Strategic approach to developing a self-microemulsifying drug delivery system to enhance antiplatelet activity and bioavailability of ticagrelor

Figures & data

Table 1 Factors and responses used in Scheffé’s mixture design

Factors	Range
	Low limit (w/w %)	High limit (w/w %)
X1: Capmul MCM (oil)	10	40
X2: Cremophor EL (surfactant)	10	80
X3: Transcutol P (cosurfactant)	10	80
Responses	Goal
Y1: Solubility (mg/mL)	Maximize
Y2: Precipitation (%)	Minimize
Y3: Droplet size (nm)	Minimize
Y4: Transmittance (%)	Maximize

Figure 1 Solubility of ticagrelor in various excipients.

Notes: (A) Oils. (B) Surfactants and cosurfactants. Values are expressed as mean ± SD (n=3).

Table 2 Evaluation of emulsification and phase separation according to the combination of preselected excipients

2:4:4 (w/w %)			Dilution media
Oil	Surfactant	Cosurfactant	Water		pH 1.2		pH 4.0		pH 6.8
			Gradea	Phase separationb	Grade	Phase separation	Grade	Phase separation	Grade	Phase separation
Capmul MCM	Labrasol	Transcutol P	D	O	C	O	C	O	C	O
		Tetraglycol	C	O	C	O	C	O	C	O
	Pluronic L64	Transcutol P	C	O	C	O	D	O	C	O
		Tetraglycol	C	X	C	O	C	O	C	O
	Tween 80	Transcutol P	A	X	B	O	B	Δ	B	O
		Tetraglycol	B	X	B	O	B	Δ	B	O
	Tween 20	Transcutol P	A	X	A	Δ	A	Δ	B	X
		Tetraglycol	B	X	B	Δ	A	Δ	B	X
	Cremophor EL	Transcutol P	A	X	A	X	A	X	A	X
		Tetraglycol	A	X	A	X	A	X	A	X

Notes:

a The grade was classified according to Table S1 (supplementary materials): grade A (excellent), grade B (good), grade C (fair), or grade D (poor).

b Phase separation: O (phase separation occurred; emulsion did not form when remixed), Δ (phase separation occurred; when remixed, emulsion was formed), X (no phase separation occurred).

Figure 2 Pseudoternary phase diagram.

Notes: (A) Capmul MCM, Cremophor EL, and Transcutol P. (B) Capmul MCM, Tween 20, and Transcutol P. Green line, red line, and blue line indicate compositions forming microemulsion of the grade A, grade B, and grade C, respectively.

Table 3 Summary of model fitting and statistical analysis

Responses	Suggested model	Model P-value	Lack-of-fit P-value	R^2	Adjusted R^2	Adequate precision
Y1: Solubility	Linear	<0.0001	0.7558	0.9646	0.9596	37.7012
Y2: Precipitation	Quadratic	0.0432	0.4149	0.7732	0.6701	8.0892
Y3: Droplet size	Cubic	0.0018	0.2559	0.9882	0.9731	23.3584
Y4: Transmittance	Special quartic	0.0022	0.0606	0.9879	0.9757	23.4122

Note: R^2, squared correlation coefficient.

Figure 3 Three-dimensional surface plots of responses.

Notes: (A) Y₁: Solubility of TCG in SMEDDS. (B) Y₂: Precipitation. (C) Y₃: Droplet size. (D) Y₄: Transmittance.

Table 4 Predicted values and actual values of optimized TCG-SM

Optimized factors	Response	Goal	Importance	95% Cl low predicted value	Predicted value	95% Cl high predicted value	Actual value	Error percentage (%)
X1: 10.0% X2: 53.8% X3: 36.2%	Y1: Solubility (mg/mL)	Maximize	+++	212.01	222.32	232.62	236.2±3.6	6.25
	Y2: Precipitation (%)	Minimize	+++	−4.0	10.4	24.7	10.6±0.3	2.50
	Y3: Droplet size (nm)	Minimize	+++	25.6	116.7	207.8	110.2±2.6	5.61
	Y4: Transmittance (%)	Maximize	+++	90.7	95.3	99.8	96.6±0.2	1.36

Note: Values are expressed as mean ± SD (n=3).

Abbreviations: TCG-SM, ticagrelor-loaded self-microemulsifying drug delivery system; CI, confdence interval.

Figure 4 (A) Desirability plot using numerical optimization. (B) Transmission electron microscopy images and an electrophoretic laser scattering measurement of optimized ticagrelor-loaded self-microemulsifying drug delivery system in corresponding distilled water. Scale bar 200 nm.

Figure 5 Dissolution profile of Brilinta^®, TCG-CE, and TCG-SM in (A) pH 1.2 media, (B) pH 4.0 media, (C) pH 6.8 media, and (D) distilled water.

Notes: Values are expressed as mean ± SD (n=3).

Abbreviations: TCG-SM, ticagrelor-loaded self-microemulsifying drug delivery system; TCG-CE, ticagrelor-loaded Cremophor EL.

Figure 6 (A) Cell viability of Caco-2 as a function of TCG corresponding concentration for raw TCG solution, blank-CE, TCG-CE, blank-SM, and TCG-SM. (B) Caco-2 cellular uptake of TCG treated with raw TCG solution, TCG-CE, and TCG-SM after incubation for 4 hours. (C) P_app of raw TCG solution, TCG-CE, and TCG-SM across Caco-2 cell monolayer.

Notes: (A) Values are expressed as mean ± SD (n=4). (B) Values are expressed as mean ± SD (n=4). *P<0.05 vs raw TCG solution, ^#P<0.05 vs TCG-CE. (C) Values are expressed as mean ± SD (n=4). *P<0.05 vs raw TCG solution, ^#P<0.05 vs TCG-CE.

Abbreviations: blank-CE, Cremophor EL without ticagrelor; blank-SM, self-microemulsifying drug delivery system without ticagrelor; CE, Cremophor EL; P_app, apparent permeability coefficients; SM, self-microemulsifying drug delivery system; TCG-CE, ticagrelor-loaded Cremophor EL; TCG-SM, ticagrelor-loaded self-microemulsifying drug delivery system; TCG, ticagrelor.

Table 5 In vivo pharmacokinetic parameters of TCG in rats after oral administration of raw TCG suspension, TCG-CE, and TCG-SM

Pharmacokinetic parameters	Samples
	Raw TCG suspension	TCG-CE	TCG-SM
Tmax (hours)	1.02±0.52	0.58±0.23	0.75±0.24
Cmax (ng/mL)	68.94±25.67	85.10±35.57	439.18±172.01*,#
AUC0–∞ (ng⋅h/mL)	443.26±147.80	427.25±153.96	2,525.29±390.27*,#
T1/2 (hours)	5.97±4.91	3.40±1.40	4.34±2.09
RBA (%) vs raw TCG suspension	–	123.4	637.1

Notes:

* P<0.05 vs raw TCG suspension,

# P<0.05 vs TCG-CE. Values are expressed as mean ± SD (n=11). “–” indicates compared samples of raw TCG suspension are equal with each other.

Abbreviations: AUC, area under the concentration–time curve; C_max, maximum plasma concentration; RBA, relative bioavailability; T_max, time to reach the maximum plasma concentration; T_1/2, half-life; TCG, ticagrelor; TCG-CE, ticagrelor-loaded Cremophor EL; TCG-SM, ticagrelor-loaded self-microemulsifying drug delivery system.

Figure 7 Mean plasma concentration–time profiles of TCG in rats after oral administration of raw TCG suspension, TCG-CE, and TCG-SM at a dose equivalent to 10 mg/kg of TCG.

Note: Values are expressed as mean ± SD (n=11).

Abbreviations: TCG, ticagrelor; TCG-CE, ticagrelor-loaded Cremophor EL; TCG-SM, ticagrelor-loaded self-microemulsifying drug delivery system.

Figure 8 In vitro concentration–response curves for TCG inhibitory actions on ADP-induced platelet aggregations of raw TCG suspension and TCG-SM.

Notes: EC₅₀ was defined as the concentration of the drug that inhibits platelet aggregation to 50%. Values are expressed as mean ± SEM (n=3).

Abbreviations: EC₅₀, 50% effective concentration; SEM, standard error of mean; TCG, ticagrelor; TCG-SM, ticagrelor-loaded self-microemulsifying drug delivery system.

Table 6 Ex vivo pharmacodynamic parameters of TCG in rats after oral administration of raw TCG suspension and TCG-SM with dose of 2, 5, and 10 mg/kg

Samples	Dose (mg/kg)	Pharmacodynamic parameters
		AUIC0–24 (%⋅hours)	ED50 (mg/kg)
			2 hours	5 hours	10 hours
Raw TCG suspension	2	119.9±86.7	5.1	4.5	8.4
	5	444.3±205.7
	10	907.0±408.8
TCG-SM	2	270.4±113.3*	3.5	2.5	4.4
	5	907.8±200.5*
	10	1,254.0±233.9*

Notes:

* P<0.05 vs raw TCG suspension of same dose. Values are expressed as mean ± SD (n=9).

Abbreviations: AUIC, area under the inhibitory curve; ED₅₀, 50% effective dose; TCG, ticagrelor; TCG-SM, ticagrelor-loaded self-microemulsifying drug delivery system.

Figure 9 (A) Ex vivo antiplatelet activity evaluation of raw TCG suspension and TCG-SM according to dose of 2, 5, and 10 mg/kg. (B) Dose–response curve for ADP-induced platelet aggregation in TCG according to time-points (2, 5, and 10 hours) after oral administration of raw TCG suspension and TCG-SM.

Notes: (A) Values are expressed as mean ± SEM (n=9). (B) ED₅₀ was defined as the dose of the drug that inhibits platelet aggregation to 50%. Values are expressed as mean ± SEM (n=9).

Abbreviations: ED₅₀, 50% effective dose; SEM, standard error of mean; TCG, ticagrelor; TCG-SM, ticagrelor-loaded self-microemulsifying drug delivery system.

Figure 10 Main design and result for the development of TCG-SM.

Abbreviations: BCS, Biopharmaceutical Classification System; blank-CE, Cremophor EL without ticagrelor; blank-SM, self-microemulsifying drug delivery system without ticagrelor; CE, Cremophor EL; TCG, ticagrelor; TCG-SM, ticagrelor-loaded self-microemulsifying drug delivery system.

Figure S1 (A) Apical-basolateral (A–B) transport of TCG across Caco-2 cell monolayer for 180 minutes. (B) Basolateral-apical (B–A) transport of TCG across Caco-2 cell monolayer for 180 minutes.

Notes: Values are expressed as mean ± SD (n=4).

Abbreviations: TCG, ticagrelor; TCG-CE, ticagrelor-loaded Cremophor EL; TCG-SM, ticagrelor-loaded self-microemulsifying drug delivery system.

Table S1 The classification standards of emulsification grade

Grade	Degree of emulsification	Droplet size (nm)	Transmittance (%)	Appearance
A	Excellent	<100	>95	Clear and transparent
B	Good	100–300	90–95	Slightly less clear
C	Fair	300–500	75–90	Bluish white
D	Poor	>500	<75	Milky

Table S2 Coefficient equations of responses according to the level of factors

Responses	Coefficient equations
Y1	88.84X1 + 163.03X2 + 321.54X3
Y2	10.13X1 + 12.67X2 + 60.77X3 + 15.91X1X2 − 96.06X1X3 − 86.73X2X3
Y3	47,658.71X1 + 53.30X2 + 965.31X3 − 90,213.26X1X2 − 85,963.82X1X3 − 1,304.24X2X3 + 97,272.71X1X2X3 − 53,069.49X1X2 (X1 − X2) − 43,814.17X1X3 (X1 − X3) + 468.88X2X3 (X2 − X3)
Y4	$100.75{\mathrm{X}}_1+99.57{\mathrm{X}}_2+48.88{\mathrm{X}}_3-28.52{\mathrm{X}}_1{\mathrm{X}}_2-47.41{\mathrm{X}}_1{\mathrm{X}}_3-62.62{\mathrm{X}}_2{\mathrm{X}}_3-1453.67{\mathrm{X}}_1^2{\mathrm{X}}_2{\mathrm{X}}_3+1025.35{\mathrm{X}}_1{\mathrm{X}}_2^2{\mathrm{X}}_3-652.61{\mathrm{X}}_1{\mathrm{X}}_2{\mathrm{X}}_3^2$

Table S3 The experimental composition and observed responses through Scheffé’s mixture design

Run	Factors			Responses
	X1	X2	X3	Y1	Y2	Y3	Y4
	Capmul MCM (w/w %)	Cremophor EL (w/w %)	Transcutol P (w/w %)	Solubility (mg/mL)	Precipitation (%)	Droplet size (nm)	Transmittance (%)
1	24.8307	65.1693	10	169.4±0.93	30.9±11.2	152.3±12.4	96.7±0.02
2	10	25.8795	64.1205	279.1±1.94	17.2±0.9	525.9±23.8	66.1±0.59
3	10	10	80	330.59±0.93	66.3±0.9	894.9±34.1	49.5±0.37
4	24.9753	46.7603	28.2644	188.7±0.91	9.4±0.4	218.3±14.8	89.2±0.11
5	38.6	38.2553	23.1447	165±3.85	13.1±0.4	306.7±25.6	79.6±0.48
6	10	10	80	316.05±3.55	67.7±0.3	1,023.6±85.1	50.3±0.52
7	10	62.3288	27.6712	211.71±2.63	21.7±0.4	98.8±5.7	98.4±0.06
8	24.9753	46.7603	28.2644	194.85±1.76	16.5±1.5	214.7±15.6	89.2±0.06
9	40	50	10	126.51±1.98	7.2±0.4	234.6±21.4	92.9±0.10
10	10	80	10	148.28±2.67	6.3±0.3	54.7±8.6	99.2±0.02
11	24.8307	65.1693	10	129.27±3.39	0.2±3.2	189.5±11.9	93.1±0.04
12	32.9684	10	57.0316	237.49±2.70	18.8±0.4	631.8±32.2	54.8±0.42
13	23.9412	27.95	48.1089	254.79±1.41	13.6±0.0	372.3±24.7	64.9±0.35
14	32.9684	10	57.0316	231.22±2.66	21±0.1	686.2±34.5	56.3±0.28
15	10	45.642	44.358	239.32±1.42	12.2±0.4	129.9±11.8	94.1±0.11
16	40	25.75	34.25	191.07±1.32	16.2±0.3	884.0±64.8	55.3±0.43
17	24.9753	46.7603	28.2644	187.13±2.60	7.0±0.2	225.8±21.0	93.2±0.07

Note: Values are expressed as mean ± SD (n=3).

TengROliverSHayesMAButlerKAbsorption, distribution, metabolism, and excretion of ticagrelor in healthy subjectsDrug Metab Dispos20103891514152120551239

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