Bioequivalence of paclitaxel protein-bound particles in patients with breast cancer: determining total and unbound paclitaxel in plasma by rapid equilibrium dialysis and liquid chromatography–tandem mass spectrometry

Figures & data

Table 1 Validation data of the analytical method used to determine total paclitaxel in human plasma

	Quality control samples
Parameters	LLOQ (5.00 ng/mL)	Low (15.0 ng/mL)	Medium (900 ng/mL)	High (12,000 ng/mL)
Accuracy and Precision
Intra-assay (%RE, %CV)	1.9~8.3, 3.3~6.2	−4.4~4.7, 2.6~3.9	−9.9~7.2, 1.1~2.8	−7.6~7.6, 1.3~5.7
Inter-assay (%RE, %CV)	5.8, 5.4	3.2, 3.2	−8.0, 2.7	−6.7, 2.0
Recovery (%)	/	119.0	106.2	110.1
%CV	/	2.0	2.3	1.4
Stability with accuracy
−20 °C 29 days (%RE, %CV)	/	−1.6, 4.4	/	−8.3, 2.2
−70 °C 88 days (%RE, %CV)	/	5.4, 5.5	/	0.6, 2.4
room temperature 6 hrs (%RE, %CV)	/	−3.9, 3.0	/	−8.6, 2.0
3 FT (−70 °C) (%RE, %CV)	/	6.2, 4.8	/	−4.3, 1.9
Recovery of IS (%RE, %CV)	105, 1.8

Abbreviations: LLOQ, lower limit of quantification; RE, relative error; CV, coefficient of variations; FT, freeze-thaw cycle; IS, internal standard.

Table 2 Validation data of the analytical method used to determine unbound paclitaxel in human plasma

	Quality control samples
Parameters	LLOQ (0.200 ng/mL)	Low (0.600 ng/mL)	Medium (8.00 ng/mL)	High (160 ng/mL)
Accuracy and Precision
Intra-assay (%RE, %CV)	−8.6~11.4, 7.2~11.6	−13.6~-4.3, 3.3~11.4	−6.1~ −3.3, 1.8~4.2	−7.0~3.1, 4.7~5.7
Inter-assay (%RE, %CV)	−1.4, 13.5	−8.6, 8.2	−4.4, 3.0	−0.9, 6.6
Recovery (%)	/	95.8	96.7	94.5
%CV	/	2.9	2.6	4.2
Stability with accuracy
−20 °C 15 days (%RE, %CV)	/	−4.0, 7.3	/	3.9, 4.6
−70 °C 15 days (%RE, %CV)	/	−13.4, 8.0	/	2.7, 3.1
room temperature 6 hrs (%RE, %CV)	/	−7.5, 4.8	/	3.1, 3.4
5 FT (−70 °C) (%RE, %CV)	/	−9.9, 5.9	/	3.4, 4.7
Recovery of IS (%RE, %CV)	96.6, 3.5

Table 3 Pharmacokinetic parameters of total and unbound paclitaxel for nab-paclitaxel (260 mg/m²) in 24 patients treated with a 30 min infusion

Parameters	Unbound Paclitaxel		Total Paclitaxel
	Mean±SD (%CV)		Mean±SD (%CV)
	Test	Reference	Test	Reference
Tmax (h)	0.5 (0.25, 0.51)	0.5(0.25, 0.52)	0.5(0.25, 0.52)	0.5(0.25, 0.52)
Cmax (ng/mL)	444.5±117.2 (26.37)	476.7±125.2 (26.26)	12,295±2370 (19.28)	12,771±2065 (16.17)
AUC0–t (h*ng/mL)	402.4±117.1 (29.11)	440.1±143.1 (32.51)	12,197±2841 (23.30)	12,619±2754 (21.83)
AUC0–∞ (h*ng/mL)	418.4±123.9 (29.62)	456.4±145.3 (31.84)	12,587±2932 (23.29)	13,078±2880 (22.02)
t1/2 (h)	21.61±9.43 (43.63)	26.74±12.59 (47.09)	24.48±4.47 (18.27)	27.32±10.10 (36.96)

Notes: T_max, the values are expressed in terms of median (range).

Abbreviations: SD, standard deviation; CV, coefficient of variation; T_max, time to peak concentration; C_max, peak plasma concentration; AUC_0–t, area under the curve to the last measurable concentration; AUC_0–∞, area under the curve extrapolated to infinity; t_1/2, elimination half-life.

Figure 1 Plasma concentration–time curves of total paclitaxel (A) and unbound paclitaxel (B) after 30 min infusion of the test formulation or the reference formulation (Abraxane®) at a dose of 260 mg/m². Data are presented as Log10 mean ± SD.

Table 4 Geometric mean ratio and statistical comparison of total paclitaxel for nab-paclitaxel (260 mg/m²) in patients treated with a 30 min infusion

Parameters	Geometric Mean			%CV	90% CI	power of test %
	Test (T)	Reference (R)	ratio(T/R)*%
Cmax (ng/mL)	11,979	12,611	94.99	6.40%	92.03~98.05	100
AUC0–t (h*ng/mL)	11,798	12,341	95.60	7.81%	91.98~99.37	100
AUC0–∞ (h*ng/mL)	12,181	12,784	95.28	8.47%	91.37~99.36	100

Notes: Statistical calculations for AUC and C_max were based on logarithmic-transformed data. Bioequivalence criteria are defined as 90% CI of the geometric mean ratios of the test/reference drug of between 80.0% and 125.0% for C_max, AUC_0–t, and AUC_0–∞.

Abbreviations: CV, coefficient of variation; CI, confidence interval; C_max, peak plasma concentration; AUC_0–t, area under the curve to the last measurable concentration; AUC_0–∞, area under the curve extrapolated to infinity.

Table 5 Geometric mean ratio and statistical comparison of unbound paclitaxel for nab-paclitaxel (260 mg/m²) in patients treated with a 30 min infusion

Parameters	Geometric Mean			%CV	90% CI	Power of test %
	Test (T)	Reference (R)	Ratio(T/R)*%
Cmax (ng/mL)	429.1	460.9	93.12	12.13%	87.70~98.86	99.49
AUC0–t (h*ng/mL)	387.7	420.6	92.16	12.20%	86.77~97.88	98.81
AUC0–∞ (h*ng/mL)	402.8	437.0	92.18	12.14%	86.81~97.88	98.88

Table 6 Summary of total paclitaxel and unbound paclitaxel pharmacokinetic parameters of nab-paclitaxel

Parameters	Data from European/American(n=14)		Data from Chinese(n=12)	Data from Chinese (n=36)		Data from this trial (test, n=24)		Data from this trial (reference, n=24)
	Total	Unbound	Total	Total	Unbound	Total	Unbound	Total	Unbound
t1/2 h (CV%)	20	—	16.93	19.43	2.82	24.48	21.61	27.32	26.74
	(21.3)	—	(28.9)	(28.3)	(30.1)	(18.27)	(43.63)	(36.96)	(47.09)
Cmax ng/mL (CV%)	19,556	1284	10,250	14,230	960	12,295	444.5	12,771	476.7
	(36.2)	(41.5)	(15.1)	(30.0)	(31.3)	(19.28)	(26.37)	(16.17)	(26.26)
AUC0–72h (h*ng/mL) (CV%)	—	—	12,130	14,130	710	12,197	402.4	12,619	440.1
	—	—	(19.8)	(26.3)	(22.5)	(23.30)	(29.11)	(21.83)	(32.51)
AUC0–∞ (h*ng/mL) (CV%)	20,324	1159	12,410	14,550	750	12,587	418.4	13,078	456.4
	(19.5)	(29.1)	(19.5)	(25.8)	(21.3)	(23.29)	(29.62)	(22.02)	(31.84)

Abbreviations: CV, coefficient of variation; t_1/2, elimination half-life; C_max, peak plasma concentration; AUC_0–72h, AUC from time 0 h to72 h.; AUC_0–∞, area under the curve extrapolated to infinity.

Table 7 %fu values of nab-paclitaxel determined by ultrafiltration method under different conditions

Ultrafiltration Conditions	Sample 1 %fu	Sample 2 %fu
2500g, 10 min, 25 °C	2.2	1.9
14000g, 10 min, 25 °C	2.7	1.4
2500g, 20 min, 25 °C	6.6	5.5
2500g, 20 min, 27 °C	6.2	5.1
2500g, 20 min, 30 °C	8.5	7.7
2500g, 20 min, 35 °C	11.1	10.6

Notes: To investigate the effects of centrifugation time, centrifugation temperature, and centrifugation speed on fu of paclitaxel, samples 1 and 2 were obtained by mixing equal volume of plasma samples collected at 1.25 and 15 h, respectively, from five patients treated with nab-paclitaxel

Abbreviations: fu, unbound fraction.