Increasing aqueous humor outflow

A method of increasing aqueous humor outflow in the eye of a human patient to treat glaucoma, the method comprising topically administering to the eye an outflow-increasing amount of an analog of ethacrynic acid having a margin of safety of at least 2.0 and having the formula ##STR1## wherein each X.sub.1 and X.sub.2, independently, is a halogen, H, or CH.sub.3, or X.sub.1 and X.sub.2 together form a substituted or unsubstituted aromatic ring; X.sub.3 is an organic group; and X.sub.4 is OH or an organic group; provided that where X.sub.1 and X.sub.2 are Cl and X.sub.4 is OH, X.sub.3 cannot be 2-methylene-1-oxobutyl; or a pharmaceutically acceptable salt thereof.

This invention relates to the treatment of disorders of the human eye, 
particularly glaucoma. 
Glaucoma is characterized by intraocular pressure resulting at least in 
part from a diminished outflow of aqueous humor through the trabecular 
meshwork. 
Epstein et al. (1982) Invest. Ophthalmol. Vis. Sci. 22, 6, 752-756 
describes experiments in which eyes from dead calves, macaques, and 
baboons were fitted with stainless-steel corneal fittings. The eyes were 
perfused, by filling the anterior chambers at 15 mm Hg and 22.degree. C., 
with a solution containing the toxic compound N-ethylmaleimide (NEM), a 
compound reactive with sulfhydryl groups. It was found that a "dosage of 
NEM of 4.7 mM or greater produced a significant increase in the facility 
of outflow in the calf eye." "NEM also caused an increase in outflow in 
the monkey eye." The paper goes on: 
Our results indicate that chemical modification of cellular --SH groups can 
also alter the egress of aqueous humor from the trabecular meshwork. 
Cellular or intercellular permeability to fluid flow in the aqueous 
outflow channels may be influenced by the state of cell membrane protein 
sulfhydryls. Trabecular --SH groups may be intimately involved in the 
normal process of aqueous outflow, especially if located at sites of 
normal resistance in the juxtacanalicular tissue or endothelium of 
Schlemm's canal. Alternatively, --SH groups may exert only a secondary 
influence on outflow through nonspecific structural changes in trabecular 
cell membranes. 
SUMMARY OF THE INVENTION 
In general, the invention features a method of increasing aqueous humor 
outflow in the eye of a human patient to treat glaucoma, which method 
comprises topically administering to the eye an outflow increasing amount 
of analogs of ethacrynic acid and their ester or amide derivatives, and 
pharmaceutically acceptable salts thereof, having a margin of safety of at 
least 2.0 and being of the general formula 
##STR2## 
wherein each X.sub.1 and X.sub.2, independently, is a halogen, H, or 
CH.sub.3, or X.sub.1 and X.sub.2 together form a substituted or 
unsubstituted aromatic ring; X.sub.3 is an organic group, preferably, a 
sulfhydryl reactive organic group; and X.sub.4 is OH or an organic group; 
provided that where X.sub.1 and X.sub.2 are Cl and X.sub.4 is OH, X.sub.3 
cannot be 2-methylene-1-oxobutyl; and where, preferably, each X.sub.1 and 
X.sub.2, independently, is H, Cl, CH.sub.3, or X.sub.1 and X.sub.2 
together form a phenyl ring; and X.sub.3 is one of 
##STR3## 
wherein a is 2-20, and b and c are, independently, 0-20. 
The invention provides effective, non-surgical treatment of glaucoma in a 
manner which increases fluid outflow while causing minimal non-fluid 
related ocular functions. 
Other features and advantages of the invention will be apparent from the 
following description of the preferred embodiments thereof, and from the 
claims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
As mentioned above, the compounds useful in the methods of the invention 
have a number of required properties, now discussed in greater detail. 
Reactivity 
Compounds of the invention are reactive with the trabecular meshwork so as 
to increase aqueous humor outflow; some are reactive with sulfhydryl 
groups of the trabecular meshwork. The reactivity of the compounds must 
not cause an unacceptable amount of swelling of the cells of the 
trabecular meshwork, particularly the inner wall endothelial cells of 
Schlemm's canal, because swelling can decrease outflow. "Unacceptable 
amount of swelling", as used herein, means an amount of swelling which 
completely counteracts the outflow increasing effects of the compounds, 
resulting in no net outflow increase. Whether swelling is caused by a 
particular compound can be determined by testing the compound in the 
system described in Epstein et al., id, and examining the trabecular 
meshwork cells morphologically. 
Sulfhydryl Reactivity 
The compounds may contain chemical groups which are capable of reacting 
with the sulfhydryl groups of the trabecular meshwork to increase aqueous 
humor outflow. Compounds which contain chemical groups capable of reacting 
with sulfhydryl groups must react with the sulfhydryl groups in a manner 
which does not cause an unacceptable amount of swelling of cells of the 
trabecular meshwork, as described above. 
Suitable sulfhydryl reactive groups include C.dbd.C, C.dbd.O, sulfhydryl, 
alkyl (e.g., methyl or ethyl) and aryl (e.g., phenyl) substituted with a 
good leaving group, e.g., halogen, tosyl, or mesyl. Preferably, in the 
case of substituted alkyl groups, substitution is primary, rather than 
secondary or tertiary, for greater reactivity. 
Toxicity and Margin of Safety 
As used herein, "margin of safety" refers to the ratio of the dosage of the 
outflow increasing compounds which causes medically unacceptable toxic 
side effects, and the dosage which causes substantial (i.e., medically 
useful) increase in aqueous humor outflow in a typical human patient with 
advanced open angle glaucoma. The margin of safety of the compounds must 
be at least 2.0, and more preferably at least 4.0. 
It is also important that the compounds not produce, at effective dosages, 
long-term deleterious changes in the eye. 
Lipophilicity 
Compounds to be administered to the eye topically must be sufficiently 
lipophilic to penetrate the corneal membrane. Sufficient lipophilicity can 
be provided by a non-polar structure, the presence of at least one aryl 
group (e.g., a substituted or unsubstituted phenyl ring), at least one 
halogen atom, and/or hydrophobic alkyl groups. For lipophilicity, it is 
also desirable that the compound not carry excessive charge; i.e., of 
absolute value greater than 2, at physiological pH. 
Lipophilicity is expressed in terms of octanol: water coefficient, 
determined by the standard technique of radiolabelling the compound and 
introducing a small amount into equal volumes of octanol and Tris buffer 
(50 mM, pH 7.4). The coefficient of the compounds is preferably at least 
0.005, and more preferably at least 0.01. 
Administration 
The outflow-increasing compounds can be administered either topically or by 
microinjection into the anterior chamber or trabecular meshwork. For 
topical administration, the compound is dissolved in a pharmaceutically 
acceptable carrier substance, e.g., physiological saline. For compounds 
having limited water solubility (e.g., the sodium salt of ethacrynic acid, 
soluble only to about 0.04M in water) the liquid carrier medium can 
contain an organic solvent, e.g., 3% methyl cellulose, in which solubility 
is greater. Methyl cellulose also provides, by its high viscosity, 
increased contact time between the compound and the eye surface, and 
therefore increased corneal penetration. Corneal penetration can also be 
increased by administering the compound mixed with an agent which slightly 
disrupts the corneal membrane, e.g., 0.001% benzalkonium chloride. 
Administration is by periodically (e.g., one time per week to ten times 
per day) applying drops of the compound in solution using an eye dropper, 
such that an effective amount of the compound is delivered through the 
cornea to the trabecular meshwork. The amount of the compound to be 
delivered in one administration will depend on individual patient 
characteristics, e.g., severity of disease, as well as characteristics of 
the compound, e.g., the specific affinity for trabecular meshwork 
sulfhydryl groups, and the magnitude of the margin of safety. Typically, 
each drop contains 50-100 microliters of a 5-10 mM solution of the 
compound, so that 0.025 to 0.10 moles of the compound are delivered to 
each eye per day. 
Direct microinjection of the solubilized compound into the anterior chamber 
or trabecular meshwork offers the advantage of concentrating the compound 
in the location where it is needed, while avoiding the possibility of side 
effects resulting from generalized exposure of the eye to the compound. 
Microinjection also provides the advantage of permitting infrequent 
periodic administration, e.g., every few weeks, months, or even years, in 
contrast to the more frequent administrations required in the case of 
topical administration. Also, direct microinjection may promote the 
washing out of the trabecular meshwork of extracellular material 
interferring with fluid outflow. Dosage for microinjection, like that for 
topical administration, varies with the above-mentioned parameters. 
Typically, microinjection dosage is such that a final concentration of the 
compound within the anterior chamber or trabecular meshwork of 0.01 to 1.0 
mM is reached. 
In Vivo Use of Ethacrynic Acid 
Ethacrynic acid (sodium salt) was used to increase aqueous humor outflow in 
cynomologous monkeys, as described below. Ethacrynic acid can be purchased 
from Merck, Sharp, and Dome, and is described in U.S. Pat. No. 3,255,241, 
hereby incorporated by reference. Ethacrynic acid has the chemical formula 
2,3-dichloro-4-(2-methylene-1-oxobutyl) phenoxy! acetic acid. Any 
suitable analog described in U.S. Pat. No. 3,255,241 or its ester or amide 
derivative can also be used as described herein; for example, the 
following compounds may be used and are available from Allergan, Inc. 
(Irvine, Calif.), as indicated by the code number below the structure. 
##STR4## 
Each animal was randomly assigned one eye for the experimental and the 
other for its control perfusion. The animals were fasted the night before 
the experiment. They were anesthetized intramuscularly with Methohexital 
Sodium 15 mg/kg and Pentobarbital Sodium 35 mg/kg. Supplemental anesthesia 
as required was carried out with Pentobarbital 10 mg/kg/hour. Needles were 
placed through the cornea into the anterior chamber and a two-step 
constant pressure perfusion method was performed in order to determine 
aqueous humor outflow facility. The basic medium for perfusion was 
Dulbecco's phosphate buffered saline with added 5.5 mM glucose. A 10 
microliter bolus of the experimental or control solution (that would 
produce the desired final concentration in the anterior chamber) was 
injected through a T shaped connector piece in the infusion line. 
Each vial of ethacrynic acid contained ethacrynate sodium powder equivalent 
to 50 mg of ethacrynic acid. The inactive ingredients were 62.5 mg 
mannitol and 0.1 milliliters thimerosol (as preservative). The powder was 
diluted with the above basic medium (Dulbecco's with added glucose) to 
yield the desired concentration. The solution was mixed at room 
temperature until dissolved, and the pH was determined (always 7.2) before 
use; the solution was filtered with a 0.2 micron filter (Nuclepore); this 
produced a solution which was stable for 24 hours. 
The control solution was composed of 9.5 mg sodium chloride (to osmotically 
balance the experimental solution), 62.5 mg mannitol and 0.1 milliliter 
thimerosol dissolved in Dulbecco's phosphate buffered saline with 5.5 mM 
added glucose to yield the desired concentration. 
During perfusion experiments, a 10 microliter bolus injection was made 
using a Hamilton syringe. Since the monkey anterior chamber is 
approximately 200 microliters, 10 microliters of 10 mM ethacrynic acid was 
infused to achieve a final concentration of 0.5 mM ethacrynic acid. 
Experiments were carried out using final ethacrynic acid concentrations in 
the aqueous humor of 0.1 mM to 0.5 mM. There were at least three animals 
and separate experiments carried out for each of the concentrations 0.1 
MM, 0.25 mM, and 0.5 mM. 
At 0.5 mM, a mean increase in fluid outflow facility of 140% due to 
ethacrynic acid was determined, compared to no change in the control 
perfused eye. At 0.25 mM, approximately half the animals perfused 
responded with a substantial increase in outflow facility due to 
ethacrynic acid and the other half did not. At lower dosages there was no 
effect. One animal was perfused at 1.0 mM and demonstrated a 355% increase 
in the experimental eye compared to an 18% increase in the control eye. 
There were no apparent corneal or crystalline lens changes. Specifically, 
there was no chronic corneal edema or opacities or cataract formation. At 
dosages above 0.25 mM some of the animals developed a dilated pupil in the 
ethacrynic treated eye. A small number of animals in both the experimental 
and control eyes developed adhesions of the iris to the peripheral cornea 
which was believed to result from the perfusion technique itself rather 
than the drug administration. 
Intraocular pressure could not be reliably taken until a few days after the 
perfusion experiments (due to the possibility of leaks in the cornea 
through the needle placements), and at that time intraocular pressure was 
symmetrical and normal in both eyes. 
For rabbit experiments Dutch-belted rabbits of either sex weighing 1.5 to 2 
kg were used for topical studies. Each animal was randomly assigned one 
eye for the experimental and the other for its control solution. 
Intraocular pressure was measured using a Digilab Pneumotonometer. Any 
animals showing asymmetry of intraocular pressure greater than 2 mm were 
excluded form the study. 
The protocol was as follows. Baseline intraocular pressure was taken in 
each eye using 0.5% proparacaine hydrochloride for topical anesthesia. 
Then a 100 microliter drop of either control solution or ethacrynic acid 
dissolved in 3% methylcellulose (Dow Corporation, lot number 14728) was 
instilled into one of the two eyes. In a half hour this was repeated. Two 
hours later intraocular pressure was measured in each eye. In some animals 
intraocular pressure was also measured five hours later and all animals 
had measurement of intraocular pressure the following day. 
Ethacrynic acid powder was dissolved in 3% methylcellulose to yield the 
desired concentration. The solution was mixed at room temperature for one 
hour and was stable for 24 hours. A similar osmotically balanced control 
solution was prepared from methyl cellulose powder dissolved in distilled 
water using low heat for several hours. The solution was refrigerated over 
night to yield a transparent, viscous fluid. The pH of the solution was 
determined by mixing one part of the control or experimental solution with 
five parts of distilled water. The pH ranged between 6.2 and 6.5 for both 
the control and experimental solutions. The 3% methylcellulose solution 
was refrigerated when not in use. 
The pressure data was as follows: for 5 mM ethacrynic acid in 3% 
methylcellulose in eight animals, two hours following instillation 
intraocular pressure in the ethacrynic treated eye had decreased from 22.4 
to 19.6 mm Hg (p less than 0.01) whereas the control eye had shown a 
slight increase from 21.5 to 23.1 mm Hg. The next day intraocular pressure 
was equal in the two eyes being 22.4 mm in the ethacrynic treated eye and 
22.7 mm in the control eye. 
In fourteen rabbits treated with 10 mM ethacrynic acid and 3% 
methylcellulose, 24 hours after instillation intraocular pressure in the 
ethacrynic eye had changed from 23.0 to 20.0 mm Hg whereas in the control 
eye it had changed from 22.9 to 24.2 mm Hg. p was less than 0.001. 
For studies at 5 mM concentration, there was slight conjunctival infection 
following administration. There were no other side effects noted. 
Following administration of 10 mM ethacrynic acid moderate conjunctival 
infection and signs of irritation were apparent. 
At higher concentrations signs of corneal toxicity (corneal edema) and 
anterior chamber inflammation were apparent for several days. However, 
these resolved without apparent sequelae. 
In vitro experiments with excised mammalian eyes indicated that the 
following ethacrynic acid analogs (available from Allergan, Inc.) 
increased aqueous humor outflow in the eyes: 
##STR5## 
Experiments similar to those described above have also been performed using 
two esters of ethacrynic acid, RCOOCH.sub.2 CH.sub.2 N(CH.sub.2 
CH.sub.3).sub.2 (hereinafter "ester A") and RCOOCH.sub.2 CH.sub.2 CH.sub.2 
N(CH.sub.2 CH.sub.3).sub.2 (hereinafter "ester B"), where R is 
2,3-dichloro-4-(2-methylene-1-oxobutyl)phenoxy!acetate. 
After topical application of 10 mM ester A, a reduction in intraocular 
pressure in the rabbit was observed. This was most apparent 24 hours after 
instillation (control eye intraocular pressure changing from 22.+-.3 to 
15.+-.2 mm Hg; n=8). In all these topical experiments 2 drops of the agent 
were given 5 minutes apart and control eyes were fully sham manipulated. 
Most of the animals demonstrated some irritation of the eyelid and a few 
to the conjunctiva and nictitating membrane, but no corneal changes were 
observed. However, not all the animals were slit lamped. No anterior 
chamber inflammation was observed that might explain the pressure 
decrease, and the external irritation of the lid seemed to clear after 24 
hours. In the two rabbits studied at 5 mM ester A, minimal if any 
irritation was observed. 
For 10 mM ester B in the rabbit, a pressure reduction only at 24 hours was 
apparent with control eyes changing from 24.+-.2 to 24.+-.2 mm Hg and 
experimental eyes from 23.+-.3 to 15.+-.3 mm Hg (n=5). Similar signs of 
external irritation to the lid and conjunctiva were apparent with ester B, 
but probably less than with ester A. At 24 hours there were clearly only 
minimal if any signs of irritation and there were no slit lamp 
observations of ocular inflammation that might explain the significant 
pressure reduction observed at 24 hours. 
In the monkey, a significant pressure reduction 24 hours after application 
of 10 mM ester A was observed (control eyes changing from 17.+-.3 to 
21.+-.2 mm Hg versus experimental eyes changing from 19.+-.1 to 5.+-.3 mm 
Hg; n=6). However, significant corneal edema was also observed in over 
half of the monkeys. This was detected by flashlight examination and 
confirmed by slit lamp examination. The corneal edema ultimately resolved 
in all animals. At 5 mM topical ester A in the monkey, corneal edema was 
likewise apparent and no pressure reduction was documented. 
Other embodiments are within the following claims.