Quantitative Determination of (R)-3-Hydroxybutyl (R)-3-Hydroxybutyrate (Ketone Ester) and Its Metabolites Beta-hydroxybutyrate, 1-3-Butanediol, and Acetoacetate in Human Plasma Using LC-MS.

Ketone ester ((R)-3-hydroxybutyl (R)-3-hydroxybutyrate) has gained popularity as an exogenous means to achieve ketosis. Regarding its potential as a therapeutic prodrug, it will be necessary to study its pharmacokinetic profile and its proximal metabolites (beta-hydroxybutyrate, 1,3-butanediol, and acetoacetate) in humans. Here we develop and validate two LC-MS methods for quantifying KE and its metabolites in human plasma. The first assay uses a C18 column to quantitate ketone ester, beta-hydroxybutyrate, and 1,3-butanediol, and the second assay uses a hydrophilic interaction liquid chromatography (HILIC) column for the quantitation of acetoacetate. The method was partially validated for intra- and inter-day accuracy and precision based on the ICH M10 guidelines. For both the assays, the intra- and inter-run accuracy was ±15% of the nominal concentration, and the precision (%CV) was <15% for all 4 molecules being quantified. The matrix effect for all molecules was evaluated and ranged from -62.1 to 44.4% (combined for all molecules), while the extraction recovery ranged from 65.1 to 119% (combined for all molecules). Furthermore, the metabolism of ketone ester in human plasma and human serum albumin was studied using the method. Non-saturable metabolism of ketone ester was seen in human plasma at concentrations as high as 5 mM, and human serum albumin contributed to the metabolism of ketone ester. Together, these assays can be used to track the entire kinetics of ketone ester and its proximal metabolites. The reverse-phase method was used to study the metabolic profile of KE in human plasma and the plasma protein binding of 1,3-BD.

Use of generally recognized as safe or dietary compounds to inhibit buprenorphine metabolism: potential to improve buprenorphine oral bioavailability.

The present study evaluated the potential of five generally recognized as safe (GRAS) or dietary compounds (α-mangostin, chrysin, ginger extract, pterostilbene and silybin) to inhibit oxidative (CYP) and conjugative (UGT) metabolism using pooled human intestinal and liver microsomes. Buprenorphine was chosen as the model substrate as it is extensively metabolized by CYPs to norbuprenorphine and by UGTs to buprenorphine glucuronide. Chrysin, ginger extract, α-mangostin, pterostilbene and silybin were tested for their inhibition of the formation of norbuprenorphine or buprenorphine glucuronide in both intestinal and liver microsomes. Pterostilbene was the most potent inhibitor of norbuprenorphine formation in both intestinal and liver microsomes, with IC50 values of 1.3 and 0.8 µM, respectively, while α-mangostin and silybin most potently inhibited buprenorphine glucuronide formation. The equipotent combination of pterostilbene and ginger extract additively inhibited both pathways in intestinal microsomes. Since pterostilbene and ginger extract showed potent CYP and/or UGT inhibition of buprenorphine metabolism, their equipotent combination was tested to assess the presence of synergistic inhibition. However, because the combination showed additive inhibition, it was not used while performing IVIVE analysis. Based on quantitative in vitro-in vivo extrapolation, pterostilbene (21 mg oral dose) appeared to be most effective in improving the mean predicted Foral and AUC∞ PO of buprenorphine from 3 ± 2% and 340 ± 330 ng*min/ml to 75 ± 8% and 36,000 ± 25,000 ng*min/ml, respectively. At a 10-fold lower dose of pterostilbene, the predicted buprenorphine Foral approximated sublingual bioavailability (~35%) and showed a 2-4 fold reduction in the variability around the predicted AUC∞ PO of buprenorphine. These results demonstrate the feasibility of using various GRAS/dietary compounds to inhibit substantially the metabolism by CYP and UGT enzymes to achieve higher and less variable oral bioavailability. This inhibitor strategy may be useful for drugs suffering from low and variable oral bioavailability due to extensive presystemic oxidative and/or conjugative metabolism.

Effects of generally recognized as safe (GRAS) and dietary compounds on phenylephrine metabolism in LS180 human intestinal cells.

Phenylephrine (PE) has low and variable oral bioavailability in humans, due in part to presystemic metabolism by sulfation. LS180 cells were used as a model of the human intestinal epithelium to examine phenylephrine metabolism and its inhibition by generally recognized as safe (GRAS) and dietary compounds. Curcumin, zingerone, resveratrol, guaiacol, pterostilbene and isoeugenol significantly inhibited phenylephrine disappearance, while vanillin, propylparaben and eugenol did not. However, when propylparaben was combined with either vanillin or eugenol, the phenylephrine disappearance was significantly inhibited. These data suggest that these compounds or combinations thereof may have potential to improve phenylephrine oral bioavailability.

Quantitative determination of buprenorphine, naloxone and their metabolites in rat plasma using hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry.

A rapid and sensitive LC-MS/MS method was developed and validated for the simultaneous determination of buprenorphine and its three metabolites (buprenorphine glucuronide, norbuprenorphine and norbuprenorphine glucuronide) as well as naloxone and its metabolite naloxone glucuronide in the rat plasma. A hydrophilic interaction chromatography column and a mobile phase containing acetonitrile and ammonium formate buffer (pH 3.5) were used for the chromatographic separation. Mass spectrometric detection was achieved by an electrospray ionization source in the positive mode coupled to a triple quadrupole mass analyzer. The calibration curves for the six analytes displayed good linearity over the concentration range 1.0 or 5.0-1000 ng/mL. The intra and inter-day precision (CV) ranged from 2.68 to 16.4% and from 9.02 to 14.5%, respectively. The intra- and inter-day accuracy (bias) ranged from -14.2 to 15.2% and from -9.00 to 4.80%, respectively. The extraction recoveries for all the analytes ranged from 55 to 86.9%. The LC-MS/MS method was successfully applied to a pharmacokinetic study of buprenorphine-naloxone combination in rats.

Inhibition of glucuronidation and oxidative metabolism of buprenorphine using GRAS compounds or dietary constituents/supplements: in vitro proof of concept.

The present study investigated the potential of generally recognized as safe (GRAS) compounds or dietary substances to inhibit the presystemic metabolism of buprenorphine and to increase its oral bioavailability. Using IVIVE, buprenorphine extraction ratios in intestine and liver were predicted as 96% and 71%, respectively. In addition, the relative fraction of buprenorphine metabolized by oxidation and glucuronidation in these two organs was estimated using pooled human intestinal and liver microsomes. In both organs, oxidation appeared to be the major metabolic pathway with a 6 and 4 fold higher intrinsic clearance than glucuronidation in intestine and liver, respectively. The oral bioavailability of buprenorphine was predicted to be 1.16%. Inhibition of 75% and 50% of intestinal and hepatic presystemic metabolism would result in an Foral of 49%, which is comparable to the bioavailability of sublingual buprenorphine. In human liver microsomes, chrysin, curcumin, ginger extract, hesperitin, magnolol, quercetin and silybin inhibited ≥50% glucuronidation, whereas chrysin, curcumin, ginger extract, 6-gingerol, pterostilbene, resveratrol and silybin exhibited ≥30% inhibition of oxidation. In human intestinal microsomes, curcumin, ginger extract, α-mangostin, quercetin and silybin inhibited ≥50% glucuronidation while chrysin, ginger extract, α-mangostin, pterostilbene and resveratrol exhibited ≥30% inhibition of oxidation. These results demonstrate the feasibility of our proposed approach of using GRAS or dietary compounds to inhibit the presystemic metabolism of buprenorphine and thus improve its oral bioavailability. An oral buprenorphine formulation containing these inhibitors or their combinations has promising potential to replace sublingual buprenorphine. Copyright © 2016 John Wiley & Sons, Ltd.

Placental ABC Transporters: Biological Impact and Pharmaceutical Significance.

The human placenta fulfills a variety of essential functions during prenatal life. Several ABC transporters are expressed in the human placenta, where they play a role in the transport of endogenous compounds and may protect the fetus from exogenous compounds such as therapeutic agents, drugs of abuse, and other xenobiotics. To date, considerable progress has been made toward understanding ABC transporters in the placenta. Recent studies on the expression and functional activities are discussed. This review discusses the placental expression and functional roles of several members of ABC transporter subfamilies B, C, and G including MDR1/P-glycoprotein, the MRPs, and BCRP, respectively. Since placental ABC transporters modulate fetal exposure to various compounds, an understanding of their functional and regulatory mechanisms will lead to more optimal medication use when necessary in pregnancy.

Analysis of a major metabolite of ursolic acid- Ursolic acid sulfate, and its quantitative determination on LC/MS in human liver homogenate.

Ursolic acid (UA) is a naturally occurring pentacyclic triterpenoid that exhibits extensive pre-systemic metabolism from in vitro studies. However, there are no available authentic metabolite standards or validated analytical methods to quantitate UA metabolites. We have identified ursolic acid sulfate (UAS) as one of the major metabolites. We were able to identify and characterize its structure via comparison to the chemically synthesized UAS. A cyano (CN, 150 × 4.6 mm, 5 µm) column along with a gradient elution of acetonitrile and 0.08% (v/v) acetic acid, pH 3.0 were employed for chromatographic separation. Negative single ion recording mode (SIR) with electron-spray ionization (ESI) source at mass-to-charge ratios of 455.3 and 535.3 were monitored for UA and UAS, respectively. UAS linearity range was 0.010-2.500 µM. The absolute values of intra-day and inter-day precision (CV, %) and accuracy (DFN, %) were all below 15%. Thus, the analytical method has been validated in the human subcellular fractions to facilitate in vitro/ in vivo DMPK and future clinical disposition studies on UA.

The Caco-2 Model: Modifications and enhancements to improve efficiency and predictive performance.

The Caco-2 cell model has been widely used to assess the permeability of drug candidates. It has provided a high throughput in vitro platform, functionally resembling the enterocytes. Since the oral route is the most preferred for drug administration, the Caco-2 cell model acts as a very important tool to elucidate the oral "druggability" of a molecule by providing a fairly reliable estimate of its permeability through the intestinal membrane. Despite its shortcomings (the lack of a mucus layer, long cultivation period, inter-lab variability, and differences in expression of enzymes, transporters, and tight junction complexes) it remains heavily used due to its reliability, predictive performance, and wide acceptance. Various modifications have been made: co-culturing with other intestinal cells, applying biosimilar mucus, reducing culturing time, combining Caco-2 monolayer with the dissolution apparatus, enhancing protein expression, and redesigning the sampling apparatus. These modifications are intended to overcome some of the shortcomings of the Caco-2 model in order to make its use easier, quicker, economical, and more representative of the intestine. The aim of this review is to discuss such modifications to enhance this model's utility, predictive performance, and reproducibility.

Functional analysis of nonsynonymous single nucleotide polymorphisms of multidrug resistance-associated protein 2 (ABCC2).

BACKGROUND: Multidrug resistance-associated protein 2 (MRP2; ABCC2) mediates the biliary excretion of glutathione, glucuronide, and sulfate conjugates of endobiotics and xenobiotics. Single nucleotide polymorphisms (SNPs) of MRP2 contribute to interindividual variability in drug disposition and ultimately in drug response. OBJECTIVES: To characterize the transport function of human wild-type (WT) MRP2 and four SNP variants, S789F, A1450T, V417I, and T1477M. METHODS: The four SNP variants were expressed in Sf9 cells using recombinant baculovirus infection. The kinetic parameters [Km, (µmol/l); V(max), (pmol/mg/min); the Hill coefficient] of ATP-dependent transport of leukotriene C(4) (LTC(4)), estradiol-3-glucuronide (E(2)3G), estradiol-17ß-glucuronide (E(2)17G), and tauroursodeoxycholic acid (TUDC) were determined in Sf9-derived plasma membrane vesicles. Transport activity was normalized for expression level. RESULTS: The V(max) for transport activity was decreased for all substrates for S789F, and for all substrates except E(2)17G for A1450T. V417I showed decreased apparent affinity for LTC(4), E(2)3G, and E(2)17G, whereas transport was similar between wild-type (WT) and T1477M, except for a modest increase in TUDC transport. Examination of substrate-stimulated MRP2-dependent ATPase activity of S789F and A1450T, SNPs located in MRP2 nucleotide-binding domains (NBDs), demonstrated significantly decreased ATPase activity and only modestly decreased affinity for ATP compared with WT. CONCLUSION: SNPs in the NBDs (S789F in the D-loop of NBD1, or A1450T near the ABC signature motif of NBD2) variably decreased the transport of all substrates. V417I in membrane spanning domain 1 selectively decreased the apparent affinity for the glutathione and glucuronide conjugated substrates, whereas the T1477M SNP in the carboxyl terminus altered only TUDC transport.

Preclinical disposition (in vitro) of novel µ-opioid receptor selective antagonists.

Recently, two novel N-heterocyclic derivatives of naltrexone [designated 17-cyclopropylmethyl-3,14ß-dihydroxy-4,5α-epoxy-6ß-[(4'-pyridyl)acetamido]morphinan (NAP) and 17-cyclopropylmethyl-3,14ß-dihydroxy-4,5α-epoxy-6α-[(3'-isoquinolyl) acetamido]morphinan (NAQ)] have been proposed as µ-opioid receptor (MOR) selective antagonists. The goal of this study was to examine their absorption and metabolism. The bidirectional transport of NAP and NAQ was determined in Caco-2 and MDCKII-MDR1 cells, and the permeability directional ratio (PDR) was estimated (PDR = P(app, B-A)/P(app, A-B), where P(app) is the apparent permeability, A is apical, and B is basolateral). Oxidative metabolism of NAQ (0.5-80 µM) and NAP (0.5-30 µM) was determined in pooled human liver microsomes. The reaction monitored the disappearance of NAQ/NAP. NAP and NAQ were quantitated by high-performance liquid chromatography-UV at 270 or 232 nm, respectively. The permeability of NAQ or NAP was similar to that of naltrexone or paracellular markers, respectively. NAP also exhibited a high PDR and was determined to be a P-glycoprotein (P-gp) substrate. Unbound fractions in human plasma for NAQ and NAP were 0.026 ± 0.019 and 0.85 ± 0.12, respectively. The metabolic oxidative reaction rates, fitted to a Michaelis-Menten model, yielded K(m) and V(max) values of 15.8 ± 5.5 µM and 192 ± 24 pmol/min for NAQ and 1.8 ± 1.5 µM and 8.1 ± 1.4 pmol/min for NAP. Intrinsic hepatic clearance was estimated to be 13 and 5 ml · min(-1) · kg(-1) for NAQ and NAP, respectively. Neither NAQ nor NAP underwent detectable glucuronidation. Thus, NAP was a P-gp substrate with low apparent permeability, whereas NAQ was not a P-gp substrate and showed better permeability. Therefore, in contrast to NAP, NAQ would be more suitable for oral absorption and penetration of the blood-brain barrier, yielding potential pharmacokinetic and pharmacodynamic advantages over naltrexone.

Therapeutic promises of 2-methoxyestradiol and its drug disposition challenges.

2-Methoxyestradiol (2MeO-E2) is an endogenous metabolite of estrogen which was initially considered to be inactive. During the last few decades it has been shown that 2MeO-E2 is a promising anticancer drug. In vitro experiments have demonstrated that it has several anticancer activities, and potential to alleviate hypertension, glomerulosclerosis, hypercholesterolemia, and other disorders. However, due to its low solubility and extensive glucuronidation, to achieve effective concentrations large doses of 2MeO-E2 would be required. Clinical studies reflected very high inter- and intrapatient variability and oral bioavailability of 1 to 2%. Thus, this review paper highlights the origin of this compound, its therapeutic promises, and possible mechanisms of action. It also discusses the pharmacokinetic properties of 2MeO-E2 as well as current developments to overcome low drug solubility and its extensive first pass metabolism.

Pulchinenosides from Pulsatilla Chinensis Increase P-Glycoprotein Activity and Induce P-Glycoprotein Expression.

Five pulchinenosides (pulchinenoside B3, pulchinenoside BD, pulchinenoside B7, pulchinenoside B10, and pulchinenoside B11) isolated from Pulsatilla chinensis (Bge) Regel saponins extract exhibited strong antitumor activities but poor gastrointestinal absorption properties. The enteric induction of P-glycoprotein (P-gp) is understood to restrict the oral bioavailability of some pharmaceutical compounds and lead to adverse drug reactions. Therefore, the present investigation was intended to delineate the impacts of pulchinenosides on cellular P-gp function and expression using Sf9 membrane vesicles and LS180 cells as a surrogate of human intestinal epithelial cells. Preliminary cytotoxic studies showed that 10 µM was an acceptable concentration for cytotoxicity and antiproliferation studies for all pulchinenosides using the alamarBlue assay. The cell cycle of LS180 cells detected by flow cytometry was not significantly influenced after 48 hours of coincubation with 10 µM of pulchinenosides. In the presence of pulchinenosides, the ATP-dependent transport of N-methyl-quinidine mediated by P-glycoprotein was stimulated significantly. The upregulation of P-glycoprotein and mRNA levels was found by Western blot and real-time PCR analysis in LS180 cells. Parallel changes indicate that all pulchinenosides are exposed to pulchinenosides-mediated transcriptional regulation. In conclusion, pulchinenosides could induce P-glycoprotein expression and directly increase its functional activity.

Megalin-targeting liposomes for placental drug delivery.

Every year, complications during pregnancy affect more than 26 million women. Some of those diseases are associated with significant morbidity and mortality, as is the case of preeclampsia, the main cause of maternal deaths globally. The ability to improve the delivery of drugs to the placenta upon administration to the mother may offer new opportunities in the treatment of diseases of pregnancy. The objective of this study was to develop megalin-targeting liposome nanocarriers for placental drug delivery. Megalin is a transmembrane protein involved in clathrin-mediated endocytic processes, and is expressed in the syncytiotrophoblast (SynT), an epithelial layer at maternal-fetal interface. Targeting megalin thus offers an opportunity for the liposomes to hitchhike into the SynT, thus enriching the concentration of any associated therapeutic cargo in the placental tissue. PEGylated (2 KDa) lipids were modified with gentamicin (GM), a substrate to megalin receptors as we have shown in earlier studies, and used to prepare placental-targeting liposomes. The ability of the targeting liposomes to enhance accumulation of a fluorescence probe was assessed in an in vivo placental model - timed-pregnant Balb/c mice at gestational day (GD) 18.5. The targeting liposomes containing 10 mol% GM-modified lipids increased the accumulation of the conjugated fluorescence probe in the placenta with a total accumulation of 2.8% of the initial dose, which corresponds to a 94 fold increase in accumulation compared to the free probe (p < .0001), and 2-4 fold accumulation compared to the non-targeting control liposomes (p < .0001), as measured by both tissue extraction assay and ex vivo imaging. Furthermore, confocal images of placental SynT cross-sections show a 3-fold increase of the targeting liposomes compared with the non-targeting liposomes. The rate and extent of uptake of a fluorescent probe encapsulated within targeting liposomes was also probed in an in vitro model of the human placental barrier (polarized BeWo monolayers) using flow cytometry. Targeting liposomes containing 5 mol% GM-modified lipids enhanced the uptake of the probe by 1.5 fold compared to the non-targeting control. An increase to 10 mol% of the modified lipid resulted in further enhancement in uptake, which was 2 fold greater compared to control. In a competition assay, inhibition of the megalin receptors resulted in a significant reduction in uptake of the fluorescence probe encapsulated in GM-modified liposomes compared to the uptake without free inhibitor (p < .0001), implicating the involvement of megalin receptor in the internalization of the liposomes. Taken together, these results demonstrate that megalin-targeted liposomes may offer an opportunity to enhance the delivery of therapeutics to the placenta for the treatment of diseases of pregnancy.

Binding of lopinavir to human alpha1-acid glycoprotein and serum albumin.

HIV protease inhibitors are an important component of highly active antiretroviral therapy used to treat pregnant women infected with HIV. They have a low placental transfer and are highly plasma protein bound. This study was carried out to determine the unbound fraction of lopinavir in cord blood, and to characterize the binding of lopinavir to alpha(1)-acid glycoprotein (AAG) and human serum albumin (HSA), and displacement by ritonavir. Serum was obtained from cord blood from placentas obtained after cesarean section of healthy, non-HIV-infected women (n = 4). The unbound fraction of lopinavir in serum obtained from this cord blood was 0.022 +/- 0.011%. The unbound fraction of lopinavir in separately obtained maternal serum samples (n = 4) was 0.89 +/- 0.12%, which was not significantly different from that observed with cord serum samples. Varying concentrations of lopinavir, AAG, and HSA in buffer solutions were then used to characterize the lopinavir binding. The data were fit to obtain the number of binding sites (N) and equilibrium dissociation constant (K(D)). Binding of lopinavir to AAG (7-23 microM) was saturable with K(D) of 5.0 +/- 1.1 microM and N of 1.2 +/- 0.2. At low HSA concentrations (15-152 microM), lopinavir binding K(D) was 24.3 +/- 8.7 microM and N was 1.1 +/- 0.4; however, at 758 microM, lopinavir binding was essentially unsaturable. Lopinavir binding to AAG and HSA was not sensitive to ritonavir, and, thus, efforts to enhance fetal exposure to lopinavir should be focused on other issues such as efflux transporters.

Formation and efflux of ATP-binding cassette transporter substrate 2,4-dinitrophenyl-S-glutathione from cultured human term placental villous tissue fragments.

Upon exposure to 1-chloro-2,4-dinitrobenzene (CDNB), the human placental tissue forms its glutathione conjugate 2,4-dinitrophenyl-S-glutathione (DNP-SG). The purpose of this study was to investigate the involvement of human placental ATP-binding cassette (ABC) transporters in the efflux of DNP-SG. Placental tissue samples were obtained from pregnant patients undergoing C-section deliveries following normal pregnancies; villous tissue was cultured in suspension, and DNP-SG formation and efflux upon exposure to 100 microM CDNB were measured by HPLC. DNP-SG efflux decreased by 69.1 (+/-11.3)%, 51.1 (+/-5.4)%, 56.7 (+/-8.3)% and 53.6 (+/-10.8)% (p < 0.05) in the presence of 5 mM sodium orthovanadate (ATPase inhibitor), 100 microM MK571 (MRP-inhibitor), 1 mM dipyridamole (BCRP/P-gp/MRP1-inhibitor) and 100 microM verapamil (P-gp/MRP1 inhibitor) respectively, without any change in DNP-SG formation, total tissue glutathione, GSH/GSSG ratio, tissue integrity or tissue viability. These data clearly established the role of ABC transporters in the human placental efflux of DNP-SG. To investigate the contribution of various ABC transporters toward DNP-SG transport, ATP-dependent transport of 3H-DNP-SG was determined in Sf9 membrane vesicles overexpressing P-gp, BCRP and the MRP proteins. MRP1-mediated DNP-SG transport was inhibited in the presence of sodium orthovanadate, MK571, dipyridamole and verapamil in the presence of glutathione. Furthermore, MRP1-mediated transport [K(t) = 11.3 +/- 1.3 microM and v(max) = 86.7 +/- 1.9 pmol/mg/min] was a high-affinity process compared to MRP2-mediated transport [K(t) = 168 +/- 7 microM and v(max) = 1367 +/- 18 pmol/mg/min]. The inhibition pattern and the kinetics of DNP-SG efflux in the placental villous tissue were consistent with MRP1-mediated DNP-SG efflux, suggesting a functional role and an apical localization for an MRP1-like transporter in the human placental syncytiotrophoblast.

Peroxynitrite and 4-Hydroxynonenal Inactivate Breast Cancer Resistance Protein/ABCG2.

Oxidative stress may arise from a variety of pathologies and results in the formation of toxic and reactive chemical species. Extensive research has been done to establish mechanisms of formation and cytotoxic effects of a number of different products of oxidation stress including peroxynitrite (PN) and 4-hydroxynonenal (4HNE). However, relatively few studies have investigated their effects on ATP-binding cassette (ABC) transporters. The objective of this investigation was to determine the effects of PN and 4HNE on BCRP/ABCG2. To eliminate the effect of metabolic enzymes, the experiments were carried out with inside-out Sf9 membrane vesicles overexpressing BCRP/ABCG2 using riboflavin as a substrate. The experiments revealed that PN produced IC50 of about 31.2 ± 2.7 µM, based upon initial concentrations. The IC50 for 4HNE was estimated to be 92 ± 1.4 µM. Preincubation of membrane vesicles with either PN or 4HNE caused the maximal rate of transport (Vmax) to drop drastically, up to 19 times, with no or much smaller effect on Km. Thus, PN and 4NE can inhibit BCRP transport activity.

Selectivity of Dietary Phenolics for Inhibition of Human Monoamine Oxidases A and B.

Monoamine oxidases (MAOs) regulate local levels of neurotransmitters such as dopamine, norepinephrine, and serotonin and thus have been targeted by drugs for the treatment of certain CNS disorders. However, recent studies have shown that these enzymes are upregulated with age in nervous and cardiac tissues and may be involved in degeneration of these tissues, since their metabolic mechanism releases hydrogen peroxide leading to oxidative stress. Thus, targeting these enzymes may be a potential anti-aging strategy. The purpose of this study was to compare the MAO inhibition and selectivity of selected dietary phenolic compounds, using a previously validated assay that would avoid interference from the compounds. Kynuramine metabolism by human recombinant MAO-A and MAO-B leads to formation of 4-hydroxyquinoline, with Vmax values of 10.2±0.2 and 7.35±0.69 nmol/mg/min, respectively, and Km values of 23.1±0.8 µM and 18.0±2.3 µM, respectively. For oral dosing and interactions with the gastrointestinal tract, curcumin, guaiacol, isoeugenol, pterostilbene, resveratrol, and zingerone were tested at their highest expected luminal concentrations from an oral dose. Each of these significantly inhibited both enzymes except for zingerone, which only inhibited MAO-A. The IC50 values were determined, and selectivity indices (MAO-A/MAO-B IC50 ratios) were calculated. Resveratrol and isoeugenol were selective for MAO-A, with IC50 values of 0.313±0.008 and 3.72±0.20 µM and selectivity indices of 50.5 and 27.4, respectively. Pterostilbene was selective for MAO-B, with IC50 of 0.138±0.013 µM and selectivity index of 0.0103. The inhibition of resveratrol (MAO-A) and pterostilbene (MAO-B) was consistent with competitive time-independent mechanisms. Resveratrol 4'-glucoside was the only compound which inhibited MAO-A, but itself, resveratrol 3-glucoside, and pterostilbene 4'-glucoside failed to inhibit MAO-B. Additional studies are needed to establish the effects of these compounds on MAO-A and/or MAO-B in humans.

Preparation of phenylephrine 3-O-sulfate as the major in vivo metabolite of phenylephrine to facilitate its pharmacokinetic and metabolism studies.

INTRODUCTION: The in vivo disposition and metabolism of phenylephrine have not been establishedby previous analytical methods and there is a lack of available standards for quantitating the metabolites. METHODS: We pursued and compared the preparation of sulfation metabolites of phenylephrine and its ethyl analog etilefrine via chemical and bio-synthesis. RESULTS: Both sulfates were obtained in higher yield and purity through chemical syntheses compared to biosynthesis. DISCUSSION: A facile method for the production of phenylephrine 3-O-sulfate and etilefrine 3-O-sulfate was established. These compounds will be useful in the development of analytical assays for studying the pharmacokinetics of phenylephrine and its main route of metabolism in the presence of formulation changes and pharmacogenetic variation.

Pharmacological characterization of 17-cyclopropylmethyl-3,14-dihydroxy-4,5-epoxy-6-[(3'-fluoro-4'-pyridyl)acetamido]morphinan (NFP) as a dual selective MOR/KOR ligand with potential applications in treating opioid use disorder.

For thousands of years opioids have been the first-line treatment option for pain management. However, the tolerance and addiction potential of opioids limit their applications in clinic. NFP, a MOR/KOR dual-selective opioid antagonist, was identified as a ligand that significantly antagonized the antinociceptive effects of morphine with lesser withdrawal effects than naloxone at similar doses. To validate the potential application of NFP in opioid addiction treatment, a series of in vitro and in vivo assays were conducted to further characterize its pharmacological profile. In calcium mobilization assays and MOR internalization studies, NFP showed the apparent capacity to antagonize DAMGO-induced calcium flux and etorphine-induced MOR internalization. In contrast to the opioid agonists DAMGO and morphine, cells pretreated with NFP did not show apparent desensitization and down regulation of the MOR. Though in vitro bidirectional transport studies showed that NFP might be a P-gp substrate, in warm-water tail-withdrawal assays it was able to antagonize the antinociceptive effects of morphine indicating its potential central nervous system activity. Overall these results suggest that NFP is a promising dual selective opioid antagonist that may have the potential to be used therapeutically in opioid use disorder treatment.

Simultaneous determination of 1-chloro-2,4-dinitrobenzene, 2,4-dinitrophenyl-S-glutathione and its metabolites for human placental disposition studies by high-performance liquid chromatography.

We developed and validated an HPLC method for determination of 1-chloro-2,4-dinitrobenzene (CDNB) and its glutathione conjugate 2,4-dinitrophenyl-S-glutathione (DNP-SG) to study the kinetics and mechanisms involved in DNP-SG formation and efflux, as a probe for human placental metabolism and transport. This method combines use of 3 microm solid phase, rapid mobile phase gradient with dual wavelength ultraviolet detection to permit determination of a lipophilic parent compound and its hydrophilic metabolites in a single short run. The selectivity, linearity, accuracy, precision, relative recovery and stability of the assay are sufficient for determining CDNB, DNP-SG and its metabolites from buffer and tissue samples to support placental drug metabolism and transport studies.