(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`(19) World Intellectual Property
`Organization
`International Bureau
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`\=
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`WIPO!IPCT
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`(81)
`
`(43) International Publication Date
`13 November 2014 (13.11.2014)
`
`GD)
`
`International Patent Classification:
`A61IP 27/02 (2006.01)
`
`(21)
`
`International Application Number:
`
`(22)
`
`International Filing Date:
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`PCT/US2014/036810
`
`(25)
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`(26)
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`(30)
`
`(71)
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`(72)
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`Filing Language:
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`Publication Language:
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`5 May 2014 (05.05.2014)
`
`English
`
`English
`
`Priority Data:
`61/819,709
`
`6 May 2013 (06.05.2013)
`
`US
`
`Applicants: KAOHSIUNG CHANG GUNG MEMORI-
`AL HOSPITAL; No. 123, Dapi Rd. Niaosng Dist, Ka-
`ohsiung City, 83301, Taiwan (TW). WU, Philip [US/US];
`11701 Park Lane Souh Apt A6B, Richmond Hill, NY
`11418 (US).
`
`Inventors: WU, Pei-Chang; No. 123, Dapi Rd. Niaosng
`Dist, Kaohsiung City, 83301 (TW). CHEN, Chueh-tan;
`No. 123, Dapi Rd. Niaosng Dist, Kaohsiung City, 83301
`(TW). TSAI, Chia-ling; No. 123, Dapi Rd. Niaosng Dist,
`Kaohsiung City, 83301 (TW).
`
`(74)
`
`Agents: DAVITZ, Michael A.ct al.; Asccnda Law Group
`PC, 84 W.SantaClara St., Ste 550, San Jose, CA 95113
`(US).
`
`(10) International Publication Number
`WO 2014/182620 Al
`
`Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY,
`BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM,
`DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT,
`HIN, HR, HU,ID,IL, IN, IR, IS, JP, KE, KG, KN, KP, KR,
`KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME,
`MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI NO, NZ,
`OM,PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA,
`SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM,
`TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM,
`ZW.
`
`(84)
`
`Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ,
`UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU,TJ,
`TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK,
`EE, ES, FI, FR, GB, GR, HR, HU,IE,IS, IT, LT, LU, LV,
`MC, MK, MT, NL, NO,PL, PT, RO, RS, SE, SI, SK, SM,
`TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW,
`KM, ML, MR,NE, SN, TD, TG).
`Published:
`
`with international search report (Art. 21(3))
`
`before the expiration ofthe time limit for amending the
`claims and to be republished in the event of receipt of
`amendments (Rule 48.2(h))
`
`(54) Title: PHARMACEUTICAL COMPOSITION AND USES THEREOF
`
`Solera
`
`
`Retina #cheseld
`SATCHEL
`
`ow chondrogenesis
`soomnde SONG Sigal —> TOF-BT
`choroid thinning
`
`Fig i.
`
`(57) Abstract: Pharmaceutical compositions containing a combination of anti-chondrogenesis agents are disclosed. Methods ofre-
`ducing scleral chondrogensis, reducing one or more ocular chondrogenic proteins, reducing inflammation induced chondrogensis
`and treating myopia by administering an effective amount of one or more anti-chondrogensis agents are also provided. The pharma-
`ceutical compositions are useful for treating myopia.
`
`wo2014/182620A1IIITNNTAIMIMTAMTYAAAAAATA
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`

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`WO 2014/182620
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`PCT/US2014/036810
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`PHARMACEUTICAL COMPOSITION AND USES THEREOF
`
`CROSS-REFERENCE TO RELATED APPLICATIONS
`
`[0001] This application claims the benefit of U.S. Application No. 61/819,709, filed on 6
`
`May 2013, the entire disclosure of which is incorporated herein by reference.
`
`TECHNOLOGYFIELD
`
`[0002] The present invention relates to pharmaceutical compositions and methods for the
`
`treating myopia,
`
`inhibiting ocular chondrogenic protein,
`
`scleral chondrogenesis and
`
`inflammation induced chondrogenesis.
`
`BACKGROUND OF THE INVENTION
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`[0003] Myopia is due to progressive clongation of the cye and stretching of the oculartissues.
`
`It is an important public health issue, as it affects approximately 25%ofthe U.S. population,
`
`and as high as 80%of the population in some Asian countries. Maculopathy of high myopia
`
`has become the leading cause of cataract, glaucoma, retinal detachment, myopic retinal
`
`degencration, visual impairment, and untrcatable blindness.
`
`[0004] Optical and laser surgical corrective techniques have been usedto alter the refractive
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`state of the myopic eye. These therapies, however, do not address the abnormal elongation
`
`of the eye and thus do not treat pathologic changes of high myopiapatients.
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`[0005] Thereis still a need for a more effective and safe treatment for myopia. The present
`
`invention addresses this need.
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`BRIEF SUMMARYOF THE INVENTION
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`[0006] Pharmaceutical
`
`compositions
`
`comprising two anti-chondrogenesis
`
`agents
`
`are
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`WO 2014/182620
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`provided herein. The pharmaceutical compositions are effective in treating myopia, reducing
`
`one or more chondrogenic proteins and reducing scleral chondrogenesis.
`
`[0007] Methods for treating myopia, comprising administering an effective amount of non-
`
`steroidal anti-inflammatory agent (NSAID) to a subject in need thereof to thereby treat
`
`myopia are provided.
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`In one embodiment, the method for treating myopia further comprises
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`administering an effective amount anti-muscarinic agent.
`
`[0008] Methods for reducing one or more ocular chondrogenic proteins, comprising
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`administering an effective amount of one or more anti-chondrogenesis agents to a subject in
`
`need thereof to reduce one or more ocular chondrogenic proteins are also provided herein.
`
`[0009] Methods for reducing scleral chondrogenesis, comprising administering an effective
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`amount of one or more anti-chondrogenesis agents to a subject in need thereof to reduce
`
`scleral chondrogenesis are also provided herein.
`
`[0010] Methods
`
`for
`
`reducing
`
`inflammation
`
`induced
`
`chondrogenesis,
`
`comprising
`
`administcring an cffective amount of one or more anti-chondrogencsis agents to a subject in
`
`need thereof to reduce inflammation induced chondrogenesis in the subject are also provided
`
`herein.
`
`[0011] The invention will become more apparent when read with the accompanying figures
`
`and detailed description which follow.
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`BRIEF DESCRIPTION OF THE DRAWINGS
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`[0012] Fig. 1 illustrates schematically a mechanism for myopia.
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`[0013] Fig. 2 is bar graph illustrating the levels of alpha smooth muscle actin (a-SMA) and
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`collagen type 2 (Col2) mRNA normalized to B-actin expression in scleral stem/progenitor
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`WO 2014/182620
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`cells (SSPCs) with or without Transforming growth factor beta (TGF-f) treatment. Data are
`
`expressed as fold change over the control sample as determined by the delta-delta Ct method.
`
`Bars, SD. * representsstatistically significant.
`
`[0014] Fig. 3 is an assembly of imagesillustrating the expression of a-SMA and Col2 in the
`
`sclera of mice with form-deprivation myopia (FDM). Panel A is a photograph of a western
`
`blot showing the scleral Col2 and o-SMA expression levels were increased in FDM eyes.
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`Panel B is a bar graph of densitometry analysis showing the levels of scleral Col2 and scleral
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`oa-SMA in FDM eyesare significantly higher than that of the control eyes.
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`[0015] Fig. 4 is a bar graph showing the levels of TGF-B1l, TGF-B2 and TGF-B3 mRNA
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`expression in the RPE-choroid complex of the FDM eyes weresignificantly higher than that
`
`of the control eyes.
`
`[0016] Fig. SA (a western blot analysis) and Fig. 5B (a bar graph) illustrate the expression
`
`profiles of Col2 and a-SMA in human SSPCtreated with 10 ng/ml TGF-f2, with or without
`
`Atropine, Ketorolac and Diclofenac.
`
`[0017] Fig. 6 is a bar graphillustrating the myopia progression rate (Diopter per year) in 11
`
`myopia subjects treated with atropine eye drops and the combined atropine and ketolorac eye
`
`drops.
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`[0018] Fig. 7 is a bar graph illustrating the myopia progression rate of a myopia subject
`
`without any treatment, followed by 3 months of ketorolac treatment.
`
`[0019] Fig. 8A is a bar graphillustrating the level of interleukin 6 (IL-6) mRNA normalized
`
`to GADPH expression in the choroids of form deprivation myopia (FDM) mice is higher
`
`than that of control mice. Fig. 8B is a bar graph illustrating the level of tumor necrosis
`
`factor-alpha (TNF-a) in the choroids is higher in the FDM mice than that of control mice.
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`TNF-a expression is suppressed by ketorolac eye drop.
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`[0020] Fig. 9A is a bar graph illustrating the suppressive effect of atropine (A), ketorolac
`
`(X1) and a pharmaceutical composition comprising atropine and ketorolac on a-SMA
`
`expression in SSPC in the presence of TGF-B2 (T2). The pharmaceutical composition
`
`comprising atropine and ketorolac has a synergistic effect on o-SMA suppression. Fig. 9B is
`
`a bar graph illustrating the suppressive effect of atropine (A), ketorolac (X1) and a
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`pharmaceutical composition comprising atropine and ketorolac on Col2 expression in SSPC
`
`in the presence of TGF-B2 (T2). The pharmaceutical composition comprising atropine and
`
`ketorolac has a synergistic effect on Col2 suppression.
`
`Definitions
`
`DETAILED DESCRIPTION
`
`[0021] As employed above and throughout
`
`the disclosure,
`
`the following terms, unless
`
`otherwise indicated, shall be understood to have the following meanings.
`39
`66
`
`[0022] As used herein, the singular forms “a,”
`
`“an,” and “the” include the plural reference
`
`unless the context clearly indicates otherwise.
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`[0023] An “effective amount,” as used herein, includes a dose of an anti-chondrogenesis
`
`agent that is sufficient to treat or ameliorate at least one symptom of myopia, or to reduce
`
`one or more ocular chondrogenic proteins, scleral chondrogenesis or inflammation induced
`
`chondrogenesis.
`
`[0024] The term “treating,” “treated,” or “treatment” as used herein refers to palliative uses
`
`or results, and/or slowing or inhibiting the advancement of myopia progression and/or
`
`myopia shift index.
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`[0025] The term “reducing” or “reduce” includes
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`slowing the formation of ocular
`
`chondrogenic protein, scleral chondrogenesis,
`
`inflammation induced chondrogenesis or
`
`myopia progression, or myopia shift, or disassembling the ocular chondrogenic proteins that
`
`have already been formed.
`
`[0026] Pharmaceutically acceptable salts of the therapeutic agent of the invention include
`
`salts derived from an appropriate base, such as an alkali metal (for example, sodium, and
`
`potassium), an alkaline earth metal (for example, calcium, and magnesium), ammonium and
`
`NX," (wherein X is C,
`
`-C4 alkyl). Pharmaccutically acceptable salts of an amino group
`
`include salts of organic carboxylic acids, such astartaric, aliphatic, cycloaliphatic, aromatic,
`
`heterocyclic, carboxylic and sulfonic classes of organic acids, such as, for example, formic,
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`glucuronic, malic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, mesylic,
`
`salicylic, hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic,
`
`ethanesulfonic, benzenesulfonic, pantothenic,
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`toluenesulfonic, 2-hydroxyethanesulfonic,
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`sulfanilic,
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`stcaric,
`
`algenic, hydroxybutyric,
`
`cyclochcxylaminosulfonic, galactaric
`
`and
`
`galacturonic acid and the like, lactobionic, fumaric, and succinic acids; organic sulfonic acids,
`
`such as methaniesulfolic, ethanesulfonic, isothionic, benzenylesulfonic and p-toluenesulfonic
`
`acids; and inorganic acids such as hydrochloric, hydrobromic, hydroiodic, nitric, carbonic,
`
`sulfuric, sulfamic and phosphoric acid and the like. Pharmaceutically acceptable salts of a
`
`compound having a hydroxy group consist of the anion of said compound in combination
`
`with a suitable cation such as Na’, NH4’ or NX," (wherein X is, for example, a C; - Ca alkyl
`
`group), Ca”, Li’, Mg”, or, K” and zinc or organic salts made from primary, secondary and
`
`tertiary amines, cyclic amines, N,N’-dibenzylethylenediamine, chloroprocaine, choline,
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`diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine and the
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`like. All of these salts may be prepared by conventional means from the corresponding
`
`compound byreacting, for example, the appropriate acid or base with the compound in free
`
`form.
`
`[0027] The term “myopia” as used herein refers to a condition associated with a refractive
`
`error of one or more eyes, wherein light rays entering the eye to focus in front of the retina
`
`rather than directly on the retina. The term “myopia” as used herein, encompasses a variety
`
`of levels (mild myopia, from0 to -3 diopters; moderate myopia, from -3 to -5 diopters; and
`
`high myopia, from -5 or lower), and types and subtypes of myopia of variousetiologies and
`
`causes, either known or unknown,including, but not limited to, simple myopia, degenerative
`
`myopia, and form deprivation myopia.
`
`[0028] The term “diopter’’ as used herein includes measurement of how much a corrective
`
`lens must bendlight to focus the light on the retina to normalize the vision. A lens that can
`
`bend parallel light rays to a focal point of 1 meter is said to have a power of | diopter
`
`(1.00D). A 2-diopters lens can focus light rays at a point 0.5 mctcrs away from itsclf.
`
`[0029] The term “‘subject” as used herein typically refers to a human or an animal subjected
`
`to the methods described herein.
`
`It is to be understood that a subject can be a patient with
`
`known or suspected myopia disorder, but subjects without known or suspected myopia
`
`disorder, such as research subjects, are also included within the scope of the term “‘subject.”’
`
`[0030] All numbers herein may be understood as modified by “about.”
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`Pharmaceutical Composition
`
`[0031] Pharmaceutical compositions for treating myopia, reducing ocular chondrogenic
`
`

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`WO 2014/182620
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`PCT/US2014/036810
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`protein, reducing scleral chondrogenesis or reducing inflammation induced chondrogenesis
`
`are provided herein. The pharmaceutical compositions comprising a combination of two
`
`anti-chondrogenesis agents preferably by advantageoussynergistic effects of the
`
`combinations.
`
`[0032] An anti-chondrogenesis agent is any agent which reduces or slows the process of
`
`chondrogenesis.
`
`In one embodiment, an anti-chondrogenesis agent in the pharmaceutical
`
`composition is a NSAID.
`
`In another embodiment, an anti-chondrogenesis agent in the
`
`pharmaceutical composition is an anti-muscarinic agent. Non limiting examples ofanti-
`
`chondrogenesis agent
`
`include a microRNA that regulates the expression of lymphoid
`
`enhancer-binding factor-1, such as miR-449a (S Paik, et al., miR-449a regulates the
`
`chondrogenesis of human mesenchymal stem cells through direct targeting of lymphoid
`
`enhancer-binding factor-1, Stem Cells Dev;21(18):3298-308, 2012), a histone deacetylase
`
`inhibitor such as valproic acid (FH Paradis et al., Exposure to valproic acid inhibits
`
`chondrogenesis
`
`and
`
`ostcogenesis
`
`in mid-organogenesis mouse
`
`limbs, Toxicol
`
`Sct; 131(1):234-41, 2013), Nicotine (Y Deng et al., Nicotine-induced retardation of
`
`chondrogenesis through down-regulation of IGF-1 signaling pathway to inhibit matrix
`
`synthesis of growth plate chondrocytes in fetal rats, Toxicol Appl Pharmacol;269(1):25-33,
`
`2013), bFGF or parathyroid hormone-like peptide (S Weiss et al., Impact of growth factors
`
`and PTHrP on early and late chondrogenic differentiation of human mesenchymal stem cells,
`
`J Cell Physiol;223(1):84-93, 2010), an agent
`
`restricting Leucine (MS Kim, Leucine
`
`restriction inhibits chondrocyte proliferation and differentiation through mechanisms both
`
`
`
`dependent of mTOR_signaling, Am J_Physiol Endocrinoland independent
`
`
`
`
`
`Metab;296(6):E1374-82, 2009), 17beta-Estradiol (S. Fushimiet al., 17beta-Estradiol inhibits
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`chondrogenesis in the skull development of zebrafish embryos, Aquat Toxicol;95(4):292-8,
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`2009), versican (Y Yang et a. The G3 domain of versican inhibits mesenchymal
`
`chondrogenesis via the epidermal growth factor-like motifs, J Biol Chem;273(49):33054-63,
`
`1998),
`
`SB203580 (a specific inhibitor of p38MAPK, D Kim et al., Alterations in the
`
`temporal expression and function of cadherin-7 inhibit cell migration and condensation
`
`during chondrogenesis of chick limb mesenchymalcells in vitro, J Cell Physiol; 221(1):161-
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`70, 2009), LiCl (an inhibitor of GSK-3beta, D Kim et al., 2009) or the like.
`
`[0033]
`
`In one exemplary embodiment, the pharmaceutical composition includes at least one
`
`NSAIDandat least one anti-muscarinic agent.
`
`[0034]
`
`In certain embodiments, NSAIDs for use in the present invention may be non-
`
`selective cyclooxygenase (COX)inhibitors, its derivatives, salts and structural analogues, i.e.,
`
`compoundsthat inhibit both COX-1 and COX-2 proteins. Non limiting examples of non-
`
`selective COX inhibitors include salicylic acid derivatives (e.g., aspirin, sodium salicylate,
`
`choline magnesium trisalicylatc, salsalatc, diflunisal, sulfasalazine and olsalazinc),
`
`indole
`
`and indene acetic acids (e.g.,
`
`indomethacin and sulindac), heteroaryl acetic acids (e.g.,
`
`tolmetin, diclofenac
`
`and ketorolac),
`
`arylpropionic
`
`acids
`
`(e.g.,
`
`ibuprofen, naproxen,
`
`flurbiprofen, ketoprofen, fenoprofen, and oxaprozin), anthranilic acids (fenamates) (e.g.,
`
`mefenamic acid and meclosfenamic acid), enolic acids (e.g., the oxicams, piroxicam and
`
`meloxicam) and alkanones(e.g., nabumetone).
`
`[0035]
`
`In certain embodiments, NSAIDs for use in the present invention may be selective
`
`COX-2 inhibitors, its derivatives, salts and structural analogues. Non limiting examples of
`
`selective COX-of selective COX-2 inhibitor include diaryl-substituted furanones (e.g.,
`
`rofecoxib), diaryl-substituted pyrazoles (e.g., celecoxib), indole acetic acids (e.g., etodolac)
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`and sulfonanilides (e.g., nimesulide). Further examples of selective COX-2 inhibitor are
`
`disclosed in U.S. Pat. No. 6,440,963 and WO 2004/054560, which are incorporated by
`
`reference in its entirety.
`
`[0036] Preferred NSAIDsfor use in the invention include, but are not limited to, Ketorolac,
`
`Diclofenac, Indomethacin, Bromfenac, Nepafenac and Flurbiprofen.
`
`[0037] Examples of anti-muscarinic agent
`
`include, but are not
`
`limited to, Atropine,
`
`Homatropine, Scopolamine, its derivatives, salts, and structural analogues.
`
`[0038] Anti-muscarinic agents may cause side effects of blurred vision and photophobia.
`
`These side effects maybe overcome by administering lower dosage of anti-muscarinic agents,
`
`in combination with one or more anti-chondrogenesis agent,
`
`to achieve the desired
`
`therapeutic effect.
`
`The observed synergistic effect of a pharmaceutical composition
`
`comprising a combination of an anti-muscarinic agent (e.g. atropine) and an NSAID(e.g.
`
`ketorolac) may afford effective treatment of myopia wherein one or even all of the lower
`
`dosages of the anti-chondrogenesis agents would not be sufficicnt to have a therapeutic cffect
`
`whenthe respective anti-chondrogenesis agent is used in monotherapy.
`
`[0039] The pharmaceutical compositions to be administered according to the methods of
`
`some embodiments provided herein can be readily formulated with, prepared with, or
`
`administered with, a pharmaceutically acceptable carrier. Such preparations may be prepared
`
`by various techniques. Such techniques include bringing into association active components
`
`(such as NSAID or anti-muscarinic agent) of the pharmaceutical compositions and an
`
`appropriate carrier.
`
`In one embodiment, pharmaceutical compositions are prepared by
`
`bringing into association active components of the pharmaceutical compositions with liquid
`
`carriers, with solid carriers, or with both.
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`[0040] The pharmaceutical compositions are administered in an aqueous suspension, an oil
`
`emulsion, water in oil emulsion and water-in-oil-in-water emulsion, and in carriers including,
`
`but not limited to, creams, gels, liposomes (neutral, anionic or cationic), lipid nanospheres or
`
`microspheres, neutral, anionic or cationic polymeric nanoparticles or microparticles, site-
`
`specific emulsions,
`
`long-residence emulsions, sticky-emulsions, micro-emulsions, nano-
`
`emulsions, microspheres, nanospheres, nanoparticles and minipumps, and with various
`
`natural or synthetic polymers that allow for sustained release of the pharmaceutical
`
`composition including anionic, neutral or cationic polysaccharides and anionic, neutral
`
`cationic polymers or copolymers, the minipumps or polymers being implanted in the vicinity
`
`of where composition delivery is required.
`
`The pharmaceutical compositions provided herein may optionally include anti-oxidants,
`
`buffers, bacteriostatic agents, suspending agents thickening agents, preservatives, co-solvents
`
`and viscosity building agents or other therapeutic ingredients. The carrier and other
`
`therapeutic ingredicnts must be acceptable in the sense of being compatible with the other
`
`ingredients of the composition and not deleterious to the recipient
`
`thereof.
`
`Suitable
`
`preservatives for ophthalmic preparations include: benzalkonium chloride,
`
`thimerosal,
`
`chlorobutanol, methyl paraben, propyl paraben, phenylethyl alcohol, edetate disodium, sorbic
`
`acid, Onamer M,or other agents knownto those skilled in the art.
`
`In one embodiment, the
`
`preservative is employedat a level of from 0.004% to 0.02%.
`
`[0041] For administration in a non-aqueouscarrier, active components of the pharmaceutical
`
`compositions provided herein are emulsified with a mineral oil or with a neutral oil such as,
`
`but not limited to, a diglyceride, a triglyceride, a phospholipid, a lipid, an oil and mixtures
`
`thereof, wherein the oil contains an appropriate mix of polyunsaturated and saturated fatty
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`acids. Examples include, but are not limited to, soybean oil, canola oil, palm oil, olive oil
`
`and myglyol, wherein the numberof fatty acid carbons is between 12 and 22 and wherein the
`
`fatty acids can be saturated or unsaturated. Optionally, charged lipid or phospholipid is
`
`suspended in the neutral oil.
`
`A suitable phospholipid is, but
`
`is not
`
`limited to,
`
`phosphatidylserine, which targets
`
`receptors
`
`on macrophages. The pharmaceutical
`
`compositions provided herein are optionally formulated in aqueous media or as emulsions
`
`using knowntechniques.
`
`[0042] The pharmaceutical compositions are administered in an amount effective to reduce
`
`ocular chondrogenic protein, reduce scleral chondrogenesis, reduce inflammation induced
`
`chondrogenesis or to induce a therapeutic response in an animal, including a human with
`
`myopia. The dosage of the pharmaceutical composition administered will depend on the
`
`severity of the condition being treated, the particular formulation, and other clinical factors
`
`such as weight and the general condition of the recipient and route of administration. In one
`
`exemplary cmbodiment,
`
`the amount of the pharmaccutical composition administered
`
`corresponds to about 0.001% to about 1% by weight atropine.
`
`In another exemplary
`
`embodiment, the amount of the pharmaceutical composition administered corresponds to
`
`about 0.005%, 0.01%, 0.015%, 0.02%, 0.025%, 0.03%, 0.035%, 0.04%, 0.045%, 0.05%,
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`0.055%, 0.06%, 0.065%, 0.07%, 0.075%, 0.08%, 0.085%, 0.09%, 0.095%, 0.1% by weight
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`atropine, or any % in between 0.001% and 1% in 0.001% increments.
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`In another exemplary
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`embodiment, the amount of the pharmaceutical composition administered corresponds to
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`about 0.05% to about 1% by weight Ketorolac.
`
`In another exemplary embodiment, the
`
`amount of the pharmaceutical composition administered corresponds to about 0.1%, 0.2%,
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`0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%,
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`0.9%, 0.95% by weight Ketorolac, or any % in between in 0.01% increments.
`
`In another
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`exemplary embodiment,
`
`the amount of the pharmaceutical composition administered
`
`corresponds to about 0.5% by weight Ketorolac.
`
`In another exemplary embodiment, the
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`amount of the pharmaceutical composition administered corresponds to from about 0.01%,
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`0.025%, 0.05%, 0.1%, 0.15% to about 0.2% of Diclofenac by weight or any % in between in
`
`0.01% increments. Useful dosages of the pharmaceutical compositions provided herein are
`
`determined by comparing their in vitro activity, and in vivo activity in animal models.
`
`Methodsfor the extrapolation of effective dosages in mice, and other animals, to humans are
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`known in the art; for example, see U.S. Pat. No. 4,938,949, which is incorporated by
`
`reference herein.
`
`[0043]
`
`In accordance with the methods provided herein, the pharmaceutical composition is
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`delivered by any of a variety of routes including, but not limited to, injection (e.g.,
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`subcutaneous, intramuscular, intravenous, intra-arterial, intraperitoneal, intradermal,
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`intravitreal); cutancously; dermally; transdermal; oral( c.g., tablet, pill, liquid medicine,
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`edible film strip); implanted osmotic pumps; suppository, aerosol spray, topical, intra-
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`articular, ocular, nasal inhalation, pulmonary inhalation, impression into skin and
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`electroporation. In one embodiment, the pharmaceutical composition of the present
`
`invention can be administered as solution in a suitable ophthalmic vehicle.
`
`[0044]
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`In forming the pharmaceutical compositions for topical ocular administration, the
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`combination comprises 0.001% to about 0.005% by weight atropine and 0.1% to about 0.5%
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`by weight Ketorolac solution in water at pH of 4.5 to 8.0, e.g. about 6.9.
`
`It is recommended
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`that the solution be topically applied by placing one drop in the affected eye once a day.
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`[0045] The pharmaceutical composition may be administered in a single dose treatment or
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`in multiple dose treatments, over a period of time appropriate to the condition being treated.
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`The pharmaceutical composition may conveniently be administered at appropriate intervals,
`
`for example, once a day, twice a day, three times a day, once every second day, once every
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`three days or once every week, over a period of at least 3 months, at least 1 year, or until
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`the symptomsand signs of myopia resolved.
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`Methods for Reducing Scleral Chondrogenesis
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`[0046] Down regulation of ocular chondrogenic proteins and/or ocular inflammation
`
`markers reduces scleral chondrogenesis.
`
`[0047]
`
`In one embodiment,
`
`the use of an effective amount of one or more anti-
`
`chondrogenesis agents or a pharmaceutical composition described herein may alter or reduce
`
`the amount of one or more ocular chondrogenic proteins in a subject in need thereof.
`
`[0048] One example of ocular chondrogenic protein is TGF-B.
`
`In one embodiment, the
`
`TGE-B protcin is sclected from the group consisting of TGF-B1, TGF-B2 and TGF-B3, all of
`
`which are located predominately in the choroid. Another example of ocular chondrogenic
`
`protein is 4-SMA. Another example of ocular chondrogenic protein is Col2. Both
`
`4 -
`
`SMAandCol? are located predominately in the sclera.
`
`[0049]
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`In another embodiment, the present invention provides the use of an effective amount
`
`of one or more anti-chondrogenesis agents or a pharmaceutical composition described herein
`
`for reducing inflammation induced chondrogenesis in a subject
`
`in need thereof.
`
`The
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`inflammation markers responsible for inducing scleral chondrogenesis include, but are not
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`limited to, IL-6 and TNF-c .
`
`[0050]
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`In yet another embodiment, the present invention provides the use of an effective
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`amount of one or more anti-chondrogenesis agents or a pharmaceutical composition
`
`described herein for reducing scleral chondrogenesis in a subject in need thereof.
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`[0051] The anti-chondrogenesis agent maybe administered concomitantly or
`
`non-
`
`concomitantly.
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`Methodsfor Treating or Reducing the Severity of Myopia
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`[0052] Without being bound by any particular theory, it was believed that the expression
`
`profiles of ocular inflammation markers and ocular chondrogenic proteins, such as TGF-,
`
`-SMAand Col?2, are correlated with scleral chondrogenesis and myopia. Fig. 1, for example
`
`and without limitation, illustrates a mechanism for the development of myopia, wherein
`
`increase levels of TGF-Bs and inflammatory markers (such as IL-6 and TNF-« ) in the
`
`choroid lead to the formation of a -SMAand Col2 in the sclera and scleral chondrogenesis.
`
`The sclera then undergo remodeling and elongation, followed by the development of myopia.
`
`[0053] The present invention provides methods for treating or reducing the severity of
`
`myopia, by administering one or more anti-chondrogenesis agents in an effective amount or
`
`the pharmaceutical composition described herein to a myopic subject in need of myopia
`
`treatment. The anti- chondrogenesis agent may be administered concomitantly or non-
`
`concomitantly. The methods also encompass research methods and uses, including in vitro
`
`and in vivo methodsoftreating, or inhibiting the progression of myopia in the subject.
`
`[0054]
`
`In one embodiment,
`
`the method for treating myopia comprises identifying a
`
`myopic subject who exhibits side effect to anti-muscarinic agent, and treating said subject
`
`with an effective amount of NSAID, without the anti-muscarinic agent or with a lower dose
`
`of anti-muscarinic agent (e.g. 0.05% of atropine).
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`[0055] Embodiments of the present invention are illustrated by the following examples,
`
`which are not to be construed in any way as imposing limitations upon the scope thereof.
`
`During the studies described in the following examples, conventional procedures were
`
`followed, unless otherwise stated.
`
`Some of the procedures are described below for
`
`illustrative purpose.
`
`Description of Materials and Methods Used in the Examples
`
`[0056] Mice: Male wild type C57BL/6 mice (Jackson Labs) were used in the examples. All
`
`procedures were performed in accordance with an institutional IACUC approved protocol as
`
`well as the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
`
`[0057] Scleral stem/progenitor cells (SSPCs) isolation and culture: The SSPCs were
`
`isolated and cultured as previously described by CL Tsai et al. (Identification of multipotent
`
`stem/progenitor cells in murine sclera. Invest Ophthalmol Vis Sci 52:5481-5487, 2011). In
`
`bricf, sclera from the mouse was obtained and was carcfully dissected away from limbus and
`
`optic disc under a dissection microscope. After retina and choroid tissues were removed,the
`
`scleral tissue was cut into small pieces and digested with 1.5 mg/ml collagenase type I
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`(Worthington Biochemical, Lakewood, USA) and 2 mg/ml of dispase (Roche, Basel,
`
`Switzerland) in PBS for 1 h at 37°C to release individual cells. Individual cells were cultured
`
`in 0-MEM(Invitrogen, Carlsbad, USA), supplemented with 20% lot-sclected FBS (Equitech-
`
`Bio, Kerrville, USA), glutamine, penicillin/streptomycin and 100 mM 2-mercaptoethanol
`
`(Invitrogen) for 8 to 10 days at 5% CO», 37C.
`
`[0058] TGF-B treatment: Different concentrations of TGF-B2 were added into 12-wells of
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`SSPCs. After 24 hrs, the images of SSPC morphology were recorded. The total RNA was
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`extracted for further analysis. A chamberslide culture for the immunofluorescence study
`
`was performed under the same condition.
`
`[0059] Induction of chondrogenic differentiation. At semiconfluence, SSPCs were
`
`trypsinized and counted to make aliquots of 2x10° cells in 2 ml growth medium which were
`
`spun downat 500g for 10 min to obtain the pellet. The pellets were incubated at 37°C, under
`
`5% CO2. Within 12—24 h of incubation, the cells formed an essentially spherical aggregate
`
`that did not adhere to the walls of the tube. Culture medium was added with 10 ng/ml TGF-
`
`B2 and the medium was changed at 2 to 3 day intervals. The pellets were then harvested at 4
`
`weeks. Subsequently, they were washed twice in PBS, fixed in 4% paraformaldehyde for 3 h
`
`at room temperature and prepared for paraffin embedment. Eight tum thick sections were
`
`obtained for immunohistochemistry,
`
`[0060] Immunohistochemistry and immunofluorescence study: Immunohistochemistry
`
`and immunofluorescence studies were performed to demonstrate the presence of 4 -SMA
`
`protein and Col2 during chondrogenesis. For immunohistochemistry, paraffin sections were
`
`treated with a 20% blocking goat serum for 30 min, then incubated with primary antibodies
`
`which were rabbit IgG anti-SMA mAb at1:200 dilution (Abcam, Temecula, CA ) and mouse
`
`IgG2aanti-type II collagen mAbbat 1:100 dilution (Abcam, Temecula, CA) at 4°C
`
`overnight. The sections were then treated with horseradish peroxidase (HRP)-conjugated
`
`secondary antibodies at 1:200 (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 hour. The
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`DABreagent (diaminobenzidine tctrahydrochloridc) was subscquently used to detect the
`
`immunoactivity. For immunofluorescence, cryostat sections and rehydrated paraffin sections
`
`were treated with blocking serum, incubated with primary antibod