PHARMACEUTICAL COMPOSITION FOR THE TREATMENT OF OCULAR NEOVASCULARISATION

Abstract

The present invention relates to a pharmaceutical composition comprising tacrolimus and a semifluorinated alkane for use in the treatment of ocular neovascularisation.

Claims

1. A pharmaceutical composition comprising tacrolimus and a semifluorinated alkane for use in a method of treatment of ocular neovascularisation.

2. The composition for the use of claim 1, wherein the ocular neovascularisation is retinal and/or choroidal neovascularisation.

3. The composition for the use of claim 1 or 2, wherein the ocular neovascularisation is caused or associated with a disease selected from age related macular degeneration, diabetic macular edema, central retinal vein occlusion, retinopathy, branch retinal vein occlusion.

4. The composition for use of any of the preceding claims, wherein the semifluorinated alkane is a semifluorinated alkane of formula (I)
CF.sub.3(CF.sub.2)n(CH.sub.2)mCH.sub.3  (I) wherein n is an integer selected from 2 to 10 and m is an integer selected from 2 to 10.

5. The composition for use of any of the preceding claims, wherein n is 3 and m is 4 or wherein n is 5 and m is 7.

6. The composition for use of any of the preceding claims, wherein tacrolimus is present at a concentration of at least 0.01% w/v with respect to the total volume of the composition.

7. The composition for use of any of the preceding claims, wherein tacrolimus is present at a concentration between about 0.01% w/v and 0.1% w/v with respect to the total volume of the composition.

8. The composition for use of any of the preceding claims, further comprising ethanol.

9. The composition for use of claim 8, wherein ethanol is comprised at a concentration of from about 0.5 to 2% w/w with respect to the total weight of the composition.

10. The composition for use of any of the preceding claims, wherein the composition is topically applied or intravitreously injected to the eye, preferably topically applied.

11. The composition for use of any of the preceding claims, wherein the ocular neovascularisation is due to uncontrolled expression of pro-angiogenic factors by inflammatory cells.

12. The composition for use of any of the preceding claims, wherein the dose of tacrolimus administered to the eye is from about 0.5 to 10 micrograms per dose per eye.

13. The composition for use according to any of the preceding claims, wherein the composition is in form of a solution.

14. The composition for use of any of claims 1 to 12, wherein tacrolimus is suspended in the semifluorinated alkane, preferably wherein tacrolimus is suspended in 1-perfluorohexyloctane.

15. A kit comprising a container for holding the composition for use of any of the claims 1 to 14 and a data carrier, wherein the container is adapted for topical application or intravitreal injection of said composition to the eye, and wherein the data carrier comprises instructions for use of said composition according to any of the claims 1 to 14.

Description

DETAILED DESCRIPTION OF THE DRAWINGS

[0075] FIG. 1: CNV lesions in fundus images and FA images. 10 post-CNV induction mice were subjected to fundus examination and FA using the Micron IV system. Images showing are representatives from three mice in each group.

[0076] FIG. 2: The effect of different treatments on CNV formation. Lesion areas were quantified based on isolectin B4 staining. Representative RPE flatmount images showing CNV in each group.

[0077] FIG. 3: the mean CNV size in each group expressed as either the absolute area or percentage of reduction compared with control non-treatment group. Data presented are Mean±SEM. * p<0.05, unpaired Student t test. Anti-VEGF, Dexamethasone 0.1% and Tacrolimus/PBS groups were compared to Blank control; 0.02% Tacrolimus/F4H5 was compared with F4H5 vehicle control.

EXAMPLES

Materials

[0078] The materials used to prepare the tacrolimus formulations listed below are: Tacrolimus (Euticals; Purity 98.2%), Ethanol (Merk, Seccosolv®, dried 0.01% H.sub.2O), 1-perfluorobutyl-pentane F4H5 (Novaliq), phosphate buffered saline (VWR, biotechnological grade)

Formulations

[0079] The following formulations were used in the experiments:

[0080] A solution of 0.02% w/v (0.2 mg/ml) Tacrolimus in 1.4% w/w Ethanol in 1-perfluorobutyl-pentane was prepared by dissolving tacrolimus in ethanol and 1-perfluorobutyl-pentane (this formulation is also referred to as Tacrolimus/SFA in Table 1, Table 2 and the Figures)

[0081] A suspension of Tacrolimus 0.02% w/v in phosphate buffered saline (PBS) was prepared (this formulation is also referred to as Tacrolimus/PBS in Table 1, Table 2 and the Figures)

[0082] Dexamethasone ophthalmic suspension Maxidex® 0.1% was purchased (Novartis Pharmaceuticals UK Ltd.).

[0083] Antimouse VEGF antibody purchased from R&D systems.

Preparation of a Suspension of Tacrolimus in F6H8

[0084] The required amount of tacrolimus (purity 100.5%; water content 2.35%) is weighed and then transferred into a grinding jar. The required volume of F6H8 is added to the jar, which is then placed into a ball mill (BM01) for one hour at 150 rpm and 10 minutes interval. The suspension is then separated from the balls by means of a pipette.

Example 1

[0085] The therapeutic effect of eyedrops of Tacrolimus 0.02% w/v in ethanol 1.4% w/w and F4H5 was tested in a mouse model of laser-induced choroidal neovascularisation (CNV) and compared with the therapeutic effect of 0.1% Dexamethasone eyedrop and with intravitreal injection of anti-mouse VEGF, respectively.

Study Design

[0086] 36 female C57BL/6 mice (12 weeks old) were purchased from Harland Laboratories UK. All mice were housed and bred in a normal experimental room and exposed to a 12-hour dark 12-hour light cycle. All procedures concerning the use of animals in this study were performed according to the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research and under the regulations of the United Kingdom Animal License Act 1986 (UK).

Laser-Induced Choroidal Neovascularisation (CNV)

[0087] The laser-induced CNV was conducted in C57BL/6 mice. Briefly, mice were anesthetized with intraperitoneal injection of 75 mg/kg ketamine and 7.5 mg/kg xylazine. The pupils were dilated with 1% tropicamide (Chauvin Pharmaceuticals Ltd, Essex, UK). Three 532 nm diode laser spots (200 mV, 100 msec, 100 μm) were applied to each fundus using Viscoelastic material Microvisc (Vision Matrix Ltd, Harrogate, UK) and a coverslip as a contact lens. The lesions were placed between retinal vessels 2 to 3 optic-disc diameter from the optic disc. Formation of a bubble at the site of laser application, which indicates rupture of Bruch's membrane, is an important factor in obtaining CNV, so only burns in which a bubble was produced were included in this study. The CNV lesion develops 2-3 days after laser treatment, peaks at day 7-12, and regresses after 14-20 days.

[0088] The laser induced choroidal neovascularisation protocol applied is also described in 1) Tobe, T., S. Ortega, J. D. Luna, H. Ozaki, N. Okamoto, N. L. Derevjanik, S. A. Vinores, C. Basilico, and P. A. Campochiaro. 1998. Targeted disruption of the FGF2 gene does not prevent choroidal neovascularization in a murine model. Am. J. Pathol. 153: 1641-1646; 2) Toma, H. S., J. M. Barnett, J. S. Penn, and S. J. Kim. 2010. Improved assessment of laser-induced choroidal neovascularization. Microvasc. Res. 80: 295-302.

Treatment Regimens

[0089] Six experimental groups were included in this study. Six mice were used in each group (Table 1). Mice were treated with either eyedrops twice daily from day 0 to day 10 or intravitreal injection of 1 μL with a concentration of 1 ng/μL anti-VEGF immediately after CNV induction. During eyedrop administration, each eye received 5 μl of eyedrop.

TABLE-US-00001 TABLE 1 In vivo treatment groups and dosing details No. of Dosing Group animals Treatment Dose route Group 1 6 None N/A N/A Group 2 6 0.02% Tacrolimus/SFA Twice daily Eye drop Group 3 6 0.02% Tacrolimus/PBS Twice daily Eye drop Group 4 6 Vehicle (SFA) Twice daily Eye drop Group 5 6 Anti-mouse VEGF One Intravitreal (1 ng/1 μl/eye) injection Group 6 6 0.1% dexamethasone Twice daily Eye drop

Clinical Examinations

[0090] The CNV lesions were examined clinically on day 10 post-CNV induction using the Micron IV (Phoenix Research Labs) system for colour fundus image and fluorescein angiography.

Sample Collection

[0091] On day 10 post-CNV induction, all mice were sacrificed by CO2 inhalation and eyes were carefully removed. Ocular tissue whole mounts were prepared using the following procedure. All eyes were fixed in 2% paraformaldehyde (Agar Scientific Ltd, Cambridge, UK) for 2 h at room temperature and then washed with PBS. To prepare retinal pigment epithelia (RPE)-choroidal whole mounts, the anterior segment of the eye including the cornea, ciliary body, iris and the lens were removed. Five vertical cuts were made in the eye cup, and the retinal tissue was then dissected off from the RPE/choroid. The extraocular tissues including conjunctiva and ocular muscles were carefully removed. The RPE/choroid whole mounts were then further processed for immunostaining.

Immunostaining of RPE Flatmounts

[0092] RPE/choroidal whole mounts were permeabilised with 0.3% triton X-100 for 1 h at room temperature. The samples were then blocked with 6% BSA for a further hour and incubated with rabbit anti mouse collagen IV (1:100, AbD Serotec, Kidlington, UK) and Biotinylated isolectin B4 (1:100, Vector Laboratories Ltd, UK) overnight at 4° C. After thoroughly washing (10 minutes×3), samples were incubated with FITC conjugated Streptavidin (Dako, Denmark) and tetramethylrhodamine goat anti-rabbit IgG (Invitrogen, UK) for 4 h at room temperature in dark. Finally, samples were washed and flatmouted on glass slides with Vectashield Mounting Medium (Vector Laboratories Ltd, Peterborough, UK) and observed by confocal microscopy. The samples were flatmounted on glass slides and examined by fluorescence microscopy (Leica DMI8).

Image and Data Analysis

[0093] An imaging software ImageJ system was used to analyse the images. The green and red channels were analysed separately. To measure the size of the CNV, the border of the CNV was outlined manually and the size was automatically calculated. The average size of CNV in each group was expressed as mean±SEM. Student's t test (unpaired, two tails) was used to detect the difference between tacrolimus/dexamethasone treated group and relevant control groups. In addition, one way ANOVA Turkey's Multiple Comparison Test was also used to detect differences among different groups.

Clinical Observations

[0094] Fundus examination and fluorescence angiography (FA) were conducted in three mice from each group on day 10 post-CNV induction. CNV lesions were detected as white spots in fundus images and hyper-fluorescent spots in FA (FIG. 1). All mice developed CNV lesions.

Immunohistological Results

[0095] A total of 216 laser burns were applied in 72 eyes (36 mice). All laser burns showed a bubble and induced CNV. Table 2 and FIGS. 2 and 3 show the results of isolectin B4 labelling. The results show that 0.02% tacrolimus/SFA eyedrop and 0.1% dexamethasone eyedrop treatment significantly suppressed CNV.

TABLE-US-00002 TABLE 2 The average size of CNV in each group as determined by isolectin B4 staining. Number of Mean CNV Group Treatment CNV (μm.sup.2) SEM 1 None 22 55394 14930 2 0.02% Tacrolimus/SFA 31 26421 2919 3 0.02% Tacrolimus/PBS 27 33837 4323 4 Vehicle (SFA) 27 55618 12391 5 Anti mouse VEGF 27 36008 5208 6 0.1% dexamethasone 28 25336 3658

[0096] The 0.02% tacrolimus/F4H5 eyedrop showed anti-angiogenic effect in the mouse model of laser-induced CNV. The anti-angiogenic effect of 0.02% tacrolimus/F4H5 was similar to 0.1% Dexamethasone eyedrop when applied twice a day.

Example 2

[0097] The antiangiogenic effect of Tacrolimus was also tested in an in vitro model of choroidal angiogenesis and compared with dexamethasone. The results of this test showed that tacrolimus at concentrations of 100 ng/mL, 20 ng/mL and 4 ng/mL suppressed choroidal angiogenesis but the effect was not dose-dependent. The suppressive effect was similar to the effect of 1 μM Dexamethasone.