Nanotechnology as a promising strategy for alternative routes of insulin delivery.

Since its discovery, insulin has been used as highly specific and effective therapeutic protein to treat type 1 diabetes and later was associated to oral antidiabetic agents in the treatment of type 2 diabetes. Generally, insulin is administered parenterally. Although this route is successful, it still has several limitations, such as discomfort, pain, lipodystrophy at the injection sites and peripheral hyperinsulinemia, which may be the cause of side effects and some complications. Thus, alternative routes of administration have been developed, namely, those based on nanotechnologies. Nanoparticles, made of synthetic or natural materials, have been shown to successfully overcome the inherent barriers for insulin stability, degradation, and uptake across the gastrointestinal tract and other mucosal membranes. This review describes some of the many attempts made to develop alternative and more convenient routes for insulin delivery.

Effects of an oral insulin nanoparticle administration on hepatic glucose metabolism assessed by ¹³C and ²H isotopomer analysis.

The purpose of this study was to evaluate hepatic glucose metabolism of diabetic induced rats after a daily oral load of insulin nanoparticles over 2 weeks. After the 2-week treatment, an oral glucose tolerance test was performed with [U-¹³C] glucose and ²H2O. Plasma glucose ²H and ¹³C enrichments were quantified and the contribution of glycogenolysis and gluconeogenesis to overall glucose production were estimated. Animals with the insulin nanoparticles displayed the lowest glycemia before the oral glucose tolerance test. In all animals, 75% of the total glucose production was from gluconeogenesis and glycogen synthesis was only detected in some animals. Gluconeogenic pathway was an active contributor to hepatic glucose production and the treatment with oral delivered insulin nanoparticles did not alter this contribution, suggesting that under this treatment, protocol hepatic glucose metabolism is not the most relevant target of insulin action but instead a more generalised effect in peripheral tissues.

Poly(epsilon-caprolactone)/eudragit nanoparticles for oral delivery of aspart-insulin in the treatment of diabetes.

Nanoparticles prepared with a blend of a biodegradable polyester (poly(epsilon-caprolactone)) and a polycationic nonbiodegradable acrylic polymer (Eudragit RS) have been used as a drug carrier for oral administration of a short-acting insulin analogue, aspart-insulin. Insulin-loaded nanoparticles, about 700 nm in diameter, encapsulated 97.5% of insulin and were able to release about 70% of their content in vitro in a neutral medium over 24 h. When administered orally to diabetic rats, insulin-loaded nanoparticles (50 IU/kg) decreased fasted glycemia for a prolonged period of time and improved the glycemic response to glucose in a time-dependent manner, with a maximal effect between 12 and 24 h after their administration. In parallel, plasma insulin levels increased. However, higher (100 IU/kg) and lower (25 IU/kg) doses of insulin did not exert any biological effect. It is concluded that polymeric nanoparticles composed of poly(epsilon-caprolactone)/Eudragit RS are able to preserve the biological activity of the insulin analogue aspart-insulin; however, the postprandial peak suppression was prolonged more than 24 h by comparison with regular insulin working only 6-8 h. This effect may be explained by the monomeric configuration of aspart-insulin, which is probably better taken up by the intestinal mucosa than regular insulin.

Nanoparticulate biopolymers deliver insulin orally eliciting pharmacological response.

The aim of this study was to characterize and evaluate a novel oral insulin nanoparticulate system based on alginate-dextran sulfate core, complexed with a chitosan-polyethylene glycol-albumin shell. Insulin-loaded nanospheres (25, 50, 100 IU/kg) administered orally to diabetic rats reduced glycemia in a dose dependent manner. This effect lasted over 24 h with a maximal effect after 14 h. Nanospheres increased insulin plasma level and improved glycemic response to an oral glucose overload. After 4 days oral administration (50 IU/kg/day), the metabolic status of diabetic rats improved with a reduction in water intake, urine excretion and proteinuria. FITC-insulin-loaded nanospheres administered to an isolated intestinal loop were taken up by the intestinal mucosa. They strongly adhered to villus apical enterocytes and markedly labeled Peyer's patches. It is concluded that nanospheres preserve insulin and exert an antidiabetic effect after oral administration. This is explained by a protective effect against proteolytic enzymes by the albumin coating, by the mucoadhesive properties of chitosan-polyethylene glycol, and by the possibility of chitosan reversibly altering tight junctions leading to an improved absorption of insulin. This formulation demonstrates beneficial effects on diabetic symptoms and will be of interest in the treatment of diabetes with oral insulin.

Polyelectrolyte biomaterial interactions provide nanoparticulate carrier for oral insulin delivery.

Nanospheres are being developed for the oral delivery of peptide-based drugs such as insulin. Mucoadhesive, biodegradable, biocompatible, and acid-protective biomaterials are described using a combination of natural polyelectrolytes, with particles formulated through nanoemulsion dispersion followed by triggered in situ gel complexation. Biomaterials meeting these criteria include alginate, dextran, chitosan, and albumin in which alginate/dextran forms the core matrix complexed with chitosan and albumin coat. Smaller size and higher albumin-based acid-protective formulation was orally administered to diabetic rats and glucose reduction and physiological response analyzed. Insulin encapsulation efficiency was 90, 82, and 66% for uncoated, chitosan-coated, and albumin-chitosan-coated alginate nanospheres, respectively. The choice of coating polymer seems to influence insulin release profile and to be crucial to prevent peptic digestion. Physiological response following oral delivery showed that insulin albumin-chitosan-coated alginate nanospheres reduced glycemia approximately 72% of basal values. Albumin serves as an important enteric coating providing acid- and protease protection enabling uptake of active drug following oral dosage.

Nanoparticle strategies for the oral delivery of insulin.

Since its discovery, insulin remains the major treatment for Type 1 diabetes and many Type 2 diabetic patients, with insulin being administered parenterally. The oral route of insulin delivery, being the most comfortable, would also be the most physiologically advantageous in taking advantage of the portal-hepatic route of absorption. However, insulin is less absorbed by the intestinal mucosa and is rapidly degraded enzymatically in the gastrointestinal tract. Polymeric biodegradable and biocompatible nanoparticles have been developed. These nanoparticles protect insulin against degradation and facilitate the uptake of insulin (associated or not associated to the nanoparticles) through a paracellular or a transcellular pathway. In this review, the physicochemical characteristics of polymer composition, in vitro release kinetics and the biological effects of insulin-loaded nanoparticles in experimental diabetes and healthy animals are discussed.

Effect of angiotensin-converting enzyme inhibition on skeletal muscle oxidative function and exercise capacity in streptozotocin-induced diabetic rats.

Since exercise capacity is related to the mitochondrial respiration rate in skeletal muscle and both parameters are potentially modulated by the onset of diabetes and by inhibition of the angiotensin-converting enzyme (ACE), we investigated whether skeletal muscle oxidative functions and exercise capacities are impaired in chronic streptozotocin-induced diabetic (STZ) rats and whether ACE inhibition could reverse such abnormalities. The ACE inhibitor perindopril (2 mg kg(-1) day(-1)) was given for a period of 5 weeks to 7-month-old STZ rats (DIA-PE, n = 8) whose haemodynamic function, skeletal muscle mitochondrial function and exercise capacity were compared with those of untreated diabetic (DIA, n = 8) and control rats (CONT, n = 8). Increased arterial blood pressure (157 +/- 12 versus 130 +/- 6 mmHg, P < 0.05) and reduced exercise capacity (29 +/- 2 versus 91 +/- 2 min, respectively, P < 0.01) were observed in DIA compared with CONT. The oxidative capacity of the gastrocnemius muscle was significantly reduced in DIA compared with CONT rats (5.4 +/- 0.5 versus 10.6 +/- 0.7 micromol O(2) min(-1)(g dry weight)(-1), respectively, P < 0.001). Moreover, the coupling between oxidation and phosphorylation was significantly impaired in DIA (-52%, P < 0.001). Angiotensin-converting enzyme inhibition (ACEi) normalized blood pressure without improving mitochondrial function (4.3 +/- 0.8 micromol O(2) min(-1) (g dry weight)(-1) in DIA-PE rats) but reduced exercise capacity to even lower levels (10 +/- 1 min, P < 0.01). Exercise capacity correlated positively with blood pressure in DIA-PE (r = 0.79, P < 0.05). In experimental type 1 diabetic rats, both skeletal muscle mitochondrial respiration and exercise capacity are impaired. The ACEi failed to restore the muscular function and worsened exercise capacity. Further studies will be useful to determine whether an inadequate muscular blood flow secondary to the reduction in mean systemic blood pressure can explain these results.

Oral delivery of insulin associated to polymeric nanoparticles in diabetic rats.

Nanoparticles prepared with a blend of a biodegradable polyester (poly(-epsilon-caprolactone)) and a polycationic non-biodegradable acrylic polymer (Eudragit RS) have been used as a drug carrier for oral administration of insulin. The rate of encapsulation of insulin was around 96%. The therapeutic efficiency of oral insulin nanoparticles (25, 50 and 100 IU/kg) in diabetic rats and the intestinal uptake of fluorescein isothiocyanate (FITC) labelled insulin were studied. When administered orally by force-feeding to diabetic rats, insulin nanoparticles decreased fasted glycemia in a dose dependant manner with a maximal effect observed with 100 IU/kg. These insulin nanoparticles also increased serum insulin levels and improved the glycemic response to an oral glucose challenge for a prolonged period of time. FITC-Insulin-loaded nanoparticles strongly adhered to the intestinal mucosa and labeled insulin, either released and/or still inside nanoparticles, was mainly taken up by the Peyer's patches. It is concluded that polymeric nanoparticles allows the preservation of insulin's biological activity. In addition, the antidiabetic effect can be explained by the mucoadhesive properties of the polycationic polymer (Eudragit) RS) allowing the intestinal uptake of insulin.

Effect of epiregulin on pancreatic beta cell growth and insulin secretion.

The aim of this study was to determine whether epiregulin, a novel member of EGF-related growth factor family, was able to affect proliferation and secretory function of rat insulinoma INS-1E and RINm5F cell lines. A 24 h treatment with epiregulin resulted in a stimulation of INS-1E and RINm5F cells proliferation; this effect was completely blocked in the presence of an anti-epiregulin antibody which did not affect basal DNA synthesis in the absence of added ligand. In acute experiments, epiregulin was able to potentiate insulin release in the presence of glucose or arginine, in the two cell lines. Finally, in the two cell lines expressing ErbB receptors, we demonstrated that only EGFR/ErbB1 was activated by epiregulin. Thus, epiregulin appears as a new growth and insulinotropic factor in pancreatic beta cell lines.

Effects of cholecystokinin octapeptide on the exocrine pancreas in a new rat model of type 2 diabetes.

We investigated the effects of increasing concentrations of cholecystokinin octapeptide (CCK-8) on the exocrine pancreas of a new model of type 2 diabetic rats due to the partial protection exerted by nicotinamide against the beta-cytotoxic effect of streptozotocin. CCK-8, administered for 8 successive days, exerted a biphasic action on the growth of the pancreas in non-diabetic and type 2 diabetic rats; however, the latter were less sensitive to CCK-8. Similar results were obtained in vitro by measuring the uptake of 5-bromo-2'-deoxyuridine (BrdU) in cultured isolated acinar cells. This effect was completely blocked by 3S(-)(N'-2,3-dihydro-1-methyl-2-oxo5-phenyl-1H-1,4-benzo-diazepin-3-yl)-1H-indole-2-carboxamide (L 364,718; a CCK(1) receptor antagonist) but not by (3R)-3[N'-(3-methylphenyl)ureido]-1,3-dihydro-1-methyl-5-phenyl-2H1,4-benzo-diazepin-2-one (L 365,260; a CCK(2) receptor antagonist), suggesting a direct effect via CCK(1) receptors. Binding studies showed that these effects were mediated by a single class of low-affinity CCK(1) receptors in diabetic rats and two classes of CCK-8 binding sites (with high and low affinity) in non-diabetic rats. Thus, in our new type 2 diabetes model, the loss of sensitivity of the pancreas to CCK-8 could be attributed to the loss of CCK(1) receptors of high affinity.