METHOD FOR THE PREPARATION OF A PHARMACEUTICAL COMPOSITION
TECHNICAL FIELD OF THE INVENTION
The present invention relates to inhibitors of anandamide transport useful for the treatment of intraocular hypertension after topical administration, as well as compositions comprising the same, and methods of use of said compositions to treat intraocular hypertension in a mammal
BACKGROUND OF THE INVENTION
Arachidonylethanolamide (AEA, figure 1 ) was the first reported endogenous hgand for the cannabinoid receptor (Science 258 1946-1949, 1992) and it was named
"anandamide" Later, various arachidonic acid-based esters, such as 2-arachιdonoyl glycerol (Biochem Pharmacol 50 83-90, 1995), palmitoylethanolamide (J Biol Chem
272 3315-3323, 1997), homo-gamma-hnolenic acid (J Med Chem 36 3032-3034, 1993) and docosatetraenoic acid (J Med Chem 36 3032-3034, 1993) were reported as endogenous gands for cannabinoid receptors (CB 1 or CB2, which are the two known subtypes of cannabinoid receptors) Later this entire group of compounds was called "anandamides" The synthetic anandamides synthesized include substitutions at the α/pbα- carbon of the ethanolamide and 2-carbon of the fatty acid (Life Sci 56 2041 - 2048, 1995), modifications of the ethanolamide chain length (Mol Pharmacol 46 516- 522, 1994) and replacement of the terminal hydroxy moiety (J Pharmacol Exp Therap
273 1172-1181, 1995) Various physiological functions such as protection against neurodegeneration and neural damage, participation as a blood pressure regulation or immune system regulator and roles in memory and cognition, have been suggested to endogenous anandamides (Eur J Pharmacol 359 1 -18, 1998)
Figure 1 The chemical structure of arachidonylethanolamide (AEA)
Recent studies have shown that anandamides decrease intraocular pressure (IOP) in rabbits after topical administration (Curr Eye Res 14: 791-797, 1995; Life Sci 63: 2181- 2188, 1998; Pharm Res 14: 1738-1743, 1997; Jpn J Ophthalmol 41: 217-220, 1997). The endogenous anandamides (arachidonylethanolamide and 2-arachidonoyl glycerol) and their analogs (modifications in the ethanolamide chain length or terminal hydroxy moiety) show an initial increase and subsequent decrease of IOP in the treated eyes after topical administration (Curr Eye Res 14: 791-797, 1995; Life Sci 63: 2181-2188, 1998). In contrast, the anandamide analogs, which have modifications at the α pbα-carbon, do not show this initial rise of IOP after ocular administration, although the hypotensive effect is clear (Pharm Res 14: 1738-1743, 1997). Blockade of the CB-1 receptor with a highly specific antagonist eliminates the hypotensive effect of α/pbα-carbon substituted anandamides and other cannabinoids (Life Sci 63: 2181-2188, 1998).
Recently, it was shown that arachidonylethanolamide is formed and hydrolyzed in the eye (Exp Eye Res 64: 707-711, 1997) and the existance of a CBl-receptor in the rat eye has also been demonstrated (Mol Brain Res 58: 240-245, 1998). The presence of arachidonylethanolamide-hydrolysing enzyme and CB-receptor in the eye suggests a physiological role of arachidonylethanolamide for controlling intraocular pressure.
DETAILED DESCRIPTION OF THE INVENTION
The present invention concerns the novel use of inhibitors of the anandamide transport mechanism. These inhibitors are useful in the treatment of intraocular hypertension after topical administration, with or without topical administration of endogenous anandamides or their analogs.
The present invention is based on the finding that arachidonylethanolamide, endogenous anandamide, is inactivated by a cellular uptake mechanism (active anandamide transport) and that inhibitors of the anandamide transport slow the inactivation of endogenous anandamides and increase the concentration of anandamides in the eye, which results in a decreased intraocular pressure (IOP).
Another object of the present invention is the use of the inhibitors of anandamide-
transport to potentiate and to prolong the IOP decreasing effect of topically administered endogenous anandamides or their analogs
The object of the present invention is thus a method for the preparation of a pharmaceutical composition for the topical treatment of intraocular hypertension, containing an active agent and optionally at least one pharmaceutically acceptable carrier or adjuvant, wherein the active agent is an anandamide transport inhibitor
An object of the invention is also a method for the topical treatment of intraocular hypertension in a mammal m need of such treatment, comprising administering to an eye of said mammal an effective amount of an anandamide transport inhibitor The anandamide transport inhibitor is preferably administered to the eye as a pharmaceutical composition containing at least one carrier or adjuvant
According to the invention, the anandamide transport inhibitor is preferably N-(4-hydro- xyphenyl)arachιdonylamιde or its equivalent analogue By an equivalent analogue is meant an analogue which inhibits the anandamide transport in the eye after topical administration
The anandamide transport inhibitor of choice, N-(4-hydroxyphenyl)arachιdonylamιde, is known per se and can be manufactured by conventional methods, from arachidonic acid chloride and 4-amιnophenol (J Med Chem 39 4515-4519, 1996)
The composition for use in the invention can be any composition which is suitable for administering the anandamide transport inhibitor topically to the eye
A topically administered composition is typically a water based solution, suspension or emulsion containing a pharmaceutically effective amount of the anandamide transport inhibitor together with pharmaceutically acceptable carriers, vehicles or adjuvants The composition can also be a lotion, a cream or ointment Generally, such compositions and their manufacture are well known to the person skilled in the art
The said compositions, especially eye drop solutions, may contain viscosity increasing
agents, for example cellulose derivatives, to increase the viscosity of the composition, and/or agents for the adjustment of the pH, for example acids, bases or buffers, and/or preservatives, for example benzoic acid derivatives, and/or agents for providing a suitable isotonic pressure. According to an advantageous embodiment, the composition is aqueous and preferably can contain one or more agents for increasing the aqueous solubility and stability of the active agent, for example cyclodextrins. A suitable amount of cyclodextrin to be used for forming a water soluble complex of the active agent is 0.5 to 40 % (m/v), advantageously 5 to 25 % (m/v).
The composition can also be in the form of a solid ocular insert containing the active agent in a suitable matrix structure, from where it is released at a suitable rate into the eye.
A suitable quantity of the anandamide transport inhibitor is a quantity that effectively lowers the intraocular pressure in the eye. A typical amount in a topically administered composition, for example an eye drop solution, can vary within wide limits, for example 0.01 to 5, preferably 0.05 to 2 % (m/v) of active agent, for administration for example 1 to 3 or 4 times daily, depending on the patient to be treated and the severity of the condition.
A suitable amount of active agent to be administered to the eye can also vary between wide limits, for example 0.0025 to 1.25 mg/eye/1 to 4 times daily, advantageously 0.0125 to 0.5 mg/eye/1 to 4 times a day.
The following example illustrates the invention without limiting the same in any way.
EXAMPLE
In this example, the effect of topical administration of 0.25% (m/v) N-(4-hydroxyphenyl) arachidonylamide (AM404) (Figure 2) on the intraocular pressure (IOP) of normotensive pigmented rabbit was studied. AM404 is a selective inhibitor of anandamide transport and although similar in structure to the arachidonoylethanolamide, does not activate the cannabinoid receptors (Science 277: 1094-1097, 1997).
More specifically, 12.5 mg of AM404 and 1250 mg of 2-hydroxypropyl-β-cyclodextrin (2-HP-β-CD) were added to distilled water, and the solution was adjusted to pH 1.4 with sodium hydroxide. Then, distilled water was added to adjust the total volume to 5.0 ml. The vehicle control contained 25% (m/v) 2-HP-β-CD.
Subsequently, 25 μl of either the AM404 or the control solution was administered unilaterally to the normotensive rabbits. IOP was measured using a BioRad (Cambridge, MA, USA) Digilab Modular One Pneumatonometer. Before each measurement , one or two drops of 0.06% (w/v) oxybuprocaine were applied to the cornea as an anesthetic before tonometry to eliminate discomfort. For each determination, at least two readings were taken from each eye. The measurements were started two hours before drug or control solution administration, and were continued for five hours after administration.
Figure 2. The chemical structure of AM404.
The results show that unilateral administration of 0.25% (m/v) AM404 significantly decreases IOP in the treated eyes in normotensive pigmented rabbits when compared to the control solution. AM404 showed a maximal IOP reduction of 4.5 mmHg two hours after topical administration. In contrast, unilateral administration of 0.25% (m/v) AM404 does not affect the IOP in the contralateral (untreated) eye when compared to the control solution.
TABLE 1
IOP changes (mmHg) at predetermined times (h) in normotensive pigmented rabbits after unilateral administration of 0 25% AM404 eyedrops (mean ± S E , n = 4)
Solution O h 0 5 h 1 h 2 h 3 h 4 h 5 h
treated eyes 0 25% AM404 0 O±O 0 - 1 4±0 9 - 0 8±1 6 - 4 5±1 3 - 3 O±l 4 - 1 8±1 0 - 3 O±O 7
Control O O±O O 1 8±0 5 2 1±0 9 0 6±0 6 1 3±0 6 2 6±1 6 1 7±0 5
untreated eyes (contralateral)
0 25% AM404 O O±O O 1 3±0 7 1 1±0 5 - 1 4±0 6 - 1 l±O 6 1±0 5 0 0±0 5
Control 0 O±O 0 - 0 7±0 5 - 0 2±0 1 - 0 7±0 4 - 0 8±0 2 0 9±0 6 0 6±0 3
Based on these results, it is apparent that AM404 acts locally within the treated eye Because AM404 does not activate cannabinoid receptors, it is apparent that AM404 inhibits the inactivation (carrier-mediated arachidonylethanolamide transport) of endogenous arachidonyl ethanolamide, which produces the hypotensive effect