WO2017013637A1 - Inhibiteurs sélectifs d'isoformes contenant alpha2 de la na,k-atpase et leur utilisation pour réduire la pression intra-oculaire - Google Patents

Inhibiteurs sélectifs d'isoformes contenant alpha2 de la na,k-atpase et leur utilisation pour réduire la pression intra-oculaire Download PDF

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WO2017013637A1
WO2017013637A1 PCT/IL2015/050741 IL2015050741W WO2017013637A1 WO 2017013637 A1 WO2017013637 A1 WO 2017013637A1 IL 2015050741 W IL2015050741 W IL 2015050741W WO 2017013637 A1 WO2017013637 A1 WO 2017013637A1
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alkyl
compound
group
moiety
isoform
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PCT/IL2015/050741
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English (en)
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Steven J.D. KARLISH
Adriana KATZ
Daniel M. TAL
Arie MARCOVICH
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Yeda Research And Development Co. Ltd.
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Priority to PCT/IL2015/050741 priority Critical patent/WO2017013637A1/fr
Priority to US15/745,441 priority patent/US10668094B2/en
Priority to EP20209796.0A priority patent/EP3907231A1/fr
Priority to PCT/IL2016/050785 priority patent/WO2017013648A1/fr
Priority to EP16747628.2A priority patent/EP3325494B1/fr
Publication of WO2017013637A1 publication Critical patent/WO2017013637A1/fr
Priority to US16/847,737 priority patent/US11077128B2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/06—Antiglaucoma agents or miotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J19/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, substituted in position 17 by a lactone ring
    • C07J19/005—Glycosides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J43/00—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton
    • C07J43/003—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton not condensed

Definitions

  • the present invention in some embodiments thereof, relates to a pharmaceutical agents and, more particularly, but not exclusively, to digoxin and digitoxin derivatives exhibiting selective inhibition of a2-containing isoforms of Na,K-ATPase, and uses thereof to reduce intraocular pressure (IOP), and/or as cardiotonic agents in a subject in need thereof.
  • IOP intraocular pressure
  • Glaucoma is a disease leading to irreversible blindness.
  • Control of intraocular pressure (IOP) is the mainstay of current therapy of glaucoma, and is achieved by various drugs, such as ⁇ -blockers, prostaglandin analogues, a.2 adrenergic receptor agonists, cholinergic agonists and carbonic anhydrase inhibitors given topically or systemically.
  • the topical route is preferable, provided the drug effectively permeates the cornea, because this minimizes systemic side-effects.
  • uncontrolled IOP in many patients eventually makes surgical intervention necessary.
  • fresh approaches to drug treatment of glaucoma are highly desirable.
  • the Na,K-ATPase is the motor for production of the aqueous humour (bodily fluid) in the ciliary body epithelium and, in principle, inhibition of the Na,K-ATPase can suppress the production of aqueous humour, and control IOP.
  • Control of IOP is the mainstay of glaucoma therapy; however, the available drugs suffer from a variety of shortcomings, particularly due to systemic adverse effects and low therapeutic index.
  • intra-venous digoxin a classical inhibitor of the Na,K-pump, typically used primarily to treat congestive heart failure, was considered for this role but was discarded due to systemic toxicity.
  • Isoforms of the Na,K-ATPase ion pump consists of a and ⁇ subunits ( ⁇ / ⁇ ) and accessory FXYD regulatory subunits. There are four isoforms of the al subunit (al-4) and three isoforms of the ⁇ subunit ( ⁇ 1-3) expressed in a tissue-specific fashion.
  • the l isoform is the common isoform that maintains Na and K gradients in all tissues, al is expressed mainly in muscle and astrocytes, and a.3 is expressed mainly in nerve cells.
  • human heart expresses l (about 70 %) and both a2 and a3 isoforms (about 30 %) and ⁇ ⁇ .
  • the ciliary epithelium in the eye is a functional syncytium consisting of apical pigmented cells (PE) oriented towards the blood and baso-lateral non-pigmented (NPE) cells oriented towards the anterior chamber of the eye.
  • PE apical pigmented cells
  • NPE baso-lateral non-pigmented cells
  • an a2-selective cardiac glycoside could be as an effective cardiotonic drug, with reduced cardiotoxicity, compared to known drugs such as digoxin.
  • Digitalis drugs such as digoxin have been used to treat heart failure for over two hundred years but are dangerous drugs with multiple side effects.
  • selective inhibition of al is especially effective in enhancing cardiac excitation-contraction coupling and mediating cardiac glycoside-mediated positive inotropy.
  • the isoform selectivity of a large number of known cardiac glycosides has been previously studied, using the yeast P. pastoris expressing Na,K-ATPase isoforms ( ⁇ ⁇ , ⁇ 2 ⁇ 1, ⁇ 3 ⁇ 1), and purified detergent- soluble isoform complexes of Na,K- ATPase [Cohen E. et al , 2005, J Biol Chem, 280(17), pp. 16610-16618; Haviv H, et al, 2007, Biochemistry, 46(44), pp.12855-12867; Lifshitz Y, et al , 2007, Biochemistry, 46(51), pp.
  • the KD of ouabain showed some preference for al over a2 and similar Ki values for all three isoforms.
  • Assays of inhibition of Na,K-ATPase activity, measured with the purified isoform protein complexes digoxin and digitoxin showed 3-4-fold lower Ki (inhibition) values for a2 compared to al, with a3 more similar to al .
  • No aglycones of any cardiac glycosides tested showed isoform selectivity.
  • digoxin derivatives with one to four digitoxose moieties the maximal a2/al selectivity was found for digoxin itself, with three digitoxose sugars.
  • Formula I including any pharmaceutically acceptable salt, hydrate, solvate, enantiomer and diastereomer thereof, and any mixtures thereof,
  • X is H or OH
  • R is represented by general Formula II:
  • A is a spacer moiety or a covalent bond
  • B is a cyclic moiety
  • B is selected from the group consisting of an alkylsulfonyl, an arylsulfonyl and a sulfonamide;
  • Ri and R 2 are each independently H or a Ci-C 4 alkyl provided that at least one of Ri and R 2 is a Ci-C 4 alkyl.
  • A is selected from the group consisting of a covalent bond, an unsubstituted Ci-C 6 alkyl, a substituted Ci-C 6 alkyl, an unsubstituted Ci-C 6 alkyl interrupted by one or more heteroatom and a substituted Ci- C 6 alkyl interrupted by one or more heteroatom.
  • B is a cyclic moiety selected from the group consisting of an unsubstituted alicyclic moiety, a substituted alicyclic moiety, an unsubstituted heterocyclic moiety, a substituted heterocyclic moiety, an unsubstituted aryl moiety, a substituted aryl moiety, an unsubstituted heteroaryl moiety and a substituted heteroaryl moiety.
  • B is an unsubstituted alicyclic moiety.
  • the unsubstituted alicyclic moiety is selected from the group consisting of cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.
  • B is a substituted alicyclic moiety.
  • the substituted alicyclic moiety is selected from the group consisting of 2,3-dimethylcyclopropane-l-yl, 3,3- dimethylcyclobutane-l-yl, 3,4-dimethylcyclopentane-l-yl and 3,5- dimethylcyclohexane-l-yl.
  • B is an unsubstituted heterocyclic moiety.
  • the unsubstituted heterocyclic moiety is selected from the group consisting of oxiranyl, aziridinyl, oxetanyl, azetidinyl, thietanyl, tetrahydrofuranyl, pyrrolidinyl, tetrahydropyranyl and piperidinyl.
  • B is an unsubstituted aryl moiety.
  • the unsubstituted aryl moiety is selected from the group consisting of cyclopentadienyl, phenyl and naphthyl.
  • B is an unsubstituted heteroaryl moiety.
  • the unsubstituted heteroaryl moiety is imidazolyl.
  • the alkylsulfonyl is selected from the group consisting of methylsulfonyl, ethylsulfonyl and isopropylsulfonyl.
  • the arylsulfonyl is selected from the group consisting of phenylsulfonyl, benzylsulfonyl and tosyl.
  • the sulfonamide is selected from the group consisting of methylsulfonamide, N-methylmethanesulfonamide and N,N-dimethylmethanesulfonamide.
  • B is -N(Et) 2 .
  • X is H.
  • A is a covalent bond and B is cyclobutyl.
  • A is -CH 2 - and B is cyclopropyl.
  • X is OH.
  • A is a covalent bond and B is selected from the group consisting of cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl.
  • A is -CH 2 - and B is selected from the group consisting of cyclopropyl, 3,3-dimethylcyclobutane-l-yl and phenyl.
  • A is -(CH 2 )2- and B is cyclopropyl.
  • R is selected from the group consisting of cyclopropyl, methylcyclopropane, ethylcyclopropane, propylcyclopropane, cyclobutyl, methylcyclobutane, methyl-3,3-dimethylcyclobutane, ethylcyclobutane, propylcyclobutane, cyclopentyl, methylcyclopentane, ethylcyclopentane, propylcyclopentane, cyclohexyl, azetidinyl, oxetanyl, thietanyl, histaminyl and benzyl.
  • R is selected from the group consisting of cyclopropyl, methylcyclopropane and cyclobutyl.
  • the compound is having an affinity to at least one isoform of Na,K-ATPase.
  • the isoform is selected from the group consisting of ⁇ , ⁇ 1 ⁇ 2, ⁇ 1 ⁇ 3, ⁇ 2 ⁇ 1, ⁇ 2 ⁇ 2, ⁇ 2 ⁇ 3, ⁇ 3 ⁇ 1, ⁇ 3 ⁇ 2, ⁇ 3 ⁇ 3, ⁇ 4 ⁇ 1, ⁇ 4 ⁇ 2 and ⁇ 4 ⁇ 3.
  • the affinity of the compound to any one of ⁇ 2 ⁇ 1, ⁇ 2 ⁇ 2 and ⁇ 2 ⁇ 3 is higher than the affinity to ⁇ , ⁇ 1 ⁇ 2, ⁇ 1 ⁇ 3, ⁇ 3 ⁇ 1, ⁇ 3 ⁇ 2, ⁇ 3 ⁇ 3, ⁇ 4 ⁇ 1, ⁇ 4 ⁇ 2 and ⁇ 4 ⁇ 3 by at least 100 %.
  • the affinity of the compound to any one of ⁇ 2 ⁇ 1, ⁇ 2 ⁇ 2 and ⁇ 2 ⁇ 3 is higher than the affinity to ⁇ by at least 300 % (4-fold).
  • the affinity of the compound to ⁇ 2 ⁇ 3 is higher than the affinity to ⁇ by at least 500 % (6-fold).
  • the compound is having a ⁇ 2 ⁇ 3 inhibition constant (Ki) lower than 10 nM.
  • a pharmaceutical composition that includes as an active ingredient a compound according to any of the embodiments of the invention and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is packaged in a packaging material and identified in print, or on the packaging material, for use in reducing intraocular pressure (IOP).
  • IOP intraocular pressure
  • the pharmaceutical composition is packaged in a packaging material and identified in print, or on the packaging material, for use in a treatment of a heart condition.
  • a method of reducing intraocular pressure (IOP) in a subject in need thereof which includes administering to the subject a therapeutically effective amount of a compound according to any of the embodiments of the invention, or a pharmaceutical composition o according to some of the embodiments of the invention.
  • IOP intraocular pressure
  • a method of treating a heart condition in a subject in need thereof that includes administering to the subject a therapeutically effective amount of a compound according to any of the embodiments of the invention, or a pharmaceutical composition according to some of the embodiments of the invention.
  • the heart condition is selected from the group consisting of atrial fibrillation, atrial flutter, mitral stenosis, chronic heart failure and congestive heart failure.
  • a pharmaceutical composition that includes as active ingredients: at least one ingredient selected from the group consisting of a prostaglandin analog, a ⁇ -blocker, an adrenergic agent, an a2-adrenergic receptor agonist, a miotic agent, a carbonic anhydrase inhibitor and a cholinergic agonist; and
  • Formula III including any pharmaceutically acceptable salt, hydrate, solvate, enantiomer and diastereomer thereof, and any mixtures thereof, and a pharmaceutically acceptable carrier,
  • X is H or OH
  • Y is O or S
  • Rl, R2 and R3 are each independently H or a Ci-C 4 alkyl
  • Ra is a Ci-C 4 alkyl
  • Rb, Rc and Rd are each independently selected from H, a Ci-C 4 alkyl and a Ci- C 4 hydroxy alkyl;
  • Re is selected from a Ci-C 4 alkyl and a Ci-C 4 hydroxyalkyl
  • A is a spacer moiety or a covalent bond
  • B is a cyclic moiety, or B is selected from the group consisting of an alkylsulfonyl, an arylsulfonyl and a sulfonamide, or B is -NR 1 R 2 , wherein Ri and R 2 are each independently H or a Ci-C 4 alkyl provided that at least one of Ri and R 2 is a Ci-C 4 alkyl,
  • the pharmaceutical composition is packaged in a packaging material and identified in print, or on the packaging material, for use in reducing intraocular pressure (IOP).
  • IOP intraocular pressure
  • an agent selected from the group consisting of a prostaglandin analog, a ⁇ -blocker, an adrenergic agent, an a2-adrenergic receptor agonist, a miotic agent, a carbonic anhydrase inhibitor and a cholinergic agonist, and
  • Formula III including any pharmaceutically acceptable salt, hydrate, solvate, enantiomer and diastereomer thereof, and any mixtures thereof,
  • X is H or OH
  • Y is O or S
  • Rl, R2 and R3 are each independently H or a Ci-C 4 alkyl
  • Ra is a Cl-C4 alkyl
  • Rb, Rc and Rd are each independently selected from H, a Ci-C 4 alkyl and a Q- C 4 hydroxy alkyl;
  • Re is selected from a Ci-C 4 alkyl and a Ci-C 4 hydroxyalkyl
  • n 0, 1 or 2;
  • R' is represented by general Formula II:
  • A is a spacer moiety or a covalent bond
  • B is a cyclic moiety, or B is selected from the group consisting of an alkylsulfonyl, an arylsulfonyl and a sulfonamide, or B is -NRiR 2 , wherein Ri and R 2 are each independently H or a Ci-C 4 alkyl provided that at least one of Ri and R 2 is a Ci-C 4 alkyl,
  • IOP intraocular pressure
  • a method of reducing intraocular pressure (IOP) in a subject in need thereof which includes co-administering to the subject a therapeutically effective amount of: an agent selected from the group consisting of a prostaglandin analog, a ⁇ - blocker, an adrenergic agent, an a2-adrenergic receptor agonist, a miotic agent, a carbonic anhydrase inhibitor and a cholinergic agonist; and
  • Formula III including any pharmaceutically acceptable salt, hydrate, solvate, enantiomer and diastereomer thereof, and any mixtures thereof,
  • X is H or OH
  • Y is O or S
  • Ri, R 2 and R 3 are each independently H or a Ci-C 4 alkyl
  • Ra is a Ci-C 4 alkyl
  • Rb, Rc and Rd are each independently selected from H, a Ci-C 4 alkyl and a Q- C 4 hydroxy alkyl;
  • Re is selected from a Ci-C 4 alkyl and a Ci-C 4 hydroxyalkyl
  • n 0, 1 or 2;
  • R' is represented by general Formula II:
  • A is a spacer moiety or a covalent bond
  • B is a cyclic moiety, or B is selected from the group consisting of an alkylsulfonyl, an arylsulfonyl and a sulfonamide, or B is -NR R 2 , wherein Ri and R 2 are each independently H or a Ci-C 4 alkyl provided that at least one of Ri and R 2 is a Q-C 4 alkyl.
  • the mode of administration is effected topically, extraocularly, intraocularly and/or intravitreally.
  • the composition according to some embodiments of the invention is formulated as an ophthalmic composition suitable for topical, extraocular, intraocular and/or intravitreal administration to the eye of the subject.
  • the composition according to some embodiments of the invention is in the form selected from the group consisting of an eye-drop solution, a spray, an eye wash solution, an ointment, a suspension, a gel, a cream and an injectable solution.
  • the intraocular pressure (IOP) is associated with glaucoma, low-tension glaucoma and normal-tension glaucoma.
  • a method of treating a heart condition in a subject in need thereof that includes co-administering to the subject a therapeutically effective amount of:
  • Formula III including any pharmaceutically acceptable salt, hydrate, solvate, enantiomer and diastereomer thereof, and any mixtures thereof,
  • X is H or OH
  • Y is O or S
  • Ri, R 2 and R 3 are each independently H or a Ci-C 4 alkyl
  • Ra is a Ci-C 4 alkyl
  • Rb, Rc and Rd are each independently selected from H, a Ci-C 4 alkyl and a Ci- C 4 hydroxyalkyl;
  • Re is selected from a Ci-C 4 alkyl and a Ci-C 4 hydroxyalkyl
  • n 0, 1 or 2;
  • R' is represented by general Formula II:
  • Formula II A is a spacer moiety or a covalent bond
  • B is a cyclic moiety, or B is selected from the group consisting of an alkylsulfonyl, an arylsulfonyl and a sulfonamide, or B is -NR R 2 , wherein Ri and R 2 are each independently H or a Ci-C 4 alkyl provided that at least one of Ri and R 2 is a Q-C 4 alkyl.
  • A is a spacer moiety or a covalent bond
  • B is a cyclic moiety, or B is selected from the group consisting of an alkylsulfonyl, an arylsulfonyl and a sulfonamide, or B is -NR1R2, wherein Ri and R 2 are each independently H or a Ci-C 4 alkyl provided that at least one of Ri and R 2 is a Ci-C 4 alkyl.
  • converting the third digitoxose moiety of digoxin or digitoxin into a dialdehyde is effected by sodium periodate (NaI0 4 ).
  • reacting the dialdehyde with a reagent represented by general formula IV is effected in the presence of NaCNBH 3 .
  • a method of determining an affinity of a compound, according to any embodiment of the invention, to at least one isoform of Na,K-ATPase includes contacting the isoform of Na,K-ATPase with the compound in an affinity measurement setup and determining the affinity.
  • a method of isolating an isoform of Na,K-ATPase of a mammal that includes: transforming yeast cells with a clone that that includes an a chain sequence and a ⁇ chain sequence of the Na,K-ATPase;
  • the a chain sequence is selected from the group consisting of al, a2, a3 and a4;
  • the ⁇ chain sequence is selected from the group consisting of ⁇ , ⁇ 2 and ⁇ 3.
  • the isoform is selected from the group consisting of ⁇ , ⁇ 1 ⁇ 2, ⁇ 1 ⁇ 3, ⁇ 2 ⁇ 1, ⁇ 2 ⁇ 2, ⁇ 2 ⁇ 3, ⁇ 3 ⁇ 1, ⁇ 3 ⁇ 2, ⁇ 3 ⁇ 3, ⁇ 4 ⁇ 1, ⁇ 4 ⁇ 2 and ⁇ 4 ⁇ 3.
  • the isoform is ⁇ 2 ⁇ 2.
  • the isoform is ⁇ 2 ⁇ 3.
  • the isolated isoform according to any embodiment of the invention, is human.
  • FIGs. 1A-D present comparative plots of IOP as a function of time, showing the dose response of a2-inhibitor compounds, according to some embodiments of the present invention, in lowering IOP in live rabbits, wherein FIG. 1A shows the results obtained for DiB, FIG. IB shows the duration of the effect of DiB while 4AP is added every 2 hours so as to maintain the raised IOP, FIG. 1C shows the results obtained for DMcP, and FIG. ID shows the results obtained for DcB;
  • FIGs. 2A-D present comparative plots of IOP as a function of time, demonstrating the capacity of the a2-inhibitor compounds, according to some embodiments of the present invention, to lower IOP below basal levels compared to a buffer control when administered topically to one eye of a rabbit, while the other eye received PBS as a control, wherein FIG. 2A shows the lack of effect of digoxin, FIG. 2B shows the lack of effect of DiB (a non-cyclic moiety inhibitor), FIG. 2C shows the notable effect of DMcP, and FIG. 2D shows the notable effect of DcB; and
  • FIGs. 3A-C present comparative plots of IOP as a function of time, demonstrating the effect of a2-inhibitor compounds, according to some embodiments of the present invention, to potentiate the drug Latanoprost in lowering IOP below basal levels, wherein FIG. 3A shows the effect of DcB alone, FIG. 3B shows the effect of Latanoprost alone, and FIG. 3C shows the effect of co-administering DcB with Latanoprost.
  • the present invention in some embodiments thereof, relates to a pharmaceutical agents and, more particularly, but not exclusively, to digoxin and digitoxin derivatives exhibiting selective inhibition of a2-containing isoforms of Na,K-ATPase, and uses thereof to reduce intraocular pressure (IOP), and/or as cardiotonic agents in a subject in need thereof.
  • IOP intraocular pressure
  • Na,K-ATPase inhibitors that exhibit selectivity towards tissue-specific isoforms of the protein present pharmaceutical advantages such as broader therapeutic window and wider scope of modes of administration.
  • Na,K-ATPase inhibitors exhibiting selectivity towards protein isoforms containing the a2 subunit offer this advantages over unselective inhibitors in treating medical conditions wherein lowering the intraocular pressure is called for.
  • R is represented by general Formula II:
  • A is a spacer moiety or a covalent bond
  • B is a cyclic moiety
  • R is represented by general Formula II:
  • A is a spacer moiety or a covalent bond
  • B is selected from the group consisting of an alkylsulfonyl, an arylsulfonyl and a sulfonamide
  • A is a spacer moiety or a covalent bond
  • B is -NR R2, wherein Ri and R 2 are each independently H or a Ci-C 4 alkyl provided that at least one of Ri and R 2 is a Ci-C 4 alkyl, namely B is a secondary amine or tertiary amine.
  • cyclic moiety refers to a group of atoms that are covalently attached to one another so as to form at least one ring of atoms.
  • Non- limiting examples of cyclic moieties include unsubstituted alicyclic moieties, substituted alicyclic moieties, unsubstituted heterocyclic moieties, substituted heterocyclic moieties, unsubstituted aryl moieties, substituted aryl moieties, unsubstituted heteroaryl moiety and substituted heteroaryl moieties.
  • a substituted cyclic moiety has one or more chemical group or atom attached to one of the atoms in the ring of atoms.
  • chemical groups or atoms include, without limitation, Ci-C 6 alkyl, hydroxyl, amine, halo, alkoxy, carboxyl, amide and the like, or a second cyclic moiety attached by a covalent bond(s) to one or two of the ring atoms of the cyclic moiety.
  • hydroxyl or "hydroxy”, as used herein, refer to an -OH group.
  • amine describes a -NR 1 R2 group where each of R 1 and R is independently hydrogen, alkyl, cycloalkyl, heteroalicyclic, aryl or heteroaryl, as these terms are defined herein.
  • the alkyl group may contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 10 carbon atoms.
  • the alkyl is a lower alkyl, including 1-6 or 1-4 carbon atoms.
  • halide refers to the anion of a halo atom, i.e. F " , CI " , Br " and ⁇ .
  • halo refers to F, CI, Br and I atoms as substituents.
  • amide as used herein encompasses C-amide and N-amide.
  • arylsulfonyl groups include p-toluenesulfonyl (tosyl; Ts), p-bromobenzenesulfonyl (brosyl; Bs ), 2- or 4- nitrobenzenesulfonyl (nosyl; Ns), methanesulfonyl (mesyl; Ms), trifluoromethanesulfonyl (triflyl; Tf), and 5-(dimethylamino)naphthalene-l-sulfonyl (Dansyl; Ds).
  • alicyclic and cycloalkyl refer to an all-carbon monocyclic or fused ring (i.e., rings which share an adjacent pair of carbon atoms), branched or unbranched group containing 3 or more carbon atoms where one or more of the rings does not have a completely conjugated pi-electron system, and may further be substituted or unsubstituted.
  • the cycloalkyl can be substituted or unsubstituted.
  • substituted alicyclic is selected from the group consisting of 2,3- dimethylcyclopropane- 1 -yl, 3 ,3-dimethylcyclobutane- 1 -yl, 3 ,4-dimethylcyclopentane- 1 - yl and 3,5-dimethylcyclohexane-l-yl.
  • heterocyclic or “heteroalicyclic”, as used herein, describe a monocyclic or fused ring group having in the ring(s) one or more atoms such as nitrogen, oxygen and sulfur.
  • the rings may also have one or more double bonds. However, the rings do not have a completely conjugated pi-electron system.
  • the heteroalicyclic may be substituted or unsubstituted. Representative examples are morpholine, piperidine, piperazine, tetrahydrofurane, tetrahydropyrane and the like.
  • the spacer moiety A can be a covalent bond, an unsubstituted Ci-C 6 alkyl, a substituted Ci-C 6 alkyl, an unsubstituted Ci-C 6 alkyl interrupted by one or more heteroatom (e.g., O, N or S) and a substituted Ci-C 6 alkyl interrupted by one or more heteroatom.
  • a spacer moiety may be substituted with one or more Ci-C 6 alkyl, hydroxyl, amine, halo, alkoxy, carboxyl, amide and the like.
  • A is a covalent bond and B an alicyclic moiety such as, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the corresponding R in Formula I is:
  • Cyclopropyl (cP) Cyclobutyl (cB) Cyclopentyl (cPe) Cyclohexyl (cHe)
  • A is a covalent bond and B a heteroalicyclic moiety such as, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the corresponding R in Formula I is:
  • A is -CH 2 - and B a cyclic moiety such as, for example, cyclopropyl, 3,3-dimethylcyclobutane-l-yl and phenyl, and the corresponding R in Formula I is:
  • X is H
  • A is -CH 2 - and B is cyclopropyl.
  • X is OH
  • A is a covalent bond
  • B is cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.
  • X is OH
  • A is -CH 2 - and B is cyclopropyl, 3,3- dimethylcyclobutane-l-yl and phenyl.
  • X is OH
  • A is -(CH 2 ) 2 - and B is cyclopropyl.
  • R of general Formula I is cyclopropyl, methylcyclopropane, ethylcyclopropane, propylcyclopropane, cyclobutyl, methylcyclobutane, methyl-3 ,3 -dimethylcyclobutane, ethylcyclobutane, propylcyclobutane, cyclopentyl, methylcyclopentane, ethylcyclopentane, propylcyclopentane, cyclohexyl and benzyl.
  • R is cyclopropyl, methylcyclopropane and cyclobutyl.
  • A is -CH 2 - and B an alkylsulfonyl such as, for example, methylsulfonyl, and the corresponding R in Formula I is:
  • A is -(CH 2 ) 2 - and B an alkylsulfonyl such as, for example, methylsulfonyl, or a solnfonamide, and the corresponding R in Formula I is:
  • DMDMcB or 4-((3S,5R,8R,9S,10S,12R,13S,14S,17R)-3-(((2S,4S,5R,6R)-5- (((2S,4S,5S,6R)-5-((4-((3,3-dimethylcyclobutyl)methyl)-2-methyl-l,4-oxazepan-7- yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-4-hydroxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)-4-hydroxy-6- methyltetrahydro-2H-pyran-2-yl)oxy)- 12, 14-dihydroxy- 10, 13-dimethylhexadecahydro- lH-cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one;
  • the compound is any one of the structures presented below:
  • the compound is any one of the structures presented below:
  • the present embodiments further encompass any enantiomers, diastereomers, optical isomers, prodrugs, solvates, hydrates, polymorphs, geometrical isomers and/or pharmaceutically acceptable salts of the compounds described herein.
  • salt encompasses both basic and acid addition salts, and include salts formed with organic and inorganic anions and cations.
  • organic or inorganic cation refers to counter-ions for an acid.
  • the counter-ions can be chosen from the alkali and alkaline earth metals, (such as lithium, sodium, potassium, barium, aluminum and calcium), ammonium and the like.
  • the term includes salts that form by standard acid-base reactions of basic groups and organic or inorganic acids.
  • Such acids include hydrochloric, hydrofluoric, hydrobromic, trifluoroacetic, sulfuric, phosphoric, acetic, succinic, citric, lactic, maleic, fumaric, cholic, pamoic, mucic, D-camphoric, phthalic, tartaric, salicylic, methanesulfonic, benzenesulfonic, p-toluenesulfonic, sorbic, picric, benzoic, cinnamic, and like acids.
  • a pharmaceutically acceptable salt of the compounds described herein may optionally be an acid addition salt comprising at least one basic (e.g., amine and/or guanidine) group of the compound which is in a positively charged form (e.g., wherein the basic group is protonated), in combination with at least one counter-ion, derived from the selected base, that forms a pharmaceutically acceptable salt.
  • at least one basic e.g., amine and/or guanidine
  • the acid addition salts of the compounds described herein may therefore be complexes formed between one or more basic groups of the compound and one or more equivalents of an acid.
  • the acid additions salts can be either mono-addition salts or poly-addition salts.
  • poly- addition salt refers to a salt in which the stoichiometric ratio between the counter-ion and the charged form of the compound is greater than 1: 1 and is, for example, 2: 1, 3: 1, 4: 1 and so on, such that the addition salt includes two or more molar equivalents of the counter-ion per one molar equivalent of the compound.
  • a pharmaceutically acceptable salt would be an ammonium cation or guanidinium cation and an acid addition salt thereof.
  • the acid addition salts may include a variety of organic and inorganic acids, such as, but not limited to, hydrochloric acid which affords a hydrochloric acid addition salt, hydrobromic acid which affords a hydrobromic acid addition salt, acetic acid which affords an acetic acid addition salt, ascorbic acid which affords an ascorbic acid addition salt, benzenesulfonic acid which affords a besylate addition salt, camphorsulfonic acid which affords a camphorsulfonic acid addition salt, citric acid which affords a citric acid addition salt, maleic acid which affords a maleic acid addition salt, malic acid which affords a malic acid addition salt, methanesulfonic acid which affords a methanesulfonic acid (mesylate) addition salt, naphthalenesulfonic acid which afford
  • enantiomer refers to a stereoisomer of a compound that is superposable with respect to its counterpart only by a complete inversion/reflection (mirror image) of each other. Enantiomers are said to have "handedness” since they refer to each other like the right and left hand. Enantiomers have identical chemical and physical properties except when present in an environment which by itself has handedness, such as all living systems.
  • a compound may exhibit one or more chiral centers, each of which exhibiting an R- or an S -configuration and any combination, and compounds according to some embodiments of the present invention, can have any their chiral centers exhibit an R- or an S -configuration.
  • diastereomers refers to stereoisomers that are not enantiomers to one another. Diastereomerism occurs when two or more stereoisomers of a compound have different configurations at one or more, but not all of the equivalent (related) stereocenters and are not mirror images of each other. When two diastereoisomers differ from each other at only one stereocenter they are epimers. Each stereo-center (chiral center) gives rise to two different configurations and thus to two different stereoisomers.
  • embodiments of the present invention encompass compounds with multiple chiral centers that occur in any combination of stereo-configuration, namely any diastereomer.
  • All stereoisomers, optical and geometrical isomers of the compounds of the present invention are contemplated, either in admixture or in pure or substantially pure form.
  • the compounds of the present invention can have asymmetric centers at one or more of the atoms. Consequently, the compounds can exist in enantiomeric or diastereomeric forms or in mixtures thereof.
  • the present invention contemplates the use of any racemates (i.e. mixtures containing equal amounts of each enantiomers), enantiomerically enriched mixtures (i.e., mixtures enriched for one enantiomer), pure enantiomers or diastereomers, or any mixtures thereof.
  • the chiral centers can be designated as R or S or R,S or d,D, 1,L or d,l, D,L.
  • prodrug refers to an agent, which is converted into the active compound (the active parent drug) in vivo.
  • Prodrugs are typically useful for facilitating the administration of the parent drug. They may, for instance, be bioavailable by oral administration whereas the parent drug is not.
  • a prodrug may also have improved solubility as compared with the parent drug in pharmaceutical compositions.
  • Prodrugs are also often used to achieve a sustained release of the active compound in vivo.
  • An example, without limitation, of a prodrug would be a compound of the present invention, having one or more carboxylic acid moieties, which is administered as an ester (the "prodrug").
  • Such a prodrug is hydrolyzed in vivo, to thereby provide the free compound (the parent drug).
  • the selected ester may affect both the solubility characteristics and the hydrolysis rate of the prodrug.
  • solvate refers to a complex of variable stoichiometry (e.g., di-, tri-, tetra-, penta-, hexa-, and so on), which is formed by a solute (the compound of the present invention) and a solvent, whereby the solvent does not interfere with the biological activity of the solute.
  • Suitable solvents include, for example, ethanol, acetic acid and the like.
  • hydrate refers to a solvate, as defined hereinabove, where the solvent is water.
  • the present invention also includes solvates of the compounds of the present invention and salts thereof.
  • “Solvate” means a physical association of a compound of the invention with one or more solvent molecules. This physical association involves varying degrees of ionic and covalent bonding, including hydrogen bonding. In certain instances the solvate will be capable of isolation.
  • “Solvate” encompasses both solution- phase and isolatable solvates.
  • suitable solvates include ethanolates, methanolates and the like.
  • “Hydrate” is a solvate wherein the solvent molecule is water.
  • the present invention also includes polymorphs of the compounds of the present invention and salts thereof.
  • polymorph refers to a particular crystalline state of a substance, which can be characterized by particular physical properties such as X- ray diffraction, IR spectra, melting point, and the like.
  • the compound is represented by general Formula III:
  • X is H or OH
  • Y is O or S; Ri, R 2 and R 3 are each independently H or a Ci-C 4 alkyl;
  • Ra is a Ci-C 4 alkyl
  • n 0, 1 or 2;
  • R' is represented by general Formula II:
  • A is a spacer moiety or a covalent bond
  • B is a cyclic moiety
  • R' is represented by general Formula II:
  • A is a spacer moiety or a covalent bond
  • B is selected from the group consisting of an alkylsulfonyl, an arylsulfonyl and a sulfonamide;
  • R' is represented by general Formula II:
  • A is a spacer moiety or a covalent bond
  • Ri and R 2 are each independently H or a Ci-C 4 alkyl provided that at least one of Ri and R 2 is a Ci-C 4 alkyl,
  • A is a spacer moiety or a covalent bond as described hereinabove.
  • the reaction of the dialdehyde is effected by reductive amination of the dialdehyde using a free amine of R (R-NH 2 ) in the presence of, e.g., NaCNBH 3 .
  • the compounds presented herein such as a compound represented by general Formula I or by general Formula III, exhibit an affinity to at least one isoform of Na,K-ATPase.
  • isoforms of Na,K- ATPase include any combination of al, a2, a3 and a4 complexed with ⁇ , ⁇ 2 and ⁇ 3; hence, isoforms of Na,K-ATPase include ⁇ , ⁇ 1 ⁇ 2, ⁇ 1 ⁇ 3, ⁇ 2 ⁇ 1, ⁇ 2 ⁇ 2, ⁇ 2 ⁇ 3, ⁇ 3 ⁇ 1, ⁇ 3 ⁇ 2, ⁇ 3 ⁇ 3, ⁇ 4 ⁇ 1, ⁇ 4 ⁇ 2 and ⁇ 4 ⁇ 3.
  • a compound represented by general Formula I or by general Formula III has a higher affinity to isoforms containing an a2 subunit, compared to its affinity to isoforms containing of the a subunits, such as al, as demonstrated in the Examples section that follows below.
  • a compound represented by general Formula I or by general Formula III is a selective inhibitor of a2-containing isoforms of Na,K-ATPase, compared to a 1 -containing isoforms of Na,K- ATPase, as demonstrated in the Examples section that follows below.
  • drugs include ⁇ -adrenergic antagonists and carbonic anhydrase inhibitors, that reduce the rate of aqueous humor production, or prostaglandin analogs, cholinergic agonists and sympathomimetics, that increase the rate of outflow through the trabecular meshwork and uveoscleral pathway.
  • the experimental results presented hereinbelow show 25-30 % higher reduction in basal IOP effected by the compounds presented herein in the rabbits, compared to Latanoprost, the current first-line drug for treatment of glaucoma.
  • the ability of the compounds presented herein, according to some embodiments, to reduce the basal IOP could be relevant not only to optical hypertension and primary open angle glaucoma but also to normotensive glaucoma for which reduction of IOP below the basal level is required.
  • ⁇ 2 ⁇ 3- selective inhibitors included not only the expectation of high potency but also of minimal local side-effects.
  • corneal swelling used to assess such adverse effect, is not observed at least over several days of topical administration of the compounds presented herein (see, e.g., Table 4). Similar evidence for safety was observed by inspection of local redness and irritation.
  • a pharmaceutical composition which includes as an active ingredient any one of the compounds represented by general Formula I and a pharmaceutically acceptable carrier.
  • the term "pharmaceutically acceptable carrier” refers to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
  • carriers are: propylene glycol, saline, emulsions and mixtures of organic solvents with water, as well as solid (e.g., powdered) and gaseous carriers.
  • excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound.
  • excipients examples include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
  • compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more pharmaceutically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the compounds presented herein into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the administration is effected topically, extraocularly, intraocularly and/or intravitreally.
  • the pharmaceutical composition is formulated as an ophthalmic composition suitable for topical, extraocular, intraocular and/or intravitreal administration to the eye of the subject.
  • the pharmaceutical composition of the invention is an ophthalmic composition which is administered topically onto the eye of a subject for facilitating effective intraocular levels of the compound and for preventing unnecessary and unintentional levels of the compound in other tissues and/or organs. Such a non-systemic, site-specific administration reduces the side effects associated with the compounds.
  • intraocular and/or intravitreal administration is effected by injecting the compound(s), or compositions and medicaments comprising the compound(s) into the eye or into a bodily tissue near the eye.
  • the composition may take the form of an injectable solution.
  • oral or otherwise systemic administration in a dosage effective for reducing the intraocular pressure is also possible.
  • the composition may be administered by a dermal patch for extended release.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP).
  • disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the compounds presented herein may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active aminoglicoside compounds doses.
  • the compounds presented herein are conveniently delivered in the form of an aerosol spray presentation (which typically includes powdered, liquefied and/or gaseous carriers) from a pressurized pack or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compounds presented herein and a suitable powder base such as, but not limited to, lactose or starch.
  • compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of the compounds preparation in water-soluble form.
  • suspensions of the compounds presented herein may be prepared as appropriate oily injection suspensions and emulsions (e.g., water-in-oil, oil-in-water or water-in-oil in oil emulsions).
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes.
  • Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran.
  • the suspension may also contain suitable stabilizers or agents, which increase the solubility of the compounds presented herein to allow for the preparation of highly concentrated solutions.
  • the compounds presented herein may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
  • compositions herein described may also comprise suitable solid of gel phase carriers or excipients.
  • suitable solid of gel phase carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin and polymers such as polyethylene glycols.
  • compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of compounds presented herein effective to prevent, alleviate or ameliorate symptoms of the disorder, or prolong the survival of the subject being treated.
  • the therapeutically effective amount or dose can be estimated initially from activity assays in animals.
  • a dose can be formulated in animal models to achieve a circulating concentration range that includes the mutation suppression levels as determined by activity assays (e.g., the concentration of the test compounds which achieves a substantial read-through of the truncation mutation). Such information can be used to more accurately determine useful doses in humans.
  • Toxicity and therapeutic efficacy of the compounds presented herein can be determined by standard pharmaceutical procedures in experimental animals, e.g., by determining the EC 50 (the concentration of a compound where 50 % of its maximal effect is observed) and the LD 50 (lethal dose causing death in 50 % of the tested animals) for a subject compound.
  • the data obtained from these activity assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl et ah, 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p. l).
  • Dosage amount and interval may be adjusted individually to provide plasma levels of the compounds presented herein which are sufficient to maintain the desired effects, termed the minimal effective concentration (MEC).
  • MEC minimal effective concentration
  • the MEC will vary for each preparation, but can be estimated from in vitro data; e.g., the concentration of the compounds necessary to achieve 50-90 % expression of the whole gene having a truncation mutation, i.e. read-through of the mutation codon. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. HPLC assays or bioassays can be used to determine plasma concentrations.
  • Dosage intervals can also be determined using the MEC value. Preparations should be administered using a regimen, which maintains plasma levels above the MEC for 10-90 % of the time, preferable between 30-90 % and most preferably 50-90 %.
  • dosing can also be a single periodic administration of a slow release composition described hereinabove, with course of periodic treatment lasting from several days to several weeks or until sufficient amelioration is effected during the periodic treatment or substantial diminution of the disorder state is achieved for the periodic treatment.
  • compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA (the U.S. Food and Drug Administration) approved kit, which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may, for example, comprise metal or plastic foil, such as, but not limited to a blister pack or a pressurized container (for inhalation).
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the pack or dispenser may also be accompanied by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions for human or veterinary administration.
  • a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions for human or veterinary administration.
  • Such notice for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.
  • Compositions comprising a compound according to the present embodiments, formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition or diagnosis, as is detailed hereinabove.
  • the compounds presented herein may be loaded into a drug-delivery device to be inserted or implanted into the eye of the subject for allowing releasing of the compound in a controlled and continuous rate, by dissolving, diffusion or leaching, thus maintaining effective therapeutic concentration over a prolonged period of time.
  • the drug-delivery device may be for example a biocompatible thin film loaded with the active agent, inserted for example beneath the lower eyelid.
  • the drug-delivery device is a contact lens or any other ophthalmic device, as these are known in the art.
  • the pharmaceutical composition is packaged in a packaging material and identified in print, or on the packaging material, for use in reducing intraocular pressure (IOP).
  • IOP intraocular pressure
  • a method of reducing intraocular pressure (IOP) in a subject includes administering to a subject in need thereof a therapeutically effective amount of a compound represented by general Formula I, or a pharmaceutical composition comprising the same.
  • IOP is associated with glaucoma, low-tension glaucoma and normal-tension glaucoma.
  • the compounds, compositions and medicaments presented herein are useful in treating, without limitation, glaucoma, low-tension glaucoma and normal- tension glaucoma.
  • treating includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.
  • the concentration of the compounds presented herein in the pharmaceutical compositions or medicaments presented herein is in the range of about 1 to about 5,000 ⁇ g/ml of composition, preferably from about 80 to about 800 ⁇ g/ml and the formulation is preferably applied in one to four doses per day wherein each dose contains about 1 to 125 ⁇ g of the compound, or from about 2 to about 20 ⁇ g of the compound.
  • a pharmaceutical composition which includes as active ingredients at least one agent (active ingredient) which is in use in reducing IOP and is not a compound represented by general Formula III, a compound represented by general Formula III, and a pharmaceutically acceptable carrier.
  • Non-limiting examples of active ingredients which are in use in reducing IOP and which are not one of the compound represented by general Formula III, include prostaglandin analogs, ⁇ -blockers, adrenergic agents, a2-adrenergic receptor agonists, miotic agents, carbonic anhydrase inhibitors and cholinergic agonists.
  • prostaglandin analogs include Latanoprost (Xalatan),
  • Bimatoprost Liigan
  • Travoprost Travatan
  • ⁇ -blockers include Timolol (Timoptic) and Betaxolol (Betoptic).
  • a non-limiting example of adrenergic agents is Brimonidine (Alphagan).
  • a non-limiting example of miotic agents is Pilocarpine (Isoptocarpine, Pilocar).
  • Non-limiting examples of carbonic anhydrase inhibitors include Dorzolamide
  • Non-limiting examples of cholinergic agonists include carbachol (Miostat), echothiophate (Phospholine) and pilocarpine (Isopto Carpine, Pilopine).
  • co-administration refers to a concomitant administration of more than one active agent (active ingredient) to a subject, whereas in the context of embodiments presented herein, the term “concomitant” means that the co-administered active agents are present in the subject (PK), or otherwise exert an effect (PD), at similar, identical, consecutive or partially overlapping periods of time.
  • PK subject
  • PD effect
  • the terms “substantially simultaneous” and “rapid succession” correspond to the term “concomitant” as used herein, namely meaning that the period of time between a first administration and a second administration of more than one active agent is sufficiently short to be regarded as a single administration event, and/or a number of administrations of different active agents takes place within 5-60 minutes or less.
  • each administration in such "rapid succession" delivers to the user a different amount or composition of one or more pharmaceutically active agents.
  • two or more of the administrations provide the same composition and amount of the one or more pharmaceutically active agents.
  • the later administration of the second active agent is performed at such timing that the first active agent of a previous administration within the same rapid succession does not yet have a significant pharmacodynamic effect or before it can be measured (pharmacokynetically) by, e.g., blood concentration thereof the first active agent.
  • a method of reducing IOP in a subject in need thereof includes co- administering to the subject a therapeutically effective amount of at least one agent which is in use in reducing IOP and is not a compound represented by general Formula III, and a therapeutically effective amount of a compound represented by general Formula III.
  • a method of reducing IOP in a subject in need thereof includes coadministering to the subject a synergistically effective amount of at least one agent which is in use in reducing IOP and is not a compound represented by general Formula III, and a synergistically effective amount of a compound represented by general Formula III.
  • a synergistically effective amount is also a therapeutically effective amount in the sense of providing the desired therapeutic effect, and is smaller than the therapeutically effective amount of a singly-administered active ingredient.
  • the selectivity of the compounds presented herein can be utilized in the treatment of other medical conditions.
  • an oil- selective inhibitor such as the compounds presented herein and represented by general Formula I, can be used as an effective cardiotonic agent, with reduced cardiotoxicity, compared to known agents such as digoxin.
  • a method of treating a heart condition which is carried out by administering to a subject in need thereof a therapeutically effective amount of a compound represented by general Formula I.
  • a pharmaceutical composition which includes as an active ingredient a compound represented by general Formula I and a pharmaceutically acceptable carrier, identified in print, or on a packaging material, for use in a treatment of a heart condition.
  • heart conditions which are relevant in the context of embodiments of the resent invention, include, without limitation, atrial fibrillation, atrial flutter, mitral stenosis, chronic heart failure and congestive heart failure.
  • the present invention provides cardiotonic compositions comprising a therapeutically effective amount of the compounds represented by general Formula I, or a pharmaceutical composition comprising the same.
  • the compounds may be formulated for oral, buccal, topical, intravenous, parenteral or rectal administration.
  • a compound represented by general Formula I or a compound represented by general Formula III can be used according to embodiments the present invention to treat a heart condition in combination with one or more other drugs for treating a heart a condition, such as, but not limited to, selective and nonselective ⁇ -blocker agents, anticoagulation agents, angiotensin-converting-enzyme inhibitors (ACE inhibitors) and angiotensin II receptor antagonists.
  • drugs such as, but not limited to, selective and nonselective ⁇ -blocker agents, anticoagulation agents, angiotensin-converting-enzyme inhibitors (ACE inhibitors) and angiotensin II receptor antagonists.
  • a method of treating a heart condition in a subject in need thereof which includes co-administering to the subject a therapeutically effective amount of an agent selected from the group consisting of a ⁇ -blocker, an anticoagulation agent, an angiotensin-converting-enzyme inhibitor and an angiotensin II receptor antagonist; and a compound represented by Formula III, as these are described herein, and a pharmaceutically acceptable carrier.
  • an agent selected from the group consisting of a ⁇ -blocker, an anticoagulation agent, an angiotensin-converting-enzyme inhibitor and an angiotensin II receptor antagonist
  • a compound represented by Formula III as these are described herein
  • Nonselective ⁇ -blocker agents include propranolol, bucindolol, carteolol, carvedilol, labetalol, nadolol, oxprenolol, penbutolol, pindolol, sotalol, timolol, eucommia bark (herb);
  • ⁇ -selective agents include acebutolol, atenolol, betaxolol, bisoprolol, celiprolol, esmolol, metoprolol, nebivolol; fil-selective, agents include butaxamine and ICI-118,551; and agents include SR 59230A.
  • Anticoagulation agents include heparin, dicumarol, Coumadin (warfarin) and aspirin.
  • ACE inhibitors include captopril, zofenopril, enalapril (vasotec, renitec), ramipril (altace, prilace, ramace, ramiwin, triatec, tritace), quinapril (accupril), perindopril (coversyl, aceon, perindo), lisinopril (listril, lopril, novatec, prinivil, zestril), benazepril (lotensin), imidapril (tanatril), trandolapril (mavik, odrik, gopten), cilazapril (inhibace) and fosinopril (fositen, monopril).
  • Angiotensin II receptor antagonists include losartan, EXP 3174, candesartan, valsartan, irbesartan, telmisartan, eprosartan, olmesartan, azilsartan and fimasartan.
  • the compound represented by general Formula I or the compound represented by general Formula III, according to embodiments of the present invention, and any one of the other drugs for treating a heart a condition can be co-formulated in a single composition, or be formulated into individual compositions.
  • a method of determining an apparent affinity of the compound represented by general Formula I to at least one isoform of Na,K-ATPase includes contacting an isoform of Na,K-ATPase with the compound in an activity measurement setup and determining the apparent affinity of the compound to the isoform.
  • a method of isolating an isoform of Na,K-ATPase of a mammal includes:
  • the a chain sequence is selected from the group consisting of al, a2, a3 and a4;
  • the ⁇ chain sequence is selected from the group consisting of ⁇ , ⁇ 2 and ⁇ 3.
  • the isolated isoform is ⁇ 2 ⁇ 2.
  • the isolated isoform is ⁇ 2 ⁇ 3.
  • the yeast-expressed ⁇ / ⁇ subunits have distinct levels of glycosylation compared to those in the human-expressed subunits.
  • the distinct yeast-expressed glycosylation pattern does not substantially affect the activity of the enzyme but may increase its stability.
  • an isolated ⁇ 2 ⁇ 2 isoform of Na,K-ATPase of a mammal having a yeast- characterizing glycosylation pattern there is provided an isolated ⁇ 2 ⁇ 2 isoform of Na,K-ATPase of a mammal having a yeast- characterizing glycosylation pattern.
  • an isolated ⁇ 2 ⁇ 3 isoform of Na,K-ATPase of a mammal having a yeast- characterizing glycosylation pattern there is provided an isolated ⁇ 2 ⁇ 3 isoform of Na,K-ATPase of a mammal having a yeast- characterizing glycosylation pattern.
  • the isolated isoforms presented herein have an amino acid sequence which identical, substantially similar or derived from any one of the isoform of human of Na,K-ATPase.
  • compositions, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
  • various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range.
  • the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts. It is expected that during the life of a patent maturing from this application many relevant genetic diseases and disorders as defined herein will be uncovered and the scope of this term is intended to include all such new disorders and diseases a priori.
  • ATPase was conducted by generation of pHil-D2 expression vector containing cDNA of human ai and HislO tagged human ⁇ was described previously [Lifshitz Y, et al., 2007, Biochemistry, 46(51), pp. 14937-14950].
  • cDNA of human ⁇ 2 and ⁇ 3 in pSD5 were a gift from K. Geering, University Lausanne, Switzerland. Open reading frames and flanking regions of human ⁇ 2 and human ⁇ 3 were amplified by PCR using primers containing Bglll and Sail cleavage sites.
  • Yeasts were grown in BMG (100 mM potassium phosphate pH 6, 1.34 % yeast nitrogen base, 4X10 "5 % biotin, 0.3 % glycerol) to OD 6-8 and expression was induced in BMM (100 mM potassium phosphate pH 6, 1.34 % yeast nitrogen base, 4X10 "5 % biotin, 0.5 % methanol added daily).
  • Pichia pastoris transformation, yeast growth, membrane preparation and His- tag purification of recombinant human ⁇ , ⁇ 2 ⁇ 1, ⁇ 2 ⁇ 2 and ⁇ 2 ⁇ 3 Na,K-ATPase were carried out essentially as described previously [Katz A, et al., 2010, Biol Chem, 285(25), pp. 19582-19592; and Katz A, et al., 2014, Biol Chem, 289(30), pp. 21153-21162].
  • Expression and purification of ⁇ , ⁇ 2 ⁇ 1, ⁇ 2 ⁇ 2 and ⁇ 2 ⁇ 3 complexes was conducted in small scale whole cell lysates that were prepared as describe in Loayza, D. et al., Mol. Cell.
  • Na,K-ATPase ⁇ , ⁇ 2 ⁇ , ⁇ 2 ⁇ 2 and ⁇ 2 ⁇ 3 complexes were reconstituted with FXYD1 and purified in a mixture of 0.1 mg/ml C12E8, 0.07 mg/ml SOPS and 0.01 mg/ml cholesterol.
  • the purified isoform complexes (0.3-0.5 mg/ml) were eluted from the BD- Talon beads in a solution containing Imidazole 170 mM, NaCl 100 mM; Tricine.HCl 20 mM pH 7.4; C12E8, 0.1 mg/ml; SOPS 0.07 mg/ml, cholesterol 0.01 mg/ml, glycerol 25 %, by gravity-column.
  • the proteins were stored at -80 °C. Protein purity was determined by gel electrophoresis and protein concentration was determined with BCA (B9643 Sigma).
  • the specific Na,K-ATPase activity was highest for ⁇ (16.4+0 ⁇ mol/mg/min) followed by ⁇ 2 ⁇ (10.9+0.6) and ⁇ 2 ⁇ 3 (10.7+1.9).
  • the ⁇ 2 ⁇ 2 isoform had the lowest activity (8.4+1.4).
  • the second significantly different parameter is the apparent K + -affinity.
  • K0.5 K + for ⁇ 2 ⁇ was 2.7+0.14 mM compared to 1.47+0.06 mM for ⁇ .
  • ⁇ 2 ⁇ 2 and ⁇ 2 ⁇ 3 had an even lower affinity than ⁇ 2 ⁇ with apparent K0.5 values of 7.4+0.19mM and 6.4+0.50mM, respectively.
  • Na-titrations revealed that the affinity for Na + -ions was not different between a ⁇ iand ⁇ 2 ⁇ whereas ⁇ 2 ⁇ 2 and ⁇ 2 ⁇ 3 had a somewhat higher Na-affinity.
  • Vanadate is a phosphate analogue that binds to the E2 conformation, mimicking the transition state E2PK 2 during dephosphorylation, thus inhibiting the enzyme. All three a 2 isoforms had a lower vanadate affinity compared to ⁇ (0.48 ⁇ ). ⁇ 2 ⁇ 2 had the lowest vanadate affinity (34 ⁇ ) followed by ⁇ 2 ⁇ 3 (19 ⁇ ) and ⁇ 2 ⁇ (3.5 ⁇ ).
  • the order of inhibition by vanadate equals the order of potassium activation (Ko . sK + and Ki vanadate ⁇ ⁇ 2 ⁇ 2 ⁇ 3 ⁇ ⁇ 2 ⁇ 3 ) supporting the hypothesis proposed above.
  • Na,K-ATPase activity of a ⁇ FXYDl complexes was measured over one hour at 37 °C in a medium containing 130 mM NaCl, 5 mM KC1, 3 mM MgCl 2 , 1 mM EGTA, 25 mM Histidine, pH 7.4 and 1 mM ATP using the PiColor Lock gold malachite green assay (Inova Biosciences).
  • the Na,K-ATPase activities were ⁇ , 21.5+5.3 ⁇ moles/min/mg; ⁇ 2 ⁇ 1, 18.7+1.8 ⁇ moles/min/mg, and ⁇ 2 ⁇ 3, 10.7+1.9 ⁇ moles/min/mg protein.
  • an important kinetic property in relation to inhibition by cardiac glycosides is Ko.5 for activation by K: ⁇ 1 ⁇ 1-1.25+0.05 mM, ⁇ 2 ⁇ 1-2.7+0.14 mM and ⁇ 2 ⁇ 3 6.4+0.50 mM, respectively.
  • ATPase activity assays as well as titrations with NaCl, KC1 and vanadate were performed as described in Lifshitz-2007 and Loayza-1998 using PiColorLockTM malachite green assay (Inova Bioscience).
  • Inhibitor assays were performed as described in Katz-2010.
  • [ 3 H]ouabain binding and K + -[ 3 H]digoxin displacement assays were performed as described in Katz-2010.
  • VCG/V0 Ki /([CG] + Ki)+ c (CG stands for cardiac glycoside). Inhibition was estimated in 3-5 separate experiments and average Ki values + standard error of the mean (SEM) were calculated. The ratios Ki ⁇ 1 ⁇ 1/ ⁇ 2 ⁇ 1, ⁇ 1 ⁇ 1/ ⁇ 2 ⁇ 2 and ⁇ 1 ⁇ 1/ ⁇ 2 ⁇ 3 was calculated for each compound.
  • Table 2 shows the Ki values and selectivity ratios (Ki ⁇ 1 ⁇ 1: ⁇ 2 ⁇ 1, Ki ⁇ 1 ⁇ 1: ⁇ 2 ⁇ 2. ⁇ ⁇ 1 ⁇ 1: ⁇ 2 ⁇ 3) for inhibition of Na,K-ATPase activity of compounds according to embodiments of the present invention, as well as some digoxin derivatives having a non-cyclic moiety, compared to digoxin and digitoxin.
  • Table 2 is arbitrarily sorted according to column "Ki ratio ⁇ 1 ⁇ 1/ ⁇ 2 ⁇ 3" marked by "*".
  • DMcP and DcB show exceptional selectivity for ⁇ 2 ⁇ 2 and ⁇ 2 ⁇ 3 over ⁇ of 22-fold and 33 -fold, respectively, and very low Ki values for inhibition of ⁇ 2 ⁇ 3 (Ki about 4 nM).
  • the full inhibition curves of DMcP and DcB emphasize the extent of the difference between ⁇ and ⁇ 2 ⁇ 3.
  • the compounds presented herein show a notable increase of the ratio of Ki's ⁇ 1 ⁇ 1: ⁇ 2 ⁇ 3 compared to ⁇ 1 ⁇ 1: ⁇ 2 ⁇ 1, which must be partly due to the reduced K- cardiac glycoside antagonism.
  • the difference for the most o ⁇ 3-selective compounds is significantly greater than that for digoxin and cannot be explained only by this factor.
  • the Ki for ⁇ is about 2-3 -fold lower than for digoxin itself but the Ki for ⁇ 2 ⁇ 3 is about 10-fold lower than for digoxin, thus raising selectivity for ⁇ 2 ⁇ 3 over ⁇ to more than 22-fold and more than 33-fold, respectively.
  • NPE cell lysates were shown to contain about 70 % a2 and 30 % al, while PE were shown to contain about 90 % al and 5-10 % a2.
  • Na,K-ATPase activity was measured and was found to be 0.195 +0.027 and 0.035 +0.008 nmoles/mg protein/min in NPE and PE cells, respectively.
  • Ouabain-sensitive fractions of total ATPase activity were about 65 % and 35 % for NPE and PE cells, respectively.
  • the cells were incubated with 0.8 mg/ml alamecithin for 30 minutes at room temperature prior to transfer to the reaction medium containing 130 mM NaCl, 5 mM KCl, 3 mM MgCl 2 , 25 mM histidine, pH 7.4, 1 mM EGTA, 1 mM sodium azide , 0.5 mM ATP, and were then incubated for 45 minutes at 37 °C, with or without the tested inhibitors as indicated, or 0.5 mM ouabain to determine the ouabain insensitive ATPase activity. The data was fitted to one or two sites inhibition model.
  • Table 3 presents Ki values for inhibition of NPE Na,K-ATPase activity by digoxin, DMe, DMcP and DcB, fitted to a single site inhibition model.
  • the Ki values of the derivatives are all lower than that of digoxin. Since NPE cells contain about 70 % a2 and 30 % al, Na,K- ATPase activity and inhibition should reflect the properties of the isoform mixture. Indeed, the detailed inhibition curve for the most ⁇ 2 ⁇ 3- selective compound, DcB, was fitted better by a two site model, compared to fitting according to a one site model.
  • IOP measurements were made with a pneumatonometer (Model 30, Reichert technologies) either after raising IOP with 4-aminopyridine (4AP; 1 drop 40 mg/ml), or on basal IOP after addition of one drop of 1 mM solution of digoxin derivatives to the right eye (RE) and one drop of PBS to the left eye (LE) that served as control.
  • a pneumatonometer Model 30, Reichert technologies
  • FIGs. 1A-D present comparative plots of IOP as a function of time, showing the dose response of a2-inhibitor compounds, according to some embodiments of the present invention, in lowering IOP in live rabbits, wherein FIG. 1A shows the results obtained for DiB, FIG. IB shows the duration of the effect of DiB while 4AP is added every 2 hours so as to maintain the raised control IOP, FIG. 1C shows the results obtained for DMcP, and FIG. ID shows the results obtained for DcB.
  • DiB having Ki ⁇ 1 ⁇ 1: ⁇ 2 ⁇ 3 ratio of 16-fold
  • the duration of the DiB effect was shows that 1 mM DiB is effective for about 8 hours before the IOP begins to rise back up to control levels, which is a significantly long effect.
  • DMcP having a Ki ⁇ 1 ⁇ 1: ⁇ 2 ⁇ 3 ratio of 22-fold
  • DcB having a Ki ⁇ 1 ⁇ 1: ⁇ 2 ⁇ 3 ratio of 33 -fold
  • IOP reduction as DiB FIG. 1A
  • low concentrations of 0.01 mM of DMcP or DcB are sufficient to prevent the 4AP-induced rise in IOP.
  • FIGs. 2A-D present comparative plots of IOP as a function of time, demonstrating the capacity of the a2-inhibitor compounds, according to some embodiments of the present invention, to lower IOP below basal levels compared to a buffer control when administered topically to one eye of a rabbit, while the other eye received PBS as a control, wherein FIG. 2A shows the lack of effect of digoxin, FIG. 2B shows the lack of effect of DiB (a non-cyclic moiety inhibitor), FIG. 2C shows the notable of effect of DMcP, and FIG. 2D shows the notable of effect of DcB.
  • FIG. 2A shows the lack of effect of digoxin
  • FIG. 2B shows the lack of effect of DiB (a non-cyclic moiety inhibitor)
  • FIG. 2C shows the notable of effect of DMcP
  • FIG. 2D shows the notable of effect of DcB.
  • both DMcP and DcB significantly reduced the basal IOP by 20-25 % (about 4mm Hg for rabbit with a basal IOP of 17mm Hg) over the test period of 4-5 hours.
  • a higher concentration of DcB (2 niM) reduces the IOP similarly to 1 mM DcB.
  • a2-inhibitor compounds can be used effectively to treat medical conditions where there in a need to lower IOP below what is co considered to be a normal pressure, such as cases of low-tension glaucoma and normal-tension glaucoma.
  • Groups of 5 rabbits were treated once a day for 5 days with Latanoprost and on the sixth day with DcB, Latanoprost, or DcB/Latanoprost combination. IOP measurements were made for the next 12 hours or over 24 hours for the group treated with both DcB and Latanoprost.
  • FIGs. 3A-C present comparative plots of IOP as a function of time, demonstrating the effect of a2-inhibitor compounds, according to some embodiments of the present invention, to potentiate the drug Latanoprost in lowering IOP below basal levels, wherein FIG. 3A shows the effect of DcB alone, FIG. 3B shows the effect of Latanoprost alone, and FIG. 3C shows the effect of co-administering DcB with Latanoprost.
  • an a2-inhibitor compound according to some embodiments of the present invention was about 25 % more effective than Latanoprost in reducing the basal (normal) levels of IOP.
  • Latanoprost was applied for 5 days prior to the day of measurement, due to reports that this pre-treatment produces optimal effects on IOP in humans, although in these experiments with rabbits it seems that this was unnecessary since the effects of Latanoprost were observed acutely and had dissipated completely after 24 hours.
  • the animals which received the treatment presented in FIGs. 3A-C were used for corneal thickness measurements after topically administering drops of DcB or Latanoprost to 4 rabbits once a day for 5 additional days, totaling 6 days of treatment with DcB or Latanoprost, and the results are presented in Table 4 below.

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Abstract

La présente invention concerne des inhibiteurs de Na,K-ATPase sélectifs d'alpha2, caractérisés en ce qu'ils présentent une fraction cyclique fixée à une digoxine ou un dérivé de digoxine, ainsi que des utilisations de ceux-ci pour abaisser la pression intraoculaire et dans le traitement du glaucome et d'affections cardiaque.
PCT/IL2015/050741 2015-07-19 2015-07-19 Inhibiteurs sélectifs d'isoformes contenant alpha2 de la na,k-atpase et leur utilisation pour réduire la pression intra-oculaire WO2017013637A1 (fr)

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PCT/IL2015/050741 WO2017013637A1 (fr) 2015-07-19 2015-07-19 Inhibiteurs sélectifs d'isoformes contenant alpha2 de la na,k-atpase et leur utilisation pour réduire la pression intra-oculaire
US15/745,441 US10668094B2 (en) 2015-07-19 2016-07-19 Selective inhibitors of Alpha2-containing isoforms of Na,K-ATPase and use thereof for reduction of intraocular pressure
EP20209796.0A EP3907231A1 (fr) 2015-07-19 2016-07-19 Inhibiteurs séléctifs de na,k-atpase contenant des isoformes alpha2 et leur utilisation pour réduire la pression intraoculaire
PCT/IL2016/050785 WO2017013648A1 (fr) 2015-07-19 2016-07-19 Inhibiteurs sélectifs des isoformes contenant alpha2 de la na,k-atpase et leur utilisation pour réduire la pression intraoculaire
EP16747628.2A EP3325494B1 (fr) 2015-07-19 2016-07-19 Inhibiteurs séléctifs de na,k-atpase contenant des isoformes alpha2 et leur utilisation pour réduire la pression intraoculaire
US16/847,737 US11077128B2 (en) 2015-07-19 2020-04-14 Selective inhibitors of Alpha2-containing isoforms of Na,K-ATPase and use thereof for reduction of intraocular pressure

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US9938316B2 (en) 2013-08-29 2018-04-10 Yeda Research And Development Co. Ltd. Selective inhibitors of α2 isoform of Na,K-ATPase and use thereof for reduction of intra-ocular pressure and as cardiotonic agents
US10668094B2 (en) 2015-07-19 2020-06-02 Yeda Research And Development Co. Ltd. Selective inhibitors of Alpha2-containing isoforms of Na,K-ATPase and use thereof for reduction of intraocular pressure

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9938316B2 (en) 2013-08-29 2018-04-10 Yeda Research And Development Co. Ltd. Selective inhibitors of α2 isoform of Na,K-ATPase and use thereof for reduction of intra-ocular pressure and as cardiotonic agents
US10668094B2 (en) 2015-07-19 2020-06-02 Yeda Research And Development Co. Ltd. Selective inhibitors of Alpha2-containing isoforms of Na,K-ATPase and use thereof for reduction of intraocular pressure
US11077128B2 (en) 2015-07-19 2021-08-03 Yeda Research And Development Co. Ltd. Selective inhibitors of Alpha2-containing isoforms of Na,K-ATPase and use thereof for reduction of intraocular pressure

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