Development of poly(lactide-co-glycolide) microparticles for sustained delivery of meloxicam.

Poly(lactide-co-glycolide) (PLGA) polymers have been widely used for drug delivery due to their biodegradability and biocompatibility. One of the objectives of encapsulating a drug in PLGA microparticles (MPs) is to achieve an extended supply of the drug through sustained release, which can range from weeks to months. Focusing on the applications needing a relatively short-term delivery, we investigated formulation strategies to achieve a drug release from PLGA MPs for two weeks, using meloxicam as a model compound. PLGA MPs produced by the traditional oil/water (O/W) single emulsion method showed only an initial burst release with minimal increase in later-phase drug release. Alternatively, encapsulating meloxicam as solid helped reduce the initial burst release. The inclusion of magnesium hydroxide [Mg(OH)2] enhanced later-phase drug release by neutralizing the developing acidity that limited the drug dissolution. The variation of solid meloxicam and Mg(OH)2 quantities allowed for flexible control of meloxicam release, yielding MPs with distinct in vitro release kinetics. When subcutaneously injected into rats, the MPs with relatively slow in vitro drug release kinetics showed in vivo drug absorption profiles consistent with in vitro trend. However, the MPs that rapidly released meloxicam showed an attenuated in vivo absorption, suggesting premature precipitation of fast-released meloxicam. In summary, this study demonstrated the feasibility of controlling drug release from the PLGA MPs over weeks based on the physical state of the encapsulated drug and the inclusion of Mg(OH)2 to neutralize the microenvironmental pH of the MPs.

Photoacoustic imaging reveals mechanisms of rapid-acting insulin formulations dynamics at the injection site.

OBJECTIVE: Ultra-rapid insulin formulations control postprandial hyperglycemia; however, inadequate understanding of injection site absorption mechanisms is limiting further advancement. We used photoacoustic imaging to investigate the injection site dynamics of dye-labeled insulin lispro in the Humalog® and Lyumjev® formulations using the murine ear cutaneous model and correlated it with results from unlabeled insulin lispro in pig subcutaneous injection model. METHODS: We employed dual-wavelength optical-resolution photoacoustic microscopy to study the absorption and diffusion of the near-infrared dye-labeled insulin lispro in the Humalog and Lyumjev formulations in mouse ears. We mathematically modeled the experimental data to calculate the absorption rate constants and diffusion coefficients. We studied the pharmacokinetics of the unlabeled insulin lispro in both the Humalog and Lyumjev formulations as well as a formulation lacking both the zinc and phenolic preservative in pigs. The association state of insulin lispro in each of the formulations was characterized using SV-AUC and NMR spectroscopy. RESULTS: Through experiments using murine and swine models, we show that the hexamer dissociation rate of insulin lispro is not the absorption rate-limiting step. We demonstrated that the excipients in the Lyumjev formulation produce local tissue expansion and speed both insulin diffusion and microvascular absorption. We also show that the diffusion of insulin lispro at the injection site drives its initial absorption; however, the rate at which the insulin lispro crosses the blood vessels is its overall absorption rate-limiting step. CONCLUSIONS: This study provides insights into injection site dynamics of insulin lispro and the impact of formulation excipients. It also demonstrates photoacoustic microscopy as a promising tool for studying protein therapeutics. The results from this study address critical questions around the subcutaneous behavior of insulin lispro and the formulation excipients, which could be useful to make faster and better controlled insulin formulations in the future.

Development of hot-melt extruded drug/polymer matrices for sustained delivery of meloxicam.

For effective resolution of regional subacute inflammation and prevention of biofouling formation, we have developed a polymeric implant that can release meloxicam, a selective cyclooxygenase (COX)-2 inhibitor, in a sustained manner. Meloxicam-loaded polymer matrices were produced by hot-melt extrusion, with commercially available biocompatible polymers, poly(ε-caprolactone) (PCL), poly(lactide-co-glycolide) (PLGA), and poly(ethylene vinyl acetate) (EVA). PLGA and EVA had a limited control over the drug release rate partly due to the acidic microenvironment and hydrophobicity, respectively. PCL allowed for sustained release of meloxicam over two weeks and was used as a carrier of meloxicam. Solid-state and image analyses indicated that the PCL matrices encapsulated meloxicam in crystalline clusters, which dissolved in aqueous medium and generated pores for subsequent drug release. The subcutaneously implanted meloxicam-loaded PCL matrices in rats showed pharmacokinetic profiles consistent with their in vitro release kinetics, where higher drug loading led to faster drug release. This study finds that the choice of polymer platform is crucial to continuous release of meloxicam and the drug release rate can be controlled by the amount of drug loaded in the polymer matrices.

The dual glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1 receptor agonist tirzepatide improves cardiovascular risk biomarkers in patients with type 2 diabetes: A post hoc analysis.

In a phase 2 trial of once-weekly tirzepatide (1, 5, 10, or 15 mg), dulaglutide (1.5 mg), or placebo, the dual glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1 receptor agonist tirzepatide dose-dependently reduced HbA1c and body weight in patients with type 2 diabetes. In this post hoc analysis, inflammation, endothelial dysfunction, and cellular stress biomarkers were measured at baseline, 4, 12, and 26 weeks to evaluate the additional effects of tirzepatide on cardiovascular risk factors. At 26 weeks, tirzepatide 10 and 15 mg decreased YKL-40 (also known as chitinase-3 like-protein-1), intercellular adhesion molecule 1 (ICAM-1), leptin, and growth differentiation factor 15 levels versus baseline, and YKL-40 and leptin levels versus placebo and dulaglutide. Tirzepatide 15 mg also decreased ICAM-1 levels versus placebo and dulaglutide, and high-sensitivity C-reactive protein (hsCRP) levels versus baseline and placebo, but not dulaglutide. GlycA, interleukin 6, vascular cell adhesion molecule 1, and N-terminal-pro hormone B-type natriuretic peptide levels were not significantly changed in any group. YKL-40, hsCRP, and ICAM-1 levels rapidly decreased within 4 weeks of treatment with tirzepatide 10 and 15 mg, whereas the decrease in leptin levels was more gradual and did not plateau by 26 weeks. In this hypothesis-generating exploratory analysis, tirzepatide decreased several biomarkers that have been associated with cardiovascular risk.

Insulin, Not the Preservative m-cresol, Instigates Loss of Infusion Site Patency Over Extended Durations of CSII in Diabetic Swine.

Insulin infusion sets worn for more than 4-5 days have been associated with a greater risk of unexplained hyperglycemia, a phenomenon that has been hypothesized to be caused by an inflammatory response to preservatives such as m-cresol and phenol. In this cross-over study in diabetic swine, we examined the role of the preservative m-cresol in inflammation and changes in infusion site patency. Insulin pharmacokinetics (PK) and glucose pharmacodynamics (PD) were measured on delivery of a bolus of regular human insulin U-100 (U-100R), formulated with or without 2.5 mg/mL m-cresol, to fasted swine following 0, 3, 5, 7, and 10 days of continuous subcutaneous insulin infusion (CSII). In a subsequent study with the same animals, biopsies were evaluated from swine wearing infusion sets infusing nothing, saline, or U-100R either with or without 2.5 mg/mL m-cresol, following 3, 7, and 10 days of CSII. Exposure to m-cresol did not impact any PK or PD endpoints. PK and PD responses dropped markedly from Days 7-10, regardless of the presence of m-cresol. Histopathology results suggest an additive inflammatory response to both the infusion set and the insulin protein itself, peaking at Day 7 and remaining stable beyond.

ß-Cell Glucagon-Like Peptide-1 Receptor Contributes to Improved Glucose Tolerance After Vertical Sleeve Gastrectomy.

Vertical sleeve gastrectomy (VSG) produces high rates of type 2 diabetes remission; however, the mechanisms responsible for this remain incompletely defined. Glucagon-like peptide-1 (GLP-1) is a gut hormone that contributes to the maintenance of glucose homeostasis and is elevated after VSG. VSG-induced increases in postprandial GLP-1 secretion have been proposed to contribute to the glucoregulatory benefits of VSG; however, previous work has been equivocal. In order to test the contribution of enhanced ß-cell GLP-1 receptor (GLP-1R) signaling we used a ß-cell-specific tamoxifen-inducible GLP-1R knockout mouse model. Male ß-cell-specific Glp-1r(ß-cell+/+) wild type (WT) and Glp-1r(ß-cell-/-) knockout (KO) littermates were placed on a high-fat diet for 6 weeks and then switched to high-fat diet supplemented with tamoxifen for the rest of the study. Mice underwent sham or VSG surgery after 2 weeks of tamoxifen diet and were fed ad libitum postoperatively. Mice underwent oral glucose tolerance testing at 3 weeks and were euthanized at 6 weeks after surgery. VSG reduced body weight and food intake independent of genotype. However, glucose tolerance was only improved in VSG WT compared with sham WT, whereas VSG KO had impaired glucose tolerance relative to VSG WT. Augmentation of glucose-stimulated insulin secretion during the oral glucose tolerance test was blunted in VSG KO compared with VSG WT. Therefore, our data suggest that enhanced ß-cell GLP-1R signaling contributes to improved glucose regulation after VSG by promoting increased glucose-stimulated insulin secretion.

Characterization and quantification of oxyntomodulin in human and rat plasma using high-resolution accurate mass LC-MS.

BACKGROUND: A thorough understanding of the biological role of oxyntomodulin (OXM) has been limited by the availability of sensitive and specific analytical tools for reliable in vivo characterization. Here, we utilized immunoaffinity capture coupled with high-resolution accurate mass LC-MS detection to quantify OXM and its primary catabolites. RESULTS: Quantification of intact OXM 1-37 in human and rat plasma occurred in pre- and post-prandial samples. Profiles for the major catabolites were observed allowing kinetic differences to be assessed between species. CONCLUSION: A validated assay in human and rat plasma was obtained for OXM 1-37 and its catabolites, 3-37 and 4-37. The value of full scan high-resolution accurate mass detection without selected reaction monitoring for low-abundance peptide quantification was also demonstrated.

Novel small molecule glucagon-like peptide-1 receptor agonist stimulates insulin secretion in rodents and from human islets.

OBJECTIVE: The clinical effectiveness of parenterally-administered glucagon-like peptide-1 (GLP-1) mimetics to improve glucose control in patients suffering from type 2 diabetes strongly supports discovery pursuits aimed at identifying and developing orally active, small molecule GLP-1 receptor agonists. The purpose of these studies was to identify and characterize novel nonpeptide agonists of the GLP-1 receptor. RESEARCH DESIGN AND METHODS: Screening using cells expressing the GLP-1 receptor and insulin secretion assays with rodent and human islets were used to identify novel molecules. The intravenous glucose tolerance test (IVGTT) and hyperglycemic clamp characterized the insulinotropic effects of compounds in vivo. RESULTS: Novel low molecular weight pyrimidine-based compounds that activate the GLP-1 receptor and stimulate glucose-dependent insulin secretion are described. These molecules induce GLP-1 receptor-mediated cAMP signaling in HEK293 cells expressing the GLP-1 receptor and increase insulin secretion from rodent islets in a dose-dependent manner. The compounds activate GLP-1 receptor signaling, both alone or in an additive fashion when combined with the endogenous GLP-1 peptide; however, these agonists do not compete with radiolabeled GLP-1 in receptor-binding assays. In vivo studies using the IVGTT and the hyperglycemic clamp in Sprague Dawley rats demonstrate increased insulin secretion in compound-treated animals. Further, perifusion assays with human islets isolated from a donor with type 2 diabetes show near-normalization of insulin secretion upon compound treatment. CONCLUSIONS: These studies characterize the insulinotropic effects of an early-stage, small molecule GLP-1 receptor agonist and provide compelling evidence to support pharmaceutical optimization.

Specific reduction of hepatic glucose 6-phosphate transporter-1 ameliorates diabetes while avoiding complications of glycogen storage disease.

D-Glucose-6-phosphatase is a key regulator of endogenous glucose production, and its inhibition may improve glucose control in type 2 diabetes. Herein, 2'-O-(2-methoxy)ethyl-modified phosphorothioate antisense oligonucleotides (ASOs) specific to the glucose 6-phosphate transporter-1 (G6PT1) enabled reduction of hepatic D-Glu-6-phosphatase activity in diabetic ob/ob mice. Treatment with G6PT1 ASOs decreased G6PT1 expression, reduced G6PT1 activity, blunted glucagon-stimulated glucose production, and lowered plasma glucose concentration in a dose-dependent manner. In contrast to G6PT1 knock-out mice and patients with glycogen storage disease, excess hepatic and renal glycogen accumulation, hyperlipidemia, neutropenia, and elevations in plasma lactate and uric acid did not occur. In addition, hypoglycemia was not observed in animals during extended periods of fasting, and the ability of G6PT1 ASO-treated mice to recover from an exogenous insulin challenge was not impaired. Together, these results demonstrate that effective glucose lowering by G6PT1 inhibitors can be achieved without adversely affecting carbohydrate and lipid metabolism.

Farnesoid X receptor agonist reduces serum asymmetric dimethylarginine levels through hepatic dimethylarginine dimethylaminohydrolase-1 gene regulation.

The farnesoid X receptor (FXR, NR1H4) is a bile acid-responsive nuclear receptor that plays critical roles in the transcriptional regulation genes involved in cholesterol, bile acid, triglyceride, and carbohydrate metabolism. By microarray analysis of hepatic genes from female Zucker diabetic fatty (ZDF) rats treated with the FXR agonist GW4064, we have identified dimethylarginine dimethylaminohydrolase-1 (DDAH1) as an FXR target gene. DDAH1 is a key catabolic enzyme of asymmetric dimethylarginine (ADMA), a major endogenous nitric-oxide synthase inhibitor. Sequence analysis of the DDAH1 gene reveals the presence of an FXR response element (FXRE) located 90 kb downstream of the transcription initiation site and within the first intron. Functional analysis of the putative FXRE demonstrated GW4064 dose-dependent transcriptional activation from the element, and we have demonstrated that the FXRE sequence binds the FXR-RXR heterodimer. In vivo administration of GW4064 to female ZDF rats promoted a dose-dependent and >6-fold increase in hepatic DDAH1 gene expression. The level of serum ADMA was reduced concomitantly. These findings provide a mechanism by which FXR may increase endothelium-derived nitric oxide levels through modulation of serum ADMA levels via direct regulation of hepatic DDAH1 gene expression. Thus, beneficial clinical outcomes of FXR agonist therapy may include prevention of atherosclerosis and improvement of the metabolic syndrome.

Reduction of hepatic and adipose tissue glucocorticoid receptor expression with antisense oligonucleotides improves hyperglycemia and hyperlipidemia in diabetic rodents without causing systemic glucocorticoid antagonism.

Glucocorticoids (GCs) increase hepatic gluconeogenesis and play an important role in the regulation of hepatic glucose output. Whereas systemic GC inhibition can alleviate hyperglycemia in rodents and humans, it results in adrenal insufficiency and stimulation of the hypothalamic-pituitary-adrenal axis. In the present study, we used optimized antisense oligonucleotides (ASOs) to cause selective reduction of the glucocorticoid receptor (GCCR) in liver and white adipose tissue (WAT) and evaluated the resultant changes in glucose and lipid metabolism in several rodent models of diabetes. Treatment of ob/ob mice with GCCR ASOs for 4 weeks resulted in approximately 75 and approximately 40% reduction in GCCR mRNA expression in liver and WAT, respectively. This was accompanied by approximately 65% decrease in fed and approximately 30% decrease in fasted glucose levels, a 60% decrease in plasma insulin concentration, and approximately 20 and 35% decrease in plasma resistin and tumor necrosis factor-alpha levels, respectively. Furthermore, GCCR ASO reduced hepatic glucose production and inhibited hepatic gluconeogenesis in liver slices from basal and dexamethasone-treated animals. In db/db mice, a similar reduction in GCCR expression caused approximately 40% decrease in fed and fasted glucose levels and approximately 50% reduction in plasma triglycerides. In ZDF and high-fat diet-fed streptozotocin-treated (HFD-STZ) rats, GCCR ASO treatment caused approximately 60% reduction in GCCR expression in the liver and WAT, which was accompanied by a 40-70% decrease in fasted glucose levels and a robust reduction in plasma triglyceride, cholesterol, and free fatty acids. No change in circulating corticosterone levels was seen in any model after GCCR ASO treatment. To further demonstrate that GCCR ASO does not cause systemic GC antagonism, normal Sprague-Dawley rats were challenged with dexamethasone after treating with GCCR ASO. Dexamethasone increased the expression of GC-responsive genes such as PEPCK in the liver and decreased circulating lymphocytes. GCCR ASO treatment completely inhibited the increase in dexamethasone-induced PEPCK expression in the liver without causing any change in the dexamethasone-induced lymphopenia. These studies demonstrate that tissue-selective GCCR antagonism with ASOs may be a viable therapeutic strategy for the treatment of the metabolic syndrome.

Hepatic and glucagon-like peptide-1-mediated reversal of diabetes by glucagon receptor antisense oligonucleotide inhibitors.

Uncontrolled hepatic glucose production contributes significantly to hyperglycemia in patients with type 2 diabetes. Hyperglucagonemia is implicated in the etiology of this condition; however, effective therapies to block glucagon signaling and thereby regulate glucose metabolism do not exist. To determine the extent to which blocking glucagon action would reverse hyperglycemia, we targeted the glucagon receptor (GCGR) in rodent models of type 2 diabetes using 2'-methoxyethyl-modified phosphorothioate-antisense oligonucleotide (ASO) inhibitors. Treatment with GCGR ASOs decreased GCGR expression, normalized blood glucose, improved glucose tolerance, and preserved insulin secretion. Importantly, in addition to decreasing expression of cAMP-regulated genes in liver and preventing glucagon-mediated hepatic glucose production, GCGR inhibition increased serum concentrations of active glucagon-like peptide-1 (GLP-1) and insulin levels in pancreatic islets. Together, these studies identify a novel mechanism whereby GCGR inhibitors reverse the diabetes phenotype by the dual action of decreasing hepatic glucose production and improving pancreatic beta cell function.

Hybrid insulin cocrystals for controlled release delivery.

The ability to tailor the release profile of a drug by manipulating its formulation matrix offers important therapeutic advantages. We show here that human insulin can be cocrystallized at preselected ratios with the fully active lipophilically modified insulin derivative octanoyl-N(epsilon)-LysB29-human insulin (C8-HI). The cocrystal is analogous to the NPH (neutral protamine Hagedorn) crystalline complex formed with human insulin, which is commonly used as the long-acting insulin component of diabetes therapy. The in vitro and in vivo release rates of the cocrystal can be controlled by adjusting the relative proportions of the two insulin components. We identified a cocrystal composition comprising 75% C8-HI and 25% human insulin that exhibits near-ideal basal pharmacodynamics in somatostatin-treated beagle dogs. The dependence of release rate on cocrystal ratio provides a robust mechanism for modulating insulin pharmacodynamics. These findings show that a crystalline protein matrix may accommodate a chemical modification that alters the dissolution rate of the crystal in a therapeutically useful way, yet that is structurally innocuous enough to preserve the pharmaceutical integrity of the original microcrystalline entity and the pharmacological activity of the parent molecule.