WO1991012004A1 - Topical use of 6-n-butyl-1,4,7,10-tetrahydro-4,10-dioxo-1,7-phenanthroline-2,8-dicarboxylic acid derivatives - Google Patents

Abstract

The application concerns a method for the prophylaxis or treatment of inflammatory conditions initiated by an immune response, by topical administration of compounds of formula (I), wherein R?1 and R2¿ independently are hydroxyl, substituted or unsubstituted amino or -OR groups, wherein R is a substituted or unsubstituted aliphatic hydrocarbon group, or a pharmaceutically acceptable salt thereof.

Description

TOPICAL USE OF

6-N-BUTYL-l,4,7,10-TETRAHYDRO-4,10-DIOXO-l,7-

PHENANTHROLINE-2,8-DICARBOΣYLIC ACID DERIVATIVES

Field of the Invention

This application is a Continuation-in-Part of U.S Application Serial No. 07/476,834, filed February 7, 1990. Th present invention relates to the topical use of 6-n-butyl 1,4,7, lθ-tetrahydro-4, 10-dioxo-l,7-phenanthroline-2,8 dicarboxylic acid derivatives. More particularly, the presen invention concerns methods for the treatment of ocular an dermatological inflammatory conditions initiated by an immun response, by topical delivery of an effective amount of suc compounds or their pharmaceutically acceptable salts thereof

Background Art 6-n-Butyl-l, 4 , 7 , l0-tetrahydro-4 , 10-dioxo-l, 7 phenanthroline-2,8-dicarboxylic acid (disodium salt: bufrolin is a known compound, which, together with othe pyridoquinolinone derivatives, is disclosed in the Unite States Patent No. 3,790,577 (UK Patent No. 1,308,787). Th compounds are described as useful in the treatment of asthma o other syndromes or diseases initiated by antigen-antibod reaction, including hay fever, urticaria and autoimmun diseases, and their pharmaceutical compositions are subject o the United States Patent No. 3,984,551. According to thes patents, the pharmaceutical compositions may be in form designed for oral or. intravenous administration or fo inhalation, and contain from 1% to 50% by weight activ ingredient.

Bufrolin's anti-allergic activity in several in vitro an in vivo models has been investigated by Evans, D.P. an Thomson, D.S., and is, for example, described in the followin publications: Evans, Nature 250. 587-593 (1974) ; Evans, Int Archs. Allergy Appl. Immun. 49. 417-427 (1975) ; and Evans an Thomson, Br. J. Pharmac♦ 53. 409-418 (1975) . Systemic anti allergic activity was tested in rats, mice and guinea pigs using various models of immediate hypersensitivity reactions The compound was administered intravenously, dissolved i saline or in a buffer immediately before initiation of the Typ I hypersensitivity reaction.

As a reference compound, disodium 5,5'-[(2 hydroxytrimethylene)dioxy]bis[4-oxo-4H-l-benzopyran-2 carboxylate] (cromolyn sodium; disodium cromoglycate; DSCG) wa employed. This is the first compound that was described t exert its activity through the stabilization of mast cel membranes, thereby preventing degranulation and release o vasoactive substances and inflammatory mediators. Mast cell are tissue cells that resemble a peripheral blood basophil an contain granules with serotonin and histamine present. Thes cells play an important role in allergic reactions. Followin the first exposure to an antigen, the target responds wit antibody formation. Cytotropic antibodies such as IgG or Ig fix to the surface of mast cells. With the second exposure, the antigen binds to these tissue-fixed antibodies and react with them at the cell surface, triggering an enzymatic cascad which causes the dissolution or expulsion of mast cel granules. Free histamine and serotonin released from mas cells act on adjacent smooth muscle and vascular endothelia cells, causing the clinical symptoms of anaphylaxis, e.g. bronchospasm, mucus secretion in the case of asthma; edema, itch and vasodilation in the condition called hay fever, etc. [see, for example, "Basic and Clinical Immunology", Fudenberg H.H., Sites, D.P., Caldwell, J.L. , and Wells, J.V. , Eds. Lang Medical Publications (1980), Chapter 22]. The discovery o DSCG (cromolyn) and its introduction into clinical practic [see, for example. Cox, Nature 216. 1328-1329 (1967)] was very significant advance in the treatment of asthma; thus ne compounds of this class are also referred to as "cromolyn-like compounds.

In the above-mentioned animal studies, bufrolin was simila (but more effective at lower doses) to disodium cromoglycate i its anti-allergic properties in the rat. However, thi similarity between the two compounds did not extend to othe species. It was, therefore, concluded that although bufroli possessed some properties in common with disodium cromoglycate it showed significant pharmacological differences, suggestin substantial underlying differences in their action mechanisms

In preclinical trials bufrolin appeared to be a promisin candidate for treating various allergic conditions, includin allergic asthma; however, clinical trials performed in t early 1970's in Europe, did not bring the desired results, a development of this compound as an anti-asthmatic agent h been discontinued (see e.g. Pharmaprojects, PJB Publicati Ltd., Richmond, Surrey, UK., Ref. No. 54867-56-0). The ma disappointment probably was bufrolin"s poor oral absorptio Pickup et al., Br. J. Clin. Pharmac. 4., 357-366 (1977) repo results of the evaluation of the pharmacokinetics of bufrol in both normal and asthmatic subjects. Tritiated compound w administered by inhalation, intravenous, oral or buccal route The radioactivity in plasma, urine, feces, sputum and exhal air was followed. These studies demonstrated that minimal or and no buccal adsorption occurred. As an average, plasma hal life of 16.1 hours was calculated for drug administration the other routes. However, the majority of the drug excreted rapidly.

It is well known that anti-allergic drugs are in great n in the eye therapy. Ocular allergic reactions affect population to varying degrees. Most patients experience m but bothersome symptoms such as itchy, watery eyes associa with systemic allergic diseases and specific ocular disea (i.e. allergic conjunctivitis or atopic conjunctivitis). significant minority are plagued with chronic forms of aller reactions (i.e. vernal._'keratoconjunctivitis, giant papill conjunctivitis, etc.) which can eventually damage the cor and compromise vision.

A 4% ophthalmic solution of sodium cromolyn (Opticro Fisons) is commercially available, for the treatment of ocu allergic symptoms. However, many patients do not benefit f Opticrom therapy, possibly due to its weak activi Therefore, there is a great need for the development of oth more efficacious compounds.

Summary of the Invention The present invention is based on the surprising discov that 6-n-butyl-l,4,7, 10-tetrahydro-4 , 10-dioxo-l, phenanthroline-2,8-dicarboxylic acid, and its es derivatives, amides and salts, can be effectively appl topically, and are highly efficient in the treatment of ocu and dermatological conditions in which mast cells are invol in the pathology. In particular, it has been discovered that topical ocular administration of this compound, its esters, amides and pharmacologically acceptable salts delivers therapeutically effective levels of these drugs to the ocular tissue; hence, these compounds are useful in the treatment of certain ocular inflammatory conditions such as, allergic conjunctivitis, atopic conjunctivitis, vernal keratoconjunctivitis, giant papillary conjunctivitis, or any other inflammatory condition in which the mast cells are known to be involved. Similarly, bufrolin and other derivatives, were found to be able to penetrate the skin, and to be highly efficient in the treatment of inflammatory skin conditions initiated by antigen-antibody reactions.

One of the usual criteria for the selection of mass cell stabilizing drugs for topical, e.g. ocular use is oral efficacy. Since the poor oral absorption of bufrolin results in inactivity in the traditional rat passive cutaneous anaphylaxis (PCA) assay, the topical (ocular or dermatological) efficacy of this compound in treating mass-cell related inflammations was unexpected.

As mentioned hereinabove, the activity of the only commercially available mast cell stabilizer known to have ophthalmic use, the chemically unrelated disodium cromoglycate (DSCG) , is not satisfactory. In experimental allergic conjunctivitis models in rats, passive ocular anaphylaxis (POA) and active ocular anaphylaxis (GPAOP) models in guinea pigs, solutions of bufrolin were found to be significantly more active than the commercially available Opticrom formulations, which were found to be either ineffective or weakly effective. It was even more surprising that in the guinea pig conjunctivitis model GPAOP, the ocular itch (pruritus) was inhibited without the inhibition of vasopermeability. The ocular topical administration of bufrolin was found to be essentially devoid of such typical ocular side-effects as changes in intraocular pressure or pupil diameter. Based upon their excellent pharmacological properties and experimental ocular safety, bufrolin and related compounds are promising candidates for the development of highly efficient drugs for the treatment of topical, in particular ocular, allergic diseases. In one aspect, the present invention relates to a metho for the prophylaxis or treatment of inflammatory condition initiated by an immune response, comprising topicall delivering to an inflammed or normal tissue susceptible t inflammation a therapeutically effective amount of a 6-n-butyl 1,4,7, 10-tetrahydro-4, 10-dioxo-l,7-phenanthroline-2,8 dicarboxylic acid derivative of formula (I)

wherein R and R independently are hydroxyl, substituted o unsubstituted amino or -OR groups, wherein R is a substitute or unsubstituted aliphatic hydrocarbon group, or pharmaceutically acceptable salt thereof. For ocular application, usually ophthalmic solutions ar used which typically contain from about 0.001% to about 10

(w/v) of a compound of formula (I) or a pharmaceuticall acceptable salt thereof.

Such ophthalmic solutions are typically administered in dose of about 1 drop/eye up to eight-times, preferably up t four-times a day.

The dermatological compositions generally are administere in the form of a solution, lotion, gel, cream, ointment suspension, emulsion or skin patch. The preferred compound of formula (I) is 6-n-butyl

1,4,7, 10-tetrahydro-4 , 10-dioxo-l,7-phenanthroline-2 , 8 dicarboxylic acid, preferably as its disodium salt.

Concomitant use of other anti-inflammatory drugs, such a antihistamines, serotonin antagonists, platelet activatin factor (PAF) antagonist, leukotriene antagonists, non-steroida anti-inflammatory agents (NSAI) or corticosteroids, may improv efficiency of treatment.

In another aspect, the present invention relates to pharmaceutical product, comprising: a container adapted to dispense its contents in meter for ; and up to about 15 ml of an ophthalmic solution in tha container, comprising from about 0.001% to about 10% (w/v) o at least one compound of formula (I) , wherein the substituent are as defined above, or a pharmaceutically acceptable sal thereof, in combination with an ophthal ically acceptabl liguid diluent.

Brief Description of Drawings Figure 1 is a diagram illustrating the summarized result of dose-response studies described in Example 1A for the 72 hour POA (passive ocular anaphylaxis) experiments (a model o Type I hypersensitivity in the rat eye lid) .

Figure 2 illustrates the results obtained with 4 solutions. The values in parenthesis represent number o studies. In every study 10 to 14 eyes were used.

Figure 3 illustrates the results obtained with 0.1 solutions in Example 1A for 72-hour POA experiments. Th values in parenthesis represent number of studies.

Figure 4 illustrates the results of the cumulative dose response test in the 72 hour Passive Ocular Anaphylaxis (POA) model (Example IB) .

Figure 5 shows the efficacy of 4% bufrolin solution relative to the time of antigen challenge.

Figures 6 and 7 depict the results of 72 hour and 24 hou comparative POA studies, respectively.

Figures 8 and 9 illustrate the effect of 4% bufroli solutions on the intraocular pressure (IOP) of rabbits.

Figure 10 shows the effect of 4% bufrolin solutions on th pupil diameter (PD) of rabbits.

Detailed Description of the Invention

Methods for preparation of 6-n-Butyl-l,4,7,10-tetrahydro

4,10-dioxo-l,7-phenanthroline-2,8-dicarboxylicacid, itsester and salts are described in the United States Patent No 3,790,577. 6-n-Butyl-l,4,7,10-tetrahydro-4,10-dioxo-l,7 phenanthroline-2,8-dicarboxylic acid can, for example, b prepared starting from n-butylbenzene. Nitration of n butylbenzene yields 2,4-dinitro-n-butylbenzene, which i reduced with hydrogen over Pd/C in ethanol to yield 2,4 diamino-n-butyl-benzene. The diamino-compound may be condense with dimethyl acetylenedicarboxylate and cyclized to affo dimethyl 6-n-butyl-4,10-dihydroxy-l,7-phenanthroline-2, dicarboxylate, which can be hydrolyzed to the desir dicarboxylic acid. Alternatively, the diamino-derivative can also be condens with diethyl oxalacetate and cyclized as before, to yie diethyl-n-butyl-4, 10-dihydroxy-l,7-phenanthroline-2, dicarboxylate which can be hydrolyzed to provide t corresponding dicarboxylic acid. The corresponding esters (R 1 and R2 represent an -OR group are either directly formed in the course of this or a simil synthesis route, or can be prepared by esterification of t appropriate carboxylic acids, or trans-esterification of oth esters, following well known methods of organic chemistr Thus, esterification can be performed with appropriate ester acid halides or anhydrides.

Similarly, technigues for the preparation of dicarboxyl acid monoesters (one of R 1 and R2 i.s hydroxyl and the other o is -OR) are known in the art. For example, monesters can prepared by alcoholysis of dicarboxylic acid anhydride partial hydrolysis of an appropriate diester, etc.

Methods for the preparation of the substituted unsubstituted carboxylic acid amides of the formula (I) a also well known in the art, and include the reaction of t corresponding acid halides or esters with ammonia or primary secondary amines.

In the definition of Rthe aliphatic hydrocarbon groups m be straight or branched chained, saturated or unsaturated, su as straight or branched chained alkyl, alkenyl or alkyn groups, usually containing up to about 12, preferably 1 to more preferably 1 to 4 carbon atoms. For ocular use, compoun in which the R alkyl groups have at least two carbon atoms, a preferred. Typical representatives of the alkyl grou include, for example, methyl, ethyl, fi- and isopropyl, n.-, se , iso- and tert-butyl, n- and isopentyl, n- and neo-hexyl, and isoheptyl, n~ and iso-octyl, etc. groups. Typical alken and alkynyl groups are vinyl, allyl, propenyl, crotyl, ethyn and propargyl.

All of these aliphatic hydrocarbon groups may carry one more identical or different substituents that may, for exampl be selected from alkyl, alkenyl or alkynyl, alkoxy, alkoxycarbonyl, or hydroxy groups, and halogen atoms. In the substituent definitions, the terms "alkyl", "alkenyl", "alkynyl", as such, or as part of other groups, have the same meanings as hereinabove defined.

The term "halogen" is used to include fluorine, chlorine, bromine and iodine, preferably chlorine.

In the definition of R¹ and R , the amino group may optionally be substituted, preferably with alkyl groups, wherein the term "alkyl" has the meanings as defined hereinabove.

If desired, an obtained compound of formula (I) , wherein R , R and R have the same meanings as defined above, is converted into a pharmaceutically acceptable salt thereof, or an obtained salt is converted into the respective free compound or into another pharmaceutically acceptable salt. Pharmaceutically acceptable salts include metal salts and acid addition salts, the metal salts being preferred.

Acid addition salts may be formed with suitable mineral or organic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, citric acid, oxalic acid, lactic acid, maleic acid, etc., and can be prepared by methods known per se. The foreseeable requirement is that they be pharmaceutically acceptable for administration to man. Metal salts of the dicarboxylic acids of formula (I) are readily formed with aqueous metal hydroxides, carbonates, hydrocarbonates, etc. in a known manner, the alkali metal (e.g. sodium, potassium) , and alkali earth metal (e.g. calcium, magnesium, copper, manganese, zinc) salts being preferred. The term "metal salt" is used in a broad sense and is intended to include all salts containing a cationic moiety including amines (e.g. tromethamine) , quaternary ammonium-containing compounds and the ammonium salts. The obtained metal salts can be readily converted back into the corresponding carboxylic acids, e.g. by aqueous mineral acids.

Although, for convenience, the active compounds of this invention are designated and illustrated as 6-n-butyl-l,4,7,10- tetrahydro-4,10-dioxo-l,7-phenanthroline-2,8-dicarboxylicacid derivatives, it will be understood that the same compounds can also exist in the enol-for . Following the known rules of keto-enol tautomerism, there is an equilibrium between the tw forms, which are both intended to be encompassed by the presen invention.

The present invention relates to the topical (ophthalmic o dermatological) application of therapeutically effectiv amounts of the compounds of formula (I) (the substituen definitions are as hereinabove defined) and thei pharmaceutically acceptable salts.

The term "therapeutically effective amount" and grammatica variations thereof, as used herein refer to sufficien quantities of the active compound that can produce the desire therapeutic effect when delivered through the skin, eye o other membrane to the site of inflammation. The ter "therapeutic effect" is used herein in a broad sense an includes prophylactic effects.

The active compounds are administered as pharmaceutica formulations designed for topical administration. It i important that such formulations provide sufficient retentio (contact) time for transmembrane and transdermal penetration o the active ingredient for purpose of exerting its therapeuti activity. If necessary, viscosity can be altered to increas contact time.

There is a direct relationship between viscosity and th efficiency of penetration. If the penetration is efficien for example due to: 1) direct property of the molecule; or 2 the use of penetration enhancers in the topical formulation the viscosity can be lower, and formulations having essentiall the same viscosity as water may be acceptable. If t penetration is slower, more viscous fomulations are required provide sufficient retention time for transmembrane transdermal penetration.

The compositions in accordance with this invention can formulated in a variety of dosage forms for topic application. Such topical formulations may include solution creams, lotions, ointments, gels, sprays, aerosols, sk patches, and the like. All of these dosage forms, along wi methods for their preparation, are well known in t pharmaceutical and cosmetic art.

For ophthalmic application, preferably solutions a prepared typically containing from about 0.001% to about 10 preferably from about 0.1% to about 6%, more preferably from about 0.1% to about 4% of active ingredient, and a physiological saline solution as a major vehicle. The pH of such ophthalmic solutions should be maintained between about 4.5 and about 8.0, preferably between about 6.0 and 7.2 with an appropriate buffer system. The formulations may also contain conventional, pharmaceutically acceptable preservatives, stabilizers and/or penetration enhancers.

The preferred vehicle that may be used in the ophthalmic solutions of the present invention is purified water, more preferably a physiological saline solution. Additional suitable vehicles include but are not restricted to, viscosity agents such as polyvinyl alcohol, povidone, hydroxypropyl methyl cellulose, poloxamers, carboxymethyl cellulose, carbomer and hydroxyethyl cellulose.

Preferred preservatives that may be used in the ophthalmic formulations of the present invention include, but are not limited to, benzalkonium chloride, chlorobutanol, thimerosal, phenylmercuric acetate and phenylmercuric nitrate. Penetration enhancers may, for example, be surface active agents; certain organic solvents, such as dimethylsulfoxide and other sulfoxides, dimethylacetamide and pyrrolidone; certain amides of heterocyclic amines, glycols (e.g. propylene glycol) ; propylene carbonate; oleic acid; alkyl amines and derivatives; various cationic, anionic, nonionic, and amphoteric surface active agents; and the like.

Tonicity adjustors may be added as needed or convenient. They include, but are not limited to, salts, particularly sodium chloride, potassium chloride, mannitol and glycerin, or any other suitable opthalmically acceptable tonicity adjustor.

Various buffers and means for adjusting pH may be used so long as the resulting preparation is ophthalmically acceptable.

Accordingly, buffers include acetate buffers, citrate buffers, phosphate buffers and borate buffers for ophthalmic use. ϊn a similar vein, an ophthalmically acceptable antioxidant for use in the present invention includes, but is not limited to, sodium metabisulfite, sodium thiosulfate, acetylcysteine, butylated hydroxyanisole and butylated hydroxytoluene.

Other excipient components which may be included in the ophthalmic preparations are chelating agents. The preferre chelating agent is edetate disodium, although other chelatin agents may also be used in place or in conjunction with it.

The ingredients are usually used in the following amounts

Ingredient Amount (% w/v) active ingredient about 0.001-6 preservative 0-1.0 vehicle 0-99.9 tonicity adjustor 0-10 buffer 0-10 pH adjustor q.s. pH 4.5-8.0 antioxidant as needed chelating agents 0-1.0% purified water as needed to make 100%

The ophthalmic formulations of the present invention ar conveniently packaged in forms suitable for metere application, such as in containers adapted to discharge thei contents dropwise. Such containers are usually made of suitable, inert, non-toxic plastic material, and may b resealable or may be designed for a single application only, i which case resealing may not be necessary. Non-resealabl containers typically hold from one to about ten, preferably on to about five, more preferably one to about three dose units where a typical dose unit is one drop (about 20-25 μl) . T resealable containers are typically equipped with a droppe to facilitate application to the eye, and generally conta between about 0.5 ml and 15 ml solution.

The topical formulations for dermatological use are usual in the form of solutions, lotions, gels, creams, ointment suspensions, or emulsions. A wide variety of dermatological acceptable inert vehicles well known in the art may employed. Typical vehicles that may be used in dermatologic formulations include, but are not limited to water, eth alcohol, stearyl alcohol, spermaciti, mineral oil, whi petrolatum and gel-producing substances.

Dermatological formulations usually contain the same typ of ingredients as the ophthalmic formulations. For exampl dermatological buffers include the ophthalmically acceptab buffers plus tris (tromethamine) and carbonate buffers. Aci or bases may be used to adjust the pH of these formulations needed .

Application forms especially designed for dermatologica use are, for example, skin or transdermal patches. Basicall there are either reservoir or matrix or monolithic devices i which delivery of the active compound to the skin is controlle by diffusion through the matrix or monolith or via a rat controlling membrane. For the latter, the device might be solution or perhaps suspension in water and glycols (e.g., propylene, polyethylene, etc.) contained in a reservoir on on side of which is an impermeable membrane. On the other side i a suitable semipermeable membrane which delivers therapeutically desirable amount of drug per hour over th desired time.

Alternatively a topical produce may be covered by occlusive or semiocclusive material to enhance th effectiveness, i.e., bioavailability. The active compound ca also be incorporated into a hydrocalloid material which i applied to the skin which by virtue of its occlusion results i high bioavailability. Bufrolin and its derivatives can also b placed in a compatible adhesive on an occlusive backing whic upon application to the area to be treated, releases the activ compound to the affected area. Typical ophthalmic an dermatological preparations of the present invention ar illustrated in the Examples. The ophthalmic preparations of the present invention ar useful in the treatment of certain ocular inflammator conditions in which mast cells are involved in the pathology Such conditions include, but are not limited to, allergic an atopic conjunctivitis, vernal keratoσonjunctivitis, gian papillary conjunctivitis, etc.

The 6-n-Butyl-l,4,7 10-tetrahydro-4, 10-dioxo-l,7 phenanthroline-2,8-dicarboxylic acid derivatives of thepresen invention work most effectively when administered before th release of mast cell mediators, therefore, their prope clinical use is very important. The most effective treatmen regimen starts before the beginning of the allergy season. I the more chronic disease cases where pretreatment is no possible, there is some time before a beneficial effect i seen. Particularly in such cases, concomitant therapy wit either antihistamine, NSAI (Ocufen* or others of this class) o corticosteroids may bring better results. In most cases, t adjunct therapy may be applied locally, however, for examp antihistamines and NSAI agents may be administered orally. T compounds of the present invention are typically administer in a daily dose of 1 drop/eye from one up to 8-time preferably up to 4-times a day, but the frequency application depends on the formulations used, and on t symptoms to be treated. The selection of optimal treatment well within the knowledge of a skilled physician. The leng of treatment is a function of the type and seriousness of t condition to be treated. The vision threatening forms ocular symptoms (vernal keratoconjunσtivitis) require chron therapy to control the disease.

The dermatological preparations of the present inventi can be used for the treatment of atopic dermatitis, acute a chronic inflammatory der atoses, urticarias and other allerg conditions. In addition, the preparations may be useful adjunct therapy in some dermatological infections and wou healing. Further details of the present invention are illustrated the following non-limiting Examples.

EXAMPLE 1 Passive Ocular Anaphylaxis (POA) in the rat conjunctiva The topical ocular efficacy of 6-n-Butyl-l,4,7,1 tetrahydro-4,10-dioxo-l,7-phenanthroline-2,8-dicarboxylicac disodium (bufrolin) was evaluated in an experimental allerg conjunctivitis model in rats.

A passive ocular anaphylaxis (POA) reaction is a passi form of an allergic response since the antibody is supplied the animal in a specific location. The basic mechanis involved in the passive' cutaneous anaphylaxis (PCA) reacti apply to this model, including passive sensitization a intravenous challenge with antigen. A. In a first set of experiments, POA reaction was elicited in the eyelid of Sprague Dawley albino rats wi a single injection of 10 microliters of murine monoclonal I antibodies (SIGMA) . 72 hours post IgE injection, the rats w challenged with antigen and Evans Blue (EB) intravenously. animals were sacrificed 30 minutes after EB injection, and amount of dye extravasated into the eyelid was determined spectrophotometrically. In most cases, both the vehicle control and the test groups contained 10 to 14 eyes.

Drugs were administered to the eye one minute before antigen challenge. A lower quantity of EB in the tissue, compared with the control animals reflects a reduction in vascular permeability due to stabilization of the conjunctival mast cells and thus a reduction in vasoactive mediator release.

Bufrolin solutions were prepared in the artificial tears, Liquifilm* (Allergan, Inc.). The concentrations were 0.5%, 1%, 2% and 4% (w/v) . Commercially available Opticrom* (Fisons, 4% sodium cromolyn, ophthalmic solution) was included as a reference in each study. In other experiments, 0.5%, 1%, 2% and 4% solutions of sodium cromolyn (prepared the same way as the respective solutions of bufrolin) were also included as a control, to provide a more direct comparison. The results are summarized in Figures 1 through 3.

As illustrated in the Figures, bufrolin inhibited the rat POA at all doses tested. All concentrations of bufrolin inhibited the leakage of Evans Blue into the eyelids. The highest dose (4%) demonstrated the most consistent and maximu effect (about 70% inhibition - see Figures 1 and 2, but even the 0.1% dose proved to be significantly more effective tha Opticrom* (See Figure 3) . These results suggest that the test compound, in particula in the form of 4% solutions, has the potential of superio efficacy in clinical situtations when topical treatment o inflammations mediated by mast cells is required.

Application of 4% solutions of bufrolin into the eyes o laboratory test animals indicates that the compound has n effect on intraocular pressure and pupil size and does no cause acute eye irritation or discomfort.

The following formulations were prepared according t methods well known to the pharmaceutical art. Unless otherwis indicated, the concentrations are in weight-by-volume.

B. In a second set of experiments, Male Sprague-Dawle rats weighing between 175-225 g were used as test animals, typically in groups of 7. Solutions of 6-n-butyl-l,4,7,10 tetrahydro-4,lθ-dioxo-1,7-phenanthroline-2,8-dicarboxylicaci disodium (bufrolin) were prepared on the day of the experiment. Water, saline or Liquifilm Tears* (Allergan, Inc.) was used a the vehicle. The pH of each solution was adjusted t neutrality.

5 μl of a monoclonal murine IgE (Sigma) solution wa injected in the lower palpebral conjunctiva of anesthertize rats to sensitize the mast cells. 72 hours later, the rat were challenged with an intravenous solution of antige (ovalbumin) and Evans Blue (EB) , both obtained from Sigma Thirty minutes after antigen challenge, the animals we sacrificed, the lower eyelid was removed, and the EB extracte overnight. The EB concentration was determine spectrophotometrically against a standard curve.

Drugs were administered to the eye one minute befo antigen challenge, in 10 μl volumes. A reduced concentration in the tissue, compared with the conrol animal represents a reduction of mast cell degranulation or antagoni of the vasoactive mediators.

Dose-response relationship

The dose-response relationship determined in th experiment is illustrated in Figure 4. The results show th bufrolin inhibited the rat POA in a dose-related manner. T amount of inhibition within one experiment demonstrated a dos response. Therefore, a cumulative dose-response figure w constructed using 22 studies, involving a total of 44 treatme groups (264 to 352 rats, 528 to 704 eyes). ED50 value of 1. was calculated from the log curve equation. As shown in Figu 4, the largest number of data points was collected for the 4% Effect of bufrolin pretreatment on the POA The duration of action of a single application of bufrol was evaluated in an indirect manner. A 4% solution of bufrol was administered various times before the intravenous challen with antigen. Approximately half of the topical ocul antiallergic activity was lost in 30 minutes (Figure 5) .

In this figure, the average inhibition from three separa studies is represented. Comparative studies

A comparison of bufrolin's in vivo antiallergic activi with cromolyn sodium and the commercially available Opticro is shown in Figures 6 and 7. The results set forth in Figure 6 summarize data obtain fro the above-described 72 hour POA protocol. Figure 7 illustrates results obtained in a test when a 24 hour sensitization period was used. The 72 hours formal was eventually selected as the screening assay since it appeared to be more reliable. Nevertheless, bufrolin demonstrated superiority over the reference compounds also in the 24 hou POA.

EXAMPLE 2 Guinea Pig Allergic Conjunctivitis This model is a form of an active allergic response since the animals are sensitized to the antigen and develop their ow antibodies which sensitize tissue mast cells.

In this experiment, guinea pigs were sensitized to chicke ovalbumin (chicken egg Grade IV or V, Sigma) with single o multiple injections. Albino, Dunkin-Hartley strain guinea pigs of either sex were used. Typically, the animals weighed 350-

550 g. Once sensitized, a standard concentration of antige was applied to the eye and several parameters were measured.

Ocular itch (pruritus) was measured by observing th animals over a 15 minute period and counting the number o scratch episodes. This method is sensitive to the external distractions, i.e. surroundings, noise, etc. and must therefor be conducted with care for reproducible results. In th present study, the animals were housed 2-3 per cage. The cage were of a standard dimension with metal grid floors. A perio of at least one month was allowed for the animals to settle i their surroundings. Temperature and humidity were regulated a

75 + 2* F and 50 + 5%, respectively.

The studies involved opening the front-access doors t allow uninterrupted observation, and to prevent access to th door-mounted feed hooper. Each animal was taken in turn fro the cage to allow administration of the stimulus.

Drugs and aqueous soluble sensory stimuli were applied t one eye in a 20μl volume. The animal was then replaced in th cage.

Itch-scratch responses were recorded over a 15 minut period beginning at the point when the last animal was replace in the cage.

A 4% solution of bufrolin effectively reduced the pruriti response to topical ovalbumin. The results are summarized i Table 1 below.

Table 1

455 I.C.I. VS Θ.SX OVALBUMIN INOUCE OCULAR PRURITUS

10 11 12 13 ROW SUM

1 VEHICLE 11 29 11 17 ιe 1β 7 1Θ 153

2 ICI 2 13 5 3 ι 4 4 2 34

14 ROW 15 ROW 16 ROW 17 ROW 18 ROW MEAN STDEV CV N SEM

1 VEHICLE 11.25 β.27 β.5β 12 1.81

2 ICI 4.5Θ 5.94 β.aa 12 1.14

EXAMPLE 3 Histamine-Induced Palpebral Vasopermeability fHIPV) This model evaluates the topical ocular activity again histamine-induced increases in palpebral vasopermeabilit Male Sprague-Dawley rats weighing between about 175 and 225 were used in groups of 7. A non-treated and/or vehicle contr group was always used.

The tissue extravasation of Evans Blue dye (Sigma) w induced by an eyelid injection of histamine (Sigma) anesthetized rats. The animals received one injection per ey 5 μl in the lower conjunctive of both eyes.

10 μl doses of the test preparations were administered minutes before histamine injection. The animals we sacrificed and the lower eyelid was removed 30 minutes aft histamine injection. The amount of dye extracted from t eyelid was a measure of vasopermeability and was determin spectrophotometrically.

Topically applied bufrolin (4% solution) failed antagonize histamine-induσed increases in vasopermeability the rat HIPV model. The mean + sem Evans Blue concentration the rat eyelid in the treated group was not different from th of the water control (26+1.2 vs 24+1.2, 14 eyes eac respectively) . These results indicate that bufrolin does n show histamine antagonism. EXAMPLE 4 Effect on Intraocular Pressure flOP) and Pupil Diameter Bufrolin dissolved in Liquifilm* (Allergan, Inc.) in the desired concentration, and Liquifilm* alone as vehicle were tested in New Zealand X Dutch Belted (NZxDB) rabbits to determine the effect on intraocular pressure (IOP) and pupil diameter (PD) .

IOP measurements were performed with a Digilab Pneumotonograph and PD was determined with an Optistick* following standard procedures.

At t=0 hour, the pupil diameter was measured. One drop of

Ophthetic* (0.5% proparacaine HC1) diluted 1:10 with physiological saline, was placed in both eyes prior to each IOP reading. The IOP was measured for each eye until a stable reading was obtained.

Immediately following the t=0 reading in both eyes, 25μl of either a 4% solution of bufrolin in Liquifilm* or Liquifilm* alone was instilled across the cornea in the test eye while th contralateral eye received 25μl of saline. IOP and P measurements were taken at 0.5, 1, 2, 3, 4 and 5 hours.

The results are illustrated in Figures 8-10. The data sho that neither the tested bufrolin solution nor Liquifilm* has a effect on IOP of PD in rabbits.

EXAMPLE 5 Ophthalmic solution

Bufrolin 0.1%

Phosphate buffer 0.02%

Physiological saline solution as needed

EXAMPLE 6

Ophthalmic Solution

Bufrolin 4.0%

Phosphate buffer 0.02% Benzalkonium chloride (Preservative) 0.001% EDTA (Chelating agent) 0.05% Physiological saline solution as needed

The White Petrolatum USP may be replaced by White Ointme

USP. Either of these may be modified as to consistency replacing some fraction of them with:

Mineral Oil USP

Light Mineral Oil NF White Wax NF

Paraffin NF

Microcrystalline Wax NF

Polyethylene NF

Hydrophilic Petrolatum USP may be modified by addition a suitable quantity of materials named in Example 6 to adju its consistency. Alternatives to Cholestrol NF and Stear Alcohol NF in the Hydrophilic Petrolatum USP that can included, both individually and in combinations: Cetyl Alcohol NF -2 „_Λ0-

Cetostearyl Alcohol NF Cetyl Esters Wax NF

Sorbitan mono and polyesters, such as Sorbita Monolaurate NF, Sorbitan Monopalmitate NF, Sorbita Monooleate, Sorbitan Trioleate, Sorbitan Sesquioleate, etc. Sucrose mono and diesters, such as Sucrose Distearate Glyceryl Monostearate NF and non-compendial Propylene Glycol Monostearate NF, and non-compendial Lanolin Alcohols NF Lanolin USP

Anhydrous Lanolin USP

Mono- and Di-Glycerides NF

Other lipophilic surface active materials

Bufrolin may first be dissolved in a suitable quantity of water, preferably to provide an approximately saturate solution.

EXAMPLE 11 Ointment-Water Soluble Bufrolin 1.0% Polyethylene Glycol Ointment NF 99.0%

The consistency may be adjusted by:

1. altering the proportions of Polyethylene Glycol (PEG) 400 and PEG 3350 in the above; 2. replacing some portion of the PEG 400 and/or PEG 335 with other PEG NF having a nominal molecular weigh between 300 and 8000. Bufrolin may first be dissolved in a suitable quantity o water as described in Example 7.

Stearyl Alcohol may be replaced by an identical quantity o another pure or mixed alkanol with C₁₄-C₂₀ of pharmaceutical o cosmetic quality. EXAMPLE 13 Emulsion-W/O

Bufrolin 1.0%

Cold Cream USPXX1 (or

Rose Water Ointment USP) 99.0%

Other examples may be found in the formularies published excipient suppliers.

EXAMPLE 14 Emulsion-O/W

Bufrolin 1.0%

Hydrophilic Ointment USP 99.0%

EXAMPLE 15 Gel Bufrolin 1.0%

Carbomer 934 NF 1.0%

NaOH NF q.s. to pH 5-7.5 Preservative q.s.

Purified Water USP q.s. ad. 100.0%

The amount of Carbomer 934 may be adjusted upward downward to achieve a more or less viscous product. Carbo 934 may be replaced by a suitable amount of: Carbomer 934P NF Carbomer 940 NF Carbomer 941 NF Carbomer 954

NaOH may be replaced by KOH NF or Trolamine NF or ot suitable inorganic or organic base (see list on page 1857 USP XXII) . The preservative may also be chosen from the l on page 1857 of USP XXII. The most preferred are: Benzyl Alcohol NF 0.2-1.5%

I idurea NF 0.2-1.5%

Methylparaben NF 0.1-1.0% Combinations of the above might include: Benzyl Alcohol and Imidurea 0.5% each

The formulation of this Example may be further stabili by the addition of Edetate Disodium USP at a concentration

0.005-0.1%. The gel could be buffered to the pH range no using any pharmaceutically acceptable buffer (see page 1857 USP XXII) . A humectant may be added (ibid.) at the level of 1- 15%.

EXAMPLE 16 Gel Bufrolin 1.0%

Hydroxypropyl Cellulose NF q.s. Preservative(s) q.s.

Buffer q.s.

Humectant q.s. Purified Water USP q.s. ad. 100.0%

The amount of Hydroxypropyl Cellulose may be varied to achieve different viscosities. Other cellulose derivatives such as Methycellulose USP, Carboxymethyl Cellulose Sodium USP, Hydroxypropyl Methylcellulose NF, or Hydroxyethyl Cellulose NF may be used at similar concentrations.

EXAMPLE 17

The quantity of Poloxamer 407 to give a desired gel is modified by the type and amount of humectant and interactions with the active ingredient.

A short chain alcohol may be added, e.g., Ethanol USP or Isopropyl Alcohol USP in a concentration of 0-20%.

A preservative, preferably selected from the list given in Example 12, may also be added. See list for Example 12.

EXAMPLE 18

The Viscosity Agent may be anything from the list on pag 1858 of USP XXII but preferably is: Carbomer 934, 034P, 940, 941 Carboxymethlcellulose Sodium Hydroxyethyl Cellulose Hydroxypropyl Cellulose Hydroxpropyl Methylcellulose Methylcellulose Xanthan Gum

EXAMPLE 21

1.0%

.0%

10.0% Preservative q.s. q.s. q.s.

Trolamine 2.0% 1.0%

Purified Water q.s. ad. 100.0%

EXAMPLE 22

Emulsion-W/O

Cream Cream

Bufrolin 1.0% 1.0%

Petrolatum 54.0% 28.0%

Sobitan Sesquioleate 6.0% 2.0%

Humectant q.s. q.s.

Polysorbate 80 NF 1.0%

Preservative q.s q.s.

Purified Water q.s. ad 100.0%

The foregoing description details specific formulations an methods that can be employed to practice the present invention. Having detailed specific compositions for the topical formulations of the present invention and specific instructions for their use in the treatment of ocular and dermatological allergic symptoms, the art skilled will well enough know how t devise other formulations and how to adapt the treatmen (formulations, doses) to a special situation. Thus, howeve detailed the foregoing may appear in text, it should not b construed as limiting the overall scope hereof; rather, th ambit of the present invention is to be governed only by th lawful construction of the appended claims.

Claims

Claims :

1. A method for the prophylaxis or treatment o inflammatory conditions initiated by an immune response comprising topically delivering to an inflammed or norma tissue susceptible to inflammation a therapeutically effectiv amount of a 6-n-butyl-l,4,7,10-tetrahydro-4,10-dioxo-l,7 phenanthroline-2,8-dicarboxylic acid derivative of formula (I

wherein R and R independently are hydroxyl, substituted o unsubstituted amino or -OR groups, wherein R is a substitute or unsubstituted aliphatic hydrocarbon group, or pharmaceutically acceptable salt thereof.

2. The method according to Claim 1, wherein said compoun of formula (I) (R , R and R are as defined in Claim 1) or pharmaceutically acceptable salt thereof, is administered t the eye as an ophthalmic solution comprising from about 0.001 to about 10% (w/v) of said compound or salt.

3. The method according to Claim 2, wherein sai ophthalmic solution comprises from about 0.1% to about 6% (w/v of said compound or salt.

4. The method according to Claim 3, wherein said compoun is 6-n-butyl-l,4,7,10-tetrahydro-4,10-dioxo-l,7-phenanthroline 2,8-dicarboxylate or the disodium salt thereof.

5. The method according to Claim 3 or Claim 4, wherei said compound of formula (I) or a pharmaceutically acceptabl salt thereof, is administered in a dose of about 1 drop/eye to eight-times a day.

6. The method according to Claim 5, wherein said compou of formula (I) or a pharmaceutically acceptable salt thereo is administered in a dose of about 1 drop/eye up to four-tim a day.

7. A pharmaceutical product, comprising: a container adapted to dispense its contents in meter form; and up to about 15 ml of an ophthalmic solution in said container, comprising from about 0.001% to about 10% (w/v) of at least one compound of formula (I)

wherein R and R independently are hydroxyl, substituted or unsubstituted amino or -OR groups, in which R is a substituted or unsubstituted aliphatic hydrocarbon group, or a pharmaceutically acceptable salt thereof, in combination with an ophthalmically acceptable liquid diluent.

8.- The pharmaceutical product according to Claim 7, wherein said ophthalmic solution comprises from about 0.1% to about 6% (w/v) of said compound or salt.

9. The pharmaceutical product according to Claim 8, wherein said compound is 6-n-butyl-l,4,7,10-tetrahydro-4,10- dioxo-l,7-phenanthroline-2,8-dicarboxylic acid or the disodium salt thereof.

10. The pharmaceutical product according to Claim 9, wherein said ophthalmically acceptable liquid diluent is a physiological saline solution.

11. The pharmaceutical product according to Claim 10, wherein said ophthalmic solution further comprises a pharmaceutically acceptable buffer adjusting the pH of said solution to between about 4.5 and about 8.0.

12. The pharmaceutical product according to Claim 11, wherein said ophthalmic solution has a substantially neutral pH.

13. The pharmaceutical product according to Claim 12, wherein said ophthalmic solution further comprises a pharmaceutically acceptable preservative.

14. The pharmaceutical product according to Claim 12, wherein said ophthalmic solution further comprises a pharmaceutically acceptable antioxidant.

15. The pharmaceutical product according to Claim 9 wherein said container is non-resealable and holds from one t about ten dose units of said solution.

16. The pharmaceutical product according to Claim 15 wherein said container holds from one to about five dose unit of said solution.

17. The pharmaceutical product according to Claim 9 wherein said container is equipped with a dropper.

18. The pharmaceutical product according to Claim 17 wherein said container holds from about 0.5 to about 10 ml o said solution.

19. The pharmaceutical product according to Claim 18 wherein said container holds from about 0.5 to about 5 ml o said solution.

20. The use of a compound of formula (I)

wherein R and R independently are hydroxyl, substituted o unsubstituted amino or -OR groups, in which R is a substitute or unsubstituted aliphatic hydrocarbon group, or pharmaceutically acceptable salt thereof, in the preparation o a pharmaceutical composition useful for treatment o prophylaxis of topical inflammatory conditions initiated by a antigen-antibody reaction, or immune reactions, comprisin admixing said compound of formula (I), wherein R¹, R² and R a as hereinabove defined, or said pharmaceutically acceptabl salt thereof, with a non-toxic, pharmaceutically acceptabl vehicle suitable for topical application.