Stabilization of Abiraterone in Human Plasma Using the Esterase Inhibitor Bis(4-nitrophenyl) Phosphate

Abstract

Background:

Abiraterone, an active metabolite of abiraterone acetate, is used for the treatment of prostate cancer. Therapeutic drug monitoring (TDM) of abiraterone could improve treatment outcomes. However, its stability in plasma for only 4 hours at room temperature, is making the TDM implementation difficult in clinical practice. Stabilization experiments were performed in our laboratory using esterase inhibitors for the stabilization of abiraterone acetate in preclinical samples. The esterase inhibitor bis(4-nitrophenyl) phosphate (BNPP) stabilizes abiraterone acetate and abiraterone as well. Therefore, we investigated whether the esterase inhibitor BNPP could stabilize abiraterone in fresh human plasma.

Methods:

BNPP at 1 and 10 mM were evaluated for its stabilizing effects on abiraterone in fresh human K₂EDTA plasma. The samples were analyzed using a validated liquid chromatography–mass spectrometry (LC-MS/MS) method. A partial validation assessed BNPP's impact on accuracy, precision, selectivity, and specificity within the fully validated LC-MS/MS method.

Results:

BNPP at 10 mM, but not 1 mM, effectively prevented abiraterone degradation in fresh human K₂EDTA plasma, maintaining stability for at least 5 days at room temperature. Partial validation confirmed that all results met the acceptance criteria of the European Medicines Agency guidelines and the US Food and Drug Administration guidance.

Conclusions:

We demonstrated that the esterase inhibitor BNPP effectively stabilizes abiraterone in fresh human K₂EDTA plasma. BNPP had no significant effect on the accuracy, precision, selectivity, or specificity of LC-MS/MS for abiraterone detection. The addition of BNPP to clinical abiraterone samples may be helpful in implementing abiraterone TDM in daily clinical practice.

INTRODUCTION

Abiraterone acetate is an abiraterone prodrug prescribed for the treatment of prostate cancer.¹ Several studies have shown an exposure–response relationship between the plasma concentrations of abiraterone and progression-free survival. Abiraterone trough concentrations (C_min) >8.4 ng/mL have been associated with an improved progression-free survival of 12.2 versus 7.4 months,² which was validated in a clinical patient cohort.³ These data, together with the high interpatient variability in abiraterone plasma concentrations,⁴ support the rationale for implementing therapeutic drug monitoring (TDM).⁵

However, the stability of abiraterone in human plasma is an obstacle to the implementation of TDM. We have previously developed and validated an assay for the TDM of abiraterone in plasma. In a validation experiment, abiraterone was stable for only 4 hours at room temperature after sample collection.⁶ Limited stability in fresh human plasma and whole blood has also been observed by Benoist et al.⁷ Interestingly, in a 2-year-old batch of human K₂EDTA plasma, abiraterone seemed to be stable for at least 48 hours at room temperature.⁶ The mechanism underlying this instability remains unknown; however, enzymatic degradation is most likely to play a role.⁶

One consequence of the short stability of abiraterone is that the samples must be shipped on dry ice to external laboratories. Most hospitals do not have the resources to set up their own liquid chromatography–mass spectrometry (LC-MS/MS) methods for abiraterone monitoring, and shipping samples on dry ice is costly. Therefore, TDM cannot be easily applied in many patients receiving abiraterone. This may result in underexposure and inadequate abiraterone treatment in a subgroup of patients.

We performed stabilization experiments with several esterase inhibitors to stabilize abiraterone acetate, the ester prodrug of abiraterone, in preclinical samples. Unexpectedly, abiraterone could be stabilized. Stabilizing clinical samples containing abiraterone could facilitate the widespread implementation of TDM. Here, we describe these stabilization experiments and the partial validation of the abiraterone assay in plasma after stabilization.

MATERIALS AND METHODS

Chemicals

Abiraterone and ²H₅-abiraterone were purchased from Alsachim (Illkirch Graffenstaden, France). Acetonitrile, methanol, water, and 99% formic acid were obtained from Biosolve Ltd (Valkenswaard, Netherlands), whereas dimethyl sulfoxide (Seccosolv grade) was sourced from Merck (Darmstadt, Germany). The esterase inhibitor bis(4-nitrophenyl) phosphate (BNPP) was procured from Sigma-Aldrich (St. Louis, MO). K₂EDTA and lithium heparin plasma were purchased from Bioreclamations LLC (Hicksville, NY). Whole blood was drawn from a healthy volunteer on the day of the stability experiment to obtain fresh human K₂EDTA plasma.

Preparation of Solutions

The stock and working solutions of abiraterone and ²H₅-abiraterone internal standards (IS) were prepared as described by van Nuland et al.⁶ The esterase inhibitor BNPP was dissolved in methanol at concentrations of 170 (500 mM) and 17 mg/mL (50 mM). Solutions were stored at −20°C.

Collection of Control Biomatrix

Approximately 5 mL of whole blood was drawn from healthy volunteers and collected in K₂EDTA tubes. After inverting the tube 10 times, the blood was centrifuged at 2500 rpm for 10 minutes at room temperature (20–25°C) and the plasma was collected.

Initial Stability Experiments in the Biomatrix

A volume of 20 µL of 50 mM BNPP or 20 µL of 500 mM BNPP was added to 970 µL of freshly collected human K₂EDTA plasma. Subsequently, 10 µL of abiraterone working solution (10,000 ng/mL) in K₂EDTA plasma was spiked to both tubes, obtaining a final concentration of 100 ng/mL abiraterone [quality control (QC) medium level] and BNPP concentrations of 1 and 10 mM, respectively. Furthermore, abiraterone working solution (10,000 ng/mL) in K₂EDTA plasma was spiked into freshly collected human K₂EDTA plasma containing 0 mM BNPP (blank) and into a 4-year-old batch of human K₂EDTA plasma, stored at −20°C without BNPP, at a final concentration of 100 ng/mL. A volume of 50 µL was collected just after addition of abiraterone (t = 0), at 4, 24, and 48 hours at room temperature (20–25°C). After sample collection at the different time points, 100 µL of acetonitrile and 20 µL of abiraterone internal standard (IS 125 ng/mL in methanol) were added to end each incubation step and further processed and analyzed as described by van Nuland et al.⁶ The abiraterone/abiraterone IS ratio at t = 0 was set to 100% and compared with other time points to investigate the stability of abiraterone over time in different batches with and without BNPP. Abiraterone was considered stable when 85%–115% of its initial concentration was recovered.

Preparation of Calibration Standards and Quality Control Samples

Calibration standards and QC samples were freshly prepared on the first day of the validation experiment as described by van Nuland et al.⁶ Aliquots of 50 µL were made and stored at −20°C until further processing.

Sample Processing and LC-MS/MS Analysis

The samples were processed and analyzed using the validated liquid chromatography–mass spectrometry (LC-MS/MS) method developed by van Nuland et al.⁶ Multiple reaction monitoring chromatograms were acquired and processed using Analyst 1.6.2 software (Sciex, Framingham, MA).

Validation Procedures

Partial validation was performed to investigate the influence of the esterase inhibitor, BNPP, on the accuracy, precision, specificity, selectivity, and short-term stability of abiraterone. The validation criteria were based on the international guidelines for bioanalytical method validation of the United States Food and Drug Administration and the European Medicines Agency.^8,9

For accuracy and precision, 5 replicates of the QC lower limit of quantification (LLOQ) and QC upper limit of quantification (ULOQ) were prepared using a 4-year-old human K₂EDTA plasma batch containing 10 mM BNPP. The samples were analyzed in a single analytical run. Samples were prepared by adding 20 µL of 500 mM BNPP in methanol to 980 µL of control human plasma. Subsequently, 50 µL of the abiraterone working solution (containing 20 and 2000 ng/mL abiraterone in control human K₂EDTA plasma) was added to 450 µL of 10 mM BNPP stabilized control human plasma, yielding abiraterone concentrations of 2 ng/mL (QC LLOQ) and 200 ng/mL (QC ULOQ), respectively. Intra-assay accuracy and precision were calculated using the equations described by Herbrink et al.¹⁰ Results were accepted when bias and coefficient of variation (CV) values were ±15% and ≤15% for QC ULOQ and ±20% and ≤20% for QC LLOQ.

To assess specificity and selectivity, 3 different batches of 4-year-old human K₂EDTA plasma containing 10 mM BNPP and 3 batches of 4-year-old human lithium heparin plasma containing 10 mM BNPP were used to prepare double blanks and QC LLOQ samples. The abiraterone and abiraterone-IS responses in the double-blank samples were compared with those in the QC LLOQ samples. Peaks should not exceed 20% of the LLOQ for abiraterone and 5% for abiraterone-IS and the mean abiraterone concentration in LLOQ samples should be within ±20% of the nominal concentration.

To demonstrate the stability of abiraterone in a 10 mM BNPP freshly collected human K₂EDTA plasma matrix, the samples were kept at room temperature (20–25°C), exposed to light at QC LLOQ and QC ULOQ concentrations, and analyzed in triplicate after 24 hours, 48 hours, and 5 days. Abiraterone was considered stable when 80%–120% and 85%–115% of the concentration was recovered for QC LLOQ and QC ULOQ, respectively.

RESULTS

Stability Experiments in the Biomatrix

The stabilizing effect of the esterase inhibitor BNPP on abiraterone is shown in Figure 1. Abiraterone was stable for only 4 hours in fresh human K₂EDTA plasma at room temperature, whereas abiraterone was stable for at least 48 hours at room temperature in a 4-year-old batch of human K₂EDTA plasma. BNPP was effective in stabilizing abiraterone in fresh human K₂EDTA plasma, and 95.5% of abiraterone was recovered after 48 hours in the presence of 10 mM BNPP. A concentration of 1 mM BNPP was less effective because 73.3% of abiraterone was recovered after 48 hours. Therefore, BNPP at a concentration of 10 mM was selected for partial validation.

FIGURE 1.

The percentage abiraterone (100 ng/mL) in fresh human K₂EDTA plasma, fresh human K₂EDTA plasma containing BNPP at a concentration of 0, 1, or 10 mM and in a 4-year-old batch human K₂EDTA plasma after incubation of 4, 24, and 48 hours at room temperature. The dotted line represents the 85% level.

Validation Procedures

The intra-assay accuracy and precision are presented in Table 1. All results were within the acceptance criteria of ±15% and ≤15% for QC ULOQ and ±20% and ≤20% for QC LLOQ. This demonstrates that BNPP has no significant effect on the accuracy and precision of the method.

TABLE 1.

Assay Performance (n = 5) and Stability (n = 3) Data of Abiraterone in Fresh Human K₂EDTA Plasma Containing 10 mM BNPP

	Nominal Concentration (ng/mL)	Intra-assay
		Accuracy (% Bias)	CV (%)
Assay performance data	2	−4.3	4.5
	200	−2.0	0.6

	Stability Conditions	Nominal Concentration (ng/mL)	Accuracy (% Bias)	CV (%)
Stability data	RT 24 h	2	−14.0	1.5
		200	−5.2	1.5
	RT 48 h	2	−9.0	6.7
		200	−4.5	1.8
	RT 5 d	2	−5.3	10.8
		200	−1.0	3.1

CV, coefficient of variation; RT, room temperature.

The mean abiraterone concentration in LLOQ samples across all tested batches of human plasma was within the required ±20% of the nominal concentration. One of the 6 batches showed an abiraterone response in a double-blank sample, but the response did not exceed 20% of the LLOQ. No abiraterone–IS response was observed in the double-blank samples. This indicates that BNPP had no effect on the selectivity and specificity of the method.⁶

The results of the stability experiments are listed in Table 1. These findings demonstrate the stability of abiraterone for at least 5 days at room temperature at QC LLOQ and QC ULOQ levels in fresh human K₂EDTA plasma stabilized with 10 mM BNPP.

DISCUSSION

Abiraterone stability was demonstrated in a long-term frozen human plasma, whereas degradation occurred in fresh human plasma. Several studies have shown a decrease in enzymes activity after storage and freeze–thaw cycles,^11–14 indicating a role for plasma enzymes in the metabolism of abiraterone. Inactivation of esterases in stored plasma has been suggested by Benoist et al.⁷ However, abiraterone does not contain any ester or amide bonds that can be hydrolyzed. Abiraterone is primarily metabolized by the liver enzyme sulfotransferase 2A1 (SULT2A1) to abiraterone sulfate, and SULT2A1 and cytochrome P450 3A4 (CYP3A4) to n-oxide abiraterone sulfate.¹⁵

Therefore, the stabilizing effect of BNPP on abiraterone was unexpected. BNPP is mainly known as an irreversible inhibitor of carboxylesterases. Carboxylesterases are involved in the metabolism of several xenobiotic compounds including the conversion of abiraterone acetate to abiraterone. They catalyze the hydrolysis of ester- or amide-containing substrates into alcohols and carboxylic acids. BNPP is commonly used in inhibition studies as a carboxylesterase inhibitor with half-maximal inhibitory concentrations in the nanomolar range.¹⁶ BNPP is not only considered a carboxylesterase inhibitor but also inhibits other esterases¹⁷ and arylacetamide deacetylase (AADAC).¹⁸ Because abiraterone does not contain any ester or amide bonds and is not metabolized by esterases or AADAC, an effect of BNPP on abiraterone stability was not expected. However, a high concentration of BNPP (10 mM) was necessary to maintain the stability of abiraterone, which probably resulted in off-target effects and the inhibition of plasma enzymes other than esterase and AADAC. The exact mechanisms underlying the stabilizing effect of BNPP on abiraterone remain unknown, but abiraterone may be metabolized by unknown enzymes.

We demonstrated that 10 mM BNPP could stabilize abiraterone in fresh human K₂EDTA plasma for at least 5 days at room temperature. BNPP had no influence on the accuracy, precision, selectivity, or specificity of the LC-MS/MS method used for the quantification of abiraterone.⁶ Therefore, clinical samples from patients using abiraterone could be stabilized with BNPP, shipped within 5 days at room temperature, and measured using a validated LC-MS/MS method.

This finding is important because TDM of abiraterone is an effective tool to identify patients who are under exposure. In the NKI/AVL, patients with C_min levels below the target of 8.4 ng/mL are recommended to take abiraterone acetate concomitantly with food. This intervention led to an increase in the abiraterone C_min levels and adequate exposure in most patients.¹⁹ Therefore, it is important to identify strategies that make TDM accessible to all patients receiving abiraterone.

The use of dried blood spots or microsampling²⁰ is a more patient-friendly strategy for TDM because it facilitates self-sampling and home sampling. Abiraterone was stable for at least 30 days in dried plasma spots,²¹ 7 days in dried blood spots,²² and 14 days in volumetric adsorptive microsampling sponges.²³ Until dried blood spots or microsampling can be applied in clinical practice, the stabilization of abiraterone in clinical samples with 10 mM BNPP is a practical and simple solution. However, further research should be conducted to investigate the feasibility of using BNPP in clinical practice, considering the use of multidrug therapy in patients and the handling of samples outside an experimental environment.

CONCLUSIONS

BNPP effectively stabilized abiraterone in fresh human K₂EDTA plasma for at least 5 days at room temperature without affecting the accuracy, precision, selectivity, or specificity of the LC-MS/MS method. Therefore, the addition of BNPP to clinical samples containing abiraterone may be a promising solution for implementing TDM of abiraterone. The feasibility in clinical practice should be further investigated, before addition of BNNP to clinical samples can be implemented.