Sustained delivery of a camptothecin prodrug – CZ48 by nanosuspensions with improved pharmacokinetics and enhanced anticancer activity

Figures & data

Figure 1 Chemical structures of CZ48 and its analogs: (A) CZ48; (B) CPT; (C) CZ44.

Abbreviation: CPT, camptothecin.

Figure 2 Response surface plot showing: (A) the influence of the concentration of CZ48 and F-108 on the particle size (nm) of CZ48 nanosuspension formulations; (B) the influence of the concentration of CZ48 and F-108 on the zeta potential (mV) of CZ48 nanosuspension formulations.

Abbreviations: wt%, % of weight; F-108, Pluronic^® F108.

Figure 3 Release profiles of optimal CZ48 nanosuspension formulations in different release media: (A) in PBS (pH 7.4) (n=6); (B) in human plasma (n=6).

Abbreviations: PBS, phosphate buffered saline; NS-L, the nanosuspension with particle size of 589.35 ± 23.27 nm; NS-S, the nanosuspension with particle size of 197.22 ± 7.12 nm.

Figure 4 Mean plasma concentration–time profiles of CZ48 (A) and its active metabolite-CPT (B) after i.v. administration of CZ48 cosolvent, NS-S, and NS-L (n=6).

Abbreviations: CPT, campotothecin; i.v., intravenous; NS-L, CZ48 nanosuspension with particle size of 589.35 ± 23.27 nm; NS-S, CZ48 nanosuspension with particle size of 197.22 ± 7.12 nm.

Figure 5 Organ distribution profiles of CZ48 and CPT from cosolvent (A and B), NS-L (C and D) and NS-S (E and F) in mice (n=6).

Figure 6 Profiles of partition coefficient (Kp, AUC_organ/AUC_plasma) of CZ48 and CPT from cosolvent (A and B), NS-L (C and D) and NS-S (E and F) in mice (n=6).

Figure 7 Average body weight of each group versus the day after the first dose. No statistical difference was observed in the body weights among different groups (n=7 in NT, CP, NP groups, n=10 in Co [5 mg/kg], NS-S-L [5 mg/kg], NS-S-M [25 mg/kg] and NS-S-H [50 mg/kg] groups).

Abbreviations: NT, no treatment; CP, cosolvent placebo; NP, nanosuspension placebo; NS-S-L, NS-S of low dose; NS-S-M, NS-S of medium dose; NS-S-H, NS-S of high dose; NS-S, CZ48 nanosuspension with particle size of 197.22 ± 7.12 nm.

Figure 8 Tumor growth versus time from the first day of dosing to day 29 of treatment period (n=7 in NT, CP, NP groups, n=10 in Co [5 mg/kg], NS-S-L [5 mg/kg], NS-S-M [25 mg/kg] and NS-S-H [50 mg/kg] groups).Abbreviations: NT, no treatment; CP, cosolvent placebo; NP, nanosuspension placebo; NS-S-L, NS-S of low dose; NS-S-M, NS-S of medium dose; NS-S-H, NS-S of high dose; NS-S, CZ48 nanosuspension with particle size of 197.22 ± 7.12 nm.

Figure 9 Percent survival in each group over time in days. The survival was expressed as % surviving from original number at time 0 (n=7 in NT, CP, NP groups, n=10 in Co [5 mg/kg], NS-S-L [5 mg/kg], NS-S-M [25 mg/kg] and NS-S-H [50 mg/kg] groups).

Abbreviations: NT, no treatment; CP, cosolvent placebo; NP, nanosuspension placebo; NS-S-L, NS-S of low dose; NS-S-M, NS-S of medium dose; NS-S-H, NS-S of high dose; NS-S, CZ48 nanosuspension with particle size of 197.22 ± 7.12 nm.

Table 1 Levels of critical influencing factors and coded correspondent values

Factor	Levels
	–α	−1	0	1	+α
X1 (CZ48, wt%)	2	4	6	8	10
X2(Tween-80, wt%)	2	22	51	80	100
X3(F-108, wt%)	2	22	51	80	100

Abbreviations: wt%, % of weight; Tween-80, polysorbate 80; F-108, Pluronic^® F108.

Table 2 Experimental responses and the results of central composite design (CCD

Formulation No.	(X1, X2, X3)	Y1: Particle size (nm)	Y2: PI	Y3: Zeta potential (mV)
1	(+1, +1, +1)	409.60	0.08	−11.20
2	(+1, +1, -1)	465.30	0.16	−13.79
3	(+1, -1, +1)	304.03	0.14	−28.25
4	(+1, -1, -1)	394.87	0.17	−28.40
5	(-1, +1, +1)	251.00	0.11	−23.00
6	(-1, +1, -1)	269.70	0.10	−38.25
7	(-1, -1, +1)	273.07	0.18	−17.00
8	(-1, -1, -1)	217.90	0.13	−21.18
9	(+α, 0, 0)	483.50	0.15	−24.40
10	(-α, 0, 0)	215.03	0.17	−23.62
11	(0, +α, 0)	286.20	0.12	−26.02
12	(0, -α, 0)	242.33	0.15	−28.98
13	(0, 0, +α)	298.03	0.14	−31.24
14	(0, 0, -α)	294.70	0.14	−30.75
15	(0, 0, 0)	221.90	0.16	−31.80
16	(0, 0, 0)	223.02	0.11	−27.46
17	(0, 0, 0)	220.93	0.12	−28.45
18	(0, 0, 0)	213.84	0.11	−28.22
19	(0, 0, 0)	218.49	0.14	−27.81
20	(0, 0, 0)	225.37	0.11	−27.21

Table 3 Predicted values and experimental results of CZ48 nanosuspension prepared under the optimal conditions

Response	Predicted value	Experimental value	Bias (%)
Y1, particle size (nm)	190.07	197.22±7.12	2.11
Y2, zeta potential (mV)	−25.76	−26.52±0.93	2.71

Table 4 Pharmacokinetic parameters of CZ48 and active metabolite CPT from cosolvent, NS-S and NS-L after i.v. administration in mice (n=6)

	Parameters	Unit	Cosolvent (5 mg/kg)	NS-S (25 mg/kg)	NS-L (25 mg/kg)
CZ48	t1/2	hrs	0.70±0.13	8.00±1.03*	5.58±1.20*^#
	C0/Dose	nM/(mg/kg)	381.66±105.31	122.29±40.14	172.48±34.39*
	Vss	L/kg	6.48±2.46	41.12±11.85*	30.61±12.28*^#
	CL	L/kg*hr	6.41±1.82	9.03±2.82*	10.19±3.47*^#
	AUC0-∝/Dose	nM*hr/(mg/kg)	385.70±81.95	440.08±54.10	362.95±63.52*^#
	Fab			1.14	0.94
CPT	t1/2	hrs	0.40±0.10	12.51±2.86*	5.81±1.71*^#
	t1/2ka	hrs	0.06±0.03	0.47±0.18	0.50±0.22
	Vss	L/kg	11,587±2143	10,570±4093	7209±1466
	CL	L/kg*hr	19,894±4300	2383±753.7*	3758±780.3*^#
	Tmax	hrs	0.20±0.04	0.67±0.10*	0.75±0.15*
	Cmax/Dose	nM/(mg/kg)	10.12±2.13	7.38±1.99*	4.46±1.10
	AUC0-∝/Dose	nM*hr/(mg/kg)	8.29±3.45	19.78±6.94*	10.95±1.33*^#
	Fab			2.39	1.32

Notes: *p<0.05 compared to that of cosolvent by t-test; ^#p<0.05 compared to that of NS-S by t-test.

Abbreviations: CPT, camptothecin; NS-L, CZ48 nanosuspension with particle size of 589.35 ± 23.27 nm; NS-S, CZ48 nanosuspension with particle size of 197.22 ± 7.12 nm; i.v., intravenous.

Table 5 CZ48 and CPT organ distribution parameters from cosolvent, NS-S and NS-L in mice after i.v. administration (n=6)

Organs	CZ48			CPT
	Cosolvent	NS-S	NS-L	Cosolvent	NS-S	NS-L
	AUC/Dose[(ng/g)*h/(mg/kg)]
Heart	296.14	378.34	169.67	18.75	32.93	23.16
Liver	690.54	15,324.07	23,288.49	99.03	181.38	189.48
Spleen	282.28	5,374.30	3,350.07	34.34	48.51	57.90
Lung	1,038.38	13,686.92	1,612.12	34.18	55.18	39.55
Kidney	604.57	332.13	355.41	57.25	57.35	59.94
Brain	59.59	34.61	34.14	8.25	8.91	8.24
Plasma	156.05	178.06	146.85	2.89	6.89	3.81
	t1/2 (hrs)
Heart	0.76	8.29	4.77	0.99	7.13	6.49
Liver	0.84	1.96	2.74	1.17	6.31	5.99
Spleen	0.93	1.64	1.92	0.81	2.99	4.58
Lung	2.00	38.21	3.07	0.77	7.28	3.81
Kidney	0.81	3.20	3.17	1.48	4.16	8.24
Brain	0.62	4.45	4.16	0.68	4.02	4.04
Plasma	0.70	5.00	5.58	0.40	12.51	5.81
	Cmax/Dose [(ng/g)*hr/(mg/kg)]
Heart	263.04	148.24	70.91	11.18	11.55	10.73
Liver	543.24	5,521.13	86,253.18	50.99	34.22	31.03
Spleen	200.65	3,230.42	2,014.29	21.46	16.15	14.40
Lung	309.97	1,689.83	5,036.33	29.26	18.71	20.95
Kidney	488.36	194.71	386.09	21.78	17.38	15.85
Brain	49.63	9.99	9.41	7.28	3.91	4.15
Plasma	154.42	49.48	69.79	3.53	2.51	1.55

Table 6 Tumor growth rate from the first day of dosing until day 11

Groups	Tumor growth rate until Day 11
NT (n=7)	0.192±0.049
CP (n=7)	0.157±0.048
NP (n=7)	0.191±0.052
Co (n=10, 5mg/kg)	0.181±0.064
NS-S-L (n=10, 5mg/kg)	0.130±0.030
NS-S-M (n=10, 25mg/kg)	0.084±0.030*^#
NS-S-H (n=10, 50mg/kg)	0.010±0.003*^#

Notes: *p<0.05 compared to placebo groups by ANOVA followed Tukey’s post hoc. ^#p<0.05 compared to cosolvent group by ANOVA followed Tukey’s post hoc.

Abbreviations: NT, no treatment; CP, cosolvent placebo; NP, nanosuspension placebo; NS-S-L, NS-S of low dose; NS-S-M, NS-S of medium dose; NS-S-H, NS-S of high dose; NS-S, CZ48 nanosuspension with particle size of 197.22 ± 7.12 nm.

Table 7 Summary of significance testing by Kaplan–Meier survival analysis among different groups

Survival comparison
	Co	NS-S-L	NS-S-M	NS-S-H
CP	0.0453	-	-	-
NP	-	0.0219	0.0002*	0.2046
Co	-	0.0309	0.0011*	0.4765
NS-S-L	-	-	0.0085*	0.9646
NS-S-M	-	-	-	0.0167

Note: *p<0.01 for a 10-way comparison in Kaplan–Meier analysis.

Abbreviations: NT, no treatment; CP, cosolvent placebo; NP, nanosuspension placebo; NS-S-L, NS-S of low dose; NS-S-M, NS-S of medium dose; NS-S-H, NS-S of high dose; NS-S, CZ48 nanosuspension with particle size of 197.22 ± 7.12 nm.

Table S1 Effects of different stabilizers and combination with Tween-80 on particle size, PI and zeta potential of nanosuspensions by media milling preparation method

Stabilizer		Particle size (nm)	PI	Zeta potential (mV)
PVP K40	H	666.0±28.4	0.118±0.022	−19.29±0.85
	M	707.6±13.3	0.132±0.027	−23.35±0.67
	L	679.6±23.5	0.103±0.032	−23.60±1.05
PVA	H	417.5±27.7	0.176±0.050	−25.20±1.06
	M	412.0±32.1	0.154±0.011	−23.52±0.25
	L	-	-	-
F-68	H	793.1±62.8*	0.178±0.034	−26.57±0.45
	M	915.7±37.3*	0.154±0.011	−27.16±1.25
	L	1,035±110.9	0.176±0.050	−27.06±0.70
F-108	H	301.3±9.50*	0.162±0.026	−31.77±1.23
	M	417.9±12.9*	0.150±0.014	−25.82±1.29
	L	578.7±10.1	0.198±0.006	−25.45±0.72
T-80	H	475.4±12.9	0.112±0.028	−32.64±0.64
	M	481.6±22.2	0.123±0.051	−32.73±0.92
	L	455.4±12.7	0.143±0.034	−28.36±1.56
PVP K40/Tween-80	(1 : 1^#)	581.9±31.22	0.128±0.049	−25.32±0.88
PVA/Tween-80		414.6±10.72	0.164±0.042	−28.80±1.11
F-68/Tween-80		413.5±22.39	0.175±0.053	−32.56±1.78
F-108/Tween-80		366.2±13.24*	0.140±0.016	−31.81±0.96

Notes: The ratios of CZ48/stabilizer were 1/1 (H), 1:0.25 (M) and 1:0.1 (L). -, : The drug could not be wetted by the stabilizer. ^#The amount of stabilizer was 10% weight of CZ48. *p<0.05 by ANOVA and Tukey’s post hoc.

Abbreviations: H, the ratio of stabilizer/CZ48 is 1:1; M, the ratio of stabilizer/CZ48 is 1:4; L, the ratio of stabilizer/CZ48 is 1:10; PVP K40, povidone 40; PVA, polyvinyl acetate; F-68, Pluronic^®F68; F-108: Pluronic^®F108; Tween-80, polysorbate 80.

Figure S1 Dependence of particle size (nm) on the milling time (hrs) (n=3), particle size decreased as milling time increase up to 24 hrs.

Table S2 Summary of central composite design (CCD) fitting parameters

Regression coefficient	Y1 (particle size, nm)		Y2 (PI)	Y3 (zeta potential, mV)
	Estimate	p-level		Estimate	p-level
b0	535.338	0.006	N/A	14.644	0.152
b1	−101.260	0.005		−9.682	0.012
b2	−2.776	0.934		−0.952	0.0001
b3	−2.705	0.010		0.140	0.214
b4	7.661	0002		0.418	0.085
b5	0.018	0.423		0.002	0.621
b6	0.031	0.055		−0.001	0.269
b7	0.617	0.049		0.118	0.0001
b8	−0.027	0.768		0.003	0.168
b9	0.166	0.313		−0.036	0.091
F-value (p<0.0001)	148			79.9
R^2	0.959			0.895