Structure-based design, SAR analysis and antitumor activity of PI3K/mTOR dual inhibitors from 4-methylpyridopyrimidinone series.

PI3K, AKT and mTOR, key kinases from a frequently dysregulated PI3K signaling pathway, have been extensively pursued to treat a variety of cancers in oncology. Clinical trials of PF-04691502, a highly potent and selective ATP competitive kinase inhibitor of class 1 PI3Ks and mTOR, from 4-methylpyridopyrimidinone series, led to the discovery of a metabolite with a terminal carboxylic acid, PF-06465603. This paper discusses structure-based drug design, SAR and antitumor activity of the MPP derivatives with a terminal alcohol, a carboxylic acid or a carboxyl amide.

Effect of ketoconazole on the pharmacokinetics of axitinib in healthy volunteers.

OBJECTIVE: Axitinib (AG-013736), an oral, potent, and selective inhibitor of vascular endothelial growth factor receptors 1, 2, and 3, is metabolized primarily by cytochrome P450 (CYP) 3A with minor contributions from CYP1A2, CYP2C19, and glucuronidation. Co-administration with CYP inhibitors may increase systemic exposure to axitinib and alter its safety profile. This study evaluated changes in axitinib plasma pharmacokinetic parameters and assessed safety and tolerability in healthy subjects, following axitinib co-administration with the potent CYP3A inhibitor ketoconazole. METHODS: In this randomized, single-blind, two-way crossover study, 32 healthy volunteers received placebo, followed by a single 5-mg oral dose of axitinib, administered either alone or on the fourth day of dosing with oral ketoconazole (400 mg/day for 7 days). RESULTS: Axitinib exposure was significantly increased in the presence of ketoconazole, with a geometric mean ratio for area under the plasma concentration-time curve from time zero to infinity of 2.06 (90% confidence interval [CI]: 1.84-2.30) and a geometric mean ratio for maximum plasma concentration (C(max)) of 1.50 (90% CI: 1.33-1.70). For axitinib alone or with ketoconazole, C(max) occurred 1.5 and 2.0 h after dosing, respectively. Adverse events were predominantly mild; the most commonly reported treatment-related adverse events were headache and nausea. CONCLUSIONS: Axitinib plasma exposures and peak concentrations were increased following concurrent administration of axitinib and ketoconazole in healthy volunteers. Axitinib alone and in combination with ketoconazole was well tolerated. These findings provide an upper exposure for expected axitinib plasma concentrations in the presence of potent metabolic inhibition.

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.

Metabolite profiling of dasatinib dosed to Wistar Han rats using automated dried blood spot collection.

To characterize and enable efficient rat pharmacokinetic (PK) screening in early drug discovery, automated sampling of blood time points are routinely employed. With the development of dried blood spot (DBS) technology for drug level quantitation, an opportunity exists for the automated collection of rat PK time points using DBS. DBS, as an alternative sample collection technique has led to the increased collection of PK study samples for the quantitative analyses of drug candidates in both pre-clinical and clinical studies. However, the feasibility of using DBS samples for drug metabolite profiling including both phase I and phase II metabolites has not been well established. This work reports the study of metabolite profiling of dasatinib dosed to Wistar Han rats using automated DBS collection. Automated DBS and plasma collection using a rat AccuSampler (VeruTech AB, Sweden) was employed using dasatinib as a model compound. The DBS and plasma samples were extracted by methanol and acetonitrile and both plasma and DBS extracts were analyzed using a Sciex API4000 Qtrap mass spectrometer coupled to a Shimazdzu HPLC system. Dasatinib and its metabolites were analyzed by multiple reaction monitoring (MRM) and MRM trigger enhanced product ion scan (MRM-EPI). Both phase I oxidative metabolites and phase II glucuronide conjugates and sulfate conjugates were detected from both rat plasma and DBS samples. Overall, comparable metabolite profiles including phase I oxidative and phase II glucuronide and sulfate conjugates were observed from both extracts of plasma and DBS samples when using the untreated DBS cards for dasatinib. Chemically treated DBS cards such as DMPK-A and DMPK-B cards may affect the dasatinib metabolites. Similar PK parameters were obtained for dasatinib from both plasma and DBS samples, after correcting for blood to plasma ratio. The results obtained from this study suggest that collection of study samples by DBS can be used for metabolite profiling, however, the availability of limited samples may be a concern for multiple injections.

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.

Quantitative analysis of PD 0332991 in mouse plasma using automated micro-sample processing and microbore liquid chromatography coupled with tandem mass spectrometry.

In the oncology therapeutic area, the mouse is the primary animal model used for efficacy studies. Often with mouse pharmacokinetic (PK) and pharmacokinetic/pharmacodynamic (PK/PD) studies, less than 20 µL of total plasma sample volume is available for bioanalysis due to the small size of the animal and the need to split samples for other measurements such as biomarker analyses. The need to conduct automated "small volume" sample processing for quantitative bioanalysis has therefore increased. An automated fit for purpose protein precipitation (PPT) method using a Hamilton MicroLab Star (Reno, NV, USA) to support mouse PK and PK/PD studies for an oncology drug candidate PD 0332991, (a specific inhibitor of cyclin-dependent kinase 4 (CDK-4) currently in development) for processing "small volumes" was developed. The automated PPT method was achieved by extracting and processing 10 µL out of a minimum sample volume of 15 µL plasma utilizing the Hamilton MicroLab Star. A 96-conical shallow well plate by Agilent Technologies, Inc (Wilmington, DE, USA) was the labware of choice used in the automated Hamilton "small volume" method platform. Analyses of a 10 µL plasma aliquot from 15 µL of plasma study samples were conducted by both automated and manual PPT method. All plasma samples were quantitated using a Sciex API 4000 triple quadrupole mass spectrometer coupled with an Eksigent Express HT Ultra HPLC system. The chromatography was achieved using an Agilent microbore C(18) Extend, 1.0 × 50 mm, 3.5 µm column at a flow rate of 0.150 mL/min with a total run time of 1.8 min. Accuracy and precision of standard and QC concentration levels were within 90-107% and <14%, respectively. Calibration curves were linear over the dynamic range of 1.0-1000 ng/mL. PK studies for PD 0332991 were conducted in female C3H mice following intravenous administration at 1mg/kg and oral administration at 2mg/kg. PK values such as area under curve (AUC), volume of distribution (Vd), clearance (Cl), half life (T(1/2)) and bioavailability (F%) demonstrated less than 11% difference between the automated Hamilton and manual PPT methods. The results demonstrate that the automated Hamilton PPT method can accurately and precisely aliquot 10 µL of plasma from 15 µL or larger volume plasma samples. The fit for purpose Hamilton PPT method is suitable for routine analyses of plasma samples from micro-sampling PK and PK/PD samples to support discovery studies.

Quantitative analysis of PD 0332991 in xenograft mouse tumor tissue by a 96-well supported liquid extraction format and liquid chromatography/mass spectrometry.

Phase II attrition of clinical candidates in the drug development cycle is currently a major issue facing the pharmaceutical industry. To decrease phase II attrition, there is an increased emphasis on validation of mechanism of action, development of efficacy models and measurement of drug levels at the site of action. PD 0332991, a highly specific inhibitor of cyclin-dependent kinase 4 (CDK-4) is currently in clinical development for the treatment of solid tumor. A clinical presurgical study will be required to better understand how PD 0332991 affects signaling pathways and how the intratumoral concentration of PD 0332991 correlates with plasma PK parameters and molecular alterations in breast cancer tissues after PD 0332991 treatment. Before conducting such a clinical study, it is important to evaluate PD 0332991 levels in tumor tissue samples from a xenograft mouse model for the determination of drug exposure at the site of action. Therefore, the objectives of this study were (1) to develop and validate a sensitive LC-MS/MS method to quantify PD 0332991 in mouse tumor tissues from MDA-MB-231-Luc human breast tumor xenografts in SCID-beige mice; (2) to quantify PD 0332991 levels in mouse tumor tissues after oral administration of PD 0332991 at 10 and 100mg/kg using the validated LC-MS/MS method. Both liquid-liquid extraction (LLE) and supported liquid extraction (SLE) in a 96-well format were developed and evaluated to achieve optimal extraction recovery with minimal matrix effects. The newly developed SLE method is more efficient (speed and ease) and demonstrates comparable recovery (93.1-100% at three different concentrations) compared to the traditional LLE method. The validated LC-MS/MS for PD 032291 in mouse tumor tissue homogenate method exhibited a linear dynamic range of 0.1-100 ng/mL with inter-day accuracy and precision within 15%. The validated method was successfully applied to measure PD 0332991 levels in tumor tissues in MDA-MB-231-Luc human breast tumor xenografts in SCID beige mice. The mean tumor concentrations at 6h post-oral PD 0332991 administration at 10 and 100mg/kg were 1793 (+/-1008) and 25,163 (+/-3959) ng/g, respectively.

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.

Azaindole hydroxamic acids are potent HIV-1 integrase inhibitors.

HIV-1 integrase (IN) is one of three enzymes encoded by the HIV genome and is essential for viral replication. Recently, HIV-1 IN inhibitors have emerged as a new promising class of therapeutics. Herein, we report the discovery of azaindole carboxylic acids and azaindole hydroxamic acids as potent inhibitors of the HIV-1 IN enzyme and their structure-activity relationships. Several 4-fluorobenzyl substituted azaindole hydroxamic acids showed potent antiviral activities in cell-based assays and offered a structurally simple scaffold for the development of novel HIV-1 IN inhibitors.