Hydrogen Peroxide-Triggered Conversion of Boronic Acid-Appended Insulin into Insulin and Its Application as a Glucose-Responsive Insulin Formulation.

p-Boronophenylmethoxycarbonyl (BPmoc) is a protecting group for amines that is removable by treatment with hydrogen peroxide (H2O2). We prepared BPmoc-modified insulin (BPmoc-Ins) and subcutaneously injected the formulation into diabetic rats. The results demonstrated that BPmoc effectively sealed the blood glucose (Glc)-lowering effects of Ins. Conversely, coinjection of BPmoc-Ins and Glc oxidase (GOx) resulted in reduced blood Glc levels, indicating that Ins was generated from BPmoc-Ins through the following reactions: oxidation of endogenous Glc by GOx; production of H2O2 accompanied by Glc oxidation; removal of BPmoc residues by H2O2. These results show the potential of BPmoc-Ins for a Glc-responsive Ins release system.

Oral Coadministration of Zn-Insulin with d-Form Small Intestine-Permeable Cyclic Peptide Enhances Its Blood Glucose-Lowering Effect in Mice.

Oral delivery of insulin remains a challenge owing to its poor permeability across the small intestine and enzymatic digestion in the gastrointestinal tract. In a previous study, we identified a small intestine-permeable cyclic peptide, C-DNPGNET-C (C-C disulfide bond, cyclic DNP peptide), which facilitated the permeation of macromolecules. Here, we showed that intraintestinal and oral coadministration of insulin with the cyclic DNP derivative significantly reduced blood glucose levels by increasing the portal plasma insulin concentration following permeation across the small intestine of mice. We also found that protecting the cyclic DNP derivative from enzymatic digestion in the small intestine of mice using d-amino acids and by the cyclization of DNP peptide was essential to enhance cyclic DNP derivative-induced insulin absorption across the small intestine. Furthermore, intraintestinal and oral coadministration of insulin hexamer stabilized by zinc ions (Zn-insulin) with cyclic D-DNP derivative was more effective in facilitating insulin absorption and inducing hypoglycemic effects in mice than the coadministration of insulin with the cyclic D-DNP derivative. Moreover, Zn-insulin was more resistant to degradation in the small intestine of mice compared to insulin. Intraintestinal and oral coadministration of Zn-insulin with cyclic DNP derivative also reduced blood glucose levels in a streptozotocin-induced diabetes mellitus mouse model. A single intraintestinal administration of the cyclic D-DNP derivative did not induce any cytotoxicity, either locally in the small intestine or systemically. In summary, we demonstrated that coadministration of Zn-insulin with cyclic D-DNP derivative could enhance oral insulin absorption across the small intestine in mice.

In Vitro and in Vivo Comparative Study of Oral Nanoparticles and Gut Iontophoresis as Oral Delivery Systems for Insulin.

Multiple daily injections of insulin for diabetes cause many hazards for diabetic patients. Oral noninvasive insulin delivery could be more convenient and less painful than parenteral route. In past decades transdermal iontophoresis had been studied for insulin delivery across the skin with or without chemical permeation enhancers. However, the results of these studies were not efficacious and serum insulin levels were not therapeutically effective. In the present study an advanced technology "gut iontophoresis" for insulin delivery across the gut wall was compared with traditional oral insulin delivery in the form of nanoparticles. In vitro application of electric current to the intestinal membrane could enhance the flux of insulin nanoparticles (3.4 fold enhancement of insulin transport) from the donor to the receptor compartment in the Franz cell. In vivo iontophoresis of insulin nanoparticles through the gut wall would produce intense hypoglycemia (57% glycemia drop in 3 h) without damage of the intestinal tissues. Cell viability assay indicated that 50-500 µg/mL nanoparticles had no toxic effect on Caco-2 cells. Nanoparticles gut iontophoresis could be a promising non-invasive technique for oral insulin delivery.

Enterically delivered insulin tregopil exhibits rapid absorption characteristics and a pharmacodynamic effect similar to human insulin in conscious dogs.

AIMS: Current therapy fails to emulate rapid (first-phase) insulin release in relation to a meal, a key defect in types 1 and 2 diabetes. We aimed to quantify the pharmacokinetic (PK) and pharmacodynamic (PD) profile of insulin tregopil, an enterically-absorbed insulin analog that restores the normal distribution of insulin between the hepatic portal and peripheral circulations. MATERIALS AND METHODS: The PK and PD profiles of insulin tregopil were studied in overnight-fasted, catheterized, conscious canines using four approaches: (1) equimolar intraportal infusions of tregopil vs human insulin; (2) escalating doses of oral tregopil; (3) identical, consecutive enteric doses of tregopil; and (4) comparison of oral tregopil to inhaled and subcutaneous human insulin administration. RESULTS: Equimolar intraportal infusions of tregopil and human insulin resulted in very similar PK profiles and PD profiles were nearly identical. Enteric delivery of tregopil brought about rapid absorption with tmax = 20 minutes in most cases. Median tmax was 20 minutes for oral tregopil and inhaled insulin and 88 minutes for subcutaneous human insulin. The time required for arterial plasma insulin levels to return to baseline was approximately 90, 210 and 360 minutes for oral tregopil, inhaled insulin and subcutaneous insulin, respectively. CONCLUSIONS: Enterically delivered tregopil is rapidly absorbed and restores a portal-to-peripheral vascular distribution. These characteristics should improve postprandial hyperglycaemia in types 1 and 2 diabetes.

Direct comparison of LC-MS/MS and RIA methods for the pharmacokinetics assessment of human insulin in preclinical development.

Insulin is an effective therapeutic for diabetes, and the level of insulin in vivo is directly related to the health of diabetic patients. Traditionally, the concentrations of insulin in vivo are determined by the radioimmunoassay (RIA) method. In this study, we developed an LC-MS/MS method for the quantification of human insulin in dog plasma and directly compared the RIA and LC-MS/MS methods. Our LC-MS/MS method exhibited superior accuracy, efficiency and cost-effective for the pharmacokinetic (PK) assessment of human insulin. The LC-MS/MS method can quantitate human insulin and canine insulin simultaneously without cross-reactivity, making the analysis more efficient. The LLOQ of our LC-MS/MS method was 38.5 pg/mL, which was necessary to fully describe the PK profiles of endogenous and exogenous insulin in vivo. The direct comparison of PK data obtained from the two methods demonstrated that LC-MS/MS could be an alternative to the RIA method and should be widely used for the quantification of insulin drugs, especially in preclinical studies.

Superior Glycemic Control With a Glucose-Responsive Insulin Analog: Hepatic and Nonhepatic Impacts.

We evaluated the hepatic and nonhepatic responses to glucose-responsive insulin (GRI). Eight dogs received GRI or regular human insulin (HI) in random order. A primed, continuous intravenous infusion of [3-3H]glucose began at -120 min. Basal sampling (-30 to 0 min) was followed by two study periods (150 min each), clamp period 1 (P1) and clamp period 2 (P2). At 0 min, somatostatin and GRI (36 ± 3 pmol/kg/min) or HI (1.8 pmol/kg/min) were infused intravenously; basal glucagon was replaced intraportally. Glucose was infused intravenously to clamp plasma glucose at 80 mg/dL (P1) and 240 mg/dL (P2). Whole-body insulin clearance and insulin concentrations were not different in P1 versus P2 with HI, but whole-body insulin clearance was 23% higher and arterial insulin 16% lower in P1 versus P2 with GRI. Net hepatic glucose output was similar between treatments in P1. In P2, both treatments induced net hepatic glucose uptake (HGU) (HI mean ± SEM 2.1 ± 0.5 vs. 3.3 ± 0.4 GRI mg/kg/min). Nonhepatic glucose uptake in P1 and P2, respectively, differed between treatments (2.6 ± 0.3 and 7.4 ± 0.6 mg/kg/min with HI vs. 2.0 ± 0.2 and 8.1 ± 0.8 mg/kg/min with GRI). Thus, glycemia affected GRI but not HI clearance, with resultant differential effects on HGU and nonHGU. GRI holds promise for decreasing hypoglycemia risk while enhancing glucose uptake under hyperglycemic conditions.

Exploration of the Key Factors for Optimizing the in Vivo Oral Delivery of Insulin by Using a Noncovalent Strategy with Cell-Penetrating Peptides.

This present study aimed to determine the optimal oral insulin delivery conditions that would maximize the utility of cell-penetrating peptides (CPPs) by using a noncovalent strategy. We first compared the effectiveness of two potential CPPs, penetratin and its analog PenetraMax, as absorption enhancers for insulin. The combined effect was evaluated under in vivo oral administration conditions. Both D-forms of CPPs were highly effective for increasing the oral absorption of insulin, and D-PenetraMax showed a more rapid onset of absorption enhancement effects compared with those of D-penetratin. However, synergistic absorption enhancement effects after combination treatment were not observed. Next, we tried a theoretical approach to establish optimized oral insulin delivery conditions. A surface plasmon resonance (SPR)-based analysis demonstrated that binding between insulin and penetratin (2 mM) might be saturated at 100-500 µM penetratin, while the bound concentration of penetratin could increase in accordance with an increased concentration of mixed insulin. To test this hypothesis, we investigated the effectiveness of different insulin doses in the gastric pH-neutralized mice. The results showed that the dissociation of noncovalent complexes of insulin and CPPs at the low gastric pH was prevented in these mice. Our findings clearly suggested that a noncovalent strategy with CPPs represents an effective approach for the L-form of CPP to increase the concentration of CPP-bound insulin to attain greater absorption of insulin, although this approach may not be appropriate for the D-form of CPP. Our findings will contribute to the development of oral dosage forms of insulin for noncovalent strategies involving CPP.

Engineering Glucose Responsiveness Into Insulin.

Insulin has a narrow therapeutic index, reflected in a small margin between a dose that achieves good glycemic control and one that causes hypoglycemia. Once injected, the clearance of exogenous insulin is invariant regardless of blood glucose, aggravating the potential to cause hypoglycemia. We sought to create a "smart" insulin, one that can alter insulin clearance and hence insulin action in response to blood glucose, mitigating risk for hypoglycemia. The approach added saccharide units to insulin to create insulin analogs with affinity for both the insulin receptor (IR) and mannose receptor C-type 1 (MR), which functions to clear endogenous mannosylated proteins, a principle used to endow insulin analogs with glucose responsivity. Iteration of these efforts culminated in the discovery of MK-2640, and its in vitro and in vivo preclinical properties are detailed in this report. In glucose clamp experiments conducted in healthy dogs, as plasma glucose was lowered stepwise from 280 mg/dL to 80 mg/dL, progressively more MK-2640 was cleared via MR, reducing by ∼30% its availability for binding to the IR. In dose escalations studies in diabetic minipigs, a higher therapeutic index for MK-2640 (threefold) was observed versus regular insulin (1.3-fold).

Quantification of Basal Insulin Peglispro and Human Insulin in Adipose Tissue Interstitial Fluid by Open-Flow Microperfusion.

BACKGROUND: Restoration of the physiologic hepatic-to-peripheral insulin gradient may be achieved by either portal vein administration or altering insulin structure to increase hepatic specificity or restrict peripheral access. Basal insulin peglispro (BIL) is a novel, PEGylated basal insulin with a flat pharmacokinetic and glucodynamic profile and altered hepatic-to-peripheral action gradient. We hypothesized reduced BIL exposure in peripheral tissues explains the latter, and in this study assessed the adipose tissue interstitial fluid (ISF) concentrations of BIL compared with human insulin (HI). METHODS: A euglycemic glucose clamp was performed in patients with type 1 diabetes during continuous intravenous (IV) infusion of BIL or HI, while the adipose ISF insulin concentrations were determined using open-flow microperfusion (OFM). The ratio of adipose ISF-to-serum concentrations and the absolute steady-state adipose ISF concentrations were assessed using a dynamic no-net-flux technique with subsequent regression analysis. RESULTS: Steady-state BIL concentrations in adipose tissue ISF were achieved by ∼16 h after IV infusion. Median time to reach steady-state glucose infusion rate across doses ranged between 8 and 22 h. The average serum concentrations (coefficient of variation %) of BIL and HI were 11,200 pmol/L (23%) and 425 pmol/L (15%), respectively. The ISF-to-serum concentration ratios were 10.2% for BIL and 22.9% for HI. CONCLUSIONS: This study indicates feasibility of OFM to measure BIL in ISF. The observed low ISF-to-serum concentration ratio of BIL is consistent with its previously demonstrated reduced peripheral action.

Evaluation of the counter-regulatory responses to hypoglycaemia in patients with type 1 diabetes during opiate receptor blockade with naltrexone.

AIMS: Hypoglycaemia is the major limiting factor in achieving optimal glycaemic control in people with type 1 diabetes (T1DM), especially intensively treated patients with impaired glucose counter-regulation during hypoglycaemia. Naloxone, an opiate receptor blocker, has been reported to enhance the acute counter-regulatory response to hypoglycaemia when administered intravenously in humans. The current study was undertaken to investigate the oral formulation of the long-acting opiate antagonist, naltrexone, and determine if it could have a similar effect, and thus might be useful therapeutically in treatment of T1DM patients with a high risk of hypoglycaemia. MATERIALS AND METHODS: We performed a randomized, placebo-controlled, double-blinded, cross-over study in which 9 intensively treated subjects with T1DM underwent a 2-step euglycaemic-hypoglycaemic-hyperinsulinaemic clamp on 2 separate occasions. At 12 hours and at 1 hour before the clamp study, participants received 100 mg of naltrexone or placebo orally. Counter-regulatory hormonal responses were assessed at baseline and during each step of the hyperinsulinaemic-clamp. RESULTS: Glucose and insulin levels did not differ significantly between the naltrexone and placebo visits; nor did the glucose infusion rates required to keep glucose levels at target. During hypoglycaemia, naltrexone, in comparison with the placebo group, induced an increase in epinephrine levels ( P = .05). However, no statistically significant differences in glucagon, cortisol and growth hormone responses were observed. CONCLUSION: In contrast to the intravenous opiate receptor blocker naloxone, overnight administration of the oral long-acting opiate receptor blocker, naltrexone, at a clinically used dose, had a limited effect on the counter-regulatory response to hypoglycaemia in intensively treated subjects with T1DM.

A comparison of the pharmacodynamic profiles of jet-injected regular human insulin versus conventionally administered insulin aspart in healthy volunteers.

AIMS: Rapid-acting insulin analogues are generally preferred over regular human insulin because of their more immediate onset of action and shorter time-action profile. However, these analogues may not always be tolerated by or universally available for people with insulin-requiring diabetes. Jet injection has been demonstrated to facilitate faster insulin absorption. We determined whether administration of regular human insulin by jet injection achieves the same pharmacological properties as that of a rapid-acting insulin analogue. METHODS: Twenty healthy volunteers received regular human insulin (0.2units/kg) by jet injection. Glucose 20% was infused intravenously to maintain euglycaemia over six hours. The glucose infusion rates (GIR) were determined to compare pharmacological profiles. These profiles were compared with data from two other studies in which a similar dose of insulin aspart was administered by conventional pen. RESULTS: Regular human insulin by jet injection had a faster onset of glucose-lowering effect compared to aspart by conventional pen (T-GIR50%, 30.8±2.9 versus 43.1±3.2min, P<0.01). There were no differences in time to maximal GIR (106.1±11.9 versus 95.8±9.2min, P=0.50), maximal GIR (8.6±0.7 versus 7.7±0.7mg/kg/min, P=0.0.33), total glucose-lowering effect (101.0±9.8 versus 87.6±7.0g, P=0.28), and time until 50% of glucose disposal (144.8±5.6 versus 151.3±5.1min, P=0.39). CONCLUSIONS: Jet-injected regular human insulin had a pharmacological profile that was essentially not dissimilar from that of aspart insulin administered by conventional pen, and can therefore be used as an alternative for conventionally administered rapid-acting insulin analogues.

Bioavailability of insulin detemir and human insulin at the level of peripheral interstitial fluid in humans, assessed by open-flow microperfusion.

AIMS: To find an explanation for the lower potency of insulin detemir observed in humans compared with unmodified human insulin by investigating insulin detemir and human insulin concentrations directly at the level of peripheral insulin-sensitive tissues in humans in vivo. METHODS: Euglycaemic-hyperinsulinaemic clamp experiments were performed in healthy volunteers. Human insulin was administered i.v. at 6 pmol/kg/min and insulin detemir at 60 pmol/kg/min, achieving a comparable steady-state pharmacodynamic action. In addition, insulin detemir was doubled to 120 pmol/kg/min. Minimally invasive open-flow microperfusion (OFM) sampling methodology was combined with inulin calibration to quantify human insulin and insulin detemir in the interstitial fluid (ISF) of subcutaneous adipose and skeletal muscle tissue. RESULTS: The human insulin concentration in the ISF was ∼115 pmol/l or ∼30% of the serum concentration, whereas the insulin detemir concentration in the ISF was ∼680 pmol/l or ∼2% of the serum concentration. The molar insulin detemir interstitial concentration was five to six times higher than the human insulin interstitial concentration and metabolic clearance of insulin detemir from serum was substantially reduced compared with human insulin. CONCLUSIONS: OFM proved useful for target tissue measurements of human insulin and the analogue insulin detemir. Our tissue data confirm a highly effective retention of insulin detemir in the vascular compartment. The higher insulin detemir relative to human insulin tissue concentrations at comparable pharmacodynamics, however, indicate that the lower potency of insulin detemir in humans is attributable to a reduced effect in peripheral insulin-sensitive tissues and is consistent with the reduced in vitro receptor affinity.

Open flow microperfusion: pharmacokinetics of human insulin and insulin detemir in the interstitial fluid of subcutaneous adipose tissue.

AIMS: To compare the time profile of insulin detemir and human insulin concentrations in the interstitial fluid (ISF) of subcutaneous adipose tissue during constant i.v. infusion and to investigate the relationship between the pharmacokinetics of both insulin molecules in plasma and the ISF of subcutaneous adipose tissue. METHODS: During a 6-h hyperinsulinaemic-euglycaemic clamp (plasma glucose level 8 mmol/l) human insulin (21 and 42 pmol/min/kg) or insulin detemir (209 and 417 pmol/min/kg) were infused i.v. in eight rats per dose level. Open flow microperfusion (OFM) was used to continuously assess interstitial insulin concentrations in subcutaneous adipose tissue. RESULTS: At the lower infusion rate, insulin detemir appeared significantly later in the ISF than in the plasma (p < 0.05) and also appeared later in the ISF relative to human insulin (p < 0.005). CONCLUSIONS: By using OFM we were able to monitor albumin-bound insulin detemir directly in the ISF of subcutaneous tissue and confirm its delayed transendothelial passage to a peripheral site of action.

Subcutaneous injection of hyaluronidase with recombinant human insulin compared with insulin lispro in type 1 diabetes.

AIMS: Prandial treatment with human regular insulin for diabetes may result in early postprandial hyperglycaemia and late hypoglycaemia due to its slow onset and long duration of action. This study compared injections of recombinant human insulin (rHI) formulated with recombinant human hyaluronidase [rHuPH20] (INSULIN-PH20) to insulin lispro for prandial treatment in subjects with type 1 diabetes (T1D). METHODS: After a 1-month run-in period using twice-daily insulin glargine (or usual basal insulin therapy for pump users) with prandial lispro, 46 subjects with T1D (42 ± 13 years; body mass index: 26 ± 4 kg/m(2); A1c: 6.8 ± 0.5%) were assigned to INSULIN-PH20 or lispro in a random sequence for two consecutive, 12-week periods as the prandial insulin in an intensive treatment regimen. RESULTS: The mean glycaemic excursion for INSULIN-PH20 (0.96 ± 2.00 mmol/l) was comparable (p = 0.322) to lispro (0.80 ± 1.95 mmol/l). The 8-point self-monitored blood glucose profiles were also comparable in the two groups. Good glycaemic control (A1c) was maintained for both treatments at 12 weeks (INSULIN-PH20: 7.0 ± 0.5%; lispro: 6.9 ± 0.6%). Overall rates of hypoglycaemia (≤ 3.9 mmol/l) were 24 events per patient per 4 weeks for INSULIN-PH20 and 22 events for lispro. There were no significant differences in adverse events or immunogenicity between treatments and both treatments were well tolerated. CONCLUSIONS: Unlike commercially available formulations of regular human insulin, a formulation of rHI with rHuPH20 was comparable to lispro for postprandial glucose excursions in a basal-bolus treatment regimen for T1D patients. Glycaemic control, safety and tolerability profiles were comparable for both treatments.

Practice tips and tools for the successful use of U-500 regular human insulin: the diabetes educator is key.

This review provides information to equip diabetes educators to instruct and guide patients in using U-500 human regular insulin (U-500R). The article includes an overview of U-500R pharmacology and clinical data, strategies for outpatient and inpatient use, and tools for patient education. U-500R is useful for treating patients with any type of diabetes who require high doses of insulin. U-500R alleviates the volume-related problems associated with high doses of U-100 insulin, making treatment with high doses of insulin more feasible (because of the need for fewer injections for patients) as well as more cost-efficient and potentially more effective. These tools can help diabetes educators feel more comfortable and confident as they advise and educate patients who receive high-dose U-500R as part of their overall diabetes care plan. The diabetes educator plays a vital role in helping patients use U-500R safely and successfully.

Molecular characterisation of long-acting insulin analogues in comparison with human insulin, IGF-1 and insulin X10.

AIMS/HYPOTHESIS: There is controversy with respect to molecular characteristics of insulin analogues. We report a series of experiments forming a comprehensive characterisation of the long acting insulin analogues, glargine and detemir, in comparison with human insulin, IGF-1, and the super-mitogenic insulin, X10. METHODS: We measured binding of ligands to membrane-bound and solubilised receptors, receptor activation and mitogenicity in a number of cell types. RESULTS: Detemir and glargine each displayed a balanced affinity for insulin receptor (IR) isoforms A and B. This was also true for X10, whereas IGF-1 had a higher affinity for IR-A than IR-B. X10 and glargine both exhibited a higher relative IGF-1R than IR binding affinity, whereas detemir displayed an IGF-1R:IR binding ratio of ≤ 1. Ligands with high relative IGF-1R affinity also had high affinity for IR/IGF-1R hybrid receptors. In general, the relative binding affinities of the analogues were reflected in their ability to phosphorylate the IR and IGF-1R. Detailed analysis revealed that X10, in contrast to the other ligands, seemed to evoke a preferential phosphorylation of juxtamembrane and kinase domain phosphorylation sites of the IR. Sustained phosphorylation was only observed from the IR after stimulation with X10, and after stimulation with IGF-1 from the IGF-1R. Both X10 and glargine showed an increased mitogenic potency compared to human insulin in cells expressing many IGF-1Rs, whereas only X10 showed increased mitogenicity in cells expressing many IRs. CONCLUSIONS: Detailed analysis of receptor binding, activation and in vitro mitogenicity indicated no molecular safety concern with detemir.

Improved postprandial glycemic control in patients with type 2 diabetes from subcutaneous injection of insulin lispro with hyaluronidase.

BACKGROUND: Coinjection of hyaluronidase has been shown to accelerate insulin absorption in healthy volunteers and patients with type 1 diabetes mellitus. This study was undertaken to compare the postprandial glycemic response of patients with type 2 diabetes mellitus (T2DM) administered insulin lispro with and without recombinant human hyaluronidase (rHuPH20) and regular human insulin (RHI) with rHuPH20. METHODS: This double-blind three-way crossover study compared the insulin pharmacokinetics and glucodynamic response to a standardized liquid meal (80 g of carbohydrate) in 21 patients with T2DM who received subcutaneous injections of individually optimized doses of lispro±rHuPH20 and RHI+rHuPH20. The optimum dose (targeting postprandial glucose [PPG] of 70-140 mg/dL) of each preparation was selected by the investigator following a fixed-dose escalation procedure in three dose-finding meals. RESULTS: Co-injection of lispro+rHuPH20 accelerated pharmacokinetics relative to lispro alone (time to peak insulin concentration, 43 vs. 74 min; P=0.0045) with increased exposure in the first hour (184% of control; P<0.0001) and reduced exposure after 2 h (67% of control; P=0.0001). These accelerated pharmacokinetics improved both total hyperglycemic excursions (area under the curve for 0-4 h >140 mg/dL, 56% of control; P=0.048) and hypoglycemic excursions (area under the curve for 0-8 h <70 mg/dL, 34% of control; P=0.033), allowing over three times as many patients to reach the American Diabetes Association's target of peak PPG <180 mg/dL without requiring glucose treatment for hypoglycemia. The mean optimum dose of lispro was reduced 8% from 0.275 U/kg without rHuPH20 to 0.254 U/kg with rHuPH20 (P=0.04). RHI+rHuPH20 had responses and optimum doses comparable to insulin lispro alone. All insulin preparations were well tolerated. CONCLUSIONS: Lispro+rHuPH20 provided superior control of glycemic excursion compared with lispro alone, with lower insulin requirements and reduced hypoglycemic excursions.

Pharmacokinetics and pharmacodynamics of high-dose human regular U-500 insulin versus human regular U-100 insulin in healthy obese subjects.

OBJECTIVE: Human regular U-500 (U-500R) insulin (500 units/mL) is increasingly being used clinically, yet its pharmacokinetics (PK) and pharmacodynamics (PD) have not been well studied. Therefore, we compared PK and PD of clinically relevant doses of U-500R with the same doses of human regular U-100 (U-100R) insulin (100 units/mL). RESEARCH DESIGN AND METHODS: This was a single-site, randomized, double-blind, crossover euglycemic clamp study. Single subcutaneous injections of 50- and 100-unit doses of U-500R and U-100R were administered to 24 healthy obese subjects. RESULTS: Both overall insulin exposure (area under the serum insulin concentration versus time curve from zero to return to baseline [AUC(0-)(t)(')]) and overall effect (total glucose infused during a clamp) were similar between formulations at both 50- and 100-unit doses (90% [CI] of ratios contained within [0.80, 1.25]). However, peak concentration and effect were significantly lower for U-500R at both doses (P < 0.05). Both formulations produced relatively long durations of action (18.3 to 21.5 h). Time-to-peak concentration and time to maximum effect were significantly longer for U-500R than U-100R at the 100-unit dose (P < 0.05). Time variables reflective of duration of action (late tR(max50), tR(last)) were prolonged for U-500R versus U-100R at both doses (P < 0.05). CONCLUSIONS: Overall exposure to and action of U-500R insulin after subcutaneous injection were no different from those of U-100R insulin. For U-500R, peaks of concentration and action profiles were blunted and the effect after the peak was prolonged. These findings may help guide therapy with U-500R insulin for highly insulin-resistant patients with diabetes.

Liposomes containing glycocholate as potential oral insulin delivery systems: preparation, in vitro characterization, and improved protection against enzymatic degradation.

BACKGROUND: Oral delivery of insulin is challenging and must overcome the barriers of gastric and enzymatic degradation as well as low permeation across the intestinal epithelium. The present study aimed to develop a liposomal delivery system containing glycocholate as an enzyme inhibitor and permeation enhancer for oral insulin delivery. METHODS: Liposomes containing sodium glycocholate were prepared by a reversed-phase evaporation method followed by homogenization. The particle size and entrapment efficiency of recombinant human insulin (rhINS)-loaded sodium glycocholate liposomes can be easily adjusted by tuning the homogenization parameters, phospholipid:sodium glycocholate ratio, insulin:phospholipid ratio, water:ether volume ratio, interior water phase pH, and the hydration buffer pH. RESULTS: The optimal formulation showed an insulin entrapment efficiency of 30% ± 2% and a particle size of 154 ± 18 nm. A conformational study by circular dichroism spectroscopy and a bioactivity study confirmed the preserved integrity of rhINS against preparative stress. Transmission electron micrographs revealed a nearly spherical and deformed structure with discernable lamella for sodium glycocholate liposomes. Sodium glycocholate liposomes showed better protection of insulin against enzymatic degradation by pepsin, trypsin, and α-chymotrypsin than liposomes containing the bile salt counterparts of sodium taurocholate and sodium deoxycholate. CONCLUSION: Sodium glycocholate liposomes showed promising in vitro characteristics and have the potential to be able to deliver insulin orally.