Study of GABA in Healthy Volunteers: Pharmacokinetics and Pharmacodynamics

Preclinical studies show that GABA exerts anti-diabetic effects in rodent models of type 1 diabetes. Because little is known about its absorption and effects in humans, we investigated the pharmacokinetics and pharmacodynamics of GABA in healthy volunteers. Twelve subjects were subjected to an open-labeled, three-period trial involving sequential oral administration of placebo, 2 g GABA once, and 2 g GABA three times/day for 7 days, with a 7-day washout between each period. GABA was rapidly absorbed (Tmax: 0.5 ~ 1 h) with the half-life (t1/2) of 5 h. No accumulation was observed after repeated oral GABA administration for 7 days. Remarkably, GABA significantly increased circulating insulin levels in the subjects under either fasting (1.6-fold, single dose; 2.0-fold, repeated dose; p \u3c 0.01) or fed conditions (1.4-fold, single dose; 1.6-fold, repeated dose; p \u3c 0.01). GABA also increased glucagon levels only under fasting conditions (1.3-fold, single dose, p \u3c 0.05; 1.5-fold, repeated dose, p \u3c 0.01). However, there were no significant differences in the insulin-to-glucagon ratio and no significant change in glucose levels in these healthy subjects during the study period. Importantly, GABA significantly decreased glycated albumin levels in the repeated dosing period. Subjects with repeated dosing showed an elevated incidence of minor adverse events in comparison to placebo or the single dosing period, most notably transient discomforts such as dizziness and sore throat. However, there were no serious adverse events observed throughout the study. Our data show that GABA is rapidly absorbed and tolerated in human beings; its endocrine effects, exemplified by increasing islet hormonal secretion, suggest potential therapeutic benefits for diabetes

Novel GLP-1 Fusion Chimera as Potent Long Acting GLP-1 Receptor Agonist

GLP-1 has a variety of anti-diabetic effects. However, native GLP-1 is not suitable for therapy of diabetes due to its short half-life (t1/2<2 min). To circumvent this, we developed a long-lasting GLP-1 receptor agonist by the fusion of GLP-1 with human IgG2 Fc (GLP-1/hIgG2). ELISA-based receptor binding assay demonstrated that GLP-1/hIgG2 had high binding affinity to the GLP-1R in INS-1 cells (Kd = 13.90±1.52 nM). Upon binding, GLP-1/hIgG2 was rapidly internalized by INS-1 cells in a dynamin-dependent manner. Insulin RIA showed that GLP-1/IgG2 dose-dependently stimulated insulin secretion from INS-1 cells. Pharmacokinetic studies in CD1 mice showed that with intraperitoneal injection (i.p.), the GLP-1/hIgG2 peaked at 30 minutes in circulation and maintained a plateau for >168 h. Intraperitoneal glucose tolerance test (IPGTT) in mice showed that GLP-1/hIgG2 significantly decreased glucose excursion. Furthermore, IPGTT performed on mice one week after a single drug-injection also displayed significantly reduced glucose excursion, indicating that GLP-1/hIgG2 fusion protein has long-lasting effects on the modulation of glucose homeostasis. GLP-1/hIgG2 was found to be effective in reducing the incidence of diabetes in multiple-low-dose streptozotocin-induced type 1 diabetes in mice. Together, the long-lasting bioactive GLP-1/hIgG2 retains native GLP-1 activities and thus may serve as a potent GLP-1 receptor agonist

Optimization of Ultrasound-mediated Anti-angiogenic Cancer Gene Therapy

Ultrasound-targeted microbubble destruction (UTMD) can be used to deliver silencing gene therapy to tumors. We hypothesized that UTMD would be effective in suppressing angiogenesis within tumors, and that modulation of the ultrasound pulsing intervals (PI) during UTMD would affect the magnitude of target knockdown. We performed UTMD of vascular endothelial growth factor receptor-2 (VEGFR2) short hairpin (sh)RNA plasmid in an heterotopic mammary adenocarcinoma model in rats, evaluating PIs of 2, 5, 10, and 20 seconds. We demonstrated that UTMD with a PI of 10 seconds resulted in the greatest knockdown of VEGFR2 by PCR, immunostaining, western blotting, smaller tumor volumes and perfused areas, and lower tumor microvascular blood volume (MBV) and flow by contrast-enhanced ultrasound (CEU) compared with UTMD-treated tumors at 2, 5, and 20 seconds, control tumors, tumors treated with intravenous shRNA plasmid and scrambled plasmid. CEU perfusion assessment using the therapeutic probe demonstrated that tumors were fully replenished with microbubbles within 10 seconds, but incompletely replenished at PI-2 and PI-5 seconds. In conclusion, for anti-VEGFR2 cancer gene therapy by UTMD, PI of 10 seconds results in higher target knockdown and a greater anti-angiogenic effect. Complete replenishment of tumor vasculature with silencing gene-bearing microbubbles in between destructive pulses of UTMD is required to maximize the efficacy of anti-angiogenic cancer gene therapy

Analytical Artefacts Preclude Reliable Isotope Ratio Measurement of Internal Water in Coral Skeletons

Internal water in cold‐water and tropical coral skeletons was extracted and measured for its oxygen and hydrogen isotope ratios. Water was extracted by crushing pieces of coral hard tissue in a percussion device connected to either a cavity ring‐down spectroscopy (CRDS) system or an isotope ratio mass spectrometry (IRMS) system. Despite most samples yielding sufficient water, each analytical system produces distinct isotope patterns. Experiments show that several characteristics specific to biominerals give rise to discrepancies and analytical artefacts that preclude the acquisition of reproducible isotope data. The main complication is that internal water in biogenic carbonates is distributed in an open interconnected micro‐network that readily exchanges with external water and potentially facilitates interaction with hydration water in the finely dispersed organic matrix in the coral skeleton. Furthermore, only an isotopically fractionated part of the internal water is released from the coral skeletons upon crushing. Altogether, isotope ratio measurement of internal water in corals with bulk crushing techniques does not give primary fluid isotope ratios useful for (palaeo‐)environmental or microbiological studies. As the resulting isotope patterns can show systematic behaviour per technique, isotope data may be erroneously interpreted to reflect the original calcifying fluid when using only a single technique to isotopically characterise internal fluids in coral skeletons.Key Points: Free water trapped inside coral skeletons was extracted and isotopically analyzed on two commonly used techniques for fluid inclusion isotope analysis. Measured oxygen and hydrogen isotope ratios do not reproduce between the techniques due to several analytical artefacts. The water extracted from coral skeletons is not of primary origin.Nederlandse Organisatie voor Wetenschappelijk Onderzoek http://dx.doi.org/10.13039/501100003246Western Indian Ocean Marine Science Association http://dx.doi.org/10.13039/50110000910

Stimulation of insulin secretion by GLP-1/<i>h</i>IgG2 in INS- cells.

<p>(A) Cells grown in 24-well plates were incubated with fresh KRB buffer devoid of glucose for 2×60 min. The cells were then treated with 2.8 or 16.8 mM glucose and various concentrations of purified GLP-1/<i>h</i>IgG2 fusion proteins in KRB buffer for 2 h. The insulin levels in the conditioned KRB buffer were measured using a rat insulin RIA kit. (B) Active GLP-1 was measured by active GLP-1 ELISA kit in the mouse serum samples incubated with recombinant GLP-1 (75 pM) or GLP-1/<i>h</i>IgG2 fusion proteins (75 pM) at 37°C for indicated time period. The reactions were terminated by adding excessive DPP-IV inhibitors and aprotinin.</p

Pharmacokinetic study of GLP-1/<i>h</i>IgG2 fusion protein in CD1 mice.

<p>CD1 mice were i.p. injected by a single-dose of GLP-1/<i>h</i>IgG2 (1 µg/mouse). Blood samples were taken from tail vein at different time points and serum GLP-1 levels were measured by GLP-1 RIA kit.</p

Intraperitoneal Glucose Tolerance Test (IPGTT) shows that GLP-1/<i>h</i>IgG2 improves glucose tolerance in CD-1 mice.

<p>After 16 h fasting, CD1 mice were i.p. injected with GLP-1/<i>h</i>IgG2 (1 µg/mouse). 30 min after the injection, IPGTT were conducted by i.p. injection of 1.5 g/kg of glucose and blood glucose levels were measured by a glucometer at 0, 10, 20, 30, 60 minutes after glucose administration. (B) 192-h after a single-dose injection of GLP-1/<i>h</i>IgG2 in CD1 mice, the mice were fasted for 16 h and IPGTT were conducted as described in (A).</p

GLP-1/<i>h</i>IgG2 improves glucose regulation in MDSD mice.

<p>(A) The intraperitoneal injections of GLP-1/<i>h</i>IgG2 (1 µg/mouse) were made every three days during the course of experiment and the first drug injection was made 3 days prior to the multiple low dose streptozotozin treatment (50 mg/kg/day for 4 consecutive i.p. injections). Glucose levels were measured by a glucometer at indicated times. (B) The glucose levels were expressed as the area under the curve (AUC). (C) Insulin tolerance test (ITT) was conducted by injecting insulin (2.0 U/kg, i.p.) and blood glucose levels were measured at indicated times. (D) IPGTT was performed in MDSD mice treated with PBS or GLP-1/<i>h</i>IgG2-Fc at the end of the experiment as shown in (A).</p

Endocytosis of GLP-1/<i>h</i>IgG2 is via a dynamin dependent manner.

<p>(A) Internalization of GLP-1/hIgG2-Fc in INS-1 cells (A) Cells were incubated with 1 µM GLP-1/<i>h</i>IgG2 at 4°C for 30 minutes, then switched to 37°C for 0, 10, 20 and 60 minutes. After fixation and blocking for non-specific binding, cells were then incubated with goat-anti-human IgG antibody (1∶500) followed by secondary Cy3-conjugated anti-goat antibody (1∶500) and Top3 dye (1∶20,000) for nuclei staining. The images were then visualized by a confocal microscope. (B) The internalization experiments were performed in cells co-transfected with either wild type dynamin or dominant negative dynamin and green fluorescent protein (GFP). 24 hours after transfection, internalization of GLP-1/<i>h</i>IgG2-Fc was conducted as described in (A). DAPI (1∶10,000) was used for nuclei staining and images were taken by a Nikon fluorescent microscope.</p