IE910394A1 - Topical pharmaceutical formulations containing¹6-n-butyl-1,4,7,10-tetrahydro-4,10-dioxo-1,¹7-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 R<2> 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

Field of the Invention This application is a Continuation-in-Part of U.S.

Application Serial No. 07/476,834, filed February 7, 1990. The present invention relates to the topical use of 6-n-butyl1,4,7,10-tetrahydro-4,10-dioxo-l, 7-phenanthroline-2, βίο dicarboxylic acid derivatives. More particularly, the present invention concerns methods for the treatment of ocular and dermatological inflammatory conditions initiated by an immune response, by topical delivery of an effective amount of such compounds or their pharmaceutically acceptable salts thereof.

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

Bufrolin's anti-allergic activity in several in vitro and in vivo models has been investigated by Evans, D.P. and Thomson, D.S., and is, for example, described in the following publications: Evans, Nature 250. 587-593 (1974); Evans, Int, Archs, Allergy AppI, Immun, 49. 417-4 27 (1975) ; and Evans and Thomson, Br, J, Pharmac. SI» 409-418 (1975). Systemic antiallergic activity was tested in rats, mice and guinea pigs, using various models of immediate hypersensitivity reactions. The compound was administered intravenously, dissolved in saline or in a buffer immediately before initiation of the Type -2I hypersensitivity reaction.

As a reference compound, disodium 5,5’-[(2hydroxytrimethylene)dioxy]bis[4-oxo-4H-1-benzopyran-2 carboxylate] (cromolyn sodium; disodium cromoglycate; DSCG) was employed. This is the first compound that was described to exert its activity through the stabilization of mast cell membranes, thereby preventing degranulation and release of vasoactive substances and inflammatory mediators. Mast cells are tissue cells that resemble a peripheral blood basophil and contain granules with serotonin and histamine present. These cells play an important role in allergic reactions. Following the first exposure to an antigen, the target responds with antibody formation. Cytotropic antibodies such as IgG or IgE fix to the surface of mast cells. With the second exposure, the antigen binds to these tissue-fixed antibodies and reacts with them at the cell surface, triggering an enzymatic cascade which causes the dissolution or expulsion of mast cell granules. Free histamine and serotonin released from mast cells act on adjacent smooth muscle and vascular endothelial 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. Lange Medical Publications (1980), Chapter 22]. The discovery of DSCG (cromolyn) and its introduction into clinical practice [see, for example, Cox, Nature 216. 1328-1329 (1967)] was a very significant advance in the treatment of asthma; thus new compounds of this class are also referred to as cromolyn-like 0 compounds.

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

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

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

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

Summary of the Invention The present invention is based on the surprising discovery that 6-n-butyl-l,4,7,10-tetrahydro-4,10-dioxo-l,7phenanthroline-2,8-dicarboxylic acid, and its ester derivatives, amides and salts, can be effectively applied topically, and are highly efficient in the treatment of ocular and dermatological conditions in which mast cells are involved -4in 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. 0 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. -5In one aspect, the present invention relates to a method for the prophylaxis or treatment of inflammatory conditions initiated by an immune response, comprising topically delivering to an inflammed or normal tissue susceptible to 5 inflammation a therapeutically effective amount of a 6-n-butyl1,4,7,10-tetrahydro-4,10-dioxo-l,7-phenanthroline-2,8dicarboxylic acid derivative of formula (I) wherein R1 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.

For ocular application, usually ophthalmic solutions are used which typically contain from about 0.001% to about 10% (w/v) of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

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

The dermatological compositions generally are administered in the form of a solution, lotion, gel, cream, ointment, suspension, emulsion or skin patch.

The preferred compound of formula (I) is 6-n-butyl1,4,7,10-tetrahydro-4,10-dioxo-l,7-phenanthroline-2,8dicarboxylic acid, preferably as its disodium salt.

Concomitant use of other anti-inflammatory drugs, such as antihistamines, serotonin antagonists, platelet activating factor (PAF) antagonist, leukotriene antagonists, non-steroidal anti-inflammatory agents (NSAI) or corticosteroids, may improve efficiency of treatment.

In another aspect, the present invention relates to a pharmaceutical product, comprising: a container adapted to dispense its contents in metered -βίο rm; and up to about 15 ml of an ophthalmic solution in that container, comprising from about 0.001% to about 10% (w/v) of at least one compound of formula (I) , wherein the substituents are as defined above, or a pharmaceutically acceptable salt thereof, in combination with an ophthalmically acceptable liquid diluent.

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

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

Figure 3 illustrates the results obtained with 0.1% solutions in Example IA for 72-hour POA experiments. The values in parenthesis represent numbet of studies.

Figure 4 illustrates the results of the cumulative dose20 response test in the 72 hour Passive Ocular Anaphylaxis (POA) model (Example IB).

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

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

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

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

Detailed Description of the Invention Methods for preparation of 6-n-Butyl-l,4,7,10-tetrahydro4,10-dioxo-l,7-phenanthroline-2,8-dicarboxylic acid, its esters 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,7phenanthroline-2,8-dicarboxylic acid can, for example, be prepared starting from n-butylbenzene. Nitration of nbutylbenzene yields 2,4-dinitro-n-butylbenzene, which is reduced with hydrogen over Pd/C in ethanol to yield 2,440 diamino-n-butyl-benzene. The diamino-compound may be condensed -710 with dimethyl acetylenedicarboxylate and cyclized to afford dimethyl 6-n-butyl-4,10-dihydroxy-l,7-phenanthroline-2,8dicarboxylate, which can be hydrolyzed to the desired dicarboxylic acid.

Alternatively, the diamino-derivative can also be condensed with diethyl oxalacetate and cyclized as before, to yield diethyl-n-buty1-4,10-dihydroxy-l,7-phenanthroline-2,8dicarboxylate which can be hydrolyzed to provide the corresponding dicarboxylic acid.

The-corresponding esters (R1 and R2 represent an -OR group) , are either directly formed in the course of this or a similar synthesis route, or can be prepared by esterification of the appropriate carboxylic acids, or trans-esterification of other esters, following well· known methods of organic chemistry. Thus, esterification can be performed with appropriate esters, acid halides or anhydrides.

Similarly, techniques for the preparation of dicarboxylic acid monoesters (one of R1 and R2 is hydroxyl and the other one is -OR) are known in the art. For example, monesters can be prepared by alcoholysis of dicarboxylic acid anhydrides, partial hydrolysis of an appropriate diester, etc.

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

In the definition of Rthe aliphatic hydrocarbon groups may be straight or branched chained, saturated or unsaturated, such as straight or branched chained alkyl, alkenyl or alkynyl groups, usually containing up to about 12, preferably 1 to 6, more preferably 1 to 4 carbon atoms. For ocular use, compounds in which the R alkyl groups have at least two carbon atoms, are preferred. Typical representatives of the alkyl groups include, for example, methyl, ethyl, q- and isopropyl, Q-, sec, iso- and tert-butyl, n- and isopentyl, n- and neo-hexyl, fland isoheptyl, n- and iso-octyl, etc. groups. Typical alkenyl and alkynyl groups are vinyl, allyl, propenyl, crotyl, ethynyl and propargyl.

All of these aliphatic hydrocarbon groups may carry one or more identical or different substituents that may, for example, -8be 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 R2, 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 R1, R2 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,4,7,10tetrahydro-4,10-dioxo-l,7-phenanthroline-2,8-dicarboxylicacid derivatives, it will be understood that the same compounds can also exist in the enol-form. Following the known rules of -9keto-enol tautomerism, there is an equilibrium between the two forms, which are both intended to be encompassed by the present invention.

The present invention relates to the topical (ophthalmic or 5 dermatological) application of therapeutically effective amounts of the compounds of formula (I) (the substituent definitions are as hereinabove defined) and their pharmaceutically acceptable salts.

The term ’’therapeutically effective amount” and grammatical 10 variations thereof, as used herein refer to sufficient quantities of the active compound that can produce the desired therapeutic effect when delivered through the skin, eye or other membrane to the site of inflammation. The term therapeutic effect” is used herein in a broad sense and includes prophylactic effects.

The active compounds are administered as pharmaceutical formulations designed for topical administration. It is important that such formulations provide sufficient retention (contact) time for transmembrane and transdermal penetration of the active ingredient for purpose of exerting its therapeutic activity. If necessary, viscosity can be altered to increase contact time.

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

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

For ophthalmic application, preferably solutions are prepared typically containing from about 0.001% to about 10%, ι -10preferably 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 10 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.

In 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 preferred -11chelating agent is edetate disodium, although other chelating agents may also be used in place or in conjunction with it.

The ingredients are usually used in the following amounts: Ingredient active ingredient preservative vehicle tonicity adjustor buffer pH adjustor antioxidant chelating agents purified water Amount (% w/v) about 0.001-6 0-1.0 0-99.9 0-10 0-10 q.s. pH 4.5-8.0 as needed 0-1.0% as needed to make 100%.

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

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

Dermatological formulations usually contain the same types of ingredients as the ophthalmic formulations. For example, dermatological buffers include the ophthalmically acceptable buffers plus tris (tromethamine) and carbonate buffers. Acids or bases may be used to adjust the pH of these formulations as -12needed.

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

Alternatively a topical produce may be covered by a occlusive or semiocclusive material to enhance the effectiveness, i.e., bioavailability. The active compound can also be incorporated into a hydrocalloid material which is applied to the skin which by virtue of its occlusion results in high bioavailability. Bufrolin and its derivatives can also be placed in a compatible adhesive on an occlusive backing which upon application to the area to be treated, releases the active compound to the affected area. Typical ophthalmic and dermatological preparations of the present invention are illustrated in the Examples.

The ophthalmic preparations of the present invention are useful in the treatment of certain ocular inflammatory conditions in which mast cells are involved in the pathology. Such conditions include, but are not limited to, allergic and atopic conjunctivitis, vernal keratoconjunctivitis, giant papillary conjunctivitis, etc.

The 6-n-Butyl-1,4,7,10-tetrahydro-4,10-dioxo-l,7phenanthroline-2,8-dicarboxylic acid derivatives of the present invention work most effectively when administered before the release of mast cell mediators, therefore, their proper clinical use is very important. The most effective treatment regimen starts before the beginning of the allergy season. In the more chronic disease cases where pretreatment is not possible, there is some time before a beneficial effect is seen. Particularly in such cases, concomitant therapy with either antihistamine, NSAI (Ocufen· or others of this class) or -13corticosteroids may bring better results. In most cases, the adjunct therapy may be applied locally, however, for example antihistamines and NSAI agents may be administered orally. The compounds of the present invention are typically administered in a daily dose of 1 drop/eye from one up to 8-times, preferably up to 4-times a day, but the frequency of application depends on the formulations used, and on the symptoms to be treated. The selection of optimal treatment is well within the knowledge of a skilled physician. The length of treatment is a function of the type and seriousness of the condition to be treated. The vision threatening forms of ocular symptoms (vernal keratoconjunctivitis) require chronic therapy to control the disease.

The dermatological preparations of the present invention can be used for the treatment of atopic dermatitis, acute and chronic inflammatory dermatoses, urticarias and other allergic conditions. In addition, the preparations may be useful as adjunct therapy in some dermatological infections and wound healing.

Further details of the present invention are illustrated in the following non-limiting Examples.

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

A passive ocular anaphylaxis (POA) reaction is a passive form of an allergic response since the antibody is supplied to the animal in a specific location. The basic mechanisms involved in the passive cutaneous anaphylaxis (PCA) reaction apply to this model, including passive sensitization and intravenous challenge with antigen.

A. In a first set of experiments, POA reaction was elicited in the eyelid of Sprague Dawley albino rats with a single injection of 10 microliters of murine monoclonal IgE antibodies (SIGMA) . 72 hours post IgE injection, the rats were challenged with antigen and Evans Blue (EB) intravenously. The animals were sacrificed 30 minutes after EB injection, and the -14£ 91394 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 5 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 0 inhibited the leakage of Evans Blue into the eyelids. The highest dose (4%) demonstrated the most consistent and maximum effect (about 70% inhibition - see Figures 1 and 2, but even the 0.1% dose proved to be significantly more effective than Opticrom® (See Figure 3).

These results suggest that the test compound, in particular in the form of 4% solutions, has the potential of superior efficacy in clinical situtations when topical treatment of inflammations mediated by mast cells is required.

Application of 4% solutions of bufrolin into the eyes of laboratory test animals indicates that the compound has no effect on intraocular pressure and pupil size and does not cause acute eye irritation or discomfort.

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

B. In a second set of experiments, Male Sprague-Dawley rats weighing between 175-225 g were used as test animals, typically in groups of 7. Solutions of 6-n-butyl-l,4,7,10tetrahydro-4,10-dioxo-l,7-phenanthroline-2,8-dicarboxylicacid disodium (bufrolin) were prepared on the day of the experiment. -15Water, saline or Liquifilm Tears· (Allergan, Inc.) was used as the vehicle. The pH of each solution was adjusted to neutrality. μΐ of a monoclonal murine IgE (Sigma) solution was 5 injected in the lower palpebral conjunctiva of anesthertized rats to sensitize the mast cells. 72 hours later, the rats were challenged with an intravenous solution of antigen (ovalbumin) and Evans Blue (EB), both obtained from Sigma. Thirty minutes after antigen challenge, the animals were sacrificed, the lower eyelid was removed, and the EB extracted overnight. The EB concentration was determined spectrophotometrically against a standard curve.

Drugs were administered to the eye one minute before antigen challenge, in 10 μΐ volumes. A reduced EB concentration in the tissue, compared with the conrol animals represents a reduction of mast cell degranulation or antagonism of the vasoactive mediators.

Dose-response relationship The dose-response relationship determined in this experiment is illustrated in Figure 4. The results show that bufrolin inhibited the rat POA in a dose-related manner. The amount of inhibition within one experiment demonstrated a doseresponse. Therefore, a cumulative dose-response figure was constructed using 22 studies, involving a total of 44 treatment groups (264 to 352 rats, 528 to 704 eyes). ED50 value of 1.4% was calculated from the log curve equation. As shown in Figure 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 bufrolin was evaluated in an indirect manner. A 4% solution of bufrolin was administered various times before the intravenous challenge with antigen. Approximately half of the topical ocular antiallergic activity was lost in 30 minutes (Figure 5).

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

Comparative studies A comparison of bufrolin's in vivo antiallergic activity with cromolyn sodium and the commercially available Opticrom· is shown in Figures 6 and 7.

The results set forth in Figure 6 summarize data obtained -16from the above-described 72 hour POA protocol illustrates results obtained in a test when 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 hour POA.

Figure 7 a 24 hour EXAMPLE 2 Guinea Pig Allergic Conjunctivitis 10 This model is a form of an active allergic response since the animals are sensitized to the antigen and develop their own antibodies which sensitize tissue mast cells.

In this experiment, guinea pigs were sensitized to chicken ovalbumin (chicken egg Grade IV or V, Sigma) with single or multiple injections. Albino, Dunkin-Hartley strain guinea pigs of either sex were used. Typically, the animals weighed 350550 g. Once sensitized, a standard concentration of antigen was applied to the eye and several parameters were measured.

Ocular itch (pruritus) was measured by observing the animals over a 15 minute period and counting the number of scratch episodes. This method is sensitive to the external distractions, i.e. surroundings, noise, etc. and must therefore be conducted with care for reproducible results. In the present study, the animals were housed 2-3 per cage. The cages were of a standard dimension with metal grid floors. A period of at least one month was allowed for the animals to settle in their surroundings. Temperature and humidity were regulated at 75 + 2* F and 50 + 5%, respectively.

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

Drugs and aqueous soluble sensory stimuli were applied to one eye in a 20μ1 volume. The animal was then replaced in the cage.

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

A 4% solution of bufrolin effectively reduced the pruritic 40 response to topical ovalbumin. The results are summarized in Table 1 below.

Table 1 4% I.C.I. VS 9.5% OVAL3U4IN INOUCE OCULAR PRURITUS 1 23 + 5 «7 39 19111213 ROW SUM VEHICLE 11 7 29 11 ICI 2 * 13 3 β 10 3 3 4 11 ,9 7 4 19 2 133 a 14 row MEAN ROW STOEV ROW CV ROW N ROW SEM VEHICLE 11.23 ICI 4.39 3.27 9.33 3.94 9.33 1.31 1.14 EXAMPLE 3 ' Histamine-Induced Palpebral Vasopermeabilitv CHIPV) This model evaluates the topical ocular activity against histamine-induced increases in palpebral vasopermeability. Male Sprague-Dawley rats weighing between about 175 and 225 g were used in groups of 7. A non-treated and/or vehicle control group was always used.

The tissue extravasation of Evans Blue dye (Sigma) was induced by an eyelid injection of histamine (Sigma) in anesthetized rats. The animals received one injection per eye: 5 μΐ in the lower conjunctive of both eyes. μΐ doses of the test preparations were administered 30 minutes before histamine injection. The animals were sacrificed and the lower eyelid was removed 30 minutes after histamine injection. The amount of dye extracted from the eyelid was a measure of vasopermeability and was determined spectrophotometrically.

Topically applied bufrolin (4% solution) failed to antagonize histamine-induced increases in vasopermeability in the rat HIPV model. The mean + sem Evans Blue concentration in the rat eyelid in the treated group was not different from that of the water control (26+1.2 vs 24+1.2, 14 eyes each, respectively). These results indicate that bufrolin does not show histamine antagonism. i -18EWLS 4 Effect on Intraocular Pressure (IOP) 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 HCI) 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, 25μ1 of either a 4% solution of bufrolin in Liquifilm· or Liquifilm· alone was instilled across the cornea in the test eye while'the contralateral eye received 25μ1 of saline. IOP and PD measurements were taken at 0.5, 1, 2, 3, 4 and 5 hours. The results are illustrated in Figures 8 -10. The data show that neither the tested bufrolin solution nor Liquifilm· has an effect on IOP of PD in rabbits. EXAMPLE 5 25 Ophthalmic solution Bufrolin 0.1% Phosphate buffer 0.02% Physiological saline solution as needed 30 EXAMPLE 6 Ophthalmic Solution Bufrolin 4.0% Phosphate buffer 0.02% Benzalkonium chloride (Preservative) 0.001% 35 EDTA (Chelating agent) 0.05% Physiological saline solution as needed -19EXAMPLE 7 Ophthalmic Ointment Bufrolin Lanolin Alcohol Chlorobutanol Mineral Oil White Petrolatum 1.0% 2.0% 0.5% 42.5% 55.0% EXAMPLE 8 Ophthalmic Aqueous Gel Bufrolin 1.0 % Carbopol 940 1.0% Benzalkonium Chloride 0.005% EXAMPLE 9 Ointment Bufrolin 1.0% White Petrolatum USP 99.0% The White Petrolatum USP may be replaced by White Ointment USP. Either of these may be modified as to consistency by replacing some fraction of them with: Mineral Oil USP Light Mineral Oil NF White Wax NF Paraffin NF Microcrystalline Wax NF Polyethylene NF EXAMPLE 10 Ointment Bufrolin 1.0% Hydrophilic Petrolatum USP 99.0% Hydrophilic Petrolatum USP may be modified by addition of a suitable quantity of materials named in Example 6 to adjust its consistency. Alternatives to Cholestrol NF and Stearyl Alcohol NF in the Hydrophilic Petrolatum USP that can be included, both individually and in combinations: Cetyl Alcohol NF -20Cetostearyl Alcohol NF Cetyl Esters Wax NF Sorbitan mono and polyesters, such as Sorbitan Monolaurate NF, Sorbitan Monopalmitate NF, Sorbitan 5 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 saturated 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 3350 with other PEG NF having a nominal molecular weight between 300 and 8000.

Bufrolin may first be dissolved in a suitable quantity of water as described in Example 7.

EXAMPLE 12 Ointment Bufrolin 1.0% Purified Water USP 5.0% Stearyl Alcohol NF 5.0% PEG Ointment NF 89.0% Stearyl Alcohol may be replaced by an identical quantity of another pure or mixed alkanol with C14-C20 of pharmaceutical or 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 by excipient suppliers.

Emulsion-O/W Bufrolin Hydrophilic Ointment Gel Bufrolin Carbomer 934 NF NaOH NF q.s. to pH Preservative Purified Water USP EXAMPLE 14 1.0% USP 99.0% EXAMPLE 15 1.0% 1.0% -7.5 q.s. q.s. ad. 100.0% The amount of Carbomer 934 may be adjusted upward or downward to achieve a more or less viscous product. Carbomer 934 may be replaced by a suitable amount of: Carbomer 934P NF 25 Carbomer 940 NF Carbomer 941 NF Carbomer 954 NaOH may be replaced by KOH NF or Trolamine NF or other suitable inorganic or organic base (see list on page 1857 of USP XXII). The preservative may also be chosen from the list on page 1857 of USP XXII. The most preferred are: Benzyl Alcohol NF 0.2-1.5% Imidurea 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 stabilized by the addition of Edetate Disodium USP at a concentration of 0.005-0.1%. The gel could be buffered to the pH range noted using any pharmaceutically acceptable buffer (see page 1857 of USP XXII). A humectant may be added (ibid.) at the level of 115%.

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 Gel Bufrolin Poloxamer 407 NF Humectant Purified Water USP q.s. ad. 1.0% .0-25.0% 1.0% 100.0% 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 Solution Bufrolin Buffer Preservative Humectant Viscosity Agent Alcohol Purified Water q.s. ad. 1.0% q.s. q.s. q.s. 0-1.0% 0-20.0% 100.0% The Viscosity Agent may be anything from the list on page -231858 of USP XXII but preferably is: Carbomer 934, 034P, 940, 941 Carboxymethlcellulose Sodium Hydroxyethyl Cellulose Hydroxypropyl Cellulose Hydroxpropyl Methylcellulose Methylcellulose Xanthan Gum EXAMPLE 19 Gel Bufrolin 1.00% DEA Oleth-3 Phosphate 6.80% Oleth-3 4.06% Oleth-5 2.72% Mineral Oil 13.60 Hexylene Glycol 3.40% Propylene Glycol 1.40% Purified Water 67.02% EXAMPLE 20 G©Z Bufrolin 1.0% Mineral Oil 10.0% 0leth-10 20.7% PEG-25 Hydrogenated Castor Oil 10.3% Propylene Glycol 8.6% Sorbitol Solution 70% 6.9% Purified Water 42.5% EXAMPLE 21 Emulsion-O/W Cream Lotion Cream Bufrolin Stearyl Alcohol Stearic Acid Mineral Oil Sorbitan Monostearate Polysorbate 60 Propylene Glycol 1.0% .0% 4.0% --- 2.0% 1.0% .20% 2.0% 2.5% 2.5% .0% 2.0% .0% . 0% -24Preservative Trolamine Purified Water q.s.

Emulsion-W/O Bufrolin Petrolatum Sobitan Sesquioleate Humectant Polysorbate 80 NF Preservative Purified Water q.s. q.s. 2.0% 1.0% ad. 100.0% EXAMPLE 22 Cream Cream 1.0% 1.0% 54.0% 28.0% 6.0% 2.0% q.s. q.s. --- 1.0% q.s q.s. q.s. ad 100.0% q.s.

The foregoing description details specific formulations and 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 0 for their use in the treatment of ocular and dermatological allergic symptoms, the art skilled will well enough know how to devise other formulations and how to adapt the treatment (formulations, doses) to a special situation. Thus, however detailed the foregoing may appear in text, it should not be construed as limiting the overall scope hereof; rather, the ambit of the present invention is to be governed only by the lawful construction of the appended claims.

Claims (19)

Claims:

1. A method for the prophylaxis or treatment of inflammatory conditions initiated by an immune response, comprising topically delivering to an inflammed or normal 5 tissue susceptible to inflammation a therapeutically effective amount of a 6-n-butyl-l,4,7,10-tetrahydro-4,10-dioxo-l,7phenanthroline-2,8-dicarboxylic acid derivative of formula (I) wherein R 1 and R 2 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.

2. The method according to Claim 1, wherein said compound of formula (I) (R 1 , R 2 and R are as defined in Claim 1) or a pharmaceutically acceptable salt thereof, is administered to 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 said 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 compound is 6-n-butyl-l,4,7,10-tetrahydro-4,10-dioxo-l,7-phenanthroline2,8-dicarboxylate or the disodium salt thereof.

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

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

7. A pharmaceutical product, comprising: a container adapted to dispense its contents in metered -26form; 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 1 and R 2 independently are hydroxyl, substituted or 15 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, 20 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-4,10dioxo-l , 7-phenanthroline-2 , 8-dicarboxylic acid or the disodium 25 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, 30 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 wherein said ophthalmic solution further pharmaceutically acceptable preservative.

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

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

16. 19. The pharmaceutical product according to Claim 18, wherein said container holds from about 0.5 to about 5 ml of 15 said solution.

17. 20. The use of a compound of formula (I) wherein R 1 and R 2 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 the preparation of 30 a pharmaceutical composition useful for treatment or prophylaxis of topical inflammatory conditions initiated by an antigen-antibody reaction, or immune reactions, comprising admixing said compound of formula (I), wherein R 1 , R 2 and R are as hereinabove defined, or said pharmaceutically acceptable 35 salt thereof, with a non-toxic, pharmaceutically acceptable vehicle suitable for topical application. -2821. The use of a compound of the formula (I), as defined in any one of claims 1 to 4, for the manufacture of a topical pharmaceutical composition for the treatment or prophylaxis of topically treatable inflammatory conditions initiated by an antigen-antibody reaction, or immune reactions .

18. 22. The use according to claim 1 wherein the inflammatory condition is an ocular inflammatory condition such as allergic conjunctivitis, atopic conjunctivitis, vernal keratoconjunctivitis, or giant papillary conjunctivitis .

19. 23. A pharmaceutical preparation substantially as hereinbefore described with reference to any of Examples 5 to 22.