Clinical Development of Imatinib: an Anticancer Drug

Figures & data

Figure 1.  Chemical structures of analyte and internal standard.

Chemical structures of (A) imatinib (B) imatinib-D4.

Figure 2.  Tandem mass spectrometric scan of analyte and internal standard.

(A) Parent ion (Q1) scan of imatinib; (B) product ion (MS/MS) scan of imatinib; (C) parent ion (Q1) scan of imatinib-D4; (D) iroduct ion (MS/MS) scan of imatinib-D4. amu: Atomic mass unit; cps: Counts per second; MS/MS: Tandem mass spectrometry.

Figure 3.  Chromatograms.

(A) Chromatogram of blank at retention time of imatinib, (B) chromatogram of blank at retention time of imatinib-D4, (C) chromatogram of subject sample predose, (D) chromatogram of subject sample predose-imatinib-D4, (E) chromatogram of LOQ – imatinib, (F) chromatogram of LOQ – imatinib-D4, (G) chromatogram of ULOQ – imatinib, (H) chromatogram of ULOQ – imatinib-D4. LQC:Low quality control; ULOQC: Upper limit of quantification.

Figure 4.  Calibration curve of imatinib.

Method was found to be linear in the range 9.57–4513.29 ng/ml.

IS: Internal standard.

Figure 5.  Pharmacokinetic profile of imatinib (test and reference) in Indian volunteers.

Circle: Reference; Rectangle: Test.

Table 1.  Demographic profile of volunteers completing the study (Indian and European).

Demographic profile	Indian volunteers (n = 24)	European volunteers (n = 30)
Gender	Male (100%)	Male (100%)
Age (years)	27.58 ± 4.92	24.7± 3.80
Height (cm)	164.23 ± 6.09	176.10 ± 6.15
BMI (kg/m^2)	21.26 ± 2.17	24.01 ± 2.52
Weight (kg)	57.30 ± 6.09	74.78 ± 11. 22

Table 2.  Optimized mass spectrometer parameters for imatinib using API–3200 mass spectrometer.

Source/gas parameter	Compound parameter (eV)
Curtain gas: 40^†	Declustering potential: 65
Collisionally activated dissociation (CAD) gas: 3^†	Entrance potential: 6
Ion spray voltage: 5000 eV	Collision energy: 38
Ion source gas1: 50^†	Collision cell exit potential: 5
Ion source gas2: 50^†	Resolution (Q1/Q3): unit/unit
Temperature: 550°C
Interface heater: on

† Typical values based on settings and scales defined in software application to control gas parameters.

Table 3.  Selectivity test for imatinib and imatinib-D4 in different lots of plasma.

Sample name	Imatinib		Imatinib-D4
	Area	Area (%)	Area	Area (%)
Blank 1	412	2.409	409	0.023
Blank 2	220	1.287	242	0.014
Blank 3	178	1.041	211	0.012
Blank 4	80	0.468	263	0.015
Blank 5	218	1.275	181	0.01
Blank 6	110	0.643	227	0.013
Blank 7^†	154	0.901	87	0.005
Blank 8^‡	163	0.953	118	0.007
LOQ–1	17559	NAP	1823001	NAP
LOQ–2	17296	NAP	1815286	NAP
LOQ–3	16403	NAP	1782262	NAP
LOQ–4	17277	NAP	1778668	NAP
LOQ–5	17164	NAP	1769828	NAP
LOQ–6	16900	NAP	1733432	NAP
Mean	17099.8	–	1783746.2	–
SD(±)	402.57	–	32523.6	–
%CV	2.35	–	1.82	–
n	6	–	6	–

† Hemolyzed blank.

‡ Lipemic blank.

CV: Coefficient of variation; NAP: Not applicable; SD: Standard deviation.

Table 4.  Intra- and inter-batch precision and accuracy of imatinib.

Quality control level	Nominal concentration (ng/ml)	Intraday (n = 12)			Interday (n = 18)
		Mean (ng/ml)	CV (%)	Accuracy (%)	Mean (ng/ml)	CV (%)	Accuracy (%)
LLOQC	9.58	9.846	3.14	102.77	9.704	3.60	101.30
LQC	23.41	22.883	1.42	97.75	22.716	1.81	97.04
MQC	1800.91	1829.983	1.86	101.61	1835.620	1.65	101.93
HQC	3601.82	3705.032	1.75	102.87	3693.256	1.78	102.54

CV: Coefficient of variation; HQC: High qulaity control; LLOQC: Quality control at limit of quantification; LQC: Low quality control; MQC: Medim quality contol.

Table 5.  Percentage recovery of imatinib and imatinib-D4 in human plasma.

QC	Imatinib	Imatinib-D4
LQC	80.86%
MQC	93.59%
HQC	87.57%
Mean recovery	87.338%	87.87%
SD(±)	6.3657
%CV	7.29
n	3

CV: Coefficient of variation; HQC: High qulaity control; IS: Internal standard; LLOQC: Quality control at limit of quantification; LQC: Low quality control; MQC: Medim quality contol.

Table 6.  Matrix effect and matrix factor for imatinib in six different lots of human plasma.

Lots of human plasma	Matrix effect				Matrix factor
	LOQQC 9.58 ng/ml^†	HQC 3601.82 ng/ml^†	LQC		MQC		HQC
	Calculated concentratin (ng/ml)	Calculated concentratn (ng/ml)	Using peak area response	Using analyte/IS peak area ratio	Using peak area response	Using analyte/IS peak area ratio	Using peak area response	Using analyte/IS peak area ratio
Matrix lot #1	9.53	3603.46	1.13	1.02	1.07	1.03	1.04	1.01
	9.10	3557.21
Matrix lot #2	9.12	3534.31	1.10	1.01	1.07	1.03	1.02	1.01
	8.95	3624.25
Matrix lot #3	10.70	3602.58	1.11	1.03	1.06	1.02	1.00	1.00
	9.11	3575.45
Matrix lot #4	9.05	3605.68	1.10	1.02	1.07	1.04	0.98	1.00
	8.97	3481.01
Matrix lot #5^‡	9.18	3506.62	1.06	1.00	1.04	1.02	0.96	0.99
	9.19	3556.62
Matrix lot #6^§	9.15	3516.40	1.02	1.01	1.02	1.02	0.98	1.00
	8.91	3515.64
Mean	9.247	3556.603	1.086	1.013	1.056	1.027	0.996	1.003
SD(±)	0.4845	46.3716	0.0400	0.0103	0.0189	0.0061	0.0306	0.0108
%CV	5.24	1.30	3.68	1.02	1.79	0.60	3.07	1.08
n	12	12	6	6	6	6	6	6
%Nominal	96.52	98.74

† Nominal concentration (ng/ml).

‡ Hemolyzed plasma lot.

§ Lipemic plasma lot.

CV: Coefficient of variation; HQC: High qulaity control; IS: Internal standard; LLOQC: Quality control at limit of quantification; LQC: Low quality control; MQC: Medim quality contol; SD: Standard deviation.

Table 7.  Sample collection process stability of imatinib.

Number of samples (n=4)	LQC		HQC
	Stability samples	Comparison samples	Stability samples	Comparison samples
	Area ratio (analyte/IS)	Area ratio (analyte/IS)	Area ratio (analyte/IS)	Area ratio (analyte/IS)
1	0.0248	0.0242	3.9210	3.9821
2	0.0245	0.0243	3.9921	3.9769
3	0.0245	0.0243	3.8916	3.9990
4	0.0239	0.0242	4.0109	3.9735
Mean	0.02443	0.02425	3.95390	3.98288
SD(±)	0.000377	0.000058	0.05678	0.011317
%CV	1.55	0.24	1.44	0.28
n	4	4	4	4
%Stability	100.72		99.27

CV: Coefficient of variation; HQC;High quality control; IS: Internal standard; LQC: Low quality control; SD: Standard deviation.

Table 8.  Different stability parameters of imatinib.

Stability parameters	Nominal concentration (ng/ml)	Stability QC samples		Comparison QC samples
Bench top stability in matrix (6.15 h)	Mean	22.480	3696.288	22.468	3787.665
	SD(±)	0.3498	113.9628	0.352	24.3493
	%CV	1.56	3.08	1.57	0.64
	%Nominal	96.03	102.62	95.81	104.98
	n	4	4	4	4
Bench top stability during extraction (9.60 h)	Mean	22.193	3564.485	22.903	3681.380
	SD(±)	0.2660	61.3054	0.8096	41.3953
	%CV	1.20	1.72	3.54	1.12
	%Nominal	94.80	98.96	97.67	102.04
	n	4	4	4	4
Freeze thaw stability (three freeze thaw cycles, below -15°C)	Mean	22.745	3695.175	22.468	3787.665
	SD(±)	0.3283	71.3907	0.352	24.3493
	%CV	1.44	1.93	1.57	0.64
	%Nominal	97.16	102.59	95.81	104.98
	n	4	4	4	4
Freeze thaw stability (three freeze thaw cycles, below -50°C)	Mean	22.715	3684.818	22.468	3787.665
	SD(±)	0.4222	42.8389	0.3520	24.3493
	%CV	1.86	1.16	1.57	0.64
	%Nominal	97.03	102.3	95.81	104.98
	n	4	4	4	4
Auto sampler stability (100.87 h)	Mean	23.023	3695.965	22.720	3508.210
	SD(±)	0.2301	87.256	0.2696	36.4595
	%CV	1	2.36	1.19	1.04
	%Nominal	98.34	102.61	96.89	97.24
	n	4	4	4	4
Long-term stability below -15°C (183 days)	Mean	23.998	3442.073	22.438	3336.62
	SD(±)	0.3456	56.969	0.0806	28.4049
	%CV	1.44	1.66	0.36	0.85
	%Nominal	102.51	95.56	95.64	92.43
	n	4	4	4	4
Long-term stability below -50°C (183 days)	Mean	23.253	3432.1	22.438	3336.62
	SD(±)	0.2916	33.309	0.0806	28.4049
	%CV	1.25	0.97	0.36	0.85
	%Nominal	99.33	95.29	95.64	92.43
	n	4	4	4	4
Stock solution stability (21 days, stock solutions were stored in refrigerator temperature 1–10°C)	%Stability for imatinib	101.63
	%Stability for imatinib-D4	99.15
Short-term stability (8.46 h at room temperature)	%Stability for imatinib	101.88
	%Stability for imatinib-D4	100.96

CV: Coefficient of variation; QC: Quality control; SD: Standard deviation..

Table 9.  Pharmacokinetic parameter of imatinib (test and reference) in Indian volunteers.

Parameters	Test	Reference
Mean Cmax (ng/ml)	2055.983 (%CV: 26.27)	1917.543 (%CV: 28.40)
AUC 0-∞	35293.60776 (%CV: 25.62)	36521.09140 (%CV: 25.46)
Mean Tmax (h)	3.0	4.50
n	24	24

CV: Coefficient of variation.

Table 10.  Confirmatory incurred sample reanalysis of imatinib.

Incurred sample reanalysis details	Imatinib
Total number of samples taken for incurred sample reanalysis	120
Number of samples meeting the acceptance criteria (i.e., % difference between the original and reanalyzed value must be within 20%)	120
Percentage of samples meeting the acceptance criteria	100.00