Self-Assembled Micellar Glutaminase Allosteric Inhibitor for Effective Therapeutic Intervention

Figures & data

Figure 1 The structures of Soluplus^® and CPD 23, and scheme illustration of CPD 23@micelles preparation.

Notes: (A) the monomeric unit of Soluplus^® and chemical structure of CPD 23; (B) scheme illustration of CPD 23@micelles preparation, and images of CPD 23@SOL micelles, blank SOL micelles and deionized water.

Table 1 The Encapsulation Capacity and Appearance of the Formulated CPD 23@SOL Micelles

CPD 23:SOL (w/w)^a	CPD 23 (mg/mL)	SOL (mg/mL)	EE (%)	DL (%)	Appearance
0:20	0	100	–	–	Light blue opalescent liquid
1:10	5	50	88.7 ± 2.8	8.13 ± 0.25	Slight precipitation in yellow brown opalescent liquid
1:20	5	100	93.4 ± 1.3	4.45 ± 0.06	Yellow brown opalescent liquid
1:30	5	150	98.2 ± 3.2	3.22 ± 0.09	Yellow brown opalescent liquid

Notes: ^aw/w is the weight of CPD 23/the weight of SOL; Mean ± SD; n=3.

Abbreviations: SOL, Soluplus^®; EE, encapsulation efficiency; DL, drug loading.

Table 2 The Average Particle Sizes, PDI and Zeta Potentials of the Formulated CPD 23@SOL Micelles

CPD 23:SOL (w/w)^a	CPD 23 (mg/mL)	SOL (mg/mL)	Particle Size (nm)	PDI (nm)	ζ (mV)
0:20	0	60	71.3 ± 3.6	0.12 ± 0.03	0.02 ± 0.003
1:20	1	20	63.7 ± 1.6	0.19 ± 0.01	−0.21 ± 0.08
1:20	2	40	100.7 ± 11.6	0.20 ± 0.02	0.54 ± 0.06
1:20	3	60	155.7 ± 8.7	0.23 ± 0.02	−0.14 ± 0.25
1:30	5	150	273.9 ± 26.3	0.58 ± 0.09	0.08 ± 0.12

Notes: ^aw/w is the weight ratio of CPD 23 to SOL; particle size: the average sizes based on Intensity; Data were presented as Mean ± SD; n ≥ 3.

Abbreviations: SOL, Soluplus^®; PDI, polydispersity index; ζ, Zeta potential.

Figure 2 The particle size characterizations of CPD 23@SOL micelles and blank SOL micelles.

Notes: The TEM images of CPD 23@SOL micelles (A) and blank SOL micelles (B) (bar scales: 100 nm and 1 μm); the particle size distribution of CPD 23@SOL micelles (C) and blank SOL micelles (D) by DLS method.

Figure 3 The identification of CPD 23 and CPD 23@SOL solid dispersion by XRPD.

Notes: The diffractograms of CPD 23 powder (1), SOL powder (2), CPD 23@SOL physical mixture (PM) (3) and CPD 23@SOL solid dispersion (SD) (4) were produced by XRPD method.

Figure 4 The in vitro inhibition of free CPD 23 and CPD 23@SOL micelles to (A) H22 mouse hepatoma cells and (B) A549 human non-small cell lung cancer cells by EZMTT assay.

Notes: Data were presented as mean ± SD; n=3.

Abbreviation: EZMTT, 2-(3-(2-methoxy-4-nitrophenyl)-2-(4-nitrophenyl)-2H-tetrazol-3-ium-5-yl)benzenesulfonate sodium salt.

Figure 5 The stability of free CPD 23 and CPD 23@SOL micelles in blood and their partitions in plasma and blood cell pellets.

Notes: Incubated with free CPD 23 and CPD 23@SOL micelles for 2 h, (A) the blood stability, (B) plasma partition and blood cell pellets partition of CPD 23 were determined; **P < 0.01, ***P < 0.001, CPD 23@SOL micelles significantly higher than free CPD 23; ^##P < 0.01, CPD 23@SOL micelles significantly lower than free CPD 23, n=3.

Table 3 The T_1/2 and Clearance Rates of Free CPD 23, CPD 23@SOL Micelles and Coumarin in Mouse Liver Microsome

Tested Samples	Fitted Equation	ke	t1/2 (h)	CL (L/h/g)
Free CPD 23	lnC = −0.2418t + 4.4704; R^2 = 0.9738	−0.2418	2.87	29.0 ± 1.2
CPD 23@SOL micelles	lnC = −0.0677t + 4.4363; R^2 = 0.9605	−0.0677	10.24	8.1 ± 0.6
Coumarin	lnC = −0.5783t + 4.6404; R^2 = 0.9922	−0.5783	1.20	69.4 ± 2.3

Note: Data were presented as Mean ± SD, n ≥ 3.

Abbreviations: k_e, the slope of the relationship of lnC (compound concentration) and t (incubation time); t_1/2, half-life; CL, Clearance of CPD in the mouse liver microsome; R², the coefficient of determination.

Figure 6 The stability of free CPD 23 and CPD 23@SOL micelles in mouse liver microsome.

Notes: Coumarin was used as the positive control. *P < 0.05, CPD 23 versus CPD 23@SOL micelles; mean ± SD, n=3.

Table 4 The Pharmacokinetic Parameters of CPD 23 Vehicle and CPD 23@SOL Micelles

PK Parameters	CPD 23 Vehicle	CPD 23@SOL Micelles
Administration	IV	IV
Dose (mg/kg)	20	20
AUC0→24 h (h·μg/mL)	6.6 ± 0.6	120.4 ± 15.3**
Cmax (μg/mL)	7.7 ± 0.3	74.0 ± 1.2***
Vd (L/kg)	1.29 ± 0.63	0.18 ± 0.09
t1/2α (h)	0.022 ± 0.001	0.124 ± 0.006**
t1/2β (h)	1.22 ± 0.15	3.55 ± 0.25**
CL (L/h/kg)	2.95 ± 0.32	0.16 ± 0.02**
Relative Exposure (%)	100	1824 ± 56

Notes: **P < 0.01; ***P < 0.00; Mean ± SD, n=3.

Abbreviations: PK, pharmacokinetics; IV, intravenous injection; AUC_{0→24 h}, the area under the curve in 0–24 h; C_max, the peak concentration of CPD in plasma; V_d, the apparent Volume of distribution; t_1/2α, distribution half-life; t_1/2β, elimination half-life; CL, the clearance of CPD in the plasma; Relative Exposure, percentage of AUC_{0→24 h(CPD 23@SOL micelles)}/AUC_{0→24 h(CPD 23 vehicle)}, taking the CPD 23 vehicle as control.

Figure 7 The AUC (Conc.-time) curves of in vivo pharmacokinetics of CPD 23 vehicle and CPD 23@SOL micelles via intravenous administration with dose of 20 mg/kg.

Notes: Conc.-time: the concentration of CPD 23 in plasma versus dosed time point; Data were presented as mean ± SD; n=3.

Abbreviation: AUC, the area under curve from 0 h to 24 h.

Figure 8 The biodistribution of CPD 23 vehicle and CPD 23@SOL micelles in tissues of (A) brain, (B) heart, (C) liver, (D) spleen, (E) lung and (F) kidney via intravenous administration at the dose of 20 mg/kg CPD 23.

Notes: The retaining CPD 23 was quantified by HPLC and presented by the ratio of the CPD 23 amount versus the weight of tissue (mean ± SD; n=3).

Figure 9 The tissue biodistributed AUC_{0-24 h} of CPD 23 vehicle and CPD 23@SOL micelles by intravenous administration of 20 mg/kg dose (CPD 23).

Notes: AUC_{0-24 h} comparison: **P < 0.01, ***P < 0.001, CPD 23@SOL micelles significantly higher than CPD 23 vehicle; ^##P < 0.01, ^###P < 0.001, CPD 23 vehicle significantly higher than CPD 23@SOL micelles; mean ± SD; n=3.

Abbreviation: AUC_{0→24 h}, the area under the curve at 0–24 h.