Repurposing levocetirizine hydrochloride loaded into cationic ceramide/phospholipid composite (CCPCs) for management of alopecia: central composite design optimization, in- silico and in-vivo studies

Figures & data

Table 1. Central composite design used for optimization of LVC-loaded CCPCs.

Factors (Independent variables)	Factor type	Levels
		(−1)		(+1)
X1: Ceramide amount (mg)	Numeric	10		30
X2: HA (mg)	Numeric	5		15
X3: DDAB amount (mg)	Numeric	5		15
Responses (Dependent variables)		Desirability Constraints
Y1: EE%		Maximize
Y2: PS (nm)		Minimize
Y3: ZP (mV)		Maximize (absolute value)

Abbreviation: HA: hyaluronic acid; EE%: entrapment efficiency percent; DDAB: dimethyldidodecylammonium bromide; LVC: levocetirizine hydrochloride; PS: particle size; ZP: zeta potential, and CCPCs; cationic ceramide/phospholipid composite.

Table 2. The atomic composition of the molecular dynamics simulated LVC-loaded CCPCs.

Solvation State	Atomic composition (atom numbers)
	LVC	PC	Ceramide III	Phytantriol	DDAB	HA	Water	Total
100% Water	1 × 52	1 × 134	1 × 112	1 × 65	1 × 83	1 × 49	3 × 3359	10,572

Abbreviation: HA: hyaluronic acid; DDAB: dimethyldidodecylammonium bromide; LVC: levocetirizine hydrochloride, PC; phospholipid, and CCPCs; cationic ceramide/phospholipid composite.

Table 3. Experimental runs, independent variables, and measured responses of LVC-loaded CCPCs.

F	Ceramide amount (mg)	HA amount (mg)	DDAB amount (mg)	EE%	PS (nm)	PDI	ZP (mV)
F1	10	5	5	88.36 ± 0.34	479.00 ± 50.34	0.377 ± 0.003	20.20 ± 1.13
F2	10	5	15	62.57 ± 0.71	342.00 ± 0.21	0.292 ± 0.061	26.40 ± 0.49
F3	10	10	10	75.51 ± 6.05	438.00 ± 13.08	0.493 ± 0.013	13.20 ± 1.13
F4	10	15	5	84.23 ± 4.86	400.00 ± 28.28	0.262 ± 0.004	15.80 ± 0.28
F5	10	15	15	69.63 ± 1.46	603.00 ± 53.74	0.530 ± 0.060	19.40 ± 0.35
F6	20	5	10	68.00 ± 4.00	756.80 ± 40.16	0.182 ± 0.0021	27.60 ± 0.91
F7	20	10	5	76.96 ± 5.16	592.45 ± 37.08	0.179 ± 0.036	10.00 ± 0.41
F8	20	10	10	61.00 ± 1.00	700.60 ± 7.84	0.271 ± 0.016	14.70 ± 1.97
F9	20	10	10	60.50 ± 0.45	720.60 ± 56.49	0.165 ± 0.024	17.50 ± 0.42
F10	20	10	10	69.61 ± 0.34	679.60 ± 20.93	0.270 ± 0.090	10.50 ± 0.84
F11	20	10	10	64.29 ± 2.19	700.60 ± 29.76	0.160 ± 0.028	12.50 ± 0.56
F12	20	10	10	69.62 ± 0.24	720.60 ± 14.14	0.260 ± 0.007	15.50 ± 0.28
F13	20	10	10	67.84 ± 0.54	679.60 ± 20.93	0.150 ± 0.010	16.90 ± 0.14
F14	20	10	15	60.35 ± 0.63	572.90 ± 12.02	0.591 ± 0.026	15.70 ± 0.49
F15	20	15	10	70.11 ± 1.43	740.70 ± 27.71	0.342 ± 0.054	16.70 ± 0.42
F16	30	5	5	80.52 ± 3.08	853.20 ± 9.33	0.325 ± 0.036	20.70 ± 0.77
F17	30	5	15	70.60 ± 3.95	536.40 ± 19.79	0.336 ± 0.030	34.40 ± 1.55
F18	30	10	10	82.55 ± 3.67	687.50 ± 26.51	0.141 ± 0.060	19.40 ± 0.28
F19	30	15	5	86.53 ± 5.16	745.80 ± 14.14	0.251 ± 0.009	7.56 ± 0.48
F20	30	15	15	89.32 ± 3.50	708.30 ± 12.94	0.556 ± 0.080	20.80 ± 0.07

Presented values are the mean ± SD (n = 3).

^aAll formulae contained 25 mg LVC and 100 mg phosphatidylcholine, in a volume of 10 ml.

Abbreviation: HA: hyaluronic acid; DDAB: dimethyldidodecylammonium bromide; EE%: entrapment efficiency percent; PS: particle size; PDI: polydispersity index; ZP: zeta potential; LVC: levocetirizine hydrochloride, and CCPCs; cationic ceramide/phospholipid composite.

Table 4. Output data of the central composite design and predicted and observed values for the optimum CCPCs.

Responses	EE%	PS (nm)	ZP (mV)
Adequate precision	14.25	22.24	20.39
Adjusted R^2	0.887	0.944	0.861
Predicted R^2	0.866	0.849	0.849
Significant factors	X1, X2, X3	X1, X2, X3	X2, X3
Predicted value of the selected formula	88.84	484.53	21.84
Observed value of the selected formula	88.36	479.00	20.20

Abbreviations: EE%, entrapment efficiency percentage; CCPCs, cationic ceramide/phospholipid composite; PS, particle size, and ZP; zeta potential.

Figure 1. Effect of formulation variables on EE% of LVC-CCPCs (A-B) and PS (C-D).

Abbreviation: EE%: Entrapment efficiency percentage, PS: Particle size, LVC: levocetirizine hydrochloride and CCPCs; cationic ceramide/phospholipid composite.

Figure 2. Effect of formulation variables on ZP of LVC-CCPCs (E-F) and desirability figure(G).

Abbreviation: ZP: zeta potential; LVC: levocetirizine hydrochloride and CCPCs; cationic ceramide/phospholipid composite.

Table 5. Stability study of the optimum CCPCs.

Parameter*	Fresh CCPCs	Stored CCPCs (3 months)
EE%	88.36 ± 0.34	87.50 ± 1.60
PS (nm)	479.00 ± 50.34	467.80 ± 10.10
PDI	0.377 ± 0.003	0.356 ± 0.001
ZP (mV)	20.20 ± 1.13	19.50 ± 0.06

* Mean ± SD (n = 3).

Abbreviation: EE%: entrapment efficiency percent; PS: particle size; PDI: polydispersity index; ZP: zeta potential, and CCPCs; cationic ceramide phospholipid composite.

Figure 3. Transmission electron micrographs of LVC-loaded CCPCs.

Abbreviation: LVC: levocetirizine hydrochloride and CCPCs; cationic ceramide/phospholipid composite.

Figure 4. Predicted docking pose of LVC-PC binding complex. Stick 3 D-representation of LVC (yellow) loaded on PC interface (green), in presence of several CCPCs additives; ceramide (cyan), DDAB (blue), Phytanriol (orange), and HA (magenta). All introduced formulation additives served as either direct or indirect connecting/supportive platforms for favored anchoring of LVC within the drugphospholipid complex. Polar interactions (hydrogen bond) are represented as black dashed lines.

Abbreviation: HA: hyaluronic acid; DDAB: dimethyldidodecylammonium bromide; PC: phospholipid; LVC: levocetirizine hydrochloride and CCPCs; cationic ceramide/phospholipid composite.

Figure 5. Time evolution conformational alterations of LVC-PC-CCPCs additive heterocomplex throughout an explicit MD simulation run at a 100% aqueous solvation system. The thermodynamic movements and stability of formulation components; LVC (yellow) loaded on PC interface (green), in presence of several CCPCs; Ceramide (cyan), DDAB (blue), Phytantriol (orange), and HA (magenta), were monitored over MD simulation trajectories being captured at different snapshots ① 0.2 ns, ② 0.4 ns; ③ 0.6 ns; ④ 0.8 ns; and ⑤ 1 ns. Polar interactions (hydrogen bond) are represented as black dashed lines.

Abbreviation: HA: hyaluronic acid; DDAB: dimethyldidodecylammonium bromide; PC: phospholipid; LVC: levocetirizine hydrochloride and CCPCs; cationic ceramide/phospholipid composite; MD: molecular dynamic simulation.

Figure 6. Overlay of LVC-PC-CCPCs additive heterocomplex across MD simulation frames (left panel) and molecular surface 3 D-representation of the inverted cone micellar configuration at 100% aqueous solvation system (right panel). Molecular surface and sticks 3 D representations were illustrated in colors being previously assigned for the optimized formulation components; yellow, green, cyan, blue, orange, and magenta for LVC, PC, Ceramide, DDAB, Phytantriol, and HA, respectively.

Abbreviation: HA: hyaluronic acid; DDAB: dimethyldidodecylammonium bromide; PC: phospholipid; LVC: levocetirizine hydrochloride and CCPCs; cationic ceramide/phospholipid composite; MD: molecular dynamic simulation.

Figure 7. Ex-vivo permeation profile of LVC from CCPCs, compared to its aqueous solution.

Abbreviation: LVC: levocetirizine hydrochloride and CCPCs; cationic ceramide/phospholipid composite.

Table 6. Permeation of LVC solution compared to the optimum CCPCs and permeation parameters.

Time (hr)	LVC solution (μg/ml)	CCPC (μg/ml)
1	3.07 ± 0.63	1.29 ± 0.33
2	3.15 ± 1.21	1.80 ± 0.09
4	3.28 ± 0.06	2.16 ± 0.20
6	3.33 ± 0.15	2.65 ± 0.38
8	3.84 ± 0.46	4.02 ± 0.64
24	15.87 ± 6.90	9.28 ± 2.96
Permeation parameters	LVC solution (μg/ml)	CCPC (μg/ml)
Q24 (mg/cm^2)	15.87 ± 6.90	9.28 ± 2.96
Dep24 (mg/cm^2)	146.47 ± 27.64	202.38 ± 14.45
Jmax (mg/h cm^2)	1.32 ± 0.57	0.77 ± 0.24
LAEI	12.04 ± 6.47	24.69 ± 8.99
LAEI ratio		2.05

* Mean ± SD (n = 3).

Q₂₄: cumulative amount permeated per unit area after 24 h, Dep₂₄: cumulative amount deposited per unit area after 24 h, J_max: maximum average flux after 24 h, LAEI: Local accumulation efficiency index; LVC: levocetirizine hydrochloride, and CCPCs: cationic ceramide/phospholipid composite.

Figure 8. In-vivo skin deposition profile of LVC from CCPCs, compared to its aqueous solution.

Abbreviation: LVC: levocetirizine hydrochloride, and CCPCs; cationic ceramide/phospholipid composite.

Figure 9. Light microscope photomicrographs showing histopathological sections (hematoxylin and eosin-stained) of rat skin in normal control (group I), rat skin treated with LVC solution (group II), and rat skin treated with optimum CCPCs (group III) with a magnification power of 16X to illustrate all skin layers (Left side) and magnification power of 40X to identify the epidermis and dermis (Right side).

Abbreviation: LVC: levocetirizine hydrochloride, and CCPCs; cationic ceramide/phospholipid composite.