WO2000067740A9 - Aminoguanidine for treating glaucomatous optic neuropathy - Google Patents

Abstract

A method for treating glaucomatous optic neuropathy using caminoguanidine is described.

Description

IN THE UNITED STATES PATENT AND TRADEMARK OFFICE

AMINOGUANIDINE FOR TREATING GLAUCOMATOUS OPTIC NEUROPATHY

This invention is directed to the use of aminoguanidine for treating glaucomatous optic neuropathy in an individual.

Background of the Invention

The glaucomas are a heterogeneous group of optic neuropathies characterized by the cupping of the optic nerve head, thinning of the retinal nerve fiber layer due to loss of retinal ganglion cells, and specific pathogenetic changes in the visual field

Although elevated intraocular pressure <IOP) is an important risk factor for the development of many common forms of glaucoma (Somner, A . et al. , Arch. Ophthalmol., 109: 1090- 1095 (1991), the phenomenon of normal tension glaucoma has been clinically established in ophthalmology (Flammer, J ., Fortschr. Ophthalmol. 87: 187( 1990). Normal tension glaucoma is characterized by an intraocular pressure which is in the normal range, i.e. not increased, but in which the optic nerve disk is pathologically excavated and the field of vision is impaired.

At the present time glaucoma including normal tension glaucoma is treated by medically and/or surgically lowering elevated pressure; however, even when IOP is maintained with in a normal range visual field loss may progress. Degeneration involving retinal ganglion cells may he related to compression of the nerve fiber bundles, excitotoxicity, ischemia, or other as yet unrecognized causitive factors. Thus, factors other than IOP may play a role in determining both the occurrence and rate of progression of retinal ganglion cell death and subsequent visual field loss.

Using laboratory models, including ischemia, optic nerve crush, optic nerve transection, and cultured retinal ganglion cells, various pharmacological agents have been tested as potential neuroprotective approaches designed to reduce retinal ganglion cell loss (Adachi, K. et al. Eur. J. Pharmacol. 350:53-57 (1998); Yoles, E. et al. , Arch. Opthalmol. 116:906-910, (1998); Di Polo, A et al., Proc. Natl. Acad.

Sci, USA 95:3978-83 (1998); Caprioli, J. et al., Invest. Ophthalmol. Vis. Sci.

37:2376-2381 (1996); Wolde ussie, E.et al., Invest. Ophthalmol. Vis. Sci. 38:S100 (1997)). These approaches have suggested that antagonism of cxcitotoxicity or supplementation of neurotrophic factors can protect retinal ganglion cells from degeneration in animal models. The use of compounds capable of reducing gluta ate toxicity (WO 94/13275) and polyamine antagonists (US Patent

No. 5,710, 165) to protect retinal ganglion cells and reduce visual field loss associated with glaucoma have been disclosed. The protective effect of MK-801 , a glutamate antagonist, in a rat model of ocular hypertension, was reported. (P.

Chaudhary et al. , Invest. Ophthalmol. Vis. Sci. 38:S813 (1997).

The role of nitric oxide (NO) in the eye has been characterized (E. Coutlier et al. , Survey of Ophthalmol. 42:71-82; Neufeld, A.H.,et al. Arch Ophthalmol. 1 15:497-

503 (1997); Wiederholt M. et l. Invest. Ophthalmol. Vis. Sci. 35:2515-2520; J. Nathanson et al. Invest. Ophthalmol. Vis. Sci. 36: 1765-73; J. Nathanson et al. Invest. Ophthalmol. Vis. Sci. 36:1774-1784; Schulman, J. Exp. Eye Res. 58:99- 105 ( 1994); Behar-Cohen, F.F. et al., J. Invest. Ophthalmol. Vis. Sci. 37: 1711-15 (1996); Nuzzi, R et al., Acta Ophthalmologica Scandinavica S224:9 (1997)) It appears that the function of NO in ocular tissue is very diverse, having actions on vascular tone, neurotransmission, immune cytotoxicity, and many others NO is an important mediator of homeostatic processes in the eye, such as, regulation of aqueous humor dynamics, retinal neurotransmission, and phototransduction.

Nitric oxide (NO) is formed from L-arginine by a family of enzymes called nitric oxide synthetases (NOS). There are three isoforms of NOS: NOS-1 (neuronal NOS), NOS-2 (inducible NOS) and NOS-3 (endothelial NOS). Increased amounts of the 3 isoforms of nitric oxide synthetase (NOS) have recently been detected in the optic nervehead of patients with primary open angle glaucoma. The increased presence of NOS-1 and the induction of NOS-2 in the astrocytes of the lamina cribrosa suggest that the glaucomatous optic nerve head is exposed to excessive levels of nitric oxide (NO), which may be neurodestructivc, locally, to the axons of the retinal ganglion cells (Neufeld, A H ,et al Arch Ophthalmol 1 15 497^" 503 (1997))

Whereas the presence of the constitutive forms of NOS-1 and NOS-3 are likely to play a role in the maintenance of homeostasis in the retina and optic nerve head, induction of NOS-2 results m the production of toxic levels of NO which is at least in part responsible tor the cytotoxicty caused by activated microglia, astrocytes and macrophages In the brain, NOS 2 is expressed in several pathological states, including tumors, trauma, demyehnation, AIDS dementia, Alzheimer's disease, and cerebral ischemia (ladecola, C J of Neuroscience 17 9157-9164 1997) It has been shown in mice lacking NOS-2 (NOS 2 knockout mouse ) that there is a decreased susceptibility to cerebral ischemia following middle cerebral artery occulsion when compared to wild type mice, suggesting that NOS-2 expression is one of the factors contributing to the expansion of the brain damage that occurs in the post-ischemic period (ladecola, C J of Neuroscience 17 9157-9164 1997). The release of NO has also been shown to exacerbate glutamate-mediated (excitotoxic) neurnnal death T he use of inhibitors of NOS to prevent glutamte neurotoxicity has been disclosed (U S Patent No 5,266,594)

Although many inhibitors of NOS are known, very few compounds are known to be selective for NOS-2 (Collins. Jon L et al , J Med Chem 41 2858-2871 (1998), Hallinan, E A et al , J Med Chem 41 775-777(1998), Southan. G J ct al Biochcm Pharmacol 54 409-417(1 97) acdonald, J Ann Rep Med Chem , 31 221 30 (1996), Shearer, B G et al. , J Med Chem 40 1901 -5 (1997), O'Neil, M J et al Eur J Pharmacology 310 1 15-122 (1996), Cuzzocrea, S Free Radical Biology and

Medicine 24 450-9 ( 1998), U S Patent No 5266594, EP 0870763 , EP 0750906, WO 9630350, WO 9738977, WO 9615120, WO 9524382, EP 759027, WO 9848826) Among the compounds that are selective inhibitors of NOS-2 is ammoguanidine (Southan, G ct al , Biochemical Pharmacology 51 383 94 (1996), Hasan, K et al , Eur J Pharmacol 249 101-6 ( 1993), Corbett, J A et al ,

Methods-Enzymol 268 398-408 ( 1996), Corbett, J A et al , Diabetes 41 552 6 ( 1992)) T he use of ammoguanidine as a NOS inhibitor for the treatment of acute or chronic inflammatory or immunologically-mediated nitric oxide mediated disease has been disclosed (U.S. Patent No. 5358969, U.S. Patent No. 5246970).^' Aminoguanidine reduced neocortical infarction following focal ischemia in a rat model of stroke (Zhang, F. et a!. , Stroke 27:317-23 ( 1996). The activity of NOS inhibitors including aminoguanidine in an LPS-induced uveitis model in rats (Allen, J.B. et al. Exp-Eye-Res. 62:21-8 (1996)) has been disclosed. Geyer has shown that nitric oxide inhibitors including aminoguiiidine protected rat retina against ischemic injury (Geyer.O. e al. , FEBS-Lett. 374:399-402( 1995).

Summary of the Invention

The present invention is directed to the use of aminoguanidine to treat glaucomatous optic neuropathy.

Description of Preferred Embodiments

Surprisingly it has been found that aminoguanidine can be used to treat glaucomatous optic neuropathy. For the purpose of this invention, equivalent compounds corresponding to aminoguanidine include biological and pharmaceutically acceptable acid addition salts. Such acid addition salts may be derived from a variety of organic and inorganic acids such as sulfuric phophoric, hydrochloric, hydrobromic, sulfamic. citric, lactic, maleic, succinic, tartaric, cinnamic, acetic, benzoic, gluconic, ascorbic and related acids.

Aminoguanidine may be administered orally using capsules or tablets or as an oral suspension or solution. Administration could take place daily and an effective quantity of the agent could range from 0.1 to 25 mg/kg. The preferred range would be from 0.5 to 10 mg/kg and the most preferred range would be 1 -5 mg/kg. Some variation in these amounts is possible

Example 1

Rat model of chronic, moderately elevated IOP. Adult, mail Wistar rats weighing approximately 250g at the beginning of the experiment were used, animals were fed ad libitum and maintained in temperature controlled rooms on a 12 hour light/dark cycle. Experiments were carried out in accordance wrdrthe RVO^Statement for thci_ sErσfAnimals irrϋphthatmic and Vision Research. All surgical procedures were under general anesthesia using a mixture of 80 mg kg ketamine (Fort Dodge Laboratories, Inc., Fort Dodge, LA) and 12 mg/kg xylaκine (Butler, Columbus. OH), given intraperitoneally.

Chronic, moderately elevated IOP was produced unilaterally in 16 rats by cautery of three, episclcral vessels; the contraiateral eye served as the comparative control. To perform the cauter, sutures were placed in the lids to keep the eye open and in the bulbar conjunctiva to manipulate the globe. Three of the 4-5 major trunks formed by limbal derived veins were exposed at the equator of the eye by incising the conjunctiva. Each vessel was lifted with a small muscle hook and cauterized by direct application of an ophthalmic, disposable cautery (Model RS201 , Roboz Surgical

Instrument Company, Inc.. Rockville, MD, USA) against the muscle hook. Immediate retraction and absence of bleeding of the cauterized ends of the vessels were noted as successful cauterization. After surgery, eyes were treated topically with bacitracin- neomycin-polymyxin (Pharmaderm Inc.. Melville. NY) for a few days during recovery.

Aminoguanidine treatment and measurements taken during treatment.

One group of 8 animals was treated with aminoguanidine. a relatively selective inhibitor of NOS-2, in the drinking water for six months; a second group of 8 animals was untreated. At the time of cauterization, the rats were divided randomly into the two groups (drug treated and untreated) and caged individually. To inhibit NOS-2 in one group, aminoguanidine (2.0 g/1) was dissolved in their drinking water, which was made up and provided fresh three times per week. The control group was not treated but rccievcd fresh drinking water, from the same source, on the same schedule. At each refilling of the drinking bottle, total volume consumed was recorded. The two groups did consume different volumes of water. On a per day basis, the group that was not pharmacologically treated drank 41.2 ^■•-/- 1 0.6 mis/day; whereas, the group that was treated with aminoguanidine drank 30.2 +/- 4.9 mis/day (pθ.01 ). Given this volume of drinking, we calculate that the treated group received 60 mg aminoguanidinc/day. Dosing was not increased as the animals gained weight during the six months of this experiment. Once a week, each animal was weighed. Once a month, each animal was anesthetized (xylazine/ketaminc) and IOP was determined bilaterally (13) using the Mentor Pneumotonometer Model Classic 30 (BioRad, Santa Ana, CA, USA). The animals were awake within 15 min. of the IOP measurement. On any given eye, 3-5 tonometer readings were taken and averaged. On a given day. mean ⁽7- SD was derived for all control and surgical (3 vessel cautery) eyes. Significant differences between surgical and control eyes were determined by chi- square analysis with Student's t test for independent means for each day on which measurements were performed.

Clinical photography of rat fundus.

After six months of unilateral, chronic, moderately elevated IOP. photographs were taken of the optic disks of each eye of anesthetized rats with a fundus camera ( 50IΛ, Topcon Corp. , Japan) through a cover slip placed on the cornea with a drop of Goniosol (CIBΛ Vision Ophthalmics, Atlanta, GA). Color photographs and red-free photographs for relief imaging were processed using ImageNet ( 1024, Topcon).

Labeling and counting of retinal ganglion cells.

One week before sacrifice, Fluoro-Gold (Fluorochromc Inc., Fnglcwood, CO, USA) was microinjcctcd bilaterally into the superior colliculi of anesthetized rats immobilized in a stereotaxic apparatus. Fluoro-Gold is taken up by the axon terminals of the retinal ganglion cells and bilaterally transported retrogradely to the so as in the retina ( 17). The Fluoro-Gold in the retina persists for at least 3 weeks without .significant fading or leakage. One week after Fluoro-Gold application, animals were sacrificed by overdose of the above anesthetic mixture and whole, flat-mounted retinas were assayed for retinal ganglion cell density. Rat eyes were enucleated and fixed in 4% parafoπrtaldchyde for 30 minutes. Eyes were bisected at the equator, ihe lens was removed, and the posterior segments prepared for flat mounts. Retinas v. ere dissected from the underlying sclera, flattened by six radial cuts (largest cut^' superior for orientation) and mounted vitreal side up on gelatin coated slides.

Labeled retinal gangfion cells were counted using fluorescence microscopy in 12 identical size fields of retina, as previously described. Noting retinal topography, six fields in two regional areas, approximately 1.0 (central) and 4.0 (peripheral) mm from the optic disk, were counted at I 25X magnification. We counted approximately 15% of the total retinal ganglion cells in each eye using digital micrography (Spot, Diagnostic Instruments. Inc.. Sterling Heights, MI) by thresholding black and white images and computer scanning for particle analysis using Optimas software (Optimas

Corporation, Bothell, WA). Changes in retinal ganglion cell densities were expressed at % loss of retinal ganglion cells comparing surgical and contralateral. control eyes from the same animal in the different retinal regions. The Wilcoxon signed-rank test was used to compare parameters between the untreated and aminoguanidine treated groups.

Histology of cross sections of rat optic nerves.

Two of the eight animals in each group were used for histology. Segments of the myelinated portion of the optic nerves were fixed in 4% paraformaldchyde overnight and post-fixed in 2% osmium tetroxide for 2 hours. Nerves were then dehydrated in alcohol, embedded in Epon, sectioned as l μm cross sections and stained with paraphenylene-diamine to identify myelin profiles.

As demonstrated in l^'igure 1 , IOP was elevated in all eyes for six months after receiving three vessel cautery (approximately 1 8 mm Hg) compared to the contralateral, control eyes (approximately 1 1.5 mm Hg). Comparing animals that were not pharmacologically treated to animals treated with aminoguanidine, the elevated lOPs in the three vessel cautery eyes were the same. The IOPs in the contralateral eyes of these two groups were also the same. Thus, aminoguanidine did not affect IOP. After six months of unilateral, chronic, moderately elevated IOP, an experienced glaucoma specialist (BB) performed ophthalmoscopy on the rat eyes in a masked manner. By observing the optic disk, he correctly identified as glaucomatous the eyes with elevated IOP in animaTsTharwcrc not treated pharmacologically. He noted cupping and pallor of the optic disk, and bending backward of the vessels over the edge of the cup as they progressed towards the center of the disk. In the contralateral eyes with normal IOP, there was no cup and the vessels were fiat along the disk. In animals treated with animoguanidine, he noted littled or no differences in the appearance of the optic disks when comparing eyes with normal IOP and eyes with elevated IOP.

Figure 2 shows the clinical appearances of the optic disks of the rats in vivo. In the eye with normal IOP, the disk is pink, the vessels emerging from (arteries), and returning to (veins), the optic disk appear to run straight and flat from the center of the optic disk. In the eye with chronic, moderately elevated IOP for six months from an animal not treated pharmacologically, the optic disk is pale and cupped and the vessels, especially the veins, appear to dip over the rim of the cup. The projections of the vessels to the center of the optic disk are behind the plane of focus. In the eye with chronic, moderately elevated IOP for six months from an animal treated with aminoguanidine (Figures 2 C in color and 2F in relief), there is no pallor or cupping and the vessels can be clearly followed to the center of the optic disk These eyes look similar, clinically, to the eyes with normal IOP.

Figure 2 also shows crois-seclions through the myelinated portion of the corresponding optic nerves. Comparing eyes with chronic, moderately elevated IOP to the contralateral eyes with normal IOP (Figure 2G), axonal degeneration is apparent in peripheral regions of the optic nerve cross section in the animal that was not pharmacologically treated (Figure 2H) but is absent in the animal treated with aminoguanidine (Figure 21).

One week before sacrifice, the retinal ganglion cells of both eyes were labeled by bilateral injection of Fluoro-Gold into the superior colliculus. Llpon enuclcation. flat mounts of paired retinae were made and the retinal ganglion cells were counted in peripheral and central retinal areas. Figure 3 dcmonstartes the loss of retinal ganglion cells by comparing the eye with elevated IOP to the contralateral, control eye in animals not treated pharmacologically and in animals treated with aminoguanidine. in the peripheral retina (Figure 3A), retinal ganglion cells in eyes with chronic, moderately elevated IOP was 35.9 +/- 5.7% at six months in untreated animals and 9.6

+/- 3.5% at six months in animals treated with aminoguanidine (p<0.01 ). In the peripheral retina, the number of retinal ganglion cells in eyes with chronic, moderately elevated IOP of animals not treated pharmacologically was significantly different than normal eyes (p<0.01). However, the number of retinal ganglion cells in eyes with chronic, moderately elevated IOP of animals treated with aminoguanidine was not significantly different than normal eyes (p<2.00). In the central retina, proximal to the optic disk, loss of retinal ganglion cells in eyes with chronic, moderately elevated IOP was not apparent (Figure 3B). The loss of retinal ganglion cells in the peripheral retina is consistent with the peripheral areas of neurodegeneration in the optic nerve cross sections (Figure 2H).

Claims

We Claim:

I . A method for treating a person suffering from glaucomatous optic

amount of aminoguanidine in a pharmaceutically acceptable carrier.