CA2300850A1 - Pharmaceutical compositions for the treatment of ocular inflammations comprising dexamethasone palmitate - Google Patents

Abstract

Ocular inflammations are treated by an oil-in-water type emulsion carrying an effective amount of a lipophilic ester of dexamethasone.

Description

PHARMACEUTICAL COMPOSITIONS FOR THE TREATMENT
OF OCULAR INFLAMMATIONS COMPRISING
DEXAMETHASONE PALMITATE
- FIELD OF TI~ INVENTION
The present invention provides a novel formulation for the treatment of ocular inflammations and its use.
BACKGROUND OF THE INVENTION
In recent years, oil-in water type emulsions increase in impor-tance as vehicles for delivery of hydrophobic drugs, for example the fat emulsion described in EP 315079. .
U.S. Patent 4,340,594 describes a parenterally administered fat emulsion containing a steroid having an anti-inflammatory activity, inter alia dexamethasone palmitate. The fat emulsions described therein are useful in the treatment of rheumatism, immunological hemolytic anemia, idiopathic thrombocytopenic purpura, Paget disease or in conjunction with kidney transplantation.
Dexamethasone palmitate works as a prodrug since it is hydro-lyzed in the body by esterases into its bioactive metabolite, dexamethasone.
Nonetheless, dexamethasone palmitate has some advantages over conven-tional water-soluble dexamethasone compositions owing to its lipid content.

SUMMARY OF THE INVENTION
The present invention is based on the new finding that fat emulsions containing dexamethasone palmitate as the active ingredient are useful in the treatment of ocular inflammations.
S Thus, the present invention relates to a pharmaceutical composition in the form of an oil-in-water emulsion for the treatment of ocular inflammations, comprising, as an active ingredient, an effective amount of a lipophilic ester of dexamethasone, e.g. dexamethasone palmitate.

Emulsion is a dispersion of one liquid in a second immiscible liquid. Since the majority of emulsions contain water as one of the phases, it is customary to classify emulsions into two types: the oil-in-water (O/W) type consisting of colloid particles dispersed in water, and the water-in-oil (W/O) 15 type in which the phases are reversed. The continuous liquid is referred as the dispersion medium and the liquid which is in the form of particles is called the disperse phase.
In order to achieve a stable emulsion, a third component - an emulsifying agent, must be present. In an O/W type emulsion for example, 20 each colloid particle has an oily core and an external layer comprising the emulsifiers and the surface active substances. A commercially available fat emulsion which contains 10% soybean oil is Intralipid~ (Pharmacia AB, Sweden).
Oil-in-water type emulsions, which thus comprise oil-based 25 particles dispersed in an aqueous medium, are capable of incorporating into their oily core or into their interfacial film, various hydrophobic drugs, and accordingly have been proposed and used as pharmaceutical carriers of such drugs. The present invention relates to such an oil-in-water type emulsion comprising a lipophilic ester of dexamethasone, as the pharmaceutical active ingredient {namely the drug), for the treatment of ocular inflammations.
The emulsion typically comprises, in addition to the active ingredient, an oil component and another component, being an emulsifier and/or a surface active agent. In addition, the aqueous phase (dispersion medium) typically comprises an osmotic pressure regulating agent dissolved therein, for raising the osmotic pressure to physiological levels.
In the following, concentrations of the ingredients of the emulsion will be given as %, meaning weight of ingredient in hundred volume units of total composition (w/v).
The composition of the invention comprises an effective amount of a lipophilic ester of dexamethasone. The term "effective amount"
should be understood as an amount sufficient to impart a therapeutic effect.
Obviously, the effective amount in the composition depends on the dosage form, on the therapeutic regime (namely whether the composition is given once daily, twice daily, etc.), the age group of the patient, the severity of the inflammation, as well as on various other factors as known per se. The artisan will have no di~culties in determining, by routine and straight-forward experimentation, as to what constitutes an effective amount in each case.
20 Typical examples of lipophilic esters of dexamethasone are esters of dexamethasone with fatty acids, typically such acids having chains of 6 to about 22 carbon atoms. Particular examples of such fatty acid are palmitic acid, oleic acid, linoleic acid, stearic acid and others. A
particularly preferred fatty acid is palmitic acid (namely the active agent is preferably dexamethasone palmitate). Dexamethasone palmitate may typically be included in the composition ranging between 0.05%, typically 0.1% and preferably 0.15% to 0.5%, typically 0.4% and preferably 0.3%.
The oil component is typically a vegetable oil. Examples of vegetable oils are oleic acid. linoleic acid, lauric acid, soybean oil, olive oil, sunflower oil, and others. Vegetable oil is typically included in a concentra-tion range of about 5% to 30% (w/v).
Typical emulsifiers are phospholipids. Examples of phospholipids are phosphatidylcholin, phosphatidylethanolamine, 5 phosphatidylinositol, phosphatidylserine and sphingomyelins. Phospholipids are preferably included in a concentration range of about 0.5%-10% (w/v), and particularly in a concentration range of I-5%.
Typical examples of surfactants are polyalkylene glycols having an average molecular weight of 1,000 to 10,000, polyoxyalkylene 10 copolymers having an average molecular weight of 1,000 to 10,000 and poly oxyethylene fatty acid esters.
Examples of osmotic pressure regulators are sucrose or glycerine.
In addition to the above ingredients, the compositions of the 15 inventions may comprise various preservation agents such as methyl, ethyl and butyl paraben, or antioxidants such as a.-tocopherol and ascorbic acid.
The pharmaceutical composition of the invention is typically provided in a suitable dosage form, e.g. in the form of eye drops, with the concentration of the active ingredient being such so that a given amount of 20 drops, e.g. 1-2 to be administered at each time.
The present invention also provides a method of treatment of ocular inflammations comprising topically applying to the eye an oil-in-water type emulsion, comprising a lipophilic ester of dexamethasone as the active ingredient.
25 Still further provided by the invention is use of a lipophilic ester of dexamethasone and an oil-in-water type emulsion, for the preparation of a topical ocular composition for the treatment of ocular inflammations.
The invention will be illustrated below, with reference to some non limiting examples.

BRIEF DESCRIPTION OF THE DRAWING
Figs. 1-3 are bar graphs (means t SD) showing the overall score of clinical examination (Fig. 1), the aqueous humor protein content (Fig. 2) and the aqueous humor cell count (Fig. 3) in a variety of treatments: control {A,B), treatment with a reference composition (C,D) and treatment with the inventive composition {E,F). In each case the left eye was injected with LPS (results are shown as columns A, C and E in each of Figs. 1-3) and the right eye was injected with saline {results shown as columns B, D and F in each of Figs. 1-3 ).
1. EXPERIMENTAL PROCEDURES
Animal Husbandry Fifteen (15) male NZW rabbits, supplied by a local breeder {Levinstein, Yokneam, Israel) and weighing 2.0-2.5 kg were used in the study.
The albino rabbit has been selected for this study, since it is widely acclaimed in reports published in the scientific literature, as a most suitable experimental model for the evaluation of newly developed and clinically applied agents for the therapy of anterior uveitis.
On arrival each animal was inspected and only suitable and healthy animals were expected for use. Animals were acclimated for 5 days following receipt during which they ware observed daily for signs of ill health. No abnormalities were detected.
Rabbits were kept within a limited access laboratory animal facility, at environmental conditions of a target temperature of 17 to 23°C, a target humidity of 30-70% and a 12-hour light/12-hour dark cycle. Temperature and relative humidity were recorded daily. No deviation from the target values were observed.

Rabbits were housed individually in stainless steel cages, mounted in batteries. The cages measured 50x45x48 cm and were fitted with perforated stainless steel floors over undertrays.
Animals were uniquely identified by ear number tattoo.
Each cage was outfitted with a cage card visible on the front and containing the study number, animal number, sex and strain of the animals and all relevant details regarding treatment.
A complete commercial pelleted rabbit diet {ANiBAR 19701) was fed without restriction and the animals were allowed free access to water supplied via a cage-side water bottle.
2. TEST AND CONTROL COMPOSITIONS
Test composition: a commercially available composition comprising an oil-in-water type emulsion containing dexamethasone palinitate at a concentration of 4 mg/ml (LIMETHASONTM, Green Cross Corporation, Japan).
Reference composition: a commercially available clear aqueous ophthalmic solution, containing 1 mg/ml dexamethasone disodium phosphate (STERODEXTM, Fischer Pharmaceutical Labs. (1975) Ltd., Israel).
3. EXPERIMENTAL DESIGN CONDITIONS
Allocation of Animals to Test Groups Animals were assigned to test groups, in a blinded fashion to prevent experimental bias on the part of the person conducting clinical and laboratory examinations. One group was treated with the Test composition, one with the Reference composition and the third group was an untreated control group.
The code detailing the respective treatment a particular test animal was subjected to, was opened only upon termination of both clinical and laboratory examinations.

Induction of Experimental Uveitis EIU was carried out in all animals of the three test groups by endotoxin lipopolysaccharide (LPS-E.coli), injected intravitreally at a dose of 2 ~,g (40 ~.1 of 50 p,l LPS/1 ml HPCD (hydroxypropyl cyclodextrin]) into the left 5 eye of each test animal. Throughout the procedure, animals were anesthetized by intramuscular injection of Ketamin HCL 30 mg/kg and Xylazine HCL 5 mg/kg.
EIU Control 10 Contralateral (right) eyes served as controls for LPS-injected eyes, and were injected intravitreally in an identical fashion, using 50 ~.1 of sterile saline.
4. TREATMENT
15 Immediately following induction of experimental uveitis, rabbits of both the Test and Reference composition groups received the first of a total of six (6) repeated ocular instillations, by applying one drop of the respective preparations into both eyes (LPS and Saline injected eyes). Subsequently, two further instillations were carried at a time interval of about 3 to 4 hours.
20 On the next day and following the 24-hour clinical examination period (see below), the remaining three installations were made in an identical fashion and at equally spaced time intervals.
5. OBSERVATIONS AND EXAMINATIONS
25 Clinical Examination Clinical examination of the rabbits' eyes were carried out 24 and 48 hours following LPS injection and based on scoring of ~conjunctival inflammatory response, iridai hyperemia and anterior chamber flare, according to the detailed grading scale shown below:

_$_ I. Gradin~Scale - Conjunctiva 0 = Normal. Vessels of the palpebral and bulbar conjunctiva easily observed. May appear blanched to reddish pink but without perilimbal injection.
1 = Flushed, reddish color predominantly confined to the palpebral conjunctiva with some perilimbal injection, primarily confined to upper and lower parts of eye.
2 = Bright red color of the palpebral conjunctiva with accompanying perilimbal injection covering at least 75% of the circumference of the perilimbal region.
3 = Dark, beefy red color with congestion of both bulbar and palpebral conjunctivae, along with pronounced perilimbal injection and the presence of petechia on the conjunctiva.
II. Grading Scale - Iris 0 = Normal. Iris without any hyperemia of iridal vessels. occasion-ally, near the pupillary borders a small area of about 1-2 mm in diameter, slightly hyperemic secondary and tertiary vessels may be discerned.
20 1 = Minimal injection of secondary vessels (but not tertiary).
Generally uniform, but may be of greater intensity at 1:00 or 6:00 o'clock positions. If visible intensive injection of vessels confined to that area, tertiary vessels are considered to be substantially hyperemic.
2 = Minimal injection of tertiary vessels and minimal to moderate injection of secondary vessels or iris stroma swelling alone.
3 = Moderate injection of secondary and tertiary vessels. . In addition, slight swelling of iris stroma (most prominent near the 3:00 and 9:00 o'clock positions).

_g_ 4 = Marked injection of the secondary and tertiary vessels with marked swelling of the iris stroma. Iris appears rugose and may be accompanied by hemorrhage in the anterior chamber.
5 III. Grading Scale - Anterior Chamber Flare 0 = Normal. Complete absence of flare.
1 = Barely detectable, faint flare.
2 = Moderate flare. Iris and lens detail clear.
3 = Marked iris flare. Iris and lens details hazy.
4 = Intense flare. Fixed coagulated aqueous humor with considerable fibrin.
The scores for all three clinical parameters, determined at each observation period, were separately totaled for each eye of individual animals.
The. scores for the 24 and 48 hour examination were added and served as "Cumulative Score ".
Aqueous Humor Differential Cell Count Following the 48-hours clinical evaluation of eyes, animals were 20 anesthetized (Ketamin HCL 100 mg/kg and Xylazine HCL 15 mg/kg by i.m.
injection) and aqueous humor was sampled from both eyes of each test animal, using an appropriate hypodermic needle and syringe. Differential cell counts were determined using a hemocytometer.
Cell count values were expressed as the total number of different cell 25 types examined (mononuclear and segmented leukocytes; erythrocytes;
epithelial cells), in both eyes of individual animals.

Measurement of Protein Content in Aqueous Humor Immediately after collection of aqueous humor samples for cell count examination, the remaining aqueous humor was centrifuged for 5 minutes in MIKRO 12/24 centrifuge (Hettich Zentrifugen). The supernatant was then submitted to protein assay, using a standard commercial assay kit (BioRad Standard III). Protein content of aqueous humor was determined for both eyes of all test animals.
Preservation of Eyes for HistopatholoQical Examination Following aqueous humor aspiration all test animals were sacrificed and their eye was enucleated and fixed in Davidson's solution.
6. RESULTS AND DISCUSSION
The results of this study are presented in Figs. 1-3 and in Table 1 below; Table 1 summarizes the data for all test groups, with respect to the evaluation criteria of clinical grading, aqueous humor protein content and cell count, for each of the three types of treatments.
Table 1: Summary of Mean Values ~ SD for EIU Parameters Measured in the Left Eye (LPS-injected) of Rabbits "Cumulative Aqueous Humor Aqueous Humor Test Groups Score" ClinicalProtein. Conc. Cell Count E~camination (mg/m1) (103gmm3) STERODEXi'M 6.2 02.49) 87.2 (t16.40) 15.6 013.48) * *

LIME'I~-IASONTM 4.0 (x-0.71) 68.0 09.51) 12.0 06.28) * *

Untreated Controls13.6 03.26) 132.5 05.89) 20.0 019.82) * Statistically significant difference from Untreated Control group using the Student t-test (p<0.05) Figs. 1-3 show the same results presented in Table 1 in a more illustrative graphic form: Fig. 1 - overall criterion of clinical examination;
Fig. 2 - aqueous humor protein content; Fig. 3 - aqueous humor cell count.
Each of the bars represented as A, B, C, D, E and F, show the mean and the standard deviation values, of the following:
A, B - untreated control; A - LPS-injected left eye and B -saline-injected right eye.
C,D - animals treated with the reference composition; C - LPS-injected left eye, D - saline-injected right eye.
E,F - animals treated with the test composition; E - LPS-injected left eye, F - saline-injected right eye.
The data pertaining to animals of the Untreated control test group clearly show the marked differences in experimental parameters between left (LPS-injected) and right (saline-injected) eyes, reflecting the well known acute inflammatory reactions to intravitreal LPS injection in the EIU model.
Thus, within 24 hours after the intravitreal injections, the left eyes of all animals of the group exhibited distinct clinical evidence of marked conjunctival and iridal congestion and anterior chamber flare and which practically remained the same at the time of the 48-hour inspection. In none of these animals, the right control eyes injected with intravitreal sterile saline showed evidence of inflammation at the two successive inspections.
Determinations of both aqueous protein content and cell count in untreated controls, confirmed the clinical observations. Particularly with respect to the former, there was a striking dii~erence between the two contralateral eyes, shown by a mean protein concentration of about 130 mg/ml in LPS-injected (left) eyes as compared to the normal level of about 2-4 mg/ml in control (right) eyes. Judging by the data . of total cell counts, and at least under the conditions of the study, this parameter seemingly was not as indicative and consistent a measure as that of protein content, towards assessing the extent of acute ocular inflammations.
Both the Test composition and the Reference Composition effectively reduced acute inflammatory response resulting from EIU evidenced in that it 5 suppressed all three examined experimental criteria. Thus, with respect to the "Cumulative Score" for grading clinical signs, the relative effects of the two compositions in reducing inflammation in left. (LPS-injected) eyes vs. corre-sponding eyes in the Untreated Control group were of statistical significance (p<.OS). Similar statistically significant (p<.OS) treatment effects was demonstrated by the reduction in the levels of aqueous humor protein - concentration by both compositions.
Regarding the comparative treatment effectiveness of both composi-tions, an apparent consistent enhanced activity by the Test composition was observed in all of the examined experimental parameters. This relative 15 increase was 'especially evident from the data pertaining to both the "Cumulative Score" of clinical grading and aqueous protein content. In terms of percentage (%) differences, the Test composition treatment caused a reduction of about 70 and 50% . in clinical severity and protein content, respectively, as compared to a corresponding reduction of about 55 and 35%
following the Reference composition. This shows that optimal concentration of the dexamethasone palmitate may be lower than that in the Test composition, e.g. 0.2% or 3%.
Treatment applied to the right saline-injected control eyes, did not reveal any obvious treatment-related effects.

Claims (5)

1. Use of a lipophilic ester of dexamethasone and an oil-in-water type emulsion for the preparation of a topical eye composition for the treatment of ocular inflammation.

2. Use according to Claim 1, wherein the dexamethasone ester is dexamethasone palmitate.

3. Use according to Claim 1 or 2, wherein said composition is in the form of eye drops.

4. A method of treatment of ocular inflammations comprising topically applying to the eye an oil-in-water type emulsion, comprising a lipophilic ester of dexamethasone as the active ingredient.

5. A method according to Claim 4, wherein the dexamethasone ester is dexamethasone palmitate.