Substituted 4-methylcoumarin inhibitors of SLC26A3 (DRA) for treatment of constipation and hyperoxaluria.

SLC26A3, also known as downregulated in adenoma (DRA), is an anion (Cl-, HCO3- and oxalate) exchanger in the luminal membrane of intestinal epithelial cells. Loss of DRA function in mice and humans causes congenital chloride-losing diarrhea and reduces urinary excretion of oxalate, a major constituent of kidney stones. Thus, inhibition of DRA is a potential treatment approach for constipation and calcium oxalate kidney stones. High-throughput screening previously identified 4,8-dimethylcoumarins (4a-4c) as DRA inhibitors, with lead candidate 4b having an IC50 of 40-50 nM for DRA inhibition. Here, we explored the effects of varying substituents at the 8-position, and replacing 8-methyl by 5-methyl (4e-4h). A focused library of 17 substituted compounds (4d-4t) was synthesized with good yield and purity. Compounds were tested for DRA inhibition potency using Fischer rat thyroid cells stably expressing DRA and a halide-sensitive YFP. Structure-activity analysis revealed that 8-bromo- (4m-4p) and 8-fluoro-coumarins (4q-4t) were slightly less potent than the corresponding 8-chloro analogs, demonstrating that the size of methyl or chloro substituents at the coumarin 8 position affects the potency. An analog containing 8-chlorocoumarin (4k) had ∼2-fold improved potency (IC50 25 nM) compared with the original lead candidate 4b. 5,8-Dimethylcoumarins were active against DRA, but with much lower potency than 4,8-disubstituted coumarins. In mice, orally administered 4k at 10 mg kg-1 reduced constipation and normalized stool water content in a loperamide-induced constipation model with comparable efficacy to 4b. Pharmacokinetic analysis of orally administered 4k at 10 mg kg-1 in mice indicated serum levels of >10 µM for at least six hours after single dose. This study expands SAR knowledge of 4,8-disubstituted coumarin inhibitors of DRA as novel drug candidates for constipation and kidney stones.

Mg2+ supplementation treats secretory diarrhea in mice by activating calcium-sensing receptor in intestinal epithelial cells.

Cholera is a global health problem with no targeted therapies. The Ca2+-sensing receptor (CaSR) is a regulator of intestinal ion transport and a therapeutic target for diarrhea, and Ca2+ is considered its main agonist. We found that increasing extracellular Ca2+ had a minimal effect on forskolin-induced Cl- secretion in human intestinal epithelial T84 cells. However, extracellular Mg2+, an often-neglected CaSR agonist, suppressed forskolin-induced Cl- secretion in T84 cells by 65% at physiological levels seen in stool (10 mM). The effect of Mg2+ occurred via the CaSR/Gq signaling that led to cAMP hydrolysis. Mg2+ (10 mM) also suppressed Cl- secretion induced by cholera toxin, heat-stable E. coli enterotoxin, and vasoactive intestinal peptide by 50%. In mouse intestinal closed loops, luminal Mg2+ treatment (20 mM) inhibited cholera toxin-induced fluid accumulation by 40%. In a mouse intestinal perfusion model of cholera, addition of 10 mM Mg2+ to the perfusate reversed net fluid transport from secretion to absorption. These results suggest that Mg2+ is the key CaSR activator in mouse and human intestinal epithelia at physiological levels in stool. Since stool Mg2+ concentrations in patients with cholera are essentially zero, oral Mg2+ supplementation, alone or in an oral rehydration solution, could be a potential therapy for cholera and other cyclic nucleotide-mediated secretory diarrheas.

"'It is very difficult in this business if you want to have a good conscience': pharmaceutical governance and on-the-ground ethical labor in Ghana": a letter to editors.

Establishing effective pharmaceutical governance is a challenge for government agencies, private enterprises, and professionals working on the ground, demanding complex ethical decisions from the actors involved, especially in a lower-middle-income country like Ghana. This letter aims to share the author's perspectives and additional considerations on the analyses of the reports in the paper "It is very difficult in this business if you want to have a good conscience": pharmaceutical governance and on-the-ground ethical labor in Ghana by Hampshire et al. The letter's authors discuss the need to advance universal health coverage in Ghana, the everyday ethics, and the disparities between the collective and individual moral consciousness of the participants, as well as other aspects of governance in the pharmaceutical sector.

Anserine is expressed in human cardiac and skeletal muscles.

We evaluated whether anserine, a methylated analog of the dipeptide carnosine, is present in the cardiac and skeletal muscles of humans and whether the CARNMT1 gene, which encodes the anserine synthesizing enzyme carnosine-N-methyltransferase, is expressed in human skeletal muscle. We found that anserine is present at low concentrations (low micromolar range) in both cardiac and skeletal muscles, and that anserine content in skeletal muscle is ~15 times higher than in cardiac muscle (cardiac muscle: 10.1 ± 13.4 µmol·kg-1 of dry muscle, n = 12; skeletal muscle: 158.1 ± 68.5 µmol·kg-1 of dry muscle, n = 11, p < 0.0001). Anserine content in the heart was highly variable between individuals, ranging from 1.4 to 45.4 µmol·kg-1 of dry muscle, but anserine content was not associated with sex, age, or body mass. We also showed that CARNMT1 gene is poorly expressed in skeletal muscle (n = 10). This is the first study to demonstrate that anserine is present in the ventricle of the human heart. The presence of anserine in human heart and the confirmation of its expression in human skeletal muscle open new avenues of investigation on the specific and differential physiological functions of histidine dipeptides in striated muscles.

Small molecule inhibitors of intestinal epithelial anion exchanger SLC26A3 (DRA) with a luminal, extracellular site of action.

The anion exchanger protein SLC26A3 (down-regulated in adenoma, DRA) is expressed in the luminal membrane of intestinal epithelial cells in colon, where it facilitates the absorption of Cl- and oxalate. We previously identified a 4,8-dimethylcoumarin class of SLC26A3 inhibitors that act from the SLC26A3 cytoplasmic surface, and demonstrated their efficacy in mouse models of constipation and hyperoxaluria. Here, screening of 50,000 new compounds and 1740 chemical analogs of active compounds from the primary screen produced five novel classes of SLC26A3-selective inhibitors (1,3-dioxoisoindoline-amides; N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)acetamides; thiazolo-pyrimidin-5-ones; 3-carboxy-2-phenylbenzofurans and benzoxazin-4-ones) with IC50 down to 100 nM. Kinetic washout and onset of action studies revealed an extracellular site of action for the thiazolo-pyrimidin-5-one and 3-carboxy-2-phenylbenzofuran inhibitors. Molecular docking computations revealed putative binding sites for these inhibitors. In a loperamide model of constipation in mice, orally administered 7-(2-chloro-phenoxymethyl)-3-phenyl-thiazolo [3,2-a]pyrimidin-5-one (3a) significantly increased stool weight, pellet number and water content. SLC26A3 inhibitors with an extracellular site of action offer the possibility of creating non-absorbable, luminally acting inhibitors with minimal systemic exposure following oral administration. Our findings also suggest that inhibitors of related SLC26 anion transporters with an extracellular site of action might be identified for pharmacological modulation of selected epithelial ion transport processes.

Physiological Roles of Carnosine in Myocardial Function and Health.

Carnosine is a pleiotropic histidine-containing dipeptide synthesized from ß-alanine and l-histidine, with the intact dipeptide and constituent amino acids being available from the diet. The therapeutic application of carnosine in myocardial tissue is promising, with carnosine playing a potentially beneficial role in both healthy and diseased myocardial models. This narrative review discusses the role of carnosine in myocardial function and health, including an overview of the metabolic pathway of carnosine in the myocardial tissue, the roles carnosine may play in the myocardium, and a critical analysis of the literature, focusing on the effect of exogenous carnosine and its precursors on myocardial function. By so doing, we aim to identify current gaps in the literature, thereby identifying considerations for future research.

Number of high-protein containing meals correlates with muscle mass in pre-frail and frail elderly.

BACKGROUND: Aging is accompanied by the inability to optimally respond to anabolic stimulus of nutrition, with consequent loss of muscle mass and functionality. It has been speculated that not only total protein intake, but also the per meal protein dose may have important implications to protein balance and, hence, muscle mass in middle-aged and older adults, but evidence is lacking in a more vulnerable population such as the frail elderly. The aim was to investigate possible associations between total protein intake and its per meal dose with multiple measures of muscle mass, strength, and functionality in a cohort of pre-frail and frail elderly individuals. METHODS: One-hundred-and-fifty-seven pre-frail and frail elderly individuals were assessed for total and per meal protein intake (food diaries), total and appendicular lean mass (DXA), vastus lateralis cross-sectional area [(CSA) B-mode ultrasound], and muscle function [leg-press and bench press 1-RM, timed-stands test, timed-up-and-go test, handgrip, and risk of falls (Biodex Balance System®)]. RESULTS: Protein intake and number of meals with either ≥20 g or ≥30 g of protein were significantly associated (after controlling for confounding factors) with greater total and appendicular lean mass and vastus lateralis CSA. CONCLUSIONS: We found that not only total protein intake but also the number of high-protein containing meals are associated with muscle mass in frail and pre-frail elderly.

Does brain creatine content rely on exogenous creatine in healthy youth? A proof-of-principle study.

It has been hypothesized that dietary creatine could influence cognitive performance by increasing brain creatine in developing individuals. This double-blind, randomized, placebo-controlled, proof-of-principle study aimed to investigate the effects of creatine supplementation on cognitive function and brain creatine content in healthy youth. The sample comprised 67 healthy participants aged 10 to 12 years. The participants were given creatine or placebo supplementation for 7 days. At baseline and after the intervention, participants undertook a battery of cognitive tests. In a random subsample of participants, brain creatine content was also assessed in the regions of left dorsolateral prefrontal cortex, left hippocampus, and occipital lobe by proton magnetic resonance spectroscopy (1H-MRS) technique. The scores obtained from verbal learning and executive functions tests did not significantly differ between groups at baseline or after the intervention (all p > 0.05). Creatine content was not significantly different between groups in left dorsolateral prefrontal cortex, left hippocampus, and occipital lobe (all p > 0.05). In conclusion, a 7-day creatine supplementation protocol did not elicit improvements in brain creatine content or cognitive performance in healthy youth, suggesting that this population mainly relies on brain creatine synthesis rather than exogenous creatine intake to maintain brain creatine homeostasis.