Selective inhibitors of α2 isoform of Na,K-ATPase and use thereof for reduction of intra-ocular pressure and as cardiotonic agents

Abstract

The present invention relates to digoxin and digitoxin derivatives that are selective inhibitors of the 2 isoform of Na,K-ATPase, and that reduce intra-ocular pressure. The invention further relates to uses of these derivatives for treating disorders associated with elevated intraocular pressure, such as glaucomas, and/or as cardiotonic agents.

Claims

1. A compound represented by the structure of general formula (I): ##STR00006## R is selected from the group consisting of CH.sub.2C(O)NH.sub.2 (compound 1), CH.sub.3 (compound 2), (CH.sub.2).sub.2C(O)NH.sub.2 (compound 3), NHC(O)NH.sub.2 (compound 4), OH (compound 6), CH(CH.sub.3)CONH.sub.2 (compound 8), CH(CH.sub.2OH)COOH (compound 9), CH(CH.sub.2OH)CONH.sub.2 (compound 10), CH.sub.2CH.sub.3 (compound 12), (CH.sub.2).sub.2CH.sub.3 (compound 13), CH.sub.2CH(CH.sub.3).sub.2 (compound 14), CH.sub.2CF.sub.3 (compound 15), CH.sub.2C(O)NHOH (compound 17), NHCSNH.sub.2 (compound 18), CH.sub.2CH.sub.2F (compound 19), and X is OH, including salts, hydrates, solvates, polymorphs, geometrical isomers, optical isomers, enantiomers, diastereomers, and mixtures thereof.

2. The compound of claim 1, being selective for 2 isoform of Na,K-ATPase over other isoforms of Na,K-ATPase.

3. The compound of claim 2, being selective for the 21, 22 and/or 23 isoform of Na,K-ATPase over the 11 isoform of Na,K-ATPase.

4. A pharmaceutical composition comprising the compound of claim 1, and a pharmaceutically acceptable carrier or excipient.

5. The composition of claim 4, being an ophthalmic composition suitable for topical application to the eye in the form of an eye-drop solution, an ointment, a suspension, a gel or a cream.

6. The composition of claim 4, for treating a condition selected from the group consisting of ocular hypertension, glaucoma and heart failure.

7. A method of treating a condition, comprising the step of administering to a subject in need of such a treatment an effective amount of the compound of claim 1 wherein said condition is selected from the group consisting of ocular hypertension, glaucoma and heart failure.

8. The method of claim 7, wherein the compound is selective for 2 isoform of Na,K-ATPase over other isoforms of Na,K-ATPase.

9. The method of claim 8, wherein the compound is selective for the 21, 22 and/or 23 isoform of Na,K-ATPase over the 11 isoform of Na,K-ATPase.

10. The method of claim 7, wherein the compound is administered in a pharmaceutical composition comprising said compound, and a pharmaceutically acceptable carrier or excipient.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 Technical features of the ocular hypertension experiments. New Zealand white rabbits were used for IOP measurements, IOP (mm Hg) of rabbits was measured using a calibrated Pneumatonometer (Model 30, Reichert technologies).

(2) FIGS. 2A-B FIG. 2A: Synthesis of perhydro-1,4-oxazepine derivatives of digoxin. FIG. 2B: Reverse phase HPLC purification of DGlyN, a representative compound of the present invention.

(3) FIGS. 3A-B Inhibition of Na,K-ATPase activity of purified isoform complexes by digoxin derivatives. Representative experiments for inhibition of Na,K-ATPase activity by DGlyN (FIG. 3A) DMe (FIG. 3B). 1/1 isoform ; 2/1 isoform .square-solid.; 3/1 isoform .Math.. Lines are the fitted curves for a one-site inhibition model (see Example 4: Experimental Section).

(4) FIG. 4 4-aminopyridine (4AP)-induced transient ocular hypertension in rabbits. Control .square-solid.; 4AP, 1 drop (40 mg/ml) ; 4AP, 2 drops (40 mg/ml) .

(5) FIGS. 5A-E Effects of DMe (FIG. 5A), DGlyN (FIG. 5B), digoxin (FIG. 5C), ouabain (FIG. 5D) and digoxigenin (FIG. 5E) on 4AP-induced ocular hypertension. Cardiac glycosides (CG's) (1 drop, 25 l) at the indicated concentrations were added to both eyes 30 min before addition of 4AP (40 mg/ml, 1 drop, 25 l). During this pre-incubation period there was little or no change in IOP. IOP was measured at the indicated times after addition of 4AP. In each experiment one rabbit was used for each concentration. The values are the mean of the IOP in both eyes.

(6) FIG. 6 Comparison of the change in IOP with different CG's. FIG. 6 depicts the change in IOP (in mmHg) after 1.5 hours administration of 4AP and 0.1 mM CG. FIG. 6 represents an average of 3 different experiments with the SEM.

(7) FIGS. 7A-C Time-course of effects of digoxin derivatives on IOP. In this experiment the IOP was elevated for 7-8 hours, by application of 4AP every 2 hours either in the absence of cardiac glycosides or after application of one drop of the cardiac glycoside (FIG. 7A and FIG. 7B), or one drop of cardiac glycoside was applied one hour after the first application of 4AP (FIG. 7C). All the other conditions and measurements are as described in FIGS. 5A-E.

(8) FIGS. 8A-C Effect of DMe, DGlyN and digoxigenin on IB-MECA-induced ocular hypertension. FIG. 8A. DMe at the indicated concentrations was added 30 minutes prior to IB-MECA. FIG. 8B. Digoxigenin, DGlyN, or DMe, 1 mM were added 1.5 hours after the first addition of IB-MECA. FIG. 8C. Digoxigenin or DGlyN, 3 mM were added 1.5 hours after the first addition of IB-MECA. 1 M IB-MECA was added at time zero and every 2 hours thereafter (arrows).

(9) FIG. 9 Dissociation of digoxigenin, digoxin, DGlyN and DMe from the 2 isoform. FIG. 9 depicts normalized data from representative experiments using the four different cardiac glycosides, obtained as described in the Methods.

(10) FIGS. 10A-D Model of Digoxin bound to the Na,K-ATPase. FIG. 10A. The model depicts the porcine 11 complex (4HYT) with bound digoxin (3B0W). FIG. 10B. Detail of residues in proximity to bound digoxin (numbering is for porcine 1 and 1). FIG. 10C and FIG. 10D depict 1 versus 3 showing 1Gln84 and Val89.

(11) FIG. 11 shows the expression of 23 and 22 as well as 21 and 11 isoform complexes in Coomassie blue stained gels. The proteins were denatured prior to treatment with PNGase.

(12) FIG. 12 K-activation of Na,K-ATPase activity of 11, 21, 22, 23 isoform complexes. FIG. 12 shows representative curves.

(13) FIG. 13 Inhibition of the Na,K-ATPase activity of 11, 21, 22, 23 by digoxin. FIG. 13 shows representative curves.

(14) FIG. 14 Inhibition of the Na,K-ATPase activity of 11, 21, 22, 23 by DIB. FIG. 14 shows representative curves.

(15) FIG. 15 Inhibition of acute ocular hypertension by DIB. FIG. 15 represents the average effects of different concentrations of DIB in four experiments.

DETAILED DESCRIPTION OF THE INVENTION

(16) Unless otherwise specified, a or an means one or more.

(17) The present invention relates to digoxin and digitoxin derivatives that are selective inhibitors of the 2 isoform of Na,K-ATPase. The compounds of the invention effectively reduce intra-ocular pressure, and are useful in the treatment of disorders associated with elevated intraocular pressure, such as glaucomas, and/or as cardiotonic agents.

(18) The term selective inhibitor of the 2 isoform of Na,K-ATPase means that the compound inhibits the 2 isoform of Na,K-ATPase to a greater degree than the other isoforms, e.g., the 1. In some embodiments, the compounds described herein are selective for the 21, 22 and/or 23 isoform of Na,K-ATPase over the 11 isoform of Na,K-ATPase. In some embodiments, the selectivity of the compound for the 2 isoform of Na,K-ATPase (e.g., 21, 22 and/or 23 isoform) is up to about 20 fold over other isoforms, e.g., up to 16 fold, 8 fold, 5 fold or 2 fold greater inhibition of the 1 isoform over other isoforms of this enzyme.

(19) Compounds

(20) According to one aspect, the present invention relates to a compound represented by the structure of general formula (I):

(21) ##STR00003## wherein R is selected from the group consisting of OH, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 haloalkyl, (CR.sup.bR.sup.c).sub.nSi(R.sup.a).sub.3, (CR.sup.bR.sup.c).sub.nC(Y)NR.sup.1R.sup.2, (CR.sup.bR.sup.c).sub.nC(Y)NHOH, (CR.sup.dR.sup.e).sub.nC(Y)COOR.sup.3; and NHC(Y)NR.sup.1R.sup.2; Y is O or S; X is H or OH; R.sup.1, R.sup.2 and R.sup.3 are each independently H or a C.sub.1-C.sub.4 alkyl; R.sup.a is a C.sub.1-C.sub.4 alkyl; R.sup.b, R.sup.c and R.sup.d are each independently selected from H, a C.sub.1-C.sub.4 alkyl and a C.sub.1-C.sub.4 hydroxy alkyl; R.sup.e is selected from a C.sub.1-C.sub.4 alkyl and a C.sub.1-C.sub.4 hydroxyalkyl; and n is 0, 1 or 2; including salts, hydrates, solvates, polymorphs, geometrical isomers, optical isomers, enantiomers, diastereomers, and mixtures thereof.

(22) According to another aspect, the present invention relates to a compound represented by the structure of general formula (IA):

(23) ##STR00004## wherein R is selected from the group consisting of OH, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 haloalkyl, (CR.sup.bR.sup.c).sub.nSi(R.sup.a).sub.3, (CR.sup.bR.sup.c).sub.nC(Y)NR.sup.1R.sup.2, (CR.sup.bR.sup.c).sub.nC(Y)NHOH, (CR.sup.dR.sup.e).sub.nC(Y)COOR.sup.3; NHC(Y)NR.sup.1R.sup.2; and (CR.sup.bR.sup.c).sub.nNH.sub.2; Y is O or S; X is H or OH; R.sup.1, R.sup.2 and R.sup.3 are each independently H or a C.sub.1-C.sub.4 alkyl; R.sup.a is a C.sub.1-C.sub.4 alkyl; R.sup.b, R.sup.c, R.sup.d and R.sup.e are each independently selected from H, a C.sub.1-C.sub.4 alkyl and a C.sub.1-C.sub.4 hydroxy alkyl; and n is 0, 1 or 2; including salts, hydrates, solvates, polymorphs, geometrical isomers, optical isomers, enantiomers, diastereomers, and mixtures thereof.

(24) In some currently preferred embodiment, the compound is selected from the group consisting of a digoxin derivative (X is OH) or a digitoxin derivative (X is H). Several preferred compounds of formula (I) or (IA) are exemplified below, with each possibility representing a separate embodiment of the present invention.

(25) A compound of formula (1), in which X is OH and R is derived from glycinamide (RCH.sub.2C(O)NH.sub.2), abbreviated herein DGlyN.

(26) A compound of formula (2), in which X is OH and R is CH.sub.3, abbreviated herein DMe.

(27) A compound of formula (3), in which X is OH and R is derived from propionamide (RCH.sub.2CH.sub.2C(O)NH.sub.2), abbreviated herein DPrN.

(28) A compound of formula (4), in which X is OH and R is derived from semicarbazide (RNHC(O)NH.sub.2), abbreviated herein DSCar.

(29) A compound of formula (5), in which X is OH and R is derived from glycine (RCH.sub.2C(O)OH), abbreviated herein DGly.

(30) A compound of formula (6), in which X and R are each is OH, abbreviated herein DOH.

(31) A compound of formula (7), in which X is OH and R is derived from glycine methyl ester (RCH.sub.2C(O)OCH.sub.3), abbreviated herein DGlyMe.

(32) A compound of formula (8), in which X is OH and R is derived from alanineamide (RCH(CH.sub.3)CONH.sub.2), abbreviated herein DAlaN.

(33) A compound of formula (9), in which X is OH and R is derived from serine (R CH(CH.sub.2OH)COOH), abbreviated herein DSer.

(34) A compound of formula (10), in which X is OH and R is derived from serinamide (RCH(CH.sub.2OH)CONH.sub.2), abbreviated herein DSerN.

(35) A compound of formula (11), in which X is OH and R is derived from ethylene diamine (RCH.sub.2CH.sub.2NH.sub.2), abbreviated herein DEtDA.

(36) A compound of formula (12), in which X is OH and R is CH.sub.2CH.sub.3 abbreviated herein DEt.

(37) A compound of formula (13), in which X is OH and R is (CH.sub.2).sub.2CH.sub.3 abbreviated herein DPr or DP.

(38) A compound of formula (14), in which X is OH and R is CH.sub.2CH(CH.sub.3).sub.2 abbreviated herein DiBu.

(39) A compound of formula (15), in which X is OH and R is derived from 2,2,2-trifluoroethyl (RCH.sub.2CF.sub.3), abbreviated herein DMeCF.sub.3.

(40) A compound of formula (17) wherein X is OH and R is CH.sub.2C(O)NHOH, abbreviated herein DGlyNHOH.

(41) A compound of formula (18) wherein X is OH and R is derived from semithiocarbazide (RNHCSNH.sub.2), abbreviated herein DSSCar.

(42) A compound of formula (19) wherein X is OH and R is CH.sub.2CH.sub.2F, abbreviated herein DCH.sub.2CH.sub.2F.

(43) A compound of formula (21) wherein X is OH and R is CH(CH.sub.3).sub.2, abbreviated herein DiPro or DIP.

(44) A compound of formula (22) wherein X is OH and R is C(CH.sub.3).sub.3, abbreviated herein DtBu.

(45) A compound of formula (23) wherein X is OH and R is methyl (trimethylsilyl) (CH.sub.2Si(CH.sub.3).sub.3), abbreviated herein DTMS.

(46) These and other representative compounds are shown hereinbelow in Table 1.

(47) The term C.sub.1-C.sub.6 alkyl group refers to any saturated aliphatic hydrocarbon, including straight-chain and branched-chain groups containing between 1 and 6 carbon atoms. The term C.sub.1-C.sub.4 alkyl group refers to any saturated aliphatic hydrocarbon, including straight-chain and branched-chain groups containing between 1 and 4 carbon atoms. Non-limiting examples of alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, t-butyl, n-pentyl, 2-pentyl, 3-pentyl, neopentyl, 1-hexyl, 2-hexyl and 3-hexyl. The alkyl group may be substituted or unsubstituted.

(48) The term halogen refers to fluoro, chloro, bromo or iodo.

(49) All stereoisomers, optical and geometrical isomers of the compounds of the instant 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, l,L or d,l, D,L. Compounds comprising amino acid residues (e.g., glycine or glycinamide) include residues of D-amino acids, L-amino acids, or racemic derivatives of amino acids.

(50) One or more of the compounds of the invention, may be present as a salt. The term salt encompasses both basic and acid addition salts, and include salts formed with organic and inorganic anions and cations. The term 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. Furthermore, 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.

(51) 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. Non-limiting examples of suitable solvates include ethanolates, methanolates and the like. Hydrate is a solvate wherein the solvent molecule is water.

(52) The present invention also includes polymorphs of the compounds of the present invention and salts thereof. The term 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.

(53) Pharmaceutical Compositions and Therapeutic Uses

(54) In some embodiments, the present invention provides a method for treating disorders associated with elevated intraocular pressure, and in particular for treating glaucoma, by administering an effective amount of a pharmaceutical compositions comprising a compound of formula I and/or IA as the active ingredient (e.g., compounds 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 21, 22 or 23) and a pharmaceutically acceptable carrier.

(55) In other embodiments, the present invention provides a method for reducing elevated intraocular pressure, by administering an effective amount of a pharmaceutical composition comprising a compound of formula I and/or IA as the active ingredient (e.g., compounds 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 21, 22 or 23) and a pharmaceutically acceptable carrier.

(56) Preferably, the pharmaceutical compositions of the invention is an ophthalmic composition which is administered topically onto the eye of a patient for facilitating effective intraocular levels of the drug and for preventing unnecessary drug level in other organs. Such a non-systemic, site-specific administration reduces the side effects associated with the drugs. However, oral or otherwise systemic administration in a dosage effective for reducing the intraocular pressure is also possible. For example, the composition may be administered by a dermal patch for extended release.

(57) When administration is topical, the pharmaceutical compositions containing the digoxin derivative of formula I or IA may be formulated in various therapeutic forms suitable for topical delivery, including solutions, suspensions, emulsions and gels. The carrier in these formulations may be any pharmaceutical acceptable carrier such as saline, buffered saline, carbopol gel, mineral oil and the like. The formulations can be prepared in accordance with known procedures for the preparation of ophthalmic formulations. Preferably, the concentration of the digoxin derivative in the pharmaceutical compositions is in the range of about 1 to about 5,000 g/ml, 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 digoxin derivative, more preferably from about 2 to about 20 g of digoxin derivative.

(58) The topical pharmaceutical compositions may be in the form of eye-drops to be applied by instillation into the eye or may be in the form of a viscous ointment, gel or cream to be applied by an ointment onto the ocular surface and may contain control release means for facilitating sustained release over a prolonged period of time.

(59) The compositions may further include non-toxic auxiliary pharmaceutically acceptable substances such as stabilizers, preservatives, chelating agents, viscosity modifying agents, buffering agents and/or pH adjusting agents. Additionally, the compositions may contain other ophthalmic active agents such as antibacterial agents, comfort enhancers, antioxidants, intra-ocular pressure (IOP)-reducing drugs and the like.

(60) In accordance with other embodiments, the digoxin/digitoxin derivative may be loaded into a drug-delivery device to be inserted or implanted into the eye of the patient for allowing releasing of the drug 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.

(61) Another possible application of an 2-selective cardiac glycoside is as an effective cardiotonic drug, with reduced cardiotoxicity, compared to known drugs such as digoxin. Thus, in other embodiments, the present invention provides cardiotonic compositions comprising a compound of formula I and/or IA as the active ingredient (e.g., compounds 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 21, 22 or 23) and a pharmaceutically acceptable carrier. In accordance with this embodiment, the compounds according to the invention may therefore be formulated for oral, buccal, topical, parenteral or rectal administration.

(62) For oral administration, the composition may be provided, for example, in the form of tablets, capsules, powders, solutions, syrups or suspensions prepared by conventional methods using acceptable diluents. For buccal administration, the composition may be provided in the form of conventionally formulated tablets or sachets.

(63) The compounds according to the invention may be formulated for parenteral administration by bolus injection or continuous infusion. Formulations for injection may be provided in the form of ampoules containing single doses or they may be provided in multiple dose containers with added preservative. The composition may be in the form of suspensions, solutions and the like.

(64) Alternatively, the active ingredient may be provided in powder form to be reconstituted before use with a suitable carrier. For topical use, the compounds according to the invention may be formulated in the conventional manner as ointments, creams, gels, lotions, powders or sprays.

(65) The principles of the invention, using an albumin conjugate isoflavone derivative bound to a bioactive moiety such as an imaging agent or a therapeutic agent for selective delivery to cells susceptible to isoflavone according to the present invention, may be better understood with reference to the following non-limiting examples.

EXAMPLES

Example 1: Synthesis and Testing of Perhydro-1,4-Oxazepine Derivatives of Digoxin

(66) Perhydro-1,4-oxazepine derivatives were prepared according to the method described in (15). FIG. 2A shows the synthetic route involving (a) selective periodate oxidation of the third digitoxose moiety and (b) reductive amination of the dialdehyde using the free amine (RNH.sub.2) plus NaCNBH.sub.3, for the case of glycinamide. Compounds were purified by HPLC, as seen for the representative example of the DGlyN derivative in FIG. 2B. Progress of both stages of the reactions as well as purification of compounds was monitored routinely by thin layer chromatography and mass spectrometry measurements. .sup.1H and 13C NMR spectra and full assignments were obtained for several derivatives and will be published elsewhere. Structures of representative compounds of the invention are shown in Table 1. For verification of the structures, masses of the purified compounds were then determined. The table shows the structures of the different amine substituents, names, and theoretical and experimentally found masses of fifteen digoxin derivatives, and also the glycine derivative of bis-digitoxose digoxigenin. Mass spectra were obtained in a Micromass ZQ 4000 spectrometer, with Electro Spray Ionization.

(67) TABLE-US-00001 TABLE 1 Structures, names and masses of perhydro-1,4-oxazepine derivatives of digoxin Theoretical Mass Derivative Name Exact Found R = Chem. Name Abbreviation Mass (M + Na.sup.+) CH.sub.2CONH.sub.2 (1) glycinamide DGlyN 820.47 843.42 CH.sub.3 (2) methylamine DMe 777.47 800.57 CH.sub.2CH.sub.2CONH.sub.2 (3) propionamide DPrN 834.49 857.30 NHCONH.sub.2 (4) semicarbazide DSCar 821.47 844.37 CH.sub.2COOH (5) glycine DGly 821.46 844.44 OH (6) hydroxylamine DOH 779.45 802.47 CH.sub.2COOCH.sub.3 (7) glycine methyl ester DGlMe 835.47 858.51 CH(CH.sub.3)CONH.sub.2 (8) alaninamide DAlaN 834.49 857.56 CH(CH.sub.2OH)COOH (9) serine DSer 851.47 874.61 CH(CH.sub.2OH)CONH.sub.2 (10) serinamide DSerN 850.48 873.59 CH.sub.2CH.sub.2NH.sub.2 (11) ethylenediamine DEtDA 806.49 829.48 CH.sub.2CH.sub.3 (12) ethylamine DEt 791.48 814.52 CH.sub.2CH.sub.2CH.sub.3 (13) propylamine DPr 805.50 828.27 CH.sub.2C(CH.sub.3).sub.2 (14) isobutylamine DiBu 819.51 842.41 CH.sub.2CF.sub.3 (15) 2,2,2-trifluoroethylamine DMeCF.sub.3 845.45 868.14 bis-CH.sub.2COOH (16) bis-glycine* DbisGly 691.39 714.40 CH.sub.2CONHOH (17) glycine hydroxamate DGlyNHOH 836.47 858.51 NHCSNH.sub.2 (18) semithiocarbazide DSSCar 821.47 844.37 CH.sub.2CH.sub.2F (19) 2-fluoroethylamine DCH.sub.2CH.sub.2F 809.47 832.46 CH(CH.sub.2).sub.3 (21) isopropylamine DiPro 805.50 828.53 C(CH.sub.3).sub.3 (22) t-butylamine DtBu 819.51 842.66 CH.sub.2Si(CH.sub.3).sub.3 (23) (Trimethylsilyl)methylamine DTMS 849.51 872.50 *bis-glycine refers to a glycine derivative of bis-digitoxose digoxigenin.

Example 2: Inhibition of Na,K-ATPase Activity

(68) FIGS. 3A-B show curves for inhibition of Na,K-ATPase activity of purified human isoforms (11, 21 and 31) of two derivatives, DGlyN (FIG. 3A) and DMe (FIG. 3B), with improved selectivity for 2 compared to digoxin itself. Table 2 provides information on the inhibitory effects of sixteen digoxin perhydro-1-4-oxazepine derivatives, in accordance with the present invention. The data in Table 2 show that the isoform selectivity ratios (Ki1/2) of several derivatives: DGlyN (7.450.46), DMe (6.470.71), DGly (5.10.54), DPrN (5.280.75) and DSCar (4.981.2) are significantly greater than that of digoxin (3.440.34). For these compounds, the Ki values for both 1 and 2 are lower than for digoxin, but the effect is greater for 2 compared to 1. Consequently, the ratio Ki 1/2 is higher for the compounds of the invention compared to digoxin. In all cases the Ki for 3 is closer to that for 1 than to 2. This feature is seen clearly for DMe and DGlyN as seen in FIGS. 3A-B, and is also applicable to the other compounds encompassed by Formula (I). Thus, it is primarily the Ki1/2 that is affected by the modification of the third digitoxose. The Ki values of several derivatives in Table 2 (e.g. DEt) are significantly lower than for digoxin itself but a differential effect between the isoforms was not observed, so that the selectivity ratio was not improved. The Ki values of several other derivatives in Table 2 (e.g. DEtDA) are significantly lower than for digoxin itself and some differential effect was observed. In other cases (e.g DOH and DSer the Ki values were higher than for digoxin and the selectivity for 2 was not improved. The result in Table 2 that the glycine derivative of bis-digitoxose digoxigenin (DbisGly) shows lower selectivity for 2 over 1 compared to the glycine derivative of the tri-digitoxose (DGly) shows that modification of the third digitoxose residues is optimal for this effect, consistent with a similar conclusion in (6). In summary, the strategy of modifying the third digitoxose moiety produced compounds with an improved ratio Ki1/2, reaching over twice the value of digoxin in the case of the most 2-selective derivatives, DGlyN and DMe.

(69) TABLE-US-00002 TABLE 2 Ki values for inhibition of Na,K-ATPase activity of isoforms 11 and 21 with selectivity ratios Ki S.E. Selectivity ratio p value, n Ki 1/2 p value CG 1 2 2 to 1 S.E. relative to digoxin Ouabain 97 4.3 90 14 1.08 0.17 Digoxigenin 139 17 130 13.5 1.07 0.17 Digoxin 189 11 55 4.4 0.0001, 12 3.44 0.34 DGlyN (1) 152 5.5 20.4 1 0.0001, 8 7.45 0.46 0.0001 DMe (2) 101 4.4 15.6 2.3 0.0001, 8 6.47 0.71 0.0001 DPrN (3) 249 37 47 7.8 0.006, 3 5.28 0.75 0.0254 DSCar (4) 102 23 20 3.6 0.014, 4 4.98 1.2 0.029 Dgly (5) 124 8.6 25 3.9 0.0003, 6 5.10 0.54 0.0167 DOH (6) 311 18.5 134 35 0.046, 3 2.32 0.62 DGlMe (7) 540 102 128 11 0.052, 3 4.22 0.88 DAlaN (8) 232 28 67 8.1 0.005, 3 3.46 0.59 DSer (9) 316 109 145 28.5 0.269, 3 2.18 0.86 DSerN (10) 242 15 144 3.5 0.033, 3 1.68 0.13 DEtDA (11) 69 10 16.7 2.1 0.003, 4 4.10 0.82 DEt (12) 53 3.4 18.5 4.9 0.0045, 5 2.88 0.78 DMeCF.sub.3 (15) 199 33 44 7 0.01, 3 4.50 1.0 DbisGly (16) 80 5.5 34.9 12 0.075, 3 2.29 0.80 Dbis* (20) 196 8.5 74 5 0.006, 3 2.65 0.81 *Dbis is digoxigenin bis digitoxide.

(70) The CG abbreviation corresponds to the following starting amine: DOH-hydroxylamine; DGly-glycine; DGlMe-glycine methyl ester; DGlyN-glycinamide; DAlaN-alaninamide; Dser-serine; DSerN-serinamide; DSCar-semicarbazide; DPrN-proprionamide; DEtDA-ethylene diamine; DMe-methylamine; DEt-ethylamine; DMeCF.sub.3-2,2,2-trifluoroethylamine; DbisGly-bis-digitoxoside glycine, p values were calculated by the t-test and denoted as *p<0.05, **P<0.01, ***p<0.001. n, number of independent experiments, p (2val) indicates the significance of differences between Ki21 and Ki11. p (v digoxin) indicates the significance of the difference of the selectivity ratio (Ki11/Ki21) compared to the (Ki11/Ki21) of digoxin.

Example 3: Reduction of Intra-Ocular Pressure by Topically Applied Digoxin and Perhydro-1,4-Oxazepine Derivatives

(71) Intra-ocular pressure in rabbits was measured using of a Reichert Model 30 Pneumatonometer after anesthetizing the cornea with local anesthetic. Two different pharmacological agents were used to induce acute elevation of IPO and determine whether topically applied glycosides of the present invention are able to counter such an effect. First, IOP elevation was induced acutely with 4-aminopyridine (4AP), which has been previously reported to acutely and transiently raise IOP in rabbits eyes by 4-8 mm Hg from a resting IOP of 22-24 mmHg (16). The mechanism of ocular hypertension induced by 4AP, which is a well-known blocker of a voltage-dependent K channel, was shown to involve release of norepinephrine from sympathetic nerves of the iris-ciliary body, leading to an increased rate of aqueous humour inflow. FIG. 4 confirms the basic effect of 4AP. One or two drops of 4AP in each eye raised the IOP by 3-6 mm Hg, and the effect was dissipated after 5 hours.

(72) Since the IOP reflects a balance of the inflow and outflow of aqueous humour, reduction of the increased 4AP-induced inflow of aqueous humour by cardiac glycosides should prevent the increase in IOP. Thus, the standard experimental design to test effects of cardiac glycosides involved topical application of the compounds (1 drop in each eye) 30 minutes prior to application of 4AP and measurement of IOP every 30 minutes over five hours. FIGS. 5A-PE show effects of DMe (FIG. 5A), DGlyN (FIG. 5B). digoxin (FIG. 5C), ouabain (FIG. 5D) and digoxigenin (FIG. 5E) on IOP using this protocol. Each experiment was done three times, but the figures depict a representative experiment using a different rabbit for each concentration of the cardiac glycoside. The plotted values represent the average pressures for both eyes although the values are similar in each eye measured separately. Digoxin (FIG. 5C) at a high concentration (1 mM) is able to prevent the 4AP-induced rise in IOP, while 0.25 mM digoxin is poorly effective. By comparison, both DMe and DGlyN (FIG. 5A and FIG. 5B respectively), the most 2-selective of the new perhydro-1,4-oxazepine derivatives, are effective at much lower concentrations (0.05-0.1 mM) than digoxin. Similarly the aglycone of digoxin, digoxigenin, effectively reduced IOP at lower concentrations than digoxin (FIG. 5E). Finally, ouabain, a widely used water-soluble cardiac glycoside, somewhat reduced IOP only at 1 mM while lower concentrations were poorly effective (FIG. 5D).

(73) FIG. 6 compares the relative effects of DGlyN, DMe, digoxigenin, digoxin and ouabain, on IOP all at 0.1 mM and one time point. The data represent the average effect SEM of the three separate experiments (i.e. 6 eyes in all) and confirm the order as DGlyNDMedigoxigenin>digoxin>ouabain.

(74) Over time, the cardiac glycosides that penetrate to the ciliary epithelium after a single application will be washed out of the eye into the general circulation and so the effect on IOP will dissipate. Although FIGS. 5A-E and 6 demonstrate that the cardiac glycosides reduce IOP with greater or less efficacy, by this experimental protocol the longevity of the effect cannot be evaluated due to the transient nature of the 4AP effect itself. Thus, additional experiments were conducted in which the effect of a single drop of digoxin, digoxigenin, DGlyN, DMe or ouabain was compared when 4AP was then added every two hours so as to maintain the IOP at the elevated level for 7-8 hours, even in the absence of the cardiac glycosides (FIGS. 7A-C). By this protocol, the reduction of IOP is indeed seen to be transient in FIGS. 7A-C. FIG. 7A shows a representative experiment with DMe that demonstrates a clear dependence of the wash-out time on concentration. IOP is held at the low level for 5.8, 4.5 and 2.5 hours for 2 mM, 0.5 mM and 0.2 mM respectively, before the IOP rises back to the elevated level with 4AP. Other experiments showed that at equal concentrations the wash-out time for DGlyN is slightly faster than for DMe. Notable differences in wash-out times were detected between DMe, DGlyN, digoxigenin, digoxin and ouabain when they were applied at equal concentrations (1 mM). As seen in FIG. 7B, DMe maintained IOP at the low level for about 5.5 hours, by comparison with 3.5 hours for DGlyN, about 2 hours for digoxigenin and only 1 hour for digoxin. Ouabain is washed out at a rate between that of digoxin and digoxigenin. The data is not shown for clarity. In short, the most 2-selective derivatives DMe (compound 2) and DGlyN (compound 1) produce the longest acting effect to reduce IOP, compared to either digoxin (a less 2-selective cardio-glycoside (CG)), or digoxigenin a non-selective CG.

(75) FIG. 7C shows that DGlyN and DMe rapidly reverse pre-established ocular hypertension. DGlyN or DMe (0.1 or 1 mM) were applied one hour after the first application of 4AP, which was added every two hours. Evidently, within 30 minutes DGlyN and DMe reversed the initial rise in IOP, indicating that the compounds permeate the cornea and bind to the pump sufficiently fast to have this effect. The normalized IOP was then maintained for at least 4 hours, as in FIG. 7B. Independently of the superior effects of 2-selective derivatives at low concentrations compared to digoxin itself, the rapid onset of the effects of DGlyN and DMe is suggestive of inhibition of 2, because 2 is known to bind cardiac glycosides much more rapidly than 1 (17).

(76) To verify that digoxin derivatives inhibits aqueous humour inflow directly and not act indirectly by, for example, interfering with the 4AP itself, topical IB-MECA was used. IB-MECA induces acute ocular hypertension by a different and well-defined mechanism. Namely, IB-MECA is a selective agonist of the A3-adenosine receptor, and rises aqueous humour inflow and IOP by activating Cl channels of the NPE cells (18, 19). A single drop of IB-MECA (1 M) induced a significant but transient increase in IOP, while repeated application each 2 hours maintained increased IOP over 4-5 hours (see FIG. 8A Control). FIG. 8A depicts the effects of DMe (0.1-1 mM) applied prior to the IB-MECA, and a similar result was obtained for DGlyN (not shown). FIG. 8B depicts effects of digoxigenin, DGlyN and DMe (1 mM) applied after the IB-MECA. The effects of digoxigenin, DGlyN and DMe at 1 mM were almost the same as seen with 4AP in FIG. 7C, obviously excluding the notion that CG's interfere with the action of 4AP itself. In addition, the duration of the effect was significantly greater for DMe and DGlyN than for digoxigenin. Furthermore, when the concentration of DGlyN and digoxigenin was raised to 3 mM, the difference in duration of the effect was greatly amplified compared to the experiment with 1 mM (see FIG. 8C).

(77) Corneal thickness was also measured after application of Digoxin (1 mM), DGlyN (0.5 mM), DMe (0.5 mM) and ouabain (1 mM) after 4AP. At least over a time scale of 4 hours, the corneal thickness, measured in microns, was not significantly affected. Thus, in this study, no change in corneal thickness was detected (Table 3), indicative of lack of local toxic effects. In addition neither redness nor local irritation were observed in the conjunctiva or cornea. Similar results were obtained with CG's applied after IB-MECA.

(78) TABLE-US-00003 TABLE 3 Pachymetry-Corneal thickness before and after application of cardiac glycosides Digoxin DGlyN DMe Ouabain 1 mM 0.5 mM 0.5 mM 1 mM Time RE LE RE LE RE LE RE LE 0 h 462 470 496 498 507 465 461 455 2 h 422 450 476 462 476 440 405 409 4 h 451 467 454 455 498 453 414 422 RE, right eye LE, left eye. Corneal thickness is given in microns. Each value represents the average of three independent measurements.

Example 4: Dissociation of Cardiac Glycosides from 21

(79) Since the principal isoform in NPE cells is 2 and dissociation from the pump is expected to affect the duration of the effects on IOP, the dissociation rates of different cardiac glycosides from the purified 21 isoform were compared. The dissociation rates of digoxin, digoxigenin, DGlyN and DMe were compared using the protocol described in (20). FIG. 9 depicts representative experiments for each of the four compounds and Table 4 shows the average rate-constants and half-times from three or four experiments. Evidently, the aglycone digoxigenin dissociates much faster than digoxin or any other glycone, and DMe and DGlyN also dissociate significantly slower than digoxin itself. The slow dissociation of DMe and DGlyN suggest their potential of a durable effect on IOP.

(80) TABLE-US-00004 TABLE 4 Rates of dissociation of cardiac glycosides from the 21 isoform complex. k SEM t.sub.1/2 SEM p CG min.sup.1, n min vs. digoxin Digoxigenin 0.645 0.189, 3 1.07 0.23 0.0006 Digoxin 0.015 0.001, 4 47.5 5.05 DGlyN 0.009 0.001, 3 78.8 8.73 0.02 DMe 0.0067 0.004, 3 103 6.12 0.001

Example 5: Digoxin Derivatives with Enhanced Selectivity for the 23 Complex

(81) Because 23 and not 21 is the major isoform complex in NPE cells, it was further investigated whether the 1, 2, or 3 isoform is an important factor.

(82) Molecular Modeling of the Digoxin Bound Na,K-ATPase

(83) A molecular insight to the interactions of the third digitoxose residue and isoform selectivity is illustrated in FIG. 10A. The figure presents a molecular model in which the digoxin molecule (co-ordinates 3B0W) was introduced onto the high affinity ouabain bound molecule (4HYT) (14) so that the lactone and steroid portions of ouabain and digoxin overlap closely, and then a minimal energy structure was obtained. The three digitoxose residues point outwards towards both and subunits. The enlarged image in FIG. 10B shows the digitoxose moiety in proximity (<3.5 A) to residues AspAspArgTrp887 in L7/8 of , the third digitoxose being close to both Trp887 and also Gln84. Support for this orientation towards the P subunit comes from an old observation that photoaffinity probes located in the third digitoxose of digitoxin label both and subunits, whereas photoaffinity probes located in other regions of cardiac glycoside molecules label only the subunit (21, 22). As suggested by the model, Trp887 is one of only four residues in extracellular loops that are different in 2 (and 3) from 1, (Gln119, Glu307, Val 881 and Trp887 in pig 1), and were inferred previously to be candidates for determining isoform selectivity (23) Close proximity to one of these four residues fits very well with the notion that interactions of the third digitoxose are important for isoform selectivity, and the present findings that derivatives of the third digitoxose can enhance isoform selectivity. In 2, Trp887 is replaced by a threonine.

(84) In addition, Gln84 in 1 is replaced by Val89 in 3 and Glu in 2 (FIGS. 10C and D). Since 23 is the major isoform complex in NPE cells it seemed that by introducing larger aliphatic groups into the perhydro-1,4-oxazepine digoxin derivatives than Me and Et which were already tried, it might be possible to produce digoxin derivatives with enhanced selectivity for 23 compared to 11 the major isoform complex in all other cells. Another possible advantage of more hydrophobic derivatives is that they could be expected to be more permeate through the cornea and thus, potentially, effective in IOP reduction at lower concentrations than DMe or DGlyN.

(85) Expression, Purification and Characterization of Human 2B3 and 22 Isoform Complexes.

(86) To develop compounds with higher selectivity to 23 human, 23 and 22 human isoform complexes were expressed as described in the Methods below. FIG. 11 shows a protein gel of the purified isoform complexes 21, 22, 23 and 11 before or after treatment with PNGase (in the denatured state), 1 has 3 glycosylation sites, 3 has two glycosylation sites while 2 has seven glycosylation sites. The mobility order on the gel, 3>2>1, fits well the predicted masses of the deglycosylated subunits of 1, 37172.6>2, 35422.2>3, 33678.9. The average Na,K-ATPase activities of the purified complexes were; 11, 19.32; 21, 18.21.6; 22, 7.71.8 and 23 9.70.26 moles/min/mg protein (n=4). Because cardiac glycosides and K ions are mutually antagonistic, an important point in relation to cardiac glycosides binding is the K0.5 K for activation of Na,K-ATPase. The term K.sub.0.5 K as used herein means the half maximal concentration of potassium ions (K) required for activation of Na,K-ATPase activity. FIG. 12 and Table 5 shows that the apparent affinities for K are significantly different between the isoform complexes, in the order 11<21<22<23. Inhibition by digoxin is more effective for the isoforms with higher K.sub.0.5 K (Ki 11>21>22>23), hence leading to a higher selectivity for 23 over 11 (FIG. 13). This increased selectivity is explained by a lower degree of K-digoxin antagonism and is expected for all the cardiac glycosides to the same extent as for digoxin.

(87) TABLE-US-00005 TABLE 5 K.sub.0.5 K for activation of Na,K-ATPase activity of 11, 21, 22 and 23 isoform complexes 11 21 22 23 K.sub.0.5K-mM K.sub.0.5K-mM K.sub.0.5 K-mM K.sub.0.5 K-mM SEM SEM SEM SEM 1.25 0.03 2.72 0.14 7.3 0.19 6.4 0.5 n = 4 n = 6 n = 6 n = 5
Synthesis and Isoform Selectivity of Aliphatic Derivatives of Digoxin.

(88) Additional set of perhydro-1,4-oxazepine digoxin derivatives with aliphatic substituents propyl (DP), iso-propyl (DIP), iso-butyl (DIB), tert-butyl (DtB) and trifluoroethyl (DMeCF3) has been synthesized and purified. Table 6 shows results of inhibition and selectivity of the most recent aliphatic derivatives for four Na,K-ATPase isoform complexes, in comparison to digoxin itself and DMe, FIG. 14 illustrates the effects of the most selective isobutyl derivative, DIB. No significant difference was observed in 11 versus 21 when compared to DMe. However, for 23, all of the aliphatic derivatives including DMe are significantly superior to digoxin and in the case of the isobutyl derivative the selectivity ratio reaches 16-fold. Generally, the curves for 22 lie between those for 23 and 21. As mentioned above, the difference in K.sub.0.5K between the 21-3 complexes should translate into a difference in Ki as seen for digoxin in the sense Ki 21>2223. However, the increase in selectivity for 23:11 is significantly higher than seen with digoxin (6.2-fold) for all these new derivatives and especially so for the isobutyl derivative, DIB (16-fold). This finding could imply that all the aliphatic derivatives, but especially DIB, interact more specifically with 23 than with 21. In any event the Ki of 5.8 nM for inhibition of 23 by DIB is lower than seen for any of the other aliphatic (or other) derivatives.

(89) The low Ki for inhibition of 23 implies that DIB could be a good inhibitor of IOP in rabbits. This was tested in experiments summarized in FIG. 15. Indeed DIB applied topically prior to 4AP effectively prevented the rise in IOP. The concentration required to fully prevent the rise in IOP (>30 M) was about 2-fold lower than required for the most effective derivative previously tested (DMe).

(90) TABLE-US-00006 TABLE 6 Selectivity of aliphatic digoxin perhydro-1,4-oxazepine derivatives for the 23 isoform complex Ki, nM SEM Selectivity CG 11 21 22 23 11/2/1 11/2/2 11/23 n Digoxin 268 13.8 58.7 5.4 58 1.9 42.8 3.0 4.5 4.6 6.2 7 DMe (2) 103 5.6 15.3 1.2 20.36 1.8 10.8 0.6 6.7 5.07 9.5 7 DEt (12) 137.9 12.6 23.2 0.9 16.4 1.6 14.4 1.27 5.9 8.3 9.5 4 DP (13) 87.7 7.9 18.3 1.68 10.5 1.8 9.8 1.1 4.8 8.3 8.8 5 DIP (21) 149 20.7 28.9 1.7 16.7 1.9 10.3 1.8 5.1 8.9 14.4 4 DIB (14) 92 8.9 20.6 1.4 10 0.8 5.8 0.6 4.4 9 16 5 DtB (22) 135 12.1 21.6 5.6 18.4 1.1 16.3 0.28 6.2 7.3 8.2 4 DTMS 108 31.5 15 7.8 3.4 7.2 13.8 2 (23) DMeCF.sub.3 119 15.0 28.6 0.9 18.1 1.9 12.4 1.5 4.1 6.5 9.6 3 (15)

(91) In conclusion, it has now been demonstrated that modification of the third digitoxose residue of digoxin can produce derivatives with increased selectivity for 2 over 1. Compared to digoxin (Ki1/2 3.44-fold), the selectivity ratio was significantly increased in the order DGlyN>DMe>DGlyDPrNDSCar, reaching a maximal value of Ki1/2=7.45 for DGlyN (Table 2).

(92) Furthermore the selectivity ratio, Ki23/11, 6.2 for digoxin itself, was significantly enhanced for all the more aliphatic derivatives DP, DIP, DIB, DtB and DMeCF.sub.3, reaching to c.16-fold for DIB.

(93) Considering the structures of the substituents in the perhydro-1-4-oxazepine ring (Tables 1, 2 and 6), it seems that the increased 2:1 selectivity (especially 2/3) is achieved with small R-groups having H-bonding potential (e.g., glycine, glycinamide, proprionamide, semicarbazide, semithiocarbazide), or small hydrophobic groups (e.g., Me, Et, Pr, iPr and t-Bu), while larger substituents (alanine, alaninamide, serine, serinamide improve selectivity to a lower extent, although these compounds may also be therapeutically useful. Important features of isoform selectivity are (a) 2-selectivity may be restricted to digitalis glycosides with -digitoxose residues since, for example, ouabain, an -rhamnoside, is slightly selective for 1 over 2 and (b) the third digitoxose residue is optimal as concluded above and also in (6).

(94) While the structures of the ouabain-bound conformations of renal Na,K-ATPase (12-14) are consistent, in general, with the observed lack of isoform selectivity of aglycones, because ouabain itself is only slightly selective for 1 over 2 (Table 2), these structures cannot explain in detail either the moderate selectivity of digoxin for 2 or increased selectivity for 1 of perhydro-1,4-oxazepine derivatives. Without wishing to be bound by any particular mechanism or theory, it is hypothesized that the relatively high selectivity of the perhydro-1,4-oxazepine derivatives of the invention (e.g., DGlyN) for 1 over 1, indicates a differential interaction with the isoform-specific residues in the exterior loops of 1 and 1. The very large difference of dissociation rates between aglycones and glycones emphasize the role of the sugars in binding to 1. Specific interactions with 1 of the modified digitoxose derivatives of DGlyN and DMe moieties are also indicated directly by the slower dissociation rates compared to digoxin (FIG. 9 and Table 4). Similarly increased selectivity of the more aliphatic derivatives in Table 6 such as DIB for the 23 complex over 11 may indicate a more specific interactions with Val89. The present findings confirm and validate the concept that modification of the third digitoxose residues can increase selectivity for the 2 isoform.

(95) In conclusion, the 2-selective digoxin derivatives described herein reduce intraocular pressure, and thus have the potential as novel drugs for control of IOP and prevention of glaucoma. When evaluated by the dose and especially duration of effects, the most 2-selective compounds DMe and DGlyN are significantly more effective than either the moderately 2-selective digoxin or non-selective digoxigenin. Furthermore when 23 selectivity is taken into account with the compounds such as DIB, superior effectiveness is observed. One important conclusion is that 23 indeed plays a major role in production of the aqueous humour, as could be predicted from its prominent expression in NPE cells.

(96) The new perhydro-1,4-oxazepine derivatives described herein may also have a favorable safety profile, making them suitable as drug candidates. Local toxicity of 23-selective cardiac glycosides, namely swelling of the cornea and lens should be minimal because corneal endothelium express 1 and a minor amount of 3 but no 2, and lens epithelium express only 1. Also, systemic cardiotoxic effects should be minimal.

(97) Lastly, perhydro-1,4-oxazepine derivatives of the more hydrophobic digitoxin may be even more effective than digoxin derivatives in reducing ocular hypertension, and/or as cardiotonic agents.

Example 5: Effects on Intra-Ocular Pressure in Rats

(98) To evaluate whether the compounds of the present invention are able to control IOP in an animal model of chronic ocular hypertension, and to assess their local and systemic toxicity, ocular hypertension is being induced in rats, for example by impeding aqueous humour outflow using microbeads (24). Digoxin derivatives are added daily and IOP changes, signs of inflammation, corneal edema or lens clarity are followed. For systemic toxicity the concentration of the digoxin derivatives in the blood is measured by a radioimmunoassay.

Example 6: Experimental Section

(99) Materials

(100) Escherichia (E.) coli XL-1 blue strain was used for propagation and preparation of plasmid constructs. Yeast Lytic Enzyme from ICN Biomedicals Inc (cat. 152270) was used for transformation of P. pastoris protease deficient strain SMD1165 (his4, prb1). DDM (cat. D310) and C12E8 (25% w/w, cat no. 0330) were purchased from Anatrace. Synthetic SOPS (sodium salt)) was obtained from Avanti Polar Lipids, and stored as a chloroform solution. BD Talon metal affinity resin (cat. 635503) was obtained from Clontech. Cholesterol, ouabain (O3125, digoxin (D6003), 4-aminopyridine, (A78403) and IB-MECA (I146)) were obtained from Sigma. Methanol HPLC grade was purchased from Baker. All the organic solvents and amines were of highest purity analytical grade.

(101) Preparation of h1h1, h1h2, h1h3, h2h1, h2h2, h2h3, h3h1 Constructs

(102) Human 1, 2 and 3 were cloned into the pHIL-D2 expression vector containing the human 1 or 2. pHIL-D2 expression vectors containing porcine (p) 1, human (h) 1 or human 2 with Hisx10 tagged porcine 1 were previously generated (7, 9). Human 1 (Accession: P05026), human 2 (Accession: P14415) and human 3 (Accession: 54709) cDNAs in pSD5 vector were a gift from K. Geering Univ. Lausanne Switzerland. The open reading frames and flanking regions of h1, h2 and h3 (in pSD5) were amplified separately by polymerase chain reaction (PCR) using synthetic primers containing BglII and SalI cleavage sites. Each one of the amplified fragments were digested with BglII and SalI and ligated to BglII and SalI treated plasmid pHIL-D2-(p1/His10p1) to generate pHIL-D2 (p1/His10h1or2or3). h1, h2 and h3 containing fragments were excised from pHIL-D2-(p1/His10h1or2or3) and subcloned into pHIL-D2-(h1/His10p1) or pHIL-D2-(h2/His10p1) to produce pHIL-D2-(h1/His10h1or2or3) and pHIL-D2-(h2/His10h1or2or3). The newly created plasmids were analyzed for correct integration and correct sequence of the insert by restriction enzymatic digestions and sequencing. DNA of each construct was prepared in large quantities in E. coli XL-1 Blue for Pichia pastoris transformation.

(103) Yeast Transformation. Expression and Purification of Human Na,K-ATPase Isoforms

(104) Methods for transformation, culture of P. pastoris clones, protein expression of Na,K-ATPase human isoforms (11, 21, 31), membrane preparation, solubilization of membranes in DDM, and purification on BD-Talon beads have been described in detail (6-9, 11, 25). In initial experiments the three 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%. In later experiments the isoforms complexes were reconstituted with purified FXYD1 on the BD-Talon beads together as described in detail in (10, 11) prior to elution of 11FXYD1, 21FXYD1 and 31FXYD1 complexes. The proteins were stored at 80 C. Protein concentration was determined with BCA (B9643 Sigma).

(105) Assay of Na,K-ATPase Activity of Purified Isoform Complexes

(106) Inhibition of Na,K-ATPase activity of the detergent-soluble 11, 21, and 31 complexes by CG's was determined as described (6) using either the or FXYD1 complexes. The presence or absence of FXYD1 does not affect inhibition of Na,K-ATPase activity by cardiac glycosides (6), but strongly stabilizes the complexes (9-11). The K.sub.0.5K was estimated by varying K concentration in a medium containing a fixed total K+choline chloride of 60 mM, and constant NaCl of 140 mM. Curves were fitted to the Hill function v=Vmax*[S].sup.n/([S].sup.n+K.sup.n), where S is the K concentration, n is the Hill coefficient and K.sup.n is K.sub.0.5K. For comparison of different curves the ratio v/Vmax for each curve was calculated and replotted. In experiments to assess inhibition of Na,K-ATPase activity by cardiac glycosides of the present invention, the percent inhibition VCG/V0 was calculated and Ki values were obtained by fitting the data to the function VCG/V0=Ki/([CG]+Ki)+ c. Inhibition was estimated in 3-8 separate experiments and average Ki values SEM were calculated. Significance of differences between Ki1 and Ki2 was calculated by the unpaired Student's t-test (p values). The ratio of Ki1/2SEM was calculated for each compound and p values were calculated by comparison with digoxin. P values <0.05 were considered significant.

(107) Dissociation Rates of Cardiac Glycosides

(108) Purified 21FXYD1 complexes (0.3-0.5 mg/ml) were incubated for 30 minutes at 37 C. in a medium containing ATP, 1 mM; NaCl 100 mM; MgCl.sub.2, 4 mM Histidine.HCl 25 mM pH 7.4 without (Control) or with 1 M of different cardiac glycosides. The enzyme solutions were then diluted 100-fold into a medium containing 100 mM NaCl, 5 mM KCl, 1 mM EDTA (Tris), 0.005 mg/ml C.sub.12E8, 0.01 mg/ml SOPS, and 0.001 mg/ml cholesterol and incubated at 37 C. for different lengths of time. Aliquots were removed at different times and Na,K-ATPase activity was measured in triplicate over 0.5 minutes (digoxigenin) or 2 minutes (other cardiac glycosides) in the standard activity medium containing 200 M ATP. The activity of test samples was divided by the activity of the control samples and the time-course for reversal of inhibition was analyzed by fitting the data to the function v.sub.t=v.sub.e.sup.kt+c. Normalized curves for comparison of different experiments (e.g. as in FIG. 9) were obtained by subtracting the constant value c from each value of the activity and re-fitting the ratio v.sub.t/v.sub.=1e.sup.kt.

(109) Synthesis of Perhydro-1,4-Oxazepine Derivatives of Digoxin

(110) The syntheses of the different digoxin perhydro-1,4-oxazepine derivatives were performed in two steps: 1) oxidation of digoxin with sodium periodate to give an open-ring dialdehyde in the third sugar moiety and 2) reductive amination with a primary amine, in the presence of NaCNBH.sub.3, closing a 7-membered ring to give the digoxin perhydro-1,4-oxazepine derivative. As an example, the synthesis of DGlyN is provided below. It is apparent to a person of skill in the art that the other compounds of the present invention may be prepared by the same or similar methods.

(111) Oxidation of Digoxin with NaIO.sub.4 (26)

(112) In a 50 ml polypropylene test tube, a solution of NaIO.sub.4 (400 mg, 1840 mol) in H.sub.2O (4 ml) was added under stirring at room temperature to a suspension of digoxin (400 mg, 512 mol) in 95% EtOH (36 ml, not fully soluble) and the mixture that immediately dissolved was allowed to stand at room temperature for 1 hr. During that time a precipitate was formed. Precipitated NaIO.sub.3 was removed, by centrifugation at 3,000g for 15 min and filtration through a syringe filter (PTFE, 0.2 um, 25 mm). The solution was concentrated in an evaporator and extracted with 40 ml CHCl.sub.3. The organic layer was washed with 28 ml water, dried over anhydrous Na.sub.2SO.sub.4, filtered and evaporated in an evaporator, and high vacuum overnight to give the dialdehyde, which is dissolved in 48 ml of absolute methanol to give a 10 mM solution of dialdehyde.

(113) Reductive Amination with Glycinamide Hydrochloride

(114) Glycinamide hydrochloride (28.2 mg, 256 moles, MW=110.54, Aldrich) was added to the digoxin dialdehyde (180 mg=240 moles) solution to give concentrations of 12 mM and 10 mM, respectively. The apparent pH was corrected to 5-6 with concentrated acetic acid in methanol, and the mixture was kept at room temperature for 5 min. The Schiff base that forms was reduced with NaCNBH.sub.3, (59.6 mg, 480 moles, MW=123.95, 20 mM) with stirring. Progress of the reaction was monitored by TLC (SiO.sub.2 with acetone/CHCl.sub.3 (3:2). The mixture was left for 1.5 h, the creation of DGlyN and disappearance of digoxin dialdehyde was confirmed by mass spectrometry, and the methanol was evaporated by rotavap and high vacuum overnight. Since side reactions can occur, such as acid or base induced hydrolysis of dialdehyde to bis-digitoxoside, the final product was purified. The DGlyN reaction mixture was dissolved in a minimal amount (5.4 ml) of 50% methanol, filtered through a syringe filter 0.2 m, PTFE, and used for purification by HPLC (FIG. 2B). HPLC purification was done on a Purospher STAR RP-18e semi-prep column, eluted with a gradient of 50-80% methanol in water in 15 column volumes at a flow rate of 4 ml/min. Other derivatives were purified using optimal gradients of methanol established in analytical HPLC runs (Chromolith RP-18e) prior to application to the semi-preparative column. The methanol was JT Baker HPLC gradient grade.

(115) Additional compounds were prepared by a similar method. Their Mass Spectral data are presented in Table 1.

(116) Digitoxin derivatives may be made by similar methods as described herein, using the digitoxin scaffold (XH) instead of the digoxin scaffold (XOH).

(117) Measurement of Intraocular Pressure in Rabbits

(118) Animals

(119) New Zealand white rabbits (3-3.5 kg) about 1 year old, of either sex, were housed individually in separate cages in animal room conditions on a reversed, 12-hour dark/light cycle. For the experiments the animals were transferred to rabbit restrainers in a quiet and calm atmosphere (FIG. 1). No ocular abnormalities were detected prior or during the experiments. Animal care and treatment were subject to the approval of the institutional committee for animal experiments, Weizmann Institute IACUC permission (no 04270911-2).

(120) Drug Preparation and Administration

(121) Stock solutions of cardiac glycosides were dissolved in ethanol, and diluted in phosphate buffer (PBS) on each day of the experiment such that the final ethanol concentration did not exceed 1%.

(122) Modeling

(123) Digoxin (co-ordinates 3B0W) was introduced manually into the structure of pig kidney Na,K-ATPase bound with ouabain (4HYT) so that the steroid and lactone moieties of ouabain and digoxin superimposed as closely as possible, see ref. The structure file with bound digoxin was then submitted to the YASARA Energy Minimization Server. The structural figure was prepared with PyMOL.

(124) Intraocular Pressure and Corneal Thickness Measurements

(125) IOP (mm Hg) of rabbits was measured using a calibrated Pneumatonometer (Model 30, Reichert technologies, FIG. 1). A local anesthetic Oxybuprocaine HCl (0.4%, 25 l) was applied to each cornea about a minute before IOP measurements. Two baseline IOP readings were taken before topical administration of the CG (or PBS as control) and after half an hour (Zero time). The readings of the two measurements were almost identical, suggesting that the CG's had no effect on the basal IOP. At zero time one drop of 4AP (40 mg/ml, 30 l) or IB-MECA (1 M, 30 l) was administered to both eyes of each rabbit IOP measurements were made at different times as indicated in each experiment. In the experiments for which the IOP was elevated for several hours, 4AP was added every 1.5 hours or IB-MECA every 2 hours. The Pneumatonometer readings were accepted when the standard deviation of the value XmmHg was between 0.1-0.4 mmHg i.e X0.1-0.4 mmHg, representing a possible error of 6-13% compared to the minimal 3 mm Hg increase and 1.6-6.7% compared to the maximal 6 mm Hg increase in IOP induced by 4AP or IB-MECA. Each experiment was repeated two or three times with similar results. In all cases the figures depict the average effect on IOP (i.e for four or six eyes) compared to control SEM. Where error bars are not seen in the figures, the errors are smaller than the symbols used. Significance of differences from the control was calculated by the unpaired Students t-test (p values), p values <0.05 were considered significant. Corneal thickness (m) was measured using an ultrasonic pachymeter (Sonogage pachometer, Cleveland, USA), before and during the experiment with CG and 4AP treatments. The values represent averages of three independent measurements for each eye.

ABBREVIATIONS

(126) IOP, intra-ocular pressure;

(127) CG, cardiac glycoside;

(128) 4AP, 4-aminopyridine.

(129) While certain embodiments of the invention have been illustrated and described, it will be clear that the invention is not limited to the embodiments described herein. Numerous modifications, changes, variations, substitutions and equivalents will be apparent to those skilled in the art without departing from the spirit and scope of the present invention as described by the claims, which follow.

REFERENCES

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