VASODILATORS FOR USE IN THE TREATMENT OF A RETINAL ISCHEMIC DISORDER

Abstract

The present invention relates to vasodilators for use in the treatment of a retinal ischemic disorder in a mammal by reducing the retinal ischemic damage for both the photoreceptors (A-wave amplitude) and the Muller and ON-bipolar cells (B-wave amplitude) by at least 10%, when measured by Electroretinography in the mammal compared to the ET-1 induced ischemia alone, at day 3 and at day 21 after the ischemic event and the vasodilator is first applied. The vasodilator may preferably be selected from the group consisting of Calcitonin gene-related peptide (CGRP), amylin, adrenomedullin, and calcitonin.

Claims

1. A vasodilator for use in the treatment of a retinal ischemic disorder in a mammal, wherein the vasodilator acts via the calcitonin receptor-like receptor (CRLR/RAMP-1) and wherein the vasodilator comprise a peptide motif in the C-terminal end having at least 50% identity to SEQ ID NO: 9.

2. A vasodilator for use according to claim 1, wherein Cys2 and Cys7 are cyclised with a disulfide bond.

3. A vasodilator for use according to claim 1, wherein the vasodilator has a Threonine (T) in amino acid position 6.

4. A vasodilator for use according to claim 1, wherein the vasodilator has at least 25% amino acid sequence similarity with a sequence selected from the group consisting of SEQ ID NO: 1-8.

5. A vasodilator for use according to claim 1, wherein the vasodilator is selected from the group consisting of Calcitonin gene-related peptide (CGRP), amylin, and adrenomedullin.

6. A vasodilator for use according to claim 1, wherein the vasodilator is CGRP, derivatives of CGRP, fragments of CGRP, any molecule containing the CGRP peptide sequence or a molecule containing a modified CGRP peptide sequence.

7. A vasodilator for use according to claim 1, wherein the vasodilator reduces the retinal ischemic damage for both the photoreceptors (A-wave amplitude) and the Muller and ON-bipolar cells (B-wave amplitude) by at least 10%, when measured by Electroretinography in the mammal compared to the ET-1 induced ischemia alone, at day 3 and at day 21 after the vasodilator is first applied to said mammal.

8. A vasodilator for use according to claim 1, wherein the vasodilator targets the ocular vasculature, the vasodilator targets the retinal vasculature, the smooth muscle cells (SMC), pericytes and/or the endothelia cells.

9. A vasodilator for use according to claim 1, wherein the retinal ischemic disorder is glaucoma or diabetic retinopathy.

10. A composition comprising a vasodilator for use according to claim 1.

11. An ocular drug delivery system comprising a vasodilator for use or a composition for use according to claim 1.

12. An ocular drug delivery system according to claim 11, wherein the ocular drug delivery system is a viral vector or a topical eye drop delivery system.

13. A CRLR/RAMP 1 agonist for use in the treatment of a retinal ischemic disorder in a mammal, wherein said CRLR/RAMP 1 agonist is a compound which (1) has a K.sub.D of less than 1 μM, and (2) causes cAMP accumulation in a cell line that expresses the CRLR/RAMP1 receptor with an EC.sub.50 of less than 10 μM.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0312] FIG. 1 shows some of the areas of injection where the invention is applied locally in the eye. (a) Intravitreal injection (b) Internal subretinal approach with vitrectomy and subretinal injection. (c) External subretinal approach: Penetrate through sclera and choroid to reach subretinal space with or without vitrectomy.

[0313] FIG. 2 shows some of the areas of application were the invention is applied locally to the eye. (a) Sub-tenon injection (b) Sub-conjunctival (c) Retrobulbar.

[0314] FIG. 3 shows the application and mechanism of the injection of a viral vector producing CGRP leading to vasodilation. The viral vector is injected in the vitreous (a) or subretinal area (b). (c) The viral vector infects the retinal pigment epithelium (d) the viral vector produces CGRP (e) CGRP dilates both the retinal and choroid vasculature.

[0315] FIG. 4 shows how a viral vector containing GFP is expressed in rat retina. GFP expression is a visual-substitute for CGRP or derivatives therefore expressed by a viral vector. (a) Subretinal injection of AAV2/8.gfp vector into rats, results in green fluorescent protein (GFP) expression in all cell-layers of the retina. (a-c,e,g,i) Retinal flatmounts and (d,f,h,j) corresponding sections of rats subretinally injected with AAV2/8.gfp. (a-c) Sclera/choroid/RPE flatmount and (d) section of the sclera/choroid/RPE flatmount showing fluorescent retinal pigment epithelium (RPE) cells. The dotted line in an outline the injected area, and the asterisk (*) marks the injection point. Arrows in a highlight GFP-expressing RPE cells outside the injected area. The square in a defines the area displayed with higher magnification in b. GFP-expressing RPE cells in c and d are located within the injected area. (e,f) Neuroretina flatmount and corresponding section display fluorescent photoreceptor cells. Arrows in f indicate photoreceptor nuclei. (g,h) Neuroretina flatmount and corresponding section show fluorescent ganglion cells and cells of the inner nuclear layer. Arrows in h indicate ganglion cells and cells of the inner nuclear layer. (i) Neuroretina flatmount displaying fluorescent axons of the ganglion cells forming the optic nerve. (j) Transverse section through the optic nerve show GFP signal in axons. Bar=100 m. AAV, adeno-associated virus. https://doi.org/10.1038/mt.2008.41

[0316] FIG. 5 shows the dilation of the ciliary artery when CGRP is applied topically to the eye of a Sprague dawley rat. (a) Fundus picture before CGRP is applied, the arrow points at the ciliary artery (b) fundus picture after CGRP is applied.

[0317] FIG. 6 shows the Electroretinogram data after CGRP (5 μl of 200 μM, n=11) or vehicle (PBS, 5 μl, n=9) has been applied intravitreally 10 minutes before ET-1 (5 μl of 500 μM). (a) The acute damage at day 3 in the photoreceptors, represented with the A-wave amplitude (b) the long-term damage at day 21, represented with A-wave amplitude.

[0318] FIG. 7 shows the Electroretinogram data after CGRP (5 μl of 200 μM, n=11) or vehicle (PBS, 5 μl, n=9) has been applied intravitreally 10 minutes before ET-1 (5 μl of 500 μM). (a) The acute damage at day 3 in the bipolar cells, represented with the B-wave amplitude (b) the long-term damage at day 21, in the bipolar cells, represented with the B-wave amplitude. Data are shown as mean±SEM. (* P<0.05, ** P<0.01, two-way ANOVA, with Bonferroni post-test).

[0319] FIG. 8 shows the dilation of the ciliary artery induced by CGRP when applied intravitreally. (a) Effect of applying the CGRP (+dilator) and the diameter of the ciliary artery after the application of 500 μM ET-1 (n=9-11). (b) Damage of the retina represented as function loss of photoreceptors cells (A-wave amplitudes) compared to the un-injected eye (n=9-11). (c) Damage of the retina represented as function loss of bipolar cells (B-wave amplitudes) compared to the un-injected eye. Data is shown as mean±SEM. (* P<0.05, ** P<0.01; Student's T-test).

[0320] FIG. 9 shows the Electroretinogram data after topical CGRP (10 μl of 200 μM, n=8) has been applied topically 10 minutes before intravitreal ET-1 (5 μl of 500 μM). (a) The acute damage at day 3 in the photoreceptors, represented with the A-wave amplitude (b) the long-term damage at day 21, represented with the A-wave amplitude. Data is shown as mean±SEM. (* P<0.05, ** P<0.01, two-way ANOVA, with Bonferroni post-test).

[0321] FIG. 10 shows the Electroretinogram data after topical CGRP (10 μl of 200 μM, n=8) has been applied topically 10 minutes before intravitreal ET-1 (5 μl of 500 μM) (a) The acute damage at day 3 in the bipolar cells, represented with the B-wave amplitude (b) the long term damage at day 21, in the bipolar cells, represented with the B-wave amplitude. Data is shown as mean±SEM. (* P<0.05, ** P<0.01, *** P<0.001, two-way ANOVA, with Bonferroni post-test).

[0322] FIG. 11 shows the dilation of the ciliary artery induced by CGRP when applied topically. (a) Effect of applying the CGRP (+dilator) after the application of 500 μM ET-1. (b) Damage of the retina represented as function loss of photoreceptors cells (A-wave amplitudes) compared to the un-injected eye. (c) Damage of the retina represented as function loss of bipolar cells (B-wave amplitudes) compared to the un-injected eye. Data is shown as mean±SEM. (* P<0.05, ** P<0.01, *** P<0.001; Student's T-test).

[0323] FIG. 12 shows the Electroretinogram data after sodium nitroprusside (5 μl of 0.5 mM, n=6) or vehicle (PBS, 5 μl, n=9) has been applied intravitreally 10 minutes before ET-1 (5 μl of 500 μM). (a) the acute damage at day 3 in the photoreceptors, represented with the A-wave amplitude (b) the long term damage at day 21, represented with the A-wave amplitude. Data is shown as mean±SEM. (* P<0.05, ** P<0.01, *** P<0.001 two-way ANOVA, with Bonferroni post-test).

[0324] FIG. 13 shows the Electroretinogram data after sodium nitroprusside (5 μl of 0.5 mM, n=8) or vehicle (PBS, 5 μl, n=9) has been applied intravitreally 10 minutes before ET-1 (5 μl of 500 μM). (a) The acute damage at day 3 in the bipolar cells, represented with the B-wave amplitude (n=6-9) (b) the long term damage at day 21, in the bipolar cells, represented with the B-wave amplitude (n=6-9). Data is shown as mean±SEM. (* P<0.05, ** P<0.01, *** P<0.001, two-way ANOVA, with Bonferroni post-test).

[0325] FIG. 14 shows the dilation of the ciliary artery induced by sodium nitroprusside (5 μl of 0.5 mM, n=6) when applied intravitreally. (a) Effect of applying the sodium nitroprusside (5 μl of 0.5 mM, +dilator) and the diameter of the ciliary artery after the application of 500 μM ET-1. (b) Damage of the retina represented as function loss of photoreceptors cells (A-wave amplitudes) compared to the un-injected eye (n=6-9). (c) Damage of the retina represented as function loss of bipolar cells (B-wave amplitudes) compared to the un-injected eye (n=6-9). Data is shown as mean±SEM. (* P<0.05, ** P<0.01, *** P<0.001; Student's T-test).

EXAMPLES

Example 1—the Vasodilator Effect in Endothelin-1 Induced Retinal Ischemia's Background

[0326] Retinal diseases such as glaucoma, diabetic retinopathy, age-related macular degeneration and retinitis pigmentosa are the major causes of blindness. A large number of experimental animal models using rodents (rats and mice) have been used for the evaluations of the pathogenesis and novel therapeutic candidates in retinal diseases.

[0327] Endothelin-1 has been implicated in the pathogenesis of ischemic diseases such as glaucoma. Glaucoma is one of the leading causes of blindness, affecting approximately 66 million patients world-wide. Previous studies have shown that endothelin-1 levels were elevated in glaucomatous eyes of humans. Previous studies with ET-1 intravitreal administration have found that it leads to retinal ganglion cell (RGC) loss, caspase-3 activation, retinal vessel vasoconstriction, electroretinography deficits, RGC and retinal nerve fiber layer (RNFL) thickness changes within one week of intravitreal injection.

[0328] Methods

[0329] Intravitreal Injections.

[0330] Rats were anesthetized with 90 mg/kg ketamine & 10 mg/kg xylazine (i.p). Oxybuprocain 0.4% Anesthetic was applied topically to the eyes.

[0331] Guided under a stereoscopic microscope to avoid lens and retinal injury, 5 μl intravitreal injection of 500 uM ET-1 (Phoenix Pharmaceuticals, Inc.) was performed using a digital controller and UltraMicroPump III, with NanoFil 10 μl syringe (WPI) connected to intraoucular injector with a 34 g bevelled needle.

[0332] Injection was performed through the sclera, inserted approximately 1 mm posterior to the corneal limbus at a 45° angle to avoid contact with the lens capsule and direct the contents, 1.5 mm into the vitreous chamber, in the right eye (FIG. 1). The needle remained in the vitreous cavity for 30 seconds after injection and was then gently withdrawn. A cotton swab was then placed on the injection site to prevent withdrawal of injected solution, and the eye ball was gently massaged to help with the distribution of compound through the vitreous. Antibiotic ointment was applied in the conjunctival sac after the intravitreal injections. The contralateral eye was used as the control eye.

[0333] Fundus imaging was performed to document any changes to the retina or vasculature. Animals recover on the heating plate and returned to the stables once awake and resuming normal behavior. The rats are tested according to the following schedule.

Day 0—ET-1 is administered intravitreally alone or in combination with the current invention. Fundus is recorded before and after injection.
Day 3—Animals are anesthetized to perform ERG Measures
Day 21—Animals are anesthetized to perform ERG Measures

[0334] Treatment Regimes [0335] A) intravitreal 5 μl PBS (vehicle) 10 min before ET-1 [0336] B) intravitreal 5 μl CGRP (200 μM) 10 min before ET-1 [0337] C) intravitreal 5 μl SNP (500 μM) 10 min before ET-1 [0338] D) topical application 10 μl CGRP (200 μM) before ET-1

[0339] ERG

[0340] After overnight dark adaptation for at least 12 hours, the animals were anesthetized with 90 mg/kg ketamine and 10 mg/kg xylazine delivered by a single intraperitoneal injection. Then, oxybuprocaine anesthetic was applied topically to the eyes and the pupils dilated with Mydriacyl and Metaoxedrin under dim red-light illumination. The body temperature was maintained with a heating pad set to 37° C. Using a Ganzfeld stimulator, white light flashes (0.0002-100 cd-s/m2) were produced under scotopic conditions. ERGs were recorded from both eyes simultaneously with a gold wire electrode loops, placed on the corneal apex, with a drop of saline applied to the corneal surface for hydration during the testing procedure. A reference electrode was placed in the mouth and a ground electrode inserted sub dermally in the tail of the animal. The scotopic A-wave amplitude and B-wave amplitude were recorded and evaluated.

[0341] Results

[0342] In order to evaluate the functional effect of ET-1 on the retina, ERG was performed at 3 and 21 days post injection. Focus was put on the damage at day 21, the long-term outcome. At day 21, photoreceptor activity, represented by the A-wave amplitude, showed a significant functional deficit when comparing ET-1 induced ischemic right eyes (5 μL 500 μM, n=9) and the control left eyes (n=9). The control left eye had an avg. max A-wave amplitude of 582±70 μV compared to 457±40 μV in the ET-1 induced ischemia right eye at day 21. The photoreceptor function deficit was less at day 21, suggesting a partial recovery, still with functional deficit in the ET-1 induced ischemia right eye (FIG. 6). The control left eye had an avg. max B-wave amplitude of 1368±209 μV compared to 804±188 μV in the ET-1 induced ischemia right eye at day 21. The photoreceptor function deficit was slightly improved at day 21, suggesting a partial recovery, still with strong functional deficit in the ET-1 induced ischemia right eye (FIG. 8).

[0343] In relation to retinal function, adding CGRP intravitreally (5 μL 200 μM, n=11) or topical (10 μL, 200 μM, n=8) had strong protective effect. Firstly, they both caused a dilation of the ciliary artery (FIG. 7 and FIG. 11). Intravitreal CGRP dilated the ciliary artery to 61±12% increase from baseline, before the injection of ET-1. Topical CGRP also increased the diameter (102±21% increase from baseline), visualized after the injection of intravitreal ET-1. After applying ET-1, the ciliary artery of the eye pretreated with intravitreal CGRP, had a diameter of 9±15% above baseline, compared to ET-1 plus vehicle where there was a contraction (−29±9% from baseline).

[0344] Applying CGRP reduced the ET-1 induced damage greatly at day 21, with the average A-wave amplitude when applied intravitreally being 545±45 μV or applied topically being 511±100 μV compared to the ET-1 ischemic eye (457±40 μV), seen in FIG. 6 and FIG. 9. Similarly, CGRP reduced the ET-1 induced damage on the bipolar cells at day 21, with the B-wave amplitude when applied intravitally being 1181±202 μV) or applied topically being 1088±411 μV compared to the ET-1 ischemic right eye (804±188 μV), seen in FIG. 7 and FIG. 10.

[0345] Sodium nitroprusside (SNP) is a well-known vasodilator. When SNP was applied intravitreally (5 μL 500 μM, n=6) it caused a vasodilation of the ciliary artery by 70±9% increase from baseline (FIG. 14). Similar to that of intravitreal CGRP injection (61±12% increase from baseline). Following the ET-1 injection, the dilation was maintained (21±14% over baseline) and the diameter was larger than that of ET-1 alone (−29±9% from baseline) and similar to the CGRP group (9±15% over baseline). However, applying SNP completely destroyed retinal function observed at day 21, with an average A-wave amplitude when applied intravitreally (61±26 μV) compared to the ET-1 control eye (457±40 μV), as seen in FIG. 12. Since there are no A-wave amplitude, no B-wave amplitude was expected, and that was indeed the case, as the B-wave amplitudes when SNP was applied intravitally was (91±86 μV) compared to the ET-1 right eye (804±188 μV), as seen in FIG. 13.

[0346] Interestingly there was an increase in a and B-wave amplitude function after applying topical CGRP. Suggesting that the left eye is slightly potentiated. This has been seen before with a potential cross talk between the eyes. Due to this the NB-wave amplitude receiver appear smaller (FIG. 11) although the absolute improvement when applying CGRP intravitreally or topically is nearly identical.

[0347] Combined, these data show that the addition of CGRP greatly preventing loss of retinal function. This is surprising as a well characterized and potent vasodilator SNP had the opposite effect, as the photoreceptor damage was further enhanced.

Example 2—Subretinal Injection of rAAV Vectors

[0348] Subretinal injection of an rAAV vector can achieve efficient transduction of RPE and other retinal cells because subretinal injection induces a bleb of concentrated virus in intimate contact with RPE cells and the neural retina (FIG. 3). In addition, the subretinal space has a relatively high degree of immunoprivilege and typically very little evidence of inflammation is seen in the vicinity of the injection site. Thus, subretinal injection is a preferred route for delivery of rAAV vectors in mammalian retina. However, other routes of delivery may be used, for example, intravitreal injection.

[0349] The subretinal injection is performed either in both eyes or unilaterally in the right eye. All procedures are performed under aseptic conditions, using sterile reagents, syringes and appropriate personal protection equipment. The eye is examined and the success of the subretinal injection is confirmed by visualization of a bleb containing fluorescein. The success of injection and the degree of retinal damage (hemorrhage) are scored.

[0350] To study the rAAV vector-induced gene transduction and cell-type specifics in the retina, the eGFP expression in retinal cross sections and RPE/retina flatmounts are examined. One approach used to identify the eGFP expressing cell types is to co-label eGFP positive cells with retinal cell markers by immunocytochemistry staining in cryosections (FIG. 4). Furthermore, production of CGRP in the vitreous and subretinal space is measured in an CGRP specific ELISA kit conforming functional transfection.

[0351] Further the outcome on ET-1 induced ischemia either acutely or long term, is tested using the electroretinogram as described in Example 1.

Example 3—Neuroprotection

[0352] Since retinal ischemia is not limited to the lack of flow but also implies that the retina of the eye is involved in the disease or disorder. The retina comprises neurons, and as such retinal disorders as such are disorders in which neuritogenesis and/or neuroprotection are desirable. The application of intravitreal CGRP, topical application of CGRP or production of CGRP from a viral vector, is combined with the intravitreal injection of a neuroprotectant, for example Glial cell-line derived neurotrophic factor (GDNF). This further enhances the retinal function as flow is restored following the CGRP application/production and neurons/retina is further protected by the neuroprotectant. This is shown with further increase in the A and B-wave amplitudes, compared to CGRP alone.

[0353] Items

1. A vasodilator for use in the treatment of a retinal ischemic disorder in a mammal.
2. A vasodilator according to item 1, wherein the vasodilator reduces the retinal ischemic damage for both the photoreceptors (A-wave amplitude) and the Muller and ON-bipolar cells (B-wave amplitude) by at least 10%, when measured by Electroretinography in the mammal compared to the ET-1 induced ischemia alone, at day 3 and at day 21 after the vasodilator is first applied to said mammal.
3. A vasodilator according to any of items 1-2, wherein the vasodilator reduces the retinal ischemic damage for both the photoreceptors (A-wave amplitude) and the Muller and ON-bipolar cells (B-wave amplitude) by at least 10%, when measured by Electroretinography in a Sprague Dawley rat compared to the ET-1 induced ischemia alone, at day 3 and at day 21 after an Endothelin-1 induced ischemic event.
4. A vasodilator according to any of items 1-3, wherein the vasodilator dilates the ciliary artery with at least 20% as visualized with fundus imaging 10 minutes after application to an albino rat.
5. A vasodilator according to any of items 1-4, wherein the vasodilator targets the ocular vasculature.
6. A vasodilator according to any of items 1-5, wherein the vasodilator targets the retinal vasculature.
7. A vasodilator according to any of items 1-6, wherein the vasodilator targets the smooth muscle cells (SMC), pericytes and/or the endothelia cells.
8. A vasodilator according to any of items 1-7, wherein the vasodilator is selected from the group consisting of Calcitonin gene-related peptide (CGRP), amylin, adrenomedullin, calcitonin, cAMP-generators, cAMP-mediators, phosphodiesterase inhibitors, potassium channel openers (hyperpolarization-mediated), calcium channel blockers (reducers of intracellular calcium), cGMP-mediators (Nitrovasodilator).
9. A vasodilator according to any of items 1-8, wherein the vasodilator targets CGRP activated vasodilation.
10. A vasodilator according to any of items 1-9, wherein the vasodilator is CGRP, derivatives of CGRP, fragments of CGRP, any molecule containing the CGRP peptide sequence or a molecule containing a modified CGRP peptide sequence.
11. A vasodilator according to any of items 1-10, wherein the retinal ischemic disorder is selected from the group consisting of diabetic retinopathy, retinitis pigmentosa, glaucoma, normotensive glaucoma, ocular hypertension, neovascularization, retinal vein occlusion, and retinal artery occlusion.
12. A vasodilator according to any of items 1-11, wherein the retinal ischemic disorder is caused by Endothelin-1.
13. A vasodilator according to any of items 1-12, wherein the retinal ischemic disorder is glaucoma.
14. A vasodilator according to any of items 1-13, wherein the retinal ischemic disorder is diabetic retinopathy.
15. A composition comprising a vasodilator according to any of items 1-14.
16. A composition according to item 15, wherein the composition further comprises a neuroprotection ingredient.
17. A composition according to item 16, wherein the neuroprotection ingredient is administered simultaneously, separately or sequentially from the vasodilator.
18. An ocular drug delivery system comprising a vasodilator or a composition according to any of items 1-17
19. An ocular drug delivery system according to item 18, wherein the ocular drug delivery system is selected from the group consisting of topical eye drop formulations, nucleic acid constructs, viral and non-viral gene therapy vectors, emulsion based formulations, suspensions, ophthalmic ointments, nanotechnology based ocular drug delivery systems (nanomicelles, nanoparticles, nanosuspensions), liposomes, monomers, multimers, dendrimers, in-situ gelling systems, contact lens, implants, slow release polymers, microneedles, microspheres, and cells capable of secreting a vasodilator according to any of items 1-14.
20. An ocular drug delivery system according to any of items 18-19, wherein the ocular drug delivery system comprises the in vivo production of a prepropeptide, where the prepropeptide is preproCGRP or a preproCGRP derivative.
21. An ocular drug delivery system according to any of items 18-20, wherein the ocular drug delivery system is an intravitreal delivery system.
22. An ocular drug delivery system according to any of items 18-20, wherein the ocular drug delivery system is a subretinal delivery system.
23. An ocular drug delivery system according to any of items 18-22, wherein the ocular drug delivery system is a viral vector.
24. An ocular drug delivery system according to any of items 18-20, wherein the ocular drug delivery system is a topical eye drop delivery system.