxy-D-glucose. Pharmacokinetic parameters were determined by non-compartmental analysis of the in vivo data. The effective blood EH-36 concentration in treated animals was 2 μM. It reduced significantly the blood glucose levels in both types of diabetic mice and also corrected the typical compensatory hyperinsulinaemia of KKAy mice. EH-36 markedly increased glucose transport in vivo into skeletal muscle and heart, but not to adipose tissue. This stimulatory effect was mediated by Thr(172) -phosphorylation in AMPK. Biochemical tests in treated animals and acute toxicological examinations showed that EH-36 was well tolerated and not toxic to the mice. These findings indicate that EH-36 is a promising prototype molecule for the development of novel antidiabetic drugs.

Thioredoxin-interacting protein (TXNIP) regulates critical biological processes including inflammation, stress and apoptosis. TXNIP is upregulated by glucose and is a critical mediator of hyperglycemia-induced beta-cell apoptosis in diabetes. In contrast, the saturated long-chain fatty acid palmitate, although toxic to the beta-cell, inhibits TXNIP expression. The mechanisms involved in the opposing effects of glucose and fatty acids on TXNIP expression are unknown. We found that both palmitate and oleate inhibited TXNIP in a rat beta-cell line and islets. Palmitate inhibition of TXNIP was independent of fatty acid beta-oxidation or esterification. AMP-activated protein kinase (AMPK) has an important role in cellular energy sensing and control of metabolic homeostasis; therefore we investigated its involvement in nutrient regulation of TXNIP. As expected, glucose inhibited whereas palmitate stimulated AMPK. Pharmacologic activators of AMPK mimicked fatty acids by inhibiting TXNIP. AMPK knockdown increased TXNIP expression in presence of high glucose with and without palmitate, indicating that nutrient (glucose and fatty acids) effects on TXNIP are mediated in part via modulation of AMPK activity. TXNIP is transcriptionally regulated by carbohydrate response element-binding protein (ChREBP). Palmitate inhibited glucose-stimulated ChREBP nuclear entry and recruitment to the Txnip promoter, thereby inhibiting Txnip transcription. We conclude that AMPK is an important regulator of Txnip transcription via modulation of ChREBP activity. The divergent effects of glucose and fatty acids on TXNIP expression result in part from their opposing effects on AMPK activity. In light of the important role of TXNIP in beta-cell apoptosis, its inhibition by fatty acids can be regarded as an adaptive/protective response to glucolipotoxicity. The finding that AMPK mediates nutrient regulation of TXNIP may have important implications for the pathophysiology and treatment of diabetes.

Diabetic kidney disease (DKD) increases the risk for mortality and is the leading cause of end-stage renal disease. Treatment with sodium-glucose cotransporter 2 inhibitors (SGLT2i) attenuates the progression of DKD, especially in patients with advanced kidney disease. Herein, we show that in diabetes, mTORC1 activity is increased in renal proximal tubule cells (RPTCs) along with enhanced tubule-interstitial fibrosis; this is prevented by SGLT2i. Constitutive activation of mTORC1 in RPTCs induces renal fibrosis and failure and abolishes the renal-protective effects of SGLT2i in diabetes. On the contrary, partial inhibition of mTORC1 in RPTCs prevents fibrosis and the decline in renal function. Stimulation of mTORC1 in RPTCs turns on a pro-fibrotic program in the renal cortex, whereas its inhibition in diabetes reverses the alterations in gene expression. We suggest that RPTC mTORC1 is a critical node that mediates kidney dysfunction in diabetes and the protective effects of SGLT2i by regulating fibrogenesis.

Diabetes is associated with increased risk for kidney disease, heart failure, and mortality. Sodium-glucose cotransporter 2 inhibitors (SGLT2i) prevent these adverse outcomes; however, the mechanisms involved are not clear. We generated a roadmap of the metabolic alterations that occur in different organs in diabetes and in response to SGLT2i. In vivo metabolic labeling with 13C-glucose in normoglycemic and diabetic mice treated with or without dapagliflozin, followed by metabolomics and metabolic flux analyses, showed that, in diabetes, glycolysis and glucose oxidation are impaired in the kidney, liver, and heart. Treatment with dapagliflozin failed to rescue glycolysis. SGLT2 inhibition increased glucose oxidation in all organs; in the kidney, this was associated with modulation of the redox state. Diabetes was associated with altered methionine cycle metabolism, evident by decreased betaine and methionine levels, whereas treatment with SGLT2i increased hepatic betaine along with decreased homocysteine levels. mTORC1 activity was inhibited by SGLT2i along with stimulation of AMPK in both normoglycemic and diabetic animals, possibly explaining the protective effects against kidney, liver, and heart diseases. Collectively, our findings suggest that SGLT2i induces metabolic reprogramming orchestrated by AMPK-mTORC1 signaling with common and distinct effects in various tissues, with implications for diabetes and aging.

Unresolved ER stress followed by cell death is recognized as the main cause of a multitude of pathologies including neonatal diabetes. A systematic analysis of the mechanisms of β-cell loss and dysfunction in Akita mice, in which a mutation in the proinsulin gene causes a severe form of permanent neonatal diabetes, showed no increase in β-cell apoptosis throughout life. Surprisingly, we found that the main mechanism leading to β-cell dysfunction is marked impairment of β-cell growth during the early postnatal life due to transient inhibition of mTORC1, which governs postnatal β-cell growth and differentiation. Importantly, restoration of mTORC1 activity in neonate β-cells was sufficient to rescue postnatal β-cell growth, and to improve diabetes. We propose a scenario for the development of permanent neonatal diabetes, possibly also common forms of diabetes, where early-life events inducing ER stress affect β-cell mass expansion due to mTOR inhibition.

Background Omacetaxine mepesuccinate is indicated in adults with chronic myeloid leukemia resistant and/or intolerant to ≥ 2 tyrosine kinase inhibitor treatments. This phase I study assessed the disposition, elimination, and safety of (14)C-omacetaxine in patients with solid tumors. Methods The study comprised a 7-days pharmacokinetic assessment followed by a treatment period of ≤ six 28-days cycles. A single subcutaneous dose of 1.25 mg/m(2) (14)C-omacetaxine was administered to six patients. Blood, urine, and feces were collected through 168 h or until radioactivity excreted within 24 h was <1 % of the dose. Total radioactivity (TRA) was measured in all matrices and concentrations of omacetaxine, 4'-desmethylhomoharringtonine (4'-DMHHT), and cephalotaxine were measured in plasma and urine. For each treatment cycle, patients received 1.25 mg/m(2) omacetaxine twice daily for 7 days. Results Mean TRA recovered was approximately 81 % of the dose, with approximately half of the radioactivity recovered in feces and half in urine. Approximately 20 % of the dose was excreted unchanged in urine; cephalotaxine (0.4 % of dose) and 4' DMHHT (9 %) were also present. Plasma concentrations of TRA were higher than the sum of omacetaxine and known metabolites, suggesting the presence of other (14)C-omacetaxine-derived compounds. Fatigue and anemia were common, consistent with the known toxicity profile of omacetaxine. Conclusion Renal and hepatic processes contribute to the elimination of (14)C-omacetaxine-derived radioactivity in cancer patients. In addition to omacetaxine and its known metabolites, other (14)C-omacetaxine-derived materials appear to be present in plasma and urine. Omacetaxine was adequately tolerated, with no new safety signals.

Laquinimod is an oral immunomodulator in clinical development to treat relapsing-remitting multiple sclerosis (RRMS). Laquinimod is in clinical development for the treatment of multiple sclerosis and Huntington Disease (HD). The objective of this study is to assess the safety, tolerability, pharmacokinetics (PK) and cytoimmunologic effects following escalating doses of laquinimod in patients with RRMS.