Glutamine Antagonist GA-607 Causes a Dramatic Accumulation of FGAR Which Can be Used to Monitor Target Engagement

Background: Metabolomic analyses from our group and others have shown that tumors treated with glutamine antagonists (GA) exhibit robust accumulation of formylglycinamide ribonucleotide (FGAR), an intermediate in the de novo purine synthesis pathway. The increase in FGAR is attributed to the inhibition of the enzyme FGAR amidotransferase (FGAR-AT) that catalyzes the ATP-dependent amidation of FGAR to formylglycinamidine ribonucleotide (FGAM). While perturbation of this pathway resulting from GA therapy has long been recognized, no study has reported systematic quantitation and analyses of FGAR in plasma and tumors. Objective: Herein, we aimed to evaluate the efficacy of our recently discovered tumor-targeted GA prodrug, GA-607 (isopropyl 2-(6-acetamido-2-(adamantane-1-carboxamido)hexanamido)-6-diazo-5-oxohexanoate), and demonstrate its target engagement by quantification of FGAR in plasma and tumors. Methods: Efficacy and pharmacokinetics of GA-607 were evaluated in a murine EL4 lymphoma model followed by global tumor metabolomic analysis. Liquid chromatography-mass spectrometry (LC-MS) based methods employing the ion-pair chromatography approach were developed and utilized for quantitative FGAR analyses in plasma and tumors. Results: GA-607 showed preferential tumor distribution and robust single-agent efficacy in a murine EL4 lymphoma model. While several metabolic pathways were perturbed by GA-607 treatment, FGAR showed the highest increase qualitatively. Using our newly developed sensitive and selective LC-MS method, we showed a robust >80- and >10-fold increase in tumor and plasma FGAR levels, respectively, with GA-607 treatment. Conclusion: These studies describe the importance of FGAR quantification following GA therapy in cancer and underscore its importance as a valuable pharmacodynamic marker in the preclinical and clinical development of GA therapies.

Chemical Stability of the Fertilizer Chelates Fe-EDDHA and Fe-EDDHSA over Time

In application conditions, the influence of environmental parameters on used fertilizer chelates and their distribution over time is important. For this purpose, the changes in the content of micronutrient ions and Fe-EDDHA and Fe-EDDHSA chelates in an aqueous medium at different pH values were studied. In the assumed time, changes in the ions content were analyzed using the voltammetry method at pH 3, 5 and 7. The content of isomers and chelate forms was analyzed by ion pair chromatography at pH 3, 5 and 7. These studies allowed us to determine the effect of pH on the stability of iron chelates over time.

Development of Ion pair Chromatography Method for Assay of Telaprevir by Response Surface Methodology

Response surface methodology approach has been utilized for the assay of Telaprevir in pure and formulation using ion-pair chromatography. In this, A risk assessment approach (Control-Noise-Experiment) has been used for identifying the risk factors, i.e. Percentage of Organic Modifier (% Acetonitrile), Buffer’s pH and flow rate of the method. The Central Composite design was applied to optimize the critical method parameters (CMPs) and to find out the Design space (DS) of the method. The coefficient of correlation (R2),%CV and Lack of fit are utilized for the evaluation of method responses (Retention time and Asymmetric factor Evaluation of model is justified by two diagnostic plots (normal probability plot of residuals and plot of residuals vs predicted values). The mobile phase is Acetate Buffer (20mM) pH 4.4: Acetonitrile (35:65) with 0.9 ml/min of Flow rate. The separation has taken placed in the Eclip Plus C-18 column (250 × 4.6 mm, 5μm) at 268 nm. The retention time of Boceprevir was found to be 4.6 min. The validation of the optimized method has performed according to ICH guideline. The method has been successfully used for routine analysis of the Telaprevir throughout the life cycle of the product.

Studying in vitro metabolism of the first and second generation of antisense oligonucleotides with the use of ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry

The aim of the present investigation was the analysis and identification of antisense oligonucleotide metabolism products after incubation with human liver microsomes regarding four different oligonucleotide modifications. Separation and detection methods based on the use of liquid chromatography coupled with quadrupole time-of-flight mass spectrometry were developed for this purpose. Firstly, the optimization of mass spectrometer parameters was done to select those which ensure the highest possible sensitivity of oligonucleotide analysis. This step was conducted for two chromatographic modes—ion pair chromatography and hydrophilic interaction liquid chromatography—due to their common application in oligonucleotide analysis. Based on sensitivity results, ion pair chromatography coupled with mass spectrometry was selected for the separation of model oligonucleotide mixtures in order to verify its selectivity for N-deleted metabolite separation. Next, the developed method was applied in the examination of oligonucleotides in vitro metabolism. First, wide optimization of incubation parameters was conducted including the concentration of the reaction buffer components. Obtained results indicated that both 3′-exonucleases and 5′-exonucleases contributed to the biotransformation of oligonucleotides. Moreover, it may be concluded that the number of metabolites depends on oligonucleotide modification and consequently its resistance to enzymatic attack. Thus, the number of the oligonucleotide metabolites decreased with the decrease of the resultant polarity of oligonucleotide caused by chemical modification.