Correction to “Discovery of AZD4831, a Mechanism-Based Irreversible Inhibitor of Myeloperoxidase, As a Potential Treatment for Heart Failure with Preserved Ejection Fraction”

Correction to “Discovery of AZD4831, a Mechanism-Based Irreversible Inhibitor of Myeloperoxidase, As a Potential Treatment for Heart Failure with Preserved Ejection Fraction

Novel Zn²⁺ Modulated GPR39 Receptor Agonists Do Not Drive Acute Insulin Secretion in Rodents

<div><p>Type 2 diabetes (T2D) occurs when there is insufficient insulin release to control blood glucose, due to insulin resistance and impaired β-cell function. The GPR39 receptor is expressed in metabolic tissues including pancreatic β-cells and has been proposed as a T2D target. Specifically, GPR39 agonists might improve β-cell function leading to more adequate and sustained insulin release and glucose control. The present study aimed to test the hypothesis that GPR39 agonism would improve glucose stimulated insulin secretion <i>in vivo</i>. A high throughput screen, followed by a medicinal chemistry program, identified three novel potent Zn²⁺ modulated GPR39 agonists. These agonists were evaluated in acute rodent glucose tolerance tests. The results showed a lack of glucose lowering and insulinotropic effects not only in lean mice, but also in diet-induced obese (DIO) mice and Zucker fatty rats. It is concluded that Zn²⁺ modulated GPR39 agonists do not acutely stimulate insulin release in rodents.</p></div

Functionalized lipid nanoparticles for subcutaneous administration of mRNA to achieve systemic exposures of a therapeutic protein

Lipid nanoparticles (LNPs) are the most clinically advanced delivery system for RNA-based drugs but have predominantly been investigated for intravenous and intramuscular administration. Subcutaneous administration opens the possibility of patient self-administration and hence long-term chronic treatment that could enable messenger RNA (mRNA) to be used as a novel modality for protein replacement or regenerative therapies. In this study, we show that subcutaneous administration of mRNA formulated within LNPs can result in measurable plasma exposure of a secreted protein. However, subcutaneous administration of mRNA formulated within LNPs was observed to be associated with dose-limiting inflammatory responses. To overcome this limitation, we investigated the concept of incorporating aliphatic ester prodrugs of anti-inflammatory steroids within LNPs, i.e., functionalized LNPs to suppress the inflammatory response. We show that the effectiveness of this approach depends on the alkyl chain length of the ester prodrug, which determines its retention at the site of administration. An unexpected additional benefit to this approach is the prolongation observed in the duration of protein expression. Our results demonstrate that subcutaneous administration of mRNA formulated in functionalized LNPs is a viable approach to achieving systemic levels of therapeutic proteins, which has the added benefits of being amenable to self-administration when chronic treatment is required

Plasma concentration–time profile of test compounds in the IPGTT using DIO mice; AZ7914 (<i>A</i>), AZ4237 (<i>B</i>), AZ1395 (<i>C</i>).

<p>Compounds were IP administered to fasted DIO mice at -30 min as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145849#pone.0145849.g008" target="_blank">Fig 8</a> legend. Data represent the value of pooled samples from 7 animals.</p

Insulin secretion assays in mouse islets.

<p>Effect on insulin secretion by each compound. Islets were treated with 1 and 10 μM of AZ7914 (<i>A</i>), AZ4237 (<i>B</i>), AZ1395 (<i>C</i>). The assays were performed in the absence or presence of 5 μM of Zn²⁺. Values were calculated as the ratio of insulin concentration compared to the basal control and expressed as the average of three separate measurements ± SEM.</p

AZ7914 (<i>A</i>), AZ4237 (<i>B</i>) and AZ1395 (<i>C</i>) ten point concentration response curves for <i>in vitro</i> screens.

<p>Concentration responses of compounds were measured with DMR, IP₁ and cAMP assays in the absence (-) or presence of 5 μM Zn²⁺ (+) as indicated in the figure. Lines represent fits to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145849#pone.0145849.e001" target="_blank">Eq 1</a>. Cells employed were HEK293s-hGPR39 (hGPR39), untransfected HEK293s (HEK), and NIT-1 cells endogenously expressing GPR39. Responses were normalized to AZ1395 in each assay and presented as % effect of control. For HEK293s and HEK293s-hGPR39 cells, individual DMR assays were run in singlicates and IP₁ and cAMP assays were run in triplicates. Values shown are means ± SEM of typically three (two to five) independent experiments. For NIT-1 cells, IP₁ assays were typically run in duplicates (one to four experiments), and values are shown as means with error bars representing range. EC₅₀ values calculated from the data in Fig 2 are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145849#pone.0145849.t001" target="_blank">Table 1</a>.</p

AZ7914 (<i>A</i>), AZ4237 (<i>B</i>), AZ1395 (<i>C</i>) and Zn²⁺ (<i>D</i>) concentration response curves in back-scattering Interferometry binding assays.

<p>Compounds were incubated with membranes from HEK293s-hGPR39 and HEK293s cells. AZ7914, AZ4237 and AZ1395 were incubated in the presence of 5 μM ZnCl₂. Signals from HEK293s membranes (reference curves) were subtracted from HEK293s-hGPR39 membrane signals at each concentration point to derive GPR39 specific binding (difference curves). Values shown are means ± SD of three (AZ7914, AZ4237 and AZ1395) or four (Zn²⁺) independent experiments. One-site binding models were used to calculate <i>K</i>_D values for AZ7914, AZ4237 and AZ1395 summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145849#pone.0145849.t001" target="_blank">Table 1</a>. For Zn²⁺, data was fitted to a two-site binding model.</p

Chemical structures of four GPR39 agonists.

<p>AZ7914, 6-(3-chloro-2-fluoro-benzoyl)-2-(2-methylthiazol-4-yl)-3,5,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-one; AZ1395, 3,4-bis(2-imidazol-1-ylethoxy)benzonitrile; AZ4237, 6-[(4-chlorophenyl)methyl]-7-hydroxy-5-methyl-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid.</p

<i>In vivo</i> exposures <i>versus in vitro</i> potencies for AZ7914 (<i>A</i>), AZ4237 (<i>B</i>), AZ1395 (<i>C</i>).

<p>C_<i>u</i>,_<i>av</i> (μM) / EC₅₀ (μM) and C_<i>u</i>,_<i>av</i> (μM) / <i>K</i>_D (μM) calculated for different compound doses (30 or 100 mg/kg) IP administered to normal C57BL/6J mice. EC₅₀ and <i>K</i>_D values were from <i>in vitro</i> assays (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145849#pone.0145849.t001" target="_blank">Table 1</a>) performed in the absence (w/o) or presence of 5 μM Zn²⁺ (+) and adjusted for protein binding. a) Compound weakly active in <i>in vitro</i> assay: Potency >33 μM, E_max ≥ 8%. b) Compound inactive in <i>in vitro</i> assay: Potency >33 μM, E_max < 2%. For bars labeled with a) and b), EC₅₀ = 33 μM has been used for calculations, resulting in overestimation of C_<i>u</i>,_<i>av</i> / EC₅₀ ratios. This is indicated with open bars. Closed bars indicate that EC₅₀ values have been determined to a defined value. ND, not determined.</p