Hongxia Lin

Targeting tumor metabolism with 2-deoxyglucose in patients with castrate-resistant prostate cancer and advanced malignancies.

A profound difference between cancer and normal tissues is the preferential utilization of glycolysis by cancer cells. To translate this paradigm in the clinic, we completed a phase I study of 2‐deoxyglucose (2DG), and assessed 2DG uptake with fluorodeoxyglucose (FDG) positron emission tomography (PET) and the autophagy substrate p62 as a marker of 2DG resistance.

Contribution of serine, folate and glycine metabolism to the ATP, NADPH and purine requirements of cancer cells

Recent observations on cancer cell metabolism indicate increased serine synthesis from glucose as a marker of poor prognosis. We have predicted that a fraction of the synthesized serine is routed to a pathway for ATP production. The pathway is composed by reactions from serine synthesis, one-carbon (folate) metabolism and the glycine cleavage system (SOG pathway). Here we show that the SOG pathway is upregulated at the level of gene expression in a subset of human tumors and that its level of expression correlates with gene signatures of cell proliferation and Myc target activation. We have also estimated the SOG pathway metabolic flux in the NCI60 tumor-derived cell lines, using previously reported exchange fluxes and a personalized model of cell metabolism. We find that the estimated rates of reactions in the SOG pathway are highly correlated with the proliferation rates of these cell lines. We also observe that the SOG pathway contributes significantly to the energy requirements of biosynthesis, to the NADPH requirement for fatty acid synthesis and to the synthesis of purines. Finally, when the PC-3 prostate cancer cell line is treated with the antifolate methotrexate, we observe a decrease in the ATP levels, AMP kinase activation and a decrease in ribonucleotides and fatty acids synthesized from [1,2-13C2]-D-glucose as the single tracer. Taken together our results indicate that the SOG pathway activity increases with the rate of cell proliferation and it contributes to the biosynthetic requirements of purines, ATP and NADPH of cancer cells.

Rationally designed treatment for solid tumors with MAPK pathway activation: a Phase I study of paclitaxel and bortezomib using an adaptive dose-finding approach

In the preclinical setting, phosphorylation and subsequent proteosomal degradation of the proapoptotic protein BIM confers resistance to paclitaxel in solid tumors with RAS/RAF/MAPK pathway activation. Concurrent administration of the proteasome inhibitor bortezomib enables paclitaxel-induced BIM accumulation, restoring cancer cell apoptosis in vitro and producing tumor regression in mice in vivo. A phase I study was conducted to determine the maximum tolerated dose (MTD) of paclitaxel and bortezomib combinatorial treatment. Sixteen patients with refractory solid tumors commonly exhibiting mitogen-activated protein kinase (MAPK) pathway activation were treated weekly with paclitaxel and bortezomib. Starting doses were 40 mg/m2 for paclitaxel and 0.7 mg/m2 for bortezomib. A modified continual reassessment method adapted for 2-drug escalation was used for MTD determination with 3-patient cohorts treated at each dose level. MTD was reached at 60 mg/m2 paclitaxel and 1.0 mg/m2 bortezomib, the recommended phase II dose. Therapy was overall well tolerated. Most frequently observed toxicities included anemia (in 43.75% of patients, one grade 3 event), fatigue (in 43.75% of patients, one grade 3 event beyond cycle 1), and neuropathy (in 31.25% of patients, one grade 3 event after cycle 1). Of 15 evaluable patients, one non–small-cell lung carcinoma (NSCLC) patient with paclitaxel exposure at the adjuvant setting had a partial response and five patients had stable disease (SD); median disease stabilization was 143.5 days; three NSCLC patients had SD lasting 165 days or longer. Thus, rationally designed weekly treatment with paclitaxel and bortezomib in solid tumors with MAPK pathway activation, including previously taxane-treated malignancies, is a tolerable regimen with preliminary signals of antitumor activity worthy of further investigation. Mol Cancer Ther; 10(8); 1509–19. ©2011 AACR.

A validated HPLC assay for the determination of R-(-)-gossypol in human plasma and its application in clinical pharmacokinetic studies

R-(-)-gossypol acetic acid (AT-101), a natural BH3 mimetic, is investigated in a Phase I/II clinical trial for the treatment of advanced solid tumor malignancies. Gossypol undergoes rapid degradation in solution phase, which causes major technical difficulty for its quantitation in plasma. We developed and validated a sensitive HPLC assay for pharmacokinetic evaluation of gossypol. Acetonitrile deproteinization method was chosen for sample preparation and Schiffs base derivative, R-(-)-gossypol-diamino-propanol (GDP), was used as internal standard. Chromatographic separation of gossypol in plasma was performed using a Zorbax Eclipse XDB column C(18) at 30 °C. The mobile phase consists of 10 mmol/L KH(2)PO(4) (pH 3.0) and acetonitrile (20:80) at 1.0 mL/min flow rate. Linearity ranged over 56-3585 ng/mL (R(2)=0.9997±0.0003, n=4), and the limit of detection was 28 ng/mL. The intra- and inter-assay precision was less than 13.7% and the bias ranged from -7.4 to 7.0%. The method was successfully applied to characterize the pharmacokinetics of AT-101 in a Phase I clinical trial. The validated assay is accurate, and sensitive with minimum loss and rapid analysis time and suitable for quantification of gossypol for pharmacokinetics evaluation.

A validated bioanalytical HPLC method for pharmacokinetic evaluation of 2-deoxyglucose in human plasma.

2-Deoxyglucose (2-DG), an analog of glucose, is widely used to interfere with glycolysis in tumor cells and studied as a therapeutic approach in clinical trials. To evaluate the pharmacokinetics of 2-DG, we describe the development and validation of a sensitive HPLC fluorescent method for the quantitation of 2-DG in plasma. Plasma samples were deproteinized with methanol and the supernatant was dried at 45°C. The residues were dissolved in methanolic sodium acetate-boric acid solution. 2-DG and other monosaccharides were derivatized to 2-aminobenzoic acid derivatives in a single step in the presence of sodium cyanoborohydride at 80°C for 45 min. The analytes were separated on a YMC ODS C₁₈ reversed-phase column using gradient elution. The excitation and emission wavelengths were set at 360 and 425 nm. The 2-DG calibration curves were linear over the range of 0.63-300 µg/mL with a limit of detection of 0.5 µg/mL. The assay provided satisfactory intra-day and inter-day precision with RSD less than 9.8%, and the accuracy ranged from 86.8 to 110.0%. The HPLC method is reproducible and suitable for the quantitation of 2-DG in plasma. The method was successfully applied to characterize the pharmacokinetics profile of 2-DG in patients with advanced solid tumors.

Comparison of LC-MS Assay and HPLC Assay of Busulfan in Clinical Pharmacokinetics Studies

Busulfan is used in preparative regimens for bone marrow transplantation and timely busulfan plasma concentration reporting is critical for subsequent dose adjustment. We compared two sensitive methods for pharmacokinetics studies including LC-MS assay and HPLC precolumn derivatization assay. Chromatographic separation was performed on a Gemini C18 column. Liquid-liquid extraction with ethyl acetate was used for plasma sample preparation. Busulfan and internal standard ([2H8]-busulfan) were detected as ammonium adducts at m/z 264.2 and 272.2 for LC-MS assay. For HPLC assay, the extraction from plasma was derivatized with 2-naphathalenethiol using synthesized internal standard (1,6-(methanesulfonyloxy)octane). The Ex and Em wavelength was 255 nm and 370 nm. The limit of detection was 15.6 ng/mL and 7.8 ng/mL for HPLC and LC-MS assay and good linearity ranging from 31.25–1000 ng/mL for HPLC and 15.6-1000 ng/mL for LC-MS assay. The intra and interday assay precision were less than 9.2% and 12.0% for LC-MS and HPLC assay. The pharmacokinetic parameters were estimated using noncompartmental pharmacokinetic model with WinNonlin. Busulfan AUClast showed an average difference of 0.7% between the two methods. The LC-MS method is accurate, reproducible, and requires less specimen, sample preparation and analysis time over the HPLC assay, making busulfan monitoring faster and easier in clinical practice.

Abstract C151: Contribution of serine, folate, and glycine metabolism to the ATP, NADPH, and purine requirements of cancer cells.

Background: Recent observations on cancer cell metabolism indicate increased serine synthesis from glucose as a marker of poor prognosis. Expression of genes in this pathway also correlate with sensitivity to the antifolate methotrexate (Vazquez A, et al, Cancer Res. 2013 Jan 15;73(2):478-82). Using a large-scale model of human cell metabolism we have predicted that serine synthesis can be routed to a pathway for ATP production. The pathway is composed by reactions from the serine synthesis, one carbon (folate) metabolism and the glycine cleavage system (SOG pathway) and its flux is predicted to increase at high proliferation rates. Results: Here we show that the SOG pathway is upregulated at the level of gene expression in a subset of human tumors and its level of expression correlates with gene signatures of cell proliferation and Myc targets activation. To investigate the activity of the SOG pathway at the level of metabolic fluxes we estimated the metabolic fluxes of the NCI60 panel of tumor derived cell lines, using previously reported exchange fluxes and a flux balance model of cell metabolism. We show that the estimated rates of reactions in the SOG pathway are highly correlated with the proliferation rates of these cell lines. We also find that the SOG pathway contributes significantly to the energy requirements of biosynthesis, the NADPH requirements of fatty acid synthesis and to the synthesis of purines. Finally, when the PC-3 prostate cancer cell line is subject to treatment with the antifolate methotrexate, we observe a decrease in the ATP levels, an inhibition of the proliferation rate and a decrease in the ribonucleotides and fatty acids synthesized from glucose. Conclusions: Taken together our results indicate that the SOG pathway activity increases with the rate of cell proliferation and it contributes to the biosynthetic requirements of purines, ATP and NADPH. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C151. Citation Format: Philip M. Tedeschi, Elke K. Markert, Murugesan Gounder, HongXia Lin, Sonia C. Dolfi, Leo Li-Ying Chan, Jean Qiu, Kim M. Hirshfield, Laszlo G. Boros, Joseph R. Bertino, Zoltan N. Oltvai, Alexei Vazquez. Contribution of serine, folate, and glycine metabolism to the ATP, NADPH, and purine requirements of cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C151.

Abstract 2759: Validated liquid chromatography-mass spectrometry assay for determination of busulfan: Application to clinical pharmacokinetics studies

Busulfan (BU), an alkylating agent, also known to have anti-leukemic efficacy, is commonly used in preparative regimens for allogenic and autologous stem cell marrow transplantation. The efficacy of BU is related to systemic levels and the area under concentration-time curve (AUC), and hence, a timely pharmacokinetic (PK) report is critical for subsequent dose adjustment. We developed two highly sensitive methods: a rapid, accurate, and sensitive liquid chromatography-mass spectrometry (LC-MS) method using direct inject tandem mass spectrometry with SIM mode, and an alternate equally sensitive high performance liquid chromatographic fluorometric (HPLC-FL) method for the quantitation of BU in human plasma. Method: BU concentrations were quantified in 50 μL of plasma spiked with [ 2 H 8 ]-busulfan, followed by liquid-liquid extraction with ethyl acetate. The solvent extract was dried under nitrogen and the residue was dissolved in mobile phase and analyzed using LC-MS. The mobile was 10mmol/L ammonium acetate and 10mL/L acetic acid in water and acetonitrile, (70:30) and flow rate was 0.5ml/min. Separation of BU was performed on a Gemini C 18 column (4.6 × 150 mm, 3μm) at 30°C. BU and IS were detected as ammonium adducts in selected-ion monitoring mode at m/z 264.2 and 272.2 at the retention time of 5.8 min. When plasma samples were analyzed by HPLC-FL method, 200μL plasma spiked with 1,6 (methanesulfonyloxy) octane was extracted in ethyl acetate and dried under air at 45°C. The residue was dissolved in 200μL ethanol and derivatized with 2-naphthelenethiol according to Nara S., et al (Analytical Sci. 2000,16, 287). The samples were analyzed on a HPLC system with fluorescence detector set at excitation wavelength 255 nm and emission wavelength 370 nm. The mobile was a mixture of methanol: acetonitrile: sodium acetate buffer (0.1M, pH 7.0) using gradient elution, and flow rate was 1.0 mL/min. Results: Both assay methods are comparable in precision, accuracy, linearity, and sensitivity. The calibration curve for LC-MS assay was linear at 31.25-1000ng/ml. The LOD of assay was 7.8ng/ml and the LOQ was 31.25ng/ml. The correlation coefficient for calibration curve was 0.997±0.003 (n =4). The intra and inter-assay precision was less than 3.0% and the accuracy ranged from 95.4% to 101.7%. Busulfan AUC last calculation comparison with HPLC-FL derivatization method showed an average difference between the assays of 7.7%. Conclusion: The method LC-MS is highly accurate, reproducible, and requires less specimen, sample preparation, and analysis time over the HPLC-FL derivitization method. The new LC-MS assay is rapid and sensitive and provides an appropriate method for quantification of BU in human plasma, making therapeutic drug monitoring of BU faster and easier in clinical practice. Acknowledgement: The study is supported by funds from Century for the Cure and U01 grant from NCI. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2759.