Thomas C. K. Chan

Discovery of a Novel Mode of Protein Kinase Inhibition Characterized by the Mechanism of Inhibition of Human Mesenchymal-epithelial Transition Factor (c-Met) Protein Autophosphorylation by ARQ 197

A number of human malignancies exhibit sustained stimulation, mutation, or gene amplification of the receptor tyrosine kinase human mesenchymal-epithelial transition factor (c-Met). ARQ 197 is a clinically advanced, selective, orally bioavailable, and well tolerated c-Met inhibitor, currently in Phase 3 clinical testing in non-small cell lung cancer patients. Herein, we describe the molecular and structural basis by which ARQ 197 selectively targets c-Met. Through our analysis we reveal a previously undisclosed, novel inhibitory mechanism that utilizes distinct regulatory elements of the c-Met kinase. The structure of ARQ 197 in complex with the c-Met kinase domain shows that the inhibitor binds a conformation that is distinct from published kinase structures. ARQ 197 inhibits c-Met autophosphorylation and is highly selective for the inactive or unphosphorylated form of c-Met. Through our analysis of the interplay between the regulatory and catalytic residues of c-Met, and by comparison between the autoinhibited canonical conformation of c-Met bound by ARQ 197 to previously described kinase domains of type III receptor tyrosine kinases, we believe this to be the basis of a powerful new in silico approach for the design of similar inhibitors for other protein kinases of therapeutic interest.

Phase I trial of piroxicam in 62 dogs bearing naturally occurring tumors

SummaryPiroxicam, a nonsteroidal antiinflammatory drug, was given to 62 dogs bearing naturally occurring tumors in a phase I clinical trial. Dose escalation was performed, with oral doses ranging from 0.5 mg/kg every 48 h (q48h) to 1.5 mg/kg q48h being tested. Dose-limiting gastromestinal irritation/ulceration occurred in all four animals that received 1.5 mg/kg q48h. The maximum tolerated dose was 1 mg/kg q48h. Subclinical renal papillary necrosis occurred in two dogs (initial dosages, 1 and 1.5 mg/kg q48h, respectively). Following dose escalation, an additional group of dogs was treated with 0.3 mg/kg piroxicam q24h per os, the accepted canine dosage prior to this trial. Inclusion of this treatment group enabled evaluation of the toxicity of and tumor response to a daily dosage regimen. No complete remissions occurred in this trial. Partial remission was documented in three of ten dogs exhibiting transitional-cell carcinoma, in three of five animals bearing squamous-cell carcinoma, in one of three dogs displaying mammary adenocarcinoma, and in the one dog that exhibited a transmissible venereal tumor. The results of this study support the additional evaluation of piroxicam in a phase II clinical trial in dogs bearing naturally occurring tumors.

A Novel Mode of Protein Kinase Inhibition Exploiting Hydrophobic Motifs of Autoinhibited Kinases DISCOVERY OF ATP-INDEPENDENT INHIBITORS OF FIBROBLAST GROWTH FACTOR RECEPTOR

Protein kinase inhibitors with enhanced selectivity can be designed by optimizing binding interactions with less conserved inactive conformations because such inhibitors will be less likely to compete with ATP for binding and therefore may be less impacted by high intracellular concentrations of ATP. Analysis of the ATP-binding cleft in a number of inactive protein kinases, particularly in the autoinhibited conformation, led to the identification of a previously undisclosed non-polar region in this cleft. This ATP-incompatible hydrophobic region is distinct from the previously characterized hydrophobic allosteric back pocket, as well as the main pocket. Generalized hypothetical models of inactive kinases were constructed and, for the work described here, we selected the fibroblast growth factor receptor (FGFR) tyrosine kinase family as a case study. Initial optimization of a FGFR2 inhibitor identified from a library of commercial compounds was guided using structural information from the model. We describe the inhibitory characteristics of this compound in biophysical, biochemical, and cell-based assays, and have characterized the binding mode using x-ray crystallographic studies. The results demonstrate, as expected, that these inhibitors prevent activation of the autoinhibited conformation, retain full inhibitory potency in the presence of physiological concentrations of ATP, and have favorable inhibitory activity in cancer cells. Given the widespread regulation of kinases by autoinhibitory mechanisms, the approach described herein provides a new paradigm for the discovery of inhibitors by targeting inactive conformations of protein kinases.

D-501036, a novel selenophene-based triheterocycle derivative, exhibits potent in vitro and in vivo antitumoral activity which involves DNA damage and ataxia telangiectasia–mutated nuclear protein kinase activation

D-501036 [2,5-bis(5-hydroxymethyl-2-selenienyl)-3-hydroxymethyl-N-methylpyrrole] is herein identified as a novel antineoplastic agent with a broad spectrum of antitumoral activity against several human cancer cells and an IC50 value in the nanomolar range. The IC50 values for D-501036 in the renal proximal tubule, normal bronchial epithelial, and fibroblast cells were >10 μmol/L. D-501036 exhibited no cross-resistance with vincristine- and paclitaxel-resistant cell lines, whereas a low level of resistance toward the etoposide-resistant KB variant was observed. Cell cycle analysis established that D-501036 treatment resulted in a dose-dependent accumulation in S phase with concomitant loss of both the G0-G1 and G2-M phase in both Hep 3B and A-498 cells. Pulsed-field gel electrophoresis showed D-501036–induced, concentration-dependent DNA breaks in both Hep 3B and A-498 cells. These breaks did not involve interference with either topoisomerase-I and topoisomerase-II function or DNA binding. Rapid reactive oxygen species production and formation of Se-DNA adducts were evident following exposure of cells to D-501036, indicating that D-501036–mediated DNA breaks were attributable to the induction of reactive oxygen species and DNA adduct formation. Moreover, D-501036–induced DNA damage activated ataxia telangiectasia–mutated nuclear protein kinase, leading to hyperphosphorylation of Chk1, Chk2, and p53, decreased expression of CDC25A, and up-regulation of p21WAF1 in both p53-proficient and p53-deficient cells. Collectively, the results indicate that D-501036–induced cell death was associated with DNA damage–mediated induction of ataxia telangiectasia–mutated activation, and p53-dependent and -independent apoptosis pathways. Notably, D-501036 shows potent activity against the growth of xenograft tumors of human renal carcinoma A-498 cells. Thus, D-501036 is a promising anticancer compound that has strong potential for the management of human cancers. [Mol Cancer Ther 2007;6(1):193–202]

Plasma and cerebrospinal fluid pharmacokinetics of cytosine arabinoside in dogs

SummaryCytosine arabinoside (ara-C) is a component of many protocols for the treatment of CNS (central nervous system) leukemia and lymphoma in humans and dogs. It is also used for the prophylaxis of CNS metastasis in acute lymphoblastic leukemia. Although ara-C enters the cerebrospinal fluid (CSF) of human cancer patients after i.v. administration, it is unclear whether a similar CNS distribution occurs in humans whose blood-brain barrier has not been compromised by invasive disease. No information on the penetration of ara-C into the CSF in dogs is available. We studied the plasma and CSF pharmacokinetics of 600 mg/m2 ara-C in ten healthy male dogs after its administration as a rapid i.v. bolus (six dogs) or as a 12-h i.v. infusion (four dogs). Ara-C concentration in blood and CSF samples was determined by high-performance liquid chromatography (HPLC). After an i.v. bolus of ara-C, the mean plasma distribution half-life was 7.1±4.5 min and the mean elimination half-life was 69±28 min. The mean plasma clearance was 227±125 ml min−1 m−2. The peak concentration of ara-C in the CSF was 29±11 μm, which occurred at 57±13 min after the ara-C bolus. The CSF elimination half-life was 113±26 min. During a 12-h infusion of ara-C (50 mg m−2 h−1), the plasma steady-state concentration was 14.1±4.2 μm, the CSF steady-state concentration was 8.3±1.1 μm, and the CSF: plasma ratio was 0.62±0.14. The plasma eleimination half-life was 64±19 min and the plasma clearance was 214±69 ml min−1 m−2. The CSF elimination half-life was 165±28 min. No clinically significant toxicity was observed over a 21-day period following drug administration in either of the treatment groups. Our data indicate that ara-C crosses the blood-brain barrier in normal dogs and that i.v. administration of this drug has potential as a treatment modality for neoplasia involving the CNS.

Synthetic methods for the preparation of ARQ 501 (β-Lapachone) human blood metabolites

ARQ 501 (3,4-dihydro-2,2-dimethyl-2H-naphthol[1,2-b] pyran-5,6-dione), a synthetic version of beta-Lapachone, is a promising anti-cancer agent currently in multiple Phase II clinical trials. Promising anti-cancer activity was observed in Phase I and Phase II trials. Metabolism by red blood cells of drugs is an understudied area of research and the metabolites arising from oxidative ring opening (M2 and M3), decarbonylation/ring contraction (M5), and decarbonylation/oxidation (M4 and M6) of ARQ 501 offer a unique opportunity to provide insight into these metabolic processes. Since these metabolites were not detected in in vitro incubations of ARQ 501 with liver microsomes and were structurally diverse, confirmation by chemical synthesis was considered essential. In this report, we disclose the synthetic routes employed and the characterization of the reference standards for these blood metabolites as well as additional postulated structures, which were not confirmed as metabolites.

Identification of the in Vitro Metabolites of 3,4-Dihydro-2,2-dimethyl-2H-naphthol[1,2-b]pyran-5,6-dione (ARQ 501; β-Lapachone) in Whole Blood

3,4-Dihydro-2,2-dimethyl-2H-naphthol[1,2-b]pyran-5,6-dione (ARQ 501; β-lapachone) showed promising anticancer activity in phase I clinical trials as monotherapy and in combination with cytotoxic drugs. ARQ 501 is currently in multiple phase II clinical trials. In vitro incubation in fresh whole blood at 37°C revealed that ARQ 501 is stable in plasma but disappears rapidly in whole blood. Our data showed that extensive metabolism in red blood cells (RBCs) was mainly responsible for the rapid disappearance of ARQ 501 in whole blood. By comparison, covalent binding of ARQ 501 and/or its metabolites to whole blood components was a minor contributor to the disappearance of this compound. Sequestration of intact ARQ 501 in RBCs was not observed. Cross-species metabolite profiles from incubating [14C]ARQ 501 in freshly drawn blood were characterized using a liquid chromatography-mass spec-trometry-accurate radioactivity counter. The results show that ARQ 501 was metabolized more rapidly in mouse and rat blood than in dog, monkey, and human blood, with qualitatively similar metabolite profiles. Six metabolites were identified in human blood using ultra-high performance liquid chromatography/time-of-flight mass spectrometry, and the postulated structure of five metabolites was confirmed using synthetic standards. We conclude that the primary metabolic pathway of ARQ 501 in human blood involved oxidation of the two adjacent carbonyl groups to produce dicarboxylic and monocarboxylic metabolites, elimination of a carbonyl group to form a ring-contracted metabolite, and lactonization to produce two metabolites with a pyrone ring to form a ring-contracted metabolite. Metabolism by RBCs may play a role in clearance of ARQ 501 from the blood compartment in cancer patients.

Measurement of NK activity in effector cells purified from canine peripheral lymphocytes

Natural killer (NK) cells spontaneously lyse a variety of tumor cells in vitro, and are believed to play an important role in host resistance to tumor growth and metastasis in vivo. As part of our work in comparative oncology, we have designed and validated a canine NK cell assay. Of several lymphocyte isolation techniques evaluated, sedimentation of whole blood through a two-step Ficoll/Hypaque gradient (sp. gr. 1.066/1.119) followed by plastic adherence of monocytes resulted in the most pure lymphocyte population (> 95% lymphocytes). Of four cell lines evaluated as targets in the NK assay, a canine thyroid adenocarcinoma (CTAC) cell line was determined to be most sensitive, and a lymphoblastoid (CT45-S) cell line was determined to be most resistant to NK lysis. A 15 h effector-target incubation period using these targets resulted in reproducible measurement of cell specific lytic activity. Passage of canine lymphocytes through nylon wool columns did not result in a significant increase in NK activity. A final sedimentation of purified lymphocytes through a 45/50% Percoll gradient concentrated NK activity into a single band of lymphocytes. Lymphocytes forming conjugates with CTAC target cells were 5.5-6.5 microns in diameter, and were characterized by a reniform nucleus and varying numbers of electron-dense cytoplasmic granules.

In vitro metabolism of β-lapachone (ARQ 501) in mammalian hepatocytes and cultured human cells

ARQ 501 (3,4-dihydro-2,2-dimethyl-2H-naphthol[1,2-b]pyran-5,6-dione, beta-lapachone) is an anticancer agent, currently in multiple phase II clinical trials as monotherapy and in combination with other cytotoxic drugs. This study focuses on in vitro metabolism in cryopreserved hepatocytes from mice, rats, dogs and humans using [(14)C]-labeled ARQ 501. Metabolite profiles were characterized using liquid chromatography/mass spectrometry combined with an accurate radioactivity counter. Ion trap mass spectrometry was employed for further structural elucidation. A total of twelve metabolites were detected in the mammalian hepatocytes studied; all of which but one were generated from phase II conjugation reactions. Ten of the observed metabolites were produced by conjugations occurring at the reduced ortho-quinone carbonyl groups of ARQ 501. The metabolite profiles revealed that glucuronidation was the major biotransformation pathway in mouse and human hepatocytes. Monosulfation was the major pathway in dog, while, in rat, it appears glucuronidation and sulfation pathways contributed equally. Three major metabolites were found in rats: monoglucuronide M1, monosulfate M6, and glucuronide-sulfate M9. Two types of diconjugation metabolites were formed by attachment of the second glycone to an adjacent hydroxyl or to an existing glycone. Of the diconjugation metabolites, glucosylsulfate M10, diglucuronide M5, and glucuronide-glucoside M11 represent rarely observed phase II metabolites in mammals. The only unconjugated metabolite was generated through hydrolysis and was observed in rat, dog and human hepatocytes. ARQ 501 appeared less stable in human hepatocytes than in those of other species. To further elucidate the metabolism of ARQ 501 in extrahepatic sites, its metabolism in human kidney, lung and intestine cells was also studied, and only monoglucuronide M1 was observed in all the cell types examined.

Facile synthesis of 5′-deoxy- and 2′,5′-dideoxy-6-thiopurine nucleosides by nucleoside phosphorylases

Abstract 5′-Deoxy-6-thioguanosine, 2′,5′-dideoxy-6-thioguanosine, 5′-deoxy-6-mercaptopurine riboside and 2′,5′-dideoxy-6-mercaptopurine riboside were synthesized enzymatically from thiopurine bases and corresponding ribosyl donors using nucleoside phosphorylase. This is the first report of trans-5′-deoxyribosylation to thiopurine bases by nucleoside phosphorylase. 5′-Deoxy-6-thioguanosine selectively blocked the growth of v-ras-transformed human bronchial epithelial cells. In addition, the in vitro antitumor cytotoxicity data for 5′-deoxy- and 2′,5′-dideoxy-6-thiopurine nucleosides were comparable to those for the corresponding thiopurine bases.