Microwave-assisted digestion and NaOH treatment of waste-activated sludge to recover phosphorus by crystallizing struvite

<p>A number of studies of waste-activated sludge (WAS) pretreatments, aimed at releasing phosphorus (P) from WAS and increasing the amount of P that can be recovered, have been performed. Here, a microwave-assisted digestion and NaOH treatment (MWs & NaOH) coupled crystallizing struvite, to promote the solubilization, transformation, and recovery of P from WAS, is proposed. Microwaves (MWs) can cause cavities to form in WAS, weakening the bonds between extracellular polymeric substances and the solid phase. Irradiating with MWs significantly increased the efficiency at which P was dissolved (i.e. transferred from the solid to the liquid phase) and the efficiency at which organic P was hydrolyzed and transformed into inorganic P when the NaOH treatment was performed. The P solubilization and transformation characteristic achieved in different treatments was examined by scanning electron microscopy and three-dimensional excitation emission matrix analysis. The MWs & NaOH method released 34.20–43.73% of total P from WAS, and 23.48–32.07% of the total P was recovered by crystallizing struvite at pH 9.5 and Mg:P ratio of 1.5:1. It would cost about USD 85–103 per ton of dry WAS to treat WAS using the MWs & NaOH method.</p

Differing roles for short chain fatty acids and GPR43 agonism in the regulation of intestinal barrier function and immune responses

<div><p>Inflammatory bowel disease (IBD) is associated with a loss of intestinal barrier function and dysregulated immune responses. It has been shown that short chain fatty acids (SCFAs) are protective in IBD and that GPR43 mediates the protective effects of SCFAs. In this study, we investigated the effects of SCFAs in comparison to highly specific GPR43 agonists on human intestinal epithelial and immune cells. Our results confirm that SCFAs are enhancers of barrier function in intestinal epithelial cells. Additionally, SCFAs also displayed potent immunoregulatory properties based upon the ability to inhibit LPS-induced cytokine production in PBMC, and human T cell proliferation and cytokine production. Unexpectedly, and in contrast to the current belief, specific GPR43 agonists failed to exhibit similar barrier enhancing and anti-inflammatory properties. These findings demonstrate that SCFA possess broad protective functions in IBD and agonizing GPR43 alone is unlikely to be beneficial in patients.</p></div

Enhancement of intestinal epithelial barrier function by butyrate.

<p>(A) C2BBe1 cells were cultured in the apical chamber of transwells in the ECIS transwell array. Barrier resistance was measured on the ECIS instrument @ 75 Hz beginning at day 2 post-addition of the test agents with resistance values normalized to those observed just prior to the addition of test agents. (B) Cells were cultured as in A for 2 days. The media was then replaced with media containing 2.5 mM EGTA (both apical and basolateral chambers) for 5 hours prior to washing and replacing with media containing test agents. Cells were monitored over the next 48 hours for barrier reformation and the plots shown are at 75 Hz with resistance values normalized to those observed just prior to the addition of test agents. The results shown are representative of 2 independent experiments.</p

Expression of GPR43 in intestinal cells.

<p>(A) The expression of GPR43 mRNA in the indicated cell lines was measured using droplet digital PCR (ddPCR). Parental CHO cells and cells transfected with GPR43 served as the negative and positive controls, respectively. (B, C) GPR43 expression in different regions of the mouse intestinal tissue (B) or purified intestinal epithelial cells (C) was measured using ddPCR. Results were expressed as copies of GPR43 per ng of isolated RNA. Plots represent the average of duplicates/triplicates with standard deviation.</p

Potency of GPR43 agonists.

<p>(A, B) Inhibition of forskolin-induced cAMP production in CHO cells stably expressing human GPR43 was measured in the presence of the SCFA (A) or GPR43 agonists (B). The data was plotted as percent of control (POC), to normalize to the levels of cAMP obtained following stimulation with forskolin alone. (C, D) GPR43 agonist-induced receptor activation in CHO-K1 cells stably expressing human GPR43 was determined by measuring the increase in cytosolic calcium concentrations. This was performed with the indicated GPR43 agonists in the absence (C) or presence (D) of 10% FBS. Plots represent the average, and the results shown are representative of 3 independent experiments.</p

Anti-inflammatory effects of butyrate on LPS-induced cytokine production.

<p>Human PBMC were incubated with the indicated concentrations of butyrate (mM range) or GPR43 agonists (μM range) for 45 minutes prior to the addition of 1 μg/mL LPS. The levels of TNFα, IL-1β and IL-6 production in the supernatants were measured 18 hours later. Inhibition curves were plotted and IC₅₀ concentrations are indicated in parenthesis when applicable. For the panel displaying cell viability, the concentrations of SCFA in mM and agonists in μM were plotted along the same axis for easier viewing. The data plotted are the values obtained following the subtraction of background levels of cytokines obtained without LPS stimulation and represent the average of duplicates with standard deviation. The results shown are representative of 2 independent experiments consisting of a total of 4 donors.</p

Optimization of GPR40 Agonists for Type 2 Diabetes

GPR40 (FFA1 and FFAR1) has gained significant interest as a target for the treatment of type 2 diabetes. TAK-875 (<b>1</b>), a GPR40 agonist, lowered hemoglobin A1c (HbA1c) and lowered both postprandial and fasting blood glucose levels in type 2 diabetic patients in phase II clinical trials. We optimized phenylpropanoic acid derivatives as GPR40 agonists and identified AMG 837 (<b>2</b>) as a clinical candidate. Here we report our efforts in searching for structurally distinct back-ups for AMG 837. These efforts led to the identification of more polar GPR40 agonists, such as AM-4668 (<b>10</b>), that have improved potency, excellent pharmacokinetic properties across species, and minimum central nervous system (CNS) penetration

Improving the Pharmacokinetics of GPR40/FFA1 Full Agonists

We recently reported the discovery of a potent GPR40 full agonist AM-1638 (<b>1</b>). Herein, we describe our efforts in improving the drug-like properties of the full agonists through the systematic introduction of polar groups in the C-, D-, and A-rings. This led to the discovery of new GPR40 full agonists with significantly improved pharmacokinetic propeties. Compound <b>8</b> and <b>20</b> also showed potent in vivo efficacy in oral glucose tolerance tests in mice in addition to the improvement in properties

Discovery and Optimization of Potent GPR40 Full Agonists Containing Tricyclic Spirocycles

GPR40 (FFAR1 or FFA1) is a target of high interest being pursued to treat type II diabetes due to its unique mechanism leading to little risk of hypoglycemia. We recently reported the discovery of AM-1638 (<b>2</b>), a potent full agonist of GPR40. In this report, we present the discovery of GPR40 full agonists containing conformationally constrained tricyclic spirocycles and their structure–activity relationships leading to more potent agonists such as AM-5262 (<b>26</b>) with improved rat PK profile and general selectivity profile. AM-5262 enhanced glucose stimulated insulin secretion (mouse and human islets) and improved glucose homeostasis in vivo (OGTT in HF/STZ mice) when compared to AM-1638

Discovery of Potent and Simplified Piperidinone-Based Inhibitors of the MDM2–p53 Interaction

Continued optimization of the N-substituent in the piperidinone series provided potent piperidinone–pyridine inhibitors <b>6</b>, <b>7</b>, <b>14</b>, and <b>15</b> with improved pharmacokinetic properties in rats. Reducing structure complexity of the <i>N</i>-alkyl substituent led to the discovery of <b>23</b>, a potent and simplified inhibitor of MDM2. Compound <b>23</b> exhibits excellent pharmacokinetic properties and substantial in vivo antitumor activity in the SJSA-1 osteosarcoma xenograft mouse model