Preclinical study of the long-range safety and anti-inflammatory effects of high-dose oral meglumine.

Meglumine is a methylamino derivative of sorbitol that is an approved drug excipient. Recent preclinical studies suggest that administration of high-dose oral meglumine can exert beneficial medicinal effects to treat diabetes, obesity, and fatty liver disease (NAFLD/nonalcoholic steatohepatitis [NASH]). Here we address gaps in knowledge about the pharmacology and toxicology of this substance administered at high concentrations to explore its medicinal potential. We observed that high-dose meglumine limited secretion of proinflammatory cytokines and cell adhesion molecules from activated human THP-1 or murine RAW264.7 monocytes. Preclinical pharmacokinetic analysis in Swiss mice confirmed that meglumine was orally available. Informed by this data, oral doses of 18 to 75 mM meglumine were administered ad libitum in the drinking water of Sprague-Dawley rats and two cohorts of C57BL/6 mice housed in different vivariums. In a 32-week study, urinary isoprostane levels trended lower in subjects consistent with the possibility of anti-inflammatory effects. In full lifespan studies, there was no detrimental effect on longevity. Heart function evaluated in C57BL/6 mice using an established noninvasive cardiac imaging system showed no detrimental effects on ejection fraction, fractional shortening, left ventricle function or volume, and cardiac output in mice up to 15-month old, with a potential positive trend in heart function noted in elderly mice consistent with earlier reported benefits on muscle stamina. Finally, in a transgenic model of inflammation-associated skin carcinogenesis, the incidence, number, and growth of skin tumors trended lower in subjects receiving meglumine. Overall, the evidence obtained illustrating the long-range safety of high-dose oral meglumine support the rationale for its evaluation as a low-cost modality to limit diabetes, hypertriglyceridemia, and NAFLD/NASH.

Cell-based assays to detect inhibitors of fungal mRNA capping enzymes and characterization of sinefungin as a cap methyltransferase inhibitor.

The m7GpppN cap at the 5' end of eukaryotic mRNAs is important for transcript stability and translation. Three enzymatic activities that generate the mRNA cap include an RNA 5'-triphosphatase, an RNA guanylyltransferase, and an RNA (guanine-7-) -methyltransferase. The physical organization of the genes encoding these enzymes differs between mammalian cells and yeast, fungi, or viruses. The catalytic mechanism used by the RNA triphosphatases of mammalian cells also differs from that used by the yeast, fungal, or viral enzymes. These structural and functional differences suggest that inhibitors of mRNA capping might be useful antifungal or antiviral agents. The authors describe several whole-cell yeast-based assays developed to identify and characterize inhibitors of fungal mRNA capping. They also report the identification and characterization of the natural product sinefungin in the assays. Their characterization of this S-adenosylmethionine analog suggests that it inhibits mRNA cap methyltransferases and exhibits approximately 5- to 10-fold specificity for the yeast ABD1 and fungal CCM1 enzymes over the human Hcm1 enzyme expressed in yeast cells.

Meglumine exerts protective effects against features of metabolic syndrome and type II diabetes.

Metabolic syndrome, diabetes and diabetes complications pose a growing medical challenge worldwide, accentuating the need of safe and effective strategies for their clinical management. Here we present preclinical evidence that the sorbitol derivative meglumine (N-methyl-D-glucamine) can safely protect against several features of metabolic syndrome and diabetes, as well as elicit enhancement in muscle stamina. Meglumine is a compound routinely used as an approved excipient to improve drug absorption that has not been ascribed any direct biological effects in vivo. Normal mice (SV129) administered 18 mM meglumine orally for six weeks did not display any gastrointestinal or other observable adverse effects, but had a marked effect on enhancing muscle stamina and at longer times in limiting weight gain. In the established KK.Cg-Ay/J model of non-insulin dependent diabetes, oral administration of meglumine significantly improved glycemic control and significantly lowered levels of plasma and liver triglycerides. Compared to untreated control animals, meglumine reduced apparent diabetic nephropathy. Sorbitol can improve blood glucose uptake by liver and muscle in a manner associated with upregulation of the AMPK-related enzyme SNARK, but with undesirable gastrointestinal side effects not seen with meglumine. In murine myoblasts, we found that meglumine increased steady-state SNARK levels in a dose-dependent manner more potently than sorbitol. Taken together, these findings provide support for the clinical evaluation of meglumine as a low-cost, safe supplement offering the potential to improve muscle function, limit metabolic syndrome and reduce diabetic complications.

Time-resolved Forster resonance energy transfer assays for the binding of nucleotide and protein substrates to p38alpha protein kinase.

We have developed assays for the binding of nucleotide and protein substrates to p38alpha protein kinase based on time-resolved Forster resonance energy transfer. p38alpha was biotinylated by addition of a sequence that targets biotin to a single lysine when coexpressed with biotin ligase in Escherichia coli, allowing formation of a complex between a streptavidin "LANCE" europium chelate conjugate and p38alpha. When this reagent was combined with M39AF, a p38 inhibitor containing a fluorescent moiety whose excitation wavelengths match the emission wavelengths of the europium chelate, a change in ratio of light emitted at 665 nm/615 nm is detected. Less than 100pM complex was detected with a signal/background ratio of >30-fold. The complex exhibits slow, tight binding kinetics where the apparent K(d) decreases with a relaxation time of 21 min at 125 pM biotin-p38alpha. Preincubating inhibitors or ATP with biotin-p38alpha and adding M39AF as a competitor yielded IC(50)s consistent with those measured by enzyme assay for the activated form of biotin-p38alpha. The same technique was also used to measure affinity of inhibitors for the unphosphorylated and catalytically inactive form of biotin-p38alpha. To measure affinity of p38alpha for its protein substrate MK2, we incubated biotin-p38alpha with a glutathione S-transferase MK2 fusion protein. Detection of the complex after incubation with streptavidin-allophycocyanin and a LANCE-conjugated anti-GST allowed measurement of affinity of MK2 for biotin-p38alpha and detection of 0.5 nM p38alpha.MK2 complex with signal/background ratio >5-fold. Competition with unbiotinylated p38alpha yielded an IC(50) value of 5 nM. Activation of either p38alpha or MK2 had no effect on the measured K(d). M39AF was found to bind in a ternary complex with p38alpha.MK2 with lower affinity than that observed in the binary complex with p38alpha alone.