Timothy J. Siford

Bio: Timothy J. Siford is an academic researcher from Science Applications International Corporation. The author has contributed to research in topics: High-performance liquid chromatography & Alvocidib. The author has an hindex of 2, co-authored 2 publications receiving 263 citations.

Determination of flavopiridol (L86 8275; NSC 649890) in human plasma by reversed-phase liquid chromatography with electrochemical detection

Abstract: Purpose: Flavopiridol is a flavone which inhibits several cyclin-dependent kinases, and exhibits potent growth-inhibitory activity against a number of human tumor cell lines both in vitro, and when grown as xenografts in mice. It is currently being evaluated in a phase I clinical trial at the National Cancer Institute. The objective of this project was to develop and validate an analytical method for the assay of flavopiridol in human plasma, with sufficient sensitivity to permit the plasma pharmacokinetics of flavopiridol to be studied during clinical trials. Methods: Flavopiridol was isolated from human plasma samples by extraction with t-butylmethyl ether following alkalinization with borate buffer (pH 8.0). The extract was evaporated, the residue was dissolved in mobile phase, and analyzed by reversed-phase high-pressure liquid chromatography. Chromatography was accomplished with a polymer-based C18 column eluted with a mobile phase consisting of methanol-phosphate buffer, pH 11.0 (53:47 v/v). Electrochemical detection (ECD) was employed. Results: Flavopiridol was recovered from human plasma with an efficiency of 85–87%. Calibration curves were linear over the concentration range 10–500 nM (4.4–219 ng/ml). Plasma standard concentrations were measured with an accuracy and precision ranging from 3.2% to 10%. Regression analysis of flavopiridol concentrations of 15 clinical trial plasma samples ranging in concentration from approximately 50 to 4000 μM quantitated by both ECD and mass spectrometry showed close agreement. The equation of the regression line was y = 1.02x + 8 with a correlation coefficient of 0.969. Continuous infusion of flavopiridol in four patients for 72 h at a rate of 50 mg/m2 per day, resulted in mean steady-state plasma concentrations of from 200 to 300 nM. Levels declined in a biexponential manner following termination of the infusion, falling to approximately 10 nM after 48 h. Conclusions: An analytical method for the assay of flavopiridol in human plasma was developed with sensitivity to at least 10 nM. The assay is accurate, precise and specific, and is suitable for determination of plasma flavopiridol concentrations for pharmacokinetic studies during clinical trials.

Cell Cycle Proteins as Promising Targets in Cancer Therapy

Abstract: Cancer is characterized by uncontrolled tumour cell proliferation resulting from aberrant activity of various cell cycle proteins. Therefore, cell cycle regulators are considered attractive targets in cancer therapy. Intriguingly, animal models demonstrate that some of these proteins are not essential for proliferation of non-transformed cells and development of most tissues. By contrast, many cancers are uniquely dependent on these proteins and hence are selectively sensitive to their inhibition. After decades of research on the physiological functions of cell cycle proteins and their relevance for cancer, this knowledge recently translated into the first approved cancer therapeutic targeting of a direct regulator of the cell cycle. In this Review, we focus on proteins that directly regulate cell cycle progression (such as cyclin-dependent kinases (CDKs)), as well as checkpoint kinases, Aurora kinases and Polo-like kinases (PLKs). We discuss the role of cell cycle proteins in cancer, the rationale for targeting them in cancer treatment and results of clinical trials, as well as the future therapeutic potential of various cell cycle inhibitors.

Cyclin-dependent kinase pathways as targets for cancer treatment.

Abstract: Cyclin-dependent kinases (cdks) are critical regulators of cell cycle progression and RNA transcription. A variety of genetic and epigenetic events cause universal overactivity of the cell cycle cdks in human cancer, and their inhibition can lead to both cell cycle arrest and apoptosis. However, built-in redundancy may limit the effects of highly selective cdk inhibition. Cdk4/6 inhibition has been shown to induce potent G1 arrest in vitro and tumor regression in vivo; cdk2/1 inhibition has the most potent effects during the S and G2 phases and induces E2F transcription factor-dependent cell death. Modulation of cdk2 and cdk1 activities also affects survival checkpoint responses after exposure to DNA-damaging and microtubule-stabilizing agents. The transcriptional cdks phosphorylate the carboxy-terminal domain of RNA polymerase II, facilitating efficient transcriptional initiation and elongation. Inhibition of these cdks primarily affects the accumulation of transcripts with short half-lives, including those encoding antiapoptosis family members, cell cycle regulators, as well as p53 and nuclear factor-kappa B-responsive gene targets. These effects may account for apoptosis induced by cdk9 inhibitors, especially in malignant hematopoietic cells, and may also potentiate cytotoxicity mediated by disruption of a variety of pathways in many transformed cell types. Current work is focusing on overcoming pharmacokinetic barriers that hindered development of flavopiridol, a pan-cdk inhibitor, as well as assessing novel classes of compounds potently targeting groups of cell cycle cdks (cdk4/6 or cdk2/1) with variable effects on the transcriptional cdks 7 and 9. These efforts will establish whether the strategy of cdk inhibition is able to produce therapeutic benefit in the majority of human tumors.

Preclinical and Clinical Development of Cyclin-Dependent Kinase Modulators

Abstract: In the last decade, the discovery and cloning of the cyclin-dependent kinases (cdks), key regulators of cell cycle progression, have led to the identification of novel modulators of cdk activity. Initial experimental results demonstrated that these cdk modulators are able to block cell cycle progression, induce apoptotic cell death, promote differentiation, inhibit angiogenesis, and modulate transcription. Alteration of cdk activity may occur indirectly by affecting upstream pathways that regulate cdk activity or directly by targeting the cdk holoenzyme. Two direct cdk modulators, flavopiridol and UCN-01, are showing promising results in early clinical trials, in which the drugs reach plasma concentrations that can alter cdk activity in vitro. Although modulation of cdk activity is a well-grounded concept and new cdk modulators are being assessed for clinical testing, important scientific questions remain to be addressed. These questions include whether one or more cdks should be inhibited, how cdk inhibitors should be combined with other chemotherapy agents, and which cdk substrates should be used to assess the biologic effects of these drugs in patients. Thus, modulation of cdk activity is an attractive target for cancer chemotherapy, and several agents that modulate cdk activity are in or are approaching entry into clinical trials.

Halogen atoms in the modern medicinal chemistry: hints for the drug design.

Abstract: A significant number of drugs and drug candidates in clinical development are halogenated structures. For a long time, insertion of halogen atoms on hit or lead compounds was predominantly performed to exploit their steric effects, through the ability of these bulk atoms to occupy the binding site of molecular targets. However, halogens in drug - target complexes influence several processes rather than steric aspects alone. For example, the formation of halogen bonds in ligand-target complexes is now recognized as a kind of intermolecular interaction that favorably contributes to the stability of ligand-target complexes. This paper is aimed at introducing the fascinating versatility of halogen atoms. It starts summarizing the prevalence of halogenated drugs and their structural and pharmacological features. Next, we discuss the identification and prediction of halogen bonds in protein-ligand complexes, and how these bonds should be exploited. Interesting results of halogen insertions during the processes of hit-to-lead or lead-to-drug conversions are also detailed. Polyhalogenated anesthetics and protein kinase inhibitors that bear halogens are analyzed as cases studies. Thereby, this review serves as one guide for the virtual screening of libraries containing halogenated compounds and may be a source of inspiration for the medicinal chemists.

Cell cycle and apoptosis.

Abstract: Apoptosis and proliferation are intimately coupled. Some cell cycle regulators can influence both cell division and programmed cell death. The linkage of cell cycle and apoptosis has been recognized for c-Myc, p53, pRb, Ras, PKA, PKC, Bcl-2, NF-kappa B, CDK, cyclins and CKI. This review summarizes the different functions of the proteins presently known to control both apoptosis and cell cycle progression. These proteins can influence apoptosis or proliferation but different variables, including cell type, cellular environment and genetic background, make it difficult to predict the outcome of cell proliferation, cell cycle arrest or cell death. These important decisions of cell proliferation or cell death are likely to be controlled by more than one signal and are necessary to ensure a proper cellular response.