Comparative pharmacokinetics and tissue distribution of primaquine enantiomers in mice.

BACKGROUND: Primaquine (PQ) has been used for the radical cure of relapsing Plasmodium vivax malaria for more than 60 years. PQ is also recommended for prophylaxis and prevention of transmission of Plasmodium falciparum. However, clinical utility of PQ has been limited due to toxicity in individuals with genetic deficiencies in glucose 6-phosphate dehydrogenase (G6PD). PQ is currently approved for clinical use as a racemic mixture. Recent studies in animals as well as humans have established differential pharmacological and toxicological properties of the two enantiomers of PQ. This has been attributed to differential metabolism and pharmacokinetics of individual PQ enantiomers. The aim of the current study is to evaluate the comparative pharmacokinetics (PK), tissue distribution and metabolic profiles of the individual enantiomers in mice. METHODS: Two groups of 21 male Albino ND4 Swiss mice were dosed orally with 45 mg/kg of S-(+)-PQ and R-(-)PQ respectively. Each of the enantiomers was comprised of a 50:50 mixture of 12C- and 13C- stable isotope labelled species (at 6 carbons on the benzene ring of the quinoline core). Three mice were euthanized from each group at different time points (at 0, 0.5, 1, 2, 4, 8, 24 h) and blood was collected by terminal cardiac bleed. Liver, spleen, lungs, kidneys and brain were removed, extracted and analysed using UPLC/MS. The metabolites were profiled by tandem mass (MS/MS) fragmentation profile and fragments with 12C-13C twin peaks. Non-compartmental analysis was performed using the Phoenix WinNonLin PK software module. RESULTS: The plasma AUC0-last (µg h/mL) (1.6 vs. 0.6), T1/2 (h) (1.9 vs. 0.45), and Tmax (h) (1 vs. 0.5) were greater for SPQ as compared to RPQ. Generally, the concentration of SPQ was higher in all tissues. At Tmax, (0.5-1 h in all tissues), the level of SPQ was 3 times that of RPQ in the liver. Measured Cmax of SPQ and RPQ in the liver were about 100 and 40 times the Cmax values in plasma, respectively. Similar observations were recorded in other tissues where the concentration of SPQ was higher compared to RPQ (2× in the spleen, 6× in the kidneys, and 49× in the lungs) than in the plasma. CPQ, the major metabolite, was preferentially generated from RPQ, with higher levels in all tissues (> 10× in the liver, and 3.5× in the plasma) than from SPQ. The PQ-o-quinone was preferentially formed from the SPQ (> 4× compared to RPQ), with higher concentrations in the liver. CONCLUSION: These studies show that in mice, PQ enantiomers are differentially biodistributed and metabolized, which may contribute to differential pharmacologic and toxicity profiles of PQ enantiomers. The findings on higher levels of PQ-o-quinone in liver and RBCs compared to plasma and preferential generation of this metabolite from SPQ are consistent with the higher anti-malarial efficacy of SPQ observed in the mouse causal prophylaxis test, and higher haemolytic toxicity in the humanized mouse model of G6PD deficiency. Potential relevance of these findings to clinical use of racemic PQ and other 8-aminoquinolines vis-à-vis need for further clinical evaluation of individual enantiomers are discussed.

Natural Products Inhibitors of Monoamine Oxidases-Potential New Drug Leads for Neuroprotection, Neurological Disorders, and Neuroblastoma.

Monoamine oxidase inhibitors (MAOIs) are an important class of drugs prescribed for treatment of depression and other neurological disorders. Evidence has suggested that patients with atypical depression preferentially respond to natural product MAOIs. This review presents a comprehensive survey of the natural products, predominantly from plant sources, as potential new MAOI drug leads. The psychoactive properties of several traditionally used plants and herbal formulations were attributed to their MAOI constituents. MAO inhibitory constituents may also be responsible for neuroprotective effects of natural products. Different classes of MAOIs were identified from the natural product sources with non-selective as well as selective inhibition of MAO-A and -B. Selective reversible natural product MAOIs may be safer alternatives to the conventional MAOI drugs. Characterization of MAO inhibitory constituents of natural products traditionally used as psychoactive preparations or for treatment of neurological disorders may help in understanding the mechanism of action, optimization of these preparations for desired bioactive properties, and improvement of the therapeutic potential. Potential therapeutic application of natural product MAOIs for treatment of neuroblastoma is also discussed.

Chronic immune polyradiculopathies: Three clinical variants of one disease?

INTRODUCTION: Chronic immune polyradiculopathies (sensory, motor, and mixed) are uncommon. METHODS: In this single-center, retrospective study, the inclusion criteria for participants were progressive sensory ataxia and/or areflexic limb weakness; tibial somatosensory evoked potential (SSEP) abnormalities of the N22 and P40 potentials with normal sensory and motor nerve conduction studies or root involvement, according to magnetic resonance imaging (MRI); and albuminocytological dissociation. RESULTS: Eight patients were included in our study. Two had weakness, two had sensory ataxia, and four had both weakness and ataxia. Patients with weakness had abnormal SSEPs and patients with sensory ataxia also had absent F waves. Electromyography showed chronic denervation. MRI scans confirmed thickening and enhancement of roots. The patients responded to corticosteroid treatment. DISCUSSION: The overlapping clinicoelectrophysiological findings and similarities in radiological and therapeutic responses suggest that these entities are clinical variants of the same disease. The terms CIS(m)P, CI(s)MP, and CISMP (for chronic immune sensory motor polyradiculopathy) could be used to denote the predominant clinical involvement.

Primaquine Loaded Solid Lipid Nanoparticles (SLN), Nanostructured Lipid Carriers (NLC), and Nanoemulsion (NE): Effect of Lipid Matrix and Surfactant on Drug Entrapment, in vitro Release, and ex vivo Hemolysis.

Primaquine (PQ), an 8-aminoquinoline antimalarial drug, has been widely used for the eradication of hypnozoites from the liver and, therefore, recognized as the radical cure of malaria. However, the clinical applications of PQ are restricted to patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency due to severe dose-related hemolytic side effects. Nanoparticle carriers have shown great potential in achieving higher PQ concentrations in the target site, thereby reducing dose-related systemic toxicity caused by non-specific exposure. This work aims to develop, compare, and evaluate three PQ-loaded lipid-based drug carriers including solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLC), and nano-emulsions (NE). The optimized PQ-SLN, PQ-NLC, and PQ-NE had a particle size of 250 nm, a PDI range of 0.1 to 0.3, a zeta potential of - 30 mV, and entrapment efficiency of ~ 90%. All lipid formulations showed sustained release in both simulated gastric and intestinal fluids over 6 h. Four empirical models - including zero-order, Higuchi, Korsmeyer-Peppas, and Hixson-Crowell models - were tested to understand the drug release mechanisms of PQ-SLN, PQ-NLC, and PQ-NE. The model fitness was found to be the highest in the Korsmeyer-Peppas model for all the PQ-loaded lipid formulations (R2: 0.88-0.94). No significant changes were observed in the entrapment efficiency, particle size, and PDI of lipid formulations throughout 1 month of storage at 4 °C and 25 °C. PQ-SLN and PQ-NLC can be further lyophilized with cryoprotectants to improve long-term stability. Finally, the treatment of erythrocytes with PQ-SLN, PQ-NLC, and PQ-NE reduced erythrocyte hemolysis by approximately 4.5-fold compared to the free drug solution.

Selective Interactions of O-Methylated Flavonoid Natural Products with Human Monoamine Oxidase-A and -B.

A set of structurally related O-methylated flavonoid natural products isolated from Senecio roseiflorus (1), Polygonum senegalense (2 and 3), Bhaphia macrocalyx (4), Gardenia ternifolia (5), and Psiadia punctulata (6) plant species were characterized for their interaction with human monoamine oxidases (MAO-A and -B) in vitro. Compounds 1, 2, and 5 showed selective inhibition of MAO-A, while 4 and 6 showed selective inhibition of MAO-B. Compound 3 showed ~2-fold selectivity towards inhibition of MAO-A. Binding of compounds 1-3 and 5 with MAO-A, and compounds 3 and 6 with MAO-B was reversible and not time-independent. The analysis of enzyme-inhibition kinetics suggested a reversible-competitive mechanism for inhibition of MAO-A by 1 and 3, while a partially-reversible mixed-type inhibition by 5. Similarly, enzyme inhibition-kinetics analysis with compounds 3, 4, and 6, suggested a competitive reversible inhibition of MAO-B. The molecular docking study suggested that 1 selectively interacts with the active-site of human MAO-A near N5 of FAD. The calculated binding free energies of the O-methylated flavonoids (1 and 4-6) and chalcones (2 and 3) to MAO-A matched closely with the trend in the experimental IC50's. Analysis of the binding free-energies suggested better interaction of 4 and 6 with MAO-B than with MAO-A. The natural O-methylated flavonoid (1) with highly potent inhibition (IC50 33 nM; Ki 37.9 nM) and >292 fold selectivity against human MAO-A (vs. MAO-B) provides a new drug lead for the treatment of neurological disorders.

Promising selective MAO-B inhibition by sesamin, a lignan from Zanthoxylum flavum stems.

Monoamine oxidase inhibition is an important therapeutic approach for various neurodegenerative disorders. Reversible MAO inhibitors selectively targeting only one isoform possess substantial merit in terms of safety, efficacy, and side effect profile. This study aimed to isolate the secondary metabolites of Zanthoxylum flavum stems and evaluate their recombinant human MAO inhibition, antimicrobial, and antiprotozoal activities. As a result, fourteen compounds were isolated and identified (nine of them were reported from Z. flavum for the first time). Compound 3 (sesamin) exhibited potent selective MAO-B inhibition (IC50 value of 1.45 ± 0.05 µM) which reported herein for the first time. Compound 2 showed selective MAO-A inhibition activity, compound 5 exhibited good trypanocidal activity, and compound 7 displayed moderate antibacterial activity. The promising MAO-B inhibitory activity of sesamin provoked us to further explore the kinetic properties, the binding mode, and the underlying mechanism of MAO-B inhibition by this lignan. This detailed investigation substantiated a reversible binding and mixed MAO-B catalytic function inhibition via sesamin (Ki: 0.473 ± 0.076 µM). Selectivity and reversibility of sesamin on MAO-B provide exciting prerequisites for further in vivo investigation to confirm its therapeutic potentiality.

Formation primaquine-5,6-orthoquinone, the putative active and toxic metabolite of primaquine via direct oxidation in human erythrocytes.

BACKGROUND: The activity and haemolytic toxicity associated with primaquine has been linked to its reactive metabolites. The reactive metabolites are thought to be primarily formed through the action of cytochrome P450-mediated pathways. Human erythrocytes generally are not considered a significant contributor to drug biotransformation. As erythrocytes are the target of primaquine toxicity, the ability of erythrocytes to mediate the formation of reactive oxidative primaquine metabolites in the absence of hepatic enzymes, was evaluated. METHODS: Primaquine and its enantiomers were incubated separately with human red blood cells and haemoglobin. Post-incubation analysis was performed with UPLC-MS/MS to identify products of biotransformation. RESULTS: The major metabolite detected was identified as primaquine-5,6-orthoquinone, reflecting the pathway yielding putative active and haematotoxic metabolites of primaquine, which was formed by oxidative demethylation of 5-hydroxyprimaquine. Incubation of primaquine with haemoglobin in a cell-free system yielded similar results. It appears that the observed biotransformation is due to non-enzymatic processes, perhaps due to reactive oxygen species (ROS) present in erythrocytes or in the haemoglobin incubates. CONCLUSION: This study presents new evidence that primaquine-5,6-orthoquinone, the metabolite of primaquine reflecting the oxidative biotransformation pathway, is generated in erythrocytes, probably by non-enzymatic means, and may not require transport from the liver or other tissues.

Selective Inhibition of Human Monoamine Oxidase B by Acacetin 7-Methyl Ether Isolated from Turnera diffusa (Damiana).

The investigation of the constituents that were isolated from Turnera diffusa (damiana) for their inhibitory activities against recombinant human monoamine oxidases (MAO-A and MAO-B) in vitro identified acacetin 7-methyl ether as a potent selective inhibitor of MAO-B (IC50 = 198 nM). Acacetin 7-methyl ether (also known as 5-hydroxy-4', 7-dimethoxyflavone) is a naturally occurring flavone that is present in many plants and vegetables. Acacetin 7-methyl ether was four-fold less potent as an inhibitor of MAO-B when compared to acacetin (IC50 = 50 nM). However, acacetin 7-methyl ether was >500-fold selective against MAO-B over MAO-A as compared to only two-fold selectivity shown by acacetin. Even though the IC50 for inhibition of MAO-B by acacetin 7-methyl ether was ~four-fold higher than that of the standard drug deprenyl (i.e., SelegilineTM or ZelaparTM, a selective MAO-B inhibitor), acacetin 7-methyl ether's selectivity for MAO-B over MAO-A inhibition was greater than that of deprenyl (>500- vs. 450-fold). The binding of acacetin 7-methyl ether to MAO-B was reversible and time-independent, as revealed by enzyme-inhibitor complex equilibrium dialysis assays. The investigation on the enzyme inhibition-kinetics analysis with varying concentrations of acacetin 7-methyl ether and the substrate (kynuramine) suggested a competitive mechanism of inhibition of MAO-B by acacetin 7-methyl ether with Ki value of 45 nM. The docking scores and binding-free energies of acacetin 7-methyl ether to the X-ray crystal structures of MAO-A and MAO-B confirmed the selectivity of binding of this molecule to MAO-B over MAO-A. In addition, molecular dynamics results also revealed that acacetin 7-methyl ether formed a stable and strong complex with MAO-B. The selective inhibition of MAO-B suggests further investigations on acacetin 7-methyl as a potential new drug lead for the treatment of neurodegenerative disorders, including Parkinson's disease.

Metabolism of primaquine in normal human volunteers: investigation of phase I and phase II metabolites from plasma and urine using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry.

BACKGROUND: Primaquine (PQ), an 8-aminoquinoline, is the only drug approved by the United States Food and Drug Administration for radical cure and prevention of relapse in Plasmodium vivax infections. Knowledge of the metabolism of PQ is critical for understanding the therapeutic efficacy and hemolytic toxicity of this drug. Recent in vitro studies with primary human hepatocytes have been useful for developing the ultra high-performance liquid chromatography coupled with high-resolution mass spectrometric (UHPLC-QToF-MS) methods for simultaneous determination of PQ and its metabolites generated through phase I and phase II pathways for drug metabolism. METHODS: These methods were further optimized and applied for phenotyping PQ metabolites from plasma and urine from healthy human volunteers treated with single 45 mg dose of PQ. Identity of the metabolites was predicted by MetaboLynx using LC-MS/MS fragmentation patterns. Selected metabolites were confirmed with appropriate standards. RESULTS: Besides PQ and carboxy PQ (cPQ), the major plasma metabolite, thirty-four additional metabolites were identified in human plasma and urine. Based on these metabolites, PQ is viewed as metabolized in humans via three pathways. Pathway 1 involves direct glucuronide/glucose/carbamate/acetate conjugation of PQ. Pathway 2 involves hydroxylation (likely cytochrome P450-mediated) at different positions on the quinoline ring, with mono-, di-, or even tri-hydroxylations possible, and subsequent glucuronide conjugation of the hydroxylated metabolites. Pathway 3 involves the monoamine oxidase catalyzed oxidative deamination of PQ resulting in formation of PQ-aldehyde, PQ alcohol and cPQ, which are further metabolized through additional phase I hydroxylations and/or phase II glucuronide conjugations. CONCLUSION: This approach and these findings augment our understanding and provide comprehensive view of pathways for PQ metabolism in humans. These will advance the clinical studies of PQ metabolism in different populations for different therapeutic regimens and an understanding of the role these play in PQ efficacy and safety outcomes, and their possible relation to metabolizing enzyme polymorphisms.

Pathway-specific inhibition of primaquine metabolism by chloroquine/quinine.

BACKGROUND: There has been some evidence to suggest that the addition of chloroquine (CQ) or quinine (QN) to 8-aminoquinoline (8-AQ) treatment regimens may increase the therapeutic efficacy of the 8-AQ and simultaneously mitigate against its haemolytic toxicity. However, both CQ and QN are considered effective, although perhaps moderate inhibitors of CYP2D6, an enzyme now regarded as necessary for primaquine (PQ) pharmacologic activity. An understanding of the influence of CQ and QN on the metabolism of PQ may shed light on the potential mechanisms of the beneficial interaction. METHODS: Differential metabolism of PQ enantiomers by recombinant human CYP2D6, monoamine oxidase A (MAO), and cryopreserved human hepatocytes in the presence/absence of CQ and QN. RESULTS: Both CQ and QN significantly inhibited the activity of CYP2D6. PQ depletion by MAO and human hepatocytes was not affected significantly by the presence of CQ and QN. CYP2D6-mediated hydroxylation was largely suppressed by both CQ and QN. The formation of the primary deaminated metabolites, including carboxyprimaquine (CPQ) and cyclized side chain derivative from the aldehyde (m/z 241), was not sensitive to the presence of CQ and QN. However, the appearance of the glucuronides of CPQ and PQ alcohol were significantly suppressed. CQ and QN also inhibited the appearance of the m/z 257 metabolite with a similar pattern, suggesting that it may be derived from the CPQ conjugate. The apparent quinone-imine of CPQ (m/z 289) was only partially suppressed by both QN and CQ, but with a differential pattern of inhibition for the two drugs. The m/z 274 (quinone-imine of a ring-hydroxylated PQ metabolite) and m/z 422 (an apparent glucose conjugate of PQ) metabolites in hepatocytes were strongly suppressed by both QN and CQ, perhaps a reflection of the 2D6 inhibition by these drugs. The formation of the carbamoyl glucuronide of PQ (m/z 480) was not affected by CQ/QN. CONCLUSION: The metabolite-specific interactions in the current studies seem at variance with earlier reports of the dependence of PQ on CYP2D6 metabolism, and enhanced PQ anti-malarial activity/reduced toxicity in the presence of CQ/QN. These results suggest a complex picture in which CQ/QN may shift metabolite pathway balances towards a profile that retains efficacy, while reducing the formation or availability of toxic metabolites to erythrocytes. Alternatively, these drugs may alter transport or distribution of PQ metabolites in a fashion that reduces toxicity while maintaining efficacy against the parasite.

Isolation of Acacetin from Calea urticifolia with Inhibitory Properties against Human Monoamine Oxidase-A and -B.

Calea urticifolia (Asteraceae: Asteroideae) has long been used as a traditional medicine in El Salvador to treat arthritis and fever, among other illnesses. The chloroform extract of the leaves of C. urticifolia showed potent inhibition of recombinant human monoamine oxidases (MAO-A and -B). Further bioassay-guided fractionation led to the isolation of a flavonoid, acacetin, as the most prominent MAO inhibitory constituent, with IC50 values of 121 and 49 nM for MAO-A and -B, respectively. The potency of MAO inhibition by acacetin was >5-fold higher for MAO-A (0.121 µM vs 0.640 µM) and >22-fold higher for MAO-B (0.049 µM vs 1.12 µM) as compared to apigenin, the closest flavone structural analogue. Interaction and binding characteristics of acacetin with MAO-A and -B were determined by enzyme-kinetic assays, enzyme-inhibitor complex binding, equilibrium-dialysis dissociation analyses, and computation analysis. Follow-up studies showed reversible binding of acacetin with human MAO-A and -B, resulting in competitive inhibition. Acacetin showed more preference toward MAO-B than to MAO-A, suggesting its potential for eliciting selective pharmacological effects that might be useful in the treatment of neurological and psychiatric disorders. In addition, the binding modes of acacetin at the enzymatic site of MAO-A and -B were predicted through molecular modeling algorithms, illustrating the high importance of ligand interaction with negative and positive free energy regions of the enzyme active site.

Enantioselective pharmacokinetics of primaquine in healthy human volunteers.

Primaquine (PQ), a racemic drug, is the only treatment available for radical cure of relapsing Plasmodium vivax malaria and blocking transmission of P. falciparum malaria. Recent studies have shown differential pharmacologic and toxicologic profiles of individual PQ enantiomers in rodent, dog, and primate animal models. This study was conducted in six healthy adult human volunteers to determine the plasma pharmacokinetic profile of enantiomers of PQ and carboxyprimaquine (cPQ), the major plasma metabolite. The individuals were orally administered PQ diphosphate, equivalent to 45-mg base, 30 minutes after a normal breakfast. Blood samples were collected at different time intervals, and plasma samples were analyzed for enantiomers of PQ and cPQ. Plasma PQ concentrations were low and variable for both parent enantiomers and peaked around 2-4 hours. Peak (-)-(R)-PQ concentrations ranged from 121 ng/ml to 221 ng/ml, and peak (+)-(S)-PQ concentrations ranged from 168 ng/ml to 299 ng/ml. The cPQ concentrations were much higher and were surprisingly consistent from subject to subject. Essentially all the cPQ detected in plasma was (-)-cPQ. The peak concentrations of (-)-cPQ were observed at 8 hours (range: 1104-1756 ng/ml); however, very high concentrations were sustained through 24 hours. (+)-cPQ was two orders of magnitude lower than (-)-cPQ, and in a few subjects it was detected but only under the limit of quantification. In vitro studies with primary human hepatocytes also suggested more rapid metabolism of (-)-PQ compared with (+)-PQ. The results suggest more rapid metabolism of (-)-PQ to (-) cPQ compared with (+)-PQ. Alternatively, (+)-PQ or (+)-cPQ could be rapidly converted to another metabolite(s) or distributed to tissues. This is the first clinical report on enantioselective pharmacokinetic profiles of PQ and cPQ and supports further clinical evaluation of individual PQ enantiomers.

Effect of living conditions on biochemical and hematological parameters of the cynomolgus monkey.

The cynomolgus monkey (Macaca fascicularis) has been increasingly used in biomedical research. Although living conditions affect behavioral and physiological characteristics in macaques, little data is available on how living conditions influence blood-based parameters in the cynomolgus monkey. We hypothesize that there are significant differences in serum biochemical and hematological parameters in single-caged versus socially housed cynomolgus monkeys, and that age and sex influence the effect of living conditions on these parameters. Sixty single-caged and 60 socially housed cynomolgus monkeys were segregated by age group (juvenile, adult) and sex. The effects of living condition, age, sex, and the interactions between these factors on commonly reported serum biochemical and hematological parameters were analyzed by a three-way analysis of variance (ANOVA). Then, the differences between single-caged and socially housed subjects were tested in each parameter by Student's t-test. Creatinine, glucose, triglyceride, alanine aminotransferase, red blood cell volume distribution width (SD, CV), median fluorescence reticulocyte percentage, white blood cell and basophil counts, and monocyte (count, %) were lower in single-caged subjects. Blood urea nitrogen and globulin were lower in single-caged juveniles and adults, respectively. Red blood cell count, hemoglobin, hematocrit, and neutrophil (count, %) were higher, and reticulocyte and lymphocyte (counts, %) were lower, in single-caged juveniles. Mean corpuscular hemoglobin concentration was higher in single-caged subjects (but more pronounced in adults). Total protein was higher in single-caged juvenile males and lower in single-caged adult females. Alkaline phosphatase was lower in single-caged juvenile females. Mean corpuscular hemoglobin was higher, and high fluorescence reticulocyte percentage was lower, in single-caged adult males. In conclusion, living conditions significantly affect several serum biochemical and hematological parameters in the cynomolgus monkey, and these effects vary by age and sex. As this macaque is commonly housed under different living conditions, these findings should aid researchers in avoiding inaccurate conclusions concerning this species.

Monoamine oxidase inhibitory constituents of propolis: kinetics and mechanism of inhibition of recombinant human MAO-A and MAO-B.

Propolis is the resinous material that bees gather from leaf buds, flowers and vegetables. Propolis extracts contain constituents with a broad spectra of pharmacological properties and are important ingredients of popular dietary supplements. Propolis extracts were evaluated in vitro for inhibition of recombinant human monoamine oxidase (MAO)-A and MAO-B. The dichloromethane extract of propolis showed potent inhibition of human MAO-A and MAO-B. Further fractionation identified the most active fractions as rich in flavonoids. Galangin and apigenin were identified as the principal MAO-inhibitory constituents. Inhibition of MAO-A by galangin was about 36 times more selective than MAO-B, while apigenin selectivity for MAO-A vs. MAO-B was about 1.7 fold. Apigenin inhibited MAO-B significantly more potently than galangin. Galangin and apigenin were further evaluated for kinetic characteristics and the mechanism for the enzymes' inhibition. Binding of galangin and apigenin with MAO-A and -B was not time-dependent and was reversible, as suggested by enzyme-inhibitor binding and dissociation-dialysis assay. The inhibition kinetics studies suggested that galangin and apigenin inhibited MAO-A and -B by a competitive mechanism. Presence of prominent MAO inhibitory constituents in propolis products suggests their potential for eliciting pharmacological effects that might be useful in depression or other neurological disorders. The results may also have important implications in drug-dietary supplement interactions.

Early-stage mapping of macromolecular content in APPNL-F mouse model of Alzheimer's disease using nuclear Overhauser effect MRI.

Non-invasive methods of detecting early-stage Alzheimer's disease (AD) can provide valuable insight into disease pathology, improving the diagnosis and treatment of AD. Nuclear Overhauser enhancement (NOE) MRI is a technique that provides image contrast sensitive to lipid and protein content in the brain. These macromolecules have been shown to be altered in Alzheimer's pathology, with early disruptions in cell membrane integrity and signaling pathways leading to the buildup of amyloid-beta plaques and neurofibrillary tangles. We used template-based analyzes of NOE MRI data and the characteristic Z-spectrum, with parameters optimized for increase specificity to NOE, to detect changes in lipids and proteins in an AD mouse model that recapitulates features of human AD. We find changes in NOE contrast in the hippocampus, hypothalamus, entorhinal cortex, and fimbria, with these changes likely attributed to disruptions in the phospholipid bilayer of cell membranes in both gray and white matter regions. This study suggests that NOE MRI may be a useful tool for monitoring early-stage changes in lipid-mediated metabolism in AD and other disorders with high spatial resolution.

Stroke-induced hexokinase 2 in circulating monocytes exacerbates vascular inflammation and atheroprogression.

BACKGROUND: Stroke accelerates inflammatory monocyte recruitment to the endothelium and consequent atheroprogression via high-mobility group box 1-receptor for advanced glycation end products signaling. Notably, Hmgb1 interacts with multiple toll-like receptors (TLRs) and promotes TLR4-mediated proinflammatory myeloid cell activation. Therefore, TLR-associated mechanism(s) within monocytes may play a role in Hmgb1-driven poststroke atheroprogression. OBJECTIVES: We aimed to elucidate the TLR-associated mechanism(s) within monocytes that contribute to stroke-induced exacerbation of atherosclerotic disease. METHODS: A weighted gene coexpression network analysis on the whole blood transcriptomes of stroke model mice identified hexokinase 2 (HK2) as a key gene associated with TLR signaling in ischemic stroke. We conducted a cross-sectional analysis of monocyte HK2 levels in patients with ischemic stroke patients. We performed in vitro and in vivo studies using high-cholesterol diet-fed myeloid-specific Hk2-null ApoE-/- (ApoE-/-;Hk2ΔMφ) mice and ApoE-/-;Hk2fl/fl controls. RESULTS: We found markedly higher monocyte HK2 levels in patients with ischemic stroke patients during the acute and subacute phases poststroke. Similarly, stroke model mice displayed a profound increase in monocyte Hk2 levels. Using aortas and aortic valve samples collected from high-cholesterol diet-fed ApoE-/-;Hk2ΔMφ mice and ApoE-/-;Hk2fl/fl controls, we found that stroke-induced monocyte Hk2 upregulation enhanced poststroke atheroprogression and inflammatory monocyte recruitment to the endothelium. Stroke-induced monocyte Hk2 upregulation induced inflammatory monocyte activation, systemic inflammation, and atheroprogression via Il-1ß. Mechanistically, we demonstrated that stroke-induced monocyte Hk2 upregulation was dependent upon Hmgb1-driven p38-dependent hypoxia-inducible factor-1α stabilization. CONCLUSION: Stroke-induced monocyte Hk2 upregulation is a key mechanism underlying poststroke vascular inflammation and atheroprogression.

Synthesis and evaluation of aplysinopsin analogs as inhibitors of human monoamine oxidase A and B.

Aplysinopsins are tryptophan-derived natural products that have been isolated from a variety of marine organisms. Previous studies have shown aplysinopsin analogs to possess a variety of biological activities, including modulation of neurotransmissions. A series of fifty aplysinopsin analogs was synthesized and assayed for monoamine oxidase A and B inhibitory activity. Three compounds displayed significant MAO inhibitory activity and selectivity. The compound (E)-5-[(6-bromo-1H-indol-3-yl)methylene]-2-imino-1,3-dimethylimidazolidin-4-one (3x) possessed an IC(50) of 5.6 nM at MAO-A and had a selectivity index of 80.24. An SAR study revealed that multiple N-methylations, one of which should be at position N-2', and bromination at C-5 or C-6 are important factors for MAO-A potency and selectivity.

Inhibition of human monoamine oxidase A and B by 5-phenoxy 8-aminoquinoline analogs.

8-Aminoquinolines (8-AQs) are important class of anti-infective therapeutics. 5-Phenoxy 8-aminoquinoline analogs have shown improved metabolic stability compared to primaquine. In view or predictive role of monoamine oxidases (MAO) in metabolism of 8-aminoquinolines the 5-phenoxy analogs were evaluated in vitro for the inhibition of recombinant human MAO-A and MAO-B. The analogs were several folds more potent inhibitors of MAO-A and MAO-B compared to primaquine, the parent drug, with selectivity for MAO-B. 5-(4-Trifluoromethylphenoxy)-4-methylprimaquine (6) Inhibited MAO-B with IC(50) value of 150 nM (626-fold more potent than primaquine). These results will have important implications in optimizing metabolic stability of 8-AQs to improve therapeutic value and also indicate scope for development of 8-AQs as selective MAO inhibitors.

Withanolide biosynthesis recruits both mevalonate and DOXP pathways of isoprenogenesis in Ashwagandha Withania somnifera L. (Dunal).

Withanolides are pharmaceutically important C(28)-phytochemicals produced in most prodigal amounts and diversified forms by Withania somnifera. Metabolic origin of withanolides from triterpenoid pathway intermediates implies that isoprenogenesis could significantly govern withanolide production. In plants, isoprenogenesis occurs via two routes: mevalonate (MVA) pathway in cytosol and non-mevalonate or DOXP/MEP pathway in plastids. We have investigated relative carbon contribution of MVA and DOXP pathways to withanolide biosynthesis in W. somnifera. The quantitative NMR-based biosynthetic study involved tracing of (13)C label from (13)C(1)-D-glucose to withaferin A in withanolide producing in vitro microshoot cultures of the plant. Enrichment of (13)C abundance at each carbon of withaferin A from (13)C(1)-glucose-fed cultures was monitored by normalization and integration of NMR signal intensities. The pattern of carbon position-specific (13)C enrichment of withaferin A was analyzed by a retro-biosynthetic approach using a squalene-intermediated metabolic model of withanolide (withaferin A) biosynthesis. The pattern suggested that both DOXP and MVA pathways of isoprenogenesis were significantly involved in withanolide biosynthesis with their relative contribution on the ratio of 25:75, respectively. The results have been discussed in a new conceptual line of biosynthetic load-driven model of relative recruitment of DOXP and MVA pathways for biosynthesis of isoprenoids. Key message The study elucidates significant contribution of DOXP pathway to withanolide biosynthesis. A new connotation of biosynthetic load-based role of DOXP/MVA recruitment in isoprenoid biosynthesis has been proposed.