Review of Preclinical Outcomes of a Topical Cationic Emulsion of Cyclosporine A for the Treatment of Ocular Surface Diseases

Figures & data

Figure 1. Principle of the use of cationic emulsion of CsA (CE-CsA) on the ocular surface. (a) Schematic structure of the CE-CsA and (b) interaction with the ocular surface.

Table 1. Similarities between the composition of the tear film and a cationic emulsion.

	Function	Healthy tear film composition	Cationic emulsion composition
Lipid phase	To prevent desiccation of the ocular surface.	<1%*	2.005%^#
Polar lipids in the lipid phase	To stabilize the lipid phase	Up to 8%^Citation50–52	0.25%

*40-90 nm of lipid layer thickness for an aqueous layer thickness of up to 4000 nm⁵¹; ^#2% medium-chain triglycerides + 0.005% CKC.

Table 2. Overview of the pharmacodynamics studies realized on the CE-CsA.

Study types	Cell/Tissue/Animal Model	Assessments	Main results	Ref.
In vitro studies	Human corneal epithelial cell line with induced of desiccating stress.	Quantification of inflammatory factors (p-NF-kB p65, p-IkBa and TNF-α).	Decrease of the inflammatory factors for CE-CsA compared to AE-CsA.	^Citation54
	Peripheral blood mononuclear cells, CD4 + T lymphocytes and human corneal epithelial cell line.	Quantification of inflammatory factors after stress exposure to stimulation with anti-CD3/anti-CD28 or lipopolysaccharide (LPS).	Decreased inflammatory gene expression (IFN-γ, IL-17A, CXCL-9 and TNF-α, THBS1 and CCL-2) in presence of CE. Decreased secretion of IL-17, TNFα, IFN-γ, IL-2, IL-6 and IL-8 in presence of CE.	^Citation55
	Human corneal epithelial cell line with scrapping area	Wound healing analysis (fluorescence microscopy)	Similar behavior of wound healing for CE-CsA, AE-CsA, and PBS.	^Citation56,Citation57
In vivo studies	Mice with spontaneous development of Sjögren’s syndrome (NOD.B10.H2^b model)	Tear volume (PRT lacrimation test), corneal staining (lissamine), conjunctival goblet cell density and IL1-β quantification	Significant increase of tear volume at days 30 and 45 for CE-CsA and nanomicelle-CsA (Cequa®) groups compared to AE-CsA group. No significant difference of corneal staining, goblet cell density and IL-1β concentration between CE-CsA, nanomicelle-CsA, and AE-CsA groups.	^Citation58
	C57BL/6 N mice with DED induced in a controlled environment room	Tear volume (PRT lacrimation test), corneal staining (fluorescein), histological analysis of the ocular surface (HES staining).	Increased tear volume for both CE-CsA and AE-CsA groups. Reduced CFS scores for CE-CsA group compared to AE-CsA group. Only 2 out 10 mice presented ocular lesions for CE-CsA group compared to 5 mice for AE-CsA group.	^Citation59
	C57BL/6 N mice with DED induced in a controlled environment room	Corneal staining (fluorescein), measurement of the expression profile of inflammatory genes	After 7 days of treatment, CFS scores were reduced by 21% and 31% with AE-CsA and CE-CsA, respectively. Decreased gene expression of IL-1α and TLR-4 for both AE-CsA and CE-CsA groups. Decreased gene expression of H2-Eb1, IL-1β, IL-1RN, IL-6, TGF-β2, TGF-β3, TLR-2 and TLR-3, only for CE-CsA group.	^Citation60
	C57BL/6 N mice with DED induced in a controlled environment room	Corneal staining (fluorescein), measurement of the expression profile of inflammatory genes	Reduced CFS scores (vs. dry eye baseline) by 9.2, 18.5 and 22.8% with lifitegrast solution (Xiidra®), CE and CE-CsA, respectively. Difference in gene expression (IL-6, CXCL-1, CXCL-10, CCL-19 and TNF) between lifitegrast solution, CE and CE-CsA groups.	^Citation61
	C57BL/6 N mice with DED induced in a controlled environment room	Tear volume (PRT lacrimation test), corneal staining (fluorescein)	Reduced CFS scores (vs. dry eye baseline) by 28, 36 and 59% with 1% methylprednisolone, CE and CE-CsA, respectively.	^Citation62,Citation63
	C57BL/6 N mice with DED induced in a controlled environment room	Tear volume (PRT lacrimation test), corneal staining (fluorescein).	Significant increased tear volume only for CE-CsA. Reduced CFS scores for both AE-CsA and CE-CsA groups.	^Citation64
	Rats with corneal scraping	Quantification of corneal wound healing and inflammatory cell infiltration by IVCM	5 days after scrapping, IVCM scores and cell infiltration were found lower for CE-CsA group compared to CE, 0.2% BAK solution or NaCl.	^Citation65

Abbreviations: AE = Anionic Emulsion; BAK = benzalkonium chloride; CCL = C-C motif Ligand; CE = Cationic Emulsion; CFS = Corneal Fluorescein Staining; CsA = Cyclosporine A; CXCL = C-X-C motif Ligand; DED = Dry Eye Disease; H2-Eb1 = H-2 class II histocompatibility antigen; HES = Hematoxylin/Eosin/Safran; IFN = Interferon; IL = Interleukin; IVCM = In Vivo Confocal Microscopy; PBS = Phosphate Buffered Solution; PRT = Phenol Red Thread; TGF = Tumor Growth Factor; TLR = Toll-like Receptor; THBS1 = Thrombospondin.

Figure 2. Main outcomes regarding pharmacodynamics of CsA cationic emulsions. (a) Effects of the cationic emulsions of cetalkonium chloride (CE-CKC) emulsions, IKK Inhibitor X (IKK Inh X), dexamethasone (DXM), and cyclosporine A (CsA) on IL-6 and IL-8 release by HCE-2 cells following lipopolysaccharide (LPS) stimulation. *p < .05; **p < .01; ***p < .001.⁵⁵ (Adapted from reference ⁵⁵). (b) Percentage of CFS score reduction after 10 days-treatment using 1% methylprednisolone (MePre), CE (CE-CsA veh) and CE-CsA on an in vivo mouse dry eye model. **p < .01; ***p < .001; ****p < .0001.⁶² (Adapted from reference ⁶²). (c) Wound closure from day 1 to day 3 after the different treatments. * p < .0001–0.02 compared to the other four groups at the corresponding times.⁵⁶ (Adapted from reference ⁵⁶). (d) Fold changes and t-test values (vs. DED untreated) for the 23 genes detected among the 34 genes followed in this study in the DED (± treatment) mice corneas. (LE, loteprednol etabonate).⁶⁰ (Adapted from reference ⁶⁰).

Table 3. Overview of the pharmacokinetics studies realized on the CE-CsA.

Study types	Cell/Tissue/Animal Model	Assessments	Main results	Ref.
Ex vivo studies	Porcine eyeballs	CsA quantification in the corneas 1 h after eye drops instillation (HPLC analysis in homogenized tissues).	Increased CsA concentration in the corneas treated with CE-CsA compared to AE- CsA. Decreased CsA concentration in the corneas treated with semifluorinated alkane-based eye drops compared to CE-CsA.	^Citation72
	Porcine eyeballs	CsA quantification in the corneas after eye drops instillation and washing (HPLC analysis in homogenized tissues).	No significant difference in the CsA concentrations for the corneas treated with CE-CsA compared to Poloxamer 407/TPGS mixed micelles.	^Citation73
In vivo studies	Pigmented rabbits	Quantification of CsA in conjunctiva and cornea at different time points.	AUC(0.5–24h) 5.4 and 3.9 times higher for CE-CsA compared to CsA hospital-compounded preparations, in the cornea and the conjunctiva, respectively.	Data on file
	Pigmented rabbits	Quantification of CsA concentrations in ocular tissues at different time points.	Higher Cmax and AUC to the cornea and conjunctiva for CE-CsA 0.1% and 0.05% compared to AE-CsA.	^Citation74

Abbreviations: AE = Anionic Emulsion; AUC = Area Under the Curve; CE = Cationic Emulsion; C_max = Maximal Concentration; HPCL = High Performance Liquid Chromatography.

Figure 3. Main outcomes regarding pharmacokinetics of CE-CsA. (a) Changes in CsA concentration with time after a single unilateral topical administration in the cornea of pigmented rabbits.⁷⁴ (Adapted from reference ⁷⁴). (b) Pharmacokinetics profile of CsA delivery after a single unilateral topical administration of CE-CsA or CsA hospital-compounded preparations (CsA-HP) in the cornea and conjunctiva of pigmented rabbits.

Table 4. Overview of the ocular toxicity and tolerance studies realized on the CE-CsA.

Study types	Cell/Tissue/Animal Model	Assessments	Main results	Ref.
In vivo studies	Mice	Non-radioactive local nymph node assay (LLNA) using 5-BrdU incorporation.	CE-CsA did not induce delayed contact hypersensitivity.	Data on file
	Mice	Phototoxicity and photosensitizing (photoallergic) skin tests.	No phototoxic or photosensitizing (photoallergic) potential observed for CE-CsA or CE.	Data on file
	New Zealand albino rabbits	28-day tolerance study. Eye irritation test (Draize test). Histological analysis	CE-CsA was found safe and well tolerated following 4 and 6 daily instillations over 28 days.	Data on file
	Rabbits	Clinical observation (IVCM), eye irritation test (Draize test)	Lowest toxicity similar to PBS for CE-CsA group.	^Citation56,Citation57
	New Zealand albino rabbits	Conjunctiva changes using slit-lamp examination, flow cytometry and impression cytology. Corneal changes using IVCM. Histological analysis	After 15 instillations at 5 min intervals, highest toxicity induced by BAK-solution. Moderate toxicity for BAK-CE and CKC-solution. Low toxicity similar to PBS for CKC-CE. Lower CD45+ cell infiltration and apoptotic cells for BAK-CE and CKC-CE compared to BAK- or CKC-solutions.	^Citation75

Abbreviations: AE = Anionic Emulsion; BrdU = Bromodeoxycytidine; CE = Cationic Emulsion; IVCM = In Vivo Confocal Microscopy.

Figure 4. Main outcomes regarding ocular toxicity and tolerance of CE-CsA. (a) Microphotographs of typical clinical features of rabbit eyes after 15 instillations at 5-min interval of 0.002% CKC or 0.02% BAK containing in a solution (sol) or a CE.⁷⁵ (b) Draize test score calculated at different time points (75 min, 4h and 1 day) after 15 instillations of CE-CsA, AE-CsA, Oil-CsA or 0.02% BAK. * p < .02 compared to PBS and p < .004 compared to 0.02% BAK. # p < .01 compared to AE-CsA, Oil-CsA, and 0.02% BAK. ♦ p < .0001 compared to PBS. $ p = .0003 compared to PBS, CE-CsA, AE-CsA, and Oil-CsA groups.⁵⁶ (Adapted from reference ⁵⁶).

Table 5. Similarities between preclinical outcomes and results of clinical studies.

	Preclinical outcomes	Results of clinical studies
Ocular inflammation	Reduced expression and secretion of pro-inflammatory factors^Citation55,Citation60	Lower expression of a cell surface inflammatory marker (HLA-DR) for the group treated with CE-CsA and CE.^Citation70,Citation71,Citation76
Tear film volume	Increased tear fluid volume (PRT: Phenol Red Thread Test)^Citation59	72.4% and 58.2% improvement in Schirmer tear test observed with CE-CsA and CE treatment, respectively.^Citation76
Tear fluid stability	Increased tear fluid elasticity and thickness^Citation49	40.5% and 22.2% improvement in TBUT observed with CE-CsA and CE, respectively.^Citation76
Corneal damage	Decreased CFS	Change over time (vs baseline) and CFS score reduction with both CE-CsA and CE. Ref 74 Greater improvement with CE-CsA over vehicle in CFS change after 6-months treatment.^Citation71,Citation76,Citation77

CE = Cationic Emulsion; CFS = Corneal Fluorescein Score; HLA-DR = Human leukocyte antigen DR; TBUT = tear breakup time.

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