Isolation of 1-(3',4'-Dihydroxyphenyl)-3-(2″,4″,6″-trihydroxyphenyl)-propan-2-ol from Grape Seed Extract and Evaluation of its Antioxidant and Antispasmodic Potential.

HPLC profiling of phenolics in grape seed extracts revealed a prominent peak of an unknown substance with concentrations up to 5.3%. Spectroscopic data allowed the identification of the compound 1 as 1-(3',4'-dihydroxyphenyl)-3-(2″,4″,6″-trihydroxyphenyl)-propan-2-ol. 1 is known to be produced from catechin and epicatechin through anaerobic bacteria from human, as well as the rat, intestines. It was hypothesized that the marc remaining after expression of juice from grapes became infested during storage, resulting in the production of 1. Because compound 1 is infrequently found in nature and has never been found in grape seeds, its presence may be considered a marker of an unwanted anaerobic bacterial process occurring during production. The antioxidant potential of 1 was determined by DPPH, ABTS, and FRAP (ferric reducing antioxidant power) assays and compared to the potential of the following compounds: phloroglucine, pyrogallol, gallic acid, catechin, and epicatechin. Furthermore, it was established that 1 significantly reduced guinea pig ileum contraction induced by histamine.

Design and synthesis of a novel series of histamine H3 receptor antagonists through a scaffold hopping strategy.

Lead compounds 5-fluoro-2-methyl-N-[2-methyl-4-(2-methyl-[1,3']bipyrrolidinyl-1'-yl)-phenyl]-benzamide (1), tetrahydro-pyran-4-carboxylic acid [((2S,3'S)-2-methyl-[1,3']bipyrrolidinyl-1'-yl)-phenyl]-amide (2), and 3,5-dimethyl-isoxazole-4-carboxylic acid [((2S,3'S)-2-methyl-[1,3']bipyrrolidinyl-1'-yl)-phenyl]-amide (3) discovered in our laboratory, displayed high histamine H3 receptor (H3R) affinity, good selectivity and weak human Ether-à-go-go-Related Gene (hERG) channel affinity with desirable overall physico-chemical and pharmacokinetic (PK) profiles. Herein, we describe the design and synthesis of a novel series of H3R antagonists utilizing a scaffold hopping strategy. Further structure-activity relationship (SAR) studies of the series culminated in the identification of ((2S,3'S)-2-methyl-[1,3']bipyrrolidinyl-1'-yl)-naphthalene-2-carboxylic acid (tetrahydro-pyran-4-yl)-amide (4c) and -[4-((2S,3'S)-2-methyl-[1,3']bipyrrolidinyl-1'-yl)-phenyl]-N-(tetrahydro-pyran-4-yl)-acetamide (4d), which exhibited good H3R affinity in vitro, good selectivity, and desirable PK properties. Compounds 4c and 4d were also assessed in cardiac safety experiments. In particular, the effects of the compounds on action potentials recorded from ventricular myocytes isolated from guinea pigs were used to screen compounds that not only displayed a low affinity towards hERG channel, but also had lower interference with other cardiac ion channels. Compound 4c did not alter the major parameters in this model system at ⩽10 µM, and no significant induction of any major haemodynamic effect when intravenously administered at 3mg/kg dose to anaesthetized mongrel dogs. Compound 4c is a new promising lead as orally potent and selective H3R antagonist belonging to a distinct structural class.

Synthesis, characterization, and biological assessment of the four stereoisomers of the H(3) receptor antagonist 5-fluoro-2-methyl-N-[2-methyl-4-(2-methyl[1,3']bipyrrolidinyl-1'-yl)phenyl]benzamide.

This Letter describes the asymmetric synthesis of the four stereoisomers (8a-8d) of a potent and highly selective histamine H3 receptor (H3R) antagonist, 5-fluoro-2-methyl-N-[2-methyl-4-(2-methyl[1,3']bipyrrolidinyl-1'-yl) phenyl]benzamide (1). The physico-chemical properties, in vitro H3R affinities and ADME of 8a-8d were determined. Stereoisomer 8c (2S,3'S) displayed superior in vitro H3R affinity over other three stereoisomers and was selected for further profiling in in vivo PK and drug safety. Compound 8c exhibited excellent PK properties with high exposure, desired brain to plasma ratio and reasonable brain half life. However, all stereoisomers showed similar unwanted hERG affinities.

Identification and profiling of 3,5-dimethyl-isoxazole-4-carboxylic acid [2-methyl-4-((2S,3'S)-2-methyl-[1,3']bipyrrolidinyl-1'-yl)phenyl] amide as histamine H(3) receptor antagonist for the treatment of depression.

Lead optimization guided by histamine H3 receptor (H3R) affinity and calculated physico-chemical properties enabled simultaneous improvement in potency and PK properties leading to the identification of a potent, selective, devoid of hERG issues, orally bioavailable, and CNS penetrable H3R antagonist/inverse agonist 3h. The compound was active in forced-swimming tests suggesting its potential therapeutic utility as an anti-depressive agent. This Letter further includes its cardiovascular and neuropsychological/behavioral safety assessments.

Discovery of aryl ureas and aryl amides as potent and selective histamine H3 receptor antagonists for the treatment of obesity (part I).

A series of structurally novel aryl ureas was derived from optimization of the HTS lead as selective histamine H3 receptor (H3R) antagonists. The SAR was explored and the data obtained set up the starting point and foundation for further optimization. The most potent tool compounds, as exemplified by compounds 2l, 5b, 5d, and 5e, displayed antagonism potencies in the subnanomolar range in in vitro human-H3R FLIPR assays and rhesus monkey H3R binding assays.

Discovery of aryl ureas and aryl amides as potent and selective histamine H3 receptor antagonists for the treatment of obesity (part II).

A novel series of histamine H3 receptor (H3R) antagonists was derived from an arylurea lead series (1) via bioisosteric replacement of the urea functionality by an amide linkage. The arylamide series was optimized through SAR studies by a broad variation of substituents in the left-hand side benzoyl residue (analogs 2a-2ag) or replacement of the benzoyl moiety by heteroarylcarbonyl residues (analogs 5a-5n). Compounds 2p and 2q were identified within the series as potent and selective H3R antagonists/inverse agonists with acceptable overall profile. Compound 2q was orally active in food intake inhibition in diet-induced obese (DIO) mice. Compound 2q represents a novel H3R antagonist template with improved in vitro potency and oral efficacy and has its merits as a new lead for further optimization.

Discovery of a potent, selective, and orally bioavailable histamine H3 receptor antagonist SAR110068 for the treatment of sleep-wake disorders.

Previous studies have shown that compound 1 displayed high affinity towards histamine H3 receptor (H3R), (human (h-H3R), K(i)=8.6 nM, rhesus monkey (rh-H3R), K(i)=1.2 nM, and rat (r-H3R), K(i)=16.5 nM), but exhibited high affinity for hERG channel. Herein, we report the discovery of a novel, potent, and highly selective H3R antagonist/inverse agonist 5a(SS) (SAR110068) with acceptable hERG channel selectivity and desirable pharmacological and pharmacokinetic properties through lead optimization sequence. The significant awakening effects of 5a(SS) on sleep-wake cycles studied by using EEG recording in rats during their light phase support its potential therapeutic utility in human sleep-wake disorders.

Characterization of primary standards for use in the HPLC analysis of the procyanidin content of cocoa and chocolate containing products.

This report describes the characterization of a series of commercially available procyanidin standards ranging from dimers DP = 2 to decamers DP = 10 for the determination of procyanidins from cocoa and chocolate. Using a combination of HPLC with fluorescence detection and MALDI-TOF mass spectrometry, the purity of each standard was determined and these data were used to determine relative response factors. These response factors were compared with other response factors obtained from published methods. Data comparing the procyanidin analysis of a commercially available US dark chocolate calculated using each of the calibration methods indicates divergent results and demonstrate that previous methods may significantly underreport the procyanidins in cocoa-containing products. These results have far reaching implications because the previous calibration methods have been used to develop data for a variety of scientific reports, including food databases and clinical studies.

Influence of Degree-of-Polymerization and Linkage on the Quantification of Proanthocyanidins using 4-Dimethylaminocinnamaldehyde (DMAC) Assay.

Proanthocyanidins (PACs) are naturally occurring flavonoids possessing health beneficial bioactivities. Their quantification often utilizes the 4-dimethylaminocinnamaldehyde (DMAC) spectrophotometric assay with the assumption that molar absorption coefficients (MACs) are similar across the various PAC species. To assess the validity of this assumption, individual PAC monomers and oligomers were examined for their absorbance response with DMAC. Our results have shown that PAC dimers and trimers with interflavan linkage variations exhibited differential absorbance response. Absence of A-type linkage between the terminal and second units in PAC molecule not only impacts absorbance intensity at 640 nm but also elicits a prominent secondary 440 nm absorbance peak. Cranberry (A-type) and cocoa (B-type) oligomeric PACs exhibited differential absorbance (MACs) relationship with degree-of-polymerization. Thus, PAC structural variations have considerable impact on the resulting MAC. The use of DMAC assay in PAC quantification, especially in comparing across specific oligomers and compositions, should not assume MACs are similar.

Cocoa extracts reduce oligomerization of amyloid-ß: implications for cognitive improvement in Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is the most common age-related neurodegenerative disorder, characterized by pathological aggregates of amyloid peptide-ß (Aß) and tau protein. Currently available therapies mediate AD symptoms without modifying disease progression. Polyphenol-rich diets are reported to reduce the risk for AD. OBJECTIVE: In the present study, we investigated the AD disease-modifying effects of cocoa, a rich source of flavanols, which are a class of polyphenols. We hypothesized that cocoa extracts interfere with amyloid-ß oligomerization to prevent synaptic deficits. METHODS: We tested the effects of three different cocoa extracts, viz. Natural, Dutched, and Lavado extracts, on Aß42 and Aß40 oligomerization, using photo-induced cross-linking of unmodified proteins technique. To assess the effects of cocoa extracts on synaptic function, we measured long term potentiation in mouse brain hippocampal slices exposed to oligomeric Aß. RESULTS: Our results indicate that cocoa extracts are effective in preventing the oligomerization of Aß, with Lavado extract being most effective. Lavado extract, but not Dutched extract, was effective in restoring the long term potentiation response reduced by oligomeric Aß. CONCLUSION: Our findings indicate that cocoa extracts have multiple disease-modifying properties in AD and present a promising route of therapeutic and/or preventative initiatives.

Inhibition of key digestive enzymes by cocoa extracts and procyanidins.

This study determined the in vitro inhibitory effects of cocoa extracts and procyanidins against pancreatic α-amylase (PA), pancreatic lipase (PL), and secreted phospholipase A(2) (PLA(2)) and characterized the kinetics of such inhibition. Lavado, regular, and Dutch-processed cocoa extracts as well as cocoa procyanidins (degree of polymerization (DP) = 2-10) were examined. Cocoa extracts and procyanidins dose-dependently inhibited PA, PL, and PLA(2). Lavado cocoa extract was the most potent inhibitor (IC(50) = 8.5-47 µg/mL). An inverse correlation between log IC(50) and DP (R(2) > 0.93) was observed. Kinetic analysis suggested that regular cocoa extract, the pentamer, and decamer inhibited PL activity in a mixed mode. The pentamer and decamer noncompetitively inhibited PLA(2) activity, whereas regular cocoa extract inhibited PLA(2) competitively. This study demonstrates that cocoa polyphenols can inhibit digestive enzymes in vitro and may, in conjunction with a low-calorie diet, play a role in body weight management.

Predicting zygoma fractures from baseball impact.

The purpose of this study is to develop injury risk functions that predict zygoma fracture based on baseball type and impact velocity. Zygoma fracture strength data from published experiments were mapped with the force exerted by a baseball on the orbit as a function of ball velocity. Using a normal distribution, zygoma fracture risk functions were developed. Experimental evaluation of these risk functions was performed using six human cadaver tests and two baseballs of different stiffness values. High speed video measured the baseball impact velocity. Post test analysis of the cadaver skulls was performed using CT imaging including three-dimensional reconstruction as well as autopsy. The developed injury risk functions accurately identify the risk of zygoma fracture as a result of baseball impact. The experimental results validated the zygoma risk functions at the lower and upper levels. The injuries observed in the post test analysis included fractures of the zygomatic arch, frontal process and the maxilla, zygoma suture, with combinations of these creating comminuted, tripod fractures of the zygoma. Tests with a softer baseball did result in injury but these had fewer resulting zygoma bone fragments and occurred at velocities 50% higher than the major league ball.

Immortalized suprachiasmatic nucleus cells express components of multiple circadian regulatory pathways.

We undertook an extensive antigenic characterization of the SCN 2.2 cell line in order to further evaluate whether the line expresses components of circadian regulatory pathways common to the hypothalamic suprachiasmatic nucleus (SCN), the central circadian clock in mammals. We found that differentiated SCN 2.2 cultures expressed a broad range of putative clock genes, as well as components of daytime, nighttime, and crepuscular circadian regulatory pathways found within the SCN in vivo. The line also exhibits several antigens that are highly expressed in a circadian pattern and/or differentially localized in the SCN relative to other hypothalamic regions. Expression of a broad complement of circadian regulatory proteins and putative clock genes further support growing evidence in recent reports that the SCN 2.2 cell line is an appropriate model for investigating the regulation of central mammalian pacemaker.

Synchronization and phase-resetting by glutamate of an immortalized SCN cell line.

SCN 2.2 cultures were stably transfected with luciferase reporter constructs driven by Ca(2+)/cAMP response element, E-box, or vasoactive intestinal peptide promoter to probe the circadian properties of this clock cell line. SCN 2.2 reporter lines displayed approximately 24-h rhythms of transcriptional activation after serum-shock. Serum-shocked cultures pulsed with glutamate exhibited phase-gated induction of phospho-CREB and of VIP, CRE, and E-box promoter activity. Glutamate-induced CRE promoter activity displayed restricted sensitivity to inhibitors of nitric oxide synthase and cGMP-dependent protein kinase. The temporal pattern of these sensitivities paralleled those of the SCN to light and glutamate during the night. Taken together, our data indicate that serum-shock can synchronize the circadian clock of SCN 2.2 cells to a state consistent with the day/night transition and, thus, establishes a temporal context for this cell line.

Spring-mediated cranial reshaping for craniosynostosis.

The evolution of modern craniofacial surgery has come full circle from the early strip craniectomies to the complete calvarial remodeling and now back to attempts at minimally invasive surgical interventions. The goal of the craniofacial surgeon has always been the correction of form and function with minimization of associated morbidity and mortality. Particularly problematic has been the ability to maintain the anatomical correction beyond the result seen on the operating room table secondary to changes with growth. The ability to improve the clinical result in a growing and developing child has been the impetus for dynamic treatment modalities. Dr Claes Lauritzen's pioneering work in this area has been particularly successful using internal springs to correct craniofacial deformities. The purpose of this study is to assess this treatment modality clinically in terms of safety and efficacy and to develop a methodology for the spring formation that would be easily reproducible. This is an institutional review board-approved prospective study of 15 children (11 male, 4 female) with non-syndromic sagittal suture synostosis. All patients were treated with a sagittal strip craniectomy and placement of 2 omega-shaped stainless steel springs at a mean age of 3.9 months. Patients were followed clinically and with cephalograms; after reossification of the intervening bone, the springs were removed at a mean age of 8.2 months. The mean force applied at initial placement of the springs was 6.9 N, and the mean spring deflection at formation was 6.87 cm. All patients completed the study protocol without any significant morbidity or any mortality. Perioperative variables, including blood loss, transfusion rate, operative time, intensive care unit stay, hospital stay, and hospital charges, were all significantly less (P < 0.05) in this study group compared with children with the same diagnosis treated with cranial vault reshaping during the same period. Furthermore, the preoperative mean cephalic index of 64.3 corrected to 77.6 after surgery and was maintained over time. Spring-mediated cranial reshaping is efficacious and safe for the treatment of sagittal synostosis. Long-term study of cranial development and clinical morphology are ongoing to validate further the effectiveness of this treatment modality.

Lateral and posterior dynamic bending of the mid-shaft femur: fracture risk curves for the adult population.

The purpose of this study was to develop injury risk functions for dynamic bending of the human femur in the lateral-to-medial and posterior-to-anterior loading directions. A total of 45 experiments were performed on human cadaver femurs using a dynamic three-point drop test setup. An impactor of 9.8 kg was dropped from 2.2 m for an impact velocity of 5 m/s. Five-axis load cells measured the impactor and support loads, while an in situ strain gage measured the failure strain and subsequent strain rate. All 45 tests resulted in mid-shaft femur fractures with comminuted wedge and oblique fractures as the most common fracture patterns. In the lateral-to-medial bending tests the reaction loads were 4180 +/- 764 N, and the impactor loads were 4780 +/- 792 N. In the posterior-to-anterior bending tests the reaction loads were 3780 +/- 930 N, and the impactor loads were 4310 +/- 1040 N. The difference between the sum of the reaction forces and the applied load is due to inertial effects. The reaction loads were used to estimate the mid-shaft bending moments at failure since there was insufficient data to include the inertial effects in the calculations. The resulting moments are conservative estimates (lower bounds) of the mid-shaft bending moments at failure and are appropriate for use in the assessment of knee restraints and pedestrian impacts with ATD measurements. Regression analysis was used to identify significant parameters, and parametric survival analysis was used to estimate risk functions. Femur cross-sectional area, area moment of inertia (I), maximum distance to the neutral axis (c), I/c, occupant gender, and occupant mass are shown to be significant predictors of fracture tolerance, while no significant difference is shown for loading direction, bone mineral density, leg aspect and age. Risk functions are presented for femur cross-sectional area and I/c as they offer the highest correlation to peak bending moment. The risk function that utilizes the most highly correlated (R2 = 0.82) and significant (p = 0.0001) variable, cross-sectional area, predicts a 50 percent risk of femur fracture of 240 Nm, 395 Nm, and 562 Nm for equivalent cross-sectional area of the 5(th) percentile female, 50(th) percentile male, and 95(th) percentile male respectively.