Efficacy of a new sustained-release microsphere formulation of exenatide, DA-3091, in Zucker diabetic fatty (ZDF) rats.

Exenatide must be administered serially by twice-daily subcutaneous (SC) injection due to its short half-life. The purpose of the present study is to develop an improved sustained-release exenatide formulation with a therapeutic efficacy comparable to serial, twice-daily injections of exenatide. A novel SR formulation of exenatide, DA-3091, was prepared by single-emulsion solvent evaporation using PLGA. It was administered by SC injection to ZDF rats in single exenatide doses of 0.1, 0.25, 0.5, 1 or 2mg/kg. On the 28th, 49th and 70th days, a 1 or 2mg/kg dose of DA-3091 was further administered to rats in dose groups of 1 or 2mg/kg. The efficacy of DA-3091 was then compared with that of serial, twice-daily SC injections of an exenatide solution for 13 weeks. NFBG and HbA1c concentrations were decreased both significantly and linearly as the exenatide dose in DA-3091 increased. In addition, food intake and body weight were suppressed both significantly and dose-dependently. In equivalent or half doses of exenatide, the efficacy of DA-3091 was comparable to that of twice-daily injections of exenatide solution for 13 weeks. In conclusion, DA-3091 has the potential to be clinically effective when administered every 3 weeks, or less frequently, which promises to significantly improve patient compliance.

Pharmacokinetics and efficacy of a biweekly dosage formulation of exenatide in Zucker diabetic fatty (ZDF) rats.

PURPOSE: To develop an improved sustained-release (SR) formulation of exenatide (a therapy for patients with type 2 diabetes mellitus) in a biweekly dosage form with therapeutic efficacy comparable to that achieved with twice-daily injections of the drug. METHODS: A SR formulation of exenatide, DA-3091, was prepared by single-emulsion solvent evaporation using poly(D,L-lactide-co-glycolide). Plasma exenatide, as well as plasma insulin, non-fasting blood glucose and HbA1c concentrations, and changes in food intake and body weight were evaluated in both Zucker diabetic fatty (ZDF) and ZDF lean control rats. RESULTS: After a single SC administration of DA-3091 (i.e., 2 mg/kg of exenatide), the plasma exenatide concentration increased and remained elevated in both groups. The concentrations of non-fasting blood glucose and HbA1c decreased significantly following a single SC injection of DA-3091 only in ZDF rats, indicating that the effects of exenatide are dependent on blood glucose concentration. On the other hand, both food intake and body weight gain were reduced in ZDF and ZDF lean control rats. A single injection of DA-3091 (i.e., 2 mg/kg of exenatide) lowered non-fasting blood glucose and HbA1c concentrations more effectively than 14 days of twice-daily administration of exenatide (i.e., 1.96 mg/kg of exenatide). CONCLUSION: DA-3091 has the potential to be used safely and efficaciously in a biweekly dosing regimen.

Functional maturation of lamina propria dendritic cells by activation of NKT cells mediates the abrogation of oral tolerance.

We previously showed that although systemic administration of alpha-galactosylceramide (alphaGalCer) or agonistic anti-CD40 induced functional maturation of dendritic cells (DC) in mesenteric lymph nodes, only the former treatment succeeded in breaking the induction of oral tolerance. In this study, we looked for the essential factor responsible for the disruption of oral tolerance. We found that lamina propria (LP)-DC was responsible for the oral OVA presentation and that Peyer's patch was not essential for the induction of oral tolerance. Therefore, we investigated the role of LP-DC. Treatment with alphaGalCer but not with anti-CD40 induced the full maturation of LP-DC at an early time point. This functional activation of LP-DC was mediated by strong activation of NKT cells that reside abundantly in the small intestinal lamina propria (SI-LP) and interferon-gamma partially contributed to the LP-DC activation. LP-DC isolated from alphaGalCer-treated OVA-fed mice induced the differentiation of naïve CD4+ T cells into Th1 and Th2 and was associated with the reduced Foxp3+ population. In contrast, LP-DC isolated from anti-CD40-treated OVA-fed mice failed to generate Th cell differentiation but induced more Foxp3+ CD4+ T cells. Our results demonstrate that triggered by NKT cells in SI-LP, functional maturation of Ag-capturing DC from SI-LP is necessary for the abrogation of oral tolerance induction.

Mediastinal lymph node CD8alpha- DC initiate antigen presentation following intranasal coadministration of alpha-GalCer.

Our previous study revealed that alpha-galactosylceramide (alpha-GalCer) is a potent nasal vaccine adjuvant inducing both potent humoral and cellular immune responses and affording complete protection against viral infections and tumors. However, the antigen-presenting cells (APC) that are activated by NKT cells and thereby initiate the immune responses following intranasal coadministration of protein antigen and alpha-GalCer are poorly understood. We assessed here where antigen presentation occurs and which APC subset mediates the early stages of immune responses when protein antigen and alpha-GalCer are intranasally administered. We show that dendritic cells (DC), but not B cells, initiated the mucosal immune responses at mediastinal lymph nodes. Of the DC subsets, the CD8alpha-B220-CD11c+ DC subset played the most prominent role in the direct and cross-presentation of protein antigen to naive T cells and in triggering the naive T cells to differentiate into effector T cells. This might be mainly caused by a relatively larger population of CD1dhigh cells of CD8alpha-B220-CD11c+ DC subset than those of other DC subsets. These results indicate that CD8alpha-B220-CD11c+ DC is the principal subset becoming immunogenic after interaction with NKT cells and abrogating tolerance to intranasally administered protein antigen when alpha-GalCer is coadministered as a nasal vaccine adjuvant.

A single intranasal immunization with inactivated influenza virus and alpha-galactosylceramide induces long-term protective immunity without redirecting antigen to the central nervous system.

alpha-Galactosylceramide (alpha-GalCer), originally isolated from a marine sponge, was known to activate natural killer T (NKT) cells through CD1d-mediated Ag presentation and induce Th1 and/or Th2 immunity. In this study, we evaluated the nasal adjuvanticity of alpha-GalCer when co-administered with formalin-inactivated influenza virus A/PR/8/34 (PR8) in BALB/c mice. A single nasal immunization of inactivated PR8 and alpha-GalCer induced brisk levels of PR8-specific IgG and IgA Abs in serum and lung washes. Antigen-specific Ab responses lasted for 3 months, providing protective immunity against challenge with live PR8. In addition, mice given alpha-GalCer also exhibited cellular immune responses including cytotoxic T lymphocyte (CTL) generation. Because it did not redirect Ags into brain, alpha-GalCer would likely pose no risk if administered as a nasal adjuvant. These results suggest for the first time that a single nasal immunization of inactivated virus and alpha-GalCer is a safe and effective means of preventing influenza infection.

Synthesis and evaluation of 1,2,3-triazole containing analogues of the immunostimulant alpha-GalCer.

alpha-GalCer is the first defined and most potent agonistic antigen of the T cell receptor of natural killer T cells. We have prepared a series of 1,2,3-triazole-containing alpha-GalCer analogues in which the lipid chain lengths have been incrementally varied. We found that this isosteric replacement of alpha-GalCer's amide moiety with triazole increases the IL-4 versus IFN-gamma bias of released cytokines. The stimulatory effect was influenced by the length of the attached chain. In particular, the long-chained triazole analogues have a comparable stimulatory effect on cytokine production as alpha-GalCer and exhibit a stronger Th2 cytokine response.