Glucose-Reducing Effect of the ORMD-0801 Oral Insulin Preparation in Patients with Uncontrolled Type 1 Diabetes: A Pilot Study

<div><p>The unpredictable behavior of uncontrolled type 1 diabetes often involves frequent swings in blood glucose levels that impact maintenance of a daily routine. An intensified insulin regimen is often unsuccessful, while other therapeutic options, such as amylin analog injections, use of continuous glucose sensors, and islet or pancreas transplantation are of limited clinical use. In efforts to provide patients with a more compliable treatment method, Oramed Pharmaceuticals tested the capacity of its oral insulin capsule (ORMD-0801, 8 mg insulin) in addressing this resistant clinical state. Eight Type I diabetes patients with uncontrolled diabetes (HbA1c: 7.5–10%) were monitored throughout the 15-day study period by means of a blind continuous glucose monitoring device. Baseline patient blood glucose behavior was monitored and recorded over a five-day pretreatment screening period. During the ensuing ten-day treatment phase, patients were asked to conduct themselves as usual and to self-administer an oral insulin capsule three times daily, just prior to meal intake. CGM data sufficient for pharmacodynamics analyses were obtained from 6 of the 8 subjects. Treatment with ORMD-0801 was associated with a significant 24.4% reduction in the frequencies of glucose readings >200 mg/dL (60.1±7.9% pretreatment vs. 45.4±4.9% during ORMD-0801 treatment; <i>p</i> = 0.023) and a significant mean 16.6% decrease in glucose area under the curve (AUC) (66055±5547 mg/dL/24 hours vs. 55060±3068 mg/dL/24 hours, <i>p</i> = 0.023), with a greater decrease during the early evening hours. In conclusion, ORMD-0801 oral insulin capsules in conjunction with subcutaneous insulin injections, well tolerated and effectively reduced glycemia throughout the day.</p><p>Trial Registration</p><p>Clinicaltrials.gov <a href="http://clinicaltrials.gov/show/NCT00867594" target="_blank">NCT00867594</a>.</p></div

Mean glucose concentrations before and during ORMD-0801 oral insulin support therapy.

<p>A. Mean blood glucose levels of six Type I diabetic subjects, continuously monitored throughout the pretreatment (blue) and ORMD-0801, oral insulin-treatment (red) phases (dotted lines represent the corresponding standard errors). The greatest reduction (21.2%) is noted between 5–7pm. B. ORMD-0801 treatment was associated with a mean 16.6% decrease in glucose area under the curve (AUC) (66055±5547 mg/dL/24 hours before treatment vs. 55060±3068 mg/dL/24 hours during ORMD-0801 treatment,*- <i>p</i> = 0.023).</p

Patient demographics and baseline characteristics.

<p>Patient demographics and baseline characteristics.</p

Study flow diagram.

<p>Study flow diagram.</p

Frequency of recordings of blood glucose concentrations below 70 mg/dL or above 200 mg/dL (% of total measurements obtained by continuous glucose monitoring).

<p>Frequency of recordings of blood glucose concentrations below 70 mg/dL or above 200 mg/dL (% of total measurements obtained by continuous glucose monitoring).</p

Inhibition of NF-κB activation is associated with an increased endocrine/total graft area ratio in the islet graft.

<p><b>A.</b> The recipient graft-bearing kidneys from normoglycemic untreated mice, and Dox- or Bortezomib (BZB)-treated animals were removed 7 days after allogeneic transplantation, fixed in formaldehyde, thin-sliced and stained with hematoxylin/eosin solution. The border between the kidney and the graft is marked (Upper panel). Paraffin sections were stained for insulin (Lower panel). <b>B.</b> Using a fixed grid, the percentage of endocrine area was calculated from the total graft area. Results are the average of at least five non-consecutive sections incorporating the whole graft area. *p = 0.033 Dox <i>vs</i> control; **p = 0.0001 BZB <i>vs</i> control n = 3.</p

Real-time PCR analysis of NF-κB target genes in islet grafts 24 hours after transplantation.

<p>CXCL-10/IP-10 (*p = 0.03 Dox <i>vs</i> control) (<b>A</b>), MCP-1 (<b>B</b>), cIAP2 (<b>C</b>)<b>,</b> iNOS (<b>D</b>), A20 (<b>E</b>) and XIAP (<b>F</b>). mRNA was extracted from ToI-β islet grafts retrieved from the kidney capsule 24 hours after syngeneic transplantation. Prior to transplantation, islet grafts were exposed to Dox in the culture media for 48 hours (D) or untreated controls (C). The right columns represent relative gene expression in isolated, untreated islets (ISLETS). Results are shown as fold induction normalized to HPRT values. Only retrieved grafts with less than 5% kidney contamination were included in the study, as assessed by expression of the kidney tissue-specific NKT (novel kidney transcript) gene. Results are the mean ± SEM of three to five independent experiments.</p

Decreased IP-10 in doxycycline- or Bortezomib-treated islet grafts.

<p>Representative anti-CXCL10/IP-10 stained graft-bearing kidney sections from normoglycemic untreated mice (control), Dox- or Bortezomib (BZB)-treated animals, 7 days after allogeneic transplantation (Upper panel- Graft). As noted in control the cytoplasmic IP-10 staining is more intense in control islets than in Dox- or Bortezomib-treated mice. The lower panel represents IP-10 staining of kidney sections of the corresponding graft adjacent areas.</p

Medium nitrite levels secreted from islets exposed in vitro to IL-1β (50 units/ml) and IFN-γ (1,000 units/ml) for 48 h in the presence or absence of Bortezomib (BZB- 100

<p> <b>nM).</b> Nitrite data are pooled from two separate experiments incorporating 5–6 repeats of each treatment, presented as the mean ± SEM. *p value = 0.023 for BZB+Cytokines <i>vs</i> Cytokines.</p

Increased lipid availability for three days reduces whole body glucose uptake, impairs muscle mitochondrial function and initiates opposing effects on PGC-1α promoter methylation in healthy subjects

<div><p>Aims</p><p>FFA and FFA metabolites cause insulin resistance and impair beta cell function. The goal of our research was to examine whether elevation of plasma FFA impairs mitochondrial function and alters PGC-1α promoter methylation.</p><p>Methods</p><p>In this uncontrolled, change from baseline study design, insulin sensitivity and glucose-stimulated insulin secretion were measured in 9 normal glucose tolerant subjects before and after 3 day lipid infusion to elevate plasma FFA concentration. Vastus lateralis muscle biopsies were obtained and mitochondrial function, PGC-1α expression, and PGC-1α promoter methylation were quantitated.</p><p>Results</p><p>Increased plasma FFA (440±93 μmol/Lto 997±242 μM, <i>p</i><0.001) decreased insulin-stimulated total glucose disposal (TGD) by 25% (<i>p</i> = 0.008), impaired suppression of endogenous glucose production (<i>p</i> = 0.01), and reduced mitochondrial ATP synthesis with complex 1 (34%, <i>p</i><0.05) and complex 2 (30%, <i>p</i><0.05) substrates. Lipid infusion had no effect on muscle PGC-1α RNA expression, total methylation or non-CpG methylation, but methylation of the alternative PGC-1α promoter decreased (1.30±0.30 to 0.84±0.15% methylated residues/patient•strand, <i>p</i> = 0.055). Within PGC-1α promoter there was demethylation of CpT residues (0.72±0.16 vs. 0.28±0.10 methylated residues/patient•strand) (<i>p</i> = 0.002), which was inversely correlated with PGC-1α mRNA expression (r = -0.94, <i>p</i><0.0001) and ATP synthesis with complex 1 (r = -0.80, p<0.01) and complex 2 (r = -0.69, <i>p</i><0.05) substrates. Lipid infusion increased DNMT-3B (methyltransferase associated with PGC-1α promoter non-CpG methylation) mRNA expression (0.87 ± 0.09 to 1.62 ± 0.22 arbitrary units, <i>p</i> = 0.005), which correlated inversely with CpT demethylation (r = 0.67, <i>p</i><0.05).</p><p>Conclusion/Interpretation</p><p>Physiologic plasma FFA elevation in NGT individuals has opposing effects on PGC-1α non-CpG residue methylation (CpT demethylation and increased DNMT-3B expression), which is correlated with changes in PGC-1α expression and skeletal muscle mitochondrial function.</p></div