PROTECTION OF OCULAR GLAND TISSUE AND FUNCTION FROM ADVERSE EXTERNAL OR INTRINSIC EFFECTS IN DRY EYE DISORDERS

RELATED APPLICATIONS

[0001] The present application claims the benefit of co-pending U.S. Provisional Patent Application Serial Number 60/938,092 filed may 15, 2007, the text of which is specifically incorporated by reference herein, and the present application claims the benefit of co-pending U.S. Provisional Patent Application Serial Number 60/944,701 filed June 18, 2007, the text of which is specifically incorporated by reference herein

FIELD OF THE INVENTION

[0002] This present invention relates generally to methods of treatment for dry eye. BACKGROUND OF THE INVENTION

[0003] Dry eye, also known genetically as keratoconjunctivitis sicca, is a common ophthalmological disorder affecting millions of Americans each year. The condition is widespread among various populations. However, it particularly affects post-menopausal women due to hormonal changes following the cessation of fertility.

[0004] As with most diseases, the symptoms are quite variable. Dry eye may afflict an individual with varying severity. In mild cases, a patient may experience burning, a feeling of dryness, and foreign body sensation of the eyes. In severe cases, vision may be substantially impaired.

[0005] Like the symptoms experienced, the causes are widespread. Various diseases, such as Sjogren's disease, several autoimmune conditions, such as rheumatoid arthritis and lupus and cicatricial pemphigoid manifest dry eye complications like a dry eye disorder. As well, trauma to the eye during surgical procedures such as lasik surgery or even photorefractive keratectomy may lead to dry eye symptoms.

[0006] Further, diseases or environmental conditions that change the quantity or components of the ocular tear fluid as well, and/or the dimension of the tear film can lead to changes in structure and/or function, irritation, damage of frontal parts of the eye such as eyelids, conjunctiva, or cornea and ultimately to vision loss Lemp et al., "Introduction. In: The dry eye. A comprehensive guide." Berlin: Springer- Verlag, 1992:1-2.

[0007] Depending on the disease causing factors which can be both external and/or intrinsic and progression of the particular dry eye disorder different outcomes/symptoms can be experienced by patients, such as, but not limited to: a. degrees of visual impairment and/or vision loss; b. occurrence of secondary infections; and/or c. development of temporary or chronic inflammation processes.

[0008] A predominant cause of these disorders are abnormalities in tear film composition including, but not limited to dysfunctions of glands, such as the lacrimal gland and the Meibomian gland; eye lid disorders, such as, but not limited to blepharitis; and various systemic eye disorders. Baum, "Clinical manifestations of dry eye states," Trans Ophthalmol Soc UK 1985, 104:415-23; Holly et al, "Tear physiology and dry eyes," Surv Ophthalmol 1977, 22:69- 87; Jones, "The lacrimal secretory system and its treatment," Am J Ophthalmol 1966; 62:47-60; Larkin et al., "The red eye," Optometry Documenta, Optometry Clinic Monograph 11, Irvine, CA: Allergan, 1979; 8-11; Lemp, "Epidemiology and classification of dry eye," Adv Exp Med Biol 1998, 438:791-803; Lemp, "Report of the National Eye Institute/Industry Workshop on Clinical Trials in Dry Eyes," CLAO J 1995, 21 :221-32; McMonnies, "Key questions in a dry eye history," J Am Optorn Assoc, 1986, 57:512-7; Pflugfelder et al., "Evaluation of subjective assessments and objective diagnostic tests for diagnosing tear-film disorders known to cause ocular irritation," Cornea 1998, 17(1) 38-56; and, Semes, "Keratoconjunctivitis sicca and ocular surface disease," In: Silbert J, ed. Anterior segment complications of contact lenses. Boston: Butterworth-Heinernann, 2000: 197-210.

[0009] The causative mechanisms underlying the different dry eye disorders are not fully known at this point and the effects of multiple etiologies may contribute to the disease. However, numerous studies have indicated that a major component in the etiology is oxidative stress where the exocrine activity of various glands is impaired.

[0010] Likewise, although it appears that dry eye may result from a number of unrelated pathogenic causes, all presentations of the complication share the common effect of the

breakdown of the pre-ocular tear film, which results in damage of the exposed outer surface and many of the symptoms outlined above.

[001 1] There are numerous curative procedures used to treat a dry eye disorder. One common approach is to supplement and stabilize the ocular tear film using so-called artificial tears instilled throughout the day. Other approaches include the use of ocular inserts that provide a tear substitute or stimulation of endogenous tear production.

[0012] Examples of the tear substitution approach include the use of buffered, isotonic saline solutions, aqueous solutions containing water-soluble polymers that render the solutions more viscous and thus less easily shed by the eye. Examples of such solutions are available from AlconLabs Inc of Fort Worth, Texas. Tear reconstitution is also attempted by providing one or more components of the tear film such as phospholipids and oils. Phospholipid compositions have been shown to be useful in treating dry eye. Another approach involves the provision of lubricating substances in lieu of artificial tears.

[0013] Although these approaches have met with some success, problems in the treatment of dry eye nevertheless remain. The use of tear substitutes, while temporarily effective, generally requires repeated application over the course of a patient's waking hours. It is not uncommon for a patient to have to apply artificial tear solution ten to twenty times over the course of the day. Such an undertaking is not only cumbersome and time consuming, but is also potentially very expensive. Transient symptoms of dry eye associated with refractive surgery have been reported to last in some cases from six weeks to six months or more following surgery.

[0014] Aside from efforts directed primarily to the alleviation of symptoms associated with dry eye, methods and compositions directed to treatment of the dry eye condition have also been pursued. Many of these compositions use steroids that have limited utility due to concomitant increases in intraocular pressure and/or development of cataracts in the eye.

[0015] Thus, there is a continuing need for an effective pharmaceutical intervention to treat the underlying causes of dry eye. An understanding of the morphological changes associated with dry eye provides a starting point for the development of appropriate pharmaceutical treatments. Analysis of the corneas of various patient populations with dry eye or dry eye symptoms show abnormalities in neuronal beds as imaged with confocal microscopy and also

show abnormalities in corneal sensitivity. The most marked change in the nerve beds is the appearance of tortuosities. A pharmaceutical formulation that may address these abnormalities in dry eye patients is, therefore, a worthwhile pursuit, such as a metal-containing catalytic antioxidant -based treatment.

[0016] While much is known of metal-containing catalytic antioxidants, no study has been performed on the use of a metal-containing catalytic antioxidant for treatment of a dry eye disorder. Researchers have, however, shown that systemic administration of a superoxide dismutase (SOD) to rats will inhibit the reverse passive Arthus reaction in the skin (Petrone et al., 1980, Proc. Natl. Acad. ScL, 77:1159; Parellada et al., 1978, Biochem. Pharm., 27:535). As well, treatment of facial lesions in patients with Crohn's disease with a topical application of liposomes containing superoxide dismutase was shown to reduce swelling (Michelson, 1982. In "Pathology of Oxygen," Academic Press, N.Y., p. 277). Other studies have shown that topical administration of a low molecular weight lipophilic copper coordination complex with superoxide dismutase-mimetic activity inhibits certain phorbol ester-induced biochemical and biological responses associated with carcinogenesis. (Kensler et al., 1983, Science, 221 :75). However, no studies have shown the effects of treatment of a dry eye disorder with a metal- containing catalytic antioxidant.

[0017] A porphyrine is a heterocyclic macrocycle made from 4 pyrrole subunits linked on opposite sides (α position) through 4 methine bridges (=CH-).

[0018] Porphyrines combine readily with metals, coordinating with them in the central cavity. Iron- (heme), magnesium- (chlorophyll), zinc-, copper-, nickel-, manganese-, and cobalt- containing porphyrines are known, and many other metals can be inserted. A porphyrine in which no metal is inserted in its cavity is called a free base. Commonly, iron-containing porphyrines are called hemes. Hemes and/or heme-containing proteins are found extensively in biochemistry in forms such as hemoglobin.

[0019] Practical known uses of porphyrines include meso-tetraphenylporphyrin iron-(III) chloride (or ClFeTPP) as a catalyst in organic chemistry, compounds for molecular memory, inks, and/or the like. However, there is not reported use of a metal containing porphyrine in the treatment of ocular disorders, such as, but not limited to a dry eye disorder. Accordingly, a

treatment regimen using a porphyrine-based compound(s) for the treatment of a dry eye disorder would most likely be a novel treatment.

[0020] A Salen is a dianionic and tetradendate ligand in an organometallic complex. Salen is a contraction for salicylic aldehyde and ethylene diamine which are the reactants for the ligand. The actual composition of the ligand depends on the various substituents. For example, and not by way of limitation, Salph is a salen ligand with a Phenyl core group from the imine forming reaction of salicylic aldehyde with o-diaminobenzene.

[0021] A macrocylic molecule is a cyclic macromolecule or a macromolecular cyclic portion of a molecule. It is understood in the art field that a macrocyclic molecule has donor atoms arranged in more fixed positions and thus there is less of an entropic effect in the binding energy of macrocycles than monodentate or bidentate ligands with an equal number of donor atoms.

[0022] Metal containing porphyrines, metal containing salens and metal containing macrocyclics can be classified as antioxidants. Antioxidants have many important biological functions. However, to date, no studies have been performed on the use of metal containing porphyrines or metal containing salens in the treatment of a dry eye disorder. Accordingly, treatments for a dry eye disorder using a at least one metal-containing catalytic antioxidant would be beneficial in therapy approaches for dry eye diseases.

SUMMARY OF THE INVENTION

[0023] Various embodiments of the present invention generally relate to methods of treating dry eye and/or dry eye symptoms. Methods and systems are disclosed for treating dry eye or a dry eye symptom with at least one formulation having at least one metal-containing catalytic antioxidant that provides enhanced benefits as compared to currently available treatments, including at least enhanced tissue regeneration. In various embodiments, the at least one formulation comprises a superoxide dismutase (SOD) mimetic. Various treatment approaches of embodiments of the present invention reduce damage to ocular gland tissue and/or allow preservation of gland function. In various embodiments, the treatment approach leads to at least one of a reduction of oxidative stress, a reduction the cellular degeneration, and/or decline in the death of acinar cells induced by oxidative stress as compared to the prior art treatments. Various

embodiments are suitable for the treatment of at least one of congenital, autoimmune conditions, chemical damage, mechanical damage, infections, and/or the like.

[0024] Various embodiments of the present invention comprise a method of treating dry eye and/or dry eye symptoms, the method comprising administering an effective amount of a formulation comprising at least one metal-containing catalytic antioxidant to a patient, wherein the administered formulation treats dry eye and/or dry eye symptoms.

[0025] Further embodiments comprise a method of protecting ocular gland tissue in a patient suffering from a dry eye syndrome or at least one of the symptoms thereof, said method comprising the steps of administering an effective amount of a formulation comprising a at least one metal-containing catalytic antioxidant to a patient, wherein the administered formulation protects ocular gland tissue.

[0026] Yet further embodiments comprise a formulation comprising an ophthalmically acceptable carrier and a therapeutically effective amount of at least one metal-containing catalytic antioxidant.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order that the manner in which the above recited and other advantages and objects of the invention are obtained, a more particular description of the invention briefly described above will be rendered by reference to specific embodiments thereof, which are illustrated, in the appended drawings. Understanding that these drawings depict only typical embodiments of the invention and are therefore not to be considered limiting of its scope, the invention will be described with additional specificity and detail through the use of the accompanying drawings in which:

[0028] Figure 1 shows Hematoxylin-Eosin stained adult NOD/LtJ (left panels, "NOD") mouse lacrimal gland tissue and wildtype C57BL/6J (right panels, "Normal") adult mouse lacrimal gland tissue.

[0029] Figures 2A-2D show micrographs of Hematoxylin-Eosin stained 12 μm thick cryo- sections of mouse lacrimal glands with or without manganese-porphyrin treatment. Control group injected with saline (PBS)(Figure 2A), Control group injected with manganese-porphyrin

(Figure 2B), NOD mice injected with saline (PBS) (Figure 2C), and NOD mice injected with manganese-porphyrin (Figure 2D).

[0030] Figure 3 is a bar graph illustrating the blood glucose concentration for control mice (C57BL6J) injected with saline, Control mice injected with manganese-porphyrin, NOD mice injected with saline, and NOD mice injected with manganese-porphyrin.

[0031 ] Figure 4 shows histochemical staining of lacrimal gland tissue from Control mice (top two panels) treated with saline alone (vehicle treated) or with MnTE-2-PyP (MnTE-2-PyP treated control) and from NOD mice (bottom two panels) treated with saline (vehicle treated NOD mice) or withMnTE-2-PyP (MnTE-2-PyP treated NOD mice).

[0032] Figure 5 is a bar graph representation of summary statistics of the immune cell infiltration for control mice, injected with saline alone (Control)and control mice injected with MnTE-2-PyP (control-inj) and for NOD mice injected with saline alone (NOD) and NOD mice injected with MnTE-2-PyP. (n=4 for each condition).

[0033] Figure 6 is a graphic representation of the histological grading of inflammatory lesions of lacrimal glands as visualized by Hematoxylin and Eosin staining in control and NOD mice untreated or treated with MnTE-2-PyP. Control mice injected with saline (Control), control mice injected with MnTE-2-PyP (Control+inj), NOD mice injected with saline (NOD), and NOD mice injected with MnTE-2-PyP (NOD+inj). Values represent mean of the group. n=4, asterisk, PO.05).

[0034] Figures 7A-7L depict micrographs of PlO mouse primary cultures acinar cells fixed after oxidative stress insult. DIC images show cells that received no treatment (Figure 7A), 100 μm of MnTMPyP (Fig. 7C), lOμm of hydrogen peroxide (Figure 7E), lOμm of hydrogen peroxide with 10 μm of MnTMPyP (Figure 7G), lOμm of hydrogen peroxide with 50 μm of MnTMPyP (Figure 71), lOμm of hydrogen peroxide with 100 μm of MnTMPyP (Figure 7K). Figures 7B, 7D, 7F, 7H, 7J, and 7L are the corresponding micrographs displaying DAPI counterstaining of the same samples. Scale bar shown in Figure 7L applies to Figures 7A-7L, 25 μm.

[0035] Figure 8 is the graphic representation of hydrogen peroxide induced cell death of lacrimal acinar cells expressed as percentage of total cell number.

[0036] Figure 9 is a bar graph comparing results of testing of various compounds for cytoprotection of lacrimal gland tissue in NOD mice.

DESCRIPTION OF THE INVENTION

[0037] The particulars shown herein are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of various embodiments of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for the fundamental understanding of the invention, the description taken with the drawings and/or examples making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

[0038] The following definitions and explanations are meant and intended to be controlling in any future construction unless clearly and unambiguously modified in the following Examples or when application of the meaning renders any construction meaningless or essentially meaningless. In cases where the construction of the term would render it meaningless or essentially meaningless, the definition should be taken from Webster's Dictionary, 3 ^ld Edition.

[0039] The following definitions and explanations are meant and intended to be controlling in any future construction unless clearly and unambiguously modified in the following examples or when application of the meaning renders any construction meaningless or essentially meaningless. In cases where the construction of the term would render it meaningless or essentially meaningless, the definition should be taken from Webster's Dictionary, 3 ^ld Edition.

[0040] As used herein, all percentages are percentages by weight, unless stated otherwise.

[0041] As used herein, the term "dismutation reaction" means and refers to a reaction in which two like-molecules react to produce two different products

[0042] As used herein, the term "dismutase" means and refers to at least one catalyst for a dismutation reaction.

[0043] As used herein, the term "effective amount" means and refers to an amount that is effective in providing at least partial relief to the condition being treated.

[0044] As used herein, a "fluid" is a continuous, amorphous substance whose molecules move freely past one another and that has the tendency to assume the shape of its container, for example, a liquid or a gas.

[0045] As used herein, the term "health care provider" is known in the art and specifically includes a physician, a person with authority to prescribe a medication (whether directly or indirectly), and a veterinarian. In certain embodiments, a health care provider includes an individual that provides a medication without prescription, such as in providing an over-the- counter medication.

[0046] As used herein, the terms "identifying subjects" and "diagnosing" are used interchangeably with regard to the detection of a "predisposition," "increased propensity," "risk," "increased risk," and the like.

[0047] As used herein, the term "intraperitoneal," "ip," and/or "i.p." means and refers to the peritoneal cavity.

[0048] As used herein, the term "intraperitoneal injection," "ip injection," and/or "i.p. injection" means and refers to at least one injection into the peritoneal cavity.

[0049] As used herein, the term "other retinal or optic nerve disease" means and refers to at least one of age-related macular degeneration, cataract, acute ischemic optic neuropathy (AION), commotio retinae, retinal detachment, retinal tears or holes, diabetic retinopathy and iatrogenic retinopathy and other ischemic retinopathies or optic neuropathies, myopia, retinitis pigmentosa, and/or the like.

[0050] As used herein, the term "administered to the subject" and/or "to the subject's site" means and refers to delivery of an effective amount of the formulation and/or medicament.

[0051] As used herein, the term "porphyrin" and/or "porphyrine" means and refers to a heterocyclic macrocycle made from 4 pyrrole subunits linked on opposite sides (α position) through 4 methine bridges (=CH~). As porphyrines follow Huckel's rule, porphyrines have aromatic properties.

[0052] As used herein, he term "subject" or "patient" refers to any invertebrate or vertebrate species. The methods of the present invention are particularly useful in the treatment of warmblooded vertebrates. Thus, in an embodiment, the invention concerns mammals and birds.

[0053] As used herein, the term "superoxide dismutase(s)" (SODs) means and refers to a family of metalloenzymes that catalyze the intra- and extracellular conversion of oxygen radicals (O ₂ .) and H ₂ into H ₂ O ₂ plus O ₂ , and represent the first line of defense against the detrimental effects of superoxide radicals. Three SODs are known and found in the human body. One is a dimeric copper- and zinc-containing enzyme (CuZn SOD) found in the cytosol of all cells. A second is a tetrameric manganese-containing SOD (Mn SOD) found within mitochondria. The third is a tetrameric, glycosylated, copper- and zinc-containing enzyme (EC-SOD) found in the extracellular fluids and bound to the extracellular matrix.

[0054] Referring to the drawings in general, it will be understood that the illustrations are for the purpose of describing a particular embodiment of the invention and are not intended to limit the invention thereto.

[0055] In some embodiments, a method of treating dry eye or dry eye symptoms may include administering a formulation of an embodiment of the present invention effectively to the eye of a patient. In various embodiments, a formulation of the present invention comprises at least one metal -containing catalytic antioxidant. In an embodiment, administration of the formulation to the patient with dry eye is capable of treating the underlying cause of dry eye.

[0056] In various embodiments, a formulation of the present invention comprises at least one superoxide dismutase (SOD) mimetic. In an embodiment, administration of the formulation to the patient with dry eye is capable of treating the underlying cause of dry eye.

[0057] In general, methods and systems are disclosed for treating dry eye or a dry eye symptom with at least one metal-containing catalytic antioxidant that provides enhanced benefits as compared to currently available treatments, including at least enhanced tissue regeneration. Various treatment approaches and/or regimens are capable of use with various embodiments of the present invention to reduce damage to ocular gland tissue and/or allow preservation of gland function.

[0058] In various embodiments, such treatment regimens are referred to as a an intervention approach, wherein of embodiments of the present invention are capable of leading to at least one of a reduction of oxidative stress, a reduction of cellular degeneration, and/or a decline in the death of acinar cells induced by oxidative stress as compared to the prior art treatments. Accordingly, various embodiments are suitable for the treatment of congenital, autoimmune conditions, chemical damage, mechanical damage, infections, and/or the like.

[0059] Metal-containing catalytic antioxidants appropriate for use in various embodiments of the present methods comprise methine (i.e., meso) substituted porphyrines, or pharmaceutically acceptable salts thereof (e.g., chloride or bromide salts). The invention includes both metal-free and metal-bound porphyrines. In the case of metal-bound porphyrines, manganic derivatives of methine (meso) substituted porphyrines are preferred, however, metals other than manganese such as iron (II or III), copper (I or II), cobalt (II or III), or nickel (I or II), can also be used. It will be appreciated that the metal selected can have various valence states, for example, manganese II, IH or V can be used. Zinc (II) can also be used even though it does not undergo a valence change and therefore will not directly scavenge superoxide. In an embodiment, the antioxidant is a SOD mimetic, such as, but not limited to a manganese porphyrine. In an alternate embodiment, the SOD mimetic is an iron porphyrine. In general, any metal complexed antioxidant can be used.

[0060] Generally, in various embodiments of mimetics of the present invention are of the Formula I:

[0061 ] or a pharmaceutically acceptable salt thereof

[0062] wherein: Ri and R ₃ are the same and are:

-H. -CF,. -COv -X.

[0063] R ₂ and R ₄ are the same and are:

[0064] Y is halogen or -CO ₂ X, each X is the same or different and is an alkyl and each R ₅ is the same or different (preferably the same) and is H or alkyl.

[0065] Preferably, Rj and R ₃ are the same and are:

[0066] R ₂ and R ₄ are the same and are:

[0067] Y is — F or -CO ₂ X each X is the same or different and is an alkyl (preferably, Ci _-4 alkyl, e.g., methyl or ethyl) and each R ₅ is the same or different (preferably the same) and is H or alkyl (preferably, Ci _-4 alkyl, e.g., -CH ₃ or -CH ₂ CH ₃ ).

[0068] Most preferably, Ri, R ₂ , R ₃ and R ₄ are the same and are

[0069] and each X is the same or different and is Ci _-4 alkyl, advantageously, methyl or ethyl, particularly, methyl.

[0070] In addition to the methine (meso) substituents described above, one or more of the pyrrole rings of the porphyrin of Formula I can be substituted at any or all beta carbons, i.e.: 2, 3, 7, 8, 12, 13, 17 or 18. Such substituents, designated P, can be hydrogen or an electron withdrawing group, for example, each P can, independently, be a NO ₂ group, a halogen (e.g., Cl, Br or F), a nitrile group, a vinyl group, or a formyl group. Such substituents alter the redox potential of the porphyrin and thus enhance its ability to scavenge oxygen radicals. For example, there can be 1, 2, 3, 4, 5, 6, 7, or 8 halogen (e.g., Br) substituents (preferably, 1-4), the remaining P's advantageously being hydrogen. When P is formyl, it is preferred that there not be more than 2 (on non-adjacent carbons), more preferably, 1, the remaining P's preferably being hydrogen.

When P is NO ₂ , it is preferred that there not be more than 4 (on non-adjacent carbons), more preferably, 1 or 2, the remaining P's being hydrogen.

[0071] Where isomers are possible, all such isomers of the herein described mimetics are within the scope of the invention.

[0072] In various embodiments, mimetics for use with varying embodiments of the present invention are capable of being selected by assaying for metal-containing antioxidant activity. Mimetics can also be screened for their ability to inhibit lipid peroxidation or scavenge ONOO ^' (as determined, for example, by the method of Szabo et al, FEBS Lett. 381 :82 (1996)). Measuring SOD activity is common in the art. In an embodiment, SOD activity is capable of being measured by the presence and absence of EDTA using the method of McCord and Fridovich (J. Biol. Chem. 244:6049 (1969)).

[0073] As such, various embodiments of the present invention comprise the use of formulations as herein disclosed as a cytoprotectant for treatment of dry eye disorders.

[0074] Various further embodiments disclose the combination therapy of more than one metal-containing catalytic antioxidant. In an embodiment, the formulation comprises at least two metal-containing catalytic antioxidants. In an alternate embodiment, the formulation comprises at least three metal-containing catalytic antioxidants. In an alternate embodiment, the formulation comprises at least four metal-containing catalytic antioxidants. In general, any number of compounds can be used.

[0075] Various further embodiments disclose the combination therapy of more than one SOD mimetic. In an embodiment, the formulation comprises at least two SOD mimetics. In an alternate embodiment, the formulation comprises at least three SOD mimetics. In an alternate embodiment, the formulation comprises at least four SOD mimetics. In general, any number of compounds can be used.

[0076] Various further embodiments comprise formulations with various additional agents. In an embodiment, the various additional agents target immune responses, infections, hydration of the ocular surface, and similar applications addressing either pathologic features of autoimmune responses and/or inflammation that are capable of being beneficial.

[0077] In various embodiments, formulations of the present invention comprise substantially pure at least one metal-containing catalytic antioxidant. In an embodiment, the concentration of metal-containing catalytic antioxidant is at least about 40%. In an alternate embodiment, the concentration of metal-containing catalytic antioxidant is at least about 50%. In an alternate embodiment, the concentration of metal-containing catalytic antioxidant is at least about 60%. In an alternate embodiment, the concentration of metal-containing catalytic antioxidant is at least about 70%. In an alternate embodiment, the concentration of metal-containing catalytic antioxidant is at least about 80%. In an alternate embodiment, the concentration of metal- containing catalytic antioxidant is at least about 90%. In an alternate embodiment, the concentration of metal-containing catalytic antioxidant is at least about 95%. In an alternate embodiment, the concentration of metal-containing catalytic antioxidant is at least about 99%. However, the concentration of metal-containing catalytic antioxidant can be varied as desired.

[0078] In various embodiments, formulations of the present invention comprise substantially pure at least one SOD mimetic. In an embodiment, the concentration of SOD mimetic is at least about 40%. In an alternate embodiment, the concentration of SOD mimetic is at least about 50%. In an alternate embodiment, the concentration of SOD mimetic is at least about 60%. In an alternate embodiment, the concentration of SOD mimetic is at least about 70%. In an alternate embodiment, the concentration of SOD mimetic is at least about 80%. In an alternate embodiment, the concentration of SOD mimetic is at least about 90%. In an alternate embodiment, the concentration of SOD mimetic is at least about 95%. In an alternate embodiment, the concentration of SOD mimetic is at least about 99%. However, the concentration of SOD mimetic can be varied as desired.

[0079] As such, in various embodiments, a method of the present invention comprises administering a formulation comprising at least about 10% metal-containing catalytic antioxidant. In various other embodiments, the metal-containing catalytic antioxidant is at least about one of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, and/or 99%.

[0080] The various formulations of the present invention comprise a pharmaceutically effective amount of a first metal-containing catalytic antioxidant. In an alternate embodiment, the formulation further comprises a pharmaceutically acceptable carrier, a first metal-containing

catalytic antioxidant and at least one other therapeutic agent. In an alternate embodiment, the formulation further comprises a pharmaceutically acceptable carrier, a first metal-containing catalytic antioxidant, a second metal-containing catalytic antioxidant, and at least one other therapeutic agent, and/or the like.

[0081] Compositions and/or formulations, in accordance with the present invention, broadly comprise a pharmaceutically acceptable carrier and a therapeutically effective amount of at least one compound having a chemical structure in accordance with various embodiments of the present invention and/or having metal-containing catalytic antioxidants..

[0082] In various embodiments of the present invention, the mode and/or route of administration of the formulation is such that a sufficient amount of the formulation is delivered to the ocular region of the patient or subject. As such, various modes of administration suitable for use with various embodiments of the present invention include those suitable for oral, rectal, nasal, topical (including transdermal, buccal, ocular, transscleral, transcorneal, and sublingual), vaginal, subcutaneous, or parenteral (including subcutaneous, intramuscular, intravenous, i.p., and intradermal) administration.

[0083] Other suitable forms of administration to the ocular region of the subject include, but are not limited to topical ocular drops or ointments; slow release devices in the cul-de-sac or implanted adjacent to the sclera or within the eye; transscleral, transcorneal, periocular, conjunctival, sub-tenons, intracameral, intravitreal, or intracanalicular injections.

[0084] Formulations suitable for oral administration may be presented as discrete units such as pills, tablets or capsules each containing a predetermined amount of active ingredient(s); as a powder or granules; as a solution or suspension. The active ingredient(s) may also be present as a bolus or paste, or may be contained within liposomes.

[0085] Formulations for rectal administration may be presented as a suppository or enema.

[0086] For parenteral administration, suitable formulations include aqueous and non-aqueous sterile injection. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed vials and ampoules, and may be stored in a freeze dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example, water prior to use.

[0087] Formulations suitable for administration by nasal inhalation include fine dusts or mists which may be generated by means of metered dose pressurized aerosols, nebulisers or insufflators.

[0088] In various embodiments, in forming compositions for topical administration to the cornea of the eye, the metal-containing catalytic antioxidant of the present invention are generally foπnulated as between about 0.0001 to about 3.0 percent by weight (wt %) solutions in an aqueous solution buffered to a pH between 4.5 to 8.0 with a suitable buffering agent. In an embodiment, the pH is between about 7.0 and about 7.6. In an alternate embodiment, formulations of the present invention are between about 0.0003 to about 0.3 wt %. In an alternate embodiment, formulations of the present invention comprise between about 0.003 and about 1.0 wt % active ingredient. In an alternate embodiment, formulations of the present invention comprise between about 0.01 and 0.1 wt % active ingredient. However, the percentage of metal-containing catalytic antioxidant can be varied as desired and/or needed for a particular application.

[0089] For making dosage units, e.g., tablets, the use of conventional additives such as fillers, colorants, polymeric binders and the like is contemplated. In general any pharmaceutically acceptable additive which does not interfere with the function of the active compounds can be used.

[0090] Further embodiments comprise formulations with additional therapeutic agents, such as prostanoids (also known as prostaglandin receptor agonists), beta-blockers (beta-adrenergic receptor antagonists), alpha-adrenergic receptor agonists, and/or carbonic anhydrase inhibitors.

[0091] In various embodiments, the additional therapeutic agents are capable of treating at least one of ocular surface pain, uveitis, scleritis, episcleritis, keratitis, surgically-induced inflammation, endophthalmitis, iritis, cystoid macular edema, diabetic macular edema, sickle cell retinopathy, optic neuropathy, exudative macular degeneration, corneal neovascularization, cyclitis, sickle cell retinopathy, pterygium, and/or other disease state.

[0092] In typical formulations, the carrier for the active agent(s) is an aqueous solution buffered to physiologically acceptable pH levels with an acidic or basic pH-adjusting agent. In various embodiments, the osmolarity of the solution would be adjusted with osmolality adjusting

agents, such as sodium chloride (NaCl) or potassium chloride (KCl), as is known in the art.

Other ingredients, excipients, and additives which may be desirable to use in the ophthalmic preparations of the present invention include anti-oxidants, co-solvents and viscosity building agents.

[0093] Likewise, an appropriate buffer system (e.g., sodium phosphate, sodium acetate, sodium citrate, sodium borate or boric acid) may be added to the compositions to prevent pH drift under storage conditions. The particular concentration will vary, depending on the substrate employed. Preferably, however, the buffer will be chosen to maintain a target pH within the range of pH 6-7.5.

[0094] Formulations of the present invention can be packaged in a single dose form or a multiple dose form. Formulations packaged in single dose form are typically for use when formulated, therefore, not requiring long storage, i.e., no preservative. However, a preservative is capable of use with various embodiments of the present invention.

[0095] Formulations packaged in multidose form generally require the addition of preservatives to prevent microbial contamination during use. In accordance with various embodiments of the present invention, the formulation comprises a naturally occurring or recombinant antimicrobial peptide. The antimicrobial peptide typically would be employed at a concentration between about 0.001% and 1.0% by weight

[0096] Viscosities greater than that of aqueous solutions may be desirable in certain cases to increase ocular absorption of the active compound, to decrease physical separation of components of the formulation, to decrease inherent variability in dispensing the formulations, and/or otherwise to improve and/or enhance the Formulation. In such cases, viscosity building agents, such as, for example, polyvinyl alcohol, polyvinyl pyrrolidone, methyl cellulose, hydroxy propyl methylcellulose, hydroxyethyl cellulose, carboxymethyl cellulose, hydroxy propyl cellulose, and/or the like can be used. Such agents are typically employed at a concentration between about 0.01% and about 2% by weight. However, the exact concentration can vary as needed for the particular application.

[0097] Typically, in a topical dosage form, a sterile solution is used. In an embodiment, the dosage range for topical (including transdermal, buccal, mocular, and sublingual) administration

is between about 0.1 and 100 micrograms (μg) per eye per day, and is most preferably between 1 and 10 μg per eye per day. While the precise regimen is left to the discretion of a health care provider, it is recommended that the resulting solution be topically applied by placing one drop in each eye one or two times a day. In various embodiments, a topical application is at least one of eye droplets, a cream, a mist, and/or the like.

[0098] In various embodiments, the formulation is administered in more than one application, such that the total dose is administered in one or more discrete applications. However, in an alternate embodiment, the formulation is administered in one dose.

[0099] For such topical administration, the compositions administered may also include various other ingredients as earners, including but not limited to surfactants, tonicity substrates, buffers, preservatives, co-solvents and viscosity building substrates.

[00100] Various tonicity substrates may be employed to adjust the tonicity of the composition, preferably to that of natural tears for ophthalmic compositions. For example, sodium chloride, potassium chloride, magnesium chloride, calcium chloride, dextrose and/or mannitol may be added to the composition to approximate physiological tonicity. Such an amount of tonicity substrate will vary, depending on the particular substrate to be added. In general, however, the compositions will have a tonicity substrate in an amount sufficient to cause the final composition to have an ophthalmically acceptable osmolality (generally about 150-450 mOsm/L, preferably 250-350 mOsm/L).

[00101] Compositions formulated for the treatment of dry eye-type diseases and disorders may also comprise aqueous carriers designed to provide immediate, short-term relief of dry eye- type conditions. Such carriers can be formulated as a phospholipid carrier or an artificial tears carrier, or mixtures of both. As used herein, "phospholipid carrier" and "artificial tears carrier" refer to aqueous compositions which: (i) comprise one or more phospholipids (in the case of phospholipid carriers) or other compounds, which lubricate, "wet," approximate the consistency of endogenous tears, aid in natural tear build-up, or otherwise provide temporary relief of dry eye symptoms and conditions upon ocular administration; and (ii) are safe. Examples or artificial tears compositions useful as artificial tears carriers include, but are not limited to, commercial products, such as Tears Naturale.RTM., Tears Naturale II. RTM., Tears Naturale Free.RTM., and

Bion Tears. RTM. (Alcon Laboratories, Inc., Fort Worth, Tex.). Examples of phospholipid carrier formulations include those disclosed in U.S. Pat. Nos. 4,804,539 (Guo et al.), 4,883,658 (Holly), 4,914,088 (Glonek), 5,075,104 (Gressel et al.), 5,278,151 (Korb et al.), 5,294,607 (Glonek et al.), 5,371 ,108 (Korb et al.), 5,578,586 (Glonek et al.); the foregoing patents are incorporated herein by reference to the extent they disclose phospholipid compositions useful as phospholipid carriers of the present invention.

[00102] Other compounds designed to lubricate, "wet," approximate the consistency of endogenous tears, aid in natural tear integrity, or otherwise provide temporary relief of dry eye symptoms and conditions upon ocular administration the eye are known in the art. Such compounds may enhance the viscosity of the composition, and include, but are not limited to: monomelic polyols, such as, glycerol, propylene glycol, ethylene glycol; polymeric polyols, such as, polyethylene glycol, hydroxypropylmethyl cellulose ("HPMC"), carboxy methylcellulose sodium, hydroxy propylcellulose ("HPC"), dextrans, such as, dextran 70; water soluble proteins, such as gelatin; and vinyl polymers, such as, polyvinyl alcohol, polyvinylpyrrolidone, povidone and carbomers, such as, carbomer 934P, carbomer 941 , carbomer 940, carbomer 974P.

[00103] In some embodiments, other compounds may also be added to the ophthalmic compositions to increase the viscosity or enhance the physical stability of the composition. Examples of viscosity enhancing substrates include, but are not limited to: polysaccharides, such as hyaluronic acid and its salts, chondroitin sulfate and its salts, dextrans, various polymers of the cellulose family; carboxy vinyl polymers such as carbomers (e.g., carbomer 974P); and acrylic acid polymers. In general, the phospholipid carrier or artificial tears carrier compositions will exhibit a viscosity of 1 to 400 centipoises ("cps").

[00104] In an embodiment, formulations of the present invention are intended for administration to a human patient suffering from dry eye or symptoms of dry eye. In an embodiment, such compounds are administered i.p. In an alternate embodiment, the formulations are administered topically. In general, the doses used for the above described purposes will vary, but will be in an effective amount to eliminate or improve dry eye conditions. Generally, 1 -2 drops of such compositions will be administered from once to many times per day.

[00105] Advantageously, the present invention may provide a formulation and method for the treatment of dry eye that addresses the need for repair of cells and tissues contributing to tear formation. Such an approach may obviate the need for costly long term use of tear replacement formulations and other palliative therapeutics.

[00106] All patents and publications referenced herein are hereby incorporated by reference. It will be understood that certain of the above-described structures, functions, and operations of the above-described embodiments are not necessary to practice the present invention and are included in the description simply for completeness of an exemplary embodiment or embodiments. In addition, it will be understood that specific structures, functions, and operations set forth in the above-described referenced patents and publications can be practiced in conjunction with the present invention, but they are not essential to its practice. It is therefore to be understood that the invention may be practiced otherwise than as specifically described without actually departing from the spirit and scope of the present invention as defined by the appended claims.

EXAMPLE 1

[00107] For in vivo studies a male NOD/LtJ mouse model of Sjogren Syndrome was utilized. A significant decrease in tear secretions is observed in the NOD/LtJ mice between 4 and 8 weeks of age that coincides with the appearance of infiltrates observed in the lacrimal glands as early as 6 to 7 weeks of age. Hence, a direct correlations exist between i) loss of secretory function, ii) increased severity of leukocytic infiltrations, and iii) the temporal changes in tear protein profiles observed for the NOD/LtJ. Histological examination of lacrimal gland tissue obtained from NOD/LtJ mice as compared to Normal (C57BL/6J) adult mice shows apparent changes in acinar cell morphology and overall histology such as the organization of acini and ducts as well as starting immune cell infiltration (Figurel).

EXAMPLE 2

[00108] Experimental validation of cytoprotective activity of Manganese(HI)- 10,15,20- tetrakis(N-ethylpyridinium-2-yl)porphyrin Pentachloride [meso tetrakis(N-ethyl-2- pyridyl)porphyrin Pentachloride; MnTE-2-PyP] is illustrated in Figure 2. Male NOD or C57BL/6J (control) mice were obtained from the Jackson laboratory (Bar Harbor ME) and were

housed under specific pathogen-free conditions in the Department of Laboratory Animal Medicine (DLAM) of the University of North Texas Health Science Center at Fort Worth (UNTHSC).

[00109] 24 of the 10 week old animals were divided equally into the following groups: vehicle (PBS) treated controls (C57BL/6J) mice, MnTE-2-PyP treated (C57BL/6J) mice, vehicle (PBS) treated NOD mice and MnTE-2-PyP treated NOD mice. For the mice in the treatment group, MnTE-2-PyP (manganese (III) meso-tetrakis (N-ethyl-2-pyridyl) porphyrin pentachloride) was injected intraperitoneally and administered every other day for 15 days at a dose of 10mg/kg body weight. The vehicle control animals received an injection of an equivalent volume of Phosphate Buffer saline (PBS). The mice were sacrificed at the end of the treatment regimen. The lacrimal glands were harvested, fixed in 4% phosphate buffered formaldehyde (ph 7.2), and prepared for histological evaluation. 12 μm cryosections were stained with hematoxylin and eosin (H&E) and color images were taken with color digital microscope camera. The histological examination of the four experimental groups: vehicle treated control ("control"); MnTE-2-PyP treated control ("control + inj"); vehicle treated NOD mice ("NOD"); MnTE-2-PyP treated NOD mice ("NOD + inj") are depicted in Figures 2A-2D. Blood glucose levels were monitored before testing to exclude the occurrence of diabetes as a complicating factor. Blood glucose levels were normal in all groups (Figure 3).

[001 10] A histochemical analysis of lacrimal gland tissue from adult NOD/LtJ and control mice with and without MnTE-2-PyP treatment was performed on the four experimental groups as in Figure 2. Immune cell infiltration and tissue damage can be seen in vehicle treated NOD mice over controls and a reduction in damage and infiltration can be seen in the MnTE-2-PyP treated NOD mice (Figure 4). Two representative sections are shown for each experimental group.

[0011 1] Figure 5 illustrates summary statistics of the immune cell infiltration data (n=4) for each condition). The groups are labeled as in Figure 2. Statistical comparison of immune cell infiltration shows that there is no statistical difference between "NOD" and "NOD + inj" groups (P > 0.5) but a statistically significant difference exists between "control" and "NOD" groups (P

< 0.05). This indicates that MnTE-2- PyP treatment has no significant effect on immune cell infiltration.

[00112] It was observed that MnTE-2-PyP treatment did not significantly reduce the infiltration with immune cells, but did reduce tissue damage and functional morphology of acinar cells, acini cells, and/or ducts in the lacrimal gland, as is illustrated in Figures 4 and 5.

EXAMPLE 3

[00113] Male NOD or C57BL/6J (control) mice were obtained from the Jackson laboratory (Bar Harbor ME) and were housed under specific pathogen-free conditions in the Department of Laboratory Animal Medicine (DLAM) of the University of North Texas Health Science Center at Fort Worth (UNTHSC). 24 of the 10 week old animals were divided equally into the following groups: vehicle (PBS) treated controls (C57BL/6J) mice, MnTE-2-PyP treated (C57BL/6J) mice, vehicle (PBS) treated NOD mice and MnTE-2-PyP treated NOD mice. For the mice in the treatment group, MnTE-2-PyP (manganese (III) meso-tetrakis (N-ethyl-2- pyridyl) porphyrin pentachloride) was injected intraperitoneally and administered every other day for 15 days at a dose of lOmg/kg body weight. The vehicle control animals received an injection of an equivalent volume of Phosphate Buffer saline (PBS). The mice were sacrificed at the end of the treatment regimen. The lacrimal glands were harvested, fixed in 4% phosphate buffered formaldehyde (ph 7.2), and prepared for histological evaluation. 12 μm cryosections were stained with hematoxylin and eosin (H&E)

[00114] Histological grading of inflammatory lesions was evaluated for each of the four experimental groups: vehicle treated control ("control"); MnTE-2-PyP treated control ("control + inj"); vehicle treated NOD mice ("NOD"); MnTE-2-PyP treated NOD mice ("NOD + inj"). Histological grading of inflammatory lesions was performed as follows: 1 = 1-5 foci composed of >20mononuclear cells per focus, 2 = >5such foci, but without significant parenchymal destruction, 3 = degeneration of parenchymal tissue, 4 = extensive infiltration of the glands with mononuclear cells and extensive parenchymal destruction, and 5 = severe destructive foci with focal fibrosis, ductal dilation, and/or fatty infiltration in addition to grade 4 lesions. Color images were taken and histological evaluation of the lacrimal gland was performed in a blinded manner. The results are depicted in Figure 6, where values represent the mean of the group .

EXAMPLE 4

[00115] Oxidative Stress Assay for pi 0 Lacrimal gland primary acinar cell cultures Isolation and Primary Culture of Acinar Cells:

C57BL/6J pups (postnatal 10 days) were used for primary acinar cell culture. The mouse lacrimal glands were carefully dissected out in Hank's balanced salt solution (HBSS) and cut into pieces. The lacrimal gland pieces were transferred to a 15 ml conical tube with trypsin (4 glands per tube) and incubated in a 37 degrees centigrade water bath for 10 minutes. After centrifugation at 300g rcf for 1 minute at room temperature, the pellet was re-suspended with 2 ml of STI solution (STI, soybean trypsin inhibitor 0.2 mg/ml in 0.5% BSA-DMEM without sodium pyruvate 2.5mM EGTA) and incubated in a 37 degrees centigrade water bath for 5 minutes and centrifuged again. The pellet was resuspended in collagenase solution (collagenase 0.4 mg/ml, in 0.5% BSA-DMEM without sodium pyruvate) and incubated for 10 minutes in a 37 degrees centigrade water bath. At this point a glass Pasteur pipette with a fire polished tip was used for mechanical dissociation of the glands. The tissue was triturated mechanically until the solution looks cloudy and then centrifuged for 2 minutes at 500g rcf. The pellet was washed with medium, centrifuged again and then resuspended with 40 ml culture media (DMEM without sodium pyruvate with 10% heat inactivated bovine growth serum (BGS) and 50U/ml penicillin and streptomycin at 37 degrees centigrade). 100 μl of cell suspension was plated per coverslip inside a 6-well plate and cells were allowed to attach to coverslips at 37 degrees centigrade for at least 2 hours. Then, 2 ml of culture media was added per well and the cell suspension was further incubated at 37 degrees centigrade in a CO ₂ /air (1 :19) atmosphere. Media was changed twice a week. The glass coverslips had been pre-coated with lOμg/ml laminin.

[001 16] After about 14 days in vitro, primary acinar cell culture was divided into four experimental groups: vehicle (PBS) treated controls, MnTE-2-PyP treated controls, hydrogen peroxide treated group and MnTE-2-PyP and hydrogen peroxide treated group. Culture media was removed from the cultured cells and 2 ml of serum-free medium with the respective treatments, 10 μm hydrogen peroxide and different concentrations of MnTE-2-PyP (10, 50, and 100 μm) or vehicle was added and incubated in a 37 degrees centigrade CO ₂ incubator for 45 minutes. After washing in serum-free media, the cells were fixed in 4% paraformaldehyde for

20 minutes at room temperature, washed in PBS twice and then mounted with mounting media containing 1.5 μg/ml of DAPI. The samples were then analyzed by standard epi-fluoresence microscopy and digital microphotography. Cells were photographed with differential interference contrast microscopy to determine parameters of cell morphology (Figure 7A-7L). Cells were considered as affected by oxidative stress when they displayed irregular round shapes, intracellular dark spots or granules, or if they were partially detached. The percentage of damaged or dead cells was calculated as the ratio over the total number of DAPI-stained cells (Figure 8). Increasing concentrations of MnTE-2-PyP (50 μM and 100 μM) were found to have a cytoprotective affect against oxidative stress.

[00117] Figure 9 is illustrates the results of testing of various compounds for cytoprotection of lacrimal gland tissue in NOD mice. The compounds tested included:

[001 18] a) Manganese(III)-5,10,15,20-tetrakis(N-ethylpyridinium-2-yl)po rphyrin Pentachloride [Mn(III) meso-tetrakis(N-ethyl-2-pyridyl)porphyrin Pentachloride; MnTE-2-PyP; molecular formula: C ₄₄ H ₂ SClFeN ₄ Oi ₂ S ₄ .

b) 5,10,15,20-Tetrakis(4-sulfonatophenyl)ρorρhyrinato Iron (III), Chloride; FeTPPS

n-manganese complex EUK-8; molecular formula: CiOH] ₄ ClMnN ₂ O ₂ .

Molecular Formula

-2H ₂ O

[00119] Cytoprotective activity was validated experimentally by measuring the area of tissue with cellular damage in lacrimal glands of 8 week old male, adult NOD/LtJ mice and comparing it to wildtype vehicle treated controls (same model as for figures 1-4). Four experimental groups for each SOD mimetic compound were analyzed:

• vehicle treated control;

• SOD mimetic treated control;

• vehicle treated NOD mice;

• SOD mimetic treated NOD mice.

[00120] The vehicle for EUK-8 was DMSO and for MnTE-2-PyP and the vehicle for FeTPPS was saline.

[00121] The respective SOD mimetic was injected i.p. at 10mg/kg every other day for two weeks.

[00122] Blood glucose levels were monitored before testing to exclude the occurrence of diabetes as a complicating factor. Blood glucose levels were normal in all groups. Lacrimal glands were fixed and sectioned and stained for histological analysis with standard hematoxylin- eosin staining.

[00123] Statistical significance between groups was calculated using a two-sample Student's t-test. Difference of mean from the wildtype vehicle treated controls at a statistical significance level of p < 0.05 is labeled with a star. Difference of mean from the NOD vehicle treated condition at a statistical significance level of p < 0.05 is labeled with a triangle. The absence of a symbol indicates no statistical difference for the respective data pairs and comparisons.

[00124] Experimental results indicated that SOD mimetics of different chemical composition (salen: EUK-8; metalloporphyrins: MnTE-2-PyP and FeTPPS) and with different complexed metals (Mn: EUK-8 and MnTE-2-PyP; Fe: FeTPPS) generate similar cytoprotection in vivo. Results indicate that cytoprotection in vivo is significant when compared to control conditions.

[00125] Approximately twenty percent of cases presented to eye care practitioners are cases involving dry eye disorders (Lemp, 1992) with a potentially even larger patient population undergoing "self-treatment" using over-the-counter pharmaceuticals. Dry eye disorders likely

affect a similar percentage and up to a quarter of the US population (Larkin and Lee, 1979). These data indicate the need for appropriate therapeutic intervention strategies for both prevention and treatment impacting both quality of life as well as economic aspects of the spectrum of disorders.

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Lemp et al. "Introduction. In: Lemp MA, Marquardt R, eds. The dry eye. A comprehensive guide" Berlin: Springer- Verlag, 1992:1-2.

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