quantitative-analysis-dietary-vitamin-metabolites-murine-ocular-non

Quantitative Analysis of Dietary Vitamin A Metabolites in Murine Ocular and Non-Ocular Tissues Using High-Performance Liquid Chromatography

G protein-coupled receptors (GPCRs) are a superfamily of transmembrane proteins that initiate signaling cascades through activation of its G protein upon ...

University of Minnesota

Endoplasmic reticulum (ER) stress underlies a variety of disorders involving inflammation, such as diabetes, neurodegenerative diseases. Guanabenz acetate (Wytensin®, GA), a clinically approved antihypertensive drug, efficiently counteracts ER stress. The entirety of clinically used GA is the E-isomer, while the Z-isomer is known to lack significant hypotensive properties. We recently discovered that the Z-isomer retains anti-ER stress activity. Coupled with its lack of sedative effects, (Z)-GA is well positioned as a potential therapeutic for a host of ER stress-related disorders. We set forth to characterize the metabolism and pharmacokinetics (DMPK) of (Z)-GA in vitro and in vivo. Toward this end, a reliable and sensitive LC-MS/MS method for simultaneous determination of the (E)- and (Z)-guanabenz was developed. Chromatographic separation of the isomers was achieved on a C18 reverse phase column with a gradient elution. Tandem mass spectrometric detection was conducted using an AB Sciex 5500 QTrap mass spectrometer with positive electrospray ionization. Full validation of the method was performed in mouse plasma with a simple and low plasma volume protein precipitation procedure. The method demonstrated good linearity, reproducibility, and accuracy over a range of 0.5-1000 nM with minimal matrix effect and excellent extraction efficiency. In addition, the developed method was successfully applied to DMPK studies of the GA isomers in vitro and in vivo. Results of these studies revealed for the first time that the DMPK profile of (Z)-guanabenz is distinct from that of (E)-guanabenz, with higher apparent volume of distribution (V) and clearance, presumably due to lower plasma protein binding.

LC-MS/MS determination of guanabenz E/Z isomers and its application to in vitro and in vivo DMPK profiling studies.

Increased oxidative stress and inflammation are implicated in the pathogenesis of Alzheimer's disease. Treatment with hydrogen sulfide (HS) and HS donors such as sodium hydrosulfide (NaSH) can reduce oxidative stress in preclinical studies, however clinical benefits of such treatments are rather ambiguous. This is partly due to poor stability and bioavailability of the HS donors, requiring impractically large doses that are associated with dose-limiting toxicity. Herein, we identified a bioavailable 3-mercaptopyruvate prodrug, sulfanegen, which is able to pose as a sacrificial redox substrate for 3-mercaptopyruvate sulfurtransferase (3MST), one of the HS biosynthetic enzymes in the brain. Sulfanegen is able to mitigate toxicity emanating from oxidative insults and the Aβ peptide by releasing HS through the 3MST pathway. When administered to symptomatic transgenic mouse model of AD (APP/PS1; 7 and 12 months) and mice that were intracerebroventricularly administered with the Aβ peptide, sulfanegen was able to reverse oxidative and neuroinflammatory consequences of AD pathology by restoring 3MST function. Quantitative neuropathological analyses confirmed significant disease modifying effect of the compound on amyloid plaque burden and brain inflammatory markers. More importantly, sulfanegen treatment attenuated progressive neurodegeneration in these mice, as evident from the restoration of TH+ neurons in the locus coeruleus. This study demonstrates a previously unknown concept that supplementation of 3MST function in the brain may be a viable approach for the management of AD. Finally, brought into the spotlight is the potential of sulfanegen as a promising AD therapeutic for future drug development efforts.

Sulfanegen stimulates 3-mercaptopyruvate sulfurtransferase activity and ameliorates Alzheimer's disease pathology and oxidative stress in vivo.

The endogenous opioid system regulates pain through local release of neuropeptides and modulation of their action on opioid receptors. However, the effect of opioid peptides, the enkephalins, is short-lived due to their rapid hydrolysis by enkephalin-degrading enzymes. In turn, an innovative approach to the management of pain would be to increase the local concentration and prolong the stability of enkephalins by preventing their inactivation by neural enkephalinases such as puromycin sensitive aminopeptidase (PSA). Our previous structure-activity relationship studies offered the S-diphenylmethyl cysteinyl derivative of puromycin () as a nanomolar inhibitor of PSA. This chemical class, however, suffered from undesirable metabolism to nephrotoxic puromycin aminonucleoside (PAN). To prevent such toxicity, we designed and synthesized 5'-chloro substituted derivatives. The compounds retained the PSA inhibitory potency of the corresponding 5'-hydroxy analogs and had improved selectivity toward PSA. In vivo treatment with the lead compound  caused significantly reduced pain response in antinociception assays, alone and in combination with Met-enkephalin. The analgesic effect was reversed by the opioid antagonist naloxone, suggesting the involvement of opioid receptors. Further, PSA inhibition by compound  in brain slices caused local increase in endogenous enkephalin levels, corroborating our rationale. Pharmacokinetic assessment of compound  showed desirable plasma stability and identified the cysteinyl sulfur as the principal site of metabolic liability. We gained additional insight into inhibitor-PSA interactions by molecular modeling, which underscored the importance of bulky aromatic amino acid in puromycin scaffold. The results of this study strongly support our rationale for the development of PSA inhibitors for effective pain management.

Modulation of endogenous opioid signaling by inhibitors of puromycin sensitive aminopeptidase.

The endogenous opioid system regulates pain through local release of neuropeptides and modulation of their action on opioid receptors. However, the effect of opioid peptides, the enkephalins, is short-lived due to their rapid hydrolysis by enkephalin-degrading enzymes. In turn, an innovative approach to the management of pain would be to increase the local concentration and prolong the stability of enkephalins by preventing their inactivation by neural enkephalinases such as puromycin-sensitive aminopeptidase (PSA). Our previous structure-activity relationship studies offered the S-diphenylmethyl cysteinyl derivative of puromycin (20) as a nanomolar inhibitor of PSA. This chemical class, however, suffered from undesirable metabolism to nephrotoxic puromycin aminonucleoside (PAN). To prevent such toxicity, we designed and synthesized 5'-chloro substituted derivatives. The compounds retained the PSA inhibitory potency of the corresponding 5'-hydroxy analogs and had improved selectivity toward PSA. In vivo treatment with the lead compound 19 caused significantly reduced pain response in antinociception assays, alone and in combination with Met-enkephalin. The analgesic effect was reversed by the opioid antagonist naloxone, suggesting the involvement of opioid receptors. Further, PSA inhibition by compound 19 in brain slices caused local increase in endogenous enkephalin levels, corroborating our rationale. Pharmacokinetic assessment of compound 19 showed desirable plasma stability and identified the cysteinyl sulfur as the principal site of metabolic liability. We gained additional insight into inhibitor-PSA interactions by molecular modeling, which underscored the importance of bulky aromatic amino acid in puromycin scaffold. The results of this study strongly support our rationale for the development of PSA inhibitors for effective pain management.

Modulation of endogenous opioid signaling by inhibitors of puromycin-sensitive aminopeptidase.

The renin-angiotensin system (RAS) is known to affect diverse physiological processes that affect the functioning of many key organs. Angiotensin-converting enzyme (ACE) modulates a variety of bioactive peptides associated with pain. ACE inhibitors (ACEis) have found applications in the treatment of cardiovascular, kidney, neurological and metabolic disorders. However, ACEis also tend to display undesirable effects, resulting in increased pain sensitization and mechanical allodynia. In this review, we provide comprehensive discussion of preclinical and clinical studies involving the evaluation of various clinically approved ACEis. With the emerging knowledge of additional factors involved in RAS signaling and the indistinct pharmacological role of ACE substrates in pain, extensive studies are still required to elucidate the mechanistic role of ACE in pain perception.

Swati More

Center for Drug Design

University of Minnesota

Quantitative Analysis of Dietary Vitamin A Metabolites in Murine Ocular and Non-Ocular Tissues Using High-Performance Liquid Chromatography

Swati More

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Center for Drug Design

University of Minnesota

Quantitative Analysis of Dietary Vitamin A Metabolites in Murine Ocular and Non-Ocular Tissues Using High-Performance Liquid Chromatography

Center for Drug Design