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Background Tetrabenazine (TBZ) activity is thought to result from four isomeric dihydrotetrabenazine (HTBZ) metabolites ([+]-α-HTBZ, [−]-α-HTBZ, [+]-β-HTBZ, [−]-β-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 (β) 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 [+]-β-HTBZ were the most abundant HTBZ isomers while [−]-β-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, [+]-β-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 [+]-β-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.

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 μM against genotype 1 polymerases and a mean 50% effective concentration (EC 50 ) of 0.075 μM 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 μM. PF-00868554 showed no cytotoxic effect in several human cell lines, up to the highest concentration evaluated (320 μM). 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.

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.

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.

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.

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, Tmax and Cmax) 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.