Design, synthesis, and evaluation of in vivo potency and selectivity of epoxysuccinyl-based inhibitors of papain-family cysteine proteases.

The papain-family cathepsins are cysteine proteases that are emerging as promising therapeutic targets for a number of human disease conditions ranging from osteoporosis to cancer. Relatively few selective inhibitors for this family exist, and the in vivo selectivity of most existing compounds is unclear. We present here the synthesis of focused libraries of epoxysuccinyl-based inhibitors and their screening in crude tissue extracts. We identified a number of potent inhibitors that display selectivity for endogenous cathepsin targets both in vitro and in vivo. Importantly, the selectivity patterns observed in crude extracts were generally retained in vivo, as assessed by active-site labeling of tissues from treated animals. Overall, this study identifies several important compound classes and highlights the use of activity-based probes to assess pharmacodynamic properties of small-molecule inhibitors in vivo.

Preclinical pharmacology of the novel antitumor agent adaphostin, a tyrphostin analog that inhibits bcr/abl.

PURPOSE: To define several pharmacological properties for the potential anticancer agent, adaphostin, in order to determine whether the compound is appropriate for clinical evaluation as an anticancer agent. METHODS: The analytical procedure involved high-performance liquid chromatography and utilized an analytical J'Sphere ODS H-80 column. RESULTS: The stability of adaphostin at two different concentrations was determined at temperatures of 37 degrees C, 4 degrees C, and -80 degrees C, in the plasma of mice, rats, dogs, and humans. The compound was most stable at the lower temperatures. At all temperatures, adaphostin was generally most stable in human plasma and least stable in dog plasma. Adaphostin bound strongly (>93%) to proteins in plasma from all four species. Following intravenous (i.v.) administration to mice (50 mg/kg; 150 mg/m(2)), plasma concentrations declined rapidly from 50 microM at 2 min to 1 microM at 2 h. Elimination was triexponential, with t (1/2) values of 1.1, 9.1, and 41.2 min. The Cl(tb) was 0.411 L/(min.m(2)), the V (dss) was 24.6 L/m(2), and the AUC was 927 microM.min. In a comparison of vehicles for intraperitoneal (i.p.) dosing, PEG 300 allowed the highest plasma concentrations of adaphostin. Bioavailability following an i.p. dose was greater than that following a subcutaneous dose, or that for a dose administered by oral gavage. For rats dosed i.v. with adaphostin (50 mg/kg; 300 mg/m(2)), plasma concentrations also decreased triexponentially, with t (1/2) values of 1.8, 10.6, and 136 min. Other pharmacokinetic values were Cl(tb) = 0.466 L/(min.m(2)), AUC = 1,161 microM.min, and V (dss)=8.0 L/m(2). Analysis of samples collected from two dogs dosed i.v. with adaphostin (7.5 mg/kg; 150 mg/m(2)) showed that plasma concentrations decreased in a biphasic manner, with individual values for t (1/2alpha) of 6.0 and 9.8 min for the distribution phase and t (1/2beta) of 40.6 and 66.2 min for the elimination phase. Other pharmacokinetic values were Cl(tb) = 0.565 and 0.852 L/(min.m(2)), AUC = 673 and 446 microM min, and V (dss) = 29.6 and 56.8 L/m(2). CONCLUSIONS: The stability of adaphostin in plasma varies with species. In mice and dogs dosed with adaphostin, plasma concentrations of the compound decreased rapidly. The clearance of adaphostin from plasma, on an m(2) basis, was equivalent for mice and rats but more rapid in dogs. These results are relevant for assessing the pharmacologic and toxicologic profiles and the antitumor activity of adaphostin in humans.

Preclinical evaluation of amino acid prodrugs of novel antitumor 2-(4-amino-3-methylphenyl)benzothiazoles.

Novel 2-(4-aminophenyl)benzothiazoles (e.g., compounds 1 and 2) possess highly selective, potent antitumor properties in vitro and in vivo. Elucidation of the mechanism of action of this structurally simple class of compounds has occurred in parallel with selection of a candidate clinical agent. Antitumor benzothiazoles induce and are biotransformed by cytochrome P 450 1A1 to putative active, as well as inactive metabolites. Metabolic inactivation of the molecule has been thwarted by isosteric replacement of hydrogen with fluorine atoms at positions around the benzothiazole nucleus. Amino acid conjugation to the exocyclic primary amine function of 2-(4-aminophenyl)benzothiazoles has been used to overcome limitations posed by drug lipophilicity. Water soluble, chemically stable prodrugs rapidly and quantitatively revert to their parent amine in mice, rats, and dogs in vivo. Plasma concentrations of 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (2) regenerated from the lysylamide prodrug (2b), sufficient to elicit cytocidal activity against ZR-75-1 and T47D human mammary carcinoma cell lines persist > 6 h. The growth of breast (MCF-7) and ovarian (IGROV-1) xenograft tumors is significantly retarded by 2b. Manageable toxic side effects are reported from preclinically efficacious doses of 2b. Cytochrome P 450 1A1 protein expression, selectively induced in sensitive carcinoma cells, was detected in MCF-7 and IGROV-1 tumors 24 h after treatment of mice with 2b (20 mg/kg). The lysyl amide prodrug of 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole is potentially suitable for clinical evaluation.