The present invention provides the fumarate salt of 4-(diethyl-3-(1-methyloctyl)-7,8,9,10-tetrahydro-6,6,9-trimethyl-6H-dibenz o[b,d]pyran-1-ol, 4-diethyl-amino)butyric acid ester, i.e. the compound having the following structure (I): ##STR1## and methods of treatment, particularly treatment of glaucoma, and pharmaceutical compositions that utilize or comprise the fumarate salt (I).

BACKGROUND OF THE INVENTION 
This invention is directed to the fumarate salt of 
4-(diethyl-3-(1-methyloctyl)-7,8,9,10-tetrahydro-6,6,9-trimethyl-6H-dibenz 
o[b,d]pyran-1-ol, 4-diethyl-amino)butyric acid ester, referred to in this 
disclosure as HGP-2 fumarate. 
The hydrochloride salt of 
4-(diethyl-3-(1-methyloctyl)-7,8,9,10-tetrahydro-6,6,9-trimethyl-6H-dibenz 
o[b,d]pyran-1-ol, 4-diethylamino)butyric acid ester (HGP-2 hydrochloride), 
and its use as a potential antiglaucoma agent, have been reported. See 
naboctate hydrochloride or simply naboctate in Pharmacological Reviews, 
38(2):75-149 (1986). 
However, salts of 4-(diethyl-3-(1 
-methyloctyl)-7,8,9,10-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran-1-o 
l, 4-diethylamino)butyric acid ester have been found to be unstable and 
therefore generally unsuitable for pharmaceutical uses. In particular, 
HGP-2 hydrochloride is a highly hygroscopic amorphous semi-solid, quite 
unstable and requires special treatment such as storage in a desiccator to 
prevent rapid decomposition. Accordingly, the compound is generally 
unsuitable for use as a pharmaceutical agent. 
Glaucoma represents a significant health problem with estimates that 
between 2 to 9 percent of the adult population worldwide suffers from 
increased intraocular pressure. Current therapy include use of so-called 
"beta-blockers". These drugs however are not effective for all patients 
and often result in undesirable side effects that include lowered pulse 
rate, asthma and gastrointestinal problems. 
It thus would be desirable to have a new means for treatment of glaucoma. 
SUMMARY OF THE INVENTION 
The present invention provides the fumarate salt of 
4-(diethyl-3-(1-methyloctyl)-7,8,9,10-tetrahydro-6,6,9-trimethyl-6H-dibenz 
o[b,d]pyran-1-ol, 4-diethyl-amino)butyric acid ester, referred to herein as 
HGP-2 fumarate and having the following structure (I): 
##STR2## 
It has been most surprisingly found that HGP-2 fumarate, unlike HGP-2 
hydrochloride and other salts, is a highly stable, substantially 
non-hygroscopic, crystalline material that shows no significant signs of 
decomposition over prolonged periods of storage at room temperature in 
open air, even periods of from 12 to 36 months or more. It has thus been 
found that HGP-2 fumarate is well suited for use as a pharmaceutical 
agent. 
It also has been found that HGP-2 fumarate is useful for treatment of 
glaucoma. Accordingly, the present invention includes methods for 
treatment and/or prophylaxis of glaucoma. The methods of invention in 
general comprise administration of a therapeutically effective amount of 
HGP-2 fumarate to a mammal, particularly a human. 
The invention also provides pharmaceutical compositions comprising a 
therapeutically effective amount of HGP-2 fumarate and a pharmaceutically 
acceptable carrier. 
The present invention includes both racemic mixtures and optically enriched 
mixtures of HGP-2 fumarate. An optically enriched mixture contains 
substantially more (e.g. about 60%, 70%, 80% or 90% or more) of one 
enantiomer or diastereoisomer than the other stereoisomer(s). 
Other aspects of the invention are disclosed infra.

DETAILED DESCRIPTION OF THE INVENTION 
HGP-2 fumarate may be suitably prepared as described in Example 1 which 
follows and which includes preparation of the HGP-2 free base and then 
formation of a fumarate salt thereof, or by other suitable route. 
Optically enriched mixtures of HGP-2 fumarate can also be prepared by the 
general procedure described in Example 1 below by making appropriate 
modifications to render the synthesis enantioselective. For example, an 
optically active 3-methylcyclohexanone (either the (R) or (S) isomer) can 
be employed (see Step 5 of Example 1 below) to introduce an optical active 
carbon to the ring structure of HGP-2 fumarate, i.e., so that optically 
active HGP-2 fumarate is provided wherein the chiral ring carbon is 
selectively either of the (R) or (S) configuration, and that HGP-2 
fumarate is substantially free of stereoisomer(s) having alternate 
configuration at the chiral ring carbon. Optically active HGP-2 fumarate 
where the chiral 1-octyl carbon atom is selectively either of the (R) or 
(S) configuration (i.e., HGP-2 fumarate substantially free of 
stereoisomer(s) having alternate configuration at the chiral 1-octyl 
carbon atom) can be prepared by e.g. employing an enantioselective 
alkylation of the 3,5-dimethoxyacetophenone or, alternatively, separation 
of the (R) and (S) enantiomers of 2-(3',5'-dimethoxyphenyl)-2-octanol such 
as by column chromatography using an optically active binding material as 
is known in the art. Substantial separation of stereoisomers of HGP-2 
fumarate having either an (R) or (S) ring carbon also could be 
accomplished by use of such a column having optically active binding 
material. Designations herein of stereoisomers having the (R) or (S) 
configuration(s) are in accordance with the Cahn-Ingold-Prelog 
nomenclature system. See Carey, F. A., Advanced Organic Chemistry, Part A, 
p. 65-66 (2d ed., Plenum Press 1984). 
As discussed above, the invention provides methods for treatment and/or 
prophylaxis of glaucoma, including chronic open-angle glaucoma, acute 
angle-closure glaucoma and corticosteroid-induced glaucoma. 
It has been found that HGP-2 fumarate exhibits a significant and prolonged 
reduction of intraocular pressure. For example, administration of a single 
drop of an aqueous treatment solution containing 0.5% w/w HGP-2 fumarate 
as the active agent to the eye of test subjects (rabbits) produced a 
significant and prolonged (up to about 5 hours) lowering of intraocular 
pressure relative to control subjects that did not receive HGP-2 fumarate. 
It also has been found that administration of a single 3.5 mg oral 
(capsule) dose of HGP-2 fumarate to each of twenty human subjects resulted 
in a reduction of intraocular pressure (IOP) of between about 30 to 50 
percent, relative to pre-administration IOP levels, for a minimum of about 
8 to 12 hours for each subject. 
While HGP-2 fumarate may be administered alone to a patient, the compound 
also may be used as part of a pharmaceutical composition. Pharmaceutical 
compositions of the invention in general comprise HGP-2 fumarate together 
with one or more acceptable carriers. The carriers must be "acceptable" in 
the sense of being compatible with other ingredients of the formulation 
and not deleterious to the recipient thereof. 
Compositions of the invention may also include, in addition to HGP-2 
fumarate, one or more other active medicaments. For example, for treatment 
of glaucoma, a composition may be administered to a subject that contains 
HGP-2 fumarate together with other therapeutic agents used for the 
treatment of glaucoma such as timolol, pilocarpine, betaxolol and the 
like. 
Pharmaceutical compositions of the invention include those for oral, 
rectal, nasal, topical (including transdermal, eye drops, buccal and 
sublingual), vaginal, or parenteral (including subcutaneous, 
intramuscular, intravenous and intradermal) administration. Generally 
preferred are oral and topical administration, particularly by eye drop or 
transdermal applications. 
The pharmaceutical compositions of the invention may be presented in unit 
dosage form, e.g., tablets and sustained release capsules, and in 
liposomes and may be prepared by any methods well known in the art of 
pharmacy. Such methods include the step of bringing into association the 
to be administered ingredients with the carrier. In general, the 
compositions are prepared by uniformly and intimately bringing into 
association the active ingredients with liquid carriers, liposomes or 
finely divided solid carriers or both, and then if necessary shaping the 
product. 
Compositions of the invention suitable for oral administration may be 
presented as discrete units such as capsules, cachets or tablets each 
containing a predetermined amount of the active ingredient; as a powder or 
granules; as a solution or a suspension in an aqueous liquid or 
non-aqueous liquid; packed in liposomes, etc. 
A tablet may be made by compression or molding, optionally with one or more 
accessory ingredients. Compressed tablets may be prepared by compressing 
in a suitable machine the active ingredient in a free-flowing form such as 
a powder or granules, optionally mixed with a binder, lubricant, inert 
diluent, preservative, dispersing agent, etc. Molded tablets may be made 
by molding in a suitable machine a mixture of the powdered compound 
moistened with an inert liquid diluent. The tablets may optionally be 
coated or scored any may be formulated so as to provide slow or controlled 
release of the active ingredient therein. 
Capsules can be made by procedures known in the art, e.g., by admixing 
HGP-2 fumarate with one or more suitable carriers that could include e.g. 
a light mineral oil, granulating the mixture and then filling gelatin 
capsules with the granulated product to provide a uniform dose per 
capsule. 
Compositions suitable for topical administration to the skin may be 
presented as ointments, creams, gels and pastes containing HGP-2 fumarate 
and a pharmaceutically acceptable carrier. A preferred composition for 
application directly to an eye of a patient comprises HGP-2 fumarate in an 
amount of from about 0.001 to 2 % w/w in an aqueous or non-aqueous (e.g. 
an oil such as a mineral oil) solution or suspension. Such treatment 
compositions may be suitably administered directly to the eye of the 
patient in form of drops. 
A further preferred topical delivery system is a transdermal patch 
containing the ingredient to be administered. Typically a transdermal 
patch delivery system of the invention will contain between about 5 and 20 
milligrams of HGP-2 fumarate within the patch matrix. Transdermal patch 
systems known in the art will be suitable for purposes of the present 
invention including a multilayer patch that comprises, in successive 
order, a backing layer, a drug reservoir layer that contains the HGP-2 
fumarate prior to use, an optional membrane layer and an adhesive layer 
for affixing the drug delivery device to the skin of a patient. 
Other compositions suitable for topical administration include lozenges, 
typically comprising the ingredients in a flavored basis. 
Compositions suitable for parenteral administration include aqueous and 
non-aqueous sterile injection solutions which may contain anti-oxidants, 
buffers, bacteriostats and solutes which render the formulation isotonic 
with the blood the intended recipient; and aqueous and non-aqueous sterile 
suspensions which may include suspending agents and thickening agents. The 
formulations may be presented in unit-dose or multi-dose containers, for 
example, sealed ampules and vials, and may be stored in a freeze dried 
(lyophilized) condition requiring only the addition of the sterile liquid 
carrier, for example water for injections, immediately prior to use. 
Extemporaneous injection solutions and suspensions may be prepared from 
sterile powders, granules and tablets of the kind previously described. 
It will be appreciated that the actual preferred amount of HGP-2 fumarate 
used in a given therapy will vary according to a number of factors as will 
be recognized by the attendant physician such as the patient's weight, 
general health, sex, etc., the particular indication being treated, the 
particular composition formulated, the mode of application, the selected 
site of administration, etc. Optimal administration rates for a given 
protocol of administration can be readily ascertained by those skilled in 
the art using conventional dosage determination tests conducted with 
regard to the foregoing guidelines. 
In general, a suitable effective dose of HGP-2 fumarate will be in the 
range of from about 0.01 to 50 milligrams per kilogram of recipient per 
day, preferably in the range of from about 0.1 to 20 milligrams per 
kilogram of recipient per day. The desired dose is suitably administered 
once daily, or more preferably several sub-doses, for example, 2 to 4 
sub-doses administered at appropriate intervals through the day, or other 
appropriate schedule. Such sub-doses may be administered as unit dosage 
forms, e.g., containing from 0.05 to 10 milligrams of HGP-2 fumarate per 
unit dosage. For an oral administration such as by capsules or tablets, 
the drug preferably will be administered no more than 1 to 3 times daily 
in an amount of about 0.1 to 5 milligrams of HGP-2 fumarate per unit 
dosage, more preferably about 0.5 to 5 milligrams of HGP-2 fumarate per 
unit dosage. In particular, unit dosages of HGP-2 fumarate of 3.5 mg, 2.5 
mg and 1.5 mg for oral administration should be useful. 
All documents mentioned herein are incorporated herein by reference. 
The present invention is further illustrated by the following examples. 
These examples are provided to aid in the understanding of the invention 
and are not to be construed as limitations thereof. 
EXAMPLE 1 
Synthesis of Fumarate Salt of 
4-(Diethyl-3-(1-methyloctyl)-7,8,9,10-tetrahydro-6,6,9-trimethyl-6H-dibenz 
o[b,d]pyran-1-ol, 4-diethyl-amino)butyric Acid Ester (HGP-2 fumarate) 
HGP-2 fumarate is suitably prepared in accordance with the following Scheme 
I and Steps 1 through 7a. In Steps 1 through 7a, references made to 1, 2, 
3, 4, 5, 6, 7, 8, 9, 10, 11, 13 and 14 refer to the corresponding 
compounds depicted in Scheme I. 
##STR3## 
Step 1. Synthesis of 3,5-Dimethoxyacetophenone (3) 
The lithium salt (2) of 3,5-dimethoxybenzoic acid was prepared from 136.0 g 
(0.747 mol) of the acid (m.p. 178.degree.-183.degree. C.; recrystallized 
from ethyl acetate) and 12.3 g (1.547 moles) of lithium hydride (Ventron) 
by adding the solid acid, in portions through a Gooch tubing, to a slurry 
of the hydride in 1280 ml of dry tetrahydrofuran (THF). A strong 
mechanical stirrer was needed to keep the reaction mixture in motion as it 
thickened. The addition was made at room temperature and the rate of 
addition was determined by the vigor of hydrogen evolution. Thirty minutes 
after the end of the addition, the reaction mixture was heated slowly to 
60.degree.-62.degree. C. and held there until evolution of hydrogen had 
stopped, at least 3 hours. 
The reaction mixture was cooled to 5.degree. C. and 0.786 mol of 
methyllithium (as a solution in diethyl ether; 500 ml of 1.3 molar and 85 
ml of 1.6 molar; Ventron) was added during 0.5 hour. The reaction was 
completed (as indicated by thin layer chromatography (TLC)) by stirring 
for 0.5 hour at 5.degree. C. and then for 1 hour at room temperature. The 
reaction mixture was poured slowly into a mixture of 250 ml of 
concentrated hydrochloric acid in ice, with vigorous stirring to assure 
complete and rapid mixing, and ice was added as needed to keep the 
solution cold. The ketone 3 was recovered from the washed- (water and 
aqueous sodium carbonate) and dried-ether layer after removal of the 
solvent in a rotary evaporator. The yield of 3 was 131.5 g (97%) as a 
light yellow solid showing essentially a single spot on TLC (1:2 ethyl 
acetate/hexane, R.sub.f =0.39). It was used in the next step without 
further purification. It can be recrystallized from petroleum ether as a 
cream-colored solid, m.p. 39.degree.-41.degree. C. This agrees with the 
published m.p. The NMR Spectrum is consistent with the proposed structure. 
Step 2. Synthesis of 2-(3',5'-dimethoxyphenol)-2-octanol (5) 
The ketone 3 was converted to the alcohol 5 in practically quantitative 
yield by reaction with the Grignard reagent 4. Thus, a solution of 131.5 g 
(0.73 mol) of 3,5-dimethoxyacetophenone (3) in 175 ml of anhydrous diethyl 
ether was added during 25 minutes to a solution of the Grignard reagent, 
prepared in the normal way from 198.4 g (1.108 mols) of 1-bromoheptane 
(Aldrich) and 24.5 g (1.008 mols) of magnesium shavings, in 940 ml of 
anhydrous diethyl ether. The initial temperature of the reaction mixture 
was 5.degree. C. and the maximum temperature was 18.degree. C. The cooling 
bath was removed at the end of the addition and the reaction mixture was 
heated at reflux for 30 minutes. By TLC (1:2 ethyl acetate/hexane, R.sub.f 
=0.47) there was no unreacted ketone at this point. 
The cooled reaction mixture was poured in a thin stream into a vigorously 
stirred slurry of ice, solid ammonium chloride and 75 ml of concentrated 
hydrochloric acid. The alcohol 5 was recovered from the washed and dried 
ether layer upon concentration in a rotary evaporator. The dark yellow oil 
(221.5 g) also contained the excess bromoheptane (17.9 g). The product 
showed two spots on TLC, the minor one being slower moving, but it was 
used in the next step without further purification. 
Step 3. Synthesis of 2-(3',5'-dimethoxyphenyl)nonane (6) 
Hydrogenolysis of the alcohol 5 was carried out in a Parr apparatus at room 
temperature and initial pressure of about 50 psig. The crude alcohol 5 
(221.5 g; 0.72 mol) was dissolved in 677 ml of glacial acetic acid, and 5 
ml of concentrated sulfuric acid (yellow solution turned red) and 25 g of 
5% palladium-on-charcoal were added. Reduction was complete in slightly 
more than 2 hours, when TLC showed the alcohol had all reacted. The 
catalyst was removed by suction filtration of the yellow reaction mixture 
through a pad of Celite and the filter cake was washed with glacial acetic 
acid. The brown oily residue remaining from concentration of the filtrate 
in a rotary evaporator was taken up in water and 1:1 diethyl 
ether/petroleum ether. The lower aqueous layer was extracted with diethyl 
ether and discarded. The acid was washed out of the combined organic 
layers with sodium bicarbonate, and the yellow organic phase was washed to 
neutrality, dried, and concentrated in a rotary evaporator. The residue of 
crude ether 6, (195.5 g) was fractionally distilled at reduced pressure, 
to give 149.5 g (77% from the ketone 3) of the nonane 6, as a colorless 
liquid, b.p. 114.degree.-120.degree. C./0.2 mm. TLC showed the forerun 
(22.3 g, b.p. 44.degree.-113.degree. C./0.2 mm) contained additional 
amounts of 6. Three very minor spots were faintly visible by TLC (1:4 
ethyl acetate/hexane) of the main fraction, along with the major one for 6 
at R.sub.f =0.58. Retention time on GLC was 4.69 minutes (2% OV-17, column 
190.degree. C.). NMR Spectra were consistent with the proposed structure. 
Step 4. 5-(1'-Methyloctyl)resorcinol (7) 
The ether 6 was demethylated by heating with strong hydriodic acid in 
acetic acid. To a solution of 149 g (0.564 mol) of 
2-(3',5'-dimethoxy)nonane (6) (b.p. 114.degree.-128.degree. C./0.2 mm) in 
317 ml of hydriodic acid (Fisher, 55% HI, d=1.7), contained in a 3-neck 
round-bottom flask carrying a short Vigreux column and condenser set for 
downward distillation, was carefully added 317 ml of acetic anhydride with 
mechanical stirring. The temperature was allowed to rise in the exothermic 
reaction. Iodomethane was then removed (vapor temperature 55.degree. C.); 
from time to time additional acetic anhydride (total 317 ml) was added 
whenever the rate of distillation slowed. After 2.5 hours the pot 
temperature had risen to 115.degree. C. and distillation had stopped. The 
temperature was raised until the vapor reached 112.degree. C. Dilution of 
the combined distillate with water caused separate of 62 ml of iodomethane 
(88% of theory). 
Acetic acid was stripped from the residue by vacuum distillation (sodium 
sulfite and water were added to assist in removal of color and acid), and 
the remainder was partioned between water and 1:1 diethyl ether/benzene. 
Additional color and acid were removed from the organic phase by washing 
with aqueous solutions of sodium sulfite and sodium bicarbonate. Then a 
wash with dilute hydrochloric acid followed by water to neutrality, and 
drying, left a dark green solution, which was concentrated in a rotary 
evaporator. Vacuum distillation of the dark red residue gave 105.8 g (79%) 
of the resorcinol 7 as a viscous orange oil, b.p. 175.degree.-185.degree. 
C./0.3 mm; showing a major spot on TLC (1:2 ethyl acetate/hexane, R.sub.f 
=0.28), along with a number of minor spots from unidentified impurities. 
The resorcinol showed retention time of 3.41 minutes of GLC (2% OV-17, 
column 220.degree. C.). NMR spectra were in agreement with its proposed 
structure, and with published data. 
Step 5. Synthesis of 
9-methyl-3-(1'-methyloctyl)-6-oxo-7,8,9,10-tetrahydro-6H-dibenzo(b,d)pyran 
-1-ol (9) 
This Pechamann reaction was most successful when the time of reaction was 
not prolonged and the temperature was kept moderate. A solution of 105.8 g 
(0.448 mol) of 5-(1'-methyloctyl) resorcinol (7) and 94.8 g (0.515 mol) of 
ethyl 4-methyl-2-cyclohexanone-1-carboxylate (8; Aldrich) in 525 ml of 
benzene was made in a flask under a nitrogen atmosphere and protected from 
moisture by a drying tube, and 82.3 g (0.537 mol) of phosphorus 
oxychloride (Baker Analyzed Reagent) was added with good magnetic 
stirring. The golden yellow reaction mixture was stirred 50 minutes at 
room temperature, then kept for 30 minutes in an oil bath at 
70.degree.-75.degree. C. After cooling and standing overnight at room 
temperature, the cherry red reaction mixture (TLC showed the absence of 
starting materials) was poured carefully into an ice-water mixture with 
vigorous mechanical stirring. Stirring was continued until the solid 
orange precipitate initially formed had changed to a creamy, tan emulsion 
(2.5 hours). Much of the water was decanted. The thick emulsion was 
collected by suction filtration, washed well with cold hexane and air 
dried. Drying was complete in a vacuum oven, over phosphorus pentoxide and 
potassium hydroxide. 
The yield of dibenzopyranol 9 was 131.3 g (82%) as a tan solid, m.p. 
136.degree.-138.5.degree. C. (capillary). Published data: (138.0.degree. 
C.). It could be recrystallized from benzene (3 ml/g) by the addition of a 
half-volume of hexane, but the m.p. was not improved. TLC showed 
essentially a single spot (1:2 ethyl acetate/hexane, R.sub.f =0.50), and 
on GLC (2% OV-17, column 295.degree. C.) the retention time (silylated 
sample) was 3.40 minutes. 
Step 6. Synthesis of 
3-(1'-Methyloctyl)-7,8,9,10-tetrahydro-6,6,9-trimethyl-6H-dibenzo(b,d)pyra 
n-1-ol (11) 
A solution of 131.3 g (0.368 mol) of 
9-methyl-3-(1'-methyloctyl)-6-oxo-7,8,9,10-tetrahydro-6H-dibenzo(b,d)pyran 
-1-ol (9) in 450 ml of dry, distilled tetrahydrofuran was added dropwise to 
a well-stirred solution of Grignard reagent, made in the usual manner from 
44.7 g (1.84 mols) of magnesium shavings and 287.4 g (2.025 mols) of 
iodomethane (Aldrich), in 127 ml of anhydrous diethyl ether. During the 
addition, more diethyl ether (1050 ml) was added without much effect, in 
an effort to enhance the fluidity of the very thick reaction mixture. The 
reaction was completed by heating at reflux for 1.5 hours and standing at 
room temperature for 72 hours. 
The reaction mixture was poured into a well-stirred mixture of ice, water, 
and hydrochloric acid, and the product was isolated from the washed and 
dried ether solution in the usual manner. Closure of the pyran ring to 
form 11 was completed by adding 2 ml of concentrated hydrochloric acid to 
the dark red concentrate when it had reached a volume of about 500 ml, and 
heating it for 15 minutes in a water bath at about 50.degree. C. Dilution 
with water, followed by washing, drying and concentrating of the organic 
layer left 138.8 g of crude pyran 11 showing a major spot on TLC at 
R.sub.f =0.52 (1:4 ethyl acetate/hexane). 
The pyran was easily purified by column chromatography through silica gel 
60 (70-230 mesh), loading ratio 10:1, eluant 2:98 ethyl acetate/hexane. 
The product consisted of 116.4 g (85%) of 11 as a dark red, very viscous 
oil, showing a single spot on TLC. The retention time of a silylated 
sample on GLC was 4.00 minutes (2% OV-17, column 260.degree. C.). A second 
peak with retention time 4.75 minutes was attributed to the presence of 
the isomeric pyran, in which the double bond of the cyclohexane ring was 
between carbon atoms 10 and 10a; it amounted to 10% of the chromatographed 
product. 
Step 7. Synthesis of 
4-(diethyl-3-(1-methyloctyl)-7,8,9,10-tetrahydro-6,6,9-trimethyl-6H-dibenz 
o[b,d]pyran-1-ol, 4-diethyl-amino)butyric acid ester Fumarate (14) 
To a solution of 20.68 g (0.0558 mol) of 
3-(1'-methyloctyl)-7,8,9,10-tetrahydro-6,6,9-trimethyl-6H-dibenzo(b,d)pyra 
n-1-ol (11) and 11.46 g (0.0586 mol) of 4-(diethylamino)butyric acid 
hydrochloride 13 (prepared as described in Step 7a below, having m.p. 
167.degree.-168.5.degree. C., and dried for 2 hours at 60.degree. C. over 
potassium hydroxide pellets in a vacuum oven prior to use) in 200 ml of 
dry dichloromethane (distilled over P.sub.2 O.sub.5) was added 12.088 g 
(0.0586 mol) of dicyclohexylcarbodiimide (DCC; Aldrich 99%) and washed in 
with 50 ml of additional dry dichloromethane. The solution was stirred 
(magnetic) at 19.degree.-20.degree. C. for 16 hours. TLC in 10% 
MeOH/chloroform showed the disappearance of pyranol; the reaction was now 
complete. The reaction mixture was cooled in an ice bath for 1.5 hours, 
filtered through a Buchner funnel and the precipitated dicyclohexylurea 
was washed with 50 ml of dichloromethane. The combined filtrate was washed 
with saturated sodium bicarbonate solution followed by saturated brine and 
then with 50 ml of water. After drying (magnesium sulfate) 1.0 g of 
decolorizing carbon was added and the mixture was filtered through a 
celite bed. The cake was washed with dichloromethane and the solvent was 
evaporated on a rotary evaporator. After pumping on the residue at 1 mm 
for about 1 hour, 200 ml of distilled hexane was added. The solution was 
left in the freezer (-5.degree. C.) for 48 hours. It was filtered through 
a sintered funnel with a celite bed. After washing the cake with hexane, 
the hexane was removed on a rotary evaporator and the residue was left 
over a high vacuum pump (0.6 mm/Hg) overnight to remove any residual 
volatile material. It gave 28.0 g (98% yield) of a viscous liquid 
(pinkish). It was pure by C,H,N elemental analysis; TLC showed a single 
spot R.sub.f =0.27 (silica, 10:90 ethanol/chloroform). By HPLC analysis 
(92%) isooctane, 8% ethanol containing 1% NH.sub.4 OH) it was &gt;99% pure. 
NMR and IR spectra were also consistent with proposed structure. 
A combined sample of the HGP-2 free base was then converted to the fumarate 
salt (14). Fumaric acid (8.23 g, 0.071 mol) was dissolved in 
tetrahydrofuran (200 ml) and added to a solution of HGP-2 free base (36.3 
g, 0.071 mol) in tetrahydrofuran (100 ml). The flasks were then rinsed 
with additional tetrahydrofuran (50 ml) to ensure complete material 
transfer. 
The reaction mixture was brought to reflux for 15 minutes. Tetrahydrofuran 
was then removed on a rotary evaporator and the residue dried at 1 mm Hg 
for a few minutes. Dry ether (250 ml) was then added and a few seed 
crystals introduced. A precipitate began to appear and the solution was 
stored in a refrigerator overnight. 
The solid was then filtered through a Buchner funnel, washed with ether and 
minimal petroleum ether. The solid was redissolved in dry THF as before 
and the procedure was repeated. The solid was dried at 60.degree. C./1 mm 
Hg for 4 hours to provide 40 g of the title compound, HGP-2 fumarate, as a 
white solid, m.p. 114.degree.-115.degree. C. 
Step 7a: Synthesis of 4-(Diethylamino) butyric acid hydrochloride (13) 
4-diethylamino butyric acid hydrochloride (13) was prepared as generally 
described by Blicke et al., J. Amer. Chem. Soc., 63:2488 (1941). Thus, a 
mixture of 4.8 g (0.035 mol) of methyl .gamma.-chlorobutyrate, 8.0 g 
(0.053 mol) of sodium iodide and 30 ml of acetone was refluxed for 8 
hours. The acetone was removed, the residue extracted with ether and the 
ether extract shaken with sodium thiosulfate solution. The ether layer was 
dried over fused Na.sub.2 SO.sub.4 and the solvent removed to provide 4.6 
g of methyl .gamma.-iodobutyrate, b.p. 80.degree.-83.degree. C. (11 mm). 
To 4.6 g of the thus formed methyl .gamma.-iodobutyrate was added 30 ml of 
benzene and five grams of diethylamine. The mixture was heated in a 
Wheaton bottle for 3 hours at 60.degree. C. The precipitated diethylamino 
hydriodide was filtered off. From the filtrate there was obtained 2.6 g 
(74%) of methyl .gamma.-diethylamino butyrate, b.p. 61.degree.-63.degree. 
C. (3 mm Hg). 
The thus formed methyl .gamma.-diethylamino butyrate was heated for 1 hour 
on a steam bath with three times its volume of 18% HCl and the mixture was 
then concentrated until crystals began to appear. Acetone was added and 
the precipitated .gamma.-diethylaminobutyric acid hydrochloride was 
recrystallized from a mixture of alcohol and ether, m.p. 166.degree. C. 
(calc. yield 2.9 g). 
EXAMPLE 2 
1. Materials 
1.1 HGP-2 fumarate 
The HGP-2 fumarate (14 in Scheme I) was stored at room temperature in a 
desiccator prior to use. 
1.2 Test Solutions 
Solutions were prepared in advance and stored in the refrigerator for the 
duration of each crossover experiment. Administered solutions containing 
HGP-2 fumarate were made by carefully weighing a precise amount of HGP-2 
fumarate into a volumetric flask, adding 100 mg of mannitol and diluting 
to 5 ml with acetate buffer with a pH of 4.0. The solution concentration 
was 0.5% HGP-2 fumarate in an acetate buffer solution. Control solutions 
were prepared by diluting 100 mg of mannitol up to a volume of 5 ml with 
the same acetate buffer, pH 4.0. For both the control and HGP-2 fumarate 
solutions, the acetate buffer solution was prepared using 2.4 ml of 
glacial acetic acid, 6.6 ml 0.94M NaOH, 37.5 ml benzyl alconium chloride 
solution (1:750 dilution) and diluting to 500 ml with distilled water and 
adjusting to pH 4.0 if necessary. 
1.3 Animals 
Eight male New Zealand white rabbits purchased from Millbrook Farms, 
Amherst, Mass. Body weight of each animal ranged from 1.8 to 2.0 1 kg. 
Each animal was identified by tattoo. The animals were individually housed 
in suspended metal cages with grid bottoms. The rabbits were housed in a 
room equipped with air conditioning equipment set to maintain the air 
temperature between 60.degree. and 75.degree. F. and a relative humidity 
between 30% and 70%. Fluorescent lighting was set to an artificial cycle 
of 12 hours light/12 hours dark during the study period including 
acclimation. 
All the rabbits were acclimated to the water-loading and intraocular 
pressure reading procedures. They were acclimated to the procedure by 
running a mock experiment using saline as the test solution. Rabbits were 
restrained in cloth bags, water-loaded with 200 ml of distilled water, and 
IOP measurements were made using an Alcon applanation pneumatonograph. 
Prior to each measurement, the animals' eyes were anesthetized with 0.5% 
tetracaine hydrochloride solution (Alcon), followed by a 0.9% saline 
rinse. Readings were taken on the same time schedule as that which was to 
be used for the study. 
A pelleted high fiber rabbit chow (Purina HF 5326) and a supply of fresh, 
potable water was available ad libitum throughout the study, including the 
acclimation period, with the exception that for approximately 16 hours 
prior to testing and during the actual text, the animals did not have 
access to food or water. 
1.4 Applanation Pneumatonograph 
An Alcon applanation pneumatonograph was used for all intraocular pressure 
(IOP) measurements. 
Each day of the study prior to use, the pneumatonograph was switched on, 
allowed to "warm up", zeroed and checked against a known pressure using an 
Alcon calibration device. 
2. Protocol Design 
The study design is outlined in Table 1 below. 
Animals were allocated to treatment using a random numbers table. Random 
allocation also determined, for unilateral administration, which eye 
received treatment with HGP-2 fumarate. 
The study utilized a double crossover design with intervening baselines 
both with and between crossover. Re-randomization to treatment groups 
occurred prior to the start of the second crossover. 
TABLE 1 
______________________________________ 
TREATMENT PROTOCOL FOR DETERMINING 
CONTRALATERAL EYE EFFECT OF 0.5% 
HGP-2 FUMARATE 
______________________________________ 
STUDY WEEK 1 
GROUP Ia (n = 4) 
GROUP IIa (n = 4) 
______________________________________ 
Day 1 0.05 ml vehicle, 
0.05 ml of 0.5% 
bilateral administra- 
HGP-2 fumarate, 
tion (crossover 
unilateral administra- 
control eyes) tion (treated eye) 
0.05 ml of vehicle; 
unilateral 
administration 
(contralateral eye) 
Day 3 0.05 ml of 0.5% 
0.05 ml vehicle, 
HGP-2 fumarate, 
bilateral administra- 
unilateral admin- 
tion (crossover 
istration (treat- 
control eyes) 
ed eye) 
0.05 ml of vehicle, 
unilateral admin- 
istration (contra- 
lateral eye) 
______________________________________ 
STUDY WEEK 2 
GROUP Ib (n = 4) 
GROUP IIb (n = 4) 
______________________________________ 
Day 8 0.05 ml vehicle, 
0.05 ml of 0.5% 
bilateral administra- 
HGP-2 fumarate, uni- 
tion (crossover 
lateral administration 
control eye) (treated eye) 
0.05 ml of vehicle, 
unilateral admin- 
istration (contra- 
lateral eye) 
Day 10 0.05 ml of 0.5% 
0.05 ml vehicle, 
HGP-2 fumarate, 
bilateral 
unilataral adminis- 
administration (cross- 
tration (treated eye) 
over control eye) 
0.052 ml of vehicle, 
unilateral admin- 
istration (contra- 
lateral eye) 
______________________________________ 
Rabbits randomly assigned to Group Ia or IIa on Day 1: Eye (left or right 
receiving HGP2 fumarate was also randomly assigned. 
Rabbits used during Week 1 were then reassigned at random, to Group Ib or 
IIb; the eye receiving HGP2 fumarate was predetermined based on Week 1 
assignment, i.e., if left eye received HGP2 during Week 1, the right eye 
received HGP2 during Week 2 and vice versa. 
3. Methods 
3.1 Control (Base Line) Measurements 
Rabbits were placed in restraining bags and allowed to settle for at least 
15 minutes prior to taking the day's control intraocular pressure 
measurements. These readings were taken for each animal before 
water-loading and dosing. Two measurements were made 30 minutes apart and 
were averaged to get a base line (pre-treatment) reading for that day. 
3.2 Water-loading and Dosing 
Each animal was water-loaded with 200 ml distilled water by oral gavage 
using a size 8, 16" feeding tube to artificially elevate intraocular 
pressure. Immediately following water-loading, each rabbit was dosed with 
50 .mu.l of dosing solution (vehicle in both eyes or 0.5% HGP-2 fumarate 
in one eye and vehicle in the contralateral eye). 
3.3 Schedule for IOP Measurements 
Intraocular pressure measurements were taken for both eyes of each animal 
at the following post-treatment intervals: 20, 60, 120, 180, 240 and 300 
minutes. At each time interval three readings were taken for each eye. An 
average of these three readings determined the value recorded for that 
time interval. 
3.4 Data Presentation 
The intraocular pressure (IOP) measurements were recorded as absolute 
pressure in mm of Hg. 
Relative intraocular pressure (mm of Hg) was calculated at each 
post-treatment time interval as follows: IOP (pretreatment)+IOP (Time t). 
A drop in pressure was assigned a negative value, a rise in pressure a 
positive value. 
3.5 Statistical Analysis 
Absolute and relative IOP values were analyzed using a pared `t` test for 
statistical significance. Tests were performed between the following 
treatment groups: 
HGP-2 fumarate vs. Crossover Control (vehicle treated) 
HGP-2 fumarate vs. Contralateral (vehicle treated) 
Contralateral (vehicle treated) vs. Crossover Control (vehicle treated) 
The null hypothesis was accepted or rejected on the basis of a two-tail 
test. 
4. Results 
The time course effects of a single unilateral instillation of 0.5% HGP-2 
fumarate in the water-loaded rabbit were as follows: 
1. Contralateral vs Crossover Control IOP Values 
(Individual Values: Tables 1a, 1b, 2a, 2b which follow). 
Group Mean Values indicate that vehicle treated contralateral and crossover 
control eyes showed a similar response to water-loading during the first 
three hours post treatment: there was a sharp rise in IOP followed by a 
gradual decrease back to pretreatment levels. At the next two intervals 
(240 and 300 minutes), however, the mean IOP of the contralateral eyes 
continued to drop while that of the crossover control eyes increased. The 
difference between the relative IOP values of the two groups was 
significant at 240 minutes (.DELTA.=3.5 mm of Hg, 0.01&lt;p&lt;0.025) and 300 
minutes (.DELTA.=3.2 mm of Hg, 0.025&lt;p&lt;0.05) post treatment. 
2. Contralateral vs. HGP-2 fumarate IOP Values 
(Individual Values: Tables 1a, 1b, 2a, 2b which follow; Group Mean Values: 
Table 3 which follows). 
Both the 0.5% HGP-2 fumarate treated eyes and the vehicle treated 
contralateral eyes showed a rise in mean IOP at 20 minutes post 
water-loading. The rise was greater, although not significantly greater 
(p.apprxeq.0.1), in the eyes which received HGP-2 fumarate. During the 
next three hours mean IOP dropped sharply in both groups but the decrease 
in the HGP-2 fumarate group was of significantly greater amplitude as 
recorded at 120 (0.025&lt;p&lt;0.05) and 180 (0.005&lt;p&lt;0.01) minutes. 
The lowest mean relative IOP readings were taken at 180 minutes in the 
HGP-2 fumarate treated group (-2.7.+-.0.8 mm Hg) and at 240 minutes in the 
contralateral eye group (-1.8.+-.1 mm Hg). Group Mean Differences were not 
statistically significant at either 240 or 300 minutes post water-loading. 
3. HGP-2 Fumarate vs Crossover Control IOP Values 
(Individual Values: Tables 1a, 1b, 2a, 2b which follow; Group Mean Values: 
Table 4 which follows). 
Mean IOP increased from 0 to 20 minutes post water-loading in both groups, 
but was significantly higher in the HGP-2 fumarate treated eyes 
(.DELTA.=6%, 0.025&lt;p&lt;0.05). During the next three hours the IOP dropped in 
both groups, with the crossover control mean values returning to baseline 
level at the 180 minute measurement interval. IOP decreased below 
pretreatment levels in the HGP-2 fumarate group, with a maximum drop 
recorded at 180 minutes. Values in the treated group were significantly 
lower (0.025&lt;p&lt;0.05) than control group values at 120 (.DELTA.=3.3 mm Hg) 
and 180 (.DELTA.=2.1 mm Hg), but not at 240 or 300 minutes post treatment. 
TABLE 1a 
__________________________________________________________________________ 
INDIVIDUAL VALUES FOR ABSOLUTE INTRAOCULAR PRESSURE, STUDY WEEK 1 
Effect of Unilateral Administration of HGP-2 Fumarate (0.5%, topical 
instillation of 50 ul drop) 
on Absolute Intraocular Pressure in the Rabbit Using the Water 
Load/Crossover Assay.sup.1 
Absolute Intraocular Pressure (mm Hg) 
Rabbit No. 
C888 C891 C892 C893 C897 C925 C926 C949 
Time.sup.3 
R.sup.2 
L.sup.2 
R L R L R L R L R L R L R L 
__________________________________________________________________________ 
Control.sup.4 
PT.sup.6 
25.1 
24.5 
24.2 
17.8 
20.3 
19.9 
27.9 
20.5 
19.2 
21.7 
20.7 
15.8 
25.0 
19.9 
23.2 
19.5 
+20 
34.5 
35.5 
28.8 
30.0 
34.3 
27.3 
32.7 
33.0 
33.0 
35.0 
35.8 
31.0 
34.3 
25.7 
33.7 
31.3 
+60 
21.0 
27.3 
40.2 
38.7 
31.2 
29.8 
35.0 
25.0 
24.0 
29.8 
23.8 
22.2 
34.0 
28.3 
29.2 
25.0 
+120 
18.5 
26.2 
28.2 
24.8 
21.0 
20.2 
21.2 
15.7 
25.2 
25.2 
22.3 
17.5 
24.5 
20.2 
25.8 
18.5 
+180 
13.3 
17.3 
22.2 
17.7 
20.5 
19.7 
20.3 
21.0 
23.2 
22.7 
22.7 
16.0 
21.2 
15.8 
19.5 
16.7 
+240 
23.0 
17.2 
33.2 
24.5 
21.7 
18.8 
30.3 
24.7 
18.5 
24.2 
24.7 
18.7 
28.7 
11.2 
23.2 
20.8 
+300 
19.5 
18.3 
31.3 
21.8 
22.0 
19.2 
23.2 
23.0 
20.8 
23.0 
24.5 
17.0 
23.8 
11.3 
24.8 
24.8 
Treated.sup.5 
PT.sup.6 
30.8 
24.1 
27.1 
25.0 
19.0 
19.3 
25.2 
17.1 
21.4 
21.8 
18.8 
19.1 
24.9 
22.7 
23.4 
20.6 
+20 
38.7 
34.7 
31.5 
31.3 
30.8 
27.5 
41.0 
28.0 
29.5 
22.5 
38.8 
34.7 
36.8 
35.2 
41.3 
38.3 
+60 
33.3 
24.8 
31.5 
34.2 
22.8 
28.8 
29.7 
23.2 
21.3 
24.0 
25.7 
23.7 
36.0 
24.5 
24.8 
25.2 
+120 
15.7 
16.0 
23.8 
29.3 
13.8 
18.7 
24.7 
20.3 
20.2 
30.0 
27.5 
20.2 
22.5 
18.7 
22.3 
22.3 
+180 
24.7 
20.2 
17.8 
22.3 
19.5 
26.2 
16.5 
18.3 
16.7 
22.7 
20.7 
15.5 
21.3 
18.2 
19.7 
16.7 
+240 
19.5 
17.2 
22.7 
17.0 
19.7 
19.5 
25.2 
15.2 
19.5 
21.2 
21.3 
19.7 
19.3 
13.7 
23.7 
20.8 
+300 
25.2 
24.2 
21.7 
19.0 
20.0 
20.3 
15.3 
15.8 
19.2 
24.2 
26.5 
24.2 
25.3 
22.2 
23.5 
18.2 
__________________________________________________________________________ 
.sup.1 Water Load/Crossover Assay: All rabbits were waterloaded with 200 
ml of water, p.o., just prior to receiving their ophthamlic drops. During 
study week one (Tables 1a and 2a), rabbits were randomly assigned to one 
of two treatment regimens: 4 rabbits (control) received vehicle in both 
eyes; another 4 (treated) received 0.5% HGP2 fumarate in one eye and 
vehicle in the contralateral eye. Two days later crossover occurred: the 
original control rabbits received HGP2 fumarate in one eye and vehicle in 
the other, while the original treated rabbits received vehicle in both 
eyes. On the second week of the study (refer to Tables 1b and 2b), animal 
were reassigned at random to treatment and the entire crossover procedure 
was repeated. This time, however, rabbits which received HGP2 fumarate in 
their left eye during week 1 received HGP2 fumarate in their right eye an 
vice versa. 
.sup.2 R = Right Eye, L = Left Eye. 
.sup.3 Time: Minutes post treatment; waterloading and administration of 
ophthalmic drops occurs at t = 0. 
.sup.4 Control: bilateral administration of vehicle. 
.sup.5 Treated: = unilateral administration of 0.5% HGP2 fumarate. = 
unilateral administration of vehicle. 
.sup.6 PT = Average of 2 pretreatment (baseline) IOP measurements. 
TABLE 1b 
__________________________________________________________________________ 
INDIVIDUAL VALUES FOR ABSOLUTE INTRAOCULAR PRESSURE, STUDY WEEK 2 
Effect of Unilateral Administration of HGP-2 Fumarate (0.5%, topical 
instillation of 50 ul drop) 
on Absolute Intraocular Pressure in the Rabbit Using the Water 
Load/Crossover Assay.sup.1 
Absolute Intraocular Pressure (mm Hg) 
Rabbit No. 
C888 C891 C892 C893 C897 C925 C926 C949 
Time.sup.3 
R.sup.2 
L.sup.2 
R L R L R L R L R L R L R L 
__________________________________________________________________________ 
Control.sup.4 
PT.sup.6 
26.5 
22.5 
22.0 
25.7 
19.8 
21.2 
24.2 
20.5 
19.9 
24.6 
21.8 
16.2 
16.6 
15.6 
23.9 
21.9 
+20 
46.3 
46.3 
38.3 
35.0 
31.5 
26.0 
34.3 
23.3 
26.2 
25.5 
28.3 
25.7 
35.3 
27.3 
36.3 
27.8 
+60 
28.8 
27.2 
37.5 
30.5 
26.7 
23.7 
31.5 
20.7 
25.2 
23.2 
32.3 
22.7 
37.0 
27.7 
25.5 
24.8 
+120 
28.7 
26.0 
35.0 
32.3 
21.0 
20.0 
23.8 
20.5 
22.7 
25.8 
29.3 
15.2 
27.2 
24.3 
23.2 
26.3 
+180 
26.2 
24.3 
25.8 
25.0 
20.8 
21.7 
24.7 
24.3 
20.0 
27.0 
20.8 
13.7 
25.0 
21.8 
22.8 
21.7 
+240 
31.3 
22.3 
29.5 
28.2 
21.5 
21.5 
23.5 
22.5 
22.7 
24.0 
18.0 
16.3 
23.7 
13.0 
19.2 
22.7 
+300 
28.0 
26.7 
19.8 
19.8 
24.0 
18.5 
30.3 
20.8 
29.2 
21.3 
24.3 
19.2 
19.2 
23.0 
27.0 
22.7 
Treated.sup.5 
PT.sup.6 
25.9 
23.7 
25.0 
20.6 
24.9 
20.7 
24.0 
14.6 
17.1 
19.7 
22.3 
17.4 
24.5 
22.0 
27.7 
22.0 
+20 
45.0 
46.3 
27.0 
32.8 
23.5 
25.8 
39.8 
36.8 
31.2 
32.0 
32.3 
25.7 
40.5 
35.3 
43.7 
36.7 
+60 
39.0 
37.8 
37.2 
26.5 
24.8 
21.3 
30.0 
22.2 
25.5 
20.3 
28.5 
21.2 
31.0 
28.5 
33.3 
24.8 
+120 
24.5 
24.5 
31.2 
17.0 
24.3 
22.0 
23.3 
19.5 
21.2 
19.7 
26.3 
27.7 
18.7 
22.0 
25.2 
19.3 
+180 
22.2 
25.3 
30.3 
21.0 
20.3 
18.5 
24.0 
15.0 
19.3 
21.0 
20.8 
19.2 
24.5 
27.7 
27.8 
19.3 
+240 
27.0 
22.7 
23.2 
18.2 
20.8 
36.2 
25.2 
23.7 
22.5 
20.8 
25.8 
16.3 
20.0 
18.7 
21.3 
21.3 
+300 
27.2 
19.0 
20.8 
17.5 
25.5 
19.3 
21.3 
14.8 
15.0 
23.0 
21.8 
17.3 
19.7 
19.3 
22.5 
21.5 
__________________________________________________________________________ 
.sup.1 Water Load/Crossover Assay: All rabbits were water loaded with 200 
ml of water, p.o., just prior to receiving their ophthamlic drops. During 
study week 1 (Tables 1a and 2a), rabbits were randomly assigned to one of 
two treatment regimens: 4 rabbits (control) received vehicle in both eyes 
another 4 (treated) received 0.5% HGP2 fumarate in one eye and vehicle in 
the contralateral eye. Two days later crossover occurred: the original 
control rabbits received HGP2 fumarate in one eye and vehicle in the 
other, while the original treated rabbits received vehicle in both eyes. 
On the second week of the study (Tables 1b and 2b), animals were 
reassigned at random to treatment and the entire crossover procedure was 
repeated. This time, however, rabbits which received HGP2 fumarate in 
their left eye during week 1 received HGP2 fumarate in their right eye, 
and vice versa. 
.sup.2 R = Right Eye; L = Left Eye. 
.sup.3 Time: Minutes post treatment; waterloading and administration of 
ophthalmic drops occurs at t = 0. 
.sup.4 Control: bilateral administration of vehicle. 
.sup.5 Treated: = unilateral administration of 0.5% HGP2 fumarate. = 
unilateral administration of vehicle. 
.sup.6 PT = Average of 2 pretreatment (baseline) IOP measurements. 
3 TABLE 2a 
INDIVIDUAL VALUES FOR RELATIVE INTRAOCULAR PRESSURE, STUDY WEEK 1 
Effect of Unilateral Administration of HGP-2 Fumarate (0.5%, topical 
instillation of 50 ul drop) on Relative Intraocular Pressure in the 
Rabbit Using the Water Load/Crossover Assay.sup.1 Relative Intraocular 
Pressure (mm Hg).sup.2 Rabbit No. C888 C891 C892 C893 C897 C925 C926 
C949 Time.sup.4 R.sup.3 L.sup.3 R L R L R L R L R L R L R L 
Control.sup.5 +20 +9.4 +11.0 +4.6 +12.2 +14.0 +7.4 +4.8 +12.5 +13.8 
+13.3 +15.1 +15.2 +9.5 +5.8 +10.5 +11.8 +60 -4.1 +2.8 +16.0 +20.9 
+10.9 +9.9 +7.1 +4.5 +4.8 +8.1 +3.1 +6.4 +9.0 +8.4 +6.0 +5.5 
+120 -6.6 +1.7 +4.0 +7.0 +0.7 +0.3 -6.7 -4.8 +6.0 +3.5 +1.6 
+1.7 -0.5 +0.3 +2.6 -1.0 +180 -11.8 -7.2 -2.0 -0.1 +0.2 -0.2 
-7.6 +0.5 +4.0 +1.0 +2.0 +0.2 -3.8 -4.1 -3.7 -2.8 +240 -2.1 
-7.3 +9.0 +6.7 +1.4 -1.1 +2.4 +4.2 -0.7 +2.5 +4.0 +2.9 +3.7 
-8.7 0.0 +1.1 +300 -5.6 -6.2 +7.1 +4.0 +1.7 -0.7 -4.7 +2.5 
+1.6 +1.3 + 3.8 +1.2 -1.2 -8.6 +1.6 +5.3 Treated.sup.6 
+20 +7.9 +10.6 +4.4 +6.3 +11.8 +8.2 +15.8 
+10.9 +8.1 +0.7 +20.0 +15.6 +11.9 +12.5 +17.9 +17.7 +60 +2.5 +0.7 
+4.4 +9.2 +3.8 +9.5 +4.5 +6.1 -0.1 +2.2 +6.9 +4.6 +11.1 +1.8 
+1.4 +4.6 +120 -15.1 -8.1 -3.3 +4.3 -5.2 -0.6 -0.5 +3.2 -1.2 
+8.2 +8.7 +1.1 -2.4 - 4.0 -1.1 +1.7 +180 -6.1 -3.9 -9.3 +2.7 
+0.5 +6.9 -8.7 +1.2 -4.7 +0.9 +1.9 -3.6 -3.6 -4.5 -3.7 -3.9 
+240 -11.3 -6.9 -4.4 -8.0 +0.7 +0.4 0.0 -1.9 -1.9 -0.6 +2.5 
+0.6 -5.6 -9.0 +0.3 +0.2 +300 -0.1 +0.1 -5.4 -6.0 +1.0 +1.0 
-9.9 -1.3 -1.3 +2.4 +7.7 +5.1 +0.4 -0.5 +0.1 
.sup.1 Water Load/Crossover Assay: All rabbits were waterloaded with 200 
ml of water, p.o., just prior to receiving their ophthalmic drops. During 
study week one (Tables 1a and 2a), rabbits were randomly assigned to one 
of two treatment regimens: 4 rabbits (control) received vehicle in both 
eyes; another 4 (treated) received 0.5% HGP2 fumarate in one eye and 
vehicle in the contralateral eye. Two days later crossover occurred: the 
original control rabbits received HGP2 fumarate in one eye and vehicle in 
the other, while the original treated rabbits received vehicle in both 
eyes. On the second week of the study (refer to Tables 1b and 2b), animal 
were reassigned at random to treatment and the entire crossover procedure 
was repeated. This time, however, rabbits which received HGP2 fumarate in 
their left eye during week 1 received HGP2 fumarate in their right eye an 
vice versa. 
.sup.2 Relative Intraocular Pressure (mm Hg): IOP (pretreatment) + IOP 
(time t); a post treatment pressure drop is (-), a rise is (+). Refer to 
Table 1a for absolute pressure values. 
.sup.3 R = Right Eye, L = Left Eye. 
.sup.4 Time: Minutes post treatment; waterloading and administration of 
ophthalmic drops occurs at t = 0. 
.sup.5 Control: bilateral administration of vehicle. 
.sup.6 Treated: = unilateral administration of 0.5% HGP2 fumarate. = 
unilateral administration of vehicle. 
3 TABLE 2b 
INDIVIDUAL VALUES FOR RELATIVE INTRAOCULAR PRESSURE, STUDY WEEK 2 
Effect of Unilateral Administration of HGP-2 Fumarate (0.5%, topical 
instillation of 50 ul drop) on Relative Intraocular Pressure in the 
Rabbit Using the Water Load/Crossover Assay.sup.1 Relative Intraocular 
Pressure (mm Hg).sup.2 Rabbit No. C888 C891 C892 C893 C897 C925 C926 
C949 Time.sup.4 R.sup.3 L.sup.3 R L R L R L R L R L R L R L 
Control.sup.5 +20 +19.8 +23.8 +16.3 +9.3 +11.7 +4.8 +10.1 +2.8 
+6.3 +0.9 +6.5 +9.5 +18.7 +11.7 +12.4 +5.9 +60 +2.3 +4.7 +15.5 
+4.8 +6.9 +2.5 +7.3 +0.2 +5.3 -1.4 +10.5 +6.5 +20.4 +12.1 +1.6 
+2.9 +120 +2.2 +3.5 +13.0 +6.6 +1.2 -1.2 -0.4 0.0 +2.8 +1.2 
+7.5 -1.0 +10.6 +8.7 -0.7 +4.4 +180 -0.3 +1.8 +3.8 -0.7 +1.0 
+0.5 +0.5 +3.8 +0.1 +2.4 -1.0 -2.5 +8.4 +6.2 -1.1 -0.2 +240 
+4.8 -0.2 +7.5 +2.5 +1.7 +0.3 -0.7 +2.0 +2.8 -0.6 -3.8 +0.1 
+7.1 -2.6 -4.7 +0.8 +300 +1.5 +4.2 -2.2 -5.9 +4.2 -2.7 +6.1 
+0.3 + 9.3 -3.3 +2.5 +3.0 +2.6 +7.4 +3.1 +0.8 Treated.sup.6 
+20 +19.1 +22.6 +2.0 +12.2 -1.4 +5.1 
+15.8 +22.2 +14.1 +12.3 +10.0 +8.3 +16.0 +13.3 +16.0 +14.7 +60 +13.1 
+14.1 +12.2 +5.9 -0.1 +0.6 +6.0 +7.6 +8.4 +0.6 +6.2 +3.8 +6.5 
+6.5 +5.6 +2.8 +120 -1.4 +0.8 +6.2 -3.6 -0.6 +1.3 +0.7 +4.9 
+4.1 0.0 +4.0 + 10.3 -5.8 0.0 -2.5 -2.7 +180 -3.7 +1.6 +5.3 
+0.4 -4.6 -2.2 0.0 +0.4 +2.2 +1.3 -1.5 +1.8 0.0 +5.7 
+0.1 -2.7 +240 +1.1 -1.0 -1.8 -2.4 -4.1 +15.5 +1.3 +9.1 +5.4 
+1.1 +3.5 -1.1 -4.5 -3.3 -6.4 -0.7 +300 +1.3 -4.7 -4.2 -3.1 
+0.6 -1.4 -2.7 +0.2 -2.1 +3.3 -0.5 -0.1 -4.8 -2.7 -5.2 
.sup.1 Water Load/Crossover Assay: All rabbits were waterloaded with 200 
ml of water, p.o., just prior to receiving their ophthalmic drops. During 
study week one (Tables 1a and 2a), rabbits were randomly assigned to one 
of two treatment regimens: 4 rabbits (control) received vehicle in both 
eyes; another 4 (treated) received 0.5% HGP2 fumarate in one eye and 
vehicle in the contralateral eye. Two days later crossover occurred: the 
original control rabbits received HGP2 fumarate in one eye and vehicle in 
the other, while the original treated rabbits received vehicle in both 
eyes. On the second week of the study (refer to Tables 1b and 2b), animal 
were reassigned at random to treatment and the entire crossover procedure 
was repeated. This time, however, rabbits which received HGP2 fumarate in 
their left eye during week 1 received HGP2 fumarate in their right eye an 
vice versa. 
.sup.2 Relative Intraocular Pressure (mm Hg): IOP (pretreatment) + IOP 
(time t); a post treatment pressure drop is (-), a rise is (+). Refer to 
Table 1a for absolute pressure values. 
.sup.3 R = Right Eye, L = Left Eye. 
.sup.4 Time: Minutes post treatment; waterloading and administration of 
ophthalmic drops occurs at t = 0. 
.sup.5 Control: bilateral administration of vehicle. 
.sup.6 Treated: = unilateral administration of 0.5% HGP2 fumarate. = 
unilateral administration of vehicle. 
TABLE 3 
______________________________________ 
EVALUATION FOR CONTRALATERAL EYE EFFECT 
IN WATER-LOADED RABBITS RECEIVING A SINGLE 
UNILATERAL DOSE OF 0.5% HGP-2 FUMARATE: 
HGP-2 FUMARATE TREATED EYE VS. 
CONTRALATERAL EYE INTRAOCULAR PRESSURE 
(IOP) RESULTS 
MEAN INTRAOCULAR PRESSURE 
(S.E.).sup.1 in mm of Hg 
CONTRALATERAL 
HGP-2 FUMARATE 
EYE.sup.3 
______________________________________ 
ABSOLUTE IOP 
Pretreatment 
22.2 (0.9) 22.3 (0.9) 
+20 Minutes 
35.3 (1.3) 33.2 (1.8) 
+60 26.6 (1.3) 28.7 (1.3) 
+120 20.8 (0.9).sup.a 
23.7 (1.1).sup.a 
+180 19.5 (0.9).sup.b 
22.6 (0.9).sup.b 
+240 22.0 (1.3) 20.5 (0.7) 
+300 20.7 (0.8) 21.2 (0.9) 
RELATIVE IOP 
+20 Minutes 
13.1 (1.2) 10.8 (1.7) 
+60 4.4 (0.8) 6.4 (1.0) 
+120 -1.4 (0.9).sup.a 
1.5 (1.5).sup.a 
+180 -2.7 (0.8).sup.c 
0.6 (1.0).sup.c 
+240 -0.2 (1.5) -1.8 (1.0) 
+300 -1.5 (0.9) -0.8 (0.8) 
______________________________________ 
Notes: 
.sup.1 Mean of 16 values (standard error of the mean), refer to Tables 1a 
1b, 2a and 2b above for individual values. 
.sup.2 HGP2 fumarate treated eye: treated with one 50 ul drop of 0.5% HGP 
fumarate (opposite eye received vehicle only). 
.sup.3 Contralateral Eye: treated with vehicle only (opposite eye receive 
one 50 ul drop of HGP2 fumarate). 
.sup.a Significantly different: 0.025 &lt; p &lt; 0.05 
.sup.b Significantly different: 0.01 &lt; p &lt; 0.025 
.sup.c Significantly different: 0.005 &lt; p &lt; 0.01 
TABLE 4 
______________________________________ 
EVALUATION FOR HYPOTENSIVE ACTIVITY OF 
A SINGLE UNILATERAL DOSE OF 0.5% FUMARATE: 
HGP-2 FUMARATE TREATED EYE VS. CROSSOVER 
CONTROL EYE INTRAOCULAR PRESSURE 
(IOP) RESULTS. 
MEAN INTRAOCULAR PRESSURE 
(S.E.).sup.1 in mm of Hg 
CROSSOVER 
HGP-2 FUMARATE 
CONTROL.sup.3 
______________________________________ 
ABSOLUTE IOP 
Pretreatment 
22.2 (0.9) 22.0 (0.9) 
+20 Minutes 
35.3 (1.3).sup.b 
31.8 (1.4).sup.b 
+60 26.6 (1.3) 28.4 (1.4) 
+120 20.8 (0.9).sup.a 
24.1 (1.1).sup.a 
+180 19.5 (0.9).sup.b 
21.6 (0.9).sup.b 
+240 22.0 (1.3) 22.5 (1.5) 
+300 20.7 (0.8) 21.7 (1.2) 
RELATIVE IOP 
+20 Minutes 
13.1 (1.2).sup.a 
9.8 (0.4).sup.a 
+60 4.4 (0.8) 6.4 (1.4) 
+120 -1.4 (0.9).sup.a 
2.1 (1.0).sup.a 
+180 -2.7 (0.8).sup.a 
-0.5 (1.0).sup.a 
+240 -0.2 (1.5) 0.5 (1.2) 
+300 -1.5 (0.9) -0.3 (1.1) 
______________________________________ 
Notes: 
.sup.1 Mean of 16 values (standard error of the mean), refer to Tables 1a 
1b, 2a and 2b above for individual values. 
.sup.2 HGP2 fumarate treated eye: treated with one 50 ul drop of 0.5% HGP 
fumarate (opposite eye received vehicle only). 
.sup.3 Crossover control eye: treated with vehicle only (opposite eye 
simultaneously received vehicle), crossover eye for treated eye. 
.sup.a Significantly different: 0.025 &lt; p &lt; 0.05 
.sup.b Significantly different: 0.01 &lt; p &lt; 0.025 
The invention has been described in detail with reference to the preferred 
embodiments thereof. However, it will be appreciated that those skilled in 
the art, upon consideration of this disclosure, may make modifications and 
improvements within the spirit and scope of the invention.