Field of Invention

The present invention relates to nitroxy derivatives of soft steroids and ophthalmic compositions that include nitroxy derivatives of soft steroids. The invention is also directed to the use of the ophthalmic compositions for treating inflammatory conditions of the palpebral or bulbar conjunctiva, cornea and anterior segment of the globe, to ameliorate inflammation associated with corneal injury, and for the treatment of dry eye.

Background of the Invention

In order to prevent and/or reduce toxicity profiles of certain drugs, the metabolic disposition of the drugs should be considered at an early stage of the drug-design process. This is true particularly if one considers the biochemical processes available within the body and certain target tissues that can chemically alter relatively robust chemical compounds. This is true even if 95% or more of a drug is excreted unchanged, for the unaccounted small portion can, and in many instances, raise toxicity concerns. "Soft drugs" can be defined as biologically active chemical compounds (drugs) which might structurally resemble known active drugs (soft analogues) or could be entirely new types of structures, but which are all characterized by a predictable in vivo destruction (metabolism) to nontoxic moieties, after they achieve their therapeutic role. The metabolic disposition of the soft drugs takes place with a controllable rate in a predictable manner, and without the incidence of unwanted side effects.

Topical steroids such as corticosteroids are commonly used for anti-inflammatory therapy of the eye, especially for treating inflammatory conditions of the palpebral or bulbar conjunctiva, cornea and anterior segment of the globe. Common therapeutic applications for steroids include allergic-conjunctivitis, ache rosacea, superficial punctate keratitis and iritis cyclitis. Steroids also are used to ameliorate inflammation associated with corneal injury due to chemical or thermal burns, or penetration of foreign bodies. Such conditions may result from surgery, injury, allergy or infection to the eye and can cause severe discomfort.

Despite their therapeutic advantages, topical ocular use of corticosteroids is associated with a number of complications, including posterior subcapsular cataract formation, elevation of intraocular pressure, secondary ocular infection, retardation of corneal wound healing, uveitis, mydriasis, transient ocular discomfort and ptosis. Numerous systemic complications also may arise from the topical ocular application of

corticosteroids. These complications include adrenal insufficiency, Cushing's syndrome, peptic ulceration, osteoporosis, hypertension, muscle weakness or atrophy, inhibition of growth, diabetes, activation of infection, mood changes and delayed wound healing.

It is known that certain glucocorticoids have a greater potential for elevating intraocular pressure ("IOP") than other compounds in this class. For example, it is known that prednisolone, which is a very potent ocular anti-inflammatory agent, has a greater tendency to elevate IOP than fluorometholone, which has moderate ocular antiinflammatory activity. It is also known that the risk of IOP elevations associated with the topical ophthalmic use of glucocorticoids increases over time. In other words, the chronic (i.e., long-term) use of these agents can increase the risk of significant IOP elevations. It is also well known that the soft topical steroid, loteprednol etabonate is used for treating inflammatory conditions of the palpebral or bulbar conjunctiva, cornea and anterior segment of the globe, and to ameliorate inflammation associated with corneal injury.

It is also known that nitric oxide donors can also reduce IOP. R. Steele et al., Bioorg. Med. Cherru Lett., Vol. 19, 6S6S-6S70 (2009). Nitric oxide donors are pharmaceutically active substances that contain a nitric oxide moiety and which donate, transfer, or release nitric oxide. Nitric oxide donors include, for example, S-nitrosothiols, nitrites, nitrates, N-oxo-N-nitrosamines, and substrates of various isozymes of nitric oxide synthase. It has been postulated that compounds containing a NO donating moiety undergo hydrolysis by the enzymes present in the eye compartments to release the core drug and NO donating moiety. However, the effects of NO donating selective glucocorticoid receptor agonists have not been previously investigated. Also, it has been recognized that NO can act as a mediator and regulator of inflammatory responses. It possesses cytotoxic properties and is produced by immune cells, including macrophages, with the aim of assisting in the destruction of pathogenic microorganisms, but it can also have damaging effects on host tissues. NO can also react with molecular oxygen and superoxide anion to produce reactive nitrogen species that can modify various cellular functions. R.

Korhonen etal., Curr. Drug Target-Inflam. & Allergy, Vol.4, 471 (2005). Furthermore, oxidative stress, occurring not only in the trabecular meshwork ("TM") but also in retinal cells, appears to be involved in the neuronal cell death affecting the optic nerve in primary open-angle glaucoma ("POAG"). A. Izzotti et al., Mutat. Res., Vol.612, No. 2, 105 (2006).

To lengthen the retention time of instilled ophthalmic drug in the eye and to enhance the bioavailability of the ophthalmic drug, various ophthalmic vehicles have been developed. Examples of such ophthalmic vehicles include various inserts, ointments, suspension, and aqueous gels. However, many of these ophthalmic vehicles do have their drawbacks. Among such ophthalmic vehicles, the so-called in situ gel-forming systems, has been particularly useful for prolonging precorneal retention time and improving ocular bioavailability of the ophthalmic drugs. Typically, in situ gel-forming systems are usually aqueous solutions that contain one or more polymers. During storage and administration, e.g., in the form of eye drops, the gel-formingsystems are low- viscosity liquids and form gels upon contact with tear fluid. The liquid-to-gel transition can be triggered by a change in temperature, pH, ionic strength, or the presence of tear proteins depending on the particular polymer system employed. For example, U.S. Patent 6,511,660 discloses a composition comprising Carbopol ^® and Pluronic ^® (a

polyoxyethylene-polyoxypropylene copolymer) formulated at pH of 4. The composition turns into a stiff gel when in contact with physiological condition (37 °C and pH of 7.4). Also, Kumar et al., /. Ocular Pharmacol., Vol. 10, 47-56 (1994), discloses an ocular drug delivery system based on a combination of Carbopol and methylcellulose, prepared at pH of 4. This system turns into a stiff gel when the pH is increased to 7.4.

Summary of the Invention

A compound of formula (I) or a pharmaceutically acceptable salt thereof

wherein

R ₁ is (a) Y-ONO ₂ group, and Y is a bivalent radical having the following meaning:

i) -(C ₁ -C ₁₀ ) alkylene, being optionally substituted with one or more of the substituents selected from the group consisting of: halogen atoms, hydroxy, sulfoxy, -ONO ₂ , and T, wherein T is -OC(O)(C ₁ - C,oalkyI)-ONO ₂ or -O(C ₁ -C ₁₀ alkyl)-ONO ₂ ;

ii) -L-(cycloalkylene) with 5 to 7 carbon atoms in the cycloalkylene ring, the ring being optionally substituted one or more of the substituents listed in a) above, and L is nothing or (C ₁ -C ₆ ) alkylene, optionally substituted with one or more C ₁ -C ₄ alkyl, halogen atoms or hydroxy; or

R ₁ is (b) L-(C ₅ -C ₁₀ )Ar, the aromatic ring being substituted with (C ₁ -C ₈ ) alkylene-ONO ₂ , or the aromatic ring being substituted with T listed in (a) above, and L is nothing or (C ₁ -C ₆ ) alkylene, optionally substituted with one or more the substituents selected from the group consisting of: C ₁ -C ₄ alkyl, halogen atoms and hydroxyl;

R ₂ is unsubstituted or substituted C ₁ -C ₁₀ alkyl or C ₂ -C ₁₀ alkenyl, the substituents being selected from the group consisting of halogen atoms, hydroxyl, phenyl, lower alkoxy, lower alkylthio, lower alkylsulfinyl, lower alkylsulfonyl;

R ₃ is hydrogen, α-hydroxy, β-hydroxy, α-methyl, β-methyl or =CH ₂ ;

R ₄ is hydrogen, fluoro or chloro;

R ₅ is hydrogen, fluoro, chloro or methyl;

X is O or S;

Z is CH ₂ , carbonyl or CH(OH); and the dotted line in ring A indicates that the 1,2 linkage is saturated or unsaturated.

The invention is also directed to ophthalmic compositions administered in the form of eye drops that include compounds of formula (I) or a pharmaceutically salt thereof, and the use of the ophthalmic compositions for treating inflammatory conditions of the palpebral or bulbar conjunctiva, cornea and anterior segment of the globe, and to ameliorate inflammation associated with corneal injury. Detailed Description of the Invention

The inactive metabolite approach to the case of the natural and synthetic

glucocorticosteroids has led to soft steroidal anti-inflammatory agents. Thus, for example, in the case of hydrocortisone, one of its major, inactive metabolites, cortienic acid, i.e., 11β,17α-dihydroxyandrost-4-en-3-one-17β-carboxylic acid, has been used as a starting point and activated by the introduction of suitable non-toxic 17α- and 17β- substituents. The activated derivatives under physiological conditions achieve their therapeutic role and then cleave in vivo to an inactive metabolite and other nontoxic moieties. The described compounds also take advantage of natural biometabolic pathways available in ocular environments to provide not only active inflammatory agents, but also the release of NO or NO adduct, which can mitigate any consequential rise in intraocular pressure in the case of many glucocorticosteroids.

In accord with this design approach, the present invention provides novel soft steroids having anti-inflammatory activity, the soft steroids having the structural formula (I).

wherein

R ₁ is (a) Y-ONO ₂ group, and Y is a bivalent radical having the following meaning:

i) -(C ₁ -C ₁₀ ) alkylene, being optionally substituted with one or more of the substituents selected from the group consisting of: halogen atoms, hydroxy, sulfoxy, -ONO ₂ , and T, wherein T is -OC(O)(C ₁ - C ₁₀ alky)-ONO ₂ or -O(C ₁ -C _l0 alkyl)-ONO ₂ ;

ii) -L-(cycloalkylene) with 5 to 7 carbon atoms in the cycloalkylene ring, the ring being optionally substituted one or more of the substituents listed in a) above, and L is nothing or (C ₁ -C ₆ ) alkylene, optionally substituted with one or more C ₁ -C ₄ alkyl, halogen atoms or hydroxy; or

R ₁ is (b) L-(C ₅ -C ₁₀ )Ar, the aromatic ring being substituted with (C ₁ -C ₈ ) alkylene-ONO ₂ , or the aromatic ring being substituted with T listed in (a) above, and L is nothing or (C ₁ -C ₆ ) alkylene, optionally substituted with one or more the substituents selected from the group consisting of: C ₁ -C ₄ alkyl, halogen atoms and hydroxyl;

R ₂ is unsubstituted or substituted C ₁ -C ₁₀ alkyl or C ₂ -C ₁₀ alkenyl, the substituents being selected from the group consisting of halogen atoms, hydroxyl, phenyl, lower alkoxy, lower alkylthio, lower alkylsulfinyl, lower alkylsulfonyl;

R ₃ is hydrogen, α-hydroxy, β-hydroxy, α-methyl, β-methyl or =CH ₂ ;

R ₄ is hydrogen, fluoro or chloro;

R ₅ is hydrogen, fluoro, chloro or methyl;

X is O or S;

Z is CH ₂ , carbonyl or CH(OH); and the dotted line in ring A indicates that the 1,2 linkage is saturated or unsaturated.

As used herein, the term "alkyl" or "alkyl group" means a linear- or branched-chain saturated aliphatic hydrocarbon monovalent group, which may be unsubstituted or substituted as stated. For example, the group may be partially or completely substituted with halogen atoms (F, CI, Br, or I). Non-limiting examples of alkyl groups include methyl, ethyl, n-propyl, l-methylethyl(isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), and the like.

As used herein, the term "alkenyl" or "alkenyl group" means a linear- or branched-chain aliphatic hydrocarbon monovalent radical containing at least one carbon-carbon double bond. This term is exemplified by groups such as ethenyl, propenyl, n-butenyl, isobutenyl, 3-methylbut-2-enyl, n-pentenyl, heptenyl, octenyl, decenyl, and the like.

As used herein, the term "alkylene" or "alkylene group" means a linear- or branched- chain saturated aliphatic hydrocarbon divalent radical having the specified number of carbon atoms. This term is exemplified by groups such as methylene, ethylene, propylene, n-butylene, and the like.

The used herein, the term "cycloalkyl" or "cycloalkyl group" means a stable aliphatic saturated or unsaturated monocyclic radical comprising primarily ring atoms of carbon, but optionally including one oxygen ring atom, or one or two nitrogen ring atoms, and in each case preferably a 5- to 7-membered monocyclic. Unless otherwise specified, the cycloalkyl ring may be attached at any carbon atom, or optionally a nitrogen atom, which results in a stable structure and, if substituted, may be substituted at any suitable carbon atom which results in a stable structure.

The used herein, the term "lower" to further describe a chemical grouping such as alkyl means a substituent with one to four carbon atoms.

A group of soft steroids of formula (I) include those compounds having the following: R ₂ is unsubstituted, or halo-substituted, C ₁ -C ₆ alkyl or C ₂ -C ₆ alkenyl; R3 is hydrogen, α- hydroxy or β-hydroxy; R ₄ and R ₅ are independently selected from hydrogen or fluoro; and X is O.

Another class of soft steroids of formula (I) include those compounds having at least one of R ₄ and R ₅ is fluoro, or R3 is hydrogen, α-methyl or β-methyl.

Another class of soft steroids of formula (I) include those compounds having the following: R ₁ is Y-ONO ₂ , and Y is a (C ₁ -C ₈ ) alkylene, being optionally substituted with one or more of the substituents selected from the group consisting of: halogen atoms, hydroxy, sulfoxy, -ONO ₂ , and T, wherein T is -OC(O)(C ₁ - C ₁₀ alkyl)-ONO ₂ or -O(C ₁ - Cioalkyl)-ONO ₂ ; R ₂ is unsubstituted, or halo-substituted, C ₁ -C ₆ alkyl or C ₂ -C ₆ alkenyl; R3 is hydrogen, α-hydroxy or β-hydroxy; R ₄ and R ₅ are independently selected from hydrogen or fluoro; and X is O. Of this class of soft steroids, many of the compounds will have Y as a (C ₁ -C ₆ ) alkylene substituted with one or more of the substituents selected from halogen atoms or T, wherein T is -OC(O)(C ₁ -C ₆ alkyI)-ONO ₂ or -O(C ₁ - C ₄ alkyl)-ONO ₂ .

Another class of soft steroids of formula (I) include those compounds having the following: R ₁ is L-(C ₅ -C ₁₀ )Ar, the aromatic ring being substituted with (C ₁ -C ₈ ) alkyl- ONO ₂ , or the aromatic ring being substituted with T as defined in paragraph [0011], and L is nothing or (C ₁ -C ₆ ) alkylene, optionally substituted with one or more the substituents selected from the group consisting of: C ₁ -C ₄ alkyl, halogen atoms and hydroxyl; R ₂ is unsubstituted, or halo-substituted, C ₁ -C ₆ alkyl or C ₂ -C ₆ alkenyl; R3 is hydrogen, α- hydroxy or β-hydroxy; R ₄ and R ₅ are independently selected from hydrogen or fluoro; and X is O. Of this class of soft steroids, many of the compounds will have R ₁ is -L- phenyl, the phenyl ring being substituted with (C ₁ -C ₈ ) alkyl-ONO ₂ , or the aromatic ring being substituted with T as defined, and L is nothing or (C ₁ -C ₆ ) alkylene, optionally substituted with one or more the substituents selected from the group consisting of: C ₁ - C ₄ alkyl, halogen atoms and hydroxyl.

Still another class of soft steroids is described by formula (II)

wherein

R ₁ is (a) Y-ONO ₂ group, and Y is -(C ₁ -C ₆ ) alkylene, being optionally substituted with T, wherein T is -OC(O)(C ₁ -C ₁₀ alkyl)-ONO ₂ or -0(C ₁ -C ₁₀ alkyl)-ONO ₂ ; or

R ₁ is (b) L-(C ₅ -C ₁₀ )Ar, the aromatic ring being substituted with (C ₁ -C ₄ ) alkyl- ONO ₂ , or the aromatic ring being substituted with T listed in (a) above, and L is nothing or (C ₁ -C ₄ ) alkylene;

R ₂ is C ₁ -C ₆ alkyl optionally substituted with halogen; R3 is hydrogen, α-hydroxy, β-hydroxy, α-methyl or β-methyl; R ₄ and R ₅ are independently selected form hydrogen or fluoro; Z is CH ₂ , carbonyl or CH(OH); and the dotted line in ring A indicates that the 1,2 linkage is saturated or unsaturated.

A group of soft steroids of formula (II) include those compounds having the following: at least one of R ₄ and R ₅ is fluoro, or R ₃ is hydrogen, α-methyl or β-methyl.

Still another class of soft steroids is described by formula (III)

wherein R ₆ is -(C ₁ -C ₆ ) alkyl-ONO ₂ ; and R ₇ is C ₁ -C ₆ alkyl optionally substituted with halogen. Also, in many instances the soft steroids of formula (ΙΠ) will have the following: R ₆ is -C ₄ H ₈ -ONO ₂ , or -C ₅ H ₁₀ -ONO ₂ ; and R ₇ is -CH ₃ , -C ₂ H ₅ , -CH ₂ C1 or - C ₂ H ₄ CI.

The soft steroid compounds of formula (I), (Π) and (ΙΠ) can generally be prepared by known methods, the method of choice being dependent on the identity of the various substituents in the desired final product More complete information on the general methods used to prepare the soft steroid compounds is found in U.S. Patent No.

4,996,335, the disclosure of which beginning on column 9, line 64 thru column 17, line 32 is incorporated herein by reference, hereafter, "Bodor".

One generally useful method for the preparation of the compounds of formula (I) wherein Z is β-hydroxymethylene and X is oxygen utilizes steroidal starting materials of the formula

wherein R3, R ₄ , Rs and the dotted line in ring A are defined as in paragraph [0011]. According to this process, a starting material of formula (AA) is reacted with R ₂ OCOCl or R ₂ OCOBr (formed by reacting R ₂ OH with COCI2 or COBr ₂ , wherein R ₂ is defined as in paragraph [0011] under anhydrous conditions, in an appropriate inert organic solvent such as dichloromethane or chloroform in the presence of a suitable acid acceptor (e.g., triethylamine, pyridine). The reaction is conveniently carried out at a temperature between 0 C and room temperature for about 1 to 6 hours thereby providing the corresponding 17β-carboxylic acid, 17α-carbonate-R ₂ . After the above-described introduction of the 17α-substituent, the compound can be converted to the corresponding acid salt. The countercation can be a suitable metal, e.g. alkali metal (such as sodium or potassium), alkaline earth metal, or ammonium, or any pharmaceutically acceptable countercation known to those in the art. Sometimes with a compound of formula (I) in which R ₂ contains a sulfinyl or sulfonyl group, the compound can be prepared from the corresponding thio-containing R ₂ derivative at a later stage in the synthetic scheme, as discussed in Bodor.

When it is desired to introduce a halo-substituted R ₁ grouping into the soft steroid, the reaction proceeds well using hexamethylphosphoramide as the solvent at lower temperatures (0 C to 10 C) and employing a R ₁ W reactant wherein W is iodine. When a non-halogen containing R ₁ grouping is desired no such restrictions need be placed on the

R ₁ -W reactant or on the solvent; thus, W can be any halogen, preferably chloro or bromo, and the usual organic solvents such as dimethylformamide, dichlormethane, acetonitrile, tetrahydrofuran or chloroform can be used. When a compound of formula (I) wherein R ₁ contains a sulfinyl or sulfonyl grouping is desired, such a grouping is generally introduced from the corresponding thio steroid described further in Bodor.

When the compounds of formula (I) wherein R ₃ is α- or β-hydroxy are desired, compounds of interest can be prepared by partial acid hydrolysis of the corresponding compounds of formula (I) wherein R ₃ is α- or β-OCOOR ₂ , in a suitable solvent medium. Use of a mild reagent, e.g., oxalic acid in methanol, is desirable. Alternatively, hydrolysis of the 16-carbonate to the 16-hydroxy compound could be carried out at an earlier stage in any synthetic scheme described herein after the introduction of the 16,17- carbonate groupings.

Another possible process for the preparation of the described soft steroid compounds of formula (I) wherein Z is β-hydroxymethylene and X is oxygen or sulfur, utilizes the 17β- carboxylic acid 17α-carbonate. This intermediate is successively treated, first with a mild acyl chloride forming agent, e.g. such as diethylchlorophosphate or oxalyl chloride, to form the corresponding novel acid chloride wherein R ₂ , R ₃ , R ₄ , R ₅ and the dotted line in ring A are defined as for formula (I), and then with R ₁ -XM in an inert solvent (e.g., THF, acetonitrile or DMF), at a temperature between about 0 C and the boiling point of the solvent, for 1 to 6 hours, to afford the corresponding compound of formula (I). The two steps of this process can be very conveniently run in the same solvent, without isolating the acid chloride intermediate formed in the first step. This process is of particular value when a compound of formula (I) wherein X is S is desired. Also, the corresponding 11-keto compounds of formula (I), formula (II), or formula (III) can be prepared by reacting the compounds with an oxidizing agent. The oxidation chemistry is usually carried out by using an oxidizing agent in an appropriate solvent using well known synthetic methods. The solvent may be any conventional solvent, for example, water, and organic acid (e.g. formic acid, acetic acid, trifluoroacetic acid), an alcohol (e.g. methanol, ethanol), a halogenated hydrocarbon (e.g. chloroform, dichloromethane), or the like. This oxidizing agent may also be any conventional agent which is effective for oxidizing a hydroxy group to a carbonyl group, for example, pyridinium chlorochromate, chromium trioxide in pyridine, hydrogen peroxide, dichromic acid, dichromates, permanganic acid, permanganates or the like. The oxidizing agent is usually used in an amount of 1 mole or more, preferably 1 to 3 mole, per mole of the compound. The reaction is usually carried out at a temperature of 0 C to 40 C, preferably at around room temperature, for about 6 to 30 hours.

As already stated, soft steroids have the potential advantage of treating inflammation without inducing elevation of intraocular pressure. In addition, soft steroids can provide the added benefit of a lower tendency to induce cataracts which may result from interaction of corticosteroids with the ocular lens proteins. With this in mind, of particular interest is the preparation of nitroxy derivatives based on the soft steroid, antiinflammatory agent, loteprednol etabonate (at times referred to as LE) by means of above described chemistry. Loteprednol etabonate ("LE") is a known soft corticosteroid based on the known inactive metabolite prednisolone acetate of the active drug prednisolone. See U.S. Pat. Nos.4,996,335 and 4,710,495.

LE is an analog of prednisolone that does not have a 20-keto group attached to the 17β- position. Instead, the 17|3-position is occupied with a metabolically-labile ester function. In biological systems, LE is hydrolysed to the inactive carboxylic acid metabolite that does not bind to glucocorticoid receptors. LE also provides superior safety by reducing the risk of steroid induced cataracts and elevation of intrα-ocular pressure. The relative inactivity of LE to enzymes located in the blood and/or liver also reduces the likelihood of systemic side effects. LE therefore provides therapeutic advantages over other corticosteroids by providing efficacy similar to its parent compound, namely, prednisolone acetate, with fewer deleterious systemic side effects. Loteprednol Etabonate ophthalmic suspension 0.2% is indicated for temporary relief of signs and symptoms of Seasonal Allergic Conjunctivitis (SAC). The recommended administration dosage is one drop to each eye (0.1 mg/eye), 4x (four times) daily for a total dosage of 0.4 mg/eye/day. An improved gel formulation over the current Alrex® formulation is described as follows. The gel formulation contains 20% less active, 0.16 wt% loteprednol etabonate vs. the 0.2 wt.% loteprednol etabonate in the Alrex® product. More importantly, a small clinical study indicates that the gel formulation (taken once daily) is more effective in reducing ocular itching for the treatment of seasonal allergic conjunctivitis than Alrex® (taken 4x per day). In other words, a once daily, drop administration of the gel formulation (0.16 wt%) is more effective than 4 x 0.2 wt% for a total administration of 0.8 wt.% of Alrex®. This is a very significant achievement as a patient has no need to administer additional drops to the eye other than once in the morning or evening, thereby significantly improving upon patient compliance and convenience. In addition, unlike the aqueous suspension Alrex® the gel formulation is non-settling, and therefore, does not require vigorous repeated shaking prior to installation, which again leads to greater patient compliance and greater convenience for the patient.

Likewise, the invention is also directed to a method of treating allergic conjunctivitis comprising instructing a person suffering from ocular itching resulting from allergic conjunctivitis to administer once daily in the form of one or more eye drops an aqueous ophthalmic composition comprising compound of formula (I), formula (Π) or formula (III), or a pharmaceutically acceptable salt thereof, described herein. The once daily administration of the composition described is likely to have greater clinical efficacy than if the same composition is administered twice or four-times daily, particularly with a non-gel formulation.

The gel formulation is sufficiently viscous (>1000 cps at 7.5 s ^-1 shear) to ensure that particles of compounds of formula (I), formula (Π) or formula (HQ, or a

pharmaceutically acceptable salt thereof, which can be based in-pait on loteprednol, are suspended in the formulation vehicle and do not settle over time. The stabilized gel formulation does not require shaking of the dosage package to re-suspend the drug particles prior to drop administration. In one aspect, the composition has a viscosity in the range from about 300 cp to about 1500 cp outside the eye. As applied herein to the present invention, viscosity is measured with a Brookfield Engineering Laboratories RVDV-III Ultra C rheometer (a cone-and-plate rheometer) with CPE-40 spindle, at 25 °C, and shear rate of 7 ± 1 sec ^'1 .

In another aspect, the present invention provides topical ophthalmic aqueous compositions comprising 0.1 wt% to 1 wt.% of a compound of formula (I), formula (II) or formula (III), or a pharmaceutically acceptable salt thereof, and polyacryclic acid. The composition has a viscosity in the range from about 300 cp to about 500 cp. In some instances, the ophthalmic composition includes a total concentration of cations of less than about 50 mM (or alternatively, less than about 40 mM, or less than about 30 raM, or less than about 20 mM, or less than about 10 mM).

The formulations administered according to the present invention may also include various other ingredients, including but not limited to surfactants, tonicity agents, buffers, preservatives, co-solvents and viscosity-building agents. Surfactants that can be used are surface-active agents that are acceptable for ophthalmic or otolaryngological uses. Useful surface active agents include but are not limited to polysorbate 80, tyloxapol, Tween ^® 80 (ICI America Inc., Wilmington, Del.), Pluronic ^® F-68 (from BASF, Ludwigshafen, Germany) and the poloxamer surfactants can also be used. These surfactants are nonionic alkaline oxide condensates of an organic compound which contains hydroxyl groups. The concentration in which the surface active agent may be used is only limited by neutralization of the bactericidal effects on the accompanying preservatives (if present), or by concentrations which may cause irritation.

An appropriate buffer system (e.g., sodium phosphate, sodium acetate, sodium citrate, sodium borate or boric acid) may be added to the formulations to prevent pH drift under storage conditions. The particular concentration will vary, depending on the agent employed.

Topical ophthalmic products are typically packaged in multidose form. Preservatives are thus required to prevent microbial contamination during use. Suitable preservatives include: biguanides, hydrogen peroxide, hydrogen peroxide producers, benzalkonium chloride, chlorobutanol, benzododecinium bromide, methyl paraben, propyl paraben, phenylethyl alcohol, edetate disodium, sorbic acid, polyquaternium-1, or other agents known to those skilled in the art. Such preservatives are typically employed at a level of from 0.001 to 1% (w/w). Unit dose formulations of the present invention will be sterile, but typically unpreserved. Such formulations, therefore, generally will not contain preservatives.

Co-solvents and viscosity-building agents may be added to the formulations to improve the characteristics of the formulations. Such materials can include nonionic water- soluble polymer. Other compounds designed to lubricate, "wet,' approximate the consistency of endogenous tears, aid in natural tear build-up, or otherwise provide temporary relief of dry eye symptoms and conditions upon ocular administration the eye are known in the art. Such compounds may enhance the viscosity of the formulation, and include, but are not limited to: monomelic polyols, such as, glycerol, propylene glycol, ethylene glycol; polymeric polyols, such as, polyethylene glycol,

hydroxypropylmethyl cellulose ("HPMC"), carboxy methylcellulose sodium, hydroxy propylcellulose ("HPC"), dextrans, such as, dextran 70; water soluble proteins, such as gelatin; and vinyl polymers, such as, polyvinyl alcohol, polyvinylpyrrolidone, povidone and carbomers, such as, carbomer 934P, carbomer 9 1, carbomer 940, carbomer 974P. Other compounds may also be added to the ophthalmic formulations of the present invention to increase the viscosity of the carrier. Examples of viscosity-enhancing agents include, but are not limited to: polysaccharides, such as hyaluronic acid and its salts, chondroitin sulfate and its salts, dextrans, various polymers of the cellulose family; vinyl polymers; and acrylic acid polymers.

A sterile soft steroid gel formulation containing a compound of formula (I), formula (Π) or formula (III), or a pharmaceutically acceptable salt thereof, and of particular interest, a gel formulation of a nitroxy derivative based in-part on loteprednol, in a polyacrylic acid-containing base can be satisfactorily produced when certain method steps are followed in its production. An aqueous polyacrylic acid suspension is made and then autoclaved under sterile conditions. This polyacrylic acid suspension is mixed with a sterile-filtrated solution of preserving agent, isotonicity agent, and chelating agent. After careful and thorough mixing of the starting materials, the addition of sterile-filtrated caustic soda solution initiates gel formation, and the gel is further subjected to agitation until it is homogenous. Meanwhile compounds of formula (I), (Π) and (ΙΠ), or its pharmaceutically acceptable salt, is sterilized. This can be accomplished by dissolving the compounds in a suitable amount of solvent, for example ethyl acetate, subjecting the solution to sterile filtration, and precipitating the active substance, for example, through the addition of sterile water with an anti-microbial agent under aseptic conditions. The microbially sterile compound is then triturated or ground to a powder with about three to ten times that amount of the gel base. The remaining amount of gel is then incorporated in the concentrate by thorough mixing. The finished gel preparation is then

conventionally decanted or drawn off under sterile conditions. In an alternative variation of this method, the microbially sterile compounds can be, to a large extent, suspended in a part of the aqueous solution of the tonicity agent. The polyacrylate gel can be made in a conventional manner with the remaining amount of isotonic agent and separately the isotonic suspension with the suspended compound can be homogenously mixed with the polyacrylate under sterile conditions.

This sterile gel is well acceptable to the patient, because its application does not have the disadvantage of known ointments and is not oily. The stability of the gel is shown to have relatively long shelf life without any change in its physical properties. In particular, no settlement of compounds from the gel upon storage (25-40 °C) is observed. In addition, no crystal growth of the active ingredient is observed. Such a sterile gel preparation represents a significantly improved form of application in the

ophthalmological field.

The invention will now be further described by way of several examples that are intended to describe but not limit the scope of the invention as defined by the claims herein.

Example 1.

To a solution of hydrocortisone (IS grams, 0.04 mol) in 120 milliliters of tetrahydrofuran and 30 milliliters of methanol at room temperature is added a warm (approximately 50 C) solution of sodium metaperiodate (25.7 grams, 0.12 mol) in 100 milliliters of water). The reaction mixture is stirred at room temperature for 2 hours, then is concentrated under reduced pressure to remove the tetrahydrofuran and methanol. The solid is triturated with 50 milliliters of water, separated by filtration, washed with water and dried in vacuo at 50 C for 3 hours. The product, 11β,17α-dihydroxyandrost-4-en-3-one- 17β-carboxylic acid (i.e., cortienic acid), melts at 231-234 C, and can be represented by the structural formula

Example 2.

To a cold solution of 11β,17α-dihydroxyandrost-4-en-3-one-17β-carboxylic acid (5% weight/volume; 1 mol) and triethylamine (4 mol) in dichloromethane is added a 50% (weight/volume) solution of methyl chloroformate (3.9 mol) in dichloromethane. The reaction mixture is allowed to warm to room temperature over a 2 hour period. The triethylamine hydrochloride precipitate which forms is removed by filtration and the filtration is washed successively with 3% sodium bicarbonate, dilute (.about.1%) hydrochloric acid and water. The organic layer is separated, dried with magnesium sulfate, and filtered. The filtrate is concentrated in vacuo to a foam. The foam is used in the next step (e.g., Example 3 below) or chromatographed and crystallized for analysis. The product, 11β-hydroxy-17α-methoxycarbonyloxyandrost-4-en-3-one-17β- carboxylic acid, melts at 198-204 C after chromatography and crystallization; ir (KBr) 3000-2800 (C-H), 1750, 1735, 1720 (C=O), 1650, 1640 (C=C— C=O) cm ¹ . The product can be represented by the structural formula

Substitution of an equivalent quantity of ethyl chloroformate for the methyl

chloroformate used above and substantial repetition of the foregoing procedure affords 17α-ethoxycarbonyloxy-11β-hydroxyan<.rost-4-en-3-one-17 β-carboxylic acid, melting at 192-195 C after chromatography and crystalization; ir (KBr) 3500 (1 lp-0~H), 3000- 2800 (C-H), 1740 (C=O), 1630 (C=C— C=O) cm ^-1 . In a similar manner, substitution of an equivalent quantity of butyl chloroformate for the methyl chloroformate used in the frist paragraph of this example and substantial repetition of the procedure there detailed affords 17α-butoxycarbonyloxy- 11β- hydroxyandrost-4-en-3-one-17β-carboxylic acid. The final product, after crystallization from tetrahydrofuran-hexane, melts at 164-166 C.

Example 3. Preparation of 4-bromobutyl nitrate

Tetrahydrofuran (12.5 g-173 mmol) is charged under nitrogen in a reactor cooled to about 5 C. Hydrogen bromide (7.0 g-86.5 mmol) is added slowly and the reaction medium is stirred over a period of 4.5 hours at about 5 C. The mixture is diluted with 22.5 g of cold water and the pH of the solution adjusted to pH=5-7 by adding 27.65% sodium hydroxide ( about 2.0 g) keeping the temperature at about 5 C. The solution is extracted twice with dichloromethane (13.25 g). The combined organic phases are washed with 25% brine (7.5 g), adjusted to pH=6-7 with 27.65% sodium hydroxide and dried over magnesium sulfate. Dichloromethane is distilled off and crude 4- bromobutanol (10.3 g-66.9 mmol) is obtained in a yield of about 77%.

In reactor cooled in ice water at 0 C, nitric acid fuming (8.5 g-135 mmol) is slowly added to a solution of 98% sulfuric acid (13.0 g-130 mmol) in dichloromethane (18.0 g-212 mmol).4-bromobutanol (10.2 g-66.6 mmol) is added to this mixture and the reaction medium is stirred at 0 C over a period of 2-5 hours. The mixture is poured into cold water ( 110 g) keeping the temperature between -5 C and 3 C. After decantation, the upper aqueous phase is extracted with dichloromethane and the combined organic phases are washed with water, adjusted to pH=6-7 by addition of 27.65% sodium hydroxide, washed with brine and dried over magnesium sulfate. Dichloromethane is distilled off under vacuum and crude 4-bromobutyl nitrate (12.7 g-64.1 mmol) is recovered in a yield of about 96%.

Example 4. Preparation of 3-f f nitroxy)methyl1phenol

3-[(Hydroxy)methyl]phenol (25 g, 0.2 mol), is dissolved in acetonitrile (300 ml) and dichloromethane (900 ml) and the resulting mixture is poured into a flask kept under argon with stirring. The solution is cooled with an ice bath and carbon tetrabromide and triphenylphosphine are added. The latter was added in small portions in order to maintain the temperature at ca. 2-3 C. The solution is stirred for 1 hour at 2-3 C followed by an additional hour at room temperature. The reaction conversion is checked by TLC, using EtOAc/Petroleum ether 3/7 as the eluent) until complete. The obtained mixture is evaporated under reduced pressure and 500 ml of petroleum ether and 500 ml of EtOAc is added to the resulting yellow thick oil in a 21 round flask. The mixture is stirred at room temperature overnight and subsequently filtered and concentrated under reduce pressure, furnishing ca. 50 g of an oily residue. The oil is purified by flash

chromatography over 600 g of silica gel, using EtOAc/Petroleum ether 2/8 as the eluent. Further purification is achieved by crystallizing the resulting bromide from petroleum ether. A white solid is obtained (24 g, 64%). HPLC purity: >98%; FT-IR (KBr, cm ^-1 ): 3252, 1589, 1479, 1392, 1270, 1208, 1155, 952, 880, 791, 741, 686.

3-[(Bromo)methyl]phenol is dissolved in 30 ml of acetonitrile and poured in the flask, kept far from light sources and stirred at 0-5 C under argon. Silver nitrate is added under these conditions, maintaining the temperature under 5 C. The reaction course is followed by TLC (EtOAc/Petroleum ether 3/7 as the eluent). After 4 hours and 30 minutes the conversion is complete. The reaction mixture is filtered, the precipitated solid is washed with ether and the filtrate iss separated in two batches. The first batch (15 ml) is kept under argon and in acetonitrile solution at -20 C. The second batch (15 ml) is worked-up as follows. The acetonitrile solution is concentrated under reduce pressure and the resulting oil is dissolved in dichloromethane (15 ml) and washed with brine (15 ml). The organic phase is separated and the aqueous phase is extracted twice with

dichloromethane (2x 25 ml). The combined organic phases are dried over MgS0 ₄ , filtered and evaporated. The residue is purified by flash chromatography over 40 g of silica gel using EtOAc/Petroleum ether 2/8 as the eluent The nitrate is obtained as an oil (0.6 g, 67%). HPLC purity: >98%; MS (ESI-): 168 (M.sup.+-1); FT-IR (neat oil, cm-'): 3365, 1632, 1599, 1459, 1282, 1160, 923, 867, 793, 757.

Example 5.

11β Hyoなxy-17α-emoxycai¾onyloxyan<irost-4-en-3-one-17β-c arboxylic acid is combined with an equivalent amount of IN sodium hydroxide in methanol and that solution is diluted to 100 times the original volume with ethyl ether. The suspension which results is refrigerated for 1 hour. Then, the crystals which form are removed by filtration, dried in an evacuated desiccator, and dissolved in hexamethylphosphoramide (10% weight/volume). A portion of the resultant solution containing 1 mole of the acid salt, i.e. of sodium 11β-hydroxy-17α-ethoxycarbonyloxyandrost-4-en-3-one-17β- carboxylate, is combined with 4 moles of 4-bromobutyl nitrate. The reaction mixture is maintained at room temperature for 3 hours, then is diluted to 10 times the original volume with ethyl acetate. The diluted reaction mixture is washed successively with 5% sodium thiosulfate, 3% sodium bicarbonate, and water. The organic layer is separated, dried with magnesium sulfate and filtered. The filtrate is concentrated in vacuo and purified by crystallization from a suitable solvent (ethyl ether or tetrahydrofuran/hexane). There is thus obtained butylnitroxy- 11β-hydroxy-17α-ethoxycarbonyloxyandrost-4-en-3- one-17β-carboxylate. The product is characterized by the structural formula

In a similar manner, substitution of an equivalent quantity of 17α-butoxycarbonyloxy- 11β-hydroxyandrost-4-en-3-one-17β-carboxylic acid for the steroidal acid used in this example and substantial repetition of the procedure there detailed affords, as the intermediate salt, sodium 17α-butoxycarbonyloxy-11β-hydroxyandrost-4-en-3-one-17β- carboxylate, and, as the final product, butylnitroxy-17α-butoxycarbonyloxy-l lp- hydroxyandrost-4-en-3-one- 17β-carboxylate.

Example 6.

11β Hydroxy-17α-emoxycarbonyloxyandrost-4-en-3-one-17β-carboxy lic acid is combined with (1.2 mmol) in chloroform (SO ml) in inert atmosphere. 3-(nitrooxy- methyl)phenol (2.4 mmol) and DMAP (cat amount) is added. The reaction is cooled at 0 C and EDAC ( 1.8 mmol) is added. The reaction was stirred at room temperature for 24 hours. The solution is treated with water, the organic layers are added to sodium sulfate and concentrated under reduced pressure. The residue is purified by flash

chromatography, eluent n-hexane/ethyl acetate 3/7. The product is characterized by the structural formula

Example 7.

11β Hydroxy- 17α-ethoxycarbonyloxyandrost-4-en-3-one-17β-carboxylic acid is combined with (1.2 mmol) in chloroform (SO ml) in inert atmosphere. 3-(bromopropyl) alcohol (0.4 g, 1.92 mmol) and DMAP (cat. amount) are added. The reaction is cooled at 0 C and EDAC (0.37 g, 1.92 mmol) is added. The reaction is stirred at room temperature for 5 hours. The solution is treated with water, the organic layers are added to sodium sulfate and concentrated under reduced pressure. The residue is purified by flash chromatography, eluent n-hexane/ethyl acetate 3/7. The elutants are collected and the volume reduced under reduce pressure to provide Compound A.

A solution of compound A (0.7 mmol) and silver nitrate (0.23 g, 1.4 mmol) in acetonitrile (50 ml) is stirred at 40 C in the dark for 4 hours. The precipitated (silver salts) are filtered and the solvent is evaporated under vacuum. The residue is purified by flash chromatography, eluent n-hexane/ethyl acetate 7/3. The product is characterized by the structural formula

Example 8.

11β Hydroxy- 17α-ethoxycarbonyloxyandrost-4-en-3-one-17β-carboxylic acid is combined with (1.2 mmol) in chloroform (SO ml) in inert atmosphere.4-bromomethyl)- benzyl alcohol (0.4 g, 1.92 mmol) and DMAP (cat amount) are added. The reaction is cooled at 0 C and ED AC (0.37 g, 1.92 mmol) is added. The reaction is stirred at room temperature for 5 hours. The solution is treated with water, the organic layers are added to sodium sulfate and concentrated under reduced pressure. The residue is purified by flash chromatography, eluent n-hexane/ethyl acetate 3/7. The elutants are collected and the volume reduced under reduce pressure to provide Compound A.

A solution of compound A (0.7 mmol) and silver nitrate (0.23 g, 1.4 mmol) in acetonitrile (50 ml) is stirred at 40 C in the dark for 4 hours. The precipitated (silver salts) are filtered and the solvent is evaporated under vacuum. The residue is purified by flash chromatography, eluent n-hexane/ethyl acetate 7/3. The product is characterized by the structural formula

Evaluation of Nitric Oxide-Mediated Activity

The formation of cyclic guanosine-3',5' monophosphate (cGMP) in cells in the eye is involved in the regulation of aqueous humor flow. Thus, elevation of cGMP levels leads to decreased aqueous humor production and reduction of intraocular pressure. One can measure the effects of test drugs on cGMP formation in a well established cell assay. Undifferentiated pheochromocytoma cells (PC12) are used. The monolayer cells are incubated for 45 min in Hank's Balanced Salt Solution enriched with 10 mM Hepes, 5 mM MgCl ₂ and 0.05% ascorbic acid at the final pH of 7.4 and containing 100 μΜ of the phosphodiesterase inhibitor, isomethyl-butyl-xanthine (IBMX), 30 μΜ of the guanylyl cyclase inhibitor, YC-1, and the test drugs at the appropriate concentration. The reaction is terminated by the removal of the incubating buffer followed by the addition of 50 L of 100% ice-cold ethanol. The plate is then dried under hot air steam and the residue dissolved, extracted and analysed using commercially available cyclic cGMP enzyme immunoassay kit. Example 9.

This prophetic example illustrates a method of making a gel according to the present invention, although the production of larger amounts of gel may be necessary to meet commercial demands. In the present example, the gel is produced with water that is suitable for injection purposes (injection grade). To produce 500 g of polyacrylate gel, 1.22 g of polyacrylic acid (packaged under the trademark Noveon ^® AA-1 Polycarbophil) is carefully suspended, with the aid of an ultrasonic apparatus, in about 700 ml water and autoclaved for 20 minutes at 121 °C and 2 bar absolute pressure (about 202 kPa). In 700 ml of sterile injection-grade water is then dissolved 0.050 g of benzalkonium chloride (BAK), 20 g sorbitol and 0.0S g of sodium EDTA dihydrate, which is then subjected to sterile filtering (Sartorius ^® cellulose nitrate filter, order no. 11307-50 ACN, 0.2 urn) into a sterile vessel. This sterile-filtered solution is then mixed, with strong agitation, into the autoclaved polyacrylic acid suspension. Sterile water in the amount of 1958.121 g is then added, and the solution is subjected to further agitation for 5 to 10 minute. Subsequently, strong sodium hydroxide in the amount of 0.465 g is dissolved in exactly 40 g of injection-grade water. This caustic soda is then introduced drop-wise under agitation over a sterile filter (Millex-GS, 0.22 urn, SLGS 025 BS der Fa Millipore). The mixture is agitated until the formation of a completely homogenous gel.

A microbially sterile form of Example 5 in the amount of 5 g (or a different amount is used for a different desired strength) is then slowly and carefully mixed with about 30 to 50 g of the gel. The gel is subjected to sterile filtration of the solution, and separation with water containing a bacteriocide under sterile conditions. The rest of the gel mixture (in total 495 g) is carefully incorporated once the compound is uniformly suspended. All method steps are carried out under aseptic conditions.

The prepared gel is likewise drawn off in tubes under aseptic conditions. By an alternative method, the microbially sterile Example 5 is suspended in a sterile-filtrated isotonic solution of 700 ml water, 0.05 g benzalkonium chloride, 20 g sorbitol and 0.05 g of disodium EDTA. This solution is then, as already described, incorporated, under strong agitation, in the autoclaved polyacrylate suspension. Further adaptation or modification of the invention, corresponding to the described production of sterile polyacrylic acid gel, falling within the scope of the following claims may occur to the skilled artisan. Mix one part (by weight) of phase Π with one part (by weight) of phase I for more than 15 minutes (and up to 10 hours) and adjust pH to 6.3-6.6 using 2N NaOH (for the foregoing formulation, about 1.6-1.7 g of 2N NaOH is adequate). The formulation has an osmolality of about 285 mOsm/kg and a viscosity of 1140 cp, as measured by Brookfield rheometer at conditions as disclosed hereinabove.

Example 10

This invention has been described by reference to certain preferred embodiments;

however, it should be understood that it may be embodied in other specific forms or variations thereof without departing from its special or essential characteristics. The embodiments described above are, therefore, considered to be illustrative in all respects and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description.