Elsevier

Journal of Chromatography B

Volume 970, 1 November 2014, Pages 95-101
Journal of Chromatography B

Quantitative determination of mithramycin in human plasma by a novel, sensitive ultra-HPLC–MS/MS method for clinical pharmacokinetic application

https://doi.org/10.1016/j.jchromb.2014.08.021Get rights and content

Highlights

Abstract

Mithramycin is a neoplastic antibiotic synthesized by various Streptomyces bacteria. It is under investigation as a chemotherapeutic treatment for a wide variety of cancers. Ongoing and forthcoming clinical trials will require pharmacokinetic analysis of mithramycin in humans, both to see if target concentrations are achieved and to optimize dosing and correlate outcomes (response/toxicity) with pharmacokinetics. Two published methods for mithramycin quantitation exist, but both are immunoassays that lack current bioanalytical standards of selectivity and sensitivity. To provide an upgraded and more widely applicable assay, a UPLC–MS/MS method for quantitation of mithramycin in human plasma was developed. Solid-phase extraction allowed for excellent recoveries (>90%) necessary for high throughput analyses on sensitive instrumentation. However, a ∼55% reduction in analyte signal was observed as a result of plasma matrix effects. Mithramycin and the internal standard chromomycin were separated on a Waters Acquity BEH C18 column (2.1 × 50 mm, 1.7 μm) and detected using electrospray ionization operated in the negative mode at mass transitions m/z 1083.5  268.9 and 1181.5  269.0, respectively, on an AB Sciex QTrap 5500. The assay range was 0.5–500 ng/mL and proved to be linear (r2 > 0.996), accurate (≤10% deviation), and precise (CV < 15%). Mithramycin was stable in plasma at room temperature for 24 h, as well as through three freeze–thaw cycles. This method was subsequently used to quantitate mithramycin plasma concentrations from patients enrolled on two clinical trials at the NCI.

Introduction

Mithramycin (plicamycin, mitracin) has antibiotic, neuroprotective, and antiproliferative properties. Though it shows potential to treat neurodegenerative diseases such as Huntington's Disease [1], Alzheimer's, and Parkinson's, it has primarily been investigated as an anticancer drug. Mithramycin has been used clinically against testicular cancer [2] and leukemia [3], and preclinical studies have shown activity against glioma [4], pancreatic cancer [5], prostate cancer [6], oral squamous cell carcinoma [7], and lung and esophageal cancer [8]. Patients receiving mithramycin often develop hepatotoxicity [9]; various analogs have been developed in an attempt to curtail dose-limiting side effects [10], [11], [12].

Mithramycin inhibits RNA synthesis by binding to GC-rich regions of DNA. The resulting complex blocks Sp1 transcription factors, leading to downregulation of multiple oncogenes [13]. The drug also sensitizes cells to apoptosis induced by TNF-related apoptosis-inducing ligand (TRAIL) [14], [15]. Another study attributed the antitumor activity of mithramycin to c-myc inhibition and p53 activation [16]. Of particular interest to the trials analyzed here, Grohar et al. showed that mithramycin has in vitro and in vivo activity against the oncogenic fusion transcription factor EWS-FLI1 and the Ewing sarcoma family of tumors it produces [17].

Despite widespread and longstanding interest in this agent's anticancer potential, only two methods for quantifying the drug have been published [18], [19]. Both are immunoassays, and only one measured mithramycin in human plasma [18]. Fujiwara et al. report a comparison of their assay to an HPLC-UV method of unspecified origin, but there are no validation data provided for this method and it has a LLOQ of 10 ng/mL, which lacks sufficient sensitivity for clinical pharmacokinetic applications following standard doses of 17–25 mg/kg.

Presented here is a fully validated UHPLC–MS/MS method for quantifying mithramycin in human plasma at concentrations as low as 0.5 ng/mL. This method uses the gold standard of tandem mass spectrometry (MS/MS) in small molecule detection, which is fast, sensitive, selective, accurate, and precise. The assay calibration range is 0.5–500 ng/mL, with the option to measure concentrations up to 5000 ng/mL via a 10-fold dilution. This method was used to generate pharmacokinetic data by analyzing samples from patients enrolled on two clinical trials at the NIH (NCT01610570, NCT01624090).

Section snippets

Materials

Mithramycin was supplied by Fermentek Ltd. (Jerusalem, Israel) to the NCI under an IND held by the CCR. Chromomycin, used as the internal standard, was purchased from Sigma–Aldrich (St. Louis, MO, USA). Mixed-mode Evolute® Express 30 mg ABN solid-phase extraction (SPE) plates, capable of extracting a wide range of acidic, basic, or neutral molecules, were purchased from Biotage (Charlotte, NC, USA). Optima-grade acetonitrile and methanol were obtained from Fisher Scientific (Fairlawn, NJ, USA)

Specificity

Fig. 2 depicts the LC–MS/MS chromatograms of a drug-free plasma extract (Fig. 2A), the internal standard only (Fig. 2B), the LLOQ at 0.5 ng/mL (Fig. 2C), and a clinical pharmacokinetic sample taken immediately following a 6-h infusion (Fig. 2D). The retention times of mithramycin and the internal standard, chromomycin, were both 1.80 min. The use of ultra-high performance liquid chromatography (UHPLC) provided sharp peaks (∼3 s peak width at 50% height) that were well resolved and separated from

Conclusions

Mithramycin is a promising option for treating numerous types of cancer. Two immunoassays for mithramycin were reported over 20 years ago, but a newer and easier method is necessary to provide support for current trials. Described here is a UPLC–MS/MS method for quantitation of mithramycin in human plasma. This method proved sensitive (LLOQ 0.5 ng/mL), selective, accurate, precise, and efficient at recovering drug from plasma (∼93% recovery). Mithramycin is stable in plasma at room temperature

Disclaimer

The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organization imply endorsement by the U.S. Government. The views in this manuscript are those of the authors and may not necessarily reflect NIH policy. No official endorsement is intended nor should be inferred.

Acknowledgements

This study has been funded in whole with federal funds from the National Cancer Institute, National Institutes of Health.

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