Development Of Novel Liposome-Encapsulated Combretastatin A4 Acylated Derivatives: Prodrug Approach For Improving Antitumor Efficacy

Figures & data

Figure 1 Molecular structure of CA4.

Abbreviation: CA4, combretastatin A4.

Figure 2 Synthesis of CA4 prodrugs with fatty chains attached to the 3′-position of the CA4 B-ring.

Abbreviation: CA4, combretastatin A4.

Table 1 Physical Properties Of The Synthesized CA4-18, CA4-16, CA4-14, CA4-10, And CA4-6

Prodrugs	CA4-18	CA4-16	CA4-14	CA4-10	CA4-6
Yield (%)	94.32	93.58	94.69	60.12	91.46
Purity (%)	97.48	98.00	98.99	97.13	97.95
State	White solid	White solid	White solid	Faint yellow oil	Faint yellow oil
Melting point (°C)	60.4–60.8	54.7–55.2	44.9–45.2	—	—

Abbreviation: CA4, combretastatin A4.

Table 2 Sizes, Zeta Potentials, And Encapsulation Efficiencies Of Liposomes

Liposome Formulation	Size (nm)	PDI	Zeta Potential (mV)	Encapsulation Efficiency (%)
CA4-6- L	147.1±0.26	0.27±0.02	−59.03±1.15	95.19±1.23
CA4-10-L	113.0±2.44	0.27±0.02	−52.50±0.93	97.23±0.92
CA4-14-L	106.7±5.84	0.28±0.01	−47.70±0.99	98.96±0.37
CA4-16-L	103.9±2.84	0.28±0.01	−48.20±1.21	98.87±0.76
CA4-18-L	90.11±4.93	0.28±0.01	−43.77±1.23	99.13±0.58

Note: Data are presented as mean±standard deviation (SD) (n≥3).

Abbreviations: PDI, polydispersity index; CA4-6-L, combretastatin A4-6 liposomes; CA4-10-L, combretastatin A4-10 liposomes; CA4-14-L, combretastatin A4-14 liposomes; CA4-16-L, combretastatin A4-16 liposomes; CA4-18-L, combretastatin A4-18 liposomes.

Figure 3 TEM micrograph of CA4-L (A), CA4-6-L (B), CA4-10-L (C), CA4-14-L (D), CA4-16-L (E), and CA4-18-L (F), respectively.

Abbreviations: TEM, transmission electron microscopy; CA4-L, combretastatin A4-liposome; CA4-6-L, combretastatin A4-6 liposomes; CA4-10-L, combretastatin A4-10 liposomes; CA4-14-L, combretastatin A4-14 liposomes; CA4-16-L, combretastatin A4-16 liposomes; CA4-18-L, combretastatin A4-18 liposomes.

Table 3 Sizes And PDI Of Liposomes In The Stability Experiments

Liposome Formulation	Day 0		Day 7		Day 14		Centrifugation
	Size	PDI	Size	PDI	Size	PDI	Size	PDI
CA4-6-L	148.3±1.87	0.25±0.03	147±0.85	0.20±0.03	148.4±0.78	0.25±0.01	147.6±0.74	0.22±0.01
CA4-10-L	112.4±2.84	0.24±0.02	114.8±0.35	0.22±0.04	112.5±1.02	0.24±0.04	114.3±1.23	0.23±0.02
CA4-14-L	106.8±2.65	0.19±0.02	107.4±0.82	0.24±0.02	107.3±0.45	0.24±0.02	107.5±0.67	0.21±0.03
CA4-16-L	101.5±1.87	0.22±0.02	101.2±1.32	0.24±0.01	103.3±0.59	0.26±0.01	104.1±1.00	0.18±0.03
CA4-18-L	89.8±1.14	0.23±0.02	90.5±0.70	0.24±0.01	91.2±0.55	0.24±0.04	91.17±0.25	0.23±0.02

Note: Data are presented as mean±SD (n=3).

Abbreviations: PDI, polydispersity index; CA4-6-L, combretastatin A4-6 liposomes; CA4-10-L, combretastatin A4-10 liposomes; CA4-14-L, combretastatin A4-14 liposomes; CA4-16-L, combretastatin A4-16 liposomes; CA4-18-L, combretastatin A4-18 liposomes.

Figure 4 (A) Cumulative release profile of CA4-6, CA4-10, CA4-14, CA4-16, and CA4-18 from CA4-6-L, CA4-10-L, CA4-14-L, CA4-16-L, and CA4-18-L, respectively. (B) The percent conversion of CA4P, CA4-6-L, CA4-10-L, CA4-14-L, CA4-16-L, and CA4-18-L after incubation with the rat plasma (n=3).

Abbreviations: CA4, combretastatin A4; CA4P, combretastatin A4 phosphate; CA4-6-L, combretastatin A4-6 liposomes; CA4-10-L, combretastatin A4-10 liposomes; CA4-14-L, combretastatin A4-14 liposomes; CA4-16-L, combretastatin A4-16 liposomes; CA4-18-L, combretastatin A4-18 liposomes.

Table 4 IC₅₀ Of CA4, CA4P, CA4-6-L, CA4-10-L, CA4-14-L, CA4-16-L, And CA4-18-L

Group	IC50 (μM)
	MCF-7	S180	HepG2
CA4	0.004±0.002	<0.001	0.064±0.006
CA4P	0.050±0.008	<0.001	0.179±0.053
CA4-6-L	0.016±0.031	0.002±0.001	0.149±0.023
CA4-10-L	0.039±0.015	0.021±0.015	0.210±0.062
CA4-14-L	0.155±0.192	0.071±0.011	0.405±0.068
CA4-16-L	0.202±0.014	0.080±0.032	0.517±0.047
CA4-18-L	0.303±0.053	0.106±0.042	0.592±0.068

Note: Data are presented as mean±SD (n=3).

Abbreviations: IC50, half maximal inhibitory concentration; CA4, combretastatin A4; CA4P, combretastatin A4 phosphate; CA4-6-L, combretastatin A4-6 liposomes; CA4-10-L, combretastatin A4-10 liposomes; CA4-14-L, combretastatin A4-14 liposomes; CA4-16-L, combretastatin A4-16 liposomes; CA4-18-L, combretastatin A4-18 liposomes.

Figure 5 Cell viability of MCF-7 (A), S180 (B), and HepG2 (C) after treatment with CA4, CA4P, CA4-6-L, CA4-10-L, CA4-14-L, CA4-16-L, and CA4-18-L for 48 h. Data are presented as mean±SD (n=3).

Abbreviations: CA4, combretastatin A4; CA4P, combretastatin A4 phosphate; CA4-6-L, combretastatin A4-6 liposomes; CA4-10-L, combretastatin A4-10 liposomes; CA4-14-L, combretastatin A4-14 liposomes; CA4-16-L, combretastatin A4-16 liposomes; CA4-18-L, combretastatin A4-18 liposomes.

Table 5 Pharmacokinetic Parameters Of CA4P And CA4-18 After Intravenous Injection Of CA4P, CA4-6-L, CA4-10-L, And CA4-18-L To SD Rats Via The Tail Vein

Parameter	Unit	CA4P	CA4-18
Cmax	μg/mL	50.16±12.03	508.79±68.79
T1/2	h	0.97±0.37	1.46±0.12*
MRT	h	0.89±0.38	1.45±0.10*
AUC0-τ	μg·(h/mL)	12.48±1.54	654.05±151.16***
AUC0-∞	μg·(h/mL)	15.22±1.05	656.24±157.58***
CL	L/h/kg	1.73±0.12	0.06±0.017***
V	L/kg	1.74±0.83	0.08±0.02**

Notes: Data are represented as mean±SD (n=6). *P<0.05, **P<0.01, and ***P<0.001, compared with CA4P.

Abbreviations: CA4P, combretastatin A4 phosphate; CA4-18-L, combretastatin A4-18 liposomes; C_max, maximum concentration; T_1/2, short half-life; MRT, mean residence time; AUC_0-τ, area under the curve from time zero to time t; AUC_0-∞, area under the curve from time zero to infinity; CL, clearance; V, volume.

Figure 6 Plasma concentration–time curve of CA4P and CA4-18-L (A) and CA4 converted from CA4P, CA4-6-L, CA4-10-L, and CA4-18-L (B) after intravenous injection to SD rats via the tail vein (n=6).

Abbreviations: CA4, combretastatin A4; CA4P, combretastatin A4 phosphate; CA4-6-L, combretastatin A4-6 liposomes; CA4-10-L, combretastatin A4-10 liposomes; CA4-18-L, combretastatin A4-18 liposomes.

Table 6 Pharmacokinetic Parameters Of CA4 Converted From CA4P, CA4-6, CA4-10, And CA4-18 After Intravenous Injection Of CA4P, CA4-6-L, CA4-10-L, And CA4-18-L To SD Rats Via The Tail Vein

Parameter	Unit	CA4P	CA4-6	CA4-10	CA4-18
Cmax	μg/mL	31.62±4.85	28.26±1.45	39.85±11.77	32.52±5.27
T1/2	h	0.51±0.17	0.34±0.01	0.34±0.04	1.35±0.08*
MRT	h	0.39±0.06	0.31±0.01	0.22±0.03	1.80±0.14***
AUC 0-τ	mg/(L·h)	6.98±0.83	3.58±0.12	3.73±0.70	58.33±9.88***
AUC 0-∞	mg/(L·h)	7.10±0.82	3.72±0.11	3.79±0.69	59.30±10.19***
CL	L/(h·kg)	2.68±0.29	5.08±0.20	5.10±0.85	0.33±0.06***
V	L/kg	1.02±0.13	1.25±0.08	1.12±0.33	0.59±0.11**

Notes: Data are represented as mean±SD (n=6). *P<0.05, **P<0.01, and ***P<0.001, compared with CA4P.

Abbreviations: CA4, combretastatin A4; CA4P, combretastatin A4 phosphate; CA4-6-L, combretastatin A4-6 liposomes; CA4-10-L, combretastatin A4-10 liposomes; CA4-18-L, combretastatin A4-18 liposomes; C_max, maximum concentration; T_1/2, short half-life; MRT, mean residence time; AUC_0-τ, area under the curve from time zero to time t; AUC_0-∞, area under the curve from time zero to infinity; CL, clearance; V, volume.

Table 7 Antitumor Effects Of CA4P, CA4-10-L, And CA4-18-L On S180 Tumor-Bearing Mice

Group	Dose	TW (g)	TIR (%)
Saline	28 mL/kg	2.16±0.74	–
CA4P	28 mg/kg	0.91±0.36	57.87
CA4-10-L	30 mg/kg	1.23±0.39	43.06
CA4-18-L	37.2 mg/kg	0.12±0.08	94.44

Note: Data are represented as mean±SD (n=9).

Abbreviations: CA4P, combretastatin A4 phosphate; CA4-10-L, combretastatin A4-10 liposomes; CA4-18-L, combretastatin A4-18 liposomes; TW, tumor weight; TIR, tumor inhibition rate.

Figure 7 Antitumor activity of CA4P, CA4-10-L, and CA4-18-L in S180 tumor-bearing mice. (A) Excised S180 tumor. (B) Tumor weights and volumes (C) of different treatment groups. Data are presented as mean±SD (n=9). ***P<0.001 compared with saline. ^▲▲▲P<0.001 compared with CA4P. ^◆◆◆P<0.001 compared with CA4-18-L.

Abbreviations: CA4P, combretastatin A4 phosphate; CA4-10-L, combretastatin A4-10 liposomes; CA4-18-L, combretastatin A4-18 liposomes.

Figure 8 Concentration of CA4P, CA4-10-L, and CA4-18-L in tissues of S180 tumor-bearing mice after intravenous administration (n=3). (A) Concentration of CA4P. (B) Concentration of CA4 converted from CA4-18-L. (C) Concentration of CA4 converted from CA4P. (D) Concentration of CA4 converted from CA4-10-L. (E) Concentration of CA4 converted from CA4P, CA4-18-L, and CA4-10-L in tumor.

Abbreviations: CA4P, combretastatin A4 phosphate; CA4-10-L, combretastatin A4-10 liposomes; CA4-18-L, combretastatin A4-18 liposomes.