PX-12 and 2-mercaptoimidazole
PX-12 is a thioredoxin inhibitor developed in Dr. Powis’s laboratory and has been undergoing early phase clinical development in the Arizona Cancer Center. Plasma concentrations of PX-12 and a major PX-12 metabolite, 2-mercaptoimidazole (2-MI), in clinical samples collected from prior clinical studies have been determined using normal phase HPLC with UV detection. PX-12 was not detected in any of the plasma samples collected from prior clinical investigation. 2-MI was found in plasma following PX-12 infusion. 2-MI is formed from the thiol/disulfide exchange between PX-12 and circulating reduced thiols and could be used as a surrogate marker for PX-12. In a recently initiated Phase Ib trial of 24-hour PX-12 infusion, Arizona Cancer Center investigators plan to monitor plasma PX-12 and 2-MI levels in samples collected from this study. Because the plasma 2-MI concentrations are expected to be low due to the slow infusion rate to be implemented in this trial and PX-12 was not detectable in prior studies using the normal phase assay with UV detection, we have developed a LC-MS/MS assay to improve the assay sensitivity and specificity. We have successfully developed a reverse phase chromatographic condition to provide adequate retention and separation of PX-12 and 2-MI. The limit of MS/MS quantification was found to be 0.02 and 0.1 ÔÅ≠g/ml for PX-12 and 2-MI, respectively, when using 0.2 ml of plasma. There were no interfering peaks from blank human plasma with the tandem mass spectrometric analysis. Figures 1A and 1B illustrate representative HPLC chromatograms for the assay.
Figure 1A - A representative HPLC chromatogram of a plasma calibration standard
Figure 1B - Detection of PX-12 and 2-MI in plasma isolated after addition of PX-12 to a fresh human whole blood preparation.
Cytochrome P450 (CYP) 3A4 is an important drug metabolizing enzyme that is responsible for the metabolism of a majority of commonly used medications, including cancer chemotherapeutic agents. Because CYP 3A4 activity is highly variable among individuals and can be affected by the usage of medications and other dietary and environmental variables, clinical assessment of CYP3A4 activity is important to predict drug response/toxicity and potential drug-drug and drug-nutrient interactions. Buspirone has been recommended as one of the preferred in vivo sensitive probe substrates for the evaluation of CYP3A4 inhibition by the U.S. Food and Drug Administration. However, its usage as a clinical probe substrate has been limited by the requirement of a sensitive assay for quantification of low buspirone plasma concentrations. We have developed an LC-MS/MS method to allow for sensitive detection of buspirone in human plasma and have applied the method to clinical assessment of CYP3A4 enzyme activity. Figures 2A and 2B illustrate representative HPLC chromatograms for the assay.
Figure 2A - A representative HPLC chromatogram of a plasma calibration standard
Figure 2B - A representative HPLC chromatogram of a human plasma sample collected 4 hrs following administration of 10mg of buspirone.
Sulindac and metabolites
Sulindac will be used in an early phase breast cancer prevention trial. The primary endpoint is drug bioavailability in the breast nipple aspirate fluid (NAF). A number of HPLC methods are available for quantification of sulindac and its metabolites in serum/plasma or urine. These methods have employed UV detection, however, with poor sensitivity. A more sensitive assay is needed for quantification of sulindac and its metabolites in NAF because of the limited sample volume. Therefore, an HPLC-mass spectrometric method has been developed in order to optimize the sensitivity of the assay. The following figure illustrates a comparison of the HPLC chromatograms of sulindac and its metabolites using UV detection and mass spectrometric detection. Injection of 0.2 ng each of sulindac and its metabolites results in peaks from UV detection being close to the limit of detection, while peaks from mass spectrometer being significant higher than the baseline/background noise. This suggests that the sensitivity of the assay is dramatically improved with mass spectrometric detection.
Figure 3. Comparisons of HPLC chromatograms from UV detection and mass spectrometric detection. I: sulindac; II: sulindac sulfone; III: sulindac sulfide; IS: internal standard, indomethacin.
Serum Bile Acids
Bile acids are the principal end products of cholesterol metabolism, and play an important role in the digestion and absorption of lipids in the small intestine. The primary bile acids, cholic acid (CHOL) and chenodeoxycholic acid (CDCA) are synthesized in the liver and secreted into bile mainly as glycine or taurine conjugates. More than 95% of the primary bile acids that enter the ileum are reabsorbed and returned to the liver via the portal vein. The non-absorbed portion passes into the colon and is metabolized by anaerobic bacteria. Metabolic processes include deconjugation of the glycine and taurine moieties to form free bile acids, followed by dehydroxylation to form the secondary bile acids, DCA and lithocholic acid (LCA). Colonic bacterial metabolism also can form keto bile acids and, with epimerization, tertiary bile acids such as ursodeoxycholic acid (UDCA), the 7-β -hydroxy epimer of CDCA. A major hypothesis of the colon carcinogenesis model is that secondary bile acids, in particular the 7-ÔÅ°-¬dehydroxylated cholic acid, DCA, act as co-carcinogens or promoters in colon carcinogenesis. As part of the colon cancer prevention program project, Arizona Cancer Center investigators plan to determine whether serum bile acid levels can be used as a predictor of colorectal adenoma recurrence. We have developed a solid phase extraction procedure and a LC-MS/MS assay condition for sensitive quantification of human serum bile acid concentrations in support of this project. The limit of quantification was found to be 2ng/ml, when using 0.25 ml of serum. Figures 3A and 3B illustrate representative chromatograms for the assay.
Figure 4A - A representative chromatogram of a calibration standard.
Figure 4B - A representative chromatogram of a human plasma sample.