Introduction
Despite therapeutic advances, prostate adenocarcinoma is the highly prevalent cancer
in men and responsible for 20% of cancer related deaths in the Western world [1 ]. Initial approach to cure the localized prostate cancer is through radiation therapy
or surgical castration or treatment with first-generation antiandrogens namely flutamide,
nilutamide, bicalutamide [2 ]. In spite of initial response, majority of the patients develop a most aggressive
form of disease called metastatic castration-resistant prostate cancer (mCRPC) that
is associated with tumor progression and survival is less than 2 years [3 ]. Consequently, in recent years, new therapy options for mCRPC with different mechanisms
of action have become available like targeting androgen receptor signaling such as
CYP17A1 inhibitor (abiraterone acetate) [4 ] and a second-generation antiandrogens namely enzalutamide ([Fig. 1a1 ]), apalutamide ([Fig. 1b ]) and darolutamide ([Fig. 1c ]) [5 ]
[6 ]
[7 ]. Treatment with these drugs showed improved overall survival and quality of life
in mCRPC patients.
Fig. 1 Structural representation of enzalutamide a1 , N-desmethylenzalutamide a2 , apalutamide b , darolutamide c and ORM-15341 d .
Apalutamide (ARN-509 or JNJ-56021927; [Fig. 1b ]) is having similar in vitro activity to enzalutamide ([Fig. 1a1 ]) but with greater in vivo activity in CRPC xenograft models [8 ]. Apalutamide ([Fig. 1b ]) weakly binds to GABAA receptors and poorly penetrates through blood-brain-barrier suggesting that the chances
of developing seizures may be less when compared with enzalutamide ([Fig. 1a1 ]) [9 ]. Recently FDA approved apalutamide ([Fig. 1b ]) based on the Phase-3 SPARTAN trial in which apalutamide ([Fig. 1b ]) reduced the risk of metastasis or death by 72% in patients with non-mCRPC [10 ]. Enzalutamide (Xtandi® ; [Fig. 1a1 ]) was approved for the treatment of mCRPC in 2012 [5 ]
[11 ]. Enzalutamide ([Fig. 1a1 ]) is metabolized by CYP3A4 and 2C8 to produce an active circulatory metabolite, N -desmethylenzalutamide ([Fig. 1a2 ]), which has similar in vitro potency to enzalutamide [12 ]
[13 ]. Darolutamide (ODM-201; [Fig. 1c ]) is an equi-mixture of 2 pharmacologically active diastereomers namely ORM-16497
and ORM-16555 (structures not shown). Darolutamide ([Fig. 1c ]) undergoes extensive Phase-I metabolism and produces active circulatory metabolite
i. e., ORM-15341 ([Fig. 1d ]). ORM-16497 (structure not shown), ORM-16555 (structure not shown) and ORM-15341
([Fig. 1d ]) exert complete antagonistic effect on bicalutamide, hydroxyflutamide, enzalutamide
([Fig. 1a1 ]) and ARN-509 mutants [14 ]
[15 ]. Unlike enzalutamide ([Fig. 1a1 ]) and apalutamide ([Fig. 1b ]), penetration of darolutamide ([Fig. 1c ]) and ORM-15341 ([Fig. 1d ]) into brain is negligible (in preclinical species) suggests that low risk of causing
seizures in patients [7 ]. It was reported that ORM-15341 plasma concentrations were higher than sum of ORM-16497
and ORM-16555 (structures not shown) plasma concentrations in cancer patients [16 ]. We have also seen the similar trend in mice post-administration of darolutamide
([Fig. 1c ]) orally and intravenously
[17 ]
[18 ]. Currently Phase-3 clinical trials are being conducted with darolutamide ([Fig. 1c ]) in non-mCRPC patients globally [7 ].
Literature search revealed that most of the bioanalytical methods published for the
quantification of second generation antiandrogens were LC-MS/MS based. Quantification
of apalutamide ([Fig. 1b ]) alone [19 ] or along with other antiandrogens was reported by us in mice plasma [18 ]. LC-MS/MS based bioanalytical methods were reported for quantification of enzalutamide
([Fig. 1a1 ]) alone [20 ] or along with its metabolites [21 ]
[22 ] and other antiandrogens [18 ]
[23 ]
[24 ] or anticancer drugs [25 ] in various biological matrices (preclinical species plasma/tissues and human plasma).
Similarly, darolutamide ([Fig. 1c ]) and ORM-15341 ([Fig. 1d ]) quantification in mice plasma was reported post-administration of darolutamide
[17 ] and along with other second-generation non-steroidal antiandrogens [18 ] using conventional achiral columns on LC-MS/MS. Apart from these achiral methods,
2 chiral LC-MS/MS methods for quantification darolutamide ([Fig. 1c ]) diastereomers alone [26 ] or along with its metabolite i. e., ORM-15341 ([Fig. 1d ]) [27 ] were reported in recent times. Of late, we have reported dried blood spot (DBS)
method for simultaneous quantification of enzalutamide ([Fig. 1a1 ]), N -desmethylenzalutamide ([Fig. 1a2 ]), darolutamide ([Fig. 1c ]) and ORM-15341 ([Fig. 1d ]) [28 ] and apalutamide ([Fig. 1b ]) [29 ] in mice blood shown the validated methods application in a mice pharmacokinetic
study.
LC-MS/MS is an expensive technique, which requires high investment in both equipment
purchase and maintenance. Many laboratories and most of the hospitals will have HPLC
systems for routine application. Only one method has been reported for quantification
of second generation antiandrogens. Recently, a HPLC-UV method has been published
for the quantification of enzalutamide ([Fig. 1a1 ]) and its active metabolite, N -desmethylenzalutamide ([Fig. 1a2 ]) trough concentration in plasma collected from mCRPC patients [30 ]. In this paper an isocratic mobile phase was used to resolve enzalutamide ([Fig. 1a1 ]) and N -desmethylenzalutamide ([Fig. 1a2 ]) along with nilutamide (structure not shown; internal standard; IS) on C18 column within 9 min run time. We tried this method for the quantification of other
second generation antiandrogens and found that apalutamide ([Fig. 1b ]) did not elute under these conditions, however darolutamide ([Fig. 1c ]) and ORM-15341 ([Fig. 1d ]) eluted very early (within 2.5 min) and there is great chance that endogenous interference
will mask these 2 analytes. Hence, we felt that there is a great need to develop and
validate a generic HPLC method for quantification of apalutamide ([Fig. 1b ]), darolutamide ([Fig. 1c ]), ORM-15341 ([Fig. 1d ]), enzalutamide ([Fig. 1a1 ]), N -desmethylenzalutamide ([Fig. 1a2 ]) in mice plasma, which will be a valuable to support pharmacokinetic studies and
clinical setup. The objective of the present study was to develop and validate a simple,
specific, sensitive and reproducible HPLC method for quantitation of apalutamide ([Fig. 1b ]), darolutamide ([Fig. 1c ]), ORM-15341 ([Fig. 1d ]), enzalutamide ([Fig. 1a1 ]) and N -desmethylenzalutamide ([Fig. 1a2 ]) in mice plasma. The validated method was successfully applied to a pharmacokinetic
study in mice.
Materials and Methods
Chemicals and reagents
Darolutamide (purity:>97%) was obtained from Angene International Limited, China.
Enzalutamide (purity:>98.3%) and N -desmethylenzalutamide (purity:>99.6%) were purchased from BioOrganics, Bangalore,
India. ORM-15341 (purity: 99.4%) and apalutamide (purity: 99.5%) were synthesized
by the Medicinal Chemistry Group, Jubilant Biosys, Bangalore, India using literature
information [31 ]
[32 ] and the compounds were characterized using chromatographic (HPLC, LC-MS/MS) and
spectral techniques (IR, UV, Mass, 1 H and 13 C-NMR) by the Analytical Research Group, Jubilant Biosys. Phenacetin (purity;>99%)
was purchased from Sigma-Aldrich (St Louis, MO, USA). HPLC grade acetonitrile and
methanol were purchased from Rankem, Ranbaxy Fine Chemicals Limited, New Delhi, India.
Analytical grade formic acid was purchased from S.D Fine Chemicals, Mumbai, India.
All other chemicals and reagents were of analytical grade and used without further
purification. Microcaps® Disposable Micropipettes (50 µL, catalogue number: 1-000-0500)
were purchased from Drummond Scientific Company, USA. The control mice K2 .EDTA plasma sample was procured from Animal House, Jubilant Biosys.
HPLC operating conditions
Apalutamide, enzalutamide, N -desmethylenzalutamide, darolutamide and ORM-15341 and the IS were assayed using Waters
2695 Alliance HPLC system (Waters, Milford, USA) equipped with performance PLUS inline
degasser along with an auto-sampler and photo diode array (PDA) detector set at λmax 250 nm for quantitation of the analytes and the IS. The chromatographic separation
apalutamide, darolutamide, ORM-15341, enzalutamide, N -desmethylenzalutamide and the IS in processed samples was achieved on a X-Terra Phenyl
column (150×3.9 mm, 5 µ; Waters Corporation, Milford, USA) maintained at 40±1°C. The
binary mobile phase system consisted of reservoir A (10 mM ammonium acetate, pH: 4.8)
and reservoir B (acetonitrile) were run as per the flow regulated gradient program
given in [Table 1 ]. The injection volume was 10 µL.
Table 1 HPLC flow-binary gradient operated-time program used for simultaneous quantitation
of apalutamide, darolutamide, ORM-15341, enzalutamide and N -desmethylenzalutamide.
Time (min)
Flow rate (mL/min)
Mobile phase A (NH4 Ac) (%)
Mobile phase B (CH3 CN) (%)
0.00
0.20
70
30
2.00
0.40
70
30
4.00
0.80
60
40
8.00
1.20
60
40
15.0
1.20
70
30
Preparation of standard solutions
Primary stock solutions of apalutamide, darolutamide, ORM-15341, enzalutamide, N -desmethylenzalutamide for preparation of calibration curve (CC) and quality control
(QC) samples were prepared from separate weighing. Individual primary stock solution
of all the analytes at 200 µg/mL was prepared in DMSO:methanol (0.2:99.8, v/v). Similarly
the primary stock solution of the IS (1000 µg/mL) was prepared in methanol. The stock
solutions of apalutamide, darolutamide, ORM-15341, enzalutamide, N -desmethylenzalutamide and the IS were stored at 2–8°C, which were found to be stable
for 45 days and successively diluted with DMSO:methanol (0.2:99.8, v/v) to prepare
appropriate working solutions to perform calibration curve (CC). Another set of working
stock solutions of apalutamide, darolutamide, ORM-15341, enzalutamide, N -desmethylenzalutamide were made in DMSO:methanol (0.2:99.8, v/v) (from primary stock)
at appropriate dilutions for preparation of QC samples. A working IS solution (500 ng/mL)
was prepared in methanol. Working stock solutions were stored approximately at 4°C
for 30 days.
Preparation of calibration curve standards and quality control samples
Calibration samples were prepared by spiking 90 µL of blank mice plasma with the mixed
working solution of analytes (10 µL) on the day of analysis. Calibration curve standard
consists of a set of 8 non-zero concentrations (209, 417, 1043, 1617, 2086, 3129,
4120 and 5125 ng/mL) for all the analytes was prepared. Samples for the determination
of precision and accuracy were prepared by spiking blank mice plasma in bulk with
mixed working stock solution of analytes at appropriate concentrations and 100 μL
aliquots were distributed into different tubes. The QCs prepared for each analyte
are: 209 ng/mL (lower limit of quantification quality control; LLOQ QC), 626 ng/mL
(low quality control; LQC), 2503 ng/mL (medium quality control; MQC) and 4172 ng/mL
(high quality control; HQC). All the samples were stored together at -80±10°C until
analysis.
Sample preparation
To an aliquot of 100 µL plasma sample, 200 µL of acetonitrile enriched with IS (500 ng/mL)
was added and vortex mixed for 3 min; followed by centrifugation for 5 min at 20,817 g in a refrigerated centrifuge (Eppendorf 5424 R) maintained at 5°C to precipitate
protein matrix and particulate matter. Clear supernatant (100 µL) was transferred
into a vial and 10 µL was injected onto HPLC system.
Validation procedures
A full validation according to the US FDA guidance was performed for the quantitation
of apalutamide, darolutamide, ORM-15341, enzalutamide and N -desmethylenzalutamide in mice plasma [33 ].
Selectivity
Selectivity of the method was determined by determining the presence of interfering
peaks from 6 individual drug-free mice plasma samples at the retention times of apalutamide,
darolutamide, ORM-15341, enzalutamide, N -desmethylenzalutamide and the IS.
Limit of quantification and carry over
The LLOQ was determined as the concentration that has a precision of<20% of the relative
standard deviation (%RSD) and accuracy between 80-120% of the theoretical value. The
auto-injector carryover was determined by injecting the highest calibration standard,
followed by injection of blank samples. The response of the blanks was then compared
to that of the LLOQ.
Recovery
The recovery of apalutamide, darolutamide, ORM-15341, enzalutamide, N -desmethylenzalutamide and the IS extraction from mice plasma was determined by comparing
the response of each analyte extracted (using simple protein precipitation by adding
acetonitrile) from replicate QC samples (n=6) with the response of analyte from neat
standards at equivalent concentrations. Recovery of apalutamide, darolutamide, ORM-15341,
enzalutamide and N -desmethylenzalutamide was determined at LQC (626 ng/mL), MQC (2503 ng/mL) and HQC
(4172 ng/mL) concentrations. Recovery of the IS was determined at a single concentration
of 500 ng/mL.
Calibration curve
Calibration samples (for all the analytes) were prepared on each validation day. Peak
area ratios of each analyte to that of the IS were used for all calculations. A least
squares linear regression (1/X
2 weighting factor) of 8 non-zero samples was used to define the calibration curve.
Precision and accuracy
The precision and accuracy of the method were evaluated by measuring the 4 QC samples
(LLOQ QC, LQC, MQC and HQC), which were prepared on each validation day (n=6 each).
Inter-day precision was assessed on 4 separate days. Inter- and intra-day precisions
were determined by calculating %RSD that should be ≤15% for all the QC levels except
for LLOQ QC where it should be ≤20%. The inter- and intra-day accuracy expressed as
percent relative error (%RE) was calculated by comparing the measured concentration
with the nominal value and deviation was limited within±15% except for LLOQ QC where
it should be ≤20%.
Stability
The stability of apalutamide, darolutamide, ORM-15341, enzalutamide and N -desmethylenzalutamide was assessed at LQC and HQC levels in 6 replicates under all
storage conditions. Freeze-thaw stability was performed following 3 freeze-thaw cycles
was evaluated (plasma samples were stored in -80±10o C between freeze/thaw cycles). Short-term temperature stability was assessed by analyzing
samples that had been kept at ambient temperature (25±1o C) for 6 h. Long-term stability was performed by analyzing samples that had been stored
at -80±10°C for 30 days. The stability of apalutamide, darolutamide, ORM-15341, enzalutamide,
N -desmethylenzalutamide and the IS in the injection solvent was determined periodically
by injecting replicate preparations of processed plasma samples for up to 24 h (in
the auto-sampler at 5°C) after the initial injection.
Dilution effect
To evaluate the effect of dilution over the calibration range, the accuracy and precision
of dilution control samples at 25,625 ng/mL (n=6; 5 times of the ULOQ) were assessed
by performing a 10-fold dilution.
Pharmacokinetic study in mice
Animal experiments were approved by Institutional Animal Ethical Committee (IAEC/JDC/2017/135).
Male BalbC mice (n =12; 27–29 g weight) were procured from Vivo Biotech, Hyderabad, India. The animals
were housed in Jubilant Biosys animal house facility in a temperature (22±2°C) and
humidity (30-70%) controlled room (15 air changes/h) with a 12:12 h light:dark cycles,
had free access to rodent feed (Altromin Spezialfutter GmbH & Co. KG., Im Seelenkamp
20, D-32791, Lage, Germany) and water for one week before using for experimental purpose.
Following 4 h fast (during the fasting period animals had free access to water) mice
received apalutamide, darolutamide and enzalutamide as a cassette dose orally at 20 mg/kg
[suspension formulation prepared using 0.1% Tween-80 with methyl cellulose (0.5% in
water); strength: 2.0 mg/mL; dose volume: 10 mL/kg]. Post-dosing blood samples (200 µL)
were collected into polypropylene tubes containing K2 .EDTA solution as an anti-coagulant at 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, 48, 72 and
96 h (sparse sampling protocol was adopted during blood collection and at each time
point blood was collected from 3 mice). Plasma was harvested by centrifuging the blood
using Biofuge (Hereaus, Germany) at 1760 g for 5 min and stored frozen at −80±10°C until analysis. Animals were allowed access
to feed 2 h post-dosing.
Thawed plasma sample were processed as mentioned in sample preparation section. Along
with plasma samples, QC samples at low, medium and high concentration (made in blank
plasma) were assayed in duplicate and were distributed among unknown samples in the
analytical run. The criteria for acceptance of the analytical runs encompassed the
following: (i) 67% of the QC samples accuracy must be within 85-115% of the nominal
concentration (ii) not less than 50% at each QC concentration level must meet the
acceptance criteria [33 ]. The pharmacokinetic parameters were calculated by using Phoenix WinNonlin software
(version 8.0; Pharsight Corporation, Mountain View, CA).
Results and Discussion
Chromatographic conditions
Selection of chromatographic conditions for the proposed method was optimized to suit
the preclinical and clinical pharmacokinetic studies. As briefed in the introduction
section we tried the enzalutamide HPLC method reported by Puszkiel et al. [30 ] for the quantification of other second generation antiandrogens and found that apalutamide
(structurally very close to enzalutamide as shown in [Fig. 1a1 ]) did not elute under these conditions, however darolutamide and ORM-15341 eluted
very early (within 2.5 min) and in comparison to our results as demonstrated in [Fig. 2 ] it is evident that endogenous interference will mask these 2 analytes. Hence we
tried different mobile phases comprising several combinations of buffers (e.g., phosphate
buffer and ammonium acetate buffer) and organic solvents (acetonitrile and methanol)
along with altered flow-rates (in the range of 0.60–1.20 mL/min) were tested to optimize
for an effective chromatographic resolution of apalutamide, darolutamide, ORM-15341,
enzalutamide, N -desmethylenzalutamide and the IS (data not shown). The best resolution of peaks was
achieved with a mobile phase comprising 10 mM ammonium acetate (pH 4.8):acetonitrile
in a flow operated binary gradient mode on an X-Terra phenyl column.
Fig. 2 HPLC chromatograms of a 10 µL injection of a mice blank plasma b blank mice plasma spiked with apalutamide, darolutamide, ORM-15341, enzalutamide
and N -desmethylenzalutamide at LLOQ (209 ng/mL) along with IS c an in vivo plasma sample collected from a mouse at 1.0 h time point following a cassette
oral administration of apalutamide, darolutamide and enzalutamide.
Recovery
The recovery (mean±S.D) for apalutamide, darolutamide, ORM-15341, enzalutamide and
N -desmethylenzalutamide at LQC, MQC and HQC and for the IS at single concentration
is shown in [Table 2 ].
Table 2 Intra- and inter-day precision and accuracy determination of apalutamide, darolutamide,
ORM-15341, enzalutamide, N -desmethylenzalutamide quality controls in mice plasma.
Concentration spiked (ng/mL)
Intra-day (%RSD)
Inter-day (%RSD)
Accuracy (%RE)
%Recovery (mean±SD)
Apalutamide
209
3.40
3.15
1.05
-
626
3.06
7.86
1.08
98.7±4.51
2503
7.51
12.4
0.92
86.8±2.86
4172
5.55
13.9
0.92
98.3±4.53
Darolutamide
209
8.88
8.22
0.88
-
626
1.30
4.21
1.01
101±4.27
2503
1.43
2.67
0.96
93.1±4.41
4172
0.61
2.06
0.99
98.4±1.24
ORM-15341
209
1.13
1.04
1.01
-
626
2.48
3.48
1.00
98.0±1.91
2503
1.12
3.66
0.97
94.0±3.68
4172
0.56
1.93
1.01
101±0.78
Enzalutamide
209
4.06
3.76
1.02
-
626
1.78
4.02
1.04
99.3±4.12
2503
1.64
3.90
0.96
95.6±4.14
4172
1.16
2.99
0.99
100±1.02
N -Desmethylenzalutamide
209
3.40
3.15
1.04
-
626
0.62
2.81
1.01
103±4.65
2503
1.81
3.69
0.96
96.2±3.29
4172
1.48
2.78
0.99
97.7±2.31
IS
500
-
-
-
95.4±8.21
RSD: relative standard deviation (SD×100/Mean)
RE: relative error (measured value/actual value)
SD: standard deviation
Selectivity
As shown in [Fig. 2 ] no interferences in the blank mice plasma traces were found from endogenous components
in drug-free mice plasma at the retention times of the apalutamide, darolutamide,
ORM-15341, enzalutamide, N -desmethylenzalutamide and the IS indicating that the method is selective. Apalutamide,
enzalutamide, N -desmethylenzalutamide, darolutamide and ORM-15341 and the IS eluted at 13.6, 11.4,
9.68, 6.11, 6.93 and 4.69 min, respectively.
Sensitivity and carry over
The lowest limit of reliable quantification for each analyte was set at the concentration
of the LLOQ. The precision (%RSD) and accuracy (%RE) at LLOQ concentration were found
to be 3.40 and 105% for apalutamide, 8.88 and 87.7% for darolutamide, 1.13 and 101%
for ORM-15341, 4.06 and 102% for enzalutamide and 3.40 and 98.8% for N -desmethylenzalutamide. There was no carry-over produced by the highest calibration
sample on the following injected mice plasma extracted sample for all the analytes.
Calibration curve
The plasma calibration curve was constructed using 8 calibration standards (viz.,
209 – 5215 ng/mL). The calibration standard curve had a reliable reproducibility over the standard
concentrations across the calibration range. Calibration curve was prepared by determining
the best fit of peak-area ratios (peak area analyte/peak area of the IS) vs. concentration,
and fitted to the y =mx +c using weighing factor (1/X
2 ). The average regression (n =4) was found to be>0.998. The lowest concentration with the RSD≤20% was taken as
LLOQ and was found to be 209 ng/mL. The accuracy observed for the mean of back-calculated
concentrations for 4 calibration curves was within 87.3−108%; while the precision
(%RE) values ranged from 0.05−12.2% for all the analytes.
Accuracy and precision
Accuracy and precision data for intra- and inter-day mice plasma samples are presented
in [Table 2 ]. The assay values on both the occasions (intra- and inter-day) were found to be
within the accepted variable limits. The data show that the method possesses adequate
accuracy and repeatability for analyzing apalutamide, darolutamide, ORM-15341, enzalutamide
and N -desmethylenzalutamide in mice plasma samples.
Stability
[Table 3 ] summarizes the results of stability studies conducted for apalutamide, darolutamide,
ORM-15341, enzalutamide and N -desmethylenzalutamide in mice plasma. The measured concentrations for these analytes
at 626 and 4172 ng/mL samples deviated within±15% of the nominal concentrations in
a battery of stability tests viz., in-injector (24 h), bench-top (6 h), repeated 3
freeze/thaw cycles and freezer stability at −80±10°C for at least for 30 days ([Table 3 ]). The results were found to be within the assay variability limits during the entire
process and demonstrated that apalutamide, darolutamide, ORM-15341, enzalutamide and
N -desmethylenzalutamide can be stored under tested conditions without compromising
the integrity of samples.
Table 3 Stability data of apalutamide, darolutamide, ORM-15341, enzalutamide and N -desmethylenzalutamide quality controls in mice plasma.
Concentration spiked (ng/mL)
Bench-top for 6 h
Long-term 30 days at -80°C
Third freeze-thaw cycle
Auto-sampler for 24 h
% RE
%RSD
% RE
%RSD
% RE
%RSD
% RE
%RSD
Apalutamide
626
0.98
7.27
1.06
3.61
1.03
3.27
0.94
8.86
4172
0.95
5.15
0.98
5.47
1.09
10.1
1.04
6.38
Darolutamide
626
0.96
2.22
0.85
5.98
0.97
4.91
0.90
3.50
4172
1.03
1.87
0.92
1.47
1.02
5.48
1.04
1.03
ORM-15341
626
0.95
2.81
0.98
2.43
0.96
5.54
0.95
1.90
4172
1.05
2.84
1.08
2.03
1.03
5.12
1.03
1.01
Enzalutamide
626
1.00
4.05
1.03
2.23
1.00
5.27
0.92
3.21
4172
1.04
3.19
0.88
2.07
0.99
4.83
0.97
0.94
N -Desmethylenzalutamide
626
0.96
2.61
0.99
4.47
0.96
3.54
0.95
2.48
4172
1.03
2.33
1.12
1.74
1.01
5.03
1.01
0.92
RSD: relative standard deviation (SD×100/Mean)
RE: relative error (measured value/actual value)
Dilution effect
The dilution integrity was confirmed for QC samples that exceeded the upper limit
of standard calibration curve. The results showed that the accuracy (within 7.5%)
and precision (≤8.7%) for 10x diluted test samples (for all the analytes), which show
the ability to dilute samples up to a dilution factor of 10 in a linear fashion.
Pharmacokinetic study
Following oral administration of apalutamide, enzalutamide and darolutamide as a cassette
dosing to mice, enzalutamide is quantifiable up to 48 h as illustrated in [Fig. 3 ]i/[1 ], N -desmethylenzalutamide (released from enzalutamide) is quantifiable up to 96 h as
shown in [Fig. 3 ]i/[1 ]. More detailed information about plasma concentrations of enzalutamide and N -desmethylenzalutamide from 0-24 h is presented in [Fig. 3 ]i/[2 ]. The apalutamide is quantifiable up to 48 h, as illustrated in [Fig. 3ii ]. Daroluatmide and ORM-15341 (released from darolutamide) are quantifiable up to
8 h and 12 h as illustrated in [Fig. 3iii ] and [Fig. 3iv ], respectively. The pharmacokinetic parameters are compiled in [Table 4 ]. Both AUC(0-∞) (area under the curve from time 0 to infinity) and Cmax (maximum concentration in plasma) were higher for ORM-15341 than darolutamide and
this data is in agreement with data reported by us [17 ]. The Cmax achieved at 0.5 h (Tmax ) for both darolutamide and ORM-15341. The half-life (T1/2 ) was found to be 4.77 and 8.53 h for darolutamide and ORM-15341, respectively. For
enzalutamide and N -desmethylenzalutamide the AUC(0-∞) and Cmax trend reported in humans [21 ] was seen in the present study. As apalutamide is structurally very close to enzalutamide
the AUC(0-∞) and Cmax are more or less similar to enzalutamide. Enzalutamide, N -desmethylenzalutamide and apalutamide have shown delayed Tmax (6–8 h). Due to their persistence in plasma for ≥48 h and low clearance the half-life
was found to be 13.2, 30.2 and 22.2 h for enzalutamide, N -desmethylenzalutamide and apalutamide, respectively. In summary the validated method
was sensitive enough to calculate the pharmacokinetics parameters of second generation
non-steroidal antiandrogens along with their active metabolites. We believe that the
current method with little or no modifications can be extended to other pre-clinical
species plasma matrix. The method can provide a lot of potential information to assist
the researchers in deciding their approach for quantitation strategy towards pharmacokinetics
and/or toxicokinetics in pre-clinical species and pharmacokinetics and/or therapeutic
drug monitoring of second generation antiandrogens in clinic.
Fig. 3 Mean plasma concentration vs. time profile for enzalutamide and N -desmethylenzalutamide (released from enzalutamide) up to 48 and 96 h, respectively
in i/1 and up to 24 h in i/2 ; for apalutamide in ii ; for darolutamide in iii and ORM-15341 (released from darolutamide) in iv in mice plasma following a cassette oral administration of apalutamide, darolutamide
and enzalutamide to mice.
Table 4 Pharmacokinetic parameters for apalutamide, darolutamide, ORM-15341, enzalutamide
and N -desmethylenzalutamide following oral administration of apalutamide, darolutamide
and enzalutamide to mice at 20 mg/Kg.
PK parameters
AUC(0-∞) (µg×h/mL)
Cmax (µg/mL)
Tmax (h)
T1/2 (h)
Apalutamide
142
3.82
6.00
22.2
Darolutamide
6.57
1.63
0.50
4.77
ORM-15341
90.6
12.3
0.50
8.53
Enzalutamide
155
5.03
6.00
13.2
N -Desmethylenzalutamide
193
4.32
8.00
30.2
