Cyclosporine Amicellar delivery system for dry eyes

Figures & data

Table 1 Particle size and transparency of different micelle solutions

Formulation	Composition (weight%)			Particle size (nm)
	Cremophor EL	Ethanol	Glycerin	Mean diameter	Transparency
Ethanol 0 system
Z1	0.01	0	2.2	–	–
Z2	0.05	0	2.2	–	–
Z3	0.5	0	2.2	–	–
Z4	1	0	2.2	–	–
Z5	3	0	2.2	17.50±0.26	✓
Z6	4	0	2.2	17.73±0.15	✓
Z7	5	0	2.2	17.27±0.31	✓
Ethanol 0.5% system
F1	0.01	0.5	2.2	359.43±6.1	×
F2	0.05	0.5	2.2	192.13±3.76	×
F3	0.5	0.5	2.2	213.93±6.97	×
F4	1	0.5	2.2	14.73±0.21	✓
F5	3	0.5	2.2	17.3±0.1	✓
F6	4	0.5	2.2	17.53±0.38	✓
F7	5	0.5	2.2	17.33±0.29	✓
Ethanol 1% system
O1	0.01	1	2.2	199.77±7.23	×
O2	0.05	1	2.2	168.1±2.4	×
O3	0.5	1	2.2	245.17±4.31	×
O4	1	1	2.2	14.53±0.4	✓
O5	3	1	2.2	17.37±0.25	✓
O6	4	1	2.2	17.53±0.06	✓
O7	5	1	2.2	17.2±0.3	✓

Notes: Z1–Z4 did not form micelle solutions in this study. Transparency was assessed visually. Data expressed as mean ± standard deviation (n=3). “✓” Transparent micelle solution; “×” non-transparent solution; “–” not formed solution.

Figure 1 Size distribution (by volume) of CsA eyedrops obtained by DLS for (A) O7 MS and (B) Restasis.

Note: Bar refers to the differential volume (%), and line graph refers to the cumulative volume (%).

Abbreviations: CsA, cyclosporine A; DLS, dynamic light scattering; MS, micelle solution.

Table 2 Changes in particle size of MS-CsA stored at room temperature and 4°C

	Ethanol 0 system			Ethanol 0.5% system			Ethanol 1% system
	Z5	Z6	Z7	F5	F6	F7	O5	O6	O7
Day	Storage at room temperature
4	18.13	18.47	19.03	18.07	18.83	18.7	18.53	18.93	18.2
7	19.43	19.97	20.27	19.73	19.47	19.57	19.83	20.27	20.93
10	18.77	18.8	18.2	19.3	18.97	19.37	18.9	19.2	19.57
14	20.33	20.07	20.1	19.97	19.8	19.83	21.73	20	19.83
21	20.8	20.73	20.8	21	20.37	20.13	20.6	20.8	20.07
30	20.8	21.03	21.17	20.63	20.53	20.37	20.4	20.1	20
60	18.53	19.27	19.47	19.07	19.63	19.5	19.4	19.5	18.93
	Storage at 4°C
4	18.2	18.47	18.7	18.37	18.7	19.07	17.97	17.9	18.77
7	18.5	18.9	18.23	18.5	19.6	19.7	20.23	19.67	19.5
10	18.17	19	19.33	19.4	19.47	19.53	19.7	19.9	19.83
14	21.73	21.87	21	21.37	21.27	21.9	21.2	20.83	19.7
21	19.53	19.33	19.2	19.37	19.73	18.8	19.63	19.5	19.3
30	20.4	21.13	21.5	20.8	20.87	20.73	20.83	21.03	21.33
60	19.27	18.5	18.07	18.03	18.8	18.63	18.97	18.33	18.47

Note: Data are expressed as mean (n=3).

Abbreviation: MS-CsA, micelle solution of cyclosporine A.

Table 3 pH and osmolarity analysis of different micelle formulations

Formulation	pH			Osmolarity (mOsm)
	Initial pH	After 60 days	Increasing ratioa	Initial osmolarity	After 60 days	Increasing ratiob
Stored at room temperature over 60 days
Z5	7.41±0.01	7.39±0.02	0.997	317.4±7.64	303.7±4.62	0.957
Z6	7.42±0.02	7.38±0.06	0.995	316.77±12.86	299.87±3.26	0.947
Z7	7.43±0.03	7.4±0.03	0.996	311±10.65	298.57±3.95	0.96
F5	7.43±0.03	7.41±0.03	0.997	305.5±3.51	296.6±13.67	0.971
F6	7.47±0.02	7.41±0.02	0.992	298.30±9.54	304.4±5.03	1.02
F7	7.44±0.01	7.4±0.01	0.995	299.63±2.68	296.6±10.82	0.99
O5	7.42±0.03	7.42±0.05	1	309.53±12.11	301.67±4	0.975
O6	7.46±0.01	7.4±0.05	0.992	311.13±4.3	297.47±2.95	0.956
O7	7.45±0.03	7.39±0.07	0.992	306.37±3.46	300.3±3.02	0.98
Stored at 4°C over 60 days
Z5	7.41±0.01	7.4±0.01	0.999	317.4±7.64	309.03±7.8	0.974
Z6	7.42±0.02	7.41±0.02	0.999	316.77±12.86	307.17±7.48	0.97
Z7	7.43±0.03	7.39±0.09	0.995	311±10.65	304.87±14.2	0.98
F5	7.43±0.03	7.41±0.08	0.997	305.5±3.51	297.7±7.57	0.974
F6	7.47±0.02	7.37±0.05	0.987	298.3±9.54	300.13±2.99	1.006
F7	7.44±0.01	7.41±0.04	0.996	299.63±2.68	303.27±10.05	1.012
O5	7.42±0.03	7.39±0.06	0.996	309.53±12.11	302±9.24	0.976
O6	7.46±0.01	7.39±0.07	0.991	311.13±4.3	298.27±3.7	0.959
O7	7.45±0.03	7.42±0.03	0.996	306.37±3.46	300.03±5.02	0.979

Notes:

a Increasing ratio = pH after 60 days/initial pH;

b increasing ratio = osmolarity after 60 days/initial osmolarity. Data expressed as mean ± standard deviation (n=3). Information about the formulation names is shown in Table 1.

Figure 2 Changes in chemical stability (remaining CsA) of MS-CsA stored at (A) room temperature and (B) 4°C at different time intervals over 60 days.

Note: Data expressed as mean ± standard deviation (n=3).

Abbreviation: MS-CsA, micelle solution of cyclosporine A.

Figure 3 (A) STT scores in the AS-treated group (control) at different time points and (B) according to CsA-formulation treatment in the rabbit dry-eye model.

Notes: *P<0.05 versus STT score of day 1; ^#P<0.05 (one-way ANOVA between the three groups). Data expressed as mean ± standard deviation (n=8).

Abbreviations: AS, atropine sulfate (1% solution); STT, Schirmer tear test; CsA, cyclosporine A; MS, micelle solution; ANOVA, analysis of variance.

Table 4 Schirmer tear test scores with cure

	Day 1	Day 3	Day 5
Group 1
AS + O7	11.23±1.1	12.13±1.84a	13.16±2.21a
AS + NS	9.85±1.78	7.78±2.16	6.81±3.08
P1	0.084	0.0007	0.0003
Tear-difference value	1.38±1.79	4.35±1.54	6.35±4.49
Group 2
AS + O4	10.76±1.86	11.16±3.12b	11.65±1.58b
AS + NS	9.93±1.59	7.25±3.14	6.75±2.45
P2	0.3497	0.0254	0.0003
Tear-difference value	0.84±1.8	3.91±4.73	4.90±2.96
Group 3
AS + Restasis	11.06±1.5	10.29±2.07	10.4±2.11c
AS + NS	9.81±2.74	7.58±3.44	6.8±1.94
P3	0.2763	0.0767	0.0032
Tear-difference value	1.25±3.55	2.71±3.12	3.6±3.27

Notes:

a P<0.05 versus AS + NS in group 1 on the same day;

b P<0.05 versus AS + NS in group 2 on the same day;

c P<0.05 versus AS + NS in group 3 on the same day. Data expressed as mean ± standard deviation (n=8). All solutions administered at 50 μL.

Abbreviations: AS, atropine sulfate (1% solution); NS, normal saline.

Table 5 Average conjunctival goblet-cell density

	Goblet-cell density	Difference
Group 1
AS + O7	94.83±8.38a,b	51.17±6.43
AS + NS	43.67±4.76
Group 3
AS + Restasis	65.17±11.51c	24.67±9.33
AS + NS	40.5±7.01

Notes:

a P<0.05 versus AS + NS in group 1;

b P<0.05 versus AS + Restasis in group 3;

c P<0.05 versus AS + NS in group 3. Data expressed as mean ± standard deviation (n=6, three separate tissues by two independent masked observers).

Abbreviations: AS, atropine sulfate (1% solution); NS, normal saline.

Figure 4 Images of histologic sections from the rabbit conjunctiva stained with H&E.

Notes: (A) Administration of normal saline only; (B) administration of 1% atropine sulfate solution only; (C) AS + NS administration in group 1; (D) AS + O7 administration in group 1; (E) AS + NS administration in group 3; (F) AS + Restasis administration in group 3. Goblet cells are stained purple and located in the superficial epithelium. Epithelia in (B), (C), and (E) were thinned with loss of goblet cells. Goblet-cell morphology in (D) was relatively well formed. Magnification 40×. All solutions administered at 50 μL.

Abbreviations: AS, atropine sulfate (1% solution); H&E, hematoxylin and eosin; NS, normal saline.