Exploring a Novel Fasudil-Phospholipid Complex Formulated as Liposomal Thermosensitive in situ Gel for Glaucoma

Figures & data

Table 1 Optimization Parameters for Preparation of Fasudil Phospholipid Complex (Data are Presented as Mean ± SD, n = 3)

Molar Ratio of Drug: Phospholipid	Reaction Temperature (°C)	Reaction Time (h)	Complexation Efficiency (%)*
1: 1	25 °C (Room temp.)	1	34.8±0.53
		2	49.2±0.21
	40 °C (Reflux)	1	62.0±0.13
		2	85.0±0.23
1: 2	25 °C (Room temp.)	1	42.5±0.31
		2	55.3±0.21
	40 °C (Reflux)	1	63.0±0.43
		2	89.4±0.37

Note: *.

Figure 1 (A) FT-IR spectra and (B) XRD patterns of Fas, PL, physical mixture and Fas/PL complex.

Table 2 Physicochemical Characterization of Different Batches of B-Lipo_disp Prepared by Methanol Injection Method (Data are Presented as Mean ± SD, n = 3)

Batch	Molar Ratio of Phospholipid: Cholesterol	Solvent/ Nonsolvent Volume Ratio	Stirring Rate (rpm)	Mean Particle Size (nm)	PdI
F1	1:0	0.6	800	422.6±3.4	0.4±0.05
F2	2:1	0.6	800	125.7±2.9	0.1±0.002
F3	4:1	0.6	800	198.6±1.8	0.3±0.04
F4	2:1	0.8	800	940.3±21.2	0.7±0.15
F5	2:1	0.6	400	320.8±12.9	0.3±0.05
F6	2:1	0.6	200	442.5±17.8	0.4±0.11

Figure 2 TEM images of liposomal formulations at magnification x25,000. Scale bars represent 200 nm.

Table 3 Gelling Properties and Viscosity Data of P407 in situ Gels and P407/HPMC in situ Gels (Data are Presented as Mean ± SD, n = 3)

In situ Gel Formula	Poloxamer % w/w	HPMC % w/v	Gelling Temperature (°C)	Gelling Time (Seconds)	Viscosity at 25°C (cP)	Viscosity at 37°C (cP)
BP407 gel	17.5	-	66.5±0.4	213.2±2.9	7.9±0.1	20.9±0.9
	20	-	36.1±0.3	22.6±0.2	23.1±0.9	951.2±7.2
	22.5	-	31.8±0.4	12.2±0.2	90.9±0.9	1620±9.6
	25	-	29.3±0.2	10.7±0.3	148.1±6.2	1980±13.4
BP407/HPMC gel	17.5	0.5	42.8±0.4	118.9±4.6	20.5±0.6	357.2±11
	20	0.5	32.8±0.3	10.7±0.3	56.1±6.8	1392±5.7
	22.5	0.5	28.5±0.3	8.5±0.4	135.2±21.1	1830±29.3
	25	0.5	27.6±0.2	6.9±0.1	219.7±23.8	2109±93.9
Fas/PL-LipoP407 gel	17.5	-	56.3±1.8	199.8±6.9	12.8±0.3	37.5±0.8
	20	-	35.5±0.2	18.2±0.6	42.2±0.4	966.5±5.6
	22.5	-	30.2±0.2	11.1±0.6	101.2±2.7	1673.5±19.3
	25	-	28.9±0.2	9.1±0.2	163.4±7.2	2029±26.8
Fas/PL-LipoP407/HPMC gel	17.5	0.5	45.6±0.7	124±6.5	54.9±1.9	374.9±15.7
	20	0.5	33.9±0.3	13.1±0.3	70.2±1.2	1256±18.1
	22.5	0.5	29.7±0.4	9.3±0.3	167.7±5.1	1759±43.8
	25	0.5	28.1±0.2	8.2±0.1	256.4±9.3	2088±75.5

Note: Shaded rows represent optimized formulations selected for further study.

Figure 3 (A) Rheogram and (B) Thixotropic behavior of in situ gel formulations at physiological temperature.

Table 4 Storage Stability Data at 4°C of Fas/PL-Lipo_disp and Fas/PL-Lipo_P407/HPMC gel (Data are Presented as Mean ± SD, n = 3)

Formulation	Fas/PL-Lipodisp		Fas/PL-LipoP407/HPMC gel
Time	Freshly Prepared	After 3 Months	Freshly Prepared	After 3 Months
Particle size (nm)	132.5±1.6	137.2±2.1	238.7±3.8	270.5±4.6
PdI	0.2±0.01	0.2±0.02	0.2±0.1	0.2±0.05
Zeta potential	− 21.6±0.93	− 24.4±0.7	27.2±1.5	25.2±2.3
% EE	78.6±0.25	75.3±0.4	77.9±0.5	74.8±0.3

Figure 4 Release profiles of tested formulations over a 24 h period in STF pH 7.4 at 50 rpm and 35 °C (n=3).

Figure 5 (A) Viscosity values of mucin dispersion in water (20% w/v), formulations, theoretical sum of formulation and mucin viscosities and the viscosity of formulation/mucin 2:1 mixtures (B) Rheological synergism of the four tested formulations upon mixing with mucin at a 2:1 ratio represented by Δη % (mean ± SD, n=3).

Table 5 Ex vivo Corneal Permeation Flux* Values

Time	Parameters	Fas Solution	Fas/PL-Lipodisp	FasP407/HPMC gel	Fas/PL-LipoP407/HPMC gel
1–3 h	Regression equation	y = 101.55x - 76.69	y = 83.628x - 63.85	y = 77.49x - 58.48	y = 64.68x - 48.22
	R^2	0.997	0.993	0.998	0.993
	Flux (µg/cm^2/h)	101.55±5.66	83.63±4.85	77.49±4.68	64.68±2.93
3–6 h	Regression equation	y = 80.912x + 6.996	y = 197.5x - 375.24	y = 206.68x - 427.1	y = 264.87x - 651.83
	R^2	0.962	0.987	0.995	0.999
	Flux (µg/cm^2/h)	80.91±4.28	197.5±2.78	206.68±3.43	264.87±3.31
Percent corneal hydration**		80.8±0.8	76.5±1.5	77.2±0.9	78.9±0.5

Notes: *Flux values were calculated from slopes of linear plots of ug permeated per cm² vs time (Figure 6A). **Measured at 6 h of the permeation runs.

Figure 6 (A) Ex vivo corneal permeation profile of fasudil from Fas/PL-Lipo_disp, Fas_{P407/HPMC gel} and Fas/PL-Lipo_{P407/HPMC gel} compared to Fas solution (mean ± SD, n=3) (B) Ex vivo corneal permeation flux values.

Figure 7 (A) Photographs of hen’s egg test-chorioallantoic membrane (HET-CAM) after treatment at room temperature to predict ophthalmic irritation potential and (B) Average grey value of pixels of test formulations analyzed using Image J software as an indicator of ocular irritation (n=4).

Figure 8 Percentage decrease in IOP after ocular application of 0.5% Fas solution, Fas/PL-Lipo_disp, Fas_{P407/HPMC gel} and Fas/PL-Lipo_{P407/HPMC gel}. Data expressed as mean ± SD (n = 3).

Table 6 Pharmacodynamic Parameters of Fas Solution, Fas/PL-Lipo_disp, Fas_{P407/HPMC Gel} and Fas/PL-Lipo_{P407/HPMC gel}

Formulation	Tmax (h)	Maximum Decrease in IOP (%) ± SD	AUC0-24h (%·h) ± SD*
Fas solution	2	38.6±0.01	215.1±9.4
Fas/PL-Lipodisp	4	41.5±1.6	439.9±24.4
FasP407/HPMC gel	4	48.8±0.7	686.1±10.3
Fas/PL-LipoP407/HPMC gel	6	52.1±1.3	761.4±27.7

Note: *Calculated from Figure 8 by the trapezoidal rule.

Figure 9 Correlation between ex vivo cumulative amount permeated (Figure 6A) and in vivo cumulative area under the pharmacodynamic curve (Figure 8) at corresponding times over 24 h.