Thomas O'Shea

Synthesis and antiplasmodial activity of novel 2,4-diaminopyrimidines.

Two sets of diaminopyrimidines, totalling 45 compounds, were synthesized and assayed against Plasmodium falciparum. The SAR was relatively shallow, with only the presence of a 2-(pyrrolidin-1-yl)ethyl group at R(2) significantly affecting activity. A subsequent series addressed high LogD values by introducing more polar side groups, with the most active compounds possessing diazepine and N-benzyl-4-aminopiperidyl groups at R(1)/R(2). A final series attempted to address high in vitro microsomal clearance by replacing the C6-Me group with CF(3), however antiplasmodial activity decreased without any improvement in clearance. The C6-CF(3) group decreased hERG inhibition, probably as a result of decreased amine basicity at C2/C4.

Evaluation of two blood microsampling approaches for drug discovery PK studies in rats.

BACKGROUND Serial sampling in discovery rat PK studies could be performed via capillary microsampling (CMS) of blood or by using the Mitra™ device to collect dried blood samples. RESULTS Blood CMS results were compared with Mitra sampling results for four test compounds dosed in rat PK studies. The PK profiles obtained from CMS sampling were found to be very similar to those obtained from the Mitra sampling. For 15-µl blood CMS samples, freezing before the dilution step was found to be acceptable. CONCLUSION Blood CMS using 15-µl glass capillary microsamples works well for serial blood sampling in rat PK studies. The Mitra microsampling device provides an alternative method for collecting 10 µl of blood as a dried blood sample.

High-throughput quantitation of amino acids in rat and mouse biological matrices using stable isotope labeling and UPLC-MS/MS analysis.

Quantifying amino acids in biological matrices is typically performed using liquid chromatography (LC) coupled with fluorescent detection (FLD), requiring both derivatization and complete baseline separation of all amino acids. Due to its high specificity and sensitivity, the use of UPLC-MS/MS eliminates the derivatization step and allows for overlapping amino acid retention times thereby shortening the analysis time. Furthermore, combining UPLC-MS/MS with stable isotope labeling (e.g., isobaric tag for relative and absolute quantitation, i.e., iTRAQ) of amino acids enables quantitation while maintaining sensitivity, selectivity and speed of analysis. In this study, we report combining UPLC-MS/MS analysis with iTRAQ labeling of amino acids resulting in the elution and quantitation of 44 amino acids within 5 min demonstrating the speed and convenience of this assay over established approaches. This chromatographic analysis time represented a 5-fold improvement over the conventional HPLC-MS/MS method developed in our laboratory. In addition, the UPLC-MS/MS method demonstrated improvements in both specificity and sensitivity without loss of precision. In comparing UPLC-MS/MS and HPLC-MS/MS results of 32 detected amino acids, only 2 amino acids exhibited imprecision (RSD) >15% using UPLC-MS/MS, while 9 amino acids exhibited RSD >15% using HPLC-MS/MS. Evaluating intra- and inter-assay precision over 3 days, the quantitation range for 32 detected amino acids in rat plasma was 0.90-497 μM, with overall mean intra-day precision of less than 15% and mean inter-day precision of 12%. This UPLC-MS/MS assay was successfully implemented for the quantitative analysis of amino acids in rat and mouse plasma, along with mouse urine and tissue samples, resulting in the following concentration ranges: 0.98-431 μM in mouse plasma for 32 detected amino acids; 0.62-443 μM in rat plasma for 32 detected amino acids; 0.44-8590μM in mouse liver for 33 detected amino acids; 0.61-1241 μM in mouse kidney for 37 detected amino acids; and 1.39-1,681 μM in rat urine for 34 detected amino acids. The utility of the assay was further demonstrated by measuring and comparing plasma amino acid levels between pre-diabetic Zucker diabetic fatty rats (ZDF/Gmi fa/fa) and their lean littermates (ZDF/Gmi fa/?). Significant differences (P<0.001) in 9 amino acid concentrations were observed, with the majority ranging from a 2- to 5-fold increase in pre-diabetic ZDF rats on comparison with ZDF lean rats, consistent with previous literature reports.

Determination of 1,25-dihydroxyvitamin D2 in rat serum using liquid chromatography with tandem mass spectrometry.

Vitamin D therapy is widely used for the treatment of hyperparathyroidism associated with chronic renal failure in renal disease patients. The vitamin D prodrug, 1α-hydroxyvitamin D(2) (1α(OH)D(2)), is used for the treatment of the end stage renal disease patients who as a result of impaired kidney function cannot convert the naturally occurring vitamin D to the active hormonal form namely 1,25-dihydroxyvitamin D(2) (1,25(OH)(2)D(2)). The systemic circulating levels of this active form are in the pg/mL range and represent a significant bioanalytical challenge for therapeutic monitoring. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) is considered the gold standard for the selective and sensitive determination of small molecule therapeutics in biological matrices. However, the reported LC-MS/MS bioanalytical assays for 1,25(OH)(2)D(2) suffer from extensive sample preparation procedures or derivatization protocols to achieve the requisite sensitivity and selectivity. In this paper, we describe an assay that employs 96-well plate solid phase extraction sample preparation combined with highly sensitive LC-MS/MS instrumentation. The utility of ultra high pressure liquid chromatography to reduce the analytical run time was also demonstrated. Employing this assay a lower limit of quantitation of 25.0 pg/mL using 300 μL sample aliquot of rat serum was achieved with linearity obtained over the range of 25.0-1000 pg/mL. Both intra-day and inter-day coefficients of variation were <15% and accuracy across the assay range was within 100±7.24%. The application of the assay was demonstrated for the analysis of 1,25(OH)(2)D(2) rat serum samples to support pharmacokinetic studies conducted at doses down to sub-microgram per kilogram of 1α(OH)D(2).

Utility of capillary microsampling for rat pharmacokinetic studies: Comparison of tail-vein bleed to jugular vein cannula sampling.

INTRODUCTION Serial sampling methods have been used for rat pharmacokinetic (PK) studies for over 20 years. Currently, it is still common to take 200-250 μL of blood at each timepoint when performing a PK study in rats and using serial sampling. While several techniques have been employed for collecting blood samples from rats, there is only limited published data to compare these methods. Recently, microsampling (≤ 50 μL) techniques have been reported as an alternative process for collecting blood samples from rats. METHODS In this report, five compounds were dosed orally into rats. For three proprietary compounds, jugular vein cannula (JVC) sampling was used to collect whole blood and plasma samples and capillary microsampling (CMS) was used to collect blood samples from the tail vein of the same animal. For the two other compounds, marketed drugs fluoxetine and glipizide, JVC sampling was used to collect both whole blood and blood CMS samples while tail-vein sampling from the same rats was also used to collect both whole blood and blood CMS samples. RESULTS For the three proprietary compounds, the blood AUC as well as the blood concentration-time profile that were obtained from the tail vein were different from those obtained via JVC sampling. For fluoxetine, the blood total exposure (AUC) was not statistically different when comparing tail-vein sampling to JVC sampling, however the blood concentration-time profile that was obtained from the tail vein was different than the one obtained from JVC sampling. For glipizide, the blood AUC and concentration-time profile were not statistically different when comparing the tail-vein sampling to the JVC sampling. For both fluoxetine and glipizide, the blood concentration profiles obtained from CMS were equivalent to the blood concentration profiles obtained from the standard whole blood sampling, collected at the same sampling site. DISCUSSION The data in this report provide strong evidence that blood CMS is a valuable small volume blood sampling approach for rats and that it provides results for test compound concentrations that are equivalent to those obtained from traditional whole blood sampling. The data also suggest that for some compounds, the concentration-time profile that is obtained for a test compound based on sampling from a rat tail vein may be different from that obtained from rat JVC sampling. In some cases, this shift in the concentration-time profile will result in different PK parameters for the test compound. Based on these observations, it is recommended that a consistent blood sampling method should be used for serial microsampling in discovery rat PK studies when testing multiple new chemical entities. If the rat tail vein sampling method is selected for PK screening, then conducting a bridging study on the lead compound is recommended to confirm that the rat PK obtained from JVC sampling is comparable to the tail-vein sampling.

Synthesis and in vitro DMPK profiling of a 1,2-dioxolane-based library with activity against Plasmodium falciparum

A 43-member 1,2-dioxolane library was synthesized by coupling a 1,2-dioxolane-3-acetic acid derivative to a range of amines. Ten compounds had EC(50)s30nM against Plasmodium falciparum 3D7 and Dd2 strains, and another 15 compounds had EC(50)s50nM against both 3D7 and Dd2. The library was then subjected to a range of in vitro DMPK assays, which revealed that side chains with a heteroatom were required for favorable solubility, LogD and membrane permeability. CYP450 inhibition was isoform dependent, with 2C19 and 3A4 particularly susceptible, and the majority of compounds tested against rat and human microsomes were metabolized rapidly.

Evaluation of 3-O-methylfluorescein as a selective fluorometric substrate for CYP2C19 in human liver microsomes.

Cytochrome P450 (P450) fluorometric high-throughput inhibition assays have been widely used for drug-drug interaction screening particularly at the preclinical drug discovery stages. Many fluorometric substrates have been investigated for their selectivity, but most are found to be catalyzed by multiple P450 isozymes, limiting their utility. In this study, 3-O-methylfluorescein (OMF) was examined as a selective fluorescence substrate for CYP2C19 in human liver microsomes (HLMs). The kinetic studies of OMF O-demethylation in HLMs using a liquid chromatography/mass spectrometry method exhibited two-enzyme kinetics with apparent Km and Vmax values of 1.14 ± 0.90 μM and 11.3 ± 4.6 pmol/mg/min, respectively, for the high affinity component(s) and 57.0 ± 6.4 μM and 258 ± 6 pmol/mg/min, respectively, for the low affinity component(s). Studies utilizing cDNA-expressed individual P450 isoforms and P450-selective chemical inhibitors showed that OMF O-demethylation to fluorescein was selective for CYP2C19 at substrate concentrations ≤1 μM. At substrate concentrations ≥10 μM, other P450 isozymes were found to catalyze OMF O-demethylation. In HLMs, analysis of the two-enzyme kinetics in the presence of P450 isozyme-selective chemical inhibitors (ticlopidine for CYP2C19, sulfaphenazole for CYP2C9, and furafylline for CYP1A2) indicated that CYP2C19 was the high affinity component and CYP2C9 was the low affinity component. Based on these findings, a fluorometric assay was developed using 1 μM OMF and 2 μM sulfaphenazole for probing CYP2C19-mediated inhibition in HLMs. The IC50 data of 13 substrates obtained from the fluorometric assay developed in this study correlated well with that reported in the literature using nonfluorescence assays.

Capillary microsampling of whole blood for mouse PK studies: an easy route to serial blood sampling

BACKGROUND Capillary microsampling (CMS) of 8 µl of blood provides a methodology that can be utilized for serial sampling in drug discovery mouse PK studies. RESULTS Blood CMS sample results were compared to plasma sample results for three compounds (with expected Cb/Cp of 1 to 2) and found to be similar. In addition, for three compounds, blood CMS results were found to be equivalent to results generated with standard whole blood sampling. In a 5-day repeated dose PK study, four mice were dosed (IV) daily and sampled on both day one and day five using blood CMS procedure. CONCLUSION Blood CMS using 8 µl glass capillary microsamples provides a straightforward and effective approach for mouse serial blood sampling.

Abstract C220: Genz‐644282, a non‐camptothecin topoisomerase one inhibitor, demonstrates a wide spectrum of in vitro and in vivo antitumor activity

Genz‐644282 (GZ) is a novel non‐camptothecin topoisomerase I (Top1) inhibitor. The in vitro and in vivo activity of GZ and its M1 and M2 metabolites were explored and compared with the activity of camptothecin Top1 inhibitors. In vitro in mouse, rat, dog, and human GZ exhibited high metabolic stability, plasma binding of 88–93% and exhibits concentration dependent partitioning into red blood cells. In vivo, GZ has a large volume of distribution and low‐to‐moderate clearance in mouse, rat and dog. In nude mice, the t 1/2 for GZ is 3.6 h (po), 10.4 h (ip) and 5.1h (iv) and longer in tumor‐bearing mice. In human HCT‐116 colon ca, HT‐29 colon ca and NCI‐H460 NSCLC cells the concentration response for Genz‐6244282, M1 and M2 are the same. Upon 72h exposure of the cells to GZ, M1 or M2 the IC 50 concentrations were 0.5‐0.65 nM and the IC 90 concentrations were 1.8–2 nM. In order to evaluate the antitumor activity of GZ as compared to several approved anticancer agents, the compound was tested in seven xenograft models: LOX‐IMVI melanoma, DLD‐1 and HCT‐15 colon, MDA‐MB‐231 breast, NCI‐H292 and NCI‐H1299 lung ca. GZ was compared against two of its metabolites, Genz‐649974 (GZ‐74) and Genz‐649975 in the HCT‐116 colon ca resulting in comparable activity with GZ‐74. GZ was administered intravenously on a QODx3 schedule for 2 cycles. The tumor growth delay, TGD, (T‐C) and increase in lifespan, ILS, (T/C) for each study are listed in the table below. All of the GZ dosages were well tolerated resulting in a maximum body weight loss of ≤20%, except for the high dosages in the HCT‐15 and NCI‐H292 in which there was a maximum body weight loss of 25.7 and 20.9%, respectively. Based on these findings and other data, GZ was selected to be a development candidate. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C220.