WO2010016043A1

WO2010016043A1 - Treatment of retinal degeneration

Info

Publication number: WO2010016043A1
Application number: PCT/IE2009/000054
Authority: WO
Inventors: Thomas Cotter; Francesca Doonan; Sebastian Clerkin; Carolyn O'driscoll
Original assignee: University College Cork, National University Of Ireland, Cork
Priority date: 2008-08-05
Filing date: 2009-08-05
Publication date: 2010-02-11
Also published as: WO2010016043A8

Abstract

The use of 7-β-hydroxyethyltheophylline, or a functional variant thereof, in the manufacture of a medicament for the treatment or prevention of a disease or condition characterised by apoptosis or degeneration of mammalian cells.

Description

TREATMENT OF RETINAL DEGENERATION

TECHNICAL FIELD

The invention relates to a method of treating or preventing a disease or condition characterised by apoptosis or degeneration of mammalian cells, especially retinal photoreceptive cells.

BACKGROUND TO THE INVENTION

The loss of retinal cells in Age-related Macular Degeneration (AMD) and Glaucoma are the two leading causes of blindness in the developed worked. Retinitis Pigmentosa (RP) is a rarer related condition that also leads to loss of sight. RP is a group of hereditary disorders of the retina caused by mutations in numerous genes involved in photoreceptor structure or function. The disease is characterized by early loss of photoreceptors leading to blindness. Glaucoma is caused by a number of different pathological mechanisms that in most cases result in elevated intraocular pressure (IOP) within the eye. Like RP, it is a multiple gene-related disease and genetic factors play a complex role in glaucoma predisposition. Over time, the increase in IOP causes damage to the optic nerve and gradual and continuous loss of retinal ganglion cells. Cell loss in AMD (Age-related Macular Degeneration) also occurs as a result of apoptosis of retinal pigment epithelial (RPE) cells followed by apoptosis of photoreceptors. A central feature of each of the above diseases is that retinal cell loss occurs by a cell death process known as apoptosis.

There are no drugs on the market for the treatment of RP. There are a small number in early stage development, but most of them rely on repeated intavitreal injections directly into the eye for their effect or a gene therapy approach. For RP, there is a market realisation that preventing apoptosis may be a treatment option. In the case of glaucoma there are several treatment options currently available for this very large market segment. Most of these rely on the delivery of the drug in the form of eye drops. The goal of such treatments is to reduce the intra ocular pressure that is a causative factor that leads to the loss of retinal ganglion cells. Like RP there are no current treatments focused on the prevention of retinal ganglion cell apoptosis. Finally, for AMD of which there are two main forms there are a number of treatment options ranging from laser therapy to the use of inhibitors that prevent blood vessel proliferation which is a characteristic of the condition and leads to loss of photoreceptors cells. Again each of these treatment options is invasive and requires repeated hospital visits.

It is an object of the invention to overcome at least one of the above problems.

BRIEF SUMMARY OF THE INVENTION

Accordingly, the invention relates to a method of treating or preventing a disease or condition characterised by apoptosis or degeneration of mammalian cells, especially retinal photoreceptive cells. The method of the invention comprises a step of treating an individual with a compound 7-β-hydroxyethyltheophylline, and functional variants thereof (hereafter "Active"). Functional variants include other N-7 substituted theophylline compounds that are capable of effecting a significant reduction of degeneration or apoptosis in cells having a phenotype characteristic of a degenerative condition such, for example, retinal photoreceptor cells from a patient with AMD or RP.

Suitably, the Active is administered in an amount effective to treat or prevent the disease or condition. When the invention relates to therapy, as opposed to prophylaxis, the individual is generally one in need of such treatment such as a patient having a retinal degenerative condition. Suitably, the disease or condition is a retinal degenerative disease, such as, for example, Retinitis Pigmentosa (RP), Glaucoma, or Age-related Macular Degeneration (AMD). In an alternative embodiment, the disease or condition is a mammalian degenerative disease, such as a neurodegenerative disease. The invention also relates to the use of the Active as a medicament. Suitably, the medicament is for treating a retinal degenerative disease, especially Retinitis Pigmentosa (RP), Glaucoma, or Age-related Macular Degeneration (AMD).

The invention also relates to the use of the Active in the manufacture of a medicament for the treatment or prevention of a disease or condition characterised by apoptosis or degeneration of mammalian cells. In particular, the invention relates to the use of the Active in the manufacture of a medicament for the treatment or prevention of a retinal degenerative condition such as a retinal dystrophy, examples being Retinitis Pigmentosa (RP), Glaucoma, or Age-related Macular Degeneration (AMD).

The invention also relates to a pharmaceutical composition formulated as a solution suitable for local delivery to the eye, the composition comprising 7-β- hydroxyethyltheophylline, or functional variants thereof, and a pharmaceutically acceptable carrier. Suitably, the pharmaceutical formulation is provided in a form selected from the group consisting of: an eye-drop solution; a solution suitable for intraocular injection; and a solution suitable for intraocular injection.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: This figure shows that sodium nitroprusside (SNP) induces apoptosis at 24 hours in the outer nuclear layer (ONL) which contains the photoreceptor cells. Photoreceptors are protected from SNP induced apoptosis by increasing concentrations of 7HT. The final panel shows that 7HT at the highest concentration used is not toxic to retinal cells.

Figure 2: This figure represents the results of 2 independent experiments. The number of TUNEL positive photoreceptor cells in 3 4Ox fields per sample was counted and graphed with error bars (+/- standard deviation - SD).

Figure 3: In this figure the study was extended from ex vivo cultures to the light damage model in vivo. In this acute model of retinal disease albino balb/c mice are exposed to excessive white light causing the photoreceptor cells in the ONL to die by apoptosis. This figure shows that mice exposed to bright white light undergo extensive apoptosis at 24 hours post light damage. 1 hour pre-treatment with 250mg/kg 7HT protects from the retinal damage observed.

Figure 4: A histogram of the data from Figure 3. TUNEL positive cells in three independent retinae were counted. Counts were performed on the central 4Ox field of the ONL and graphed with error bars (+/- standard deviation-SD). The graph indicates approx 25% protection from light induced cell death in the central retina at 24 hours.

DETAILED DESCRIPTION OF THE INVENTION

The therapeutic method, and therapeutic products, of the invention are directed against diseases or conditions characterised by apoptosis or degeneration of mammalian cells. In one embodiment of the invention, the disease or condition characterised by apoptosis or degeneration of mammalian cells is an ocular disease or condition, especially a retinal degenerative condition or disease such as a retinal dystrophy. In one embodiment of the invention, the disease or condition characterised by apoptosis or degeneration of mammalian cells, is a neurodegenerative disease. Typically, the neurodegenerative disease is selected from the group comprising: motor neurone disease (ALS) or variants thereof including primary lateral sclerosis and spinal muscular atrophy; prion disease; Huntington's disease; Parkinson's disease; Parkinson's plus; Tauopathies; Chromosome 17 dementias; Alzheimer's disease; Multiple sclerosis (MS); hereditary neuropathies; and diseases involving cerebellar degeneration. The term should also be taken to include Central Nervous System (CNS) injury such as that caused by ischemia or trauma (i.e. stroke), and neurological complications associated with diabetes in a diabetic individual (i.e. diabetic neuropathy and diabetic retinopathy).

In a preferred embodiment of the invention, the retinal degenerative condition is selected from the group comprising: RP; Glaucoma; retinopathies; and AMD.

In this specification the term "amount effective" should be taken to mean an amount which results in a clinically significant reduction of degeneration or apoptosis in cells having a phenotype characteristic of a degenerative condition (i.e. retinal photoreceptor cells from a patient with AMD or RP). Suitably, the Active is administered at a dose of between 1 microgram and 10 miligrams per ml, preferably between 10 micrograms and 5 miligrams per ml, more preferably between 100 micrograms and 2 miligrams per ml. Typically, it is given as a bolus dose. However, when continuous infusion is used, such as by intrathecal pump, the Active may be administed at a dosage rate of between 5 and 20 μg/kg/minute, preferably between 7 and 15 μg/kg/minute. In the context of the therapeutic aspects of the present invention, the term "individual in need thereof shall be taken to mean an individual who is afflicted with a disease or condition which involves apoptosis or degeneration of mammalian cells, especially apoptosis or degeneration of the photoreceptive cell. Retinal degenerative conditions or diseases such as RP, Glaucoma, Retinopathies, and AMD, and variants thereof as described herein, are examples of such diseases or conditions.

In this specification, the term "functional variants" should be taken to include NT- substituted theophylline compounds, such as, for example, Etophylline, Acefylline, Proxyphylline, 7-chloroethyl theophylline, Doxofylline, Diprophylline, Theophylline, 7-acetylaminobutanol, Lomifylline, Theophylline-2-aminoethanol, Chinoin 170 or Dihydro- 1 ,3-dimethyl-7-[(5-methyl- 1 ,2,4-oxadiazol-3-yl)methyl]- 1 H-purine-2,6- dione , Fluprophylline or 7-(3-(4-(p-fluorobenzoyl)piperidino)propyl, Mepifiline, Neobiphylline, or 7-(l,3-Dithiolan-2-ylmethyl)-l,3-dimethylxanthine, N2-substituted compounds such as Theophylline-2-aminoethanol, N8-substituted compounds such as 8-phenyltheophylline or 8-bromotheophylline or N3 -substituted compounds such as Furafylline or Enprofylline.

In one embodiment of the invention, an individual in treated with the Active by direct delivery of the Active by a means selected from the group: intravenous delivery; intrperitoneal delivery; oral delivery; intramuscular delivery; intrathecal delivery; and inhaled delivery. Methods for achieving these means of delivery will be well known to those skilled in the art of drug delivery. Specific examples are provided below: • Delivered intrathecially by mini-osmotoc pump, (ref: Ignacio et al., Ann. N. Y. Acad. Sci. 2005, 1053: 121-136). • Intramuscular- delivery directly into muscle(s) by syringe or mini osmotic pump (Azzouz et al., Nat Med. 2005;l l(4):429-33).

• Intraperitoneal- for systemic administration. Directly administered to peritoneum by syringe or mini osmotic pump (Kieran et al., Nat Med 2004; 10(4):402).

• Subcutaneous- for systemic administration. Directly administered below the skin by syringe (Reinholz et al., Exp Neurol. 1999;159(l):204-16).

• Intraventricular- direct administration to the ventricles in the brain, by injection or using small catheter attached to an osmotic pump.(Sathasivam et al., 2005 Neuropath App Neurobiol; 31(5): 467)

• Implant- Active can be prepared in an implant (eg small silicon implant) that will release the active. Implant can be placed at muscles or directly onto the spinal cord (Kieran and Greensmith, 2004 Neurosci 125(2):427-39).

In one embodiment of the therapy of the invention, the Active is linked to a coupling partner, e.g. an effector molecule, a label, a drug, a toxin and/or a carrier or transport molecule. Techniques for coupling the Active of the invention to both peptidyl and non-peptidyl coupling partners are well known in the art.

The invention provides methods of treatment and prevention of diseases or conditions characterized by apoptosis or degeneration of mammalian cells, especially photoreceptive cells, by administration to a subject in need of such treatment of a therapeutically or prophylactically effective amount of the Active. The subject is preferably an animal, including, but not limited to, animals such as monkeys, cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably human..

Apart from the specific delivery systems embodied below, various delivery systems are known and can be used to administer the Active of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules. Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The Active may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the Active of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

In a specific embodiment, it may be desirable to administer the Active of the invention locally to the area in need of treatment; this may be achieved, for example, by means of eye drops, intraocular injection, or an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.

In another embodiment, the Active can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)

In yet another embodiment, the Active can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed., Eng. 14:201 (1987); Buchwald et al., Surgery 88:75 (1980); Saudek et al., N. Engl. J. Med. 321 :574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, FIa. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71 :105 (1989)). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990)). The present invention also provides pharmaceutical compositions comprising the Active. Such compositions comprise a therapeutically effective amount of the Active, and a pharmaceutically acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the Active is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like.

The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.

The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. Such compositions will contain a therapeutically effective amount of the therapeutic, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration. In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to, ease pain at the, site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

The Active of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2- ethylamino ethanol, histidine, procaine, etc.

The amount of the Active of the invention which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vivo and/or in vitro assays may optionally be employed to help predict optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

Experimental

Ex-vivo Methods

Retinal explant culture: Eyes from postnatal day (P) 10, C57BL/6 mice were removed and cleaned with 70% ethanol. The anterior segment, vitreous body, and sclera were removed and the retina mounted on Millicell nitrocellulose inserts (Millipore, Billerica, MA) photoreceptor-side down. Explants were cultured without retinal pigment epithelium (RPE) in 1.2 ml of Rl 6 specialised media (from Dr. P. A. Ekstrom, Wallenberg Retina Centre, Lund University, Lund, Sweden) without additional serum. Treated explants were cultured in medium containing 300μM of the nitric oxide donor SNP for 24 h. Pre-treatment with 20μM 7-β- (hydroxylethyl)theophylline (7HT) was for 1 hour. Figure 1 shows that photoreceptors are protected from SNP induced apoptosis by increasing concentrations of 7HT. The final panel in Figure 1 shows that 7HT at the highest concentration used in not toxic to retinal cells.

Apoptosis detection by Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL): Retinal explants were fixed in 10% neutral buffered formalin overnight at 4°C, followed by cryoprotection in 25% sucrose overnight at 4°C. Frozen sections (7μm) were incubated with terminal deoxynucleotidyl transferase (MSC, Dublin, Republic of Ireland) and fluorescein- 12- dUTP (Roche, Lewes, UK) according to manufacturers' instructions at 37°C for Ih. Sections were mounted and viewed under a fluorescence microscope (Leica DM LB2; Leica, Nussloch, Germany) using an FITC filter. Figure 2 shows that 7HT protects photoreceptive cells from SNP induced cell death in an ex-vivo retinal explant model.

In-vivo Methods

Light damage model: Balb/c mice were dark adapted for 18 h prior to exposure to constant light. Mice were injected intraperitoneally with the Active 1 hour prior to light damage. Immediately prior to light exposure their pupils were dilated with 0.5% cyclopentolate under red light. Retinal light damage was induced by exposure to 2 h of cool white fluorescent light at an illumination of 5000 lux. Following exposure to constant light, animals were placed in the dark for 24 hours then killed immediately by cervical dislocation. TUNEL staining was performed as described above. Figures 3 and 4 show that 2 hrs light damage induces apoptosis after 24 hours in the ONL. Photoreceptors are protected by IP injection of 250mg/kg of 7HT.

The invention is not limited to the embodiment hereinbefore described which may be varied in construction and detail without departing from the spirit of the invention.

Claims

1. Use of 7-β-hydroxyethyltheophylline, or a functional variant thereof, in the manufacture of a medicament for the treatment or prevention of a disease or condition characterised by apoptosis or degeneration of mammalian cells.

2. Use as claimed in Claim 1 in which the disease or condition characterized by apoptosis or degeneration of mammalian cells is a retinal dystrophy.

3. Use as claimed in Claim 2 in which the retinal dystrophy is selected from the group consisting of: Retinitis Pigmentosa (RP); Glaucoma; or Age-related Macular Degeneration (AMD).

4. Use as claimed in Claim 1 in which the disease or condition characterized by apoptosis or degeneration of mammalian cells is a neurodegenerative disease.

5. Use as claimed in Claim 4 in which the neurodegenerative disease is selected from the group: motor neurone disease (ALS) or variants thereof including primary lateral sclerosis and spinal muscular atrophy; prion disease; Huntington's disease; Parkinson's disease; Parkinson's plus; Tauopathies; Chromosome 17 dementias; Alzheimer's disease; Multiple sclerosis (MS); hereditary neuropathies; and diseases involving cerebellar degeneration.

6. Use as claimed in Claim 6 in which 7-β-hydroxyethyltheophylline, or a functional variant thereof; is locally administered to the eye.

7. Use as claimed in any preceding Claim in which 7-β-hydroxyethyltheophylline, or a functional variant thereof is administered by means of application of eye drops, intravitreal injection or intraocular injection.

8. A pharmaceutical composition formulated as a solution suitable for local delivery to the eye, the composition comprising 7-β-hydroxyethyltheophylline, or a functional variant thereof, and a pharmaceutically acceptable carrier A pharmaceutical formulation as claimed in Claim 8 in a form selected from the group consisting of: an eye-drop solution; a solution suitable for intraocular injection; and a solution suitable for intravitreal injection.