Elsevier

Journal of Chromatography B

Volume 1000, 1 September 2015, Pages 181-186
Journal of Chromatography B

Development and validation of a sensitive HPLC–MS/MS method for determination of chidamide (epidaza), a new benzamide class of selective histone deacetylase inhibitor, in human plasma and its clinical application

https://doi.org/10.1016/j.jchromb.2015.07.001Get rights and content

Highlights

  • A HPLC–MS/MS assay for chidamide, the newest listed HDACi drug, was developed.

  • The assay is simple and sensitive with a broad calibration range of 1–1000 ng/mL.

  • The method is successfully applied to samples in an ongoing phase Ib trial.

Abstract

Chidamide (epidaza), a new oral isotype-selective histone deacetylase inhibitor (HDACi), which is just approved in China for the treatment of recurrent or refractory peripheral T-cell lymphoma (PTCL) in December 2014, is the first listed benzamide class of HDACi in the world, and is currently undergoing global clinical trials for solid tumor treatments. Here, we report a sensitive, rapid and robust HPLC–MS/MS method for determination of chidamide in human plasma. Plasma sample was subjected to a simple acetonitrile protein precipitation containing MS-275 used as an internal standard (IS). Chromatography was performed on a Hypersil GOLD C18 analytical column, using a gradient methanol/water mobile phase containing 0.1% formic acid. A tandem mass spectrometer equipped with electrospray ionization source was used as detector and operated in the positive-ion mode. Selected reaction monitoring (SRM) using the precursor/ product transitions (m/z) of 391.1/265.1 for chidamide and 377.1/359.2 for IS were used for quantification, respectively. Good linearity was obtained in the range of 1–1000 ng/mL. The method gave R.S.D.% values for precision always lower than 13.8% and R.E.% values for accuracy between −3.7 and 9.1%. In addition, the specificity, recovery, stability and matrix effect were satisfactory too. The method is now being successfully applied to plasma samples as part of an ongoing chidamide phase Ib clinical trial in patients with solid tumors, and had demonstrated consistent AUClast and t1/2 results with the published phase I pharmacokinetic data, which was also analyzed by this method, thus further confirming the reproducibility and accuracy during its clinical application. Considering the excellent performance of this method, it will continue being utilized for future clinical developments of chidamide and for routine monitoring of plasma exposure of chidamide during its clinical therapy.

Introduction

Over time, an appreciation of the importance and complexity of epigenetic events, such as histone post-translational modifications, has fueled interest in many new areas of cancer treatment research [1], [2], [3], [4]. Reversible acetylation of histone and non-histone proteins is one of the most abundant post-translational modifications in eukaryotic cells and is controlled by the antagonistic actions of two types of enzymes [5], histone acetyltransferases (HATs) and histone deacetylases (HDACs). To date, 18 human HDACs have been identified and structurally grouped into four classes (Class I: HDAC1–3, 8; Class II: HDAC4–7, 9, 10; Class III: SIRT1–7; Class IV: HDAC11) [6]. Although the precise biological role of individual HDACs is largely unknown, it is well accepted that HDACs act as transcription repressors to control cell survival, proliferation, angiogenesis and immunity [7]. More importantly, there are often over-expression of HDACs, especially class I HDACs, in multiple human cancers, resulting in decrease in histone acetylation, and thus the silencing of tumor suppressor genes or activation of oncogenes [8].

Because of this, HDACs have emerged as promising targets in cancer therapeutics, and the development of HDAC inhibitors (HDACi), a rapidly evolving area of clinical anticancer therapy [9], [10], [11], [12]. HDACi can be subdivided into four different classes according to their chemical structures, hydroxamic acid derivatives, benzamides, cyclic peptides and short-chain fatty acids [8]. Several HDACis are currently being evaluated for treatment of various human cancers and there is accumulating evidence that HDACi is effective for the treatment of hematological malignancies and solid tumors [13], [14], [15], [16], [17]. To date, the FDA has in total approved three HDACi (Vorinostat, 2006; Romidepsin, 2009; Belinostat, 2014) drugs for the treatment of lymphoid malignancies. But none of them belong to benzamide type.

Chidamide (epidaza) is a new oral isotype-selective HDACi (selectively inhibiting HDAC1, 2, 3 and 10) drug [18] which belongs to benzamide class with significant anti-tumor activities [19], [20], [21], [22], [23]. It has been confirmed that chidamide can increase the acetylation levels of histone H3 and to inhibit the PI3K/Akt and MAPK/Ras signaling pathways, thus resulting in arresting cancer cells at the G1 phase of the cell cycle and promoting apoptosis [24], [25]. Chidamide has just been approved by the Chinese Food and Drug Administration for the treatment of patients with recurrent or refractory peripheral T-cell lymphoma (PTCL) in December 2014. It is the first listed benzamide class of HDACi in the world and is currently undergoing global clinical trials in the United States and China for treatments of solid tumors such as non small cell lung cancer, breast cancer or prostate cancer [26].

An analytical method capable of detecting chidamide sensitive enough in biological fluids is essential during its clinical development. Because chidamide was a low nanomolar inhibitor of HDAC1–3 and 10 [18], its clinical dosage was as low as to micromole level, thus resulting in the clinical requirements of this analytical method sensitive enough for sample analysis. Wang et al. [27] had established a liquid chromatography coupled single mass spectrometry (LC–MS) method for determining chidamide in rat plasma using selective ion monitoring (SIM) mode, which had been applied to a rat pharmacokinetic study of chidamide following a relatively very high of a 30 mg/kg oral dose, but the unsatisfactory sensitivity of a lower limit of quantification (LLOQ) of 10 ng/mL couldn’t meet the needs of chidamide in its clinical development, probably because its detection was performed by a single mass spectrometry rather than the more sensitive mode of tandem mass spectrometry. In order to support the clinical development of chidamide, we had developed a sensitive and robust high performance liquid chromatography tandem mass spectrometry (HPLC–MS–MS) method for determining chidamide in human plasma. A structural analog, MS-275, was used as internal standard (IS). Fig. 1 shows the chemical structures of chidamide and IS, respectively. The method had already been applied in clinical pharmacokinetic analysis of chidamide in a phase I study in patients with solid tumors or lymphoma [26], as well as a phase II study in patients with PTCL (the results of the phase II study will be reported in the near future). This work describes the detailed method development, validation and its current application to plasma samples as part of an ongoing phase Ib clinical trial of chidamide in patients with solid tumors.

Section snippets

Reagents and materials

Chidamide (≥98%) and MS-275 (IS, ≥98%) were both provided by Chipscreen Bioscience, Ltd (Shenzhen, China). HPLC-grade methanol, acetonitrile and formic acid were all purchased from Fisher Scientific (NJ, USA). Distilled water was purified using a SZ-93A auto-double distillation system (YaRong Corp., Shanghai, China). Argon (≥99.999%) and liquid nitrogen (≥99.999%) were both purchased from the Beijing Capital Oxygen Manufactory (Beijing, China). Blank human plasma was obtained from the Blood

Method development

To establish sample pretreatment conditions, considering that chidamide is a benzamide compound with a small molecular weight of only about 390.1 Da protein precipitation method, which is quite simple and timesaving, was chosen for the plasma sample pretreatment, using acetonitrile as the precipitating solvent. In order to improve the method sensitivity to better fit the clinical analysis requirements, we managed to decrease the volume ratio of precipitating solvent: sample from a commonly used

Conclusion

Chidamide (epidaza) is the newest drug in the benzamide class of HDACi approved for PTCL treatment in China in December 2014, and is currently undergoing global clinical trials with solid tumor therapy, however, no full HPLC–MS/MS assay has been reported for this drug. This work described herein details the development and validation of a HPLC–MS/MS method for the measurement of chidamide in human plasma. The method features simple sample preparation, rapid analysis, excellent accuracy and

Acknowledgement

This work was partly supported by the Chinese National key Technology Program of Pharmacokinetics Research (Grant 2012ZX09301003-001-007).

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