Increased Aurora A expression occurs in a variety of human cancers and induces chromosomal abnormalities during mitosis associated with tumor initiation and progression. MLN8054 is a selective smallmolecule Aurora A kinase inhibitor that has entered Phase I clinical trials for advanced solid tumors. MLN8054 inhibits recombinant Aurora A kinase activity in vitro and is selective for Aurora A over the family member Aurora B in cultured cells. MLN8054 treatment results in G2/M accumulation and spindle defects and inhibits proliferation in multiple cultured human tumor cells lines. Growth of human tumor xenografts in nude mice was dramatically inhibited after oral administration of MLN8054 at well tolerated doses. Moreover, the tumor growth inhibition was sustained after discontinuing MLN8054 treatment. In human tumor xenografts, MLN8054 induced mitotic accumulation and apoptosis, phenotypes consistent with inhibition of Aurora A. MLN8054 is a selective inhibitor of Aurora A kinase that robustly inhibits growth of human tumor xenografts and represents an attractive modality for therapeutic intervention of human cancers.cancer ͉ mitosis ͉ apoptosis

ABSTRACT:Apparent intrinsic clearance (CL int,app ) of 7-ethoxycoumarin, phenacetin, propranolol, and midazolam was measured using rat and human liver microsomes and freshly isolated and cryopreserved hepatocytes to determine factors responsible for differences in rates of metabolism in these systems. The cryopreserved and freshly isolated hepatocytes generally provided similar results, although there was greater variability using the latter system. The CL int,app values in hepatocytes are observed to be lower than that in microsomes, and this difference becomes greater for compounds with high CL int,app . This could partly be attributed to the differences in the free fraction (f u ). The f u in hepatocyte incubations (f u,hep-inc ) was influenced not only by the free fraction of compounds in the incubation buffer (f u,buffer ) but also by the rate constants of uptake (k up ) and metabolism (k met ). This report provides a new derivation for f u,hep-inc , which can be expressed as, where the C hep , C buffer , V hep , and V buffer represent the concentrations of a compound in hepatocytes and buffer and volumes of hepatocytes and buffer, respectively. For midazolam, the f u,hep-inc was calculated, and the maximum metabolism rate in hepatocytes was shown to be limited by the uptake rate.The determination of in vitro intrinsic clearance (CL int ) for drug candidates in the early discovery stage is a common practice in the pharmaceutical industry (Houston, 1994;Lave et al., 1997;Obach et al., 1997). The CL int values of drug candidates can help to confirm whether metabolism is the main clearance pathway when it is compared with the total body clearance in vivo. It is also helpful in rank-ordering drug candidates based on their metabolic stabilities, assessing species and gender differences in metabolic clearance, and projecting the metabolic clearance of drug candidates in humans. The in vitro CL int may be derived from enzyme kinetic data such as V max /K m (Lin et al., 1996;Tan and Pang, 2001;Griffin and Houston, 2004) or from the in vitro t 1/2 values where subK m substrate concentrations are used (Lave et al., 1997, Obach, 1999Lau et al., 2002;Jones and Houston, 2004). The CL int can be calculated from the experimental apparent intrinsic clearance, CL int,app , by correcting for free fraction of test compounds in the incubations. To further predict the in vivo hepatic clearance from the in vitro intrinsic clearance, a well stirred model is often used (Naritomi et al., 2001;Ito and Houston, 2004). A survey of literature revealed that in hepatocyte incubations, the free fraction of test compound has not been well defined. Simply assuming a steady state where the intracellular free concentration equals the extracellular free concentration may allow one to roughly estimate CL int for some compounds. However, clearance, after a dose in vitro or in vivo, is actually a dynamic system such that at any given time the amount of compound getting into a cell typically equals the amount of compound leaving the cell by diffus...

This article is available online at http://dmd.aspetjournals.org ABSTRACT:1-Aminobenzotriazole (ABT) has been extensively used as a nonspecific inhibitor of cytochromes P450 (P450s) in animals for mechanistic studies, and antipyrine (AP) has been used as a probe for hepatic oxidative metabolic capacity determination in vivo. The method of use of ABT has been variable from lab to lab due largely to unknown pharmacokinetics of ABT itself and incomplete information on various P450s inhibited. The oral pharmacokinetic profiles of ABT were generated in rats, dogs, and monkeys in the dose range of 5 to 200 mg/kg. The results showed that after single oral doses of 50 mg/kg in rats, and 20 mg/kg in dogs and monkeys, the plasma concentrations were high and were sustained for over 24 h. In vitro, inhibition of various expressed P450s upon 30-min preincubation with ABT (0-500 M) showed that CYP1A2, 2B6, 2C9, 2C19, 2D6, and 3A4 were inhibited in a dose-dependent manner. The intravenous pharmacokinetics of AP also was affected in a dose-dependent manner in all species, treated 2 h earlier with ABT. Thus, the plasma clearance of AP was inhibited by 88% in rats pretreated with 50 mg/kg ABT and 96% in dogs and 83% in monkeys pretreated with 20 mg/kg ABT. Based on these data in rats, dogs, and monkeys, and the established safety profile of ABT in rats dosed up to 100 mg/kg, a pretreatment at 2 h with a single oral dose of ABT at 100 mg/kg in rats (providing 93% inhibition) and 20 mg/kg in dogs and monkeys effectively inhibited the clearance of the probe compound. ABT1 has been extensively used as a nonspecific inhibitor of cytochromes P450 in animals for mechanistic studies (Mico et al., 1988;Huijzer et al., 1989;Mugford and Tarloff, 1995;Su et al., 1998;Constan et al., 1999;Marczylo and Ioannides, 1999). The method of use of ABT has been quite variable from lab to lab, for example, single versus multiple dosing of animals prior to the test compound, p.o. versus i.v. route of administration, different pretreatment times, and different dose levels. Moreover, the pharmacokinetics of ABT in different species have not been reported. Also there is incomplete information in the literature on the inhibition of P450s by ABT (Knickle and Bend, 1992;Su et al., 1998;Di Re et al., 1999). To provide a guideline for the pretreatment regimen of ABT, 1) single dose oral pharmacokinetic studies were conducted in rats, dogs, and monkeys at several dose levels; 2) in vitro inhibition of various recombinant P450s by ABT was determined; and 3) the effect of selected doses of ABT on the intravenous pharmacokinetics of antipyrine, a probe for measuring efficiency of hepatic oxidative metabolism, was determined. The results from these studies are described in this report. Materials and MethodsABT and antipyrine (AP) were obtained from Sigma-Aldrich (St. Louis, MO). Single oral dose pharmacokinetics of ABT, in 0.5% aqueous methylcellulose, were studied in fasted male Sprague-Dawley rats at 10, 50, and 200 mg/kg (n Ն 3) and in Beagle dogs and Cynomolus m...

The high-throughput screening in drug discovery for absorption, distribution, metabolism and excretion (ADME) properties has become the norm in the industry. Only a few years ago it was ADME properties that were attributed to more failure of drugs than efficacy or safety in the clinic trials. With the realization of new techniques and refinement of existing techniques better projections for the pharmacokinetic properties of compounds in humans are being made, shifting the drug failure attributes more to the safety and efficacy properties of drug candidates. There are a tremendous number of tools available to discovery scientists to screen compounds for optimization of ADME properties and selection of better candidates. However, the use of these tools has generally been to characterize these compounds rather than to select among them. This report discusses applications of the available ADME tools to better understand the clinical implication of these properties, and to optimize these properties. It also provides tracts for timing of studies with respect to the stage of the compound during discovery, by means of a discovery assay by stage (DABS) paradigm. The DABS provide the team with a rationale for the types of studies to be done during hit-to-lead, early and late lead optimization stages of discovery, as well as outlining the deliverables (objectives) at those stages. DABS has proven to be optimal for efficient utilization of resources and helped the discovery team to track the progress of compounds and projects.

A series of highly potent, structurally novel, non-nucleoside RT inhibitors has been described. Low nanomolar concentrations of 5-chloro-3-(phenylsulfonyl)-indole-2-carboxamide (1) inhibit the HIV-1 RT enzyme in vitro and HTLVIIIb viral spread in MT-4 human T-lymphoid cells. Good oral bioavailability was observed in rhesus monkeys upon oral dosing of 1 as a suspension in methocel. When compared to other non-nucleoside inhibitors (e.g. 15-18), 1 possesses improved inhibitory potency with respect to the wild-type RT, as well as the K103N and Y181C mutant enzymes. Additional studies within this class of inhibitors are in progress.