Accepted Manuscript Title: Simultaneous quantification of lenalidomide, ibrutinib and its active metabolite PCI-45227 in rat plasma by LC-MS/MS: application to a pharmacokinetic study Author: Sridhar Veeraraghavan Srikant Viswanadha Satheeshmanikandan Thappali Babu Govindarajulu Swaroopkumar Vakkalanka Manivannan Rangasamy PII: DOI: Reference:
S0731-7085(14)00577-9 http://dx.doi.org/doi:10.1016/j.jpba.2014.11.041 PBA 9828
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Revised date: Accepted date:
26-9-2014 19-11-2014 23-11-2014
Please cite this article as: S. Veeraraghavan, S. Viswanadha, S. Thappali, B. Govindarajulu, S. Vakkalanka, M. Rangasamy, Simultaneous quantification of lenalidomide, ibrutinib and its active metabolite PCI-45227 in rat plasma by LCMS/MS: application to a pharmacokinetic study, Journal of Pharmaceutical and Biomedical Analysis (2014), http://dx.doi.org/10.1016/j.jpba.2014.11.041 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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*Graphical Abstract
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*Highlights (for review)
1. Highly selective method for the determination of ibrutinib, PCI-45227 and lenalidomide in plasma 2. This is the first LC-MS/MS- based method for simultaneous determination of ibrutinib, PCI-45227 and lenalidomide in plasma
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3. Plasma sample clean-up involves a simple liquid-liquid extraction procedure
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4. The developed LC-MS/MS based method was applied successfully for pharmacokinetic study
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Simultaneous quantification of lenalidomide, ibrutinib and its active metabolite PCI-45227 in rat plasma by LC-MS/MS: application to a pharmacokinetic study
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Sridhar Veeraraghavana,c, Srikant Viswanadhaa, Satheeshmanikandan Thappalia , Babu
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Govindarajulua, Swaroopkumar Vakkalankaa, Manivannan Rangasamyb,
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Professional affiliations:
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a
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Incozen Therapeutics Private Limited,
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450, Alexandira Knowledge Park, Turkapplly
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Hyderabad - 500078, Andhra Pradesh, India.
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b
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Dept of Pharmaceutics
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Annai JKK Sampoorani Ammal College of Pharmacy
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Komarapalayam, Namakkal - 638 183, Tamilnadu, India
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c
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CRD, PRIST University,
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Vallam, Thanjavur 613403. Tamilnadu, India
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Current affiliations:
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Sridhar Veeraraghavan
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Incozen Therapeutics Private Limited,
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450, Alexandria Knowledge Park, Turkapplly
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Hyderabad - 500078, Andhra Pradesh, India.
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Corresponding author:
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Tel: +91 40 2348 0336
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E-mail addresses:
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[email protected] (Sridhar V)
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Abstract:
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Efficacy assessments using a combination of ibrutinib and lenalidomide necessitate the
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development of an analytical method for determination of both drugs in plasma with
4
precision. A high performance liquid chromatography-tandem mass spectrometry (LC-
5
MS/MS) method was developed for the simultaneous determination of lenalidomide,
6
ibrutinib, and its active metabolite PCI45227 in rat plasma. Extraction of lenalidomide,
7
ibrutinib, PCI45227 and tolbutamide (internal standard; IS) from 50 µl rat plasma was carried
8
out by liquid-liquid extraction with ethyl acetate: dichloromethane (90:10) ratio.
9
Chromatographic separation of analytes was performed on YMC pack ODS AM (150 mm ×
10
4.6 mm, 5 µ m) column under gradient conditions with acetonitrile: 0.1% formic acid buffer
11
as the mobile phases at a flow rate of 1 ml/min. Precursor ion and product ion transition for
12
analytes and IS were monitored on a triple quadrupole mass spectrometer, operated in the
13
selective reaction monitoring with positive ionization mode. Method was validated over a
14
concentration range of 0.72-183.20 ng/ml for ibrutinib, 0.76-194.33 ng/ml for PCI-45227 and
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1.87-479.16 ng/ml for lenalidomide. Mean extraction recovery for ibrutinib, PCI-45227,
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lenalidomide and IS of 75.2%, 84.5%, 97.3% and 92.3% were consistent across low,
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medium, and high QC levels. Precision and accuracy at low, medium and high quality control
18
levels were less than 15% across analytes. Bench top, wet, freeze-thaw and long term
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stability was evaluated for all the analytes. The analytical method was applied to support a
20
pharmacokinetic study of simultaneous estimation of lenalidomide, ibrutinib, and its active
21
metabolite PCI-45227 in Wistar rat. Assay reproducibility was demonstrated by re-analysis of
22
18 incurred samples.
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Key words: ibrutinib, PCI-45227, lenalidomide, Plasma, LC-MS/MS
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1. Introduction
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The B-cell receptor (BCR) pathway dictates the proliferation, survival, apoptosis, and other
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phenomena required for normal B-cell functioning [1]. Several B-cell malignancies such as
4
chronic lymphocytic leukemia (CLL), Mantle Cell Lymphoma (MCL), and Non-Hodgkin’s
5
Lymphoma (NHL) are often associated with dysregulated BCR signalling [2-3]. Historically,
6
treatment of B-cell malignancies has involved the use of chemotherapeutic agents
7
(fludaribine, cyclophosphamide, chlorambucil, doxorubicin, vincristine, prednisolone, and
8
rituximab) in different permutations and combinations in a bid to increase patient survival [4-
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7] Because chemotherapeutic drugs work by killing dividing cells irrespective of their health
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status, prolonged and indiscriminate use of these agents could potentially precipitate the
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disease condition thereby reducing the quality of life. While lenalidomide is a promising
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therapy for haematological malignancies concerns remain with respect to its usage given the
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tumor flare induction and associated cytokine release [8]. Addition of a targeted agent to
14
lenalidomide is therefore desirable to negate the deleterious effects besides reducing the
15
dosage and duration of chemotherapeutic treatment.
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Within the Tec family of kinases , the Burton Tyrosine Kinase (BTK) lies downstream to the
17
BCR and dictates B-cell functioning including the regulation of Akt [9] extracellular signal–
18
regulated kinase [10], and nuclear factor kappa light chain enhancer of activated B cells [11]
19
pathways. In addition, BTK is essential to chemokine-mediated homingand adhesion of B
20
cells. ibrutinib (PCI-32765) is potent BTK inhibitor that binds to the active site of the kinase
21
in an irreversible manner [12]. Data from clinical trials in patients affected with a variety of
22
B-cell malignancies including MCL, follicular lymphoma, and CLL demonstrated significant
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activity with durable remissions for ibrutinib [13-15]. Ibrutinib is metabolized primarily by
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cytochrome P450, CYP3A, and to a minor extent by CYP2D6. The active metabolite, PCI-
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45227, is a dihydrodiol metabolite with inhibitory activity towards BTK approximately 15
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times lower than that of ibrutinib. A Phase 1 clinical trial evaluating the effect of the
3
combination of ibrutinib and lenalidomide in CLL is currently ongoing (NCT01886859).
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Given the importance of BTK in B-cell functions inclusive of chemokine-mediated homing
5
and adhesion, combination of lenalidomide with ibrutinib serves as an attractive therapeutic
6
strategy in B-cell malignancies.
7
To best of our knowledge, no published LC-MS/MS and HPLC based methods are reported
8
for the quantification of ibrutinib and its active metabolite PCI-45227 in plasma. Methods for
9
the determination of lenalidomide in biological fluids by HPLC-UV or LC-MS/MS have been
10
reported [16-20]. However, reports describing a LC-MS/MS- based method for simultaneous
11
determination of lenalidomide, ibrutinib, and its metabolite PCI-45227 in plasma are not
12
available. Simultaneous detection of lenalidomide, ibrutinib and its metabolite PCI-45227 in
13
plasma would help establishing a pharmacokinetic and pharmacodynamic co-relation in
14
animal models that require administration of both drugs to achieve maximal efficacy. In the
15
current article, we describe a highly sensitive, selective, and rapid LC-MS/MS method that
16
was developed and fully validated for simultaneous estimation of lenalidomide, ibrutinib, and
17
its metabolite PCI-45227 in rat plasma. This method offers a small turnaround time for
18
analysis and utilizes only 50 µl rat plasma for sample processing using liquid-liquid
19
extraction. Translation of this methodology to pharmacokinetic studies is also demonstrated
20
by re-analysis of incurred sample.
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2. Experimental
22
2.1. Chemicals and reagents
23
Ibrutinib and lenalidomide were obtained from Selleckchem, Houston, TX. Ammonium
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acetate and tolbutamide was obtained from sigma-aldrich Germany. PCI-45227 was obtained
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from Incozen Therapeutics Pvt Ltd, Hyderabad, India. Methanol and Acetonitrile (HPLC
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gradient grade) were procured from RCI Lab Scan, Thailand. Ethyl acetate and
3
Dichloromethane (HPLC gradient grade) were procured from Merck specialities Pvt Ltd
4
Mumbai, India. Ultra pure water of 18 MOhms*cm was obtained from Milli-Q purification
5
system, Millipore, MA, USA.
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2.2. Liquid chromatographic and mass spectrometric conditions
9
HT system (Shimadzu Corporation, Japan). Separation of analytes and IS was performed on
10
YMC Pack ODS AM (150 mm × 4.6 mm, 5 µm) analytical column (YMC®-PACK,
11
JAPAN), maintained at 40°C in a column oven (CTO-10ASVP). Five microliters of each
12
sample were loaded on the column, separated, and eluted using a gradient mobile phase
13
consisting of acetonitrile (A): 0.1% formic acid buffer (B); (minutes, % mobile phase A): (0,
14
25), (2.0, 90), (5.0, 90), (5.2, 25), (7.5, 25). For gradient elution, the flow rate of the mobile
15
phase was kept at 1.0 ml/min with 70% flow split after post column elution. Flow was
16
directed to the ion spray interface. Autosampler (SIL20ACHT) temperature was maintained at
17
10°C. Mass spectrometric detection of analytes and IS was carried out on a triple quadrupole
18
mass spectrometer (Thermo Scientific - Finnigan TSQ Quantum Ultra, San Jose, CA, USA),
19
equipped with a heated electrospray ionization and operated in a positive ionization mode.
20
All analyses were carried out in positive ionization mode with HESI probe. HESI and
21
capillary temperature was set at 350°C, and the ion spray needle voltage was adjusted to
22
4500V. The transitions monitored were m/z 441.18/138.08, 475.20/304.07, 260.08/149.09,
23
and 271.10/91.12 for ibrutinib, PCI-45227, lenalidomide and tolbutamide respectively
24
(Fig.1.). Sheath gas and auxiliary gas were set at 40 kPa respectively. Skimmer offset was set
25
at 0V. Argon gas collision induced dissociation was used at a pressure of 1.5 mTorr.
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Reverse phase chromatographic analysis of analytes was achieved on a Shimadzu SIL-20 AC
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Collision energy (V) was set at 27, 31, 17 and 34 ibrutinib, PCI-45227, lenalidomide and
2
tolbutamide. Tube lens (eV) was set at 94, 122, 83 and 83 ibrutinib, PCI-45227, lenalidomide
3
and tolbutamide.
4
Selective Reaction Monitoring (SRM) mode was used for data acquisition. Peak integration
5
and calibration were carried out using LC Quan 2.5.2 software (Thermo- Scientific).
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2.3. Calibration standard and quality control samples
9
accurately and transferred into a 10 ml volumetric flask. Analytes were dissolved in 5 ml of
10
methanol and volume was made up to 10 ml with methanol. Final concentration of ibrutinib,
11
PCI-45227, lenalidomide and tolbutamide were 305.33 µg/ml, 239.58 µg/ml, 323.88 µg/ml
12
and 1.10 mg/ml. From the stock solution, 150 µl of ibrutinib, 150 µl of PCI-45227 and 500 µl
13
of lenalidomide were transferred into a 10 ml volumetric flask and volume was made up to 10
14
ml with methanol: water (50:50, v/v). The final mixed intermediate working standard
15
concentration of ibrutinib, PCI-45227 and lenalidomide were 4.58 µg/ml, 4.86 µg/ml and
16
11.98 µg/ml. From the mixed intermediate stock solution, analytical working standards were
17
prepared with methanol: water (50:50, v/v) by serial dilutions.
18
Calibration standards (CSs) and quality control (QC) samples were made by spiking blank
19
plasma with appropriate volumes of working solutions. Final calibration standard
20
concentrations for ibrutinib/ PCI-45227/ lenalidomide were 0.72/0.76/1.87, 1.43/1.52/3.74,
21
2.86/3.04/7.49, 5.72/6.07/14.97, 11.45/12.15/29.95, 22.90/24.29/59.90, 45.80/48.58/119.79,
22
91.60/97.16/239.58, and 183.20/194.33/479.16 ng/ml respectively. The QC samples were
23
prepared at four concentration levels; 137.40/145.75/359.37 ng/ml (HQC, high quality
24
control), 68.70/72.87/179.69 ng/ml (MQC, medium quality control), 2.15/2.28/5.62 ng/ml
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(LQC, low quality control) and 0.72/0.76/1.87 ng/ml (LLOQ QC, lower limit of
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Ibrutinib, PCI-45227, lenalidomide and tolbutamide working standards were weighed
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quantification quality control), for the ibrutinib/ PCI-45227/ lenalidomide combination.
2
tolbutamide (IS) stock solution was diluted with methanol: water (50:50) to achieve a final
3
concentration of 2000 ng/ml. Standard stock and working solutions were stored at 2-8°C until
4
further use.
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2.4. Extraction procedure
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microliters of tolbutamide (2µg/ml) and 750 µl of extraction solvent (ethyl acetate:
9
dichloromethane 90:10) were added to an aliquot of 50 µl plasma and sample was vortexed
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Analytes were extracted from rat plasma by liquid-liquid extraction. Briefly, Twenty five
for 3 min. After centrifugation of the sample at 12,000 rpm at 4°C for 5 min, supernatant was
11
removed and evaporated under nitrogen stream at 50°C for an additional 7 min (nitrogen
12
evaporator, Caliper Instruments USA). Residue was reconstituted with 150 µl (methanol:
13
water 90:10) and 5 µl was injected in the chromatographic system.
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2.5. Validation procedures
17
mixture of analytes and IS at the start of each batch. System performance was assessed by
18
injecting one extracted blank (without analytes and IS) and one extracted LLOQ sample with
19
IS at the beginning of each analytical batch. Autosampler carryover was evaluated by
20
sequentially injecting extracted blank plasma → ULOQ sample → two extracted blank
21
plasma sample → LLOQ sample → extracted blank plasma at the start and end of each batch.
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Selectivity of the method was assessed for potential matrix interferences in six batches of
23
blank rat plasma by extraction and inspection of the resulting chromatograms for interfering
24
peaks. Cross- talk of selective reaction monitoring for analytes and IS was checked using
25
highest standard on calibration curve and working solution of IS. Nine non-zero
26
concentrations were used to determine linearity. A quadratic, 1/x 2, least-squares regression
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System suitability was determined by injecting six consecutive samples of aqueous standard
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algorithm was used to plot the peak area ratio (analyte/ IS) from selective reaction monitoring
2
versus concentration. Linear equations were used to calculate the predicted concentrations in
3
all samples within the analytical runs. Correlation coefficient for each calibration curve was
4
set at ≥0.998 for all analytes. Re-injection reproducibility for extracted samples was checked
5
by injection of an entire analytical run after storage at 10°C. Intra-day accuracy and precision
6
were evaluated by replicate analysis of plasma samples on the same day. The analytical run
7
consisted of a calibration curve and four replicates of LLOQ, LQC, MQC and HQC samples.
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Inter-day accuracy and precision were assessed by analysis of three precision and accuracy
9
batches on three consecutive validation days. Precision (% CV) at each concentration level
10
from the nominal concentration was set at < 15%. Similarly, values for the mean accuracy
11
were set at 85-115%, except for the LLOQ where the allowed range was 80-120% of the
12
nominal concentration. Stability results in plasma were evaluated by measuring the area ratio
13
response (analyte/IS) of stability samples against freshly prepared comparison standards with
14
identical concentration. Auto sampler (wet extract), bench top (at room temperature), freeze–
15
thaw (at -70°C) and long term stability (at -70°C) was performed at LQC and HQC level
16
using four replicates. Stability data were acceptable if the % CV of the replicate
17
determinations did not exceed 15% and the mean accuracy value was within ±15% of the
18
nominal value. To demonstrate the dilution integrity of analyte, From the stock solution, 15
19
µl of ibrutinib, 15 µl of PCI-45227 and 50 µl of lenalidomide were transferred into a 1 ml
20
micro centrifuge tube and volume was made up to 1 ml with rat blank plasma. From this
21
working solution, 131 µl of ibrutinib, 123.5 µl of PCI-45227 and 83.5 µl of lenalidomide
22
were transferred into a 1 ml micro centrifuge tube and volume was made up to 1 ml with rat
23
blank plasma. The final mixed concentration of ibrutinib, PCI-45227 and lenalidomide were
24
600 ng/ml, 600 ng/ml and 1000 ng/ml. From this a pre-determined aliquot was diluted with
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rat plasma (1:4 and 1:8) and analyzed.
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2.6. In vivo pharmacokinetic study:
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Healthy 6-8 weeks male Wistar rats weighing 180 ± 30 g were obtained from Mahaveera
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Enterprises, Hyderabad and housed at Incozen Therapeutics Pvt. Ltd., Hyderabad in
4
appropriate cages. Animals were maintained under standard laboratory conditions with
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regular 12 h day-night cycle in well-ventilated rooms with an average temperature of 24-
6
27°C and relative humidity of 40-60%. Standard pellet laboratory chow diet (Provimi Animal
7
Nutrition India Pvt. Ltd., Bengaluru, India) and water were allowed ad libitum to rats. Study
8
protocol was approved by the Institutional Animal Ethics and Care Committee at Incozen. All
9
applicable national and international ethical guidelines for maintenance and experimental
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studies with Wistar rats were followed.
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Oral formulations were prepared in suspension form by triturating an accurately weighed
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amount of powdered compound in methyl cellulose (0.5%, w/v water) in a gravimetric
13
dilution pattern. Oral doses of 10 and 5 mg/kg for ibrutinib and lenalidomide were
14
administered using a gavage needle at 10 ml/kg to rats after an overnight fast (12 hr). Feed
15
was offered 4 h after dosing. Blood samples (0.15 ml) were collected from retro-orbital sinus
16
at Predose, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h post dose in K2EDTA (di-potassium ethylene di
17
amine tetra acetic acid) tubes and were kept on ice till further processing. Plasma was
18
separated by centrifugation at 4°C for 10 min at 4000 rpm and stored at -70°C till further
19
analysis.
20
Pharmacokinetic parameters such as the maximum plasma concentration (Cmax), area under
21
the concentration-time curve (AUC), time to reach the maximum concentration (Tmax), half-
22
life (t1/2) and elimination constant (Kel) were estimated by means of a non-compartmental
23
analysis using Phoenix WinNonlin (Pharsight Inc., USA, version 6.1). Statistical parameters
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like mean, standard deviation and % C.V were calculated by using MS-Excel 2007
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(Microsoft®).
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3. Results and discussion
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To determine the most sensitive ionization mode for the components studied, ESI positive
7
and negative were tested with various combinations of components of the mobile phase, i.e.
8
methanol/acetonitrile and water / 2 mM ammonium acetate buffer / 0.1% formic acid. Signal
9
intensity for [M+H]+ ions in ESI positive ion mode were 4-10-fold higher for all components
10
analyzed using acetonitrile: 0.1% formic acid buffer compared to experiments run with ESI
11
negative ion mode. Precursor and product ions were optimized by infusing 250 ng/ml
12
solutions in the mass spectrometer between m/z 100-500 range. The Q1 MS full scan spectra
13
for the analytes and IS predominantly contained protonated precursor [M+H]+ ions at m/z
14
260.08, 441.18, 475.20, and 271.10 for lenalidomide, ibrutinib, PCI-45227 and tolbutamide
15
respectively. The most abundant and consistent product ions in product ion spectra were
16
observed at m/z 149.09, 138.08, 304.07, and 91.12 for lenalidomide, ibrutinib, PCI-45227 and
17
tolbutamide by applying 17, 27, 31, and 34 eV of collision energy respectively (Fig. 1).
18 19 20
3.2 Liquid chromatography
21
sensitivity, better peak shape, and resolution. 0.1% formic acid buffer was required to achieve
22
acceptable peak width, shapes, and acceptable ionisation. Samples were run using a reverse
23
phase C18 column (150 mm×4.6 mm i.d., 5 µm) (YMC-PACK®, Japan) with 0.1% formic
24
acid: acetonitrile in a gradient mode. All components eluted between 2.2-5.0 min.
25
Representative chromatograms of extracted blank rat plasma, blank plasma with analytical
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3.1 Mass spectrometry
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Page 12 of 30
standards and IS and rat plasma sample 1 hr after single dose administration are shown in
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Fig. 2.
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3.3 Calibration standard accuracy and precision, LLOQ and LOD
4
The three calibration curves were linear over the concentration range of 0.72-183.20 ng/ml
5
for ibrutinib, 0.72-183.20 ng/ml for PCI-45227 and 1.87-479.16 ng/ml for lenalidomide, with
6
a correlation coefficient (r2) ≥ 0.9981 for the analytes. Mean linear equations obtained for
7
ibrutinib, PCI-45227 and lenalidomide were y = (0.00688± 0.0007) x + (0.0006 ± 0.0002), y
8
= (0.00743 ± 0.00016) x + (-0.00049 ± 0.00032) and y = (0.00338 ± 0.00005) x + (-0.00110
9
± 0.00073) respectively. Accuracy and precision (% CV) for the calibration curve standards
10
ranged from 92.5 to 111.5% and 0.1 to 1.3% for ibrutinib, 98.3 to 104.7% and 0.5 to 3.8% for
11
PCI-45227, 92.6 to 104.3% and 0.2 to 6.1% for lenalidomide respectively. The lower limit of
12
quantitation (S/N ≥ 20) and limit of detection (LOD, S/N ≥ 5) were 0.72 ng/ml and 0.25
13
ng/ml for ibrutinib, 0.76 ng/ml and 0.25 ng/ml for PCI-45227, and 1.87 ng/ml and 0.50 ng/ml
14
for lenalidomide respectively.
15
3.4 Intra- and inter-batch accuracy and precision
16
Intra-batch and inter-batch precision and accuracy were established from validation runs
17
performed at four QC levels (Table 1). The within-batch precision (% CV) ranged from 3.3 to
18
11.6 for ibrutinib, 2.7 to 8.5 for PCI-45227, 6.7 to 9.1 for lenalidomide while the accuracy
19
was within 96.0-105.7% for ibrutinib, 95.7-103.1% for PCI-45227 and 99.1-100.7% for
20
lenalidomide. Similarly, for the between-batch experiments, the precision varied from 0.3 to
21
2.1 for ibrutinib, 0.3 to 4.0 for PCI-45227 and 0.2 to 2.2 for lenalidomide while the accuracy
22
was within 95.5-103.5% for ibrutinib, 97.1-102.7% for PCI-45227, and 99.5-103.0% for
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lenalidomide.
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3.5 Stability results and dilution reliability
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Stock solutions of analytes and IS for short term were stable at room temperature up to 6 h
3
respectively. Analytes in control rat plasma (bench top) were stable for 6 h at 25°C. Extracted
4
quality control samples were stable up to 24 h at 10°C. Long term stability of the spiked
5
quality control samples was unaffected up to 30 days at -70°C. Detailed results for stability
6
experiments are presented in Table 2. Precision (% CV) values for reliability of 1/4 and 1/8th
7
dilution were between 1.2 and 3.5%, while the accuracy results were within 97.6-103.5% for
8
ibrutinib, PCI-45227 and lenalidomide respectively Results are within the acceptance limit of
9
15% for precision (% CV) and 85-115% for accuracy as shown in Table 3.
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3.5 Recovery & matrix effect
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Recovery of ibrutinib, PCI-45227 and lenalidomide from plasma was estimated at their
12
respective low, medium, and high quality control levels. Plasma samples (in quadruplets)
13
containing all analytes at low, medium and high quality control concentrations were also
14
spiked with respective internal standards. Results comparing the peak responses of the post-
15
extraction, spiked samples, with those of the pure standards prepared in methanol: water
16
(90:10, v/v) for low, medium, and high quality control levels, indicated that the ratios of the
17
peak responses were within acceptable limits. Absolute recoveries ranged from 73.2 to
18
77.5%, 81.0 to 87.5%, and 95.6 to 98.8%, for ibrutinib, PCI-45227 and lenalidomide
19
respectively (Table 4). The recovery of IS was 92.3%. Matrix effect was determined by
20
comparing analyte and internal standard area ratios of the extracted low and high QC in
21
matrix with the analyte and internal standard area ratio obtained from the neat solution
22
prepared at similar concentration levels (Table 5). Percent CV of the area ratios at low,
23
medium, and high quality control levels were lesser than 15% across the analytes.
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3.6 Application of the method in pharmacokinetic study and incurred sample analysis
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time profile of ibrutinib, PCI-45227 and lenalidomide in plasma following oral administration
5
of ibrutinib and lenalidomide.
6
Absorption was rapid and with maximum plasma concentrations of 0.70, 1.06 µg/ml at 0.5,
7
and 1.0 h after oral administration of ibrutinib, and lenalidomide. Formation of PCI-45227
8
was rapid with maximum plasma concentration of 0.32 µg/ml at 1.0 h after oral
9
administration of ibrutinib, and lenalidomide. Absorbed ibrutinib, PCI-45227 and
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The described analytical method was applied to generate the plasma concentration versus
lenalidomide were eliminated with a half life of 1.43, 2.25, and 1.50 h respectively. Area
11
under plasma curve (AUC0-24) was 1.42, 1.20 and 2.38 µg.h/ml for ibrutinib, PCI-45227 and
12
lenalidomide. Plasma concentrations were observed up to 24.0 hr for ibrutinib, PCI-45227
13
and lenalidomide after oral administration of the combination (Fig. 3).
14
In the current study, ISR was performed on 18 plasma samples from six different rats at
15
Cmin, Cmax, and the time point covering the phase of elimination. As per the acceptance
16
criterion, at least two-thirds of the original results and repeat results should be within 20%
17
(Table 6). Pharmacokinetic parameters of ibrutinib, PCI-45227 and lenalidomide are
18
presented in Table 7. Data demonstrated the adaptability and successful translation of the
19
validated analytical method for estimation of ibrutinib, PCI-45227 and lenalidomide to an in
20
vivo setting.
21 22 23
3.7 Comparison with reported methods
24
detection and a few LC-MS/MS based assays are also reported [16-20]. Published methods
25
indicate longer run times with a high plasma volume requirement besides issues with low
26
sensitivity [16-20]. The aim of present investigation was to develop and validate a simple
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Available methods for estimation of lenalidomide are based on the use of HPLC-UV
13
Page 15 of 30
LC-MS/MS method using a gradient mode with sufficient accuracy and precision for
2
simultaneous estimation of ibrutinib, PCI-45227 and lenalidomide and its subsequent use in
3
pharmacokinetic studies in rats. The present method involves liquid-liquid extraction
4
procedure with good sensitivity and a gradient reverse-phase LC-MS/MS analysis for all
5
analytes of interest. This method is specific for ibrutinib, PCI-45227 and lenalidomide with
6
good linearity, accuracy and precision. This method involves 50 µl plasma followed by a
7
single step liquid-liquid extraction procedure. Furthermore, this extraction procedure
8
decreases both the cost and duration of the assay. The chromatographic conditions of this
9
method were optimized for a 7.5 min run time on LC-MS/MS.
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1
4. Conclusion
13
lenalidomide in rat plasma was fully validated as per USFDA guidelines [21]. The proposed
14
method has a much higher sensitivity for the analytes compared to other reported methods
15
either as a single analyte or with combination in different biological matrices. Efficiency of
16
liquid-liquid extraction and chromatographic run time of 7.5 min per sample renders the
17
method useful in high-throughput bioanalysis. Absence of matrix interference is effectively
18
shown by the precision (% CV) values for the calculated slopes of calibration curves in
19
different plasma sources. The validated method showed acceptable data for all the validation
20
parameters, with adequate sensitivity and selectivity for their simultaneous quantification in a
21
clinical setting. Moreover, this is the first combination method for estimation of ibrutinib,
22
PCI-45227 and lenalidomide in rat plasma. Further, incurred sample reanalysis of 18 samples
23
authenticates the reproducibility of the proposed method.
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The developed LC-MS/MS method for the quantitation of ibrutinib, PCI-45227 and
24 25 14
Page 16 of 30
Acknowledgement
2
Financial support for this research was provided by Incozen Therapeutics Pvt Ltd,
3
Hyderabad, India. Authors would like to thank management of Incozen Therapeutics Pvt Ltd
4
for extending the research facility and providing the necessary resources including reagents
5
and instruments access in this project.
6 7 8
References:
9
Reviews Immunology, 2 (2002) 945-956.
ip t
1
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cr
[1] H. Niiro, E.A. Clark, Regulation of B-cell fate by antigen-receptor signals, Nature
[2] M. Gururajan, C.D. Jennings, S. Bondada, Cutting edge: constitutive B cell receptor
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multiple downstream signaling pathways via PLC Gamma 2 in B cells, BMC Immunology, 2
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[11] J.B. Petro, S.J. Rahman, D.W. Ballard, W.N. Khan, Bruton's tyrosine kinase is required
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engagement, The Journal of Experimental Medicine, 191 (2000) 1745-1754.
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[12] L.A. Honigberg, A.M. Smith, M. Sirisawad, E. Verner, D. Loury, B. Chang, S. Li, Z.
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Pan, D.H. Thamm, R.A. Miller, J.J. Buggy, The Bruton tyrosine kinase inhibitor PCI-32765
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blocks B-cell activation and is efficacious in models of autoimmune disease and B-cell
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[15] C. Harrison, Nature Reviews Drug Discovery, Trial watch: BTK inhibitor shows
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[16] M.S. Hasnain, S. Rao, M.K. Singh, N. Vig, A. Gupta, A. Ansari, P. Sen, P. Joshi, S.A.
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Ansari, Development and validation of LC-MS/MS method for the quantitation of
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lenalidomide in human plasma using Box-Behnken experimental design, Analyst, 138 (2013)
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1581-1588.
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[17] N.Y. Khalil, I.A. Darwish, T.A. Wani, A.R.A. Al-Majed, Trace determination of
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lenalidomide in plasma by non-extractive HPLC procedures with fluorescence detection after
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pre-column derivatization with fluorescamine, Chemistry Central Journal, 7 (2013) 52-57.
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[18] Q. Liu, K.L. Farley, A.J. Johnson, N. Muthusamy, C.C. Hofmeister, K.A. Blum, L.J.
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Schaaf, M.R. Grever, J.C. Byrd, J.T. Dalton, M.A. Phelps, Development and validation of a
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highly sensitive liquid chromatography/mass spectrometry method for simultaneous
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quantification of lenalidomide and flavopiridol in human plasma, Therapeutic Drug
18
Monitoring, 30 (2008) 620-627.
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[19] J.A. Muscal, Y. Sun, J.G. Nuchtern, R.C. Dauser, L.H. McGuffey, B.W. Gibson, S.L.
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Berg, Cancer Chemotherapy and Pharmacology, Plasma and cerebrospinal fluid
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pharmacokinetics of thalidomide and lenalidomide in nonhuman primates, 69 (2012) 943-
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947.
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[20] S. Veeraraghavan, S. Thappali, S. Viswanadha, S. Nalla, S. Chennupati, M. Golla, S.
24
Vakkalanka, M. Rangasamy, Simultaneous quantification of idelalisib, fludarabine and
25
lenalidomide in rat plasma by using high-performance liquid chromatography coupled with
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Page 19 of 30
heated electrospray ionization tandem mass spectrometry, J. Chromatogr. B, Analyt Technol
2
Biomed Life Sci. 949-950 (2014) 63-69.
3
[21] Guidance for Industry, Bioanalytical Method Validation, US Department of
4
Health and Human Services, Food and Drug Administration Centre for Drug
5
Evaluation and Research (CDER) May 2001.
6
Figure Legend
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1
Fig.1. Product ion chromatogram of (a) ibrutinib (b) PCI-45227 (c) lenalidomide (d) tolbutamide (internal standard)
13 14
Fig.3. Time verses plasma concentration plot after single dose oral administration of ibrutinib, and lenalidomide in six wistar rats.
cr
7 8 9 10 11 12
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Fig.2. Chromatograms of (a) lenalidomide (b) PCI-45227 (c) tolbutamide (internal standard) (d) ibrutinib for (I) blank plasma sample (II) extracted rat plasma (III) extracted rat plasma at 1 hr after single dose of 10mg/kg of ibrutinib and 5mg/kg of lenalidomide
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Page 20 of 30
cr
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Table 1
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Table 1: summary of precision and accuracy from QC samples in wistar rat plasma
Within batch (n=4)
105.7
11.6
2.2
2.1±0.1
96.0
5.0
68.7
66.9±2.2
97.4
135.1±5.3
0.8
% Accuracy
% C.V 2.1
2.1±0.0
95.5
0.5
3.3
67.1±0.2
97.7
0.3
98.4
3.9
132.8±2.3
96.6
1.8
0.8±0.1
103.1
8.3
0.8±0.0
102.7
0.3
2.3
2.2±0.2
97.7
8.5
2.2±0.0
98.1
0.5
72.9
70.0±4.7
96.0
6.8
70.8±0.8
97.1
1.1
145.8
139.5±3.8
95.7
2.7
145.3±5.8
99.7
4.0
1.9
1.9±0.1
99.7
6.7
1.9±0.0
99.9
0.2
5.6
5.6±0.5
99.1
9.1
5.6±0.0
99.5
0.4
179.7
178.1±12.9
99.1
7.2
181.7±3.6
101.1
2.0
359.4
362.0±31.1
100.7
8.6
370.3±8.3
103.0
2.2
ep te
Ac c
Lenalidomide
% C.V
Measured concentration (ng/ml) (mean ± SD) 0.8±0.0
%
103.5
137.4
PCI-45227
% Accuracy
an
0.7
Measured concentration (ng/ml) (mean ± SD) 0.8±0.1
M
Ibrutinib
Spiked concentration (ng/ml)
d
Drug
Between batch (n=3)
Page 21 of 30
cr
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Table 2
Nominal
Sample condition
Bench top stability a % % Accuracy CV
concentration
30 days storage stability d % Accuracy
% CV
2.2
102.2
8.4
109.2
11.1
105.1
7.3
97.4
12.0
137.4
96.6
5.2
100.1
4.2
96.3
4.6
93.1
3.8
2.3
96.8
5.9
98.9
5.3
97.9
4.2
104.5
8.4
101.3
7.8
103.9
6.2
93.6
7.6
102.8
6.8
101.6
8.0
95.0
6.6
102.3
10.2
103.9
7.6
95.7
2.0
98.2
1.3
94.1
3.9
106.8
1.9
Ibrutinib
PCI-45227
ep te
145.8 5.6
359.4
b
c
Exposed at ambient temperature (25°C) for 6h, Kept at autosampler temperature (10°C) for 24h, After three freeze-thaw cycles, d Stored at -70°C
Ac c
a
Freeze-thaw stability c % % Accuracy CV
d
M
(ng/ml)
Lenalidomide
Autosampler stability b % % Accuracy CV
an
Drug
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Table 2. Stability in rat plasma (n=4)
Page 22 of 30
cr
ip t
Table 3
ibrutinib 600 ng.ml-1 1/4 dilution
1/8 dilution
( ng.ml-1)
150
75
Mean ± SD ( n=4)
148.2 ± 11.8
( ng.ml )
1/4 dilution
1/8 dilution
150
75
250
125
155.2 ± 7.9
77.5 ± 3.9
252.8 ± 9.9
128.1 ± 3.9
97.60 2.3
103.46 1.5
103.33 3.5
101.12 1.2
102.48 2.5
M
1/8 dilution
73.2 ± 4.8
98.80 1.6
Ac c
% Nominal % CV
lenalidomide 1000 ng.ml-1
1/4 dilution
ep te
-1
PCI-45227 600 ng.ml-1
d
Dilution concentration
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Table 3. Dilution integrity evaluation of ibrutinib, PCI-45227 and lenalidomide in wistar rat plasma
Page 23 of 30
Table 4
Table 4: Extraction recovery in rat plasma (n=4)
PCI-45227
% C.V
77.5 75.0 73.2 87.5 85.0 81.0 97.5 95.6 98.8
3.5 2.5 1.2 1.5 2.5 1.2 5.7 3.0 3.0
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Lenalidomide
Recovery (%)
ip t
Ibrutinib
Concentration (ng/ml) 2.15 68.70 137.40 2.28 72.87 145.75 5.62 179.69 359.37
cr
Drug
Page 24 of 30
Table 5
Table 5: Matrix effect in rat plasma (n=4)
Ibrutinib PCI-45227
2.15 137.40 2.28 145.75 5.62 359.37
Average Post spiked area ratio 0.023 0.725 0.036 1.112 0.024 1.257
Average aqueous standard area ratio
% C.V
0.024 0.750 0.038 1.134 0.025 1.298
4.5 3.4 5.6 2.2 4.9 1.3
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Lenalidomide
Conc (ng/ml)
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Drug
Page 25 of 30
Table 6
Table 6. Incurred sample reanalysis of ibrutinib, PCI-45227, and lenalidomide.
PCI-45227
lenalidomide
No. of total samples taken for ISR
18
18
18
No. of samples meeting the acceptance criteria (i.e., % difference between original and reanalyzed value must be within 20%)
16
17
17
% of samples meeting the acceptance criteria
88.9%
94.4%
ip t
ibrutinib
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94.4%
Page 26 of 30
Table 7
Table 7: Pharmacokinetic parameters (Mean ± S.D.) after single dose oral administration of ibrutinib and lenalidomide simultaneously in wistar rats Units
ibrutinib
PCI-45227
lenalidomide
C max AUC 0-24 AUC 0-inf T max t½ K el
µg/ml µg.h/ml µg.h/ml h h h -1
0.70±0.16 1.42±0.22 1.44±0.22 0.50±0.13 1.43±1.35 0.49±0.30
0.32±0.11 1.20±0.11 1.25±0.12 1.00±0.00 2.25±2.11 0.31±0.15
1.06±0.43 2.38±0.20 2.40±0.20 0.50±0.00 1.50±1.43 0.46±0.22
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Parameters
Page 27 of 30
Ac ce p
te
d
M
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Fig 1
Page 28 of 30
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Fig 1
Page 29 of 30
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Fig 3
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S0731-7085(14)00577-9 http://dx.doi.org/doi:10.1016/j.jpba.2014.11.041 PBA 9828
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Revised date: Accepted date:
26-9-2014 19-11-2014 23-11-2014
Please cite this article as: S. Veeraraghavan, S. Viswanadha, S. Thappali, B. Govindarajulu, S. Vakkalanka, M. Rangasamy, Simultaneous quantification of lenalidomide, ibrutinib and its active metabolite PCI-45227 in rat plasma by LCMS/MS: application to a pharmacokinetic study, Journal of Pharmaceutical and Biomedical Analysis (2014), http://dx.doi.org/10.1016/j.jpba.2014.11.041 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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*Graphical Abstract
Page 1 of 30
*Highlights (for review)
1. Highly selective method for the determination of ibrutinib, PCI-45227 and lenalidomide in plasma 2. This is the first LC-MS/MS- based method for simultaneous determination of ibrutinib, PCI-45227 and lenalidomide in plasma
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3. Plasma sample clean-up involves a simple liquid-liquid extraction procedure
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4. The developed LC-MS/MS based method was applied successfully for pharmacokinetic study
Page 2 of 30
Simultaneous quantification of lenalidomide, ibrutinib and its active metabolite PCI-45227 in rat plasma by LC-MS/MS: application to a pharmacokinetic study
4
Sridhar Veeraraghavana,c, Srikant Viswanadhaa, Satheeshmanikandan Thappalia , Babu
5
Govindarajulua, Swaroopkumar Vakkalankaa, Manivannan Rangasamyb,
6
Professional affiliations:
7
a
8
Incozen Therapeutics Private Limited,
9
450, Alexandira Knowledge Park, Turkapplly
10
Hyderabad - 500078, Andhra Pradesh, India.
11
b
12
Dept of Pharmaceutics
13
Annai JKK Sampoorani Ammal College of Pharmacy
14
Komarapalayam, Namakkal - 638 183, Tamilnadu, India
15
c
16
CRD, PRIST University,
17
Vallam, Thanjavur 613403. Tamilnadu, India
18 19
Current affiliations:
20
Sridhar Veeraraghavan
21
Incozen Therapeutics Private Limited,
22
450, Alexandria Knowledge Park, Turkapplly
23
Hyderabad - 500078, Andhra Pradesh, India.
24
Corresponding author:
25
Tel: +91 40 2348 0336
26
E-mail addresses:
27
[email protected] (Sridhar V)
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1
Page 3 of 30
Abstract:
2
Efficacy assessments using a combination of ibrutinib and lenalidomide necessitate the
3
development of an analytical method for determination of both drugs in plasma with
4
precision. A high performance liquid chromatography-tandem mass spectrometry (LC-
5
MS/MS) method was developed for the simultaneous determination of lenalidomide,
6
ibrutinib, and its active metabolite PCI45227 in rat plasma. Extraction of lenalidomide,
7
ibrutinib, PCI45227 and tolbutamide (internal standard; IS) from 50 µl rat plasma was carried
8
out by liquid-liquid extraction with ethyl acetate: dichloromethane (90:10) ratio.
9
Chromatographic separation of analytes was performed on YMC pack ODS AM (150 mm ×
10
4.6 mm, 5 µ m) column under gradient conditions with acetonitrile: 0.1% formic acid buffer
11
as the mobile phases at a flow rate of 1 ml/min. Precursor ion and product ion transition for
12
analytes and IS were monitored on a triple quadrupole mass spectrometer, operated in the
13
selective reaction monitoring with positive ionization mode. Method was validated over a
14
concentration range of 0.72-183.20 ng/ml for ibrutinib, 0.76-194.33 ng/ml for PCI-45227 and
15
1.87-479.16 ng/ml for lenalidomide. Mean extraction recovery for ibrutinib, PCI-45227,
16
lenalidomide and IS of 75.2%, 84.5%, 97.3% and 92.3% were consistent across low,
17
medium, and high QC levels. Precision and accuracy at low, medium and high quality control
18
levels were less than 15% across analytes. Bench top, wet, freeze-thaw and long term
19
stability was evaluated for all the analytes. The analytical method was applied to support a
20
pharmacokinetic study of simultaneous estimation of lenalidomide, ibrutinib, and its active
21
metabolite PCI-45227 in Wistar rat. Assay reproducibility was demonstrated by re-analysis of
22
18 incurred samples.
23
Key words: ibrutinib, PCI-45227, lenalidomide, Plasma, LC-MS/MS
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1. Introduction
2
The B-cell receptor (BCR) pathway dictates the proliferation, survival, apoptosis, and other
3
phenomena required for normal B-cell functioning [1]. Several B-cell malignancies such as
4
chronic lymphocytic leukemia (CLL), Mantle Cell Lymphoma (MCL), and Non-Hodgkin’s
5
Lymphoma (NHL) are often associated with dysregulated BCR signalling [2-3]. Historically,
6
treatment of B-cell malignancies has involved the use of chemotherapeutic agents
7
(fludaribine, cyclophosphamide, chlorambucil, doxorubicin, vincristine, prednisolone, and
8
rituximab) in different permutations and combinations in a bid to increase patient survival [4-
9
7] Because chemotherapeutic drugs work by killing dividing cells irrespective of their health
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1
status, prolonged and indiscriminate use of these agents could potentially precipitate the
11
disease condition thereby reducing the quality of life. While lenalidomide is a promising
12
therapy for haematological malignancies concerns remain with respect to its usage given the
13
tumor flare induction and associated cytokine release [8]. Addition of a targeted agent to
14
lenalidomide is therefore desirable to negate the deleterious effects besides reducing the
15
dosage and duration of chemotherapeutic treatment.
16
Within the Tec family of kinases , the Burton Tyrosine Kinase (BTK) lies downstream to the
17
BCR and dictates B-cell functioning including the regulation of Akt [9] extracellular signal–
18
regulated kinase [10], and nuclear factor kappa light chain enhancer of activated B cells [11]
19
pathways. In addition, BTK is essential to chemokine-mediated homingand adhesion of B
20
cells. ibrutinib (PCI-32765) is potent BTK inhibitor that binds to the active site of the kinase
21
in an irreversible manner [12]. Data from clinical trials in patients affected with a variety of
22
B-cell malignancies including MCL, follicular lymphoma, and CLL demonstrated significant
23
activity with durable remissions for ibrutinib [13-15]. Ibrutinib is metabolized primarily by
24
cytochrome P450, CYP3A, and to a minor extent by CYP2D6. The active metabolite, PCI-
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Page 5 of 30
45227, is a dihydrodiol metabolite with inhibitory activity towards BTK approximately 15
2
times lower than that of ibrutinib. A Phase 1 clinical trial evaluating the effect of the
3
combination of ibrutinib and lenalidomide in CLL is currently ongoing (NCT01886859).
4
Given the importance of BTK in B-cell functions inclusive of chemokine-mediated homing
5
and adhesion, combination of lenalidomide with ibrutinib serves as an attractive therapeutic
6
strategy in B-cell malignancies.
7
To best of our knowledge, no published LC-MS/MS and HPLC based methods are reported
8
for the quantification of ibrutinib and its active metabolite PCI-45227 in plasma. Methods for
9
the determination of lenalidomide in biological fluids by HPLC-UV or LC-MS/MS have been
10
reported [16-20]. However, reports describing a LC-MS/MS- based method for simultaneous
11
determination of lenalidomide, ibrutinib, and its metabolite PCI-45227 in plasma are not
12
available. Simultaneous detection of lenalidomide, ibrutinib and its metabolite PCI-45227 in
13
plasma would help establishing a pharmacokinetic and pharmacodynamic co-relation in
14
animal models that require administration of both drugs to achieve maximal efficacy. In the
15
current article, we describe a highly sensitive, selective, and rapid LC-MS/MS method that
16
was developed and fully validated for simultaneous estimation of lenalidomide, ibrutinib, and
17
its metabolite PCI-45227 in rat plasma. This method offers a small turnaround time for
18
analysis and utilizes only 50 µl rat plasma for sample processing using liquid-liquid
19
extraction. Translation of this methodology to pharmacokinetic studies is also demonstrated
20
by re-analysis of incurred sample.
21
2. Experimental
22
2.1. Chemicals and reagents
23
Ibrutinib and lenalidomide were obtained from Selleckchem, Houston, TX. Ammonium
24
acetate and tolbutamide was obtained from sigma-aldrich Germany. PCI-45227 was obtained
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Page 6 of 30
from Incozen Therapeutics Pvt Ltd, Hyderabad, India. Methanol and Acetonitrile (HPLC
2
gradient grade) were procured from RCI Lab Scan, Thailand. Ethyl acetate and
3
Dichloromethane (HPLC gradient grade) were procured from Merck specialities Pvt Ltd
4
Mumbai, India. Ultra pure water of 18 MOhms*cm was obtained from Milli-Q purification
5
system, Millipore, MA, USA.
6 7 8
2.2. Liquid chromatographic and mass spectrometric conditions
9
HT system (Shimadzu Corporation, Japan). Separation of analytes and IS was performed on
10
YMC Pack ODS AM (150 mm × 4.6 mm, 5 µm) analytical column (YMC®-PACK,
11
JAPAN), maintained at 40°C in a column oven (CTO-10ASVP). Five microliters of each
12
sample were loaded on the column, separated, and eluted using a gradient mobile phase
13
consisting of acetonitrile (A): 0.1% formic acid buffer (B); (minutes, % mobile phase A): (0,
14
25), (2.0, 90), (5.0, 90), (5.2, 25), (7.5, 25). For gradient elution, the flow rate of the mobile
15
phase was kept at 1.0 ml/min with 70% flow split after post column elution. Flow was
16
directed to the ion spray interface. Autosampler (SIL20ACHT) temperature was maintained at
17
10°C. Mass spectrometric detection of analytes and IS was carried out on a triple quadrupole
18
mass spectrometer (Thermo Scientific - Finnigan TSQ Quantum Ultra, San Jose, CA, USA),
19
equipped with a heated electrospray ionization and operated in a positive ionization mode.
20
All analyses were carried out in positive ionization mode with HESI probe. HESI and
21
capillary temperature was set at 350°C, and the ion spray needle voltage was adjusted to
22
4500V. The transitions monitored were m/z 441.18/138.08, 475.20/304.07, 260.08/149.09,
23
and 271.10/91.12 for ibrutinib, PCI-45227, lenalidomide and tolbutamide respectively
24
(Fig.1.). Sheath gas and auxiliary gas were set at 40 kPa respectively. Skimmer offset was set
25
at 0V. Argon gas collision induced dissociation was used at a pressure of 1.5 mTorr.
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Reverse phase chromatographic analysis of analytes was achieved on a Shimadzu SIL-20 AC
5
Page 7 of 30
Collision energy (V) was set at 27, 31, 17 and 34 ibrutinib, PCI-45227, lenalidomide and
2
tolbutamide. Tube lens (eV) was set at 94, 122, 83 and 83 ibrutinib, PCI-45227, lenalidomide
3
and tolbutamide.
4
Selective Reaction Monitoring (SRM) mode was used for data acquisition. Peak integration
5
and calibration were carried out using LC Quan 2.5.2 software (Thermo- Scientific).
6 7 8
2.3. Calibration standard and quality control samples
9
accurately and transferred into a 10 ml volumetric flask. Analytes were dissolved in 5 ml of
10
methanol and volume was made up to 10 ml with methanol. Final concentration of ibrutinib,
11
PCI-45227, lenalidomide and tolbutamide were 305.33 µg/ml, 239.58 µg/ml, 323.88 µg/ml
12
and 1.10 mg/ml. From the stock solution, 150 µl of ibrutinib, 150 µl of PCI-45227 and 500 µl
13
of lenalidomide were transferred into a 10 ml volumetric flask and volume was made up to 10
14
ml with methanol: water (50:50, v/v). The final mixed intermediate working standard
15
concentration of ibrutinib, PCI-45227 and lenalidomide were 4.58 µg/ml, 4.86 µg/ml and
16
11.98 µg/ml. From the mixed intermediate stock solution, analytical working standards were
17
prepared with methanol: water (50:50, v/v) by serial dilutions.
18
Calibration standards (CSs) and quality control (QC) samples were made by spiking blank
19
plasma with appropriate volumes of working solutions. Final calibration standard
20
concentrations for ibrutinib/ PCI-45227/ lenalidomide were 0.72/0.76/1.87, 1.43/1.52/3.74,
21
2.86/3.04/7.49, 5.72/6.07/14.97, 11.45/12.15/29.95, 22.90/24.29/59.90, 45.80/48.58/119.79,
22
91.60/97.16/239.58, and 183.20/194.33/479.16 ng/ml respectively. The QC samples were
23
prepared at four concentration levels; 137.40/145.75/359.37 ng/ml (HQC, high quality
24
control), 68.70/72.87/179.69 ng/ml (MQC, medium quality control), 2.15/2.28/5.62 ng/ml
25
(LQC, low quality control) and 0.72/0.76/1.87 ng/ml (LLOQ QC, lower limit of
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Ibrutinib, PCI-45227, lenalidomide and tolbutamide working standards were weighed
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Page 8 of 30
quantification quality control), for the ibrutinib/ PCI-45227/ lenalidomide combination.
2
tolbutamide (IS) stock solution was diluted with methanol: water (50:50) to achieve a final
3
concentration of 2000 ng/ml. Standard stock and working solutions were stored at 2-8°C until
4
further use.
5 6 7
2.4. Extraction procedure
8
microliters of tolbutamide (2µg/ml) and 750 µl of extraction solvent (ethyl acetate:
9
dichloromethane 90:10) were added to an aliquot of 50 µl plasma and sample was vortexed
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Analytes were extracted from rat plasma by liquid-liquid extraction. Briefly, Twenty five
for 3 min. After centrifugation of the sample at 12,000 rpm at 4°C for 5 min, supernatant was
11
removed and evaporated under nitrogen stream at 50°C for an additional 7 min (nitrogen
12
evaporator, Caliper Instruments USA). Residue was reconstituted with 150 µl (methanol:
13
water 90:10) and 5 µl was injected in the chromatographic system.
14 15 16
2.5. Validation procedures
17
mixture of analytes and IS at the start of each batch. System performance was assessed by
18
injecting one extracted blank (without analytes and IS) and one extracted LLOQ sample with
19
IS at the beginning of each analytical batch. Autosampler carryover was evaluated by
20
sequentially injecting extracted blank plasma → ULOQ sample → two extracted blank
21
plasma sample → LLOQ sample → extracted blank plasma at the start and end of each batch.
22
Selectivity of the method was assessed for potential matrix interferences in six batches of
23
blank rat plasma by extraction and inspection of the resulting chromatograms for interfering
24
peaks. Cross- talk of selective reaction monitoring for analytes and IS was checked using
25
highest standard on calibration curve and working solution of IS. Nine non-zero
26
concentrations were used to determine linearity. A quadratic, 1/x 2, least-squares regression
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System suitability was determined by injecting six consecutive samples of aqueous standard
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Page 9 of 30
algorithm was used to plot the peak area ratio (analyte/ IS) from selective reaction monitoring
2
versus concentration. Linear equations were used to calculate the predicted concentrations in
3
all samples within the analytical runs. Correlation coefficient for each calibration curve was
4
set at ≥0.998 for all analytes. Re-injection reproducibility for extracted samples was checked
5
by injection of an entire analytical run after storage at 10°C. Intra-day accuracy and precision
6
were evaluated by replicate analysis of plasma samples on the same day. The analytical run
7
consisted of a calibration curve and four replicates of LLOQ, LQC, MQC and HQC samples.
8
Inter-day accuracy and precision were assessed by analysis of three precision and accuracy
9
batches on three consecutive validation days. Precision (% CV) at each concentration level
10
from the nominal concentration was set at < 15%. Similarly, values for the mean accuracy
11
were set at 85-115%, except for the LLOQ where the allowed range was 80-120% of the
12
nominal concentration. Stability results in plasma were evaluated by measuring the area ratio
13
response (analyte/IS) of stability samples against freshly prepared comparison standards with
14
identical concentration. Auto sampler (wet extract), bench top (at room temperature), freeze–
15
thaw (at -70°C) and long term stability (at -70°C) was performed at LQC and HQC level
16
using four replicates. Stability data were acceptable if the % CV of the replicate
17
determinations did not exceed 15% and the mean accuracy value was within ±15% of the
18
nominal value. To demonstrate the dilution integrity of analyte, From the stock solution, 15
19
µl of ibrutinib, 15 µl of PCI-45227 and 50 µl of lenalidomide were transferred into a 1 ml
20
micro centrifuge tube and volume was made up to 1 ml with rat blank plasma. From this
21
working solution, 131 µl of ibrutinib, 123.5 µl of PCI-45227 and 83.5 µl of lenalidomide
22
were transferred into a 1 ml micro centrifuge tube and volume was made up to 1 ml with rat
23
blank plasma. The final mixed concentration of ibrutinib, PCI-45227 and lenalidomide were
24
600 ng/ml, 600 ng/ml and 1000 ng/ml. From this a pre-determined aliquot was diluted with
25
rat plasma (1:4 and 1:8) and analyzed.
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Page 10 of 30
2.6. In vivo pharmacokinetic study:
2
Healthy 6-8 weeks male Wistar rats weighing 180 ± 30 g were obtained from Mahaveera
3
Enterprises, Hyderabad and housed at Incozen Therapeutics Pvt. Ltd., Hyderabad in
4
appropriate cages. Animals were maintained under standard laboratory conditions with
5
regular 12 h day-night cycle in well-ventilated rooms with an average temperature of 24-
6
27°C and relative humidity of 40-60%. Standard pellet laboratory chow diet (Provimi Animal
7
Nutrition India Pvt. Ltd., Bengaluru, India) and water were allowed ad libitum to rats. Study
8
protocol was approved by the Institutional Animal Ethics and Care Committee at Incozen. All
9
applicable national and international ethical guidelines for maintenance and experimental
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studies with Wistar rats were followed.
11
Oral formulations were prepared in suspension form by triturating an accurately weighed
12
amount of powdered compound in methyl cellulose (0.5%, w/v water) in a gravimetric
13
dilution pattern. Oral doses of 10 and 5 mg/kg for ibrutinib and lenalidomide were
14
administered using a gavage needle at 10 ml/kg to rats after an overnight fast (12 hr). Feed
15
was offered 4 h after dosing. Blood samples (0.15 ml) were collected from retro-orbital sinus
16
at Predose, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h post dose in K2EDTA (di-potassium ethylene di
17
amine tetra acetic acid) tubes and were kept on ice till further processing. Plasma was
18
separated by centrifugation at 4°C for 10 min at 4000 rpm and stored at -70°C till further
19
analysis.
20
Pharmacokinetic parameters such as the maximum plasma concentration (Cmax), area under
21
the concentration-time curve (AUC), time to reach the maximum concentration (Tmax), half-
22
life (t1/2) and elimination constant (Kel) were estimated by means of a non-compartmental
23
analysis using Phoenix WinNonlin (Pharsight Inc., USA, version 6.1). Statistical parameters
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Page 11 of 30
1
like mean, standard deviation and % C.V were calculated by using MS-Excel 2007
2
(Microsoft®).
3 4 5
3. Results and discussion
6
To determine the most sensitive ionization mode for the components studied, ESI positive
7
and negative were tested with various combinations of components of the mobile phase, i.e.
8
methanol/acetonitrile and water / 2 mM ammonium acetate buffer / 0.1% formic acid. Signal
9
intensity for [M+H]+ ions in ESI positive ion mode were 4-10-fold higher for all components
10
analyzed using acetonitrile: 0.1% formic acid buffer compared to experiments run with ESI
11
negative ion mode. Precursor and product ions were optimized by infusing 250 ng/ml
12
solutions in the mass spectrometer between m/z 100-500 range. The Q1 MS full scan spectra
13
for the analytes and IS predominantly contained protonated precursor [M+H]+ ions at m/z
14
260.08, 441.18, 475.20, and 271.10 for lenalidomide, ibrutinib, PCI-45227 and tolbutamide
15
respectively. The most abundant and consistent product ions in product ion spectra were
16
observed at m/z 149.09, 138.08, 304.07, and 91.12 for lenalidomide, ibrutinib, PCI-45227 and
17
tolbutamide by applying 17, 27, 31, and 34 eV of collision energy respectively (Fig. 1).
18 19 20
3.2 Liquid chromatography
21
sensitivity, better peak shape, and resolution. 0.1% formic acid buffer was required to achieve
22
acceptable peak width, shapes, and acceptable ionisation. Samples were run using a reverse
23
phase C18 column (150 mm×4.6 mm i.d., 5 µm) (YMC-PACK®, Japan) with 0.1% formic
24
acid: acetonitrile in a gradient mode. All components eluted between 2.2-5.0 min.
25
Representative chromatograms of extracted blank rat plasma, blank plasma with analytical
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3.1 Mass spectrometry
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Acetonitrile, rather than methanol, was chosen as the organic modifier due to its high
10
Page 12 of 30
standards and IS and rat plasma sample 1 hr after single dose administration are shown in
2
Fig. 2.
3
3.3 Calibration standard accuracy and precision, LLOQ and LOD
4
The three calibration curves were linear over the concentration range of 0.72-183.20 ng/ml
5
for ibrutinib, 0.72-183.20 ng/ml for PCI-45227 and 1.87-479.16 ng/ml for lenalidomide, with
6
a correlation coefficient (r2) ≥ 0.9981 for the analytes. Mean linear equations obtained for
7
ibrutinib, PCI-45227 and lenalidomide were y = (0.00688± 0.0007) x + (0.0006 ± 0.0002), y
8
= (0.00743 ± 0.00016) x + (-0.00049 ± 0.00032) and y = (0.00338 ± 0.00005) x + (-0.00110
9
± 0.00073) respectively. Accuracy and precision (% CV) for the calibration curve standards
10
ranged from 92.5 to 111.5% and 0.1 to 1.3% for ibrutinib, 98.3 to 104.7% and 0.5 to 3.8% for
11
PCI-45227, 92.6 to 104.3% and 0.2 to 6.1% for lenalidomide respectively. The lower limit of
12
quantitation (S/N ≥ 20) and limit of detection (LOD, S/N ≥ 5) were 0.72 ng/ml and 0.25
13
ng/ml for ibrutinib, 0.76 ng/ml and 0.25 ng/ml for PCI-45227, and 1.87 ng/ml and 0.50 ng/ml
14
for lenalidomide respectively.
15
3.4 Intra- and inter-batch accuracy and precision
16
Intra-batch and inter-batch precision and accuracy were established from validation runs
17
performed at four QC levels (Table 1). The within-batch precision (% CV) ranged from 3.3 to
18
11.6 for ibrutinib, 2.7 to 8.5 for PCI-45227, 6.7 to 9.1 for lenalidomide while the accuracy
19
was within 96.0-105.7% for ibrutinib, 95.7-103.1% for PCI-45227 and 99.1-100.7% for
20
lenalidomide. Similarly, for the between-batch experiments, the precision varied from 0.3 to
21
2.1 for ibrutinib, 0.3 to 4.0 for PCI-45227 and 0.2 to 2.2 for lenalidomide while the accuracy
22
was within 95.5-103.5% for ibrutinib, 97.1-102.7% for PCI-45227, and 99.5-103.0% for
23
lenalidomide.
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Page 13 of 30
3.5 Stability results and dilution reliability
2
Stock solutions of analytes and IS for short term were stable at room temperature up to 6 h
3
respectively. Analytes in control rat plasma (bench top) were stable for 6 h at 25°C. Extracted
4
quality control samples were stable up to 24 h at 10°C. Long term stability of the spiked
5
quality control samples was unaffected up to 30 days at -70°C. Detailed results for stability
6
experiments are presented in Table 2. Precision (% CV) values for reliability of 1/4 and 1/8th
7
dilution were between 1.2 and 3.5%, while the accuracy results were within 97.6-103.5% for
8
ibrutinib, PCI-45227 and lenalidomide respectively Results are within the acceptance limit of
9
15% for precision (% CV) and 85-115% for accuracy as shown in Table 3.
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3.5 Recovery & matrix effect
11
Recovery of ibrutinib, PCI-45227 and lenalidomide from plasma was estimated at their
12
respective low, medium, and high quality control levels. Plasma samples (in quadruplets)
13
containing all analytes at low, medium and high quality control concentrations were also
14
spiked with respective internal standards. Results comparing the peak responses of the post-
15
extraction, spiked samples, with those of the pure standards prepared in methanol: water
16
(90:10, v/v) for low, medium, and high quality control levels, indicated that the ratios of the
17
peak responses were within acceptable limits. Absolute recoveries ranged from 73.2 to
18
77.5%, 81.0 to 87.5%, and 95.6 to 98.8%, for ibrutinib, PCI-45227 and lenalidomide
19
respectively (Table 4). The recovery of IS was 92.3%. Matrix effect was determined by
20
comparing analyte and internal standard area ratios of the extracted low and high QC in
21
matrix with the analyte and internal standard area ratio obtained from the neat solution
22
prepared at similar concentration levels (Table 5). Percent CV of the area ratios at low,
23
medium, and high quality control levels were lesser than 15% across the analytes.
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Page 14 of 30
1 2 3
3.6 Application of the method in pharmacokinetic study and incurred sample analysis
4
time profile of ibrutinib, PCI-45227 and lenalidomide in plasma following oral administration
5
of ibrutinib and lenalidomide.
6
Absorption was rapid and with maximum plasma concentrations of 0.70, 1.06 µg/ml at 0.5,
7
and 1.0 h after oral administration of ibrutinib, and lenalidomide. Formation of PCI-45227
8
was rapid with maximum plasma concentration of 0.32 µg/ml at 1.0 h after oral
9
administration of ibrutinib, and lenalidomide. Absorbed ibrutinib, PCI-45227 and
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The described analytical method was applied to generate the plasma concentration versus
lenalidomide were eliminated with a half life of 1.43, 2.25, and 1.50 h respectively. Area
11
under plasma curve (AUC0-24) was 1.42, 1.20 and 2.38 µg.h/ml for ibrutinib, PCI-45227 and
12
lenalidomide. Plasma concentrations were observed up to 24.0 hr for ibrutinib, PCI-45227
13
and lenalidomide after oral administration of the combination (Fig. 3).
14
In the current study, ISR was performed on 18 plasma samples from six different rats at
15
Cmin, Cmax, and the time point covering the phase of elimination. As per the acceptance
16
criterion, at least two-thirds of the original results and repeat results should be within 20%
17
(Table 6). Pharmacokinetic parameters of ibrutinib, PCI-45227 and lenalidomide are
18
presented in Table 7. Data demonstrated the adaptability and successful translation of the
19
validated analytical method for estimation of ibrutinib, PCI-45227 and lenalidomide to an in
20
vivo setting.
21 22 23
3.7 Comparison with reported methods
24
detection and a few LC-MS/MS based assays are also reported [16-20]. Published methods
25
indicate longer run times with a high plasma volume requirement besides issues with low
26
sensitivity [16-20]. The aim of present investigation was to develop and validate a simple
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Available methods for estimation of lenalidomide are based on the use of HPLC-UV
13
Page 15 of 30
LC-MS/MS method using a gradient mode with sufficient accuracy and precision for
2
simultaneous estimation of ibrutinib, PCI-45227 and lenalidomide and its subsequent use in
3
pharmacokinetic studies in rats. The present method involves liquid-liquid extraction
4
procedure with good sensitivity and a gradient reverse-phase LC-MS/MS analysis for all
5
analytes of interest. This method is specific for ibrutinib, PCI-45227 and lenalidomide with
6
good linearity, accuracy and precision. This method involves 50 µl plasma followed by a
7
single step liquid-liquid extraction procedure. Furthermore, this extraction procedure
8
decreases both the cost and duration of the assay. The chromatographic conditions of this
9
method were optimized for a 7.5 min run time on LC-MS/MS.
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4. Conclusion
13
lenalidomide in rat plasma was fully validated as per USFDA guidelines [21]. The proposed
14
method has a much higher sensitivity for the analytes compared to other reported methods
15
either as a single analyte or with combination in different biological matrices. Efficiency of
16
liquid-liquid extraction and chromatographic run time of 7.5 min per sample renders the
17
method useful in high-throughput bioanalysis. Absence of matrix interference is effectively
18
shown by the precision (% CV) values for the calculated slopes of calibration curves in
19
different plasma sources. The validated method showed acceptable data for all the validation
20
parameters, with adequate sensitivity and selectivity for their simultaneous quantification in a
21
clinical setting. Moreover, this is the first combination method for estimation of ibrutinib,
22
PCI-45227 and lenalidomide in rat plasma. Further, incurred sample reanalysis of 18 samples
23
authenticates the reproducibility of the proposed method.
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The developed LC-MS/MS method for the quantitation of ibrutinib, PCI-45227 and
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Page 16 of 30
Acknowledgement
2
Financial support for this research was provided by Incozen Therapeutics Pvt Ltd,
3
Hyderabad, India. Authors would like to thank management of Incozen Therapeutics Pvt Ltd
4
for extending the research facility and providing the necessary resources including reagents
5
and instruments access in this project.
6 7 8
References:
9
Reviews Immunology, 2 (2002) 945-956.
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[12] L.A. Honigberg, A.M. Smith, M. Sirisawad, E. Verner, D. Loury, B. Chang, S. Li, Z.
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Pan, D.H. Thamm, R.A. Miller, J.J. Buggy, The Bruton tyrosine kinase inhibitor PCI-32765
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blocks B-cell activation and is efficacious in models of autoimmune disease and B-cell
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malignancy, Proceedings of the National Academy of Sciences, 107 (2010) 13075-13080.
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[13] L. Alinari, B. Christian, R.A. Baiocchi, Novel targeted therapies for mantle cell
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Buggy, D. Loury, A. Hamdy, in: ASH Annual Meeting Abstracts, 2010, pp. 964.
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[15] C. Harrison, Nature Reviews Drug Discovery, Trial watch: BTK inhibitor shows
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positive results in B cell malignancies, 11 (2012) 96-97.
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[16] M.S. Hasnain, S. Rao, M.K. Singh, N. Vig, A. Gupta, A. Ansari, P. Sen, P. Joshi, S.A.
8
Ansari, Development and validation of LC-MS/MS method for the quantitation of
9
lenalidomide in human plasma using Box-Behnken experimental design, Analyst, 138 (2013)
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1581-1588.
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[17] N.Y. Khalil, I.A. Darwish, T.A. Wani, A.R.A. Al-Majed, Trace determination of
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lenalidomide in plasma by non-extractive HPLC procedures with fluorescence detection after
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pre-column derivatization with fluorescamine, Chemistry Central Journal, 7 (2013) 52-57.
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Schaaf, M.R. Grever, J.C. Byrd, J.T. Dalton, M.A. Phelps, Development and validation of a
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highly sensitive liquid chromatography/mass spectrometry method for simultaneous
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quantification of lenalidomide and flavopiridol in human plasma, Therapeutic Drug
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Monitoring, 30 (2008) 620-627.
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[19] J.A. Muscal, Y. Sun, J.G. Nuchtern, R.C. Dauser, L.H. McGuffey, B.W. Gibson, S.L.
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pharmacokinetics of thalidomide and lenalidomide in nonhuman primates, 69 (2012) 943-
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3
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Figure Legend
ip t
1
Fig.1. Product ion chromatogram of (a) ibrutinib (b) PCI-45227 (c) lenalidomide (d) tolbutamide (internal standard)
13 14
Fig.3. Time verses plasma concentration plot after single dose oral administration of ibrutinib, and lenalidomide in six wistar rats.
cr
7 8 9 10 11 12
an
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Fig.2. Chromatograms of (a) lenalidomide (b) PCI-45227 (c) tolbutamide (internal standard) (d) ibrutinib for (I) blank plasma sample (II) extracted rat plasma (III) extracted rat plasma at 1 hr after single dose of 10mg/kg of ibrutinib and 5mg/kg of lenalidomide
Ac
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18
Page 20 of 30
cr
ip t
Table 1
us
Table 1: summary of precision and accuracy from QC samples in wistar rat plasma
Within batch (n=4)
105.7
11.6
2.2
2.1±0.1
96.0
5.0
68.7
66.9±2.2
97.4
135.1±5.3
0.8
% Accuracy
% C.V 2.1
2.1±0.0
95.5
0.5
3.3
67.1±0.2
97.7
0.3
98.4
3.9
132.8±2.3
96.6
1.8
0.8±0.1
103.1
8.3
0.8±0.0
102.7
0.3
2.3
2.2±0.2
97.7
8.5
2.2±0.0
98.1
0.5
72.9
70.0±4.7
96.0
6.8
70.8±0.8
97.1
1.1
145.8
139.5±3.8
95.7
2.7
145.3±5.8
99.7
4.0
1.9
1.9±0.1
99.7
6.7
1.9±0.0
99.9
0.2
5.6
5.6±0.5
99.1
9.1
5.6±0.0
99.5
0.4
179.7
178.1±12.9
99.1
7.2
181.7±3.6
101.1
2.0
359.4
362.0±31.1
100.7
8.6
370.3±8.3
103.0
2.2
ep te
Ac c
Lenalidomide
% C.V
Measured concentration (ng/ml) (mean ± SD) 0.8±0.0
%
103.5
137.4
PCI-45227
% Accuracy
an
0.7
Measured concentration (ng/ml) (mean ± SD) 0.8±0.1
M
Ibrutinib
Spiked concentration (ng/ml)
d
Drug
Between batch (n=3)
Page 21 of 30
cr
ip t
Table 2
Nominal
Sample condition
Bench top stability a % % Accuracy CV
concentration
30 days storage stability d % Accuracy
% CV
2.2
102.2
8.4
109.2
11.1
105.1
7.3
97.4
12.0
137.4
96.6
5.2
100.1
4.2
96.3
4.6
93.1
3.8
2.3
96.8
5.9
98.9
5.3
97.9
4.2
104.5
8.4
101.3
7.8
103.9
6.2
93.6
7.6
102.8
6.8
101.6
8.0
95.0
6.6
102.3
10.2
103.9
7.6
95.7
2.0
98.2
1.3
94.1
3.9
106.8
1.9
Ibrutinib
PCI-45227
ep te
145.8 5.6
359.4
b
c
Exposed at ambient temperature (25°C) for 6h, Kept at autosampler temperature (10°C) for 24h, After three freeze-thaw cycles, d Stored at -70°C
Ac c
a
Freeze-thaw stability c % % Accuracy CV
d
M
(ng/ml)
Lenalidomide
Autosampler stability b % % Accuracy CV
an
Drug
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Table 2. Stability in rat plasma (n=4)
Page 22 of 30
cr
ip t
Table 3
ibrutinib 600 ng.ml-1 1/4 dilution
1/8 dilution
( ng.ml-1)
150
75
Mean ± SD ( n=4)
148.2 ± 11.8
( ng.ml )
1/4 dilution
1/8 dilution
150
75
250
125
155.2 ± 7.9
77.5 ± 3.9
252.8 ± 9.9
128.1 ± 3.9
97.60 2.3
103.46 1.5
103.33 3.5
101.12 1.2
102.48 2.5
M
1/8 dilution
73.2 ± 4.8
98.80 1.6
Ac c
% Nominal % CV
lenalidomide 1000 ng.ml-1
1/4 dilution
ep te
-1
PCI-45227 600 ng.ml-1
d
Dilution concentration
an
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Table 3. Dilution integrity evaluation of ibrutinib, PCI-45227 and lenalidomide in wistar rat plasma
Page 23 of 30
Table 4
Table 4: Extraction recovery in rat plasma (n=4)
PCI-45227
% C.V
77.5 75.0 73.2 87.5 85.0 81.0 97.5 95.6 98.8
3.5 2.5 1.2 1.5 2.5 1.2 5.7 3.0 3.0
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Lenalidomide
Recovery (%)
ip t
Ibrutinib
Concentration (ng/ml) 2.15 68.70 137.40 2.28 72.87 145.75 5.62 179.69 359.37
cr
Drug
Page 24 of 30
Table 5
Table 5: Matrix effect in rat plasma (n=4)
Ibrutinib PCI-45227
2.15 137.40 2.28 145.75 5.62 359.37
Average Post spiked area ratio 0.023 0.725 0.036 1.112 0.024 1.257
Average aqueous standard area ratio
% C.V
0.024 0.750 0.038 1.134 0.025 1.298
4.5 3.4 5.6 2.2 4.9 1.3
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Lenalidomide
Conc (ng/ml)
ip t
Drug
Page 25 of 30
Table 6
Table 6. Incurred sample reanalysis of ibrutinib, PCI-45227, and lenalidomide.
PCI-45227
lenalidomide
No. of total samples taken for ISR
18
18
18
No. of samples meeting the acceptance criteria (i.e., % difference between original and reanalyzed value must be within 20%)
16
17
17
% of samples meeting the acceptance criteria
88.9%
94.4%
ip t
ibrutinib
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cr
94.4%
Page 26 of 30
Table 7
Table 7: Pharmacokinetic parameters (Mean ± S.D.) after single dose oral administration of ibrutinib and lenalidomide simultaneously in wistar rats Units
ibrutinib
PCI-45227
lenalidomide
C max AUC 0-24 AUC 0-inf T max t½ K el
µg/ml µg.h/ml µg.h/ml h h h -1
0.70±0.16 1.42±0.22 1.44±0.22 0.50±0.13 1.43±1.35 0.49±0.30
0.32±0.11 1.20±0.11 1.25±0.12 1.00±0.00 2.25±2.11 0.31±0.15
1.06±0.43 2.38±0.20 2.40±0.20 0.50±0.00 1.50±1.43 0.46±0.22
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Parameters
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Ac ce p
te
d
M
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cr
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Fig 1
Page 28 of 30
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Fig 1
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Fig 3
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