Eplerenone nanocrystals engineered by controlled crystallization for enhanced oral bioavailability

Figures & data

Table 1. Variables and their levels and constraints in D-optimal combined mixture process design.

	Independent variables	Lower level	Upper level
Mixture components (X=A+B)	A=solvent (mL)	1	9
	B=antisolvent (mL)	1	9
Process factors	C=cryoprotectant concentration (%)	3	15
	D=freezing method	Rapid	Slow
	Dependent variables	Constraints
Response 1	Nanocrystal size (nm)	Minimum
Response 2	Dissolution efficiency (%)	Maximum

Table 2. The experimental runs generated by the optimal design and measured responses for the prepared EPL-NCs.

Run	Factor 1 (A+B)		Factor 2	Factor 3	Response 1	Response 2
	A: solvent (mL)	B: antisolvent (mL)	C: cryoprotectant concentration (%)	D: freezing method	Nanocrystal size (nm)	Dissolution efficiency (%)
1	1	9	3	Rapid	80.1 ± 1.4	70.0 ± 1.3
2	1	9	3	Slow	1076.0 ± 2.7	47.0 ± 0.9
3	1	9	15	Slow	955.0 ± 3.6	55.0 ± 1.1
4	1	9	3	Slow	1316.0 ± 2.9	46.0 ± 1.4
5	1	9	15	Slow	1020.0 ± 3.8	51.0 ± 1.0
6	3	7	15	Rapid	45.5 ± 0.8	95.0 ± 1.6
7	5	5	3	Rapid	54.4 ± 1.1	89.0 ± 2.5
8	5	5	3	Slow	62.5 ± 0.9	75.0 ± 0.7
9	5	5	15	Slow	60.9 ± 0.7	86.0 ± 1.1
10	9	1	15	Rapid	61.3 ± 1.3	77.0 ± 0.8
11	9	1	15	Slow	228.8 ± 2.1	67.0 ± 0.4
12	9	1	15	Slow	349.0 ±1.7	65.0 ± 1.6
13	9	1	15	Rapid	61.6 ± 0.6	78.0 ± 2.2
14	9	1	3	Slow	541.0 ± 1.2	64.0 ± 0.9
15	9	1	3	Slow	635.0 ± 0.9	59.0 ± 0.6

The amount of EPL was 200 mg in all formulations. For the responses, data are presented as mean ± S.D. (n = 3).

Table 3. Statistical analysis of the experimental data with ANOVA and the predicted and observed results of the optimized EPL-NCs.

Response	F-value	Model p value	Lack of fit	Lack of fit p value	R^2	Adjusted R^2	Predicted mean value	Range for mean values at 95% confidence	Obtained value	Error (%)
Nanocrystal size (nm)	151.24	<.0001	1.18	.4053	0.9913	0.9847	45.2	42.94–47.46	46.8 ± 0.5	3.5
Dissolution efficiency (%)	112.46	<.0001	0.516	.6254	0.9912	0.9824	95.8	91.01–100.59	96.0 ± 1.5	0.2

The polynomial equations generated after the statistical analysis are as follow:

$\text{Nanocrystal size}=+5.23A + 5.68B-5.97AB-0.3330AC-0.7776AD-1.31BD+3.54ABD.$$$

$\text{Dissolution efficiency}=+69.33A + 61.05B+91.33AB+2.43AC+5.39AD +3.01BC+11.12BD +13.20ABC.$

p < .05 represents statistically significant difference. For the obtained values, data are presented as mean ± S.D. (n = 3).

Error (%)=(obtained value – predicted value)/predicted value × 100.

Figure 1. 2D contour plots presenting the effects of independent variables on size (A, B) and dissolution efficiency (C, D) of EPL-NCs.

Figure 2. Particle size distribution curve (A) and scanning electron microscopic image (B) of the optimized EPL-NCs.

Figure 3. Chemical structure of EPL (A) and FTIR spectra (B) of EPL (a), mannitol (b), physical mixture (c), and EPL-NCs (d).

Figure 4. DSC thermograms (A) and PXRD patterns (B) of EPL (a), mannitol (b), physical mixture (c), and EPL-NCs (d).

Figure 5. Saturation solubility (A) and dissolution profile (B) of the optimized EPL-NCs. Data are presented as mean ± S.D. (n = 3). *p<.001 vs. EPL powder.

Figure 6. Average plasma level vs. time curve after oral administration of EPL-NCs and EPL powder to rats at a dose equivalent to 10 mg/kg of EPL. Data are expressed as mean ± S.D. (n = 3).

Table 4. Non-compartmental pharmacokinetic parameters after oral administration of EPL-NCs and EPL powder to rats at a dose equivalent to 10 mg/kg of EPL.

Parameters	EPL	EPL-NCs
AUC0→t (μg·h/mL)	6.66 ± 0.93	10.50 ± 0.42*
AUC0→∞ (μg·h/mL)	7.19 ± 1.03	10.96 ± 0.43*
MRT (h)	5.10 ± 0.10	3.90 ± 0.04
Cmax (µg/mL)	1.31 ± 0.14	2.97 ± 0.09*
Tmax (h)	2.0 ± 0.00	1.00 ± 0.00*
t1/2 (h)	3.12 ± 0.06	2.60 ± 0.03
Kel (h^–1)	0.22 ± 0.003	0.27 ± 0.003
Frel (%)	–	151.0 ± 37.1

Data are expressed as mean ± S.D. (n = 3).

* p ˂ .01 vs. EPL.

Figure 7. Organ-to-body weight index (A) and histological microphotographs of liver, kidney and heart tissues of mice (B) at day 14 after treatment with EPL-NCs. In histological micrographs, (1) hepatocytes, (2) hepatic artery, (3) sinusoids, (4) central vein, (5) bile duct, (6) bowman capsule, (7) glomerulus, (8) renal tubule, (9) proximal tubule, (10) distal tubule, and (11) myocardial fibers. Organ-to-body weight indices are presented as mean ± S.D. (n = 6).

Figure 8. Blood hemolysis by the optimized EPL-NCs indicated with real-time photographs of RBCs samples (A) and quantified with spectroscopic analysis of hemoglobin at 541 nm (B). The RBCs were mixed with Triton X-100 solution (positive control), isotonic PBS (negative control), 20, 40, 60, 80, and 100 µg/mL of EPL (D-1 to D-5) and 20, 40, 60, 80, and 100 µg/mL of EPL-NCs (F-1 to F-5), respectively. For the quantification of hemolysis (%), data are presented as mean ± S.D. (n = 3).

Figure 9. DSC thermograms (A) and PXRD patterns (B) of EPL-NCs at day 0 (a) and after 90 days (b) of storage stability period.

Table 5. Storage stability of optimized EPL-NCs at 40 °C for 90 days.

	Day 0	Day 90	Change (%)
Particle size (nm)	46.8 ± 0.5	49.9 ± 2.3	+6.62
Dissolution efficiency (%)	96.0 ± 1.5	93.5 ± 1.0	–2.60

Data are presented as mean ± S.D. (n = 3).