Use of calpain inhibitors to treat ocular neural pathology

The invention provides pharmaceutical compositions containing calpain inhibitors and methods of using these calpain inhibitors to prevent or ameliorate ocular neural tissue disease or damage.

BACKGROUND OF THE INVENTION
 The present invention relates generally to the field of ocular
 neuroprotectants and more specifically to the use of inhibitors of
 calcium-stimulated proteases to treat ocular neurodegeneration.
 Evidence from various studies suggests that one of the earliest events in
 the chain of reactions leading to neuronal death caused by ischemia or
 excitatory amino acid (e.g., glutamate) toxicity is an increase in
 intracellular free calcium. This intracellular free calcium increase is
 the consequence of extracellular Ca.sup.2+ channel opening, release of
 calcium from intracellular stores and/or energy depletion. Increases in
 intracellular calcium activate a number of cellular responses and
 processes that are thought to mediate cytotoxicity, including the
 activation of phospholipases, kinases, nitric oxide synthases,
 endonucleases and proteases, such as neutral proteases collectively
 referred to as the calpains. Additionally, an increase in intracellular
 calcium is also thought to induce changes in gene expression, such as the
 up-regulation of calpains.
 Calpains are a family of calcium activated cysteine (thiol) proteases which
 are present in the cytoplasm of many tissues. It is known that there are
 two distinct classes of isozymes, calpain I and calpain II. These enzymes
 require .mu.M and mM levels of calcium, respectively, for their optimal
 enzymatic activation. Hence, calpain I is also known as .mu.-calpain and
 calpain II is also known as m-calpain. Both calpains predominantly exist
 within cells in the form of an inactive precursor (Suzuki,
 Calcium-activated neutral protease and its endogenous inhibitor, FEBS
 Letters, volume 220, number 2, pages 271-277 (1987)). The precursor is
 converted into its active form in the presence of calcium through a
 self-digestion process which occurs at the N-terminal of the protein
 (Zimmerman, Two-stage autolysis of the catalytic subunit initiates
 activation of calpain I, Biochimica et Biophysica Acta, volume 1078, pages
 192-198 (1991)). Calpain II is the predominant form, but calpain I is
 found at synapses and is thought to be the form involved in long term
 potentiation and synaptic plasticity.
 Activated calpain hydrolyzes cellular proteins such as cytoskeletal
 proteins (e.g. spectrin, fodrin, talin, filamin, .alpha.-actinin,
 microtubule-associated proteins), membrane receptors (e.g. epidermal
 growth factor receptor, estrogen receptor, progesterone receptor,
 glucocorticoid receptor, platelet-derived growth factor receptor), cell
 adhesion molecules (e.g. integrin, cadherin, N-CAM), ion transporters
 (e.g. calcium-ATPase), calmodulin-binding proteins, guanyl
 nucleotide-binding regulatory proteins (G proteins), kinases (e.g. protein
 kinase C, myosin light chain kinase, calmodulin-dependent kinase, pp60
 src), phosphatases (e.g. calcineurin), phospholipases (e.g., phospholipase
 C), xanthine oxidase and transcription factors (e.g. Fos, Jun). (See,
 Saido, Calpain: new perspectives in molecular diversity and
 physiological-pathological involvement, FASEB Journal, volume 8, pages
 814-822 (1994).)
 It is clear that calpain participates in the control of many inter- and
 intracellular signal transduction systems. Thus, abnormal activation of
 calpain can have serious effects on cellular functions and viability
 (Murachi, Intracellular Regulatory System Involving Calpain And
 Calpastatin, Biochemistry International, volume 18, number 2, pages
 263-294 (1989); Nixon, Calcium-Actrvated Neutral Proteinases as Regulators
 of Cellular Function, Annals of the New York Academy of Sciences, volume
 568, pages 198-208 (1989)). Indeed, rapid activation of calpain, which
 occurs during ischemia and during treatment with excitatory amino acids,
 causes an acute neurotoxicity, apoptosis and neuronal cell death in brain
 tissues (Siman, Calpain I Activation Is Specifically Related to Excitatory
 Amino Acid Induction of Hippocampal Damage, Journal of Neuroscience,
 volume 9(5), pages 1579-1590 (1989); Lee, Inhibition of proteolysis
 protects hippocampal neurons from ischemia, Proceedings of the National
 Academy of Sciences USA, volume 88, pages 7233-7237 (1991)).
 Calpain activation has been associated with several neurodegenerative
 conditions, including those caused by Alllheimer's Disease, Parkinson's
 Disease, Pick's Disease, traumatic brain injury, subarachnoid hemorrhage,
 HIV-induced neuropathy, stroke, hypoxia, ischemia, lesions, and exposure
 to toxins (Wang, Calpain inhibition. an overview of its therapeutic
 potential, Trends in Pharmacological Sciences, volume 15, pages 412-419
 (1994); Saido, FASEB Joumal, volume 8, pages 814-822 (1994)).
 Calpain inhibitors have been shown to reduce neuronal damages induced by
 hypoxia or by amino acid excitotoxicity in brain tissue (Minami, Effects
 of inhibitors of protein kinase C and calpain in experimental delayed
 cerebral vasospasm, Journal of Neurosurgy, volume 76, pages 111-118
 (1992); Caner, Attenuation of AMPA-induced neurotoxicity by a calpain
 inhibitor, Brain Research, volume 607, pages 354-356 (1993); Rami,
 Protective effects of calpain inhibitors against neuronal damage caused by
 cytotoxic hypoxia in vitro and ischemia in vivo, Brain Research, volume
 609, pages 67-70 (1993); Hiramatsu, improved Posthypoxic Recovery of
 Synaptic Transmission in Gerbil Neocortical Slices Treated With a Calpain
 Inhibitor, Stroke, volume 24, pages 1725-1728 (1993); Hong,
 Neuroprotection With a Calpain Inhibitor in a Model of Focal Cerebral
 Ischemia, Stroke, volume 25, pages 663-669 (1994); and Bartus, Calpain
 Inhibitor AK295 Protects Neurons From Focal Brain Ischemia, Stroke, volume
 25, pages 2265-2270 (1994). Bartus et al. (WO 92/11850) describe several
 classes of calpain inhibitors and methods for identifying calpain
 inhibitors in which the enzymatic activity of calpain was assayed by the
 detection of spectrin breakdown products through Western blot analysis
 using a spectrin-specific antibody. Various publications in the scientific
 and patent literature have described numerous chemical classes of calpain
 inhibitors.
 There is increasing evidence which suggests that calpain is present in the
 retina (Karisson, Slow axonal transport of soluble proteins and calpain in
 retinal ganglion cells of aged rabbits, Neuroscience Letters, volume 141,
 pages 127-129 (1992); Persson, Immunohistochemical localization of
 calpains and calpastatin in the rabbit eye, Brain Research, volume 611,
 pages 272-278 (1993); Azarian, Characterization of calpain II in the
 retina and photoreceptor outer segments, Journal of Cell Sciences, volume
 105, pages 787-798 (1993); Azarian, Calpain activity in the retinas of
 normal and RCS rats, Current Eye Research, volume 14, pages 731-735
 (1995)).
 SUMMARY OF THE INVENTION
 The present invention is directed to the use of calpain inhibitors to
 protect ocular neural tissue. In particular the present invention is
 directed to compositions containing calpain inhibitors and methods of
 using these compositions to prevent ocular cell damage or diseases due to
 neurodegenerative disorders of the retina. Such neurodegerative disorders
 may be caused by ischemia, hypoxia, edema, oxidative injury, metabolic
 insufficiency, excitotoxicity, trauma and apoptotic cell death. Examples
 of neurodengenerative disorders of the retina include ischemic
 retinopathies or optic neuropathies (e.g. AION), commotio retinae,
 glaucoma, macular degeneration, retinitis pigmentosa, retinal detachment,
 retinal tears or holes, diabetic retinopathy and iatrogenic retinopathy.
 The calpain inhibitors are believed to protect the retinal neurons by
 inhibiting the destructive action of activated calpain, as described
 above. The calpain inhibitors may be administered by various means such as
 orally, parenterally, intraocularly or topically. Various compositions
 necessary for these various pharmaceutical applications are set forth
 below. Since there is no currently accepted treatment of the
 above-mentioned retinopathies, the calpain inhibitors provide a novel
 means to prevent or reduce retina and optic nerve head damage related to
 various ocular pathologies.
 DETAILED DESCRIPTION OF THE INVENTION
 The present invention is directed to compositions and methods of using
 calpain inhibitors to prevent or ameliorate ocular neuronal tissue damage
 or pathologies resulting from various cellular insults. As stated above,
 calpain is believed to be a critical factor involved in the cell's
 cytotoxic response to ischemia, hypoxia, amino acid excitotoxicity or
 other attacks on the ocular neuronal tissue.
 Calpain is activated by increases in intracellular calcium, which can be
 effected by all of the above pathological conditions. For example,
 excessive synaptic levels of glutamate (the major excitatory
 neurotransmitter in the retina) activate ionotropic excitatory amino acid
 receptors (which are themselves sodium and calcium ion channels) in the
 postsynaptic membrane, producing a sustained and elevated influx of sodium
 and calcium ions into the cell. Sodium influx induces membrane
 depolarization which opens voltage-gated calcium channels and causes
 further calcium influx, thus; further increasing the intracellular calcium
 concentration.
 In addition, trauma and ischemia in neuronal tissues can also increase
 intracellular calcium. At least two different mechanisms are involved. One
 involves excitotoxicity (resulting from excessive presynaptic release and
 impaired reuptake of glutamate), thus leading to calcium mobilization.
 Another mechanism involves the depletion of cellular ATP and the
 subsequent loss of ion transport regulation across intracellular
 organelles, as well as across the plasma membrane. These events directly
 and indirectly increase intracellular calcium and stimulate protease
 activity of calpain.
 Based on calpain's role in neuronal cell death following cellular stress,
 it is believed that calpain is also involved in various forms of
 retinopathy or optic nerve pathology related to glaucoma, ischemia, edema
 or other traumas. It is also believed that an inhibitor of the enzymatic
 activity of calpain will have therapeutic value in the prevention or
 treatment of the above-mentioned ocular diseases. As indicated above, the
 protease activity of calpain is responsible for the hydrolysis of many
 intracellular proteins which in turn interferes with cellular functions.
 This interference of normal cellular functions eventually leads to cell
 death. The present invention provides the use of calpain inhibitors for
 the protection of ocular neuronal tissue, i.e., the retina and optic
 nerve.
 Molecules from various chemical classes can be used as calpain inhibitors.
 As used herein, the term "calpain inhibitor" refers to those molecules
 which retard or inhibit the catalytic action of calpain.
 Calpain inhibitors of the present invention are known and have been
 described in numerous scientific and patent literature. For example, U.S.
 Pat. No. 5,081,204 (Higuchi), U.S. Pat. No. 5,486,623 (Zimmerman), U.S.
 Pat. No. 5,498,616 (Mallamo), U.S. Pat. No. 5,506,243 (Ando), and U.S.
 Pat. No. 5,514,694 (Powers) describe a variety of different chemical
 entities for the inhibition of calpain including: N-substituted peptidyl
 compounds, peptidyl ketone heterocyclic ethers, heterocyclic-N-heteroatom
 methyl ketones, sulfonamide pyrolidines, and peptidyl ketoamides,
 respectively. Additional examples of calpain inhibitors in the patent
 literature include WIPO Publication Nos. WO 92/11850 (Cortex
 Pharmaceutical), WO 94/00095 (Cortex) and WO 95/00535 (Akermes Inc.) which
 disclose peptide keto compounds, peptide aldehyles and .alpha.-ketoamides,
 respectively. Other examples of calpain inhibitors have been published in
 European Patent Application Publications. Still other calpain inhibitors
 in the scientific literature include .alpha.-mercaptoacrylic acids,
 disclosed in Proc. Natl. Acad. Sci. USA, volume 93, pages 6687-6692
 (1996).
 Preferred calpain inhibitors include the following peptide keto-compounds,
 all of which are disclosed in WIPO Patent Application No. WO 94/00095:
 Compound 1 (Example PKC 131)
 PhCH.sub.2 OCO-leucine-norvaline-CONH--CH.sub.2 -2-pyridyl;
 Compound 2 (Example PKC 145)
 Ph.sub.2 CHCO-leucine-.alpha.-aminobutyric acid-CONH--CH.sub.2 -2-pyridyl;
 Compound 3 (Example PKC146)
 Ph.sub.2 CHCO-leucine-.alpha.-aminobutyric acid-CONH--(CH.sub.2).sub.3
 -4-morpholinyl;
 Compound 4 (Example PKC104)
 PhCH.sub.2 OCO-leucine-.alpha.-aminobutyric acid-CONH--CH.sub.2 -2-pyridyl;
 and
 Compound 5 (Example PKC78)
 PhCH.sub.2 OCO-leucine-.alpha.-aminobutyric acid-CONH--CH.sub.2 --CH(OH)Ph.
 The most preferred calpain inhibitor is PD 150606, which is disclosed in
 Wang, Proc. Natl. Acad. Sci. USA, volume 93, pages 6687-6692 (1996),
 described above.
 The calpain inhibitors of the present invention may also be determined by
 various assays described in the literature. The following publications
 teach various methods for calpain inhibitor elucidation, the entire
 contents of which are incorporated herein by reference:
 1) Wang, An alpha-mercaptoacrylic acid derivative is a selective nonpeptide
 cell-permeable calpain inhibitor and is neuroprotective, Proc. Natl. Acad.
 Sci. USA, volume 93, pages 6687-6692 (1996);
 2) WIPO Publication No. WO 95/00535 (Alkermes, Inc.), Page 47; and
 3) WIPO Publication No. WO 92/11850 (Cortex Pharmaceuticals, Inc),
 Substitute Sheet, Page 49.
 The compositions and methods of the present invention use agents which
 inhibit calpain, for preventing or protecting the retina and optic nerve
 head from diseases or damages caused by glaucoma, ischemia, trauma or
 edema.
 Calpain inhibitors may be administered systemically, topically, by
 intraocular injection, intraocular perfusion, periocular injection or
 retrobulbar injection. When calpain inhibitors are delivered by systemic
 administration, including oral administration, intramuscular injection,
 subcutaneous injection, intravenous injection, transdermal administration
 and transmucosal administration, the daily dosage of calpain inhibitors
 will range between about 0.01 and 100 milligrams per kilogram body weight
 per day (mg/kg/day), preferably between about 0.1 and 10 mg/kg/day.
 The exact dosage of one or more calpain inhibitor(s) to be administered to
 the patient will vary, but will be determined by skilled clinicians in the
 art. Various factors affecting the dosage amount include the actual
 disease to be treated, the severity of condition, the health of the
 patient, the potency and specific efficacy of the calpain inhibitor, and
 so on. The amount dosed, however, will be an "effective amount." As used
 herein, the term "effective amount" is an amount which inhibits calpain at
 a level effective for therapy.
 The calpain inhibitors of the present invention may be contained in various
 types of ophthalmic compositions, in accordance with formulation
 techniques known to those skilled in the art For example, the compounds
 may be included in solutions, suspensions and other dosage forms adapted
 for topical, intravitreal or intracameral use.
 The ophthalmic compositions of the present invention will include one or
 more calpain inhibitor(s) of the present invention and a pharmaceutically
 acceptable vehicle. Aqueous solutions are generally preferred, based on
 ease of formulation and physiological compatibility. However, the calpain
 inhibitors of the present invention may also be readily incorporated into
 other types of compositions, such as suspensions, viscous or semi-viscous
 gels or other types of solid or semi-solid compositions. The ophthalmic
 compositions of the present invention may also include various other
 ingredients, such as buffers, preservatives, co-solvents and viscosity
 building agents.
 An appropriate buffer system (e.g., sodium phosphate, sodium acetate or
 sodium borate) may be added to prevent pH drift under storage conditions.
 Ophthalmic products are typically packaged in multidose form. Preservatives
 are thus required to prevent microbial contamination during use. Suitable
 preservatives include: benzalkonium chloride, thimerosal, chlorobutanol,
 methyl paraben, propyl paraben, phenylethyl alcohol, edetate disodium,
 sorbic acid, polyquatemium-1, or other agents known to those skilled in
 the art. Some of these preservatives, however, may be unsuitable for
 particular applications, (e.g., benzalkonium chloride may be unsuitable
 for intraocular injection). Such preservatives are typically employed at a
 level of from is 0.001 to 1.0% weight/volume ("% w/v").
 While at the present time there are no effective methods to effect back of
 the eye treatment of chronic conditions via topical administration, it is
 contemplated that such methods will be developed. If topical
 administration of calpain inhibitors becomes feasible, the dosage
 generally will range between about 0.001 and 5% weight/volume ("w/v"),
 preferably between 0.1 and 1% (w/v). Solutions, suspensions, ointments,
 gels, jellies and other dosage forms adapted for topical administration
 are preferred. Additionally, calpain inhibitors may be delivered slowly,
 over time, to the afflicted tissue of the eye through the use of contact
 lenses. This regimen is generally performed by first soaking the lenses in
 a calpain inhibitor solution, and then applying the contact lenses to the
 eye for normal wear.
 As used herein, the term "pharmaceutically acceptable carrier" refers to
 any formulation which is acceptable, i.e., safe and provides the
 appropriate delivery for the desired route of administration, of an
 effective amount of at least one calpain inhibitors of the present
 invention.

The compositions of the present invention are further illustrated in the
 following formulation examples, calpain inhibitors of the present
 invention are represented generically in the examples as "Calpain
 Inhibitor."
 EXAMPLE 1
 A topical ophthalmic composition useful for treating ocular neural tissue:

Amount per Tablet
 Ingredient (mg)
 Calpain Inhibitor 200
 Cornstarch 50
 Lactose 145
 Magnesium stearate 5
 EXAMPLE 4
 An systemic injectable solution useful for treating ocular neural tissue:

Ingredient Amount
 Calpain Inhibitor 200 mg
 0.4 M KH.sub.2 PO.sub.4 solution 2 ml
 1 N KOH solution q.s. to pH 7.0
 Water for injection q.s. to 20 ml