Comparative efficacy of cyclosporine eye drop formulations in a mouse model of dry eye

Abstract

Purpose:Dry eye disease (DED) is a complex multifactorial disease with both inflammation and tear film instability recognized as its core mechanism. Topical steroids are effective in treating inflammation but may have important secondary effects. The aim of the present study was to compare the efficacy of various CsA formulations in a mouse model of dry eye with severe corneal epithelium lesions. Methods:Eight to 12-week-old female C57BL6 mice with tail patches of scopolamine (replaced every other days) were housed in controlled environment room to induce dry eye. At day three, following dry eye confirmation by corneal fluorescein staining (CFS, score 0-15) and phenol red thread (PRT) lacrimation test, the mice (n=10/gp) were either treated 3 times a day in both eyes with: 0.05% CsA (Restasis, Allergan, USA), 0.1% CsA (Ikervis, Santen, France), 1% CsA oil solution, and 0.5% loteprednol etabonate (Lotemax, Baush+Lomb, USA), or left untreated. Aqueous tear production and CFS score were assessed during the treatment period, while CD4+ and CD11b+ counts on flat mounted cornea were evaluated at the end of the experiment (at day 10). Results:The PRT lacrimation test confirmed the scopolamine-induced decrease in tear secretion, even though the tear reduction was significant only for the Ikervis group. After 7 days of treatment, the CFS score was reduced (vs. dry eye baseline) by 21, 31, and 44% with Restasis, Ikervis, and the 1% CsA oil solution, respectively. CFS scores at day 3 decreased to 7.8±1.3, 7.3±1.4, and 5.9±1.3 at day 10 for Restasis, Ikervis and the 1% CsA oil solution, respectively. By contrast 0.5% Loteprednol etabonate was not able to decrease CFS. No statistically significant dose dependent CFS reduction was observed with the three CsA formulations. CD11b+ and CD4+ cell counts were slightly decreased both in central and peripheral cornea by Restasis and Ikervis when compared to the untreated control. Conclusions:This study indicates that the three CsA formulations are effective at reducing corneal epithelium lesions in a mouse model of severe DED, and represents good treatment options for the management of DED in patients. The study also suggests that no strict dose dependent relationship exists, since Ikervis (0.1% CsA) is not statistically different from the 1% CsA oil solution.

Full text

D0130
Abstract D0130 presented at the Annual Meeting of the Association of Research for Vision and Ophthalmology (ARVO); May 1–5, 2016; Seattle, WA, USA
Comparative efcacy of cyclosporine A eye drop formulations in a mouse model of dry eye
Jean-Sebastien Garrigue1, Philippe Daull1, Laurence Feraille2, Stefano Barabino3
1Novagali Innovation Center, Santen SAS, Evry, France; 2Iris Pharma, La Gaude, France; 3Clinica Oculistica, Di.N.O.G.M.I., Azienda Ospedaliera Universitaria San Martino-IST, Genoa, Italy
INTRODUCTION
Dry eye disease (DED) is a complex
multifactorial disease with both inammation
and tear lm instability recognized as its core
mechanisms.1 Topical steroids are effective
in treating inammation but may have
important secondary effects.2 Cyclosporine A
(CsA), a potent anti-inammatory molecule
with a good safety prole, is the most
commonly used prescription treatment for the
management of DED signs and symptoms.3,4
PURPOSE
The aim of this study was to compare the efcacy of a new cationic emulsion5
of CsA (CsA CE, Ikervis®)6 to that of other available CsA formulations (anionic
emulsion and oily solution) in a mouse model of dry eye with severe corneal
epithelial lesions.7
Table 1. Summary of the physicochemical characteristics of CsA
emulsions8
Parameters Anionic emulsion
Restasis®
Cationic emulsion
Ikervis®
Aspect White opaque to slightly translucent
pH 6.5–8.0 6.0
Osmolality (mOsmol/kg) 230–320 270
Mean droplet size, nm (%)a24 (6), 170 (85), 880 (9) 170 (100)
Zeta potential (mV)bNegative (–60 to –40) Positive (+20 to +40)
Sterility Sterile
CsA (%) 0.05 0.1
aMean droplet size was determined by dynamic light scattering (HPPS, Malvern Instruments).
bzeta potential was determined by electrophoretic mobility measurement (Zetasizer 2000,
Malvern Instruments).
CsA, cyclosporine A.
METHODS
To induce dry eye, female C57BL/6 mice (8–12 weeks old) were housed in
a controlled environment room (CER) and tail patches of scopolamine were
replaced every 2 days.7 Dry eye was conrmed at Day 3 by corneal uorescein
staining (CFS; score 0–15) and the phenol red thread (PRT) lacrimation test. Mice
(10 per cohort) were treated three times a day in both eyes with 0.05% CsA
(Restasis®, Allergan, USA), 0.1% CsA (Ikervis®, Santen, France; Table 1), 1% CsA
oil solution, or 0.5% loteprednol etabonate (Lotemax®, Bausch+Lomb, USA)
or left untreated. Aqueous tear production and CFS score were assessed for 7
days and changes from baseline (Day 3) to Day 10 were calculated. CD4+ and
CD11b+ cell counts were evaluated at Day 10 on at-mounted cornea.
RESULTS
Figure 1. Tear secretion over time (A; n=20 eyes per group) and lacrimation improvement at Day 10 (B; n=20 eyes per group) in mice with induced dry eye treated
with different eye drops
Untreated Lotemax®Restasis®Ikervis®1% CsA oil
0
2
4
6
Untreated Lotemax®Restasis®Ikervis®1% CsA oil
0
20
60
80
40
100
B
A
PRT (mm)
Lacrimation improvement (%)
Baseline Day 3 – before treatment Day 6 Day 10
***
****
**p=0.001, ****p<0.0001.
Data are mean + standard deviation.
CsA, cyclosporine A; PRT, phenol red thread.
Figure 2. Mean CFS score over time (A) and mean reduction in CFS score at Day 10 (B) following induction of dry eye (vs Day 3)
Untreated Lotemax®Restasis®Ikervis®1% CsA oil
0
5
10
15
Untreated Lotemax®Restasis®Ikervis®1% CsA oil
–100
–50
0
50
B
A
Mean CFS score
Reduction in CFS score (%)
Baseline Day 3 – before treatment Day 6 Day 10
****
****
********
****
****
****
****
****
****
****
****
***
***
**
NS NS
n=202020 20 20 20 19 19 19 20 20 20 19 20 20 20 20 20 20 20 20 n=19 n=19 n=20 n=20
**p=0.001, ***p=0.0001, ****p<0.0001. Numbers within or below data columns indicate the number of eyes evaluated for each group. Data are mean + standard deviation.
CFS, corneal uorescein staining; CsA, cyclosporine A; NS, not signicant.
Figure 3. CD11b+ (A) and CD4+ (B) cell densities in at-mounted cornea from mice treated with Restasis® (n=10) or Ikervis® (n=8) or left untreated (n=10) at Day 10
0
10
20
30
40
50
60
70
80
90
100
110
Restasis®Ikervis®
Untreated control
Flat-mounted cornea CD11b+ density Flat-mounted cornea CD4+ density
Restasis®Ikervis®
Untreated control
Peripheral zone Central zone Peripheral zone Central zone
0
50
100
150
200
B
A
CD11b+
density (positive cells/mm
2)
CD4+
density (positive cells/mm
2)
SUMMARY
nThe PRT lacrimation test conrmed the scopolamine-induced decrease in
aqueous production by the lacrimal gland (Figure 1)
nAfter 7 days of treatment, mean CFS scores (Figure 2) reduced by 31% with
Ikervis®; CFS score at Day 3 before treatment was 10.8±1.7 vs 7.3±1.4 at
Day10
nThe anionic emulsion, Restasis®, and the 1% CsA oil solution reduced CFS by
21% and 45%, respectively (Figure 2)
nSurprisingly, Lotemax® did not perform well in this model and resulted in a
11% increase in mean CFS score (Figure 2)
nNo statistical difference in CFS reduction between 1% CsA oil and Ikervis®
was observed; however, a trend toward better efcacy for the 10-fold higher
CsA dose was observed
nA slight reduction, although not signicant, in CD11b+ and CD4+ cell density
was also observed in at-mounted cornea from the Ikervis® group when
compared with the untreated group (Figure 3)
nNo differences between Restasis® and Ikervis® were observed in CD11b+ or
CD4+ cell counts
CONCLUSIONS
nThe three tested CsA formulations
effectively reduced corneal epithelial lesions
in a mouse model of DED, and this study
provides preclinical data in support of these
treatment options for the management of
severe keratitis in DED patients
nNo clear evidence of a CsA-dose-dependent
relationship was observed among the
different CsA formulations in this study
nThese data support the mechanism of
action of the cationic emulsion of CsA
(Ikervis®) recently approved for the
treatment of severe keratitis in patients
with DED6,8,9
REFERENCES 1. DEWS. Ocul Surf 2007;5:75–92. 2. Jung HH et al. Chonnam Med J 2015;51:26–32. 3. Kashani S,
Mearza AA. Expert Opin Drug Saf 2008;7:79–89. 4. Lallemand F, Daull P, Benita S, Buggage R, Garrigue JS. J Drug Deliv
2012;2012:604204. 5. Bague S et al. US Patent 2007-0248645. Santen, 2007. 6. European Medicines Agency. Ikervis
Authorisation. January 2015. 7. Barabino S et al. Invest Ophthalmol Vis Sci 2005;46:2766–2771. 8. Daull P et al. Cornea
2013;32:345–534. 9. Daull P, Lallemand F, Garrigue JS. J Pharm Pharmacol 2014;66:531–541.
Note: In March 2015, IKERVIS® (IKERVIS® 1 mg/mL eye drops, emulsion) was granted Marketing Authorization
by the European Commission.
IKERVIS®, once a day at bed time, is indicated for “treatment of severe keratitis in adult patients with dry eye
disease, which has not improved despite treatment with tear substitutes.”6
ACKNOWLEDGEMENTS/DISCLOSURES
J.-S. Garrigue and P. Daull are employees of Santen SAS (E). L. Feraille is an employee of Iris Pharma (E). S. Barabino
(placeholder for disclosures)