Preclinical characterization of PF-00868554, a potent nonnucleoside inhibitor of the hepatitis C virus RNA-dependent RNA polymerase.

PF-00868554 is a nonnucleoside inhibitor of the hepatitis C virus (HCV) RNA polymerase, which exerts its inhibitory effect by binding to the thumb base domain of the protein. It is a potent and selective inhibitor, with a mean 50% inhibitory concentration of 0.019 microM against genotype 1 polymerases and a mean 50% effective concentration (EC(50)) of 0.075 microM against the genotype 1b-Con1 replicon. To determine the in vitro antiviral activity of PF-00868554 against various HCV strains, a panel of chimeric replicons was generated, in which polymerase sequences derived from genotype 1a and 1b clinical isolates were cloned into the 1b-Con1 subgenomic reporter replicon. Our results indicate that PF-00868554 has potent in vitro antiviral activity against a majority (95.8%) of genotype 1a and 1b replicons, with an overall mean EC(50) of 0.059 microM. PF-00868554 showed no cytotoxic effect in several human cell lines, up to the highest concentration evaluated (320 microM). Furthermore, the antiviral activity of PF-00868554 was retained in the presence of human serum proteins. An in vitro resistance study of PF-00868554 identified M423T as the predominant resistance mutation, resulting in a 761-fold reduction in susceptibility to PF-00868554 but no change in susceptibility to alpha interferon and a polymerase inhibitor that binds to a different region. PF-00868554 also showed good pharmacokinetic properties in preclinical animal species. Our results demonstrate that PF-00868554 has potent and broad-spectrum antiviral activity against genotype 1 HCV strains, supporting its use as an oral antiviral agent in HCV-infected patients.

Allosteric inhibitors of hepatitis C polymerase: discovery of potent and orally bioavailable carbon-linked dihydropyrones.

The discovery and optimization of a novel class of carbon-linked dihydropyrones as allosteric HCV NS5B polymerase inhibitors are presented. Replacement of the sulfur linker atom with carbon reduced compound acidity and greatly increased cell permeation. Further structure-activity relationship (SAR) studies led to the identification of compounds, exemplified by 23 and 24, with significantly improved antiviral activities in the cell-based replicon assay and favorable pharmacokinetic profiles.

Differences in Dihydrotetrabenazine Isomer Concentrations Following Administration of Tetrabenazine and Valbenazine.

BACKGROUND: Tetrabenazine (TBZ) activity is thought to result from four isomeric dihydrotetrabenazine (HTBZ) metabolites ([+]-α-HTBZ, [-]-α-HTBZ, [+]-ß-HTBZ, [-]-ß-HTBZ). Each isomer has a unique profile of vesicular monoamine transporter 2 (VMAT2) inhibition and off-target binding. Previously published data only report total isomer (α) and (ß) concentrations. We developed a method to quantify the individual HTBZ isomers in samples from patients with Huntington's disease receiving TBZ. For comparison, concentrations of [+]-α-HTBZ, the single active metabolite shared by valbenazine (VBZ) and TBZ, were determined in samples from patients with tardive dyskinesia receiving VBZ. METHODS: A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for quantitation of the four individual HTBZ isomers. Concentrations were determined in serum from patients with Huntington's disease administered TBZ and plasma from patients with tardive dyskinesia administered VBZ once daily. RESULTS: In patients administered TBZ, [-]-α-HTBZ and [+]-ß-HTBZ were the most abundant HTBZ isomers while [-]-ß-HTBZ and [+]-α-HTBZ were present as minor metabolites. Only [+]-α-HTBZ was observed in patients administered VBZ. CONCLUSIONS: Based on relative abundance and potency, [+]-ß-HTBZ appears to be the primary contributor to VMAT2 inhibition by TBZ, a finding in contrast with the generally held assertion that [+]-α-HTBZ is the major contributor. [-]-α-HTBZ, the other abundant TBZ metabolite, has much lower VMAT2 inhibitory potency than [+]-ß-HTBZ, but increased affinity for other CNS targets, which may contribute to off-target effects of TBZ. In contrast, pharmacological activity for VBZ is derived primarily from [+]-α-HTBZ. Individual HTBZ isomer concentrations provide a more clinically relevant endpoint for assessing on- and off-target effects of TBZ than total isomer concentrations.

A Plasma-Based Protein Marker Panel for Colorectal Cancer Detection Identified by Multiplex Targeted Mass Spectrometry.

INTRODUCTION: Colorectal cancer (CRC) testing programs reduce mortality; however, approximately 40% of the recommended population who should undergo CRC testing does not. Early colon cancer detection in patient populations ineligible for testing, such as the elderly or those with significant comorbidities, could have clinical benefit. Despite many attempts to identify individual protein markers of this disease, little progress has been made. Targeted mass spectrometry, using multiple reaction monitoring (MRM) technology, enables the simultaneous assessment of groups of candidates for improved detection performance. MATERIALS AND METHODS: A multiplex assay was developed for 187 candidate marker proteins, using 337 peptides monitored through 674 simultaneously measured MRM transitions in a 30-minute liquid chromatography-mass spectrometry analysis of immunodepleted blood plasma. To evaluate the combined candidate marker performance, the present study used 274 individual patient blood plasma samples, 137 with biopsy-confirmed colorectal cancer and 137 age- and gender-matched controls. Using 2 well-matched platforms running 5 days each week, all 274 samples were analyzed in 52 days. RESULTS: Using one half of the data as a discovery set (69 disease cases and 69 control cases), the elastic net feature selection and random forest classifier assembly were used in cross-validation to identify a 15-transition classifier. The mean training receiver operating characteristic area under the curve was 0.82. After final classifier assembly using the entire discovery set, the 136-sample (68 disease cases and 68 control cases) validation set was evaluated. The validation area under the curve was 0.91. At the point of maximum accuracy (84%), the sensitivity was 87% and the specificity was 81%. CONCLUSION: These results have demonstrated the ability of simultaneous assessment of candidate marker proteins using high-multiplex, targeted-mass spectrometry to identify a subset group of CRC markers with significant and meaningful performance.

Validation of a soy food-frequency questionnaire and evaluation of correlates of plasma isoflavone concentrations in postmenopausal women.

BACKGROUND: Soy foods may have various health benefits, but little is known about the patterns and correlates of soy consumption among postmenopausal women in the United States. OBJECTIVE: We assessed the reliability and validity of a soy food-frequency questionnaire (FFQ) and examined demographic, lifestyle, and dietary correlates of plasma isoflavone concentrations in postmenopausal women. DESIGN: In this cross-sectional study, soy isoflavone intake and plasma isoflavone concentration were analyzed in 96 postmenopausal women aged 50-79 y; the data were obtained at 2 visits that were 1 wk apart. Intake was determined with a 20-item soy FFQ and a comprehensive FFQ that included questions about tofu and soymilk. Fasting plasma daidzein and genistein concentrations were determined with liquid chromatography-mass spectrometry. RESULTS: Intraclass correlations between week 1 and week 2 values were >0.98 for both the soy and comprehensive FFQs. Median reported isoflavone intake was <2 mg/d. Pearson's product-moment correlation coefficients relating isoflavone intakes with plasma isoflavone concentrations ranged from 0.35 to 0.43. Plasma isoflavone concentrations were positively associated with age, fiber consumption, servings of fruit and vegetables, and dietary supplement use and were inversely associated with caffeine consumption; no associations with body mass index, education, dietary beliefs, activity level, alcohol intake, or fat intake were observed. CONCLUSIONS: Within a population with low soy consumption, the soy FFQ and comprehensive FFQ showed good reliability and moderate validity. Associations of plasma isoflavone concentrations with other dietary behaviors suggest that these compounds may serve as biomarkers of health behaviors in populations with low soy consumption.

Dietary determinants of plasma enterolactone.

Enterolactone is a lignan produced by fermentation of dietary precursors in the human gut. Because lignan precursors are uniquely found in plant foods, plasma enterolactone concentration may serve as a biological marker of plant food consumption. This cross-sectional study examined associations of dietary intake with plasma enterolactone concentration. Weight-stable, 20-40-year-old volunteers (115 women and 78 men in Seattle, Washington) reporting intake of < or =2.5 or > or =4.5 fruit and vegetable servings/day and no antibiotic use for > or =3 months completed a food frequency questionnaire and 3-day food record. Time-resolved fluoroimmunoassay was used to measure plasma enterolactone. Based on diet records, plasma enterolactone was positively correlated with daily vegetable servings (r = 0.17; P < 0.05), fiber (r = 0.36; P < 0.0001), alcohol (r = 0.24; P < 0.001), caffeine (r = 0.21; P < 0.001), and daily botanical group servings [Chenopodiaceae (r = 0.15; P < 0.05), Juglandaceae (r = 0.15; P < 0.05), Leguminosae (r = 0.20; P < 0.001), Pedaliaceae (r = 0.20; P < 0.001), and Vitaceae (r = 0.20; P < 0.001)]. Fat-related variables were not correlated with plasma enterolactone. Based on linear regression models, plasma enterolactone increased by 37.0% (SE = 2.3%) for each 10-g increase in fiber and by 6.6% (SE = 0.2%) for each 50-mg serving of caffeine. Participants consuming 0.5-1 alcoholic drink/day had plasma enterolactone concentrations that were 131.4% (SE = 37.6%) higher than those of nondrinkers. Although plasma enterolactone may be useful as a biological measure of exposure to lignan-containing foods, it may be of limited use as a specific biomarker of fruit and vegetable or plant food intake because coffee, tea, and alcoholic beverages also significantly increase its plasma concentration.

Overnight urinary isoflavone excretion in a population of women living in the United States, and its relationship to isoflavone intake.

Dietary isoflavones are biologically active in humans, but few observational data exist on the relationship between isoflavone intake and excretion in Western populations. We examined associations between self-reported soy intakes and overnight urinary isoflavone excretion in a population-based sample of western Washington State women, and we investigated the usefulness of one versus two overnight urine samples, collected 48 h apart, as a biomarker of intake. Isoflavones (genistein, daidzein, O-desmethylangolensin, and equol) were measured in two overnight urine collections from 363 women recruited from a health maintenance organization. Soy food intakes were assessed using two 1-day diet records completed on each day prior to the urine collections and a food frequency questionnaire (FFQ) that had been completed by 312 of the women with regard to their dietary habits 3.5 years (range, 2-5 years) before the urine collections. Twenty-one percent of the women consumed soy on either day of the diet recall, and 13% and 34% of the women consumed soy at least once a week or at least once a month, respectively, according to the FFQ. Women who consumed soy at either of the two diet recalls or at the FFQ (at least once a week or at least once a month) had a significantly higher urinary excretion of isoflavones than women who did not consume soy (P < 0.01). Among women who consumed soy at either of the two diet recalls or at the FFQ (soy consumed at least once a month), isoflavone intake and excretion correlated significantly (P < 0.01). Excretion of the individual isoflavones correlated significantly between the two urine samples collected 48 h apart (genistein, r = 0.41 and P < 0.001; daidzein, r = 0.30 and P < 0.001; O-desmethylangolensin, r = 0.46 and P < 0.001; equol, r = 0.60 and P < 0.001). Differences between soy consumers and nonconsumers and associations between intakes and excretion remained significant whether one or both urine collections were considered. Measuring isoflavone excretion in one overnight urine collection serves as a biomarker of recent or past isoflavone intake, even in populations whose intake of soy foods is relatively low.

Urinary equol excretion in relation to 2-hydroxyestrone and 16alpha-hydroxyestrone concentrations: an observational study of young to middle-aged women.

Approximately one-third to one-half of individuals harbor the colonic bacteria that are capable of metabolizing the soy isoflavone daidzein to equol. Results of prior studies suggest beneficial effects of producing equol in relation to breast cancer risk, potentially through effects on endogenous hormones. High urinary excretion of 2-hydroxyestrone (2-OH E(1)) relative to 16alpha-hydroxyestrone (16alpha-OH E(1)) has been associated with a reduced risk of breast cancer. In this pilot study we examined associations between urinary excretion of equol and 2-OH E(1), 16alpha-OH E(1), and their ratio, and investigated whether excretion of these estrogen metabolites differed between two samples collected 48h apart. Isoflavones (genistein, daidzein, O-desmethylangolensin (ODMA), and equol) were measured in two overnight urines from 126 women. Excretion of 2-OH E(1) and 16alpha-OH E(1) were measured in the first overnight urine from all 126 women and in the second overnight urine from 30 of these women; there were no significant differences between samples collected 48h apart in excretion of 2-OH E(1) or 16alpha-OH E(1) (P=0.75 and 0.17, respectively). Among all women, correlations between total isoflavone excretion (sum of genistein, daidzein, ODMA, and equol) and estrogen metabolites were non-significant (P>0.05). Among women with detectable levels of equol, total isoflavone excretion was significantly positively correlated with 16alpha-OH E(1) (r=0.32, P=0.02), but was not correlated with 2-OH E(1) or 2-OH E(1):16alpha-OH E(1) ratio (r=0.21, P=0.14, and r=-0.05, P=0.70, respectively). Equol excretion (adjusted for other isoflavone excretion) was significantly positively correlated with 2-OH E(1):16alpha-OH E(1) ratio (r=0.38, P=0.005), but was not correlated with 2-OH E(1) or 16alpha-OH E(1) (r=0.15, P=0.29, and r=-0.17, P=0.24, respectively). The finding that equol excretion, but not total isoflavone excretion, correlated positively with the 2-OH E(1):16alpha-OH E(1) ratio suggests that the colonic bacterial profile associated with equol production may be involved in estrogen metabolism, and may therefore possibly influence breast cancer risk.

Validation of a soy food frequency questionnaire with plasma concentrations of isoflavones in US adults.

OBJECTIVE: To validate assessment of soy intake using food frequency questionnaires (FFQs) compared with plasma isoflavone (genistein and daidzein) concentrations. DESIGN: Cross-sectional analysis of soy isoflavone intake and plasma analysis of isoflavones. SUBJECTS: 77 men and women, age range 20 to 40 years, recruited from the Seattle metropolitan area. MAIN OUTCOME MEASURES: Isoflavone intake was determined from responses to a 40-item soy FFQ and from tofu and soymilk intake assessed as part of a comprehensive FFQ used for the Women's Health Initiative (WHI FFQ). Isoflavone concentrations in fasting blood samples were determined by liquid chromatography-mass spectrometry. STATISTICAL ANALYSES: Correlation coefficients were calculated for: a) isoflavone intake assessed by the soy FFQ and the WHI FFQ, b) intake assessed by the soy FFQ and plasma isoflavone concentrations, and c) intake assessed by the WHI FFQ and plasma isoflavone concentrations. RESULTS: Isoflavone intake was highly correlated between the soy FFQ and the WHI FFQ (r = 0.84). Genistein and daidzein intakes determined by the soy FFQ were significantly correlated with plasma concentrations (r = 0.53 and 0.45, respectively). Isoflavone intake assessed from the WHI FFQ was also correlated with plasma concentration (r = 0.46 and 0.45). Soymilk and tofu were the two major contributors to isoflavone intake (38.6%). CONCLUSIONS: A soy-specific, 40-item FFQ assessed isoflavone intake with good validity. Isoflavone intake assessed by the WHI FFQ (tofu and soymilk) had lower correlations with plasma concentrations compared with the soy FFQ. Nonetheless, assessment of the two foods is a reasonably good marker for soy food consumption in this sample.

Discovery pharmacokinetic studies in mice using serial microsampling, dried blood spots and microbore LC-MS/MS.

BACKGROUND: Initial pharmacokinetic (PK) studies of discovery compounds are conducted in mice to demonstrate exposure prior to conducting efficacy studies. PK information obtained from a single mouse by serial blood microsampling, dried blood spot collection and analyses using microbore (1 mm internal diameter column) LC-MS/MS is presented. Ex vivo blood to plasma concentration ratios (BPRs) from mouse PK studies were compared with in vitro BPRs for 15 compounds. RESULTS: Two compounds were orally dosed and blood was collected at time points via serial blood sampling. The calculated PK parameters (AUC, T(max) and C(max)) were comparable across liquid blood, dried blood spot and plasma matrices. The BPR results from both methods were comparable. CONCLUSION: Serial blood microsampling has led to reduced animal and compound usage with improved PK data. Ex vivo BPR is suitable in a discovery setting. Microbore LC-MS/MS is well suited in instances where sample volume is limited, and enables faster analyses, reduced solvent use, and less frequent MS source cleaning.

Discovery of (R)-6-cyclopentyl-6-(2-(2,6-diethylpyridin-4-yl)ethyl)-3-((5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)methyl)-4-hydroxy-5,6-dihydropyran-2-one (PF-00868554) as a potent and orally available hepatitis C virus polymerase inhibitor.

The HCV RNA-dependent RNA polymerase has emerged as one of the key targets for novel anti-HCV therapy development. Herein, we report the optimization of the dihydropyrone series inhibitors to improve compound aqueous solubility and reduce CYP2D6 inhibition, which led to the discovery of compound 24 (PF-00868554). Compound 24 is a potent and selective HCV polymerase inhibitor with a favorable pharmacokinetic profile and has recently entered a phase II clinical evaluation in patients with genotype 1 HCV.