Validation of a method for quantifying enzalutamide ...

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Validation of a method for quantifying enzalutamide and its major metabolites in human plasma by LC–MS/MS Background: Enzalutamide is an androgen receptor inhibitor that targets multiple steps in the androgen receptor signaling pathway. Oral enzalutamide was recently approved by the US FDA and health authorities in other regions for the treatment of patients with metastatic castration-resistant prostate cancer who previously received docetaxel. The objective of this study was to validate a method for quantification of enzalutamide and its two major metabolites in human plasma. Results: The analytes were extracted from plasma by an LLE procedure, separated by reversed phase HPLC and detected by MS/MS in positive mode ESI. The quantitation range was 0.0200–50.0 µg/ml. Conclusion: The method proved to be rapid and simple, and met FDA validation criteria. Enzalutamide acts on different steps in the androgen receptor (AR) signaling pathway. It has been shown to competitively inhibit androgen binding to AR, inhibit AR nuclear translocation and inhibit AR interaction with DNA [1]. Enzalutamide decreased proliferation and induced cell death of prostate cancer cells in vitro, and decreased tumor volume in a mouse prostate cancer xenograft model. Enzalutamide (XTANDI®) is approved in the USA, Canada and Europe for the treatment of patients with metastatic castrationresistant prostate cancer who have previously received docetaxel. The efficacy and safety of enzalutamide were assessed in a randomized, placebo-controlled, multicenter Phase III clinical trial (AFFIRM; clinical trial registry number NCT00974311) [2]. A mass balance and biotransformation study in male volunteers demonstrated that N-desmethyl enzalutamide and a carboxylic acid metabolite (Figure 1) are major metabolites in human plasma [Gibbons JA, Ouatas T, Krauwinkel W

plasma. This article reports the results of the validation study.

et al. Clinical pharmacokinetics of enzalutamide (2013),

Calibration

Experimental Reagents & chemicals Critical reagents are summarized in Table 1. Medivation, Inc. (CA, USA) provided enzalutamide, N-desmethyl enzalutamide, the carboxylic acid metabolite and their respective 13 CD3-analogs, which served as IS. Preparation

of standard solutions Stock solutions of enzalutamide and its metabolites were prepared in acetonitrile at 6 mg/ml. Working solutions from 0.400 to 1000 µg/ml were prepared in acetonitrile by dilution of the stock solutions. IS stock solutions were prepared in acetonitrile at 0.1 mg/ml and the IS working solution in acetonitrile was prepared from the primary IS stock solutions at 0.2 µg/ml. All solutions were stored at 4°C in silanized amber glass and equilibrated to room temperature before use.

The first-in-human study showed that plasma concentrations of enzalutamide and its metabolites each reach peak concentrations of up to approximately 50 μg/ml with daily dosing, and definition of the terminal phase after a single oral dose requires concentration determinations down to approximately 0.02 µg/ml [3]. To support further clinical PK studies, an LC–MS/MS method was developed to measure enzalutamide, N-desmethyl enza lutamide and the carboxylic acid metabolite in human

standards & QC samples Calibration standards and QC samples were prepared by diluting the standard solutions 20-fold with blank plasma. Before validation, the desired range of the assay for enzalutamide and its two major metabolites was established to be 0.02 to 50 µg/ml. Therefore, the calibration standards consisted of each of the three analytes in K 2EDTA human plasma at 0.0200, 0.0500, 0.100, 0.500, 1.00, 2.00, 5.00, 10.0 and 50.0 µg/ml. QC samples (i.e., K 2EDTA human plasma spiked with each of the analytes) were

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Bioanalysis (Epub ahead of print)

Submitted].

Daniel Bennett1, Jacqueline A Gibbons*2 , Roelof Mol3, Yoshiaki Ohtsu4 & Clark Williard1 inVentiv Health Clinical Lab, Inc, 301D College Road East, Princeton, NJ 08540, USA 2 Medivation, Inc., 525 Market Street, 36th Floor, San Francisco, CA 94105, USA 3 Astellas Pharma Europe B.V., Sylviusweg 62, PO Box 344, 2300 AH Leiden, The Netherlands 4 Astellas Pharma Inc. 2-1-6, Kashima,Yodogawa-ku, Osaka 532-8514, Japan *Author for correspondence: Tel.: +1 415 829 4140 Fax: +1 415 543 3411 E-mail: jackie.gibbons@ medivation.com 1

ISSN 1757-6180

R apid Communication | Enzalutamide

N-desmethyl enzalutamide

NC S

F3C

Bennett, Gibbons, Mol, Ohtsu & Williard

N

NC

F

F 3C

N

N H

NC S

O

O

Carboxylic acid metabolite

*

209 (+H)

N O

F

S

O N *

F3C

NH2

N O

195 (+H)

F O

N

OH * 196 (+H)

Figure 1. Structures of enzalutamide, N-desmethyl enzalutamide and the carboxylic acid metabolite, and the corresponding product ions. *Notes the position of the 13CD3 in the IS.

prepared at three concentrations, one three-times the low end of the range, one in the midrange and one close to the high end of the range. This corresponded to low, mid and high QC sample concentrations of 0.0600, 1.00 and 40.0 µg/ml, respectively. Method

Key Terms Androgen receptor: Type of

nuclear receptor that is activated by binding of the androgenic hormones, testosterone and dihydrotestosterone. Binding of androgens to androgen receptor occurs in the cytoplasm and is followed by translocation of androgen receptor into the nucleus.

Metastatic castrationresistant prostate cancer:

Form of prostate cancer in which the cancer cells metastasize (spread) from the prostate to other parts of the body, particularly the bones and lymph nodes.

Mass balance and biotransformation study:

Study in which subjects are dosed with a radiolabeled form of a drug, and plasma, urine and feces samples are collected until a majority of the radioactive dose is recovered in the excreta. The ex vivo samples are measured for total radioactivity and radiolabeled products, such as the parent molecule and metabolites. Based on these data, the total fate of drug-related material is obtained, including routes of excretion and metabolic pathways.

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optimization The analytical method was adopted from a nonclinical assay that supported animal PK assessments of enzalutamide independently of its metabolites. Essentially no optimization occurred prior to validation in human plasma because the existing method was found to be adequate for measuring enzalutamide and its major metabolites within the desired concentration ranges, and also because there was urgency in providing a validated method to support clinical studies. Sample

preparation A 50 µl volume of each plasma sample was added to the appropriate well of a 96-well polypropylene plate. A 25 µl volume of IS working solution was added to all samples except for the doubleblank samples, which received 25 µl acetonitrile. All samples (inclusive of the double-blanks) were spiked with 200 µl of an aqueous solution of 5% sodium bicarbonate, briefly vortexed, spiked with approximately 1 ml methyl-t-butyl ether and mixed thoroughly. As the human mass balance and biotransformation study showed no evidence of acyl glucuronides or isobaric metabolites in human plasma (i.e., forms that could potentially convert to the carboxylic acid metabolite), sample preparation did not require acidification procedures. The plate was left undisturbed for approximately 3 min to enable the organic and aqueous phases to separate, and the top organic layer was transferred to a new plate and fully evaporated under a gentle stream of nitrogen at 40°C. Samples were reconstituted with 250 µl of 0.1% formic Bioanalysis (Epub ahead of print)

acid in 40/60 (v/v) methanol/water, briefly vortexed and approximately 15 µl was injected into the LC–MS/MS system for analysis. LC–MS/MS

analysis The analysis was performed on the LC–MS/MS equipment summarized in Table 1. The HPLC column was maintained at ambient temperature. The mobile phase was a mixture of 0.1% formic acid in water (A) and 0.1% formic acid in methanol (B). The flow rate was 0.300 ml/min. The elution gradient started with 50% of B with linear increase to 80% over 1.5 min, kept constant for 0.5 min and then increased within 0.1 min to 100% for 1 min, before returning to 50% for re-equilibration. During the 1-min wash and reequilibration, blank reconstitution solution was injected to minimize any potential carryover from the previous injection. The selected MRM transitions, dwell times and instrument settings are summarized in Table 2 . Data acquisition and processing (integration) involved Analyst software v1.4.1 (Applied Biosystems/MDS Sciex, Concord, Canada). Validation

of the method The method validation was performed according to the US FDA guidance [4]. The components of the validation included precision, accuracy, sensitivity, extraction recovery, selectivity, matrix effects, carryover, dilution integrity, hemolysis effects and stability. Results & discussion LC–MS/MS analysis An LC–MS/MS method was developed for the quantification of enzalutamide, N-desmethyl enzalutamide and the carboxylic acid metabolite. Representative mass chromatograms are provided in Figure 2 . future science group

Validation of a method for quantifying enzalutamide & its major metabolites

| Rapid Communication

Table 1. Critical reagents and equipment. Reagents/equipment

Manufacturer

Location

Acetonitrile, HPLC grade Dipotassium EDTA human plasma

EMD Chemicals Bioreclamation Valley Biomedical EMD Chemicals EMD Chemicals Sigma-Aldrich EMD Chemicals EMD Chemicals Applied Biosystems/MDS Sciex Agilent Technologies LEAP Technologies Tomtec Advanced Chromatography Technologies

Gibbstown, NJ, USA Hicksville, NY, USA Winchester, VA, USA Gibbstown, NJ, USA Gibbstown, NJ, USA St. Louis, MO, USA Gibbstown, NJ, USA Gibbstown, NJ, USA Concord, Ontario, Canada Wilmington, DE, USA Carrboro, NC, USA Hamden, CT, USA Chadds Ford, PA, USA

Formic acid (≥98% pure) Methanol, HPLC grade Sodium bicarbonate (≥99% pure) Water, HPLC grade Methyl-t-butyl ether (>99% pure) MDS Sciex API 3000 equipped with a Turbo Ionspray® Agilent 1100 series LC pumps CTC Analytics HTS PAL system Tomtec Quadra 96 Model 196–320 ACE 5 C18 HPLC column 5 µm, 2.1 × 30 mm

The maximum pressure in the liquid chromatography system during the elution gradient was 0.9957.

analytes, inter- and intra-assay precision (RSD) was ≤7.24%, and accuracy (%bias) ranged from -8.17% to 9.00%. RSD was defined as (SD/mean concentration) × 100. Percentage bias was defined as ([mean measured concentration - theoretical concentration]/theoretical concentration) × 100. To assess sensitivity, experiments were performed with ‘LLOQ samples’, which corresponded to the lowest calibration standard (0.0200 µg/ml in K 2EDTA human plasma; n = 6). As shown in Table 3, for all three analytes in the LLOQ samples, precision was ≤4.87% and accuracy ranged from -0.500 to 9.00%.

Precision,

Extraction

Calibration

accuracy & sensitivity Precision and accuracy were assessed with low, mid and high QC samples. Intra- and inter-assay precision and accuracy were assessed by calculating individual run and overall mean concentration values for six replicates assayed in four analytical sequences (runs) (Table 3). For all three

recovery, selectivity & matrix effects To assess extraction recovery, standard sample preparation procedures were used to extract low, mid and high QC samples. Control samples were prepared by extracting blank K 2EDTA human plasma, and then adding the appropriate

Table 2. MRM transitions per analyte and MS settings.

Key Terms

Target compound†

Precursor ion‡ (m/z)

Product ion (m/z)

Dwell time (ms)

ENZA

465.1

209.2

100

ENZA-13CD3 (IS) N-DM-ENZA N-DM-ENZA-13CD3 (IS) ENZA-CAM ENZA-CAM-13CD3 (IS)

469.1 451.1 455.1 452.1 456.1

213.2 195.2 199.2 196.1 200.1

50 100 50 100 50

†

For all target compounds, the MS parameters were as follows: electron-spray ionization mode: ESI (Turbo Ion Spray) positive; source temperature: 500ºC; collision gas: 8 arbitrary units; curtain gas, gas 1, and gas 2: 50 psi; ion spray voltage: 4500 V; declustering potential: 65 V; entrance potential: 10 V; collision energy: 30 eV; collision cell exit potential: 16 V. ‡ Precursor ions are reported in the (M+H) + ionized form. ENZA: Enzalutamide; ENZA-13CD3: Enzalutamide 13CD3 analog; ENZA-CAM: Enzalutamide carboxylic acid metabolite; ENZA-CAM-13CD3: Enzalutamide carboxylic acid metabolite 13CD3 analog; N-DM-ENZA: N‑desmethyl enzalutamide; N-DM-ENZA -13CD3: N-desmethyl enzalutamide 13CD3 analog.

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Carryover: Appearance of an analyte signal in blank sample after the analysis of samples with a high analyte concentration.

Dilution integrity:

Demonstration that dilution does not affect accuracy and precision. This is especially relevant for samples with concentrations higher than the quantitative range of the analytical method, which require dilution to bring them within the working range of the method.

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Enzalutamide; RT = 1.70 min; k´ = 3.4 1.70

Intensity (cps)

2000 1500 1000 500

2.81

0 0.5

Intensity (cps)

3000

1.0

1.5 2.0 2.5 Time (min) N-desmethyl enzalutamide; RT = 1.62 min; k´ = 3.2 1.62

2500 2000 1500 1000 500

1.76

0

Intensity (cps)

0.5

800 700 600 500 400 300 200 100 0

2.93 2.72

1.0

1.5 2.0 2.5 Time (min) Carboxylic acid metabolite; RT = 1.79 min; k´ = 3.6 1.62

1.79

1.96 0.5

1.0

1.5 2.0 Time (min)

2.5

Figure 2. Mass chromatograms of enzalutamide, N-desmethyl enzalutamide and the carboxylic acid metabolite at the LLOQ (0.0200 µg/ml) for all analytes. For the carboxylic acid metabolite, the peak at RT = 1.62 min corresponds to the precursor and product ions of N-desmethyl enzalutamide, which is within 1.0 Dalton of the carboxylic acid metabolite and thus contributes to the isotopic distribution of mass spectral signal in the carboxylic acid metabolite mass spectral channel. The chromatographic peak at RT = 1.79 min is the only peak integrated for quantification of the carboxylic acid metabolite. k´: Retention factor; RT: Retention time.

amounts of each analyte and respective IS. Recovery corresponded to the IS-normalized response (i.e., analyte peak area/IS peak area) for the extracted QC samples divided by the IS-normalized response for the controls. The extraction recoveries of the three analytes were constant over the concentration range tested and similar to those of the respective IS (Table 4). The extraction recovery of the carboxylic acid metabolite was low (≤22.1%), most likely 10.4155/BIO.13.325


because sample extraction was performed under basic conditions. The selectivity and specificity of the method was established using six different lots of K2EDTA human plasma. In the selectivity experiment, low QC samples (each analyte at 0.0600 µg/ml) were prepared in each of the six lots. For all three analytes, the precision was ≤4.17% and accuracy ranged from -1.83 to 3.50% (Table 4). In the specificity experiment, blank samples from each of the six lots were submitted for analysis by the LC–MS/MS method, and no significant level of interfering substances was observed at the retention times for the analytes and IS. Matrix effects were evaluated to assess possible suppression or enhancement of ionization of the analytes by the presence of components in a human plasma [5]. The analytes (1.00 µg/ml) and their respective IS were added to Solution 1, in which matrix ions were present (namely, postextracted K 2EDTA human plasma blanks) and Solution 2, in which matrix ions were absent (namely, neat solutions). Matrix effects were computed as the ratio of the IS-normalized response in Solution 1/Solution 2. The mean (± SD; n = 6) matrix effect ratios for enzalutamide, N‑desmethyl enzalutamide and the carboxylic acid metabolite were 1.01 (± 0.0134), 0.979 (± 0.0109) and 1.01 (± 0.0400), respectively (Table 4). As these ratios were marginally different from 1.00, ionization of the analytes appeared not to be affected in any important way by components in human plasma. Possible interference by drugs commonly used in prostate cancer was assessed. Low QC samples were spiked with these drugs and their respective major metabolites (where applicable) at concentrations that matched or exceeded their maximum plasma concentrations based on published data. The testing included the following: abiraterone at 10 µg/ml, abiraterone sulfate at 100 µg/ml, abiraterone N-oxide sulfate at 100 µg/ml, dexamethasone at 100 ng/ml, docetaxel at 3,000 ng/ml, prednisolone at 100 ng/ml, prednisone at 100 ng/ml, R-warfarin at 10 ng/ml and S-warfarin at 10 ng/ml. The potential interference of minor metabolites of enzalutamide was tested in a similar fashion. All data were within the acceptance criteria (±15.0% bias) (data not shown). Carryover

To assess carryover, a blank sample was injected after the highest calibration standard. Carryover was deemed insignificant because no signal >20% of the LLOQ signal was observed at any of the retention times for the analytes or IS. future science group



Table 3. Precision and accuracy. ENZA

N-DM-ENZA

ENZA-CAM

3.08 3.76 4.53 4.67 4.33 3.95 2.31 2.99 2.35 2.49 2.61 4.37 4.87

2.10 3.13 3.01 3.60 2.79 2.76 3.56 4.82 3.64 4.01 6.39 3.47 4.37

3.92 2.81 5.75 5.77 4.50 3.65 7.24 3.39 2.92 3.93 4.38 6.25 4.72

5.75 5.69 3.09

4.68 4.99 4.61

5.33 5.61 4.47

1.00 -6.83 -3.17 2.33 8.00 -0.30 -0.50 0.00 -3.25 -0.250 -3.00 -5.00 1.00

-1.67 -6.83 -8.17 -2.33 4.00 -1.10 2.00 8.00 6.00 8.75 8.00 4.75 9.00

-1.67 2.00 -3.00

-4.67 3.00 6.75

Intra-assay precision (RSD) QC1†, run 1 QC1†, run 2 QC1†, run 3 QC1†, run 4 QC2‡, run 1 QC2‡, run 2 QC2‡, run 3 QC2‡, run 4 QC3 § , run 1 QC3 § , run 2 QC3 § , run 3 QC3 § , run 4 LLOQ Inter-assay precision (RSD) QC1†, runs 1–4 QC2‡, runs 1–4 QC3 § , runs 1–4

Intra-assay accuracy (% bias) QC1†, run 1 QC1†, run 2 QC1†, run 3 QC1†, run 4 QC2‡, run 1 QC2‡, run 2 QC2‡, run 3 QC2‡, run 4 QC3 § , run 1 QC3 § , run 2 QC3 § , run 3 QC3 § , run 4 LLOQ

1.33 -6.83 -7.00 1.33 9.00 -3.30 0.00 3.00 -3.50 -2.75 -5.50 -2.75 -0.50

Inter-assay accuracy (% bias) QC1†, runs 1–4 QC2‡, runs 1–4 QC3 § , runs 1–4

-2.83 2.00 -3.75

QC1: 0.0600 µg/ml (n = 6). QC2: 1.00 µg/ml (n = 6). § QC3: 40.0 µg/ml (n = 6). QC samples (K 2EDTA human plasma spiked with each of the analytes) are used to assess accuracy and precision. LLOQ is 0.0200 µg/ml (n = 6). ENZA: Enzalutamide; ENZA-CAM: Enzalutamide carboxylic acid metabolite;N-DM-ENZA: N‑desmethyl enzalutamide. † ‡

Dilution

integrity To assess dilution integrity, over-range concentrations were obtained by assaying replicate samples (n = 6) that contained 100 µg/ml of each analyte in K 2EDTA human plasma. The samples were diluted 50-fold with K 2EDTA human plasma. For all three analytes, the precision was ≤4.63% and accuracy ranged from 9.00 to 12.0%. future science group

Hemolysis

effect Data from low and high QC samples (0.0600 and 40.0 µg/ml, respectively; n = 3) that were spiked with 2% human K 2EDTA whole blood showed that hemolysis does not affect the precision or accuracy for any of the analytes [6]. For all three analytes, the precision was ≤9.42% and accuracy ranged from -0.500% to -6.50%. www.future-science.com

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Table 4. Extraction recovery and matrix effects. ENZA

N-DM-ENZA

ENZA-CAM

63.6 67.3 70.0 66.7

67.9 69.9 72.7 69.6

18.1 15.5 22.1 13.3

2.63

3.65

4.17

-1.83

0.333

3.50

1.01

0.97

1.01

Extraction recovery (%) QC1† QC2‡ QC3 § IS Selectivity precision (RSD) QC1†

Selectivity accuracy (% bias) QC1† Matrix effect (ratio) QC2

QC1: 0.0600 µg/ml. ‡ QC2: 1.00 µg/ml. § QC3: 40.0 µg/ml. QC samples (K 2EDTA human plasma spiked with each of the analytes) are used to assess accuracy and precision. Reported values are the means of replicate determinations as follows: n = 3 for extraction recovery, and n = 6 for matrix effect and selectivity. ENZA: Enzalutamide; ENZA-CAM: Enzalutamide carboxylic acid metabolite; N-DM-ENZA: N‑desmethyl enzalutamide. †

Stability

As summarized in Table 5, stability of enza lutamide, N-desmethyl enzalutamide and the carboxylic acid metabolite was demonstrated in K 2EDTA human whole blood, plasma and plasma extracts, as well as in acetonitrile (i.e., the solvent used in the stock solutions). Stability during processing of K 2EDTA human whole blood samples was assessed in two experiments with each of the three analytes at 0.0600 µg/ml [7]. In experiment 1, whole blood

samples were incubated on ice for 1 h and centrifuged to isolate the plasma fraction. After centrifugation, the tubes were placed on ice for 3 h, and the plasma fraction was separated and submitted for analysis by the LC–MS/MS method. In experiment 2, whole blood samples were incubated at approximately 37°C for 20 min and centrifuged to isolate the plasma fraction. The plasma fraction was removed, stored at ambient temperature for 2 h and submitted for analysis by the LC–MS/MS method. Data from these two

Table 5. Stability of enzalutamide, N-desmethyl enzalutamide and the carboxylic acid metabolite. Temperature

ENZA

N-DM-ENZA

ENZA-CAM

Ice water Ambient

4h 2h

4h 2h

4h 2h

-20ºC -70ºC Ambient -20ºC -70ºC

5 cycles 5 cycles 23 h 730 days 1106 days



Ambient

151 h

151 h

151 h

4ºC

359 days

359 days

359 days

Sample handling Experiment 1 Experiment 2 Plasma Freeze–thaw Freeze–thaw Short-term Long-term Long-term Plasma extracts Postpreparative Stock solution Long-term

Whole blood: human K 2EDTA whole blood; all three analytes were tested in whole blood at 0.0600 µg/ml; details of experiments 1 and 2 are provided in the text. Plasma: K 2EDTA human plasma; all three analytes were tested in plasma at 0.0600 µg/mL and 0.400 µg/ml (long-term stability tests only). Plasma extracts: extracts of K 2EDTA human plasma; all three analytes were tested in plasma extracts at 0.0600, 1.00, and 40.0 µg/ml; experimental details are provided in the text. Stock solution: 1000 µg/ml of each analyte in acetonitrile. ENZA: Enzalutamide; ENZA-CAM: Enzalutamide carboxylic acid metabolite; N-DM-ENZA: N‑desmethyl enzalutamide.

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Incurred sample reanalysis On completion of the validation, incurred sample reanalysis was performed in all cases where the method supported a clinical trial [5]. This involved reanalysis of ≥10% of the study samples for ≤1000 samples and reanalysis of ≥5% for >1000 samples. All data met the acceptance criteria (i.e., reanalysis results within 20% of their mean for at least 67% of the repeats). future science group

3000

Enzalutamide; RT = 1.53 min 1.53

Intensity (cps)

2500 2000 1500 1000 500 0

0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 Time (min)

400,000

N-desmethyl enzalutamide; RT = 1.44 min 1.44

350,000

Intensity (cps)

300,000 250,000 200,000 150,000 100,000 50,000 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 Time (min) 90,000

Carboxylic acid metabolite; RT = 1.66 min 1.44

80,000 70,000 Intensity (cps)

experiments demonstrated that enzalutamide, N‑desmethyl enzalutamide and the carboxylic acid metabolite are stable in human K 2EDTA whole blood under conditions that emulate the handling of PK samples at clinical sites. Stability in human plasma was assessed by conventional procedures [4]. Experiments with low and high QC samples (each analyte at 0.0600 and 40.0 µg/ml, respectively) established freeze–thaw stability at both -20 and -70°C after 5 cycles and short-term stability up to 23 h at ambient temperature. Experiments with low and high QC samples also established long-term stability up to 730 and 1106 days at -20 and ‑70°C, respectively. As clinical trials can last for several years, demonstration of long-term stability for up to 1106 days (3 years) provides the flexibility to defer sample analysis until the end of the study. Experiments with plasma extracts assessed stability of the analytes and their respective IS under typical conditions of postpreparative handling, including residence time in the autosampler. The sequences of low, mid and high QC samples (each analyte at 0.0600, 1.00 and 40.0 µg/ml, respectively; n = 6) and duplicate calibration standard curves in K 2EDTA human plasma, were extracted and promptly injected on the LC–MS/MS system. The polypropylene plate containing the extracts was stored at ambient temperature for a period of 151 h and then reinjected on the system. Data from the LC–MS/MS analysis showed that accuracy and precision were maintained after 151 h at ambient temperature, both when an entire run containing calibration standards was reinjected and when individual samples were reinjected independently of calibration standards. Based on these results, in the event of instrument failure, it is acceptable to reinject and reanalyze entire analytical runs or individual samples, as long as the reinjection occurs within 151 h of sample extraction. Solutions of the analytes at 1000 µg/ml in acetonitrile were demonstrated to be stable at 4°C in silanized amber glass containers for 359 days.


60,000

1.66

50,000 40,000 30,000 20,000 10,000 0

0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 Time (min)

Figure 3. Mass chromatograms of a PK plasma sample from a prostate cancer patient treated with enzalutamide (160 mg/day) for 13 weeks in the AFFIRM trial (clinical trial registry number NCT00974311) [2] . RT: Retention time. Data taken from [Gibbons JA, Outas T, Krauwinkel W et al. Clinical pharmacokinetics of enzalutamide (2013), Submitted].

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Conclusion As demonstrated by data from validation experiments, the LC–MS/MS method for the determination of enzalutamide and its major metabolites in K 2EDTA human plasma is precise, accurate, sensitive and selective. This method is rapid, simple and robust, and meets FDA validation criteria [4]. The concentration range of the assay (0.0200 to 50 μg/ml) favorably supports single-dose PK studies, in which an LLOQ of 0.0200 µg/ml enables plasma concentrations to be measured for several half-lives. The assay range also favorably supports steady-state (multipledose) clinical studies, in which the majority of PK samples have plasma concentrations of the drug and metabolites in the range of 10 to 30 µg/ml and peak concentrations rarely exceed 50 μg/ml [Gibbons JA, Ouatas T, Krauwinkel W et al. (2013), Submitted].To date, the validated method has successfully

supported nearly a dozen clinical trials, including the analysis of more than 4000 PK samples from the AFFIRM trial (Figure 3) [2]. Future perspective The availability of a rapid and efficient method for analyzing enzalutamide in human plasma will enable clinical investigators to explore the possible benefits of this new drug in additional diseases. While oral enzalutamide was recently approved for the treatment of patients with metastatic

castration-resistant prostate cancer who previously received docetaxel, it is currently being investigated in other stages of prostate cancer, as well as breast cancer. A major trend in clinical oncology research is to combine cancer drugs; therefore, an increasing number of clinical trials are expected to investigate enzalutamide in combination with other drugs. Optimizing the dose and schedule of enzalutamide in these settings will require the use of PK determinations and these will be made possible by the bioanalytical method described in this article. Financial & competing interests disclosure D Bennett and C Williard are employees of inVentiv Health Clinical Lab, Inc. (NJ, USA). J Gibbons is an employee of Medivation (CA, USA). All other authors are employees of Astellas (Leiden, The Netherlands and Osaka, Japan). The research described here was funded by Medivation and Astellas, and individuals from these organizations were involved in the design and conduct of the studies, collection of the data, and analysis and interpretation of the data. The authors take full responsibility for the content of the paper. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Executive summary

A method was validated for quantification of enzalutamide and its two major metabolites in human plasma.

The analytes were extracted from plasma by an LLE procedure, separated by reversed phase HPLC and detected by MS/MS.

The method proved to be rapid and simple, and met US FDA validation criteria.

The concentration range of the assay (0.0200 to 50 µg/ml) favorably supports PK determinations in clinical studies. To date, the method has been used to support the analysis of thousands of samples in nearly a dozen clinical trials.

References Papers of special note have been highlighted as: n of interest 1

2

3

Tran C, Ouk S, Clegg NJ et al. Development of a second-generation antiandrogen for treatment of advanced prostate cancer. Science 324(5928), 787–790 (2009). Scher HI, Fizazi K, Saad F et al. AFFIRM investigators. Increased survival with enzalutamide in prostate cancer after chemotherapy. N. Engl. J. Med. 367(13), 1187–1197 (2012). Scher HI, Beer TM, Higano CS et al. Prostate Cancer Foundation/Department of Defense Prostate Cancer Clinical Trials Consortium. Antitumour activity of enzalutamide in castration-resistant prostate cancer: a phase

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1–2 study. Lancet 375(9724), 1437–1446 (2010). 4

n

5

US Department of Health and Human Services, US FDA, Center for Drug Evaluation and Research, Center for Veterinary Medicine. Guidance for Industry Bioanalytical Method Validation. Center for Drug Evaluation and Research, Rockville, MD, USA (2001).

6

n

7

Excellent resource for details on how to validate a bioanalytical method. Viswanathan CT, Bansal S, Booth B et al. Quantitative bioanalytical methods validation and implementation: best practices for chromatographic and ligand binding assays. Pharm. Res. 24(10), 1962–1973 (2007).


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Hughes NC, Bajaj N, Fan J, Wong EY. Assessing the matrix effects of hemolyzed samples in bioanalysis. Bioanalysis 1(6), 1057–1066 (2009). Describes how to assess the impact of hemolysis on bioanalytical measurements. Lowes S, Jersey J, Shoup R et al. Recommendations on: internal standard criteria, stability, incurred sample reanalysis and recent 483s by the Global CRO Council for Bioanalysis. Bioanalysis 3(12), 1323–1332 (2011). Discusses the potential for co-administered drugs present in clinical samples to affect the stability of analytes subject to bioanalytical measurement. future science group