Liquid chromatography–mass spectrometric assay for the quantitation in human plasma of ABT-888, an orally available, small molecule inhibitor of poly(ADP-ribose) polymerase

doi:10.1016/j.jchromb.2008.07.032

Journal of Chromatography B

Volume 872, Issues 1–2, 1 September 2008, Pages 141-147

https://doi.org/10.1016/j.jchromb.2008.07.032 Get rights and content

Abstract

ABT-888, a poly(ADP-ribose) polymerase (PARP) -inhibitor in clinical trials, potentiates DNA-damaging agents. We developed and validated, according to FDA guidelines, an LC–MS assay for sensitive, accurate and precise quantitation of ABT-888 and its metabolite M8 in 0.2 mL human plasma. After ethyl acetate extraction, separation is achieved with a hydro-Synergi column and a 0.1% formic acid in acetonitrile/water-gradient. Detection uses electrospray, positive-mode ionization mass spectrometry. Between 10 (LOQ) and 1000 ng/mL, accuracy was 95.5–98.5% for ABT-888 and 91.4–100.9% for M8, and precision was 0.1–4.9% for ABT-888 and 0–13.7% for M8. The assay is being applied to samples generated in several clinical trials.

Introduction

Poly-(ADP-ribosyl)ation is involved in many cellular processes, including: differentiation; gene regulation; protein degradation; replication; transcription; overall maintenance of genomic stability [1]. A family of 18 poly(ADP-ribose) polymerases (PARPs) has been identified, but only the most abundant, PARP-1 and PARP-2, which are both nuclear enzymes, are activated by DNA damage [1]. PARP-1 and PARP-2 play a critical role in the DNA damage response process by regulating a variety of DNA repair mechanisms. Elevated levels of PARP in cancer cells compared to normal cells have been linked to drug resistance and the overall ability of cancer cells to survive genotoxic stress [2].

Inhibition of PARP sensitizes tumor cells to cytotoxic agents that cause DNA damage that would normally be repaired by the base excision repair system. These cytotoxics include: alkylating agents, such as temozolomide and cyclophosphamide; platinum analogues, such as cisplatin, carboplatin and oxaliplatin; topoisomerase I poisons, such as irinotecan and topotecan [3], [4], [5], [6]. Furthermore, PARP inhibition sensitizes cancer cells to radiation [2], [3], [7]. Consequently, PARP inhibition may improve the efficacy of DNA-damaging cytotoxic therapies [2].

ABT-888 is a novel, orally active PARP-inhibitor, currently in clinical trials [6]. ABT-888 has an oral bioavailability of 56–92% in mice, rats, dogs, and monkeys [3] and is excreted primarily via the urine, with approximately 50% as intact parent compound and another 15% as the inactive lactone metabolite M8 (Fig. 1) [4].

In a phase 0 clinical study, ABT-888 doses of 10–25 mg were associated with approximately 85% inhibition of PARP in tumor tissues and peripheral blood mononuclear cells [6].

As ABT-888 is undergoing more extensive clinical development, there is a need to evaluate its pharmacokinetics. To facilitate such an evaluation, we developed a simple, rapid and sensitive LC–MS assay for the quantitation of ABT-888 and M8 in human plasma and validated it according to the most recent FDA guidelines for bioanalytical method validation [8].

Section snippets

Chemicals and reagents

ABT-888 (A-861695) (D₀/D₃ > 99.8%), the internal standard ([D₃]-ABT-888, D₃/D₀ > 99.9%) and A925088 (the M8 metabolite) were graciously provided by Abbott Laboratories (Abbott Park, IL, USA). Acetonitrile (HPLC grade) and ethyl acetate (HPLC grade) were purchased from Fisher Scientific (Fairlawn, NJ, USA). Water was purified using a Q-gard^® 1 Gradient Milli-Q system (18.2 MΩ cm, Millipore, Billerica, MA, USA). Formic acid was purchased from Sigma–Aldrich (St. Louis, MO, USA). Control human plasma was

Chromatography

ABT-888 and the internal standard [D₃]-ABT-888 had an identical retention time of approximately 8.0 min, corresponding to a capacity factor of 7.0 (with a void time of 1 min). M8 eluted slightly later at a retention time of 8.9 min, corresponding to a capacity factor of 7.9. Representative chromatograms of ABT-888, M8 (at the LLQ), and internal standard in plasma are displayed in Fig. 2.

Calibration curve and LLQ

According to the FDA guidelines for bioanalytical method validation [8], the calibration curve describes the

Conclusion

Our objective was to develop and validate an analytical method for the quantitation of ABT-888 and its inactive metabolite M8 in human plasma. We accomplished this by using reversed phase chromatography equipped with single quadrupole mass spectrometric detection.

The method presented here allows the quantitation of ABT-888 and M8 in human plasma and, to our knowledge, is the first validated assay for ABT-888 and M8 published to date. Rodriguez et al. reported concentrations of ABT-888 in plasma

Acknowledgements

Supported by grants UO1CA099168 and 2P30CA47904, and NIH/NCRR/CTSA Grant UL1 RR024153 from the NIH. We thank the University of Pittsburgh Cancer Institute Hematology/Oncology Writing Group for constructive suggestions regarding the manuscript and Susan Christner for analytical assistance.

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Prediction of clinical drug-drug interactions of veliparib (ABT-888) with human renal transporters (OAT1, OAT3, OCT2, MATE1, and MATE2K)
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Veliparib (ABT-888) is largely eliminated as parent drug in human urine (70% of the dose). Renal unbound clearance exceeds glomerular filtration rate, suggesting the involvement of transporter-mediated active secretion. Clinically relevant pharmacokinetic interactions in the kidney have been associated with OAT1, OAT3, OCT2, MATE1, and MATE2K. In the present study, interactions of veliparib with these transporters were investigated. Veliparib inhibited OAT1, OAT3, OCT2, MATE1, and MATE2K with IC₅₀ values of 1371, 505, 3913, 69.9, and 69.5 μM, respectively. The clinical unbound maximum plasma concentration of veliparib after single oral dose of 50 mg (0.45 μM) is manyfold lower than IC₅₀ values for OAT1, OAT3, OCT2, MATE1, or MATE2K. These results indicate a low potential for drug–drug interaction (DDI) with OAT1/3, OCT2, or MATE1/2 K. Additional studies demonstrated that veliparib is a substrate of OCT2. In Oct1/Oct2 double-knockout mice, the plasma exposure of veliparib was increased by 1.5-fold, and the renal clearance was decreased by 1.8-fold as compared with wild-type mice, demonstrating that organic cation transporters contribute to the renal elimination in vivo. In summary, the in vitro transporter data for veliparib predicts minimal potential for an OAT1/3-, OCT2-, and MATE1/2 K-mediated DDI given the clinical exposure after single oral dose of 50 mg.
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A reversed-phase liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) method was developed and validated for simultaneous determination of ABT-888 and its major metabolite (M8) in human plasma. Sample preparation involved a liquid–liquid extraction by the addition of 0.25 ml of plasma with 10 μl of 1 M NaOH and 1.0 ml ethyl acetate containing 50 ng/ml of the internal standard zileuton. The analytes were separated on a Waters XBridge C₁₈ column using a gradient mobile phase consisting of methanol/water containing 0.45% formic acid at the flow rate of 0.2 ml/min. The analytes were monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the ABT-888 and M8 concentration ranges of 1–2000 ng/ml in human plasma. The lower limits of quantitation (LLOQ) were 1 ng/ml for both ABT-888 and M8 in human plasma. The accuracy and within- and between-day precisions were within the generally accepted criteria for bioanalytical method (<15%). This method was successfully employed to characterize the plasma concentration–time profile of ABT-888 after its oral administration in cancer patients.

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Liquid chromatography–mass spectrometric assay for the quantitation in human plasma of ABT-888, an orally available, small molecule inhibitor of poly(ADP-ribose) polymerase

Abstract

Introduction

Section snippets

Chemicals and reagents

Chromatography

Calibration curve and LLQ

Conclusion

Acknowledgements

Expert Rev. Mol. Med.

Clin. Cancer Res.

Proc. Am. Assoc. Cancer Res.

Proc. Annu. Meet Am. Assoc. Cancer Res.